Self-assembled composite ultrasmall peptide-polymer hydrogels

Abstract

The present invention relates to composite hydrogels comprising at least one non-peptidic polymer and at least one peptide having the general formula: Z(X).sub.m(Y).sub.nZ.sub.p, wherein Z is an N-terminal protecting group; X is, at each occurrence, independently selected from an aliphatic amino acid, an aliphatic amino acid derivative and a glycine; Y is, at each occurrence, independently selected from a polar amino acid and a polar amino acid derivative; Z is a C-terminal protecting group; m is an integer selected from 2 to 6; n is selected from 1 or 2; and p is selected from 0 or 1. The present invention further relates to methods of producing the composite hydrogels, to uses of the composite hydrogels for the delivery of drugs and other bioactive agents/moieties, as an implant or injectable agent that facilitates tissue regeneration, and as a topical agent for wound healing. The present invention further relates to devices and pharmaceutical or cosmetic compositions comprising the composite hydrogels and to medical uses of the composite hydrogels.

Claims

1. A composite hydrogel comprising at least one non-peptidic polymer and at least one peptide, wherein said peptide is selected from the group consisting of Z-LIVAGDD-Z.sub.p (SEQ ID NO: 1), Z-LIVAGDE-Z.sub.p(SEQ ID NO: 2), Z-LIVAGED-Z.sub.p(SEQ ID NO: 3), Z-LIVAGEE-Z.sub.p(SEQ ID NO: 4), Z-LIVAGKC-Z.sub.p(SEQ ID NO: 5), Z-LIVAGSC-Z.sub.p(SEQ ID NO: 6), Z-AIVAGKC-Z.sub.p(SEQ ID NO: 7), Z-AIVAGSC-Z.sub.p(SEQ ID NO: 8), Z-LIVAGC-Z.sub.p(SEQ ID NO: 9), Z-LIVAGD-Z.sub.p(SEQ ID NO: 10, Z-AIVAGD-Z.sub.p(SEQ ID NO: 14), Z-LIVAGE-Z.sub.p(SEQ ID NO: 15), Z-LIVAGK-Z.sub.p(SEQ ID NO: 16), Z-LIVAGS-Z.sub.p(SEQ ID NO: 17), Z-ILVAGS-Z .sub.p(SEQ ID NO: 18), Z-AIVAGS-Z.sub.p(SEQ ID NO: 19), Z-LIVAGT-Z.sub.p(SEQ ID NO: 20), Z-AIVAGT-Z.sub.p(SEQ ID NO: 21), Z-ILVAGK-Z.sub.p(SEQ ID NO: 49), Z-ILVAG(Orn)-Z.sub.p(SEQ ID NO: 50), Z-ILVAG(Dab)-Z.sub.p(SEQ ID NO: 51), Z-ILVAG(Dap)-Z.sub.p(SEQ ID NO: 52), Z-ILVAGS-Z.sub.p(SEQ ID NO: 53), Z-AIVAGK-Z.sub.p(SEQ ID NO: 55), Z-AIVAG(Orn)-Z .sub.p(SEQ ID NO: 56), Z-AIVAG(Dab)-Z .sub.p(SEQ ID NO: 57), Z-AIVAG(Dap)-Z.sub.p(SEQ ID NO: 58), Z-LIVAG(Orn)-Z.sub.p(SEQ ID NO: 59), Z-LIVAG(Dab)-Z.sub.p(SEQ ID NO: 60), and Z-LIVAG(Dap)-Z.sub.p(SEQ ID NO: 61; wherein Z is an N-terminal protecting group wherein said N-terminal protecting group has the general formula C(O)R, wherein R is selected from the group consisting of H, and alkyl; Z is a C-terminal protecting group wherein said C-terminal protecting group is an amide group or an ester group; p is selected from 0 or 1; wherein when the non-peptidic polymer is a cationic polymer comprising chitosan or poly (amido amine) dendrimers a polar amino acid or a polar amino acid derivative of the peptide adjacent to Z consists of an acidic polar amino acid or an acidic polar amino acid derivative; wherein when the non-peptidic polymer is a neutral polymer comprising polyethylene glycol (PEG), then the polar amino acid or polar amino acid derivative of the peptide adjacent to Z consists of a neutral polar amino acid, a neutral polar amino acid derivative, a basic polar amino acid or a basic polar amino acid derivative; and wherein when the non-peptidic polymer is an anionic polymer comprising a nucleic acids, chondroitin sulphate or hyaluronic acid, then the polar amino acid or polar amino acid derivative of the peptide adjacent to Z consists of a basic polar amino acid or a basic polar amino acid derivative.

2. The composite hydrogel of claim 1, wherein the hydrophobicity decreases from the N-terminus to the C-terminus of said peptide.

3. The composite hydrogel of any of claim 1, wherein said acidic polar amino acid and acidic polar amino acid derivative are selected from the group consisting of aspartic acid (Asp, D), glutamic acid (Glu, E), and cysteine (Cys, C), and wherein said basic polar amino acid and basic polar amino acid derivative are selected from the group consisting of serine (Ser, S), threonine (Thr, T), allo-threonine, lysine (Lys, K), ornithine (Orn), 2,4-diaminobutyric acid (Dab), and 2,4-diaminopropionic acid (Dap.

4. The composite hydrogel of claim 1, wherein said acidic polar amino acid and acidic polar amino acid derivative are selected from aspartic acid (Asp, D) and glutamic acid (Glu, E), or wherein said basic polar amino acid and basic polar amino acid derivative are selected from the group consisting of lysine (Lys, K), ornithine (Orn), 2,4-diaminobutyric acid (Dab), and 2,4-diaminopropionic acid (Dap).

5. The composite hydrogel of claim 1, wherein said cationic polymer is a hydrophilic or amphiphilic polymer, and said non-peptidic polymer is a linear or branched polymer, wherein said branched polymer is a dendrimer.

6. The composite hydrogel of claim 1, wherein said cationic polymer is selected from the group consisting of chitosan, and poly(amido amine) dendrimers, said anionic polymer is selected from the group consisting of nucleic acids, comprising RNA and DNA, said neutral polymer is polyethylene glycol (PEG), wherein said PEG is selected from diethylene glycol diacrylate (DEGDA), bi-functional PEG, N-hydroxysuccinimide-PEG-maleimide and bis(succinimidyl) polyethylene glycol (BS-PEG).

7. The composite hydrogel of claim 1, wherein the composite hydrogel further comprises at least one bioactive agent, wherein said at least one bioactive agent is selected from the group consisting of nucleic acids, (poly)peptides, virus particles, oligosaccharides, polysaccharides, vitamins, sialic acids, antigens, antibiotics, anti-inflammatory molecules, vaccines, drugs, prodrugs, nanoparticles and other organic or inorganic compounds.

8. The composite hydrogel of claim 7, wherein said at least one bioactive agent is selected from a growth factor or cell adhesion molecule, a nucleic acid encoding a growth factor and a cell adhesion molecule, wherein said growth factor comprises a cytokine, and said nucleic acid encodes a cytokine.

9. The composite hydrogel of claim 7, wherein said at least one bioactive agent is encapsulated by said composite hydrogel, or wherein said at least one bioactive agent is conjugated to said composite hydrogel, wherein said at least one bioactive agent is coupled to at least one functional group present on said non-peptidic polymer or to at least one functional group present on said peptide, wherein said at least one functional group is selected from the group consisting of amines, carboxylic acids, thiols, alcohols, carbohydrates, amides, imines, imides, azides, nitriles, peroxides, esters, thioesters, phosphates, aryls, aldehydes, ketones, sulfates, sulfites, nitrates, nitrites, phosphonates, silanes, alkanes, alkenes and alkynes.

10. The composite hydrogel of claim 7, wherein said at least one bioactive agent is a negatively charged bioactive agent, and said non-peptidic polymer is a cationic polymer, or wherein said at least one bioactive agent is a positively charged bioactive agent, and said non-peptidic polymer is an anionic polymer, or wherein said at least one bioactive agent is a neutral bioactive agent, and said non-peptidic polymer is a neutral polymer.

11. The composite hydrogel of claim 1, wherein said nucleic acid comprises an oligonucleotide, polynucleotide, DNA, RNA, modified and artificial nucleic acids and nucleic acid analogues, or combinations thereof, wherein said nucleic acid is selected from the group consisting of oligonucleotide, polynucleotide, plasmid DNA, aptamers, mRNA, microRNA, siRNA and short hairpin RNA.

12. The composite hydrogel of claim 11, wherein said nucleic acid is a bioactive agent or encodes a bioactive agent, a growth factor, or a cell adhesion molecule, and/or wherein said nucleic acid is conjugated to a bioactive agent.

13. The composite hydrogel of claim 1, wherein said at least one non-peptidic polymer is present at a concentration of 50% (w/w) or less, with respect to the total weight of said composite hydrogel, or wherein said at least one non-peptidic polymer is present at a concentration of 40% (w/w) or less, with respect to the total weight of said composite hydrogel, or wherein the total nucleic acid content of said composite hydrogel is 50% or less by charge ratio.

14. A pharmaceutical or cosmetic composition comprising a composite hydrogel of claim 1.

15. A method of producing a composite hydrogel of claim 1, comprising the steps of: preparing an aqueous solution of a mixture of said at least one non-peptidic polymer of claim 1 and said at least one peptide of claim 1; or treating a preformed hydrogel comprising said at least one peptide with a solution of said at least one non-peptidic polymer, optionally further comprising at least one of the steps of: adding at least one bioactive agent; adding an ultraviolet (UV) photoinitiator to said aqueous solution and exposing said aqueous solution to UV irradiation; adding at least one coupling reagent to facilitate the formation of covalent linkages; adding at least one compound acting as gelation enhancer; adding at least one buffer, preferably at least one physiologically acceptable buffer.

16. A method comprising culturing cells with a 3-D scaffold of the composite hydrogel of claim 1, wherein the scaffold allows for the embedding of the cells.

17. The method of claim 16, wherein said cells are stem cells, including embryonic stem cells, human induced pluripotent stem cells (iPS), progenitor cells, adult stem cells, cord blood stem cells, mesenchymal stem cells, adipose-derived stem cells, and hematopoietic stem cells.

18. A method for delivery of a drug or a bioactive agent to a subject, comprising administering to the subject the composite hydrogel of claim 1 that comprises the drug or bioactive agent.

19. The method of claim 18, wherein the delivery is for sustained or controlled release, or as an implant or as an injectable agent that gels in situ.

20. A 3-D scaffold for culturing cells comprising a composite hydrogel of claim 1, wherein said cells are preferably stem cells, including embryonic stem cells, human induced pluripotent stem (iPS) cells, progenitor cells and adult stem cells, cord blood stem cells, mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells.

21. An implant or injectable agent comprising a composite hydrogel of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Schematic of ultrasmall peptide polymer composite hydrogel formation.

(2) Composite hydrogels are formed by mixing self-assembling ultrashort peptides with linear and branched polymers. The polymeric component can be incorporated into the mixture during the peptide self-assembling process or post-assembly. The polymeric component can interact with the peptides electrostatically, or by van der Waals interaction, hydrogen bondings or covalent bonding, thereby forming cross-links between peptide fibers, and/or providing avenues for the attachment for bioactive moieties of interest.

(3) FIG. 2. Ultrashort peptide-cationic polymer composite hydrogels

(4) Ultrashort peptides with acidic polar head groups form stable composite hydrogels when mixed with cationic polymers. As a proof-of-concept, chitosan (A), a biocompatible natural polysaccharide soluble at low pH, was used as the cationic polymeric component. The presence of primary amine groups on the polymer can potentially react with the carboxylic acid group on the acidic polar head group and will facilitate conjugation chemistry for the attachment of bioactive moieties. (B) Composite AcLD.sub.6-chitosan hydrogels (on the right) are formed by mixing AcLD.sub.6 (L) peptide (Ac-LIVAGD; SEQ ID NO: 65; dissolved in sodium hydroxide) with 1 kDa water soluble chitosan. At the same concentration, the peptide component dissolved in sodium hydroxide (control sample on the left) does not form hydrogels. (C) Hydrogel formation using different compositions of peptide and chitosan.

(5) FIG. 3. Composite hydrogels retain the nanofibrous microarchitecture of pure peptide hydrogels, as observed under field emission scanning microscopy.

(6) Composite hydrogel retains porous honeycomb structure, though nanoscale fibers are less defined. In comparison, the polymeric component did not demonstrate any fibrous architecture.

(7) FIG. 4. Mechanical properties of composite hydrogel.

(8) (A, B) Increasing chitosan concentration results in an increase in ability to tolerate strain (elasticity) with a concomitant slight decrease in mechanical strength. (C) Increasing polymer length, wherein W300 is longer than W5, leads to slight compromise in mechanical strength and increase in ability to tolerate strain.

(9) FIG. 5. Mechanical properties of composite hydrogel as compared to those of nucleus pulposus (NP).

(10) Composite peptide-polymer hydrogels (A) have comparable mechanical stiffness to that of human NP(C). Peptide hydrogels are significantly stronger, but not as elastic. Thus the polymeric component can be used to modulate the mechanical properties to match that of native tissue. Porcine NP (B) is 20 times weaker than human (C). This could be attributed to differences in instruments used for measurements and experimental techniques.

(11) FIG. 6. Ultrashort peptide-neutral polymer composite hydrogels amendable to photo-initiated crosslinking.

(12) Ultrashort peptides with neutral polar head groups form stable composite hydrogels when mixed with neutral polymers. As a proof-of-concept, diethylene glycol diacrylate (DEGDA) mixed with AcAS.sub.6 (L; Ac-AIVAGS; SEQ ID NO: 69) formed hydrogels at various compositions. To stimulate photo-initiated crosslinking, a photoinitiator such as Irgacure 2959 (B) was added to the mixture. In the presence of ultraviolent light, the photoinitiator initiated a free-radical reaction, leading to the vinyl group of DEGDA being reactive towards neighbouring species.

(13) FIG. 7. Mechanical properties of composite peptide-DEGDA hydrogels, with and without photo-initiated crosslinking.

(14) The addition of DEGDA (with and without photoinitiated crosslinking) enhanced the mechanical properties of AcAS.sub.6, particularly the stiffness.

(15) FIG. 8. Ultrashort peptide-neutral polymer composite hydrogels that can be covalently crosslinked.

(16) Ultrashort peptides with basic polar head groups form stable composite hydrogels when mixed with neutral polymers. As a proof-of-concept, linear bi-functional PEG and branched multi-functional PEG were mixed with AcLK.sub.6 (L; Ac-LIVAGK; SEQ ID NO: 66) peptides (A) to form composite peptide-polymer hydrogels at various compositions (B). The N-hydroxysuccinimide (NHS) functionality will react with the primary amine group on AcLK.sub.6. Crosslinking between peptides are observed, as HPLC-MS analysis revealed the presence of the crosslinked species peptide-PEG.sub.5-peptide (C).

(17) FIG. 9. Mechanical properties of composite peptide-PEG hydrogels.

(18) The mechanical properties of the composite peptide-polymer (NHS-PEG) hydrogel are comparable to that of the peptide alone (25 mg/mL AcLK.sub.6, i.e. Ac-LIVAGK; SEQ ID NO: 66).

(19) FIG. 10. Conjugation strategies for the immobilization of bioactive moieties such as growth factors.

(20) Bioactive moieties can be immobilized onto composite peptide-polymer hydrogel by either mixing pre-conjugated polymer with self-assembling ultrashort peptides (A) or addition of the bioactive moiety post-gelation (B), where it will react with the relevant functional groups on the polymer. As a proof-of-concept, functional groups such as maleimide and primary amine groups can be exploited for conjugation using maleimide-thiol and NHS-amine chemistry.

(21) FIG. 11. Functionalized composite hydrogels formed by mixing functionalized polymers with ultrashort peptides.

(22) As a proof-of-concept, a bioactive moiety (here: L-DOPA) was conjugated onto a linear bi-functional PEG polymer, bis(succinimidyl)PEG (BS-PEG), using NHS-amine chemistry (A). In lieu of the bi-functional BS-PEG, COOH-PEG.sub.n-COOH can also be activated using carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). In this example, DOPA-PEG.sub.5-DOPA (C) was the functionalized polymer, and can be purified by centrifugation followed by dialysis, HPLC, or gel permeation chromatography (GPC). The completion of the reaction was determined using HPLC-MS. The functionalized polymer formed composite hydrogels with AcLK.sub.6 (L; Ac-LIVAGK; SEQ ID NO: 66) at different compositions (B).

(23) FIG. 12. Composite peptide polymer hydrogels functionalized with growth factor post-assembly.

(24) As a proof-of-concept, a bioactive moiety in the form of a growth factor FGF (needs to be defined?) was used to functionalize a composite (Ac-LIVAGK)-PEG composite hydrogel post-assembly. Hetero bi-functional PEG in the form of NHS-PEG-Maleimide (A) was used to form a composite peptide-polymer hydrogel either by mixing during hydrogelation or addition to a Ac-LIVAGK (SEQ ID NO: 66) hydrogel. Growth factors containing surface thiol groups (not needed for bioactivity), such as FGF are then added to the composite hydrogel. The resulting functionalized composite hydrogel was evaluated as a synthetic cell culture substrate. The functionalization with FGF enhanced the attachment of pluripotent embryonic stem cells (B). As compared to unmodified Ac-LIVAGK hydrogels, more but smaller colonies attached to the FGF functionalized composite hydrogel. Furthermore, more of the cells maintained their pluripotency, as demonstrated by the higher hOct4-GFP (GFP needs to be defined?) expression (hOct4 is a marker for pluripotency). Such functionalized composite hydrogels can be further used to encapsulate bioactive small molecules for controlled sustained release. Applications include use as synthetic cell culture substrates, particularly for the large-scale or three-dimensional culture of stem cells (C). These multi-functionalized hydrogel scaffolds can potentially be used to maintain stem cell pluripotency (or multipotency) or to direct the differentiation into specific cell lineages, depending on the bioactive moieties immobilized and encapsulated.

(25) FIG. 13. The (pure) peptide and composite peptide polymer hydrogels can take on different forms (sol hydrogels, viscous gels and dried membranes) and can be molded into different three-dimensional shapes and sizes.

(26) Small molecules such as dyes can be incorporated into the composite hydrogels as the solution mixture is poured into moulds. Following the gelation process, sol hydrogels are obtained. Moulds can be used to cast the hydrogels into specific three-dimensional shapes and sizes. When a sheet (or disc) of hydrogel is dried, membranes are obtained.

(27) FIG. 14. Peptide stability studies.

(28) TGA studies demonstrate high thermal stability of peptides (A) even in the presence of salts (B). Evaluation by .sup.1H-NMR shows that peptides, exemplified by AcID.sub.3, i.e. Ac-IVD (SEQ ID NO: 72), (C) and AcLD.sub.6, i.e. Ac-LIVAGD (SEQ ID NO: 65), (D) do not decompose when UV treated (2 h, 254 nm) or autoclaved (30 min, 120 C.). HPLC-MS analysis indicates that the degradation of AcID.sub.3 i.e. Ac-IVD (SEQ ID NO: 72), (E) and AcLD.sub.6 i.e. Ac-LIVAGD (SEQ ID NO: 65), (F) following UV and autoclave treatment was minimal.

(29) FIG. 15. In vivo biocompatibility of peptides.

(30) In vivo biocompatibility was evaluated in C57BL/6 mice by implanting various hydrogels subcutaneously for up to two months (A). The hydrogel implant was stable and could still be detected after 2 months, as shown by the amorphous eosinophilic (pink) foreign body material beneath the skeletal muscle layer (B, C). Minimal to mild inflammatory reaction to subcutaneous hydrogel implants was observed, in the form of several multi-nucleated giant cells found at the peripheral of the implant (D). However, this is attributed to the surgery.

(31) FIG. 16. In vivo scaffold for adipose tissue regeneration Fat pad observed under the skeletal muscle layer in the region of the human adipose stem cell (hASC) implant 45 days post-implantation (A-C). The transplanted cells have the appearance of normal mature adipose tissue (D)adipocytes with dark nuclei at edge, cells filled with fat.

(32) FIG. 17. Composite peptide-oligonucleotide hydrogels.

(33) Various peptides (AcLD.sub.6, i.e. Ac-LIVAGD, SEQ ID NO: 65; AcLK.sub.6, i.e. Ac-LIVAGK, SEQ ID NO: 66; AcLS.sub.6, i.e. Ac-LIVAGS, SEQ ID NO: 67) were used to encapsulate plasmid DNA, forming composite peptide-DNA hydrogels (A). In particular the peptide AcLK.sub.6 was extremely efficient in encapsulating and trapping the DNA, possibly through electrostatic interactions. There was minimal release of the DNA, as observed over the course of several days. AcLK.sub.6 also effectively protected the DNA against nuclease degradation (B). When the composite hydrogels were incubated with DNAse, digested DNA fragments were not observed, compared to the naked DNA control. Compared to the original DNA fragment used to form the composite hydrogel, the composite hydrogel fragments did not migrate out of the well during electrophoresis, indicating that the peptide strongly interacts with oligonucleotides, increasing the mass and thereby hindering migration through the agarose gel. Potential applications include synthetic cell culture substrates that mediate sustained DNA, mRNA, siRNA and short hairpin RNA delivery (C).

EXAMPLES

(34) In the course of our studies, different ultrasmall peptide and polymer combinations were used to demonstrate the feasibility and properties of the composite hydrogels. The formulation of each combination can further be optimized to alter the physical properties for different applications.

Example 1

Ultrasmall Peptide-cationic Polymer Composite Hydrogels

(35) Ultrasmall peptides with an acidic polar head group, such as AcLD.sub.6 (L) (i.e. Ac-LIVAGD; SEQ ID NO: 65), AcAD.sub.6 (L) (i.e. Ac-AIVAGD; SEQ ID NO: 76) and AcID.sub.3 (L) (i.e. Ac-IVD; SEQ ID NO: 72) form particularly stable composite hydrogels when mixed with (linear and branched) cationic polymers, such as chitosan and poly(amido amine) dendrimers (see Example 2 for more information). Gel formation is observed at higher pH compared to peptide only hydrogels. The stability of these composite hydrogels, as determined by their mass loss, in high salt conditions is also significantly improved. This observation suggests that the cationic polymers stabilize the hydrogels.

(36) 1. Formulation

(37) As a proof of concept, the linear cationic polymer chitosan is used to demonstrate the feasibility of forming composite hydrogels with ultrasmall peptide AcLD.sub.6 (L) (i.e. Ac-LIVAGD; SEQ ID NO: 65). The formation of the composite hydrogel depends on many factorspeptide concentration, polymer concentration, molecular weight and charge of the polymer, the presence of other salts and solvents.

(38) Increasing peptide and chitosan concentration increases the ease of gel formation (FIG. 2). Chitosan also stabilizes gel formation at low peptide concentration. Furthermore, more stable composite hydrogels are formed when the molecular weight and concentration of the chitosan is increased. These gels require repeated washing in high salt conditions under agitation, for complete dissociation.

(39) 2. Microstructure of the Composite Scaffold

(40) The microstructure of lyophilized composite hydrogels is observed under field-emission scanning microscopy (FESEM) as shown in FIG. 3. The composite hydrogel has a porous honeycomb microstructure, though the nanoscale fibres are less well-defined compared to peptide only hydrogels.

(41) 3. Mechanical Properties of the Composite Scaffold

(42) The mechanical properties of the composite hydrogels are demonstrated in FIG. 4. The composite hydrogels are slightly weaker than peptide hydrogels, as reflected by a lower storage modulus. However, this is compensated by a dramatic increase in elasticity, from 0.5% strain to 5%. The gel strength decreases with increasing chitosan concentration. Increasing the polymer length further increases the elasticity of the composite hydrogel.

(43) The mechanical properties of the composite hydrogel can be tuned by varying the composition. This is attractive for clinical applications, in which the gel strength can be varied to match that of tissue to be repaired, such as that of the nucleus pulposus (FIG. 5) to design an implant or an injectable therapeutic agent for degenerative disc disease or a dermal filler injected intradermally.

(44) 4. Moulding into Three-Dimensional Macroshapes and Casting Membranes

(45) While the composite mixture is still in liquid phase, it can be poured into moulds before gelation occurs. The resulting composite hydrogel adopts the shape of the mould, enabling three-dimensional macrostructures such as discs and cubes, as shown in FIG. 13. As the surface of the moulds have been treated, the composite hydrogels can be easily unmolded without compromising their macroshape. When discs of composite hydrogels are dried, membranes are obtained.

(46) 5. Incorporating Bioactive Therapeutics

(47) Bioactive therapeutics such as growth factors, cell adhesion molecules and prodrugs can be incorporated into the composite hydrogel by encapsulation (in the bulk phase) or by conjugation. Cationic polymers enhance the encapsulation of negatively charged molecules, such as oligonucleotides (plasmid DNA, messenger RNA, small interfering RNA); thereby increasing the loading capacity of the hydrogels. To conjugate bioactive molecules onto the polymer, functional groups, such as primary amines, on the polymer can exploited. Polyethylene glycol (PEG) crosslinkers, such as bi-functional or multi-functional polyethylene glycol (PEG), can be used to conjugate the bioactive moiety of interest to either the peptide or the polymer. The use of such linkers will enable the flexible movement of the conjugated moiety, facilitating cell recognition or allowing the moiety to adopt its ideal confirmation for therapeutic efficacy. FIG. 10 demonstrates a schematic for immobilizing growth factors onto the composite hydrogel. These gels can be subsequently deployed as drug delivery devices, cell culture substrates and implants for tissue regeneration.

Example 2

Ultrasmall Peptide-functionalized Polymer Composite Hydrogels Incorporating Branched Polymers

(48) Branched polymers can also be used to formulate stable composite hydrogels when mixed with different ultrasmall peptides. In particular, synthetic, functionalized water-soluble polymers such as dendrimers offer unique properties and advantages with respect to functional group variation, molecular weights, polarity, and structural diversity. The possibility of adding different functional end groups, such as amines, carboxylic acids, thiols, alcohols, and carbohydrates offer attractive material and chemical properties, as well as conjugation strategies to immobilize bioactive molecules. The number of functional groups can easily be adjusted, and therefore the concentration of the therapeutic molecules of interest within the gel can also easily be modified and fine-tuned.

(49) Dendrimers belong to a large class of molecules with discrete structural diversities, characterized by the specific dendrimer generation. This diversity can range over several generations, seen by the amount of functional groups that increases per generation. Each generation is characterized by its initiator core. The amount of functional groups is determined by the structure of the initiator core. Starting with two functional groups in the first generation, the number of functional groups increases with 2.sup.n, where n corresponds to the type of generation. Hence, the second generation has four functional groups, and so forth. The characteristic branching of dendrimers limits the rate at which the therapeutic molecule will leach out of the gel, enabling controlled and sustained release.

Example 3

Ultrasmall Peptide-neutral Polymer Composite Hydrogels

(50) Ultrasmall peptides with various (acidic, basic, neutral) polar head groups formed stable composite hydrogels when mixed with uncharged polymers such as polyethylene glycol (PEG).

(51) 1. Formulation

(52) As a proof-of-concept, di(ethylene glycol)diacrylate (DEGDA) forms composite hydrogels with ultrasmall peptides AcAS.sub.6 (L) (i.e. Ac-AIVAGS; SEQ ID NO: 69), AcLS.sub.6 (L) (i.e. Ac-LIVAGS; SEQ ID NO: 67), AcLT.sub.6 (L) (i.e. Ac-LIVAGT; SEQ ID NO: 68), and AcLD.sub.6 (L) (i.e. Ac-LIVAGD; SEQ ID NO: 65). The formation of these composite hydrogels depends on peptide concentration, PEG concentration and molecular weight, and the presence of other solvents.

(53) Photoinitiators such as Irgacure 2959 and 369 (from BASF) can be incorporated into the composite AcAS.sub.6-DEGDA (i.e. Ac-AIVAGS; SEQ ID NO: 69), hydrogels (FIG. 6). Following UV irradiation, the gels change in appearance from clear/translucent to opaque.

(54) The mechanical properties of the AcAS.sub.6-DEGDA Ac-AIVAGS; (i.e. Ac-AIVAGS; SEQ ID NO: 69), composite hydrogel are comparable to that of AcAS.sub.6 (i.e. Ac-AIVAGS; SEQ ID NO: 69), peptide hydrogels (FIG. 7).

(55) By pouring the liquid mixture of peptide and PEG into moulds prior to gelation, three-dimensional composite hydrogels can be obtained.

(56) Using moulds that permit UV transmission, composite scaffolds can be formed by irradiating peptide-DEGDA composite gels containing photoinitiator. The resulting structures retain their shapes and do not collapse into membranes even after the removal of the water by evaporation.

(57) 2. Formulation of Chemically/Covalently Crosslinked Composite Hydrogels

(58) As a proof-of-concept, bi-functional PEG with two (terminal) N-hydroxysuccinimide (NHS) groups forms composite hydrogels with ultrasmall peptides AcLK.sub.6 (L) (i.e. Ac-LIVAGK; SEQ ID NO: 66), AcIK.sub.6 (L) (i.e. Ac-ILVAGK; SEQ ID NO: 71), AcAK.sub.6 (L) (i.e. Ac-AIVAGK; SEQ ID NO: 70), AcIK.sub.3 (L) (i.e. Ac-IVK; SEQ ID NO: 73), AcLK.sub.3(L) (i.e. Ac-LVK; SEQ ID NO: 74) and AcAK.sub.3(L) (i.e. Ac-AVK; SEQ ID NO: 75). The extent of crosslinking depends on the ratio of polymer to peptide, as well as the number of functional groups available for reaction. The extent of crosslinking can be detected and quantified using mass spectrometry.

(59) 3. Formulation of Composite Hydrogels with Immobilized Growth Factors

(60) As a proof-of-concept, a solution of hetero bi-functional NHS-PEG-maleimide polymer was added to pre-formed Ac-LIVAGK (L) (SEQ ID NO: 66) hydrogels (FIGS. 8 to 10). After allowing for the polymer to diffuse into the gel and for the NHS functional group to react with the primary group on Ac-LIVAGK (L), -FGF was added (FIG. 12). The maleimide on the polymer would then react with a free sulfhydryl group on the surface of the growth factor. The resulting hydrogel demonstrated improved cell adhesion characteristics, resulting in greater numbers of embryonic stem cell colony attachment.

Example 4

Ultrasmall Peptide-Oligonucleotide Composite Hydrogels

(61) Ultrasmall peptides of different amino acid sequences and (acidic, basic, neutral) polar head groups formed stable composite hydrogels when mixed with aptamers and oligonucleotides, such as plasmid DNA, messenger RNA and small interfering RNA (siRNA). These composite hydrogels are unique in that the polymer is also the bioactive molecule of interest.

(62) 1. Formulation

(63) Plasmid DNA is used to demonstrate the feasibility of forming composite hydrogels with different peptide sequences. The peptide sequence (particularly the polar head group) and concentration plays a significant role in determining the maximum amount of DNA that can be incorporated into the composite hydrogel. Peptides with basic (C-terminal) head groups, such as AcLK.sub.6 (L) (i.e. Ac-LIVAGK; SEQ ID NO: 66), could integrate the highest concentration of DNA. Peptides with acidic head groups such as AcLD.sub.6 (L) (i.e. Ac-LIVAGD; SEQ ID NO: 65) had the lowest threshold for DNA loading and further increases in DNA concentration resulted in incomplete or non-gelation.

(64) 2. Release of the Oligonucleotides

(65) Dissociation of the composite hydrogel, with the concurrent release of the DNA was minimal, as demonstrated in FIG. 17. Even after 10 days, at least 80% of the DNA loaded remained in composite hydrogel. In particular, the Ac-LIVAGK (L)-DNA hydrogel retained 95% of the DNA added. The release of the nucleic acid can potentially be modulated by the peptide composition. The DNA was also protected from degradation, which bodes well for the incorporation of the more labile messenger RNA. As the physical and chemical properties of DNA, messenger RNA and siRNA are comparable, it is reasonable to conclude that the incorporation efficiency and dissociation characteristics will be similar.

(66) 3. Applications

(67) The present invention offers a package solution for patients on demand for therapies using tissue engineering, i.e. a package solution for patient-tailored cell therapy. The self-assembled composite ultrasmall peptide-polymer hydrogels of the invention provides an overall solution, starting from taking patient's healthy tissue, such as fibroblasts, and change it to the cell type where a therapy is needed.

(68) By using messenger RNA that encodes for the Yamanaka factors in the composite hydrogel, re-programming of the somatic cells can be achieved without the use of viral vectors or repeated transfection. Messenger RNA encoding growth factors and transcription factors can be used to direct the differentiation of stem cells cultured on the composite hydrogel.

(69) For clinical applications, messenger RNA encoding growth factors (to enhance regeneration of the tissue) or cytokines (to reduce inflammation) will facilitate recovery and localize the therapeutic effect, reducing undesirable systemic side effects.

(70) According to FIG. 1, which shows the hypothesis of hydrogel formation, when the pH of acidic peptide hydrogels, such as hydrogels based on AcLD.sub.6 (i.e. Ac-LIVAGD; SEQ ID NO: 65), is raised, some peptides ionize and diffuse away. The rationale of creating composite hydrogels is thus to capture the ionized peptides with cationic polymers, thereby stabilizing the hydrogel. As a proof of concept, the inventors have chosen chitosan. The inventors found that gelation depends on many factors, of which the most important is the type of chitosan used. Screening seven different chitosans, the inventors identified three candidates for further rheological evaluation. The first was a water soluble chitosan with molecular weight around 1 kDa. The latter two candidates were mid and high-molecular weight chitosans. These three chitosans formed composite hydrogels which were more stable in PBS, though with repeated washing steps, they also dissociated. This suggests that the change in ionic environment can be applied to retrieve cells cultured in the hydrogel, eliminating the use of enzymes.

(71) According to FIG. 4, the composite hydrogels were slightly weaker than peptide hydrogels and the storage modulus decreased with increasing chitosan concentration. However, there was a dramatic increase in elasticity, from 0.5% strain to 5%, making them more attractive for in vivo applications. Increasing the polymer length further increased the elasticity of the composite hydrogel.

(72) For tuning the properties of their peptide hydrogels to that of native tissue, the inventors first determined the mechanical properties of nucleus pulposus. Using the pig as a large animal model, the inventors found that the storage modulus of porcine nucleus pulposus is 20 weaker than published data for humans (FIG. 5). This could be attributed to humans being upright and hence our spinal column would experience more compressive forces. The inventors also found that the age of the animals did not make a significant difference in their elastic properties, unlike for humans. The hydrogels of the present invention are significantly stronger, though much less elastic with yield points of 0.5% strain compared to 2% for porcine tissue.

Example 5

Injection Therapy and Implants

(73) The composite polymer-peptide hydrogels can form the basis of an injectable therapy wherein the mixture injected as a solution will gel in situ upon injection into the body. For instance, for degenerative disc disease, the treatment will be applied to the nucleus pulposus; while for urinary incontinence, the injection will be applied to the wall of the urethra. Such treatments are minimally invasive (not requiring surgery) and provide mechanical support to the degenerated tissue.

(74) Subcutaneous implantations of AcLK.sub.6 (i.e. Ac-LIVAGK; SEQ ID NO: 66) hydrogels seeded with human derived adipose stem cells into nude mice, resulted in the formation of fat pads under the implant site after 45 days (FIG. 16). This suggests that the hydrogels can be applied as dermal fillers and scaffolds for adipose tissue transplantation/regeneration.

(75) Subcutaneous implants of various peptide hydrogels in mice did not cause any significant immunogenic or physiological reactions. There were no behavioral changes or weight loss following implantation for up to 2 months, and there was no significant difference in red or white blood cell counts or blood chemistry (liver and kidney function) between mice implanted with hydrogels and sham operated mice.

(76) Using a guinea pig model, Kligman maximization assays were performed (by a contract research organization) for 12 ultrashort peptide candidates to date. Topical applications and intradermal injections of the various peptide hydrogels did not stimulate any irritation or allergic reactions after 24 hours. Subsequent immunologic challenges at day 27 also elicited no immune or allergic response. Likewise, no systemic toxicity was observed. This indicated that the peptides tested were non-immunogenic and the potential for stimulating an allergic response with repeated use is low.

(77) TABLE-US-00001 TABLE 1 Pilot study with 4 mice (10 days) implant = 30 L Ac-LIVAGK (L) gel 5 mm diameter, 1 mm height un- normal range treated SCID 1 SCID 2 normal neutrophils (K/uL) 0.1-2.4 2.16 2.1 0.77 1.6 lymphocytes (K/uL) 0.9-9.3 0.89 0.88 0.9 3.76 monocytes (K/uL) 0-0.4 0.78 0.22 0.12 0.42 eosinophils (K/uL) 0-0.2 0.17 0.31 0.03 0.11 basophils (K/uL) 0-0.2 0.21 0.08 0 0.02 thrombocytes (M/uL) 6.36-9.42 1.01 0.607 0.978 0.833 erythrocytes (M/uL) 0.592-2.972 9.72 9.29 11.33 10.65

(78) TABLE-US-00002 TABLE 2 Second study in mice (45 days): Liver enzyme levels and electrolyte composition Fairly consistent across the group ALB ALP ALT AMY TBIL BUN TP GLOB CRE g/dL U/L U/L U/L mg/dL mg/dL g/dL g/dL mg/dL Mouse 1 2.6 7 145 0 0.3 18 3.7 1.1 0 Mouse 2 2.2 17 202 497 0.3 10 3.5 1.3 0 Mouse 3 2.8 35 52 617 0.3 15 4.3 1.6 0 Mouse 4 2.8 15 50 637 0.2 14 4.1 1.2 0 Mouse 5 2.5 24 126 602 0.3 11 4.1 1.6 0 Average 2.58 19.60 115.00 470.60 0.28 13.60 3.94 1.36 0.00 Stdev 0.25 10.53 64.78 268.58 0.04 3.21 0.33 0.23 0.00

(79) Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

(80) Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

(81) Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

(82) The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

(83) The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

(84) Throughout this specification, unless the context requires otherwise, the word comprise or variations such as comprises or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as comprises, comprised, comprising and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean includes, included, including, and the like; and that terms such as consisting essentially of and consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

(85) Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.