Apolipoprotein-based hydrogels

Abstract

The invention relates to novel molecules capable of forming hydrogels, which comprise a peptide-based portion at least part of which is capable of forming an amphipathic alpha-helix, and a hydrogel-stabilizing portion. The invention also relates to hydrogels formed thereby, compositions comprising the hydrogels, and associated methods and uses.

Description

[0001] APOLIPOPROTEIN-BASED HYDROGELS

[0002] FIELD OF THE INVENTION

[0003] The invention is broadly in the field of self-assembling hydrogels and uses thereof, and in particular relates to the self-assembly and hydrogelation of proteins or peptides, and their therapeutic applications, such as local drug delivery, controlled drug release, wound healing, tissue engineering, or vaccination.

[0004] BACKGROUND

[0005] Hydrogels are three-dimensional networks formed from supramolecular mono- or polymers that retain significant amounts of water. These materials have demonstrated potential in various therapeutic applications, including local drug delivery, controlled drug release, wound healing, tissue engineering, and vaccination.

[0006] The current landscape of hydrogel technologies is diverse, encompassing platforms based on different materials and mechanisms. Polymer-based hydrogels, derived from either natural polymers like alginate, chitosan, and hyaluronic acid or synthetic polymers like polyethylene glycol (PEG) and polyacrylamide, are among the most widely used. While versatile, PEG and similar polymeric materials present known issues, including toxicity and anti-PEG immune responses. Additionally, these hydrogels often face challenges in fine-tuning their mechanical properties and degradation profiles for specific applications. Small organic molecule gelators represent another category, relying on low-molecular-weight compounds that self-assemble into gel networks. Although these materials offer unique responsiveness to external stimuli, their scalability and biocompatibility remain limiting factors. Inorganic hydrogels, typically formed from metal oxides or other inorganic compounds, exhibit excellent mechanical strength but suffer from limited biocompatibility and lack a desired degradation mechanism, restricting their therapeutic utility.

[0007] Emerging hydrogel platforms include DNA-based hydrogels, which provide highly specific molecular recognition capabilities. However, mechanical and thermal stability may not be sufficient for certain therapeutic applications. Additionally, the incorporation of therapeutic payloads within a DNA-hydrogel network poses significant challenges. Peptide-based hydrogels, constructed from short peptides that self-assemble into nanofibrous networks, hold promise for biomedical applications due to their biocompatibility and tunability. However, many peptide-based hydrogels rely on unnatural aromatic groups or beta-sheet-forming sequences, which can limit their applicability.

[0008] While significant advancements have been made in hydrogel technology, existing platforms fail to address the combined requirements of biocompatibility, tunability, and broadapplicability across diverse tissues and therapeutic modalities. This highlights the urgent need for innovative hydrogel systems that can overcome these limitations and provide transformative solutions for local drug delivery, controlled release, and immunotherapeutic applications.

[0009] SUMMARY

[0010] A first aspect of the invention provides a molecule capable of self-assembling into a three-dimensional network to form a hydrogel, wherein said molecule comprises:

[0011] a) a peptide-based portion at least part of which is capable of forming an amphipathic alpha-helix; and

[0012] b) a hydrogel-stabilizing portion wherein the hydrogel-stabilizing portion promotes the formation of the hydrogel, wherein the hydrogel formation involves intermolecular interaction between the amphipathic alpha-helices.

[0013] A further aspect provides a hydrogel comprising the molecule.

[0014] Another aspect provides a pharmaceutical or cosmetic composition comprising the molecule or the hydrogel.

[0015] A further aspect provides the molecule, the hydrogel or the pharmaceutical composition for use as a medicament.

[0016] A further aspect provides the molecule, the hydrogel, or the pharmaceutical composition for use in vaccination, transplantation and / or the treatment of a disease, such as a neoplastic disease or an infectious disease.

[0017] A further aspect provides a cosmetic use of the molecule, the hydrogel, or the cosmetic composition.

[0018] A further aspect provides in vitro or ex vivo use of the molecule or the hydrogel in cell or tissue culture.

[0019] These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.Fig. 1. Overview of N-terminal modifications on the 2F peptide mimetic that were synthesized and tested in the initial screening approach (A) and gelation heatmap upon gelation of different alkyl N-terminal derivatizations at 0.5-4 w / v% peptide concentration in 1 M Phosphate buffer (pH 7.4) (B) Subset of unsaturated derivatizations at 0.5-4 w / v% peptide concentration in 1 M Phosphate buffer (pH 7.4) (C) Subset of amino acid based derivatizations at 0.5-4 w / v% peptide concentration in 1 M Phosphate buffer (pH 7.4) (D) Fig. 2. Micrograph of C6-2F, C14-2F, C18-2F and C22-2F at 500 pM concentration, at 28.000x magnification (top) and 92.000x magnification (bottom) forming fibers with approximately 4 nm diameter.

[0022] Fig. 3. Rheology measurement based on time sweep for 24 hours (plateau reached) of derivatized peptides at 4 w / v% in 1.0 M Phosphate buffer at 21 °C.

[0023] Fig. 4. Overview of modification in 2F sequence, with modification 2 illustrated (top) and relevance of positioning depicted in the helical wheel diagram of 2F (bottom)

[0024] Fig 5. Gelation heatmap of a subset of ApoA-1 mimetic peptides and their derivatizations at 0.5-4 w / v% peptide concentration in 1M phosphate buffer (pH 7.4).

[0025] Fig. 6. Cumulative release of dye, peptide-dye, derivatized peptide-dye or protein (mVenus) from 2w / v% C18 gel (top), 4 w / v% C18 gel (middle) and 2 w / v% Fmoc gel (bottom).

[0026] Fig. 7. PET-CT images of mice injected with89Zr-DFO(-2F) either subcutaneously or intraperitoneally. Imaged at 0 and 48 hours post injection. Subcutaneous gels show barely any release of payload after 48 hours, whereas intraperitoneal injections shows gradual release and uptake in the lymph nodes.

[0027] Fig. 8. % of dose remaining at injection site after 48hours (top) and biodistribution after 48 hours (bottom)

[0028] Fig. 9. (A) Average tumor growth of B16F10 tumor model over time of PBS and empty gel control groups compared to local and distant injection of 10 ug 2F-rapamycin in 2 w / v% C18-2F gels. (B) corresponding survival over time of control and treatment groups.

[0029] DESCRIPTION OF EMBODIMENTS

[0030] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0031] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes”, “containing”, or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms alsoencompass “constituted of”, “consists in”, “consisting of”, and “consists of”, and also the terms “consisting essentially of”, “consisting essentially in” and “consists essentially of”, which enjoy well-established meanings in patent terminology.

[0032] The recitation of numerical ranges by endpoints includes all intervening values between the lower and upper endpoints, as well as the recited endpoints. Intervening values may be integers or, where applicable, fractions, i.e., more broadly any real numbers such as any rational numbers. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to...” or the expression “between... and...” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, each sub-range between any stated value in a stated range and any other stated value in that stated range is also specifically disclosed. Each sub-range between any stated value in a stated range and either the lower endpoint or the upper endpoint of the stated range is also specifically disclosed. The stated value may be an isolated value or an endpoint of a range subsumed by or overlapping with the stated range. For example, for a stated range with lower endpoint L1 and upper endpoint U1 (i.e., stated range L1-LI1) and a stated sub-range nested within the stated range with lower endpoint L2 and upper endpoint U2 (i.e., stated sub-range L2-LI2), also specifically disclosed are the subranges L1-L2, L1-U2, L2-U1, and U2-U1.

[0033] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0034] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0035] Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear perse, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to allsaid members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0036] As used herein, the term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0037] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0038] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0039] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. , also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0040] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0041] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner,as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0042] Similarly, it should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0043] In the present description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. Parenthesized or emboldened reference numerals affixed to respective elements merely exemplify the elements by way of example, with which it is not intended to limit the respective elements. Unless otherwise indicated, all figures and drawings in this document are not to scale and are chosen for the purpose of illustrating different embodiments of the invention. In particular the dimensions of the various components are depicted in illustrative terms only, and no relationship between the dimensions of the various components should be inferred from the drawings, unless so indicated.

[0044] The inventors provide a novel hydrogel platform based on amphipathic alpha-helix forming proteins or peptides.

[0045] The hydrogels of the present invention are highly versatile, can entrap any kind of payload, ranging from small molecule drugs to peptide drugs, proteins such as interleukins and cytokines, and RNAs, including mRNA, but also nanomedicines (e.g., apolipoprotein nanoparticles), and can be used for several therapeutic applications, including, but not limited vaccination, local drug release (e.g., localized cytokine therapy or local delivery of checkpoint inhibitor antibodies), and systemic exposure via endogenous lipoproteins. The hydrogels of the present invention can be used to improve drugs’ safety profiles, efficacy and applicability beyond their current use. Moreover, the payload can be chemically modified with an apolipoprotein or apolipoprotein mimetic, such as an apoA1 peptide, which improves its incorporation in the hydrogel. Moreover, the additional advantage of modifying the payload with an apolipoprotein or apolipoprotein mimetic, such as an apoA1 peptide, is the affinity for the endogenous lipoprotein fraction in a subject’s body. Therefore, the present hydrogels canoptionally make their payloads “hitch hike” to the myeloid compartment following their release.

[0046] In view thereof, a first aspect of the invention provides a molecule capable of self-assembling into a three-dimensional network to form a hydrogel, wherein said molecule comprises: a) a peptide-based portion at least part of which is capable of forming an amphipathic alpha-helix; and

[0047] b) a hydrogel-stabilizing portion wherein the hydrogel-stabilizing portion promotes the formation of the hydrogel, wherein the hydrogel formation involves intermolecular interaction between the amphipathic alpha-helices.

[0048] The term “self-assembling” as used herein, such as in the context of a “self-assembling molecule”, refers to a molecule that can perform self-assembly. The term “self-assembly” as used herein refers to a process in which a disordered system of pre-existing molecules, such as proteins or peptides, forms an organized structure or pattern as a consequence of specific, local interactions (e.g. hydrophobic interactions, hydrogen bonds, van der Waals forces, etc.) among the polypeptides themselves, without external direction or trigger although external factors might influence speed and nature of self-assembly. This particularly means that when two or more disordered and / or unfolded molecules are brought into contact, they interact with each other and consequently form a three dimensional structure. The change from a disordered system to an organized structure or pattern during self-assembly can lead to a transition from a fluid state to a gelatinous and / or solid state and a corresponding increase in viscosity. Methods for monitoring the transition from a fluid state to a gelatinous or solid state are well-established in the art. These include measurement of light scattering, which can detect changes in the optical properties of the system, and rheological measurements, which assess changes in the material's flow properties and viscoelasticity. For transitions to a solid state, optical methods can be employed, including visual observation and various spectroscopic techniques. In turn, “three-dimensional network” generally refers to the interconnected structure that results from the self-assembly process.

[0049] The term “hydrogel” generally refers to a three-dimensional network of molecules, such as polymers, macromolecules, or small molecules, that display hydrophilic properties, so that the hydrogel can absorb and retain a significant amount of water or aqueous solutions while maintaining its structure. The unique properties of hydrogels arise from the interplay between the hydrophilic components of the network, which have a high affinity for water, and the intermolecular interactions that prevent complete dissolution. This balance results in a material that combines the properties of both solids and liquids: the structural integrity of asolid with the high water content and diffusive properties similar to a liquid. Hydrogels typically have a soft, gel-like consistency when hydrated.

[0050] The term “protein” generally encompasses macromolecules comprising one or more polypeptide chains. The term “polypeptide” generally encompasses linear polymeric chains of amino acid residues linked by peptide bonds. A “peptide bond”, “peptide link” or “amide bond” is a covalent bond formed between two amino acids when the carboxyl group of one amino acid reacts with the amino group of the other amino acid, thereby releasing a molecule of water. Especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably to denote such a protein. The terms are not limited to any minimum length of the polypeptide chain. Polypeptide chains consisting essentially of or consisting of 50 or less (< 50) amino acids, such as < 45, < 40, < 35, < 30, < 25, < 20, < 15, < 10 or < 5 amino acids may be commonly denoted as a “peptide”. In the context of proteins, polypeptides or peptides, a “sequence” is the order of amino acids in the chain in an amino to carboxyl terminal direction in which residues that neighbour each other in the sequence are contiguous in the primary structure of the protein, polypeptide or peptide. The terms may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins, polypeptides or peptides. Hence, for example, a protein, polypeptide or peptide can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or a protein, polypeptide or peptide can be recombinant, i.e. , produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Peptide synthesis can be achieved through various established techniques, including solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis, and chemical ligation, which allow for the controlled assembly of amino acid sequences to form peptides. Without limitation, a protein, polypeptide or peptide can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis. The terms also encompasses proteins, polypeptides or peptides that carry one or more co-or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, lipidation, acetylation, amidation, phosphorylation, sulphonation, methylation, pegylation (covalent attachment of polyethylene glycol typically to the N-terminus or to the side-chain of one or more Lys residues), ubiquitination, sumoylation, cysteinylation, glutathionylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or prehormones into active forms, etc. Such co- or post-expression-type modifications may beintroduced in vivo by a host cell expressing the proteins, polypeptides or peptides (co- or post-translational protein modification machinery may be native to the host cell and / or the host cell may be genetically engineered to comprise one or more (additional) co- or post-translational protein modification functionalities), or may be introduced in vitro by chemical (e.g., pegylation) and / or biochemical (e.g., enzymatic) modification of the isolated proteins, polypeptides or peptides. In certain embodiments acetylation of the free alpha amino group at the N-terminus of chemically synthesized peptides and / or the amidation of the free carboxyl group at the C-terminus of chemically synthesized peptides may be opted for to alter the overall charge of the peptides and / or to stabilize the resulting peptides and enhance their ability to resist enzymatic degradation by exopeptidases.

[0051] The term “amino acid” encompasses naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D- and L-stereoisomers, provided their structure allows such stereoisomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A “naturally encoded amino acid” refers to an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy-lysine or selenocysteine. The 20 common amino acids are: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or lie), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T orThr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). A “non-naturally encoded amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy-lysine or selenocysteine. The term includes without limitation amino acids that occur by a modification (such as a post-translational modification) of a naturally encoded amino acid, but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex, as exemplified without limitation by N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Further examples of non-naturally encoded, un-natural or modified amino acids include 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine, beta-Aminopropionic acid, 2-Aminobutyric acid, 4-Aminobutyric acid, piperidinic acid, 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4 Diaminobutyric acid, Desmosine, 2,2’-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, homoserine, homocysteine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-lsoleucine, N-Methylglycine, N-Methylisoleucine, 6-N-Methyllysine, N-Methylvaline, Norvaline, Norleucine, or Ornithine. A further example of such an amino acid is citrulline. Also included are amino acid analogues, in which one or more individual atoms have been replaced either with a different atom, an isotope of the same atom, or with a different functional group. Also included are un-natural amino acids and amino acid analogues described in Ellman et al. Methods Enzymol. 1991, vol. 202, 301-36. The incorporation of non-natural amino acids into proteins, polypeptides or peptides may be advantageous in a number of different ways. For example, D-amino acid-containing proteins, polypeptides or peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing counterparts. More specifically, D-amino acid-containing proteins, polypeptides or peptides may be more resistant to endogenous peptidases and proteases. The term “peptide-based portion” refers to a molecular segment composed of amino acid residues linked by peptide bonds, forming a continuous chain. This portion is characterized by its chemical backbone, which includes the repetitive sequence of amide linkages between the carboxyl group of one amino acid and the amino group of the next. It may be linear or include branched structures, cyclic portions, or other non-linear arrangements, as long as the fundamental peptide bond linkages are present. The “peptide-based” designation refers to the structural composition of the segment, irrespective of the overall length of the chain. This portion thus embodies the chemical nature and backbone structure typical of peptides, polypeptides, or proteins, without being constrained by specific length limitations.

[0052] In particular embodiments, the peptide-based portion may comprise at least one, such as one, two or three, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten, unnatural amino acids (UAAs) such as one or more beta-(homo)-amino acids. Advantageously, beta-amino acids are capable of forming alpha-helices with a near-perfect 3 residue turn, such as described in Aguilar and Yarovsky Quest for New Generation Biocompatible Materials: Tailoring p-Peptide Structure and Interactions via Synergy of Experiments and Modelling. Journal of Molecular Biology 2024 volume 436:168646.

[0053] At least part of the peptide-based portion of the molecule is capable of forming an amphipathic alpha-helix. In certain embodiments, the peptide-based portion may contain one or more distinct parts, stretches or segments that are each capable of forming an amphipathic alpha-helix. For example, in some embodiments, the peptide-based portion may include a single stretch capable of forming an amphipathic alpha-helix, providing a straightforward approach to introducing amphipathic characteristics. In other embodiments, the peptide-based portion may comprise two or three such stretches, allowing for multiple regions ofamphipathic interaction that can enhance the molecule’s ability to engage in intermolecular interactions. In certain embodiments, the part, stretch, or segment of amino acids within the peptide-based portion that forms the amphipathic alpha-helix can correspond to varying percentages of the overall length of the peptide-based portion. For instance, in some embodiments, the amphipathic alpha-helix forming part may encompass >10% of the total length of the peptide-based portion, such as >25%, >50%, >75%, >80%, >85%, >90%, >95%, or may even span 100% of the peptide-based portion.

[0054] The term “amphipathic alpha-helix” as used herein refers to a helical structure in peptides, polypeptides or proteins stabilized by hydrogen bonds between the carbonyl group of the i-thamino acid residue and the amide group of the (i+4)-thamino acid residue, typically featuring 3.6 residues per turn, wherein i can be any integer. The side chains of the amino acid residues project outward in a radial arrangement, providing a functional interface for interaction with other molecules and / or membranes. The alpha-helix can be defined as being amphipathic if the hydrophobic and hydrophilic amino acid residues of the helix are arranged in such a way as to create opposing polar and nonpolar faces oriented along the long axis of the helix. This duality allows the alpha-helix to interact with both aqueous environments and hydrophobic environments such as lipid membranes. Examples of amino acids that can contribute to the hydrophilic face of an amphipathic helix include polar uncharged amino acids such as serine (Ser), threonine (Thr), asparagine (Asn), and glutamine (Gin), as well as charged amino acids like lysine (Lys) and arginine (Arg). Histidine (His) can also contribute to the hydrophilic face, particularly when positively charged. While charged amino acids like aspartic acid (Asp) and glutamic acid (Glu) can sometimes be found on the hydrophilic face, they are less common in typical amphipathic helices due to their negative charge and potential for disrupting the helical structure. Examples of amino acids that can contribute to the hydrophobic face of the amphipathic helix include alanine (Ala), valine (Vai), leucine (Leu), isoleucine (lie), phenylalanine (Phe), methionine (Met), and to some extent, tryptophan (Trp). Tyrosine (Tyr), while containing a polar hydroxyl group, often aligns with the hydrophobic face due to its aromatic ring.

[0055] In particular embodiments, the length of the amphipathic alpha-helix forming part is about 4 to about 40 or more residues, such as about 5 to 15 residues, about 15 to 25 residues, or about 25 to 40 residues or more, such as more typically a length of 6 to 14, 7 to 13 or 8 to 12 residues, such as 8, 9, 10, 11 or 12 residues. In particular embodiments, the peptide-based portion comprises a part which is capable of forming an amphipathic alpha-helix comprising at least 2.0 consecutive (i.e. uninterrupted) turns, preferably at least 2.75 consecutive turns, such as at least 3.0 or at least 4.0 consecutive turns. In particular embodiments, the amphipathic alpha-helix is not a broken alpha-helix. For example, glycineor proline, which can potentially destabilize the helical structure, may be avoided, or the presence of amino acids with different configurations (L vs D) within the helix may be avoided, in order to prevent formation of a broken alpha-helix.

[0056] In particular embodiments, the peptide-based portion comprises or consists of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive amino acids. In particular embodiments, the peptide-based portion comprises or consists of between 6 and 400, between 6 and 380, between 6 and 360, between 8 and 350, between 8 and 300, between 8 and 250, between 8 and 200, between 8 and 150, between 8 and 100, between 10 and 80, or between 10 and 60 consecutive amino acids, preferably between 10 and 40 consecutive amino acids. In certain embodiments, the amphipathic alpha-helix forming part and the peptide-based portion may be coterminous, i.e., the ‘part’ and the ‘portion’ are the same element. In other embodiments, the amphipathic alpha-helix forming part is of a shorter length than the peptide-based portion and is encompassed by this portion. In certain embodiments, the C-terminus of the amphipathic alpha-helix forming part may coincide with or match the C-terminus of the peptide-based portion, while the portion may extend beyond the N-terminus of the part. In other embodiments, the N-terminus of the amphipathic alpha-helix forming part may coincide with or match the N-terminus of the peptide-based portion, while the portion may extend beyond the C-terminus of the part. In yet other embodiments, the N- and C-termini of the amphipathic alpha-helix forming part respectively coincide with or match the N- and C-termini of the peptide-based portion. In yet other embodiments, the peptide-based portion may extend beyond both the N-terminus and the C-terminus of the amphipathic alpha-helix forming part.

[0057] In certain embodiments, where the hydrogel-stabilizing portion is provided N-terminally relative to the peptide-based portion, the C-terminus of the peptide-based portion may be equipped with suitable functional handles. Alternatively, where the hydrogel-stabilizing portion is provided C-terminally relative to the peptide-based portion, the N-terminus of the peptide-based portion may be equipped with suitable functional handles. Such handles may provide for any desired functionalities, such as inter alia allowing to link a payload thereto, for example by established chemistries as such as click chemistry reactions, orthogonal protecting group strategies, or bioorthogonal conjugation methods. These functional handles may include, but are not limited to, alkyne-containing amino acids, azide-containing amino acids, or amino acids with selectively cleavable protecting groups. The modification may enable subsequent conjugation with payloads through copper-catalyzed or strain-promoted azide-alkyne cycloadditions, thiol-maleimide coupling, or other chemoselective ligation techniques. Such strategies provide versatile means for site-specific attachment of variousmolecules, including but not limited to therapeutic agents, imaging probes, or other functional entities.

[0058] In particular embodiments, the peptide-based portion comprises, consists essentially of or consists of an apolipoprotein, apolipoprotein mimetic, or a fragment or variant thereof.

[0059] Apolipoproteins are specialized proteins that bind lipids such as triglycerides, cholesterol, and phospholipids to form lipoproteins. They play a role in transporting these lipids, along with fat-soluble vitamins, through blood, cerebrospinal fluid, and lymph. The lipid components of lipoproteins are inherently insoluble in water. However, due to their amphipathic (detergent-like) properties, apolipoproteins can surround these lipids, creating water-soluble lipoprotein particles that can be carried through aqueous body fluids like blood and lymph. In addition to their transport function, apolipoproteins serve several other important roles: they act as cofactors for enzymes involved in lipoprotein metabolism, function as ligands for cellsurface receptors facilitating the cellular uptake and clearance of lipoproteins, and some, like apolipoprotein A-l, play a role in reverse cholesterol transport. There are several classes of apolipoproteins, including A, B, C, D, E, and others, each with specific functions in lipid metabolism. The structure of apolipoproteins typically includes amphipathic helices, which allow them to interact with both lipids and aqueous environments.

[0060] While there are different classes of amphipathic helices in apolipoproteins, the classification and nomenclature of these helices can vary in the literature. Typically, amphipathic helices in apolipoproteins are classified into several distinct classes, with the most commonly recognized being Class A, Class G*, and Class Y. Class A, the most prevalent in exchangeable apolipoproteins, is characterized by positively charged residues at the polar-nonpolar interface and negatively charged residues at the center of the polar face. Class G* is similar to Class A but with a narrower angle of the polar face, while Class Y has a wide nonpolar face and positively charged residues on the polar face. Further classifications have been proposed in the literature, including subclasses A1 and A2 of Class A, differentiated by the positioning of positively charged residues.

[0061] In lipid transport, apolipoproteins serve multiple functions: they act as structural components of lipoprotein particles, ligands for cell-surface receptors, lipid transport proteins, and cofactors for enzymes. Different lipoprotein particles contain specific combinations of apolipoproteins, which determine their function and metabolism. An example is apolipoprotein A-l (apoA1), the major structural protein of high-density lipoproteins (HDL), although it is also present in smaller amounts in other lipoproteins. ApoA1 is a 28 kDa protein composed of several amphipathic alpha-helical repeats, typically 11 or 22 amino acids long and often separated by proline residues. These helices have a hydrophilic face that interactswith the aqueous environment and a hydrophobic face that interacts with lipids, enabling apoA1 to bind lipids and stabilize lipoprotein particles. This structure allows apoA1 to facilitate lipid transport and participate in reverse cholesterol transport. Certain oligopeptides, such as synthetic oligopeptides, known as apolipoprotein mimetics can replicate some properties of apoA1. These peptides, typically ranging from 16 to 38 amino acids in length, mimic the amphipathic helical structure of apoA1.

[0062] In particular embodiments, the apolipoprotein is selected from the group consisting of apoA-I, apoA-l Milano, apoA-ll, apoA-IV, apoA-V, apoB-48, apoB-100, apoC-l, apoC-ll, apoC-lll, apoC-IV, apoD, apoE, apoF, apoH, apoJ, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, apoM, and apoO. While Roman numerals are typically used in these designations, Arabic numerals are also in common use, for example, apoA-l may be commonly designated as apoA1. In certain preferred embodiments, the apolipoprotein is apoA1. In certain embodiments, the peptide based portion comprises, consists essentially of, or consists of a mimetic of any one of these apolipoproteins. In certain embodiments, the peptide based portion comprises, consists essentially of, or consists of a fragment or variant of any one of these apolipoproteins or apolipoprotein mimetics. In particular embodiments, the apolipoprotein is apoA1 or ApoE. An apolipoprotein mimetic is a peptide, polypeptide, or protein, for example produced synthetically or by recombinant technologies, designed to replicate specific functional and / or structural features of natural apolipoproteins. These mimetics can share primary, secondary, or tertiary structural elements with their native counterparts, such as peptide sequences, alpha-helices, beta sheets, or overall three-dimensional conformations. Functionally, they may mimic the ability of apolipoproteins to bind specific receptors, interact with (phospho)lipids, promote cholesterol efflux, or exhibit anti-inflammatory and / or antioxidant properties. Characteristics of apolipoprotein mimetics may include several features. Many mimetics are engineered with a lipid-binding capacity, efficiently binding phospholipids and other lipids to form lipid-protein complexes similar to natural lipoproteins. In terms of cellular interactions, some mimetics can interact with specific cell types in a manner analogous to their natural counterparts, such as apoA-l mimetics binding to myeloid cells with comparable affinity to native apoA-l. Mimetics may also show therapeutic potential, being explored for treating various lipid disorders and cardiovascular diseases due to their ability to modulate lipid metabolism and transport. Additionally, many apolipoprotein mimetics, particularly those mimicking apoA-l, exhibit an amphipathic nature, featuring helices that facilitate interaction with both lipids and aqueous environments.

[0063] Various apolipoprotein mimetics are known in the art, such as the apoA1, apoE and apoC-ll mimetics described in Wolska et al. (Cells. 2021 Mar; 10(3): 597). For example, apoA1mimetic peptides have largely been designed based on their ability to efflux cholesterol from cells. As this process seems to mainly rely on the amphipathic helical structures comprised by apoA1 , many if not most apoA1 mimetic peptides mimic the amphipathic helix secondary structure without necessarily displaying primary amino acid identity or similarity to apoA1. In particular embodiments, the peptide-based portion comprises, consists essentially of, or consists of an apoA1 mimetic peptide, such as a mimetic peptide selected from the group consisting of 2F, 3F, 4F, 5F, 6F, and 7F, ATI-5261, ESP-2418, ELK peptides, FAMP, i-FAMP, 18A, R18A, 37pA, and combinations thereof, such as 2F, 3F, 4F, 5F, 6F, and 7F, ELK peptides, FAMP, i-FAMP, 18A, R18A, 37pA, and combinations thereof. In certain embodiments, the peptide-based portion comprises, consists essentially of, or consists of 2F, or 4F, or a combination thereof. Amino acid sequences of certain exemplary apoA1 mimetics are as follows (herein and elsewhere in the specification “Ac” denotes N-terminal acetylation, and “NH2” denotes C-terminal amidation, modifications which are typically included in such peptides, but depending on the context, may be optional): 18A: DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1); 2F: AC-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 2); 4F: Ac-DWFKAFYDKVAEKFKEAF-NH2 (SEQ ID NO: 3); ATI-5261: Ac- EVRSKLEEWFAAFREFAEEFLARLKS-NH2 (SEQ ID NO: 56); ESP-2418: Ac-PVLDLFRELLNELLEALKQKLK-NH2 (SEQ ID NO: 57); 5F: Ac-DWLKAFYDKVFEKFKEFF-NH2 (SEQ ID NO: 4); 6F: AC-DWLKAFYDKFFEKFKEFF-NH2 (SEQ ID NO: 5); 7F: Ac-DWFKAFYDKFFEKFKEFF-NH2 (SEQ ID NO: 6); 37pA: DWLKAFYDKVAEKLKEAFPDWLKAFYDKVAEKLKEAF (SEQ ID NO: 7). The ELK peptides are a class of apoA1 mimetics composed of only glutamic acid (E), leucine (L), and lysine (K), and occasionally alanine, with variations in their specific sequences and with varying degrees of amphipathicity and net charge. Exemplary ELK peptide sequences include: ELK EKLKELLEKLLEKLKELL-P-EKLKELLEKLLEKLKELL (SEQ ID NO: 8); ELK-3E3K3A: EELKAKLEELKAKLEEKL-P-EELKAKLEELKAKLEEKL (SEQ ID NO: 9); ELK-2A EKLKALLEKLLAKLKELL-P-EKLKALLEKLLAKLKELL (SEQ ID NO: 10), and others, for example as described in D’Souza et al. Structure-function relationships of apolipoprotein A-l mimetic peptides: implications for anti-atherogenic activities of high density lipoprotein. Circ Res. 2010, vol. 107(2), 217-227. FAMP (Fukuoka University ApoA-l Mimetic Peptide) and its improved version i-FAMP are more recent developments. The sequences of various exemplary FAMP types are as follows: FAMP type 1 (FAMP1) ALEHLFTLSEKAKKALEDLLKKLL (SEQ ID NO: 11); FAMP type 2 (FAMP2) ALEHLFTLSEKALKALEDLLKKLL (SEQ ID NO: 12); FAMP type 3 (FAMP3) ATEHLSTLSEKALKAFEDLLKKLL (SEQ ID NO: 13); FAMP type 4 (FAMP4) ATEHLSTLSEKAKPALEDLLKKLL (SEQ ID NO: 14); FAMP type 5 (FAMP5)ALEHLFTLYEKALKALEDLLKKLL (SEQ ID NO: 15); FAMP type 6 (FAMP6) ALEHLFTLWEKALKALEDLLKKLL (SEQ ID NO: 16); FAMP type 12 (FAMP12) YALEHLFTLSEKALKALEDLLKKLL (SEQ ID NO: 17) (Uehara et al. FAMP, a Novel ApoA-l Mimetic Peptide, Suppresses Aortic Plaque Formation Through Promotion of Biological HDL Function in ApoE-Deficient Mice. J Am Heart Assoc. 2013, vol. 2(3): e000048). i-FAMP types include inter alia i-FAMP-D1, -D2, -D3 and -D4 with the following sequences: i-FAMP-D1: ALEHFTLYEKALKALEDLLKKLLD-A (SEQ ID NO: 18), i-FAMP-D2: D-A-LEHLFTLYEKALKALEDLLKKLL (SEQ ID NO: 19), i-FAMP-D3: ALEHLFTLYEKALKALEDD-L-LKKLL (SEQ ID NO: 20), i-FAMP-D4: ALEHLFTLYEKALKALEDLLKKD-LL (SEQ ID NO: 21) where D-A and D-L are alanine and leucine with D configuration respectively, as disclosed by Suematsu et al. Anti-atherosclerotic effects of an improved apolipoprotein A-l mimetic peptide. Int J Cardiol. 2019, vol. 297, HI-117. Preferably, the FAMP is acetylated at its N-terminus, such as when it is linked to a hydrogel-stabilizing portion being an aliphatic chain.

[0064] ApoE plays a role in lipid metabolism and exhibits several atheroprotective functions. To harness these beneficial properties, various apoE mimetic peptides have been developed. These peptides are designed to emulate functional aspects of the full-length apoE protein, with a primary focus on facilitating the hepatic clearance of apoB-containing lipoproteins. The design of apoE mimetic peptides typically involves two components: a receptor-binding motif derived from the N-terminal domain of apoE, and a lipid-binding region. This lipid-binding segment is often based on the C-terminal domain of apoE or may incorporate alternative sequences that promote lipid association. The inclusion of both receptor-binding and lipid-binding elements in existing apoE mimetics was followed, as apoE can only interact with its receptors when bound to lipids. In particular embodiments, the peptide-based portion comprises, consists essentially of, or consists of an apoE mimetic peptide, such as a mimetic peptide selected from the group consisting of ApoE(130-149), Ac-hE18A-NH2, COG133, CQG1410, COG112, ApoE(141-155)2, and combinations thereof. Exemplary sequences of these peptides include: ApoE(130-149): Ac-TEELRVRLASHLRKLRKRLL-NH2 (SEQ ID NO: 22); Ac-hE18A-NH2: AC-LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 23), COG133: AC-LRVRLASHLRKLRKRLL-NH2 (SEQ ID NO: 24); CQG1410: Ac-AS(Aib)LRKL(Aib)KRLL-NH2 (SEQ ID NO: 25), where Aib is aminoisobutyric acid; and ApoE(141-155)2: LRKLRKRLLRDADDL-LRKLRKRLLRDADDL-NH2 (SEQ ID NO: 26) (e.g. as described in Croy et al. Two apolipoprotein E mimetic peptides, ApoE(130-149) and ApoE(141 -155)2, bind to LRP1. Biochemistry 2004, vol.43, 7328-7335).

[0065] ApoC-ll mimetic peptides have been developed using two primary design strategies to replicate the functions of native apoC-ll. One approach involves linking a shortened first helix(18A) to the LPL-activation domain of apoC-ll, exemplified by the C-ll-a peptide. This design combines an amphipathic helix for lipoprotein binding and cholesterol efflux with the last helix of apoC-ll for LPL activation. The second strategy utilizes both the first and second helices based on native apoC-ll sequences, incorporating amino acid substitutions to enhance bihelical binding to lipoproteins. An example of this approach is the D6PV peptide, a 41-amino acid bihelical peptide with the sequence DYLKEVFEKLRDLYEKFTPAVSTYTGIFTDQVLSVLKGEE (SEQ ID NO: 27). This peptide effectively activates LPL and antagonizes apoC-lll, demonstrating significant potential in lowering plasma triglycerides. Recent advancements in the field include the development of hydrocarbon-stapled apoC-ll mimetic peptides, which offer improved potency and resistance to proteolysis. In particular embodiments, the peptide-based portion comprises, consists essentially of, or consists of an apoC-ll mimetic peptide, such as D6PV.

[0066] The term “fragment” as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and / or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 5%, at least about 10%, at least about 20%, at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 5 consecutive amino acids, > 6 consecutive amino acids, > 8 consecutive amino acids, or> 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, >300, >350, > 360, > 370, > 380, or > 390, consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein. The terms encompass fragments arising by any mechanism, in vivo, in vitro, or by synthesis, such as, without limitation, by alternative transcription or translation, exo- and / or endo-proteolysis, exo- and / or endo-nucleolysis, or degradation of the peptide, polypeptide, protein or nucleic acid encoding the peptide, polypeptide or protein, such as, for example, by physical, chemical and / or enzymatic proteolysis or nucleolysis. In the present context, a fragment of any recited peptide, polypeptide, or protein may optionally be produced synthetically.

[0067] Reference to “fragment or variant” or “variant or fragment” of any peptide, polypeptide, protein or nucleic acid, also encompasses fragments of variants of such peptide, polypeptide, protein or nucleic acid, and variants of fragments of such peptide, polypeptide, protein or nucleic acid.Particularly envisaged are “functionally active” or “functional” fragments and / or variants, denoting that the fragment and / or variant at least partly retains the intended functionality of the respective or corresponding peptide, polypeptide or protein, such as in the present context the ability to participate in hydrogel formation to at least some extent, and ideally to an extent comparable to or the same as or even greater than the corresponding peptide, polypeptide, or protein.

[0068] By means of an example, the human ApoA1 protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_001304947.1 (isoform 1 preproprotein), and Uniprot (www.uniprot.org) accession number P02647.1. In particular embodiments, the ApoA1 is wild-type ApoA1 (e.g. as derived from the human precursor of ApoA1 as defined by SEQ ID NO: 39, of which the first 18 amino acids form the signal peptide). In particular embodiments, the ApoA1 is wild-type human ApoA1 as defined by SEQ ID NO: 55.

[0069] By means of an example and not limitation, human apolipoprotein A-l comprises 243 amino acid residues, typically encoded by two exons wherein the first 43 residues are encoded by exon-3 and the 44-243 region is encoded by exon-4. A fragment of human apoA1 may denote any portion of apoA1 which comprises at least one stretch capable of forming an amphipathic alpha helix. For example, such fragment may comprise one or more of the following alpha helix forming stretches of apoA1: 44-65 (“helix 1”), 66-87 (“helix 2”), 88-98 (“helix 3”), 99-120 (“helix 4”), 121-142 (“helix 5”), 143-164 (“helix 6”), 165-187 (“helix 7”), 188-208 (“helix 8”), 209-219 (“helix 9”), and 220-243 (“helix 10”), wherein the positions of the helix-forming regions are indicated with reference to SEQ ID NO: 55, as described in Mei Xiaohu and Atkinson David, Lipid-free Apolipoprotein A-l Structure: Insights into HDL Formation and Atherosclerosis Development, Archives of Medical Research, Vol. 46, Issue 5, July 2015, pages 351-360. For example, such fragment may comprise one or more of the following alpha helix forming stretches of apoA1: aa8-23 (“ helix 1”), aa24-45 (“ helix 2”), aa46-56 (“ helix 3”), aa57-78 (“ helix 4”), aa79-100 (“ helix 5”), aa101-122 (“ helix 6”), aa123-144 (“ helix 7”), aa145-166 (“ helix 8”), aa167-177 (“ helix 9”), aa186-201 (“ helix 10”). The location as the alpha helix forming stretches of apoA1 are known in the art, such as described on the RCSB protein data base (https: / / www.rcsb.org / ). In certain embodiments, a fragment of human apoA1 may also retain the myeloid-binding activity of apoA1 to at least some extent, and ideally to an extent comparable to or the same as or even greater than apoA1.

[0070] The term “variant” of a protein, polypeptide or peptide generally refers to proteins, polypeptides or peptides the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, or peptide, e.g.,at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the recited protein, polypeptide or peptide. Preferably, a variant may display such degrees of identity to a recited protein, polypeptide, or peptide when the whole sequence of the recited protein, polypeptide, or peptide is queried in the sequence alignment (i.e., overall sequence identity). Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2, penalty for a mismatch = -2, reward for a match = 1, gap x_dropoff = 50, expectation value = 10.0, word size = 28; or for the BLASTP algorithm: matrix= Blosum62 (Henikoff etal., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3).

[0071] An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (BI2seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). If the two compared sequences share homology, then the output will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100. The percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view foreach segment of alignment as outputted by BI2seq already conveniently includes the percentage of identities.

[0072] A variant of a protein, polypeptide or peptide may be a homologue (e.g., orthologue or paralogue) of said protein, polypeptide or peptide. As used herein, the term “homology” generally denotes structural similarity between two macromolecules from same or different taxons, wherein said similarity is due to shared ancestry.

[0073] A variant of a protein, polypeptide, or peptide may comprise one or more amino acid additions, deletions, or substitutions relative to (i.e., compared with) the corresponding protein or polypeptide. For example, a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 70 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70) conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide; and / or a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 20 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19) non-conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide.

[0074] A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0075] Alternatively or in addition, for example, a variant (deletion variant) of a protein, polypeptide, or peptide may lack up to 20 amino acid segments (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 segments) relative to (i.e., compared with) the corresponding protein or polypeptide. The deletion segment(s) may each independently consist of one amino acid, two contiguous amino acids or three contiguousamino acids. The deletion segments may be non-contiguous, or two or more or all of the deletion segments may be contiguous.

[0076] In particular embodiments, the variant comprises at least one, such as one, two or three, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten, alpha-methylated and / or beta-methylated substitutions. For example, an apolipoprotein ApoA-l mimetic peptide may be modified as to comprise six alpha-methylated amino acids, such as described in Islam et al. Incorporation of alphamethylated amino acids into Apolipoprotein A-l mimetic peptides improves their helicity and cholesterol efflux potential. Biochem Biophys Res Commun. 2020, vol. 526(2), 349-354. In particular embodiments, the variant comprises at least one, such as one, two or three, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten, N-methylated amino acid substitutions. A non-limiting example of an N-methylated amino acid is sarcosine.

[0077] Preferably, the peptide-based portion comprises, consists essentially of or consists of apolipoprotein mimetic 2F or a variant thereof. The apolipoprotein mimetic 2F mimics apoATs lipid binding features and cholesterol efflux properties, and has a class A alphahelical secondary structure, with defined hydrophobic and hydrophilic domains.

[0078] In particular embodiments, the apolipoprotein, apolipoprotein mimetic, or fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to - or differing by no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or by no amino acids from - the amino acid sequence DWLKX1FYDKVX2EKLKEAF (SEQ ID NO: 28), wherein Xi and X2can be any amino acid, preferably wherein Xi is E or A and X2is K, more preferably wherein Xi is A and X2is K. In particular embodiments, where the hydrogel-stabilizing portion is provided N-terminally relative to the peptide-based portion, the C-terminus of the peptide-based portion comprising, consisting essentially of or consisting of SEQ ID NO: 28 as defined above may be amidated. In particular embodiments, where the hydrogel-stabilizing portion is provided C-terminally relative to the peptide-based portion, the N-terminus of the peptide-based portion comprising, consisting essentially of or consisting of SEQ ID NO: 28 as defined above may be acetylated. In particular embodiments, the apolipoprotein, apolipoprotein mimetic, or fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to - or differing by no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or by no amino acids from - amino acid sequenceDWLKAFYDKVXiEKLKEAF (SEQ ID NO: 29), wherein Xi is a positively charged amino acid, preferably wherein Xi is K.

[0079] In particular embodiments, the apolipoprotein, apolipoprotein mimetic, or fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to - or differing by no more than 5, no more than 4, no more than 3, no more than 2, no more than 1, or by no amino acids from - amino acid sequence DWLKAFYDKVAEKLKEAF (SEQ ID NO: 30), DWLKEFYDKVKEKLKEAF (SEQ ID NO: 31), or DWLKAFYDKVKEKLKEAF (SEQ ID NO: 32). The person skilled in the art will understand that substitution of a charged amino acid residue into the hydrophobic side of the helix of apolipoprotein mimetic 2F is expected to prevent gelation. Comparably, replacing a charged amino acid residue with a hydrophobic one is expected to lead to precipitation. In particular embodiments, the variant of apolipoprotein mimetic 2F may comprise one or more amino acid substitutions by histidine, such as no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 amino acid substitution by histidine.

[0080] In particular embodiments, the apolipoprotein, apolipoprotein mimetic, or fragment or variant thereof comprises, consists essentially of, or consists of an amino acid sequence DWLKAFYDKVAEKLKEAF, DWLKEFYDKVKEKLKEAF, or DWLKAFYDKVKEKLKEAF, or an amino acid sequence comprising at most 4, at most 3, at most 2 or at most 1 amino acid substitution vis-a-vis DWLKAFYDKVAEKLKEAF, DWLKEFYDKVKEKLKEAF or DWLKAFYDKVKEKLKEAF. In particular embodiments, the peptide-based portion of the molecule as taught herein comprises, consists essentially of, or consists of an amino acid sequence DWLKAFYDKVAEKLKEAF, DWLKEFYDKVKEKLKEAF, or DWLKAFYDKVKEKLKEAF, or an amino acid sequence comprising at most 4, at most 3, at most 2 or at most 1 amino acid substitution vis-a-vis DWLKAFYDKVAEKLKEAF, DWLKEFYDKVKEKLKEAF or DWLKAFYDKVKEKLKEAF.

[0081] In certain embodiments, the hydrogel-stabilizing portion of the molecule includes a moiety capable of participating in various intermolecular interactions, which promote the formation and stabilization of the hydrogel network. These interactions occur between the hydrogelstabilizing portions of different molecules, each contributing to the formation of the larger network structure.

[0082] The terms “hydrogel-stabilizing” and “promote the formation of the hydrogel” are employed broadly to describe a portion of the molecule that contributes to the molecule’s ability to form a hydrogel. This portion ensures that the molecule either forms the hydrogel or facilitates its formation in a manner that is more thermodynamically stable and / or kinetically favorablecompared to a molecule lacking this portion. Thermodynamically, the hydrogel-stabilizing portion may be capable of lowering the system’s Gibbs free energy, thereby promoting a more stable hydrogel state. This stabilization can occur through enhanced intermolecular interactions, which reduce the overall energy of the assembled structure. Kinetically, the presence of the hydrogel-stabilizing portion may be able to accelerate the rate of hydrogel formation by lowering activation energy barriers or facilitating the assembly pathways, thus allowing the hydrogel to form more rapidly and efficiently. These terms encompass scenarios where a molecule lacking the hydrogel-stabilizing portion either fails to form a hydrogel entirely, with the stabilizing portion enabling or inducing its formation, or forms a hydrogel that is less stable and / or slower to form. The hydrogel-stabilizing portion may therefore play a role in either one or both of the structural integrity and the efficiency of hydrogel formation. By means of an example and without limitation, to evaluate the thermodynamic and kinetic advantages conferred by the hydrogel-stabilizing portion of the molecule, several experimental techniques can be employed. These methods aim to quantify changes in properties such as free energy, assembly rates, and structural stability of the hydrogel. Thermodynamic assessment may involve for example Differential Scanning Calorimetry (DSC). This technique can be used to measure the heat flow associated with the phase transitions of the hydrogel. It allows the determination of melting temperatures and enthalpy changes, providing insights into the stability of the hydrogel network, as larger negative enthalpy changes often correlate with stronger interactions and potentially enhanced gel stability. Isothermal Titration Calorimetry (ITC) can be employed to directly measure the heat released or absorbed during the self-assembly process. The data can be used to calculate changes in free energy (AG), enthalpy (AH), and entropy (AS), helping to elucidate the thermodynamic favorability of hydrogel formation. Rheological measurements or rheometry can be used to assess the mechanical stability and viscoelastic properties of the hydrogel. A more stable hydrogel typically exhibits a higher storage modulus (G’) compared to the loss modulus (G’), with G’ dominating across a range of frequencies. Both moduli should display minimal frequency dependence, indicating a robust, well-structured network that maintains its mechanical integrity under varying conditions. These parameters can be determined for example by a frequency sweep test or an amplitude sweep test.

[0083] Kinetic assessment may involve Time-Resolved Spectroscopy. Techniques such as UV-Vis or fluorescence spectroscopy can be used to monitor changes in absorbance or fluorescence intensity over time, providing real-time insights into the rate of hydrogel formation. Faster assembly rates will be indicated by quicker changes in spectral properties. Dynamic Light Scattering (DLS) can be employed to monitor the formation of hydrogels by measuring changes in the size and distribution of particles or polymer clusters over time. An increase inaverage particle size and a broadening of the size distribution often indicate the progression of the assembly process, with significant changes observed near the sol-gel transition point. Atomic force microscopy (AFM) and confocal microscopy can be used to visualize and quantify the evolving microstructure of hydrogels during formation. AFM provides high-resolution topographical imaging and mechanical property measurements, while confocal microscopy offers 3D visualization of network development, both allowing real-time monitoring of gelation kinetics and structural changes at different time points.

[0084] The hydrogel-stabilizing portion can facilitate various intermolecular interactions, which in certain embodiments may include hydrophobic interactions, intermolecular beta-sheet formation, TT-TT stacking, or amphiphilic interactions. Without wishing to be limited by any hypothesis, the inventors believe that these play a central role in the self-assembly process, contributing to the structural integrity and functionality of the resulting hydrogel.

[0085] In some embodiments, the hydrogel-stabilizing portion may contain only a single moiety, for example may consist of a single moiety, designed to engage in a specific type of intermolecular interaction, such as a hydrophobic moiety facilitating hydrophobic interactions. Alternatively, in other embodiments, the hydrogel-stabilizing portion may include multiple moieties, each capable of participating in either the same type of interaction or different types. For example, a combination of hydrophobic and aromatic moieties could enable both hydrophobic interactions and -TT stacking, providing a multifaceted approach to stabilization and assembly. This versatility in the configuration of the hydrogel-stabilizing portion allows for the design of molecules tailored to specific applications, where the choice and combination of moieties can be optimized to achieve desired properties or functional characteristics of the hydrogel, such as mechanical properties, porosity, swelling capacity, biocompatibility, biodegradability, drug release profile, pH and temperature sensitivity, and the like.

[0086] Hydrophobic interactions involve the association of nonpolar moieties, which can drive the self-assembly process by minimizing exposure to aqueous environments, thereby promoting the formation of a stable hydrogel network. The propensity for hydrophobic interactions can be suitably assessed by examining the structure of a moiety. A) presence of nonpolar groups: alkyl chains, aromatic rings, and other hydrocarbon-rich structures; B) LogP value, or the octanol-water partition coefficient, is a measure of a compound’s lipophilicity or hydrophobicity. It quantifies the distribution of a neutral (uncharged) molecule between two immiscible phases, typically octanol (representing a lipid phase) and water. The value is calculated as the logarithm of the ratio of the compound’s concentrations in these two phases and the higher the positive LogP value, the more hydrophobic the compound. C) Molecularsurface area: Larger nonpolar surface areas contribute to stronger hydrophobic interactions. Experimentally, hydrophobic interactions in hydrogel-forming molecules may be evaluated for example by fluorescence spectroscopy using hydrophobicity-sensitive probes, surface tension measurements, ITC, or reverse-phase HPLC.

[0087] Intermolecular beta-sheet formation involves the alignment of peptide strands from different molecules, creating an extended hydrogen-bonding network. The propensity for beta-sheet formation can be assessed by examining a peptide sequence. Alternating hydrophobic and hydrophilic residues often promote beta-sheet formation. Amino acids like valine, isoleucine, and threonine have high beta-sheet propensities. Proline and glycine tend to disrupt betasheets. Experimentally, beta-sheet formation in hydrogel-forming peptides can be determined through inter alia Circular Dichroism (CD) spectroscopy - characteristic negative band at 218 nm and positive band at 195 nm indicate beta-sheet structure, Fourier Transform Infrared (FTIR) spectroscopy: amide I band at -1620-1640 cm-1 is indicative of beta-sheets, Thioflavin T (ThT) fluorescence assay - increased fluorescence upon binding to beta-sheet structures, and / or X-ray diffraction, which can reveal characteristic cross-beta patterns. Pi-pi (TT-TT) stacking refers to attractive, non-covalent interactions between aromatic rings. In hydrogel assembly, these interactions can contribute to self-assembly and network stabilization by promoting the association of aromatic moieties. The propensity for pi-pi stacking can be assessed by examining several key features of the molecular structure. First, the presence of aromatic rings, such as benzene, pyridine, or other conjugated systems, facilitates these interactions. The electronic nature of these aromatics also plays a role; electron-rich or electron-poor systems, influenced by their substituents, can significantly affect the strength and orientation of the stacking. For instance, electron-withdrawing groups can enhance pi-pi interactions by increasing the polarization of the aromatic system. The planarity of the aromatic moieties is another critical factor, as flat systems typically stack more efficiently due to maximized overlap of their pi orbitals. Computational methods, such as density functional theory (DFT) calculations, can be employed to predict the electron density distribution and planarity of these systems, providing insights into their potential for pi-pi stacking. Additionally, molecular dynamics simulations can offer valuable predictions about the likelihood and strength of these interactions in various environments, helping to anticipate their role in hydrogel formation and stability. Experimentally, pi-pi stacking in hydrogelforming molecules can be determined through various spectroscopic and crystallographic techniques. UV-Vis spectroscopy can reveal characteristic shifts in absorption bands, indicating changes in the electronic environment of aromatic systems due to stacking interactions. Fluorescence spectroscopy offers insights through alterations in emission spectra or quantum yield, which often occur when aromatic molecules engage in pi-pistacking. NMR spectroscopy provides valuable information by showing upfield shifts of aromatic protons, a phenomenon attributed to ring current effects in stacked systems. For a more direct structural observation, X-ray crystallography can be employed to visualize stacking arrangements in the solid state, although this method may not always reflect the exact configuration in solution or within the hydrogel network. These complementary techniques, when used in combination, can provide a comprehensive understanding of pi-pi stacking interactions in hydrogel-forming molecules.

[0088] Amphiphilic interactions arise from molecules that possess both hydrophilic and hydrophobic regions. The propensity for amphiphilic interactions can be assessed by examining the molecular structure, particularly the balance and distribution of polar and nonpolar moieties within the molecule. Hydrophilic groups, such as charged or polar functionalities, promote water solubility and hydrogen bonding, while hydrophobic segments, often consisting of alkyl chains or aromatic rings, drive self-assembly through the hydrophobic effect. The relative sizes and positions of these contrasting regions influence the molecule’s behavior in aqueous environments. Computational methods, including molecular dynamics simulations and calculations of hydrophobicity scales, can predict the amphiphilic character and potential for self-assembly. Experimentally, amphiphilic interactions in hydrogel-forming molecules can be probed through various techniques. Surface tension measurements provide insights into the molecule's ability to reduce water surface tension, a characteristic of amphiphiles. Fluorescence spectroscopy, using environment-sensitive probes, can detect the formation of hydrophobic microdomains. Small-angle X-ray or neutron scattering (SAXS / SANS) can reveal the nanoscale structures formed by amphiphilic self-assembly. Additionally, cryotransmission electron microscopy (cryo-TEM) allows direct visualization of the supramolecular structures resulting from amphiphilic interactions within the hydrogel network.

[0089] In certain embodiments, the hydrogel-stabilizing portion, such as optionally the hydrogelstabilizing portion that facilitates hydrophobic interactions, comprises an aliphatic chain, such as a C4-C22 aliphatic chain, or a C4-C20 aliphatic chain. An aliphatic chain refers to a linear or branched chain of carbon atoms that are saturated (alkanes), unsaturated (alkenes or alkynes), or a combination of saturated and unsaturated bonds. Aliphatic chains are characterized by their open-chain structure, as opposed to aromatic rings, and can be part of larger organic molecules, contributing to hydrophobic interactions due to their nonpolar nature. These chains are typically hydrocarbons, meaning they consist entirely of carbon (C) and hydrogen (H) atoms. The notation “C4-C22” specifies the number of carbon atoms present in the aliphatic chain. A C4 chain has four carbon atoms, while a C22 chain has twenty-two carbon atoms. This range indicates the number of carbon atoms that the aliphaticchain in the hydrogel-stabilizing portion can have, influencing the chain’s length and its hydrophobic properties. In certain embodiments, the aliphatic chain may be a short aliphatic chain, typically classified as C4-C8, or a medium aliphatic chain, typically classified as 09-C14, or a long aliphatic chain, typically classified as C15-C22. In certain embodiments, the hydrogel-stabilizing portion comprises an aliphatic chain with a carbon length that can be specifically chosen among C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22 allowing for tailored hydrophobic interactions within the hydrogel network. In certain embodiments, the aliphatic chain is an alkyl chain.

[0090] In certain embodiments, the aliphatic chain is acetylated at its C1-tail, i.e. the C of the aliphatic chain closest to the peptide-based portion. In certain embodiments, the aliphatic chain within the hydrogel-stabilizing portion may be part of the residue of a fatty acid, such as specifically a C4-C22 fatty acid or C4-C20 fatty acid. Fatty acids are carboxylic acids with long aliphatic chains, which can be either saturated or unsaturated. When a fatty acid is incorporated into a molecule, it often forms an acyl residue, which is the portion of the fatty acid remaining after the removal of the hydroxyl group (-OH) from the carboxyl group (-COOH). Incorporating an acyl residue provides a means to introduce hydrophobic interactions, as the aliphatic chain contributes to the molecule’s overall lipophilicity. This can enhance the self-assembly behavior and stability of the hydrogel network. An acyl group can be conveniently linked to a reminder of a molecule by inter alia an ester bond, a thioester bond, or an amide bond. In certain embodiments, the fatty acid may be selected from the group consisting of stearic acid, undecanoic acid, hexanoic acid, palmitic acid, lauric acid, oleic acid, myristic acid, linoleic acid, and arachidic acid. In certain embodiments, the fatty acid may be stearic acid, undecanoic acid, or hexanoic acid.

[0091] In certain embodiments, the hydrogel-stabilizing portion comprises an aliphatic chain having a carbon length selected from C4-C22, C4-C20, C6-C22, C8-C22, or C8-C20, such as C12-C20 or C12-C18. In certain embodiments, the aliphatic chain is a C4-C20 or C4-C22 aliphatic chain, such as a C4-C22 fatty acid (e.g. fatty acid residue). In certain embodiments, the fatty acid (residue) is a stearic acid, an undecanoic acid, or a hexanoic acid.

[0092] In certain embodiments, the hydrogel-stabilizing portion comprises, consists essentially of or consists of, one or more collagen-derived peptide motifs, such as, for example, one or more motifs selected from GLY-PRO-X and GLY-X-HYP, wherein X is any amino acid other than GLY, PRO and HYP.

[0093] In certain embodiment, the hydrogel-stabilizing portion , comprises, consists essentially of or consists of, a polyproline-rich peptide or polyproline, such as, e.g. P9 (PPPPPPPPP (SEQ ID NO: 33)). A “polyproline” or “polyproline-rich peptide” refers to a peptide consisting ofproline amino acid residues. The polyproline may consist of 2 (P2), 3 (P3), 4 (P4; SEQ ID NO: 34), 5 (P5; SEQ ID NO: 35), 6 (P6; SEQ ID NO: 36), 7 (P7; SEQ ID NO: 37), 8 (P8; SEQ ID NO: 38), 9 (P9; SEQ ID NO: 33) or more proline amino acid residues.

[0094] In certain embodiments, the hydrogel-stabilizing portion comprises an aromatic moiety capable of TT-TT stacking. These interactions occur between the aromatic rings of different molecules, contributing to the cohesive energy and structural integrity of the hydrogel. One example of such a moiety is the fluorenyl ring system, known for its effective stacking properties. Additionally, other aromatic moieties can be utilized to achieve similar effects, including the phenyl ring, which serves as a basic yet versatile option for TT-TT stacking. The naphthyl ring system, with its fused aromatic structure, offers a larger surface area for enhanced interactions. Similarly, the anthracenyl ring system, comprising three linearly fused benzene rings, provides extended stacking potential. The pyrenyl ring system, recognized for its extensive conjugated Tr-system, ensures robust stacking interactions. The biphenyl ring system, consisting of two phenyl rings connected by a single bond, allows for flexible stacking configurations. For even more extensive TT-TT interactions, the perylenyl ring system, with its extended aromatic framework, is an option. The carbazolyl ring system introduces heteroaromatic characteristics that can further strengthen TT-TT stacking interactions. By incorporating these aromatic moieties, the hydrogel-stabilizing portion can be tailored to optimize the hydrogel’s stability and self-assembly properties. In certain embodiments, the moiety comprises or consists of a fluorenyl moiety, optionally a fluorenylmethoxycarbonyl (Fmoc) moiety. In certain embodiments, the hydrogel-stabilizing portion comprises an aromatic moiety capable of TT-TT stacking, comprises, consists essentially of or consists of F orW.

[0095] In certain embodiments, the hydrogel-stabilizing portion comprises, consists essentially of or consists of a fluorenyl moiety, optionally a fluorenylmethoxycarbonyl (Fmoc) moiety, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0096] In certain embodiments, the hydrogel-stabilizing portion comprises a dipeptide selected from FP or WP. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion. In certain embodiments, the dipeptide is N-terminally acetylated. The hydrogel-stabilizing portion may be directly or indirectly (such as through a suitable linker or spacer that is compatible with the hydrogel formation promoting function of that portion) linked, preferably covalently linked, to the peptide-based portion of the molecule. Non-limiting examples of suitable linkers or spacers include amino acid based spacers, poly-ethylene-glycol (PEG)-based linkers and alkyl linkers. In certain embodiments, the hydrogel-stabilizingportion may be linked, preferably covalently linked, to the peptide-based portion of the molecule via an amino acid based spacer, such as via one or more (preferably one or two) amino acids selected from G, P and A, or a combination thereof. For example, the hydrogelstabilizing portion may be linked, preferably covalently linked, to the peptide-based portion of the molecule via a dipeptide consisting of amino acids selected from G, P and A such as, for example, GP or GG. In certain preferred embodiments, the bond may be a direct covalent bond. There are various established ways how to provide for such a covalent bond, for example but without limitation, an ester bond, a thioester bond, an amide bond, a thioamide bond, thiol-maleimide chemistry, hydrazone linkage, and the like. The choice of linking method depends on factors such as the specific functional groups available.

[0097] In certain embodiments, the hydrogel-stabilizing portion comprising, consisting essentially of or consisting of F or W is linked, preferably covalently linked, to the peptide-based portion of the molecule via a spacer consisting of G, P, A, GP, GA, PA, AG, AP or PG. In certain embodiments, if the hydrogel-stabilizing portion comprises, consists essentially of or consists of F or W, the hydrogel-stabilizing portion does not comprise a charged amino acid, such as K or E N- and / or C-terminally of the hydrogel-stabilizing portion.

[0098] In certain embodiments, if the hydrogel-stabilizing portion comprises, consists essentially of or consists of W, the hydrogel-stabilizing portion does not comprise a G N-terminally of the hydrogel-stabilizing portion.

[0099] In certain embodiments, the hydrogel-stabilizing portion comprises, consists essentially of or consists of WP, FP, EWP, KWP, KFP or EFP and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion.

[0100] In certain embodiments, the hydrogel-stabilizing portion is provided N-terminally of the peptide-based portion. In certain embodiments, the hydrogel-stabilizing portion is N-terminally adjacent to the peptide-based portion, for example it is linked directly to the N-terminus of the peptide-based portion.

[0101] In other embodiments, the hydrogel-stabilizing portion is provided C-terminally of the peptide-based portion. In certain embodiments, the hydrogel-stabilizing portion is C-terminally adjacent to the peptide-based portion, for example it is linked directly to the C-terminus of the peptide-based portion.

[0102] In certain embodiments, the hydrogel-stabilizing portion comprises, consists essentially of or consists of an aliphatic chain having a carbon length selected from C6-C22, C8-C22, or C8-C20, such as C12-C20 or C12-C18 and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0103] In certain embodiments, the hydrogel as taught herein comprises 2.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of an aliphatic chain having a carbon length of C6 and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0104] In certain embodiments, the hydrogel as taught herein comprises at least 2.0% (w / v), such as from 2.0% (w / v) to 8.0% (w / v), or from 2.0% (w / v) to 6.0% (w / v), preferably from 2.0% (w / v) to 4.0% (w / v), of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of an aliphatic chain having a carbon length of C8, C11, C16, C18, C20 or C22, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0105] In certain embodiments, the hydrogel as taught herein comprises at least 1.0% (w / v), at least 2.0% (w / v), such as from 1.0% (w / v) to 8.0% (w / v), from 1.0% (w / v) to 6.0% (w / v), or from 1.0% (w / v) to 4.0% (w / v), preferably from 2.0% (w / v) to 4.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of an aliphatic chain having a carbon length of C18, optionally wherein the fatty acid is an oleic acid or a linoleic acid, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F or 4F, more preferably 2F.

[0106] In certain embodiments, the hydrogel as taught herein comprises at least 1.0% (w / v), such as from 1.0% (w / v) to 8.0% (w / v) or from 1.0% (w / v) to 6.0% (w / v), preferably from 1.0% (w / v) to 4.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of an aliphatic chain having a carbon length of C12, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0107] In certain embodiments, the hydrogel as taught herein comprises at least 0.5% (w / v), such as from 0.5% (w / v) to 8.0% (w / v), or from 0.5% (w / v) to 6.0% (w / v), preferably from 0.5% (w / v) to 4.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of an aliphatic chain having a carbon length of C14, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.In certain embodiments, the hydrogel as taught herein comprises at least 2.0% (w / v), such as from 2.0% (w / v) to 8.0% (w / v), or from 2.0% (w / v) to 6.0% (w / v), preferably from 2.0% (w / v) to 4.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of a Fmoc moiety, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F.

[0108] In certain embodiments, the hydrogel as taught herein comprises at least 0.5% (w / v), such as from 0.5% (w / v) to 1% (w / v) of a molecule as taught herein comprising a hydrogelstabilizing portion comprising, consisting essentially of or consisting of WP, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion.

[0109] In certain embodiments, the hydrogel as taught herein comprises at least 2.0% (w / v), such as from 2.0% (w / v) to 3.0% (w / v), for example about 2.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of FP, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion. The hydrogel-stabilizing portion may comprise a G N-terminally of the dipeptide FP.

[0110] In certain embodiments, the hydrogel as taught herein comprises at least 4.0% (w / v), such as from 4.0% (w / v) to 8.0% (w / v), or from 4.0% (w / v) to 6.0% (w / v), preferably about 4.0% (w / v) of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of KWP, KFP or EFP, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion.

[0111] In certain embodiments, the hydrogel as taught herein comprises at least 2.0% (w / v), such as from 2.0% (w / v) to 8.0% (w / v), or from 2.0% (w / v) to 6.0% (w / v), preferably from 2.0% (w / v) to 4.0% (w / v), of a molecule as taught herein comprising a hydrogel-stabilizing portion comprising, consisting essentially of or consisting of EWP, and the peptide-based portion comprises, consists essentially of or consists of an ApoA-1 mimetic, preferably ApoA-1 mimetic 2F. The P may act as a spacer between the hydrogel-stabilizing portion and the peptide-based portion.

[0112] In certain embodiments, the peptide-based portion of the present molecules, and insofar the hydrogel-stabilizing portion is also peptide-based, the whole molecule, can be prepared bystandard peptide synthesis method, such as solid phase peptide synthesis. Where the hydrogel-stabilizing portion is not peptide-based or not entirely peptide-based, its addition, such as to the N-terminus of the peptide-based portion, may also be done on-resin in the peptide synthesis process. The molecules can then be purified by established methods, such as standard preparative HPLC. Gelation can be achieved by standard approaches, such as dissolving the molecules in a suitable buffer, and heating to a suitable temperature, such as about 50°C, for 5-30 min, and cooling whereupon gelation occurs. Payloads, if desired, may be added just before cooling.

[0113] A further aspect provides a hydrogel comprising the molecule capable of self-assembling into a three-dimensional network to form a hydrogel as taught herein.

[0114] In particular embodiments, the hydrogel comprises at least 0.5% (w / v), at least 1.0 (w / v), at least 1.5 (w / v), at least 2.0 (w / v), at least 2.5 (w / v) or at least 3.0 (w / v) of one or more of the molecules as taught herein. The molecules as taught herein within the hydrogel may all be the same or different, e.g. different types of peptide-based portions and / or hydrogelstabilizing portions.

[0115] In particular embodiments, the viscoelasticity of the hydrogel ranges from 10 to 15000 Pa, such as from 10 to 10000 Pa, from 20 to 8000 Pa, from 30 to 6000 Pa or from 40 to 5000 Pa, optionally as measured at a temperature of 21°C. The viscoelasticity of the hydrogel may be determined by any methods known in the art, such as by rheology and a rheometer. Preferably, the molecule or hydrogel as taught herein is biodegradable, biocompatible, non-immunogenic and / or non-inflammatory.

[0116] In particular embodiments, the hydrogel as taught herein comprises a payload. In particular embodiments, the payload is a therapeutic agent or an imaging agent.

[0117] When the term “therapeutic agent” is used, it refers to the desired pharmacological, immunogenic and I or physiological properties when administered to an organism (e.g. human or non-human animal) by local and I or systemic effects. Any synthetic or naturally occurring biologically active compound or composition that elicits an effect is included. Thus, this term encompasses compounds or chemicals that are conventionally recognized as drugs (i.e. medicaments), vaccines, and biopharmaceuticals, including molecules such chemical compounds, peptides, proteins, nucleic acids or nucleic acid analogs, biologies, lipids and nanoparticles e.g. apolipoprotein nanoparticles as described in International patent application WO2022 / 268913, WO2023 / 046931, WO 2023 / 233042, WO 2023 / 227682, WO 2024 / 200824 or WO 2024 / 200824 , optionally comprising a payload such as mRNA.For example, the therapeutic agent may be a large molecule (e.g. such as with a molecular weight ranging between 5 and 500 kDa, preferably between 5 and 200 kDa or between 5 and 150 kDa), such as cytokines or antibody-based biologies. The presence of such payload does not negatively impact the gelation properties of the molecule or hydrogel as taught herein.

[0118] For example, the payload may be a nucleic acid or a nucleic acid analog. Examples may be but are not limited to mRNA, siRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA. The nucleic acid analogue may be peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA), or mixtures or combinations thereof.

[0119] Alternatively, the payload may be a small organic compound such as a small molecule drug. Generally, the small organic compound is synthesized. The therapeutic may for example be an anticancer agent or anticancer therapy such as a chemotherapy. For example, the payload may be rapamycin or a structurally or functionally related analog, such as everolimus, temsirolimus, or ridaforolimus, or other well-known anticancer agents. Alternatively, the payload may be a biologic. When used herein, the term biologic is used to indicate a biopharmaceutical, also known as a biologic(al) medical product, and can be any pharmaceutical drug product manufactured in, extracted from, or semi-synthesized from biological sources. Biologies can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or may be living cells or tissues.

[0120] In certain embodiments, the payload may be an imaging agent. The imaging agent may be selected from a group consisting of fluorescent dyes, radioactive isotopes, magnetic resonance imaging (MRI) contrast agents, positron emission tomography (PET) tracers, single-photon emission computed tomography (SPECT) agents, ultrasound contrast agents, and X-ray contrast agents. Such imaging agents may include, but are not limited to, fluorophores, lanthanide chelates, gadolinium-based compounds, superparamagnetic iron oxide nanoparticles, radionuclides, iodinated contrast media, microbubbles, and barium sulfate. The selection of the specific imaging agent may depend on the intended imaging modality and the particular application.

[0121] In certain embodiments, the payload may be trapped within the hydrogel network, for example without providing the payload with a structure, such as a peptide tail, to incorporate it into the supramolecular structure. In other embodiments, the payload may be modified with a structure, such as a peptide tail, which facilitates its incorporation into the supramolecular structure of the hydrogel.In particular embodiments, for further tuning the release properties, the payload can be chemically modified with an apolipoprotein or apolipoprotein mimetic or a variant or fragment thereof, such as an apoA1 peptide, preferably 2F, which improves the incorporation of the payload in the gel. The additional advantage of modifying the payload with an apolipoprotein or apolipoprotein mimetic or a variant or fragment thereof is the affinity for the endogenous lipoprotein fraction in our body. Therefore, the hydrogels as taught herein have the option to make their payload “hitch hike” to the myeloid compartment following their release. In certain embodiments, the 2F is a variant of 2F designed for covalent conjugation of the payload. For example, in particular embodiments, 2F is C-terminally modified to 2F-propargyl, 2F-Mal or 2F-Azide.

[0122] In particular embodiments, such as when the hydrogel as taught herein is used for vaccination, the payload is an antigen (e.g. ovalbumin, OT-1 peptide) and / or one or more adjuvants (e.g. squalene, monophosphoryl-lipid A (MPLA) and / or telratolimod).

[0123] In particular embodiments, such as when the hydrogel as taught herein is used for transplantation, the therapeutic agent is tacrolimus and / or rapamycin or a CD40(L) inhibitor, such as an anti-CD40L antibody.

[0124] Therapy relying on intravenously administered antibody-based drugs can cause very serious immune-related side effects and autoimmunity. In addition, checkpoint inhibitor drugs are very costly and benefit a minority of cancer patients. To overcome these challenges and unlock untapped opportunities, locally administering checkpoint inhibitors such as by use of the molecule and / or hydrogel as taught herein would be advantageous. The hydrogel as taught herein can gradually release an immunotherapeutic molecule locally, rather than spreading over the whole body. Furthermore, local checkpoint inhibition using the hydrogel as taught herein has the potential to promote cytotoxic T cell activation, albeit using considerably less of these potent drugs with much lower expected adverse effects as result. As a result, this would mean less hospital visits and less side effects for the patient.

[0125] Accordingly, in particular embodiments, the therapeutic agent is an immunotherapeutic molecule.

[0126] In particular embodiments, the therapeutic agent is a checkpoint inhibitor (e.g. a programmed cell death protein 1 (PD-1) inhibitor, a programmed cell death-ligand 1 (PD-L1) inhibitor or a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor), a histone deacetylate (HDAC) inhibitor or a cytokine (e.g. interleukin 2 (IL-2) or interleukin 4 (IL-4)). Non-limiting examples of PD-1 inhibitors include Nivolumab, Pembrolizumab, Cemiplimab, Tislelizumab, Dostarlimab, Retifanlimab and Toripalimab. Non-limiting examples of PD-L1 inhibitors include Atezolizumab, Avelumab, Durvalumab, and Cosibelimab. Non-limiting xamples ofCTLA-4 inhibitors include Ipilimumab and tremilumumab. Non-limiting HDAC inhibitors include Vorinostat, Romidepsin, Belinostat and Panobinostat.

[0127] The hydrogel as taught herein can be used for local immunoregulation and restoring T cell effector function, more particularly, the hydrogel as taught herein can be used for overcoming myeloid-driven immunosuppression and activating T cells in the tumor microenvironment. In particular embodiments, the therapeutic agent is an agent capable of activating cytotoxic T cells, such as IL-2, IL-4, an anti-PD-L1 antibody (e.g. anti-PD-L1 nanobody), an anti-PD1 antibody (e.g. anti-PD1 nanobody), an anti-CTLA-4 antibody (e.g. anti-CTLA-4 nanobody), or mRNA encoding these.

[0128] In particular embodiments, the therapeutic agent is a apolipoprotein nanoparticle as described in International patent application WO2022 / 268913, WO2023 / 046931, WO 2023 / 233042, WO 2023 / 227682, WO 2024 / 200824 or WO 2024 / 200824 , optionally comprising mRNA encoding IL-2 as a payload.

[0129] The hydrogel as taught herein may be prepared by any methods known in the art, such as by use of solid-phase peptide synthesis.

[0130] In an exemplary method for preparing the hydrogel as taught herein, the apolipoprotein mimetic peptide is synthesized and modified with a lipophilic tail on the N-terminus using Solid Phase Peptide Synthesis. Subsequently, this peptide is purified to >99% purity using preparative LCMS to yield apo-lip. Next, the resulting molecule as taught herein is dissolved in a combination of phosphate buffer (pH 7.3) and NaCI. By briefly warming to 50° C and letting the mixture cool to room temperature, the molecule as taught herein forms a hydrogel at concentrations larger than 1 w / v% (10 mg / mL). After warming up the dissolved peptide, a payload can be incorporated in these gels for gradual release.

[0131] A further aspect provides a hydrogel obtained by or obtainable by the method for preparing a hydrogel as taught herein.

[0132] A further aspect provides a pharmaceutical composition comprising the molecule as taught herein or the hydrogel as taught herein, and optionally a pharmaceutical carrier. The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0133] A further aspect provides a cosmetic composition comprising the molecule as taught herein or the hydrogel as taught herein, and optionally a cosmetically acceptable carrier. The cosmetically acceptable carrier may comprise one or more components selected from the group consisting of water, alcohols, oils, emollients, humectants, thickeners, emulsifiers, andcombinations thereof. In certain embodiments, the carrier may include, without limitation, purified water, ethanol, glycerin, propylene glycol, mineral oil, silicone oils, fatty acid esters, cetyl alcohol, stearic acid, carbomers, and polysorbates. The cosmetic composition may be formulated in various forms, including but not limited to creams, lotions, gels, serums, masks, sprays, or powders. In some embodiments, the molecule or hydrogel as taught herein may be present in the cosmetic composition in an amount effective to provide a desired cosmetic benefit. The present disclosure further provides a cosmetic use of the molecule, the hydrogel or the cosmetic composition. Such cosmetic uses may include, but are not limited to, improving skin hydration and moisture retention, reducing the appearance of fine lines and wrinkles, enhancing skin elasticity and firmness, promoting a more even skin tone, providing a protective barrier for the skin, delivering active ingredients to targeted skin layers, and temporary filling or plumping of skin areas. The cosmetic composition may be applied topically to the skin, hair, or nails, with the frequency and method of application varying based on the specific cosmetic use and product form.

[0134] A further aspect provides the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein for use as a medicament. In a related aspect, provided herein is the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein for use in the manufacture of a medicament. A further aspect provides the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein for use in vaccination, transplantation, and / or the treatment (e.g. treatment and / or prevention) of a disease, such as an infectious disease or a neoplastic disease, like cancer, such as by immunotherapy (e.g. localized immunotherapy), such as by vaccination. In a related aspect, provided herein is the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein for use in the manufacture of a medicament for vaccination, transplantation and / or the treatment (e.g. treatment and / or prevention) of a neoplastic disease, like cancer, such as by immunotherapy (e.g. vaccination).

[0135] A related aspect provides a method of vaccination, transplantation and / or treating (e.g. treating and / or preventing a neoplastic disease, like cancer, such as by immunotherapy (e.g. vaccination), in a subject, comprising administering a prophylactically or therapeutically effective amount of the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein to said subject.

[0136] A related aspect relates to use of the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein for vaccination, transplantation and / ortreating (e.g. treating and / or preventing) a neoplastic disease, like cancer, such as by immunotherapy (e.g. vaccination).

[0137] The treatment of an infectious disease can for example encompass the delivery and release of antimicrobials, topically, such as for example to skin lesions and wounds (wound healing), or into the body. In certain embodiments, the antimicrobial agent may be selected from a group consisting of antibiotics, antifungals, antivirals, and antiparasitics. The delivery method may include, but is not limited to, topical application, transdermal delivery, oral administration, parenteral injection, or implantation of a drug-eluting device. For topical applications, the antimicrobial agent may be formulated to facilitate adherence to the skin or wound surface and promote sustained release. The release profile of the antimicrobial agent may be modulated through various techniques, including pH-responsive release, enzymatic degradation, or stimuli-responsive systems, to optimize therapeutic efficacy and minimize potential side effects.

[0138] Except when noted, the terms “subject” or “patient” can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms “subject” or “patient” include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly a bone-related disease or disorder. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and / or those in who said condition is to be prevented.

[0139] The term “prophylactically effective amount” refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the pharmaceutical formulation as taught herein.

[0140] The term “neoplastic disease” generally refers to any disease or disorder characterized by neoplastic cell growth and proliferation, whether benign (not invading surrounding normal tissues, not forming metastases), pre-malignant (pre-cancerous), or malignant (invading adjacent tissues and capable of producing metastases). The term neoplastic diseasegenerally includes all transformed cells and tissues and all cancerous cells and tissues. Neoplastic diseases or disorders include, but are not limited to abnormal cell growth, benign tumors, premalignant or precancerous lesions, malignant tumors, and cancer. Examples of neoplastic diseases or disorders are benign, pre-malignant, or malignant neoplasms located in any tissue or organ, such as in the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, or urogenital tract.

[0141] In certain embodiments of the methods or uses as taught herein, the neoplastic disease may be a tumor or may be characterized by the presence of a tumor.

[0142] As used herein, the terms “tumor” or “tumor tissue” refer to an abnormal mass of tissue that results from excessive cell division. A tumor or tumor tissue comprises tumor cells which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign, pre-malignant or malignant, or may represent a lesion without any cancerous potential. A tumor or tumor tissue may also comprise tumor-associated non-tumor cells, e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue.

[0143] In certain embodiments of the methods or uses as taught herein, the neoplastic disease is cancer.

[0144] As used herein, the term “cancer” refers to a malignant neoplasm characterized by deregulated or unregulated cell growth. The term “cancer” includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject’s body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor). The term “metastatic” or “metastasis” generally refers to the spread of a cancer from one organ or tissue to another non-adjacent organ or tissue. The occurrence of the neoplastic disease in the other non-adjacent organ or tissue is referred to as metastasis.

[0145] Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include without limitation: squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung and large cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as CNS cancer, melanoma, head and neck cancer, bone cancer, bone marrow cancer, duodenum cancer, esophageal cancer, thyroid cancer, or hematological cancer. In particular embodiments, the cancer is melanoma, colorectal cancer or ovarian cancer, such as epithelial ovarian cancer.

[0146] Other examples of cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Urethra, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Glioblastoma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, IntraocularMelanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Non-melanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo- / Malignant Fibrous Sarcoma, Osteosarcoma / Malignant Fibrous Histiocytoma, Osteosarcoma / Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumour, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraganglioma, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm / Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Urethra Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Urethra, Transitional Renal Pelvis and Urethra Cancer, Trophoblastic Tumours, Urethra and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, or Wilms’ Tumour.

[0147] In particular embodiments, the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein is being administered to the subject by essentially any rout of administration, such as without limitation, parenteral administration (such as, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal or intrasternal injection or infusion). In this way, the prophylactic or therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc. depending of the specific needs of a given application. In particular embodiments, the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein is being administered to the subject subcutaneously, intraperitoneal and / or intratumoral.A further aspect provides the in vitro or ex vivo use of the molecule as taught herein, the hydrogel as taught herein or the pharmaceutical composition as taught herein in cell or tissue culture.

[0148] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and scope of the appended claims.

[0149] The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0150] EXAMPLES

[0151] Example 1. Testing of several types of hydrogel-stabilizing portions.

[0152] 1.1 Materials and methods

[0153] All chemicals and solvents were purchased from commercial sources (VWR, Biosolve, Iris Biotech) and used as received, unless stated otherwise.

[0154] Solid phase peptide synthesis

[0155] 2F peptide (comprising an amino acid sequence DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), optionally wherein its N-terminus is acetylated and its C-terminus is amidated) and its peptidic derivatives were synthesized using Fmoc-based solid-phase peptide synthesis (SPPS) using an automated peptide synthesizer with scales ranging from 50 pmol to 5 mmol on a Rink amide MBHA resin (Iris Biotech).

[0156] N-terminal peptide modification was performed on resin. N-terminal acetylation was performed using 20:20:60 v / v% pyridine:acetic anhydride: DM F for 1 hour. N-terminal labeling with an alkyl tail (e.g. C18) was performed on resin using stearic acid:HCTU:DIPEA (3:2:6 equivalents, in comparison to resin) for 1 hour. The person skilled in the art will understand that, in view of the N-terminal labeling, the 2F peptide within the molecule as disclosed herein is not N-terminally acetylated, whereas the C-terminus may be amidated.

[0157] After the synthesis, resin was washed and dried under reduced pressure. Removal of protecting groups and cleavage of the resin was performed by incubation in a mixture of trifluoracetic acid (TFA), H2O, and triisopropylsilane (TIS) (96.5:2.5:1) for 2 hours withcontinuous agitation, followed by precipitation in excess of ice-cold diethyl ether (Et20), dried under compressed air, dissolved in Milli-Q and lyophilized to obtain the crude peptide. All peptides were purified using preparative HP-LC. This was performed using an alkyl-modified column (e.g. C18 column) using ultrapure water with 0.1% formic acid (FA) and acetonitrile with 0.1% FA with various gradients. Correct mass and purity of peptides was identified using LC-MS.

[0158] LC-MS lontrap Analysis

[0159] Analytical chromatography coupled with mass-spectrometry (LC-MS), performed on a C4 Jupiter SuC4300A 150 x 2.0 mm column using ultrapure water with 0.1% FA and formic acid with 0.1% FA. In general, a gradient of 5% to 100% acetonitrile over 10 minutes was used, connected to a Thermo Fischer LCQ Fleet Ion Trap Mass Spectrometer. The purity of the samples was assessed using a UV detector.

[0160] Circular Dichroism

[0161] CD spectra were measured on a JASCO J-815 CD Spectrometer equipped with a Peltier temperature controller. Measurements were performed at 20 °C in Quartz cuvettes with a pathlength of 1 cm and a concentration of 85 pM. A scanning rate of 100 nm / min, a bandwidth for monitoring of 1.0 nm, a response time of 1 s, a data pitch of 0.1 nm, triple accumulation and a scanning range of 350-190 nm were employed.

[0162] Nile Red

[0163] Fluorescence spectroscopy with Nile Red was measured on a Cary Eclipse Fluorescence Spectrophotometer. Measurements were performed at 20 °C. The sample was excited at 550 ± 10 nm and the emission was recorded at 565-800 nm with fast scan control, medium PMT detector voltage, averaging 5 scansions.

[0164] CryoTEM

[0165] Glacios

[0166] Vitrified thin films for cryo-TEM analysis were prepared using an automated vitrification robot (FEI Vitrobot Mark IV) by plunge vitrification in liquid ethane. Before vitrification, a 200-mesh copper grid covered with a Quantifoil R 2 / 2 holey carbon film (Quantifoil Micro Tools GmbH,DE) was surface plasma treated for 40 seconds using a Cressington 208 carbon coater. CryoTEM imaging was carried out on a Glacios (Thermo Fisher, NL), equipped with a field emission gun (X-FEG), Ceta 16M camera and a Falcon 4i direct electron detector. The microscope was operated at 200 kV acceleration voltage in bright-field TEM at a nominal magnification of 6.500* and a dose rate of 2 e- / A2s; or at 24.000* magnification and a dose rate of 4 e- / A2s; both with a 1s image acquisition time.

[0167] Talos

[0168] 3 uL of sample was applied to glow-discharged 200 mesh gold Quantifoil grids (Electron Microscopy Sciences, USA) and vitrified in liquid ethane using an automated vitrification robot (FEI Vitrobot Mark IV). Imaging was performed using a TALOS F200C-G2 transmission electron microscope (Thermo Fisher, NL) operated at 200 kV, equipped with a Falcon 4i direct electron detector to visualize fibers at a magnification of 28.000x or 92.000x.

[0169] Gelation into hydrogel

[0170] Freeze-dried peptide was weighed into 1.5 mL clean, separate glass vials. Then, the solids were dissolved in adequate amount of 1 M Phosphate Buffer (pH 7.4). The samples were heated for 15 minutes at 50 °C. After heating, the samples were cooled down to room temperature. After overnight gelation, the state of the solution is visually described, based on a range from dissolved to precipitation, via hydrogel (with hydrogel being considered as a hit). Investigated w / v% of the hydrogel range from 0.5 till 10 w / v%, with 100 pL volumes hydrogel.

[0171] In addition to 1 M Phosphate Buffer pH 7.4, also other buffers were investigated (including but not limited to HEPES, TRIS, PBS). Results indicated that variations in the buffer are possible and can influence the properties of the hydrogels.

[0172] Based on visual inspection, formulations were ranked as either solution, viscous, runny gel, gel, turbid gel or precipitate (see Fig. 1).

[0173] Rheology

[0174] Rheological measurements were performed to determine the mechanical properties of the hydrogel. Hydrogels were prepared according to the hydrogel sample prep and heated to 50 °C on a heating plate. The samples were then pipetted onto the rheometer. All measurements were performed in triplo and measured at 21 °C. A solvent trap was used to minimize sample drying during the measurements. Hydrogel formation was followed for 12 h (plateau wasreached), by measuring the storage and loss moduli (G’ and G” respectively) by applying an oscillating deformation of amplitude y 0.01 at a frequency of 1 rad s-1. Strain sweep experiments were measured at strains ranging from 0.1 to 100%, at a constant frequency of 1 rad S’1. Frequency sweeps were performed at frequencies ranging from 0.1 to 100 rad s-1, at a constant strain of 1%. Stress relaxation test were performed under 0.5% strain with a strain rise time of 0.01 s. The released stress was quantified after 1000 s.

[0175] Results

[0176] All peptides described in Example 1 (Fig. 1A) were successfully synthesized and purified, as confirmed by LCMS analysis. All peptides dissolved at 0.5, 1, 2, 4 w / v% were inverted and scored based on the property of the content (Fig. 1, B-D). Where acetylated (e.g. N-terminally acetylated) 2F peptide dissolves at all concentrations, the introduction of an alkyl tail resulted in the formation of a gel. A trend can be seen between tail length and concentration needed for gelation, where a C14 tail can gelate at 1 w / v%, and both longer and shorter tails lead to a gradual need for higher concentrations to form a gel. The introduction of an unsaturated bond and thus an angle in the lipid tail (Linolenic acid) did not differ much from the saturated C18 variant. The Fmoc protecting group formed a clear gel at both 2 and 4 w / v%.

[0177] Peptides derivatized with amino acid tails were also successfully synthesized, purified and analyzed. Strikingly, FP and WP showed gelation at 2 w / v% (FP) or 0.5 and 1 w / v% (WP) (Fig. 1, D). The introduction of a charge (K / E) on the N-terminus to both WP and FP resulted in runny gels at 4 w / v%.

[0178] Samples of 2F with varying alkyl tails were dissolved at 500 pM for CryoTEM analysis. Fig.

[0179] 2 shows fibers forming at this concentration, with a different bundling profile for different tail lengths, but all composed of fibers with a constant diameter of 3.7 ± 0.2 nm.

[0180] Rheological measurements were performed 4 w / v% of varying tails. The storage (G’ ) and loss (G”) moduli are plotted in Fig. 3. Within these gels, a broad range of moduli could be achieved. Besides C6, all derivatives showed a tan(8) below 1, confirming their gel-like properties. These results indicate that modularity of the rheological properties is possible by changing derivatization.

[0181] Example 2. Variations of the peptide-based portion

[0182] 2.1 Materials and methods

[0183] Peptides used, including N-terminal derivatisation ( e.g. with an alkyl tail such as C18) and subsequent purification, were synthesized as described in Example 1. Accordingly, thesequences shown in Fig. 4 are N-terminally linked to an alkyl tail, more particularly C18, and are C-terminally amidated.

[0184] Gelation into hydrogel was performed as described in Example 1, resulting in a heatmap. The rheological properties of the hydrogels were determined as described in Example 1.

[0185] 2.2 Results

[0186] Systematic changes in the sequence of 2F and the result of that change on the gelation properties is depicted in Fig. 4. The 3D configuration of the 2F derivative is important. This is highlighted by the introduction of a proline (which has a fixed angle) in the middle or at the end of the 2F sequence. As illustrated in the top of Fig. 4, a proline in the middle of the sequence breaks the a-helical nature of the peptide and this indeed prevents gelation. The amount of charges is also an important aspect in the gelation property of 2F. The peptide has 4 positive and 4 negative charges (4+ / 4-), but when these are partially replaced by noncharged amino acids (final charge ratio of 3+ / 3-) in 4 different replacements they all precipitate. When introducing a net charge (5+ / 4-) the placement of this extra charge is important. An introduced lysine at the interface of the hydrophilic and hydrophobic domain of 2F resulted in a 2F-derivative which could form a gel at an even lower concentration than the unmodified 2F derivative.

[0187] ApoA1 mimetic peptides ATI-5261, Ac-FAMP, 4F, 37pA and ESP-2418 were screened as acetyl, C18 and Fmoc derivative and Ac-FAMP, C18-4F and Ac-37pA readily formed a gel at 4 w / v% (Fig 5).

[0188] Example 3. (In vivo) release of a payload from the hydrogel

[0189] 3.1 Materials and methods

[0190] Synthesis

[0191] Peptides used, including N-terminal derivatisation (e.g. C18) and subsequent purification, were synthesized as described in Example 1.

[0192] Additionally, variants of the used 2F were designed for covalent conjugation of payload. The different C-terminally modified peptides are described below.

[0193] 2F-propargyl (Ac-DWLKAFYDKVAEKLKEAFGG(propargyl) SEQ ID NO: 40) Propargylglycine was introduced as non-natural amino acid on the C terminus for copper-click functionalization with payload.- 2F-Mal (Ac-DWLKAFYDKVAEKLKEAFGK(alloc) SEQ ID NO: 41)

[0194] A lysine with an alloc orthogonal protecting group was introduced on the C-terminus. This protecting group was selectively cleaved using Pd-tetrakis, followed by NHS- PEG4-Mal coupling to give final product 2F-Mal.

[0195] - 2F-Azide (Ac-DWLKAFYDKVAEKLKEAFGK(N3) SEQ ID NO: 42)

[0196] Azido-lysine was introduced as non-natural amino acid on the C terminus for strain- promoted click functionalization with payload.

[0197] Azido-sCy5 (Lumiprobe) (0.91 pmol, 1.1 eq, 0.695 mg) was added from a 10 mg / mL stock in DMSO to peptide dissolved in phosphate buffer (0.83 pmol, 1 eq, 2.1 mg) supplemented with THPTA:CUSO4 (2:1)(12.5 pmol , 15 eq, 13 mg) and ascorbic acid (25 pmol, 30 eq, 4.4 mg). This mixture was reacted for two hours and directly purified using prep-HPLC as described in Example 1.

[0198] Comparable protocols were followed for the functionalization with azido-Cy3 (Lumiprobe), DFO-thiol (with only phosphate buffer) and DFO-DBCO (Macrocyclics, also only phosphate buffer)

[0199] (In vitro) release

[0200] Of azido-sCy5, 2F-sCy5, C18-2F-sCy5, mVenus protein and Fmoc-2F-sCy5, 500 pM stocks were made. 10 pL of stock was added to 90 pL of heated gel and placed on an insert with semipermeable membrane (8pm membrane pore size, GBO ThinCerts). These dye-loaded gels were let to gelate overnight and subsequently placed in 600 pL release buffer (PBS supplemented with 0.5 mg / mL HDL and 40 mg / mL BSA). At set timepoints, the release buffer was collected and replaced with fresh buffer. The collected samples were quantified by fluorescence using a Tecan MC Spark platereader and a calibration curve based on the dye stock, diluted in release buffer.

[0201] PK and Biodistribution

[0202] DFO and DFO-2F was radiolabeled with89Zr using a previously described protocol. In brief, a89Zr oxalate solution in 1 M oxalic acid was neutralized using a 1 M sodium carbonate solution until a pH between 6.8-7.4 was reached (total volume < 25 pL). The neutralized89Zr solution was added to DFO(-2F) dissolved in Milli-Q water (0.5 ml) and the mixture wasincubated at 37 °C using a thermomixer (300 r.p.m.) for 60 min. Completion of the reaction was confirmed by radio-TLC (Typhoon 7000IP plate reader, GE Healthcare).

[0203] Female C57BL / 6 mice (n=5 per group) were intravenously administered with89Zr-DFO(-2F) in 80-100 pl of 4w / v% C18-2F gel via subcutaneous or intraperitoneal injection. At various time points after injection (1 min, 15 min, 30 min, 1h, 2 h, 4h, 24h and 48h) a blood sample was collected and the emitted y radiation was measured by a gamma counter (2480 WIZARD2 Automatic Gamma Counter, PerkinElmer). Radioactivity values were corrected for decay and normalized to tissue weight to express radioactivity concentration as %ID per gram.

[0204] Injection site quantification was performed by measuring the radioactivity of the carcass after organ removal, leaving the injection site intact and correcting for decay.

[0205] PET-CT

[0206] Positron emission tomography imaging was performed 48 h after89Zr-DFO(-2F) injection using an IRIS PET-CT (Inviscan). Mice were anesthetized with a gas mixture of 2% isoflurane and 5% oxygen. A 10 min static whole-body PET scan was conducted using an energy window between 250-750 keV followed by a 20 s whole-body CT scan (energy 80 kV, 0.9 mA, 576 projections, voxel size 160 pm). Reconstruction of the PET images was achieved with the 3D Ordered Subsets Expectation Maximization (3D-OSEM-MC) algorithm (eight subsets and eight iterations) using decay, random and dead-time correction.

[0207] 3.1 Results

[0208] Release in buffer

[0209] The amount of released dye from gels into release buffer could be reliably measured in duplo and quantified. In all cases, unmodified gel released from the gel the fastest, whereas functionalization of dye with 2F peptide resulted in a slower release from gels. The release of dye from 2 and 4 w / v% were comparable, where 2w / v% Fmoc had a much slower release (Fig. 6). These results indicate that variations in derivatization enable modularity of the platform, making it possible to choose the desired release rate from the library of derivatives.

[0210] Release in vivo

[0211] 4 w / v% C18-2F was used for all groups, while varying sc and ip injection, as well as DFO and 2F-functionalized DFO between groups. After 48 hours, all subcutaneous gels were still in place as shown on PET-CT (Fig. 7) with minimal signal grom other organs. Intraperitoneal gels showed some distribution to lymph nodes, and visually appeared to be released furtherthan s.c. injected gels. These results show the applicability of this platform towards therapeutic use, with facile handling prior to injection, and a gel structure that is stable in vivo. After 48 hours the majority of DFO was found to still be present at the injection site, as shown in Fig. 8. S.c. injections indeed seem to be slower than i.p., and functionalizing DFO with 2F appears to have a retaining effect, comparable to the results from release in buffer. Biodistribution of DFO after 48 hours gave a very promising profile, with significant signal from immune-related organs such as spleen and lymph nodes, confirming the accumulation seen on PET-CT.

[0212] Example 4. In vivo therapeutic study

[0213] 4.1 Methods and materials

[0214] Rapamycin-2F conjugate

[0215] 100 mg (0.109 mmol, 1 eq) of rapamycin was dissolved in 5 mL dry dichloromethane (DOM) and to this solution, 27.6 mg (0.327 mmol, 3 eq) of azidoacetic acid and 66 mg (0.327 mmol, 3 eq) of dicyclohexylcarbodiimide (DCC) was added. Upon dissolution, 1.1 mg (0.09 mmol, 0.083 eq) of 4-dimethylaminopyridine (DMAP) was added and the solution was flushed with nitrogen. After 3 hours the reaction mixture was filtered and purified through a 4 gram silica column with an ethyl acetate / hexane gradient from 40-60% ethyl acetate.

[0216] 13.3 mg of 2F-azide (5.5 umol, 1 eq) as described in Example 3 was dissolved in 2 mLof 1M phosphate buffer pH 7.4. In parallel, 5.2 mg (5.2 umol, 0.95 eq) of rapamycin-azide was dissolved in 1.5 mL of acetonitrile (ACN) and added to the dissolved peptide. 24.4 mg of Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) was dissolved in milli-Q water (MQ) and added to 11.4 mg of CuSCL in 1 mL of MQ. The copper complex was added to the reaction mixture and directly after 28.4 mg of ascorbic acid was added in 1 mL of MQ. After 2 hours, the reaction mixture was purified using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) with an mass spectrometry (MS) detector and a 5-100% acetonitrile (ACN) gradient

[0217] B16F10 tumor inoculation and treatment regimen

[0218] Female C57BL / 6J mice were inoculated with 1*105B16F10 (ATCC) cells in 100 pL PBS via subcutaneous injection in the right flank on day -7. Mice without palpable tumor on day 0 were excluded from the experiment, and the included mice were allocated to treatment and control groups. Treatment consisted of subcutaneous administration of 100 pL 2 w / v% C18-2F hydrogel containing 2F-rapamycin (10 pg rapamycin equivalent, 0.5 mg / kg) eitherperitu moral ly or in the flank opposite of the tumor, control groups consisted of 100 pL of either PBS or empty 2 w / v% C18-2F hydrogel subcutaneously administered peritumorally. Mice were euthanized in accordance with Dutch animal welfare regulations once they reached the predefined humane endpoint criteria, which included a tumor volume of 1500 mm3or greater, or the occurrence of tumor ulceration or observable signs of distress.

[0219] 4.2 Results

[0220] Daily tumor measurements showed a rapid increase in tumor volume, typical for B16F10 melanoma. Despite this rapid growth, a difference can be seen between rapamycin-containing gels and PBS / empty gel controls (Figure 9A). The low dose is able to achieve an effect within the timeframe of this model, as is also illustrated by the survival plot (Figure 9B). after 12 days, an increased survival is achieved by both the local (peritumoral) and distant (other flank) subcutaneously administered gels.

Claims

CLAIMS1. A molecule capable of self-assembling into a three-dimensional network to form a hydrogel, wherein said molecule comprises:a) a peptide-based portion at least part of which is capable of forming an amphipathic alpha-helix; andb) a hydrogel-stabilizing portion wherein the hydrogel-stabilizing portion promotes the formation of the hydrogel, wherein the hydrogel formation involves intermolecular interaction between the amphipathic alpha-helices.

2. The molecule according to claim 1, wherein the peptide-based portion is between 6 and 400 consecutive amino acids.

3. The molecule according to claim 1 or 2, wherein the peptide-based portion is between 10 and 40 consecutive amino acids.

4. The molecule of any one of claims 1 to 3, wherein the peptide-based portion comprises an apolipoprotein, an apolipoprotein mimetic peptide, or a fragment or variant thereof.

5. The molecule according to claim 4, wherein the apolipoprotein is ApoA-1 orApoE, or the apolipoprotein mimetic is selected from the group consisting of ApoA-1 mimetic peptides, including 2F, 3F, 4F, 5F, 6F, and 7F, ATI-5261, ESP-2418, ELK peptides, FAMP, i-FAMP, 18A, R18A, and 37pA; ApoE mimetic peptides, including ApoE(130-149), Ac-hE18A-NH2, COG133, COG1410, COG112, and ApoE(141-155)2; and apo C-ll mimetic peptides, including D6PV.

6. The molecule according to any one of claims 1 to 5, wherein the hydrogel-stabilizing portion comprises a moiety capable of intermolecular hydrophobic interactions, intermolecular beta-sheet formation, intermolecular TT-TT stacking, or intermolecular amphiphilic interaction.

7. The molecule according to any one of claims 1 to 6, wherein the hydrogel-stabilizing portion comprises a C4-C22 aliphatic chain, such as a C4-C22 fatty acid, optionally a stearic acid, an undecanoic acid, or a hexanoic acid.

8. The molecule according to any one of claims 1 to 6, wherein the hydrogel-stabilizing portion comprises an aromatic moiety capable of TT-TT stacking, such as a fluorenyl moiety, optionally a fluorenylmethoxycarbonyl (Fmoc) moiety.

9. The molecule according to any one of claims 1 to 6, wherein the hydrogel-stabilizing portion comprises a dipeptide selected from FP or WP, optionally wherein the dipeptide is N-terminally acetylated.

10. The molecule according to any one of claims 1 to 9, wherein the hydrogel-stabilizing portion is N-terminally adjacent to the peptide-based portion.

11. A hydrogel comprising the molecule according to any one of claims 1 to 10.

12. The hydrogel according to claim 11, comprising at least 0.5% (w / v) of the molecule according to any one of claims 1 to 10.

13. The hydrogel according to claim 11 or 12, further comprising a payload, preferably wherein said payload is a therapeutic agent.

14. A pharmaceutical or cosmetic composition comprising the molecule according to any one of claims 1 to 10 or the hydrogel according to any one of claims 11 to 13.

15. The molecule according to any one of claims 1 to 10, the hydrogel according to any one of claims 11 to 13 or the pharmaceutical composition according to claim 14 for use as a medicament.

16. The molecule according to any one of claims 1 to 10, the hydrogel according to any one of claims 11 to 13 or the pharmaceutical composition according to claim 14 for use in vaccination, transplantation and / or the treatment of a disease such as a neoplastic disease or an infectious disease.

17. A cosmetic use of the molecule according to any one of claims 1 to 10, the hydrogel according to any one of claims 11 to 13 or the cosmetic composition according to claim 14.

18. In vitro or ex vivo use of the molecule according to any one of claims 1 to 10 or the hydrogel according to any one of claims 11 to 13 in cell or tissue culture.

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