Use of a cholesterol-binding peptide to improve the cellular uptake of a peptide coacervate delivery system

Abstract

The present invention provides methods for enhancing cellular binding or entry of a peptide coacervate composition utilising a peptide that comprises a cholesterol binding domain / motif. The invention also covers novel peptide coacervates that comprise a peptide that comprises a cholesterol binding domain / motif, optionally peptide coacervates that also comprises a payload, such as an active agent to be delivered into the cell, and their use in therapy or diagnosis.

Description

[0001] USE OF A CHOLESTEROL-BINDING PEPTIDE TO IMPROVE THE CELLULAR UPTAKE OF A PEPTIDE COACERVATE DELIVERY SYSTEM

[0002] TECHNICAL FIELD

[0003] The present invention lies in the field of targeted delivery of active agents using peptide coacervates.

[0004] BACKGROUND

[0005] Biomacromolecules, including peptides (Jin et al., Theranostics.10:10141 , 2020; Zou et al., Biomat. Sci.8:4975, 2020), proteins (Guillard et al., 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.

[0006] 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)

[0007] 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.

[0008] 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). 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. However, there still exists a need for means of enhancing the cellular uptake / binding of coacervate compositions.

[0009] Cholesterol is an integral component of eukaryotic cell membranes. Cholesterol concentration can reach up to 25 mol% in plasma membranes. The interaction of this membrane component with proteins is usually governed by ‘’interfacial” amino acids like K (Lysine), R (Arginine), W (Tryptophan), Y (Tyrosine). In the scientific literature, one of the most widely recognized cholesterol-binding domains is Cholesterol Recognition / lnteraction Amino Acid Sequnce (CRAC) with sequence L / VX(i-5>YX(i.5)K / R, where X(i-5> represents a motif containing 1 to 5 of any residues), as well as its inverted version, CARC, Frontiers in physiology 4 (2013): 31. This domain can be found in membrane-bound proteins and even exploited by cellular pathogens to increase the binding to the membrane and hijack cellular processes (Cholesterol Binding and Cholesterol Transport Proteins: Structure and Function in Health and Disease, 77-108).

[0010] SUMMARY

[0011] The present invention is based on the inventors' finding that coacervate compositions incorporating or treated with a peptide with a cholesterol binding motif (a cholesterol binding peptide or CBP) increases the cell membrane binding and can also increase subsequent uptake of the coacervate composition.

[0012] The present invention provides, inter alia, methods of preparing coacervate compositions, that may comprise one or more payloads, the coacervate compositions per se, and their uses, e.g., in methods of treatment or methods of diagnosis. Such coacervate compositions can be used to deliver active agents onto the cell, or into the cell and release it directly in the cytosol. Such payloads / active agents may be useful in various applications, including bioinspired protoceils and smart drug-delivery systems.

[0013] According to a first aspect of the invention there is provided an ex vivo method for increasing the binding or uptake of a coacervate composition by one or more cells comprising contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding motif.

[0014] Suitably, the cholesterol binding motif can be any sequence which is capable of binding to cholesterol. A suitable cholesterol binding motif is a CRAC motif, an inverted CRAC motif or a tilted domain motif.

[0015] Suitably, the cells are red blood cells, such as human red blood cells.

[0016] The present invention is particularly suited to means for increasing the binding of coacervate compositions to cells where increasing uptake is not applicable, such as red blood cells.

[0017] The present invention is particularly suited to means for increasing the binding of coacervate compositions to red blood cells. According to the second aspect of the invention there is provided a coacervate composition comprising at least one peptide comprising a cholesterol binding motif.

[0018] According to the third aspect of the invention, there is provided a method for preparing a coacervate composition that comprises a peptide comprising a cholesterol binding motif, the method comprising (1)(a) providing an aqueous solution of coacervate forming peptides; (b) combining the aqueous solution of coacervate-forming peptides with a peptide comprising a cholesterol binding motif; and (c) inducing coacervate formation, optionally, wherein step (b) further includes combining with a payload molecule; or (2) mixing a peptide comprising a cholesterol binding motif with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition, optionally, wherein the preformed coacervate composition comprises a payload molecule.

[0019] According to the fourth aspect of the invention there is provided the coacervate composition of the second aspect of the invention or the coacervate composition produced by the method of the third aspect of the invention for use in therapy or diagnosis.

[0020] According to the fifth aspect of the invention there is provided the use of a peptide comprising a cholesterol binding motif to enhance the binding or uptake of a coacervate composition into a cell.

[0021] According to the sixth aspect of the invention there is provided a peptide comprising a cholesterol binding motif for use in enhancing the binding or uptake of a coacervate composition into a cell.

[0022] According to the seventh aspect of the invention there is provided a composition for the delivery of an active agent that comprises or is comprised in a peptide coacervate composition, wherein the composition comprises one or more coacervate forming peptides, a peptide comprising a cholesterol binding motif, and an active agent recruited in the peptide coacervate.

[0023] According to the eighth aspect of the invention there is provided a composition for the delivery of a diagnostic agent that comprises or is comprised in a peptide coacervate composition, wherein the composition comprises one or more coacervate forming peptides, a peptide comprising a cholesterol binding motif, and a diagnostic agent recruited in the peptide coacervate. As diagnostic tools, the following agents could be potentially incorporated in coacervates: 1 Enzymes: specific enzymes indicating the presence of certain substances

[0024] 2 Fluorescent molecules that bind to a target analyte.

[0025] 3. Antibodies

[0026] 4. DNA and RNA aptamers that bind to specific targets and can be modified to have a detectable signal when bound to their target

[0027] 5. Nanoparticles, particularly gold nanoparticles, quantum dotes and magnetic nanoparticles

[0028] 6. Environment-sensitive dyes

[0029] A few examples from scientific literature demonstrate the versatility and potential of coacervate for diagnostics. A study on peptide-based RNA sensors using tetraphnenylene- conjugated peptides that undergo LLPS and have increased luminosity in the presence of RNA demonstrated potential for intracellular RNA imaging and theranostics (Yang at al., ACS / Vano 2023, 17, 9, 8195-8203).

[0030] Another study explores DNA coacervates triggered by miRNA-21 and ions for miRNA imaging and mitochondrial targeting in breast cancer cells. This approach showcases the coacervated ability to inhibit mitochondrial functions, suggesting their potential use in cancer diagnostics (Sun et al., Ana / . Chem. 2023, 95, 37, 14101-14110).

[0031] One more example is oligopeptide-based coacervates with a focus in enzyme interaction, particularly with the enzymes involved in the coronavirus life cycle. Exploring enzyme kinetics, the research provides insights into potential applications of enzyme responsive coacervates, (Jin et al., ACS / Vano 2023, 17, 17, 16980-16992).

[0032] According to the ninth aspect of the invention there is provided the use of a peptide comprising a cholesterol binding motif in the formation of a coacervate composition.

[0033] 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 and drawings, and from the claims. The embodiments recited for any aspect of the invention can be applied to the other aspects mutatis mutandis.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. FACS analysis of HBpep coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC peptide. No increase of uptake was observed with a scrambled CRAC sequence confirming that increased binding to the membrane resulted in enhanced cellular uptake.

[0035] Figure 2. Fluorescent micrographs of HBpep coacervates without (top) and with adding CRAC peptide (bottom). Cell nuclei are stained with Hoechst33342 (blue channel), and HBpep coacervates are loaded with EGFP (green channel). More coacervates are seen to be associated (bound and internalized) with cells when CRAC peptide is added to the coacervates.

[0036] Figure 3. FACS analysis of HBpep-SP coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC peptide. No increase of uptake was observed with a scrambled CRAC sequence.

[0037] Figure 4. FACS analysis of HBpep-SP coacervates internalization after 15 min of incubation time, demonstrating an increase in uptake with the addition of CRAC peptide.

[0038] Figure 5. FACS analysis of HBpep-SP coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC peptide in the cell culture media.

[0039] Figure 6. FACS analysis of HBpep-SP coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC-14 peptide.

[0040] Figure 7. FACS analysis demonstrating an increase in HBpep-27W coacervates internalization with the addition of CRACLA / peptide versus the CRAC scrambled sequence after 30 min of incubation.

[0041] Figure 8. FACS analysis of the Alexa-488 transferrin uptake in the presence of CRAC peptide by HeLa cells, demonstrating no significant changes in uptake. The data are normalized to control (no CRAC) and shown as a mean ± SD, N = 3.

[0042] Figure 9. FIB-SEM z-stack images, in series, of cell uptake of HBpep-SP coacervates with the addition of CRAC peptide, showing internalized and dissolving coacervates (black arrowheads). Two locations are shown, showing pores in the dissolving coacervates. Top dark area: extracellular space. Figure 10. Live cell imaging of cellular uptake of HBpep-SP coacervates with the addition of CRAC peptide. Time-lapse confocal images of coacervates showing that the majority underwent internalization and movement inside the cells (white arrowheads).

[0043] DETAILED DESCRIPTION

[0044] The inventors found that incorporating a peptide that comprises a CRAC motif (a cholesterol binding motif) into a coacervate composition (exemplified using coacervates comprised of standard artificial peptides derived from histidine-rich beak peptide (HBpep)), resulted in significantly increased cellular uptake of the coacervate. The inventors demonstrate that modulating the adhesion of coacervates to the cell membrane by recruiting cholesterol-binding peptides within the coacervates enhanced cell uptake, likely via actin and cholesteroldependent non-classical endocytosis.

[0045] The use of one or more peptides capable of binding to cholesterol in coacervate systems allows for enhanced cellular uptake or binding for coacervates. Coacervates can be used to deliver therapeutic, diagnostic or other payloads into cells and so this invention also provides means for preparing payload delivering coacervates which may have therapeutic uses, and such coacervates for use in therapy or diagnosis.

[0046] According to various aspects of the invention, in particular the first aspect and fifth aspect of the invention, a peptide comprising a cholesterol-binding motif is used to promote enhanced binding and attachment of a peptide coacervate composition to a selected cell type. Any type of cell that presents or displays cholesterol on its surface can be employed. Red blood cells which are characterized by an elevated cholesterol content in its plasma membrane (Cooper R.A., Journal of supramolecular structure. 8.4, 413-430, 1978) are particularly suitable and used in the examples herein.

[0047] In a preferred embodiment, such enhanced binding is exploited to improve the encapsulated drug delivery efficiency. It has been demonstrated that red blood cells serve effectively as vehicles for drug delivery (Brenner, J.S. et al., Nature communications. 9(1), 2684, 2018), thereby further substantiating the use of the present invention in therapeutic applications.

[0048] In a preferred embodiment, the cells according to the first and fifth aspects of the invention are ceils which possess cholesterol in its plasma membrane and / or display cholesterol on the cell surface. Cells with elevated membrane cholesterol content include, but not limited to, macrophages, hepatocytes, neurons, adipocytes, enterocytes, and immune cells, such as T cells, B cells and dendritic cells, where cholesterol is presented in lipid rafts and general membrane bilayer. hi particular embodiments, the cells according to the first and fifth aspects of the invention are selected from the list consisting of: red blood cells, macrophages, hepatocytes, neurons, adipocytes, enterocytes, and immune cells, such as T cells, B cells and dendritic cells.

[0049] In a preferred embodiment, the ceils according to the first and fifth aspects of the invention are red blood cells.

[0050] Definitions:

[0051] 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 cell” also covers the method on a population of cells (which covers 2 or more cells, but typically involves many thousands of cells). 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.

[0052] 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.%.

[0053] “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.

[0054] As used herein, the term “ex vivo’’ means that the method takes place outside of a human or animal body. Suitably, the cells used in said ex vivo setting / method may have previously been taken from a living organism. Alternatively, the cells may be from a repository and / or may have been subjected to prior manipulation. In the present context, the term ex vivo also includes in vitro; thus, the method of the first aspect could be classified as an ex vivo or in vitro method.

[0055] “Histidine-rich proteins”, as used herein, relates to proteins that include at least one histidine residue and overall have a comparably high number 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%, relative to the total number of amino acids in the peptide sequence.

[0056] 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 or produced chemically such via solid phase peptide synthesis, 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.

[0057] 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.

[0058] 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.

[0059] The term “self-immolative (SR) moiety”, as used herein, refers to a moiety that is selfcleaving 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.

[0060] The term “(amino acid) residue”, as used herein, relates to one or more amino acids which are considered as part of the peptide. The term “peptide”, as used herein, rebates 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 14 amino acids to 100, preferably to 80, 70, 60 or 50 amino acids in length, for example, at least 14, 15,16, 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 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.

[0061] 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-naturally) 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.

[0062] The term “about”, as used herein, in connection with a numerical value, means said value ± 10 %, for example, ± 5 %.

[0063] According to a first aspect of the invention there is provided an ex vivo method for increasing the binding or uptake of a coacervate composition by one or more cells comprising contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding motif.

[0064] Suitably, the binding of the coacervate composition by one or more cells is increased in the method of the first aspect.

[0065] Suitably, the uptake of the coacervate composition by one or more cells is increased in the method of the first aspect.

[0066] Suitably, the binding of the coacervate composition by one or more cells is increased in cells where increasing the uptake is not applicable, for example red blood cells. Suitably, both the binding and the uptake of the coacervate composition by one or more cells is increased in the method of the first aspect.

[0067] Suitably, the increase in binding or uptake of a coacervate composition by one or more cells is an increase in cellular binding or uptake of the coacervate composition by a population of cells.

[0068] The cells, the peptide comprising the cholesterol binding motif and the coacervates (or coacervate forming peptides) can be brought together in various orders. For example,

[0069] (i) the cells can be contacted with the peptide comprising the cholesterol binding motif for a period of time before contacting with the coacervate mixture.

[0070] (ii) the peptide comprising the cholesterol binding motif can be mixed with coacervate forming peptide so that it becomes integrated into the coacervates in the coacervate composition; this can then be contacted with the cells.

[0071] (iii) the peptide comprising the cholesterol binding motif can be contacted with preformed coacervate composition so that it can become associated with, e.g. attached to or coated onto, the coacervates in the coacervate mixture; this can then be contacted with the cells.

[0072] (iv) the peptide comprising the cholesterol binding motif, the coacervate mixture and the cells can be brought into contact with each other simultaneously, e.g. by adding the peptide comprising the cholesterol binding motif and the coacervate composition to the culture medium that the cells are in.

[0073] It will be appreciated that the coacervate composition and cells should be in contact with each other (e.g. incubated together in appropriate medium) for a sufficient time and / or under conditions to permit entry into the cell by the coacervates.

[0074] Any suitable medium, e.g. tissue culture medium, can used. Suitably, the medium is a culture medium suitable for growth of the particular cells being used. Dulbecco’s Modified Eagle Medium (DMEM), a widely used cell culture medium for mammalian cell culture. Other mammalian ceil culture media include Roswell Park Memorial Institute (RPMI) 1640, Dulbecc”s Modified Eagle Medium Nutrient Mixture F-12 (DMEM F12), Eagles Minimum Essential Medium (EMEM), Minimum Essential Medium (MEM), reduced serum Optimem media (Gibco).

[0075] Suitably, the peptide comprising a cholesterol binding motif has been incorporated into the coacervates in the coacervate composition.

[0076] In particular embodiments, the peptide comprising a cholesterol binding motif is mixed with the peptides used to form the coacervate composition prior to formation of the coacervate composition so that when formed the coacervate composition comprises the peptide comprising a cholesterol binding motif. In particular embodiments, the peptide comprising a cholesterol binding motif and the coacervate forming peptides are mixed in a, 1 :5 - 1 :100 mass ratio, such as a 1:10 - 1:60 mass ratio.

[0077] Suitably, the peptide comprising a cholesterol binding motif is contacted for a period of time with the coacervate composition before contacting with the cell. Here the term contacting simply means that the peptide comprising a cholesterol binding motif and the coacervate composition are mixed together, typically in solution in a vessel such as a test tube.

[0078] In particular embodiment, the peptide comprising a cholesterol binding motif is mixed with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition.

[0079] In particular embodiments, the peptide comprising a cholesterol binding motif and the preformed coacervate composition are mixed in a 1 :10 - 1:60 mass ratio.

[0080] Suitably, the peptide comprising a cholesterol binding motif and coacervate composition are allowed to mix for at least 5 minutes, such as at least 10, 15, 30, or more minutes, prior to contacting with the cells.

[0081] In one embodiment, the cell is (or) cells are contacted with the peptide comprising a cholesterol binding motif for a period of time before contacting with the coacervate composition. Suitably, the cell is contacted with the peptide comprising a cholesterol binding motif for at least 15 minutes, such as at least 30, 40, 50, 60, 90, 120, 150 or more minutes, prior to contacting with the coacervate composition.

[0082] Here the term contacting typically involves incubation of the cell(s) with the peptide comprising a cholesterol binding motif added to the culture medium.

[0083] In particular embodiments, the peptide comprising a cholesterol binding motif is contacted with the cells by adding the peptide comprising a cholesterol binding motif to the culture medium comprising the cells at a concentration of at least 20 pg / ml.

[0084] According to particular embodiments, the cell is incubated in culture medium comprising the coacervate composition for at least 15 minutes, such as 20, 30, 60, 90, 120, 150, 240 or more minutes. In one embodiment, the coacervate composition has been previously contacted with the peptide comprising a cholesterol binding motif. Suitably, the peptide comprising a cholesterol binding motif is attached to the surface of the coacervates in the coacervate composition. In another embodiment, the cell has been previously contacted with the peptide comprising a cholesterol binding motif. Suitably, an increase in the uptake of the coacervate composition is an increase in the amount of coacervates taken up by a cell or population of cells or the amount of cells that take up the coacervates, e.g. intracellular delivery efficiency. This can also be referred to as "uptake efficiency” or “intracellular delivery efficiency”. Suitably, an increase in the binding of the coacervate composition is an increase in the amount of coacervates bound to or by a cell or population of cells or the amount of cells that bound the coacervates. This can also be referred to as "binding efficiency”.

[0085] The presence of / use of the peptide comprising a cholesterol binding motif in the method of the first aspect of the invention facilitates increased binding or uptake of the coacervate by the cell(s). Indeed, as can be seen in the Examples herein, uptake increases of at least 20%, e.g., 50% (as determined by using the reporter protein) have been observed. An increase in uptake is a relative term and can be measured by the amount / number of cells that have taken up the coacervate composition, or by quantifying the mean fluorescent intensity per cell.

[0086] According to particular embodiments, the increase in binding or uptake efficiency as at least 20%, such as at least 30%, 40%, 50% or more, compared to binding or uptake efficiency in the absence of the peptide comprising a cholesterol binding motif or when using a control peptide. Suitably, the control peptide is a scrambled peptide that lacks a functional cholesterol binding motif.

[0087] The efficiency of the uptake of a coacervate composition by a cell (uptake efficiency) can be determined by any appropriate technique known to the person skilled in the art. In a particular embodiment, the uptake efficiency is assessed by fluorescence-activated cell sorting analysis (FACS) and fluorescence microscopy.

[0088] Suitably the uptake efficiency is evaluated by FACS after 10 minutes contacting / culturing, such as after 15, 20, 30, 40, 50, 60 or more minutes of contacting / incubation.

[0089] The method of the invention facilitates increased binding or uptake of a coacervate composition by a cell. The cell can be any eukaryotic cells (or cells). In a particular embodiment, the cell is a eukaryotic cell, such as a mammalian cell. In a particular embodiment, the cell is a human cell. In a particular embodiment, the cell is a red blood cell.

[0090] Coacervates and coacervate compositions

[0091] The Humboldt squid beak indudes a hard biomolecular composite made of chitin and proteins. The squid beak proteins were recently isolated and sequenced and two families of proteins, chitin binding beak proteins (DgCBPs) and 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 liquid-liquid 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 cross-linking 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-term-nal 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-repeats (GAGFA (SEQ ID NO:17), GHGXX7X" (SEQ ID NO:18) or GHGXY (SEQ ID NO:19), where X represents a hydrophobic residue, X' usually represents tyrosine and X" represents either glycine or alanine. The C-terminal domain motifs were found to be responsible for DgHBPs selfcoacervation properties (Cai et al. (2017) Soft Matter 13 (42), 7740-7752).

[0092] The term "coacervate", as used herein, has the meaning as commonly understood in the art. Coacervates are dense microdroplets formed in a process called liquid-liquid phase separation (LLPS). Coacervates can form from two or more different types of molecules (complex coacervates) or one type of macromolecule through self-coacervation (simple coacervates). The peptide-rich coacervate phase is also referred to herein as “coacervates”, “peptide coacervate (micro)droplets”, “coacervate particles”, or "condensates".

[0093] A self-coacervating peptide is one that is able to form simple coacervates.

[0094] In a particular embodiment the coacervate for use in any aspect of the invention is a complex coacervate. Complex coacervates can form from two or more oppositely charged macromolecules (peptides, proteins, nucleic acids, polymers).

[0095] In a particular embodiment the coacervate for use in any aspect of the invention is a simple coacervate, such as those formed from a self-coacervating peptide.

[0096] An example of a biomacromolecule which exhibit self-coacervation include the histidine-rich beak peptide (HBpep). HBpep is derived from the Humbolt squid (Dosidicus gigas) beak protein and its self-coacervation property plays an essential role in the formation of the mechanical gradient of squid beaks (Tan et al., Nat. Chem. Bid., 2015, 11 (7), 488). HBpep is characterized by a low sequence complexity consisting of only 5 copies of the tandem repeat GHGXY (SEQ ID NO:19) (where X could be leucine (L), proline (P), or valine (V)) and a single C-terminal Trp (W) residue. Further, a key feature of the HBpep is the presence of 5 His (H) residues in the 5 repeat sequence motifs GHGXY (SEQ ID NO: 19) that confer pH- responsivity LLPS behaviour (Gabryelczyk, B. et ai., Nat. Comms.10:5465, 2019). Notably, this allows the HBpep to remain in a monomeric state at a low pH, but to quickly phase separate or self-coacervate into coacervate microdroplets at neutral pH and to concomitantly recruit various macromolecules from the solution during the process.

[0097] 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 recently demonstrated to be able to cross the cell membrane via a non-classical endocytosis 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).

[0098] Self-coacervating peptides that are derivatives of the wild-type HBpep are known (e.g, US2019 / 0388357 and WO2021 / 246961), and any of these can be utilised in the present invention.

[0099] 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 particular the Humboldt squid (Dosidicus gigas).

[0100] In particular embodiments, the self-coacervating peptides used to prepare coacervate compositions in accordance with the present invention comprise the amino acid sequence: (GHGX1Y)a[(GX2GX3A)n(GHGLX4)c(GFA)d]f(GHGX1Y)a (SEQ ID NO: 1) wherein X1is valine (V), leucine (L) or proline (P) , X2is alanine (A) or proline (P); X3is phenylalanine (F) or tyrosine (Y), X4is leucine (L), histidine (H), tyrosine (Y) or glycine (G) , each a is 0 or an integer >1; each b is 0 or an integer >1; each c is 0 or an integer >1 ; each d is 0 or an integer >1; f is an integer >1; with the sum of all a being >2 and the sum of all a+b+c+d is >4.

[0101] Such peptides are disclosed in US 2019 / 0388357. hi particular embodiments, the self-coacervating peptides used to prepare coacervate compositions in accordance with the present invention comprise the amino acid sequence:

[0102] (GHGXY)n K (GHGXY)m Z (SEQ ID NO: 2),

[0103] (GHGXY K)n(GHGXY)m Z (SEQ ID NO: 3), or

[0104] (GHGXY)n (K GHGXY)m Z (SEQ ID NO: 4), wherein

[0105] X is valine (V), leucine (L) or proline (P),

[0106] Z is tryptophan (W) or absent, n is 0, 1 , 2, 3, 4 or 5, m is 0, 1, 2, 3, 4 or 5, n+m is 3, 4 or 5, preferably 5.

[0107] Non-limiting isolated peptides comprise or consist of an amino acid sequence, such as but not limited to:

[0108] (i) K GHGXY GHGXY GHGXY GHGXY GHGXY W (SEQ ID NO:5),

[0109] (ii) GHGXY K GHGXY GHGXY GHGXY GHGXY W (SEQ ID NO:6),

[0110] (iii) GHGXY GHGXY K GHGXY GHGXY GHGXY W (SEQ ID NO:7),

[0111] (iv) GHGXY GHGXY GHGXY K GHGXY GHGXY W (SEQ ID NO:8),

[0112] (v) GHGXY GHGXY GHGXY GHGXY K GHGXY W (SEQ ID NO:9),

[0113] (vi) GHGXY GHGXY GHGXY GHGXY GHGXY W K (SEQ ID NO: 10),

[0114] (vii) K GHGVY GHGVY GHGPY GHGPY GHGLY W (SEQ ID NO: 11),

[0115] (viii) GHGVY K GHGVY GHGPY GHGPY GHGLY W (SEQ ID NO:12),

[0116] (ix) GHGVY GHGVY K GHGPY GHGPY GHGLY W (SEQ ID NO: 13),

[0117] (x) GHGVY GHGVY GHGPY K GHGPY GHGLY W (SEQ ID NO: 14), (xi) GHGVY GHGVY GHGPY GHGPY K GHGLY W (SEQ ID NO: 15), or

[0118] (xii) GHGVY GHGVY GHGPY GHGPY GHGLY W K (SEQ ID N0:16).

[0119] In various embodiments, the lysine residue (K) is modified at an epsilon (s)- amino group with a self-immolative moiety. 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.

[0120] 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.

[0121] 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(=0)-0- (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.

[0122] “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(=0)R1-0C(=0)R1-CC(=0O0RR1, halogen, such as fluorine, chlorine and bromine, -N3, with R1being selected from unsubstituted or halo-substituted C1.

[0123] 4 alkyl or alkenyl, unsubstituted or halo-substituted C5.6 cycloalk(en)yl, or unsubstituted or halo-substituted Ce-u aryl. It can be preferred that the substituent is not a charged group.

[0124] “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.

[0125] “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.

[0126] “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. hi various embodiments, the self-immolative (SR) moiety includes a disulfide 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.

[0127] In various non-limiting embodiments, the self-immolative moiety has the formula such as but not limited to: -C(=0)-0-(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, for example 1 , 2, 3, 4, or 5.

[0128] In various non-limiting embodiments, R may be a group of the formula such as but not limited to: -(CH2)n-0-C(=0)-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.

[0129] Such peptides are disclosed in WO2021 / 246961.

[0130] In particular embodiments, the coacervate forming peptide has a minimum length of 10 amino acids, suitable coacervate forming peptides have a length of at least 16 amino acids, for example 17 amino acids, and comprise at least three sequence motifs, e.g,, GHGXY, optionally a lysine with a self-immolative (SR) moiety attached and / or optionally a tryptophan (W) at or close to the C-terminus. By way of a non-limiting example, the sequence motif may include at least one sequence motif GHGVY (SEQ ID NO:20), at least one sequence motif GHGPY (SEQ ID NO:21), and one sequence motif GHGLY (SEQ ID NO:22). As a further example, the isolated peptides (hBpep-K) may include at least four copies, or may include five copies of the sequence motif GHGXY (SEQ ID NO:19), Z, and K. In various embodiments, the isolated peptides (hBpep-K) may include, for example, two copies of the sequence motif GHGVY (SEQ ID NO:20), two copies of the sequence motif GHGPY (SEQ ID NO:21) and one copy of the sequence motif GHGLY (SEQ ID NO:22). The C-terminal amino acid, Z, which may represent tryptophan (T p or W), may be present or may be absent.

[0131] The upper limit in peptide length of the isolated coacervate forming peptides may be 50 amino adds, for example, up to 40, up to 35 or up to 30 amino adds. In various embodiments, the isolated coacervate forming peptides may be 25-30 amino acids long, such as 27 amino acids long.

[0132] In various embodiments, the isolated coacervate forming peptides are derived from histidine- rich proteins.

[0133] The self-coacervating peptides identified above (and, e g., in US2019 / 0388357 and WO2021 / 246961), form coacervates readily, in particular under neutral conditions, i.e. pH values of around 7. 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.

[0134] 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.

[0135] In various embodiments, the peptide coacervate, i.e., the coacervate phase formed, can be covalently crosslinked. The crosslinking may be achieved by use of a suitable crosslinker. As the peptides disclosed herein typically comprise at least one tyrosine residue, the hydroxyl groups thereof may be used for crosslinking. Suitable crosslinking agents include, without limitation, those that comprise catechol moieties, for example and without limitation 4- methylcatechol (4-MC). In such cases, crosslinking may occur by a redox reaction between the catechol moieties and aromatic hydroxyl groups, such as the tyrosine hydroxyl groups, heteroaryl groups, such as histidine imidazole groups, and amine groups, such as lysine, arginine, asparagine or glutamine amine groups. In one specific embodiment, the peptide coacervate is crosslinked with 4-methylcatechol (4-MC) and sodium periodate (NalO 4).

[0136] 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.

[0137] 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 the peptides derived from histidine-rich proteins and the active agent; and (2) a dilute aqueous phase.

[0138] The coacervates formed in the above-described methods 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.

[0139] 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. For exampie, release of the recruited pharmaceutical or diagnostic agent may be facilitated by exposure to condition which disrupts the disulfide bond resulting in the autocatalytic cleavage of the self-immolative (SR) moiety, restoration of the charged lysine side chain, and resulting dissolution of the peptide coacervates.

[0140] Additional release mechanisms may include the use of surfactants or denaturing agents that disrupt the formed phases.

[0141] 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.

[0142] Peptide comprising a cholesterol binding motif.

[0143] A peptide comprising a cholesterol binding motif can be any peptide which is capable of specifically and / or selectively binding to cholesterol. Such peptide will typically comprise a cholesterol binding motif sequence (motif sequence is also known as a consensus sequence). Numerous cholesterol binding consensus sequences are known.

[0144] In a particular embodiment, the peptide comprising a cholesterol binding motif is an affine non-cytotoxic cholesterol-binding peptide (CBP). By affine, we mean that the peptide binds cholesterol with high selectivity. By non-cytotoxic we mean it does not cause any significant toxic effects at the concentration used. In the context of a therapeutic agent this covers any agent that has received regulatory approval (i.e., where any benefit outweighs any toxic side effects at the dosages and treatment regime authorised by the health authority) The peptide comprising a cholesterol binding motif for use in the invention can be of any suitable length, such as between 8 and 50 amino acids in length. In particular embodiments, the peptide comprising a cholesterol binding motif is at least 8 amino acids in length.

[0145] Suitably, the peptide comprising a cholesterol binding motif is from 10 - 30 amino acids in length, such as between 12 and 20 amino acids in length.

[0146] The cholesterol binding motif can be any sequence which is capable of binding to cholesterol. A suitable cholesterol binding motif is a CRAC motif, an inverted CRAG motif (also known as CARC) or a tilted domain motif.

[0147] For details of cholesterol recognition / interaction amino acid consensus (CRAC) sequences, see, Jamin et al., Characterization of the Cholesterol Recognition Amino Acid Consensus Sequence of the Peripheral-Type Benzodiazepine Receptor. Mol Endocrinol. 19(3):588-594, 2005; and, Li et aL, Cholesterol binding at the cholesterol recognition / interaction amino acid consensus (CRAC) of peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV-TAT-CRAC peptide. Proc Natl Acad Sci. 98(3): 1267-1272, 2001. Epand, Richard M. “"Proteins and cholesterol-rich domains”" Biochimica et Biophysica Acta (BBA)-Biomembranes 1778.7-8 (2008): 1576-1582.

[0148] For details of CRAC motif, inverted CRAC (or CARC) motif or tilted domain motif sequences, see, Fantini and Barrantes (How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains. Frontiers I Physiology. 4(31): 1-9, 2013).

[0149] In particular embodiments, the cholesterol binding motif has the CRAC motif: V / L - [X]i-s- Y / W-[X]I-5-R / K, wherein V is valine, L is leucine, X is any amino acid, Y is tyrosine, W is tryptophan, R is arginine and K is lysine. [X]i-s means there can be 1-5 (inclusive) amino acids.

[0150] In particular embodiments, the cholesterol binding motif has the CRAC motif:

[0151] V / L - [X]i-5- Y -[X]I-5-R / K , wherein V is valine, L is leucine, X is any amino acid, Y is tyrosine, W is tryptophan, R is arginine and K is lysine. [X]i-s means there can be 1-5 (inclusive) amino acids.

[0152] In particular embodiments, the cholesterol binding motif has the CRAC motif: V / L - [X]i-s- W -[X]I-5-R / K, wherein V is valine, L is leucine, X is any amino acid, Y is tyrosine, W is tryptophan, R is arginine and K is lysine. [X]i-s means there can be 1-5 (inclusive) amino acids. In particular embodiments, the cholesterol binding motif has the CRAC motif: V / L - [X]2-4- Y / W-[X]I-3-R / K, wherein V is valine, L is leucine, X is any amino acid, Y is tyrosine, W is tryptophan, R is arginine and K is lysine. [X]2-4or [X]i-3 means there can be 2-4 or 1-3 amino acids, respectively.

[0153] In particular embodiments, the cholesterol binding motif has the CRAC motif: L-[X]2-4-Y-[X]I-3- R / K, wherein L is leucine, X is any amino acid, Y is tyrosine, R is arginine and K is lysine. [X]2-4 or [X]i-3 means there can be 2-4 or 1-3 amino acids, respectively.

[0154] In particular embodiments, the cholesterol binding motif has the CRAC motif: L-[X]2-4-W-[X]I. 3-R / K, wherein L is leucine, X is any amino acid, Y is tyrosine, R is arginine and K is lysine. [X]2-4 or [X]i-3 means there can be 2-4 or 1-3 amino acids, respectively.

[0155] In particular embodiments, the cholesterol binding motif has the inverted CRAC motif: R / K- [X]I-5-Y / W-[X]I-5-V / L, or R / K-[X]I-5-Y / F-[X]I.6-V / L (both options together being: R / K-[X]I-5- Y / F / W-[X]I.5-V / L), wherein V is valine, L is leucine, X is any amino acid, Y is tyrosine, W is tryptophan, R is arginine and K is lysine. [X]i-s means there can be 1-5 (inclusive) amino acids.

[0156] In particular embodiments, the cholesterol binding motif has the inverted CRAC motif: R / K- [X]I.3-Y-[X]2-4-V / L, wherein L is leucine, X is any amino acid, Y is tyrosine, R is arginine and K is lysine. [X]2-4 or [X]i-3 means there can be 2-4 or 1-3 amino acids, respectively.

[0157] In particular embodiments, the peptide comprising a CRAC motif comprises or consists of one of the following sequences:

[0158] (i) FDRARMLEEYSKRFKKFGY (SEQ ID NO:23)

[0159] (ii) FDRARMVEEYSKRFKKFGY (SEQ ID NO:24)

[0160] (iii) FDRARM LEEWSKRFKKFGY (SEQ I D NO:25)

[0161] (iv) FDRARM LEEGYSKRFKKFGY (SEQ ID NO:26)

[0162] (v) FDRARM LEEYGSKRFKKFGY (SEQ ID NO:27)

[0163] (vi) ARMLEEYSKRFKKF (SEQ ID NO:28)

[0164] In particular embodiments, the peptide comprising an inverted CRAC motif comprises or consists of the sequence YGFKKFRKSYEELMRARDF (SEQ ID NO:29).

[0165] Payload

[0166] Coacervates can be used to introduce a payload (e.g., active agent or diagnostic) into cells and release them in the cytosol. The present invention can be employed for any coacervate composition and so can be used for any coacervate composition that includes a payload. Suitable payloads include proteins or peptides, nucleic acids, or large or small molecule compounds.

[0167] In particular embodiments of any aspects of the present invention, the coacervate composition comprises a payload (e g., active agent or diagnostic). Suitably, the payload is a protein or peptide, a nucleic acid, or a small molecule compound. In a particular embodiment, the payload is an antibody or a fragment thereof. In a particular embodiment, the payload is an antigen, such as a polypeptide. In a particular embodiment, the payload is a vaccine. In a particular embodiment, the payload is a nucleic acid, such as a plasmid DNAs, RNA oligonucleotides or variants thereof, small interfering RNAs, microRNAs, messenger RNAs, long non-coding RNAs, and other RNA oligonucleotides such as those used in CRISPR / Cas9 or other genome-editing systems, or a gene therapy construct / vector.

[0168] In various embodiments, the active agent is a protein or polypeptide. Suitable proteins and polypeptides include antibodies, antibody fragments, antibody variants and antibody-like molecules. “Antibodies”, as used herein, refers to immunoglobins comprising antigen-binding site(s) and includes monoclonal and polyclonal antibodies comprising the various isotypes IgG, IgM, IgD, IgA, IgE. In some embodiments, antibodies may be or include, but is not limited to, recombinant antibodies or recombinant antibody fragments, such as Fab or scFv fragments. Suitably, the antibody is a monoclonal antibody, or a fragment thereof.

[0169] In various embodiments, the active agent is a nanoparticle. 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.

[0170] In various embodiments, the active agent is a chemical compound, in particular a small molecule chemical compound. Diagnostic payloads, such as radiolabelled molecules can also be chemical compounds.

[0171] “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.

[0172] 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.

[0173] In various embodiments, a volume ratio of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent may be greater than 1 : 5, for example, in the range of 1 : 5 to 1 : 20. In some embodiments, the volume ratio of the aqueous solution of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent is between 1 : 8 to 1 : 10, for example, at about 1 : 9, or at about 1 : 9.5.

[0174] Generally, the payload may be encapsulated alone or co-encapsulated together with a release agent that facilitates release of the payload (e g., active agent) from the coacervate. Examples of such release agents are disclosed in US2019 / 0388357 and WO2021 / 246961 and include magnetic nanoparticles and glucose oxidase in a non-limiting embodiment. Depending on the type of release agent used, the release mechanism may differ. One type of release agents leads to an acidification of the environment of the colloidal phase, with the lowering of the pH triggering the dissolution of the coacervate phase. The acidification may be dependent on the presence of an initiator or substrate, in the case of glucose oxidase, glucose. The addition of glucose or the increase in glucose concentration may thus lead to sufficient acidification to facilitate release of the encapsulated agent, for example insulin. Other types of release agents cause heat development in the vicinity of the colloidal phase that also effects release of the encapsulated agent. Such heat development may, for example, be achieved by magnetic nanoparticles and exposure to a magnetic field.

[0175] Further release mechanisms, such as peptide degradation by use of a peptidase, may be possible and can be selected by those skilled in the art dependent on the intended use.

[0176] Generally, the release may be a burst release where essentially the total load of the active agent is released over a short time span or may be a sustained release where the release occurs over a prolonged period of time. In general, the release may occur within several minutes up to several days or weeks. The release may also be step-wise in that upon exposure to certain conditions the release starts but stops when the conditions are no longer met. It can then start again once the conditions for release are again met. Such conditions that may be varied to facilitate a step-wise or need dependent release may include, but are not limited to, glucose concentration and magnetic field exposure.

[0177] Thus, in various embodiments, the coacervate composition comprising the payload / active agent is adapted for administration to a mammalian subject, for example, a human being.

[0178] In various embodiments, the composition comprises a pharmaceutical or diagnostic formulation for administration to a mammalian subject, for example a human being. Such formulations may additionally comprise all the known and accepted additional components for such applications. These include auxiliaries, carriers and excipients that are pharmaceutically or diagnostically acceptable, for example various solvents, preservatives, dyes, stabilizers and the like. Such formulations may additionally comprise further active agents that are not recruited in the peptide 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, i.e. 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

[0179] In various embodiments, the self-immolative moiety of the peptide coacervate autocatalytically cleaves itself upon exposure to specific conditions selected from the group such as but not limited to: pH changes, redox changes, exposure to release agents, and combinations thereof. In some embodiments, the release agent is glutathione (GSH), specifically, cell endogenous GSH, which is ubiquitous in cells.

[0180] In general, the release of the active agent may for example, be a burst release where essentially the total load of the act-ve agent is released over a short time frame, or may be a sustained release where release occurs over a prolonged duration. Generally, the release occurs within several minutes but may take up to several weeks or days. The release may also be step-wise such that upon exposure to specific conditions, the release starts but stops once said conditions are removed. It may then re-start again once those conditions for release are again met. Such conditions may be tailored to facilitate a step-wise, or need dependent release and are not limited to pH changes, redox changes, and / or exposure to release agents (e.g. reducing agents, such as ceil endogenous GSH). In various embodiments, it is preferred that intracellular release may be a burst or sustained release in the presence of reducing agent GSH, i.e., cell endogenous GSH According to the second aspect of the invention there is provided a coacervate composition comprising at least one peptide comprising a cholesterol binding motif.

[0181] In this context the at least one peptide means at least one type of peptide, e.g. a peptide having the same amino acid sequence, as opposed to a single peptide molecule.

[0182] In one embodiment, molecules of a single peptide sequence are used.

[0183] In one embodiment, molecules of distinct peptide sequences are used, so that when formed the coacervates are a heterogenous mix of peptides. Suitably, peptide comprising 2, 3, 4 or more distinct peptide sequences are used.

[0184] In this aspect, the peptide comprising a cholesterol binding motif and the coacervate composition are as described herein for employment in the method of the first aspect of the invention.

[0185] As described above, to produce the coacervate composition of the second aspect of the invention, the peptide comprising the cholesterol binding motif can be mixed with coacervate forming peptide so that it becomes integrated into the coacervates in the coacervate composition.

[0186] In a particular embodiment, the at least one peptide comprising a cholesterol binding motif is formed into the coacervates.

[0187] Alternatively, as described above, to produce the coacervate composition of the second aspect of the invention, the peptide comprising the cholesterol binding motif can be contacted with pre-formed coacervate composition so that it can become associated with, e.g., attached to or coated onto, the coacervates in the coacervate mixture.

[0188] In a particular embodiment, the at least one peptide comprising a cholesterol binding motif is coated on the surface of the coacervates (coacervate particles) and / or bound to the surface of the coacervates (coacervate particles).

[0189] In a particular embodiment, the coacervate composition of the second aspect of the invention comprises a therapeutic payload or a diagnostic payload. Suitably, the payload is a protein or peptide, an antibody or fragment thereof, a nucleic acid, a small molecule compound, or a vaccine as described herein.

[0190] According to the third aspect of the invention, there is provided a method for preparing a coacervate composition that comprises a peptide comprising a cholesterol binding motif, the method comprising (1)(a) providing an aqueous solution of coacervate forming peptides; (b) combining the aqueous solution of coacervate-forming peptides with a peptide comprising a cholesterol binding motif; and (c) inducing coacervate formation, optionally, wherein step (b) further includes combining with a payload molecule; or (2) mixing a peptide comprising a cholesterol binding motif with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition, optionally, wherein the preformed coacervate composition comprises a payload molecule.

[0191] In particular embodiments, the coacervate forming peptides, the peptide comprising a cholesterol binding motif and the optional payload are as described herein.

[0192] In one embodiment, the pH of the aqueous solution of the coacervate-forming peptides is >1 and <7.

[0193] In one embodiment, the concentration of the coacervate-forming peptides provided in the aqueous solution ranges from about 0.3 mg / mL to about 100 mg / mL.

[0194] The embodiments recited for the first aspect of the invention can be applied to this third aspect mutatis mutandis.

[0195] According to another aspect of the invention, there is provided an ex vivo or in vitro method for increasing the binding or uptake efficiency of a coacervate composition by a population of cells comprising:

[0196] A

[0197] (i) incubating a population of cells for a first period of time in a culture medium comprising a peptide comprising a cholesterol binding motif;

[0198] (ii) adding a coacervate composition to the culture medium and incubating for second period of time under conditions to permit binding or uptake of the coacervate / coacervates into or onto the cells; or

[0199] B incubating a population of cells in a culture medium comprising a peptide comprising a cholesterol binding motif and a coacervate composition under conditions to permit binding or uptake of the coacervate into the cells.

[0200] In a particular embodiment of this aspect of the invention, the uptake efficiency is the amount of coacervate composition that is taken up by each cell / a population of cell.

[0201] The embodiments recited for the first aspect of the invention can be applied to this aspect also. According to the ninth aspect of the invention there is provided the use of a peptide comprising a cholesterol binding motif in the formation of a coacervate composition. The embodiments recited for the first aspect of the invention can be applied to this aspect also.

[0202] Medical or diagnostic use

[0203] Advantageously, peptide coacervates present a novel and safe delivery platform for both the intracellular delivery and direct cytosolic release of a large palette of biomacromolecular therapeutics or diagnostics Critically, the recruitment process of a therapeutic agent is carried out under aqueous environments, thereby preventing the loss of bioactivity of said therapeutic agent and enhancing safety. Redox-responsive peptide coacervates remain stable at neutral conditions, i e., neutral pH, enabling intracellular delivery of therapeutic agents which take advantage of extracellular and intracellular GSH gradients. The versatility of cargo recruitment and release makes this intracellular delivery platform a promising candidate for the treatment of disease, such as cancer, metabolic, and / or infectious diseases. The versatility of cargo recruitment and release makes this intracellular delivery platform a promising candidate for delivery and release of a diagnostic agent, e.g., radiolabelled compound, specific enzyme, fluorescently labelled molecule, aptamer, nanoparticle or imaging agent.

[0204] 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.

[0205] 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.

[0206] The administration may be systemic or localized, e.g., topically.

[0207] 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.

[0208] The conditions that trigger the release of the pharmaceutical or diagnostic agent may generally be selected from those disclosed above for the delivery methods. The subject may be a mammal, for example a human.

[0209] In non-limiting embodiments of these methods for the treatment of a disease or disorder, the subject is a human afflicted by a disease or disorder that the active agent / payload is designed to treat.

[0210] In further non-limiting embodiments, the subject is a human afflicted by cancer, wherein the pharmaceutical agent is doxorubicin, wherein the coacervate further comprises encapsulated magnetic nanoparticles, and wherein release is facilitated by exposure of the subject to a magnetic field resulting in a temperature increase in the coacervate. In such embodiments, the subject or a body region of the subject may be exposed to magnetic fields that lead to a temperature increase in the vicinity of the magnetic particles and as a result loss of the structural integrity of the coacervate phase that then releases the doxorubicin.

[0211] Additional applications of the compositions and methods will be identifiable by the person skilled in the art.

[0212] According to the fourth aspect of the invention there is provided the coacervate composition of the second aspect of the invention or the coacervate composition produced by the method of the third aspect of the invention for use in therapy.

[0213] According to a particular aspect of the invention there is provided the coacervate composition of the second aspect of the invention or the coacervate composition produced by the method of the third aspect of the invention for use in diagnosis. Suitably, the diagnosis is one carried out on the human or animal body, e.g. wherein the coacervate is administered to the subject.

[0214] According to the fifth aspect of the invention there is provided the use of a peptide comprising a cholesterol binding motif to enhance the binding or uptake of a coacervate composition into a cell.

[0215] Suitably, the use of a peptide comprising a cholesterol binding motif is to increase the binding of a coacervate composition to a cell. Suitably, the use of a peptide comprising a cholesterol binding motif is to increase the uptake of a coacervate composition into a cell.

[0216] Suitably, the use of a peptide comprising a cholesterol binding motif is to increase the binding and uptake of a coacervate composition into a cell.

[0217] Suitably, the use of a peptide comprising a cholesterol binding motif is to increase the binding of a coacervate composition to a cell where increasing the uptake is not applicable, such as a red blood cell.

[0218] According to the sixth aspect of the invention there is provided a peptide comprising a cholesterol binding motif for use in enhancing the binding or uptake of a coacervate composition into a cell.

[0219] Suitably, the peptide comprising a cholesterol binding motif is for use in enhancing the binding of a coacervate composition to a cell.

[0220] Suitably, the peptide comprising a cholesterol binding motif is for use in enhancing the uptake of a coacervate composition into a cell.

[0221] Suitably, the peptide comprising a cholesterol binding motif is for use in enhancing the binding and uptake of a coacervate composition into a cell.

[0222] Suitably, the peptide comprising a cholesterol binding motif is for use in enhancing the binding of a coacervate composition into cell where increasing the uptake is not applicable, for example a red blood cell. According to the seventh aspect of the invention there is provided a composition for the delivery of an active agent that comprises or is comprised in a peptide coacervate composition, wherein the composition comprises one or more coacervate forming peptides, a peptide comprising a cholesterol binding motif, and an active agent recruited in the peptide coacervate.

[0223] The embodiments recited for the first aspect of the invention can be applied to this aspect also.

[0224] According to the eighth aspect of the invention there is provided a composition for the delivery of a diagnostic agent that comprises or is comprised in a peptide coacervate composition, wherein the composition comprises one or more coacervate forming peptides, a peptide comprising a cholesterol binding motif, and a diagnostic agent recruited in the peptide coacervate. The embodiments recited for the first aspect of the invention can be applied to this eighth aspect also.

[0225] The embodiments recited for the first aspect of the invention can be applied to the other aspect mutatis mutandis.

[0226] Additional applications of the compositions and methods will be identifiable by the person skilled in the art.

[0227] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Detailed Description, and Examples is hereby incorporated herein by reference.

[0228] The compositions and methods herein disclosed are further illustrated in the following statements and examples, which are provided by way of illustration and are not intended to be limiting the scope of the present disclosure.

[0229] Statements:

[0230] 1 . An ex vivo method for increasing the binding or uptake of a coacervate composition by one or more cells comprising contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding motif.

[0231] 2. The method according to statement 1 , wherein the cell(s) are contacted with the peptide comprising a cholesterol binding motif for a period of time before contacting with the coacervate composition.

[0232] 3. The method according to statement 2, wherein the cell(s) are contacted with the peptide comprising a cholesterol binding motif for at least 30 minutes, such as at least 40, 50, 60, 90, 120, 150 or more minutes, prior to contacting with the coacervate composition.

[0233] 4. The method according to statement 1 , wherein the peptide comprising a cholesterol binding motif is contacted for a period of time with the coacervate composition before contacting with the cell(s).

[0234] 5. The method according to any one of statements 1 - 4, wherein the cell(s) are contacted in a culture medium comprising the coacervate composition for at least 5 minutes, such as 15, 30, 60 or more minutes.

[0235] 6. The method according to any one of the preceding statements, wherein the increase in uptake of a coacervate composition by the one or more cells is an increase in cellular uptake of the coacervate composition by a population of cells. The method according to any one of the preceding statements, wherein the increase in uptake or binding efficiency is at least 20%, such as at least 30%, 40%, 50% or more, compared to uptake or binding efficiency in the absence of the peptide comprising a cholesterol binding motif or when using a control peptide. The method according to statement 1, wherein uptake efficiency is assessed by fluorescence-activated cell sorting analysis (FACS) and fluorescence microscopy. The method according to statement 7, wherein the control peptide is a scrambled peptide that lacks a functional cholesterol binding motif. The method according to statement 1 , wherein uptake efficiency is evaluated by FACS 10 minutes after contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding, such as after 15, 20, 30, 40, 50, 60 or more minutes. The method according to any one of the preceding statements, wherein the cholesterol binding motif is a CRAC motif, an inverted CRAC motif or a tilted domain motif. The method according to statement 11 , wherein the CRAC motif is V / L - [X]i-s- Y / W-[X]I-5-R / K, wherein X is any amino acid. The method according to statement 11 or 12, wherein the CRAC motif is V / L - [X]2-4- Y / W-[X]I-3-R / K. The method according to statement 11 , 12 or 13, wherein the CRAC motif is L- [X]2.4-Y-[X]1.3-R / K The method according to statement 11 , wherein the inverted CRAC motif is R / K- [X]I.5-Y / W-[X]I.5-V / L or R / K-[X]I.5-Y / F-[X]I.5-V / L, wherein X is any amino acid. The method according to statement 11 or 15, wherein the inverted CRAC motif is R / K-[X]I.3-Y-[X]2-4- / L, wherein X is any amino acid. The method according to statement 11 , wherein the peptide comprising a CRAC motif comprises one of the following sequences:

[0236] (i) FDRARMLEEYSKRFKKFGY (SEQ ID NO:23)

[0237] (ii) FDRARMVEEYSKRFKKFGY (SEQ ID NO:24)

[0238] (iii) FDRARMLEEWSKRFKKFGY (SEQ ID NO:25)

[0239] (iv) FDRARMLEEGYSKRFKKFGY (SEQ ID NO:26)

[0240] (v) FDRARMLEEYGSKRFKKFGY (SEQ ID NO:27)

[0241] (vi) ARMLEEYSKRFKKF (SEQ ID NO:28) The method according to statement 11 , wherein the peptide comprising the inverted CRAC motif comprises the sequence YGFKKFRKSYEELMRARDF (SEQ ID NO: 29) The method according to any one of the preceding statements, wherein the coacervate composition comprises one or more self-coacervating peptides derived from HBpep. The method according to statement 19, wherein at least one of the selfcoacervating peptides comprise the amino acid sequence (GHGX1Y)4(GX2GX3A)b(GHGLX4)c(GFA)d]i(GHGX1Y)awherein X1is valine (V), leucine (L) or proline (P);

[0242] X2is alanine (A) or proline (P);

[0243] X3is phenylalanine (F) or tyrosine (Y),

[0244] X4is leucine (L), histidine (H), tyrosine (Y) or glycine (G), each a is 0 or an integer >1 ; each b is 0 or an integer >1 ; each c is 0 or an integer >1 ; each d is 0 or an integer >1 ; f is an integer >1 ; with the sum of all a being >2 and the sum of all a+b+c+d is >4. The method according to statement 19, wherein at least one of the selfcoacervating peptide comprises the amino acid sequence: (GHGXY)n K (GHGXY)m Z,

[0245] (GHGXY K)n(GHGXY)m Z, or (GHGXY)n (K GHGXY)m Z, wherein X is valine (V), leucine (L) or proline (P), Z is tryptophan (W) or absent, n is 0, 1 , 2, 3, 4 or 5, m is 0, 1 , 2, 3, 4 or 5, n+m is 3, 4 or 5, preferably 5. The method according to any one of the preceding statements, wherein the coacervate composition comprises a payload. The method according to statement 22, wherein the payload is a protein or peptide, a nucleic acid, or a small molecule compound. The method according to statement 22 or 23, wherein the payload is an antibody or a fragment thereof. The method according to any one of the preceding statements, wherein the peptide comprising a cholesterol binding motif is at least 8 amino acids in length. The method according to any one of the preceding statements, wherein the peptide comprising a cholesterol binding motif, optionally a CRAG or inverted CRAC motif, is from 10 - 30 amino acids in length, such as between 12 and 20 amino acids in length. The method according to statement 1 , wherein the peptide comprising a cholesterol binding motif is an affine non-cytotoxic cholesterol-binding peptide (CBP). The method according to any one of the preceding statements, wherein the peptide comprising a cholesterol binding motif is mixed with self-coacervating peptides prior to formation of the coacervate composition so that when formed the coacervate composition comprises the peptide comprising a cholesterol binding motif. The method according to statement 28, wherein the peptide comprising a cholesterol binding motif and the self-coacervating peptides are mixed in a 1 :10 — 1 :50 mass ratio prior to coacervate formation. The method according to any one of statements 1 - 27, wherein the peptide comprising a cholesterol binding motif is mixed with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition. The method according to any one of the preceding statements, wherein the peptide comprising a cholesterol binding motif is contacted with the cell(s) by adding the peptide comprising a cholesterol binding motif to culture medium comprising the cell(s), optionally wherein the peptide comprising a cholesterol binding motif is added to the culture medium at a concentration of at least 20 ug / ml. The method according to statement 31 , wherein the cell(s) and peptide comprising a cholesterol binding motif are incubated for at least 10 minutes, such as at least 15 min or 30 min, before adding the coacervate composition. The method according to statement 32, wherein the cell(s), peptide comprising a cholesterol binding motif and coacervate composition are incubated for at least 10 minutes, such as at least 15, 30, 60, 90, 120 or more minutes. The method according to any one of the preceding statements, wherein the cell(s) is / are eukaryotic cell(s), such as mammalian cell(s). The method according to any one of the preceding statements, wherein the cell(s) is / are human cell(s), such as red blood cell(s). A coacervate composition comprising at least one peptide comprising a cholesterol binding motif. The coacervate composition according to statement 36, wherein the at least one peptide comprising a cholesterol binding motif is formed into the coacervates in the coacervate composition. The coacervate composition according to statement 36, wherein the at least one peptide comprising a cholesterol binding motif is coated on the surface of the coacervates and / or bound to the surface of the coacervates in the coacervate composition. The coacervate composition according to any one of statements 36-38, wherein the coacervates therein comprise a therapeutic or diagnostic payload. The coacervate composition according to statement 39, wherein the payload is a protein or peptide, a nucleic acid, or a small molecule compound. The coacervate composition according to statement 40, wherein the payload is an antibody or a fragment thereof or a vaccine. The coacervate composition according to any one of statements 36 to 41 , wherein the cholesterol binding motif is a CRAC motif, an inverted CRAC motif or a tilted domain motif. The coacervate composition according to statement 42, wherein the CRAC motif is V / L - [X]i-5- Y / W-[X]i-s-R / K, wherein X is any amino acid. The coacervate composition according to statement 42 or 43, wherein the CRAC motif is V / L - [Xk-4- Y / W-[X]I-3-R / K, wherein X is any amino acid. The coacervate composition according to statement 42, 43 or 44, wherein the CRAC motif is L-[X]2-4-Y-[X]I-3-R / K, wherein X is any amino acid. The coacervate composition according to statement 42, wherein the inverted CRAC motif is R / K-[X]I.5-Y / W-[X]I.5-V / L or R / K-[X]I.5-Y / F-[X]I.5- / L, wherein X is any amino acid. The coacervate composition according to statement 42 or 46, wherein the inverted CRAC motif is R / K-[X]I-3-Y-[X]2-4-V / L, wherein X is any amino acid. The coacervate composition according to statement 42, wherein the peptide comprising a CRAC motif comprises one of the following sequences:

[0246] (i) FDRARMLEEYSKRFKKFGY (SEQ ID NO:23)

[0247] (ii) FDRARMVEEYSKRFKKFGY (SEQ ID NO:24)

[0248] (iii) FDRARM LEEWSKRFKKFGY (SEQ I D NO:25)

[0249] (iv) FDRARM LEEGYSKRFKKFGY (SEQ ID NO:26)

[0250] (v) FDRARM LEEYGSKRFKKFGY (SEQ ID NO:27)

[0251] (vi) ARMLEEYSKRFKKF (SEQ ID NO:28) The coacervate composition according to statement 42, wherein the peptide comprising the inverted CRAC motif comprises the sequence YGFKKFRKSYEELMRARDF (SEQ ID NO: 29) The coacervate composition according to any one of statements 36 to 49, wherein the coacervate composition comprises one or more self-coacervating peptides derived from HBpep. The coacervate composition according to any one of statements 36 to 50, wherein at least one of the self-coacervating peptides comprise the amino acid sequence (GHGX1Y)a[(GX2GX3A)b(GHGLX4)c(GFA)d]:(GHGX1Y)a wherein X1is valine (V), leucine (L) or proline (P);

[0252] X2is alanine (A) or proline (P);

[0253] X3is phenylalanine (F) or tyrosine (Y);

[0254] X4is leucine (L), histidine (H), tyrosine (Y) or glycine (G), each a is 0 or an integer >1 ; each b is 0 or an integer >1 ; each c is 0 or an integer >1 ; each d is 0 or an integer >1 ; f is an integer >1 ; with the sum of all a being >2 and the sum of all a+b+c+d is >4. The coacervate composition according to any one of statements 36 - 51, wherein at least one of the self-coacervating peptide comprises the amino acid sequence: (GHGXY)nK (GHGXY)m Z,

[0255] (GHGXY K)n (GHGXY)m Z, or (GHGXY)n (K GHGXY)m Z, wherein X is valine (V), leucine (L) or proline (P), Z is tryptophan (W) or absent, n is 0, 1 , 2, 3, 4 or 5, m is 0, 1 , 2, 3, 4 or 5, n+m is 3, 4 or 5, preferably 5, The coacervate composition according to any one of statements 36 to 52, wherein the coacervate composition comprises a payload. The coacervate composition according to statement 53, wherein the payload is a protein or peptide, a nucleic acid, or a small molecule compound. The coacervate composition according to statement 53 or 54, wherein the payload is an antibody or a fragment thereof, or a vaccine. The coacervate composition according to any one of statements 36 to 55, wherein the peptide comprising a cholesterol binding motif is at least 8 amino acids in length. The coacervate composition according to any one of statements 36 to 56, wherein the peptide comprising a cholesterol binding motif, optionally a CRAC or inverted CRAC motif, is from 10 - 30 amino acids in length, such as between 12 and 20 amino acids in length. The coacervate composition according to one of statements 36 to 57, wherein the peptide comprising a cholesterol binding motif is an affine non-cytotoxic cholesterol-binding peptide (CBP). A method for producing a coacervate composition according to one of statements 36 to 58, the method comprising mixing the peptide comprising a cholesterol binding motif with the self-coacervating peptides prior to formation of the coacervate composition so that when formed the coacervate composition comprises the peptide comprising a cholesterol binding motif. The method according to statement 59, wherein the peptide comprising a cholesterol binding motif and the coacervating peptide are mixed in a 1 :10 - 1 :50 mass ratio prior to coacervate formation. A method for producing a coacervate composition according to any one of statements 36 to 58, the method comprising mixing a peptide comprising a cholesterol binding motif with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition. Use of a peptide comprising a cholesterol binding motif to enhance the uptake or binding of a coacervate composition into a cell. A peptide comprising a cholesterol binding motif for use in enhancing the uptake of a coacervate composition into a cell. A peptide comprising a cholesterol binding motif for use in enhancing the binding of a coacervate composition to a cell. The coacervate composition according to any one of statements 36 - 58, for use in therapy. The coacervate composition according to any one of statements 36 - 58, for use in diagnosis. EXAMPLES

[0256] The inventors have found that adhesion of coacervates was cholesterol dependent. Furthermore, that treating cells with the cholesterol-depleting molecule MpCD (5 mM for 50 min prior to addition of coacervates), abolished coacervate uptake with the cell membrane unable to engulf the coacervates. Cholesterol is an integral component of eukaryotic cell membranes and is a key molecule controlling membrane fluidity. Cholesterol concentration within the cell membranes can range anywhere from < 5 mol% in mitochondrial membranes to > 25 mol% in plasma membranes (van Meer et al., Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112-124, 2008). To evaluate the role of cholesterol on coacervate attachment, Giant Unilamellar Vesicles (GUVs) prepared from the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and containing 10 to 50 mol% cholesterol were incubated with HBpep coacervates. Interestingly, HBpep coacervates exhibited an appreciable increase in attachment only for POPC GUVs containing 20 mol% cholesterol (0.75 ± 0.19 coacervates / pm of GUV), whereas the attachment to POPC GUVs containing 10, 30, 40 and 50 mol% cholesterol decreased. At higher cholesterol levels (above 30%), coacervate attachment decreased to below 0.40 / pm, suggesting that cholesterol plays a key role in coacervate- mem brane binding.

[0257] Since the findings from GUV data indicated that the adhesion of coacervates was cholesterol-dependent, the inventors hypothesized that adding a cholesterol-binding molecule within the coacervates would enhance their uptake. The inventors designed coacervates containing recruited cholesterol-binding peptide and examined their uptake and progressive internalization with high-temporal resolution. The inventors chose a CRAC peptide (with sequence L / VX(i-5)YX(i-5)K / R, where X<i-5) represents a motif containing 1 to 5 of any residues) (Koufos et al., Use of a Cholesterol Recognition Amino Acid Consensus Peptide To Inhibit Binding of a Bacterial Toxin to Cholesterol. Biochemistry 55, 4787-4797, 2016) because it has been reported to bind cholesterol with a high affinity, does not disrupt lipid packing, and is not cytotoxic.

[0258] The peptide coacervate delivery system was prepared from HBpep, or HBpep-SP peptide, loaded with the enhanced green fluorescent protein (EGFP). HBpep and HBpep-SP peptides are simple coacervates enriched in histidine (His) and tyrosine (Tyr) residues, in addition to bearing one tryptophan (Trp) at the C-terminus. HBpep does not bear other charged residues (Table 1) but HBpep-SP has one extra lysine (Lys) that is conjugated with a redox- responsive and self-immolative moiety (Sun et al., Nat. Chem. 14, 274-283, 2022). An affine noncytotoxic peptide bearing a cholesterol recognition / amino acid consensus (CRAC) sequence (Table 1) (Koufos et al. Biochemistry 55, 4787-4797, 2016) was added to (mixed with) HBpep or HBpep-SP peptides before coacervate preparation in a 1 :10-1 :60 mass ratio, or directly in the cell culture media in a 40 or 60 mg / ml concentration, before transfecting HeLa cells with the coacervates loaded with EGFP. To verify the presence of CRAG peptide in the coacervates, CRAG labelled with the red fluorophore TAMRA was mixed with 10 mg mL“1of HBpep-SP (at various ratios from 1 :10 - 1:60, typically 1:25 mass ratio) in 10 mM acetic acid and pipetted into phosphate buffer (6.5 pH, 100 mM ionic strength) containing EGFP at a volume ratio of 1 :9. Fluorescence microscopy visualized TAMRA-labelled CRAC peptide encapsulation and confirmed uniform distribution within the coacervates. Cellular uptake efficiency of EGFP-loaded HBpep and HBpep-SP coacervates with the CRAC peptide was assessed using fluorescence-activated cell sorting analysis (FACS) and fluorescent microscopy after a short time, namely 15 or 30 min incubation. Cells were briefly washed with 0.5 M 1-Ethyl-3-methylimidazolium chloride [EMIM]CI to dissolve coacervates bound to the outer cell membrane but not internalized. As a control, the uptake efficiency was also evaluated for the coacervates with a control CRAC peptide wherein the consensus cholesterol binding sequence was scrambled so as to not have affinity to cholesterol.

[0259] Table 1. The amino acid sequence of HBpep, HBpep-SP, CRAC peptide, CRAC scrambled, CRAC-14 (shorter peptide), CRACL / V peptides.

[0260] Adding CRAC peptide to HBpep coacervates significantly increased cellular uptake (by at least 50%) as evaluated by FACS within 30 min incubation time (Figure 1). In contrast, adding a peptide with a scrambled CRAC sequence did not increase uptake. These data demonstrate that the uptake increase is sequence-specific and is most likely due to enhanced interaction of the CRAC-loaded coacervates with cholesterol and increased membrane binding.

[0261] On fluorescent micrographs, more HBpep coacervates were observed to be associated with cells when adding CRAC peptide (Figure 2).

[0262] FACS analysis of GFP-containing HBpep-SP coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC peptide. In a control experiment where the CRAC sequence was scrambled, no effect on cell uptake was observed, confirming that cholesterol binding plays a key role in mediating coacervate uptake (Figure 3)

[0263] FACS analysis of cells treated with GFP-containing HBpep-SP coacervates after just a 15 min incubation time also showed an accelerated uptake with the CRAC peptide recruited within the coacervates (Figure 4).

[0264] A similar effect was observed when the CRAC peptide was added directly to the cell culture media in a 40 pg / ml concentration before adding the coacervates (Figure 5). This result can be explained by the adsorption of the CRAC peptide on the surface of the coacervates.

[0265] FACS analysis of HBpep-SP coacervates internalization after 30 min of incubation time, demonstrating an increase in uptake with the addition of CRAC-14 peptide (Figure 6). Here the CRAC-14 peptide was recruited within the coacervate.

[0266] FACS analysis demonstrating an increase in HBpep-27W coacervates internalization with the addition of CRACLA / peptide versus the CRAC scrambled sequence after 30 min of incubation (Figure 7). It is also possible to absorb the CRAC peptide to already formed coacervates, by incubating the coacervate composition with CRAC peptide for a suitable period of time before contacting with the cells.

[0267] Furthermore, the CRAC peptide did not affect the uptake of Alexa 488-transferrin, which is known to enter cells by clathrin-dependent endocytosis (Figure 8).

[0268] These results indicate that cholesterol-mediated adhesion to the bilayer - which can have an additive effect to specific adhesion caused by ligand-receptor interactions - improves coacervate uptake and that enhancing binding to the cell membrane may be an efficient strategy to optimize cell uptake for therapeutic applications.

[0269] To further confirm the internalization of HBpep-SP coacervates with the added CRAC peptide, the inventors used Focus Ion Beam (FIB)-SEM. FIB-SEM combines the benefits of TEM and SEM, enabling the visualization of intracellular structure and the acquisition of volume information through serial sectioning. After 3 h of incubation of HeLa cells with HBpep-SP coacervates loaded with both CRAC and EGFP, FIB-SEM imagery showed internalized and partially disassembled coacervates inside the cells (Figure 9).

[0270] To dynamically visualize internalization of HBpep-SP coacervates with the added CRAC peptide, the inventors performed live cell imaging using tubulin imaging dyes. HeLa cells of 3 x 105were seeded in 35 mm glass bottom dishes (Mattel, USA) and cultured for 24 h and stained with tubulin stain (Spirochrome, Germany) for 2 h, then rinsed four times with PBS. The PBS was replaced with live cell imaging solution (Thermofisher scientific, USA), and the dishes were mounted in the observation chamber with temperature and CO2 control. HBpep- SP coacervates were loaded to the dishes and kept for 2 min before imaging until coacervates settled on the cell surfaces. Time-lapse Z-stack images were then collected with 5-min time interval for 20 or 30 min across the cell with 0.3 urn slices, using high-speed spinning-disc confocal microscope (Nikon Ti2-E) equipped with a 100 x 1.45NA PlanApo objective lens and an ORCA-Fusion sCMOS camera (Hamamatsu Photonics). GFP, Cy5, and mCherry fluorescence channels were used. Images were processed using Image J (NIH, USA) software. The HBpep-SP coacervates with the CRAC peptide displayed a variety of behaviours, where the majority underwent fast uptake at the cell periphery and their movements were observed within the cells (Figure 10).

[0271] These studies show that by increasing the adhesion of HBpep-SP coacervates to the cell membrane by recruiting a cholesterol binding peptide within the coacervates enables modulating cellular uptake. In summary, the results demonstrate that adding various CRAC peptide sequences to a coacervate delivery system can improve cellular uptake through the enhanced binding to the membrane. This method can be applied to other coacervate delivery systems, particularly weakly charged and weakly adhering to the cell membrane, and lead to more efficient cell delivery systems.

[0272] MATERIALS AND METHODS

[0273] Peptides. HBpep was purchased from GL Biochem (Shanghai) Ltd, China, and subjected to an additional purification step by High Performance Liquid Chromatography (HPLC) using a C8 column. HBpep-SP was synthesized according to the protocol described previously (Sun et al., Nat. Chem. 14, 274-283, 20222). To prepare the coacervates, 10 mg / mL of HBpep or HBpep-SP in 10 mM acetic acid was pipetted into phosphate buffer (pH 6.5, 100 mM ionic strength) at a volume ratio of 1 :9 as previously described (Sun et al., MRS Bull. 45:1039- 1047, 2020). CRAC and CRAC scrambled peptides were synthesized in a solid-phase peptide synthesizer (CEM Liberty Blue) using general protocols. Firstly, Wang resin, which supports the growth of peptide, was swollen in N,N’-dimethylformamide (DMF) for 30 mins before being transferred into the reaction vessel. Fluorenylmethyloxycarbonyl (Fmoc) protected peptides, activator N,N’-dimethylcarboimide (DIC), activator base ethyl cyanohydroxyiminoacetate (oxyma), and piperidine were dissolved in DMF, and transferred into the synthesizer. Peptide and piperidine were injected into a heated reaction vessel to remove Fmoc functional group. Then DIC and oxyma were injected, and heat was applied to the reaction vessel to couple the deprotected amino acid with peptide chain. The two steps repeated until all amino acids were connected. After synthesis, the resin was washed by dichloromethane (DCM) and DMF. Then a cocktail solution containing 95% trifluoric acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% H2O was added into the resin to cleave the peptides from the resin. The resin was soaked in the cocktail for 2 hours, and cold diethyl ether was added to precipitate peptides. The precipitates were collected after centrifugation. After drying in nitrogen, they were dissolved in 5% acetic acid and purified by high- performance liquid chromatography (HPLC). The products were isolated by lyophilization.

[0274] Giant unilamellar vesicles (GUVs) preparation for studies of coacervate attachment with varying lipid charge and cholesterol. GUVs were prepared via gel-assisted formation on PVA. Briefly, 5% (w / w) solution of Polyvinyl alcohol (PVA) was prepared by stirring PVA in water at 90°C. 100 pL of PVA solution was added onto an ozone-cleaned microscope coverslip, which was then dried in an oven at 50°C for 45 min. 25 pL of lipids (1 mg / mL) was subsequently spread onto the dried PVA film and placed under vacuum for 30 min until the solvent evaporated. 350 pL of sucrose solution (187 mM) was added to the PVA film and incubated for 45 min to allow GUV formation. The GUVs were pipetted into an Eppendorf tube and stored at 4°C until further use. For coacervates- GUV interaction studies, 5 pL of the GUV solution was added to 200 L of phosphate buffer and incubated for 5 min at room temperature. 20 pL of EGFP-loaded coacervates was then added to the GUVs for interaction studies. Fluorescence microscopy images were acquired using a Delta vision elite inverted epifluorescence microscope with Olympus IX-71 base fitted with 10x / 0.40, 20x / 0.75, 40x70.65-1.35 oil objectives (Olympus, Tokyo, Japan), DAPI, TRITC and FITC Semrock filters (New York, NY), a mercury lamp (Intensilight C-HGFIE, Nikon Corporation, Tokyo, Japan), and a high-precision motorized stage. Images were collected using Softworx 4.1.0 (Applied Precision, Inc., Issaquah, WA) and processed using ImageJ.

[0275] Cell culture. HeLa and HepG2 cells (ATCC, USA) were cultured on DMEM (Gibco) and EMEM (ATCC) media supplemented with 10% FBS and penicillin / streptomycin solution (Gibco) in a humidified atmosphere at 37C° and 5% CO2. Cells were routinely tested for mycoplasma using Mycostrip kit (Invivogen).

[0276] Coacervate preparation for cell uptake experiments. For cell uptake experiments, reduced serum Optimem media (Gibco) was used. Coacervates were formed by mixing one part of peptide stock solution in 10 mM acetic acid (10 mg / ml for HBpep-SP or 20 mg / ml for HBpep) with 9 parts of 10 mM sodium phosphate buffer with 100 mM sodium chloride, pH 7.5 for HBpep and 6.5 for HBpep-SP containing EGFP, 0.1 mg / ml concentration. The media in cell culture dishes or flasks were replaced with Optimem, and coacervate mixtures were gently pipetted in. For HBpep, the final peptide concentration in Optimem was 0.2 or 0.4 mg / ml, for HBpep-SP and HBpep-GW27 - 0.1 mg / ml. To prepare the coacervates with CRAC peptide, 10 mg / mL HBpep-SP mixed with CRAC (1 :25 mass ratio) in 10 mM acetic acid was pipetted into phosphate buffer (6.5 pH, 100 mM ionic strength) containing EGFP at a volume ratio of 1 :9.

[0277] Cell uptake analysis.

[0278] Fluorescence-activated cell sorting (FACS). Cells were seeded in 12 well or 24 well plates (1.5*105or 1*105cells / well) and grown for 24 h. For coacervate uptake studies, the media was substituted with Optimem. Then, HBpep or HBpep-SP coacervates loaded with CRAC peptides and EGFP were added (the final peptide concentration in Optimem for HBpep-SP - 0.05 mg / ml or 0.1 mg / ml) and incubated for specified amount of time. In the case when CRAC peptide was added to the cell culture media, cells were pre-incubated with CRAC peptide for specified amount of time followed by incubation with EGFP-loaded coacervates. Cells were washed trice with cold PBS to remove the non-internalized coacervates, detached with Accutase (Invitrogen, USA), washed with FACS buffer (PBS containing 2% FBS), resuspended in 350 pl FACS buffer. FACS analysis was performed on BD LSRFortessa X-20 Cll Analyzer (BD Biosciences, USA). The data were processed in Flojo software (Flojo, USA). Fluorescent microscopy. For fluorescent imaging, the cells were grown on coverslips in 12-well plates, treated with coacervates containing CRAC peptide and EGFP as described above, fixed with 4% paraformaldehyde, and observed by fluorescent microscope (Nikon).

Claims

CLAIMS1. An ex vivo method for increasing the binding or uptake of a coacervate composition by one or more cells comprising contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding motif.

2. The method according to claim 1, wherein the cell(s) are contacted with the peptide comprising a cholesterol binding motif for a period of time before contacting with the coacervate composition.

3. The method according to claim 1 , wherein the peptide comprising a cholesterol binding motif is contacted with the coacervate composition for a period of time before contacting with the cell(s).

4. The method according to any one of claims 1 to 3, wherein the increase in uptake or binding of a coacervate composition by the one or more cells is an increase in cellular uptake of the coacervate composition by a population of cells, optionally wherein the increase in uptake efficiency is at least 20%, such as at least 30%, 40%, 50% or more, compared to uptake efficiency in the absence of the peptide comprising a cholesterol binding motif or when using a control peptide.

5. The method according to any one of claims 1 to 4, wherein uptake efficiency is evaluated by FACS at least 10 minutes after contacting the cell(s) with the coacervate composition and a peptide comprising a cholesterol binding motif, such as after 15, 20, 30, 40, 50, 60 or more minutes.

6. The method according to any one of claims 1 to 3, wherein the binding of the coacervate composition by one or more cells is increased.

7. The method according to any one of claims 1 to 3 or 6, wherein the binding of the coacervate composition by one or more cells is increased in cells where increasing the uptake is not applicable.

8. The method according to any one of the preceding claims, wherein the cholesterol binding motif is a CRAG motif, an inverted CRAC motif or a tilted domain motif.

9. The method according to claim 8, wherein the CRAC motif is V / L - [X]i-5- Y / W-[X]i-s-R / K, wherein X is any amino acid.

10. The method according to claim 8, wherein the inverted CRAC motif is R / K-[X]I-5-Y / W-[X]I. s-V / L or R / K-[X]I-5-Y / F-[X]I-5-V / L, wherein X is any amino acid.

11. The method according to any one of the preceding claims, wherein the coacervate composition comprises one or more self-coacervating peptides derived from HBpep.

12. The method according to any one of the preceding claims, wherein at least one of the self-coacervating peptides comprise the amino acid sequence(GHGX1Y)a[(GX2GX3A)b(G HG LX4)C(G FA)d]f(G H GX1Y)awherein X1is valine (V), leucine (L) or proline (P);X2is alanine (A) or proline (P);X3is phenylalanine (F) or tyrosine (Y):X4is leucine (L), histidine (H), tyrosine (Y) or glycine (G), each a is 0 or an integer >1 ; each b is 0 or an integer >1 ; each c is 0 or an integer >1 ; each d is 0 or an integer >1 ; f is an integer >1; with the sum of all a being >2 and the sum of all a+b+c+d is >4.

13. The method according to any one of claims 1 - 12, wherein at least one of the selfcoacervating peptides comprises the amino acid sequence:(GHGXY)n K (GHGXY)m Z, (GHGXY K)n(GHGXY)m Z, or (GHGXY)n (K GHGXYJm Z, wherein X is valine (V), leucine (L) or proline (P), Z is tryptophan (W) or absent, n is 0, 1 , 2, 3, 4 or 5, m is 0, 1, 2, 3, 4 or 5, n+m is 3, 4 or 5, preferably 5.

14. The method according to any one of the preceding claims, wherein the coacervate composition comprises a payload, optionally wherein the payload is a protein or peptide,an antibody or a fragment thereof, a nucleic acid, a small molecule compound or a vaccine15. The method according to any one of the preceding claims, wherein the peptide comprising a cholesterol binding motif is at least 8 amino acids in length, such as from 10 - 30 amino acids in length, such as from 12 - 20 amino acids in length, inclusive.

16. The method according to any one of the preceding claims, wherein the peptide comprising a cholesterol binding motif is mixed with self-coacervating peptides prior to formation of the coacervate composition so that when formed the coacervate composition comprises the peptide comprising a cholesterol binding motif, optionally wherein the peptide comprising a cholesterol binding motif and the self-coacervating peptides are mixed in a 1:10 - 1:60 mass ratio prior to coacervate formation.

17. The method according to any one of claims 1 to 16, wherein the peptide comprising a cholesterol binding motif is mixed with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervates in the coacervate composition.

18. The method according to any one of the preceding claims, wherein the cell(s) is / are eukaryotic cell(s), such as mammalian cell(s), optionally wherein the cell(s) is / are human cell(s); further optionally wherein the cell(s) is / are red blood cell(s).

19. A coacervate composition comprising at least one peptide comprising a cholesterol binding motif.

20. The coacervate composition according to claim 19, wherein the at least one peptide comprising a cholesterol binding motif is formed into the coacervates in the coacervate composition.

21. The coacervate composition according to claim 19, wherein the at least one peptide comprising a cholesterol binding motif is coated on the surface of the coacervates and / or bound to the surface of the coacervates in the coacervate composition.

22. The coacervate composition according to any one of claims 19 to 21 , wherein the cholesterol binding motif is a CRAC motif, an inverted CRAC motif or a tilted domain motif.

23. The coacervate composition according to any one of claims 19 to 22, wherein the coacervate composition comprises a payload, optionally wherein the payload is a protein or peptide, an antibody or a fragment thereof, a nucleic acid, a small molecule compound or a vaccine.

24. A method for producing a coacervate composition according to one of claims 19 to 23, the method comprising mixing the peptide comprising a cholesterol binding motif with self-coacervating peptides prior to formation of the coacervate composition so that when formed the coacervate composition comprises the peptide comprising a cholesterol binding motif.

25. A method for producing a coacervate composition according to any one of claims 19 to 23, the method comprising mixing a peptide comprising a cholesterol binding motif with a preformed coacervate composition so that the peptide comprising a cholesterol binding motif attaches to the surface of the coacervate particles in the coacervate composition.

26. Use of a peptide comprising a cholesterol binding motif to enhance the binding of a coacervate composition to a cell or the uptake of a coacervate composition into a cell.

27. Use of a peptide comprising a cholesterol binding motif to enhance the binding of a coacervate composition to a cell into a cell where binding is not applicable.

28. The use of claim 27, wherein the cell is a red blood cell.

29. The coacervate composition according to any one of claims 19 to 23, for use in therapy or diagnosis.

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