Coacervate compositions

Abstract

The present invention relates to an isolated peptide comprising the amino acid sequence of Formula (I): (O)n-K-(O)m-Z; wherein K is lysine optionally modified with a self-immolative moiety Z is tryptophan or is absent; O is GHGX1Y (SEQ ID NO: 1); G is glycine, H is histidine, Y is tyrosine; each X1 is independently selected from alanine, glycine, serine, asparagine, histidine (H), arginine (R), aspartic acid, tyrosine, and proline; n is 0 – 5; m is 0 – 5; n+m is 3, 4, 5, 6, 7 or 8, and optionally, wherein not all of the X1 residues are proline (P). Such peptide is a coacervate forming peptide. The present invention also relates to methods of preparing coacervate compositions comprising the isolated peptide, optionally also comprising one or more biomacromolecules (payloads). The present invention further relates to the coacervate compositions per se, and their uses, e.g. in methods of treatment or diagnosis.

Description

[0001] COACERVATE COMPOSITIONS

[0002] TECHNICAL FIELD

[0003] The present invention lies in the field of targeted delivery of active agents using peptide coacervates including isolated peptides, methods of peptide coacervate formation, and active agent recruitment and delivery using the peptide coacervates. Such coacervates may be used in diagnostic or therapeutic approaches.

[0004] BACKGROUND

[0005] Biomacromolecules, including peptides (Jin et al., Theranostics.10:10141 , 2020; Zou et al., Biomat. Sci.8:4975, 2020), proteins (Guillard et a , Trends in Biotech. 33:163, 2015; Fuet al., Bioconjugate Chem. 25:1602, 2014; Nelson et al., Nat. Reviews Drug Disc. 9:767, 2010) and RNAs (Dowdy et al., Nat. Biotech., 35:222, 2017; Jackson et al. New Eng. J. Med. 383:1920, 2020), offer promising therapeutic prospects for the treatment of various diseases owing to key advantages such as high potency, specificity, or safety (Du et al., J. Am. Chem. Soc. 140:15986, 2018). However, their full therapeutic potential has not been fulfilled because of their poor cell membrane permeability and / or endosomal trapping that limits their intracellular release (Goswami et al., Trends in Pharmacol. Sci., 41 : 743, 2020).

[0006] A key challenge in developing efficient intracellular delivery systems is achieving high cellular uptake. Possible strategies include using nano-size delivery carriers and / or carriers with a high positive charge. Such techniques may result in reduced encapsulation efficiency of some biomacromolecules or higher cell toxicity.

[0007] Coacervate delivery systems are novel promising drug delivery and transfection agents with low toxicity and high encapsulation efficiency (e g., see Johnson & Wang. Coacervate delivery systems for proteins and small molecule drugs. Expert Opin. Drug Deliv. 11 , 1829-1832; 2014; Lim et al. Magnetically responsive peptide coacervates for dual hyperthermia and chemotherapy treatments of liver cancer. Acta Biomater. 110, 221-230; 2020; Sun et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat. Chem. 14, 274-283, 2022; and WO20190388357 - Nanyang Technical University).

[0008] Coacervate compositions are able to cross the cell membrane, are not trapped inside endosomal vesicles, and so can directly deliver the biomacromolecule into the cell.

[0009] Coacervates can thus be employed to deliver biomacromolecules (e.g. active agents or payloads) into cells. The Humboldt squid (Dosidicus gigas) beak includes a hard biomolecular composite made of chitin and proteins. The squid beak proteins were recently isolated and sequenced and two families of proteins, Dosidicus gigas chitin binding beak proteins (DgCBPs) and Dosidicus gigas histidine-rich beak proteins (DgHBPs) were discovered within the beak. DgCBPs likely bind to chitin to form a chitin-DgCBPs scaffold, while DgHBPs exhibit self-coacervation ability, a liquidliquid phase separation (LLPS) process resulting in the formation of highly concentrated protein microdroplets. DgHBP coacervates have been hypothesized to infiltrate the chitin-DgCBPs scaffold (Tan et al. (2015) Nat. Chem. Biol. 11 (7), 488-95) followed by interchain covalent crosslinking during maturation, with the very high cross-link density imparting the beak with its impressive mechanical properties (Miserez et al. (2007) Acta Biomater. 3 (1), 139-49; Miserez et al. (2010) J. Biol. Chem. 285 (49), 38115-24). The DgHBPs identified have been sequenced and have been found to exhibit a two-domain organisation. The N-terminal domains contain non- repetitive, long stretches of Alanine (Ala) and Histidine (His)-rich regions, whereas the C-terminal domains includes tandem His- and Gly-rich penta-repeat motifs. The C-terminal domain motifs were found to be responsible for DgHBPs self-coacervation properties (Cai et al. (2017) Soft Matter 13 (42), 7740-7752). Coacervates can be generated from these histidine-rich beak protein peptides (HBpep).

[0010] A previous study by the inventors has shown that HBpep coacervates have the ability to recruit various biomacromolecules with high efficiency of above 95%, and exhibit low toxicity (Lim, Z.W. et al., Bioconjugate Chem., 2018, 29, 2176). HBpep coacervates were also demonstrated to be able to cross the cell membrane via an endocytosis-free pathway (Lim, Z.W. et al., Acta Biomat, 2020, 110, 221). It has therefore been suggested that self- coacervating HBpeps may be potential candidates for intracellular delivery of therapeutics. Preliminary attempts to use HBpep coacervates to recruit and deliver proteins resulted in successful transmembrane delivery. For example, the inventors observed that HBpep coacervates successfully recruited biomacromolecules such as insulin and doxorubicin, and delivered said coacervates intracellularly (US 2019 / 0388357).

[0011] WO2021246961 discloses peptides derived from DgHB protein sequence capable of forming coacervates and recruiting therapeutic macromolecules within the peptide coacervates at pH of about 6.5. Such peptides are based on the tandem repeat GHGXY (SEQ ID NO: 25, where X could be valine (V), proline (P), or leucine (L) [HBpepVPL]). However, such peptide forming coacervates are unsuitable for recruiting macromolecules that can only be solubilised at higher alkali pH.

[0012] Therefore, there still exists a need for a novel and safe delivery platform for both the intracellular delivery and direct cytosolic release of a large variety of biomacromolecules (e.g. therapeutics), in particular for those that solubilise at pH above about 7.0, such as between, 7.0 and 9.0, 7.0 and 8.0 or 8.0 and 9.0. Such platforms would have promising potential in the treatment of cancers, metabolic diseases, or as vaccines or diagnostics.

[0013] SUMMARY

[0014] The inventors have found that the previously existing drawbacks of delivery platforms based on HBpepVPL coacervates could be overcome by using modified peptides, as described herein, for coacervate formation.

[0015] Thus, the present invention is based on the inventors' finding that peptide coacervates formed from the (modified) isolated peptides described herein can be used for the recruitment of therapeutic macromolecules that are soluble at pH above 6.5, e g. in the pH range of about 7.0- 9.0. The isolated peptide coacervates formed may co-recruit one, two or more active agents to be applicable and effective in the management and / or treatment of diseases or disorders, such as cancer, or for diagnostic applications. Additionally, the inventors’ findings provide general guidelines and concepts for designing isolated peptide coacervates with LLPS ability for direct cytosolic release of the active agents which may be applicable in various applications, including bio-inspired protocells and smart drug-delivery systems.

[0016] The present invention provides, inter alia, coacervate forming peptides, methods of preparing coacervate compositions, that may comprise one or more biomacromolecules (payloads), the coacervate compositions per se, and their uses, e g. in methods of treatment or diagnosis. Such coacervate compositions can be used to deliver active agents into the cell and release it directly in the cytosol. Such payloads / active agents may be useful in various applications, including bioinspired protocells and smart drug-delivery systems.

[0017] According to a first aspect of the invention there is provided an isolated peptide comprising the amino acid sequence of Formula (I):

[0018] (O)n-K-(O)m-Z

[0019] (I) wherein K is lysine optionally modified with a self-immolative moiety

[0020] Z is tryptophan (W) or is absent

[0021] O is GHGX1Y (SEQ ID NO: 1)

[0022] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0023] In a particular embodiment, not all of the X1residues are proline (P).

[0024] In particular embodiments, the lysine residue is located at the 1 st, 6th, 11th, 16th, 21 st, or 26thposition from the N-terminus.

[0025] In a particular embodiment, n is 3 and m is 2.

[0026] Suitably, the peptide of Formula I is a peptide of Formula (II):

[0027] [O]a-[B]b-K-[B]c-[O]d-[O]e-Z,

[0028] (II) wherein

[0029] O is GHGX1Y (SEQ ID NO: 27)

[0030] B is GHGPY (SEQ ID NO: 2)

[0031] G is glycine (G), H is histidine (H), Y is tyrosine (Y) and P is proline (P); each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);

[0032] K is lysine (K) optionally modified with a self-immolative moiety; and

[0033] Z -is tryptophan (W) or is absent, a, b, c, d, and e are each independently 0, 1 or 2 and the sum of a+b+c+d+e is 3-8, suitably the sum of a+b+c+d+e is 5.

[0034] In particular embodiments, the isolated peptide comprises or consists of an amino acid sequence, such as but not limited to:

[0035] (i) GHGAY GHGAY GHGPY K GHGPY GHGAY W (SEQ ID NO: 3)

[0036] (ii) GHGGY GHGGY GHGPY K GHGPY GHGGY W (SEQ ID NO: 4)

[0037] (iii) GHGSY GHGSY GHGPY K GHGPY GHGSY W (SEQ ID NO: 5)

[0038] (iv) GHGNY GHGNY GHGPY K GHGPY GHGNY W (SEQ ID NO: 6)

[0039] (v) GHGHY GHGHY GHGPY K GHGPY GHGHY W (SEQ ID NO: 7) (vi) GHGRY GHGRY GHGPY K GHGPY GHGRY W (SEQ ID NO: 8)

[0040] (vii) GHGDY GHGDY GHGPY K GHGPY GHGDY W (SEQ ID NO: 9)

[0041] (viii) GHGYY GHGYY GHGPY K GHGPY GHGYY W (SEQ ID NO: 10)

[0042] (ix) GHGRY GHGRY GHGPY K GHGPY GHGYY W (SEQ ID NO: 11)

[0043] (x) GHGDY GHGDY GHGPY K GHGPY GHGYY W (SEQ ID NO: 12);

[0044] Optionally, wherein the lysine residue (K) is modified at an epsilon (s)-amino group with a self- immolative moiety, such as (SP) group.

[0045] According to a second aspect of the invention there is provided a composition for delivery of an active agent, the composition comprising a peptide coacervate, wherein the peptide coacervate comprises:

[0046] (i) one or more isolated peptides according to the first and / or eighth aspects of the invention; and

[0047] (ii) an active agent recruited in the peptide coacervate.

[0048] In particular embodiments, the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.

[0049] According to a third aspect of the invention there is provided a method for the recruitment of an active agent in a peptide coacervate, the method comprising:

[0050] (i) providing an aqueous solution of coacervate-forming peptides, said coacervateforming peptides comprising one or more isolated peptides of the first and / or eighth aspects of the invention;

[0051] (ii) combining the aqueous solution of the coacervate-forming peptides with an aqueous solution of an active agent; and

[0052] (iii) inducing coacervate formation.

[0053] Suitably, the pH of (a) the aqueous solution of the coacervate-forming peptides; and / or (b) the aqueous solution of an active agent; and / or (c) the aqueous solution of the aqueous solution of the coacervate-forming peptides mixed with the aqueous solution of an active agent is above 6.5, such as above 7.0, such as above 7.5, above 8.0, above 8.5, or in the range 7.0-9.0, including 7.0-8.0 and 8.0-9.0.

[0054] Suitably, the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and < about 9.0, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

[0055] According to a fourth aspect of the invention there is provided a method for the delivery of an active agent, said method comprising:

[0056] (i) providing a composition including a peptide coacervate, wherein the peptide coacervate comprises: a. one or more isolated peptides of the first and / or eighth aspects of the invention, optionally wherein the lysine residue (K) is modified at an epsilon (s)-amino group with a self-immolative moiety; b. an active agent, wherein the active agent is recruited in the peptide coacervate; and

[0057] (ii) exposing the peptide coacervate to conditions that trigger the release of the active agent from the peptide coacervate.

[0058] The conditions that trigger the release of the active agent may be selected from those disclosed above for the composition for the delivery of the active agent.

[0059] According to a fifth aspect of the invention there is provided a method for treating or diagnosing a condition or disease in a subject in need thereof, comprising:

[0060] (i) administering a composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides according to the first and / or eighth aspects of the invention, optionally wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self- immolative moiety, b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate, and

[0061] (ii) exposing the peptide coacervate to conditions that trigger the release of the pharmaceutical or diagnostic agent from the peptide coacervate.

[0062] In a particular embodiment of any of the first, second, third, fourth or fifth aspects of the invention the lysine residue (K) in the coacervate-forming peptide is modified at an epsilon (E)- amino group with a self-immolative moiety.

[0063] In a particular embodiment of any of the second, third, fourth or fifth aspects of the invention the peptide coacervate is comprised of two or more distinct types of peptide. At least one, but optionally more, of the distinct types of peptide are an isolated peptide according to the first and / or eighth aspects of the invention.

[0064] According to a sixth aspect of the invention there is provided the use of an isolated peptide of the first and / or eighth aspect of the invention for preparing a coacervate composition. Suitably, there is provided the use of an isolated peptide of the first aspect of the invention in conjunction with an active agent for preparing a coacervate composition comprising the active agent.

[0065] According to a seventh aspect of the invention there is provided the isolated peptide of the first and / or eighth aspects of the invention or the composition of the second aspect of the invention for use in therapy or for use in a method of diagnosis practised on a mammal, such as a human.

[0066] According to an eighth aspect of the invention there is provided an isolated peptide comprising the amino acid sequence of Formula (IV):

[0067] (O)n-X2-(O)m-Z

[0068] (IV) wherein X2is lysine optionally modified with a self-immolative moiety or absent

[0069] Z is tryptophan (W) or is absent

[0070] O is GHGX1Y (SEQ ID NO: 1)

[0071] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), proline (P), lysine (K) and glutamine (E). n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0072] Suitably, the peptide of Formula (IV) is a peptide that comprises the amino acid sequence of Formula (V):

[0073] (O)n-X2-(O)m-Z

[0074] (V) wherein X2is lysine optionally modified with a self-immolative moiety or absent wherein Z is tryptophan (W) or is absent

[0075] O is GHGX1Y (SEQ ID NO: 1)

[0076] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from arginine (R), tyrosine (Y), lysine (K) and glutamine (E) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0077] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects and advantages will be apparent from the description (including the examples) and drawings, and from the claims.

[0078] BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Optical microscopic images of HBpep(VPL)-SP at various pHs.

[0080] Figure 2 Optical microscopic images of HBpep(AP)-SP and HBpep(GP)-SP at various pHs.

[0081] Figure 3 Phase diagrams of HBpep-SP variants at various pHs.

[0082] Figure 4 (a) Particle size and (b) zeta potential of HBpep-SP variants measured by DLS.

[0083] Figure 5 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep- SP variant coacervates for 10 minutes, 4 hours and 24 hours.

[0084] Figure 6 Fluorescence microscopy images of HeLa cells treated with R-PE-loaded HBpep- SP variant coacervates for 24 hours.

[0085] Figure 7 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(HP)-SP and HBpep(RP)-SP coacervates for 10 minutes and 4 hours.

[0086] Figure 8 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(DP)-SP coacervates for 4 hours and 24 hours.

[0087] Figure 9 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(NP)-SP coacervates for 4 hours and 24 hours.

[0088] Figure 10 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(YP)-SP coacervates for 4 hours and 24 hours. Figure 11 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(RPY)-SP coacervates for 10 minutes and 4 hours.

[0089] Figure 12 Fluorescence microscopy images and FACS of HeLa cells treat with EGFP- loaded HBpep(VPL)-SP, HBpep(HP)-SP HBpep(RPY)-SP coacervates for 2 and 4 hours.

[0090] Figure 13 Fluorescence microscopy images of HeLa cells treated with R-PE-loaded HBpep- SP variant coacervates for 24 hours.

[0091] Figure 14 Fluorescence microscopy images of HeLa cells treated with AF-IgG-loaded HBpep-SP variant coacervates for 24 hours.

[0092] Figure 15 Relative cell viabilities of HeLa cells treated with saporin-loaded HBpep-SP variant coacervates at various saporin concentrations for 24 hours.

[0093] Figure 16 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(DPY)-SP coacervates for 4 hours and 24 hours.

[0094] Figure 17 Fluorescence microscopy images of primary human fibroblast cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)- SP (right) coacervates for 24 hours.

[0095] Figure 18 Fluorescence microscopy images of Jurkat (immortalized T lymphocyte) cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)-SP (right) coacervates for 4 hours.

[0096] Figure 19 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)-SP (right) coacervates in the presence of 10% FBS for 4 hours.

[0097] Figure 20 Fluorescence microscopy images and FACS of HeLa cells treated with FAM-Smac- loaded HBpep-SP variant coacervates for 4 hours.

[0098] Figure 21 In vitro transfection of efficiencies of luciferase-encoded pDNA mediated by HBpep-SP variant coacervates in HeLa cells.

[0099] Figure 22 Fluorescence microscopy images and FACS of HeLa cells transfected with EGFP- encoded pDNA, EGFP-encoded mRNA and FAM-siRNA mediated by HBpep-SP variant coacervates for 24 hours.

[0100] Figure 23 Analysis of indel frequency at the HBB locus in HeLa cells treated with (a) HBB targeted pDNA loaded HBpep-SP variant coacervates compared to Lipofectamine 2000, (b) HBB targeted mRNA / sgRNA loaded HBpep-SP variant coacervates compared to Lipofectamine 3000, and (c) HBB targeted Cas9 RNP loaded HBpep- SP variant coacervates compared to Lipofectamine CRISPRMAX.

[0101] Figure 24 Cell viability of HBpep-SP variant coacervates at the concentrations of 0.1 mg / mL in HeLa cells.

[0102] Figure 25 Fluorescence microscopy images of RAW264.7 (immortalized macrophage) cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)-SP (right) coacervates for 4 hours.

[0103] Figure 26 The sequence of sgRNA targeting HBB and EGFP, and primers for amplifying HBB locus.

[0104] Figure 27 Illustration of SFA measurements and force-distance (F-D) curves between cross-cylinders of coacervate variants, with F normalized by the radius of the cylindrically curved surfaces.

[0105] Figure 28 Internalization of EGFP-loaded coacervates by HeLa cells at different timepoint from 0 to 60 min measured by FACS. Data are presented as the mean ± SD of n = 3 independent experiments.

[0106] Figure 29 Mean fluorescence intensity changes from 0 to 4 hours due to cargo release from EGFP-loaded coacvertaes measured by FACS. Data are presented as the mean ± SD of n = 3 independent experiments. All fluorescence micrographs were imaged on live cells.

[0107] Figure 30 FACS measurements of HeLa cells treated with R-PE-loaded coacervates variants for 24 hours compared to PULSin. Data are presented as the mean ±SD of n = 3 independent experiments; two-sided Student’s t-test, *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0108] Figure 31 FACS measurements of HeLa cells treated with EGFP-loaded coacervates variants for 24 hours compared to the commercial reagent PULSin. Data are presented as the mean ±SD of n = 3 independent experiments; two-sided Student’s t-test, *P < 0.05, **P < 0.01 , ***P < 0.001 .

[0109] Figure 32 Intracellular delivery into hard-to transfect cells mediated by the HBpep- SP coacervate variants. FACS measurements of Human Foreskin Fibroblast (HFF) cells, Jurkat T-cells, and macrophage RAW264.7 cells treated with EGFP-loaded CMs variants for 4 hours compared to PULSin. Data are presented as the mean ±SD of n = 3 independent experiments; two-sided Student’s t-test, *P < 0.05, **P < 0.01 , ***P < 0.001 compared to commercial reagents. Figure 33 Sequence and self -coacervation of HBpep variants and derivates. (a) Peptide sequences of HBpep and HBpep-Kspderivates modified by a self- immolative moiety on the lysine (K) side chain. The abbreviations refer to the X positions in the pentapeptide repeat GHGXY. (b) Representative optical micrographs of variants (50 pM, pH = 7.0, IS = 100 mM). (c) Phase diagram of variants at the ionic strength (IS) of 100 mM, with the region of coacervation or aggregation shadowed in blue and grey, respectively.

[0110] Figure 34 Complex coacervation of HBpep variants and derivates. (a) Representative optical micrographs of variant mixtures (total concentration = 50 pM, mix ratio = 1 :1 , pH = 7.0, IS = 100 mM). (b-e) Phase diagram of variant mixtures including RP:YP = 1 :1 (b), RP-Ksp:YP-Ksp= 1 :1 (c), RP-Ksp:YP-Ksp= 4:1 (d), and RP- Ksp:YP = 4:1 (e) at the ionic strength (IS) of 100 mM, with the region of coacervation or aggregation shadowed in blue and grey, respectively, (f) Phase diagram of RP-Ksp:YP-Kspmixed at different ratios. With increased YP-Ksppercentage, the coacervation region (blue-shadowed) expands and then becomes aggregation (grey-shadowed), (g) Normalized force-distance (F / R vs D) curves measured by SFA during the approach (A) and separate (S) of two cross-cylinder mica surfaces with RP-KSP(50 pM in PBS) and RP-Ksp:YP-Ksp= 1 :1 (total concentration = 50 pM, mix ratio = 1 :1 , in PBS) in between, (h) Optical micrographs of aggregates formed by YP-KSPevolve with / without RP-KSPaddition. The arrows indicate the formation of coacervates after adding the RP- Ksp.

[0111] Figure 35 Molecular interactions in RP-Ksp / YP-Kspcomplex coacervation, (a) Normalized force-distance (F / R vs D) curves measured by SFA during the approach (A) and separation (S) of two cross-cylinder mica surfaces coated with RP-KSPor YP-KSPlayers. Measurements were conducted in buffer at pH = 7.0 and IS = 100 mM with (red) or without (grey) the addition of 100 mM TMA. (b) Snapshots of the slab simulation of RP-Ksp, RP-Ksp / YP-Kspmixture, and YP-KSP, indicate that the mixture and YP-KSPform a single stable cluster, whereas RP-Kspstays as isolated oligomers, (c) Quantification of contacts between different residues normalized by the number of residue-residue pairs in RP-Ksp / YP-Kspmixture systems, (d-e) The density of peptides (d) and water (e) in RP-KSP, RP-KSP / YP-KSPmixture, and YP-KSPclusters, analyzed from slab simulations, (f) Concentration decay of RP-KSPand YP-KSPpeptide in the RP- Ksp:YP-Ksp= 1 :1 complex coacervates reduced by 1 mM GSH over time, (g) Concentration decay in natural logarithmic scale plotted as a function of time. The reaction rate constant k was obtained from the slopes of the fitted lines. Data are presented as the mean ±SD of n = 3 independent experiments.

[0112] Figure 36 Delivery and release of macromolecules from complex coacervates stabilized by cation-n interactions, (a-c) Fluorescence micrographs (a) and FACS measurements (b-c) of HeLa cells treated with EGFP-loaded complex coacervates for 4 hours, (d) Fluorescence micrographs of HeLa cells treated with EGFP-loaded complex coacervates formed by RP-Ksp / YP with various ratios for 1 and 2 hours, (e) Mean fluorescence intensity vs time from 0 to 4 hours due to EGFP release from complex coacervates formed by RP-KSF7YP at various ratios measured by FACS. Data are presented as the mean ± SD of n = 3 independent experiments, (f-g) Fluorescence micrographs (f) and FACS measurements (g) of HeLa cells treated with EGFP mRNA-loaded complex coacervates formed by RP-KSF7YP at various ratios for 24 hours. Data are presented as the mean ± SD of n = 3 independent experiments.

[0113] Figure 37 The responsivity of cation-n stabilized complex coacervates to different concentrations of proteins, (a) Schematic illustrations of cationic and aromatic peptides interact in low and high concentrations of external proteins. At low protein concentrations, proteins are recruited within the network formed by peptides. However, at high protein concentrations, the protein competes to interact with peptides, disrupting the formation of peptide clusters and further coacervation, (b-c) Turbidity measurements of RP:YP = 1 :1 (a) and RP-Ksp:YP- Ksp= 1 :1 (b) at zero, low (0.01 %) and high (10%) protein concentrations.

[0114] Figure 38 Macrophage engineering mediated by RP-Ksp / YP complex coacervates.

[0115] (a) Fluorescence micrographs and FACS measurements of RAW264.7 cells treated with EGFP-loaded complex coacervates formed by RP-Ksp / YP with various ratios for 4 hours compared to the commercial reagent PULSin. Data are presented as the mean ±SD of n = 3 independent experiments, (b) Fluorescence micrographs (left), merged images of the fluorescence channel and optical widefield channel (middle), and FACS analysis (right) of RAW264.7 cells treated with AF-IgG-loaded complex coacervates formed by RP- Ksp:YP = 4:1 for 4 hours, (c) Fluorescence micrographs (left), merged images of the fluorescence channel and optical widefield channel (middle), and FACS analysis (right) of RAW264.7 cells treated with R-PE-loaded complex coacervates formed by RP-Ksp:YP = 4:1 for 4 hours, (d) Fluorescence micrographs and FACS measurements of RAW264.7 cells treated with EGFP mRNA-loaded complex coacervates formed by RP-Ksp / YP with various ratios for 24 hours compared to the commercial reagent Lipofectamine MessengerMax (MMax). Data are presented as the mean ±SD of n = 3 independent experiments, (e-f) Analysis of indel frequency at the SIRPa locus in RAW264.7 cells treated with SIRPa-targeted Cas9 RNP-loaded complex coacervates formed by RP-Ksp:YP = 4:1 (e) and SIRPa-targeted Cas9 mRNA / sgRNA-loaded complex coacervates formed by RP-Ksp:YP = 8:1 (f). The increase in the number of bands from 1 to 3 is due to T7EI cleavage of the amplicon from the edited genomic locus.63(g) The expression of SIRPa on edited RAW264.7 cells measured by FACS compared to the negative control (NC) group, which did not undergo editing.

[0116] DETAILED DESCRIPTION

[0117] Compositions for delivery of an active agent, such as a pharmaceutical or diagnostic agent, may include a peptide coacervate, said peptide coacervate comprising peptides derived from histidine-rich proteins and comprising the amino acid sequence of Formula I II, III, IV or V disclosed herein, and said active agent, wherein the active agent is encapsulated in the coacervate, as well as methods of manufacture thereof and methods of use thereof.

[0118] Definitions:

[0119] It is to be understood that the disclosures are not limited to particular compositions or methods, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. So a method reciting “a peptide” also covers the method on a population of peptides (typically the same type). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing, specific examples of appropriate materials and methods are described herein.

[0120] “Histidine-rich proteins”, as used herein, relates to proteins that include at least one histidine residue and overall have a comparably high amount of residues of the amino acid histidine (His or H). This may mean that the histidine content of a given protein is above 3%, for example greater than 5% or greater than 10% or greater than 12% or greater than 15%, relative to the total number of amino acids in the peptide sequence. As used herein, “peptides derived from histidine-rich proteins” generally refers to peptides that represent fragments or variants or both of histidine-rich proteins, in particular histidine-rich proteins that naturally occur, for example in the Humboldt squid. The peptides may be produced by genetic engineering techniques as known to those skilled in the art. The peptides thus artificially produced may represent amino acid stretches of the proteins they are derived from but do not encompass the full native protein sequence. In various embodiments, the derived peptides are N- and / or C-terminally truncated fragments of the respective histidine-rich protein. Additionally, the peptides may also comprise amino acid substitutions, deletions or insertions relative to the protein sequence they are derived from. As the peptides include fragments and variants of histidine-rich proteins that do not occur in nature and have typically been artificially produced, the peptides are, in various embodiments, artificial peptides, such as those created by genetic engineering techniques. Suitable synthesis methods are well-known to those skilled in the art and may be selected using their routine knowledge.

[0121] The term “coacervate", as used herein, has the meaning as commonly understood in the art. Coacervates are two-phase liquid compositions, i.e. exhibiting liquid-liquid phase separation (LLPS), comprising or consisting of a concentrated macromolecule-rich (or coacervate) phase and a dilute macromolecule-depleted phase. The two phases of the peptide coacervates are one peptide-rich coacervate phase and one dilute peptide-depleted phase. The peptide-rich coacervate phase is also referred to herein as “peptide coacervate (micro)droplets”.

[0122] A self-coacervating peptide is one that is capable of exhibiting coacervation (or LLPS).

[0123] Complex coacervation is a form of liquid-liquid phase separation (LLPS), whereby two (or more) types of macromolecules (e.g. peptides) self-assemble into dense microdroplets driven by weak molecular interactions. Suitably, the two or more peptides possess opposite net charges to facilitate assembly into dense microdroplets driven by weak molecular interactions.

[0124] Suitably, the peptides capable of forming coacervates for use in the present invention are derived from histidine-rich proteins, in particular derived from the histidine-rich proteins of the beak of a squid, in particularthe Humboldt squid (Dosidicus gigas).

[0125] As used herein, HBpep(VPL) is the nomenclature of the coacervate forming peptide having the following sequence: GHGVY-GHGVY-GHGPY-K-GHGPY-GHGLY-W (SEQ ID NO: 13).

[0126] As used herein, HBpep(VPL)-SP is the nomenclature of the coacervate forming peptide having the following sequence: GHGVY-GHGVY-GHGPY-K(SP)-GHGPY-GHGLY-W (SEQ ID NO: 14).

[0127] SP is the SR immolative group that possesses a phenyl terminal group. HBpep(VPL) and HBpep(VPL)-SP can be used as reference or control coacervate forming peptides. HBpep(VPL)-SP may be the preferred control or reference peptide when comparing to a variant peptide that also has an SP attached to the lysine residue.

[0128] The coacervates may be referred to herein based on the substitutions in the HBpep peptide; for example HBpep(RPY)-SP coacervates may be referred to as RPY coacervates or RPY variant.

[0129] The term “aqueous solution”, as used herein, means that the dilute phase is mainly water, i.e. comprises at least 50 vol.% water. In various embodiments, the composition may use water as the only solvent, i.e. no additional organic solvents, such as alcohols, are present. In other embodiments, the composition is an aqueous composition that additionally contains one or more solvents other than water, with water however being the major constituent, i.e. being present in an amount of at least 50, at least 60, at least 70, at least 80, at least 90, at least 95 or 99 vol.%.

[0130] “Encapsulate”, as used herein in relation to the active agent, means that the active agent is entrapped in the peptide coacervate phase, for example the coacervate droplets formed by the peptides. Said entrapment may be such that the active agent is completely surrounded by peptides forming the coacervate phase but also includes embodiments, where the active agent is at least partially exposed on the surface of the respective coacervate phase, for example by being tethered to the colloidal phase via a certain group or moiety.

[0131] The term “protein", as used herein, relates to polypeptides, i.e. polymers of amino acids connected by peptide bonds, including proteins that comprise multiple polypeptide chains. A polypeptide typically comprises more than 50, for example, 100 amino acids or more.

[0132] The term “peptide”, as used herein, relates to polymers of amino acids, typically short strings of amino acids. In various non-limiting embodiments, the peptides may include only amino acids selected from the 20 proteinogenic amino acids encoded by the genetic code, namely, glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, tyrosine, tryptophan, histidine, arginine, lysine, aspartic acid, glutamic acid, cysteine, and methionine. These amino acids are also designated herein by their three or one letter code (as above). Generally, peptides may be dipeptides, tripeptides or oligopeptides of at least 4 amino acids in length. The typical length for the peptides of the invention may range from at least about 16 amino acids to 150, preferably to 80, 70, 60 or 50 amino acids in length, for example, at least 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In particular embodiments, the upper limit for example, being 50, 40 or 35 amino acids. Generally, it may be preferred to use peptides as short as possible without impairing their functionality.

[0133] Accordingly, the term “peptide(s)”, as used herein, refers to a unique polymer of amino acids, in accordance with various embodiments. Unless indicated otherwise, the standard single letter amino acid code is used herein; with “X” being used to allow for any amino acid. By “isolated peptide” we mean one that has been isolated or purified from its natural environment or produced artificially (e.g. on a peptide synthesiser). As used herein, a method reciting “a peptide” also covers the method on a population of peptides (typically the same type).

[0134] The term “self-immolative (SR) moiety”, as used herein, refers to a moiety that is self-cleaving upon encountering a certain triggering stimulus, such as a change in pH or redox potential. “Self- immolative” and “self-cleaving” are thus used interchangeably herein. In response to such a stimulus, the molecule autocatalytically cleaves itself to release the functional group, typically in form of a harmless by-product, such that the unmodified side chain amino group of the lysine residue (K) is reformed. In various embodiments disclosed herein, the self-immolative (SR) moiety comprises or includes a disulfide bond (-S-S-), i.e. disulfide bridge with a covalent bond between the two sulfur (S) atoms. As used herein, the term (SA) refers to the immolative moiety that possesses an acetyl group at the extremity of the self-immolative moiety (generically referred to as HBpep-SA) and the term (SP) refers to the immolative moiety that possesses a phenyl group at the extremity of the self-immolative moiety (generically referred to as HBpep- SP). HBpep-SA and HBpep-SP are collectively referred to as HBpep-SR.The term “isolated”, as used herein, relates to the fact that the referenced peptide is at least partially separated from other components it may (naturally or non-natu rally) associate with, for example other molecules, cellular components and cellular debris. Said isolation may be achieved by purification protocols for proteins and peptides well known to those skilled in the art. The term isolated peptides also applies to non-natural peptides which are synthesised or produced synthetically (e.g. in vitro).

[0135] The term “about”, as used herein, in connection with a numerical value, means said value ± 10 %, for example, ± 5 %.

[0136] According to a first aspect of the invention there is provided an isolated peptide comprising the amino acid sequence of Formula (I):

[0137] (O)n-K-(O)m-Z

[0138] (I) wherein K is lysine optionally modified with a self-immolative moiety

[0139] Z is tryptophan (W) or is absent

[0140] O is GHGX1Y (SEQ ID NO: 1)

[0141] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0142] Each O may be referred to herein as a penta-peptide motif.

[0143] In a particular embodiment, not all of the X1residues are proline (P).

[0144] In particular embodiments, the lysine residue is located at the 1 st, 6th, 11th, 16th, 21 st, or 26thposition from the N-terminus. Suitably, the lysine residue is at the 16thposition from the N- terminus (of the peptide).

[0145] In a particular embodiment, n is 3 and m is 2.

[0146] Suitably, the peptide of Formula I is a peptide that comprises the amino acid sequence of Formula (II):

[0147] [O]a-[B]b-K-[B]c-[O]d-[O]e-Z,

[0148] (II) wherein

[0149] O is GHGX1Y (SEQ ID NO: 27)

[0150] B is GHGPY (SEQ ID NO: 2)

[0151] G is glycine (G), H is histidine (H), Y is tyrosine (Y) and P is proline (P); each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);

[0152] K is lysine (K) optionally modified with a self-immolative moiety; and

[0153] Z -is tryptophan (W) or is absent, a, b, c, d, and e are each independently 0, 1 or 2 and the sum of a+b+c+d+e is 3-8.

[0154] In a particular embodiment, the sum of a+b+c+d+e is 5.

[0155] Each O and B is a pentamotif. It will be appreciated that when X1is proline (P) within any of the pentamotifs in Formula II, the pentamotif is “B” motif.

[0156] The isolated peptide of the invention may be composed of multiple repeat units of the Formula I or II amino acid sequence, i.e. wherein the amino acid sequence of Formula I or II is repeated 2, 3, 4, 5 or 6 times to create a longer peptide. Thus, for example, with the amino acid sequence of Formula I when n+m is 5 or with the amino acid sequence of Formula II when the sum of a+b+c+d+e is 5, then with the lysine (K) the nominal peptide can be 26 amino acids in length if the optional tryptophan (W) is absent, or 27 amino acids in length if the optional tryptophan (W) is present; so, for example, the isolated peptide that consists of two repeats could be 52 or 54 amino acids in length or the isolated peptide that consists of four repeats could be 104 or 108 amino acids in length.

[0157] Suitably, the peptide of Formula I is a peptide that comprises the amino acid sequence of Formula (III):

[0158] ([O]-[O]-[B]-K-[B]-[O]-z)n

[0159] (HI) wherein

[0160] O is GHGX1Y (SEQ ID NO: 1)

[0161] B is GHGPY (SEQ ID NO: 2)

[0162] Wherein G is glycine (G), H is histidine (H), Y is tyrosine (Y) and P is proline (P); each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);

[0163] K is lysine (K) optionally modified at an epsilon (E)- amino group with a self-immolative moiety; and z -is tryptophan (W) or is absent, n is 1 to 4; optionally wherein at least two of the X1residues are different to each other.

[0164] The following embodiments apply to any peptide of the first aspect of the invention, including any peptide that comprises the amino acid sequence of Formula I, II or III.

[0165] The following embodiments also apply to any isolated peptide of the eighth aspect of the invention, including any peptide that comprises the amino acid sequence of Formula IV or V.

[0166] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 7.0.

[0167] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 7.5.

[0168] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 8.0.

[0169] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 8.5. In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 9.0.

[0170] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates in the pH range 7.0 - 9.0.

[0171] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates in the pH range 7.5 - 9.0.

[0172] In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates in the pH range 8.0 - 9.0.

[0173] In a particular embodiment, the amino acid residue at X1is the same at each X1position.

[0174] In particular embodiments, all the residues at the X1positions are the same and are selected from: A, G, and H. Examples of such peptides are the HBpep(AP)-SP, HBpep(GP)-SP and HBpep(HP)-SP variants. Such peptides form coacervates with enhanced cellular uptake compared to HBpep(VPL)-SP. Such peptides form coacervates capable of enhanced gene transfection compared to HBpep(VPL)-SP.

[0175] In a particular embodiment, at least two of the X1residues are different to each.

[0176] In particular embodiments, the amino acid residue at each X1position is different to the residue at the other X1positions.

[0177] In a particular embodiment, the two N-terminal X1amino acid residues and the C-terminal X1amino acid residue are the same as each other. Examples of such peptides include the HBpep(AP)-SP, HBpep(GP)-SP, HBpep(HP)-SP, HBpep(SP)-SP, HBpep(NP)-SP, HBpep(RP)- SP and HBpep(DP)-SP variants.

[0178] In a particular embodiment, the two N-terminal X1amino acid residues are the same as each other and the C terminal X1amino acid residue is different to the two N-terminal X1amino acids. Examples of such peptides include the HBpep(RPY)-SP and HBpep(DPY)-SP variants.

[0179] In particular embodiments, at least one of the residues at an X1position is selected from: A, G, H and R.

[0180] In particular embodiments, at least one of the residues at an X1position is selected from: H and R. Such peptides form coacervates with enhanced cellular uptake and increased cargo release compared to HBpep(VPL)-SP. In particular embodiments, all the residues at the X1positions are the same and are selected from: H and R. Such peptides form coacervates with enhanced cellular uptake and increased cargo release compared to HBpep(VPL)-SP.

[0181] In particular embodiments, the isolated peptide comprises the Z tryptophan. In other embodiments, the isolated peptide lacks the Z tryptophan.

[0182] Exemplary peptides encompassed by the first aspect of the invention are described below.

[0183] In particular embodiments, the isolated peptide of the first aspect of the invention comprises or consists of an amino acid sequence, such as but not limited to:

[0184] (i) GHGAY GHGAY GHGPY K GHGPY GHGAY W (SEQ ID NO: 3)

[0185] (ii) GHGGY GHGGY GHGPY K GHGPY GHGGY W (SEQ ID NO: 4)

[0186] (iii) GHGSY GHGSY GHGPY K GHGPY GHGSY W (SEQ ID NO: 5)

[0187] (iv) GHGNY GHGNY GHGPY K GHGPY GHGNY W (SEQ ID NO: 6)

[0188] (v) GHGHY GHGHY GHGPY K GHGPY GHGHY W (SEQ ID NO: 7)

[0189] (vi) GHGRY GHGRY GHGPY K GHGPY GHGRY W (SEQ ID NO: 8)

[0190] (vii) GHGDY GHGDY GHGPY K GHGPY GHGDY W (SEQ ID NO: 9)

[0191] (viii) GHGYY GHGYY GHGPY K GHGPY GHGYY W (SEQ ID NO: 10)

[0192] (ix) GHGRY GHGRY GHGPY K GHGPY GHGYY W (SEQ ID NO: 11)

[0193] (x) GHGDY GHGDY GHGPY K GHGPY GHGYY W (SEQ ID NO: 12);

[0194] Optionally, wherein in any of (i) - (x), the lysine residue (K) is modified at an epsilon (s)-amino group with a self-immolative moiety, such as (SP) group.

[0195] In the above sequences and all sequences disclosed herein, amino acids are typically identified by their one letter code. Thus, G stands for glycine, H stands for histidine, A stands for alanine, L stands for leucine, Y stands for tyrosine, etc. The peptides are also shown in the conventional way, i.e. in the N- to C-terminal orientation. The individual amino acids are covalently coupled to each other by peptide bonds.

[0196] In particular embodiments, the peptides of the invention are capable of LLPS formation in a wide range of alkaline pH. When forming coacervates, the pH of the aqueous solution comprising the peptide of the first aspect of the invention may be below 9, for example below 8.5, or below 8.0, or below 7.5, or below 7.0.

[0197] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is able to form coacervates at a pH between about 6.5 and 9.0, such as between about 7.0 and 9.0 or 7.0 and 8.0.

[0198] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) at a pH of 7.0.

[0199] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) at a pH of 8.0.

[0200] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) at a pH of 9.0.

[0201] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) in the pH range of about 7.0 to about 9.0.

[0202] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) in the pH range of about 7.0 to about 8.0.

[0203] In a particular embodiment, the peptide is capable of undergoing liquid-liquid phase separation (LLPS) in the pH range of about 8.0 to about 9.0.

[0204] In particular embodiments, the isolated peptide of the first aspects of the invention is able to form coacervates that can enter the cell more efficiently than coacervates formed from HBpep(VPL)- SP.

[0205] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is able to form coacervates that are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP. The kinetics of disassembly of such coacervates is thus more efficient when in the cell cytosol.

[0206] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is used to form coacervates composed solely one type of isolated peptide of the first or eighth aspects of the invention.

[0207] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is able to form coacervates with at least one other type of peptide. The one other type of peptide could be a different isolated peptide of the first or eighth aspects of the invention or any other different coacervate-forming peptide.

[0208] The efficiency (of release) can be measured by a variety or means, such as measurement of fluorescence intensity of cargos over short time period, or viabilities of cells treated with toxic protein loaded coacervates (as shown in Figure 12 and Figure 15).

[0209] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is less sensitive to serum during cellular uptake than coacervates formed from HBpep(VPL)-SP.

[0210] Serum can impede the ability of certain coacervates to enter the cell, and thus deliver its payload, if present. For example, as can be seen from Figure 19, delivery of EGFP payload into the cell by the known VPL coacervates is significantly impaired.

[0211] By “less sensitive to serum during cellular uptake” we mean being able to deliver 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200% or more greater payload to the cell compared to control coacervate composition (e.g. HBpep(VPL)-SP).

[0212] The ability to measure the relative effect serum has on cellular entry / delivery can be determined as described in the Examples herein (see Example 2.2), e g. by the measuring effect 10% fetal calf serum (FCS) has on cellular entry of the coacervate formed from peptides of the first aspect of the invention compared to a suitable control (e g. HBpep(VPL)-SP).

[0213] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention displays faster cargo release than coacervates formed from HBpep(VPL)-SP. In particular embodiments, increased cargo release occurs within 4 hours, such as within 2 hours of administration. Increased cargo release can be an increase of 10%, 20%, 30%, 40%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500% or more, relative to HBpep(VPL)SP.

[0214] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention displays enhanced transfection efficiency than coacervates formed from HBpep(VPL)-SP. Enhanced transfection can be measured by an increase in the amount of payload delivered into the cell (e.g., a nucleic acid, such as a gene, siRNA or ASO; a ribonucleoprotein complex, like CRISPR / CAS9; a polypeptide or protein, like an antibody, and the like). Enhanced transfection efficiency can be an increase of 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500% or more, relative to HBpep(VPL)-SP.

[0215] Certain properties of coacervates formed from the various peptides of the first aspect of the invention are show in Table 3.2 herein. In various embodiments, the lysine residue (K) (in Formula I, II or III, or when present in Formula IV or V) is modified at an epsilon (E)- amino group with a self-immolative moiety (also referred to herein as SR). For example, the self-immolative (SR) moiety may be conjugated to the amine, i.e. NH2 group of the lysine residue (K), in other words, conjugated to the E- nitrogen (N) of the lysine side chain.

[0216] In various embodiments, the self-immolative modification is a modification by an organic moiety. Said modification may serve to adjust phase separation behaviour, for example by masking the charge of the lysine residue (K) and / or increasing hydrophobicity.

[0217] In various embodiments, the self-immolative (SR) moiety is an organic group with up to 20 carbon atoms. In various embodiments, it comprises the group of the formula -C(=O)-O-(CH2)n- S-S-R, with the carbonyl C being attached to the epsilon N of the lysine side chain and n being an integer from 1 to 10, preferably 1 , 2, 3, 4 or 5, in particular 2 or 3. In such embodiments, R may include, or may be any organic moiety with 1 to 20 carbon atoms, such as, without limitation substituted or unsubstituted alkyl, alkenyl, cycloalk(en)yl, and aryl.

[0218] “Alkyl”, as used herein, relates to a linear or branched alkyl group with 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, such as, without limitation, methyl, ethyl, n-propyl, isopropyl, t- butyl, n-butyl, and 2-butyl. If substituted, the substituent may be selected from the group consisting of -OR1, -C(=O)R1-OC(=O)R1-C(=O)OR1, halogen, such as fluorine, chlorine and bromine, -N3, with R1being selected from unsubstituted or halo-substituted C1.4 alkyl or alkenyl, unsubstituted or halo-substituted C5-6 cycloalk(en)yl, or unsubstituted or halo-substituted Ce- 14 aryl. It can be preferred that the substituent is not a charged group.

[0219] “Alkenyl”, as used herein, refers to the alkyl groups that comprise at least one C-C double bond, such as, without limitation, ethenyl (vinyl), 2-propenyl (allyl), and 2-butenyl. If substituted, the substituents are defined as for alkyl above.

[0220] “Cycloalk(en)yl”, as used herein, refers to cyclic, non-aromatic alkyl or alkenyl groups, such as without limitation, cyclohexyl. If substituted, the substituents are defined as for alkyl above.

[0221] “Aryl”, as used herein, refers to cyclic aromatic groups with 6 to 14 carbon atoms, such as phenyl. If substituted, the substituents are defined as for alkyl above.

[0222] In various embodiments, the self-immolative (SR) moiety comprises or includes a disulfide moiety / bond (-S-S-), i.e. disulfide bridge with a covalent bond between the two sulfur (S) atoms. Said disulfide bond may provide a biologically relevant precursor to engineer specific intracellular release of the cargo upon exposure to specific conditions. For example, the disulfide bond may be reduced in a reducing environment, such that the disulfide bond is reduced to two thiols (-SH), i.e. dithiols, and trigger the autocatalytic cleavage of the self-immolative (SR) moiety. In various embodiments, the self-immolative (SR) moiety thus comprises a disulfide group that separates upon reduction into two thiols, with one being still attached to the lysine side chain and the other being released. The remaining one thiol group on the lysine side chain then autocatalytically cleaves itself off such that the amino group of the lysine residue (K) is reformed, and the resulting restoration of the charged lysine residue (K) destabilizes the peptide coacervate leading to the subsequent dissolution of the coacervate phase and, if present, release of the recruited active agent / payload.

[0223] In various non-limiting embodiments, the self-immolative moiety has the formula such as but not limited to: -C(=O)-O-(CH2)n-S-S-R, wherein R is selected from: substituted or unsubstituted alkyl, alkenyl, cycloalk(en)yl, and aryl, and n is an integer from 1 to 10, suitably n is 1 , 2, 3, 4, or 5.

[0224] In various non-limiting embodiments, R may be a group of the formula such as but not limited to: -(CH2)n-O-C(=O)-R’, wherein n is 1 , 2, 3, 4, or 5, and wherein R’ is selected from: C1-C4 alkyl, aryl, preferably phenyl, said alkyl or aryl group optionally substituted with halogen. Suitably, the R’ is as set out as the -R in Route 1 shown in the Examples.

[0225] In the above peptides, there are at least 3 pentapeptide motifs (n+m is 3, or a+b+c+d+e is 3) with a lysine (K) residue and an optional tryptophan (W) after the C-terminal position as shown in Formula I II or III. Accordingly, the minimum sequence length of a peptide of the invention is 16 amino acids or 17 when Z is present. A preferred peptide has 5 pentapeptide motifs and so this would have 26 amino acids or 27 when Z is present. However, the isolated peptide of the invention can comprise up to 4 consecutively linked peptide units and so the length of the isolated peptide can be as much as 108 amino acids long. In addition to the above sequence motifs, the peptides may comprise additional amino acids on their N- or C-terminal end or on both, for example 1-10 or 1-5 additional amino acids. In various non-limiting embodiments, the peptides may include only 1-5 additional amino acids on their termini in addition to the above sequence motifs. Accordingly, by way of example, the isolated peptide of the invention can be in the range of 26 to about 120 amino acids in length.

[0226] In particular embodiments, the isolated peptide of the first or eighth aspects of the invention is 15 - 45 or 16-45 amino acids long. Suitably, the isolated peptide is 25 or 26 or 27 amino acids long.

[0227] All peptides disclosed herein may be additionally modified by non-amino acid moieties, such as lipid or carbohydrate or other organic or inorganic moieties, including PEGylation, farnesylation, and the like. These modifications may impart additional desirable properties, for example increased hydrophobicity and the like.

[0228] In non-limiting embodiments, the peptides may include, consist essentially of, or consist of the amino acid sequences set forth above. The peptides may be synthesized using any conventional method known for peptide synthesis, including chemical synthesis and recombinant production. Suitable methods are well-known to those skilled in the art and may be selected using their routine knowledge.

[0229] According to an eighth aspect of the invention there is provided an isolated peptide comprising the amino acid sequence of Formula (IV):

[0230] (O)n-X2-(O)m-Z

[0231] (IV) wherein X2is lysine optionally modified with a self-immolative moiety or absent

[0232] Z is tryptophan (W) or is absent

[0233] O is GHGX1Y (SEQ ID NO: 1)

[0234] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), proline (P), lysine (K) and glutamine (E). n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0235] Suitably, the peptide of Formula (IV) is a peptide that comprises the amino acid sequence of Formula (V):

[0236] (O)n-X2-(O)m-Z

[0237] (V) wherein X2is lysine optionally modified with a self-immolative moiety or absent wherein

[0238] Z is tryptophan (W) or is absent

[0239] O is GHGX1Y (SEQ ID NO: 1)

[0240] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from arginine (R), tyrosine (Y), lysine (K) and glutamine (E). n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0241] Exemplary peptides encompassed by the eighth aspect of the invention are described below.

[0242] In particular embodiments, the isolated peptide of the eighth aspect of the invention comprises or consists of an amino acid sequence, such as but not limited to:

[0243] (i) GHGRY GHGRY GHGPY GHGPY GHGRY W (SEQ ID NO: 28)

[0244] (ii) GHGYY GHGYY GHGPY GHGPY GHGYY W (SEQ ID NO: 29)

[0245] (iii) GHGKY GHGKY GHGPY GHGPY GHGKY W (SEQ ID NO: 30)

[0246] (iv) GHGEY GHGEY GHGPY GHGPY GHGEY W (SEQ ID NO: 31)

[0247] (v) GHGRY GHGRY GHGPY K GHGPY GHGRY W (SEQ ID NO: 18)

[0248] (vi) GHGYY GHGYY GHGPY K GHGPY GHGYY W (SEQ ID NO: 21)

[0249] Optionally, wherein in either of (v) and (vi), the lysine residue (K) at position 16 is modified at an epsilon (s)-amino group with a self-immolative moiety, such as (SP) group.

[0250] In the above peptides, there are at least 3 pentapeptide motifs (n+m is 3, or a+b+c+d+e is 3) with an optional lysine (K) residue (at X2) and an optional tryptophan (W) (at Z) as shown in Formula IV or V. Accordingly, the minimum sequence length of a peptide of the invention is 15 amino acids or 16 when X2or Z is present or 17 when X2and Z are present. A preferred peptide has 5 pentapeptide motifs and so this would have 25 amino acids or 26 when X2or Z is present or 27 when X2and Z are present. However, the isolated peptide of the invention can comprise up to 4 consecutively linked peptide units and so the length of the isolated peptide can be as much as 108 amino acids long. In addition to the above sequence motifs, the peptides may comprise additional amino acids on their N- or C-terminal end or on both, for example 1-10 or 1-5 additional amino acids. In various non-limiting embodiments, the peptides may include only 1-5 additional amino acids on their termini in addition to the above sequence motifs. Accordingly, by way of example, the isolated peptide of the invention can be in the range of 26 to about 120 amino acids in length.

[0251] As appropriate, the embodiments that are applicable to the isolated peptide of the first aspect of the invention apply mutatis mutandis to the isolated peptide of the eighth aspect of the invention. Coacervates and coacervate compositions

[0252] It has been found that the peptides of the present invention form coacervates readily, in particular under neutral to alkaline conditions, i.e. pH values of 7 and higher. Stable solutions of these peptides without any distinct phase separation can be formed at low pH, for example less than 4. In various embodiments, the peptides may be prepared as stock solutions in slightly acidic solutions, such as 1-100 mM, for example about 10 mM acetic acid or other suitable weak acids.

[0253] In various embodiments, the pH of the composition is 7.0 or higher, for example ranging from about 7.4 to about 9.5. These pH values ensure that the colloidal phase remains stable.

[0254] In particular embodiments of any aspect of the invention where a peptide coacervate is formed (including the second, third, fourth, and fifth aspects of the invention) the peptide coacervate is formed by self-coacervation whereby a single type of peptide is formed in the coacervate composition (excluding any payload or ancillary peptide).

[0255] In particular embodiments of any aspect of the invention where a peptide coacervate is formed (including the second, third, fourth and fifth aspects of the invention) the peptide coacervate is formed by complex-coacervation whereby two or more coacervate-peptides are formed in the coacervate composition (excluding any payload or ancillary peptide). Suitably the ratio of the different types of peptide used to form the coacervate can be adjusted to alter the properties of the coacervate.

[0256] In these contexts, an ancillary peptide could be a peptide with a targeting motif, such as one having a CRAG sequence capable of binding to cholesterol.

[0257] Suitably, two of the types of peptide used to form the coacervates posses opposite net charges. In such situations the peptides can assemble into coacervates driven by weak molecular interactions.

[0258] The inventors have found that strong interactions between the cationic and aromatic peptides can be formed which facilitate complex coacervation and inhibit or even reverse aggregation. These results suggest a potential strategy to mitigate or reverse aggregation by harnessing cation-ir interactions in aggregation-prone peptides.

[0259] Suitably, at least one of the types of coacervate peptide used to form the coacervate possesses an aromatic amino acid (e.g., Tyr) at the X1position and another type of coacervate peptide possesses a cationic amino acid (e.g., Lys or Arg) at the X1position. By “at the X1position” we mean at the same or different X1positions when comparing the positions of the two types of peptide. In such situations the peptides can assemble into coacervates driven by non-covalent

[0260] T1 interactions such as cation-n interactions. In particular embodiments the peptide with an aromatic amino acid at the X1position and the peptide with an cationic amino acid at the X1position are mixed in a ratio of between 1 :20 and 20:1 , such as between 1 :15 and 15:1 , 1 :10 and 10:1 , or 1 :8 and 8:1 (e.g. between 1 :1 and 1 :10). Coacervates formed from mixtures comprising two type of peptide wherein one of the peptides possesses Tyr at the X1position and another possesses Arg at the X1position are particularly preferred.

[0261] In a preferred embodiment the coacervate is formed from RP-KSF7YP-KSPpeptides to form a RP- KSP / YP-KSPcomplex (coacervate). In particular embodiments the RP-Kspand YP-KSPpeptides are mixed in a ratio of between 1 :20 and 20:1 , such as between 1 :15 and 15:1 , 1 :10 and 10:1 , or 1 :8 and 8:1.

[0262] Active agent

[0263] The isolated peptides of the first aspect of the invention are capable of encapsulating or recruiting one or more biomacromolecules (also referred to herein as “active agent” or “payload”). Suitably, the active agent may be or include, but is not limited to, proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, chemical compounds and nanoparticles. Suitable nanoparticles may be or include, but not limited to, metal nanoparticles, metal oxide nanoparticles, and combinations thereof. The nanoparticles may be magnetic nanoparticles. Suitably, the active agent is a pharmaceutical or diagnostic agent.

[0264] Suitable nanoparticles include those, such as but not limited to, metal nanoparticles, metal oxide nanoparticles and combinations thereof. The nanoparticles may be magnetic nanoparticles. “Nanoparticles”, as used herein, refer to particles that have dimensions, such as ESD, in the nanometer range, typically up to 500 nm, for example up to 250 or up to 100 nm. The nanoparticles may be substantially spherical in shape in a non-limiting embodiment. “Chemical compounds”, as used in this context, relates in particular to small molecules, i.e. organic compounds with a molecular weight of 1000 g / mol or less, such as 750 g / mol and less. This group of compounds includes, for example, known small molecule pharmaceutical compounds, such as doxorubicin. A pharmaceutical agent from the group of (poly)peptides includes insulin and other peptide hormones.

[0265] In various embodiments, the pharmaceutical or diagnostic agent may be or include, but not limited to, RNA oligonucleotides or variants thereof, such as those used in CRISPR / Cas9 or other genome-editing systems, small molecules, antibodies or antibody-like molecules, and the like.

[0266] General methods for forming coacervates that encapsulate an active agent are known. For the encapsulation, an aqueous solution of the coacervate-forming peptides can be used. As described above, it is possible to dissolve the peptides in an aqueous solution if the pH is low enough. Accordingly, the peptides can be dissolved in aqueous acetic acid, for example of a concentration of 1 to 100 mM, such as 10 mM. Other acids may be equally suitable, as long as they do not hydrolyse the peptide bonds or are used in concentrations low enough to avoid hydrolysis of the peptides. In these embodiments, the pH of the aqueous solution of the coacervate-forming peptides may be below 7, for example below 6.5 or below 6.0 or below 5.5 or below 5.0 or below 4.5 or below 4.0. The pH is however, in various embodiments, higher than 0, for example 1 or higher, such as 2 or higher.

[0267] For forming the coacervate and at the same time encapsulating the active agent, the solution of the coacervate-forming peptides is combined with the active agent and coacervate formation is induced. The induction of coacervate formation is typically induced by increasing the pH of the resulting solution containing both the coacervate-forming peptides and the active agent, as well as optional additional components and auxiliaries. The pH may be increased to values of 6.0 or more, 6.5 or more, 7.0 or more, but, in various embodiments, not higher than 10.0. The pH increase may be achieved by adding an alkaline agent to the solution. In case the active agent is provided in form of an aqueous solution, too, said solution may have a pH >7 and thus effect coacervate formation. To maintain the pH in a range high enough upon combination of the two solutions, the solution of the active agent may be buffered with suitable buffering agents, such that the combined aqueous solutions of the active agent and the coacervate-forming peptides retain a pH >7.

[0268] The concentration of the coacervate-forming peptides in the aqueous solution may range from 2 to 100 mg / mL. To allow efficient coacervate formation, in various embodiments, the concentration of the coacervate-forming peptides in the aqueous solution after addition of the active agent is greater than 0.3 mg / mL.

[0269] After the coacervate has been formed, it may be an aqueous liquid two phase formulation, as described above, i.e. a composition comprising (1) a coacervate colloidal phase comprising one or more peptides of the first aspect of the invention and the active agent; and (2) a dilute aqueous phase.

[0270] The coacervates formed in the above-described processes may have the form of droplets, for example microdroplets, having a substantially spherical shape with a diameter ranging from about 0.2 to about 5 pm, or may take the form of a condensed hydrogel.

[0271] According to a second aspect of the invention there is provided a composition for delivery of an active agent, the composition comprising a peptide coacervate, wherein the peptide coacervate comprises:

[0272] (i) one or more isolated peptides according to the first and / or eighth aspects of the invention; and

[0273] (ii) an active agent recruited in the peptide coacervate.

[0274] In particular embodiments of this second aspect of the invention the one or more isolated peptides in step (i) comprise a self-immolative moiety which autocatalytically cleaves itself upon exposure to specific conditions selected from the group consisting of: pH changes, redox changes, exposure to release agents, and combinations thereof.

[0275] In particular embodiments, the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.

[0276] In particular embodiments, the peptide coacervate is formed from the same type of peptide (i.e. one having the same amino acid sequence)

[0277] In particular embodiments, the peptide coacervate is formed from a combination of distinct type of peptide (i.e. two or more peptides having distinct amino acid sequences). Suitably, the peptide coacervate is formed from a combination of two distinct types of peptide. Suitably the two types of peptide have opposite net charges. Suitably the peptide coacervate is formed from a combination of a peptide having an aromatic amino acid at an X1position and a distinct peptide having a cationic amino acid at an X1position in the same relative position (i.e. relative to the position in the other type of peptide). Suitably, the aromatic amino acid is tyrosine (Y) and the cationic amino acid is arginine (R).ln particular embodiments, two distinct types of coacervateforming peptide are used in a ratio between 20:1 and 1 :20, such as between 10:1 and 1 :10.

[0278] Suitably, the active agent is a pharmaceutical or diagnostic agent.

[0279] In particular embodiments, the active agent is a protein or (poly)peptide. Suitably, the protein or (poly)peptide is an antibody, antibody variant, antibody fragment or peptide.

[0280] An example of a diagnostic active agent (payloads) for inclusion in the coacervates of the invention could be a diagnostic enzyme that has bioactivity in certain diseased cells, or a peptide with Nanoclick™ or FREP functions that can respond to molecules / enzyme on certain cells to give luminescence or fluorescence signals. The coacervates of the invention are particularly suitable for intracellular delivery of a diagnostic moiety that cannot be uptaken by cells on its own.

[0281] The coacervates formed of the isolated peptides of the invention and an active agent may be formulated as compositions. The composition may be a pharmaceutical or diagnostic formulation for administration to a subject. In various embodiments it can thus comprise one or more pharmaceutically or diagnostically acceptable excipients (such as diluents, auxiliaries, carriers and the like). Such formulations may additionally comprise further active agents that are not encapsulated in the coacervate phase. In various embodiments, such compositions are liquid compositions, including gels and pastes. “Liquid”, as used herein, particularly refers to compositions that are liquid under standard conditions (20° C. and 1013 mbar). In various embodiments, such liquid compositions are pourable. The compositions may be in single dose or multi dose form. Suitable forms and packaging options are well known to those skilled in the art.

[0282] The pharmaceutical or diagnostic formulation can be adapted for and so may be suitable for human or veterinary use.

[0283] Accordingly, the subject may be a mammal, such as horse, dog, cat, cow, sheep, goat, monkey, ape (including man) or any other mammal. Suitably, the subject is a human.

[0284] In particular embodiments, the pH of the composition is > 5.0 and < 9.5, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0. Most suitably, the pH of the composition is, in various embodiments, 7.0 or higher, for example in the range of 7.4 to 9.5.

[0285] In particular embodiments, the coacervates (in the composition) are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.

[0286] In particular embodiments, the coacervates (in the composition) are capable of enhanced cellular uptake compared to coacervates formed from HBpep(VPL)-SP.

[0287] In particular embodiments, the coacervates (in the composition) are capable of enhanced transfection efficiency compared to coacervates formed from HBpep(VPL)-SP.

[0288] According to a third aspect of the invention there is provided a method for the recruitment of an active agent in a peptide coacervate, the method comprising:

[0289] (i) providing an aqueous solution of coacervate-forming peptides, said coacervateforming peptides comprising one or more isolated peptides of the first and / or eighth aspects of the invention;

[0290] (ii) combining the aqueous solution of the coacervate-forming peptides with an aqueous solution of an active agent; and

[0291] (iii) inducing coacervate formation.

[0292] Suitably, the pH of (a) the aqueous solution of the coacervate-forming peptides; and / or (b) the aqueous solution of an active agent; and / or (c) the aqueous solution of the aqueous solution of the coacervate-forming peptides mixed with the aqueous solution of an active agent is above 7.0, such as above 7.5, above 8.0, above 8.5, or in the range 7.0 - 9.0, including 7.0-8.0 and 8.0-9.0.

[0293] Suitably, the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and < about 9.0, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

[0294] In particular embodiments, the peptide coacervate is formed from the same type of coacervateforming peptide (i.e. one having the same amino acid sequence)

[0295] In particular embodiments, the peptide coacervate is formed from a combination of distinct type of coacervate-forming peptide (i.e. two or more peptides having distinct amino acid sequences). Suitably, the peptide coacervate is formed from a combination of two distinct types of coacervate-forming peptide. Suitably the two types of peptide have opposite net charges. Suitably the peptide coacervate is formed from a combination of a peptide having an aromatic amino acid at an X1position and a distinct peptide having a cationic amino acid at an X1position in the same relative position (i.e. relative to the position in the other type of peptide). Suitably, the aromatic amino acid is tyrosine (Y) and the cationic amino acid is arginine (R).

[0296] In particular embodiments, two distinct types of coacervate-forming peptide are used in a ratio between 20:1 and 1 :20, such as between 10:1 and 1 :10.

[0297] In particular embodiments, the volume ratio of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent is between 1 : 5 and 1 : 20.

[0298] In various embodiments, the active agents in the combined aqueous solution are also in the form of an aqueous solution. Said aqueous solution may have a pH >7.0, such as >8.0 and, in some embodiments, is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides obtained in the combined aqueous solution has a pH >7.0, such as >8.0, as disclosed elsewhere herein.

[0299] In various embodiments, the combination of the aqueous solution with the active agent changes the pH of the solution in which the peptide for coacervate forming is solved to greater than 7 and thus initiates coacervate formation.

[0300] In various embodiments, the concentration of the coacervate-forming peptides in the provided aqueous solution is greater than about 0.3 mg / mL and may, for example, range from about 0.3 to about 100 mg / mL. The combined solution after coacervate formation may be an aqueous liquid two phase formulation, comprising (1) a coacervate colloidal phase comprising the peptides derived from histidine-rich proteins and the active agent; and (2) a dilute aqueous phase.

[0301] In certain embodiments, the coacervate colloidal phase comprises the peptides of the first and / or eighth aspects of the invention in a form of colloids encapsulating the active agent. In some embodiments, the colloidal phase has the form of droplets having a substantially spherical shape with a diameter ranging from about 0.2 to about 5 pm. The diameter of the substantially spherical shape may be the “equivalent spherical diameter (ESD)” referring to the diameter of a perfect sphere of equivalent volume as the potentially irregularly shaped droplet. For example, the droplet may have an ellipsoid shape, and the equivalent spherical diameter would then be the diameter of a perfect sphere of exactly the same volume. Each of the droplets is made up of the coacervate-forming peptides and, in various embodiments, is homogeneous in that it has no distinct core-shell morphology, but rather is a colloidal particle with no peptide gradient over its radius. In alternative or additional embodiments, the coacervate phase may take the form of a condensed hydrogel.

[0302] The dilute aqueous phase may include a water-based liquid, as described above. It is peptide- depleted in that the majority of the peptides are located in the colloidal phase, e.g. 80 wt.-% or more of the peptides are present in the colloidal phase, for example at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99 wt.-%. The dilute phase may thus contain residual amounts of peptides in the coacervate composition. As the coacervate formation is an equilibrium reaction, the exchange of peptides from the colloidal phase to the dilute phase and vice versa may be dynamic. However, in various embodiments, the above distribution applies.

[0303] Thus, according to certain embodiments, the coacervates formed may have the form of droplets having substantially spherical shape with a diameter ranging from about 0.2 to about 5 pm.

[0304] In an embodiment of the third aspect of the invention, there is a provided a coacervate composition comprising an active agent produced by the method of the third aspect of the invention.

[0305] According to a fourth aspect of the invention there is provided a method for the delivery of an active agent, said method comprising:

[0306] (i) providing a composition including a peptide coacervate that comprises: a. one or more isolated peptides of the first and / or eighth aspects of the invention, optionally wherein the lysine residue (K), when present, is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. an active agent, wherein the active agent is recruited in the peptide coacervate; and

[0307] (ii) exposing the peptide coacervate to conditions that trigger the release of said active agent from the peptide coacervate.

[0308] In this context the lysine residue (K), when present, refers to the lysine that, when present, is the one located outside of the pentameric motif [i.e. not within the “O” motif, GHGX1Y (SEQ ID NO: 1) motif or not at the X2position].

[0309] In a preferred embodiment, the one or more isolated peptides of the first aspect of the invention in (a.) have the lysine residue (K) modified at an epsilon (E)- amino group with a self-immolative moiety. In another preferred embodiment, the one or more isolated peptides of the eighth aspect of the invention in (a.) have the lysine residue (K) modified at an epsilon (E)- amino group with a self-immolative moiety. In another preferred embodiment, the one or more isolated peptides of the eighth aspect of the invention in (a.) lack the lysine at X2.

[0310] The conditions that trigger the release of the active agent may be selected from those disclosed above for the composition for the delivery of the active agent.

[0311] In various embodiments of the above methods, the conditions that trigger the release of the active agent may be or include, but not limited to, elevated temperatures, pH changes, exposure to release agents and combinations thereof.

[0312] In the methods for the delivery of an active agent, such as a pharmaceutical or diagnostic agent, the provided compositions comprising a peptide coacervate may be exposed to or subjected to conditions that facilitate the release of the active agent from the coacervate phase. Said release may be facilitated by dissolution of the peptides of the coacervate phase, for example reversing the formation process by decreasing the pH, or degradation or disruption of the coacervate phase by suitable means. Some of the release mechanisms have been described above. Additional release mechanisms may include the use of surfactants or denaturing agents that disrupt the formed phases.

[0313] In various embodiments, the conditions that trigger the release of the pharmaceutical or diagnostic agent may be or include, but not be limited to, elevated temperatures, pH changes, exposure to release agents, such as enzymatic agents that degrade peptides, denaturing agents or surfactants, and combinations thereof. Methods of treatment / diagnosis

[0314] Methods for treating or diagnosing a condition or disease or disorder in a subject in need thereof is also disclosed, wherein the compositions described above may be used in the treatment and / or diagnosis. Such methods of treatment also include methods where a disease, condition or disordered is managed, for example in that the symptoms or effects are alleviated.

[0315] According to the fifth aspect of the invention there is provided a method for treating or diagnosing a condition or disease in a subject in need thereof, comprising:

[0316] (i) administering a composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides according to the first and / or eighth aspects of the invention, in particular wherein the lysine residue (K), when present, is modified at an epsilon (£)- amino group with a self-immolative moiety; and b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate; and

[0317] (ii) exposing the peptide coacervate to conditions that trigger the release of the pharmaceutical or diagnostic agent from the peptide coacervate.

[0318] The conditions that trigger the release of the pharmaceutical or diagnostic agent may be selected from those disclosed above for the delivery methods.

[0319] Suitably, the composition is administered to a subject in a therapeutically effective amount.

[0320] In such methods, the compositions described herein and comprising a peptide coacervate and a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is encapsulated in the coacervate are administered to said subject. The administration may make use of any suitable administration route including oral administration or parenteral administration, for example intravenous, intramuscular, subcutaneous, epidural, intracerebral, intracerebroventricular, nasal, intraarterial, intraarticular, intracardiac, intradermal, intralesional, intraocular, intraosseous, intravitreal, intraperitoneal, intrathecal, intravaginal, transdermal, transmucosal, sublingual, buccal, and perivascular.

[0321] The administration may be systemic or localized, e.g. topically.

[0322] After administration, the release of said pharmaceutical or diagnostic agent from the coacervate may be facilitated by exposing the coacervate to conditions that trigger the release of the pharmaceutical or diagnostic agent. Said exposure may occur automatically due to conditions in the body of the patient, such as metabolic action, or may be triggered externally by applying a stimulus to the patient that leads to release of the encapsulated agents, such as exposure to a magnetic field.

[0323] The conditions that trigger the release of the pharmaceutical or diagnostic agent may generally be selected from those disclosed above for the delivery methods.

[0324] The subject may be a mammal, for example a human.

[0325] According to a sixth aspect of the invention there is provided the use of an isolated peptide of the first and / or eighth aspect of the invention for preparing a coacervate composition, optionally wherein the coacervate composition comprises an active agent. Suitably, there is provided the use of an isolated peptide of the first and / or eighth aspects of the invention in conjunction with an active agent for preparing a coacervate composition comprising the active agent.

[0326] According to a seventh aspect of the invention there is provided the isolated peptide of the first and / or eighth aspects of the invention or the composition of the second aspect of the invention for use in therapy or for use in a method of diagnosis practised on a mammal.

[0327] According to a ninth aspect of the invention there is provided a peptide coacervate composition comprised of two types of coacervate-forming peptides wherein the two types of coacervateforming peptides are capable of interacting with each other via at least one ir-cationic interaction.

[0328] According to a tenth aspect of the invention there is provided a peptide coacervate composition comprising two types of coacervate-forming peptides whose amino acid sequences differ from one another, and wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one TT-cationic interaction.

[0329] Suitably, in the ninth or tenth aspect, the ir-cationic interaction is formed between an aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type.

[0330] Suitably, in the ninth or tenth aspect, the aromatic amino acid is selected from tyrosine (Y), histidine (H), phenylalanine (F) or tryptophan (W).

[0331] Suitably, in the ninth or tenth aspect, the cationic amino acid is selected from arginine (R), lysine (K), or ornithine (Orn).

[0332] Suitably, in the ninth or tenth aspect, the at least one n-cationic interaction is formed between arginine (R) and tyrosine (Y) amino acids. Suitably, in the ninth or tenth aspect, the aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type are in the same relative location on the respective peptides.

[0333] Suitably, in the ninth or tenth aspect, the aromatic amino acid in one coacervate-forming peptide type and the cationic amino acid in the other coacervate-forming peptide are at the same X1 position of an isolated coacervate-forming peptide having the Formula (I), (II), (III), (IV) or (V).

[0334] In an eleventh aspect, there is provided a method for creating a peptide coacervate composition comprising mixing two types of coacervate-forming peptides wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one ir-cationic interaction.

[0335] Suitably, the first coacervate-forming peptide has a different amino acid sequence to the second coacervate-forming peptide.

[0336] Additional applications of the compositions and methods will be identifiable by the person skilled in the art. The compositions and methods herein disclosed are further illustrated in the following examples and the accompanying Figures, which are provided by way of illustration and are not intended to be limiting the scope of the present disclosure.

[0337] List of preferred embodiments:

[0338] 1 . An isolated peptide comprising the amino acid sequence of Formula (I):

[0339] (O)n-K-(O)m-Z

[0340] (I) wherein K is lysine optionally modified with a self-immolative moiety

[0341] Z is tryptophan (W) or is absent

[0342] O is GHGX1Y

[0343] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other; optionally wherein not all of the X1residues are proline (P).

[0344] 2. The isolated peptide of embodiment 1 , wherein the lysine residue is located at the 1 st, 6th, 11th, 16th, 21st, or 26thposition from the N-terminus.

[0345] 3. The isolated peptide of embodiment 1 or 2, wherein n is 3 and m is 2.

[0346] 4. The isolated peptide of embodiment 1 , wherein the peptide of Formula (I) is a peptide of Formula (II):

[0347] [O]a-[B]b-K-[B]c-[O]d-[O]e-Z

[0348] (II) wherein

[0349] O is GHGX1Y (SEQ ID NO: 27)

[0350] B is GHGPY (SEQ ID NO: 2)

[0351] G is glycine (G), H is histidine (H), Y is tyrosine (Y) and P is proline (P); each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);

[0352] K is lysine (K) optionally modified with a self-immolative moiety; and

[0353] Z -is tryptophan (W) or is absent, a, b, c, d, and e are each independently 0, 1 or 2 and a+b+c+d+e is 3-8, optionally wherein a+b+c+d+e is 5.

[0354] 5. The isolated peptide of any one of the preceding embodiments, wherein the isolated peptide is 16 - 45 amino acids long.

[0355] 6. The isolated peptide of any one of the preceding embodiments, wherein the peptide is 26 or 27 amino acids long

[0356] 7. The isolated peptide of any one of the preceding embodiments, wherein the amino acid residue at X1is the same at each X1position.

[0357] 8. The isolated peptide of any one of embodiments 1 to 6, wherein the two N-terminal X1amino acid residues are the same as each other and the C terminal X1amino acid residue is different to the two N-terminal X1amino acids. 9. The isolated peptide of any one of embodiments 1 to 6, wherein the two N-terminal X1amino acid residues and the C-terminal X1amino acid residue are the same as each other.

[0358] 10. The isolated peptide of any one of the preceding embodiments, wherein at least one of the residues at an X1position is selected from: A, G, H and R.

[0359] 1 1 . The isolated peptide of claim 7, wherein all the residues at the X1positions are the same and are selected from: A, G, and H.

[0360] 12. The isolated peptide of any one of the preceding embodiments, wherein at least one of the residues at an X1position is selected from: H and R.

[0361] 13. The isolated peptide of embodiment 7, wherein all the residues at the X1positions are the same and are selected from: H and R.

[0362] 14. The isolated peptide of any one of the preceding embodiments, wherein the peptide comprises the Z tryptophan

[0363] 15. The isolated peptide of any one of embodiments 1 to 13, wherein the peptide lacks the Z tryptophan.

[0364] 16. The isolated peptide of any one of embodiments 1 to 14, wherein the isolated peptide comprises or consists of an amino acid sequence selected from the group consisting of:

[0365] (i) GHGAY GHGAY GHGPY K GHGPY GHGAY W (SEQ ID NO: 3)

[0366] (ii) GHGGY GHGGY GHGPY K GHGPY GHGGY W (SEQ ID NO: 4)

[0367] (ill) GHGSY GHGSY GHGPY K GHGPY GHGSY W (SEQ ID NO: 5)

[0368] (iv) GHGNY GHGNY GHGPY K GHGPY GHGNY W (SEQ ID NO: 6)

[0369] (v) GHGHY GHGHY GHGPY K GHGPY GHGHY W (SEQ ID NO: 7)

[0370] (vi) GHGRY GHGRY GHGPY K GHGPY GHGRY W (SEQ ID NO: 8)

[0371] (vii) GHGDY GHGDY GHGPY K GHGPY GHGDY W (SEQ ID NO: 9)

[0372] (viii) GHGYY GHGYY GHGPY K GHGPY GHGYY W (SEQ ID NO: 10)

[0373] (ix) GHGRY GHGRY GHGPY K GHGPY GHGYY W (SEQ ID NO: 11) (x) GHGDY GHGDY GHGPY K GHGPY GHGYY W (SEQ ID NO: 12);

[0374] Optionally, wherein the lysine residue (K) is modified at an epsilon (s)- amino group with a self- immolative moiety.

[0375] 17. The isolated peptide of any one of embodiments 1 to 16 or 24 to 40, which is able to form coacervates at a pH between about 6.5 and 9.0, such as between about 7.0 and 9.0 or 7.0 and 8.0.

[0376] 18. The isolated peptide of any one of embodiments 1 to 17 or 24 to 40, which is able to form coacervates that can enter the cell more efficiently than coacervates formed from HBpep(VPL)- SP.

[0377] 19. The isolated peptide of any one of embodiments 1 to 18 or 24 to 40, which is able to form coacervates that are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.

[0378] 20. The isolated peptide of any one of embodiments 1 to 19 or 24 to 40, wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety.

[0379] 21 . The isolated peptide of embodiment 20, wherein the self-immolative moiety comprises a disulfide (-S-S-) moiety.

[0380] 22. The isolated peptide of embodiments 20 or 21 , wherein the self-immolative moiety has the formula -C(=O)-O-(CH2)n-S-S-R, wherein n is 1 , 2, 3, 4, or 5, and wherein R is selected from: substituted or unsubstituted alkyl, alkenyl, cycloalk(en)yl, and aryl.

[0381] 23. The isolated peptide of embodiment 22, wherein R is -(CH2)n-O-C(=O)-R' wherein n is 1 , 2, 3, 4, or 5, and wherein R’ is selected from: C1.4 alkyl, Ce-aryl, preferably phenyl, optionally substituted with halogen. . An isolated peptide comprising the amino acid sequence of Formula (IV): (O)n-X2-(O)m-Z

[0382] (IV) wherein X2is lysine optionally modified with a self-immolative moiety or absent

[0383] Z is tryptophan (W) or is absent

[0384] O is GHGX1Y (SEQ ID NO: 1)

[0385] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), proline (P), lysine (K) and glutamine (E). n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0386] 25. The isolated peptide of embodiment 24, wherein the peptide of Formula (IV) is a peptide of Formula (V):

[0387] (O)n-X2-(O)m-Z

[0388] (V) wherein X2is lysine optionally modified with a self-immolative moiety or absent wherein

[0389] Z is tryptophan (W) or is absent

[0390] O is GHGX1Y (SEQ ID NO: 1)

[0391] G is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from arginine (R), tyrosine (Y), lysine (K) and glutamine (E) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other.

[0392] 26. The isolated peptide of embodiment 24 or 25, wherein X2is lysine optionally modified with a self-immolative moiety.

[0393] 27. The isolated peptide of embodiment 26, wherein the lysine residue is located at the 1 st, 6th, 11th, 16th, 21st, or 26thposition from the N-terminus.

[0394] 28. The isolated peptide of embodiment 24, 25 or 26, wherein n is 3 and m is 2.

[0395] 29. The isolated peptide of any one of the embodiments 24 to 28, wherein the isolated peptide is 15 - 45 amino acids long. 30. The isolated peptide of any one of embodiments 24 to 29, wherein the peptide is 25, 26 or 27 amino acids long

[0396] 31 . The isolated peptide of any one of embodiments 24 to 30, wherein the amino acid residue at X1is the same at each X1position.

[0397] 32. The isolated peptide of any one of embodiments 24 to 30, wherein the two N-terminal X1amino acid residues are the same as each other and the C terminal X1amino acid residue is different to the two N-terminal X1amino acids.

[0398] 33. The isolated peptide of any one of embodiments 24 to 30, wherein the two N-terminal X1amino acid residues and the C-terminal X1amino acid residue are the same as each other.

[0399] 34. The isolated peptide of any one of embodiments 24 to 34, wherein at least one of the residues at an X1position is selected from: A, G, H and R.

[0400] 35. The isolated peptide of claim 34, wherein all the residues at the X1positions are the same and are selected from: A, G, and H.

[0401] 36. The isolated peptide of any one of embodiments 24 to 35, wherein at least one of the residues at an X1position is selected from: H and R.

[0402] 37. The isolated peptide of embodiment 34, wherein all the residues at the X1positions are the same and are selected from: H and R.

[0403] 38. The isolated peptide of any one of embodiments 24 to 37, wherein the peptide comprises the Z tryptophan

[0404] 39. The isolated peptide of any one of embodiments 24 to 37, wherein the peptide lacks the Z tryptophan.

[0405] 40. The isolated peptide of any one of embodiments 24 to 39, wherein the isolated peptide comprises or consists of an amino acid sequence selected from the group consisting of:

[0406] (i) GHGRY GHGRY GHGPY GHGPY GHGRY W (SEQ ID NO: 28)

[0407] (ii) GHGYY GHGYY GHGPY GHGPY GHGYY W (SEQ ID NO: 29)

[0408] (vii) GHGKY GHGKY GHGPY GHGPY GHGKY W (SEQ ID NO: 30)

[0409] (viii) GHGEY GHGEY GHGPY GHGPY GHGEY W (SEQ ID NO: 31) 41 . A composition for delivery of an active agent, the composition comprising a peptide coacervate, wherein the peptide coacervate comprises:

[0410] (i) one or more isolated peptides of any one of embodiments 1 to 40; and

[0411] (ii) an active agent recruited in the peptide coacervate.

[0412] 42. The composition of embodiment 41 , wherein the peptide comprises a self-immolative moiety which autocatalytically cleaves itself upon exposure to specific conditions selected from the group consisting of: pH changes, redox changes, exposure to release agents, and combinations thereof.

[0413] 43. The composition of embodiment 41 or 42, wherein the peptide coacervate is formed of a single type of isolated peptide.

[0414] 44. The composition of embodiment 41 or 42, wherein the peptide coacervate is formed of two or more types of isolated peptide.

[0415] 45. The composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide that posses opposite net charges.

[0416] 46. The composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide that posses opposite net charges.

[0417] 47. The composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide wherein one peptide wherein one of the types of coacervate peptide possesses an aromatic amino acid (e.g., Tyr) at the X1position and other type of coacervate peptide possesses a cationic amino acid (e g., Lys or Arg) at the X1position.

[0418] 48. The composition of embodiment 47, wherein the peptide with an aromatic amino acid at the X1position and the peptide with an cationic amino acid at the X1position are mixed in a ratio of between 1 :20 and 20:1 .

[0419] 49. The composition of any one of embodiments 41 to 48, wherein the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.

[0420] 50. The composition of any one of embodiments 41 to 49, wherein the active agent is a pharmaceutical or diagnostic agent. 51 . The composition of any one of embodiments 41 to 50, wherein the active agent is a protein or (poly)peptide.

[0421] 52. The composition of embodiment 51 , wherein the protein or (poly)peptide is an antibody, antibody variant, antibody fragment or peptide.

[0422] 53. The composition of any one of embodiments 41 to 52, wherein the composition is a pharmaceutical or diagnostic formulation for administration to a subject.

[0423] 54. The composition of any one of embodiments 41 to 53, wherein the pH of the composition is > 5.0 and < 9.5, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

[0424] 55. The composition of any one of embodiments 41 to 54, wherein the coacervates formed are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.

[0425] 56. The composition of any one of embodiments 41 to 55, wherein the coacervates formed are capable of enhanced cellular uptake compared to coacervates formed from HBpep(VPL)-SP.

[0426] 57. The composition of claim any one of embodiments 41 to 56, wherein the coacervates formed are capable of enhanced transfection efficiency compared to coacervates formed from HBpep(VPL)-SP.

[0427] 58. A method for the recruitment of an active agent in a peptide coacervate, the method comprising:

[0428] (i) providing an aqueous solution of coacervate-forming peptides, wherein the coacervateforming peptides are selected from the isolated peptides of any one of embodiments 1 to 40;

[0429] (ii) combining the aqueous solution of the coacervate-forming peptides with an aqueous solution of an active agent; and

[0430] (iii) inducing coacervate formation.

[0431] 59. The method of embodiment 58, wherein the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and < about 9.0, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

[0432] 60. The method of embodiment 58 or 59, wherein a volume ratio of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent is between 1 : 5 and

[0433] 1 : 20. 61 . A method for the delivery of an active agent, the method comprising:

[0434] (i) providing a composition comprising a peptide coacervate, wherein the peptide coacervate comprises: a. one or more isolated peptides selected from the peptides of any one of embodiments 1 to 40 wherein, when present, the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. an active agent, wherein the active agent is recruited in the peptide coacervate; and

[0435] (ii) exposing the peptide coacervate to conditions that trigger the release of the active agent from the peptide coacervate.

[0436] 62. A method for treating or diagnosing a condition or disease in a subject in need thereof, comprising:

[0437] (i) administering a composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides selected from the peptides of any one of embodiments 1 to 40 wherein, when present, the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate; and

[0438] (ii) exposing the peptide coacervate to conditions that trigger the release of the pharmaceutical or diagnostic agent from the peptide coacervate.

[0439] 63. Use of an isolated peptide of any one of embodiments 1 - 40 for preparing a coacervate composition.

[0440] 64. The isolated peptide of any one of embodiments 1 to 40 for use in therapy.

[0441] 65. The isolated peptide of any one of embodiments 1 to 40 for use in a method of diagnosis practised on a mammal, such as a human.

[0442] 66. The composition of any one of embodiments 41 to 57 for use in therapy.

[0443] 67. The composition of any one of embodiments 41 to 57 for use in a method of diagnosis practised on a mammal, such as a human. 68. A peptide coacervate composition comprised of two types of coacervate-forming peptides wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one iT-cationic interaction.

[0444] 69. A peptide coacervate composition comprising two types of coacervate-forming peptides whose amino acid sequences differ from one another, and wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one TT- cationic interaction.

[0445] 70. The peptide coacervate composition of embodiment 68 or 69, wherein the n-cationic interaction is formed between an aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type.

[0446] 71 . The peptide coacervate composition of any one of embodiments 68 to 70, wherein the aromatic amino acid is selected from tyrosine (Y), histidine (H), phenylalanine (F) or tryptophan (W)

[0447] 72. The peptide coacervate composition of any one of embodiments 68 to 71 , wherein the cationic amino acid is selected from arginine (R), lysine (K), or ornithine (Orn).

[0448] 73. The peptide coacervate composition of any one of embodiments 68 to 72, wherein the at least one n-cationic interaction is formed between arginine (R) and tyrosine (Y) amino acids.

[0449] 74. The peptide coacervate composition of any one of embodiments 68 to 73, wherein the aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type are in the same relative location on the respective peptides.

[0450] 75. The peptide coacervate composition of any one of embodiments 68 to 74, wherein the aromatic amino acid in one coacervate-forming peptide type and the cationic amino acid in the other coacervate-forming peptide are at the same X1 position of an isolated coacervateforming peptide having the Formula (I), (II), (III), (IV) or (V).

[0451] 76. A method for creating a peptide coacervate composition comprising mixing two types of coacervate-forming peptides wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one ir-cationic interaction.

[0452] 77. The method according to embodiment 76, wherein the first coacervate-forming peptide has a different amino acid sequence to the second coacervate-forming peptide. Examples

[0453] Example 1 - HBpep-SP peptide coacervates.

[0454] Experimental Methodology

[0455] Peptide Synthesis and Purification

[0456] The peptides used in this study were synthesized using a Liberty Blue microwave peptide synthesizer using the solid phase peptide synthesis (SPPS) technique [4]. All amino acids in the peptide sequence were coupled onto the resin by performing coupling / deprotection cycles from the C-terminals of the peptides to the N-terminals. After the synthesis, peptides were cleaved from the resins by using the cocktail containing 95% of trifluoroacetic acid (TFA), 2.5% of H2O and 2.5% of triisopropylsilane (TIPS). The supernatants were concentrated by using nitrogen flow and precipitated into 50 mL of cold diethyl ether. After centrifugation, the pellets were dried under vacuum and re-dissolved by using 90% of 10 mM acetic acid and 10% acetonitrile for HPLC purification. The purified peptides were isolated by lyophilization from certain HPLC elutes.

[0457] Self-immolative Moiety Synthesis

[0458] The amine reactive moieties with self-immolative property used in this study were designed based on the literature [5], The synthesis routes were shown below.

[0459] Route 1 Synthesis routes of amine reactive NHS-SS-R.SA is the SR immolative group that possesses an acetyl terminal group (*-R = ‘-CHa).

[0460] SP is the SR immolative group that possesses a phenyl terminal group (*-R = )

[0461] First, to synthesize the intermediate product HO-SS-R, 2-hydroxyethyl disulfide (1 equiv, 10 mmol) was dissolved in 10 mL acetonitrile (ACN), and another 10 mL THF containing carboxylic acid reactants such as benzoic acid (0.9 equiv, 9 mmol) was added. Then, 12 mmol of N,N’- Diisopropylcarbodiimide (DIC) was added into the reaction mixtures dropwise under the ice bath. The reaction was kept at the ice bath for 0.5 hours before being moved to room temperature. After the overnight reaction, the supernatants were collected by filtration and evaporated under reduced pressure. The raw products were purified by using silica gel chromatography with ethyl acetate / hexane (1 / 4) as elute. The purified products were isolated by rotary evaporation.

[0462] Then, the amine reactive NHS-SS-R was synthesized by coupling HO-SS-R and N- hydroxysuccinimide (NHS) by using triphosgene. In detail, HO-SS-R (1 equiv, 10 mmol), and 4- dimethylaminopyridine (DMAP, 0.1 equiv, 1 mmol) was dissolved in 15 mL of THE. Then, triphosgene (0.37 equiv, 3.7 mmol) in 10 mL THF was added into the mixture dropwise under an ice bath. After another 0.5 hours of the ice bath, the reactions were moved to 40 °C water bath for 4 hours reaction. Then the reaction mixture was evaporated under reduced pressure to remove excess phosgene before adding NHS (1.5 equiv, 7.5 mmol) in 20 mL THF and DIEPA (1 .5 equiv, 7.5 mmol) orderly. The reactions were kept at 40 °C for 24 hours. The raw products were collected by evaporation and then purified by silica gel chromatography with ethyl acetate / hexane (1 / 3) as elute. The purified products were isolated by rotary evaporation. The amine-reactive products NHS-SS-Ac (labelled as “SA”) and NHS-SS-Ph (labelled as “SP”) were synthesized from acetic acid and benzoic acid.

[0463] Peptide Modification

[0464] The redox-responsive peptides were synthesized by the reaction between the s-amine of lysine in N-terminal protected peptide and the amine reactive NHS-SS-R, followed by deprotection of Fmoc. The HBpep-K peptide (1 equiv, 10 pmol) was dissolved in 3.9 mL of DMF containing DIPEA (15 equiv, 150 pmol). Then, NHS-SS-R (1.5 equiv, 15 pmol) in 0.1 mL of DMF was added into the prior solution. The reaction was conducted at room temperature overnight before adding 1 mL of piperidine. The mixture was stirred at room temperature for 2 hours for Fmoc deprotection. The raw products were collected from the precipitates after adding 30 mL of cold diethyl ether, and purified by HPLC. The final products were isolated by lyophilization as white solids.

[0465] Microscopy of Peptide Coacervates

[0466] The optical microscope image of coacervates was collected by inverted microscopy (AxioObserver.ZI , Zeiss). To induce phase separation, peptide stocks (10 mg peptide in 10 mM acetic acid) were added into the coacervation buffer with various pHs. Then the mixture was observed by using the inverted microscope.

[0467] Surface force apparatus (SFA) measurements

[0468] The viscoelastic property of coacervate variants was measured using an SFA 2000 (SurForce LLC, Santa Barbara). As described in previous studies (Wu,. X., et al. , Communications Chemistry 7, 5 (2024); Deepankumar, K. et al. Adv. Mater. 34, 2103828 (2022)) freshly cleaved mica with a 55 nm silver layer deposited on its back was glued on the glass disks. Then, 2 pL of freshly prepared coacervates (0.3 mM in buffer, pH 6.5 for PL and pH 7.0 for others) was mixed with 18 L of PBS and injected in the gap between two mica surfaces. The distance D between two surfaces was measured and calculated based on the fringes of equal chromatic order (FECO) technique. After the sample was injected into the gap, the system was equilibrated for 30 min by keeping two surfaces in contact with a bridging coacervate film. Then, the two surfaces started to approach followed by separation. The measured force F was normalized by the effective radius of the surface R.

[0469] Fluorescence recovery after photobleaching (FRAP) experiments

[0470] To perform FRAP measurements, HBpep-SP peptide variants were labeled with Alexa Fluor 488 NHS ester on the N-terminus. The labeled peptides were mixed with pristine ones at a ratio of 0.5:99.5 to prepare stock solutions with a final concentration of 3 mM. The Coacervates were prepared by mixing the stock solution with the pH 6.5 buffer (VPL) or pH 7.0 buffer (GP and RPY) at a ratio of 1 :9. The experiment started by applying a 488 nm laser pulse at the power of 50% on the chosen area within a single coacervate from the confocal microscope (Eclipse Ti2, Nikon) to bleach its fluorescence. The confocal microscope then took images of the sample every 5 s. The fluorescence intensity was quantified by using Imaged software and normalized by the intensity before the photobleaching.

[0471] Atomic force microscopy (AFM) measurements

[0472] After an initial immobilization of coacervates on mica (Ted Pella, Inc.) for a duration of 5 min and removal of unbound droplets via buffer (PBS) exchange, an AFM tip (SNL, Bruker) was brought in proximity to the coacervates. Single coacervates were localized by imaging in tapping mode (frequency about 30 kHz, amplitude setpoint about 90%). Generally, images of individual coacervates were acquired at a scan rate of 0.2-0.4 Hz and a resolution of 64 points per line. After localization, “Point & Shoot” function (NanoScope, Bruker) was used to place the AFM tip on the apex of selected coacervates before applying indentation using ramp mode. As a control, points on solid surface near selected coacervates were also indented. The coacervates (and the control point on solid) were indented up to a threshold force value of about 2 nN at a preset frequency of 0.5 Hz. Indented coacervates were imaged again in tapping mode to detect signs of degradation, drift, or displacement due to tip lateral forces. The height of individual Coacervates before and after indentation were calculated by placing a trace line over the particles on images previously flattened using Gwyddion 2.47 Necas, D. et al., Central European Journal of Physics 10, 181-188 (2012). For elasticity evaluation, we proceeded as follows: deflection versus piezo displacement was converted to force versus tip-surface separation (or indentation distance) using protocols written in Igor Pro (Wavemetrics) (Butt, H.-J ., et. al. Surf. Sci. Rep. 59, 1-152 (2005)). Fit to the force versus indentation distance curve in the tip-coacervate contact region was used to calculate modulus based on Sneddon model (NanoScope, Bruker). Correction to the modulus was applied using bottom effect cone correction (BECC) (Gavara, N. et al. Nat. Nanotechno!. 7, 733-736 (2012)). Ten indentations per coacervate were used forthe evaluations. Average modulus of GP coacervates was calculated from at triplicate independent preparations. Additionally, GP and RPY coacervates were investigated using softer tips (MLCT-Bio, Bruker). Prior to the measurements, the AFM tip was cleaned using UV-ozone (Novascan). Dimension FastScan (Bruker) was used in all the measurements. The calibrated tip parameters included stiffness (about 0.2 N / m for SNL and 0.04 N / m for MLCT-Bio) using the thermal method, and optical lever sensitivity using cantilever-mica hard contact (set point equal to 0.4 V) (Butt, H.-J., et. al. Surf. Sci. Rep. 59, 1-152 (2005)).

[0473] Molecular dynamics (MD) simulations

[0474] Molecular dynamics (MD) simulations were carried out for two peptides GP and RPY to understand how intermolecular interactions modulate the properties of coacervates. For each peptide, systems containing 4, 10, and 30 peptide molecules were simulated, respectively. Each system was subject to 3 replicates of simulations, with each replicate running for 1 s, resulting in a total simulation time of 18 s. In each simulation, the required number of peptide molecules was randomly placed in a cubic box and solvated with water molecules. To be consistent with in vitro experimental conditions, 0.16 M NaCI was added to each system. Each system was initially subject to 500 steps using steep descent energy minimization. Subsequently, a 100 ps of MD simulation in the NVT ensemble was carried out, followed by production simulations in the NPT ensemble that proceeded in three stages. The first stage involved a 300 ns of simulation at 300 K, during which the peptide was observed to aggregate and form irregular clusters. In the second stage, a 200 ns simulated annealing simulation was applied to accelerate equilibration. Finally, a 500 ns production run at 300 K was carried out. The details of each system are summarized in Table S2. The number of hydrogen bonds, proximal radial distribution functions (pRDF) (Lin, B. et al., The Journal of Chemical Physics 134 (2011) and Sementa, D. et al. Angewandte Chemie International Edition 62, e202311479 (2023)), solvent accessible surface area (SASA), and the number of TT-TT and cation-ir interaction pairs were calculated using the combination of the last 300 ns of the three replicates.

[0475] In all simulations, the peptides were modelled using the AMBER14sb force field and water was described by the TIP3P model (Maier, J.A. et al. J. Chem. Theory Com put. 11 , 3696-3713 (2015) and Jorgensen W. L., The Journal of Chemical Physics 79, 926-935 (1983). Parameters of the unnatural amino acid KSP were obtained using the antechamber module of AMBER 20 package (Case, D.A. et al. AmberTools. J. Chem. Inf. Model. 63, 6183-6191 (2023)). Lennard-Jones and short-range electrostatic interactions were computed using a cutoff of 0.9 nm, while long-range electrostatic interactions were calculated using PME (Essmann, U. et al. The Journal of Chemical Physics 103, 8577-8593 (1995)). All simulations were carried out in the NPT ensemble with temperature and pressure maintained at 300 K and 1 bar except forthe simulated annealing simulations, which were conducted at a temperature of 400 K. All simulations were carried out using GROMACS 2021 patched with Plumed-2.9 (Abraham, M.J. et al. SoftwareX 1-2, 19-25 (2015) and Tribello, G.A., et al. Comput. Phys. Commun. 185, 604-613 (2014)).

[0476] Interactions between lipid bilayers and Coacervates

[0477] The giant unilamellar vesicles (GUVs) were prepared from 99.5% of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) and 0.5% of 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rhod-PE) using the gel-assisted method described in previous studies (Weinberger, A. et al. Biophys. J. 105, 154- 164 (2013). To investigate the interactions between GUVs and coacervate variants, 100 pL of freshly prepared GP or RPY coacervates (0.3 mM peptide, containing 0.5% Alexa Fluor 488 labeled peptide) were diluted in 900 pL of PBS containing GUVs. The mixture was visualized using a confocal microscope (Eclipse Ti2, Nikon).

[0478] The adhesion of Coacervates on the supported lipid bilayers (SLBs) was evaluated by quartz crystal microbalance (QCM). First, the SLBs were deposited on the silica sensor by solvent exchange method (Ferhan, A.R. et al. Nat. Protoc. 14, 2091-2118 (2019)). Then, PBS flowed into the QCM chamber at a flow rate of 0.05 mL / min until Af< 2 Hz in 10 min. Afterthe system reached equilibrium, the mixture of 100 pL freshly prepared GP or RPY coacervates (0.3 mM) and 900 pL of PBS flowed in at the flow rate of 0.05 mL / min for 15 min, followed by 20 min of rinse with PBS.

[0479] Cell internalization mechanism study

[0480] Based on the literature, (Mout, R et al., 2017; Lin, Q et al., 2014; Xu, C., et al., 2015) various inhibitors were used to study the pathway of the coacervates internalization. HeLa cells were treated with chlorpromazine (CPM, 30 pM), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA, 30 pM), methyl-p-cyclodextrin (MpCD, 2.5 mM) orwortmannin (Wort, 350 mM) separately for 1 hour. Then 100 pL of EGFP-loaded coacervates (0.3 mM HBpep-SP variants, 0.1 mg / mL EGFP) was added. After another 2 hours of incubation, the cells were washed twice with a pH 5.0 phosphate buffer followed by PBS twice. Then, the treated cells were dissociated by trypsin for FACS. For the 4°C treated group, the cells were pre-incubated for 1 hour and kept at low temperature during the 2 hours of uptake process. The mean fluorescence intensity of treated HeLa cells measured by FACS was normalized to the control group that was treated only with EGFP-loaded Coacervates in the absence of inhibitors.

[0481] Cargo recruitment

[0482] The recruitment of biomacromolecule cargos by peptide coacervates was performed simultaneously with inducing the LLPS. Cargos were first dissolved or diluted in the optimal buffers to achieve the required concentration. Then, the peptide stock solutions were mixed with the cargo-contained buffer with the volume ratio of 1 / 9 to induce coacervation and cargo recruitment.

[0483] Protein and Peptide Delivery

[0484] For protein and peptide delivery, 105HeLa cells were cultured in a 35 cm2culture dish with 1 mL of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum, 100 units / mL of penicillin and 100 pg / mL of streptomycin. After overnight incubation, the medium was replaced with 900 pL of fresh Opti-MEM. Then, 100 pL of freshly prepared protein- or peptide-loaded HBpep-SP coacervate suspensions (0.1 mg / mL of protein or 0.05 mg / mL of peptide, 1 mg / mL of HBpep-SP) were added into the Opti-MEM. After 4 hours of cellular uptakes, the medium was removed and cells were washed with PBS twice before adding 1 mL of the full medium (DMEM, 10% FBS, antibiotics). The cells were incubated for another 20 hours before being imaged under a fluorescence microscope and analysed by FACS (LSR Fortessa X20, BD Biosciences) to evaluate the 24 h release efficiencies of coacervate variants. The commercially available protein transfection reagent PULSin (Polyplus) was used as a comparison according to protocols from the manufacturers. The peptide delivery efficiencies of coacervate variants were compared with octa-arginine conjugated Smac (R8-Smac) at the same concentration of 50 pM. All fluorescence micrographs were imaged on live cells.

[0485] Plasmid DNA Transfection

[0486] Two reporter genes encoding enhanced green fluorescent protein (EGFP) and luciferase were used to evaluate the pDNA transfection efficiencies of HBpep-SP peptides. Before transfection, HeLa cells were cultured in 96-wells plate with a density of 104cells per well overnight. Then the medium was replaced with 90 pL of Opti-MEM, followed by adding 10 pL of freshly prepared pDNA-loaded coacervate suspensions (10 pg / mL of pDNA, 1 mg / mL of HBpep-SP). After 4 hours of uptake, the medium was removed and the cells were washed with PBS twice before adding 100 pL of the full medium. Then transfection was continued for another 20 hours before being imaged under a fluorescence microscope (for EGFP), or tested the luminescence by using Nano- Glo® Dual-Luciferase® kit and a microplate reader. Additionally, the transfection efficiency for EGFP was quantified by fluorescence-activated cell sorting (FACS).

[0487] Two siRNAs including FAM labeled anti-PCSK9 and unlabeled anti-EGFP siRNA were used to evaluate the delivery efficiency of HBpep-SP peptides. HeLa or HeLa-EGFP cells were cultured in 35 cm2dishes with full medium before the transfection. Then, the medium was replaced with 900 pL of Opti-MEM and 100 pL of freshly prepared siRNA-loaded coacervates (0.3 mM HBpep- SP variants, 200 nM siRNA). After 4 h of uptake, the cells treated with FAM-siRNA were imaged by fluorescence microscopy and the delivery efficiency was quantified by FACS. On the other hand, to measure the knock-down efficiency, after 4 h of uptake, the HBpep-SP peptides containing medium was removed. The cells were washed with PBS twice and then cultured in 1.5 mL of full medium for another 20 h. The PCSK9 mRNA knock-down efficiency was measured by reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR) and normalized by comparing it to glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA. Meantime, by delivering anti-EGFP siRNA into HeLa-EGFP cells, the protein level knock-down efficiency could be visualized by fluorescence microscopy and quantified by FACS. All fluorescence micrographs were imaged on live cells.

[0488] To deliver all three types of CRISPR / Cas9 genome editing modalities, HeLa cells were cultured in 35 cm2dishes until reaching 40% confluence. Then the medium was replaced with 900 pL of Opti-MEM and 100 pL of cargo-loaded HBpep-SP peptides. The final cargo concentration is 2 pg / mL for the all-in-one pDNA, 2 and 1 pg / mL forthe mRNA and sgRNA mixture, and 2 and 1 pg / mL for the Cas9 nuclease and sgRNA complex. After 4 h of uptake, the medium was discarded. The cells were washed with PBS twice and cultured in full media for another 44 h. The efficiency of 48 h of transfection can be evaluated by using the T7 Endonuclease 1 (T7EI) assay. First, the genomic DNA was extracted by using a DNeasy blood and tissue kit (QIAGEN). The target genomic locus was amplified by PCR using Q5 Hot Start high-fidelity 2X master mix (NEB) and primers listed in Figure 26 and purified by PureLink PCR purification kit (Thermo Fisher Scientific). Then, 200 ng of PCR products were digested by T7EI and analyzed by 2% agarose gels before imaging with the gel documentation system. The gray level of digested bands and undigested bands was measured by Imaged. The indel percentage could be calculated by the following formula (Guschin, D.Y. et al. in Engineered Zinc Finger Proteins: Methods and Protocols, (eds. J.P. Mackay & D.J. Segal) 247-256 (Humana Press, Totowa, NJ; 2010) and Wan, T., et al., Science Advances 7, eabe2888 (2021)):

[0489] [1 — (1 -fraction cleaved)1'2] x o where fraction cleaved = the sum of each digested band intensity / (the sum of each digested band intensity + undigested band intensity).

[0490] The knock-out efficiency was also quantified by delivering Cas9 mRNA and EGFP-targeting siRNA into HeLa-EGFP cells following prior protocols. After 48 h of transfection, treated HeLa- EGFP cells were imaged under a fluorescence microscope, and their EGFP intensity decrease was measured by FACS. All fluorescence micrographs were imaged on live cells.

[0491] Cytotoxicity study

[0492] The cytotoxicity of HBpep-SP peptides was determined by using the MTT assay [6], In detail, 104HeLa cells in 100 pL of the full medium were cultured in 96-wells plates and incubated overnight. Then the media were replaced with 90 pL of Opti-MEM and 10 pL of coacervate suspensions (1 mg / mL of HBpep-SP). After 4 hours of cellular uptake, the media were removed and the cells were washed with PBS twice before adding 100 pL of the full medium. The cells were incubated for another 20 hours before 10 pL of 5mg / mL MTT dissolved in PBS was added. The media were removed after 4 hours of incubation, and the cells were washed with PBS twice. After that, 100 L of DMSO per well was added for absorbance measurement at 570 nm by using a microplate reader. Additionally, the cytotoxicity of HBpep-SP variants and saporin-loaded HBpep-SP variants was evaluated using the Cell Counting Kit-8 (CCK-8). As described previously (Cai, L. et al. ACS Omega 4, 12036-12042 (2019)), cells were cultured in 96-well plates with 100 pL of full media and incubated for 24 h. The medium was then replaced with 100 pL of Opti-MEM containing saporin-loaded coacervates (various concentrations of saporin, 0.3 mM HBpep-SP variants) or various concentrations of HBpep-SP variants. After 4 h of uptake, the medium was removed, and the cells were washed with PBS twice and cultured in 100 pL of fresh full medium. The cells were incubated for another 20 h before changing the medium to the full medium containing 10% CCK- 8 solution. After 4 h of incubation, the cells were measured for absorbance at 460 nm using a microplate reader (Infinite M200 Pro, Tecan). The relative cell viability for both the MTT assay and the CCK-8 were calculated as below. At, Ab and Ac represent the absorbance of tested cells, no cells and control cells, respectively.

[0493] Cell cultures of “hard-to-transfect” cell lines.

[0494] Primary human foreskin fibroblast (HFF), and RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin under typical conditions (37 °C and 5% CO2). Jurkat cells were cultured in RPMI-1640 Medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin.

[0495] For HFF, the subculture started by detaching the cells with trypsin treatment, followed by centrifugation (1000 rpm, 5 min) to collect the cells. Then the pellets were resuspended with fresh media for subculture or experiments. The Jurkat cells are suspension cells, the subculture was conducted by dilution of cell culture in fresh media to achieve the required cell density. For RAW 264.7 cells, the cells were detached from the culture flask using a cell scraper (Corning) and collected by centrifugation (1000 rpm, 5 min). Then the pellets were resuspended with fresh media for subculture or experiments.

[0496] Data and Results

[0497] Coacervation Behavior of HBpep-SP variants

[0498] To decrease the overall hydrophobicity of the HBpep-SP, as shown in Table 2.1 , the original V and L residues in the X position (see Table 2.1 - X position is shown in bold) were replaced with less hydrophobic but non-polar residues, namely alanine (A) and glycine (G). As a result of decreased hydrophobic interactions, HBpep(AP)-SP and HBpep(GP)-SP (which are named after the residues in their X positions) could form coacervates over a broader pH range. Compared to the original HBpep(VPL)-SP, which only forms coacervates at pH 6.5 (Figure 1), the AP variant forms microdroplets in a pH range from 7.0 to 8.0 (Figure 2). Furthermore, changing V and L to G expands the coacervation pH to 9.0 without the formation of aggregates (Figure 2).

[0499] Table 2.1 Sequence of HBpep-SP variants with non-polar residues in the X position. The variable X positions are shown in bold.

[0500] Peptide Sequence

[0501] HBpep(VPL)-SP G H G VY-G HGVY-G HG PY- K(SP)-G HG PY-G HGLY- W (SEQ ID NO: 14)

[0502] HBpep(AP)-SP GHGAY-GHGAY-GHGPY-K(SP)-GHGPY-GHGAY-W (SEQ ID NO: 15)

[0503] HBpep(GP)-SP GHGGY-GHGGY-GHGPY-K(SP)-GHGPY-GHGGY-W (SEQ ID NO: 16)

[0504] Based on these promising results, we further explored other types of residues such as the positively charged histidine (His, H) and arginine (Arg, R), the negatively charged aspartic acid (Asp, D), the non-charged and polar asparagine (Asn, N), the aromatic tyrosine (Tyr, Y) residues, and their combinations in 3 different X positions such as RPY and DPY. The sequences are shown in Table 2.2. All HBpep-SP variants show coacervation over a range of pHs, with a general pattern that the more hydrophilic peptides can form microdroplets at the higher pH and in a broader pH range (Figure 3). Coacervate microdroplets formed by these variants showed similar size and surface charges as shown in Figure 4.

[0505] Table 2.2 Sequence of HBpep-SP variants with positively charged (H and R), negatively charged (D), non-charged and polar (N) and aromatic (Y) residues in the X position. The variable X positions are shown in bold.

[0506] Peptide Sequence

[0507] HBpep(HP)-SP GHGHY-GHGHY-GHGPY-K(SP)-GHGPY-GHGHY-W

[0508] (SEQ ID NO: 17)

[0509] HBpep(RP)-SP GHGRY-GHGRY-GHGPY-K(SP)-GHGPY-GHGRY-W

[0510] (SEQ ID NO: 18)

[0511] HBpep(DP)-SP GHGDY-GHGDY-GHGPY-K(SP)-GHGPY-GHGDY-W

[0512] (SEQ ID NO: 19)

[0513] HBpep(NP)-SP GHGNY-GHGNY-GHGPY-K(SP)-GHGPY-GHGNY-W

[0514] (SEQ ID NO: 20)

[0515] HBpep(YP)-SP GHGYY-GHGYY-GHGPY-K(SP)-GHGPY-GHGYY-W

[0516] (SEQ ID NO: 21)

[0517] HBpep(RPY)-SP GHGRY-GHGRY-GHGPY-K(SP)-GHGPY-GHGYY-W

[0518] (SEQ ID NO: 22)

[0519] HBpep(DPY)-SP GHGDY-GHGDY-GHGPY-K(SP)-GHGPY-GHGYY-W

[0520] (SEQ ID NO: 23)

[0521] Mechanical and biophysical properties of HBpep-SP coacervate variants

[0522] Having established the LLPS tunability of the HBpep-SP variants, we delved deeper into the mechanical and biophysical properties of coacervates. Utilizing the surface force apparatus (SFA) technique (Hwang, D.S. et al. Soft Matters, 3232-3236 (2010) and Israelachvili, J. et al. Reports on Progress in Physics 73, 036601 (2010)), we first assessed their viscoelastic characteristics. The SFA employs two mica surfaces affixed on perpendicularly oriented cylindrical discs, between which the samples are injected. The surfaces coated with the coacervates were approached with sub-nm distance resolution and then retracted, while the normal force between the cylinders was monitored with a few mN force sensitivity. During separation, coacervates formed by NP and GP (Figure 27) exhibited mechanical instability - indicated by a sudden “jump-out” on the normalized force-distance (F-D) curves- suggesting gellike properties (Wu, X., et al., Communications Chemistry 7, 5 (2024)). Conversely, VPL, HP, and RPY coacervates exhibited F-D curves with continuous retraction forces, indicative of viscous liquid bridges between the surfaces (Kim, S. et al.. Proceedings of the National Academy of Sciences 113, E847-E853 (2016)). Further evidence of the gel versus liquid behavior was evident from the hysteresis during approach / separation cycles, which were markedly present for HP and RPY coacervates but minimal for GP and NP. Fluorescence recovery after photobleaching (FRAP) was used to further explore the molecular mobility within single coacervates Goetz, S.K. et al., Dev. Cell 55, 97-107 (2020) and Zheng, J., et al., Aggregate 5, e449 (2024)) which was carried out by mixing fluorescently-labelled HBpep- SP variants with unlabeled peptides in a 0.5:99.5 ratio to minimize the influence of the fluorophore on the phase behavior. GP coacervates exhibited the slowest recovery rate among the three tested coacervates, indicating a reduced molecular mobility, which is consistent with SFA measurements that denoted gel-like properties. Conversely, RPY coacervates displayed higher fluidity with the fastest recovery rate.

[0523] To substantiate the differences in viscoelastic properties, we carried out AFM nanoindentation measurements of coacervates (Methods). For GP coacervates, on the approach cycle, the force increased in the contact region with a slope that was significantly shallower compared with the force curve on the solid substrate. Furthermore, the retraction cycle showed hysteresis due to energy dissipation processes inside the coacervates. Using the (Sneddon model, Sneddon, I.N. International Journal of Engineering Science 3, 47-57 (1965) and Gavara, N., et al., Nat. Nanotechnol. 7, 733-736 (2012)), the average elastic moduli of GP coacervates was 2.4 ± 1.4 MPa for droplets deep enough to alleviate substrate effects. On the contrary, for RPY coacervates the force traces were essentially identical on the droplets and the solid substrate, indicating that the AFM tip penetrated through RPY coacervates without resistance, until it reached the substrate whereupon a contact force was detected, an observation that corroborates the liquid-like nature of RPY coacervates.

[0524] To understand the differences in viscoelastic response at the molecular level, we conducted independent molecular dynamics (MD) simulations of 4, 10 and 30 chains of GP and RPY variants in aqueous conditions for 1000 ns (Methods). At equilibrium, both GP and RPY coacervates contained large amounts of water molecules in their clusters, however, a significantly higher number of peptide-water hydrogen bonds were seen in RPY, mainly arising from R4, R9, and Y25, compared to G4, G9, and G25 in GP clusters. Consequently, we attribute the liquid-like behavior of RPY coacervates compared to GP coacervates to their higher molecular hydration (see Figure 3).

[0525] Cellular uptake and release kinetics of HBpep-SP coacervate variants determined by their material properties

[0526] Since we could regulate the mechanical properties of the coacervates, we postulated that such variations could be exploited to control interactions with the membrane, and in turn cellular uptake efficacy. Giant unilamellar vesicles (GUVs) prepared from 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC) were utilized as a surrogate model bilayer to examine mechanical interactions between lipid bilayer and coacervates. Interactions between GUV and GP coacervates led to bending of the GUV’s lipid bilayer, likely induced by the higher rigidity of GP coacervates, which may lead to increased energy barrier during internalization and consequently decreased uptake rates (Wei, Q. et al. Adv. Mater. 30, 1707464 (2018)). Conversely, the more fluidic RPY coacervates were deformed upon interaction with GUVs and wetted the lipid bilayer. Although the mechanical properties of GUVs differ from those of real cells due to the absence of the cytoskeleton components, these results corroborate the differences in mechanical properties and wetting behaviors between GP and RPY coacervates, consistent with the findings from SFA, FRAP, and AFM measurements.

[0527] Next, we assessed adhesive interactions between coacervates and lipid bilayer membranes by carrying out quartz crystal microbalance (QCM) measurements of coacervates on supported lipid bilayers (SLBs). In the normal mode, wherein the SLB is situated at the bottom of the flow cell, both GP and RPY coacervates displayed high initial binding as evidenced by the decreased frequency (f), followed by rapid recovery upon rinsing. Compared to GP, RPY coacervates were more resistant to wash-off during the rinsing step, suggesting stronger membrane binding. In order to alleviate the effect of gravitational sedimentation from the dense coacervates, measurements were also carried out in the inverted mode (Gudlur, S. et al. Frontiers in Soft Matter 3 (2024)), whereby the flow cell was flipped upside down and SLBs oriented in the downward direction. RPY Coacervates showed significantly higher interaction with SLBs than GP Coacervates, persistently adhering to the bilayer even after 20 min of rinsing and causing a Afof - 21 Hz.

[0528] The inventors used various endocytic inhibitors and did not observe significant changes in the internalizations pathway as a result of residue mutations. The cell uptake of three tested coacervates, including GP, VPL, and RPY, was affected by macropinocytosis inhibitors such as 5-(N-Ethyl-N-isopropyl)amiloride (El PA) and Wortmannin (Wort), whereas the clathrin-mediated endocytosis inhibitor Chlorpromazine (CPZ) did not have any effect on cell uptake. The 4°C and methyl-p-cyclodextrin (MpCD) treatments also significantly reduced the internalization of all coacervates suggesting that coacervate uptake occurs by a macropinocytosis-like pathway involving cytoskeleton remodeling. Despite VPL coacervates displaying liquid-like properties, they exhibited lower uptake rates than GP coacervates, which we attribute to incomplete phase separation caused by their more hydrophobic nature. Indeed, fewer coacervates were formed at pH 6.5 as evidenced by half the count of coacervates for VPL compared to GP and RPY in FACS measurements.

[0529] Coacervate disassembly (and concomitant release of cargos) is activated by GSH-induced disulfide bond reduction, which leads to cleavage of the sidechain grafted to the Lys residue of HBpeps (Sun, Y. et al. ACS Nano 17, 16597-16606 (2023)). Thus, the disassembly kinetics can be monitored by measuring the concentration decay of non-reduced HBpep-SPs upon incubation with GSH by high-performance liquid chromatography (HPLC). This decay could be adequately fitted by a first-order kinetic model and occurred faster in RPY coacervates compared to GP coacervates, with a 1 8-fold higher rate constant. Since the grafted moiety is identical for both peptides, we attribute this disparity to the internal structure and mechanical properties of the coacervates. Owing to their higher molecular mobilities, we reason that the liquid-like RPY coacervates enabled faster diffusion of GSH, thus accelerating the chemical reduction and initiating faster coacervate disassembly. This was further substantiated by fluorescence microscopy and FACS measurements of HeLa cells treated with EGFP-loaded coacervates described below.

[0530] Protein delivery mediated by HBpep-SP variants

[0531] Following the characterization of the coacervation properties of the variants, their ability to intracellularly deliver proteins and peptides were evaluated using model proteins and peptides. Enhanced green fluorescent protein (EGFP) was first employed as cargo to study the cellular uptake and protein delivery efficiency of HBpep-SP variants. As shown in Figure 5, AP and GP variants showed a better cellular uptake after 10 minutes, as more EGFP-loaded coacervates were observed inside the cells. Also, with decreased hydrophobicity, HBpep-SP variants exhibit slower release rates. Compared to the original VPL peptide, the GP variant showed more fluorescence puncta (attributed to intact coacervates) compared to the well-distributed signal inside the cytoplasm. This pattern is more obvious when delivering larger proteins like R- phycoerythrin (R-PE) using coacervates formed by HBpep-SP variants. As shown in Figure 6, all three variants could deliver R-PE into HeLa cells, but GP showed much less cargo release compared to VPL.

[0532] Compared to those with non-polar and hydrophobic X residues, the variants with charged or polar sidechain behaved in a more complex fashion. The HP variant showed both enhanced cellular uptake and cargo release properties. Within only 10 minutes, the cells treated with EGFP-loaded HBpep(HP)-SP showed substantial uptake (Figure 7), which is significantly higher than the original VPL peptide (Figure 5). In addition, the HP coacervates released most of the EGFP cargo within 4 hours, also significantly faster than VPL. Compared to H, R has a higher pKa, resulting in HBpep(RP)-SP to be more charged and hydrophilic at neutral pH. Therefore, the HBpep(RP)-SP coacervates tend to pre-maturely release the cargo before cellular internalization. As shown in Figure 7, the HeLa cells treated with EGFP-loaded HBpep(RP)-SP coacervates showed homogenous but weak fluorescence signals.

[0533] The HBpep(DP)-SP, with negatively charged D in the X position, is the only variant that could phase separate at acidic pH. However, similar to RP, due to its charge and hydrophilicity, coacervates formed by this variant also pre-maturely dissembled and showed no EGFP delivery at all after 4 and 24 hours incubation times (Figure 8). On the other hand, cells treated with EGFP-loaded NP coacervates showed generally weak signals at 4 hours and 24 hours (Figure 9), suggesting the NP variant without charges is hydrophobic enough to prevent the pre-maturely disassemble but its cargo recruitment ability may decrease due to the increase of hydrophilicity. For the YP variant which has the aromatic residue tyrosine in its X position, it could deliver EGFP into the cells but cannot release it (Figure 10). An explanation is that YP has stronger hydrophobic interactions and TT-TT stacking compared to VPL, which is the main driven force for coacervation. Therefore, even after the reduction and removal of the self-immolative side chain, HBpep(YP)-K could still maintain phase separation, and the redox-triggered disassembly did not occur in this case. Hence the HBpep(YP)-SP does not have a cargo release mechanism.

[0534] These results show that the variants with highly charged residues, like R, can achieve fast release but may prematurely dissemble even before entering cells due to the small area of the two-phase region. Meanwhile, YP variants showed strong coacervation / aggregation ability but cannot release cargos. Therefore, a new variant was designed with two R and one Y in the X position to combine the advantages of RP and YP variants. This peptide, namely RPY, showed remarkable delivery and release abilities. The HBpep(RPY)-SP coacervates could enter the cell and release the EGFP cargo even faster than the HP variant (Figures 7, 11 , 28 and 29). This enhanced release ability of HBpep(RPY)-SP coacervates could be evidently observed in shorter times of incubations such as 2 and 4 hours. As shown in Figure 12, both fluorescence and FACS data show increased intensity of EGFP in cells treated with EGFP loaded RPY coacervates. While most cells internalized coacervates after 1 h regardless of the peptide variant, there was a clear correlation between peptide variant and release kinetics as assessed by the mean fluorescence intensities (MFI) that directly reflects EGFP release in the cytosol. In the more liquid-like coacervates, release kinetics increased in the order RPY > HP > VPL. The release of larger proteins such R-PE (a red fluorescence protein also known as R-phycoerythrin) and IgG was also enhanced using RPY coacervates (Figures 13, 14, and 30). After 24 hours of incubation, in addition to the homogeneous red fluorescence signal of released R-PE, there are obvious punta in cells treated with VPL and HP coacervates loaded with R-PE, suggesting the existence of cargo trapped inside coacervates. But in the case of R-PE delivery mediated by RPY coacervates, the signal of R-PE is more homogeneously dispersed inside cells with much less punta (Figure 13). Similarly, there are more IgG releases from RPY coacervates compared to VPL and HP. The enhanced release property of RPY could result in better therapeutic efficiency. Using saporin, a toxic protein, as cargo, RPY coacervates showed higher anticancer effects at the same cargo concentration compared to VPL or HP (Figure 15). In all cases, the transfection efficiency followed the same performance trend of RPY > HP > VPL > GP > NP.

[0535] This strategy can also be used to improve other variants with poor or no delivery ability such as the DP variant. To avoid the premature disassembly of the DY variant (Figure 8), a new variant with two D and one Y in its X positions was prepared, namely DPY The Y residue provided extra hydrophobicity to help stabilize the formed coacervates during dilution. As shown in Figure 16 and 31, DPY coacervates managed to deliver EGFP into HeLa cells; however its efficiency was inadequate compared to VPL. These results demonstrate that simple modifications of the residue in the X position can tune the resultant peptide coacervation properties as well as the release kinetics, from non-release to slow release to fast release. The cellular uptake can also be enhanced. In addition, based on the case study of the RPY and DPY variants, it has been demonstrated that there are abundant possibilities to design this delivery system by strategically adjusting the X position to a certain amino acid residue or a combination of multiple residues, making it possible to customize the coacervates to deliver specific cargos into specific cells or tissues.

[0536] The enhanced cellular uptake and delivery efficiencies of variant coacervates by mutations in X position have also been observed in “hard-to-transfect” cell lines such as primary cells, T cells and macrophage cells. As shown in Figures 17, 18,25, and 32, primary fibroblast, Jurkat and RAW264.7 cells treated with EGFP-loaded HP and RPY coacervates showed higher EGFP signals and less punta compared to those treated with EGFP-loaded VPL coacervates. This could be valuable for further applications including immunotherapy and tissue engineering.

[0537] Another drawback of HBpep(VPL)-SP coacervates is its sensitivity to serum during cellular uptake. In the presence of 10% FBS, VPL coacervates lost the great ability to deliver EGFP into the cell (Figure 19 and 31). However, variants with positively charged residues in their X- position, such as HP and RPY, showed improved tolerance to serum. This tackles one of the major issues that may obstruct the in vivo application of coacervates-based delivery system.

[0538] Peptide delivery mediated by HBpep-SP variants

[0539] Other than large proteins, these variants could also be used for the delivery of small peptides. The fluorescein isothiocyanate (FITC) labelled second mitochondria derived activator (Smac, AVPIAQK (SEQ ID NO: 24)) was employed as a model peptide to assess the delivery efficiency of HBpep-SP variants. As shown in Figure 20, both HP and RPY variants showed better peptide delivery efficiency compared to VPL and the cell penetrating peptide octaarginine conjugated Smac (R8Smac). These results indicate that the variants are capable of universally delivering different types of biomacromolecular therapeutics just like VPL, as demonstrated in our previous study [3].

[0540] Gene transfection and editing mediated by HBpep-SP variants

[0541] Nucleic acids are another type of important biomacromolecular therapeutics. However, their therapeutic efficiency heavily relies on the delivery system. To test the nucleic acid delivery efficiency of HBpep-SP variants, two plasmids encoding for EGFP and luciferase reporter genes were used. As shown in Figure 21 , all three variants including AP, GP, and HP showed better transfection efficiency for luciferase-encoded pDNAthan the original VPL peptide. In particular, HP and RPY variants demonstrated excellent efficiencies, transfecting 91 .3% and 94.3% of cells for pDNA, respectively. The good transfection results were also visualized by delivering EGFP- encoded pDNA (Figure 22). More interestingly, even though NP performed poorly in the delivery of EGFP, it showed good pDNA transfection efficiency. This may be due to the lower cargo concentration used for pDNA transfection (1 pg / mL) compared to EGFP delivery (10 pg / mL). In this case, the decreased cargo recruitment efficiency of NP would not have significant impacts on the delivery efficiency. The HBpep(NP)-SP could deliver sufficient amount of pDNA into the cell, triggered by the reducing environment of the cytosol to release pDNA just like other variants.

[0542] The HBpep-SP variant coacervates could also deliver other nucleic acid therapeutics like mRNA or siRNA. As shown in Figure 22, the GP, HP and RPY coacervates all have better mRNA transfection efficiency than VPL and commercially available reagent Lipofectamine 3000 and transfected 95% (with a 1 .4-fold higher MFI compared to VPL), 99.5%, and 98.7% of cells, respectively. The increased efficiency for GP coacervates compared to VPL in delivering nucleic acids can be attributed to the higher peptide concentration required to achieve full cargo recruitment for GP, which indicates weaker peptide-nucleic acid interactions, in turn leading to enhanced release. Furthermore, the high nucleic acid recruitment of GP resulted in a prolonged release kinetics profile. While RPY coacervates exhibited superior mRNA transfection efficiency at 4 and 24 hours, its efficiency decayed after a few more days, whereas GP coacervates maintained approximately 90% efficiency even 96 hours post-transfection. This result indicates that GP may offer distinct advantages as a delivery vehicle for applications requiring slow release, particularly for nucleic acid therapeutics, which function at lower concentrations than protein therapeutics (Gupta, A., et al., Adv. Drug Delivery Rev. 178, 113834 (2021)). Additionally, in Jurkat T-cells and RAW264.7 macrophage cell lines, HP and RPY coacervates showed much better EGFP positivity for mRNA than Lipo3000 and VPL coacervates.

[0543] For siRNA delivery, HeLa cells treated with FAM-siRNA loaded coacervates displayed increasing rates of positive cells and MFI in the order RPY > HP >VPL > GP >NP, where HP and RPY coacervates showed higher cellular uptake and fluorescence intensities of compared to those treated with FAM-siRNA loaded VPL coacervates and VPL, HP, and RPY all outperforming the highly specialized lipofectamine RNAiMAX (LipoRMAX) (Figure 22). Using quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify mRNA degradation induced by anti-PCSK9 siRNA, the siRNA-loaded HP and RPY coacervates achieved mRNA knockdown rates of 74.2% and 86.5%, surpassing 50.3% mediated by LipoRMAX. Furthermore, delivering anti-EGFP siRNA into HeLa cells expressing EGFP provided insights into the knockdown efficiency at the protein level. siRNA transfection mediated by HP and RPY coacervates decreased the EGFP signal significantly more than LipoRMAX-mediated transfection.

[0544] In addition, the coacervates could deliver all three types of CRISPR / Cas9 genome editing modalities including all-in-one pDNA encoding both Cas9 nuclease and sgRNA sequences, the complex of sgRNA and mRNA encoding Cas9 nuclease, and the Cas9 ribonucleoprotein (RNP) complex (Figure 23). Variants like GP, HP and RPY showed better delivery efficiency and caused higher insertion-deletion (indel) frequencies compared to three commercially available reagents specialized for different modalities in delivering the all-in-one pDNA and mRNA / sgRNA gene editing machineries (Figure 23). The delivery system of all three variants showed excellent knockout efficacy where they all reduced the EGFP signal more efficiently than Lipo3000 (Figure 23b). These results again confirm that the HBpep-SP variants have great potential in delivering different types of therapeutic and could be customized for specific cargos.

[0545] Cytotoxicity study

[0546] To verify that these variants are not cytotoxic as previously demonstrated for HBpep(VPL)-SP [3], the MTT assay and CCK-8 assay were used to test the viability of cells treated with coacervates formed by HBpep-SP variants. As shown in Figure 24, all tested HBpep-SP variants showed near 100% viability with the MTT assay and with the CCK-8 assay the variants showed negligible cytotoxicity in all four cell lines including HeLa, HFF, Jurkat and RAW 264.7, proving these peptide coacervates are safe delivery systems for functional biomacromolecules.

[0547] Conclusion

[0548] In summary, the X position in the GHGXY (SEQ ID NO: 1) repeats of HBpep-SP backbone could be adjusted to all types of amino acid residues and their combinations (Table 3.1). Tuning the properties of the X residues not only expand the pH window for the peptide to phase separate, but also improved its performance as a delivery platform, including improved cellular uptake, cargo release and gene transfection efficiencies (Table 3.2). All variants showed improvements in at least one of these listed properties except YP. But the Y residue could be helpful when combined with other hydrophilic residues such as R and D to create phase separating peptides with even better performances, such as RPY shown above. Therefore, current data suggest multiple possibilities to customize the coacervates for any specific cargos with controllable release profiles by simply changing a few residues in the primary sequence of the phase separating peptides.

[0549] Table 3.1 Summary of HBpep-SP variants with different types of mutation in the X position in the GHGXY(SEQ ID NO: 1).

[0550] Name Sequence Description

[0551] HBpep(VPL)-SP GHGVY-GHGVY-GHGPY-K(SP)-GHGPY-GHGLY-W (SEQ ID NO: 14). HBpep(AP)-SP GHGAY-GHGAY-GHGPY-K(SP)-GHGPY-GHGAY- Hydrophobic W (SEQ ID NO: 15). HBpep(GP)-SP GHGGY-GHGGY-GHGPY-K(SP)-GHGPY-GHGGY- W Special

[0552] (SEQ ID NO: 16). HBpep(SP)-SP GHGSY-GHGSY-GHGPY-K(SP)-GHGPY-GHGSY-W (SEQ ID NO: 26). HBpep(NP)-SP GHGNY-GHGNY-GHGPY-K(SP)-GHGPY-GHGNY- Hydrophilic W (SEQ ID NO: 20). HBpep(HP)-SP GHGHY-GHGHY-GHGPY-K(SP)-GHGPY-GHGHY- W (SEQ ID NO: 17). Positively-charged HBpep(RP)-SP GHGRY-GHGRY-GHGPY-K(SP)-GHGPY-GHGRY- W (SEQ ID NO: 18). HBpep(DP)-SP GHGDY-GHGDY-GHGPY-K(SP)-GHGPY-GHGDY-

[0553] W Negatively-charged

[0554] (SEQ ID NO: 19). HBpep(YP)-SP GHGYY-GHGYY-GHGPY-K(SP)-GHGPY-GHGYY-W Aromatic (SEQ ID NO: 21). HBpep(RPY)-SP GHGRY-GHGRY-GHGPY-K(SP)-GHGPY-GHGYY-

[0555] W

[0556] (SEQ ID NO: 22).

[0557] Hybrid HBpep(DPY)-SP GHGDY-GHGDY-GHGPY-K(SP)-GHGPY-GHGYY-

[0558] W (SEQ ID NO: 23). Table 3.2 Summary of properties changes of HBpep-SP variants compared to HBpep(VPL)- SP induced by X position mutations.

[0559] Name LLPS pH Cellular uptake Cargo release Gene transfection

[0560] Example 2 - Complex coacervate combinations

[0561] Preparation and Coacervation Study of Peptide Variants Derived from HBpep and HBpep- SP

[0562] To investigate the effect of residue substitutions on the coacervation of phase-separating peptides, four variants were synthesized by mutating the 4th, 9th, and 24th positions of the HBpep sequence (Figure 33a). Three variants containing charged residues, namely RP, KP, and EP, did not undergo phase separation at concentrations up to 100 pM across a broad pH range of 5 to 9 (Figures 33b-c). Conversely, the Y-containing variant YP, tended to form aggregates due to strong hydrophobic interactions and TT-TT stacking (Figures 33b-c).

[0563] The complex coacervation behavior of peptide variants was then assessed. Unlike the favorable electrostatic interactions typically seen in classic poly-lysine and poly-glutamic acid systems,35'37neither RP nor KP could engage in strong interactions with EP to drive the formation of complex coacervates (Figure 34a). A likely explanation is that the presence of only three charged residues per peptide is insufficient to establish stable inter-peptide interactions, while simultaneously weakening hydrophobic interactions. As a result, these variants not only failed to undergo selfcoacervation but are also unable to form complex coacervates driven by electrostatic interactions.

[0564] In contrast, RP and YP readily formed complex coacervates at a 1 :1 molar ratio across a broad range of concentrations and pH values, as shown in Figure 34a-b. However, KP failed to prevent the aggregation of YP (Figure 34a). Further investigations across a wide range of KP concentrations showed no impact on the coacervation of RP-KSPor the aggregation of YP-Ksp, highlighting that the Lys-Tyr cation-rr pair is insufficient to modulate phase behaviors of peptide variants. In contrast, the cation-ir interactions emerging from Arg-Tyr pairs is much stronger and can compete with TT-TT stacking between Tyr-Tyr pairs, resulting in intermolecular force balance that enables the peptides to form stable complex coacervates.38-40Similar to their parent peptides, the RP-Kspand YP-Kspmixtures also formed coacervates under these conditions, but began to aggregate at pH 8 due to the hydrophobic nature of the Kspmoiety (Figure 34c). However, this aggregation could be further mitigated by increasing the proportion of RP-KSPin the mixture (Figure 34d). Moreover, RP-KSPand YP-KSPcan also interact with their less hydrophobic parent peptides, RP and YP, and influence the threshold concentration required to induce complex coacervation (Figure 34e). Based on this design, the phase behavior of the complexes could be fine-tuned by adjusting the cationic-to-aromatic peptide ratio, resulting in various phase behaviors ranging from no phase separation to complex coacervation and, ultimately, aggregation (Figure 34f). This ability to modulate the phase behavior highlights the versatility of this peptide family to form complex coacervates under varying conditions.

[0565] To further validate the formation of complex coacervates, we evaluated the viscoelastic characteristics of RP-KSF7YP-KSPcomplex coacervates using the surface force apparatus (SFA). The SFA employs two mica surfaces affixed on perpendicularly oriented cylindrical discs of radii R, where the coacervates form a liquid bridge in between. The cross-cylinders are approached with sub-nm distance resolution and then retracted, while the normal force between the cylinders is monitored with N force sensitivity.41-42Upon retraction, negative normalized forces (F / R) correspond to attractive interactions between the cross-cylinders. As shown in Figure 34g, RP- Kspby itself did not exhibit adhesive force, indicating the absence of coacervate formation, which is consistent with its weak self-coacervation ability. However, the RP-Ksp / YP-Kspcomplex demonstrated a strong adhesive force of - 8.78 mN / m, as well as clear hysteresis between approach and separation, both hallmark signatures of coacervates.43-44

[0566] Intrigu ingly, the cationic RP-KSPnot only prevented the aggregation of aromatic YP-KSP, but could also dissolve the pre-formed peptide aggregates. Figure 34h shows time-lapse microscopy images of YP-Kspaggregates changing in size and texture, which evolved over time into coacervate microdroplets, whereas the aggregates remained intact in the absence of RP-Ksp. Structural analyses using attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR) revealed that YP-KSPformed p-turn structures, characterized by the amide I peak centered at 1676 coacervate-1.45In contrast, the amide I peak of the RP-KSP / YP-KSPmixture at the molar ratio of 1 :1 shifted to 1646 coacervate-1, indicative of disordered structures similar to homotypic RP-KSP.46These findings suggest strong interactions between the cationic and aromatic peptides, which facilitate complex coacervation and inhibit or even reverse aggregation. This is particularly stimulating, as misfolding of proteins into amyloid aggregates are central to many protein-related diseases.47-48These results suggest a potential strategy to mitigate or reverse aggregation by harnessing cation-ir interactions in aggregation-prone peptides.

[0567] Cation-TT Interactions Modulate Complex Coacervates Formation

[0568] To elucidate the role of weak non-bonded inter-molecular interactions between cationic and aromatic variants and derivatives in complex coacervation, we employed SFA measurements and molecular dynamics (MD) simulations. Symmetric SFA measurements -whereby the same peptide layer is coated on both cylinders- were conducted for RP-KSPand YP-KSPto quantitatively evaluate interactions between the same peptides. Notably, the adhesion force between YP-KSPlayers was approximately 1 4-fold higher compared to that between RP-KSPlayers (Figure 35a). Moreover, the force-distance curves of YP-Ksplayers were not affected by the addition of tetramethylammonium hydroxide (TMA), an inhibitor of cation-n interactions, likely due to the absence of positively charged residues in its sequence. In contrast, the average adhesion force between RP-KSPlayers slightly decreased from 7.34 ± 0.10 mN / m to 6.67 ± 0.93 mN / m upon TMA addition, suggesting weak perturbation of cation-ir interactions likely arising between Arg and Tyr of the GHGRY repeats as well as Arg and the C-terminal Trp (Figure 35a). Asymmetric SFA measurements between RP-KSPand YP-KSPlayers exhibited the strongest adhesion force of 11 .69 ± 0.34 mN / m, which significantly decreased by 55% with the addition of 100 mM TMA (Figure 35a). This result suggests that strong cation-n interactions occur between these peptide variants, which may prevent YP-KSPfrom aggregating and instead favor the formation ofYP-Ksp / RP-Kspcomplex coacervates.

[0569] MD simulations provided further insights into peptide assembly and peptide / peptide interactions at the molecular level. Simulations using both slab and cubic boxes indicated low propensity to form a compact cluster for RP-KSPpeptides, whereas YP-KSPpeptides and the 1 :1 mixture of RP-KSF7YP-KSPpeptides rapidly collapsed into stable clusters (Figure 35b). The residue contact map of the RP-KSF7YP-KSPmixture and the total number of cation-iT interactions confirmed strong cation-iT interactions for the pairs Arg-Tyr and Arg-Trp (Figure 35c), consistent with the SFA measurements (Figure 35a). Additionally, interactions between pairs such as His / Tyr, His / Trp, Tyr / Tyr, Tyr / Trp, and Trp / Trp were also observed, consistent with our previous study,23which established hydrogen bonding and TT-TT stacking as the primary driving forces for the coacervation of HBpeps. In this process, increasing the pH above the pKa of His induces deprotonation of His residues, triggering coacervation through H-bonding between the deprotonated imidazole side chain and the hydroxyl group of Tyr. This pH responsiveness is maintained in the HBpep variants and derivatives examined in this study, as most peptides and their mixtures form coacervates or aggregates at pH values above 6, where deprotonation of His residues begin to occur.

[0570] From simulations using a slab box, the peptide density profile showed a single peak for both YP- Kspand RP-Ksp / YP-Kspmixture, while multiple small peaks appeared for RP-Ksp, corresponding to several small clusters (Figures 35d-e). Within the peptide coacervates, water density decreased but did not drop to zero, indicating the presence of significant numbers of "internal" water molecules. Further analysis of the RP-Ksp / YP-Kspmixture revealed distinct spatial distributions, with YP-KSPpreferentially located in the interior of the coacervates and RP-KSPpositioned at the coacervates-water interface (dashed line in Figure 35d). This distribution arises from differences in hydrophobicity since RP-Kspis more hydrophilic than YP-Kspand thus prefers to be exposed to the coacervates-water interface. The magnitude of the peaks in the proximal radial distribution function (pRDF) profiles for water molecules relative to the peptide surface supports these findings. Consequently, RP-Ksp / YP-Kspcoacervates occupy a slightly larger volume and possess a greater surface area compared to YP-KSPcoacervates, suggesting enhanced hydration.

[0571] The different microenvironments in the coacervates of RP-Kspand YP-Kspcould influence the chemical cleavage of Kspmodifications, which is triggered by the reduction of its disulfide bond by GSH. As shown in Figures 35f-g, both RP-Kspand YP-Ksppeptides in the complex coacervates (formed at 1 :1 ratio) exhibited a concentration decay in the presence of GSH (as measured by HPLC), which was well-fitted by first-order reaction kinetics. However, RP-KSPhad a 3.2-fold higher reaction rate coefficient compared to YP-KSP. This indicates that the reduction rate of the disulfide bond in the Kspside chain, and consequently the disassembly of the complex coacervates, can be tuned by adjusting the RP-Kspto YP-Kspratio, thereby enabling controlled kinetics of cargo release concomitant with the disassembly of coacervates.

[0572] Intracellular Delivery and Release of Macromolecules Mediated by Complex Coacervates

[0573] Our previous studies have demonstrated that homotypic coacervates hold significant potential for the intracellular delivery of macromolecular therapeutics.30'31 49HBpep variants and their derivatives applicable as intracellular delivery vehicles for functional macromolecules that can form complex coacervates were further explored. First, the delivery of EGFP proteins into HeLa cells was tested. Unlike typical complex coacervates driven by electrostatic interactions, which often have limited protein release capabilities,50'51the complex coacervates formed by RP and YP at the ratio of 1 :1 and 2:1 , without the self-immolative Kspmodification, successfully delivered and released EGFP within 4 hours, as evidenced by well-distributed fluorescence signals throughout the cells (Figure 36a). However, decreasing the relative YP content resulted in low cell uptake and decreased mean fluorescence intensity (MFI) (Figure 36b), suggesting that the complex coacervates became unstable and prematurely disassembled before entering the cells. Coacervates formed by the mixture of RP and YP-KSPexhibited high uptake rate but low MFI (Figure 36b). This result may be due to the increased hydrophobicity of YP-KSPcompared to YP, which stabilizes the coacervates but also hinders efficient disassembly and cargo release.

[0574] The release of cargo proteins from RP / YP complex coacervates is likely due to the disruption of cation-TT interactions by the high concentration of proteins in the crowded cellular environment, which competitively interact with RP and YP (Figure 37a). To test this hypothesis, turbidity measurements were employed to evaluate coacervates formation in the presence of bovine serum albumin (BSA) to mimic the cytosolic environment. As shown in Figures 37b-c, at a low BSA concentration (0.01%), the change in turbidity with peptide concentration was similar to that of the control group without BSA, suggesting that low concentrations of protein cargos do not significantly affect complex coacervation or cargo recruitment. However, when the BSA concentration reached 10%, similar to the total protein concentration inside cells,52'53the turbidity of both RP / YP and RP-KSF7YP-KSPcoacervates significantly decreased, reaching nearly 0% at the peptide concentration of 200 pM. This suggests that the cationic and aromatic peptide variants cannot form coacervates effectively under protein-rich conditions such as the cytosol.54'55This disruption of the cation-p interactions can thus trigger disassembly of cationic / aromatic peptide coacervates and subsequent cargo release.

[0575] Although the RP / YP system demonstrated potential for protein delivery and release, its efficiency was not satisfactory and was limited to a narrow RP / YP ratio range. To address this, the mixtures of RP-KSPwith either YP or YP-KSPwere tested, as the Kspmodification introduces additional hydrophobicity to the cationic peptide, potentially enhancing the stability of the complex coacervates without compromising cargo release. As shown in Figures 36a and c, using RP-KSPas the cationic peptide significantly improved both uptake efficiency and cargo release. Within just 4 hours, HeLa cells treated with EGFP-loaded RP-Ksp / YP or RP-Ksp / YP-Kspcoacervates exhibited over 90% uptake, along with higher MFI compared to the best-performing RP / YP coacervates. This excellent improvement surpassed our previous efforts using self-coacervating peptides with the same self-immolative modification, which only achieved substantial cargo release after 24 hours.31The accelerated release observed here may be attributed to both the chemical cleavage of the self-immolative modification and the responsiveness of cation-n interactions in the crowded cellular environment. Both RP-KSF7YP and RP-KSP / YP-KSPcoacervates showed increased MFI with a rising RP-KSPratio, peaking at an optimal 4:1 ratio before slightly decreasing due to coacervate instability caused by excessive cationic peptides (Figure 36c). The improvement in delivery efficiency is likely driven by the change of the hydrophobic core formed by aromatic peptides. Similar results were observed when comparing RP-Ksp / YP and RP-Ksp / YP-Kspat the same cationic / aromatic peptide ratio, where RP-Ksp / YP system performed better. Due to its weaker hydrophobicity, YP allows the complex coacervates to have improved access to GSH and proteins in the crowded cellular environment, which serve as triggers for cargo release.

[0576] The unique properties of this complex coacervate system provide a potential strategy to control cargo release kinetics simply by adjusting the mixing ratio of the two peptides. To further explore this idea, we conducted a detailed evaluation of the release profiles of complex coacervates formed with various cationic / aromatic peptide ratios, focusing on the best-performing RP-Ksp / YP system. As shown in Figures 36d-e, cells treated with coacervates at lower RP-Ksp / YP ratios, such as 1 :1 and 2:1 , exhibited relatively slower release rates, with EGFP continuously released over the 4-hour measurement period. In contrast, coacervates with higher RP-Kspcontent reached maximum release within 2 hours, indicating full release of EGFP, which is also evidenced by the disappearance of fluorescence puncta inside the cytoplasm (Figure 36d).

[0577] In addition to proteins, the complex coacervate system can also deliver mRNA, another macromolecular therapeutic that has gained significant attention, particularly for its role in the development of COVID-19 vaccines.56'57As shown in Figures 36f-g, the RP-Ksp / YP coacervates with varying mix ratios effectively transfected HeLa cells with a reporter mRNA encoding EGFP, demonstrating a similar pattern to protein delivery: transfection efficiency increased as the YP content decreased. The coacervates achieved the highest transfection efficiency at an 8:1 ratio, successfully transfecting 95% of treated HeLa cells. Combined with their lower cytotoxicity compared to commercially available reagents including PULSin and Lipo MMax, this robust and versatile delivery capability of the complex coacervates offers significant potential for various applications, including cell therapy, vaccine development, and drug screening. Although we recently demonstrated that similar transfection results can be achieved using self-coacervates formed by homotypic peptides with minimal cytotoxicity,58this approach has certain limitations. First, the peptide backbone must be carefully designed and validated for different cargos and cell lines, which is both time- and labour-intensive. Additionally, the release profiles of different peptide coacervates are fixed for a given peptide, unlike the complex coacervates in this study which allow for facile adjustment of cargo release kinetics simply by altering the cationic / aromatic peptide ratios. Most importantly, this study introduces a distinct approach using complex coacervates made from cationic and aromatic variants derived from well-established selfcoacervating peptide sequences, offering new molecular design and applications of peptide- based coacervates.

[0578] Macrophage Engineering Mediated by Complex Coacervates

[0579] Macrophage cells are increasingly studied for their pivotal roles in disease-related bioactivities, such as immune response, cancer development, and inflammation.59'61However, their intrinsic resistance to foreign material transfections poses a significant challenge in developing targeted therapeutics.62To overcome this drawback, we explored the potential of RP-Ksp / YP coacervates for delivering various functional macromolecules into macrophages. Using EGFP as a model protein, all coacervates with RP-KSF7YP ratios ranging from 1 :1 to 8:1 successfully transfected over 90% of RAW264.7 macrophages, significantly outperforming the 23.3% transfection efficiency achieved using PULSin, a commercially available reagent commonly used for protein delivery (Figure 38a). Additionally, RP-Ksp / YP (4:1) coacervates effectively delivered high molecular weight proteins including Alexa Fluor 488-labeled Immunoglobulin G (150 kDa) and R- phycoerythrin (250 kDa), achieving over 99% delivery efficiency (Figures 38b-c). Moreover, the RP-KSF7YP coacervates demonstrated excellent mRNA transfection capabilities in RAW264.7 cells, particularly at an 8:1 mix ratio, achieving 83.4% efficiency, significantly higher than the 22.8% efficiency obtained using the highly optimized Lipofectamine MessengerMax reagent (Figure 38d).

[0580] The delivery of more complex therapeutics, namely the clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR associated protein 9 (Cas9) genome editing tools, were explored. The delivery of CRISPR / Cas9 tools is considered challenging as it involves delivering two components: the Cas9 nuclease and single guided RNA (sgRNA).64The most common way to tackle this is to deliver an all-in-one plasmid that encodes both the sequence of Cas9 and sgRNA.65However, plasmid based editing tools are associated with risks of genome integration and off-target effects.66To mitigate these risks, we employed RP-KSF7YP coacervates for the delivery of two plasmid-free CRISPR / Cas9 tools: a Cas9 mRNA / sgRN A mixture and Cas9 / sgRNA ribonucleoprotein (RNP), both targeting the signal-regulatory protein alpha (SIRPa) gene. SIRPa is a cell surface receptor primarily expressed on macrophages that interacts with CD47 to deliver a "don't eat me" signal, preventing macrophages from engulfing healthy cells. However, many tumor cells overexpress CD47 to evade immune detection, making the SIRPa- CD47 axis a prime target for cancer immunotherapies. Disrupting this interaction can enhance macrophage-mediated elimination of cancer cells.33'34The T7 Endonuclease I (T7EI) assay confirmed successful editing at the SIRPa locus, with insertion-deletion (indel) frequencies exceeding 40% at optimal cargo concentrations (Figures 38e-f). Additionally, flow cytometry analysis demonstrated a significant decrease in SIRPa expression in the edited cells (Figure 38g), highlighting the potential of coacervate-mediated delivery systems for macrophage cell therapies.

[0581] Conclusion

[0582] The inventors developed a novel strategy for designing a complex coacervate-based intracellular delivery system using the HBpep family of peptides. By incorporating cationic, anionic, and aromatic residues into the HBpep sequence, the inventors systematically investigated how these substitutions affect both self- and complex coacervation behaviors. The findings indicate that a low density of oppositely charged residues is unable to establish stable electrostatic interactions, resulting in homogeneous solutions of KP and EP and failure to form complex coacervates. Conversely, the strong cation-rr interactions between RP and YP prevent RP dissolution and YP aggregation, instead promoting and stabilizing the formation of complex coacervates. Notably, the cation-TT interactions are disrupted by the protein-rich cellular environment, serving as a trigger for coacervate disassembly and cargo release in cells.

[0583] By introducing Lys and a self-immolative disulfide moiety into the peptide sequences, the inventors engineered complex coacervates capable of responding to intracellular redox conditions, thereby enhancing the controlled release of cargos. Adjusting the ratio of cationic to aromatic peptides allowed precise control over the phase behavior and release kinetics of cargos from the coacervates, facilitating the efficient delivery of a diverse range of macromolecular therapeutics, including proteins, mRNA, and CRISPR / Cas9 gene editing tools. Notably, this approach proved effective at genetically editing macrophages, a cell type notoriously resistant to transfection, highlighting the versatility and adaptability of our peptide-based complex coacervate system for immune cell therapies.

[0584] These findings emphasize the importance of cation-TT interactions in designing complex coacervates with tunable stability and cargo release profiles, offering a new avenue for the development of advanced intracellular drug delivery vehicles. This strategy not only enhances the performance of coacervates as delivery vehicles but also offers potential solutions to broader biomedical challenges, such as those related to protein aggregation.

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Claims

CLAIMS1. An isolated peptide comprising the amino acid sequence of Formula (I):(O)n-K-(O)m-Z(I) wherein K is lysine optionally modified with a self-immolative moietyZ is tryptophan (W) or is absentO is GHGX1YG is glycine (G), H is histidine (H), Y is tyrosine (Y) each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P) n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X1residues are different to each other; optionally wherein not all of the X1residues are proline (P).

2. The isolated peptide of claim 1 , wherein the lysine residue is located at the 1st, 6th, 11th, 16th, 21st, or 26thposition from the N-terminus.

3. The isolated peptide of claim 1 or 2, wherein n is 3 and m is 2.

4. The isolated peptide of claim 1 , wherein the peptide of Formula (I) is a peptide ofFormula (II):[O]a-[B]b-K-[B]c-[O]d-[O]e-Z(II) whereinO is GHGX1Y (SEQ ID NO: 27)B is GHGPY (SEQ ID NO: 2)G is glycine (G), H is histidine (H), Y is tyrosine (Y) and P is proline (P);each X1is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);K is lysine (K) optionally modified with a self-immolative moiety; andZ -is tryptophan (W) or is absent, а, b, c, d, and e are each independently 0, 1 or 2 and a+b+c+d+e is 3-8, optionally wherein a+b+c+d+e is 5.

5. The isolated peptide of any one of the preceding claims, wherein the isolated peptide is 16 - 45 amino acids long. б. The isolated peptide of any one of the preceding claims, wherein the peptide is 26 or 27 amino acids long7. The isolated peptide of any one of the preceding claims, wherein the amino acid residue at X1is the same at each X1position.

8. The isolated peptide of any one of claims 1 to 6, wherein the two N-terminal X1amino acid residues are the same as each other and the C terminal X1amino acid residue is different to the two N-terminal X1amino acids.

9. The isolated peptide of any one of claims 1 to 6, wherein the two N-terminal X1amino acid residues and the C-terminal X1amino acid residue are the same as each other.

10. The isolated peptide of any one of the preceding claims, wherein at least one of the residues at an X1position is selected from: A, G, H and R.11 . The isolated peptide of claim 7, wherein all the residues at the X1positions are the same and are selected from: A, G, and H.

12. The isolated peptide of any one of the preceding claims, wherein at least one of the residues at an X1position is selected from: H and R.

13. The isolated peptide of claim 7, wherein all the residues at the X1positions are the same and are selected from: H and R.

14. The isolated peptide of any one of the preceding claims, wherein the peptide comprises the Z tryptophan15. The isolated peptide of any one of claims 1 to 13, wherein the peptide lacks the Z tryptophan. in16. The isolated peptide of any one of claims 1 to 14, wherein the isolated peptide comprises or consists of an amino acid sequence selected from the group consisting of:(i) GHGAY GHGAY GHGPY K GHGPY GHGAY W (SEQ ID NO: 3)(ii) GHGGY GHGGY GHGPY K GHGPY GHGGY W (SEQ ID NO: 4)(iii) GHGSY GHGSY GHGPY K GHGPY GHGSY W (SEQ ID NO: 5)(iv) GHGNY GHGNY GHGPY K GHGPY GHGNY W (SEQ ID NO: 6)(v) GHGHY GHGHY GHGPY K GHGPY GHGHY W (SEQ ID NO: 7)(vi) GHGRY GHGRY GHGPY K GHGPY GHGRY W (SEQ ID NO: 8)(vii) GHGDY GHGDY GHGPY K GHGPY GHGDY W (SEQ ID NO: 9)(viii) GHGYY GHGYY GHGPY K GHGPY GHGYY W (SEQ ID NO: 10)(lx) GHGRY GHGRY GHGPY K GHGPY GHGYY W (SEQ ID NO: 11)(x) GHGDY GHGDY GHGPY K GHGPY GHGYY W (SEQ ID NO: 12);Optionally, wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self- immolative moiety.

17. The isolated peptide of any one of claims 1 to 16, which is able to form coacervates at a pH between about 6.5 and 9.0, such as between about 7.0 and 9.0 or 7.0 and 8.0.

18. The isolated peptide of any one of claims 1 to 17, which is able to form coacervates that can enter the cell more efficiently than coacervates formed from HBpep(VPL)-SP.

19. The isolated peptide of any one of claims 1 to 18, which is able to form coacervates that are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.

20. The isolated peptide of any one of claims 1 to 19, wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety.21 . The isolated peptide of claim 20, wherein the self-immolative moiety comprises a disulfide (- S-S-) moiety.

22. The isolated peptide of claim 20 or 21 , wherein the self-immolative moiety has the formula -C(=O)-O-(CH2)n-S-S-R, wherein n is 1 , 2, 3, 4, or 5, and wherein R is selected from: substituted or unsubstituted alkyl, alkenyl, cycloalk(en)yl, and aryl.

23. The isolated peptide of claim 22, wherein R is -(CH2)n-O-C(=O)-R' wherein n is 1 , 2, 3, 4, or 5, and wherein R’ is selected from: C alkyl, Ce-aryl, preferably phenyl, optionally substituted with halogen.

24. A composition for delivery of an active agent, the composition comprising a peptide coacervate, wherein the peptide coacervate comprises:(i) one or more isolated peptides of any one of claims 1 to 23; and(ii) an active agent recruited in the peptide coacervate.

25. The composition of claim 24, wherein the peptide comprises a self-immolative moiety which autocatalytically cleaves itself upon exposure to specific conditions selected from the group consisting of: pH changes, redox changes, exposure to release agents, and combinations thereof.

26. The composition of claim 24 or 25, wherein the peptide coacervate is formed of a single type of isolated peptide.

27. The composition of claim 24 or 25, wherein the peptide coacervate is formed of two or more types of isolated peptide.

28. The composition of claim 27, wherein the peptide coacervate is formed of two types of isolated peptide that posses opposite net charges.

29. The composition of claim 27, wherein the peptide coacervate is formed of two types of isolated peptide wherein one peptide wherein one of the types of coacervate peptide possesses an aromatic amino acid (e.g., Tyr) at the X1position and other type of coacervate peptide possesses a cationic amino acid (e g., Lys or Arg) at the X1position.

30. The composition of claim 29, wherein the peptide with an aromatic amino acid at the X1position and the peptide with an cationic amino acid at the X1position are mixed in a ratio of between 1 :20 and 20:1.31 . The composition of any one of claims 24 to 30, wherein the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.

32. The composition of any one of claims 24 to 31 , wherein the active agent is a pharmaceutical or diagnostic agent.

33. The composition of claim 31 or 32, wherein the agent is a protein or (poly)peptide.

34. The composition of claim 33, wherein the protein or (poly)peptide is an antibody, antibody variant, antibody fragment or peptide.

35. The composition of any one of claims 31 to 34, wherein the composition is a pharmaceutical or diagnostic formulation for administration to a subject.

36. The composition of any one of claims 31 to 35, wherein the pH of the composition is > 5.0 and < 9.5, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

37. The composition of any one of claims 31 to 36, wherein the coacervates formed are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.

38. The composition of any one of claims 31 to 37, wherein the coacervates formed are capable of enhanced cellular uptake compared to coacervates formed from HBpep(VPL)-SP.

39. The composition of claim any one of claims 31 to 38, wherein the coacervates formed are capable of enhanced transfection efficiency compared to coacervates formed from HBpep(VPL)- SP.

40. A method for the recruitment of an active agent in a peptide coacervate, the method comprising:(i) providing an aqueous solution of coacervate-forming peptides, wherein the coacervateforming peptides are selected from the isolated peptides of any one of claims 1 to 23;(ii) combining the aqueous solution of the coacervate-forming peptides with an aqueous solution of an active agent; and(iii) inducing coacervate formation.

41. The method of claim 40, wherein the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and < about 9.0, such as > about 6.5 and < about 9.0, or >about 7.0 and < about 8.0.

42. The method of claim 40 or 41 , wherein a volume ratio of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent is between 1 : 5 and 1 : 20.

43. A method for the delivery of an active agent, the method comprising:(i) providing a composition comprising a peptide coacervate, wherein the peptide coacervate comprises: a. one or more isolated peptides selected from the peptides of any one of claims 1 to 23 wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. an active agent, wherein the active agent is recruited in the peptide coacervate; and(ii) exposing the peptide coacervate to conditions that trigger the release of the active agent from the peptide coacervate.

44. A method for treating or diagnosing a condition or disease in a subject in need thereof, comprising:(i) administering a composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides selected from the peptides of any one of claims 1 to 23 wherein the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate; and(ii) exposing the peptide coacervate to conditions that trigger the release of the pharmaceutical or diagnostic agent from the peptide coacervate.

45. Use of an isolated peptide of any one of claims 1 to 23 for preparing a coacervate composition.

46. The isolated peptide of any one of claims 1 to 23 for use in therapy.

47. The isolated peptide of any one of claims 1 to 23 for use in a method of diagnosis practised on a mammal, such as a human.

48. The composition of any one of claims 24 to 39 for use in therapy.

49. The composition of any one of claims 24 to 39 for use in a method of diagnosis practised on a mammal, such as a human.

50. A peptide coacervate composition comprised of two types of coacervate-forming peptides wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one ir-cationic interaction.51 . A peptide coacervate composition comprising two types of coacervate-forming peptides whose amino acid sequences differ from one another, and wherein the two types of coacervateforming peptides are capable of interacting with each other via at least one ir-cationic interaction.

52. The peptide coacervate composition of claim 50 or 51 , wherein the ir-cationic interaction is formed between an aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type.

53. The peptide coacervate composition of any one of claims 50 to 52, wherein the aromatic amino acid is selected from tyrosine (Y), histidine (H), phenylalanine (F) or tryptophan (W)54. The peptide coacervate composition of any one of claims 50 to 53, wherein the cationic amino acid is selected from arginine (R), lysine (K), or ornithine (Orn).

55. The peptide coacervate composition of any one of claims 50 to 54, wherein the at least one TT-cationic interaction is formed between arginine (R) and tyrosine (Y) amino acids.

56. The peptide coacervate composition of any one of claims 50 to 55, wherein the aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type are in the same relative location on the respective peptides.

57. The peptide coacervate composition of any one of claims 50 to 56, wherein the aromatic amino acid in one coacervate-forming peptide type and the cationic amino acid in the other coacervate-forming peptide are at the same XI position of an isolated coacervate-forming peptide having the Formula (I), (II), (III), (IV) or (V).

58. A method for creating a peptide coacervate composition comprising mixing two types of coacervate-forming peptides wherein the two types of coacervate-forming peptides are capable of interacting with each other via at least one rr-cationic interaction.

59. The method according to claim 58, wherein the first coacervate-forming peptide has a different amino acid sequence to the second coacervate-forming peptide.

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