Coacervate-forming reagents and methods

Abstract

The invention relates to coacervate-forming polymers having defined structural formulae. The invention also relates solutions comprising the coacervate-forming polymers and methods of making solutions comprising the coacervate-forming polymers. The invention also relates to polymer coacervates, comprising the coacervate-forming polymers within the coacervates, including polymer coacervates comprising cargo compounds, and methods for making such polymer coacervates. The coacervates of the invention are capable of penetrating into cells and thereby delivering cargo compounds into cells. The invention further relates to compositions, including pharmaceutical compositions, comprising the polymer coacervates. The invention also relates to uses of the coacervate-forming polymers, including medical uses.

Description

[0001] COACERVATE-FORMING REAGENTS AND METHODS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to coacervate-forming polymers having defined structural formulae. The invention also relates solutions comprising the coacervate-forming polymers and methods of making solutions comprising the coacervate-forming polymers. The invention also relates to polymer coacervates, comprising the coacervate-forming polymers within the coacervates, including polymer coacervates comprising cargo compounds, and methods for making such polymer coacervates. The coacervates of the invention are capable of penetrating into cells and thereby delivering cargo compounds into cells. The invention further relates to compositions, including pharmaceutical compositions, comprising the polymer coacervates. The invention also relates to uses of the coacervate-forming polymers, including medical uses.

[0004] BACKGROUND TO THE INVENTION

[0005] Over the last several decades biomacromolecules have emerged as one of the two main classes of therapeutic agents, alongside small molecule drugs. Biomacromolecular drug modalities, sometimes also referred to as “biologies”, include proteins (Porello I, Cellesi F. Intracellular delivery of therapeutic proteins. New advancements and future directions. Front Bioeng Biotechnol. 2023; 11: 1211798), peptides (Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. Nat Rev Drug Discov. 2021 Apr;20(4):309-325), and nucleic acids (Kulkami JA, Witzigmann D, Thomson SB, Chen S, Leavitt BR, Cullis PR, van der Meel R. The current landscape of nucleic acid therapeutics. Nat Nanotechnol. 2021 Jun;16(6):630- 643). Monoclonal antibodies have proven to be a particularly successful class of biologic drugs (Sharma P, Joshi RV, Pritchard R, Xu K, Eicher MA. Therapeutic Antibodies in Medicine. Molecules. 2023 Sep 5;28(18):6438; Wang Z, Wang G, Lu H, Li H, Tang M, Tong A. Development of therapeutic antibodies for the treatment of diseases. Mol Biomed. 2022 Nov 22;3(1):35), but therapeutic oligonucleotides (Mangla P, Vicentini Q, Biscans A. Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking. Cells. 2023 Sep 11 ; 12(18):2253) and RNA (Damase TR, Sukhovershin R, Boada C, Taraballi F, Pettigrew RI, Cooke JP. The Limitless Future of RNA Therapeutics. Front Bioeng Biotechnol. 2021 Mar 18;9:628137) are also gaining in importance.

[0006] Many biomacromolecules are not intrinsically cell-penetrating due to their large size and, for some, surface charge. The penetration of the cell membrane is therefore a common requirement for making therapeutic use of such agents, at least when targeting intracellular processes. Moreover, for cell penetration to be effective, the entry mechanism needs to evade endosomal entrapment (Tian Y, Tirrell MV, LaBelle JL. Harnessing the Therapeutic Potential of Biomacromolecules through Intracellular Delivery of Nucleic Acids, Peptides, and Proteins. Adv Healthc Mater. 2022 Jun;l l(12):e2102600). Indeed, although many small molecule drugs can enter cells to some degree directly due to their small size, their bioavailability can typically be improved by active cell delivery approaches (Vargason AM, Anselmo AC, Mitragotri S. The evolution of commercial drug delivery technologies. Nat Biomed Eng. 2021 Sep;5(9):951-967). Importantly, the constraint of poor cell membrane permeability has limited the use of monoclonal antibodies largely to extracellular targets (Slastnikova TA, Ulasov AV, Rosenkranz AA, Sobolev AS. Targeted Intracellular Delivery of Antibodies: The State of the Art. Front Pharmacol. 2018 Oct 24;9: 1208).

[0007] A currently emerging concept for therapeutic intervention is the use of gene editing tools (such as CRISPR) in support of in vivo or ex vivo gene therapy approaches. These scenarios, too, are faced with the challenge of conveying biomacromolecules - in this case typically protein-RNA complexes or DNA - across the cell membrane (Lotfi M, Morshedi Rad D, Mashhadi SS, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Farrokhi S, Hashemi M, Lotfi M, Ebrahimi Warkiani M, Abbaszadegan MR. Recent Advances in CRISPR / Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells. Stem Cell Rev Rep. 2023 Nov;19(8):2576-2596).

[0008] Possible solutions to the challenges of efficient cell membrane penetration and endosomal / lysosomal evasion of macromolecular cargoes have been sought for several decades. Physical methods, such as electroporation and injection, involve the temporary breaching of the cell membrane and are typically not amenable to clinical translation. The delivery of genetic material, in particular, is open to biological delivery approaches, for example viral transduction (Naso MF, Tomkowicz B, Perry WL 3rd, Strohl WR. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017 Aug;31(4):317-334). However, chemical nanocarriers are more generally applicable to different types of macromolecular cargoes, including proteins, peptides, and nucleic acids. These cytosolic delivery methods include, amongst others, cell-penetrating- peptides (Shoari A, Tooyserkani R, Tahmasebi M, Lbwik DWPM. Delivery of Various Cargos into Cancer Cells and Tissues via Cell-Penetrating Peptides: A Review of the Last Decade. Pharmaceutics. 2021 Sep 2; 13(9): 1391), inorganic, polymeric and lipid- based nanoparticles (Scaletti F , Hardie J , Lee YW , Luther DC , Ray M , Rotello VM . Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies. Chem Soc Rev. 2018 May 21;47(10):3421-3432), liposomes, and exosomes (He X, Xiong S, Sun Y, Zhong M, Xiao N, Zhou Z, Wang T, Tang Y, Xie J. Recent Progress of Rational Modified Nanocarriers for Cytosolic Protein Delivery. Pharmaceutics. 2023 May 29; 15(6): 1610).

[0009] Whilst some of these widely different types of nanocarriers show promise for future translation into clinical practice, especially for specialised applications, they share one or more of several common problems. Prominent amongst these is the problem that the cargo gets entrapped in the endosomal / lysosomal pathway following cell entry (Du S, Liew SS, Li L, Yao SQ. Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins. J Am Chem Soc. 2018 Nov 28;140(47):15986-15996). Immunogenicity and toxicity are other areas of concern from some of the proposed approaches (Brock DJ, Kondow-McConaghy HM, Hager EC, Pellois JP. Endosomal Escape and Cytosolic Penetration of Macromolecules Mediated by Synthetic Delivery Agents. Bioconjug Chem. 2019 Feb 20;30(2):293-304). Moreover, harsh manufacturing processes or the need for covalent conjugation for some nanocarriers can negatively impact on the bioavailability or activity of biomacromolecular cargoes (Lee YW, Luther DC, Kretzmann JA, Burden A, Jeon T, Zhai S, Rotello VM. Protein Delivery into the Cell Cytosol using Non-Viral Nanocarriers. Theranostics. 2019 May 18;9(11):3280- 3292). These drawbacks of the currently available cargo delivery approaches highlight the need for novel solutions that combine different characteristics, including effective delivery of multiple different cargo types, improved biosafety, evasion of endosomal entrapment and preservation of bioactivity of the therapeutic payload.

[0010] Liquid-liquid phase separation (LLPS) is the physicochemical mechanism by which homogeneous solutions of macromolecules (typically polymers) can de-mix into two distinct phases - a concentrated macromolecule-rich phase (also referred to as condensates, biomolecular condensates (BMCs) or coacervates), and a macromoleculepoor diluted phase (Flory PJ. 1941. Thermodynamics of high polymer solutions. J Chem Phys 9: 660). The phenomenon of LLPS has relatively recently been proposed to be implicated in a variety of biological processes, often through the formation of nonstoichiometric assemblies of proteins and nucleic acids called biomolecular condensates (Ditlev JA, Case LB, Rosen MK. Who's In and Who's Out-Compositional Control of Biomolecular Condensates. J Mol Biol. 2018 Nov 2;430(23):4666-4684). When found inside cells and of a sufficiently large size (on the order of micrometers), these structures are also classed as membraneless organelles (Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. 2017 May;18(5):285-298) and are implicated in a variety of physiological processes, including cellular stress response, compartmentalization of biochemical reactions, mechanical regulation and cell signalling (Alberti S, Gladfelter A, Mittag T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell. 2019 Jan 24; 176(3):419-434). Importantly, the dynamic interactions required to control cellular processes in these contexts is afforded by the selective enrichment or exclusion of biomolecules from the BMCs (Mitrea DM, Mittasch M, Gomes BF, Klein IA, Murcko MA. Modulating biomolecular condensates: a novel approach to drug discovery. Nat Rev Drug Discov. 2022 Nov;21(l l):841-862).

[0011] The principal driving forces for the formation of biomolecular condensates are provided by comparatively weak but multivalent interactions between particular proteins, often in combination with RNA molecules (Boeynaems S, Holehouse AS, Weinhardt V, Kovacs D, Van Lindt J, Larabell C, Van Den Bosch L, Das R, Tompa PS, Pappu RV, Gitler AD. Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties. Proc Natl Acad Sci U S A.

[0012] 2019 Apr 16;116(16):7889-7898). The interactions driving LLPS in this biological context include charge-charge interactions, pi-pi and pi-cation interactions, hydrophobic contacts and hydrogen bonding (Dignon GL, Best RB, Mittal J. Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties. Annu Rev Phys Chem. 2020 Apr 20;71:53-75). Many of the proteins involved in BMC-formation encompass intrinsically disordered regions (IDRs) (Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, Tompa P, Fuxreiter M. Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol. 2018 Jun;28(6):420-435), which are characterised by a low sequence complexity and a lack of the well-defined secondary and tertiary structure found, for example, in globular proteins. A “stickers-and-spacers” model has been put forward to rationalise the patterning of adhesive and flexible non-adhesive resides / domains within IDRs (Choi JM, Holehouse AS, Pappu RV. Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions. Annu Rev Biophys.

[0013] 2020 May 6;49: 107-133; Wang J, Choi JM, Holehouse AS, Lee HO, Zhang X, Jahnel M, Maharana S, Lemaitre R, Pozniakovsky A, Drechsel D, Poser I, Pappu RV, Alberti S, Hyman AA. A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell. 2018 Jul 26;174(3):688-699.el6).

[0014] The study of BMCs in the cellular context of membraneless organelles is primarily concerned with highly complex macromolecular assemblies, typically involving many dozens of different protein and RNA species. However, the underlying LLPS process has also been invoked in the formation of comparatively simple assemblies of small synthetic polymers, such as peptides. In the scientific literature these are variously, and often without sharp distinction, referred to as condensates or coacervates (the latter term implying charge-charge interactions as the predominant adhesive force). Throughout the present disclosure, the terms condensates, biomolecular condensates (BMCs), membraneless organelles (MLOs) and coacervates are used interchangeably. Peptides of many different sequences and sizes have been shown to form coacervates under appropriate conditions (including their concentration and the presence of the other polymers such as RNA, as well as the pH, ionic strength, and temperature of the solution). Some of these coacervating peptides are informed by the amino acid sequence of naturally occurring IDRs, presumably recapitulating the adhesive forces that govern the LLPS events that lead to the formation of the corresponding MLOs in vivo, thus serving as reductionist models for the latter (Liu J, Zhorabek F, Dai X, Huang J, Chau Y. Minimalist Design of an Intrinsically Disordered Protein-Mimicking Scaffold for an Artificial Membraneless Organelle. ACS Cent Sci. 2022 Apr 27;8(4):493-500; Alshareedah I, Thurston GM, Banerjee PR. Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates. Biophys J. 2021 Apr 6; 120(7): 1161-1169; Alshareedah I, Moosa MM, Pham M, Potoyan DA, Banerjee PR. Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides. Nat Commun. 2021 Nov 16; 12(l):6620). Moreover, certain peptide coacervates have been shown to dynamically recruit and release other molecules (including pharmaceutically active agents) as “cargoes”. This ability has led to their application in various therapeutic applications (Ma L, Fang X, Wang C. Peptide-based coacervates in therapeutic applications. Front Bioeng Biotechnol. 2023 Jan 4; 10: 1100365; Lim ZW, Ping Y, Miserez A. Glucose-Responsive Peptide Coacervates with High Encapsulation Efficiency for Controlled Release of Insulin. Bioconjug Chem. 2018 Jul 18;29(7):2176-2180). In addition, other studies have shown that specific cargo-loaded peptide coacervates can penetrate cell membranes and deliver their payload to the cytosol of their target cells (Sun Y, Lau SY, Lim ZW, Chang SC, Ghadessy F, Partridge A, Miserez A. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat Chem. 2022 Mar;14(3):274-283).

[0015] Notwithstanding the present state of the art, additional reagents and methods are required to promote coacervate formation, and to facilitate coacervate-mediated cell penetration and cargo delivery in a manner that avoids endosomal / lysosomal depletion of coacervates and their cargoes. SUMMARY OF THE INVENTION

[0016] The invention provides, inter alia, a coacervate-forming polymer comprising or consisting of a structure according to formula (I):

[0017] Rl-N[Pl-P2-P3-P4]nC (I) wherein:

[0018] A. Ri is occupied by one or more aromatic molecules;

[0019] B. N is the N-terminal end of the polymer;

[0020] C. [P1-P2-P3-P4] is a structural unit of the polymer;

[0021] D. n is the number of structural units of the polymer, wherein n is an integer of 2 or more;

[0022] E. C is the C-terminal end of the polymer;

[0023] F. P is an amino acid residue position or a spacer position in the polymer; and

[0024] G. each one of Pi, P2, P3 and P4consists of one or more amino acid residue positions and / or one or more spacer positions, wherein: i. one or more of said positions is occupied by a molecule selected from the group Al consisting of arginine (R), histidine (H) and lysine (K); and ii. one or more additional position(s) is occupied by a molecule selected from the group A2 consisting of tyrosine (Y), phenylalanine (F), and tryptophan (W); and iii. one or more further additional positions are occupied by a molecule selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G-G-G), glycine- glycine-glycine-glycine (G-G-G-G), or any spacer.

[0025] A complete summary of all aspects of the invention is provided in the section below: Aspects of the Invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1

[0027] FAM-[RGYGG]5C forms coacervates when added to tissue culture media regardless of the concentration at which the liquid-liquid phase separation (LLPS) occurs, or whether the dilution curve is performed before or after LLPS. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0028] Figure 2

[0029] FAM-[RGYGG]5C forms coacervates in vitro in different cell culture media.

[0030] Liquid-Liquid Phase Separation occurs when FAM-[RGYGG]5C is added to common tissue culture media. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0031] Figure 3

[0032] FAM-[RGYGG]5C coacervates formed in different tissue culture media can enter mammalian cells.

[0033] HEK293 cells labelled homogenously with FAM-[RGYGG]5C. The composite images show the nuclei in blue (Hoechst 34580), and FAM-[RGYGG]5C in green. Sytox Deep Red was used to label dead or dying cells and is represented in purple in the Composite images. Brightfield images show the cells and the coacervates (dark dots). All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0034] Figure 4

[0035] FAM-[RGYGG]5C forms coacervates and can enter different mammalian cell types. HEK293, U20S and HeLa cells labelled homogenously with FAM- [RGYGG]5C. The composite images show the nuclei in blue (Hoechst 34580), and FAM-[RGYGG]5C in green. Free FAM fluorophore was used as negative control. All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0036] Figure 5

[0037] FAM-[RGYGG]5C coacervates can incorporate molecular cargoes (protein).

[0038] FAM-[RGYGG]5C forms complex coacervates with different proteins. The composite images show the coacervates containing FAM-[RGYGG]5C in green, and the client protein in red. Brightfield images show the coacervates. All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0039] Figure 6

[0040] FAM-[RGYGG]5C coacervates can incorporate molecular cargoes (antibody).

[0041] FAM-[RGYGG]5C forms complex coacervates with different antibodies. The composite images show the coacervates containing FAM-[RGYGG]5C in green, and the client antibody in either red or magenta. Brightfield images show the coacervates. All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0042] Figure 7

[0043] FAM-[RGYGG]5C coacervates can incorporate molecular cargoes (ssDNA oligo).

[0044] FAM-[RGYGG]5C forms complex coacervates with ssDNA. The composite images show the coacervates containing FAM-[RGYGG]5C in green, and the client ssDNA in magenta. Brightfield images show the coacervates. All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 gm.

[0045] Figure 8

[0046] FAM-[RGYGG]5C coacervates loaded with cargo can enter HEK293 cells (antibody).

[0047] Cargo delivery: 20 μ FMAM-[RGYGG]5C (vehicle) and 5 gg / ml AF594 anti- Nucleophosmin Antibody (cargo). The composite images (A and C) show the nuclei in blue (Hoechst 34580), FAM-[RGYGG]5C in green, and the antibody in purple. The white arrow in D highlights the presence of antibody in the nucleolus. Brightfield images show the cells and the coacervates (dark dots). All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0048] Figure 9

[0049] FAM-[RGYGG]5C coacervates loaded with cargo can enter HEK293 cells (antibody).

[0050] Vehicle-only control: 20 pM FAM-[RGYGG]5C (vehicle). The composite images (A and C) show the nuclei in blue (Hoechst 34580), FAM-[RGYGG]5C in green. Brightfield images show the cells and the coacervates (dark dots). All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 gm.

[0051] Figure 10

[0052] FAM-[RGYGG]5C coacervates loaded with cargo can enter HEK293 cells (antibody).

[0053] Cargo-only control: 5 gg / ml AF594 anti-Nucleophosmin Antibody. The composite images (A and C) show the nuclei in blue (Hoechst 34580). Brightfield images show the cells. All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 gm.

[0054] Figure 11

[0055] FAM-[RGYGG]5C coacervates loaded with cargo can enter mammalian cells (mRNA).

[0056] HEK293 expressing mScarlet when treated with FAM-[RGYGG]5C. The composite images show the nuclei in blue (Hoechst 34580), and FAM-[RGYGG]5C in green. Sytox Deep Red was used to label dead or dying cells and is represented in purple in the Composite images. Brightfield images show the cells and the coacervates (dark dots). All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Positive control with 8 gg / ml of Lipofectamine, negative control with no vehicle used. Scale bar = 10 gm.

[0057] Figure 12

[0058] FAM-[RGYGG]5C coacervates loaded with cargo can enter mammalian cells (ssDNA oligo). ssDNA enters HEK293 cells when these are treated with FAM-[RGYGG]5C. The composite images show the nuclei in blue (Hoechst 34580), and FAM- [RGYGG]5C in green. ssDNA oligo labelled with Cy5 is represented in purple in the Composite images. Brightfield images show the cells and the coacervates (dark dots). All images were normalised to the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Negative controls with either no vehicle, or no cargo used. Scale bar = 20 pm.

[0059] Figure 13

[0060] FAM-[RGYGG]nC polymers can form coacervates if n = 2 or higher. LLPS of the polymers FAM-[RGYGG]2C, FAM-[RGYGG]3C, FAM- [RGYGG]4C, FAM-[RGYGG]5C, FAM-[RGYGG]6C and FAM-[RGYGG]8C, after addition to OptiMEM tissue culture media. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0061] Figure 14

[0062] FAM-[RGYGG]nC polymers of different lengths can enter cells.

[0063] HEK293 cells treated with FAM-[RGYGG]3C, FAM-[RGYGG]4C, or FAM- [RGYGG]5C. The composite images show the nuclei in blue (Hoechst 34580), and the polymers in green. Sytox Deep Red was used to label dead or dying cells and is represented in purple. Brightfield images show the cells and the coacervates (dark dots). All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0064] Figure 15

[0065] FAM-[RGYGG]5C coacervate formation and redox state.

[0066] LLPS of the polymers DMSO-treated FAM-[RGYGG]5C, un-treated FAM- [RGYGG]5C, FAM-[RGYGG]5, [RGYGG]5C-FAM, and FAM-[RGYGG]5C + 2.5 mM TCEP, after addition to OptiMEM tissue culture media. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0067] Figure 16

[0068] Relationship between redox state of FAM-[RGYGG]5C and its ability to enter cells.

[0069] HEK293 cells incubated with DMSO-treated FAM-[RGYGG]5C, un-treated FAM-[RGYGG]5C, FAM-[RGYGG]5, or FAM-[RGYGG]5C + 2.5 mM TCEP. The composite images show the nuclei in blue (Hoechst 34580), and the polymers in green. Sytox Deep Red was used to label dead or dying cells and is represented in purple. Brightfield images show the cells and the coacervates (dark dots). All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0070] Figure 17

[0071] Ri-[RGYGG]5C polymers form coacervates with different aromatic headgroups in position “Ri”.

[0072] Images of the coacervates obtained at different polymer concentrations and the corresponding head groups containing aromatic rings. The blue “SP” block represents the location where the head group is covalently linked with the small polymer. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 gm.

[0073] Figure 18

[0074] Ri-[RGYGG]5C polymers form coacervates with different aromatic headgroups in position “Ri”.

[0075] Representations of the different non-aromatic head groups tested that do not promote LLPS in the concentrations tested (320 to 5 μ)M. The bluμeM “SP” block represents the location where the head group is covalently linked with the small polymer.

[0076] Figure 19

[0077] Ri-[RGYGG]5C polymers form coacervates with different aromatic headgroups in position “Ri”.

[0078] Relationship between the Hydrophobicity of the whole molecule, the Aromaticity of the head group, and the ability of a molecule to form coacervates. Figure 20

[0079] FAM-[PiGYGG]5C polymers can form coacervates if Pi is any of R or H, but not if it is A or N.

[0080] Liquid-Liquid Phase Separation occurs when FAM-[PiGYGG]5C polymers are added to OptiMEM, if Pi is any of R or H, but not if it is A or N. Brightfield images acquired with CX7 LZR PRO (Thermo), using a 40x objective. Scale bar = 10 pm.

[0081] Figure 21

[0082] FAM-[RGP3GG]5C polymers can form coacervates if P3 is any of Y, F or W, but not if it is A or N.

[0083] Liquid-Liquid Phase Separation occurs when FAM-[RGP3GG]5C polymers are added to OptiMEM, if P3 is any of Y, F or W, but not if it is A or N. Brightfield images acquired with CX7 LZR PRO (Thermo), using a 40x objective for FAM-[RGYGG]5C; FAM-[RGWGG]5C, and FAM-[RGAGG]5C, and a 20x objective for FAM- [RGFGG]5C. Scale bar = 10 pm.

[0084] Figure 22

[0085] FAM-[PIGP3GG]5C polymers, where Pi is any of R, or H, but not A or N, and where P3 is any of Y, F or W, but not A or N, can form coacervates regardless of the position of Pi or P3 in the molecule.

[0086] Liquid-Liquid Phase Separation occurs when the polymers FAM-[RGYGG]5C or FAM-[YGRGG]5C are added to OptiMEM. Brightfield images acquired with CX7 LZR PRO (Thermo), using a 40x objective. Scale bar = 10 pm.

[0087] Figure 23

[0088] FAM-[RP2YGG]5C polymers can form coacervates if P2 is a spacer of any length. Images of the coacervates obtained at different polymer concentrations and the corresponding spacers: Glycine (A), 0- Alanine (B), y-Aminobutyric acid (C), £- Amino valeric acid (D). Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0089] Figure 24

[0090] FAM-[RP2YP4]5C polymers can form coacervates if P2 and P4 are any of A, G, GG, or GGG spacers.

[0091] Images of the coacervates obtained at different polymer concentrations and the corresponding spacers. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0092] Figure 25

[0093] FAM- [peptide] C polymers can form coacervates if the peptide part of the polymer is constituted by amino acid residues which can be referred to as “stickers”, as well as “spacers”. In this particular example, each structural unit of the coacervateforming polymer comprises at least 2 amino acid residues, wherein at least one amino acid residue is selected from the group R, H and K and is R, and wherein at least one further amino acid residue is selected from the group Y, F and W and is Y. In addition, each structural unit of the coacervate-forming polymer comprises at least two molecules selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G- G), glycine-glycine-glycine (G-G-G), or any spacer, and wherein the at least two molecules are G.

[0094] Images of the coacervates obtained at different polymer concentrations and the corresponding spacers. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm. Figure 26

[0095] WW-[SU-SU-SU-SU-SU]C molecules can form coacervates if the molecule is constituted by 5 structural units (SU), where a peptide structural unit consists of 5 amino acids, where the first amino acid is any of R, or H, or K, where the second amino acid is any spacer, where the third amino acid is any of Y, or F or W, and where the fourth and fifth amino acid is any spacer.

[0096] Images of the coacervates obtained at different polymer concentrations. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0097] Figure 27

[0098] WW-[SU-SU-SU-SU-SU]C molecules can form complex coacervates and deliver RNA cargo to myotubes if the molecule is constituted by 5 structural units, where a peptide structural unit consists of 5 amino acids, where the first amino acid is any of R, or H, or K, where the second amino acid is any spacer, where the third amino acid is any of Y, or F or W, and where the fourth and fifth amino acid is any spacer.

[0099] C2C12 myotubes treated with the different peptides (Figure 27 A). The mCherry images show the expression levels of mCherry elicited by the mRNA cargo. Brightfield images show the cells. All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 50 pm.

[0100] The graph (Figure 27B) shows the normalized transfection efficiency (as determined by the area successfully transfected in each well) and the normalized fluorescence intensity. The normalization of both parameters was performed in relation to the positive control (lipofectamine).

[0101] Figure 28

[0102] WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) coacervates loaded with DNA cargo can transfect myocytes and promote protein expression. C2C12 myotubes treated with WW-HGYGG-HGWGG-HGFGG-KGYGG- KGWGG-C (SP-1540) (Figure 28A). The eGFP images show the expression levels of GFP elicited by the different DNA cargos. Brightfield images show the cells. All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 50 pm.

[0103] The graphs (Figure 28B) show the normalized transfection efficiency (as determined by the area successfully transfected in each well) and the normalized fluorescence intensity. The normalization of both parameters was performed in relation to the positive internal control.

[0104] Figure 29

[0105] WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) and WW- KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643) peptides can form complex coacervates with proteins and peptides, enter cells, and deliver the cargo.

[0106] U2OS cells treated with WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG- C (SP-1540) or WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643). The composite images show the nuclei in blue (Hoechst 34580), the FAM-labelled peptides in green, or the AF594 - anti-Nuclear Pore Complex Protein Antibody in red. Brightfield images show the cells and the coacervates (dark dots). All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 20 pm.

[0107] Figure 30

[0108] In the molecule WW-[SU-SU-SU-SU-SU]C (SU - structural units), replacing a R, H, or K with an alternative amino acid, specifically Pip IF (2-Amino-4-(piperidin-l- yl)propanoic acid), or replacing a F, W, or Y with an alternative amino acid, specifically TIC (l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), result in molecules that can form coacervates. Images of the coacervates obtained at different polymer concentrations. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm.

[0109] Figure 31

[0110] In the molecule WW-[SU-SU-SU-SU-SU]C (SU - structural units), replacing a R, H, or K with an alternative amino acid, specifically Pip IF (2-Amino-4-(piperidin-l- yl)propanoic acid), or replacing a F, W, or Y with an alternative amino acid, specifically TIC (l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), result in molecules that can form complex coacervates and deliver RNA cargo to myotubes.

[0111] C2C12 myotubes treated with the different peptides (Figure 31 A). The mCherry or eGFP images show the expression levels of mCherry or eGFP elicited by the mRNA cargo. Brightfield images show the cells. All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 50 pm.

[0112] The graph (Figure 3 IB) shows the normalized transfection efficiency (as determined by the area successfully transfected in each well) and the normalized fluorescence intensity. The normalization of both parameters was performed in relation to the positive control (lipofectamine).

[0113] Figure 32

[0114] FAM-[rgygg]5c D-peptide can form coacervates.

[0115] Images of the coacervates obtained at different polymer concentrations. Brightfield images acquired with CX7 LZR PRO (Thermo). Scale bar = 10 pm. Figure 33

[0116] FAM-[RGYGG]5C polymers can form coacervates and enter cells regardless of whether the C-terminus of the polymer is a Carboxylic Acid group or a Carboxamide group.

[0117] HEK293 cells treated with FAM-[RGYGG]5C carboxylic acid (FAM- [RGYGG]5C-carb) or with FAM-[RGYGG]5C carboxamide (FAM-[RGYGG]5C- amide). The composite images show the nuclei in blue (Hoechst 34580), and the polymers in green. Sytox Deep Red was used to label dead or dying cells and is represented in purple. Brightfield images show the cells and the coacervates (dark dots). All images were modified with the same levels of brightness and contrast. Images acquired with CX7 LZR PRO (Thermo). Scale bar = 20 pm.

[0118] DESCRIPTION OF THE SEQUENCES

[0119] SEQ ID NOS: 1 to 291 are provided in the accompanying sequence listing, which forms part of the description.

[0120] DETAILED DESCRIPTION OF THE INVENTION

[0121] The inventors have identified new classes of coacervate-forming polymers. The polymers of the invention are able to promote the formation of coacervates, as described and defined further herein. The invention further relates to polymer coacervates produced using the polymers of the invention, and methods for making such polymer coacervates. The polymer coacervates are capable of acting as carrier vehicles for cargo compounds, such as therapeutic proteins, nucleic acids, small molecules, and large biological molecules such as antibodies, etc. The polymer coacervates are capable of penetrating cells and delivering cargo compounds into cells. Polymer coacervates as described and defined further herein can find wide-ranging uses, particularly where the delivery of cargo compounds can affect biological processes.

[0122] Aspects of the invention are described in more detail below. Polymer Formulae and Terminology

[0123] A coacervate-forming polymer of the invention comprises or consists of a structure according to the formula (I):

[0124] Rl-N[Pl-P2-P3-P4]nC (I) wherein:

[0125] A. Ri is occupied by one or more aromatic molecules;

[0126] B. N is the N-terminal end of the polymer;

[0127] C. [P1-P2-P3-P4] is a structural unit of the polymer;

[0128] D. n is the number of structural units of the polymer, wherein n is an integer of 2 or more;

[0129] E. C is the C-terminal end of the polymer;

[0130] F. P is an amino acid residue position or a spacer position in the polymer; and

[0131] G. each one of Pi, P2, P3 and P4consists of one or more amino acid residue positions and / or one or more spacer positions, wherein: i. one or more of said positions is occupied by a molecule selected from the group Al consisting of arginine (R), histidine (H) and lysine (K); and ii. one or more additional position(s) is occupied by a molecule selected from the group A2 consisting of tyrosine (Y), phenylalanine (F), and tryptophan (W); and iii. one or more further additional positions are occupied by a molecule selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G-G-G), glycine- glycine-glycine-glycine (G-G-G-G), or any spacer.

[0132] In the context of the present invention a polymer is taken to mean a molecule having multiple (“poly”) units (“mers”). In the context of the polymers of the present invention, a single unit (“mer”) is a structural unit (SU) as further defined herein. Accordingly, a structural unit or “mer” or “SU” is represented by the designation [Pi- P2-P3-P4]. Thus, a polymer according to the invention is a molecule having multiple such structural units, where the number of units is defined by the term “n”, wherein n is an integer of 2 or more. A polymer of the invention may therefore have e.g. five structural units ([Pi-P2-P3-P4]s, i.e. a 5-mer). As will be apparent from the discussion herein, the structural units of the polymer may have the same or different structure, such as amino acid sequence, and the structural units may be ordered so as to have, in the direction N-terminal to C-terminal, the same or different structure.

[0133] The polymer of the invention may also take the form of a “dimer” comprising or consisting of two “monomers”. In this context the polymer is referred to as a “dimeric polymer”. In this context, the term “monomer” of a dimeric polymer according to the invention refers to a polymer comprising or consisting of multiple structural units as described and defined further herein. Thus, for example, a dimeric polymer may comprise one monomer having five structural units represented by the designation [Pi- P2-P3-P4]s, i.e. a first polymer of five structural units, and may comprise a further monomer also having five structural units represented by the designation [Pi-P2-P3-P4]s, i.e. a second polymer of five structural units, and wherein first and second polymer “monomers” are joined together, such as via a chemical bond, such as a covalent bond, e.g. a disulphide bond. In this example, the dimeric polymer will have 10 structural units.

[0134] As defined above, each one of Pi, P2, P3 and P4 consists of one or more amino acid residue positions and / or one or more spacer positions.

[0135] The molecules arginine (R), histidine (H) and lysine (K) constituting the group Al and the molecules tyrosine (Y), phenylalanine (F), and tryptophan (W) constituting the group A2 may be referred to as “stickers”. The molecules glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G-G-G), glycine-glycine-glycine- glycine (G-G-G-G), or “any spacer” may be referred to as “spacers”.

[0136] The “sticker-spacer” terminology is used extensively in the scientific literature in relation to the functional attributes of coacervate-forming polymers and are used as terms of art to those familiar with this technical field (see e.g. Tom, J. A. K and Deniz, A. K., Complex Dynamics of Multicomponent Biological Coacervates. Current Opinion in Colloid & Interface Science. 2021, vol. 56, 101488). A “sticker” can refer to a molecule or functional group in the polymer which can form functional interactions with other molecules in the environment and thereby drive liquid-liquid phase separation and coacervate formation. Such interactions can include charge-charge, cation-pi, pi-pi, and hydrophobic interactions. A “spacer” can refer to a molecule which can tether the stickers together on the polymer and which may itself form functional interactions with other molecules.

[0137] Without wishing to be bound by theory, the inventors believe that the repeating nature of the “sticker” molecules selected from the groups Al and A2 and the “spacer” molecules of the group A3 in the polymers of the invention play an important role, together with the associated aromatic headgroup at position Ri, in driving liquid-liquid phase separation and coacervate formation.

[0138] In the case of consecutive amino acid residues at adjacent positions, the residues are typically connected via conventional peptide bonds. For example, in the case of the structural unit [R-G-Y-G-G], each amino acid residue may be connected by a peptide bond. In this context “-” signifies a peptide bond.

[0139] Thus, in any one of the polymers of the invention “-” may signify a peptide bond between consecutive amino acid residues in the polymer.

[0140] An amino acid residue may occupy a position in the polymer adjacent to a consecutive spacer. For example, in the case of the structural unit [R-0-alanine-Y-G- G], the arginine and tyrosine residues occupy positions in the polymer adjacent to the consecutive spacer which in this example is a 0-alanine molecule. In such cases, the relevant amino acid residue may typically be connected to the spacer by a bond which is appropriate for connecting the amino acid to the spacer, typically an amide bond. In this context the character “-” signifies an appropriate bond, typically an amide bond, to facilitate the connection between the amino acid and the spacer. Thus, in any one of the polymers of the invention the character “-” may signify an appropriate bond between consecutive amino acid residues in the polymer, typically a peptide bond, or an appropriate bond between an amino acid residue and a spacer, typically an amide bond.

[0141] The polymers of the invention may also be represented by formulae in which there is an absence of the character “-” within the structural unit definition, for example the formula 5-FAM-[RGYGG]. In accordance with the conventional definition and understanding of amino acid sequence listings the sequence RGY GG is understood to relate to a sequence of five consecutive amino acids, wherein each amino acid is connected to another adjacent consecutive amino acid by a peptide bond.

[0142] In the case of structural units comprising one or more spacers, the structural unit may be designated without the character between amino acids and spacers, e.g. [R0- alaYGG]. In this designation, in accordance with conventional definition and understanding, adjacent amino acids are still connected by an appropriate bond, typically a peptide bond, and an adjacent amino acid and a spacer are still connected by an appropriate bond, typically an amide bond.

[0143] Accordingly, the absence of the character within the structural unit definition is not to be interpreted as the absence of a peptide or amide bond. On the contrary, the sequence is to be interpreted in accordance with the conventional definition and understanding of amino acid sequence and spacer listings wherein amino acids are connected together via peptide bonds or wherein amino acids and spacers are connected together via amide bonds.

[0144] In certain instances involving a structural unit comprising one or more spacers, the spacer may be demarcated from the adjacent positions using square brackets, i.e. For example, structural unit no. 6 in Table Al is represented may also be represented as R-0-ala-Y-G-G, [R-0-ala-Y-G-G], or [R[0-ala]YGG]. The same structural unit may also be represented as R{0-ala}YGG, R- {0-ala}-Y-G-G, [R{0-ala}YGG] and [R-{0-ala}-Y-G-G]. All of these designations are equivalent and refer to the same structural unit having the same molecular structure. In any of these designations, adjacent amino acids are still connected by an appropriate bond, typically a peptide bond, and the spacer and an adjacent amino acid are still connected by an appropriate bond, typically an amide bond.

[0145] The structural units of all polymers of the invention are defined as having an N- terminal to C-terminal directionality according to standard peptide / polypeptide nomenclature. For example, in the formula Ri-N[Pi-P2-P3-P4]cn, N denotes the N- terminal end of the polymer and C denotes the C-terminal end of the polymer. It will be appreciated that the polymer may not actually have a free amino group (+NHs) at the N- terminal end, since it will possess an Ri group. Furthermore, the polymer may not have a free carboxyl group (COO") at the C-terminal end, since it may possess an R2 group. Thus, in the context of the polymers of the present invention the terms “N” and “C” are provided merely to indicate the standard directionality of a peptide or polypeptide according to conventional nomenclature concerning the positioning of the amine and carboxylic acid groups. Accordingly, a skilled person will readily be able to ascertain the directionality of the polymer.

[0146] The structural units of certain polymers of the invention may include an R2 group / position at the C-terminal end of the polymer, represented by formula (II):

[0147] Rl-N[Pl-P2-P3-P4]nC-R2 (II).

[0148] In certain embodiments the R2 group may be a cysteine amino acid residue, i.e. Ri-N[Pi-P2-P3-P4]nC-C. In this formula cysteine, represented by the upper-case character “C”, is distinguished from “c”, which denotes the c-terminal end of the last structural unit of the polymer, because the latter is presented in subscript.

[0149] Polymers may be referred to herein without the “N” and “c” nomenclature by way of shorthand notation, e.g. FAM-[RGYGG]5. Such formulae are intended to be interpreted in accordance with standard amino acid sequence listings, whereby the N- terminal end of the structural unit is at the left-hand side of the unit, i.e. in this case “R”, and the C-terminal end of the structural unit is at the right-hand side of the unit, i.e. in this case the final “G”. In the formula FAM-[RGYGG]5, there is no R2 group. In the formula FAM-[RGYGG]5C, an R2 group is present and is cysteine.

[0150] As explained previously, P is an amino acid residue position or a spacer position in the polymer. For the avoidance of doubt, the designation “P” in this context is not to be confused with the single letter designation for the amino acid proline. This will be clear in the context of the present disclosure, since the specific designation of each one of the four P positions in the relevant formulae is indicated with a subsequent number displayed in subscript, i.e. Pi, P2, P3 and P4. Accordingly, wherever reference is made to Pi, P2, P3 and / or P4, this corresponds to an amino acid residue position or a spacer position in the polymer. Similarly, the designations Ri or R2 are not to be confused with the single letter designation for the amino acid arginine. Accordingly, wherever reference is made to Ri, this corresponds to the one or more aromatic molecules or “headgroup”. Wherever reference is made to R2, this corresponds to any chemical group or molecule that is capable of forming a bond, preferably a covalent bond, more preferably a disulphide bond, with a further R2 group of a second polymer according to the invention, preferably cysteine.

[0151] In the context of the formulae presented herein, the designations N and C, representing N-terminal and C-terminal respectively, are not to be confused with the single letter designations for the amino acids asparagine and cysteine respectively. This will be clear in the context of the present disclosure, since the specific designations of N and C, representing N-terminal and C-terminal respectively, in the relevant formulae are displayed in subscript. Accordingly, wherever reference is made to N and C presented in subscript, this corresponds to N-terminal and C-terminal respectively.

[0152] Polymer Structural Units

[0153] Structural Unit Notation

[0154] The coacervate-forming polymers of the invention are defined according to formulae (I) and (II) and further herein:

[0155] Ri-N[Pi-P2-P3-P4]nc (I); and

[0156] Rl-N[Pi-P2-P3-P4]nC-R2 (II).

[0157] As used herein, a structural unit is demarcated by square brackets. Accordingly, a structural unit is represented by [P1-P2-P3-P4]. Square brackets are used purely for illustrative purposes to denote the unit. For example [P1-P2-P3-P4HP1-P2-P3-P4] denotes two structural units. This can also be represented by [P1P2P3P4HP1P2P3P4] or [P1P2P3P4][P1P2P3P4]. In one illustrative embodiment [R-G-Y-G-G]-[R-G-Y-G-G] denotes two structural units. This can also be represented by [RGYGG]-[RGYGG], [RGYGG][RGYGG] and [RGYGG]2. Where used, the dash between amino acid residue or spacer positions and the square brackets are used purely for illustration and information purposes only. In each case the polymer comprises the stated amino acid and spacer positions in sequential order connected by the appropriate and relevant bonds.

[0158] Number of and Orientation of Structural Units

[0159] All coacervate-forming polymers according to the invention have at least two structural units. The number of structural units of the polymer is denoted by the character “n”, where n is an integer of 2 or more. In a polymer according to the invention n may be an integer of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more. In preferred embodiments, n is 5 or more, preferably 5.

[0160] In applications of the polymers of the invention, particularly in the formation of coacervates in solution, the number of structural units of the polymer may be chosen by the user so as to optimise the functional effect of the polymer in a given context. In this sense, the number of structural units of the polymer can be regarded as a “tuneable” variable.

[0161] In a polymer according to the invention, any two structural units of the polymer, or all structural units of the polymer, may be arranged, in the direction N-terminal to C- terminal, to have the same positional orientation, i.e. [P1-P2-P3-P4HP1-P2-P3-P4], or [P4- P3-P2-P1HP4-P3-P2-P1]. Alternatively, in a polymer according to the invention, any two structural units of the polymer, or all structural units of the polymer, may be arranged, in the direction N-terminal to C-terminal, to have opposing positional orientations i.e. [P1-P2-P3-P4 P4-P3-P2-P1], or [P4-P3-P2-P1HP1-P2-P3-P4].

[0162] Accordingly, in any one of the polymers according to the invention having the formula: Rl-N[Pl-P2-P3-P4]nC (I); or

[0163] Rl-N[Pl-P2-P3-P4]nC-R2 (H); in the direction N-terminal to C-terminal:

[0164] A. all structural units of the polymer may be ordered in the sequence [P1-P2-P3-P4];

[0165] B. the first structural unit at the N-terminal end of the polymer may be ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer may be ordered in the sequence [P4-P3-P2-P1];

[0166] C. the first structural unit at the N-terminal end of the polymer may be ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer may be ordered in the sequence [P1-P2-P3-P4];

[0167] D. the last structural unit at the C-terminal end of the polymer may be ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer may be ordered in the sequence [P4-P3-P2-P1];

[0168] E. the last structural unit at the C-terminal end of the polymer may be ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer may be ordered in the sequence [P1-P2-P3-P4]; or

[0169] F. all structural units of the polymer may be ordered in the sequence [P4-P3-P2-P1].

[0170] In applications of the polymers of the invention, particularly in the formation of coacervates in solution, the orientation of structural units of the polymer may be chosen by the user so as to optimise the functional effect of the polymer in a given context. In this sense, the orientation of (as well as the number of) structural units of the polymer can be regarded as a “tuneable” variable.

[0171] In certain embodiments, a coacervate-forming polymer may be represented herein by an abridged formula such as FAM-[RGYGG]5C as presented in certain examples herein. As explained and defined further herein, such a polymer consists of a FAM Ri headgroup, followed by five consecutive structural units, and ending with a C- terminal cysteine R2 group, wherein each structural unit has the sequence RGYGG. For the avoidance of any doubt, unless specifically stated to the contrary or otherwise apparent from the context, a formula such as FAM-[RGYGG]5C represents a polymer wherein each one of the structural units is ordered in the same orientation in the direction N-terminal to C-terminal, i.e. FAM-[RGYGG]-[RGYGG]-[RGYGG]- [RGYGG]-[RGYGG]-C.

[0172] Isolated Polymer

[0173] A polymer according to the invention may also be referred to as an “isolated polymer”.

[0174] As used herein, the term "isolated" refers to a polymer that has been (1) separated from at least some of the components which it may have been associated with when initially produced, and / or (2) produced, prepared, and / or manufactured by the hand of man. An isolated polymer may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the other components with which they were initially associated. An isolated polymer may be about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% pure. As used herein, an isolated polymer is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of an isolated polymer should not include excipients (e.g., buffer, solvent, water, etc.).

[0175] To the best of the applicant’s knowledge and belief, the polymers described and defined herein are produced, prepared and manufactured by the hand of man and do not occur in nature. Accordingly, any polymer of the invention may be referred to as a “non naturally-occurring” or “artificial” polymer or “isolated non naturally-occurring” or “isolated artificial” polymer.

[0176] Insoluble Polymer Forms

[0177] Any polymer according to the invention as described and defined herein may exist in an insoluble form. Any polymer according to the invention as described and defined herein may exist in a dried form, e.g. dried by air, dried using vacuum concentrator system or dried under an inert gas.

[0178] Any polymer according to the invention as described and defined herein may exist in a lyophilized (freeze-dried) form.

[0179] Polymer Solutions & Methods of Making Polymer Solutions

[0180] Any polymer according to the invention as described and defined herein may exist in a soluble form, i.e. in solution.

[0181] Any polymer according to the invention as described and defined herein may exist dissolved in water, dissolved in 100% of a solvent, optionally an organic solvent, or dissolved in a mixture of any percentage of a solvent, optionally an organic solvent in water.

[0182] Any one of the polymers according to the invention as described and defined herein may be dissolved in a solvent or solution to provide a polymer solution, optionally wherein the solvent is an organic solvent, such as DMSO (Dimethyl sulfoxide).

[0183] Accordingly, there is provided a method of making a polymer solution, the method comprising providing any polymer according to the invention as described and defined herein, contacting the polymer with a solvent or solution, dissolving the polymer in the solvent or solution, thereby creating the polymer solution, optionally wherein the solvent is an organic solvent, such as DMSO (Dimethyl sulfoxide).

[0184] Suitable solvents and solutions include water or an organic solvent such as DMSO (Dimethyl sulfoxide), e.g. 100% water; 100% organic solvent such as DMSO, or any mixture of organic solvent such as DMSO and water, in which the percentage of the organic solvent varies between 1% and 99%. The choice of concentration ratio of water to organic solvent can readily be chosen by the user depending upon the solubility of the particular polymer being used. Suitable concentrations include the following:

[0185] Aromatic Molecule (Ri “Headgroup”)

[0186] All coacervate-forming polymers of the invention comprise an Ri group which is one or more aromatic molecules. An Ri group may also be referred to herein as a “headgroup”. Ri may be referred to as a “position”, i.e. position Ri is occupied by a “headgroup” or otherwise referred to as an “aromatic molecule”. All polymers of the invention comprise at least one aromatic molecule at the Ri position. Typically, polymers of the invention may have a headgroup that consists of one aromatic molecule at the Ri position. Polymers of the invention may have a headgroup that consists or comprises two or more aromatic molecules at the Ri position. An aromatic molecule as defined herein is a molecule which has at least one aromatic ring. Any suitable aromatic molecule or molecules may be incorporated at the Ri position, provided that the polymer is a coacervate-forming polymer. Aromatic molecules may include e.g. benzene, indole, pyrrole, pyridine, naphthalene and anthracene.

[0187] The headgroup may be further defined as having an aromaticity value. The aromaticity value of an aromatic molecule or molecules used in a coacervate-forming polymer according to the invention is scored by the number of atoms in the aromatic molecule(s) engaged in aromatic bonds as defined by Hueckel’s rule (calculated using public access cheminformatics tool OpenBabel 3.0.0; N M O'Boyle, M Banck, C A James, C Morley, T Vandermeersch, and G R Hutchison. "Open Babel: An open chemical toolbox." J. Cheminf. (2011), 3, 33. DOI:10.1186 / 1758-2946-3-33). Accordingly, the aromatic molecule (if there is only one, individually) or aromatic molecules (if there is a group of two or more combined) which occupies position Ri may have an individual total or combined total aromaticity value of 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more or 35 or more. The aromatic molecule(s) which occupies position Ri may preferably have an individual total or combined total aromaticity value of between 5 and 35. In particularly preferred embodiments, the individual total or combined total aromaticity value is 18 or 20.

[0188] In applications of the polymers of the invention, particularly in the formation of coacervates in solution, the nature of the headgroup(s) of the polymer may be chosen by the user so as to optimise the functional effect of the polymer in a given context. The nature of the headgroup(s) of the polymer may be chosen in combination with the choice of the sequence of amino acid residues and spacers in any given structural unit, as well as the number of structural units and the orientation of the structural units relative to each other. In this sense, the nature of the headgroup of the polymer can be regarded as a “tuneable” variable.

[0189] In the context of the formation of coacervates in solution, and without wishing to be bound by theory, it is believed that the aromaticity of the headgroup in the polymers of the invention may contribute to the formation of coacervates by promoting relevant functional interactions such as Pi-interactions, e.g. Pi-Pi interactions, such as Pi-stacking between aromatic rings of headgroups and / or aromatic amino acid side chains of the structural units of the polymer, and / or Pi-cation interactions between aromatic groups and cations in solution.

[0190] Suitable exemplary headgroups which may occupy position Ri include the following:

[0191] Amino Acid Residues

[0192] A coacervate-forming polymer of the invention comprises or consists of a structure according to the formula:

[0193] Ri-N[Pi-P2-P3-P4]nc (I); or

[0194] Rl-N[Pl-P2-P3-P4]nC-R2 (II); wherein in either of the two formulae:

[0195] A. Ri is occupied by one or more aromatic molecules;

[0196] B. N is the N-terminal end of the polymer;

[0197] C. [P1-P2-P3-P4] is a structural unit of the polymer; D. n is the number of structural units of the polymer, wherein n is an integer of 2 or more;

[0198] E. C is the C-terminal end of the polymer; F. P is an amino acid residue position or a spacer position in the polymer; and

[0199] G. each one of Pi, P2, P3 and P4 consists of one or more amino acid residue positions and / or one or more spacer positions, wherein: i. one or more of said positions is occupied by a molecule selected from the group Al consisting of arginine (R), histidine (H) and lysine (K); and ii. one or more additional position(s) is occupied by a molecule selected from the group A2 consisting of tyrosine (Y), phenylalanine (F), and tryptophan (W); and iii. one or more further additional positions are occupied by a molecule selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G-G-G), glycine- glycine-glycine-glycine (G-G-G-G), or any spacer.

[0200] In the case of a polymer further defined as formula (II), R2 is further defined herein.

[0201] The identity of the amino acid residues selected from the groups Al and A2, as well as the identity of molecules or spacers selected from the group A3 may be chosen by the user to provide an appropriate functional effect in a given context, particularly in the formation of coacervates in solution. Accordingly, these selections provide further “tuneable” variables for the selection and optimisation of coacervate-forming polymers.

[0202] An amino acid homolog, analog or derivative may be used as an alternative to an amino acid. For example, an arginine homolog, such as homoarginine, may be used as an alternative to arginine. A cysteine homolog, such as homocysteine, may be used as an alternative to cysteine. Accordingly, in any of the polymers described and defined herein an amino acid homolog, analog or derivative may be used as an alternative to any given specified amino acid.

[0203] In the group Al consisting of R, K and H, any one of these residues may alternatively be replaced with an analog having one of the molecules listed in the table below:

[0204] In the group A2 consisting of Y, F and W, any one of these residues may alternatively be replaced with an analog having any one of the molecules listed in the table below:

[0205] In any of the polymers described and defined herein, either of the L- (Levorotatory) and D- (Dextrorotatory) isomers of amino acids or amino acid homologs may be used. In the examples described below, the inventors tested primarily L isomers. However, it is also shown in Example 22 that in the case of 5-FAM- [RGYGG]5C, when D isomers of the amino acids were used in the structural units of the polymer, the polymer was readily able to form coacervates. Accordingly, it is expected that D and L isomers of amino acids or amino acid homologs are fully functionally interchangeable. Accordingly, in any of the polymers described and defined herein, one or more of the amino acids or amino acid homologs of the polymer may be in the L- (Levorotatory) isomeric form. In any of the polymers described and defined herein, one or more of the amino acids or amino acid homologs of the polymer may be in the D- (Dextrorotatory) isomeric form.

[0206] Spacer

[0207] The term “spacer” is a well-understood term of art in the field of peptide / polypeptide chemistry.

[0208] In certain embodiments described and defined herein, certain positions of a structural unit of the polymer may be occupied by a molecule selected from the group A3 and which is a spacer. Accordingly, a “spacer” of a structural unit is any chemical structure that can provide a connection or linkage to any relevant amino acid residue of a structural unit of the polymer or to any other spacer.

[0209] In addition, the headgroup which occupies position Ri may be connected to the first molecule of the first structural unit of the polymer at the N-terminal end of the polymer by a “spacer”. Accordingly, a “spacer” is also any chemical structure that can provide a connection or linkage between the headgroup at position Ri and the first molecule of the first structural unit of the polymer at the N-terminal end of the polymer.

[0210] The connection or linkage may be between the two relevant functional groups. Within a structural unit of the polymer the connection or linkage may be between the two relevant functional groups of the amino acids (i.e. the amine group and carboxylic acid group).

[0211] Between the headgroup at position Ri and the first molecule of the first structural unit of the polymer at the N-terminal end of the polymer, the connection or linkage may be between the amine group of the amino acid, and the aromatic group of the headgroup at position Ri. Other linkages, e.g. covalent linkages, could be used.

[0212] A “spacer” may be a “heterobifunctional linker” or a “heterobifunctional spacer”.

[0213] A “spacer” may be a “homobifunctional linker” or a “homobifunctional spacer”.

[0214] A glycine amino acid residue, or a plurality of consecutive glycine amino acid residues may be referred to as a “spacer”, e.g. G-G, G-G-G, G-G-G-G or more than 4 Gs. An alanine amino acid residue, or a plurality of consecutive alanine amino acid residues may also be referred to as a “spacer”. Glycine and alanine residues are used extensively in the art as spacers. Such spacers may be used to provide flexible linkages. Without wishing to be bound by theory, the inventors consider that such spacers are unlikely to actively engage in the types of low-afflnitiy / high- valency interactions that promote liquid-liquid demixing and coacervate formation.

[0215] In any of the polymers described and defined herein, the spacer may be an “alkyl spacer” (z.e. a chain of CH2 groups).

[0216] In any of the polymers described and defined herein, the spacer may be a “hetero-alkyl spacer” (i.e. a chain of CH2 groups interspaced by O, N or S atoms).

[0217] In any of the polymers described and defined herein, the spacer may be a molecule comprising 3 carbon bonds (C3 spacer), e.g. 0-alanine.

[0218] In any of the polymers described and defined herein, the spacer may be a molecule comprising 4 carbon bonds (C4 spacer), e.g. 4-aminobutyric acid or two glycine residues.

[0219] In any of the polymers described and defined herein, the spacer may be a molecule comprising 5 carbon bonds (C5 spacer), e.g. 5 -amino valeric acid.

[0220] In any of the polymers described and defined herein, the spacer may be a molecule comprising 6 carbon bonds (C6 spacer), e.g. 6-aminohexanoic acid.

[0221] In any of the polymers described and defined herein, the spacer may be a polyethylene glycol (PEG) spacer, e.g. a PEG2 spacer (8-amino-3,6-dioxaoctanoic acid), a PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), or a PEG4 spacer (15- amino-4,7, 10, 13-tetraoxapenta-decanoic acid). R2 Group

[0222] Any of the coacervate-forming polymers described and defined herein may comprise a molecule which occupies position R2, also referred to as an “R2 group”.

[0223] A molecule comprising two such coacervate-forming polymers joined via a bond between R2 groups is referred to as a “dimeric coacervate-forming polymer” as further described and defined herein, wherein the dimeric coacervate-forming polymer comprises two coacervate-forming polymers, each referred to individually as a “monomer”.

[0224] An R2 group may be any chemical group or molecule that is capable of forming a bond with a further R2 group attached to a second coacervate-forming polymer as described and defined herein. Preferably, an R2 group may be any chemical group or molecule that is capable of forming a covalent bond with a further R2 group. More preferably, an R2 group may be any chemical group or molecule that is capable of forming a disulphide bond with a further R2 group.

[0225] An R2 group may be a thiol group or a molecule having a thiol group such as cysteine or homocysteine. The thiol group may be connected directly to the C-terminus of the polymer, such as via an amino acid side chain, as in the case of cysteine. The thiol group may alternatively be connected directly to the C-terminus of the polymer, such as via a C-terminal 2-mercapto-ethylamine.

[0226] A preferred R2 group is cysteine. In Example 13 below, the inventors demonstrated that the polymer represented by the formula 5-FAM-[RGYGG]5C exhibits good performance in terms of coacervate formation. However, the C-terminal cysteine was shown not to be essential, since the polymers represented by the formula 5-FAM-[RGYGG]5, which lacks the C-terminal cysteine, and [RGYGG]5C-FAM, where the FAM was conjugated directly to the cysteine side chain, were demonstrated to still be capable of promoting coacervate formation, albeit at higher concentrations.

[0227] In any of the polymers of the invention described and defined herein, including polymers which comprise a C-terminal cysteine at position R2, the C-terminus of the polymer may comprise a carboxylic acid group or a carboxamide group. Redox State

[0228] In Example 13 below, the inventors demonstrated that the polymer represented by the formula 5-FAM-[RGYGG]5C exhibits good performance in terms of coacervate formation when present in an oxidised state. However, the oxidation state was shown not to be essential, since the same polymer was demonstrated still to be capable of promoting coacervate formation in the reduced state.

[0229] Accordingly, any of the polymers as described and defined herein may be in an oxidised redox state. Any of the polymers as described and defined herein may be in a reduced redox state. Preferably, polymers as described and defined herein are in an oxidised redox state.

[0230] Methods of Making Solutions of Oxidised Polymer

[0231] The invention also provides methods of making a solution of an oxidised polymer.

[0232] A method of making a solution of an oxidised polymer may comprise providing any polymer as described and defined herein, and dissolving the polymer in a solvent comprising an oxidising agent or in a solution comprising an oxidising agent. The oxidising agent may be any suitable oxidising agent such as potassium ferricyanide, iodine, N-chlorosuccinimide (NCS), 2,2'-dithiopyridine (DPDS) and dimethyl sulfoxide (DMSO). A preferred oxidising agent is DMSO.

[0233] A method of making a solution of an oxidised polymer may comprise providing any polymer as described and defined herein, and dissolving the polymer in a solution comprising oxidising agent (in water), optionally 30% oxidising agent (in water), such as DMSO (in water), optionally 30% DMSO (in water).

[0234] A method of making a solution of an oxidised polymer may comprise providing a polymer in solution wherein the polymer is any polymer as described and defined herein, and subjecting the polymer solution to oxidising conditions. The step of subjecting the polymer solution to oxidising conditions may comprises exposing the polymer solution to atmospheric air for 2 or more hours, e.g. from 2 hours to 24 hours, preferably in dark conditions at room temperature.

[0235] The step of subjecting the polymer solution to oxidising conditions may comprises contacting the polymer solution with an oxidising agent. The oxidising agent may be any suitable oxidising agent such as potassium ferricyanide, iodine, N- chlorosuccinimide (NCS), 2,2'-dithiopyridine (DPDS) or dimethyl sulfoxide (DMSO), preferably DMSO.

[0236] Contacting the polymer solution with oxidising agent may comprise forming a solution of polymer in oxidising agent. For example, contacting the polymer solution with DMSO may comprise forming a solution of polymer in DMSO.

[0237] Contacting the polymer solution with oxidising agent may comprise adding oxidising agent to the polymer solution. For example, contacting the polymer solution with DMSO may comprise adding DMSO to the polymer solution.

[0238] Contacting the polymer solution with oxidising agent may comprise adding oxidising agent to the polymer solution to create a solution of polymer in oxidising agent. For example, contacting the polymer solution with DMSO may comprise adding DMSO to the polymer solution to create a solution of polymer in DMSO.

[0239] In any of the aforementioned methods involving oxidising agent, such as DMSO, the concentration of oxidising agent in the final solution may be 30%. The concentration of oxidising agent in the final solution may be chosen by the user as appropriate, and may be as set out in any of the embodiments in the table below.

[0240] Methods of Making Solutions of Reduced Polymer

[0241] The invention also provides methods of making a solution of a reduced polymer.

[0242] A method of making a solution of a reduced polymer may comprise providing any polymer as described and defined herein, and dissolving the polymer in a solvent comprising a reducing agent or in a solution comprising a reducing agent. The reducing agent may be any suitable reducing agent such as ethanedithiol (EDT), 0- mercaptoethanol, dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). A preferred reducing agent is TCEP.

[0243] A method of making a solution of a reduced polymer may comprise providing any polymer as described and defined herein, and dissolving the polymer in a solution comprising a reducing agent, such as TCEP.

[0244] A method of making a solution of a reduced polymer may comprise providing a polymer in solution wherein the polymer is any polymer as described and defined herein, and subjecting the polymer solution to reducing conditions.

[0245] The step of subjecting the polymer solution to reducing conditions may comprise contacting the polymer solution with a reducing agent. The reducing agent may be TCEP. Contacting the polymer solution with reducing agent may comprise forming a solution of polymer in reducing agent. For example, contacting the polymer solution with TCEP may comprise forming a solution of polymer in TCEP.

[0246] Contacting the polymer solution with reducing agent may comprise adding reducing agent to the polymer solution. For example, contacting the polymer solution with TCEP may comprise adding TCEP to the polymer solution.

[0247] Contacting the polymer solution with reducing agent may comprise adding reducing agent to the polymer solution to create a solution of polymer in reducing agent. For example, contacting the polymer solution with TCEP may comprise adding TCEP to the polymer solution to create a solution of polymer in TCEP.

[0248] In any of the aforementioned methods involving reducing agent, the concentration of reducing agent in the final solution may be 2.5mM. For example, in any of the aforementioned methods involving TCEP, the concentration of TCEP in the final solution may be 2.5mM. The concentration of reducing agent in the final solution may be chosen by the user as appropriate, and may alternatively be as set out in any of the embodiments in the table below.

[0249] Dimeric Coacervate-Forming Polymers

[0250] Any coacervate-forming polymer of the invention may also take the form of a “dimer” comprising or consisting of two “monomers”. Such a polymer is referred to as a “dimeric” coacervate-forming polymer.

[0251] In a dimeric coacervate-forming polymer according to the invention, the term “monomer” refers to any one of the coacervate-forming polymers described and defined herein and comprising or consisting of a structure according to formula (II):

[0252] Rl-N[Pl-P2-P3-P4]nC-R2 (II).

[0253] A dimeric coacervate-forming polymer therefore comprises or consists of two monomers, wherein the first monomer is any one of the coacervate-forming polymers described and defined herein comprising or consisting of a structure according to formula (II):

[0254] Ri-N[Pi-P2-P3-P4]nc-R2 (II); and wherein the second monomer is also any one of the coacervate-forming polymers described and defined herein comprising or consisting of a structure according to formula (II):

[0255] Rl-N[Pl-P2-P3-P4]nC-R2 (II).

[0256] Thus, for example, a dimeric coacervate-forming polymer may comprise one monomer which is a coacervate-forming polymer as further described and defined herein, and having five structural units (i.e. [Pi-P2-P3-P4]s), and a further monomer which is also a coacervate-forming polymer as further described and defined herein also having five structural units (z'.e. [Pi-P2-P3-P4]s), and wherein the first and second polymer “monomers” are joined together, such as via a disulphide bond. In this illustrative example, the dimeric polymer will have 10 structural units.

[0257] Each monomer of the dimer may have the same number of structural units. Alternatively, the two monomers of the dimer may have a different number of structural units.

[0258] The structural units of each monomer of the dimer may have the same amino acid residue / spacer structure. Alternatively, the two monomers of the dimer may have different amino acid residue / spacer structures.

[0259] The structural units of each monomer of the dimer may be in the same orientation as defined further herein. Alternatively, the structural units of each monomer of the dimer may be in a different orientation as defined further herein.

[0260] The structural units of each monomer of the dimer may have the same aromatic molecule at position Ri. Alternatively, the structural units of each monomer of the dimer may have different aromatic molecules at position Ri.

[0261] Coacervates & Polymer Coacervates

[0262] Coacervates

[0263] As noted previously, liquid-liquid phase separation (LLPS) is the physicochemical mechanism by which homogeneous solutions of macromolecules (typically polymers) can undergo a process of demixing. Two distinct phases are produced - concentrated macromolecule-rich phases and a macromolecule-poor diluted phase. The concentrated macromolecule-rich phases, when formed in solution or inside cells adopt a spheroidal appearance and can be described interchangeably as “globules”, “globular foci” or “particles”. These structures are also referred to as condensates, biomolecular condensates (BMCs), membraneless organelles, complex coacervates or merely coacervates. All of these terms can be used interchangeably and refer to the same structures. The term “coacervate” is used primarily herein for the purposes of consistency. Detection and monitoring of coacervates can be performed using any suitable method, as well as the methods set out in the examples below. Exemplary methods include microscopy, light scattering, flow cytometry, and microfluidic methods.

[0264] Coacervates can be detected using microscopy, e.g., differential interference contrast or fluorescence microscopy, to directly observe the particles at high magnification. With the aid of a computer, microscope images can be automatically obtained and analyzed. Additionally, microscopy can allow for continual or frequent monitoring of at least a portion of a mixture containing coacervates.

[0265] Coacervates can be detected using flow cytometry. In flow cytometry, one or more beams of light, e.g., each of a single wavelength, are directed onto a hydrodynamically-focused stream of fluid. Suspended coacervates passing through the beams scatter the light, and fluorescent chemicals found in the coacervates or attached to the coacervates may be excited. The scattered and / or fluorescent light is analysed by detectors within the device, from which information about coacervate size and fluorescence can be determined. Modem flow cytometers can analyze several thousand coacervates every second, in "real time", and can actively separate and isolate coacervates having specified properties.

[0266] Coacervates can be detected using cytometry methods, devices, and systems as disclosed e.g. in US patent application publication nos. US2009 / 0079963 and US2010 / 0179068, and international patent application publication no. W02009 / 112594.

[0267] Coacervates can be detected using microfluidic methods, devices, and systems. For example, coacervates can be detected using a lab-on-a-chip device or system, or the like (see, e.g., US patent application publication nos. US2009 / 0326903 and US2009 / 0297733).

[0268] Coacervates may typically be about 0.5-20 pm in size, e.g., between about any two sizes selected from 0.5, 1,1.5, 2, 2.5, 3,4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 pm (e.g., about 1-10 pm in size).

[0269] Coacervates may be smaller than about 0.5 pm in size. Phase-separated coacervates, including those that are smaller than about 0.5 pm in size, may be detected by changes in the turbidity of the solution. Changes in the turbidity of solutions can be measured by standard means and can be quantified typically according to the Formazin Turbidity Unit (FTU) or Formazin Nephelometric Unit (FNU). Other methods include size exclusion chromatography, including multiangle light scattering (SEC-MALS). However, Coacervates may be much larger in size, such as described in certain examples herein.

[0270] Coacervates may be larger than about 20 pm in size.

[0271] The concentration of coacervates may be approximately 10 to 5000 coacervates / nl, e.g., between any two numbers of coacervates selected from 10, 20, 50,100, 200, 500, 1000, 2000, and 5000 coacervates per nl, may be detected (e.g. about 100-500 coacervates per nl). The concentration of coacervates may be approximately 200-400 coacervates per nanolitre.

[0272] Coacervates can be concentrated using standard techniques such as centrifugation, and then resuspended to a given fixed concentration, e.g. as appropriate for a given purpose.

[0273] Polymer coacervates

[0274] The polymers of the invention are coacervate-forming polymers as described and defined further herein.

[0275] In certain examples described herein, coacervate formation was observed to occur in a simple system comprising merely of a solution of a polymer of the invention. In the majority of examples an exemplary polymer of the invention was used having 5- FAM as the exemplary Ri headgroup. Since 5-FAM is a fluorophore, it can be used to visualise the polymer under suitable conditions permitting fluorescence detection. By virtue of these visualisation techniques, the polymer was seen to strongly co-localise with and into the coacervates, indicating that the polymer localizes to the concentrated macromolecule-rich phase during the demixing process.

[0276] The observation that the polymers of the invention strongly co-localise with and into the coacervates under suitable conditions leads to the definition of a novel structural entity which is referred to herein as a polymer coacervate. A polymer coacervate comprises any one of the polymers of the invention as described and defined herein within the coacervate, wherein the polymer is capable of forming a polymer coacervate in an aqueous solution. By “within the coacervate” it is meant that the polymer is highly concentrated within any part of the coacervate structure, and conversely it is observed to be poorly concentrated and diffuse within the macromolecule-poor diluted phase of the demixed solution outside of the coacervate structure. Polymer coacervates may be detected by the same means for detecting coacervates as described above.

[0277] The size and appearance of the polymer coacervates which comprise any one of the polymers of the invention as described and defined herein may vary depending upon the structure of the polymer, e.g. including the exact amino acid sequence of the structural units and nature of the headgroup at position Ri.

[0278] Accordingly, any one of the coacervate-forming polymers of the invention as described and defined herein, may be capable of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate.

[0279] Any coacervate-forming polymer of the invention according to the formulae described herein may be tested to demonstrate its capability of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate. Accordingly, any polymer may be tested to determine if it is a coacervate-forming polymer according to the invention. A suitable test to determine that a polymer is a coacervate-forming polymer is described as follows.

[0280] 1) dissolve the polymer in mammalian tissue culture compatible media (TC media) such as DMEM (Dulbecco's modified Eagle's medium; Dulbecco R, Freeman G (1959). "Plaque production by the polyoma virus". Virology. 8 (3): pp 396-397);

[0281] 2) create a concentration dilution series of 320pM, 160pM, 80pM, 40pM, 20pM, 10pM, and 5pM of the polymer in TC media;

[0282] 3) transfer a 20pl aliquot of each TC media-polymer solution in the series into a separate well of an imaging plate;

[0283] 4) subject the imaging plate to centrifugation at 137g for 2 minutes at room temperature; 5) subject the imaging plate to brightfield microscopy and count the number of coacervates which are visible in an area of 218 pm x 218 pm, when observed with a 40x magnification objective lens;

[0284] 6) establish that a polymer is a coacervate-forming polymer when in an aliquot of TC media-polymer solution at any concentration in the series 10 or more coacervates are counted in the magnification area, preferably when 50 or more coacervates are counted in the magnification area, more preferably when 100 or more coacervates are counted in the magnification area.

[0285] Any coacervate-forming polymer of the invention according to the formulae described herein may be tested to demonstrate its capability of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate, and wherein the polymer coacervate is capable of penetrating into a eukaryotic cell upon contact with the cell in an aqueous solution. An aqueous solution may preferably be a mammalian tissue culture compatible media (TC media) such as DMEM (Dulbecco's modified Eagle's medium; Dulbecco R, Freeman G (1959). "Plaque production by the polyoma virus". Virology. 8 (3): pp 396-397.). A suitable test is described as follows.

[0286] 1) (a) if a head group (Ri group) of the polymer is fluorescent, then dissolve the polymer (the “test” polymer) in mammalian tissue culture compatible media (TC media) such as DMEM (Dulbecco's modified Eagle's medium;

[0287] Dulbecco R, Freeman G (1959). "Plaque production by the polyoma virus". Virology. 8 (3): pp 396-397.) to form a polymer solution; or (b) if the head group of the test polymer is not fluorescent, then mix the polymer with a “homologue” polymer, wherein the homologue polymer is identical to the test polymer except that a fluorophore is attached to the molecule which occupies the last position in the polymer at the C-terminal end of the polymer, wherein the fluorophore is preferably FAM, provided that the fluorophore and the head group are not the same and are distinguishable; then dissolve both the test polymer and the homologue polymer in TC media to form a mixed polymer solution, wherein the homologue polymer is present in the mixed polymer solution at a concentration such that it would be incapable of forming a polymer coacervate in an aqueous solution when present alone, but at a concentration at which it can be measurably detected, e.g. wherein the homologue polymer is present in the mixed polymer solution at a concentration of about IpM;

[0288] 2) create a concentration dilution series of 400pM, 200pM, lOOpM, 50pM, and 25 pM of the polymer solution or mixed polymer solution in TC media ;

[0289] 3) transfer a 1 Opl aliquot of each TC media-polymer solution in the series into a separate well of an imaging plate containing cultured mammalian cells, such as HEK293 cells, in 40pl of the same TC media;

[0290] 4) subject the imaging plate containing the cultured mammalian cells and the TC media-polymer solution to centrifugation at 137g for 1 minute at room temperature;

[0291] 5) subject the imaging plate containing the cultured mammalian cells and the TC media-polymer solution to 15 minutes to 2 hours of incubation in a mixture of air + 5% CO2 at 37°C;

[0292] 6) wash the imaging plate wells containing the cultured mammalian cells and the TC media-polymer solution 3 times, by adding and removing 50pl of fresh TC media;

[0293] 7) subject the cultured mammalian cells treated with the TC media-polymer solution to a cell impermeable DNA fluorescent dye (such as Sytox) e.g. between the concentrations 2.5 - 0.25 pM, to distinguish dead from live cells, and a cell permeable DNA fluorescent dye (such as Hoechst) e.g. between the concentrations of 0.5 - 5 pg / ml;

[0294] 8) subject the imaging plate wells containing the cultured mammalian cells treated with the TC media-polymer solution and the DNA dyes, to 3 washes, by adding and removing 50pl of fresh TC media;

[0295] 9) subject the imaging plate to fluorescent microscopy and: a. using the fluorescent properties of the two DNA dyes, identify the live cells visible in an area of 218 pm x 218 pm, when observed with a 40x magnification objective lens; b. using the fluorescent properties of the fluorescent head group of the test polymer, count the number of live cells that are fluorescent in an area of 218 pm x 218 pm, when observed with a 40x magnification objective lens; and c. if using a homologue polymer, using the fluorescent properties of the fluorophore of the homologue polymer, count the number of live cells that are fluorescent in an area of 218 pm x 218 pm, when observed with a 40x magnification objective lens; and

[0296] 10) establish that a polymer is a coacervate-forming polymer capable of penetrating into a eukaryotic cell, when, in an aliquot of the TC media- polymer solution at any concentration in the series, at least one live cell in the magnification area is identified and which emits fluorescence which is dependent upon the fluorescent properties of the head group or which is dependent upon the fluorescent properties of the fluorophore of the homologue polymer if used, preferably 50% or more of the live cells in the magnification area emit fluorescence which is dependent upon the fluorescent properties of the head group or which is dependent upon the fluorescent properties of the fluorophore of the homologue polymer if used, more preferably 100% of the live cells in the magnification area emit fluorescence which is dependent upon the fluorescent properties of the head group or which is dependent upon the fluorescent properties of the fluorophore of the homologue polymer if used.

[0297] The invention further provides a polymer coacervate, wherein the polymer coacervate comprises any polymer of the invention as described and defined herein within the coacervate.

[0298] Methods for Making Polymer Coacervate Aqueous Solutions and Compositions Accordingly, the invention further provides a method of making a polymer coacervate aqueous solution, the method comprising providing a stock solution comprising any polymer of the invention as described and defined herein and contacting a volume of the stock solution with a volume of an aqueous diluent solution thereby forming the polymer coacervate solution. The invention further provides a polymer coacervate aqueous solution, wherein the polymer coacervate aqueous solution is obtained by performing this method. The stock solution as described herein comprises any polymer of the invention dissolved in a coacervate incompetent solution, as described and defined further herein. The aqueous diluent solution as described herein is a coacervate competent solution, as described and defined further herein.

[0299] The invention further provides a composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises any polymer of the invention as described and defined herein within the coacervate.

[0300] The invention further provides a method of making a composition comprising a plurality of polymer coacervates in aqueous solution, the method comprising providing a stock solution of any polymer of the invention as described and defined herein; contacting a volume of the stock solution with a volume of an aqueous diluent solution, thereby forming a plurality of polymer coacervates in the aqueous diluent solution; and optionally concentrating the coacervates in the aqueous diluent solution and adjusting the concentration of the aqueous diluent solution to provide a defined amount of coacervates per volume of aqueous diluent solution. The invention further provides a composition comprising a plurality of polymer coacervates in aqueous solution, wherein the composition is obtained by performing this method.

[0301] In any of the aforementioned methods, the stock solution / coacervate incompetent solution may comprise an oxidising agent, such as dimethyl sulfoxide (DMSO), e.g. 30% DMSO in water.

[0302] In any of the aforementioned methods, the stock solution / coacervate incompetent solution may comprise a reducing agent, such as Tris(2- carboxyethyljphosphine hydrochloride (TCEP), e.g. 2.5mM TCEP in water. In any of the aforementioned methods, the aqueous diluent solution / coacervate competent solution may comprise an aqueous salt solution, a phosphate-buffered saline (PBS) solution, cell culture media or tissue culture media.

[0303] An aqueous salt solution salt may comprise one, more or all of sodium chloride, potassium chloride, sodium acetate and potassium acetate wherein the final salt concentration is about 20mM to about 500mM, and wherein the final pH is about 6.0 to about 9.0. The solution may further comprise divalent cations or other cofactors, but these are not necessary for coacervate formation.

[0304] A PBS solution may have the following final (lx) composition: NaCl at a concentration of 137 mmol / L, KC1 at a concentration of 2.7 mmol / L, Na2HPO4 at a concentration of 10 mmol / L and KH2PO4 at a concentration of 1.8 mmol / L, pH 7.4.

[0305] The cell / tissue culture media that are considered appropriate include the following

[0306] • Gibco DMEM (ThermoFisher)

[0307] • Gibco OptiMEM (ThermoFisher)

[0308] • Gibco EMEM (ThermoFisher)

[0309] • Gibco RPMI1640 (ThermoFisher)

[0310] • ExpiCHO Expression Medium (ThermoFisher)

[0311] • Gibco McCoy’s (ThermoFisher).

[0312] Also provided is a composition comprising a plurality of polymer coacervates according to the invention in a dried form, e.g. dried by air, dried using a vacuum concentrator system or dried under an inert gas, such as using a spray drier, or a drum drier, or a pulse combustion drier. A composition comprising a plurality of polymer coacervates according to the invention may exist in a lyophilized (freeze-dried) form. Any one of the compositions comprising a plurality of polymer coacervates according to the invention as described and defined herein may be provided in dried or lyophilized form, including compositions comprising a plurality of polymer coacervates wherein the polymer coacervates further comprise one or more cargo compounds within the coacervates, and further including pharmaceutical formulations comprising these compositions, as described and defined further herein. Absence of a Cofactor Requirement

[0313] An advantage of the coacervate-forming polymers of the invention as described and defined herein is that they are capable of forming coacervates independently, i.e. without the need for any additional molecule(s) to be present in order to form the coacervates (i.e. without the need for any additional “cofactor” or “cofactor(s)”).

[0314] For example, prior publications have noted a requirement for the presence of a crowding agent in the formation of coacervates in a number of systems (e.g. see Biswas, S. et al. Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates. Biomacromolecules 2023, 24, 11, pp4771- 4782.). Relevant crowding agents in this respect include sucrose, polyethylene glycol (PEG) and free RNA. In the examples provided herein, the inventors have demonstrated coacervate formation with the coacervate-forming polymers of the invention in the complete absence of any additional cofactors of the type described above.

[0315] Accordingly, any one of the coacervate-forming polymers of the invention as described and defined herein may be a coacervate-forming polymer wherein a cofactor is not required for coacervate formation mediated by the coacervate-forming polymer, or wherein coacervate formation mediated by the polymer excludes the use of a coacervate-forming cofactor. Any one of the coacervate-forming polymers of the invention as described and defined herein may be a coacervate-forming polymer wherein a crowding agent is not required for coacervate formation mediated by the coacervate-forming polymer, or wherein coacervate formation mediated by the polymer excludes the use of a crowding agent. Any one of the coacervate-forming polymers of the invention as described and defined herein may be a coacervate-forming polymer wherein a crowding agent such as RNA, sucrose and / or PEG is not required for coacervate formation mediated by the coacervate-forming polymer, or wherein coacervate formation mediated by the polymer excludes the use of a crowding agent such as RNA, sucrose and / or PEG. In any method described and defined herein, the process of coacervate formation mediated by the coacervate-forming polymers of the invention may be a process whereby a cofactor is excluded from the method. In any method described and defined herein, the process of coacervate formation mediated by the coacervate-forming polymers of the invention may be a process whereby a crowding agent is excluded from the method. In any method described and defined herein, the process of coacervate formation mediated by the coacervate-forming polymers of the invention may be a process whereby a crowding agent such as RNA, sucrose and / or PEG is excluded from the method.

[0316] Coacervates as Cargo Delivery Vehicles (Polymer-Cargo Coacervates)

[0317] As detailed in the examples below, the polymer of the invention, as described and defined herein, is seen to strongly co-localise with and into coacervates, thus forming polymer coacervates. This is demonstrated to occur in a wide range of aqueous environments, highlighting the generalised nature of this particular property. In addition, polymer coacervates are demonstrated in the examples below to be capable of penetrating into cells. This is demonstrated to occur in a wide variety of cells, also highlighting the generalised nature of this particular property. The inventors have further demonstrated that cargo compounds / molecules may be collocated into the polymer coacervates of the invention. This has been demonstrated for a wide range of different types of cargo compound / molecule, again also highlighting the generalised nature of this particular property.

[0318] The observation that the polymers of the invention and cargo compounds / molecules strongly co-localise with and into coacervates under suitable conditions leads to the definition of a further novel structural entity which is referred to herein as a polymer-cargo coacervate. A polymer-cargo coacervate comprises any one of the polymers of the invention as described and defined herein, and any one or more species of cargo compound / molecule within the coacervate. A cargo may be any suitable compound (also referred to as a molecule) that is desired to be comprised within a polymer coacervate, such as for delivery purposes. By one or more species of cargo compound it is meant that the polymer coacervate may contain one species of cargo compound, such as e.g. one particular antibody, or at least two species of cargo compound, such as e.g. two different antibodies, or e.g. an antibody and a DNA molecule.

[0319] Accordingly, the invention provides a polymer coacervate, wherein the polymer coacervate is a coacervate comprising any polymer of the invention as described and defined herein within the coacervates; and wherein the polymer coacervate further comprises one or more cargo compounds within the coacervates.

[0320] Types of Cargo Compound

[0321] In the examples below the inventors have demonstrated the capability of forming polymer coacervates with a wide range of different types of cargo compound / molecule, and that these polymer-cargo coacervates can penetrate into cells. In the examples, the inventors have demonstrated the feasibility of creating polymer- cargo coacervates with proteins, antibodies, DNA and mRNA. It is therefore expected that there is no particular restriction on the nature of the compound / molecule that can be incorporated within a polymer-cargo coacervate.

[0322] Accordingly, in any of the polymer coacervates described and defined herein involving a cargo compound, or in any one of the methods described and defined herein involving a cargo compound, the cargo compound may be a peptide, a polypeptide, a protein, an antibody or fragment thereof, a therapeutic protein, a DNA including ssDNA and dsDNA, an RNA including ssRNA and dsRNA, a modified nucleic acid, am enzyme, a carbohydrate, a lipid, a liposome, a nanoparticle and / or a small molecules.

[0323] Suitable types of cargo compound include the following:

[0324] Peptides, including:

[0325] • Peptide complexes

[0326] • Peptide conjugates

[0327] • Peptide-drug conjugates

[0328] • Peptide-antibody fusions (peptibodies) Cyclic peptides, including:

[0329] • Cyclic peptide complexes

[0330] • Cyclic peptide conjugates

[0331] • Cyclic peptide-drug conjugates

[0332] Bicyclic peptides, including:

[0333] • Bicyclic peptide complexes

[0334] • Bicyclic peptide conjugates

[0335] • Bicyclic peptide-drug conjugates

[0336] Polypeptides, including:

[0337] • Polypeptide complexes

[0338] • Polypeptide conjugates

[0339] • Polypeptide-drug conjugates

[0340] Proteins, including:

[0341] • Protein complexes

[0342] • Protein conjugates

[0343] • Protein-drug conjugates

[0344] • Designed ankyrin repeat proteins (DARPins)

[0345] • Fusion proteins

[0346] Antibodies, including:

[0347] • Full-length antibodies

[0348] • Bispecific antibodies

[0349] • Bispecific T cell Engagers (BiTEs)

[0350] • Antibody fragments, such as fragments consisting or comprising scFvs, diabodies, F(ab’)2, nanobodies such as sdAb and VHH

[0351] • Antibody-conjugates and antibody fragment-conjugates • antibody-drug conjugates and antibody fragment-drug conjugates

[0352] • Fc fusions

[0353] Therapeutic proteins, including hormones and enzymes, including (Dimitrov Methods Mol Biol. 2012; 899: 1-26):

[0354] • Insulin (blood glucose regulator)

[0355] • Pramlintide acetate (glucose control)

[0356] • Growth hormone GH (growth failure)

[0357] • Pegvisoman (growth hormone receptor antagonist)

[0358] • Mecasermin (IGF1, growth failure)

[0359] • Factor VIII (coagulation factor)

[0360] • Factor IX (coagulation factor)

[0361] • Protein C concentrate (anti-coagulation)

[0362] • al -proteinase inhibitor (anti-trypsin inhibitor)

[0363] • Erythropoietin (stimulates erythropoiesis)

[0364] • Filgrastim (granulocyte colony-stimulating factor, G-CSF; stimulates neutrophil proliferation)

[0365] • Sargramostim36, 37 (granulocytemacrophage colony-stimulating factor, GM-CSF)

[0366] • Oprelvekin (interleukin 11 , IL 11 )

[0367] • Human follicle-stimulating hormone (FSH)

[0368] • Human chorionic gonadotropin (HCG)

[0369] • Lutropin-a (human luteinizing hormone)

[0370] • Interleukin 2 (IL2)

[0371] • Denileukin diftitox (fusion of IL2 and Diphtheria toxin)

[0372] • Interferon alfacon 1 (consensus interferon)

[0373] • Interferon-a2a (IFNa2a)

[0374] • Interferon-a2b (IFNa2b)

[0375] • Interferon-an3 (IFNan3) • Interferon-pia (rIFN-0)

[0376] • Interferon-pib (rIFN-0)

[0377] • Intcrl'cron-v lb (IFNy)

[0378] • Salmon calcitonin (32-amino acid linear polypeptide hormone)

[0379] • Teriparatide (part of human parathyroid hormone 1-34 residues)

[0380] • Exenatide (Incretin mimetic with actions similar to glucagon-like peptide 1)

[0381] • Octreotide (octapeptide that mimics natural somatostatin)

[0382] • Dibotermin-a (recombinant human bone morphogenic protein 2)

[0383] • Recombinant human bone morphogenic protein 7

[0384] • Histrelin acetate (gonadotropin-releasing hormone; GnRH)

[0385] • Palifermin (keratinocyte growth factor, KGF)

[0386] • Becaplermin (platelet-derived growth factor, PDGF)

[0387] • Nesiritide (recombinant human B-type natriuretic peptide)

[0388] • Lepirudin (recombinant variant of hirudin, another variant is Bivalirudin)

[0389] • Anakinra (interleukin 1 (IL1) receptor antagonist)

[0390] • Enfuvirtide (an HIV- 1 gp41 -derived peptide)

[0391] DNA-based cargo compounds, including:

[0392] • ssDNA;

[0393] • dsDNA;

[0394] • DNA plasmids;

[0395] • DNA oligonucleotides;

[0396] • DNA aptamers;

[0397] • antisense DNA

[0398] • DNA acidzymes

[0399] • DNA-based probes such as molecular beacons, FISH probes such as single molecule FISH (smFISH) probes, oligopaint probes

[0400] • DNA-based hybrid chain reaction (HCR) hairpins

[0401] • Morpholines RNA-based cargo compounds, including,

[0402] • Messenger RNA (mRNA) including mRNA vaccines;

[0403] • Antisense RNA (asRNA);

[0404] • RNA interference (RNAi)

[0405] • Small interfering RNA (siRNA);

[0406] • Micro RNA (miRNA);

[0407] • RNA activation (RNAa)

[0408] • Small activating RNAs (saRNAs)

[0409] • Self-amplifying RNA (saRNA); and

[0410] • RNA aptamers

[0411] • Riboswitches

[0412] • Ribozymes

[0413] Modified nucleic acid cargo compounds, including:

[0414] • Peptide nucleic acid (PNA)

[0415] • Locked nucleic acid (LNA)

[0416] • Unlocked nucleic acid (UNA)

[0417] • Bridged nucleic acid (BNA)

[0418] Carbohydrates

[0419] Lipids

[0420] Liposomes

[0421] Nanoparticles

[0422] Small molecules

[0423] Proteolysis targeting chimeras (PROTACs)

[0424] Proteins and complexes for gene targeting, editing and modification, including:

[0425] • Zinc finger nucleases (ZFNs)

[0426] • Transcription activator-like effector nucleases (TALENs) • Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related proteins and / or guide RNA (or DNA encoding CRISPR and CRISPR-related proteins and / or guide RNA) including the following proteins or derivatives thereof:

[0427] • Casl3

[0428] • Cas3

[0429] • Cas8a / Cas5

[0430] • Cas8b

[0431] • Cas8c

[0432] • CaslOd

[0433] • Csel, Cse2

[0434] • Csyl / Csy2 / Csy3

[0435] • GSU0054

[0436] • CaslO

[0437] • Csm2

[0438] • Cmr5

[0439] • CaslO / Csxl l

[0440] • CsxlO

[0441] • Csfl

[0442] • Cas9 Csn2

[0443] • Cas4 Casl2

[0444] • Casl2a (Cpfl) Casl2b (C2cl)

[0445] • Casl2c (C2c3)

[0446] • Casl2d (CasY)

[0447] • Casl2e (CasX)

[0448] • Casl2f (Casl4, C2cl0)

[0449] • Casl2g

[0450] • Casl2h

[0451] • Casl2i

[0452] • Casl2k (C2c5) • C2c4 / C2c8 / C2c9

[0453] • Casl3

[0454] • Casl3a (C2c2)

[0455] • Casl3b

[0456] • Casl3c

[0457] • Casl3d

[0458] • Casl3x.l

[0459] Linkage of Polymer and Cargo

[0460] In any of the polymer-cargo coacervates described and defined herein, the cargo compound(s) / molecule(s) may be present within the polymer coacervate without having any physical linkage with the polymer. The inventors have demonstrated in the examples that polymer-cargo coacervates can be created by the simple mixing of polymer and cargo, and polymer-cargo coacervates can thus be formed without the polymer and cargo needing to be physically connected.

[0461] Nevertheless, in any of the polymer-cargo coacervates described and defined herein, or in any one of the methods described and defined herein involving a cargo compound, the cargo compound(s) / molecule(s) may indeed have a physical linkage with the polymer.

[0462] Accordingly, a cargo compound may be connected to a polymer of the invention within a polymer-cargo coacervate.

[0463] A cargo compound may be connected to a polymer of the invention within a polymer-cargo coacervate by a chemical bond, such as a covalent bond.

[0464] A cargo compound may be connected to a polymer of the invention within a polymer-cargo coacervate by a linker.

[0465] Examples of linkers are any of the “spacers” described and defined herein.

[0466] A linker may be connected to the cargo at one end of the linker by a covalent bond, and the linker may be connected to the polymer at the opposite end also by a covalent bond. The cargo may be connected to the polymer at any suitable location, for example via the headgroup at position Ri, or via any suitable amino acid side chain or spacer position in the structural unit of the polymer.

[0467] One general method of connecting a cargo compound and a polymer is via “click chemistry”. Several types of click chemistry are available. For example, a free amino group may be provided on the cargo compound and a terminal azide group may be provided on the polymer. Alternatively, a free amino group may be provided on the polymer and a terminal azide group may be provided on the cargo compound. The free amino group can react, via an amidation reaction, e.g. with an N-hydroxysuccinimide (NHS) functionalised bicyclononyne (BCN) to create a BCN-functionalised position. The azide group can then be covalently attached, e.g. via a copper-free click chemistry, to the BCN-functionalised position. DBCO (dibenzocyclooctyne) click chemistry provides alternative methods. In these methods, a DBCO compound can be used to activate (functionalise) one molecule, and this is then reacted with the other molecule which has been activated / functionalised with an azide group. Reaction leads to the conjugation of the two molecules which become joined via a triazole linker. Click chemistry is used extensively to conjugate molecules for a wide range of biological applications and has wide and versatile applicability.

[0468] A linker which connects a cargo compound and a polymer may be a cleavable linker. Examples of suitable cleavable linkers are included in the table below.

[0469]

[0470] A linker which connects a cargo compound and a polymer may be a peptide linker. Examples of suitable peptide linkers are included in the table below.

[0471] Methods for Making Polymer-Cargo Coacervate Aqueous Solutions and

[0472] Compositions The inventors have devised methods for making polymer-cargo coacervate aqueous solutions and compositions.

[0473] Any one of the coacervate-forming polymers of the invention as described and defined herein may be dissolved in an aqueous solution and stored as a stock solution, wherein the said solution on its own is not competent to promote liquid-liquid demixing and coacervate formation. When a coacervate-forming polymer of the invention is dissolved in such a solution, no coacervates are formed. Such a solution is referred to herein as a coacervate incompetent solution. Examples of such solutions include polymers dissolved in water or an organic solvent such as DMSO (Dimethyl sulfoxide), e.g. 100% water; 100% organic solvent such as DMSO, or any mixture of organic solvent such as DMSO and water, as described further herein.

[0474] In contrast, any one of the coacervate-forming polymers of the invention may be contacted with an aqueous solution / diluent solution which is competent to promote liquid-liquid demixing and coacervate formation. When a coacervate-forming polymer of the invention is contacted with such a solution, coacervates are formed. Such a solution is referred to herein as a coacervate competent solution. Examples of such solutions include an aqueous salt solution, a phosphate-buffered saline (PBS) solution, cell culture media or tissue culture media. Any one of the coacervate-forming polymers of the invention may be contacted with a coacervate competent solution by contacting a suitable amount the polymer in dried form with a suitable volume of the coacervate competent solution such that coacervate formation occurs. Any one of the coacervateforming polymers of the invention may be contacted with a coacervate competent solution by contacting a suitable amount of the polymer dissolved in a coacervate incompetent solution, as described above, with a suitable volume of a coacervate competent solution, as described above, such that coacervate formation occurs. In these methods the cargo compounds may be mixed with the polymer in the coacervate incompetent solutions.

[0475] Accordingly, the invention provides a method of making a polymer coacervate aqueous solution comprising polymer coacervates comprising one or more cargo compounds, the method comprising:

[0476] A. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;

[0477] (ii) contacting a volume of the cargo solution with any one of the polymers of the invention as described and defined herein to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting the polymer is in a dried form, or is dissolved in a coacervate incompetent solution; and

[0478] (iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates;

[0479] B. (i) providing a polymer solution of any one of the polymers of the invention as described and defined herein, wherein the solution is a coacervate incompetent solution;

[0480] (ii) contacting a volume of the polymer solution with one or more cargo compounds to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting the cargo compounds(s) is in a dried form or is dissolved in a coacervate incompetent solution; and

[0481] (iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates;

[0482] C. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;

[0483] (ii) contacting a volume of the cargo solution with a volume of a coacervate competent solution to form a further cargo solution;

[0484] (iii) contacting a volume of the further cargo solution with any one of the polymers of the invention as described and defined herein, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprises the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting the polymer is in a dried form or is dissolved in a coacervate incompetent solution;

[0485] D. (i) providing one or more cargo compounds in a dried form; (ii) contacting the one or more cargo compounds with a volume of a coacervate competent solution to form a cargo solution;

[0486] (iii) contacting a volume of the cargo solution with any one of the polymers of the invention as described and defined herein, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting, the polymer is in a dried form, or is dissolved in a coacervate incompetent solution;

[0487] E. (i) providing a polymer solution of any one of the polymers of the invention as described and defined herein, wherein the solution is a coacervate incompetent solution;

[0488] (ii) contacting a volume of the polymer solution with a volume of a coacervate competent solution thereby forming a solution comprising a plurality of polymer coacervates;

[0489] (iii) contacting a volume of the solution comprising a plurality of polymer coacervates with one or more cargo compounds, whereupon the cargo compound(s) localise into the polymer coacervates thereby forming polymer coacervates comprising the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting the cargo compound(s) is in a dried form or is dissolved in a coacervate incompetent solution;

[0490] F. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is any one of the polymers of the invention as described and defined herein, and wherein the coacervates comprises the polymer within the coacervates, optionally wherein the composition is lyophilised; (ii) providing a cargo compound solution comprising one or more cargo compounds in a coacervate competent solution;

[0491] (iii) contacting a volume of the composition with a volume of the cargo compound solution, whereupon the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates; or

[0492] G. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is any one of the polymers of the invention as described and defined herein, and wherein the coacervates comprise the polymer within the coacervates, optionally wherein the composition is lyophilised;

[0493] (ii) providing one or more cargo compounds in a dried form; and

[0494] (iii) (a) contacting a volume of the composition with a volume of a coacervate competent solution, followed by contacting the said solution with the one or more cargo compounds;

[0495] (b) contacting the one or more cargo compounds with a volume of a coacervate competent solution, followed by contacting the said solution with a volume of the composition; or

[0496] (c) simultaneously contacting the one or more cargo compounds and a volume of the composition with a volume of a coacervate competent solution; whereupon in each of (a), (b) and (c) the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates.

[0497] In any of the above described methods, the one or more cargo compounds may be any one or more cargo compound as described and defined herein. The invention also provides a method of making a composition in aqueous solution comprising a plurality of polymer coacervates comprising one or more cargo compounds, the method comprising making a polymer coacervate aqueous solution by performing any one of the above described methods of making a polymer coacervate aqueous solution, concentrating the coacervates in the solution and adjusting the concentration of the solution to provide a defined amount of coacervates per volume of solution.

[0498] In any of the aforementioned methods, the coacervate incompetent solution may comprise an oxidising agent, such as dimethyl sulfoxide (DMSO), e.g. 30% DMSO in water.

[0499] In any of the aforementioned methods, the coacervate incompetent solution of the polymer may comprise a reducing agent, such as Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), e.g. 2.5mM TCEP in water.

[0500] In any of the aforementioned methods, the coacervate competent solution may comprise an aqueous salt solution, a phosphate-buffered saline (PBS) solution, cell culture media or tissue culture media.

[0501] An aqueous salt solution salt may comprise one, more or all of sodium chloride, potassium chloride, sodium acetate and potassium acetate wherein the final salt concentration is about 20mM to about 500mM, and wherein the final pH is about 6.0 to about 9.0. The solution may further comprise divalent cations or other cofactors, but these are not necessary for coacervate formation.

[0502] A PBS solution may have the following final (lx) composition: NaCl at a concentration of 137 mmol / L, KC1 at a concentration of 2.7 mmol / L, Na2HPC>4 at a concentration of 10 mmol / L and KH2PO4 at a concentration of 1.8 mmol / L, pH 7.4.

[0503] The cell / tissue culture media that are considered appropriate include the following

[0504] • Gibco DMEM (ThermoFisher)

[0505] • Gibco OptiMEM (ThermoFisher)

[0506] • Gibco EMEM (ThermoFisher)

[0507] • Gibco RPMI1640 (ThermoFisher)

[0508] • ExpiCHO Expression Medium (ThermoFisher) Gibco McCoy’s (ThermoFisher).

[0509] Pharmaceutical

[0510] Polymer coacervates of the invention may be formulated into pharmaceutical compositions.

[0511] A pharmaceutical composition comprising a polymer coacervate of the invention may be formulated as a non-frozen liquid coacervate suspension.

[0512] A pharmaceutical composition comprising a polymer coacervate of the invention may be formulated as a frozen liquid (cryopreserved) coacervate suspension.

[0513] A formulation may include one or more pharmaceutically acceptable excipient such as buffers, salts, sugars or polyols, stability enhancers, polysaccharides, amino acids, nucleosides, antioxidants, organic solvents and proteins or polypeptides.

[0514] A buffer may be included in a formulation to provide for pH control and tonicity. Examples include tris, histidine, sodium acetate

[0515] Salt may be included in a formulation to provide for tonicity and stabilization. Examples include sodium chloride, potassium chloride, magnesium chloride.

[0516] Sugars or polyols may be included in a formulation to provide for tonicity, stabilization and cryoprotection (if relevant). Examples include trehalose, sucrose, glucose (reducing sugar), penta-isomaltose and mannitol.

[0517] Stability enhancers may be included in a formulation such as glycerol, ethylene glycol, dextran, hydroxycellulose, lactobionic acid and disaccharides.

[0518] Polysaccharides may be included in a formulation to provide for collapse temperature modification (if relevant). Examples include dextran and hydroxyethyl starch.

[0519] Amino acids may be included in a formulation to provide for stabilization, tonicity, pH control and cryoprotection (if relevant). Examples include cell culture media components.

[0520] Nucleosides may be included in a formulation to provide for stabilization.

[0521] Examples include adenosine and guanosine. Antioxidants may be included in a formulation to provide for oxidation prevention. Examples include methionine and sodium edetate.

[0522] Organic solvents may be included in a formulation to provide for cryoprotection (if relevant). Examples include glycerol, ethylene glycol and DMSO.

[0523] Proteins or polypeptides may be included in a formulation to provide for stabilization; cryoprotection (if relevant). Examples include HSA, FCS, human plasma, poly-L-lysine.

[0524] Pharmaceutical formulations may be provided in a unit dose form.

[0525] Cargo Delivery, Uses and Medical Uses

[0526] The invention provides a method for delivering one or more cargo compounds into eukaryotic cells, the method comprising providing any one of the polymer coacervate aqueous solutions comprising one or more cargo compounds as described and defined herein, or any one of the compositions comprising a plurality of polymer coacervates comprising one or more cargo compounds as described and defined herein, or any one of the pharmaceutical formulation as described and defined herein, and introducing the solution or composition into an aqueous environment which comprises a plurality of eukaryotic cells, whereupon the polymer coacervates penetrate the plurality of cells and thereby release and deliver the one or more cargo compounds into the eukaryotic cells.

[0527] The aqueous environment which comprises the plurality of eukaryotic cells may be an in vitro or ex vivo cell culture environment. The aqueous environment which comprises the plurality of eukaryotic cells may be an in vivo cell culture or in vivo environment.

[0528] Accordingly, any one of the polymers as described and defined herein, any one of the polymer coacervate aqueous solutions as described and defined herein, any one of the polymer coacervate aqueous solutions comprising one or more cargo compounds as described and defined herein, or any one of the compositions comprising a plurality of polymer coacervates comprising one or more cargo compounds as described and defined herein, or any one of the pharmaceutical formulation as described and defined herein may be provided for a wide range of uses, including the following.

[0529] • Delivery of gene editing components into cells and performing the steps of gene editing. Gene editing components may comprise a CRISPR / Cas or equivalent nuclease, one or more guide RNAs for targeting the CRISPR / Cas or equivalent nuclease to a target nucleic acid sequence of interest, and optionally a template nucleic acid for facilitating homologous recombination with the target acid sequence of interest.

[0530] • Delivery of therapeutic molecules into cells for therapeutic purposes, such as delivery of small molecule therapeutic agents, protein or peptide therapeutic agents, therapeutic antibodies or antibody fragments.

[0531] • Delivery of protein encoding nucleic acids, such as mRNA or DNA templates, for the purpose of expressing therapeutic proteins in the target cells. Expressed proteins may include antibodies, antigens, or enzyme replacement products. Any one of the polymers as described and defined herein, any one of the polymer coacervate aqueous solutions as described and defined herein, any one of the polymer coacervate aqueous solutions comprising one or more cargo compounds as described and defined herein, or any one of the compositions comprising a plurality of polymer coacervates comprising one or more cargo compounds as described and defined herein, or any one of the pharmaceutical formulation as described and defined herein may be provided for use in the treatment of a disease in a subject in need thereof, the methods may comprise administering an effective amount of the polymer, the polymer coacervate, the polymer coacervate aqueous solution or the composition to the subject.

[0532] The invention further provides a method of treating a disease or a disorder in an individual in need of treatment, the method comprising administering to the individual an effective amount of any one of the polymers as described and defined herein, any one of the polymer coacervate aqueous solutions as described and defined herein, any one of the polymer coacervate aqueous solutions comprising one or more cargo compounds as described and defined herein, or any one of the compositions comprising a plurality of polymer coacervates comprising one or more cargo compounds as described and defined herein, or any one of the pharmaceutical formulation as described and defined herein.

[0533] Exemplary Embodiments

[0534] A number of individual exemplary structural units and embodiments are listed in the tables below.

[0535] Tables Al, A2 and A3 - Exemplary Structural Units and Polymers.

[0536] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural unit, or in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0537] A. position Pi is occupied by a molecule selected from the group Al;

[0538] B. position P2 is occupied by a molecule selected from the group A3;

[0539] C. position P3 is occupied by a molecule selected from the group A2; and

[0540] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in any one or more structural unit, or in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table Al below.

[0541] Table Al - Exemplary Structural Units.

[0542] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein: A. position Pi is occupied by a molecule selected from the group Al;

[0543] B. position P2 is occupied by a molecule selected from the group A3; C. position P3 is occupied by a molecule selected from the group A2; and

[0544] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table A2 below.

[0545] Table A2 - Exemplary Polymers.

[0546] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein: A. position Pi is occupied by a molecule selected from the group Al;

[0547] B. position P2 is occupied by a molecule selected from the group A3;

[0548] C. position P3 is occupied by a molecule selected from the group A2; and

[0549] D. position P4 is occupied by a molecule selected from the group A3; and further wherein the polymer has the structure of any one of the polymers identified in Table A3 below. Table A3 - Further Exemplary Polymers.

[0550] Tables Bl and B2 - Exemplary Structural Units and Polymers.

[0551] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural unit, or in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0552] A. position Pi is occupied by a molecule selected from the group A2;

[0553] B. position P2 is occupied by a molecule selected from the group A3;

[0554] C. position P3 is occupied by a molecule selected from the group Al; and D. position P4 is occupied by a molecule selected from the group A3; and further wherein in any one or more structural unit, or in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table Bl below. Table Bl - Exemplary Structural Units.

[0555] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0556] A. position Pi is occupied by a molecule selected from the group A2;

[0557] B. position P2 is occupied by a molecule selected from the group A3;

[0558] C. position P3 is occupied by a molecule selected from the group Al; and

[0559] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table B2 below.

[0560] Table B2 - Exemplary Polymers.

[0561] Tables Cl and C2 - Exemplary Structural Units and Polymers.

[0562] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural unit, or in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0563] A. position Pi is occupied by a molecule selected from the group Al;

[0564] B. position P2 is occupied by a molecule selected from the group A2;

[0565] C. position P3 is occupied by a molecule selected from the group A3; and

[0566] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in any one or more structural unit, or in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table Cl below. Table Cl - Exemplary Structural Units. A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0567] A. position Pi is occupied by a molecule selected from the group Al;

[0568] B. position P2 is occupied by a molecule selected from the group A2; C. position P3 is occupied by a molecule selected from the group A3; and

[0569] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table C2 below. Table C2 - Exemplary Polymers. Tables DI and D2 - Exemplary Structural Units and Polymers.

[0570] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural unit, or in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0571] A. position Pi is occupied by a molecule selected from the group A2;

[0572] B. position P2 is occupied by a molecule selected from the group Al;

[0573] C. position P3 is occupied by a molecule selected from the group A3; and

[0574] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in any one or more structural unit, or in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table DI below.

[0575] Table DI - Exemplary Structural Units.

[0576] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in all structural units, each one of Pi, P2, P3 and P4 consists of one position, wherein:

[0577] A. position Pi is occupied by a molecule selected from the group A2;

[0578] B. position P2 is occupied by a molecule selected from the group Al; C. position P3 is occupied by a molecule selected from the group A3; and

[0579] D. position P4 is occupied by a molecule selected from the group A3; and further wherein in all structural units of the polymer, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table D2 below.

[0580] Table D2 - Exemplary Polymers.

[0581] Table E - Exemplary Structural Units

[0582] A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units:

[0583] A. position Pi is occupied by two amino acid residues, wherein: i. the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; and

[0584] B. position P2: i. is occupied by one amino acid residue, which is a molecule selected from the group Al; or ii. is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; or iii. is occupied by one amino acid residue, which is a molecule selected from the group A2; and

[0585] C. position P3 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; and D. position P4 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G-G; and further wherein in any one or more structural units, or in all structural units, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table El below. Table El - Exemplary Structural Units.

[0586] Any of the polymers having a structural unit identified in Table El above may have the specific polymer structure identified in Table E2 below. Table E2 - Exemplary Polymers.

[0587] Table F - Exemplary Structural Units and Polymers A coacervate-forming polymer of the invention may be a coacervate-forming polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units:

[0588] A. position Pi: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; and

[0589] B. position P2: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; and

[0590] C. position P3: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; and

[0591] D. position P4: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; and further wherein in any one or more structural units, or in all structural units, positions Pi, P2, P3 and P4 are occupied by the molecules identified in Table Fl below. Table Fl - Exemplary Structural Units.

[0592] Any of the polymers having a structural unit identified in Table Fl above may have the specific polymer structure identified in Table F2 below.

[0593] Table F2 - Exemplary Polymers.

[0594] General Definitions

[0595] As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0596] References to items in the singular are also to be interpreted as references to items in the plural, unless the context clearly dictates otherwise. For example, in general references to “a polymer”, “a cargo compound” and “a coacervate” should be interpreted as references to a plurality / multitude of polymers, cargo compounds and coacervates unless the context clearly dictates otherwise. Aspects of the Invention

[0597] 1. A coacervate-forming polymer comprising or consisting of a structure according to formula (I):

[0598] Rl-N[Pl-P2-P3-P4]nC (I) wherein:

[0599] A. Ri is occupied by one or more aromatic molecules;

[0600] B. N is the N-terminal end of the polymer;

[0601] C. [P1-P2-P3-P4] is a structural unit of the polymer;

[0602] D. n is the number of structural units of the polymer, wherein n is an integer of 2 or more;

[0603] E. C is the C-terminal end of the polymer;

[0604] F. P is an amino acid residue position or a spacer position in the polymer; and

[0605] G. each one of Pi, P2, P3 and P4consists of one or more amino acid residue positions and / or one or more spacer positions, wherein: i. one or more of said positions is occupied by a molecule selected from the group Al consisting of arginine (R), histidine (H) and lysine (K); and ii. one or more additional position(s) is occupied by a molecule selected from the group A2 consisting of tyrosine (Y), phenylalanine (F), and tryptophan (W); and iii. one or more further additional positions are occupied by a molecule selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G-G-G), glycine- glycine-glycine-glycine (G-G-G-G), or any spacer.

[0606] 2. A polymer according to aspect 1, wherein the spacer in group A3 comprises:

[0607] A. a molecule comprising 3 carbon bonds (C3 spacer), e.g. 0-alanine;

[0608] B. a molecule comprising 4 carbon bonds (C4 spacer), e.g. 4-aminobutyric acid or two glycine residues; C. a molecule comprising 5 carbon bonds (C5 spacer), e.g. 5-aminovaleric acid;

[0609] D. a molecule comprising 6 carbon bonds (C6 spacer), e.g. 6-aminohexanoic acid; or

[0610] E. a polyethylene glycol (PEG) spacer, e.g. a PEG2 spacer (8-amino-3,6- dioxaoctanoic acid), a PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), or a PEG4 spacer (15-amino-4,7,10,13-tetraoxapenta-decanoic acid).

[0611] 3. A polymer according to aspect 1 or aspect 2, wherein in the direction N-terminal to C-terminal:

[0612] A. all structural units of the polymer are ordered in the sequence [P1-P2-P3-P4];

[0613] B. the first structural unit at the N-terminal end of the polymer is ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer is ordered in the sequence [P4-P3-P2-P1];

[0614] C. the first structural unit at the N-terminal end of the polymer is ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer is ordered in the sequence [P1-P2-P3-P4];

[0615] D. the last structural unit at the C-terminal end of the polymer is ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer is ordered in the sequence [P4-P3-P2-P1];

[0616] E. the last structural unit at the C-terminal end of the polymer is ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer is ordered in the sequence [P1-P2-P3-P4]; or

[0617] F. all structural units of the polymer are ordered in the sequence [P4-P3-P2-P1].

[0618] 4. A polymer according to any one of the preceding aspects, wherein n is an integer of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more, preferably wherein n is 5 or more.

[0619] 5. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein: A. position Pi is occupied by a molecule selected from the group Al;

[0620] B. position P2 is occupied by a molecule selected from the group A3;

[0621] C. position P3 is occupied by a molecule selected from the group A2; and

[0622] D. position P4 is occupied by a molecule selected from the group A3.

[0623] 6. A polymer according to aspect 5, wherein in any one or more structural units, or in all structural units:

[0624] 1. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G;

[0625] 2. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;

[0626] 3. a) position Pi is occupied by H; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;

[0627] 4. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by W; and d) position P4 is occupied by G-G; a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by F; and d) position P4 is occupied by G-G; a) position Pi is occupied by R; b) position P2 is occupied by a spacer which is: i. P-alanine; ii. y-aminobutyric acid; or iii. 5-aminovaleric acid; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G; a) position Pi is occupied by R; b) position P2 is occupied by a spacer which is y-aminobutyric acid; c) position P3 is occupied by Y ; and d) position P4 is occupied by a spacer which is y-aminobutyric acid; a) position Pi is occupied by R; b) position P2 is occupied by A; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G; a) position Pi is occupied by R; b) position P2 is occupied by G-G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G; a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G-G; or a) position Pi is occupied by R; b) position P2 is occupied by G-G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G; a) position Pi is occupied by H; b) position P2 is occupied by G; c) position P3 is occupied by F; and d) position P4 is occupied by G-G; a) position Pi is occupied by H; b) position P2 is occupied by G c) position P3 is occupied by W; and d) position P4 is occupied by G-G; a) position Pi is occupied by K; b) position P2 is occupied by G c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G; or 15. a) position Pi is occupied by K; b) position P2 is occupied by G c) position P3 is occupied by W; and d) position P4 is occupied by G-G.

[0628] 7. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:

[0629] A. position Pi is occupied by a molecule selected from the group A2;

[0630] B. position P2 is occupied by a molecule selected from the group A3;

[0631] C. position P3 is occupied by a molecule selected from the group Al; and

[0632] D. position P4 is occupied by a molecule selected from the group A3.

[0633] 8. A polymer according to aspect 7, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by Y ; b) position P2 is occupied by G; c) position P3 is occupied by R; and d) position P4 is occupied by G-G.

[0634] 9. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:

[0635] A. position Pi is occupied by a molecule selected from the group Al;

[0636] B. position P2 is occupied by a molecule selected from the group A2;

[0637] C. position P3 is occupied by a molecule selected from the group A3; and

[0638] D. position P4 is occupied by a molecule selected from the group A3. 10. A polymer according to aspect 9, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by R; b) position P2 is occupied by Y ; c) position P3 is occupied by G; and d) position P4 is occupied by G.

[0639] 11. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:

[0640] A. position Pi is occupied by a molecule selected from the group A2;

[0641] B. position P2 is occupied by a molecule selected from the group Al;

[0642] C. position P3 is occupied by a molecule selected from the group A3; and

[0643] D. position P4 is occupied by a molecule selected from the group A3.

[0644] 12. A polymer according to aspect 11, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by Y ; b) position P2 is occupied by R; c) position P3 is occupied by G; and d) position P4 is occupied by G.

[0645] 13. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units:

[0646] A. position Pi is occupied by two amino acid residues, wherein: i. the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; and

[0647] B. position P2: i. is occupied by one amino acid residue, which is a molecule selected from the group Al; or ii. is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; or iii. is occupied by one amino acid residue, which is a molecule selected from the group A2; and

[0648] C. position P3 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; and

[0649] D. position P4 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G-G.

[0650] 14. A polymer according to aspect 13, wherein n=3, and wherein in the direction N- terminal to C-terminal:

[0651] A. in the first structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; ii. position P2 is occupied by one amino acid residue, which is a molecule selected from the group Al and is R; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G; and

[0652] B. in the second structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G; and

[0653] C. in the third structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; ii. position P2 is occupied by one amino acid residue which is a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G.

[0654] 15. A polymer according to any one of aspects 1-4, wherein in any one or more structural units, or in all structural units:

[0655] A. position Pi: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; and

[0656] B. position P2: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; and

[0657] C. position P3: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; and

[0658] D. position P4: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G.

[0659] 16. A polymer according to aspect 15, wherein n=3, and wherein in the direction N- terminal to C-terminal:

[0660] A. in the first structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by a molecule selected from the group A3 and is G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G; and

[0661] B. in the second structural unit: i. position Pi is occupied by a molecule selected from the group A3 and is G-G; ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G; and

[0662] C. in the third structural unit: i. position Pi is occupied by a molecule selected from the group A3 and is G-G; ii. position P2 is occupied by a molecule selected from the group A3 and is G; iii. position P3 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; and iv. position P4 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y.

[0663] 17. A polymer according to any one of the preceding aspects, wherein Ri is one or more aromatic molecules, wherein Ri has a total aromaticity value of 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more or 35 or more, preferably the aromaticity value is between 5 and 35, more preferably the aromaticity value is 18 or 20.

[0664] 18. A polymer according to any one of the preceding aspects, wherein Ri comprises or consists of any one or more of the molecules:

[0665] A. fluorescein or a fluorescein amidite (FAM), preferably 5 -FAM (5- carboxyfluorescein);

[0666] B. 5-FAM-PEG2;

[0667] C. Rhodamine B;

[0668] D. Hexachloro-fluorescein (HEX);

[0669] E. naphtyl-alanyl-4-naphtyl butanoic acid;

[0670] F. pyrenebutyric acid;

[0671] G. fluorenylmethoxycarbonyl protecting group (FMOC)-PEG2;

[0672] H. FMOC;

[0673] I. carboxytetramethylrhodamine (TAMRA);

[0674] J. 4-naphthalen-2-yl-butanoic acid;

[0675] K. anthracene propanoic acid;

[0676] L. naproxyl;

[0677] M. phenylbutanoic acid;

[0678] N. 5 -(piperazin- l-yl)pyrazine-2-carboxylic acid; or

[0679] O. an aromatic amino acid, such as phenylalanine, tyrosine, histidine, or tryptophan.

[0680] I l l 19. A polymer according to aspect 16, wherein Ri consists of two or more of the molecules A-O, wherein the two or more molecules are the same molecules or different molecules.

[0681] 20. A polymer according to any one of the preceding aspects, wherein the aromatic molecule Ri is attached to the N-terminus of the first structural unit of the polymer by a linker, optionally wherein the linker is any one of the spacers (A) to (E) defined in aspect 2.

[0682] 21. A polymer according to aspect 5, wherein at least one structural unit of the polymer has the amino acid sequence:

[0683] 1. [R-G-Y-G];

[0684] 2. [R-G-Y-G-G];

[0685] 3. [H-G-Y-G-G];

[0686] 4. [R-G-W-G-G];

[0687] 5. [R-G-F-G-G];

[0688] 6. [R-P-alanine-Y-G-G];

[0689] 7. [R-y-aminobutyric acid-Y-G-G];

[0690] 8. [R-5-aminovaleric acid-Y-G-G];

[0691] 9. [R-y-aminobutyric acid-Y-y-aminobutyric acid];

[0692] 10. [R-A-Y-G-G];

[0693] 11. [R-G-G-Y-G-G];

[0694] 12. [R-G-Y-G-G-G];

[0695] 13. [R-G-G-Y-G];

[0696] 14. [H-G-F-G-G];

[0697] 15. [H-G-W-G-G];

[0698] 16. [K-G-Y-G-G]; or

[0699] 17. [K-G-W-G-G]; optionally wherein: A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / or

[0700] B. all structural units of the polymer have the same amino acid sequence.

[0701] 22. A polymer according to aspect 7, wherein at least one structural unit of the polymer has the amino acid sequence:

[0702] [Y-G-R-G-G]; optionally wherein:

[0703] A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / or

[0704] B. all structural units of the polymer have the same amino acid sequence.

[0705] 23. A polymer according to aspect 9, wherein at least one structural unit of the polymer has the amino acid sequence:

[0706] [R-Y-G-G]; optionally wherein:

[0707] A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / or

[0708] B. all structural units of the polymer have the same amino acid sequence.

[0709] 24. A polymer according to aspect 11, wherein at least one structural unit of the polymer has the amino acid sequence:

[0710] [Y-R-G-G]; optionally wherein:

[0711] A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / or

[0712] B. all structural units of the polymer have the same amino acid sequence.

[0713] 25. A polymer according to any one of aspects 22-25, wherein: n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10; or n=10 or more, preferably wherein n=5. 26. A polymer according to aspect 13, wherein:

[0714] A. n=3

[0715] B. the first structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [RYRGGGGG];

[0716] C. the second structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [YRYRGGGGG]; and

[0717] D. the third structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [YRYGGGGG].

[0718] 27. A polymer according to aspect 15, wherein:

[0719] A. n=3

[0720] B. the first structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [RYRYGGG];

[0721] C. the second structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [GGRYRYGG]; and

[0722] D. the third structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [GGGRYRY],

[0723] 28. A polymer according to any one of the preceding aspects, wherein position Ri is occupied by FAM or two tryptophan amino acids (WW).

[0724] 29. A polymer according to any one of the preceding aspects, wherein formula (I) is further defined as formula (II):

[0725] Rl-N[Pi-P2-P3-P4]nC-R2 (II) wherein R2 is any chemical group or molecule that is capable of forming a bond, preferably a covalent bond, more preferably a disulphide bond, with a further R2 group of a second polymer according to any one of the preceding aspects, preferably wherein the molecule at position R2 is cysteine. 30. A dimeric coacervate-forming polymer comprising or consisting of two monomers, wherein each monomer comprises or consists of a coacervate-forming polymer according to any one of aspects 1 to 28, wherein each monomer has a structure further defined by formula (II):

[0726] Rl-N[Pi-P2-P3-P4]nC-R2 (II); and wherein the two monomers are linked by a bond between each R2 group, preferably a covalent bond, more preferably a disulphide bond, optionally wherein the two monomers have the same structure, or wherein the two monomers have different structures.

[0727] 31. A polymer according to any one of the preceding aspects, wherein the polymer is capable of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate.

[0728] 32. A polymer according to any one of aspects 1 to 30, wherein the polymer is capable of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate, and wherein the polymer coacervate is capable of penetrating into a viable eukaryotic cell upon contact with the cell in an aqueous solution supporting the viability of the cell.

[0729] 33. A polymer according to any one of the preceding aspects, wherein a cofactor, such as a crowding agent, e.g. RNA, sucrose or polyethylene glycol (PEG), is not required for coacervate formation mediated by the polymer.

[0730] 34. A method of making a polymer solution, the method comprising providing a polymer, contacting the polymer with a solvent or solution, and dissolving the polymer in the solvent or solution, thereby creating the polymer solution, wherein the polymer is defined according to any one of aspects 1 to 33. 35. A method of making a solution of an oxidised polymer, the method comprising providing a polymer in solution, wherein the polymer is defined according to any one of aspects 1 to 33, and subjecting the polymer solution to oxidising conditions.

[0731] 36. A method according to aspect 35, wherein the subjecting the polymer solution to oxidising conditions comprises exposing the polymer solution to air for 2 or more hours, e.g. from 2 hours to 24 hours, preferably in dark conditions at room temperature, or wherein the oxidising conditions comprise adding an oxidising agent to the polymer solution, optionally wherein the oxidising agent is dimethyl sulfoxide (DMSO).

[0732] 37. A solution of an oxidised polymer, wherein the solution is obtained by performing the method of aspect 35 or aspect 36.

[0733] 38. A polymer coacervate, wherein the polymer coacervate comprises a polymer as defined according to any one of aspects 1 to 33 within the coacervate.

[0734] 39. A method of making a polymer coacervate aqueous solution, the method comprising providing a stock solution comprising a polymer as defined according to any one of aspects 1 to 33, and contacting a volume of the stock solution with a volume of an aqueous diluent solution thereby forming the polymer coacervate solution.

[0735] 40. A polymer coacervate aqueous solution, wherein the polymer coacervate aqueous solution is obtained by performing the method of aspect 39.

[0736] 41. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises a polymer as defined according to any one of aspects 1 to 33 within the coacervate.

[0737] 42. A method of making a composition comprising a plurality of polymer coacervates in aqueous solution, the method comprising providing a stock solution of a polymer as defined according to any one of aspects 1 to 33, contacting a volume of the stock solution with a volume of an aqueous diluent solution thereby forming a plurality of polymer coacervates in the aqueous diluent solution; and optionally concentrating the coacervates in the aqueous diluent solution and adjusting the concentration of the aqueous diluent solution to provide a defined amount of coacervates per volume of aqueous diluent solution.

[0738] 43. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein the composition is obtained by performing the method of aspect 42.

[0739] 44. A polymer coacervate, wherein the polymer coacervate is a coacervate comprising a polymer as defined according to any one of aspects 1 to 33 within the coacervates; and wherein the polymer coacervate further comprises one or more cargo compounds within the coacervates.

[0740] 45. A method of making a polymer coacervate aqueous solution comprising polymer coacervates comprising one or more cargo compounds, the method comprising:

[0741] A. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;

[0742] (ii) contacting a volume of the cargo solution with a coacervate-forming polymer according to any one of aspects 1 to 33 to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting, the polymer is in a dried form, or is dissolved in a coacervate incompetent solution; and

[0743] (iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates; B. (i) providing a polymer solution comprising a coacervate-forming polymer according to any one of aspects 1 to 33, wherein the solution is a coacervate incompetent solution;

[0744] (ii) contacting a volume of the polymer solution with one or more cargo compounds to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting, the cargo compounds(s) is in a dried form or is dissolved in a coacervate incompetent solution; and

[0745] (iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates;

[0746] C. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;

[0747] (ii) contacting a volume of the cargo solution with a volume of a coacervate competent solution to form a further cargo solution;

[0748] (iii) contacting a volume of the further cargo solution with a coacervate-forming polymer according to any one of aspects 1 to 33, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting the polymer is in a dried form or is dissolved in a coacervate incompetent solution;

[0749] D. (i) providing one or more cargo compounds in a dried form;

[0750] (ii) contacting the one or more cargo compounds with a volume of a coacervate competent solution to form a cargo solution; (iii) contacting a volume of the cargo solution with a coacervate-forming polymer according to any one of aspects 1 to 33, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting, the polymer is in a dried form, or is dissolved in a coacervate incompetent solution;

[0751] E. (i) providing a polymer solution of a coacervate-forming polymer according to any one of aspects 1 to 33, wherein the solution is a coacervate incompetent solution;

[0752] (ii) contacting a volume of the polymer solution with a volume of a coacervate competent solution thereby forming a solution comprising a plurality of polymer coacervates;

[0753] (iii) contacting a volume of the solution comprising a plurality of polymer coacervates with one or more cargo compounds, whereupon the cargo compound(s) localise into the polymer coacervates thereby forming polymer coacervates comprising the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting, the cargo compound(s) is in a dried form or is dissolved in a coacervate incompetent solution;

[0754] F. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is a coacervate-forming polymer according to any one of aspects 1 to 33, and wherein the coacervates comprise the polymer within the coacervates, optionally wherein the composition is lyophilised;

[0755] (ii) providing a cargo compound solution comprising one or more cargo compounds in a coacervate competent solution;

[0756] (iii) contacting a volume of the composition with a volume of the cargo compound solution, whereupon the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates; or

[0757] G. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is a coacervate-forming polymer according to any one of aspects 1 to 33, and wherein the coacervates comprise the polymer within the coacervates, optionally wherein the composition is lyophilised;

[0758] (ii) providing one or more cargo compounds in a dried form; and

[0759] (iii) (a) contacting a volume of the composition with a volume of a coacervate competent solution, followed by contacting the said solution with the one or more cargo compounds;

[0760] (b) contacting the one or more cargo compounds with a volume of a coacervate competent solution, followed by contacting the said solution with a volume of the composition; or

[0761] (c) simultaneously contacting the one or more cargo compounds and a volume of the composition with a volume of a coacervate competent solution; whereupon in each of (a), (b) and (c) the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates.

[0762] 46. A polymer coacervate aqueous solution comprising one or more cargo compounds, wherein the polymer coacervate aqueous solution is obtained by performing the method of aspect 45.

[0763] 47. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein polymer coacervates of the plurality comprise a polymer as defined according to any one of aspects 1 to 33 within the coacervates; and further comprise one or more cargo compounds within the coacervates.

[0764] 48. A method of making a composition in aqueous solution comprising a plurality of polymer coacervates comprising one or more cargo compounds, the method comprising making a polymer coacervate aqueous solution by performing the method of aspect 45, concentrating the coacervates in the solution and adjusting the concentration of the solution to provide a defined amount of coacervates per volume of solution.

[0765] 49. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein polymer coacervates of the plurality comprise polymer and one or more cargo compounds within the coacervates, and wherein the composition is obtained by performing the method of aspect 48.

[0766] 50. A method according to any one of aspects 39, 42, 45 or 48, or a polymer coacervate aqueous solution according to aspect 29 or aspect 35; wherein the coacervate incompetent solution comprises water, optionally water and dimethylsulfoxide (DMSO), preferably a solution of 30% DMSO in water.

[0767] 51. A method according to any one of aspects 39, 42, 45 or 48, or a polymer coacervate aqueous solution according to aspect 40 or aspect 46; wherein the coacervate competent solution comprises a cell culture media.

[0768] 52. A polymer coacervate according to aspect 44, a method according to any one of aspects 45(a), 48(a), 50, or 51, a polymer coacervate aqueous solution according to aspect 46, or a composition according to aspect 47 or aspect 49; wherein the cargo compound is linked to the polymer, preferably wherein the cargo compound is linked to the polymer via a covalent bond or via a linker, optionally a cleavable linker.

[0769] 53. A polymer coacervate according to aspect 44 or aspect 52, a method according to any one of aspects 45, 48, 50, 51 or 52; a polymer coacervate aqueous solution according to any one of aspects 46, 50, 51 or 52; or a composition according to any one of aspects 47, 49 or 52; wherein the one or more cargo compounds comprises any one or more of a peptide, a polypeptide; a polypeptide complex; an antibody or fragment thereof; a polypeptide-drug conjugate; an antibody-drug conjugate; a nucleic acid, such as a single-stranded or double-stranded DNA or a single-stranded or double-stranded RNA; a peptide nucleic acid (PNA); a locked nucleic acid (LNA); an unlocked nucleic acid (UNA); a bridged nucleic acid (BNA); a carbohydrate, a lipid, a nanoparticle or a small molecule.

[0770] 54. A method for delivering one or more cargo compounds into eukaryotic cells, the method comprising providing a polymer coacervate aqueous solution comprising one or more cargo compounds as defined according to any one of aspects 46, 50 or 51, or a composition comprising a plurality of polymer coacervates comprising one or more cargo compounds as defined according to any one of aspects 47, 45 or 49, and introducing the solution or composition into an aqueous environment which comprises a plurality of eukaryotic cells, whereupon the polymer coacervates penetrate the plurality of cells and thereby release and deliver the one or more cargo compounds into the eukaryotic cells.

[0771] 55. A method according to aspect 54, wherein the aqueous environment which comprises the plurality of eukaryotic cells is an in vitro or ex vivo cell culture environment.

[0772] 56. A method according to aspect 54, wherein the aqueous environment which comprises the plurality of eukaryotic cells is an in vivo environment.

[0773] 57. The use of a polymer according to any one of aspects 1 to 33 in the formation of a polymer coacervate, wherein the coacervate comprises the polymer within the coacervate. 58. The use of a polymer according to any one of aspects 1 to 33 in the production of a composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises the polymer within the coacervate.

[0774] 59. The use of a polymer according to any one of aspects 1 to 33 and one or more cargo compounds in the formation of a polymer coacervate, wherein the coacervate comprises the polymer and the one or more cargo compounds within the coacervate.

[0775] 60. The use of a polymer according to any one of aspects 1 to 33 and one or more cargo compounds in the production of a composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises the polymer and the cargo compound within the coacervate.

[0776] 61. The use according to aspect 59 or aspect 60, wherein the one or more cargo compounds comprises any one or more of the compounds defined in aspect 53.

[0777] 62. The use according to any one of aspects 57 to 61, wherein a cofactor, such as a crowding agent, e.g. RNA, sucrose or polyethylene glycol (PEG), is not required for coacervate formation mediated by the polymer.

[0778] 63. A pharmaceutical formulation comprising a pharmaceutically acceptable vehicle and a polymer according to any one of aspects 1 to 33, a polymer coacervate according to any one of aspects 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of aspects 40, 46, 52 or 53, or a composition according to any one of aspects 41, 43, 47, 49, 52 or 53.

[0779] 64. A pharmaceutical formulation according to aspect 63, wherein the formulation is in a unit dose form. 65. A polymer according to any one of aspects 1 to 33, a polymer coacervate according to any one of aspects 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of aspects 40, 46, 52 or 53, a composition according to any one of aspects 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to aspect 63 or aspect 64 for use as a medicament.

[0780] 66. The use of a polymer according to any one of aspects 1 to 33, a polymer coacervate according to any one of aspects 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of aspects 40, 46, 52 or 53, a composition according to any one of aspects 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to aspect 63 or aspect 64 in the manufacture of a medicament.

[0781] 67. A polymer according to any one of aspects 1 to 33, a polymer coacervate according to any one of aspects 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of aspects 40, 46, 52 or 53, a composition according to any one of aspects 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to aspect 63 or aspect 64 for use in the treatment of a disease in a individual in need thereof, the method comprising administering an effective amount of the polymer, the polymer coacervate, the polymer coacervate aqueous solution or the composition.

[0782] 68. A method of treating a disease or a disorder in an individual in need of treatment, the method comprising administering to the individual an effective amount of a polymer according to any one of aspects 1 to 33, a polymer coacervate according to any one of aspects 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of aspects 40, 46, 52 or 53, a composition according to any one of aspects 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to aspect 63 or aspect 64. EXAMPLES

[0783] The invention is illustrated by the following Examples. The Examples are provided to illustrate the invention but not to limit the invention.

[0784] Example 1. General Procedures

[0785] Polymer oxidation

[0786] Polymers containing a C-terminal cysteine perform better when oxidation is actively promoted with this protocol. Polymers that have not undergone the “Polymer Oxidation” procedure are referred as “un-treated”.

[0787] Materials

[0788] • Dry polymer in 1.5 ml centrifuge tube.

[0789] • 30% DMSO solution (in water).

[0790] • 1.5 ml centrifuge tube stand.

[0791] Aluminum foil.

[0792] Method

[0793] 1. The polymer is resuspended in 30% DMSO solution up to a stock concentration of 5 mM to 10 mM.

[0794] 2. The centrifuge tube is left open to the air, in a tube stand, and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[0795] 3. The volume of solvent lost during the procedure is replaced with water.

[0796] 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C. Imaging plate preparation

[0797] The plates used in all the examples were black with clear bottoms and coated with poly-D-Lysine. They were prepared in sterile conditions inside a cell culture hood, using standard mammalian cell culture procedures and best practices.

[0798] Materials

[0799] • 384 well black plate with optically clear polystyrene bottom for imaging.

[0800] • Poly-D-Lysine (PDL) solution, sterile for tissue culture.

[0801] • lx PBS, sterile for tissue culture.

[0802] • Water, sterile for tissue culture.

[0803] Methods

[0804] 1. Dilute Poly-D-Lysine (PDL) in sterile lx PBS to prepare a 50 pg / ml working solution.

[0805] 2. Fill one third of the volume of the wells of the imaging plate with the PDL solution.

[0806] 3. Leave at room temperature for 1 hour or overnight at 4 °C.

[0807] 4. Remove the PDL and tap the plate dry with paper towels.

[0808] 5. Wash plate with sterile water 3 times.

[0809] 6. Keep at 4 °C for up to 1 week.

[0810] Coacervate formation and imaging Materials

[0811] Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[0812] • Polymer (DMSO-treated or un-treated).

[0813] • Tissue culture media.

[0814] • Centrifuge tubes or PCR plates.

[0815] • Molecular biology grade water.

[0816] • Plate centrifuge.

[0817] • Microscope with brightfield capabilities.

[0818] Methods

[0819] Coacervates are microscopic structures visible in brightfield with a microscope, that result from liquid-liquid demixing and phase separation (LLPS). The procedure to make coacervates consists, in simple terms, in mixing the polymer under appropriate buffer conditions, e.g. by mixing with tissue culture media.

[0820] When performing dilution curves to test different final or assay concentrations (Cf) (e.g. 320, 160, 80, 40, 20, 10 or 5 pM), these can be done in water before LLPS is induced (by mixing the polymer with buffer, such as tissue culture medium), or directly, in tissue culture media (that is, after induction of LLPS) (Table 1). In either case, use centrifuge tubes or PCR plates to perform the dilution curves by adding the polymer to either water or to tissue culture media. Furthermore, LLPS can be induced by adding the polymer to tissue culture media at 5x to lx the final polymer concentration of the assay, and mixing by pipetting, at which point the coacervates form (Table 1). At this stage, coacervates are allowed to pre-incubate in solution for 0 to 60 minutes. Table 1 summarises several variations on the procedure for coacervate formation of the polymers. Table 1. Method examples for coacervate formation. Cf - final or assay concentration; LLPS - liquid-liquid phase separation; C - concentration; DC - dilution curve; TC media - tissue culture media; LLPS C - concentration at which LLPS occurs; Top DC C - top concentration of the dilution curve.

[0821] Method Variations #1 #2 #3 #4 #5 #6

[0822] LLPS @ 5x Cf 2x Cf lx Cf 5x Cf 2x Cf lx Cf

[0823] 1stLLPS, 1stLLPS, 1stLLPS, 1stDilution 1stDilution 1stDilution 2ndDilution 2ndDilution 2ndDilution Curve, Curve, Curve,

[0824] °rderCurve Curve Curve 2ndLLPS 2ndLLPS 2ndLLPS

[0825] DC

[0826] , . TC media TC media TC media Water Water Water solvent

[0827] Top

[0828] Assay C Highest concentration tested

[0829] (pM) g

[0830] LLPS C 5x Top 2x Top lx Top 5x Top 2x Top lx Top

[0831] ° (pM) Assay C Assay C Assay C Assay C Assay C Assay C

[0832] Top DC C 5x Top 2x Top lx Top 50x Top 30x Top 25x Top

[0833] (pM) Assay C Assay C Assay C Assay C Assay C Assay C

[0834] Mix polymer with TC media in the

[0835] „fwells of the top row of a 96W PCR Mix polymer in water in the wells of eP plate. the top row of a 96W PCR plate

[0836] LLPS is induced at this stage.

[0837] Perform a 1 :2 dilution series by transferring half of the coacervate mixture in the wells of the top row of Perf°rma L2 dilution series by a 96W PCR plate, into the same transferring half of the volume of the

[0838] © volume of TC media in the wells of polymer in the wells of the top row of

[0839] ® Step 2 the adjacent row of the PCR plate. a 96W PCR plate, into the same

[0840] S Repeat step until the dilution series isv°lumeof water in the wells of the completed adjacent row of the PCR plate. Repeat step until the dilution series is

[0841] The complex coacervates can be pre- completed incubated in solution for 0 to 60 minutes.

[0842] Add 1 Add 1 Add Add 1 Add 1 Add 1

[0843] Ste 3v°ume°f volume of coacervate volume of volume of volume of coacervate coacervate mixture the polymer the polymer the polymer mixture into mixture into the to 9 to 14 to 25 the wells of into the empty volumes of volumes of volumes of an imaging wells of an wells of the TC media TC media TC media plate imaging imaging containing plate plate 4 volumes containing LLPS is induced at this stage. The ofTC 1 volume coacervates can be pre-incubated in media ofTC solution for 0 to 60 minutes media

[0844] Add 1

[0845] Add 1 volume of volume of coacervate Add coacervate mixture coacervate mixture into the mixture into the wells of an into the

[0846] Step 4 wells of an imaging empty imaging plate wells of the plate containing imaging containing 4 volumes plate 1 volume of ofTC TC media media

[0847] Once ready, the imaging plate containing the coacervates is centrifuged at standard conditions compatible with mammalian cell culture. The plate is loaded into the microscope and images of the coacervates are acquired. Figure 1 shows the coacervates formed by LLPS of FAM-[RGYGG]5C, using the different method variations described above. All the various methods resulted in the formation of coacervates.

[0848] Mixed coacervate formation and imaging

[0849] Mixed coacervates result from the liquid-liquid demixing and phase separation of a mixture of two or more molecules or polymers. The molecule or polymer that promotes LLPS is designated as vehicle, whereas the molecule that admixes with the vehicle, but that is unable to undergo LLPS by itself, is designated as cargo. Materials

[0850] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[0851] • Polymer or vehicle (DMSO-treated or un-treated).

[0852] • Cargo (e.g. protein, antibody, ssDNA, capped mRNA, other).

[0853] • Tissue culture media.

[0854] • Centrifuge tubes or PCR plates.

[0855] • Molecular biology grade water.

[0856] • Plate centrifuge.

[0857] • Microscope.

[0858] Methods

[0859] The procedure for mixed coacervation is similar to the methods previously described for coacervate formation in Table 1, with the following differences:

[0860] 1. Mix 1 volume of the vehicle at 50x the final concentration (Cf) with 1 volume of the cargo at 50x the final concentration in a centrifuge tube.

[0861] 2. Add 8 volumes or 23 volumes of tissue culture media to the vehicle and cargo mixture and mix by pipetting. At this point the complex coacervates form at either 5x or 2x the Cf, respectively.

[0862] 3. The complex coacervates can be pre-incubated in solution for 0 to 60 minutes.

[0863] 4. If coacervation is induced at 5x Cf, then one volume of coacervate mixture is added to 4 volumes of tissue culture media contained in the well of the imaging plate. If coacervation is induced at 2x Cf, then one volume of coacervate mixture is added to 1 volume of tissue culture media contained in the well of the imaging plate.

[0864] 5. The mixture of mixed coacervates is centrifuged at standard conditions compatible with mammalian cell culture.

[0865] 6. The imaging plate is loaded into the microscope and images of the mixed coacervates are acquired.

[0866] Coacervate formation and cell entry

[0867] The entire procedure is performed in a cell culture hood, using standard mammalian cell culture procedures and best practices. Mammalian cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[0868] Cell entry and diffusion can be defined by presence of peptide diffused in the cytosol, diffused in the nucleus, or diffused in both. Cell entry can also be defined by the presence of peptide in cytosolic puncta.

[0869] Materials

[0870] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL) previously seeded with mammalian cells.

[0871] • Polymer (DMSO-treated or un-treated).

[0872] • Tissue culture media.

[0873] • Centrifuge tubes or PCR plates.

[0874] • Molecular biology grade water.

[0875] • Plate centrifuge.

[0876] • Microscope. Methods

[0877] As with the procedure for coacervate formation and imaging described in Table 1, the methods used to form coacervates for cell entry assays, encompass dilution curves performed in water before LLPS, dilution curves performed in tissue culture after LLPS, and LLPS occurring at 5x or 2x the final assay concentration. Table 2 contains instructions on different variations to the procedure to determine if a polymer can enter mammalian cells.

[0878] The coacervate formation and cell entry procedure starts by washing the cells and replacing their conditioning media with either 4 volumes or 2 volumes of the same tissue culture media used to induce LLPS.

[0879] Table 2. Method examples for coacervate production to be used in cell entry assays. Cf - final concentration; LLPS - liquid-liquid phase separation; C - concentration; DC - dilution curve; TC media - Tissue culture media. LLPS C - concentration at which LLPS occurs; Top DC C - top concentration of the dilution curve. LLPS is induced at this stage.

[0880] Perform a 1 :2 dilution series by transferring half of the coacervate mixture in the wells of the top row of Perform a 1 :2 dilution series by a 96W PCR plate, into the same transferring half of the volume of the volume of TC media in the wells of polymer in the wells of the top row of Step 2 ^e adjacent row of the PCR plate. a 96W PCR plate, into the same

[0881] Repeat step until the dilution series is volume of water in the wells of the completed. adjacent row of the PCR plate.

[0882] Repeat step until the dilution series is

[0883] The complex coacervates can be precompleted incubated in solution for 0 to 60 minutes.

[0884] Add 1 volume of Add 1 volume of Add 1 volume of Add 1 volume of coacervate coacervate the polymer to 9 the polymer to 14 mixture into the mixture into the volumes of TC volumes of TC wells of an wells of an media media

[0885] Step 3 imaging plate imaging plate containing 4 containing 1 LLPS is induced at this stage. The volumes of TC volume of TC coacervates can be pre-incubated in media media solution for 0 to 60 minutes

[0886] Add 1 volume of Add 1 volume of coacervate coacervate mixture into the mixture into the wells of an wells of an

[0887] Step 4 imaging plate imaging plate containing 4 containing 1 volumes of TC volume of TC media media The imaging plate is centrifuged at standard conditions for mammalian cell culture. The cells are incubated in standard conditions of temperature and CO2 for mammalian cell culture, between 15 minutes to 4 hours, preferably between

[0888] 15 minutes to 2 hours, preferably for 1 hour. The mixture of coacervates is washed from the cells with Tissue Culture media adequately supplemented for the mammalian cell type in use. The imaging plate is loaded into the microscope and images are taken of the cells. Mixed coacervate formation and cargo delivery

[0889] The entire procedure is performed in a cell culture hood, using standard mammalian cell culture procedures and best practices. Mammalian cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[0890] Cell delivery can be defined by the presence of the cargo diffused in the cytosol, diffused in the nucleus, or diffused in both. Cell delivery can also be defined by the presence of fluorescent cytosolic puncta. Cell delivery can also be defined by the observation of the expected physiological outcome of the cargo entering the cell (e.g. expression of protein coded by mRNA delivered as cargo).

[0891] Materials

[0892] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL) previously seeded with mammalian cells.

[0893] • Polymer or vehicle (DMSO-treated or un-treated).

[0894] • Cargo (Protein, Antibody, ssDNA, capped mRNA).

[0895] • Tissue culture media.

[0896] • Centrifuge tubes or PCR plates.

[0897] • Molecular biology grade water.

[0898] • Plate centrifuge.

[0899] • Microscope.

[0900] Methods

[0901] The procedure for cargo delivery into mammalian cells build from the processes of coacervate formation and cell entry and mixed coacervate formation and imaging. 1. This procedure starts by washing the cells and replacing their conditioning media with either 4 volumes or 2 volumes of the same tissue culture media used to induce LLPS.

[0902] 2. Mix 1 volume of the vehicle at 50x the final concentration (Cf) with 1 volume of the cargo at 50x the final concentration in a centrifuge tube.

[0903] 3. Add 8 volumes or 23 volumes of tissue culture media to the vehicle and cargo mixture and mix by pipetting. At this point the mixed coacervates form at either 5x or 2x the Cf, respectively.

[0904] 4. The complex coacervates can be pre-incubated in solution for 0 to 60 minutes.

[0905] 5. If coacervation is induced at 5x Cf, then one volume of coacervate mixture is added to 4 volumes of tissue culture media contained in the well of the imaging plate. If coacervation is induced at 2x Cf, then 2 volumes of coacervate mixture are added to 2 volumes of tissue culture media contained in the well of a clear bottom plate.

[0906] 6. The imaging plate is centrifuged at standard conditions for mammalian cell culture.

[0907] 7. The cells are incubated in standard conditions of temperature and CO2 for mammalian cell culture, between 15 minutes to 4 hours, preferably between 15 minutes to 2 hours, preferably for 1 hour.

[0908] 8. The mixture of coacervates is washed from the cells with Tissue Culture media adequately supplemented for the mammalian cell type in use.

[0909] 9. The imaging plate is loaded into the microscope and images are taken of the cells. Example 2. FAM-1RGYGG15C forms coacervates in vitro in different cell culture media.

[0910] Purpose and summary of experiment

[0911] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C will undergo liquid-liquid phase separation (LLPS) and form coacervates in a broad representative range of tissue culture media, regardless of supplementation with Foetal Bovine Serum (FBS). Un-supplemented Tissue Culture media is defined as any media formulation that is used in tissue culture to maintain mammalian cell lines, that has not been supplemented with any Serum, such as Foetal Bovine Serum.

[0912] Three different tissue culture media that are commonly used in mammalian cell culture were tested, with and without the addition of FBS, up to 10% final concentration.

[0913] Three different final concentrations of the polymer FAM-[RGYGG]5C were tested (40 pM, 20 pM, and 10 pM).

[0914] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[0915] Materials and methods

[0916] • FAM-[RGYGG]5C.

[0917] • OptiMEM tissue culture media.

[0918] • DMEM tissue culture media.

[0919] • EMEM tissue culture media.

[0920] • Foetal Bovine Serum, heat inactivated (FBS).

[0921] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[0922] • CX7 LZR PRO Microscope. Peptide oxidation

[0923] 1. Resuspend FAM-[RGYGG]5C in 30%DMSO (in water) up to a stock concentration of 5 mM, in a centrifuge tube.

[0924] 2. The tube is left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[0925] 3. Replace the volume of solvent lost during the procedure with water.

[0926] 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C.

[0927] Prepare the tissue culture media

[0928] 1. Reserve half of the volume of OptiMEM, DMEM, and EMEM.

[0929] 2. Add FBS to the other half of the volume of OptiMEM, DMEM, and EMEM up to a final volume of 10% FBS.

[0930] 3. The resultant un-supplemented tissue culture media are: a. OptiMEM b. DMEM c. EMEM.

[0931] 4. The resultant supplemented tissue culture media are: a. DMEM + 10%FBS b. OptiMEM + 10%FBS c. EMEM + 10%FBS.

[0932] Coacervate formation and imaging

[0933] 1. Add 4 volumes of Tissue Culture media to the respective wells of the 384W plate. 2. In a centrifuge tube, add FAM-[RGYGG]5C to the Tissue Culture media to 5x final concentration.

[0934] 3. Mix FAM-[RGYGG]5C into the Tissue Culture media by pipetting, at which point the coacervates form.

[0935] 4. Add 1 volume of coacervate mixture to the 4 volumes of fresh Tissue Culture media contained in the well of a clear bottom plate. Match like with like: e.g. coacervates made in OptiMEM added to wells containing OptiMEM.

[0936] 5. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[0937] 6. Load the plate containing the coacervates into the CX7 LZR PRO microscope and acquire images of the coacervates.

[0938] Results and conclusion

[0939] FAM-[RGYGG]5C will undergo LLPS and form coacervates when mixed into all tissue culture media tested, whether these were supplemented with 10% FBS or not. Furthermore, all final concentrations of FAM-[RGYGG]5C tested (40 pM, 20 pM, and 10 pM) resulted in the formation of coacervates.

[0940] Figure 2 shows the brightfield images obtained for the coacervates produced in this example.

[0941] Example 3. FAM-1RGYGG15C coacervates formed in different tissue culture media can enter mammalian cells.

[0942] Purpose and summary of experiment

[0943] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C will form coacervates, and enter and diffuse inside HEK293 cells, regardless of the tissue culture media used, or their supplementation with Foetal Bovine Serum (FBS). Three different tissue culture media that are commonly used in mammalian cell culture were tested, with and without the addition of FBS, up to 10% final concentration.

[0944] Three different final concentrations of the polymer FAM-[RGYGG]5C were tested (40 pM, 20 pM, and 10 pM).

[0945] After induction of LLPS, the coacervates were added to HEK293 cells, previously seeded into 384W imaging plates. The cells we incubated with the coacervates and observed on a microscope.

[0946] Hoechst 34580 was used to stain and identify the nuclei, and Sytox Deep Red was used to identify dead or permeabilised cells.

[0947] Materials and methods

[0948] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[0949] • FAM-[RGYGG]5C.

[0950] • Hoechst 34580.

[0951] • Sytox Deep Red.

[0952] • Sterile OptiMEM tissue culture media.

[0953] • Sterile DMEM tissue culture media.

[0954] • Sterile EMEM tissue culture media.

[0955] • Sterile Foetal Bovine Serum, heat inactivated (FBS).

[0956] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[0957] • HEK293 cells.

[0958] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[0959] • CX7 LZR PRO Microscope. Peptide oxidation

[0960] 1. Resuspend FAM-[RGYGG]5C in 30% DMSO (in water) up to a stock concentration of 5 mM, in a centrifuge tube.

[0961] 2. The tube is left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[0962] 3. Replace the volume of solvent lost during the procedure with water.

[0963] 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C.

[0964] Cell culture

[0965] HEK293 cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[0966] Prepare the tissue culture media

[0967] 1. Reserve half of the volume of OptiMEM, DMEM, and EMEM.

[0968] 2. Add FBS to the other half of the volume of OptiMEM, DMEM, and EMEM up to a final volume of 10% FBS.

[0969] 3. The resultant un-supplemented tissue culture media are: o OptiMEM o DMEM o EMEM.

[0970] 4. The resultant supplemented tissue culture media are: o DMEM + 10%FBS o OptiMEM + 10%FBS o EMEM + 10%FBS. Coacervate formation and cell delivery

[0971] 1. Wash HEK293 cells and replace the old media with 4 volumes of the new tissue culture media previously prepared.

[0972] 2. In a centrifuge tube, add FAM-[RGYGG]5C to the Tissue Culture media, to 5x final concentration.

[0973] 3. Mix FAM-[RGYGG]5C into the Tissue Culture media by pipetting, at which point the coacervates form.

[0974] 4. Add 1 volume of coacervate mixture to the 4 volumes of fresh Tissue Culture media containing the mammalian cells in the well of a clear bottom 384W plate. Match like with like: e.g. coacervates made in OptiMEM added to wells containing cells in OptiMEM.

[0975] 5. Centrifuge the imaging plate for Imin at 137xg.

[0976] 6. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for 2h.

[0977] 7. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[0978] 8. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.25 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[0979] 9. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[0980] 10. Centrifuge the imaging plate for Imin at 137xg.

[0981] 11. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times: Channel Mode Dye Light Source Emission Filter

[0982] #1 Widefield Hoechst 405 nm 446 / 37 nm

[0983] #2 Confocal FAM 488 nm 524 / 46 nm

[0984] #3 Widefield Sytox 647 nm 677 / 34 nm

[0985] #4 BF N / A N / A N / A

[0986] Results and conclusion

[0987] For the purposes of this example, significant cell entry is defined as a diffuse pattern of FAM-elicited fluorescence, evenly distributed throughout the cell. Bright green puncta inside of the cells, could be interpreted as undissolved coacervates, and will also be considered as cell entry.

[0988] FAM-[RGYGG]5C entered the cells and diffused through both the cytosol and nucleus in all tissue culture media tested, whether it was supplemented or not with 10%FBS.

[0989] Figure 3 shows both the composite and the brightfield images obtained for the cells and coacervates assayed this example. The nuclei of the cells are identified by the colour blue in the composite images. FAM-[RGYGG]5C is represented in green and presents a diffuse and even distribution inside of the cells. Sytox Deep Red was used to identify dead or permeable cells and is represented by the colour purple in the composite images. The absence of Sytox nuclear staining of green cells (positive for FAM-[RGYGG]5C) indicates that the polymer coacervates entered healthy cells without damaging or permeabilising them.

[0990] FAM-[RGYGG]5C polymer coacervates formed and entered cells in all conditions tested. The ability of FAM-[RGYGG]5C polymer coacervates to enter cells varied with the concentration of the molecule, the tissue culture media used, and whether it was supplemented with 10% FBS or not (Figure 3). These results are summarized in Table 3. Table 3. Summary of results on cell entry for 40 pM and 20 pM FAM-[RGYGGJ 5 C on different cell culture media.

[0991] Media 40 ll XIFAM-[RGYGG] 5C 20 pM FAM-[RGYGG] 5C n fMFM Good cell entry - diffuse FAM- Cell entry - diffuse FAM-

[0992] P1[RGYGG]5C in majority of cells [RGYGG]5C in subset of cells

[0993] OptiMEM + Cell entry - diffuse FAM- Reduced cell entry - puncta in

[0994] 10% FBS [RGYGG]5C in subset of cells cytosol Good cell entry - diffuse Good cell entry - dittuse FAM- •

[0995] DMEM ror-vr-rur - u r n FAM- RGYGG 5 C in

[0996] [RGYGG] 5 C in majority of cells . fc..

[0997] J Jmajority of cells

[0998] DMEM + 10% Good cell entry - diffuse FAM- Cell entry - diffuse FAM-

[0999] FBS [RGYGG] 5 C in majority of cells [RGYGG] 5 C in subset of cells Good cell entry - diffuse Good cell entry - diffuse FAM- r* w •

[1000] EMEM ror-vr-rur - u r n FAM- RGYGG 5 C in

[1001] [RGYGG] 5 C in majority of cells . fc..

[1002] J Jmajority of cells

[1003] EMEM + 10% Cell entry - diffuse FAM- Reduced cell entry - puncta in

[1004] FBS [RGYGG] 5 C in subset of cells cytosol

[1005] Example 4. FAM- [RGYGG] 5C forms coacervates and can enter different mammalian cell types.

[1006] Purpose and summary of experiment

[1007] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C will form coacervates, enter and diffuse inside different types of mammalian cells.

[1008] Three different human cell types that are commonly used in research were tested, HEK293, U2OS and HeLa.

[1009] After induction of LLPS, the coacervates were added to the cells, previously seeded into 384W imaging plates. The cells were incubated with the coacervates and observed on a microscope. Hoechst 34580 was used to stain and identify the nuclei, and Sytox Deep Red was used to identify dead or permeabilised cells.

[1010] Materials and methods

[1011] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1012] • FAM-[RGYGG]5C.

[1013] • Hoechst 34580.

[1014] • Sytox Deep Red.

[1015] • Sterile OptiMEM tissue culture media.

[1016] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1017] • HEK293_NPMl:mScarlet cells.

[1018] • U2OS cells.

[1019] • HeLa cells.

[1020] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1021] • CX7 LZR PRO Microscope.

[1022] Cell culture

[1023] HEK293, U2OS and HeLa cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[1024] Coacervate formation and cell delivery

[1025] 1. Wash cells and replace the old media with 4 volumes of the new tissue culture media previously prepared. 2. In a centrifuge tube, add FAM-[RGYGG]5C to OptiMEM, to 50 pM (5x final concentration). Mix by pipetting, at which point the coacervates form.

[1026] 3. Add 1 volume of coacervate mixture to the 4 volumes of fresh OptiMEM containing the mammalian cells in the well of the imaging plate (Cf = 10 pM).

[1027] 4. Centrifuge the imaging plate for Imin at 137xg.

[1028] 5. Incubate the cells in standard conditions of temperature and CO2 for mammalian cell culture for 4h.

[1029] 6. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.25 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1030] 7. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1031] 8. Centrifuge the imaging plate for Imin at 137xg.

[1032] 9. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1033] Channel Mode Dye Light Source Emission Filter

[1034] #1 Widefield Hoechst 405 nm 446 / 37 nm

[1035] #2 Widefield FAM 488 nm 542 / 27 nm

[1036] #3 Widefield mScarlet 561 nm 600 / 41 nm

[1037] #4 Widefield Sytox 647 nm 677 / 34 nm

[1038] #5 Brightfield N / A N / A N / A

[1039] Results and conclusion

[1040] For the purposes of this example, significant cell entry is defined as a diffuse pattern of FAM-elicited fluorescence, evenly distributed throughout the cell. Bright green puncta inside of the cells, could be interpreted as undissolved coacervates, and will also be considered as cell entry.

[1041] FAM-[RGYGG]5C coacervates entered and diffused through both the cytosol and nucleus of all three human cell types tested. Figure 4 shows both the composite and the FAM images obtained for the cells assayed in this example. The nuclei of the cells are identified by the colour blue in the composite images. FAM-[RGYGG]5C is represented in green and presents a diffuse and even distribution inside of the cells. Sytox Deep Red was used to identify dead or permeable cells and is represented by the colour purple in the composite images. The absence of Sytox nuclear staining of green cells (positive for FAM-[RGYGG]5C) indicates that the polymer coacervates entered healthy cells without damaging or permeabilizing them.

[1042] Example 5. FAM-1RGYGG15C coacervates can incorporate molecular cargoes (protein).

[1043] Purpose and summary of experiment

[1044] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C can incorporate molecular cargos such as proteins when forming coacervates in tissue culture media.

[1045] Three different fluorescent protein cargos were tested:

[1046] 1. mScarlet

[1047] 2. NPM1: mScarlet

[1048] 3. NPMkmCardinal.

[1049] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope. The partition ratio of each cargo was calculated by normalising the fluorescence level inside of the coacervates to the fluorescence level outside of the coacervates.

[1050] Materials and methods

[1051] • FAM-[RGYGG]5C.

[1052] • mScarlet.

[1053] • NPM 1 :mScarlet.

[1054] • NPM 1 :mCardinal.

[1055] • OptiMEM tissue culture media.

[1056] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1057] • CX7 LZR PRO Microscope.

[1058] Peptide oxidation

[1059] 1. Resuspend FAM-[RGYGG]5C in 30% DMSO (in water) up to a stock concentration of 5 mM, in a centrifuge tube.

[1060] 2. The tube is left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1061] 3. Replace the volume of solvent lost during the procedure with water.

[1062] 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C.

[1063] Coacervate formation and imaging

[1064] 1. In a centrifuge tube, mix 1 pl of FAM-[RGYGG]5C at 250 pM (25x final concentration) with 1 pl of client protein at 25x final concentration (50 pM mScarlet; 25 pM NPM 1: mScarlet; 25 pM NPM 1 :mCardinal). 2. Final concentrations of FAM-[RGYGG]5C was 10 pM.

[1065] 3. Final concentrations of the client / cargo were 2 pM for mScarlet, 1 pM for NPMl:mScarlet, and for 1 pM NPMl:mCardinal.

[1066] 4. Add OptiMEM to each centrifuge tube up to 25 pl final volume. Mix by pipetting, at which point the coacervates form.

[1067] 5. Add 20 pl of the coacervate mix to the wells of an imaging plate.

[1068] 6. Centrifuge the plate for 2 min at 137xg.

[1069] 7. Load the plate containing the coacervates into the CX7 LZR PRO microscope and acquire images of the coacervates. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1070] Channel Mode Fluorophore Light Source Emission Filter

[1071] #1 BF N / A N / A N / A

[1072] #2 Widefield FAM 488 nm 524 / 46 nm

[1073] #3 Confocal mScarlet 561 nm 600 / 41 nm

[1074] #4 Confocal mCardinal 594 nm 646 / 74 nm

[1075] Results and conclusion

[1076] A molecule will be considered the cargo of a coacervate if its partition rate (PR) is greater than 1. Furthermore, the higher the PR, the better the molecule is being incorporated into the coacervates, and the more effective a cargo it will be.

[1077] All proteins tested successfully clientised into the coacervates formed by 10 pM of FAM-[RGYGG]5C. Table 4 summarises the results for the partition ratios calculated for the three proteins tested. Figure 5 shows the complex coacervates formed by the mixture of FAM-[RGYGG]5C with the cargos. Table 4. Partition ratios for the three tested proteins.

[1078] Vehicle Client / Cargo PR (Avg ± SD)

[1079] 10 pM FAM-[RGYGG]5C 2 pM mScarlet 1.438 ± 0.335

[1080] 10 pM FAM-[RGYGG]5C 1 pM NPMl-mScarlet 4.367 ± 1.271

[1081] 10 pM FAM-[RGYGG]5C 1 pM NPMl-mCardinal 3.966 ± 1.144

[1082] 10 pM FAM-[RGYGG]5C None 1.003 ± 0.117

[1083] Example 6. FAM-1RGYGG15C coacervates can incorporate molecular cargoes (antibody).

[1084] Purpose and summary of experiment

[1085] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C can incorporate molecular cargos such as antibodies when forming coacervates in tissue culture media.

[1086] Three different antibodies labelled with fluorophores were tested:

[1087] • AlexaFluor-594 anti-Nucleophosmin Antibody (mouse IgG)

[1088] • AlexaFluor-647 anti-Nuclear Pore Complex Proteins Antibody (mouse IgG)

[1089] • Atto 594 anti-GFP nanobody (VHH).

[1090] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope. The partition ratio of each cargo was calculated by normalising the fluorescence level inside of the coacervates to the fluorescence level outside of the coacervates.

[1091] Materials and methods

[1092] • FAM-[RGYGG]5C. • AlexaFluor-594 anti-Nucleophosmin Antibody (mouse IgG).

[1093] • AlexaFluor-647 anti-Nuclear Pore Complex Proteins Antibody (mouse IgG).

[1094] • Atto 594 anti-GFP nanobody (VHH).

[1095] • OptiMEM tissue culture media.

[1096] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1097] • CX7 LZR PRO Microscope.

[1098] Peptide oxidation

[1099] 1. Resuspend FAM-[RGYGG]5C in 30% DMSO (in water) up to a stock concentration of 5 mM, in a centrifuge tube.

[1100] 2. The tube is left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1101] 3. Replace the volume of solvent lost during the procedure with water.

[1102] 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C.

[1103] Coacervate formation and imaging

[1104] 1. In a centrifuge tube, mix 1 pl of FAM-[RGYGG]5C at 250 pM (25x final concentration) with 1 pl of client antibody at 125 pg / ml (25x final concentration).

[1105] 2. Final concentrations of FAM-[RGYGG]5C was 10 pM.

[1106] 3. Final concentrations of the antibodies were 5 pg / ml.

[1107] 4. Add OptiMEM to each centrifuge tube up to 25 pl final volume. Mix by pipetting, at which point the coacervates form.

[1108] 5. Add 20 pl of the coacervate mix to the wells of an imaging plate.

[1109] 6. Centrifuge the plate for 2 min at 137xg. 7. Load the plate containing the coacervates into the CX7 LZR PRO microscope and acquire images of the coacervates. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1110] Channel Mode Fluorophore Light Source Emission Filter

[1111] #1 BF N / A N / A N / A

[1112] #2 Widefield FAM 488 nm 524 / 46 nm

[1113] #3 Confocal Alexa594 / Atto594 594 nm 646 / 74 nm

[1114] #4 Confocal Alexa647 647 nm 677 / 34 nm

[1115] Results and conclusion

[1116] A molecule will be considered the cargo of a coacervate if its partition rate (PR) is greater than 1. Furthermore, the higher the PR, the better the molecule is being incorporated into the coacervates, and the more effective a cargo it will be.

[1117] All antibodies tested successfully clientised into the coacervates formed by 10 pM of FAM-[RGYGG]5C. Table 5 summarises the results for the partition ratios calculated for the three proteins tested. Figure 6 shows the complex coacervates formed by the mixture of FAM-[RGYGG]5C with the cargos.

[1118] Table 5. Partition ratios for the three tested antibodies

[1119] Vehicle Client / Cargo PR (Avg ± SD)

[1120] 5 pg / ml AlexaFluor-594 anti-

[1121] 10 pM FAM-[RGYGG]5C 3.813 ± 1.186 Nucleophosmin Ab

[1122] 5 pg / ml AlexaFluor-647 anti-Nuclear

[1123] 10 pM FAM-[RGYGG]5C 5.102 ± 1.665 Pore Complex Proteins Ab

[1124] 10 pM FAM-[RGYGG]5C 5 pg / ml Atto 594 anti-GFP nanobody 1.619 ± 0.258

[1125] 10 pM FAM-[RGYGG]5C None 1.003 ± 0.117 Example 7. FAM-1RGYGG15C coacervates can incorporate molecular cargoes (ssDNA oligo).

[1126] Purpose and summary of experiment

[1127] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C can incorporate molecular cargos such as ssDNA when forming coacervates in tissue culture media.

[1128] A random 28 nucleotide ssDNA oligo labelled with Cy5 was tested.

[1129] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope. The partition ratio of each cargo was calculated by normalising the fluorescence level inside of the coacervates to the fluorescence level outside of the coacervates.

[1130] Materials and methods

[1131] • FAM-[RGYGG]5C.

[1132] • ssDNA oligo - Cy5.

[1133] • OptiMEM tissue culture media.

[1134] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1135] • CX7 LZR PRO Microscope.

[1136] Peptide oxidation

[1137] 1. Resuspend FAM-[RGYGG]5C in 30% DMSO (in water) up to a stock concentration of 5 mM, in a centrifuge tube.

[1138] 2. The tube is left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1139] 3. Replace the volume of solvent lost during the procedure with water. 4. The DMSO-treated molecule is aliquoted and stored @ -20 °C.

[1140] Coacervate formation and imaging

[1141] 1. In a centrifuge tube, mix 1 pl of FAM-[RGYGG]5C at 250 pM (25x final concentration) with 1 pl of client ssDNA at 25 pM (25x final concentration).

[1142] 2. Final concentrations of FAM-[RGYGG]5C was 10 pM.

[1143] 3. Final concentrations of the cargo was 1 pM.

[1144] 4. Add OptiMEM to each centrifuge tube up to 25 pl final volume. Mix by pipetting, at which point the coacervates form.

[1145] 5. Add 20 pl of the coacervate mix to the wells of an imaging plate.

[1146] 6. Centrifuge the plate for 2 min at 137xg.

[1147] 7. Load the plate containing the coacervates into the CX7 LZR PRO microscope and acquire images of the coacervates. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1148] Channel Mode Fluorophore Light Source Emission Filter

[1149] #1 BF N / A N / A N / A

[1150] #2 Widefield FAM 488 nm 524 / 46 nm

[1151] #3 Confocal Cy5 647 nm 677 / 34 nm

[1152] Results and conclusion

[1153] A molecule will be considered the cargo of a coacervate if its partition rate (PR) is greater than 1. Furthermore, the higher the PR, the better the molecule is being incorporated into the coacervates, and the more effective a cargo it will be.

[1154] The single stranded DNA molecule successfully clientised into the coacervates formed by 10 pM of FAM-[RGYGG]5C. Table 6 summarises the results for the partition ratios calculated for the three proteins tested. Figure 7 shows the complex coacervates formed by the mixture of FAM-[RGYGG]5C with the cargo.

[1155] Table 6. Partition ratios for tested cargo.

[1156] Vehicle Client / Cargo PR (Avg ± SD)

[1157] 10 pM FAM-[RGYGG]5C 1 pM ssDNA oligo - Cy5 6.020 ± 1.915

[1158] 10 pM FAM-[RGYGG]5C None 1.003 ± 0.117

[1159] Example 8. FAM-1RGYGG15C coacervates loaded with cargo can enter HEK293 cells (antibody).

[1160] Purpose and summary of experiment

[1161] This experiment was performed to test the cell delivery capability of the FAM- [RGYGG]5C vehicle, specifically for an antibody cargo delivered to HEK293 cells. The antibody is fluorescently-labelled and specific for nucleophosmin, a protein that is enriched in the nucleoli of cells. It has been used as a proxy for the antibody and large protein class of therapeutic cargos.

[1162] This example demonstrates that an antibody cargo can be delivered into human cells by the FAM-[RGYGG]5C vehicle, without requiring covalent conjugation of the cargo to the vehicle.

[1163] Materials and methods

[1164] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1165] • FAM-[RGYGG]5C.

[1166] • Alexa Fluor 594 conjugated Mouse IgGlK anti-Nucleophosmin Antibody, BioLegend Catalog number 686803. • Hoechst 34580.

[1167] • Sytox Deep Red.

[1168] • Sterile OptiMEM tissue culture media..

[1169] • Sterile DMEM tissue culture media.

[1170] • Sterile Foetal Bovine Serum, heat inactivated (FBS).

[1171] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1172] • HEK293 cells.

[1173] • Sterile 384W plates, black with clear optical bottom, pre-coated with poly-D- Lysine.

[1174] • CX7 LZR PRO Microscope.

[1175] Cell culture

[1176] HEK293 cells were seeded at a density of 7000 cells per well into 384W clear optical bottom, black plates (sterile) in 10% serum-supplemented DMEM medium using standard mammalian cell culture procedures. The cells were incubated overnight at 37°C and 5% CO2.

[1177] Coacervate formation and cell delivery

[1178] 1. Wash HEK293 cells 3 times to replace the old media with optiMEM medium. In this case, 50uL of optiMEM was applied and removed from cells containing 40uL of medium per well.

[1179] 2. In a centrifuge tube, mix FAM-[RGYGG]5C and AF594 anti-nucleophosmin antibody such that they will reach a 2x final concentration in 1 volume of medium, in this case 40uM FAM-[RGYGG]5C and lOug / mL AF594 anti-nucleophosmin antibody. In this case, 3.6uL of ImM FAM-[RGYGG]5C and 1.8uL of 0.5 mg / mL AF594 anti-nucleophosmin antibody.

[1180] 3. Negative control experiments are vehicle-only and cargo-only variants of this mixture. . Other antibody cargos that have successfully been tested in this manner include:

[1181] 5. Alexa Fluor-594 anti-Nuclear Pore Complex Proteins Antibody, BioLegend 682202

[1182] 6. Alexa Fluor-647 anti-Nuclear Pore Complex Proteins Antibody, BioLegend 682203

[1183] 7. Generate coacervates containing cargo by adding medium to the correct volume to make a 2X concentrated coacervate mix. In this case, 90uL of optiMEM.

[1184] 8. Transfer an equal volume of this 2X coacervate mix to the cells. In this case, 40uL coacervate mix to 40uL of cells. The final assay concentration in this case is 20uM FAM-[RGYGG]5C and 5 ug / mL AF594 anti-nucleophosmin antibody.

[1185] 9. Centrifuge the mixture of the coacervates and mammalian cells at standard conditions for mammalian cell culture.

[1186] 10. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for minimum 1 hour.

[1187] 11. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.4 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1188] 12. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1189] 13. Centrifuge the cells at standard conditions for mammalian cell culture.

[1190] 14. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1191] Channel Mode Dye Light Source Emission Filter

[1192] #1 Confocal Hoechst 405 nm 446 / 37 nm

[1193] #2 Confocal FAM 488 nm 542 / 25 nm

[1194] #3 Confocal AF594 594 nm 646 / 74 nm

[1195] #4 widefield AF647 647 nm 677 / 34 nm

[1196] #5 Brightfield none White Brightfield Results and conclusion

[1197] Figure 8 shows that the Alexa Fluor 594-conjugated anti-nucleophosmin antibody cargo is successfully delivered into cells in the presence of the FAM- [RGYGG5]C vehicle. Figure 8D is a higher magnification single-channel view of this result showing robust diffuse distribution of the antibody in the cell cytoplasm (magenta), with the arrowhead pointing to a nucleolar enrichment of this antibody, suggesting that the antibody retains its intracellular function in binding to its in vivo target protein.

[1198] Figure 9 is the vehicle-only control, which demonstrates that the signal observed for the antibody label in Figure 8 is not due to an imaging or experimental artefact of cross-talk. Figure 10 is the cargo-only control, which demonstrates that the antibody cannot enter the cell without the presence of the vehicle. There are further controls that are not depicted in these figures, namely, the untreated cells, and the sytox label that shows the overall cell health of all the cells in this assay was good, and that cell entry and cargo delivery observed here are not artefacts of permeabilised or unhealthy cells. Additionally, as mentioned in the materials and methods, other antibodies have been tested for functional cell delivery, such as the anti-nuclear pore antibodies, which also successfully label their target cellular location (the nuclear envelope).

[1199] From these data, we conclude that the FAM-[RGYGG5]C vehicle is able to deliver antibody cargos into the cell and furthermore to their correct intracellular location.

[1200] Example 9. FAM-1RGYGG15C coacervates loaded with cargo can enter mammalian cells (mRNA).

[1201] Purpose and summary of experiment

[1202] This experiment was performed to test the cell delivery capability of the FAM- [RGYGG]5C vehicle, specifically for a mRNA cargo delivered to HEK293 cells. The mRNA used was capped and expressed in vitro and coded for the fluorescent protein mScarlet. Lipofectamine was used as the positive control at the suggested concentration of 8 pg / ml. As the negative control, no vehicle was used, and the mRNA was added directly to the cells.

[1203] This example demonstrates that mRNA can be delivered into human cells by FAM-[RGYGG]5C, without requiring covalent conjugation of the cargo to the vehicle, and that the delivered cargo maintains its function.

[1204] Materials and methods

[1205] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1206] • FAM-[RGYGG]5C.

[1207] • mScarlet capped mRNA.

[1208] • Lipofectamine 2000.

[1209] • Hoechst 34580.

[1210] • Sytox Deep Red.

[1211] • Sterile OptiMEM tissue culture media.

[1212] • Sterile DMEM tissue culture media.

[1213] • Sterile Foetal Bovine Serum, heat inactivated (FBS).

[1214] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1215] • HEK293 cells.

[1216] • Imaging plate - Sterile 384W plates, black with clear optical bottom, pre-coated with poly-D-Lysine.

[1217] • CX7 LZR PRO Microscope Cell culture

[1218] HEK293 cells were seeded at a density of 5000 cells per well into an imaging plate (sterile) in 10% serum-supplemented DMEM medium using standard mammalian cell culture procedures. The cells were incubated at 37°C and 5% CO2 until use.

[1219] Coacervate formation and cell delivery

[1220] 1. Wash HEK293 cells 3 times to replace the old media with OptiMEM medium.

[1221] In this case, 50 pL of OptiMEM was applied and removed from wells containing 40 pL of medium.

[1222] 2. In a centrifuge tube, mix FAM-[RGYGG]5C and capped mScarlet mRNA such that they will reach 5x final concentration in 1 volume of medium. a. Negative control experiments are vehicle-only and cargo-only variants of this mixture. b. Positive control experiments contain Lipofectamine at final concentration of 8 pg / ml, used as per manufacturer’s instructions.

[1223] 3. Add OptiMEM to the cargo and vehicle mixture, up to 1 volume, and mix by pipetting. At this stage the coacervates form.

[1224] 4. Transfer 1 volume of this 5x coacervate mix to the cells in the plate wells containing 4 volumes of fresh OptiMEM.

[1225] 5. Centrifuge the imaging plate at standard conditions for mammalian cell culture.

[1226] 6. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for 1 hour.

[1227] 7. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1228] 8. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.4 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1229] 9. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS. 10. Centrifuge the cells at standard conditions for mammalian cell culture.

[1230] 11. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1231] Channel Mode Dye Light Source Emission Filter

[1232] #1 Widefield Hoechst 405 nm 446 / 37 nm

[1233] #2 Confocal FAM 488 nm 524 / 46 nm

[1234] #3 Confocal mScarlet 561 nm 612 / 69 nm

[1235] #4 Widefield Sytox 647 nm 677 / 34 nm

[1236] #5 Brightfield none White Brightfield

[1237] Results and conclusion

[1238] Capped mRNA produced in vitro was successfully delivered to HEK293 culture cells by FAM-[RGYGG5]C. Figure 11 shows that FAM-[RGYGG5]C can be used as a vehicle to deliver mRNA into mammalian cells. Furthermore, this mRNA is functional, allowing the cells to produce the coded protein, mScarlet.

[1239] Example 10. FAM-1RGYGG15C coacervates loaded with cargo can enter mammalian cells (ssDNA oligo).

[1240] Purpose and summary of experiment

[1241] This experiment was performed to test the cell delivery capability of the FAM- [RGYGG]5C vehicle, specifically for a ssDNA cargo delivered to HEK293 cells. The ssDNA used was labelled with the fluorophore Cy5. As the negative control, no vehicle was used, and the ssDNA was added directly to the cells.

[1242] This example demonstrates that ssDNA can be delivered into human cells by FAM-[RGYGG]5C, without requiring covalent conjugation of the cargo to the vehicle. Materials and methods

[1243] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1244] • FAM-[RGYGG]5C.

[1245] • Cy5 - ssDNA (28 nucleotides) oligo.

[1246] • Hoechst 34580.

[1247] • Sytox Deep Red.

[1248] • Sterile OptiMEM tissue culture media.

[1249] • Sterile DMEM tissue culture media.

[1250] • Sterile Foetal Bovine Serum, heat inactivated (FBS).

[1251] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1252] • HEK293 cells.

[1253] • Imaging plate - Sterile 384W plates, black with clear optical bottom, pre-coated with poly-D-Lysine.

[1254] • CX7 LZR PRO Microscope.

[1255] Cell culture

[1256] HEK293 cells were seeded at a density of 5000 cells per well into an imaging plate (sterile) in 10% serum-supplemented DMEM medium using standard mammalian cell culture procedures. The cells were incubated at 37°C and 5% CO2 until use.

[1257] Coacervate formation and cell delivery

[1258] 1. Wash HEK293 cells 3 times to replace the old media with OptiMEM medium. At the end of the washes there should be 4 volumes of OptiMEM in each well. In a centrifuge tube, mix FAM-[RGYGG]5C and Cy5-ssDNA such that they will reach 5x final concentration in 1 volume of medium. Negative control experiments are vehicle-only and cargo-only variants of this mixture. Add OptiMEM to the cargo and vehicle mixture, up to 1 volume, and mix by pipetting. At this stage the coacervates form. Transfer 1 volume of the 5x coacervate mix to the cells in the plate wells containing 4 volumes of fresh OptiMEM. Centrifuge the imaging plate at standard conditions for mammalian cell culture. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for 2 hours. W ash the cells 3x with phenol-free T issue Culture media supplemented with FBS . Add Hoechst 34580 (final concentration = 5 pg / mL) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS. Centrifuge the cells at standard conditions for mammalian cell culture. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1259] Channel Mode Dye Light Source Emission Filter

[1260] #1 Widefield Hoechst 405 nm 446 / 37 nm

[1261] #2 Widefield FAM 488 nm 524 / 46 nm

[1262] #3 Widefield Sytox 647 nm 677 / 34 nm

[1263] #4 Brightfield none White Brightfield Results and conclusion ssDNA labelled with a fluorophore was successfully delivered to HEK293 culture cells by FAM-[RGYGG] 5C. Figure 12 shows that FAM-[RGYGG5]C can be used as a vehicle to deliver ssDNA oligos into mammalian cells. Cells treated with the coacervates formed by FAM-[RGYGG]5C mixed with ssDNA oligo displayed bright puncta in the cytosol, indicating cell entry of the mix coacervates.

[1264] Example 11. FAM-[RGYGG]nC peptides can form coacervates if n = 2 or higher.

[1265] Purpose and summary of experiment

[1266] This experiment was performed to understand the importance of the length of the peptide part of the polymers FAM-[RGYGG]nC, where n = 2, 3, 4, 5, 6 or 8 in the phenomenon of liquid-liquid phase separation and coacervate formation in unsupplemented tissue culture media.

[1267] Different final concentrations of the polymers were tested (320 pM, 160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1268] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1269] Materials and methods

[1270] • FAM-[RGYGG]2C.

[1271] • FAM-[RGYGG]3C.

[1272] • FAM-[RGYGG]4C.

[1273] • FAM-[RGYGG]5C.

[1274] • FAM-[RGYGG]6C. • FAM-[RGYGG]8C.

[1275] • OptiMEM tissue culture media.

[1276] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1277] • CX7 LZR PRO Microscope.

[1278] Peptide oxidation

[1279] 1. Resuspend the polymers (FAM-[RGYGG]2C, FAM-[RGYGG]3C, FAM- [RGYGG]4C, FAM-[RGYGG]5C, FAM-[RGYGG]6C, and FAM-[RGYGG]8C) in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1280] 2. The tubes are left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1281] 3. Replace the volume of solvent lost during the procedure with water.

[1282] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1283] Coacervate formation and imaging

[1284] 1. Add 4 volumes of OptiMEM to the wells of a 384W clear bottom, black plate, pre-coated with poly-D-Lysin.

[1285] 2. In centrifuge tubes, add the polymers (FAM-[RGYGG]2C, FAM-[RGYGG]3C, FAM-[RGYGG]4C, FAM-[RGYGG]5C, FAM-[RGYGG]6C, and FAM- [RGYGG]8C) to OptiMEM to 5x final concentration.

[1286] 3. Mix the polymers into OptiMEM by pipetting, at which point the coacervates form.

[1287] 4. Add 1 volume of coacervate mixture to the 4 volumes of OptiMEM contained in the wells of the 384W plate.

[1288] 5. Centrifuge the mixture of the coacervates for 2 min at 137xg. 6. Load the plate containing the coacervates into the CX7 microscope and acquire images of the coacervates.

[1289] Results and conclusion

[1290] Polymers constituted by FAM-[RGYGG]nC where n was equal or greater than 1 were able to undergo significant LLPS to form coacervates. This ability was dependent not only on the number of RGYGG repeats and consequently the length of the peptide, but also dependent on the concentration of the polymer in solution. The ability to undergo significant LLPS and form coacervates increased with the length of the peptide. Longer peptides were able to form coacervates up to lower concentrations than shorter peptides. Furthermore, the size and number of the coacervates formed increased with increasing concentrations of the polymer in solution. Figure 13 shows the outcomes of this experiment and the results are summarised in Table ?.

[1291] Table 7. Summary of results on coacervate formation.

[1292] [Polymer]

[1293] 320 μM 160 μM 80 μM 40 μM 20 μM 10 μM 5μM

[1294] FAM-

[1295] [RGYGGJ2CYeS YeS N°N°N°N°N°

[1296] FAM- Not .. .. .. ,T,T,T

[1297] |RGYGG|3C testedYes Yes Yes No No No

[1298] FAM- Not Not

[1299] < < .i < < .i Yes Yes Yes Yes Yes

[1300] [RGYGG] 4C tested tested

[1301] FAM- Not Not Not .. .. .. ..

[1302] [RGYGG] 5C tested tested testedYeS YeS YeS YeS

[1303] FAM- Not Not Not .. .. .. ..

[1304] [RGYGG] 6C tested tested testedYeS YeS YeS YeS

[1305] FAM- Not Not Not .. .. .. ..

[1306] [RGYGG] 8C tested tested testedYeS YeS YeS YeS While FAM-[RGYGG]2C formed coacervates at the highest concentrations tested, 320 pM and 160 pM, FAM-[RGYGG]3C formed coacervates at 160, 80, and 40 pM. FAM-[RGYGG]4C FAM-[RGYGG]5C, FAM-[RGYGG]6C, and FAM- [RGYGG]8C were able to form coacervates at all of the concentrations tested.

[1307] Example 12. FAM-[RGYGG]nC peptides of different lengths can enter cells.

[1308] Purpose and summary of experiment

[1309] This experiment was performed to understand the importance of the length of the peptide part of the polymers FAM-[RGYGG]nC, where n was equal or larger than 2, in entering and diffusing inside HEK293 cells.

[1310] Five different final concentrations of the polymers were tested (160 pM, 80 pM, 40 pM, 20 pM, and 10 pM) and their ability to enter and diffuse in mammalian cells was determined.

[1311] After induction of LLPS in OptiMEM, the coacervates were added to HEK293 cells, previously seeded into 384W imaging plates. The cells we incubated with the coacervates and observed on a microscope.

[1312] Hoechst 34580 was used to stain and identify the nuclei, and Sytox Deep Red was used to identify dead or permeabilised cells.

[1313] Materials and methods

[1314] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1315] • FAM-[RGYGG]2C.

[1316] • FAM-[RGYGG]3C.

[1317] • FAM-[RGYGG]4C.

[1318] • FAM-[RGYGG]5C. • Hoechst 34580.

[1319] • Sytox Deep Red.

[1320] • Sterile OptiMEM tissue culture media.

[1321] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1322] • HEK293 cells.

[1323] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1324] • CX7 LZR PRO Microscope.

[1325] Peptide oxidation

[1326] 1. Resuspend the polymers (FAM-[RGYGG]2C, FAM-[RGYGG]3C, FAM- [RGYGG]4C, FAM-[RGYGG]5C) in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1327] 2. The tubes are left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1328] 3. Replace the volume of solvent lost during the procedure with water.

[1329] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1330] Cell culture

[1331] HEK293 cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[1332] Coacervate formation and cell delivery

[1333] 1. Wash HEK293 cells and replace the old media with 4 volumes of OptiMEM. 2. In centrifuge tubes, add the polymers (FAM-[RGYGG]2C, FAM-[RGYGG]3C, FAM-[RGYGG]4C, FAM-[RGYGG]5C) to OptiMEM to 5x final concentration. Mix by pipetting, at which point the coacervates form.

[1334] 3. Add 1 volume of coacervate mixture to the 4 volumes of OptiMEM containing the mammalian cells in the well of a clear bottom 384W plate.

[1335] 4. Centrifuge the imaging plate for 1 min at 137xg.

[1336] 5. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for 2h.

[1337] 6. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1338] 7. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.25 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1339] 8. W ash the cells 3x with phenol-free T issue Culture media supplemented with FBS .

[1340] 9. Centrifuge the imaging plate for Imin at 137xg.

[1341] 10. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1342] Channel Mode Dye Light Source Emission Filter

[1343] #1 Widefield Hoechst 405 nm 446 / 37 nm

[1344] #2 Confocal FAM 488 nm 524 / 46 nm

[1345] #3 Widefield Sytox 647 nm 677 / 34 nm

[1346] #4 BF N / A N / A N / A

[1347] Results and conclusion

[1348] For the purposes of this example, significant cell entry is defined as a diffuse pattern of FAM-elicited fluorescence, evenly distributed throughout the cell. Bright green puncta inside of the cells, could be interpreted as undissolved coacervates, and will also be considered as cell entry.

[1349] Polymers constituted by FAM-[RGYGG]nC where n was equal or greater than 2 were able to enter HEK293 cells. This capability was dependent not only on the number of RGYGG repeats (structural units) and consequently the length of the peptide, but also dependent on the concentration of the polymer in solution.

[1350] The proficiency of the polymer to significantly enter and diffuse inside HEK293 cells increased with the length of the peptide. Longer peptides were able to do so at lower concentrations than shorter peptides. The results are shown in Figure 14 and summarised in Table 8.

[1351] Table 8. Summary of results on cell entry and diffusion.

[1352] [Polymer] 160 μM 80 μM 40μM 20 μM 10 μM

[1353] Reduced cell

[1354] Cell entry -

[1355] FAM- entry - faint No cell puncta in No cell entry No cell entry [RGYGGJ2C puncta in entry cytosol cytosol

[1356] Cell entry -

[1357] Reduced cell bright puncta Cell entry -

[1358] FAM- entry -faint No cell in cytosol; puncta in No cell entry [RGYGGJ3C puncta in entry

[1359] High cytosol cytosol background

[1360] Good cell Good cell entry - entry - Reduced

[1361] Cell entry -

[1362] FAM- diffuse diffuse cell entry -

[1363] Not tested puncta in [RGYGGJ4C polymer in polymer in faint puncta cytosol majority of majority of in cytosol cells cells

[1364] Good cell Good cell entry - entry -

[1365] Cell entry -

[1366] FAM- diffuse diffuse

[1367] Not tested Not tested puncta in [RGYGGJ5C polymer in polymer in cytosol majority of majority of cells cells Both FAM-[RGYGG]2C and FAM-[RGYGG]3C entered the cells at high concentrations and showed a punctate pattern in the cytosol. FAM-[RGYGG]4C had good cell entry at 80 pM and 40 pM, while FAM-[RGYGG]5C showed good cell entry at 40 pM and 20 pM.

[1368] Example 13. FAM-1RGYGG15C proficiency for coacervate formation depends on its redox state.

[1369] Purpose and summary of experiment

[1370] This experiment was performed to understand the importance of the redox state of FAM-[RGYGG]5C, through the oxidation of the Cysteine of two neighbouring polymer molecules and the formation of a thiol bond, in the phenomenon of liquidliquid phase separation and coacervate formation in OptiMEM tissue culture media.

[1371] Different redox states of FAM-[RGYGG]5C were assayed, and the importance of a C-terminal Cysteine for LLPS and coacervate formation in OptiMEM was determined:

[1372] • FAM-[RGYGG]5C purposefully oxidized by 30% DMSO treatment

[1373] • FAM-[RGYGG]5C un-treated (mixed redox state)

[1374] • FAM-[RGYGG]5C reduced by 2.5 mM TCEP

[1375] • FAM-[RGYGG]5 without a cysteine and therefore unable to be oxidized.

[1376] • [RGYGG]5C-FAM where the FAM was conjugated directly to the cysteine side chain, and therefore unable to be oxidized.

[1377] Different final concentrations of the polymers were also tested (160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM). After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1378] Materials and methods

[1379] • FAM-[RGYGG]5C DMSO-treated.

[1380] • FAM- [RGYGG] 5 C un-treated.

[1381] • FAM-[RGYGG]5

[1382] • [RGYGG]5C-FAM

[1383] • TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)

[1384] • OptiMEM tissue culture media.

[1385] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL)

[1386] • CX7 LZR PRO Microscope

[1387] Peptide oxidation

[1388] 1. Resuspend the polymer FAM-[RGYGG]5C and [RGYGG]5C-FAM in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1389] 2. The tubes are left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1390] 3. Replace the volume of solvent lost during the procedure with water.

[1391] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1392] Prepare OptiMEM supplemented with 2,5 mM TCEP (OptiMEM + 2,5 mM TCEP)

[1393] Dilute the reducing agent TCEP into OptiMEM to final concentration of 2.5 mM. Coacervate formation and imaging

[1394] 1. Add 4 volumes of OptiMEM or OptiMEM + 2.5mM TCEP to the wells of a 384W clear bottom, black plate, pre-coated with poly-D-Lysine.

[1395] 2. Resuspend a new aliquot of FAM-[RGYGG]5C (recently synthesized) in water up to a stock concentration of 5 mM, in a centrifuge tube. This sample will be from now on referred to “FAM-[RGYGG]5C un-treated”.

[1396] 3. In centrifuge tubes, add the polymers FAM-[RGYGG]5C DMSO-treated, FAM- [RGYGG]5C un-treated, FAM-[RGYGG]5, and [RGYGG]5C-FAM to OptiMEM to 5x the final concentration. Add FAM-[RGYGG]5C un-treated to OptiMEM + 2.5mM TCEP to 5x the final concentration.

[1397] 4. Mix the polymers into the tissue culture buffers by pipetting, at which point the coacervates form.

[1398] 5. Add 1 volume of coacervate mixture to the 4 volumes of OptiMEM or OptiMEM + 2.5mM TCEP contained in the wells of the imaging plate. Match like with like, that is coacervates made in OptiMEM added to wells containing OptiMEM, coacervates made in OptiMEM + 2.5mM TCEP added to wells containing OptiMEM + 2.5mM TCEP.

[1399] 6. Centrifuge the imaging plate for 2 min at 137xg.

[1400] 7. Load the plate containing the coacervates into the CX7 microscope and acquire images of the coacervates.

[1401] Results and conclusion

[1402] FAM-[RGYGG]5C requires a C-terminal Cysteine able to form a thiol bond with a neighbouring molecule in order to undergo significant LLPS and form coacervates. This ability is dependent on the redox state of the molecule, and on the concentration of the polymer in solution. The capability to undergo significant LLPS and form coacervates increased with level of oxidation of FAM-[RGYGG]5C.

[1403] Figure 15 shows the outcomes of this experiment, and the results are summarised in Table 9. Freshly reconstituted and un-treated FAM-[RGYGG]5C will form coacervates in all the tested concentrations (40, 20 10, and 5 pM), but these are smaller than the coacervates resulting from DMSO-treated FAM-[RGYGG]5C peptide in the same conditions. Furthermore, when the cysteine in FAM-[RGYGG]5C is reduced by 2.5 mM TCEP, the polymer will only undergo LLPS at concentrations greater than 20 pM, and the coacervates formed are smaller and less numerous.

[1404] The molecule FAM-[RGYGG]5, which lacks a C-terminal cysteine, can only form coacervates at final concentrations greater than 40 pM, and these are morphologically different than the coacervates formed by the DMSO-treated FAM- [RGYGG]5C. Furthermore, when the C-terminal cysteine side chain is unavailable to form thiol bonds, as is the case of [RGYGG]5C-FAM, significant LLPS only occurred at final concentrations greater than 40 pM (Figure 15).

[1405] Table 9. Summary of results on coacervate formation.

[1406] DMSO-treated Un-treated FAM- rncvcci^r

[1407] [Polymer] FAM- FAM- [RGYGGJ5C

[1408] 160 pM Not tested Not tested Not tested Yes Not tested

[1409] 80 pM Not tested Not tested Not tested Yes Yes

[1410] 40 pM Yes Yes Yes Yes Yes

[1411] 20 pM Yes Yes Yes No No

[1412] 10 pM Yes Yes No No No

[1413] 5 pM Yes Yes No No No Example 14. Relationship between the ability of FAM-1RGYGG15C to enter cells and its redox state.

[1414] Purpose and summary of experiment

[1415] This experiment was performed to understand the relationship of the redox state of the C-terminal cysteine in the polymer FAM-[RGYGG]5C, in the ability this molecule to enter mammalian cells.

[1416] Different redox states of FAM-[RGYGG]5C were assayed, and the importance of a C-terminal Cysteine for coacervate formation in OptiMEM, cell entry and diffusion was determined:

[1417] • FAM-[RGYGG]5C purposefully oxidized by 30% DMSO treatment.

[1418] • FAM-[RGYGG]5C un-treated (mixed redox state).

[1419] • FAM-[RGYGG]5C reduced by 2.5 mM TCEP.

[1420] • FAM-[RGYGG]5 without a cysteine and therefore unable to be oxidized.

[1421] Different final concentrations of the polymers were also tested (160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM).

[1422] After induction of LLPS, the coacervates were added to imaging plates previously seeded with HEK293 cells, incubated for 2h in standard conditions of temperature and CO2, and observed on a microscope.

[1423] Materials and methods

[1424] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1425] • FAM-[RGYGG]5C DMSO-treated. • FAM- [RGYGG] 5 C un-treated.

[1426] • FAM-[RGYGG]5.

[1427] • TCEP (Tris(2-carboxyethyl)phosphine hydrochloride).

[1428] • Hoechst 34580.

[1429] • Sytox Deep Red.

[1430] • Sterile OptiMEM tissue culture media.

[1431] • Sterile Phenol-free Tissue Culture media supplemented with FBS.

[1432] • HEK293 cells.

[1433] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1434] • CX7 LZR PRO Microscope.

[1435] Peptide oxidation

[1436] 1. Resuspend the polymer FAM-[RGYGG]5C in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1437] 2. The tubes are left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1438] 3. Replace the volume of solvent lost during the procedure with water.

[1439] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1440] Cell culture

[1441] HEK293 cells were seeded into the imaging plate using standard mammalian cell culture procedures. Prepare OptiMEM supplemented with 2,5 mM TCEP (OptiMEM + 2,5 mM TCEP)

[1442] Dilute the reducing agent TCEP into OptiMEM to final concentration of 2.5 mM.

[1443] Coacervate formation and cell

[1444] 1. Wash HEK293 cells and replace the old media with 4 volumes of OptiMEM or OptiMEM + 2.5 mM TCEP.

[1445] 2. In centrifuge tubes, add the polymers FAM-[RGYGG]5C DMSO-treated, FAM- [RGYGG]5C un-treated, and FAM-[RGYGG]5 to OptiMEM to 5x the final concentration. Add FAM-[RGYGG]5C un-treated to OptiMEM + 2.5mM TCEP to 5x the final concentration.

[1446] 3. Mix the polymers into the tissue culture media by pipetting, at which point the coacervates form.

[1447] 4. Add 1 volume of coacervate mixture to the 4 volumes of OptiMEM or OptiMEM + 2.5mM TCEP contained in the wells of the imaging plate. Match like with like, that is coacervates made in OptiMEM added to wells containing OptiMEM, coacervates made in OptiMEM + 2.5mM TCEP added to wells containing OptiMEM + 2.5mM TCEP.

[1448] 5. Centrifuge the imaging plate for Imin at 137xg.

[1449] 6. Incubate the HEK293 cells in standard conditions of temperature and CO2 for mammalian cell culture for 2h.

[1450] 7. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1451] 8. Add Hoechst 34580 (final concentration = 5 pg / mL) and Sytox Deep Red (final concentration = 0.25 pM) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1452] 9. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS. 10. Centrifuge the imaging plate for Imin at 137xg.

[1453] 11. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1454] Channel Mode Dye Light Source Emission Filter

[1455] #1 Widefield Hoechst 405 nm 446 / 37 nm

[1456] #2 Confocal FAM 488 nm 524 / 46 nm

[1457] #3 Widefield Sytox 647 nm 677 / 34 nm

[1458] #4 Brightfield N / A N / A N / A

[1459] Results and conclusion

[1460] For the purposes of this example, significant cell entry is defined as a diffuse pattern of FAM-elicited fluorescence, evenly distributed throughout the cell. Bright green puncta inside of the cells, could be interpreted as undissolved coacervates, and will also be considered as cell entry.

[1461] Polymers containing a C-terminal Cysteine free to be oxidized were able to enter HEK293 cells and diffuse evenly throughout. This capability was dependent not only on the redox state of the molecule, but also dependent on the concentration of the polymer in solution.

[1462] The proficiency of the polymer to significantly enter and diffuse inside HEK293 cells increased with the redox level of the peptide. DMSO-treated FAM-[RGYGG]5C significantly enter the cells when tested at 40 and 20 pM. Untreated FAM-[RGYGG]5C (with mixed redox state) and the presence of 2.5 mM TCEP shows cell entry in the same conditions, but the cells have a dimer stain. The absence of C-terminal Cysteine meant that FAM-[RGYGG]5 was visible in the cells mainly in a punctate pattern and at much higher assay concentrations. Figure 16 shows the outcomes of this experiment, and the results are summarised in Table 10. Table 10. Summary of results on cell entry and diffusion.

[1463] DMSO-treated FAM-

[1464] Untreated FAM- FAM-

[1465] [Polymer] FAM- [RGYGG]5C + [RGYGG]5C [RGYGGJ5 [RGYGG]5C 2.5 mM TCEP

[1466] Cell entry -

[1467] 160 pM Not tested Not tested Not tested Bright puncta; High background

[1468] Cell entry -

[1469] 80 pM Not tested Not tested Not tested Puncta

[1470] Good cell entry -

[1471] Cell entry - Cell entry - Faint Reduced cell diffuse polymer

[1472] 40 pM diffuse polymer diffuse polymer; entry - Faint in majority of in subset of cells Puncta puncta cells

[1473] Good cell entry -

[1474] Cell entry - Faint diffuse polymer Reduced cell

[1475] 20 pM diffuse polymer No cell entry in majority of entry - Puncta in some cells cells

[1476] Cell entry - Faint Reduced cell

[1477] Reduced cell

[1478] 10 pM diffuse polymer; entry - Faint No cell entry entry - Puncta

[1479] Bright puncta puncta Example 15. R1-RGYGG5C peptides form coacervates with different aromatic headgroups in position “Ri”.

[1480] Purpose and summary of experiment This experiment was performed to understand the importance of the headgroup covalently linked to the N-terminal side of the peptide [RGYGG]5C, in the phenomenon of liquid-liquid phase separation and coacervate formation in OptiMEM tissue culture media.

[1481] Different headgroups, with varying degrees of aromaticity and / or hydrophobicity were assayed, and the importance of these two parameters for LLPS and coacervate formation in OptiMEM was determined. Different final concentrations of the polymers were tested (320 pM, 160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM). After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1482] Materials and methods

[1483] • Polymers:

[1484] Table 11. List of compounds tested in this example.

[1485] Polymer N-terminal group

[1486] FAM-[RGYGG]5C FAM

[1487] WW-[RGYGG]5C WW (Tryptophan-Tryptophan)

[1488] [RGYGG]5C N / A

[1489] FMOC-[RGYGG]5C FMOC

[1490] Chloroalkane- [RGYGG] 5 C Chloroalkane

[1491] Azidohexanoic acid- [RGYGG] 5 C Azidohexanoic acid

[1492] TAMRA-[RGYGG]5C TAMRA

[1493] Pyrenebutyric acid- [RG Y GG] 5 C Pyrenebutyric acid

[1494] Phenylbutanoic acid-[RGYGG]5C Phenylbutanoic acid

[1495] Anthracyl-[RGYGG]5C Anthracyl

[1496] N aproxyl- [RG Y GG] 5 C Naproxyl

[1497] Butyric acid- [RGYGG] 5 C Butyric acid

[1498] Myristic acid- [RGYGG] 5 C Myristic acid

[1499] 2-Methoxyethoxyacetic acid-[RGY GG]5C 2-Methoxyethoxyacetic acid 5 -(piperazin- l-yl)pyrazine-2-carboxylic acid - 5 -(piperazin- 1 -yl)pyrazine-2-carboxylic [RGYGG]5C acid

[1500] HEX-[RGYGG]5C HEX

[1501] RhodamineB-[RGY GG] 5 C RhodamineB

[1502] FMOC-PEG2-[RGYGG]5C FMOC-PEG2

[1503] PEG2-[RGYGG]5C PEG2 4-Naphthalen-2-yl-butanoic acid -

[1504] 4-Naphthalen-2-yl-butanoic acid [RGYGG]5C

[1505] Naphtyl-alanyl-4-naphtyl butanoic acid-

[1506] Naphtyl-alanyl-4-naphtyl butanoic acid [RGYGG]5C

[1507] FAM-PEG2-[RGYGG]5C FAM-PEG2

[1508] • Centrifuge for tubes or PCR plates.

[1509] • Molecular biology grade water.

[1510] • OptiMEM tissue culture media.

[1511] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1512] • CX7 LZR PRO Microscope. oxidation

[1513] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1514] 2. The tubes are left open to the air and covered with aluminum foil (to protect from light) up to 24h to oxidize.

[1515] 3. Replace the volume of solvent lost during the procedure with water.

[1516] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1517] Coacervate formation and imaging

[1518] 1. Using the top wells of a PCR plate (row A), dilute the polymers in water to 4 mM concentration and a final volume of 4 pl.

[1519] 2. Fill the subsequent wells in the plate (row B to row F) with 2 pl of molecular biology grade water. 3. Perform a 1 :2 serial dilution, by taking 2 jxl of the top wells (row A) into the wells of row B and mix by pipetting. Repeat the step 6 times, all the way to row F.

[1520] 4. Add 24 pl of OptiMEM to the wells of a different PCR plate. Fill rows A to G.

[1521] 5. Add 1 of the polymer dilution series to the 24 pl of OptiMEM in the PCR plate. Mix the polymers by pipetting, at this point the coacervates form.

[1522] 6. Add 20 pl of the coacervate mix to the wells of the imaging plate.

[1523] 7. Centrifuge the imaging plate for 2 min at 137 xg.

[1524] 8. Load the plate containing the coacervates into the CX7 microscope and acquire images of the coacervates in brightfield.

[1525] Results and conclusion

[1526] The hydrophobicity of the whole molecule was characterized by the parameter cLogP. Higher positive cLogP values correspond to higher hydrophobicity, whereas more negative cLogP values correspond to higher hydrophilicity.

[1527] The aromaticity was scored by the number of atoms within the headgroup engaged in aromatic bonds as defined by Hueckel’s rule (calculated using public access cheminformatics tool OpenBabel 3.0.0; N M O'Boyle, M Banck, C A James, C Morley, T Vandermeersch, and G R Hutchison. "Open Babel: An open chemical toolbox." J. Cheminf. (2011), 3, 33. DOI: 10.1186 / 1758-2946-3-33).

[1528] NH2-[RGYGG]5C represents the peptide-only part of the molecule (without any headgroup at its N-terminus), and it is characterised by an aromaticity of 30 and a cLogP of -1.4946. Higher aromaticity values and / or positive cLogP values correspond to more aromatic and / or more hydrophobic molecules due to the addition of the specified head group.

[1529] Table 12 summarises the results for coacervate formation obtained in this example, and Figure 17, Figure 18, and Figure 19 depict the relationship between the molecules’ hydrophobicity, aromaticity and their ability to form coacervates. Overall, hydrophobic head groups that contained one or more aromatic rings promoted LLPS and the formation of coacervates. Polymers with head groups that contained linear hydrophobic molecules (e.g. Myristic acid, Chloroalkane, Azidohexanoic acid) or linear hydrophilic molecules (eg. PEG2, 2-Methoxyethoxyacetic acid) failed to undergo LLPS and form coacervates at the concentrations tested.

[1530] Table 12. Summary of the results on coacervate formation. N-terminal groups highlighted in bold. XI = 5-(piperazin-l-yl)pyrazine-2-carboxylic acid. X2 = 4- Naphthalen-2-yl-butanoic acid. X3 = Naphtyl-alanyl-4-naphtyl butanoic acid. Hydrophobicity assessed by cLogP of the whole molecule. The aromaticity value indicates the number of atoms within the headgroup engaged in aromatic bonds as defined by Hueckel’s rule (calculated using public access cheminformatics tool OpenBabel 3.0.0; N M O'Boyle, M Banck, C A James, C Morley, T Vandermeersch, and G R Hutchison. "Open Babel: An open chemical toolbox. " J. Cheminf. (2011), 3, 33. DOI:10.1186 / 1758-2946-3-33). Cmin= Minimum concentration for coacervate formation.

[1531] „ , . . . .. Coacervate Cmin (in ,k,

[1532] Polymer Aromaticity cLogP , .sMorphology formation pM)

[1533] NH2-[RGYGG]5C 0 -1.4946 No N / A N / A

[1534] FAM-[RGYGG]5C 18 1.6465 Yes 5 Round, opaque

[1535] WW-[RGYGG]5C 20 2.331 Yes 10 Round, clear

[1536] FMOC-[RGYGG]5C 12 1.776 Yes 10 Round, clear

[1537] " 0.4732 No N / A N / A

[1538] Azidohexanoic acid- „

[1539] 0-0.32304 No N / A N / A

[1540] [RGYGGJ5C

[1541] TAMRA-[RGYGG]5C 18 2.3673 Yes 20 Round, clear

[1542] Pyrenebutyric acid- „ „„„„ , rD r. vr r i^193.2738 Yes 10 Round, clear

[1543] [KCj Y (jupC

[1544] Phenylbutanoic acid- , .. Round, clear,

[1545] 6 0.3764 Yes 40

[1546] [RGYGG]5C small

[1547] Anthracene propanoic „ .. ,

[1548] • . 16 2.2927 Yes 40 Round, clear acid-[RGYGG]5C

[1549] Naproxyl-[RGYGG]5C 11 1.319 Yes 80 Butyric acid-

[1550] 0 -0.8464 No N / A N / A [RGYGG]5C

[1551] Myristic acid-

[1552] 0 3.0546 No N / A N / A

[1553] [RGYGG]5C -Methoxyethoxyacetic

[1554] 0 -1.6359 No N / A N / A acid-[RGYGG]5C

[1555] . Round, clear,

[1556] X1-[RGYGG]5C 16 -1.7393 Yes160very sma 1l1l

[1557] HEX-[RGYGG]5C 18 5.5669 Yes 5 Round, small

[1558] RhodamineB-

[1559] 21 3.3838 Yes 5 Round [RGYGG]5C

[1560] FMOC-PEG2-

[1561] 12 1.3163 Yes 10 Round [RGYGG]5C

[1562] PEG2-[RGYGG]5C 0 -1.9543 No N / A N / A

[1563] X2-[RGYGG]5C 11 1.5296 Yes 20 Round, clear

[1564] X3-[RGYGG]5C 22 3.8012 Yes 5 Round, clear

[1565] FAM-PEG2-

[1566] 18 1.1868 Yes 5 Round, opaque [RGYGG]5C

[1567] Example 16. FAM-[PiGYGG]5C peptides can form coacervates if Pi is any of R, or

[1568] H, but not if it is A or N.

[1569] Purpose and summary of experiment

[1570] This experiment was performed to understand the importance of the amino acid side chain in the position Pi in the peptide FAM-[PiGYGG]5C, in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The amino acids tested for this position were R, H, A or N.

[1571] Different final concentrations of the polymers were tested (320 pM, 160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed. After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1572] Materials and methods

[1573] • FAM-[RGYGG]5C.

[1574] • FAM-[HGYGG]5C.

[1575] • FAM-[AGYGG]5C.

[1576] • FAM-[NGNGG]5C.

[1577] • OptiMEM tissue culture media.

[1578] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1579] • CX7 LZR PRO Microscope.

[1580] Peptide oxidation

[1581] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1582] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1583] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1584] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1585] Coacervate formation and imaging

[1586] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[1587] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1588] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate. 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1589] 5. Load the plate into the CX7 microscope and acquire images of the coacervates.

[1590] Results and conclusion

[1591] Polymers constituted by FAM-[PiGYGG]5C where Pi = R or H, but not if it is A or N were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution, and on which amino acid side chain was present in position Pi.

[1592] The ability to undergo significant LLPS and form coacervates increased with the amino acid side chain in the position Pi according to the following series: R > H.

[1593] It was found that replacing R with an A or an N in Ai caused the polymer to form non-coacervate aggregates in aqueous solutions.

[1594] Figure 20 shows the outcomes of this experiment, and the results are summarised in Table 13.

[1595] Table 13. Summary of results on coacervate formation. NCA - N on-coacervate aggregates.

[1596] [Polymer]

[1597] 320 μM 160 μM 80μM 40μM 20μM 10μM 5μM

[1598] FAM- mrvrrirr Not tested Not tested Yes Yes Yes Yes Yes

[1599] |KAJ Y 5C

[1600] FAM- lurvrrur Not tested Not tested Yes Yes Yes Yes Yes

[1601] [nG i GG]5C

[1602] FAM- NCA NCA NCA NCA NCA NCA NCA

[1603] [AG i GGpL

[1604] FAM-r'CAi* „ NCA NCA NCA NCA NCA NCA NCA

[1605] [IM (JIM CJCJJ 3 V Example 17. FAM-1RGP3GG15C peptides can form coacervates if P3 is any of Y. F or W, but not if it is A or N.

[1606] Purpose and summary of experiment

[1607] This experiment was performed to understand the importance of the amino acid side chain in the position A3 in the peptide FAM-[RGP3GG]5C, in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The amino acids tested for this position were Y, F, W, A or N.

[1608] Different final concentrations of the polymers were tested (320 pM, 160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1609] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1610] Materials and methods

[1611] • FAM-[RGYGG]5C

[1612] • FAM-[RGFGG]5C

[1613] • FAM-[RGWGG]5C

[1614] • FAM-[RGAGG]5C

[1615] • FAM-[NGNGG]5C

[1616] • OptiMEM tissue culture media.

[1617] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL)

[1618] • CX7 LZR PRO Microscope

[1619] Peptide oxidation

[1620] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes. 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1621] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1622] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1623] Coacervate formation and imaging

[1624] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[1625] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1626] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[1627] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1628] 5. Load the plate into the CX7 microscope and acquire images of the coacervates.

[1629] Results and conclusion

[1630] Polymers constituted by FAM-[RGP3GG]5C where P3 = Y, F or W, but not if it is A or N were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution, and on which amino acid was present in position P3.

[1631] The ability to undergo significant LLPS and form coacervates increased depending on the amino acid in the position P3 according to the following series: W > Y > F.

[1632] Replacing Y with an A in P3 prevented the formation of coacervates at the concentrations tested. Replacing Y with an N in P3 led the polymer to form noncoacervate aggregates in aqueous solutions.

[1633] Figure 21 shows the outcomes of this experiment, and the results are summarised in Table 14. Table 14. Summary of results on coacervate formation. NCA - non-coacervate aggregates. Example 18. FAM-[PIGP3GG]5C peptides, where Pi is any of R, or H, but not K, A or N, and where P3 is any of Y, F or W, but not A or N, can form coacervates.

[1634] Purpose and summary of experiment This experiment was performed to understand if the amino acid side chains in the positions Ai and A3 in the peptide FAM-[PIGP3GG]5C, were interchangeable in the phenomenon of liquid-liquid phase separation and coacervate formation in unsupplemented tissue culture media.

[1635] Different final concentrations of the polymers were tested (320 pM, 160 pM, 80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1636] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope. Materials and methods

[1637] • FAM-[RGYGG]5C.

[1638] • FAM-[YGRGG]5C.

[1639] • FAM-[RGRGG]5C.

[1640] • OptiMEM tissue culture media.

[1641] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1642] • CX7 LZR PRO Microscope.

[1643] Peptide oxidation

[1644] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1645] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1646] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1647] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1648] Coacervate formation and imaging

[1649] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[1650] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1651] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[1652] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1653] 5. Load the plate into the CX7 microscope and acquire images of the coacervates. Results and conclusion

[1654] Polymers constituted by FAM-[PIGP3GG]5C where either Pi = Y and P3 = R, or Pi = R and P3 = Y, were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution and did not varied significantly between the two polymers.

[1655] Replacing both Pi and P3 with R resulted in the loss of coacervate formation at the concentrations tested.

[1656] Figure 22 shows the outcomes of this experiment, and the results are summarised in Table 15.

[1657] Table 15. Summary of results on coacervate formation.

[1658] [Polymer]

[1659] 320 μM 160 μM 80 μM 40μM 20μM 10 μM 5μM

[1660] FAM- mrvrrnr Not tested Not tested Yes Yes Yes Yes Yes

[1661] [KAJ Y VJVJ J > C

[1662] FAM- ivrnrrnr Not tested Not tested Yes Yes Yes Yes Yes

[1663] [ Y VJKYJLJ] 5C

[1664] FAM- NoNo No No No No No

[1665] [KAJK.VJVJ J 3 V

[1666] Example 19. FAM-[RP2YGG]5C peptides can form coacervates if P2 is a spacer of any length.

[1667] Purpose and summary of experiment

[1668] This experiment was performed to understand the importance of the length of the spacer in the position A2 in the peptide FAM-[RP2YGG]5C, in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The spacers tested for this position were Glycine (C2), 0- Alanine (C3), y-Aminobutyric acid (C4), c-Aminovalcric acid (C5). Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1669] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1670] FAM-[R{y-ABA}Y {y-ABA}]5C, a molecule in which both spacers (G, GG) were replaced by y- Aminobutyric acid (C4) was also tested in the same conditions.

[1671] Materials and methods

[1672] • FAM-[RGYGG]5C.

[1673] • FAM-[R{0-Ala}YGG]5C (0-Alanine replacement).

[1674] • FAM-[R{y-ABA}YGG]5C (y-Aminobutyric acid replacement).

[1675] • FAM-[R{s-AVA}YGG]5C (s-Aminovaleric acid replacement).

[1676] • FAM-[R{y-ABA}Y jy-ABA }]5C (Two y-Aminobutyric acid replacement)

[1677] • OptiMEM tissue culture media.

[1678] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1679] • CX7 LZR PRO Microscope.

[1680] Peptide oxidation

[1681] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1682] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1683] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1684] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C. Coacervate formation and imaging

[1685] 1. Add 20 gl of OptiMEM to the wells of the 384W imaging plate.

[1686] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 gl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1687] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[1688] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1689] 5. Load the plate into the CX7 microscope and acquire images of the coacervates.

[1690] Results and conclusion

[1691] Polymers constituted by FAM-[RP2YGG]5C where P2 = Glycine (G), 0-Alanine (0-Ala), y-Aminobutyric acid (y-ABA), c-Aminovalcric acid (E-AVA) were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution, and on which spacer was present in position P2.

[1692] The ability to undergo significant LLPS and form coacervates increased depending on the length of the spacer in the position P2 according to the following series: G < 0-Ala < y-ABA = s-AVA.

[1693] Furthermore, replacing both “spacers” y- Aminobutyric acid (C4) also resulted in a molecule capable of undergoing significant LLPS, further demonstrating the flexibility in the length of “spacers” that can be used.

[1694] Figure 23 shows the outcomes of this experiment, and the results are summarised in Table 16. Table 16. Summary of results on coacervate formation.

[1695] [Polymer]

[1696] 80 pM 40 pM 20 pM 10 pM 5 pM

[1697] FAM-[RGYGG]5C Yes Yes Yes Yes Yes

[1698] FAM-[R{p-Ala}YGG]5C Yes Yes Yes Yes Yes

[1699] FAM-[R{y-ABA}YGG]5C Yes Yes Yes Yes Yes

[1700] FAM-[R{E-AVA}YGG]5C Yes Yes Yes Yes Yes

[1701] FAM-[R{y-ABA}Y{y-ABA}]5C Yes Yes Yes Yes Yes

[1702] Example 20. FAM-[RP2YP4]5C peptides can form coacervates if P2 and P4 are any of A, G. GG. or GGG spacers.

[1703] Purpose and summary of experiment

[1704] This experiment was performed to understand the importance of the type of the spacer in the position P2 and P4 in the peptide FAM-[RP2YP4]5C, in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The spacers tested for this position were Alanine (A), Glycine (G), Glycine-Glycine (GG), and Glycine-Glycine-Glycine (GGG).

[1705] Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1706] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1707] Materials and methods

[1708] • FAM-[RAYGG]5C.

[1709] • FAM-[RGYGG]5C. • FAM-[RGGYGG]5C.

[1710] • FAM-[RGYG]5C.

[1711] • FAM-[RGYGGG]5C.

[1712] • FAM-[RGGYG]5C.

[1713] • OptiMEM tissue culture media.

[1714] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1715] • CX7 LZR PRO Microscope.

[1716] Peptide oxidation

[1717] 1. Resuspend the polymers in 30% DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1718] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1719] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1720] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1721] Coacervate formation and imaging

[1722] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[1723] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1724] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[1725] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1726] 5. Load the plate into the CX7 microscope and acquire images of the coacervates. Results and conclusion

[1727] Polymers constituted by FAM-[RP2YP4]5C where P2 and / or P4 = Alanine (A), Glycine (G), Glycine-Glycine (GG), and Glycine-Glycine-Glycine (GGG) were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution, and on which spacer was present in positions P2 and P4.

[1728] The ability to undergo significant LLPS and form coacervates was increased when spacer in the positions P2 and P4 = Glycine (G), Glycine-Glycine (GG), and Glycine-Glycine-Glycine (GGG), than when A4 = Alanine (A).

[1729] Figure 24 shows the outcomes of this experiment, and the results are summarised in Table 17. Table 17. Summary of results on coacervate formation.

[1730] [Polymer]

[1731] 80μM 40 μM 20μM 10 μM 5μM

[1732] FAM-[RGYGG]5C Yes Yes Yes Yes Yes

[1733] FAM-[RAYGG]5C Yes Yes Yes Yes Yes

[1734] FAM-[RGGYGG]5C Yes Yes Yes Yes Yes

[1735] FAM-[RGYG]5C Yes Yes Yes Yes Yes

[1736] FAM-[RGYGGG]5C Yes Yes Yes Yes Yes

[1737] FAM-[RGGYG]5C Yes Yes Yes Yes Yes

[1738] Example 21. FAM- [peptide] C molecules can form coacervates if the peptide part of the molecule is constituted by blocks of “stickers” and “spacers”, where a block of “stickers” is made of at least 2 amino acids of any of R, or H, and Y, or F or W, but not K, A or N, and where a block of “spacers” is made of at least 2 elements of any of A, G, B- Alanine, y-Aminobutyric acid, E- Aminovaleric acid, PEG.

[1739] Purpose and summary of experiment

[1740] This experiment was performed to understand the role of “sticker” - “spacer” blocks in a molecule type FAM-[peptide]C, in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The “sticker” blocks tested were Arginine-Tyrosine (RY), Arginine-Tyrosine- Arginine (RYR), Tyrosine-Arginine-Tyrosine (YRY), Arginine-Tyrosine-Arginine-Tyrosine (RYRY), and Tyrosine -Arginine-Tyrosine -Arginine (YR YR). The “spacers” tested were Glycine-Glycine (GG), and Glycine-Glycine-Glycine-Glycine-Glycine (GGGGG).

[1741] Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1742] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1743] Materials and methods

[1744] • FAM-[RGYGG]5C (FAM-RGYGGRGYGGRGYGGRGYGGRGYGGC)

[1745] • FAM-[RYGG]5C (FAM-RYGGRYGGRYGGRYGGRYGGC)

[1746] • FAM-RYR[G]5YRYR[G]5YRY[G]5C (FAM- RYRGGGGGYRYRGGGGGYRYGGGGGC)

[1747] • FAM-RYRY[G]5RYRY[G]5RYRYC (FAM- RYRYGGGGGRYRYGGGGGRYRYC)

[1748] • FAM-[RY]5C (FAM-RYRYRYRYRYC)

[1749] • OptiMEM tissue culture media. Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1750] CX7 LZR PRO Microscope.

[1751] Peptide oxidation

[1752] 1. Resuspend the polymers in 30%DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1753] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1754] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1755] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1756] Coacervate formation and imaging

[1757] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[1758] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[1759] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[1760] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[1761] 5. Load the plate into the CX7 microscope and acquire images of the coacervates.

[1762] Results and conclusion

[1763] Polymers constituted by FAM-[peptide]C where the peptide part of the molecule is constituted by blocks of “stickers” and “spacers”, were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution. Furthermore, FAM-[RY]5C, a molecule constituted by only “spacers” failed to undergo LLPS, forming instead non-coacervate aggregates, which demonstrates the need for both “stickers” and “spacers” for the formation of coacervates.

[1764] Figure 25 shows the outcomes of this experiment, and the results are summarised in Table 18.

[1765] Table 18. Summary of results on coacervate formation. NCA - non-coacervate aggregates.

[1766] [Polymer]

[1767] 0μM 40 μM 20 μM 10μM 5μM

[1768] FAM-[RGYGG]5C Yes Yes Yes Yes Yes

[1769] FAM-[RYGG]5C Yes Yes Yes Yes Yes

[1770] FAM-RYR[G] 5YRYR[G] 5YRY[G] 5C Yes Yes Yes Yes Yes

[1771] FAM-RYRY[G] 5RYRY [G] 5RYRYC Yes Yes Yes Yes Yes FAM-[RY]5C \[CA NCA NCA NCA NCA

[1772] Example 22. WW-[SU-SU-SU-SU-SU]C molecules can form coacervates if the molecule is constituted by 5 blocks of peptide structural units, where a peptide structural unit consists of 5 amino acids, where the first amino acid is any of R, or H, or K, where the second amino acid is any spacer, where the third amino acid is any of Y, or F or W, and where the fourth and fifth amino acid is any spacer.

[1773] Purpose and summary of experiment

[1774] This experiment was performed to understand the behaviour of molecule type WW-[SU-SU-SU-SU-SU]C (“SU” - structural unit), in the phenomenon of liquidliquid phase separation and coacervate formation in un-supplemented tissue culture media. The blocks tested were HGYGG, HGWGG, HGFGG, KGYGG, KGWGG, RGWGG, RGFGG. Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1775] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1776] Materials and methods

[1777] • WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540)

[1778] • WW-RGWGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1567)

[1779] • WW-RGFGG-HGYGG-HGWGG-HGFGG-KGYGG-C (SP-1571)

[1780] • WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643)

[1781] • OptiMEM tissue culture media.

[1782] • Plate centrifuge

[1783] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1784] • CX7 LZR PRO Microscope.

[1785] Peptide oxidation

[1786] 1. Resuspend the polymers in 30%DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1787] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1788] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1789] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C. Coacervate formation and imaging

[1790] 1. Using the top wells of a PCR plate (row A), dilute the polymers in water to 4 mM concentration and a final volume of 4 pl.

[1791] 2. Fill the subsequent wells in the plate (row B to row E) with 2 pl of molecular biology grade water.

[1792] 3. Perform a 1 :2 serial dilution, by taking 2 pl of the top wells (row A) into the wells of row B and mix by pipetting. Repeat the step 5 times, all the way to row E.

[1793] 4. Add 24 pl of OptiMEM to the wells of a different PCR plate. Fill rows A to F.

[1794] 5. Add 1 of the polymer dilution series to the 24 pl of OptiMEM in the PCR plate.

[1795] Mix the polymers by pipetting, at this point the coacervates form.

[1796] 6. Add 20 pl of the coacervate mix to the wells of the imaging plate.

[1797] 7. Centrifuge the imaging plate for 2 min at 137 xg.

[1798] 8. Load the plate containing the coacervates into the CX7 microscope and acquire images of the coacervates in brightfield.

[1799] Results and conclusion

[1800] Polymers constituted by WW-[SU-SU-SU-SU-SU]C where the molecule is constituted by the structural units HGYGG, HGWGG, HGFGG, KGYGG, KGWGG, RGWGG, RGFGG in random positions, were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution.

[1801] Figure 26 shows the outcomes of this experiment, and the results are summarised in Table 19. Table 19. Summary of results on coacervate formation. NCA - non-coacervate aggregates.

[1802] [Polymer]

[1803] 80 pM 40 pM 20 pM 10 pM 5 pM

[1804] WW-HGYGG-HGWGG-HGFGG-

[1805] KGYGG-KGWGG-C (SP-1540)YeS YeS YeS YeS N°

[1806] WW-RGWGG-RGFGG-HGYGG-v

[1807] HGWGG-HGFGG-C (SP-1567)YeS YeS YeS YeS N°

[1808] WW-RGFGG-HGYGG-HGWGG-

[1809] HGFGG-KGYGG-C (SP-1571)YeS YeS YeS YeS N°

[1810] WW-KGYGG-RGFGG-HGYGG-

[1811] HGWGG-HGFGG-C (SP-1643)YeS YeS YeS N°N°

[1812] Example 23. WW-[SU-SU-SU-SU-SU]C molecules can form complex coacervates and deliver RNA cargo to myotubes if the molecule is constituted by 5 structural units, where a peptide structural unit consists of 5 amino acids, where the first amino acid is any of R, or H, or K, where the second amino acid is any spacer, where the third amino acid is any of Y, or F or W, and where the fourth and fifth amino acid is any spacer.

[1813] Purpose and summary of experiment This experiment was performed to understand the behaviour of a molecule type

[1814] WW-[SU-SU-SU-SU-SU]C, in the phenomenon of liquid-liquid phase separation, complex coacervate formation in un-supplemented tissue culture media , and RNA cargo delivery to myotubes. The structural units tested were HGYGG, HGWGG, HGFGG, KGYGG, KGWGG, RGWGG, RGFGG. The polymers were tested at a final concentration of 80 pM. The cargo, mCherry mRNA, was tested at a final concentration of 5 ng / pl. After induction of LLPS, the coacervates were added to C2C12 myotubes, previously seeded into 96W imaging plates. The cells we incubated for 24h and then observed on a microscope.

[1815] Materials and methods

[1816] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1817] • WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540)

[1818] • WW-RGWGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1567)

[1819] • WW-RGFGG-HGYGG-HGWGG-HGFGG-KGYGG-C (SP-1571)

[1820] • WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643)

[1821] • CleanCap mCherry mRNA (Tebubio, L-7203)

[1822] • Sterile, phenol-free, serum-free DMEM tissue culture media.

[1823] • Myotube differentiation tissue culture media (DMEM + 2% Horse Blood Serum)

[1824] • Myotubes differentiated from C2C12 cell line.

[1825] • Imaging plate (96 well black plate with optically clear polystyrene bottom, precoated with collagen).

[1826] • CX7 LZR PRO Microscope.

[1827] Cell culture

[1828] C2C12 cells were seeded into the imaging plate, and myotubes were differentiated using standard mammalian cell culture procedures. Coacervate formation and cell delivery

[1829] 1. Wash myotubes and replace the old media with 5 volumes of DMEM (phenol- free, serum-free).

[1830] 2. In a centrifuge tube, mix the polymers with the cargo (CleanCap mCherry mRNA). Induce condensation by adding DMEM (phenol-free, serum-free) to 3.5x final concentration, and mix by pipetting.

[1831] 3. Add 2 volumes of coacervate mixture to the 5 volumes of DMEM (phenol-free, serum-free) containing the myotubes in the well of the imaging plate.

[1832] 4. Centrifuge the imaging plate for Imin at 137xg.

[1833] 5. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for Ih.

[1834] 6. Wash the cells 3x with Myotube Differentiation Tissue Culture media.

[1835] 7. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for 24h.

[1836] 8. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1837] Channel Mode Dye Light Source Emission Filter Notes

[1838] #1 BF N / A N / A N / A

[1839] 80% Acquisition time

[1840] #2 Widefield mCherry 594 nm 646 / 74 nm of positive control

[1841] #3 Widefield mCherry 594 nm 646 / 74 nm Results and conclusion

[1842] All the tested peptides formed complex coacervates with the cargo (RNA) and successfully transfected post-mitotic myotubes differentiated from C2C12 cells, shown coacervates loaded with DNA cargo can transfect myocytes and promote protein expression.

[1843] Purpose and summary of experiment

[1844] This experiment was performed to demonstrate that the polymer WW-HGYGG- HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) will form coacervates, enter and release cargo inside myotubes differentiated from C2C12. The cargos consist of different types of DNA (nanoplasmid and linear dsDNA).

[1845] Three different final concentrations of the cargos Nanoplasmid - eGFP and linear dsDNA - GFP (Doggybone) were tested (20, 10 and 5 ng / |il) with the same final concentration of polymer (80 |1M). After induction of LLPS, the coacervates were added to C2C12 myotubes, previously seeded into 96W imaging plates. The cells we incubated for 48h and then observed on a microscope.

[1846] Materials and methods

[1847] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1848] • WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540).

[1849] • pALD-Nanoplasmid-CMV-EGFP (Aldevron; 5202FS)

[1850] • CMV-TE-eGFP Doggybone (Touchlight, RD.100-028.212.2)

[1851] • Sterile, phenol-free, serum-free DMEM tissue culture media.

[1852] • Myotube differentiation tissue culture media (DMEM + 2% Horse Blood Serum)

[1853] • Myotubes differentiated from C2C12 cell line.

[1854] • Imaging plate (96 well black plate with optically clear polystyrene bottom, precoated with collagen).

[1855] • CX7 LZR PRO Microscope.

[1856] Cell culture

[1857] C2C12 cells were seeded into the imaging plate, and myotubes were differentiated using standard mammalian cell culture procedures.

[1858] Coacervate formation and cell delivery

[1859] 1. Wash myotubes and replace the old media with 5 volumes of DMEM (phenol- free, serum-free). 2. In a centrifuge tube, mix the polymer WW-HGYGG-HGWGG-HGFGG- KGYGG-KGWGG-C (SP-1540) with the cargo (pALD-Nanoplasmid-CMV- EGFP or CMV-TE-eGFP Doggybone). Induce condensation by adding DMEM (phenol-free, serum- free) to 3.5x final concentration, and mix by pipetting.

[1860] 3. Add 2 volumes of coacervate mixture to the 5 volumes of DMEM (phenol-free, serum-free) containing the myotubes in the well of the imaging plate.

[1861] 4. Centrifuge the imaging plate for Imin at 137xg.

[1862] 5. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for Ih.

[1863] 6. Wash the cells 3x with Myotube Differentiation Tissue Culture media.

[1864] 7. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for 48h.

[1865] 8. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1866] Channel Mode Dye Light Source Emission Filter Notes

[1867] #1 BF N / A N / A N / A

[1868] #2 Widefield eGFP 488 nm 542 / 27 nm 80% Acquisition time of positive control

[1869] #3 Widefield eGFP 488 nm 542 / 27 nm

[1870] Results and conclusion

[1871] The peptide WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) formed complex coacervates with the cargo (DNA) and successfully transfected postmitotic myotubes differentiated from C2C12 cells, shown in Figure 28. Results summarised in Table 21. Table 21. Summary of results on myotube transfection.

[1872] Cargo (ng / pl) 20 10 5

[1873] Nanoplasmid- %Norm Transfection Efficiency 102.5 109.2 42.3

[1874] EGFP %Norm Fluorescence Intensity 69.9 84.3 11.0 eGFP %Norm Transfection Efficiency 57.5 71.8 15.7

[1875] Doggybone %Norm Fluorescence Intensity 36.1 39.4 5.7

[1876] Example 25. WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) and WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643) peptides can form complex coacervates with proteins and peptides, enter cells, and deliver the cargo.

[1877] Purpose and summary of experiment

[1878] This experiment was performed to demonstrate that the polymers WW- HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540) and WW-KGYGG- RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643) will form complex coacervates, enter and release cargo inside U2OS cells. The cargos consist of protein (AF594 - anti- Nuclear Pore Complex Protein Antibody) or F AM-labelled peptides.

[1879] The polymers were tested at a final concentration of 20 (SP-1540) or 10 pM (SP-1643). The cargos were tested at a final concentration of 3 ng / pl (AF594-antibody), 5 pM (F AM-labelled peptide 1), and 10 pM (F AM-labelled peptide 2).

[1880] After induction of LLPS, the coacervates were added to U2OS cells, previously seeded into 384W imaging plates. The cells were incubated for 4h and then observed on a microscope. Materials and methods

[1881] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1882] • WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP-1540).

[1883] • WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643).

[1884] • AF594-antibody.

[1885] • FAM-labelled peptide 1.

[1886] • FAM-labelled peptide 2.

[1887] • Hoechst 34580.

[1888] • Sterile OptiMEM tissue culture media.

[1889] • U2OS cells.

[1890] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1891] • CX7 LZR PRO Microscope.

[1892] Cell culture

[1893] U2OS cells were seeded into the imaging plate using standard mammalian cell culture procedures.

[1894] Coacervate formation and cell delivery

[1895] 1. Wash U2OS cells and replace the old media with 5 volumes of OptiMEM.

[1896] 2. In a centrifuge tube, mix the polymer WW-HGYGG-HGWGG-HGFGG- KGYGG-KGWGG-C (SP-1540) or and WW-KGYGG-RGFGG-HGYGG- HGWGG-HGFGG-C (SP-1643) with the cargos (AF594-antibody, FAM-labelled peptide 1 or 2). Induce condensation by adding OptiMEM to 3.5x final concentration and mix by pipetting. 3. Add 1 volume of coacervate mixture to the 5 volumes of OptiMEM containing the mammalian cells in the well of the imaging plate.

[1897] 4. Centrifuge the imaging plate for Imin at 137xg.

[1898] 5. Incubate the U2OS cells in standard conditions of temperature and CO2 for mammalian cell culture for Ih.

[1899] 6. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1900] 7. Incubate the U2OS cells in standard conditions of temperature and CO2 for mammalian cell culture for 3h.

[1901] 8. Add Hoechst 34580 (final concentration = 1 pg / mL) to the cells and incubate for 20 minutes in standard conditions of temperature and CO2.

[1902] 9. Wash the cells 3x with phenol-free Tissue Culture media supplemented with FBS.

[1903] 10. Centrifuge the imaging plate for Imin at 137xg.

[1904] 11. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1905] Channel Mode Dye Light Source Emission Filter

[1906] #1 BF N / A N / A N / A

[1907] #2 Widefield Hoechst 405 nm 446 / 37 nm

[1908] #3 Widefield FAM 488 nm 542 / 27 nm

[1909] #4 Widefield AF594 594 nm 646 / 74 nm

[1910] Results and conclusion

[1911] The polymers WW-HGYGG-HGWGG-HGFGG-KGYGG-KGWGG-C (SP- 1540) and WW-KGYGG-RGFGG-HGYGG-HGWGG-HGFGG-C (SP-1643) formed complex coacervates with the cargos (AF594-labelled antibody; F AM-labelled peptides 1 and 2) and successfully delivered them to U20S cells, shown in Figure 29. Results summarised in Table 22.

[1912] Table 22. Summary of results on cell entry and diffusion.

[1913] AF594-Antibody FAM-labelled Peptides

[1914] WW-HGYGG-HGWGG-HGFGG-

[1915] KGYGG-KGWGG-C (SP-1540)YeS YeS

[1916] WW-KGYGG-RGFGG-HGYGG-

[1917] HGWGG-HGFGG-C (SP-1643)

[1918] Example 26. In the molecule WW-1SU-SU-SU-SU-SU1C (SU - structural units), replacing a R, H, or K with an alternative non-natural amino acid, specifically PiplF (2-Amino-4-(piperidin-l-yl)propanoic acid), or replacing a F, W, or Y with an alternative non-natural amino acid, specifically TIC (1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid), result in molecules that can form coacervates.

[1919] Purpose and summary of experiment

[1920] This experiment was performed to understand the impact of using non-natural amino acid chains in molecules of the type WW-[SU-SU-SU-SU-SU]C (“SU” - structural unit), in the phenomenon of liquid-liquid phase separation and coacervate formation in un-supplemented tissue culture media. The alternative amino acids tested were PiplF and TIC. The molecules tested were WW-(PiplF)GYGG-RGFGG- HGYGG-HGWGG-HGFGGGG-C (SP-1780), and WW-HGYGG-HGWGG- HG(TIC)GG-KGYGG-KGWGG-C (SP- 1793).

[1921] Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1922] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope. Materials and methods

[1923] • WW-(PiplF)GYGG-RGFGG-HGYGG-HGWGG-HGFGGGG-C (SP-1780).

[1924] • WW-HGYGG-HGWGG-HG(TIC)GG-KGYGG-KGWGG-C (SP-1793).

[1925] • OptiMEM tissue culture media.

[1926] • Plate centrifuge

[1927] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1928] • CX7 LZR PRO Microscope.

[1929] Peptide oxidation

[1930] 1. Resuspend the polymers in 30%DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1931] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[1932] 3. Replace the volume of solvent lost during the procedure with molecular grade water.

[1933] 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[1934] Coacervate formation and imaging

[1935] 1. Using the top wells of a PCR plate (row A), dilute the polymers in water to 4 mM concentration and a final volume of 4 pl.

[1936] 2. Fill the subsequent wells in the plate (row B to row E) with 2 pl of molecular biology grade water.

[1937] 3. Perform a 1 :2 serial dilution, by taking 2 pl of the top wells (row A) into the wells of row B and mix by pipetting. Repeat the step 5 times, all the way to row E.

[1938] 4. Add 24 pl of OptiMEM to the wells of a different PCR plate. Fill rows A to F. 5. Add 1 of the polymer dilution series to the 24 pl of OptiMEM in the PCR plate. Mix the polymers by pipetting, at this point the coacervates form.

[1939] 6. Add 20 pl of the coacervate mix to the wells of the imaging plate.

[1940] 7. Centrifuge the imaging plate for 2 min at 137 xg. 8. Load the plate containing the coacervates into the CX7 microscope and acquire images of the coacervates in brightfield.

[1941] Results and conclusion Polymers constituted by WW-[SU-SU-SU-SU-SU]C where either R, K, or H, was replaced by PiplF, or W, F, or Y was replaced by TIC, were able to undergo significant LLPS to form coacervates. This ability was dependent on the concentration of the polymer in solution.

[1942] Figure 30 shows the outcomes of this experiment, and the results are summarised in Table 23.

[1943] Table 23. Summary of results on coacervate formation. NCA - non-coacervate aggregates.

[1944] [Polymer]

[1945] 80 pM 40 pM 20 pM 10 pM 5 pM

[1946] WW-(PiplF)GYGG-RGFGG-HGYGG-

[1947] Yes Yes Yes Yes No

[1948] HGWGG-HGFGGGG-C (SP-1780)

[1949] WW-HGYGG-HGWGG-HG(TIC)GG-

[1950] Yes Yes Yes Yes No

[1951] KGYGG-KGWGG-C (SP-1793) Example 27. In the molecule WW-1SU-SU-SU-SU-SU1C (SU - structural units), replacing a R, H, or K with an alternative non-natural amino acid, specifically PiplF (2-Amino-4-(piperidin-l-yl)propanoic acid), or replacing a F, W, or Y with an alternative non-natural amino acid, specifically TIC (1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid), result in molecules that can form complex coacervates and deliver RNA cargo to myotubes.

[1952] Purpose and summary of experiment

[1953] This experiment was performed to understand the impact of using non-natural amino acid chains in molecules of the type WW-[SU-SU-SU-SU-SU]C (“SU” - structural unit), in the phenomenon of liquid-liquid phase separation, complex coacervate formation in un-supplemented tissue culture media, and RNA cargo delivery to myotubes. The alternative amino acids tested were PiplF and TIC. The molecules tested were WW-(PiplF)GYGG-RGFGG-HGYGG-HGWGG-HGFGGGG-C (SP- 1780), and WW-HGYGG-HGWGG-HG(TIC)GG-KGYGG-KGWGG-C (SP-1793).

[1954] The polymers were tested at a final concentration of 80 pM. The cargo, mCherry mRNA or eGFP mRNA, was tested at a final concentration of 5 ng / pl.

[1955] After induction of LLPS, the coacervates were added to C2C12 myotubes, previously seeded into 96W imaging plates. The cells we incubated for 24h and then observed on a microscope.

[1956] Materials and methods

[1957] The entire protocol for this example was performed in a cell culture hood, using standard mammalian cell culture procedures and best practices.

[1958] • WW-(PiplF)GYGG-RGFGG-HGYGG-HGWGG-HGFGGGG-C (SP-1780).

[1959] • WW-HGYGG-HGWGG-HG(TIC)GG-KGYGG-KGWGG-C (SP-1793).

[1960] • CleanCap mCherry mRNA (Tebubio, L-7203)

[1961] • eGFP mRNA N 1 -methyl-pseudouridine (Genscript, SC2346) • Sterile, phenol-free, serum-free DMEM tissue culture media.

[1962] • Myotube differentiation tissue culture media (DMEM + 2% Horse Blood Serum)

[1963] • Myotubes differentiated from C2C12 cell line.

[1964] • Imaging plate (96 well black plate with optically clear polystyrene bottom, precoated with collagen).

[1965] • CX7 LZR PRO Microscope.

[1966] Cell culture

[1967] C2C12 cells were seeded into the imaging plate, and myotubes were differentiated using standard mammalian cell culture procedures.

[1968] Coacervate formation and cell delivery

[1969] 1. Wash myotubes and replace the old media with 5 volumes of DMEM (phenol- free, serum-free).

[1970] 2. In a centrifuge tube, mix the polymers with the cargo (mRNA). Induce condensation by adding DMEM (phenol-free, serum-free) to 3.5x final concentration, and mix by pipetting.

[1971] 3. Add 2 volumes of coacervate mixture to the 5 volumes of DMEM (phenol-free, serum-free) containing the myotubes in the well of the imaging plate.

[1972] 4. Centrifuge the imaging plate for Imin at 137xg.

[1973] 5. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for Ih.

[1974] 6. Wash the cells 3x with Myotube Differentiation Tissue Culture media.

[1975] 7. Incubate the myotubes in standard conditions of temperature and CO2 for mammalian cell culture for 24h. 8. Load the plate into the CX7 LZR PRO microscope and acquire images of the cells. All images were acquired using the same combination of lasers, filters, and image acquisition times:

[1976] Channel Mode Dye Light Source Emission Filter Notes

[1977] #1 BF N / A N / A N / A eGFP / 488nm / 594 524 / 46 nm / 80% Acquisition time mCherry nm 646 / 74 nm of positive control , , eGFP / 488nm / 594 524 / 46 nm /

[1978] #3 Widefield mCherry nm 646 / 74 nm

[1979] Results and conclusion

[1980] Polymers constituted by WW-[SU-SU-SU-SU-SU]C where either R, K, or H, was replaced by PiplF, or W, F, or Y was replaced by TIC, were able to form complex coacervates with the cargo (RNA) and successfully transfected post-mitotic myotubes differentiated from C2C12 cells, shown in Figure 31. Results summarised in Table 24.

[1981] Table 24. Summary of results on myotube transfection.

[1982] %Norm %Norm

[1983] Transfection Fluorescence

[1984] Efficiency Intensity

[1985] WW-(PiplF)GYGG-RGFGG-HGYGG-

[1986] HGWGG-HGFGGGG-C (SP-1780)89 5 8 0 5 Example 28. FAM-[rgygg]5c D-peptide can form coacervates.

[1987] Purpose and summary of experiment

[1988] This experiment was performed to determine if a peptide constituted by D-amino acids and the sequence FAM-[rgygg]5c (lower case denoting D-amino acids) was capable of undergoing liquid-liquid phase separation and form coacervate in unsupplemented tissue culture media.

[1989] Different final concentrations of the polymers were tested (80 pM, 40 pM, 20 pM, 10 pM, and 5 pM) and their ability to undergo LLPS and form coacervates in OptiMEM was accessed.

[1990] After induction of LLPS, the coacervates were added to imaging plates and observed on a microscope.

[1991] Materials and methods

[1992] • FAM-[RGYGG]5C (upper case denoting L-peptide).

[1993] • FAM-[rgygg]5c (lower case denoting D-peptide).

[1994] • OptiMEM tissue culture media.

[1995] • Imaging plate (384 well black plate with optically clear polystyrene bottom, precoated with PDL).

[1996] • CX7 LZR PRO Microscope.

[1997] Peptide oxidation

[1998] 1. Resuspend the polymers in 30%DMSO (in water) up to a stock concentration of 5 mM, in centrifuge tubes.

[1999] 2. The tubes are left open to the air and covered with aluminium foil (to protect from light) up to 24h to oxidize.

[2000] 3. Replace the volume of solvent lost during the procedure with molecular grade water. 4. The DMSO-treated molecules are aliquoted and stored @ -20 °C.

[2001] Coacervate formation and imaging

[2002] 1. Add 20 pl of OptiMEM to the wells of the 384W imaging plate.

[2003] 2. In centrifuge tubes, add the polymers to OptiMEM to a final volume of 10 pl and at 5x their final concentration. Mix the polymers by pipetting, at which point the coacervates form.

[2004] 3. Add 5 pl of coacervate mixture to the wells of the 384W imaging plate.

[2005] 4. Centrifuge the mixture of the coacervates for 2 min at 137xg.

[2006] 5. Load the plate into the CX7 microscope and acquire images of the coacervates.

[2007] Results and conclusion

[2008] Polymers constituted by FAM-[RGYGG]5C were able to undergo significant LLPS to form coacervates, regardless of whether they were constituted by L-amino acids or D-amino acids. This ability was dependent on the concentration of the polymer in solution.

[2009] Figure 32 shows the outcomes of this experiment, and the results are summarised in Table 25.

[2010] Table 25. Summary of results on coacervate formation.

[2011] [Polymer]

[2012] 80 pM 40 pM 20 pM 10 pM 5 pM

[2013] FAM-[RAYGG]5C (L-peptide) Yes Yes Yes Yes Yes

[2014] FAM-[rgygg]5c (D-peptide) Yes Yes Yes Yes Yes Example 29. FAM-1RGYGG15C peptides can form coacervates and enter cells regardless of whether the C-terminus of the polymer is a Carboxylic Acid group or a Carboxamide group.

[2015] Purpose and summary of experiment

[2016] This experiment was performed to demonstrate that the polymer FAM- [RGYGG]5C will form coacervates, enter and diffuse inside HEK293 cells, regardless of whether the C-terminus of the polymer is a carboxylic acid group (FAM- [RGYGG]5C-carb) or a Carboxamide group (FAM-[RGYGG]5C-amide). These two different C-terminal end groups result from using different resins to synthesize the polymer.

[2017] Three different final concentrations of the polymers FAM-[RGYGG]5C-carb and FAM-[RGYGG]5C-amide were tested (40 pM, 20 pM, and 10 pM).

[2018] After induction of LLPS, the coacervates were added to HEK293 cells, previously seeded into 384W i...

Claims

1. CLAIMS1. A coacervate-forming polymer comprising or consisting of a structure according to formula (I):Rl-N[Pl-P2-P3-P4]nC (I) wherein:A. Ri is occupied by one or more aromatic molecules;B. N is the N-terminal end of the polymer;C. [P1-P2-P3-P4] is a structural unit of the polymer;D. n is the number of structural units of the polymer, wherein n is an integer of 2 or more;E. C is the C-terminal end of the polymer;F. P is an amino acid residue position or a spacer position in the polymer; andG. each one of Pi, P2, P3 and P4consists of one or more amino acid residue positions and / or one or more spacer positions, wherein: i. one or more of said positions is occupied by a molecule selected from the group Al consisting of arginine (R), histidine (H) and lysine (K); and ii. one or more additional position(s) is occupied by a molecule selected from the group A2 consisting of tyrosine (Y), phenylalanine (F), and tryptophan (W); and iii. one or more further additional positions are occupied by a molecule selected from the group A3 consisting of glycine (G), alanine (A), glycine-glycine (G-G), glycine-glycine-glycine (G- G-G), glycine-glycine-glycine-glycine (G-G-G-G), or any spacer.

2. A polymer according to claim 1, wherein the spacer in group A3 comprises:A. a molecule comprising 3 carbon bonds (C3 spacer), e.g. 0-alanine;B. a molecule comprising 4 carbon bonds (C4 spacer), e.g. 4-aminobutyric acid or two glycine residues;C. a molecule comprising 5 carbon bonds (C5 spacer), e.g. 5-aminovaleric acid;D. a molecule comprising 6 carbon bonds (C6 spacer), e.g. 6-aminohexanoic acid; orE. a polyethylene glycol (PEG) spacer, e.g. a PEG2 spacer (8-amino-3,6- dioxaoctanoic acid), a PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), or a PEG4 spacer (15-amino-4,7,10,13-tetraoxapenta-decanoic acid).

3. A polymer according to claim 1 or claim 2, wherein in the direction N-terminal to C-terminal:A. all structural units of the polymer are ordered in the sequence [P1-P2-P3-P4];B. the first structural unit at the N-terminal end of the polymer is ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer is ordered in the sequence [P4-P3-P2-P1];C. the first structural unit at the N-terminal end of the polymer is ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer is ordered in the sequence [P1-P2-P3-P4];D. the last structural unit at the C-terminal end of the polymer is ordered in the sequence [P1-P2-P3-P4], and at least one further structural unit of the polymer is ordered in the sequence [P4-P3-P2-P1];E. the last structural unit at the C-terminal end of the polymer is ordered in the sequence [P4-P3-P2-P1], and at least one further structural unit of the polymer is ordered in the sequence [P1-P2-P3-P4]; orF. all structural units of the polymer are ordered in the sequence [P4-P3-P2-P1].

4. A polymer according to any one of the preceding claims, wherein n is an integer of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more, preferably wherein n is 5 or more.

5. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:A. position Pi is occupied by a molecule selected from the group Al;B. position P2 is occupied by a molecule selected from the group A3;C. position P3 is occupied by a molecule selected from the group A2; andD. position P4 is occupied by a molecule selected from the group A3.

6. A polymer according to claim 5, wherein in any one or more structural units, or in all structural units:

1. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G;2. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;3. a) position Pi is occupied by H; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;4. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by W; and d) position P4 is occupied by G-G;5. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by F; and d) position P4 is occupied by G-G;6. a) position Pi is occupied by R; b) position P2 is occupied by a spacer which is: i. P-alanine; ii. y-aminobutyric acid; or iii. 5-aminovaleric acid; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;7. a) position Pi is occupied by R; b) position P2 is occupied by a spacer which is y-aminobutyric acid; c) position P3 is occupied by Y ; and d) position P4 is occupied by a spacer which is y-aminobutyric acid;8. a) position Pi is occupied by R; b) position P2 is occupied by A; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G;9. a) position Pi is occupied by R; b) position P2 is occupied by G-G; c) position P3 is occupied by Y ; andd) position P4 is occupied by G-G;10. a) position Pi is occupied by R; b) position P2 is occupied by G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G-G-G;11. a) position Pi is occupied by R; b) position P2 is occupied by G-G; c) position P3 is occupied by Y ; and d) position P4 is occupied by G;12. e) position Pi is occupied by H; f) position P2 is occupied by G; g) position P3 is occupied by F; and h) position P4 is occupied by G-G;13. e) position Pi is occupied by H; f) position P2 is occupied by G g) position P3 is occupied by W; and h) position P4 is occupied by G-G;14. e) position Pi is occupied by K; f) position P2 is occupied by G g) position P3 is occupied by Y ; and h) position P4 is occupied by G-G; ore) position Pi is occupied by K; f) position P2 is occupied by G g) position P3 is occupied by W; and h) position P4 is occupied by G-G.

7. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:A. position Pi is occupied by a molecule selected from the group A2;B. position P2 is occupied by a molecule selected from the group A3;C. position P3 is occupied by a molecule selected from the group Al; andD. position P4 is occupied by a molecule selected from the group A3.

8. A polymer according to claim 7, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by Y ; b) position P2 is occupied by G; c) position P3 is occupied by R; and d) position P4 is occupied by G-G.

9. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:A. position Pi is occupied by a molecule selected from the group Al;B. position P2 is occupied by a molecule selected from the group A2;C. position P3 is occupied by a molecule selected from the group A3; andD. position P4 is occupied by a molecule selected from the group A3.

10. A polymer according to claim 9, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by R; b) position P2 is occupied by Y ; c) position P3 is occupied by G; and d) position P4 is occupied by G.

11. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units each one of Pi, P2, P3 and P4 consists of one position, and wherein:A. position Pi is occupied by a molecule selected from the group A2;B. position P2 is occupied by a molecule selected from the group Al;C. position P3 is occupied by a molecule selected from the group A3; andD. position P4 is occupied by a molecule selected from the group A3.

12. A polymer according to claim 11, wherein in any one or more structural units, or in all structural units: a) position Pi is occupied by Y ; b) position P2 is occupied by R; c) position P3 is occupied by G; and d) position P4 is occupied by G.

13. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units:A. position Pi is occupied by two amino acid residues, wherein: i. the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; orii. the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; andB. position P2: i. is occupied by one amino acid residue, which is a molecule selected from the group Al; or ii. is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2, and the second of said two amino acid residues is occupied by a molecule selected from the group Al; or iii. is occupied by one amino acid residue, which is a molecule selected from the group A2; andC. position P3 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; andD. position P4 is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G-G.

14. A polymer according to claim 13, wherein n=3, and wherein in the direction N- terminal to C-terminal:A. in the first structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; ii. position P2 is occupied by one amino acid residue, which is a molecule selected from the group Al and is R; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G; andB. in the second structural unit:i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G; andC. in the third structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y, and the second of said two amino acid residues is occupied by a molecule selected from the group Al and is R; ii. position P2 is occupied by one amino acid residue which is a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by a molecule selected from the group A3 and is G-G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G-G.

15. A polymer according to any one of claims 1-4, wherein in any one or more structural units, or in all structural units:A. position Pi: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; orii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G; andB. position P2: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; andC. position P3: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G; andD. position P4: i. is occupied by two amino acid residues, the first of said two amino acid residues is occupied by a molecule selected from the group Al, and the second of said two amino acid residues is occupied by a molecule selected from the group A2; or ii. is occupied by one spacer residue position, which is a molecule selected from the group A3 and is G-G.

16. A polymer according to claim 15, wherein n=3, and wherein in the direction N- terminal to C-terminal:A. in the first structural unit: i. position Pi is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ;ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by a molecule selected from the group A3 and is G; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G; andB. in the second structural unit: i. position Pi is occupied by a molecule selected from the group A3 and is G-G; ii. position P2 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; iii. position P3 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; and iv. position P4 is occupied by a molecule selected from the group A3 and is G-G; andC. in the third structural unit: i. position Pi is occupied by a molecule selected from the group A3 and is G-G; ii. position P2 is occupied by a molecule selected from the group A3 and is G; iii. position P3 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y ; andiv. position P4 is occupied by two amino acid residues, wherein the first of said two amino acid residues is occupied by a molecule selected from the group Al and is R, and the second of said two amino acid residues is occupied by a molecule selected from the group A2 and is Y.

17. A polymer according to any one of the preceding claims, wherein Ri is one or more aromatic molecules, wherein Ri has a total aromaticity value of 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more or 35 or more, preferably the aromaticity value is between 5 and 35, more preferably the aromaticity value is 18 or 20.

18. A polymer according to any one of the preceding claims, wherein Ri comprises or consists of any one or more of the molecules:A. fluorescein or a fluorescein amidite (FAM), preferably 5 -FAM (5- carboxyfluorescein);B. 5-FAM-PEG2;C. Rhodamine B;D. Hexachloro-fluorescein (HEX);E. naphtyl-alanyl-4-naphtyl butanoic acid;F. pyrenebutyric acid;G. fluorenylmethoxycarbonyl protecting group (FMOC)-PEG2;H. FMOC;I. carboxytetramethylrhodamine (TAMRA);J. 4-naphthalen-2-yl-butanoic acid;K. anthracene propanoic acid;L. naproxyl;M. phenylbutanoic acid;N. 5 -(piperazin- l-yl)pyrazine-2-carboxylic acid; orO. an aromatic amino acid, such as phenylalanine, tyrosine, histidine, or tryptophan.

19. A polymer according to claim 16, wherein Ri consists of two or more of the molecules A-O, wherein the two or more molecules are the same molecules or different molecules.

20. A polymer according to any one of the preceding claims, wherein the aromatic molecule Ri is attached to the N-terminus of the first structural unit of the polymer by a linker, optionally wherein the linker is any one of the spacers (A) to (E) defined in claim2.

21. A polymer according to claim 5, wherein at least one structural unit of the polymer has the amino acid sequence:

1. [R-G-Y-G];2. [R-G-Y-G-G];3. [H-G-Y-G-G];4. [R-G-W-G-G];5. [R-G-F-G-G];6. [R-P-alanine-Y-G-G];7. [R-y-aminobutyric acid-Y-G-G];8. [R-5-aminovaleric acid-Y-G-G];9. [R-y-aminobutyric acid-Y-y-aminobutyric acid];10. [R-A-Y-G-G];11. [R-G-G-Y-G-G];12. [R-G-Y-G-G-G];13. [R-G-G-Y-G];14. [H-G-F-G-G];15. [H-G-W-G-G];16. [K-G-Y-G-G]; or17. [K-G-W-G-G]; optionally wherein:A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / orB. all structural units of the polymer have the same amino acid sequence.

22. A polymer according to claim 7, wherein at least one structural unit of the polymer has the amino acid sequence:[Y-G-R-G-G]; optionally wherein:A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / orB. all structural units of the polymer have the same amino acid sequence.

23. A polymer according to claim 9, wherein at least one structural unit of the polymer has the amino acid sequence:[R-Y-G-G]; optionally wherein:A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / orB. all structural units of the polymer have the same amino acid sequence.

24. A polymer according to claim 11, wherein at least one structural unit of the polymer has the amino acid sequence:[Y-R-G-G]; optionally wherein:A. in the direction N-terminal to C-terminal all structural units of the polymer are ordered in the sequence P1-P2-P3-P4; and / orB. all structural units of the polymer have the same amino acid sequence.

25. A polymer according to any one of claims 22-25, wherein: n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10; or n=10 or more, preferably wherein n=5.

26. A polymer according to claim 13, wherein:A. n=3B. the first structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [RYRGGGGG];C. the second structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [YRYRGGGGG]; andD. the third structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [YRYGGGGG].

27. A polymer according to claim 15, wherein:A. n=3B. the first structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [RYRYGGG];C. the second structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [GGRYRYGG]; andD. the third structural unit of the polymer has the amino acid sequence in the direction N-terminal to C-terminal [GGGRYRY],28. A polymer according to any one of the preceding claims, wherein position Ri is occupied by FAM or two tryptophan amino acids (WW).

29. A polymer according to any one of the preceding claims, wherein formula (I) is further defined as formula (II):Rl-N[Pi-P2-P3-P4]nC-R2 (II) wherein R2 is any chemical group or molecule that is capable of forming a bond, preferably a covalent bond, more preferably a disulphide bond, with a further R2 group of a second polymer according to any one of the preceding claims, preferably wherein the molecule at position R2 is cysteine.

30. A dimeric coacervate-forming polymer comprising or consisting of two monomers, wherein each monomer comprises or consists of a coacervate-forming polymer according to any one of claims 1 to 28, wherein each monomer has a structure further defined by formula (II):Rl-N[Pi-P2-P3-P4]nC-R2 (II); and wherein the two monomers are linked by a bond between each R2 group, preferably a covalent bond, more preferably a disulphide bond, optionally wherein the two monomers have the same structure, or wherein the two monomers have different structures.

31. A polymer according to any one of the preceding claims, wherein the polymer is capable of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate.

32. A polymer according to any one of claims 1 to 30, wherein the polymer is capable of forming a polymer coacervate in an aqueous solution, wherein the polymer coacervate comprises the polymer within the coacervate, and wherein the polymer coacervate is capable of penetrating into a viable eukaryotic cell upon contact with the cell in an aqueous solution supporting the viability of the cell.

33. A polymer according to any one of the preceding claims, wherein a cofactor, such as a crowding agent, e.g. RNA, sucrose or polyethylene glycol (PEG), is not required for coacervate formation mediated by the polymer.

34. A method of making a polymer solution, the method comprising providing a polymer, contacting the polymer with a solvent or solution, and dissolving the polymer in the solvent or solution, thereby creating the polymer solution, wherein the polymer is defined according to any one of claims 1 to 33.

35. A method of making a solution of an oxidised polymer, the method comprising providing a polymer in solution, wherein the polymer is defined according to any one of claims 1 to 33, and subjecting the polymer solution to oxidising conditions.

36. A method according to claim 35, wherein the subjecting the polymer solution to oxidising conditions comprises exposing the polymer solution to air for 2 or more hours, e.g. from 2 hours to 24 hours, preferably in dark conditions at room temperature, or wherein the oxidising conditions comprise adding an oxidising agent to the polymer solution, optionally wherein the oxidising agent is dimethyl sulfoxide (DMSO).

37. A solution of an oxidised polymer, wherein the solution is obtained by performing the method of claim 35 or claim 36.

38. A polymer coacervate, wherein the polymer coacervate comprises a polymer as defined according to any one of claims 1 to 33 within the coacervate.

39. A method of making a polymer coacervate aqueous solution, the method comprising providing a stock solution comprising a polymer as defined according to any one of claims 1 to 33, and contacting a volume of the stock solution with a volume of an aqueous diluent solution thereby forming the polymer coacervate solution.

40. A polymer coacervate aqueous solution, wherein the polymer coacervate aqueous solution is obtained by performing the method of claim 39.

41. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises a polymer as defined according to any one of claims 1 to 33 within the coacervate.

42. A method of making a composition comprising a plurality of polymer coacervates in aqueous solution, the method comprising providing a stock solution of a polymer as defined according to any one of claims 1 to 33, contacting a volume of thestock solution with a volume of an aqueous diluent solution thereby forming a plurality of polymer coacervates in the aqueous diluent solution; and optionally concentrating the coacervates in the aqueous diluent solution and adjusting the concentration of the aqueous diluent solution to provide a defined amount of coacervates per volume of aqueous diluent solution.

43. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein the composition is obtained by performing the method of claim 42.

44. A polymer coacervate, wherein the polymer coacervate is a coacervate comprising a polymer as defined according to any one of claims 1 to 33 within the coacervates; and wherein the polymer coacervate further comprises one or more cargo compounds within the coacervates.

45. A method of making a polymer coacervate aqueous solution comprising polymer coacervates comprising one or more cargo compounds, the method comprising:A. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;(ii) contacting a volume of the cargo solution with a coacervate-forming polymer according to any one of claims 1 to 33 to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting, the polymer is in a dried form, or is dissolved in a coacervate incompetent solution; and(iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates;B. (i) providing a polymer solution comprising a coacervate-forming polymer according to any one of claims 1 to 33, wherein the solution is a coacervate incompetent solution;(ii) contacting a volume of the polymer solution with one or more cargo compounds to form a mixed solution of cargo compounds(s) and polymer in the coacervate incompetent solution, wherein prior to said contacting, the cargo compounds(s) is in a dried form or is dissolved in a coacervate incompetent solution; and(iii) contacting a volume of the mixed solution with a volume of a coacervate competent solution, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates;C. (i) providing a cargo solution of one or more cargo compounds, wherein the solution is a coacervate incompetent solution;(ii) contacting a volume of the cargo solution with a volume of a coacervate competent solution to form a further cargo solution;(iii) contacting a volume of the further cargo solution with a coacervate-forming polymer according to any one of claims 1 to 33, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting the polymer is in a dried form or is dissolved in a coacervate incompetent solution;D. (i) providing one or more cargo compounds in a dried form;(ii) contacting the one or more cargo compounds with a volume of a coacervate competent solution to form a cargo solution;(iii) contacting a volume of the cargo solution with a coacervate-forming polymer according to any one of claims 1 to 33, thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting, the polymer is in a dried form, or is dissolved in a coacervate incompetent solution;E. (i) providing a polymer solution of a coacervate-forming polymer according to any one of claims 1 to 33, wherein the solution is a coacervate incompetent solution;(ii) contacting a volume of the polymer solution with a volume of a coacervate competent solution thereby forming a solution comprising a plurality of polymer coacervates;(iii) contacting a volume of the solution comprising a plurality of polymer coacervates with one or more cargo compounds, whereupon the cargo compound(s) localise into the polymer coacervates thereby forming polymer coacervates comprising the polymer and the cargo compound(s) within the coacervate, and wherein prior to said contacting, the cargo compound(s) is in a dried form or is dissolved in a coacervate incompetent solution;F. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is a coacervate-forming polymer according to any one of claims 1 to 33, and wherein the coacervates comprise the polymer within the coacervates, optionally wherein the composition is lyophilised;(ii) providing a cargo compound solution comprising one or more cargo compounds in a coacervate competent solution;(iii) contacting a volume of the composition with a volume of the cargo compound solution, whereupon the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates; orG. (i) providing a composition comprising a plurality of polymer coacervates in dried form, wherein the polymer is a coacervate-forming polymer according to any one of claims 1 to 33, and wherein the coacervates comprise the polymer within the coacervates, optionally wherein the composition is lyophilised;(ii) providing one or more cargo compounds in a dried form; and(iii) (a) contacting a volume of the composition with a volume of a coacervate competent solution, followed by contacting the said solution with the one or more cargo compounds;(b) contacting the one or more cargo compounds with a volume of a coacervate competent solution, followed by contacting the said solution with a volume of the composition; or(c) simultaneously contacting the one or more cargo compounds and a volume of the composition with a volume of a coacervate competent solution; whereupon in each of (a), (b) and (c) the polymer coacervates are rehydrated and the cargo compound(s) localise into the polymer coacervates thereby forming a plurality of polymer coacervates in solution, wherein the polymer coacervates comprise the polymer and the cargo compound(s) within the coacervates.

46. A polymer coacervate aqueous solution comprising one or more cargo compounds, wherein the polymer coacervate aqueous solution is obtained by performing the method of claim 45.

47. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein polymer coacervates of the plurality comprise a polymer as defined according to any one of claims 1 to 33 within the coacervates; and further comprise one or more cargo compounds within the coacervates.

48. A method of making a composition in aqueous solution comprising a plurality of polymer coacervates comprising one or more cargo compounds, the method comprising making a polymer coacervate aqueous solution by performing the method of claim 45, concentrating the coacervates in the solution and adjusting the concentration of the solution to provide a defined amount of coacervates per volume of solution.

49. A composition comprising a plurality of polymer coacervates in aqueous solution, wherein polymer coacervates of the plurality comprise polymer and one or more cargo compounds within the coacervates, and wherein the composition is obtained by performing the method of claim 48.

50. A method according to any one of claims 39, 42, 45 or 48, or a polymer coacervate aqueous solution according to claim 29 or claim 35; wherein the coacervate incompetent solution comprises water, optionally water and dimethylsulfoxide (DMSO), preferably a solution of 30% DMSO in water.

51. A method according to any one of claims 39, 42, 45 or 48, or a polymer coacervate aqueous solution according to claim 40 or claim 46; wherein the coacervate competent solution comprises a cell culture media.

52. A polymer coacervate according to claim 44, a method according to any one of claims 45(a), 48(a), 50, or 51, a polymer coacervate aqueous solution according to claim 46, or a composition according to claim 47 or claim 49; wherein the cargo compound is linked to the polymer, preferably wherein the cargo compound is linked to the polymer via a covalent bond or via a linker, optionally a cleavable linker.

53. A polymer coacervate according to claim 44 or claim 52, a method according to any one of claims 45, 48, 50, 51 or 52; a polymer coacervate aqueous solution according to any one of claims 46, 50, 51 or 52; or a composition according to any one of claims 47, 49 or 52; wherein the one or more cargo compounds comprises any one or more of a peptide, a polypeptide; a polypeptide complex; an antibody or fragment thereof; a polypeptide-drug conjugate; an antibody-drug conjugate; a nucleic acid, such as a single-stranded or double-stranded DNA or a single-stranded or double-stranded RNA; a peptide nucleic acid (PNA); a locked nucleic acid (LNA); an unlocked nucleic acid (UNA); a bridged nucleic acid (BNA); a carbohydrate, a lipid, a nanoparticle or a small molecule.

54. A method for delivering one or more cargo compounds into eukaryotic cells, the method comprising providing a polymer coacervate aqueous solution comprising one or more cargo compounds as defined according to any one of claims 46, 50 or 51, or a composition comprising a plurality of polymer coacervates comprising one or more cargo compounds as defined according to any one of claims 47, 45 or 49, and introducing the solution or composition into an aqueous environment which comprises a plurality of eukaryotic cells, whereupon the polymer coacervates penetrate the plurality of cells and thereby release and deliver the one or more cargo compounds into the eukaryotic cells.

55. A method according to claim 54, wherein the aqueous environment which comprises the plurality of eukaryotic cells is an in vitro or ex vivo cell culture environment.

56. A method according to claim 54, wherein the aqueous environment which comprises the plurality of eukaryotic cells is an in vivo environment.

57. The use of a polymer according to any one of claims 1 to 33 in the formation of a polymer coacervate, wherein the coacervate comprises the polymer within the coacervate.

58. The use of a polymer according to any one of claims 1 to 33 in the production of a composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises the polymer within the coacervate.

59. The use of a polymer according to any one of claims 1 to 33 and one or more cargo compounds in the formation of a polymer coacervate, wherein the coacervate comprises the polymer and the one or more cargo compounds within the coacervate.

60. The use of a polymer according to any one of claims 1 to 33 and one or more cargo compounds in the production of a composition comprising a plurality of polymer coacervates in aqueous solution, wherein a polymer coacervate of the plurality comprises the polymer and the cargo compound within the coacervate.

61. The use according to claim 59 or claim 60, wherein the one or more cargo compounds comprises any one or more of the compounds defined in claim 53.

62. The use according to any one of claims 57 to 61, wherein a cofactor, such as a crowding agent, e.g. RNA, sucrose or polyethylene glycol (PEG), is not required for coacervate formation mediated by the polymer.

63. A pharmaceutical formulation comprising a pharmaceutically acceptable vehicle and a polymer according to any one of claims 1 to 33, a polymer coacervate according to any one of claims 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of claims 40, 46, 52 or 53, or a composition according to any one of claims 41, 43, 47, 49, 52 or 53.

64. A pharmaceutical formulation according to claim 63, wherein the formulation is in a unit dose form.

65. A polymer according to any one of claims 1 to 33, a polymer coacervate according to any one of claims 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of claims 40, 46, 52 or 53, a composition according to any one of claims 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to claim 63 or claim 64 for use as a medicament.

66. The use of a polymer according to any one of claims 1 to 33, a polymer coacervate according to any one of claims 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of claims 40, 46, 52 or 53, a composition according to any one of claims 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to claim 63 or claim 64 in the manufacture of a medicament.

67. A polymer according to any one of claims 1 to 33, a polymer coacervate according to any one of claims 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of claims 40, 46, 52 or 53, a composition according to any one of claims 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to claim 63 or claim 64 for use in the treatment of a disease in a individual in need thereof, the method comprising administering an effective amount of the polymer, the polymer coacervate, the polymer coacervate aqueous solution or the composition.

68. A method of treating a disease or a disorder in an individual in need of treatment, the method comprising administering to the individual an effective amount of a polymer according to any one of claims 1 to 33, a polymer coacervate according to any one of claims 38, 44, 52 or 53, a polymer coacervate aqueous solution according to any one of claims 40, 46, 52 or 53, a composition according to any one of claims 41, 43, 47, 49, 52 or 53, or a pharmaceutical formulation according to claim 63 or claim 64.

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