A series of injectable hydrogels self-assembled from short peptides for various biomedical applications

Abstract

Peptides that can be self-assembled into hydrogels are described herein. The peptide includes an amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y), wherein each hydrophobic amino acid is independently selected from isoleucine (I), valine (V) and leucine (L), each hydrophilic amino acid is independently selected from arginine (R), lysine (K), glutamic acid (E) and aspartic acid (D), at least one hydrophilic amino acid is selected from arginine and lysine, at least one hydrophilic amino acid is selected from glutamic acid and aspartic acid, and the amino acid sequence includes at least 8 amino acids. A composition is further described, which includes a hydrogel or a dry form of a hydrogel formed by a peptide and water of a β-sheet configuration. Hydrogels can be used for growing cells. On the other hand, hybrid hydrogels prepared by IIK 12 (IRIKIEIEIRIK) and IK8L (IRIKIRIK) can be used to treat bacterial and / or fungal infections.

Description

Technical Field

[0001] The present invention relates to peptides that self-assemble into hydrogels. Background Art

[0002] Hydrogels are 3D polymer networks capable of storing large amounts of water and have been extensively studied and used as matrices for biomedical applications [1]. In particular, they are often chosen for regenerative medicine applications due to their biocompatibility, inherent structural similarity to the extracellular matrix (ECM), and ability to provide a suitable biochemical environment [2,3].

[0003] Many types of hydrogels have been reported in the literature, using both natural and synthetic materials. Artificial basement membranes [4,5], collagen [6,7], hyaluronic acid [8], and gelatin [9] have been widely used to form hydrogel-based scaffolds for tissue engineering because of their advantages of being specifically recognized by host cells. However, the use of animal-derived scaffolds is often limited due to the uncertainty or inconsistency of molecular length and structure, as well as potential immunogenicity risks

[10] . Polymers based on poly(ethylene glycol) [10,11], poly(vinyl alcohol)

[12] , poly(L-lactide) (PLLA)

[13] , poly(lactide-co-glycolide) (PLGA)

[14] , and poly(hydroxyethyl methacrylate) (HEMA)

[15] have also been widely used to produce hydrogels. However, synthetic polymer hydrogels are biochemically inert in nature and cannot interact with cells, and therefore have difficulty in allowing cells to proliferate within the matrix

[10] .

[0004] Recently, peptide hydrogels have received much attention. Peptide-based hydrogels derived from natural amino acids have advantages such as biocompatibility, biodegradability, and non-immunogenicity [16,17]. With the advancement of solid-phase peptide synthesis, the production cost has also been greatly reduced. In addition, each amino acid coupling step is precisely controlled, so that well-defined sequences, molecular lengths, and reproducibility can be easily obtained. In the early 1990s, Zhang and co-workers discovered a natural yeast protein motif, EAK16-II (AEAEAKAKAEAEAKAK) (SEQ ID No. 1), which is characterized by alternating repeats of ionic hydrophilic and hydrophobic amino acids

[17] . The peptide adopts a β-sheet configuration with distinct hydrophobic and hydrophilic surfaces and produces self-assembled nanofibers in the presence of salt. Since then, a series of peptide self-assembly systems have been designed and developed to form 3D hydrogels with nanofibrous structures. The most widely studied peptide, RADA16-I (AcN-RADARADARADARADA-CONH2) (SEQ ID No. 2)

[16] (PuraMatrix TM) and RADA-II (AcN-RARADADARARADADA-CONH2) (SEQ ID No. 3)

[18] are composed of periodically alternating hydrophilic arginine residues and hydrophobic alanine (A) residues. Peptide scaffolds self-assembled from RADA16-I and RADA16-II form hydrogels (99% water) with nanofibers (diameter 10-20 nm) in physiological solution. These peptide hydrogels are used as scaffolds for 3D cell culture, accelerated wound healing, and neural repair [19-21]. For example, the scaffolds can support neurite outgrowth and synapse formation

[21] . In addition, RADA16 hydrogels have been used to deliver proteins and active cytokines in a sustained release manner

[22] .

[0005] Schneider, Pochan and colleagues developed another type of peptide with a central tetrapeptide II' type β turn (V D PPT) is flanked by alternating valine (hydrophobic) and lysine (hydrophilic) residues [23, 24]. Triggered by changes in pH, ions, and temperature, peptides such as MAX1 (VKVKVKVK-V D PPT-VKVKVKVK) (SEQ ID No. 4) transforms from a random coil to a β-hairpin configuration and self-assembles into a β-sheet-rich hydrogel. MAX1 has been shown to be non-hemolytic to human erythrocytes and supports the attachment of NIH3T3 cells [24, 25]. However, MAX1 hydrogels are not ideal for 3D cell encapsulation due to their slow gelation kinetics

[26] .

[0006] Recently, Hauser et al. reported a new series of ultrashort linear peptides with 3-7 natural aliphatic amino acids that self-assemble into supramolecular helical fibers [27, 28]. The peptide motif contains an aliphatic amino acid tail with reduced hydrophobicity and a hydrophilic polar head. As the concentration increases to its critical gelation concentration, the peptide undergoes a secondary conformational transition from random coil to α-helix to β-sheet. The peptide self-assembles into fibers through α-helical intermediates and subsequently condenses into fibrils to form hydrogels

[27] . The resulting peptides entrap up to 99.9% of water and resemble collagen fibers. The mechanical stiffness ranges from 10 3 Up to 10 5 Pa, which can be modulated by peptide concentration and the use of salt

[28] . These peptides were shown to be biocompatible with human mesenchymal stem cells (hMSC) and rabbit retinal epithelial cells

[28] . The lead peptide Ac-ILVAGK-NH2 (SEQ ID No. 5) was evaluated for its application in wound healing

[29] . In contrast, the peptide hydrogel promoted the healing of partial-thickness burn wounds. However, when immersed in large amounts of water, the fibers became diluted, causing the peptide hydrogel to lose its integrity

[30] .

[0007] Recently, the use of hydrogels to combat infections associated with medical devices and wound healing has been reported. Diabetic foot ulcer (DFU) infections are a major cause of morbidity, hospitalization, and the leading cause of amputation

[31] . Chronic CFU is extremely difficult to treat because it involves a wide variety of bacteria (including Gram-positive and Gram-negative bacteria, especially multidrug-resistant bacteria) and biofilms

[32] . Most existing antibiotics are ineffective in treating such infections. Silver ions or silver nanoparticles have been introduced into wound dressings to prevent DFU infections [33, 34]. Long-term use of such products can lead to silver accumulation in the skin, resulting in blue or gray discoloration of the skin (argyria). Therefore, for the prevention and treatment of DFU infections, there is an urgent need for a biodegradable antimicrobial hydrogel that has a broad spectrum of activity against multidrug-resistant microorganisms and can disperse biofilms without developing resistance. Summary of the invention

[0008] In a first aspect of the present invention, a peptide is provided, comprising an amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y), wherein each hydrophobic amino acid is independently selected from isoleucine (I), valine (V) and leucine (L), each hydrophilic amino acid is independently selected from arginine (R), lysine (K), glutamate (E) and aspartic acid (D), at least one hydrophilic amino acid is selected from arginine and lysine, at least one hydrophilic amino acid is selected from glutamate and aspartic acid, and the amino acid sequence contains at least 8 amino acids.

[0009] Amino acids generally refer to L-amino acids, but D-amino acids may also be used. In particular, the peptides described herein may be produced from all L-amino acids or all D-amino acids. Unless otherwise indicated, the peptide sequences described herein include all L-amino acid sequences and all D-amino acid enantiomers, which are known to have the same properties except for the ability to rotate plane polarized light.

[0010] Peptides also include two or more amino acid sequences connected by a linker. Such peptides may also form β-sheets in water and self-assemble into hydrogels at lower peptide concentrations. The linker can be any conventional organic compound linker and can be connected to the respective C-terminus and N-terminus.

[0011] Preferably, the amino acid sequence is not IRVEIEVK.

[0012] Preferably, the amino acid sequence has an even number of amino acids. Advantageously, this allows the peptide to maintain chemical complementarity, in other words, an equal number of hydrophilic and hydrophobic amino acid residues. For example, the amino acid sequence may have 8 or 12 amino acids. The latter are believed to be more likely to form hydrogels at lower concentrations.

[0013] Preferably, there is at least one arginine and at least one lysine in the amino acid sequence.

[0014] In an example, the four sequentially arranged hydrophilic amino acids (Y1, Y2, Y3 and Y4) in the amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y) are selected such that Y1 and Y2 are each independently selected from glutamic acid and aspartic acid, and Y3 and Y4 are each independently selected from arginine and lysine. Advantageously, this provides a peptide with a (--++) arrangement of hydrophilic amino acid residues.

[0015] As used herein, the phrase "sequentially arranged hydrophilic amino acids" refers to consecutive hydrophilic amino acids in an alternating sequence of hydrophilic amino acids (Y) and hydrophobic amino acids (X). For example, four sequentially arranged (hydrophilic) amino acids in a peptide means that the peptide comprises the sequence Y1X1Y2X2Y3X3Y4. A peptide with 8 amino acids will have the sequence Y1X1Y2X2Y3X3Y4X4, and this sequence is contained in peptides with 8 or more amino acids. The terms Y1, Y2, Y3 and Y4 represent the sequential order of hydrophilic amino acids. The same applies to any number of sequentially arranged amino acids.

[0016] At a pH of about 7 (or 7.4) or physiological conditions, arginine and lysine are positively charged, while glutamic acid and aspartic acid are negatively charged. Thus, the selection of hydrophilic amino acids provides a peptide with localized charge properties that allow the properties of the peptide to be modulated and can improve the resulting properties of the hydrogel formed by the peptide.

[0017] In an example, the four sequentially arranged hydrophilic amino acids (Y1, Y2, Y3 and Y4) in the amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y) are selected such that Y1 and Y3 are each independently selected from arginine and lysine, and Y2 and Y4 are each independently selected from glutamic acid and aspartic acid. Advantageously, this provides a peptide with a (+-+-) arrangement of hydrophilic amino acid residues.

[0018] In an example, the six sequentially arranged hydrophilic amino acids (Y1, Y2, Y3, Y4, Y5 and Y6) in the amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y) are selected such that Y1, Y3 and Y5 are each independently selected from arginine and lysine, and Y2, Y4 and Y6 are each independently selected from glutamic acid and aspartic acid. Advantageously, this provides a peptide having a (+-+-+-) arrangement of hydrophilic amino acid residues, and can be viewed as an extension of the example of having four sequentially arranged amino acids having the same alternating charged species arrangement.

[0019] In an example, the six sequentially arranged hydrophilic amino acids (Y1, Y2, Y3, Y4, Y5 and Y6) in the amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y) are selected such that Y1, Y2 and Y3 are each independently selected from glutamic acid and aspartic acid, and Y4, Y5 and Y6 are each independently selected from arginine and lysine. Advantageously, this provides a peptide with a (---+++) arrangement.

[0020] In an example, the six sequentially arranged hydrophilic amino acids (Y1, Y2, Y3, Y4, Y5 and Y6) in the amino acid sequence of alternating hydrophobic amino acids (X) and hydrophilic amino acids (Y) are selected such that Y1, Y2, Y5 and Y6 are each independently selected from arginine and lysine, and Y3 and Y4 are each independently selected from glutamic acid and aspartic acid. Advantageously, this provides a peptide with a (++--++) arrangement.

[0021] Additionally, the reverse sequence of the hydrophilic amino acid residues in the above examples are also described and included herein.

[0022] Preferably, the hydrophilic amino acids are selected so that the peptide has a net neutral charge or a net positive charge. For example, the peptide may have a net positive charge of +2. Advantageously, peptides with a net positive charge may more easily form hydrogels without the need for salts to reduce gelation time and at lower concentrations, resulting in reduced costs for preparing hydrogels. This may be due to enhanced beta sheet hydrogen bonding in peptides with a net positive charge.

[0023] Preferably, at least half of the hydrophobic amino acids in the amino acid sequence are isoleucine or leucine.

[0024] Preferably, there are 12 amino acids in the amino acid sequence, and the hydrophobic amino acid residues are each independently selected from isoleucine and valine.

[0025] In an example, the amino acid sequence is any one of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25 and SEQ ID No. 26. In addition, the peptide may consist of two or more of these sequences connected by a linker, in particular the sequences have the same SEQ ID No.

[0026] Preferably, the peptide is amidated at the C-terminus of the amino acid sequence and / or acetylated at the N-terminus of the amino acid sequence. The C-terminus is preferably amidated in all peptides. The hydrophilic amino acid residue at the N-terminus may be acetylated, while the hydrophobic amino acid at the N-terminus may not require functionalization of the N-terminus. Other functionalizations of the peptide termini may also be used. Advantageously, this minimizes charge repulsion at the peptide termini.

[0027] In a second aspect of the present invention, a composition is provided, comprising a hydrogel or a dry form of a hydrogel formed by a plurality of peptides according to the first aspect in a β-sheet configuration and water. Advantageously, the formation of β-sheets forms a supramolecular structure with rigid viscoelastic properties, which can act as a hydrogel that can contain water and other compounds and can be used in different applications.

[0028] The dried form of the hydrogel can be obtained by freeze drying to increase stability and storage time, and can be reconstituted back into a hydrogel by adding water. If the dried hydrogel contains a therapeutic agent, such as the second peptide of SEQ ID No. 17 or SEQ ID No. 18, the dried hydrogel can be reconstituted by, for example, a patient or a medical professional at the time of use.

[0029] Preferably, the concentration of the plurality of peptides is at least 0.6% w / v. The remainder of the hydrogel will be water and will form the bulk of the hydrogel.

[0030] Preferably, the concentration is at most 10% w / v, at most 5% w / v. More preferably, the concentration is at most 4% w / v, or less than 4% w / v, or at most 3% w / v.

[0031] Preferably, the composition further comprises a salt. The salt may be phosphate buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM) or minimal essential medium (MEM). Salts such as sodium or potassium salts may also be used, and are preferably neutral or weakly acidic or alkaline salts, such as sodium chloride and sodium acetate.

[0032] In examples, the peptide has an amino sequence of SEQ ID No.12, SEQ ID No.15, SEQ ID No.16, SEQ ID No.22, SEQ ID No.25 or SEQ ID No.26.

[0033] Preferably, the composition or hydrogel comprises a therapeutic agent. More preferably, the composition or hydrogel comprises a second peptide of SEQ ID No. 17 or SEQ ID No. 18 (which is used as a therapeutic agent). The molar ratio of the peptide to the second peptide may be in the range of 3: 1 to 1: 3 (or alternatively 3 to 0.33). Advantageously, the composition or hydrogel may be used as a medicament. In particular, as a medicament for bacterial and / or fungal infections. In addition, the composition or hydrogel may be used to manufacture a medicament for treating bacterial and / or fungal infections. In an example, the fungal infection may be caused by yeast.

[0034] In a third aspect, a method for treating bacterial and / or fungal infections is provided, the method comprising administering to a subject suffering from bacterial and / or fungal infections a therapeutically effective amount of a composition or hydrogel comprising a plurality of peptides according to the first aspect and a second peptide of SEQ ID No. 17 or SEQ ID No. 18.

[0035] In a fourth aspect, there is provided a method of forming a hydrogel, the method comprising mixing the peptide according to the first aspect in water to form a hydrogel; and isolating the hydrogel.

[0036] Preferably, the stirring step is performed in the presence of a salt.

[0037] Preferably, the stirring step is performed at a temperature of 20°C to 40°C.

[0038] Preferably, the method further comprises drying the hydrogel to form a dried hydrogel suitable for reconstitution into a hydrogel.

[0039] Preferably, the concentration of the plurality of peptides is at least 0.6% w / v. The remainder of the hydrogel will be water, which forms the bulk of the hydrogel.

[0040] Preferably, the concentration is at most 10% w / v, at most 5% w / v. More preferably, the concentration is at most 4% w / v, or less than 4% w / v, or at most 3% w / v, or at most 2% w / v,

[0041] Preferably, the composition further comprises a salt. The salt may be phosphate buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM) or minimal essential medium (MEM). Salts such as sodium or potassium salts may also be used, and are preferably neutral or weakly acidic or alkaline salts, such as sodium chloride and sodium acetate.

[0042] In a fifth aspect, an in vitro method for growing cells is provided, the method comprising providing a mixture of the peptide according to the first aspect, a cell culture medium and a cell population to form a gel containing the cell population; and incubating the gel under suitable conditions to grow the cell population.

[0043] Preferably, the cell is selected from any one of the following: healthy cells, stem cells for treating or growing tissues, and cancer cells for growing tumors for in vitro and in vivo studies. In an example, the stem cell can be a human stem cell, and the hydrogel formed can be used as a culture medium to cultivate (or grow) stem cells for use. The stem cell is preferably an adult stem cell, such as human mesenchymal stem cells (hMSC). The hydrogel can also be used to cultivate cancer cells, which are used for in vitro and / or in vivo testing of therapeutic agents for cancer cells. Many cancer cell lines can be used and can be used together with the hydrogel to cultivate cell lines for further use, and include breast cancer cell lines, such as MCF-7 and BT-474.

[0044] In the figure:

[0045] FIG1 shows the CD spectrum of a peptide amphiphile consisting of 8 amino acids. Figure 1A Corresponds to IVK8; Figure 1B Corresponds to ILK8, and Figure 1C Corresponds to IIK8. Measured at 1.0 mg / mL in water or PBS (pH 7.4) at room temperature (-23-24°C).

[0046] FIG2 shows the CD spectrum of a peptide amphiphile consisting of 12 amino acids. Figure 2A Corresponds to IRVKIEVEIRVK(IVK12); Figure 2B Corresponds to IRVEIRVEIRVE(IRV12), and Figure 2C Corresponds to IEVEIEVKIRVK (IEV12).Measured at 1.0 mg / mL in water or PBS (pH 7.4) at room temperature (-23-24°C).

[0047] FIG3 shows a peptide amphiphile composed of 8 amino acids that self-assembles into a supramolecular hydrogel. Figure 3A It shows how the hydrophobicity of the peptide increases when the hydrophobic amino acid residue Val is replaced by Ile, resulting in faster gelation. The peptide was dissolved in water and incubated at room temperature (~23-24°C) for 30 min; Figure 3B Peptide hydrogel formation initiated by the addition of DMEM is shown; Figure 3C Shown are SEM images of IVK8 hydrogels prepared at a concentration of 1.5% in water and incubated overnight at 37 °C.

[0048] FIG. 4 shows the rheological behavior of IVK8 in water at a concentration of 1.5 (w / v)%. Figure 4A The effect of frequency sweep on storage modulus (G') and loss modulus (G") is shown; Figure 4B Flow scans showing the viscosity as a function of shear rate are shown; Figure 4C Dynamic step strain amplitude tests are shown (strain = 0.5% or 100%).Hydrogels were prepared at room temperature (~23-24°C).

[0049] FIG. 5 shows the rheological behavior of IVK8 in DMEM at a concentration of 1.5 (w / v)%. Figure 5A The frequency sweeps of the storage modulus (G') and loss modulus (G") are shown; Figure 5B Flow scans showing the viscosity as a function of shear rate are shown; Figure 5C Dynamic step strain amplitude tests (strain = 0.5% or 100%) are shown; Figure 5D The effect of DMEM content on peptide hydrogel stiffness is shown (peptide concentrations were 1.5%, 1.2% and 1.0% for 0, 20 and 30% DMEM, respectively).Hydrogels were prepared at 37°C.

[0050] FIG. 6 shows the rheological behavior of IIK12 (2.0 (w / v) %) in water. Fig. 6A The frequency sweeps of the storage modulus (G') and loss modulus (G") are shown; Figure 6B Flow scans showing the viscosity as a function of shear rate are shown; Figure 6C Dynamic step strain amplitude tests are shown (strain = 0.5% or 100%).Hydrogels were prepared at 37°C.

[0051] Figures 7A-7C Confocal images of hMSCs on the surface of IVK8 hydrogels and hMSCs encapsulated in peptide hydrogels are shown, with cells grown in a confocal chamber used as a control. Figures 7A-7C In the image, living cells are seen as white dots on a black background.

[0052] Fig. 8A The proliferation of MCF-7 cells on the IVK8 hydrogel surface is shown. Figure 8B Shown is the proliferation of BT-474 cells within IVK8 hydrogel (3D).

[0053] Fig. 9 Shown are SEM images of IIK12 and IIK12 / IK8D hybrid hydrogels prepared at 1.0% IIK12 concentration in water with and without IK8D (molar ratio of IIK12:IK8D = 1:1) and incubated overnight at 37°C.

[0054] FIG. 10 shows the rheological behavior of IIK12 / IK8L hybrid hydrogel in water with 0.8 (w / v)% IIK12 and 0.8 (w / v)% IK8L. Fig. 10A The frequency sweeps of the storage modulus (G') and loss modulus (G") are shown; Fig. 10BFlow scans showing the viscosity as a function of shear rate are shown; Fig. 10C Dynamic step strain amplitude tests are shown (strain = 0.5% or 100%). Gels were prepared at 37°C.

[0055] Figure 11 shows the antimicrobial activity of IIK12 / IK8L hybrid hydrogel against various microorganisms, including Fig.11A Gram-positive bacteria Staphylococcus aureus (S. aureus), Fig. 11B Gram-negative bacteria Escherichia coli (E. coli) and Fig. 11C The yeast Candida albicans (C.albicans) in the experiment. The CFU were counted at the end of the experiment. · means no colonies were found.

[0056] Fig.12 The change in the count of viable Staphylococcus aureus cells in contact with IIK12 / IK8L hybrid hydrogels over time is shown. The hydrogels were prepared using IK8L at a concentration of 2.56 mg / mL (ie, 0.256%). X indicates that no colonies were observed.

[0057] FIG. 13 shows the antimicrobial activities of IK8L, IIK12 / IK8L, and IIK12 / IK8D hybrid hydrogels evaluated by disk diffusion assay (DDA). Fig.13A Sterile discs containing different concentrations of IK8L are shown, with water used as a control; Fig. 13B IIK8 / IK8L hybrid hydrogels are shown. IIK12_1% / IK8L_0.5% represents 1% IIK12 and 0.5% IK8L in the hydrogel formulation. IIK12 hydrogel was used as a control; Fig. 13C IIK8 / IK8D hybrid hydrogels are shown, and IIK12_1% / IK8D_0.8% indicates 1% IIK12 and 0.8% IK8D in the hydrogel formulation. 40 μL of Staphylococcus aureus suspension was added at 10 6 CFU / mL were plated on agar plates. The plates were incubated at 37°C for 24 h. The lines indicate the areas where the gel was applied.

[0058] Fig.14 The hemolytic activity of IIK12 / IK8D hybrid hydrogels prepared with different concentrations of IK8D is shown. The experiment was repeated three times and the data are presented as mean ± standard deviation.

[0059] Figure 15 shows the viability of human dermal fibroblasts after 24 h of treatment with various formulations, as measured by MTT assay. Fig.15A The viability of the peptide solution using IK8L is shown; Fig. 15B The viability using IIK12 / IK8L hybrid hydrogel is shown; Fig. 15C Viability with mupirocin cream is shown; Fig.15D Viability using polymyxin B is shown.

[0060] Figure 16 shows the viability of human primary keratinocytes after 24 h treatment with various formulations, as measured by MTT assay. Fig.16A The viability of the peptide solution using IK8L is shown; Fig. 16B The viability using IIK12 / IK8L hybrid hydrogel is shown; Fig. 16C Viability with mupirocin cream is shown.

[0061] In the following description, many specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the present invention. However, it will be appreciated by those skilled in the art that embodiments of the present invention may be implemented without some or all of these specific details. Embodiments described in the context of one of the methods or devices are similarly applicable to other methods or devices. Similarly, embodiments described in the context of the method are equally valid for the device, and vice versa.

[0062] Features described in the context of an embodiment may be correspondingly applicable to the same or similar features in other embodiments. Features described in the context of an embodiment may be correspondingly applicable to other embodiments, even if not explicitly described in these other embodiments. In addition, additions and / or combinations and / or substitutions described for features in the context of an embodiment may be correspondingly applicable to the same or similar features in other embodiments.

[0063] For the purpose of the specification and claims, the terms "agent" and "drug" as used herein mean a chemical compound, a mixture of chemical compounds, a biomacromolecule, or an extract made from biological material such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues suspected of having therapeutic properties. An agent or drug may be purified, substantially purified, or partially purified.

[0064] The term "physiologically acceptable" defines a carrier or diluent that does not eliminate the biological activity and properties of the compound. The pharmaceutical compositions described herein can be administered to human patients by themselves, or in pharmaceutical compositions in which they are mixed with other active ingredients (such as in combination therapy) or suitable carriers or one or more excipients. The formulation and administration techniques of the compounds of the present application can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990. The term "physiological conditions" refers to conditions that are normally present in organisms and cells, and generally refers to conditions where the temperature ranges from 20-40°C, the atmospheric pressure is 1, the pH is 6-8, the glucose concentration is 1-20mM, and the atmospheric oxygen concentration.

[0065] In another aspect, the present invention relates to a pharmaceutical composition comprising a physiologically acceptable surfactant, carrier, diluent, excipient, lubricant, suspending agent, film-forming material and coating aid, or a combination thereof; and a compound disclosed herein. The pharmaceutical composition facilitates administration of the compound to an organism. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), the entire contents of which are incorporated herein by reference. Preservatives, stabilizers, dyes, sweeteners, aromatics, flavoring agents, etc. may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and parabens may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated fatty alcohols, and the like can be used as surfactants; sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium aluminate metasilicate, synthetic aluminum silicate, calcium carbonate, sodium carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like can be used as excipients; magnesium stearate, talc, hardened oil, and the like can be used as lubricants; coconut oil, olive oil, sesame oil, peanut oil, and soybeans can be used as suspending agents or lubricants; cellulose acetate phthalate, which is a derivative of carbohydrates such as cellulose or sugar, or methyl acetate-methacrylate copolymer, which is a derivative of polyethylene, can be used as suspending agents; and plasticizers such as phthalates, and the like can be used as suspending agents.

[0066] Additional therapeutic or diagnostic agents may be introduced into the pharmaceutical composition.Alternatively or additionally, the pharmaceutical composition may be combined with other compositions comprising other therapeutic or diagnostic agents.

[0067] It should be understood that the compositions provided herein can be in any form that allows the composition to be administered to the patient. For example, the composition can be in the form of a solid, liquid or gas (e.g., an aerosol). Suitable routes of administration include, but are not limited to, enteral (e.g., oral or rectal), topical, parenteral (e.g., sublingual, oral, sublingual, vaginal or intranasal). The term parenteral as used herein includes subcutaneous injection, intravenous, intraarterial, intradermal, intramuscular, intrasternal, intracavernous, intrathecal, intraperitoneal, intraocular injection or infusion techniques. The pharmaceutical composition is formulated so that when the composition is administered to the patient, the active ingredient contained therein is bioavailable. The composition administered to the patient takes the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container of one or more compounds of the present invention in the form of an aerosol can accommodate multiple dosage units. The compound can also be administered in a sustained release or controlled release dosage form, including an accumulated injection, an osmotic pump, a pill, a transdermal (including electromigration) patch, etc., for extending and / or timing, pulse administration at a predetermined rate.

[0068] Dosage amount and interval can be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the modulatory effect or minimum effective concentration (MEC). The MEC for each compound will vary, but can be estimated based on in vitro data. The dosage required to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0069] The pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

[0070] Pharmaceutical compositions suitable for administration include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal (including humans) being treated, and the physical characteristics of the particular animal under consideration. The dose can be adjusted to achieve the desired effect, but will depend on factors such as body weight, diet, concurrent medication, and other factors that will be recognized by those skilled in the art of medicine. More specifically, a therapeutically effective amount refers to the amount of a compound that effectively prevents, alleviates, or improves symptoms of a disease or prolongs the survival of a subject being treated. The determination of a therapeutically effective amount is entirely within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Typically, human clinical applications of a product begin with a lower dose level, with an increase in the dose level until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to determine the useful dose and route of administration of the composition identified by the present method using established pharmacological methods.

[0071] In non-human animal studies, the use of potential products is started at a higher dose level, and the dose is gradually reduced until the desired effect is no longer achieved or the adverse side effects disappear. The dosage range may be wide, depending on the desired effect and the therapeutic indication. Generally, the dosage can be between about 10 micrograms / kg and 100 mg / kg body weight, preferably between about 100 micrograms / kg and 10 mg / kg body weight. Alternatively, as will be appreciated by those skilled in the art, the dosage can be based on or calculated from the surface area of ​​the patient.

[0072] The exact formulation, route of administration and dosage of the pharmaceutical composition of the present invention can be selected by the individual physician according to the patient's condition. (See, for example, "Goodman & Gilman's The Pharmacological Basis of Therapeutics" 13 th Edition 2017, the entire contents of which are incorporated herein by reference) Generally, the dosage range of the composition administered to the patient can be about 0.5 to 1000 mg / kg patient body weight. Depending on the needs of the patient, the dosage can be a single or a series of two or more administered over the course of one or more days. In cases where human doses of compounds have been determined for at least some conditions, the present invention will use those same doses, or doses between about 0.1% and 500%, more preferably between about 25% and 250%, of the determined human doses. In cases where human doses have not been determined, such as in the case of newly discovered drug compounds, suitable human doses can be inferred from ED50 or ID50 values ​​or other appropriate values ​​derived from in vitro or in vivo studies, which are qualified by animal toxicity studies and efficacy studies.

[0073] It should be noted that the attending physician knows how and when to terminate, interrupt or adjust administration due to toxicity or organ dysfunction. On the contrary, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The magnitude of the dosage for the treatment of the target disease will vary with the severity of the disease to be treated and the route of administration. For example, the severity of the disease can be assessed in part by standard prognostic assessment methods. In addition, dosage and possible dosage frequency will also vary according to the age, body weight and response of individual patients. Programs comparable to the programs discussed above can be used in veterinary medicine.

[0074] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injections, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. In various aspects, the formulation may be administered therapeutically; that is, administered to treat an existing disease or condition. In other various aspects, the formulation may be administered prophylactically; that is, administered to prevent a disease or condition. In one aspect, administration of a tablet refers to oral administration.

[0075] As used herein, the term "immediate release" refers to a property indicating that a desired substance is released relatively immediately into its target environment. In one aspect, an "immediate release" tablet releases more than about 40% of the desired substance within hours after administration, as measured under a Tablet Dissolution Test.

[0076] As used herein, the term "controlled release" refers to an attribute indicating that a desired substance, such as a drug (e.g., a magnesium salt), is released into its target environment (e.g., a subject) in a controlled manner rather than immediately. Thus, a "controlled release" formulation releases no more than about 40% of the desired substance within 1 hour after administration, as measured under a tablet dissolution test. "Controlled release" includes both "delayed release" and "sustained release" formulations. In one aspect, "controlled release" does not include "immediate release" formulations; however, it is envisioned that certain "controlled release" formulations may include an immediate release aspect. For example, a formulation with an immediate release controlled core and an enteric coating would not be referred to as an "immediate release" formulation; such a formulation may be referred to as a "controlled release" formulation and a "delayed release" formulation, but not a "sustained release" formulation. Examples of "controlled release" tablets include "delayed release" tablets, "sustained release" tablets, and "delayed / sustained release" tablets.

[0077] As used herein, the term "delayed release" refers to an attribute indicating that a desired substance, such as a drug (e.g., a magnesium salt), is not immediately released to its target environment (e.g., a subject) after administration. In one aspect, the dosage form controls the rate at which the drug is released into the gastrointestinal tract, releasing most of the drug in a portion of the gastrointestinal tract away from the duodenum. This can reduce the incidence or severity of gastrointestinal side effects. In addition, this can increase the amount of drug absorbed into the blood. In another aspect, a "delayed release" preparation releases no more than about 5% of the desired substance within 2 hours after administration. In yet another aspect, a "delayed release" preparation releases no more than about 5% of the desired substance within 2 hours after administration, and releases no more than about 40% of the desired substance within 3 hours after administration. In a further aspect, a "delayed release" preparation releases no more than about 5% of the desired substance within 2 hours after administration, releases no more than about 40% of the desired substance within 3 hours after administration, and releases no more than about 80% of the desired substance within 8 hours after administration. In a further aspect, a "delayed release" formulation releases no more than about 5% of the desired substance within 2 hours after administration, releases no more than about 40% of the desired substance within 4 hours after administration, and releases about 50 to about 80% of the desired substance within 8 hours after administration. In another aspect, substantially all drugs are released within 12 hours. "Delayed release" is a subset of "controlled release". The FDA guide also refers to "delayed release" tablets as solid dosage forms that release drugs (or multiple drugs) at a time that is not immediate after administration. Enteric-coated products are delayed release dosage forms. The term includes both "delayed release" tablets and "delayed / sustained release" tablets.

[0078] As used herein, the term "delayed / sustained release" refers to an indication that a desired substance such as a drug (e.g., a magnesium salt) is released to its target environment (e.g., a subject) at a time other than immediately after administration, and is maintained at a desired time interval to release to its target environment at a desired dose. In one aspect, the dosage form controls the rate at which the drug is released into the gastrointestinal tract, releasing most of the drug in a portion of the gastrointestinal tract away from the duodenum. This can reduce the incidence or severity of gastrointestinal side effects. In addition, this can increase the amount of drug absorbed into the blood. In another aspect, the dosage form controls the drug release rate to target the distal small intestine. In yet another aspect, the dosage form controls the drug release rate to target the distal small intestine, thereby increasing the amount of magnesium available for interaction with TRPM6 and / or TRPM7 cation channels. In one aspect, the dosage form controls the drug release rate to reduce the frequency of administration. This can maintain the desired blood level of the drug without relying on the frequency of administration. This can also increase the patient's compliance with a given treatment regimen. In another aspect, the "delayed / sustained release" formulation releases no more than about 5% of the desired substance within 2 hours after administration, and releases no more than about 40% of the desired substance within 3 hours after administration. In a further aspect, a "delayed / sustained release" formulation releases no more than about 5% of the desired substance within 2 hours after administration, no more than about 40% of the desired substance within 3 hours after administration, and no more than about 80% of the desired substance within 8 hours after administration. In a further aspect, a "delayed / sustained release" formulation releases no more than about 5% of the desired substance within 2 hours after administration, no more than about 40% of the desired substance within 3 hours after administration, and about 50% to about 80% of the desired substance within 8 hours after administration. In another aspect, substantially all of the entire drug is released within 12 hours. "Delayed / sustained release" is a subset of "controlled release." "Delayed / sustained release" is a subset of "delayed release." "Delayed / sustained release" is a subset of "sustained release."

[0079] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired result or to have an effect on an adverse condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or to have an effect on an adverse symptom but generally not sufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex and diet; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; the drugs used in combination or simultaneously with the specific compound used, and similar factors well known in the medical field. For example, it is entirely within the technical scope of the art to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple doses for administration purposes. Therefore, a single dose composition may contain such an amount or a submultiple thereof to constitute a daily dose. If there are any contraindications, the individual physician may adjust the dose. The dose may vary and may be administered in one or more doses per day for one or more days. For a given type of drug, guidance on appropriate doses can be found in the literature. In additional aspects, the formulations can be administered in a "prophylactically effective amount," ie, an amount effective to prevent a disease or disorder.

[0080] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injection solutions or dispersions before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. For example, appropriate fluidity can be maintained by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions and by using surfactants. These compositions can also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc., prevention of the effects of microorganisms can be ensured. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable drug forms can be brought about by containing agents that delay absorption (such as aluminum monostearate and gelatin). Injectable accumulation forms are prepared by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide, poly(orthoester) and poly(anhydride). The release rate of the drug can be controlled according to the ratio of the drug to the polymer and the properties of the specific polymer used. Accumulated injectable preparations can also be prepared by embedding the drug in liposomes or microemulsions compatible with body tissues. Injectable preparations can be sterilized, for example, by filtering through a bacteria-retaining filter or by introducing a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use. Suitable inert carriers can include sugars, such as lactose.

[0081] As used herein, the term "immortalized cell" refers to a cell that reproduces indefinitely. The cell has escaped the normal growth restriction of a finite number of division cycles. The term does not include malignant cells.

[0082] As used herein, the term "normal cell" refers to a cell with growth restriction, i.e., a limited number of division cycles (Hayflick limit); therefore, a non-tumorigenic cell. Normal cells include primary cells, i.e., non-immortalized cells or cell lines taken directly from a living organism.

[0083] As used herein, the term "cell growth" refers to an increase in the size of a cell population.

[0084] As used herein, the term "cell division" refers to mitosis, the process by which cells reproduce.

[0085] As used herein, the term "proliferation" means the growth and division of cells. "Actively proliferating" means cells that are actively growing and dividing.

[0086] As used herein, the term "inhibiting cell proliferation" refers to slowing and / or preventing the growth and division of cells. Cells can be further designated as being arrested in a particular cell cycle phase: G1 (interphase 1), S phase (DNA synthesis), G2 (interphase 2), or M phase (mitosis).

[0087] As used herein, the term "preferentially inhibiting cell proliferation" refers to slowing down and / or preventing the growth and division of cells compared to normal cells.

[0088] The term "purified" does not require absolute purity; on the contrary, it is intended as a relative definition. It is explicitly considered that the starting material or natural material is purified to at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude. The term "purified" is further used herein to describe polypeptides or polynucleotides of the present invention that have been separated from other compounds (including but not limited to polypeptides or polynucleotides, carbohydrates, lipids, etc.). The term "purified" can be used to specify the separation of monomeric polypeptides of the present invention from oligomeric forms (such as homo- or heterodimers, trimers, etc.). The term "purified" can also be used to specify the separation of covalently closed (i.e., circular) polynucleotides from linear polynucleotides. Substantially pure polypeptides or polynucleotides generally account for about 50%, preferably 60 to 90% weight / weight, more usually about 95%, and preferably more than about 99% pure, but can be specified as any percentage integer between 50 and 100. Polypeptide and polynucleotide purity or homogeneity is indicated by a variety of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of the sample, followed by observation of individual bands after staining the gel. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art. As an alternative embodiment, the purification of the polypeptides and polynucleotides of the present invention can be expressed as "at least" percent purity relative to heterologous polypeptides and polynucleotides (DNA, RNA or both). As a preferred embodiment, the polypeptides and polynucleotides of the present invention are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99% or 100% pure relative to heterologous polypeptides and polynucleotides, respectively. As a further preferred embodiment, the polypeptides and polynucleotides have a purity to the thousandth place in the range of 90% to 100% relative to the heterologous polypeptide or polynucleotide (e.g., at least 99.995% pure polypeptide or polynucleotide), respectively, or as a weight / weight ratio relative to all compounds and molecules other than those present in the vector. Each figure expressing a percentage purity to the thousandth place can be stated as a single substance purity.

[0089] The terms "polypeptide" and "protein" used interchangeably herein refer to polymers of amino acids, regardless of the length of the polymer; therefore, peptides, oligopeptides and proteins are included in the definition of polypeptides. The term also does not explicitly indicate or exclude chemical or post-expression modifications of the polypeptides of the present invention, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Thus, for example, modifications to polypeptides (including covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups, etc.) are explicitly included in the term polypeptide. In addition, polypeptides with these modifications may be designated as single substances included or excluded in the present invention. Natural or other chemical modifications (such as those listed in the examples above) may occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains and amino or carboxyl termini. It should be understood that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. In addition, a given polypeptide may contain multiple types of modifications. Polypeptides may be branched, for example, due to ubiquitination, and they may be cyclic with or without branches. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to protein such as arginylation, and ubiquitination. [See, e.g., Creighton, (1993), Posttranslational Covalent Modification of Proteins, WH Freeman and Company, New York BC Johnson, Ed., Academic Press, New York 1-12; Seifter, et al., (1990) Meth Enzymol 182:626-646; Rattan et al., (1992) Ann NY Acad Sci 663:48-62]. Also included in the definition are polypeptides comprising one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids that naturally occur only in unrelated biological systems, modified amino acids from mammalian systems, etc.), polypeptides with substituted linkages, and other modifications known in the art, including both naturally occurring and non-naturally occurring ones.

[0090] In this study, we aimed to generate a new class of short peptides (X1Y1X2Y2) n , where X1 and X2 are hydrophobic residues (Ile, Val or Leu), Y1 and Y2 are hydrophilic residues (Arg, Lys Glu or Asp), and n is a repeating unit, but the same amino acid does not have to be selected, as is apparent from the examples herein. Peptide amphiphiles self-assemble into hydrogels with tunable mechanical strength and sustainable stability for a range of biomedical applications. To the best of our knowledge, these peptide sequences have not been reported in the literature. The main features of these peptides include the regular alternation of hydrophobic and hydrophilic amino acids, including arginine (R) and lysine (K), with the charges arranged in a pattern (--++). R was selected because it has a higher tendency to gel, while K was used in combination with R because it has lower cytotoxicity to provide a positively charged portion in the peptide under physiological conditions. Peptide amphiphiles consisting of 8 amino acid residues exist in a random coil configuration in water and form peptide hydrogels (~99% water) at high concentrations. Interestingly, these peptides self-assemble into a β-sheet configuration in salt or physiological conditions and form stronger hydrogels. The hydrophobic residues were systematically adjusted to study their effects on the mechanical strength of the peptide hydrogels. The biocompatibility of the peptide hydrogels was evaluated. In addition, another type of short peptide amphiphiles consisting of 12 natural amino acids, IRIKIEIEIRIK (IIK12) and IRVKIEVEIRVK (IVK12), have been designed with alternating hydrophobic and hydrophilic amino acids. The charge arrangement follows the pattern (++--++), where the central peptide IEIE is flanked by IRIK. IIK12 self-assembles into a β-sheet configuration and forms a hydrogel at a very low peptide concentration (0.6 (w / v)%) in aqueous solution. It further complexes with an antimicrobial peptide (IRIKIRIK (IK8)) that forms a β-sheet through electrostatic interactions to form a hybrid antimicrobial hydrogel. This hybrid antimicrobial hydrogel is designed for the prevention and treatment of DFU infections. The effect of peptide concentration on the physicochemical properties of the hydrogel was studied. The antimicrobial activity of the hybrid hydrogel against a variety of pathogenic microorganisms was evaluated. The release of antimicrobial peptides from hybrid peptide hydrogels was analyzed using disk diffusion assay.

[0091] In addition, peptides having sequences in the reverse order of those described in the examples may also have similar or identical properties, and the ends of these peptides may be functionalized to avoid ion formation and thereby reduce or eliminate electrostatic interactions (especially repulsion) at the ends, for example, the N-terminus may be acetylated and the C-terminus may be amidated. The peptides described herein comprise 8 and 12 amino acids, and two or more of these peptides may be connected by a linker and are capable of forming β-sheets and hydrogels as described herein. The linker may be any suitable component, length or portion, and may be connected to the C or N-terminus of the peptide by methods known in the art to synthesize peptides of longer lengths. In addition, peptides having longer amino acid sequences, for example, in additional blocks of four amino acids having alternating hydrophobic amino acids and hydrophilic amino acids, may still be capable of forming β-sheets and hydrogels as described herein.

[0092] method

[0093] Material

[0094] The designed peptides were synthesized by GL Biochem (Shanghai, China) and purified to 95% purity using analytical reverse phase high performance liquid chromatography (RP-HPLC). The molecular weight of the synthetic peptides was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Autoflex II, Bruker Daltonics Inc., USA) using saturated α-cyano-4-hydroxycinnamic acid (4-HCCA) (Sigma-Aldrich, Singapore) in acetonitrile / water mixture (1:1 volume ratio) as a matrix. 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide (MTT) and acetonitrile were purchased from Sigma-Aldrich and used as is. Human bone marrow mesenchymal stem cells (hMSC) and mesenchymal stem cell culture medium (MSCGM) were purchased from Lonza (USA). Human breast cancer BT-474 cell line, MCF-7, 4T1 cells were purchased from ATCC, USA, and cultured according to the recommendations of ATCC. Cells were cultured in Roswell Park Memorial Institute (RPMI1640) medium enriched with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin (HyClone, USA) at 37°C and 5% CO2. Human dermal fibroblasts and human epidermal keratinocytes were obtained from Cordlabs Singapore and cultured according to the manufacturer's instructions. Microbial growth medium was prepared using Muller Hinton Broth (MHB) powder (BD Diagnostics). Staphylococcus aureus (ATCC No. 6538), Escherichia coli (ATCC No. 25922), Pseudomonas aeruginosa (ATCC No. 9027) and Candida albicans (ATCC No. 10231) were obtained from ATCC, USA.

[0095] Biophysical research

[0096] CD measurements were performed using a Jasco Model J-810 spectropolarimeter (Jasco, Great Dunmow, Essex, UK) using a quartz cuvette with a 1 mm optical path (Hellma, Germany). Spectra were recorded from 240 to 190 nm at a scan rate of 50 nm / min (average of 3 scans). Ellipticity was derived from the formula: [θ] λ =(M / (N-1)×θ λ ) / (d×c), where θ λ represents the observed ellipticity (degrees) at wavelength λ, d represents the path length (cm), c represents the concentration (g / mL), and [θ] λ Represents the residue ellipticity (deg cm2 dmol -1 The thermal scans were measured using a Jasco J-810 spectropolarimeter equipped with a Peltier temperature controller. The solutions were allowed to equilibrate at room temperature for 10 minutes prior to testing.

[0097] Preparation of hydrogel

[0098] The peptide powder is dissolved in water or other aqueous solutions with a desired concentration. Alternatively, the required salt can be added after the preparation of the peptide aqueous solution. The hydrogel is formed by mixing the peptide aqueous solution and the salt (if necessary). For example, the solution is vortexed, and then incubated to form a hydrogel. The hydrogel formed can be stored as formed, or can be dried, for example, by freeze drying. Alternatively, a hydrogel can be formed as needed.

[0099] The concentration of solutes (e.g., peptides and salts) herein may be provided as a weight / volume percentage (and may be abbreviated herein as (% w / v), (w / v%), or (%), and is equivalent to the mass of solute (in grams) dissolved in 100 mL of solvent. Alternatively, wt% (or wt.%) may be used, since the solvent is water and a density of 1 g / cm is assumed. 3 .

[0100] Rheological study of hydrogels

[0101] The gelation time was determined by the vial tilt method. When the sample showed no flow, it was considered to be a gel. Rheological experiments were performed at room temperature using a controlled strain rheometer (ARES G2, TA Instruments). The rheometer was equipped with two sensitive force transducers with a torque range of 0.05 μN.m to 200 mN.m. The peptide powder was dissolved in water at a concentration of 1.0 to 2.0 (w / v)%. The solution was vortexed and then incubated to form a hydrogel. The gel was placed on a parallel plate geometry (8 mm in diameter). The dynamic storage modulus (G′) and loss modulus (G″) were examined for changes in frequency from 1 to 100 rad / s. Measurements were performed at strain amplitude (γ) to ensure the linearity of viscoelasticity. Flow scans (viscosity changes with shear rate) were also performed to study the shear thinning properties of the hydrogel.

[0102] Scanning electron microscopy (SEM)

[0103] The hydrogels were cut and frozen in liquid nitrogen. The samples were then freeze-dried and the morphology of the hydrogels was observed using a field emission SEM (JEOL JSM-7400F) operated at an accelerating voltage of 10.0 kV and a working distance of 8.0 mm.

[0104] Culture of hMSCs

[0105] hMSCs were cultured in MSCGM medium (Lonza, USA) and incubated at 37°C, 5% CO2. The medium was changed every three days. The cells were harvested with PBS containing 0.025 (w / v)% trypsin and 0.01% EDTA, centrifuged and subcultured in MSCGM to the 4th passage for 2D culture on the hydrogel surface or 3D hydrogel encapsulation as described below.

[0106] Cell culture on peptide hydrogel surfaces

[0107] IEVEIRVK (IVK8) powder was dissolved in 300 mM sucrose solution at a peptide concentration of 1.5 (W / V)%. It should be mentioned that sucrose was used to maintain physiological osmotic pressure. 35 μL of peptide solution was transferred to a 96-well plate. A soft gel was formed and then 15 μL of DMEM complete medium was added. The gel was incubated at 37 °C for further solidification for 10 min. For 2D cell proliferation on the hydrogel surface, 100 μL of MCF-7 cells in RPMI medium were seeded onto the peptide hydrogel surface. The culture medium was changed every 2-3 days. Cell proliferation on the hydrogel was quantified using the MTT assay.

[0108] Cell culture in 3D peptide hydrogels

[0109] IVK8 powder was dissolved in 300mM sucrose solution at a peptide concentration of 1.5 (w / v)% and kept at 4 ° C. 70 μL of peptide solution was transferred to the open end of a 1 mL syringe. It should be noted that the open end of the syringe should be cut before the solution is transferred. BT-474 cells were suspended in DMEM at 3 million / mL. 30 μL of cell suspension was added to the peptide solution. The syringe was transferred to an incubator and incubated at 37 ° C for 20 min. Afterwards, the peptide gel was transferred to a 24-well plate for long-term culture. The culture medium was replaced every 2-3 days. The viability of cells in the hydrogel was quantified by the MTT assay. The gel was collected at predetermined time intervals and homogenized with a tissue disruptor (Qiagen, USA). The results are expressed as a percentage of the absorbance on the first day.

[0110] Confocal laser scanning microscopy

[0111] The morphology and viability of hMSCs on the hydrogel surface and inside the 3D peptide hydrogel environment are observed by an inverted confocal laser scanning microscope (CLSM) (CLSM, Carl Zeiss, Germany). Live / dead cell staining assay (Invitrogen) is used. Briefly, the cells cultured on the hydrogel surface in the chamber cover glass (NUNC, Denmark) are washed with PBS and incubated in 0.5mL of PBS containing dye for 10min, then visualized by CLSM at an excitation wavelength of 488nm and 532nm. All observations are carried out under the same conditions. For cells grown in 3D hydrogels, constructs are cut, and then staining agents are added. Gel is transferred to a cover glass, and then visualized by CLSM using the same method as described for cells on the hydrogel surface.

[0112] Antimicrobial activity

[0113] 1. Peptide MIC Measurement

[0114] Bacterial and fungal samples were grown in MHB at 37°C and room temperature, respectively, with shaking (100 rpm). They were then incubated overnight to enter the logarithmic growth phase. The MIC of each peptide was determined using the broth microdilution method. Each microbial suspension (100 μL) was inoculated into each well of a 96-well plate (3 × 10 5 CFU ml -1 ), to which 100 μL of broth containing different concentrations of peptides was added. The plate was then incubated at 37°C for 18 h under shaking (100 rpm). MIC was considered to be the lowest peptide concentration that completely inhibited microbial growth observed with a microplate reader (TECAN, Switzerland). A negative control (broth containing only microorganisms and not treated with peptides) was used. Six replicates were repeated for each experiment. Note that Candida albicans cells were grown in yeast medium broth (YMB, BD) at room temperature.

[0115] 2. Antimicrobial activity of peptide hydrogels

[0116] Hydrogels for antimicrobial assays were prepared in 96-well tissue culture plates (NUNC). Briefly, IIK12 (1%) with different contents of antimicrobial peptides was dissolved in water and vortexed. 50 μL of the solution was transferred to the wells. Gelation occurred overnight at 37°C. Microbial growth on the hydrogels was measured using the broth dilution method. Briefly, 30 μL of microbial suspension (3.5×10 5 CFU / mL) were introduced into the hydrogel, and a 96-well plate without antimicrobial peptides and peptide hydrogels were used as controls. 600nmMonitor the optical density readings of the bacterial solution. The assay was repeated four times for each sample, and the experiment was repeated at least three times. Antimicrobial activity was further tested by the diffusion plate method. In brief, the hydrogel was attacked with a microbial solution, and at the end of the treatment, the microbial suspension on the surface of the hydrogel was taken out and diluted in sequence, and then spread on a 1.5% LB agar plate. The plate was incubated at 37°C for 24 hours (Candida albicans was incubated at room temperature for 48 hours). Microbial colonies were formed and counted. The experiment was performed in triplicate and repeated three times.

[0117] 3. Killing kinetics test

[0118] Microorganisms were treated with peptide hydrogels as described above. At different time periods of treatment, microbial samples were diluted for plating (LB Agar, 1st Base). Incubation conditions: Bacteria were 18 h at 37°C. Colony forming units were counted after the incubation period. The experiment was repeated 3 times in independent settings.

[0119] 4. Disc Diffusion Assay

[0120] The antimicrobial activity was further tested by disk diffusion assay. Briefly, 3.5 × 10 6 CFU / mL (40 μL) of microbial suspension (Staphylococcus aureus, ATCC No. 6538) was spread on 1.5% LB agar plates. Discs (Sigma) were prepared to contain 50 μL of peptides at different concentrations by dropping 50 μL of peptide solution onto sterile discs. The discs were air-dried for several minutes and then placed on different areas of the agar plate. The hydrogel (100 μL) was placed on the LB agar plate. The plate was incubated at 37 ° C for 24 h.

[0121] 5. Hemolysis Assay

[0122] Fresh rat erythrocytes were washed 3 times with PBS. A suspension of erythrocytes (4% by volume) in 100 μL PBS was placed on the hydrogel surface in each well of a 96-well plate, and then 100 μL PBS was added to the wells. The plate was incubated at 37°C for one hour. The 96-well plate was centrifuged at 2000 rpm for 5 min. An aliquot (100 μL) of the supernatant was transferred to a new 96-well plate. Hemoglobin release was measured at 576 nm using an ELISA reader (TECAN). Erythrocytes in PBS were used as a negative control. The absorbance of the wells with erythrocytes lysed with 0.2% triton X-100 was taken as 100% hemolysis. The hemolysis percentage was calculated using the following formula: Hemolysis (%) = [(OD in sample 576nm -OD in PBS 576nm ) / (OD in 0.2% triton X-100 576nm -OD in PBS576nm ) × 100. Data are presented as the mean and standard deviation of four replicates and the test was repeated three times.

[0123] 6. In vitro cytotoxicity study of antimicrobial peptide hydrogels

[0124] 1000 μL HDF or keratinocytes (10 5 Cells) were inoculated into 12-well Transwell plates (Corning). IIK12 (1%) with different content of antimicrobial peptides was dissolved in water and incubated overnight at 37°C, and 100 μL of gel was transferred to the insert of the Transwell plate. 1 mL of fresh culture medium was added to the insert. Before performing the MTT assay, the plate was incubated for 24 h. It is worth noting that cells without any treatment and mupirocin (GSK) were used as controls. Data are expressed as the mean and standard deviation of three replicates and the test was repeated 3 times.

[0125] Results and discussion

[0126] Peptide design and characterization

[0127] In this study, short amphipathic peptides consisting of 4-12 natural amino acid residues were designed. Preferably, the peptides have an even number of amino acid residues to maintain the chemical complementarity of the peptides. These peptide sequences are characterized by the periodic repetition of charged hydrophilic and hydrophobic amino acids, where the hydrophobic residues are isoleucine (I), valine (V) and leucine (L), and the hydrophilic residues are arginine (R), lysine (K), glutamic acid (E) and aspartic acid (D). Arginine and lysine are positively charged under physiological conditions or at pH 7 to 7.4 at 25°C, while glutamic acid and aspartic acid are negatively charged. Isoleucine (I), valine (V) and leucine (L) were selected as hydrophobic residues because they have a strong β-sheet folding tendency. It is worth noting that arginine (R) was selected because of its faster and stronger gelation properties, while lysine (K) was used in combination with R because of its lower cytotoxicity. In order to minimize charge repulsion, the peptides were amidated at the C-terminus. When the N-terminus has a hydrophobic residue, it is not necessary to acetylate the N-terminus, but it can be performed by known methods. If the N-terminus has a hydrophilic residue, it can be functionalized by known methods such as acetylation to minimize charge repulsion. RP-HPLC showed that the purity of the peptide was greater than 95%. MS was used to verify the molecular weight of the peptide. As shown in Table 1, the molecular weight measured by MS is consistent with the theoretically calculated molecular weight of the peptide, indicating that the peptide was successfully synthesized.

[0128] In addition, peptides with reverse sequences to SEQ ID No. 9-16 are likely to be able to have similar, substantially similar or identical functions to the corresponding hydrogels. These peptides are identified as SEQ ID No. 19-26 in Table 1, respectively, and can be prepared by methods known in the art. These peptides with reverse sequences should preferably be acetylated at the N-terminus and amidated at the C-terminus. This minimizes the charge repulsion between the two ends of the peptide.

[0129] Peptides containing both positively and negatively charged amino acids may be able to form hydrogels at lower concentrations than peptides with only positively charged amino acids. For example, peptides with only positively charged amino acids typically form gels at 8% w / v or higher concentrations, while peptides with both positively and negatively charged amino acids form hydrogels at 0.6% w / v and higher concentrations. In addition, peptides with only negatively charged amino acids will not self-assemble to form hydrogels.

[0130] CD studies of peptides

[0131] like Figure 1A As shown, the CD spectrum of IVK8 in aqueous solution shows a minimum at 198 nm, indicating that it adopts a random coil structure. This is attributed to the intermolecular electrostatic repulsion between the protonated R and K residues. However, in the presence of salt such as PBS, IVK8 folds and self-assembles into a β-sheet configuration, characterized by a maximum at ∼197 nm and a strong negative ellipticity at ∼219 nm. Salt triggers the self-assembly of peptides because it screens and neutralizes charges, with hydrophilic residues E, R, and K located on the same side with complementary ionic interactions from positively and negatively charged ionic groups. Similar observations were also reported by Zhang et al., where EAK16 and EAK12 self-assembled into a β-sheet secondary structure after salt addition [17, 35]. However, EAK8 (AEAEAKAK) did not form a β-sheet secondary structure under the same conditions

[35] . Unlike EAK8, our peptide IEVEIRVK (IVK8) self-assembled into a β-sheet secondary configuration after salt addition. One possible explanation is that the use of hydrophobic isoleucine and valine on the hydrophobic peptide face increases the hydrophobicity of the peptide, thereby stabilizing the β-sheet configuration of IVK8 by enhancing the hydrophobic interactions between isoleucine and valine. Based on the above studies, IVK8 was identified as a peptide with an ideal length and sequence, and its hydrophobicity was therefore further altered to study its effects on biophysical properties. Replacing Val with Leu or Ile in IVK8 produced the peptide sequences ILK8 and IIK8, respectively. Similar to IVK8, ILK8 and IIK8 adopt a random configuration in aqueous solution, but fold into a β-sheet structure after the addition of salt ( Figure 1AInterestingly, replacement of Val with Leu or Ile results in a greater degree of β-sheet folding, as indicated by the molecular ellipticity at the minimum point (θ M ). It can be seen that the θ of IVK8, ILK8 and IIK8 M They are -11.2, -21.4 and -28.9 deg.cm respectively 2 dmol -1 , indicating that IIK8 has the largest β-sheet configuration compared to ILK8 and IVK8. This is because Ile and Leu are more hydrophobic than Val, which increases the hydrophobicity of the peptide on the nonpolar side and causes the β-sheet to be more stable and increases the peptide β-sheet configuration [28, 36]. It is generally believed that peptides with more repeating units of alternating hydrophilic and hydrophobic amino acid residues tend to have a high tendency to form a β-sheet configuration. Therefore, a new class of 12 amino acid peptide amphiphiles composed of (X1Y1X2Y2)3 was designed, in which X1 and X2 are hydrophobic residues (Ile or Val), Y I and Y2 are hydrophilic residues (Arg, Lys or Glu) (Table 1). Interestingly, these peptide amphiphiles folded and assembled into β-sheet structures in water, as shown by the strong negative ellipticity at 218 nm (Figure 2). The CD spectra of peptide amphiphiles composed of 12 amino acids were further studied in the presence of salt. The minimum negative ellipticity at 218 nm for peptide solutions with 150 mM PBS indicated that they also adopted a β-sheet-rich secondary structure. Notably, the β-sheet content of the peptide amphiphile IVK12 in the presence of salt was similar to that in the absence of salt ( Figure 2A ). This is because IVK12 has a net positive charge (+2) in PBS. Electrostatic repulsion leads to a lower degree of β-sheet configuration for the peptide amphiphile IVK12. Similar phenomena were observed for IRV12 and IEV12 (Figure 2). Although IRV12 and IEV12 have different arrangements of charged species, they have similar net charges and exhibit similar gelation behavior, so the net charge may play a more important role than the specific arrangement of hydrophilic residues. Advantageously, peptides with a net positive charge can more easily form β sheets and hydrogels even in the absence of salt, which may be necessary for certain applications of hydrogels and also leads to reduced costs.

[0132] Rheological properties of peptide hydrogels

[0133] It is generally believed that peptide amphiphiles have a strong tendency to form α-helical or β-sheet structures. Under appropriate conditions, the resulting α-helical or β-sheet structures can further self-assemble into supramolecular structures, such as hydrogels that embed large amounts of water ( Figure 3A and 3B )

[16] . Typical SEM images of hydrogels are shown in Figure 2. Figure 3C As shown in Figure 2, it can be clearly seen that the hydrogel is highly porous. Peptide gelation can be regulated by adjusting intrinsic factors such as amino acid sequence, number of repeating units, peptide concentration, and external environment such as temperature, pH, and salt concentration

[23] .

[0134] Effects of peptide length and structure

[0135] Peptide amphiphiles consisting of 4-6 amino acid residues were unable to form hydrogels at concentrations up to 2% (Table 2). These results are consistent with previous findings that peptide length determines the strength of intermolecular and intramolecular interactions. The design of peptide amphiphiles containing 8 amino acid residues is shown in Table 2. First, the positions of the hydrophilic ionic residues have been systematically adjusted to evaluate their effects on peptide gelation. No gel formation (up to 2%) was observed for IRK8, in which the hydrophilic amino acid residues were arranged in a (+--+) pattern. Possibly, IRK8 can form gels at higher temperatures (e.g., 37°C) and / or concentrations. However, gelation was observed for IRE8, in which the hydrophilic amino acid residues were arranged in a (+-+-) pattern. It should be noted that the gelation kinetics were slow, for example, IRE8 required 3 days at room temperature to form a gel at a concentration of 2%. When the temperature was increased to 37°C, the gelation time was significantly reduced to 1 day. Similarly, it is expected that 12 amino acid peptides can form hydrogels at room temperature with extended gelation times and / or higher concentrations.

[0136] Notably, IVK8 achieved rapid gelation, in which the hydrophilic amino acid residues were arranged in a (--++) pattern. For example, at 2%, IVK8 gelled within 60 minutes at room temperature (23-24°C), while at 37°C it formed a gel within 30 minutes. Using IVK8 as the basic peptide sequence, its hydrophobic amino acid residue Val was replaced with Ile or Leu, while the hydrophilic amino acid residues and arrangement remained unchanged. Under the same conditions, replacement of Val with Ile or Leu resulted in faster gelation (Table 2). For example, when incubated at 37°C, IIK8 gelled within 15 minutes at 1.5%. It was also observed that an increase in peptide length to 12 amino acids resulted in a decrease in gel concentration. Notably, IIK12 and IVK12 were the best performing peptides due to ease of gelation, and they self-assembled into hydrogels at concentrations as low as 0.6 (w / v)%. These findings indicate that peptide chain length and peptide structure play an important role in gelation. Figure 3A In FIG. 1 , hydrogels formed from 1.5% w / v IVK8 and 1.5 and 2.0% w / v IIK8 are shown, where the white bar is the surface 10 of the gel in the vial.

[0137] The rate of gel formation can be increased by adjusting at least one of the following: peptide concentration, the presence of salt, and increasing temperature. Peptides with 12 amino acid residues generally form gels more readily than peptides with 8 amino acids. In addition, peptides with a net positive charge (but containing negatively charged hydrophilic amino acids) tend to form hydrogels at lower concentrations due to enhanced beta-sheet hydrogen bonding.

[0138] Effects of peptide concentration and peptide hydrophobicity on mechanical properties

[0139] The effect of peptide concentration on rheological properties was investigated using dynamic mechanical analysis. The peptide amphiphiles self-assembled into supramolecular hydrogels that exhibited rigid viscoelastic properties. Typical frequency sweep measurements such as Figure 4A and Figure 5A As shown, the storage modulus (G') is much larger than the loss modulus (G") value. In addition, the G' value is independent of the frequency from 0.1 to 50 rad / s. The G' value at 1 Hz (6.28 rad / s) is listed in Table 3 and used for gel stiffness comparison. It was found that increasing the concentration of peptide IVK8 from 1.0% to 1.75% significantly increased the stiffness. For example, the G' value at 1.75% was 10 times that at 1.0% (Table 3). The results indicate that peptide gel stiffness is strongly affected by peptide concentration (i.e., the amount of peptide in the aqueous solution). This is because more peptide molecules are involved in physical cross-linking at higher concentrations. In order to study the effect of the hydrophobicity of amino acid residues on gel strength, all Val in IVK8 were replaced with Ile or Leu. This modification produces peptide II K8 and ILK8. The G' values ​​of IVK8, IIK8 and ILK8 were 84, 125 and 332 Pa, respectively (Table 3), indicating that the increased hydrophobicity of the non-polar amino acid residues in the peptide structure resulted in a harder gel. It is reported that Ile and Leu have similar hydrophobicity. However, compared with IIK8, the gel strength formed by ILK8 is greater. This is because Leu may stack more effectively than Ile during self-assembly and gelation. The maximum peptide concentration may be about 3-5 w / v%, up to 5 w / v%, up to 4 w / v% or up to 3 w / v%. The presence of excess peptide may make the hydrogel too hard for certain applications, and the optimal peptide concentration will depend in part on the peptide structure and the use of the peptide. This can be further adjusted by the presence of salt as shown below.

[0140] Effects of Salt

[0141] In aqueous solution, peptide amphiphiles composed of 8 amino acids exist in a random coil configuration. However, after the addition of salt, they self-assemble into a β-sheet structure and form a rigid hydrogel. For example, in the presence of PBS, IVK8 has a G' of ~900Pa at 1.5%, which is almost twice that in the absence of salt. A similar phenomenon was also observed in DMEM. When DMEM cell culture medium is added to the aqueous peptide solution, the peptide forms a rigid hydrogel. For example, in DMEM, IVK8 has a G' of ~2400Pa at 1.5%, which is almost 5 times that in the absence of DMEM. Notably, in PBS, IVK8 obtained an extremely hard hydrogel of 10kPa at 2.5%. In particular, rapid gelation was achieved at 37°C. This is crucial for tissue engineering applications because the gelation kinetics must be fast enough to ensure uniform cell distribution within the matrix. In addition, a harder gel was formed at 37°C than at room temperature (Table 3).

[0142] In addition, gels with high stability in a cell culture environment were obtained under optimal conditions (by adjusting the peptide concentration and DMEM volume) (Table 4). Table 4 shows the initial concentrations of IVK8 used (1.0, 1.2 and 1.5% w / v), which were adjusted by adding DMEM to achieve the ratios shown in Table 4. It can be observed that for most hydrogels, the prepared hydrogels were stable for at least 7 days, while for higher concentrations of peptide hydrogels, they were stable for at least 15 days.

[0143] The significant increase in the strength of peptide hydrogels in the presence of salt is attributed to the fact that salt shields charges and promotes intra- and intermolecular interactions (hydrogen bonds and van der Waals interactions), thereby forming a highly cross-linked network. It would be ideal if hydrogel constructs containing drugs and / or cells could be injected into the body and remain intact during administration. For peptides with 8 amino acids, the presence of salt in the solution increases the stiffness of the hydrogel and reduces the peptide concentration required to form a gel. In the absence of salt, IVK8, IIK8, and ILK8 have been found to form gels at a minimum concentration of 1.0% w / v. In the presence of salt, the concentration of these peptides can be reduced to 0.6% w / v, thereby reducing the cost of preparing the gel. In addition, salt greatly reduces the gelation time, both at room temperature and at higher temperatures (e.g., 37°C).

[0144] Flow scanning was performed to investigate whether the peptide hydrogels were injectable. Figure 4B The viscosity of IVK8 decreased significantly with increasing shear rate, indicating the shear-thinning property of the hydrogel. A similar phenomenon was observed for IVK8 in the presence of DMEM ( Figure 5B ). The gelation of IVK8 peptide solution was triggered by the addition of DMEM, where DMEM was located within the peptide hydrogel network.

[0145] The decrease in viscosity is attributed to the disruption of the physical cross-links between peptide molecules upon application of shear stress. For the delivery of peptide gel constructs containing drugs and / or cells via syringe, it is critical that the ruptured gel with low viscosity can quickly recover to a stiff gel when the shear stress is terminated

[26] . The recovery ability of peptide hydrogels was studied using a dynamic step strain amplitude test, in which strains of 0.5% and 100% were periodically applied to the peptide hydrogels. Figure 4C As shown, when 0.5% strain was applied, the initial G' value of IVK8 was ~50Pa. However, when subjected to high strain (100%), the G' value quickly dropped ~250 times to 0.2Pa, further demonstrating the shear thinning property. After 30s of continuous high strain, the strain was reduced to 0.5%. Interestingly, the G' value was found to recover immediately, indicating that the rheological behavior was reversible. This reversibility of the peptide hydrogel in the presence of DMEM was further evaluated under the same conditions. Figure 5C It shows that when a strain of 0.5% is applied, the initial G' value is ~2000Pa. When subjected to a high strain of 100%, the G' value is significantly reduced by ~2000 times to ~1Pa. It is worth noting that when the strain is reduced to 0.5%, the G' value is immediately restored. A similar phenomenon was also observed for IIK12 hydrogels, where the storage modulus of IIK12 hydrogels increased with increasing peptide concentration (Table 5). In addition, IIK12 hydrogels exhibited reversible rheological behavior in response to dynamic step strain amplitude measurements (Figure 6). This rheological behavior of peptide hydrogels provides great advantages for use as injectable matrices to deliver therapeutic agents, such as drugs and cells. The rapid recovery kinetics can be explained by the fact that the physical crosslinking of the gel network is immediately restored after the high strain is terminated.

[0146] In vitro biocompatibility of peptide hydrogels

[0147] The biocompatibility of peptide hydrogels and their effects on cell proliferation were investigated using different cell types. First, hMSCs were seeded on the surface of peptide hydrogels. As shown in Figure 7, most cells were still alive after 24 hours of attachment to the surface of peptide hydrogels (live cells were seen as white dots on a black background), indicating that peptide hydrogels are biocompatible. Next, we analyzed the proliferation rate of MCF-7 cells on IVK8 hydrogels. Fig. 8AAs shown, the proliferation rate depends on the cell seeding density. MCF-7 cultured on peptide hydrogels with lower cell density grew much faster than those with higher cell density. This is because cell growth is limited by the limited space of two-dimensional culture. Fortunately, this can be overcome by cell culture in a 3D environment. Hydrogels are attractive substrates for cell growth in 3D environments due to their ability to capture high water content. In addition, hydrogels have high permeability to oxygen and nutrients through their high water content matrix, which is ideal for cell growth and tissue engineering. The peptides were dissolved in an aqueous solution containing 300mM sucrose and formed into a malignant solution at 4°C. Gelation was triggered by the addition of cell culture medium containing hMSCs. The cell viability of the encapsulated hMSCs was evaluated to assess the cytotoxicity of the peptide hydrogels. At 1 day after encapsulation, viability was assessed by live / dead staining using confocal microscopy. It was found that most of the encapsulated hMSCs were still alive in IVK8 hydrogels. BT-474 cells were also encapsulated in IVK8 hydrogels, and cell growth was quantified using the MTT assay. Figure 8B The number of cells in the hydrogels on day 4 was almost double that on day 1, indicating that the peptide hydrogels are suitable substrates for 3D cell culture. In conclusion, injectable peptide hydrogels with tunable mechanical properties provide a promising biocompatible system for cell growth and tissue engineering.

[0148] Peptide hydrogels containing β-sheets form synthetic antimicrobial oligopeptides for the prevention and treatment of bacterial infections.

[0149] We have previously developed a series of synthetic cationic oligopeptides that form β-sheets and exhibit broad-spectrum antimicrobial activity and high selectivity against a variety of microorganisms, including Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and yeast Candida albicans [37, 38]. Peptides formed from D-amino acids exhibited stronger antimicrobial activity. The lead antimicrobial peptides IK8L (IRIKIRIK SEQ ID No. 17) and IK8D (D-isomer, SEQ ID No. 18) effectively killed a variety of clinically isolated multidrug-resistant (MDR) microorganisms, including MRSA, VRE, multidrug-resistant Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa, and yeast Cryptococcus neoformans (C. neoformans)

[37] . For example, the MICs of IK8L against MRSA, A. baumannii, VRE, P. aeruginosa, and C. neoformans were 31.3, 12.5, 15.6, 7.8, and 7.8 mg / L, respectively. The MICs of IK8D against MRSA, A. baumannii, VRE, P. aeruginosa, and C. neoformans were 3.9, 31.3, 3.9, 15.6, and 7.8 mg / L, respectively. In particular, repeated treatment of E. coli and S. aureus with the peptide mitigated the development of resistance. IK8L also exhibited strong antifungal activity and could effectively remove fungal biofilms formed in vitro and in a fungal biofilm-induced keratitis mouse model without causing significant toxicity to the eye

[39] . However, IK8D / IK8L formed hydrogels at higher peptide concentrations (e.g., 8 w / v%), which would induce cytotoxicity at gelling concentrations, making it unsuitable for therapeutic use alone. However, adding these antimicrobial peptides to IIK12 hydrogels via electrostatic interactions resulted in the formation of hybrid antimicrobial peptide hydrogels. We systematically evaluated the antimicrobial activity of the hybrid hydrogels against pathogenic microorganisms.

[0150] Rheological behavior of antimicrobial hybrid hydrogels

[0151] Table 6 shows the gelation of 0.5% IIK12 and IK8L or IK8D in water at 37°C. IIK12 alone was unable to form a hydrogel. However, the addition of IK8D or IK8L promoted the gelation (Table 7). SEM images showed that the IIK12 / IK8D gel had more pores and smaller sizes than the IIK12 gel ( Fig. 9). In order to study the effect of IIK12 concentration on the strength of hybrid hydrogels, we performed dynamic frequency scans on hybrid hydrogels with different IIK12 concentrations. It was observed that the stiffness of IIK12 / IK8L increased with increasing IIK12 concentration (Table 8). It was found that at lower IIK12 concentrations, the stiffness of the hybrid hydrogels was comparable to that of the peptide hydrogels in the absence of IK8L (Table 5), indicating that the presence of IK8L does not affect the mechanical strength of gels at lower concentrations. However, the G' value of IIK12 / IK8L was slightly lower than that of the IIK12 hydrogel with 1.0% IIK12. It is worth noting that when IIK12 was kept at a concentration of 1%, gelation of the hybrid hydrogel was observed even at 0.8% IK8L. This further proves that the introduction of IK8L does not significantly affect the gelation of IIK12. Similar to IIK12 hydrogels, IIK12 / IK8L hybrid hydrogels exhibit shear thinning and recovery rheological behaviors (Figure 10). When shear stress was applied, the viscosity of IIK12 / IK8L decreased rapidly, indicating that it was injectable. The rheological behavior of IIK12 / IK8L was further evaluated by dynamic step strain amplitude testing. At 0.5% strain, the G' value of IIK12 / IK8L was ~200 Pa. When 100% strain was applied to the gel, the stiffness decreased significantly. When the strain was reduced to 0.5%, the hybrid hydrogel quickly self-healed and recovered its initial stiffness. This property allows the hydrogel to be delivered via syringe without the gel strength being affected by the injection process. The reversibility of the rheological behavior is also very useful for topical applications.

[0152] Antimicrobial activity of IIK12 / IK8 hybrid hydrogels

[0153] The antimicrobial activity of the hybrid hydrogels against a representative panel of clinically relevant microorganisms, including Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and the yeast Candida albicans, was investigated. Common bacterial pathogens associated with DFU infection are reported to be Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and Pseudomonas aeruginosa. Staphylococcus aureus is the main pathogen, and Escherichia coli and Pseudomonas aeruginosa occur in approximately 10–20% of patients

[32] . Hybrid hydrogels were prepared with 1% IIK12. At the same time, the concentration of IK8L was varied to study its effect on the antimicrobial activity of the hybrid hydrogels. Each hydrogel surface was incubated with a cell density of 10 5CFU / mL of three pathogens were attacked. Microbial proliferation was evaluated by optical density (OD) measurement, and the live cells on the surface of the treated hydrogel were quantified by agar plate. As shown in Figure 11, the control gel IIK12 was ineffective in killing microorganisms, as shown in the OD measurement and agar plate results. The incorporation of the antimicrobial peptide IK8L imparted antimicrobial activity. Hybrid hydrogels containing 0.128% (1.28 mg / mL) or 0.256% (2.56 mg / mL) IK8L were able to kill Staphylococcus aureus, Escherichia coli and Candida albicans. Importantly, the hybrid hydrogels showed 100% lethality when in contact with Staphylococcus aureus, Escherichia coli and Candida albicans. Cationic antimicrobial peptides are released from the hydrogel and are attracted to and interact with the anionic cell membrane of the microorganism through electrostatic interactions. In the presence of microbial cell membranes, IK8L readily folds into a secondary β-sheet structure stabilized by electrostatic interactions and then inserts its hydrophobic residues into the lipid bilayer of the microorganism, leading to the physical disruption of the microbial cell membrane.

[0154] The ability of the hybrid hydrogel to kill Staphylococcus aureus was further investigated by analyzing live microorganisms in contact with IIK12 / IK8L at different exposure times. Fig.12 As shown, at 2.56 mg / mL of IK8L (i.e., 0.256%), S. aureus cells were completely killed after half an hour of exposure to the hybrid gel surface, indicating rapid killing kinetics. It is worth noting that the Log (CFU) values ​​for the control medium and control gel IIK12 after 24 hours of incubation were 8.76 and 9.28, respectively. However, no CFU was observed on the surface of the hybrid hydrogel, indicating that 100% of the microorganisms were killed after contact with the hybrid hydrogel. This finding further demonstrates that IK8L plays a key role in the effective antimicrobial effect of the hybrid hydrogel.

[0155] The antimicrobial activity of the peptides and peptide hydrogels in preventing microbial colony formation was further investigated using the disk diffusion assay technique. First, sterile filter disks containing different concentrations of IK8L were placed on agar plates inoculated with fresh Staphylococcus aureus. Fig.13A As shown, IK8L effectively inhibited S. aureus colony formation on agar plates, showing an inhibition zone around the filter disc containing the peptide. Notably, the inhibition zone area increased with increasing peptide concentration. The IIK12 / IK8L hybrid hydrogel was then cast on agar plates inoculated with S. aureus and incubated at 37°C for one day. Bacterial overgrowth was observed on the agar and surrounding areas in contact with the IIK12 gel, indicating its ineffectiveness in preventing S. aureus colony formation ( Fig. 13BIn contrast, in the case of IIK12 / IK8L, the bottom agar in contact with the gel remained transparent, demonstrating its ability to prevent S. aureus colony formation ( Fig. 13B ). The inhibition zone of the hybrid hydrogel was also observed. Since IK8D has stronger antimicrobial activity than IK8L, the inhibition zone of IIK12 / IK8D hybrid hydrogel is larger ( Fig. 13C ). These findings suggest that antimicrobial peptides can be released from the hybrid hydrogels.

[0156] In vitro biocompatibility of IIK12 / IK8 hybrid hydrogels

[0157] Hemolysis is one of the major side effects caused by many cationic peptides and polymers. The hemolytic behavior of the hybrid hydrogels was evaluated after incubation with rat erythrocytes. We previously found that IK8L and IK8D exhibited low hemolytic activity, with HC 10 The values ​​(the lowest concentrations that induce 10% or more hemolysis) were 2000 and 1750 μg / mL, respectively. Similar to previous findings on IK8L and IK8D, their corresponding hybrid hydrogels had little hemolytic effect on rat erythrocytes even at IK8D concentrations as high as 10.24 mg / mL (1.024%). Fig.14 ).

[0158] The in vitro biocompatibility of the peptide gel was further evaluated using human primary dermal fibroblasts (HDFs) and keratinocytes. We first evaluated the cytotoxicity of peptide IK8L in HDFs. Cell viability exceeded 85% ( Fig.15A However, when the concentration of IK8L was increased to 1000 μg / mL, the cell viability decreased to 22%. Notably, HDFs exposed to IIK12 / IK8L hydrogels showed more than 95% cell viability at 1000 μg / mL of IK8L, indicating that the hybrid hydrogels were not cytotoxic at the tested concentrations ( Fig. 15B Under the same test conditions, the in vitro biocompatibility of mupirocin and polymyxin B, which are commonly used in antimicrobial creams, was compared with IIK12 / IK8L ( Fig. 15C Mupirocin exhibited good biocompatibility for HDFs, as demonstrated by cell viability exceeding 80% even at 2000 μg / mL. Polymyxin B had similar cytotoxicity profiles as IK8L in HDFs. In the case of keratinocytes, IK8L exhibited dose-dependent cytotoxicity. When exposed to 125 μg / mL of IK8L, more than 70% of keratinocytes remained viable ( Fig.16A). At 125 μg / mL of IK8L, IIK12 / IK8L showed higher cell viability (close to 100%) than IK8L ( Fig. 16B ). The viability of keratinocytes after exposure to mupirocin was 80-90% ( Fig. 16C ).

[0159] Advantageously, the short peptides described herein self-assemble into hydrogels at lower concentrations than previously prepared hydrogels. In addition, the peptide hydrogels are biocompatible and non-cytotoxic at the concentrations tested and used. The peptide hydrogels can be used in a variety of biomedical applications, such as as scaffolds for 2D and / or 3D cell culture, cell delivery, and matrices for controlled release of therapeutic agents.

[0160] Although short peptide hydrogels alone have no antimicrobial activity, they can be combined with previously reported IK8L / IK8D to form hybrid hydrogels that combine the desirable properties of both peptides. In particular, the biocompatibility, lack of cytotoxicity, and reversibility of their rheological behavior (properties) of the peptide hydrogels are described herein. Thus, these new peptide hydrogels can be used as carriers to deliver therapeutic agents (e.g., drugs). On the other hand, the IK8L / IK8D peptides contribute to the antimicrobial activity while forming hybrid hydrogels at much lower concentrations (e.g., 8 (w / v)%) than previously reported, thereby alleviating the cytotoxicity issues when using IK8L / IK8D alone (e.g., 2 (w / v)%).

[0161] In particular, hybrid antimicrobial hydrogels (i.e., IIK12 / IK8L) are designed for the prevention and treatment of infection, wherein IIK12 serves as a drug carrier and IK8L serves as an antimicrobial agent. Advantageously, IIK12 forms a hydrogel at a relatively low concentration. In addition, it is biocompatible and non-cytotoxic. In addition, the IIK12 / IK8L hybrid hydrogel delivers IK8L to the DFU site to eliminate infection at an effective concentration, thereby killing microorganisms while minimizing cytotoxicity to mammalian cells and tissues. Similarly, the other hydrogels described herein are expected to similarly form hybrid hydrogels having similar therapeutic properties while minimizing cytotoxicity.

[0162] in conclusion

[0163] In this study, a series of short synthetic peptides with alternating hydrophobic residues (X) and hydrophilic residues (Y) were designed. The hydrophobic residues (X) were selected from Ile, Val and Leu, and the hydrophilic residues (Y) were selected from Arg, Lys, Glu and Asp. The peptide amphiphiles self-assembled into hydrogels with tunable mechanical strength, reversible rheological behavior and sustainable stability. Peptide hydrogels self-assembled from peptides with 8 amino acids have been shown to be biocompatible with a range of cells including hMSCs and show great potential for use as cell delivery vehicles to support cell proliferation. Peptide amphiphiles including 12 amino acids self-assembled into a β-sheet configuration and formed peptide hydrogels in aqueous solutions with low peptide concentrations (0.6% w / v). Antimicrobial peptides forming β-sheets were introduced into the hydrogels through electrostatic interactions to form hybrid antimicrobial peptide hydrogels. The hybrid hydrogels exhibited shear-thinning and recovery rheological behaviors. The hybrid hydrogels exhibited broad-spectrum antimicrobial activity against various clinically relevant microorganisms. In addition, they demonstrated in vitro biocompatibility. These antimicrobial hybrid peptide hydrogels show great potential in the prevention and treatment of bacterial and fungal infections, including DFU infections.

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[0204] surface

[0205] Table 1. Peptide design and molecular weight characterization.

[0206]

[0207]

[0208] The peptides of SEQ ID No. 6 to 16 are amidated at the C-terminus. The peptides of SEQ ID No. 19 to 26 are amidated at the C-terminus and N-acetylated at the N-terminus.

[0209] Table 2. Effects of different charge distributions and hydrophobicity on gel properties.

[0210]

[0211]

[0212] Table 3. G′ (storage modulus) and G″ (loss modulus) values ​​of peptide hydrogels self-assembled from IVK8, IIK8 and ILK8 at a frequency of 1 Hz.

[0213]

[0214]

[0215] Table 4. Stability of peptide hydrogels self-assembled by IVK8 in the presence of DMEM. Hydrogels were formed at 37°C. For entries with an initial peptide concentration of 1.0 (w / v)%, the final peptide concentrations were 0.8%, 0.7%, 0.6% and 0.5% for ratios of 80:20, 70:30, 60:40 and 50:50, respectively. The same applies to other initial concentrations.

[0216]

[0217]

[0218] Table 5. G′ (storage modulus) and G″ (loss modulus) values ​​of peptide hydrogels self-assembled from IIK12 at a frequency of 1 Hz.

[0219]

[0220] The hydrogels were incubated at 37 °C overnight.

[0221] Table 6. Gelation of antimicrobial peptide hybrid hydrogels in water.

[0222]

[0223]

[0224] Table 7. Gelation of antimicrobial peptide hybrid hydrogels.

[0225]

[0226] Final salt concentration in hydrogel: 150 mM

[0227] Table 8. G' (storage modulus) and G" (loss modulus) values ​​of peptide hydrogels self-assembled from antimicrobial peptide hydrogels at a frequency of 1 Hz.

[0228]

[0229]

[0230] The hydrogels were incubated at 37 °C overnight. Sequence Listing <110> Agency for Science, Technology and Research <120> A series of injectable hydrogels self-assembled from short peptides for various biomedical applications <130> 20200868 <150> SG 11201906759W <151> 2019-07-22 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> N-acetylated carboxamide <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (16) <223> Amidation <400> 2 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> N-acetylated carboxamide <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (16) <223> Amidation <400> 3 Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala 1 5 10 15 <210> 4 <211> 20 <212> PRT <213> Artificial sequence <220> <223> A central tetrapeptide II' type β-turn flanked by alternating valine (hydrophobic) and lysine (hydrophilic) residues <220> <221> SITE <222> (9)..(9) <223> Xaa (X) is D-valine <220> <221> TURN <222> (9)..(12) <223> Type II' β-turn <400> 4 Val Lys Val Lys Val Lys Val Lys Xaa Pro Pro Thr Val Lys Val Lys 1 5 10 15 Val Lys Val Lys 20 <210> 5 <211> 6 <212> PRT <213> Artificial sequence <220> <223> N-acetylated carboxamide <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (6) <223> Amidation <400> 5 Ile Leu Val Ala Gly Lys 1 5 <210> 6 <211> 4 <212> PRT <213> Artificial sequence <220> <223> 4 amino acid peptide <220> <221> MOD_RES <222> (4) <223> Amidation <400> 6 Ile Arg Val Glu 1 <210> 7 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 6 amino acid peptide <220> <221> MOD_RES <222> (6) <223> Amidation <400> 7 Ile Arg Val Glu Ile Lys 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IRK8 <220> <221> MOD_RES <222> (8) <223> Amidation <400> 8 Ile Arg Val Glu Ile Glu Val Lys 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IRE8 <220> <221> MOD_RES <222> (8) <223> Amidation <400> 9 Ile Arg Val Glu Ile Lys Val Glu 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IVK8 <220> <221> MOD_RES <222> (8) <223> Amidation <400> 10 Ile Glu Val Glu Ile Arg Val Lys 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IIK8 <220> <221> MOD_RES <222> (8) <223> Amidation <400> 11 Ile Glu Ile Glu Ile Arg Ile Lys 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> ILK8 <220> <221> MOD_RES <222> (8) <223> Amidation <400> 12 Ile Glu Leu Glu Ile Arg Leu Lys 1 5 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> IRV12 <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 13 Ile Arg Val Glu Ile Arg Val Glu Ile Arg Val Glu 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <220> <223> IEV12 <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 14 Ile Glu Val Glu Ile Glu Val Lys Ile Arg Val Lys 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220> <223> IVK12 <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 15 Ile Arg Val Lys Ile Glu Val Glu Ile Arg Val Lys 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> IIK12 <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 16 Ile Arg Ile Lys Ile Glu Ile Glu Ile Arg Ile Lys 1 5 10 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IK8L <400> 17 Ile Arg Ile Lys Ile Arg Ile Lys 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IK8D, all D-amino acids <220> <221> SITE <222> (1)..(8) <223> All D-amino acids <400> 18 Ile Arg Ile Lys Ile Arg Ile Lys 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Reverse IRE8 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (8) <223> Amidation <400> 19 Glu Val Lys Ile Glu Val Arg Ile 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Reverse IVK8 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (8) <223> Amidation <400> 20 Lys Val Arg Ile Glu Val Glu Ile 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial sequence <220> <223> Reverse IIK8 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (8) <223> Amidation <400> twenty one Lys Ile Arg Ile Glu Ile Glu Ile 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial sequence <220> <223> Reverse ILK8 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (8) <223> Amidation <400> twenty two Lys Leu Arg Ile Glu Leu Glu Ile 1 5 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> Reverse IRV12 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> twenty three Glu Val Arg Ile Glu Val Arg Ile Glu Val Arg Ile 1 5 10 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> Reverse IEV12 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> twenty four Lys Val Arg Ile Lys Val Glu Ile Glu Val Glu Ile 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Reverse IVK12 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 25 Lys Val Arg Ile Glu Val Glu Ile Lys Val Arg Ile 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Reverse IIK12 <220> <221> MOD_RES <222> (1) <223> Acetylation <220> <221> MOD_RES <222> (12)..(12) <223> Amidation <400> 26 Lys Ile Arg Ile Glu Ile Glu Ile Lys Ile Arg Ile 1 5 10

Claims

1. A peptide, the amino acid sequence of which is any one of the following: SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No.

16.

2. The peptide according to claim 1, wherein The amino acid sequence is any one of the following: SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No.

16.

3. A composition comprising a hydrogel formed from a plurality of peptides according to claim 1 or 2 in a β-sheet configuration and water, or a dried form of the hydrogel.

4. The composition according to claim 3, wherein The concentration of the plurality of peptides is at least 0.6% w / v.

5. The composition according to claim 4, wherein The concentration is up to 5% w / v.

6. The composition of claim 3, comprising a salt selected from the group consisting of PBS, DMEM, MEM, potassium salts and sodium salts.

7. The composition according to claim 3, wherein The peptide is shown as SEQ ID No. 12, SEQ ID No. 15 or SEQ ID No.

16.

8. The composition of claim 3, comprising a therapeutic agent.

9. The composition according to claim 3, comprising the second peptide of SEQ ID No. 17 or SEQ ID No.

18.

10. Use of the composition according to any one of claims 3 to 9 in the preparation of a medicament for treating bacterial infection and / or fungal infection.

11. A method for forming a hydrogel, the method comprising mixing the peptide according to claim 1 or 2 in water to form a hydrogel; and isolating the hydrogel.

12. The method according to claim 11, wherein: The mixing step is performed in the presence of a salt, or wherein the mixing step is performed at a temperature of 20°C to 40°C, or the method further comprises drying the hydrogel to form a dried hydrogel suitable for reconstitution into the hydrogel.

13. An in vitro method for growing cells, the method comprising providing a mixture of a peptide according to claim 1 or 2, a cell culture medium and a cell population to form a gel containing the cell population; and incubating the gel under suitable conditions to grow the cell population, wherein the cells are selected from any one of the following: healthy cells, stem cells for treating or growing tissues, and cancer cells for growing tumors for in vitro and in vivo studies.

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