Compositions and methods of use thereof

Abstract

The present invention relates to compositions and methods of promoting wound healing, inhibiting or eradicating infection, and avoiding the pain associated with conventional wound dressing and treatment.

Description

COMPOSITIONS AND METHODS OF USE THEREOF

[0001] The application claims priority from U.S. Provisional Application No. 63 / 617,303 filed on January 03, 2024, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.GOVERNMENT INTERESTS

[0004] This invention was made with government support under R21 AG060430, R01 GM136874, and R21 AG066119 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0005] The present invention relates to compositions and methods of promoting wound healing and tissue-regeneration.BACKGROUND OF THE INVENTION

[0006] An important factor for the rapid healing of wounds is the maintenance of a moist environment, as this can facilitate high oxygen permeation and wound exudate removal. Therefore, therapeutics should promote these processes while also supporting proliferation and tissue remodeling. Naturally occurring polymers, such as chitosan, alginate, elastin, cellulose, fibrin, hyaluronic acid, pectin, dextran, and collagen, are biopolymers that are generally used in wound dressings. However, these natural polymers have poorer stability, structural heterogeneity, and mechanical properties than their synthetic counterparts, which also possessother merits, such as ease of application, appropriate firmness and elasticity, and superior water retention capacity.SUMMARY OF THE INVENTION

[0007] Aspects of the invention are drawn towards a compound according to Formula (I) or Formula (II):(Formula I); wherein Ri is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, H, or R; aas is selected from the group consisting of D, E, L, H, or R; and R4 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic group, or a hydroxylated derivative thereof; or(Formula II); wherein Ri is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, H, or R; aas is selected from the group consisting of D, E, L, H, or R; aai is selected from the group consisting of A, V, I, L, M, F, Y, or W; and R2 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic, or a hydroxylated derivative thereof.

[0008] The compound of claim 1, wherein the R2-C(O)- group or R4-C(O)- group are selected from the group consisting of 10-Hydroxy decan oyl, 3-Hydroxydecanoyl, 10-Hydroxydec-2- enoyl, 12-Hydroxydodecanoyl, 12-Hydroxy octadecenoyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, docosahexaenoyl, docosahexaenoyl, docosapentaenoyl, prostaglandin El, eicosapentaenoyl, arachidonoyl, linolenoyl, linoleicoyl, octadecenoyl, hexacosanoyl, a C4-40 saturated fatty carbonyl, or a hydroxylated derivative thereof.

[0009] In embodiments, the compound is:C12-ILD-0H;C8-LLD-NH2;-hydroxyl dodecanoic acid-LD-NIb;-hydroxyl octadecanoic acid-ID-NH2;-hydroxyl octadecanoic acid-LD-NH2;-hydroxyl octadecanoic acid-IK-NH2;-hydroxyl octadecanoic acid-LK-NH2;12-hydroxyl octadecanoic acid-IK-NEE; or12-hydroxyl octadecanoic acid-LK-NH2.

[0010] Aspects of the disclosure are drawn towards a hydrogel comprising one or more compounds described herein. In embodiments, the hydrogel further comprising a buffer or cell culture media. In embodiments, the buffer is selected from the group consisting of a saline buffer, a phosphate buffer, PBS (Phosphate Buffered Saline), DPBS (Dulbecco's Phosphate Buffered Saline), HBSS (Hank's Balanced Salt Solution), or any combination thereof. In embodiments, the cell culture media is selected from the group consisting of MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI-1640, IMDM (Iscove's Modified Dulbecco's Medium), or any combination thereof. In embodiments, the saline buffer comprises a phosphate-buffered saline solution.

[0011] In embodiments, the has a viscoelasticity of about 0.1-15 kPa. In embodiments, comprises about 0.05 % w / v to about 10% w / v of one or more compounds described herein. In embodiments, the hydrogel comprises a pH of about 2 to about 10.5. In embodiments, the hydrogel comprises a swelling ratio of about 100% to about 400%. In embodiments, the hydrogel further comprises an additional active agent. In embodiments the additional active agent comprises an antiviral agent, an antimicrobial agent, an analgesic agent, an antiinflammatory agent, or any combination thereof.

[0012] Aspects of the disclosure are drawn towards a method of promoting wound healing and / or tissue regeneration, the method comprising administering the hydrogel described herein to a wound. In embodiments, the administration comprises topical administration or parenteral administration. In embodiments, the wound comprises a bum wound, an infected wound, apressure ulcer, a diabetic ulcer, a venous ulcer, an arterial ulcer, or any combination thereof. In embodiments, they hydrogel increases the healing rate of the wound by about 1.5 times to about 20 times.

[0013] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 shows a non-limiting, exemplary schematic of general Fmoc / tBu-based solid-phase peptide synthesis (SPPS). a) For peptides with a C-terminal amide (-NH2), Rink amide resin was used (for compounds 1-4 and 9-15). b) For peptides with a C-terminal acid (- OH), Wang resin was used (for compounds 5-8).

[0015] FIG. 2 shows a non-limiting, exemplary NMR spectra of caprylic acid-isoleucine- leucine-aspartic acid amide conjugate [C8 acid-ILD-NH2(2)]. a)13C NMR (100 MHz), b) 'H NMR (400 MHz), c)13C DEPT-135 NMR (100 MHz). Data were acquired for purified (2) (30 mg) dissolved in 0.7 mL DMSO-d6.

[0016] FIG. 3 shows non-limiting, exemplary LC-MS / MS of caprylic acid-isoleucine- leucine-aspartic acid amide conjugate [C8 acid-ILD-NH2(2)]. a) Deprotonated molecular structure of C8 acid-ILD-NH2(2) and interpretative illustration of its MS / MS fragment ions, b) A typical CIS-reversed phase chromatogram from LC-MS / MS analysis of (2). c) LC- MS / MS spectrum of (2) and interpretation of its MS / MS fragment ions (right). The LC-MS / MS system consisted of an Agilent 1100 LC and QTRAP 6500+quadruple-linear trap mass spectrophotometer with electrospray ionization.

[0017] FIG. 4 shows a non-limiting, exemplary data comprising olecular structure, gelable concentration, and pH-related stability of the hydrogel self-assembled from C8-ILD-NH2(2). (a) Molecular structure of (2). (b) Swelling ratio at 1.5% and 3% (w / v). (c) Gelation at different concentrations of (2) (w / v) in PBS at pH 7.4: 0.05%, no gelation; 0.1%, no gelation; 0.25%, gelated; 0.5%, gelated; 1.0%, gelated. (d) Photos showing the gel stability of C8-ILD-NH2(2) (1.5% w / v) at different pH: pH 2-8, gelated and stable; pH 10, no gelation. Results are presented as mean ± SD (n = 3) with *p < 0.05, **p < 0.01. NS: no significant difference.

[0018] FIG. 5 shows a non-limiting, exemplary data. The injectable and rheological properties of C8-ILD-NH2(2) hydrogel, (a) Photo showing the injectable nature: injection by syringe. The hydrogel re-gelated quickly along the vial neck after injection, (b) A zoom-inphoto of the re-gelated hydrogel after injection, (c) Shear strain amplitude sweep experiment of (2) with constant frequency of 10 rads / s. Arrow marks the flow point where G’ and G” crosses over, (d) Frequency sweep experiment with a constant shear strain of 1%. (e) Timesweep curing experiment with a constant strain 10% and frequency 1 Hz; Arrow marks the crossover point of G’ and G”. (f) Thixotropic test with imposition of a gel-endurable shear strain of 0.1% and a gel-breaking shear strain of 200%. The rheological tests were conducted using (2) at 3.0% (w / v) in PBS at pH 7.4 and 37°C.

[0019] FIG. 6 shows a non-limiting, exemplary data. Surface structures of the fibrous networks from freeze-dried C8-ILD-NH2(2) hydrogel were observed using field emission scanning electron microscopy at different magnifications. Secondary electrons (SE) were detected using an upper detector (U), with a working distance of 2.2 mm and acceleration voltage of -3.0 kV. a) Image at * 10,000 b) Image at *35,000.

[0020] FIG. 7 shows a non-limiting, exemplary data. C8-ILD-NH2(2) hydrogel accelerated the healing of third-degree burn wounds in mice, a) Left, typical photographs of wounds. The thin transparent ruler in the focal plate was used as the scale bar. Right, hydrogel formed by (2) promoted wound closure, b) Hydrogel formed by (2) accelerated epithelialization. Left, representative micro-images of hematoxylin / eosin-stained wound sections; right, epithelial gaps. Results are presented as mean ± SEM with *p < 0.05, **p < 0.01 (t test).

[0021] FIG. 8 shows a non-limiting, exemplary schematic of embodiments described herein.

[0022] FIG. 9 shows a non-limiting, exemplary *H NMR of compound 6 in DMSO-d6.

[0023] FIG. 10 shows a non-limiting, exemplary13C NMR of compound 6 in DMSO-d6.

[0024] FIG. 11 shows a non-limiting, exemplary13C DEPT NMR of compound 6 in DMSO-d6.

[0025] FIG. 12 shows a non-limiting, exemplary13C NMR of compound 7 in DMSO-d6.

[0026] FIG. 13 shows a non-limiting, exemplary13C NMR of compound 7 in DMSO-d6.

[0027] FIG. 14 shows a non-limiting, exemplary13C DEPT NMR of compound 7 in DMSO-d6.

[0028] FIG. 15 shows a non-limiting, exemplary1H NMR of compound 8 in DMSO-d6.

[0029] FIG. 16 shows a non-limiting, exemplary13C NMR of compound 8 in DMSO-d6.

[0030] FIG. 17 shows a non-limiting, exemplary13C DEPT NMR of compound 8 in DMSO-d6.

[0031] FIG. 18 shows a non-limiting, exemplary1HNMR of compound 9 in DMSO-d6.

[0032] FIG. 19 shows a non-limiting, exemplary13C NMR of compound 9 in DMSO-d6.

[0033] FIG. 20 shows a non-limiting, exemplary13C DEPT NMR of compound 9 in DMSO-d6.

[0034] FIG. 21 shows a non-limiting, exemplary1HNMR of compound 10 in DMSO-d6.

[0035] FIG. 22 shows a non-limiting, exemplary13C NMR of compound 10 in DMSO-d6.

[0036] FIG. 23 shows a non-limiting, exemplary13C DEPT NMR of compound 10 in DMSO-d6.

[0037] FIG. 24 shows a non-limiting, exemplary1HNMR of compound 11 in DMSO-d6.

[0038] FIG. 25 shows a non-limiting, exemplary13C NMR of compound 11 in DMSO-d6.

[0039] FIG. 26 shows a non -limiting, exemplary13C DEPT NMR of compound 11 in DMSO-d6.

[0040] FIG. 27 shows a non-limiting, exemplary1HNMR of compound 12 in DMSO-d6.

[0041] FIG. 28 shows a non-limiting, exemplary13C NMR of compound 12 in DMSO-d6.

[0042] FIG. 29 shows a non-limiting, exemplary13C DEPT NMR of compound 12 (PH91) in DMSO-d6.

[0043] FIG. 30 shows a non-limiting, exemplary1H NMR of compound 13 in DMSO-d6.

[0044] FIG. 31 shows a non-limiting, exemplary13C NMR of compound 13 in DMSO-d6.

[0045] FIG. 32 shows a non-limiting, exemplary13C DEPT NMR of compound 13 in DMSO-d6.

[0046] FIG. 33 shows a non-limiting, exemplary *H NMR of compound 14 in DMSO-d6.

[0047] FIG. 34 shows a non-limiting, exemplary13C NMR of compound 14 in DMSO-d6.

[0048] FIG. 35 shows a non -limiting, exemplary13C DEPT NMR of compound 14 in DMSO-d6.

[0049] FIG. 36 shows a non-limiting, exemplary schematic of the general strategy for Fmoc / tBu-based solid-phase peptide synthesis (SPPS) of a new amphiphiles from its moiety via peptide bond linkage under the molecular sequence template as hydroxyl fatty acid-amino acid 1 -amino acid 2-amino acid 3 (hfa-aal-aa2-aa3).

[0050] FIG. 37 [fig 1] shows a non-limiting, exemplary data. The molecular structures of typical hfa-aal-aa2-aa3 amphiphiles, hda-ILD and hdaa-ILD, made via SPPS organic synthesis were confirmed by LC-MS / MS analysis. A) Queen bee acid (hda) was covalently bonded to tri-peptide isoleucine (I)-leucine (L)-aspartic acid (D) (ILD), forming hda-ILD amphiphilic molecule. It was analyzed by Cl 8 LC-MS / MS as we did previously for caprylic- peptide conjugates81. B) LC-MS / MS chromatogram of hda-ILD. C) LC-MS / MS spectrum of hda-ILD. D) The hdaa bonded to ILD forming hdaa-ILD amphiphilic molecule. E) LC-MS / MS chromatogram of hdaa-ILD. F) LC-MS / MS spectrum of hdaa. Sciex Qtrap 6500+MS / MS was used.

[0051] FIG. 38 shows non-limiting, exemplary data of new fatty acid-peptide covalently bonded conjugates described herein. Notes (a) in the structure column C8, C12, C14, and C16 denote the corresponding carbon chain length; letters I, L, D, A, and V denote amino acids; I - isoleucine, L - leucine, D - aspartic acid, A - alanine, V - valine; -NH2 : C-terminus amide group; and -OH: C-terminus carboxylic acid group; (b) MS / MS ion acquired by LC MS / MS, single charged (z = 1, m / z = Daltons); (c) conjugate: 1.5 % w / w in PBS saline, pH 6.5-7.8.

[0052] FIG. 39 shows a non-limiting, exemplary data described herein. Hydrogels formed from compound 1 and 3 are stable in PBS at pH 2 to 10 and 2 to 8, respectively. (A) Photos showing that compound 1 gelated and remained gelated in PBS at pH 2 (left) and 10 (right). (B) Photos showing that compound 3 gelated and remained gelated in PBS at pH 2 (left) and 8 (middle). The hydrogel was generated from compound 1 or 3 at 1.5% w / v in PBS, and transformed to solution phase at pH 11.5 or 10, respectively.

[0053] FIG. 40 shows a non-limiting, exemplary experimental data. Gelation and rheological and injectable tests of hda-ILD-NH2 (Compound 3) and hdaa-ILD-NH2 (Compound 1) gels. For hda-ILD-NFt (3) gels: A) Shear strain amplitude sweep test with a constant frequency of 10 rads / s; B) Frequency sweep test; C) Thixotropic test; D) Photos: vial- inversion test for gelation (upper) and injectability test (lower). For hdaa-ILD-NFt (1) gels: E) Shear strain amplitude sweep test with a constant frequency of 10 rads / s; F) Frequency sweep test with a constant shear strain of 1%.; G) Thixotropic test with imposition of a gel-endurable shear strain of 0.1% and a gel-breaking shear strain of 200%; H) Photo of vial-inversion test for gelation. Gels were self-assembled from hda-ILD-NFC (3) or hdaa-ILD-NFF (1) at 3% (w / v) in PBS at pH 7.4 at 23°C, and then tested on an Anton-Paar MCR 092 model rheometer or injected from a syringe to surface at 37°C.

[0054] FIG. 41 shows a non-limiting, exemplary images. Fibrous structures of hda-ILD- NH2 (3) was determined using transmission electron microscopy (TEM). The TEM image was at x 60000 magnification.

[0055] FIG. 42 shows a non-limiting, exemplary data described herein. The amphiphile hdaa-ILK-NH2 (6) modified from hdaa with tripeptide ILK-NH2 by peptide bonding inhibited S. aureus in Kirby-Bauer disc diffusion assay. S. aureus was inoculated and 5 mg hdaa-ILK- NH2 (6) was added to the well. The inhibition zone of S. aureus was photographed and measured to be 25 mm in diameter.DETAILED DESCRIPTION OF THE INVENTION

[0056] Aspects described herein provide for compositions and methods of promoting wound healing and tissue-regeneration.

[0057] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0058] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with theterm “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0059] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0060] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0061] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0062] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower). In embodiments, the term “about” can be denoted

[0063] As used herein, the term “substantially the same” or “substantially” can refer to variability typical for a particular method is taken into account.

[0064] The terms “sufficient” and “effective”, as used interchangeably herein, can refer to an amount (e.g., mass, volume, dosage, concentration, and / or time period) needed to achieve one or more desired result(s).

[0065] Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The disclosure can be used for other embodiments or of being practiced or carried out in various ways. Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent toone having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages can be included within this description, and be within the scope of the disclosure.

[0066] Aspects of the invention are drawn towards a compound according to Formula (I) or Formula (II):(Formula I); whereinRi is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, H, or R; aas is selected from the group consisting of D, E, L, H, or R; andR4 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic group, or a hydroxylated derivative thereof; or(Formula II); whereinRi is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, H, or R; aa3 is selected from the group consisting of D, E, L, H, or R; aai is selected from the group consisting of A, V, I, L, M, F, Y, or W; and R2 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic, or a hydroxylated derivative thereof.

[0067] As used herein, the amino acids described herein can be referred to by their single letter abbreviation or three letter abbreviation as known by one of ordinary skill in the art. For example, “A” can refer to alanine, “R” can refer to arginine, “N” can refer to asparagine, “D” can refer to aspartic acid, “C” can refer to cysteine, “Q” can refer to glutamine, “E” can refer to glutamic acid, “G” can refer to glycine, “H” can refer to histidine, “I” can refer to isoleucine, “L” can refer to leucine, “K” can refer to lysine, “M” can refer to methionine, “F” can refer tophenylalanine, “P” can refer to proline, “S” can refer to serine, “T” can refer to threonine, “W” can refer to tryptophan, “ Y” can refer to tyrosine, or “V” can refer to valine.

[0068] In embodiments, the hydrogel described herein can have a viscoelasticity of about 0.1- 15 kPa. For example, the hydrogel can have a viscoelasticity of less than 0.1 kPa, about 0.1 kPa, about 0.2 kPa, about 0.3 kPa, about 0.4 kPa, about 0.5 kPa, about 0.6 kPa, about 0.7 kPa, about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3.0 kPa, about 3.5 kPa, about 4.0 kPa, about 4.5 kPa, about 5.0 kPa, about 5.5 kPa, about 6.0 kPa, about 6.5 kPa, about 7.0 kPa, about 7.5 kPa, about 8.0 kPa, about 8.5 kPa, about 9.0 kPa, about 9.5 kPa, about 10.0 kPa, about 10.5 kPa, about 11.0 kPa, about 11.5 kPa, about 12.0 kPa, about 12.5 kPa, 13.0 kPa, 13.5 kPa, about 14.0 kPa, about 14.5 kPa, about 15.0 kPa, or greater than about 15.0 kPa.

[0069] In embodiments, the hydrogel can be at a pH of about 2 to about 10.5. For example, the hydrogel can be less than about 2, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or greater than about 12.0.

[0070] As used herein, the term “swelling ratio” can refer to the water intake capacity of the hydrogels and is calculated as the percentage of water intake weight relative to the initial weight of hydrogel. Hydrogel comprises a swelling ratio of about 100% to about 400%. For example, the swelling ratio can be less than about 25%, about 25%, about 50%, about 75%, about 100%, about 125%, about 150%, about 175%, about 200%, about 225%, about 250%, about 275%, about 300%, about 325%, about 350%, about 375%, about 400%, about 425%, about 450%, about 475%, about 500%, or greater than about 500%.

[0071] In embodiments, the R2-C(O)- group or R4-C(O)- group are selected from the group consisting of 10-Hydroxydecanoyl, 3 -Hydroxy decanoyl, 10-Hydroxydec-2-enoyl, 12- Hydroxydodecanoyl, 12-Hydroxy octadecenoyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, docosahexaenoyl, docosahexaenoyl, docosapentaenoyl, prostaglandin El, eicosapentaenoyl, arachidonoyl, linolenoyl, linoleicoyl, octadecenoyl, hexacosanoyl, a C4-40 saturated fatty carbonyl, or a hydroxylated derivative thereof. For example, the fatty acid can be any fatty acid known in the art.

[0072] In embodiments, the compound is:C8-ILD-0H;C8-IVD-NH2;C8-LID-NH2;2-hydroxyl dodecanoic acid-ID-NIb;-hydroxyl dodecanoic acid-LD-NIb;-hydroxyl octadecanoic acid-ID-Nhh;-hydroxyl octadecanoic acid-LD-Nhh;2-hydroxyl octadecanoic acid-IK-NIb;-hydroxyl octadecanoic acid-LK-NH2;-hydroxyl octadecanoic acid-IK-NH2; or12-hydroxyl octadecanoic acid-LK-NH2.

[0073] In embodiments, the compounds can comprise a mono-peptide compound. In embodiments, the mono-amino-acid containing compound of the following structure:H, wherein R2-C(O)- group is selected from the group consisting of: 10- Hydroxydecanoyl, 3-Hydroxydecanoyl; 10-Hydroxydec-2-enoyl; 12-Hydroxydodecanoyl; 12- Hydroxy octadecenoyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, docosahexaenoyl, docosahexaenoyl, docosapentaenoyl, prostaglandin El, eicosapentaenoyl, arachidonoyl, linolenoyl, linoleicoyl, octadecenoyl, any other hydroxylated fatty acid moiety, or any other fatty carbonyl (Cn-, n = 4-40); wherein aal (animo acid 1): A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, or R; and wherein Ri: -NH2or -OH.

[0074] Aspects of the disclosure are drawn towards a hydrogel comprising one or more compounds described herein. In embodiments, the gel comprises about 0.05 % w / v to about 10% w / v of one or more compounds described herein. For example, the compounds described herein can be present in about less than 0.1 % w / v, about 0.1 % w / v, 0.25 % w / v, about 0.5 % w / v, about 0.75 % w / v, about 1.0 % w / v, about 1.25 % w / v, about 1.5 % w / v, about 1.75 % w / v, about 2.0 % w / v, about 2.5 % w / v, about 3.0 % w / v, about 3.5 % w / v, about 4.0 % w / v, about 4.5 % w / v, about 5.0 % w / v, about 5.5 % w / v, about 6.0 % w / v, about 6.5 % w / v, about 7.0 % w / v, about 7.5 % w / v, about 8.0 % w / v, about 8.5 % w / v, about 9.0 % w / v, about 9.5 % w / v, about 10.0 % w / v, about 11.0 % w / v, about 12.0 % w / v, about 13.0 % w / v, about 14.0 % w / v, about 15.0 % w / v, about 16.0 % w / v, about 17.0 % w / v, about 18.0 % w / v, about 19.0 % w / v, about 20.0 % w / v, or greater than about 20.0 % w / v.

[0075] In embodiments, the hydrogel can comprise a saline, buffer or cell culture media known in the art. For example, the saline can be a sodium chloride saline. For example the buffer can be a saline buffer, a phosphate buffer, PBS (Phosphate Buffered Saline), DPBS (Dulbecco'sPhosphate Buffered Saline), HBSS (Hank's Balanced Salt Solution), or any combination thereof. For example, the cell culture media can be MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI-1640, IMDM (Iscove's Modified Dulbecco's Medium), or any combination thereof. In embodiments, the saline buffer comprises a phosphate-buffered saline solution.

[0076] In embodiments, the hydrogel can further comprise an additional active agent. As used herein, the term “additional active agent” can refer to an additional substance that functions alongside any one or more of the compounds described herein. For example, the additional active agent can be an antiviral agent (such as Oseltamivir for influenza, Remdesivir for COVD-19, and Acyclovir for herpesviruses) an antimicrobial agent (such as antibiotics, antifungals, antiseptics), an analgesic agent (such buprenorphine), an anti-inflammatory agent such as Nonsteroidal Anti-Inflammatory Drugs, Corticosteroids, and / or Methotrexate), or any combination thereof. However, the antiviral agent, the antimicrobial agent, the analgesic agent, or anti-inflammatory agent can be any known in the art.

[0077] Aspects of the disclosure are drawn towards a method of promoting wound healing or tissue regeneration, the method comprising administering the hydrogel described herein to a wound. For example, the hydrogels can be applied topically to wounds or skin by coating with swabs or gauze, dropping with pipettes or syringes, spraying with sprayers, or injected into skin or wounds with needled-syringes. As used herein, the term “promoting wound healing” can refer to accelerate reepithelization, vascularization, collagen deposition, and wound closure, as well as increase wound breaking strength. As used herein, the term “tissue regeneration” can refer to regrow cells to restore the lost tissue caused by wounding or diseases.

[0078] In embodiments, the administration can comprise topical administration or parenteral administration. For example, the compounds and / or hydrogels described herein can be applied topically to wounds or skin by coating with swabs or gauze, dropping with pipettes or syringes, spraying with sprayers. In embodiments, the compounds and / or hydrogels described herein can be injected into skin or wounds with needled-syringes.

[0079] As used herein, the term “wound” can refer to an injury to the body. For example, the wound can involve a break, damage, or destruction of the tissue and / or cells in the skin or other tissues. For example, the wound can be caused by accidents, diseases, and / or surgery. In embodiments, the wound can comprise a burn wound, an infected wound, a pressure ulcer, a diabetic ulcer, a venous ulcer, an arterial ulcer, surgical wounds, abrasions, crush injuries, or any combination thereof. In embodiments, the compounds or hydrogels described herein canincrease the healing rate of the wound by about 1.5 times to about 20 times. For example, the wound healing rate can be increased by about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.25 times, about 2.5 times, about 2.75 times, about 3.0 times, about3.25 times, about 3.5 times, about 3.75 times, about 4.0 times, about 4.25 times, about 4.5 times, about 4.75 times, about 5.0 times, about 5.25 times, about 5.5 times, about 5.75 times, about 6.0 times, about 6.25 times, about 6.5 times, about 6.75 times, about 7.0 times, about7.25 times, about 7.5 times, about 7.75 times, about 8.0 times, about 8.5 times, about 9.0 times, about 9.5 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 20 times, about 25 times, or greater than about 25 times. For example, in some embodiments, the compounds and / or hydrogels described herein can increase the rate of healing about 6.3 times at about 7 days post-burn. For example, the compounds and hydrogels described herein can increase the rate of healing about 1.2 times at about 14 days post-bum.

[0080] The term "alkyl" refers to the radical of saturated aliphatic groups, including straightchain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkylsubstituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0081] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims can include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0082] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein can refer to an alkyl group, as defined herein, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl"have similar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent described herein as alkyl can be a lower alkyl.

[0083] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl can include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.

[0084] The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined herein for alkyl groups.

[0085] The term "alkylthio" refers to an alkyl group, as defined herein, having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, and ethylthio. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined herein for alkyl groups.

[0086] The terms "alkenyl" and "alkynyl", refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but that contain at least one double or triple bond respectively. For example,

[0087] The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined herein, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether," for example, can be two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O- alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described herein for alkyl.

[0088] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:wherein R9, Rio, and Rio’ each independently represent a hydrogen, an alkyl, an alkenyl, - (CH2)m- Rs or R9 and Rio taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rs represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9 or Rio can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide. In still other embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9 and Rio represents a carbonyl. In additional embodiments, R9 and Rio (and optionally Rio ) each independently represent a hydrogen, an alkyl or cycloalkyl, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein can refer to an amine group, as defined herein, having a substituted (as described hereinfor alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9 and Rio is an alkyl group.

[0089] As used herein, the term “imide” can refer to -C(O)NR’R”, wherein R’ and R” are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclyl alkyl group as defined herein.

[0090] As used herein, the term “halogen” can refer to -F, -Cl, -Br or -I; the term "sulfhydryl" can refer to -SH; the term "hydroxyl" can refer to -OH; and the term "sulfonyl" can refer to -SO2-.

[0091] The term “substituted” as used herein, refers to permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups. As used herein in reference to an “R” group, the name used to describe said “R” group can be the chemical name prior to the removal of a hydrogen. For example, wherein “R” is described as an “alkane” can refer to an “alkyl” group.

[0092] Heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0093] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. The heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.

[0094] In various aspects, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl,ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.

[0095] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carb oxami doaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0096] Aspects of the invention are drawn towards a pharmaceutical composition comprising a compound described herein or combination of compounds, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0097] Pharmaceutically acceptable esters can include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.

[0098] Pharmaceutically acceptable enol ethers can include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocyclyl. Pharmaceutically acceptable enol esters can include, but are not limited to, derivatives of formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl ar heterocyclyl.

[0099] Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

[0100] A reference to a compound of the disclosure and sub-groups thereof also includes ionic forms, salts, solvates, isomers, tautomers, esters, prodrugs, isotopes and protected forms thereof; such as, the salts or tautomers or isomers or solvates thereof; and more advantageously, the salts or tautomers or solvates thereof. As used herein, the term “isomer” can refer to molecules or polyamtoic ions with identical molecular formulas, but distinct arrangements of atoms in space. For example, constitutional isomers and stereoisomers of compounds describedherein are also embodiments of the invention. For example, the enantiomers and diastereomers of compounds described herein can be aspects of the invention. For example, if (R ,S) of a compound is described herein, (R, R), (S, R), and (S, S) can also be aspects of the invention. As used herein, the term “enantiomer” can refer to molecules which are nonsuperimposable mirror images of each other. As used herein, the term “diastereomer” can refer to a stereoisomer of a compound having two or more chiral centers that is not a mirror image of another stereoisomer of the same compound.

[0101] While the compounds described herein can be administered without formulation, they can also be formulated as pharmaceutical compositions. Aspects of the invention are drawn towards a pharmaceutical composition comprising a compound of any one of the compounds described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0102] The formulations or pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and / or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like. For example, the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the present invention are those which render the compounds of the invention amenable to intranasal delivery, oral delivery, parenteral delivery, intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery and the like. Moreover, the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition / formulation packaged and / or delivered therewith.

[0103] In embodiments, compounds and compositions described herein can be administered via oral administration. In some embodiments, the disclosed compositions are formulated in a pharmaceutically acceptable oral dosage form. Oral dosage forms can comprise oral liquid dosage forms (such as tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like) and oral solid dosage forms. The pharmaceutical compositions can also be prepared as formulations suitable for parenteral administration, intramuscular, subcutaneous, intraperitoneal, or intravenous injection, comprising physiologically acceptable sterile aqueousor non-aqueous solutions, dispersions, suspensions or emulsions, liposomes, and sterile powders for reconstitution into sterile injectable solutions or dispersions.

[0104] “Parenteral administration” can refer to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration. For example, compounds and compositions described herein can be administered via intraperitoneal injection (I.P.), intravenous injection (I.V.), and intramuscular injection (I.M.).

[0105] For oral preparations, the composition or pharmaceutical composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0106] Oral solid dosage can comprise lozenges, troches, tablets, capsules, caplets, powders, pellets, multiparticulates, beads, spheres, and / or any combinations thereof. Oral solid dosage forms can be formulated as immediate release, controlled release, sustained release, extended release, or modified release formulations. Accordingly, in some embodiments, the disclosed oral solid dosage forms can be in the form of a tablet (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation can be in the form of a powder. In still other embodiments, the pharmaceutical formulation can be in the form of a tablet, including a fast-melt tablet. Additionally, pharmaceutical formulations can be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation can be administered in two, three, four, or more capsules or tablets.

[0107] Oral solid dosage forms can contain pharmaceutically acceptable excipients such as fillers, diluents, lubricants, surfactants, glidants, binders, dispersing agents, suspending agents, disintegrants, viscosity-increasing agents, film-forming agents, granulation aid, flavoringagents, sweetener, coating agents, solubilizing agents, and combinations thereof. Oral solid dosage forms also can comprise one or more pharmaceutically acceptable additives such as a compatible carrier, complexing agent, ionic dispersion modulator, disintegrating agent, surfactant, lubricant, colorant, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, alone or in combination, as well as supplementary active compound(s).

[0108] Oral liquid dosage forms include tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like. These oral liquid dosage forms may be formulated with any pharmaceutically acceptable excipient known to those of skill in the art for the preparation of liquid dosage forms, and with solvents, diluents, carriers, excipients, and the like chosen as appropriate to the solubility and other properties of the active agents and other ingredients. Solvents may be, for example, water, glycerin, simple syrup, alcohol, medium chain triglycerides (MCT), and combinations thereof.

[0109] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. Pharmaceutical formulations can be prepared as liquid suspensions or solutions using a sterile liquid, such as but not limited to, an oil, water, an alcohol, and combinations of these pharmaceutically suitable surfactants, suspending agents, emulsifying agents, can be added for oral or parenteral administration. Liquid formulations also may be prepared as single dose or multi-dose beverages. Suspensions may include oils. Such oils include peanut oil, sesame oil, cottonseed oil, com oil, and olive oil. Suitable oils also include carrier oils such as MCT and long chain triglyceride (LCT) oils. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Suspension formulations may include alcohols, (such as ethanol, isopropyl alcohol, hexadecyl alcohol), glycerol, and propylene glycol. Ethers, such as polyethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water may also be used in suspension formulations. Suspension can thus include an aqueous liquid or a non-aqueous liquid, an oil-in-water liquid emulsion, or a water- in-oil emulsion.

[0110] In some embodiments, formulations are provided comprising the disclosed compositions and at least one dispersing agent or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained. The aqueous dispersion cancomprise amorphous and non-amorphous particles consisting of multiple effective particle sizes such that a drug is absorbed in a controlled manner over time.

[0111] Supplementary active compounds include preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents. Preservatives can be used to inhibit microbial growth or increase stability of the active ingredient thereby prolonging the shelf life of the formulation. Suitable preservatives are known in the art and include EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include vitamin A, vitamin C (ascorbic acid), vitamin E, tocopherols, other vitamins or provitamins, and compounds such as alpha lipoic acid.

[0112] Unit dosage forms for oral administration, such as syrups, elixirs, and suspensions, can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration can comprise the composition or pharmaceutical composition in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[0113] Embodiments of the composition or pharmaceutical composition can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0114] Embodiments of the composition or pharmaceutical composition can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the composition or pharmaceutical composition can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0115] Embodiments of the composition or pharmaceutical composition can be formulated in an injectable composition in accordance with the disclosure. For example, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation can also be emulsified or the active ingredient (triamino-pyridine derivative and / or the labeled triamino-pyridine derivative) encapsulated in liposome vehicles in accordance with the disclosure.

[0116] In an embodiment, the composition or pharmaceutical composition can be formulated for delivery by a continuous delivery system. The term “continuous delivery system" is used interchangeably herein with "controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0117] Embodiments of the composition or pharmaceutical composition can be administered to a subject in one or more doses. For example, at 5 to 100 pl hydrogel per 1 cm2of wound area topically and at concentration 0.1 to 10% w / v, and applied weekly, daily, or whenever needed. Those of skill will readily appreciate that dose levels can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0118] In an embodiment, multiple doses of the composition or pharmaceutical composition are administered. The frequency of administration of the composition or pharmaceutical composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the composition or pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (ad), twice a day (qid), three times a day (tid), or four times a day. As discussed above, in an embodiment, the composition or pharmaceutical composition is administered 1 to 4 times a day over a 1 to 10- day time period.

[0119] The duration of administration of the composition or pharmaceutical composition analogue, e.g., the period of time over which the composition or pharmaceutical composition is administered, can vary, depending on any of a variety of factors, including patient response. For example, the composition or pharmaceutical composition in combination or separately, can be administered over a period of time of about one day to one week, about one day to two weeks.

[0120] Embodiments of the composition or pharmaceutical composition can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. Routes of administration can include, but are not limited to, enteral administration, parenteral administration, or inhalation.

[0121] Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the disclosure.

[0122] The term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. For example, subjects to which compounds of the disclosure can be administered include animals, such as mammals. Non-limiting examples of mammals include primates, such as humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted above or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject. As used herein, "pharmaceutically acceptable derivatives" of a compound can include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.

[0123] As used herein, the term "administering" can refer to introducing a substance, such as the compounds described herein, or derivatives thereof, isomers thereof, or a combination thereof into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration. In embodiments, "administering" can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can beby way of carriers or vehicles, such as injectable solutions, including sterile aqueous or nonaqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.EXAMPLES

[0124] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0125] Development of a New Covalently Bonded Conjugate of Caprylic Acid Tripeptide (Tsoleucine-leucine-Aspartic Acid) for Wound-compatible and Injectable Hydrogel to Accelerate Healing

[0126] Non-limiting Description of the Invention

[0127] Third-degree burn injuries pose a significant health threat. Safer, easier-to-use, and more effective techniques are urgently needed for their treatment. Described herein are covalently bonded conjugates of fatty acids and tripeptides that can form wound-compatible hydrogels that can accelerate healing. We first designed conjugated structures as fatty acid- aminoacidl-amonoacid2-aspartate amphiphiles (Cn acid-AAl-AA2-D), potentially capable of self-assembling into hydrogels according to the structure and properties of each moiety. We then generated 14 new conjugates based on this design by using two Fmoc / tBu solid-phase peptide synthesis techniques; we verified their structures and purities through liquid chromatography with tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Of them, 13 conjugates formed hydrogels at low concentrations (> 0.25% w / v), but C8 acid-ILD-NH2 showed the best hydrogelation and was investigated further. Scanning electron microscopy revealed that C8 acid-ILD-NEE formed fibrous network structures and rapidly formed hydrogels that were stable in phosphate-buffered saline (pH 2-8, 37°C), atypical pathophysiological condition. Injection and rheological studies revealed that the hydrogels manifested important wound-treatment properties, including injectability, shear thinning, rapid re-gelation, and wound-compatible mechanics (e.g., moduli G" and G', ~0.5- 15 kPa). The C8 acid-ILD-NH2(2) hydrogel markedly accelerated healing of third-degree burn wounds on C57BL / 6J mice. Taken together, our findings demonstrated the potential of the Cn fatty acid-AAl-AA2-D molecular template to form hydrogels capable of promoting the wound healing of third-degree bums.

[0128] Introduction

[0129] Third-degree bum injuries involve the destruction of full-thickness skin, posing a significant health threat [1-3], The typical treatment for burn wounds involves debridement, followed by the application of topical and antimicrobial agents to support skin rebuilding and vascular network formation [4], In general, the traditional clinical methods to treat wounds, including third-degree burns, are anti-infection, vacuum suction, oxygen therapy, and the use of dressings such as gauze, foams, bandages, hydrocolloids, iodine and silver dressings, and films. Some of third-degree bum wounds are also repaired using skin grafts by harvesting healthy skin from other parts of the body. However, graft harvesting itself results in new wounds, thereby compromising the function of the donor sites. The overall graft procedure is lengthy, risky, and costly and can result in debilitation, thereby necessitating the development of a safer, easier-to-use, and more effective technique for the repair of third-degree bums.

[0130] The most important factor for the rapid healing of wounds is the maintenance of a moist environment, as this can facilitate high oxygen permeation and wound exudate removal. Therefore, therapeutics should promote these processes while also supporting proliferation and tissue remodeling [5], Naturally occurring polymers, such as chitosan, alginate, elastin, cellulose, fibrin, hyaluronic acid, pectin, dextran, and collagen, are biopolymers that are generally used in wound dressings [6-14], However, these natural polymers have poorer stability, structural heterogeneity, and mechanical properties than their synthetic counterparts, which also possess other merits, such as ease of application, appropriate firmness and elasticity, and superior water retention capacity

[0015] , Therefore, biomaterials are usually incorporated into hydrogels for wound dressings; various polymer-based hydrogels, such as polyethylene glycol or polyvinyl alcohol, can serve as inert supports / scaffolds

[0016] , Hydrogels are used to promote wound healing because they can hold large amounts of water or biological fluid, thereby structurally mimicking the three-dimensional (3D) network structure of the natural extracellular matrix [17-23], Hydrogels fabricated from natural tissuecomponents, such as collagens, fibrins, and cellular dermal matrices, have been used successfully to promote wound repair and regeneration [24-26]; however, tissue-derived natural biomaterials have several disadvantages, including high cost, poor reproducibility, low availability, and the potential risk of disease transmission [24-26], To overcome these shortcomings, synthetic biomaterials that can be fabricated into hydrogels are highly desirable, as they can mimic the structural, mechanical, and chemical properties of skin.

[0131] Peptide-based hydrogels generated through organic synthesis are inherently biocompatible and biodegradable because they can be broken down to individual nutrient amino acids by enzymes present in tissues

[0027] , Concomitantly, peptide-incorporated hydrogels have found enormous applications in the biochemical and biomedical fields as materials for sustained release of drugs and biomolecules

[0028] , cell culture substrates, tissue engineering

[0029] , and (pertinent to this report) scaffolds for wound healing [30,31], Peptides containing aromatic amino acids, including NFGAIL

[0032] , DFNKF

[0033] , KLVFFAE

[0034] , and FDFSFDFS

[0035] tend to assume a P-sheet conformation due to TI~ TI stacking that drives fibril formation. The pentapeptide KYFIL forms a stimulus-responsive and highly stable injectable hydrogel for tissue engineering applications

[0036] . Similarly, an ultrashort peptide-based hydrogel showing high mechanical properties has been used for the healing of critical bone defects in rabbits

[0037] . Peptide amphiphiles containing a hydrophobic alkyl tail and a hydrophilic peptide domain, such as LIVAGD, can spontaneously self-assemble into hydrogels in an aqueous solution to form robust hydrogels via P-sheet assembly

[0038] ,

[0132] Described herein is a covalently bonded conjugate of fatty acid and tripeptide would form a wound-compatible hydrogel capable of accelerating healing. Described herein is a hydrogelator that is as simple, as small as the reported ultrashort peptide hydrogelators, easily made, and effective in promoting the healing of third-degree burns. In embodiments, the hydrogel should have the following properties: be compatible with burn wounds in terms of viscoelasticity (0.1-15 kPa [39,40]), injectable to ensure easy application to wounds, stable in wounds for more than a week (with a pH of 2-8); have the osmolarity of saline; exert no systematic or local toxicity; and should be degradable when the wounds are healed. The advantages of using fatty acids are that they are safe as they naturally exist in the human body, can be easily linked to the N-terminal amino groups of peptides via amide bonds, and can participate in hydrogelation through their hydrophobic carbon chains as a moiety of the hydrogelator. No hydrogelator with this composition has yet been reported. We targeted a tripeptide as a moiety of the hydrogelator. Additionally, longer peptides have more amidebonds, which without wishing to be bound by theory, can not only increase the preparation cost but also increase the sites for undesired enzymatic degradation or nonenzymatic hydrolysis of the peptides, thus impacting the stability of the peptides. Shorter peptides (dipeptides) can have lower gelability than longer peptides because of the lower intermolecular interaction (hydrogen bonds and hydrophobic force) [32-35],

[0133] We have developed new covalently bonded conjugates of caprylic acid- tripeptide (isoleucine-leucine-aspartic acid) compounds capable of hydrogelation. These compounds were designed and synthesized using Fmoc / tBu-based solid-phase peptide synthesis (SPPS) strategies and characterized using liquid chromatography with tandem mass spectrometry (LC-MS / MS) and NMR spectroscopic techniques. The hydrogelation was undertaken in saline, followed by concentration and pH studies, and swelling ratio (%) for a representative compound (2). The new hydrogel from (2) has injectable properties and its mechanical properties revealed that 2 possesses wound- compatible storage modulus and excellent shear thinning ability. The compound 2 can form wound-compatible and injectable hydrogel capable of accelerating wound healing of third-degree burn wounds.

[0134] Materials and Methods

[0135] 2.1. Materials

[0136] Fmoc-protected amino acids — Fmoc-Ile-OH (CAS No. 71989-23-6, 98%),Fmoc-Ala-OH (CAS No. 35661-39-3, 99.92%), Fmoc-Val-OH (CAS No. 68858-20-8, 99.56%), Fmoc-Leu-OH (CAS No. 35661-60-0, 99.78%), Fmoc-Asp(OtBu)-OH (CAS No. 71989-14-5, 99.92%) and fatty acids, namely n-octanoic acid (CAS No. 124-07-2, 99.56%) and tetradecanoic acid (CAS No. 544-63-8, 99%) — were purchased from BLD Pharmatech (Cincinnati, USA) and used without further purification. Lauric acid (CAS No. 143-07-7) and palmitic acid (CAS No. 57-10-3, 99%) were purchased from Sigma Chemicals (St. Louis, MO, USA). Fmoc-rink amide resin (0.57 mmol / g, 100-200 mesh), Wang resin (0.9 mmol / g, 100- 200 mesh), the Kaiser test kit (Catalog no. KGZ001), and O-benzotriazole-N, N, N’ and N’- tetramethyluronium-hexafluoro-phosphate (CAS No. 94790-37-1) were purchased from Aapptec, LLC (Louisville, KY, USA). Hydroxybenzotriazole (HOBt, CAS No. 2592-95-2, 98.75%) was purchased from Apexbio (Houston, TX, USA). Trifluoroacetic acid (TFA; CAS No. 76-05-1, 99%) was purchased from Honeywell Research Chemicals (Muskegon, MI, USA). Piperidine (CAS No. 110-89-4, 99%) was purchased from Sigma-Aldrich LLC (St. Louis, MI, USA). N, N-diisopropylethylamine (DIPEA; CAS No. 7087-68-5, 99%) was purchased from TCI America (Portland, OR, USA). N, N-dimethylformamide (DMF; CAS No.68-12-2), diethyl ether (DE; CAS No. 60-29-7), and dichloromethane (DCM; CAS No. 75-09- 2) were obtained from Thermo-Scientific (Ward Hill, MA, USA).

[0137] 2.2. Organic synthesis and structure of fatty acid-conjugated tripeptides

[0138] The fatty acid-conjugated peptides were synthesized manually using Fmoc / tBu-based SPPS strategies using PolyPrep columns obtained from Bio-Rad Laboratories (Hercules, CA, USA). The synthesis was conducted on a 0.1 mmol scale on the Fmoc-Rink amide resin (for the C-terminus amide) and Wang resin (for the C-terminus acid).

[0139] 2.2.1. General procedure for synthesis of C-terminus amide conjugates. TheFmoc-Rink amide resin (175 mg) was swollen in DCM (2.0 mL) in a Bio-Rad column for 30 min. The solvent was then pushed out with positive pressure and replaced with 20% piperidine in DMF (2.0 mL), and the mixture was shaken for 20 min. The solvent was removed and the resin was washed with DMF (3 ^ 2 mL) and DCM (3 x 2 mL) (confirmed by a positive Kaiser test). The Fmoc-Asp(tBu)-OH (164 mg) was placed in a scintillation vial and dissolved in DMF (2.0 mL) together with HBTU (152 mg), HOBt (54 mg), and DIPEA (0.2 mL). The mixture was sonicated for 1 min and then added to a resin column, which was shaken on a vortex mixer for 6 h. The solvent was removed from the column and the column was washed with DMF (3 x 2 mL) and DCM (3 x 2 mL) (confirmed by a negative Kaiser test). The resin was endcapped by adding 5 mL of acetic anhydride:pyridine solution (3:2, v / v) and rocking the resin for 1 h, followed by washing with DMF (3 x 2 mL) and DCM (3 x 2 mL) (confirmed by a negative Kaiser test).

[0140] The Fmoc group was removed by adding 20% piperidine in DMF (2.0 mL) and shaking for 20 min. The solvent was removed and the column was washed with DMF (3 x 2 mL) and DCM (3 x 2 mL) (confirmed by a positive Kaiser test). In a 10-mL scintillation vial, the second amino acid (4 equiv), HBTU (152 mg), HOBt (54 mg), and DIPEA (0.2 mL) were dissolved in DMF (2.0 mL). This mixture was shaken well for 10 min and added to the column, and the column was vortexed for 6 h. The solvent was removed from the column, and the column was washed with DMF (3 x 2 mL) and DCM (3 x 2 mL) (confirmed by a negative Kaiser test). This procedure was repeated for coupling the third amino acid and fatty acid, each of which was also added at 4 equiv.

[0141] The cleavage of the fatty acid-peptide conjugate from resin was conducted by adding a mixture of TFA:H2O:TIPS (5.0 mL; 95:2.5:2.5 v / v) to the resin, and the mixture was stirred at room temperature for 2 h. The solution containing TFA was filtered from the column, and the filtrate was evaporated on a rotary evaporator to remove excess TFA. Di-isopropylether was added to the resulting crude product to yield a white solid precipitate. The ether was removed by decantation, and the precipitated compound was washed three times with diethyl ether. The resulting compound was dried in a rotary evaporator, purified by lyophilization for 24 h in a freeze-dryer (Thermo Savant, Holbrook, NY, USA), and validated using LC-MS / MS.

[0142] 2.2.2. General procedure for synthesizing C-terminus acid conjugates. The synthesis of C-terminus carboxylic acid derivatives was similar to the procedure described for synthesizing the C-terminus amide, except for the resin choice and the first coupling step, as described below. The Wang resin was placed in a Bio-Rad column and swelled in DCM (2.0 mL) for 30 min, and the solvent was pushed out with positive pressure. The first amino acid (Fmoc-Asp(tBu)-OH) was placed in a scintillation vial, dissolved in DMF (2.0 mL), and DIC (100 mg), HOBt (54 mg), and DMAP (5 mg) were added to the vial. This mixture was sonicated for dissolution, added to the resin, and shaken on a vortex mixer for 6 h. The solvent was removed from the column and the column was washed with DMF (3 ^ 2 mL) and DCM (3 x 2 mL) (confirmed by a negative Kaiser test). We then followed the same protocol described in Section 2.2.1.

[0143] 2.2.3. Procedures to remove trifluoracetic acid counterions from fatty acid- peptide conjugates by counteranion exchange. The trifluoroacetic acid counteranion was replaced with HC1 by dissolving the white precipitate obtained after ether trituration in 5.0 mL of 0.1 M HC1 solution, stirring for 15 min, and then adding 5 mL of acetonitrile. The soluble mixture was then dried in a dry ice bath and lyophilized overnight to yield a dry solid powder.

[0144] 2.2.4. Determination of the molecular structures and quantities of compounds. The reagents and the fatty acid-amino acid / peptide conjugates were analyzed using an LC-MS / MS system consisting of an Agilent 1100 LC system (HPLC-DAD- autosampler, Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 6500+quadruple- linear trap mass spectrophotometer with electrospray ionization (Sciex.com, Framingham, MA, USA). The nuclear magnetic resonance (NMR) analysis was conducted using a Brucker 400-MHz NMR instrument, DMSO-de (CAS No. 2206-27-1, 99.9 atom% D, Thermo- scientific, Fair Lawn, NJ, USA) as a solvent, and Topspin 4.3.0 version software (Bruker.com). The 'H NMR (400 MHz),13C NMR (100 MHz) and13C DEPT data were acquired using a purified compound (30 mg) dissolved in 0.7 mL DMSO-de in a 5 mm diameter NMR tube. DEPT 135 was used to determine the multiplicity of carbon atoms, CH2 groups showed inverted signals, whereas CH and CH3 groups were upright. The quaternary carbon (C) did not show any signal.

[0145] 2.3. Hydrogel formation

[0146] 2.3.1. Hydrogel preparation. Lyophilized fatty acid-peptide conjugates were dissolved in phosphate-buffered saline (PBS) at a final concentration of 0.5% (5.0 mg in 1 mL of PBS), 1.5% (15.0 mg in 1 mL of PBS), and 3% (30.0 mg in 1 mL of PBS). The pH of the peptide solutions was increased to 9.0 by adding 0.1 M NaOH to dissolve the compounds and readjusted to pH 2-8 by drop-wise addition of 0.1 N HC1, followed by sonication. Most compounds formed hydrogels immediately after sonication; some were maintained overnight at room temperature for gel formation. The gel formation was confirmed by the vial inversion method, and photographs were taken.

[0147] 2.3.2. Hydrogel sterilization. PBS, 0.1 N HC1, 0.1 M NaOH, pipette tips, andEppendorf tubes were autoclaved (Steris, AMSCO 250LS) at 130°C for 45 min. The other procedures for hydrogel formation were performed under pathogen-free conditions inside a BSL-2 hood.

[0148] 2.4. Rheological studies

[0149] Tests were performed on 50 pL hydrogel samples using an Anton Paar MCR 092 rheometer (Anton Paar USA, Houston, TX, USA) with a 20 mm cone plate at a measuring gap of 39 pm. The effects of concentration on gel strength and viscoelastic behavior were assessed by conducting amplitude / strain sweep experiments for all gels using oscillatory shearing strain. Storage (G') and loss (G") moduli were measured as a function of strain (ranging from 0.01 to 100%) at a constant frequency of 10 rad / s. The mechanical stability of the peptide hydrogels was tested by running frequency sweep experiments at angular frequencies ranging from 1 to 100 rad / s at a constant 1% strain, which was under the limit of the linear viscoelastic region obtained from the amplitude sweep test. The hydrogel structure survived the tests under this strain. Time-sweep curing experiment was conducted with a constant strain 10% and frequency 1 Hz. We studied the thixotropic properties to understand the time-dependent shear thinning of gels under 4 min of constant high shear strain (200%) that liquefied the gel. We then followed the re-gelation after the shear strain returned to the low shear strain of 0.1% used for the initial 2 min test. The G' and G" values represent the elastic gel-like and viscous liquid behaviors of our samples, respectively

[0041] ,

[0150] 2.5. Hydrogel swelling ratio (%)

[0151] The swelling ratio assay was used to determine the water intake capacity of the hydrogels. First, different gel concentrations (1.5% and 3.0% w / v) were formed in PBS in preweighed vials (250 pL of gel in each vial). Next, 2 mL of PBS was added to the vials, andthe gels were left to swell at different times. The excess PBS was then removed from the vial, and the vials were weighed. The swelling ratio (%) of the hydrogel was calculated using the following equation:

[0152] Swelling ratio (%) = [(Wt - Wi) / Wi] x 100

[0153] where Wt is the weight of the swollen hydrogel at a specific time point t and Wi is the initial weight of the hydrogel.

[0154] 2.6. Field emission scanning electron microscopy

[0155] The 3% w / v hydrogel was freeze-dried at -80°C and then lyophilized under vacuum to obtain a fine powder. Field emission scanning electron microscopy (FESEM) analysis of the lyophilized hydrogel was conducted on an S4800 field emission scanning electron microscope (Hitachi, Santa Clara, CA, USA) under a high vacuum to assess the surface structure of the freeze-dried peptide-based hydrogel according to published and widely used procedures [42,43], The following parameters were used: stage distance, 12 mm; acceleration voltage, -3.0 kV; and working distance, 2.2 mm. The sample was initially sputter coated with a thin carbon layer for increased conductivity. High magnification allowed the observation of the fibrous network on the top layer of the hydrogel samples.

[0156] 2.7. Hydrogel treatment of third-degree bum wounds generated on mice and histological study of treated wounds

[0157] The animal use protocol was authorized and approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center, New Orleans, and followed the ARRIVE guidelines

[0044] , Briefly, full-thickness bum wounds (6 mm diameter) were generated in the dorsal skin on both sides along the midline of C57BL / 6J mice (female, 18 months old, Jackson Laboratory, Bar Harbor, ME, USA) at 0 day postburn (dpb), similar to the procedures performed by us and others previously [1,45-49], Each treatment group included four mice. We excised a 6-mm-diameter circle of coagulated or necrotic fullthickness skin at the center of each bum wound at 48 h (2 dpb) and filled each excisiongenerated space with hydrogel (30 pL) by injection. The wounds were covered with Tegaderm waterproof dressings to protect the tissue and hydrogel in the wounds and to prevent water loss. Each wound was covered with two pieces of a 50.8 mm long and 30 mm wide Tegaderm waterproof film dressing (www.3m.com) (cut from a roll of the product 50.8 mm wide x 10058 mm long, catalog number 16002) to protect the tissue and hydrogel in the wounds and to prevent water loss, one was on dorsal side, the other was on the abdominal side. Each two ends of four ends of these two film pieces overlap each other, forming a cylinder-shape adhesivebelt wrapping around the skin of mouse body trunk. This film belt adhered to the skin, including the wound margins, from the dorsal to abdominal side, all around the mouse body trunk. This method of film application allows the film to reduce skin contraction, facilitating wound closure through re-epithelialization, as described in prior studies [50-52], thus better resembling wound healing in humans. We used swabs to clean the area of skin surface to allow the film to stick to the skin surface tightly. The adhesion of a film end to the end of another film is much stronger than adhesion of film to the skin. In turn, our method of wrapping wounds with Tegaderm film allowed the film to adhere to skin around wounds for the intended duration even when the mice were active, which is likely to be more effective and durable than those in references [50-52] for wound contract control using Tegaderm film. The film was changed at 2 dpb for the excision / debridement of necrotic tissue of the bum wound. To clearly photograph the wound area, we also changed the film and hydrogel at Day 7 post-burn. As the renewal only took a few minutes, it should not affect the control of wound contraction. The excision mimics the practice of debriding severely burned skin. The same type of wounds without hydrogel treatment were used as the control. The mice were anesthetized with ketamine and xylazine (100 and 10 mg / kg, respectively, i.p.) prior to any wounding. Sustained-release buprenorphine was also injected (s.c., 1 mg / kg) for analgesia. We monitored mouse health and behavior, including drinking, foraging, grooming, and eating, and we examined all wounds twice per day to ensure that the hydrogel and dressing stayed in place. Wound healing was assessed as described previously [53-58], The wounds were photographed, and their areas were calculated using NIH ImageJ software. The wound closure was reported as the percentage of the closed wound area compared with the initial bum-wound area. The mice were euthanized, and bum wounds with 3-mm skin rims were excised and histologically studied after hematoxylin-eosin staining as we did previously [59, 60], The livers, kidneys, and spleens were also collected, weighed, and measured for size to obtain a gross assessment of toxicity. The sections stained with hematoxylin-eosin were photographed using an OLYMPUS scanning microscope. The epithelial gap — i.e., the distance between the neoepithelium emerging from the edges of the wound area — was measured using OLYMPUS OlyVIA software, as described previously but with some modifications [53,60,61],

[0158] 2.8. Statistical analysis

[0159] Statistical analysis was conducted using t tests or ANOVA via GraphPad Prism 9.0 software, and p < 0.05 was considered statistically significant. Data are presented as mean ± standard error of mean (SEM) or standard derivation (SD).

[0160] Results and Discussion

[0161] 3.1. Design, synthesis, and hydrogelability tests of new covalent fatty acid- tripeptide conjugates

[0162] Described herein is our development of amphiphilic conjugates as innovative hydrogelators, with each hydrogel containing a fatty acid covalently bonded to a peptide and capable of self-assembling into a hydrogel with the hydrophobic tail from the linear carbon chain of fatty acid and the hydrophilic head from the peptide domain. To this effect, we designed and synthesized a large panel of compounds through SPPS, including peptides inspired by the pentapeptide KYFIL

[0036] , The amphiphilic conjugates of these peptides and fatty acids were also synthesized. Unfortunately, these compounds were unable to form hydrogels. Based on our findings from these unreported trials and the inspiration from the LIVAGD hydrogelator

[0038] , we synthesized a fatty acid-conjugated tripeptide possessing a molecular template of a fatty acid (with n carbons) coupled to an amino acidl-amino acid2- aspartic acid tripeptide (Cn acid-AAl-AA2-D). This coupling generates new amphiphilic molecules that can self-assemble into hydrogels within the living pathophysiological niche. AA1 and AA2 are hydrophobic amino acids such as alanine (A), valine (V), isoleucine (I), or leucine (L) that play a role in self-assembly. Aspartic acid (D), an amino acid with a hydrophilic carboxylic acid side chain, forms the hydrophilic head at the tripeptide terminus and offers polarity and aqueous solubility. The C-terminus polar hydrophilic end (carboxyl or amidated carboxyl) then improves solubility in aqueous media, while the side chain carboxylic acid group further increases the hydrophilic character of the hydrogel in solution. The fatty acid, either octanoic (C8) acid, dodecanoic / lauric (C12) acid, tetradecanoic / myristic (C14) acid, or hexadecanoic / palmitic (Cl 6) acid, was predicted to participate in self-assembly due to its hydrophobicity and the shape of the long carbon chain. Shorter fatty acids (such as C6 acid) are likely not to be hydrophobic enough for hydrogelation.

[0163] 3.1.1. Design, synthesis, and hydrogelability tests of Cn fatty acid-ILD-NH2 -type conjugates. We tested this design by initially synthesizing compound (1) (H-ILD- NH2) with the C-terminal carboxyl bonded to an amide (-NH2) but with the N-terminal intact (H) using Rink amide resin -based SPPS (Fig. 1 panel a, Fig. 38) to find if the tripeptide itself undergoes gelation. We observed that it did not gelate in PBS.

[0164] We then synthesized compound 2 [C8-ILD-NH2(2)] with a C8 fatty acid linked to the N-terminus of (1) (tripeptide ILD-NH2) via an amide bond (Fig. 1 panel a). The molecular structure and purity of the synthesized C8-ILD-NH2 (2) were determined usingNMR and LC-MS / MS. The NMR spectra illustrated in Fig. 2 indicate chemical shifts 5 and coupling constants J that clearly verified the structure of (2) as the following:13C NMR (100 MHz, DMSO-de) (Fig. 2a): 5 172.49, 172.29, 171.95, 171.70, 171.47, 56.94, 51.21, 49.42, 40.28 (DMSO-de), 35.98, 35.95, 35.08, 31.22, 28.52, 28.45, 25.36, 24.48, 24.04, 23.05, 22.06, 21.44, 15.39, 13.95, 10.81. ’H NMR (400 MHz, DMSO-de) (Fig. 2b): 5 12.27 (brs, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.97 (dd, J = 12.2, 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 4.42 (dd, J = 8.4, 6.8 Hz, 1H), 4.25 (dd, J = 8.4, 6.0 Hz, 1H), 4.12 (t, J = 6.0 Hz, 1H), 2.64 (dd, J = 8.4, 6.0 Hz, 1H), 2.54 (d, J= 7.2 Hz, 1H), 2.51 (dd, J = 8.4, 3.7 Hz, 1H), 2.17-2.10 (m, 2H), 1.73-1.69 (m, 1H), 1.61-1.56 (m, 1H), 1.51-1.40 (m, 5H), 1.29-1.24 (m, 8H), 1.12-1.06 (m, 1H), 0.87 (s, 6H), 0.82 (s, 9H);13C DEPT-135 NMR (100 MHz, DMSO-de) (Fig. 2c): 56.94 (CaH), 51.21 (CaH), 49.42 (CaH), 40.28 (CH2), 35.98 (CH), 35.95 (CH2), 35.08 (CH2), 31.22 (CH2), 28.52 (CH2), 28.45 (CH2), 25.36 (CH2), 24.48 (CH2), 24.04 (CH), 23.05 (CH3), 22.06 (CH2), 21.44 (CH3), 15.39 (CH3), 13.95 (CH3), 10.81 (CH3).

[0165] The LC-MS / MS spectrum confirmed a molecular mass M of 484 Daltons, while the fragment ions m / z (single charge, z = 1): 114, 131, 142, 165, 207, 224, 242, 350, 368, 421, 439, 465, and 483 [M - H+] offered fingerprints for the structure of C8-ILD-NH2(2) (Fig. 3). The fragment ions corresponding to the details of the molecular structure are interpreted in Fig. 3a. The LC-MS analysis also indicated that (2) was highly pure (> 96%). When we dissolved C8-ILD-NH2(2) into PBS by raising the pH to pH 9 and then reducing it to pH 7.4, it formed hydrogels (Fig. 38), while it also formed hydrogel if PBS was replaced with deionized water. These results supported the suitability of our synthetic route and the procedures illustrated in Fig. la for synthesizing Cn acid-AAl-AA2-D-NH2type compounds, while demonstrating their potential hydrogelability.

[0166] Compounds 3 [C12-ILD-NH2)] and 4 [C16-ILD-NH2], with C12 and C16 fatty acids, respectively, linked to the N-terminus of (1) (tripeptide ILD-NH2) by an amide bond, were then synthesized, as illustrated in Fig. la. We determined the hydrogelability of both compounds following the same protocol used for (2) after the verification of their structures and purity (>95%) through LC-MS / MS. Both (3) and (4) formed hydrogels similar to (2) in PBS buffer at > 0.25% (w / v) concentration and remained as a solution below 0.25% (w / v) (Fig. 4c). The key difference in the gelation observed between (2), (3), and (4) was that(2) instantly formed a hydrogel upon sonication, whereas (3) and (4) took more time under the same conditions. Moreover, when shaken by hand, hydrogel (2) was more stable than either(3) or (4), suggesting a more compact packing of shorter C8 carboxylic acid. The selectedcompound C8-ILD-NH2(2) gelates in 2 min after the pH was adjusted to 7.4, whereas most of the other compounds in Fig. 38 form gels immediately within 2-15 min after the pH was adjusted to 7.4. The gelation time increased as the carbon of Cn fatty acids increased from 8 to 16 carbons.

[0167] 3.1.2. Design, synthesis, and hydrogelability tests of Cn fatty acid-ILD-OH-type conjugates. The C-terminal carboxyl of compounds (2), (3), and (4) is amidated with a primary -NH2 group. We questioned whether the hydrogelability would change if this carboxyl was not amidated, and we addressed this possibility by synthesizing compounds 5[C8-ILD- OH(5)], 6[C12-ILD-OH)(6)], 7[C14-ILD-OH(7)], and 8[C16-ILD-OH(8)] at high purity (> 95%) via Wang resin-based SPPS (Fig. lb) with C8, C12, C14, and C16 fatty acids, respectively, bonded to the N-terminus of ILD-OH. The representative NMR and LC-MS / MS data, which confirmed the molecular structures of these compounds, are as follows:

[0168] C12-ILD-OH(6). 'II NMR (in DMSO-d6): 5 12 5 (brs, 2H), 8 06 (d, J = 8.4Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 4.50 (dd, J = 7.5, 6.6 Hz, 1H), 4.36 (dd, J= 8.4, 7.0Hz, 1H), 4.16 (t, J = 7.5 Hz, 1H), 2.68-2.55 (m, 2H), 2.53-2.51 (m, 1H), 2.16- 2.07 (m, 2H), 1.72-1.69 (m, 1H), 1.62-1.56 (m, 1H), 1.48-1.42 (m, 4H), 1.29-1.24 (brs, 16H), 1.11-1.04 (m, 1H), 0.87 (t, J = 7.5 Hz, 6H), and 0.82 (t, J = 7.5 Hz, 9H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-d6(Figs. S2 and S3): 5 172.2, 172.1, 171.6, 171.6, 171.6, 171.0, 56.6, 50.6, 48.4, 40.8, 36.1, 35.8, 35.1, 31.3, 29.0, 28.9, 28.8, 28.7, 28.5, 25.4, 24.3, 24.0, 23.0,22.1, 21.4, 15.3, 13.9, and 10.7. MS / MS fragmentation ions verified the molecular mass and structure (Fig. 38).

[0169] C14-ILD-OH(7) 'll NMR ((in DMSO-d6): 5 12.51 (brs, 2H), 8.07 (d, J = 9.0Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 4.52 (q, J = 7.6Hz, 1H), 4.34 (dd, J = 7.6Hz, 1H), 4.16 (t, J = 7.6 Hz, 1H), 2.68-2.52 (m, 2H), 2.53-2.51 (m, 1H), 2.16-2.09 (m, 2H), 1.75-1.69 (m, 1H), 1.62-1.55 (m, 1H), 1.47-1.43 (m, 4H), 1.25 (brs, 20 H), 1.11-1.04 (m, 1H), 0.87 (t, J = 7.5 Hz, 6H), and 0.82 (t, J = 7.5 Hz, 9H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-de: 5 172.2, 172.1, 171.6, 171.6, 171.6, 171.0, 56.6, 50.6, 48.4, 40.8,36.1, 35.8, 35.1, 31.3, 29.0, 29.0, 28.9, 28.8, 28.7, 28.5, 25.4, 24.3, 24.0, 23.0, 22.1, 21.4, 15.3,13.9, and 10.7. MS / MS fragmentation ions confirmed a molecular mass and structure (Fig. 38).

[0170] C16-ILD-OH(8). 'll NMR (in DMSO-d6) (Fig. S7): 5 12.50 (brs, 2H), 8.06(d, J= 8.0Hz, 1H), 7.91 (d, J= 8.0Hz, 1H), 7.83 (d, J = 8.6Hz, 1H), 4.52 (q, J= 7.6Hz, 1H), 4.34 (dd, J = 7.6 Hz, 1H), 4.16 (t, J = 7.6 Hz, 1H), 2.68-2.55 (m, 2H), 2.53-2.51 (m, 1H), 2.17-2.07 (m, 2H), 1.74-1.68 (m, 1H), 1.61-1.56 (m, 1H), 1.48-1.43 (m, 4H), 1.24 (brs, 28H), 1.11-1.04 (m, 1H), 0.87 (t, J = 7.5 Hz, 6H), and 0.82 (t, J = 7.5 Hz, 9H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-de: 5 172.2, 172.1, 171.6, 171.6, 171.0, 56.6, 50.6, 48.4, 40.9, 36.1, 35.9, 35.1, 31.3, 29.0, 29.0, 28.9, 28.8, 28.7, 28.5, 25.4, 24.3, 24.0, 23.0, 22.1, 21.4, 15.4, 13.9, and 10.7. MS / MS fragmentation ions verified the molecular mass and structure (Table 1).

[0171] C8-ILD-OH(5), the C-terminal carboxylic acid counterpart of C8-ILD-NH2(2), was unable to form a hydrogel at all, unlike (2) (Fig. 38). Hydrogels were formed from the other tested Cn acid-ILD-OHs (C12-ILD-OH(6), C14-ILD-OH(7), and C16-ILD-OH(8) with C12, C14, and C16 fatty acids, respectively, bonded to the N-terminus of ILD-OH). However, these hydrogels were weaker than those formed by Cn acid-ILD-NH2 with the same Cn acid when shaken by hand. Thus, the primary amide offers better gelation than its carboxylic acid counterpart when attached to the C-terminus of the Cn acid-ILD molecular framework. Of note, MS / MS fragmentation ions verified the molecular masses and were structured as (5), (6), (7), and (8) (Fig. 38).

[0172] 3.1.3. Design, synthesis, and hydrogelability of more new C8 fatty acid-AA1-AA2-D-NH1 -type conjugates. We also synthesized different analogs of C8-ILD- NH2(2), in which the first 2 amino acids I (AAi) and L (AA2) were swapped with each other or replaced by other hydrophobic amino acids (e.g., alanine (A) and valine (V)). The same Rink amide resin-based SPPS outlined in Fig. 1 panel a for the organic synthesis of (2) was used for the generation of these new analogs. The representative NMR and LC -MS / MS data that verified the molecular structures and purities (> 95%) of these analogs are as follows:

[0173] C8-IAD-NH2 (9) 'll NMR (400 MHz, DMSO-d6): 5 12 18 (brs, 1H), 8 85 (d, J = 9.8 Hz, 1H), 8.38 (t, J = 7.7 Hz, 1H), 8.08 (d, J = 6.8 Hz, 1H), 7.88 (t, J = 7.6 Hz, 2H), 7.12 (d, J = 5.3 Hz, 2H), 4.42 (q, J = 7.2 Hz, 1H), 4.24-4.16 (m, 1H), 4.13 (t, J = 7.95 Hz, 1H), 2.63 (dd, J = 7.2, 6.0Hz, 1H), 2.55 (d, J = 7.2 Hz, 1H), 2.20-2.08 (m, 2H), 1.74-1.69 (m, 1H), 1.53-1.40 (m, 3H), 1.28-1.24 (m, 9H), 1.21 (d, J = 7.1 Hz, 4H), 1.14-1.05 (m, 1H), 0.86 (t, J = 6.9 Hz, 3H), and 0.82 (t, J = 6.9 Hz, 6H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-de: 5 172.5, 172.3, 171.9, 171.3, 144.5, 143.1, 126.2, 56.8, 49.3, 48.5, 36.2, 35.9, 35.0, 31.2, 28.5, 28.4, 25.3, 24.4, 22.0, 17.6, 15.4, 13.9, and 10.9; MS / MS fragmentation ions verified the molecular mass and structure of (9) (Fig. 38).

[0174] C8-IVD-NH2 (10) 'll NMR (400 MHz, DMSO-d6): 5 12 29 (brs, 1H), 8 05 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.10 (brs, 2H), 4.45 (d, J = 7.5 Hz, 1H), 4.18 (t, J = 8.1 Hz, 1H), 4.10 (t, J = 7.4 Hz, 1H), 2.64 (dd, J = 7.4, 5. 7 Hz,1H), 2.56-2.49 (m, 1H), 2.17-2.10 (m, 2H), 1.96-1.94 (m, 1H), 1.75-1.69 (m, 1H), 1.51-1.40 (m, 3H), 1.23 (brs, 8H), 1.12-1.06 (m, 1H), 0.87 (s, 6H), and 0.80 (s, 9H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-de: 5 172.3, 172.1, 171.8, 171.5, 170.6, 57.8, 56.8, 49.3, 35.9, 35.8, 35.0, 31.2, 30.3, 28.5, 28.4, 25.4, 24.4, 22.0, 19.1, 18.0, 15.4, 13.9, and 10.7; MS / MS fragmentation ions verified the molecular mass and structure of (10) (Fig. 38).

[0175] C8-ALD-NH2 (11). 'll NMR (400 MHz, DMSO-d6): 5 12 27 (brs, 1H), 7 97 (dd, J = 12.2, 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 4.42 (dd, J = 8.4, 6.8 Hz, 1H), 4.25 (dd, J = 8.4, 6.0 Hz, 1H), 4.12 (t, J = 6.0 Hz, 1H), 2.64 (dd, J = 8.4, 6.0 Hz, 1H), 2.54 (d, J = 7.2 Hz, 1H), 2.51 (dd, J = 8.4, 3.7 Hz, 1H), 2.17-2.10 (m, 2H), 1.73-1.69 (m, 1H), 1.61-1.56 (m, 1H), 1.51-1.40 (m, 5H), 1.29-1.24 (m, 8H), 1.12-1.06 (m, 1H), 0.87 (s, 6H), and 0.82 (s, 9H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-d6: 5 172.8, 172.4, 172.3, 171.9, 171.7, 51.4, 49.3, 48.2, 40.3, 35.8, 35.0, 31.1, 28.5, 28.4, 25.1, 24.0, 23.0, 22.0, 21.5, 17.6, and 13.9. MS / MS fragmentation ions verified the molecular mass and structure of (11) (Fig. 38).

[0176] C8-LLD-NH2 (12). 'll NMR (400 MHz, DMSO-d6): 5 8 09-8 02 (m, 2H), 7.54 (brs, 1H), 7.00 (brs, 2H), 4.36-4.28 (m, 2H), 4.18 (q, J = 7.1 Hz, 1H), 4.04 (q, J = 7.1 Hz, 1H), 2.41-2.39 (m, 1H), 2.13-2.09 (m, 1H), 1.64-1.56 (m, 2H), 1.49-1.45 (m, 4H), 1.24 (brs, 8H), 1.12-1.06 (m, 1H), and 0.89-0.86 (m, 12H);13C NMR (100 MHz) and13C DEPT NMR, DMSO-de: 5 172.8, 172.4, 172.3, 171.9, 171.7, 51.4, 49.3, 48.2, 40.3, 35.8, 35.0, 31.1, 28.5, 28.4, 25.1, 24.0, 23.0, 22.0, 21.5, 17.6. The MS / MS fragmentation ions verified the molecular mass and structure of (12) (Fig. 38).

[0177] C8-VLD-NH2 (13), C8-IID-NH2(14), and C8-LID-NH2(15) structures were verified using the same NMR and LC-MS / MS methods as for other Cn acid-AAl-AA2-D compounds above.

[0178] The Cn acid-AAl-AA2-D-NH2 compounds (9-15) can all self-reassemble into hydrogels in PBS at a 1.5% w / v concentration when subjected to gelation conditions. All formed hydrogels (Fig. 38).

[0179] 3.2. Determination of wound compatibility, injectability, and rheological properties of a new hydrogel formed from a selected Cn fatty acid-AAl-AA2-D conjugate

[0180] We explored the suitability of our new hydrogels for accelerating the healing of third-degree burn wounds by exploring the biocompatibility, toxicity, and biodegradability of C8-ILD-NH2(2) as a representative compound chosen from the 13 hydrogelating Cn acid- AA1-AA2-D type conjugates compiled in Fig. 38. We chose (2) because it showed more rapidgelation and formed a more stable hydrogel in saline compared with the other conjugates, based on our visual observations (Fig. 38, Fig. 4). Swelling in aqueous environments is an important characteristic of hydrogels and is mostly determined using the pore size, the intermolecular forces inside the hydrogel network structure, and the hydrophilic nature of the hydrogel

[0062] , Hydrogels with large pore sizes and sufficient hydrophilicity can take up large amounts of water (liquid / fluid) and swell to weights multiple times greater than their own weight, and hydrogels with these qualities have been successfully applied for full-thickness skin wound healing and tissue regeneration [62,63] The strong swelling capacity is useful for absorbing leaked blood, exudate, and body fluids and for transferring nutrients and metabolites. The swelling ratios of hydrogel (2) at concentrations of 1.5% (red) and 3% (blue) (w / v) were 197.3 and 232.6%, respectively, after 12 h of PBS absorption at a physiological pH of 7.4 (Fig. 4 panel b). These percentages continuously increased to 276.0% and 364.0%, respectively, at 36 h. However, the swelling behavior subsequently reached a plateau of 276.0-283.3% and 364.0- 366.0%, respectively, from 36 h to 48 h. Hydrogel (2) would clearly be able to take up unwanted discharge / exudate from wounds to promote wound healing.

[0181] The gelability of C8-ILD-NH2 (2) at concentrations from 0.05% to 1% (w / v) using a vial inversion test (Fig. 4 panel c). C8-ILD-NH2(2) gelated in PBS at concentrations >0.25% (w / v), but remained a solution at lower concentrations. We also tested the C8-ILD- NH2(2) gel from pH 2 to pH 10 to reflect the pH changes in human wounds under various treatments [64,65], C8-ILD-NH2(2) formed a hydrogel and stayed gelated from pH 2 to pH 8 (Fig. 4 panel d), confirming its ability to form a stable hydrogel in PBS at a concentration as low as 0.25% and within a pathophysiologically relevant pH range.

[0182] The injectability of a hydrogel determines its capability to precisely fill 3D surfaces and cavities as a liquid and then solidify onsite as an elastic matrix. The matrix can be engineered to comply with mechanical and metabolic needs, including the delivery of drugs or stem cells for the recovery of locally damaged cells and tissues. For these purposes, a hydrogel will be more versatile and have minimal potential to induce itching or pain in wound treatment if it is injectable and compliant with the internal strain from cell / tissue regeneration or remodeling, as well as any external strains that the wound can experience, such as compression or shearing between the wound surface and wound dressing. Gelation time is an important parameter for injectability, which was used to further evaluate our hydrogels (Fig. 38). C8- ILD-NH2(2) gelated in 2 min after the pH was adjusted to 7.4 as shown in Video SI, while other compounds in Fig. 38 gelated within 2-15 min after the pH was adjusted to 7.4. Gelationtimes for C11-ILD-NH2 type compounds increased from 2 to 10 min when the n of Cn fatty acid increased from 8 to 16 carbons. Gelation times for Cn-ILD-OH type compounds increased from 7 to 15 min when the n of Cn fatty acid increased from 12 to 16 carbons, but when the n reduced from 12 to 8 [i.e. from Cn-ILD-OH(6) to C8-ILD-OH(5)], the gelability lost (Table 1). These results indicate that n needs to be more than 8 for Cn-ILD-OH type compounds to gelate. With the increase in the carbon chain length Cn, there was an increase in the hydrophobicity in the molecule. The increased hydrophobicity resulted in poor water retention and took more time for gelation. Gelation times were longer for C12-ILD-OH(6) (7 min) and C16-ILD-OH(8) (15 min) respectively than for C12-ILD-NH2(3) (6 min) and C16-ILD-NH2(4) (10 min) when Cn acid is the same (Fig. 38). For C8-AAl-AA2-D-NH2 type compounds, the gelation times were in an increased order from left to right as follows: C8-ILD-NH2(2) (2 min) = C8-IAD-NH2(9) (2 min) < C8-IVD-NH2(10) (3 min) = C8-ALD-NH2(11) (3 min) < C8- LLD-NH2(12) (5 min) = C8-IID-NH2(14) (5 min) = C8-LID-NH2(15) (5 min) < C8-VLD- NH2(13) (6 min) (Fig. 38).

[0183] The injectability of the C8-ILD-NH2 (2) hydrogel was studied by tapping the vial rapidly and repeatedly on a table surface until the hydrogel liquefied. We then drew the liquid into a 1-mL syringe through a 25-gauge needle and quickly injected the liquid into the target site. We observed spontaneous re-gelation, as shown in the photos (Fig. 5 panel a and 5 panel b) of this process.

[0184] The amplitude sweep test of the C8-ILD-NH2(2) hydrogel showed that it had a larger storage modulus G’ than loss modulus G” in the limit of the linear viscoelastic region (on the left of the chart in Fig. 5 panel c), indicating the presence of an elastic gel structure. When the shear strain increase just surpassed 10.56%, the G’ and G” curves crossed over each other (cross-over point G’ = G”), so that G’ < G”, indicating a transition from the gel state to a viscous liquid state. Thus, the shear strain that promotes gel flow (i.e., the flow point) is 10.56%. The frequency sweep test (0.1-100 rad / s) of the C8-ILD-NH2(2) hydrogel at the 1% strain showed G’ (-10000 Pa) > G” (2-3 kPa), indicating a gel status under this condition (Fig. 4 panel d). It also showed that G” and G’ readouts were independent of the frequency, reflecting the high stability of hydrogel (2). Hydrogels formed from 1%, 1.5%, and 3% C8-ILD-NH2(2) in PBS our study (Fig. 38, Fig. 4) showed elastic moduli comparable to those of the dermis or skin (0.5-18 kPa) [39,40], supporting the suitability of our hydrogels for wound treatment. The isothermal time-sweep curing test for gel formation (Fig. 5 panel e) from G’ < G” sol state toG’ > G” gel state showcased the gel formation of (2) and a cross-over point at 3.87 min (G’ = G”) under a constant 10% shear strain (amplitude) and constant 1 Hz frequency.

[0185] We further explored the mechanism underpinning the injectability of C8-ILD- NH2 (2) hydrogel by studying its thixotropic properties (Fig. 5 panel f). The shear strain imposed on the gel started at 0.5 min and 0.1%, which the gel tolerated, but was changed at 2 min to 200%, which broke the gel. The shear strain was reduced to 0% at 6.3 min and then returned to 0.1% at 7.5 min. The G’ and G” curves switched in approximately 10 s from G’ > G” to G” < G’ and the values of both G’ and G” dropped from approximately 3000-12000 Pa to approximately 5-50 Pa upon changing the strain from 0.1% to 200%. These changes revealed the rapid shear-thinning property of C8-ILD-NH2(2) hydrogel, as the gel was liquefied by the increased shear strain. Moreover, the G’ and G” values completely switched back to the values observed at 0.1% strain at 0.5-2 min, when the shear strain was returned to 0.1% at 7.5 min, indicating the complete regeneration of the gel. The shear-thinning and regenerative properties of the C8-ILD-NH2(2) hydrogel provided mechanistic insights into its potential for injectability and were confirmed as completely repeatable by a thixotropic test repeated at 18 min (Fig. 5 panel f).

[0186] 3.3. Fibrous networks in hydrogels self-assembled from a selected Cn fatty acid-AAl-AA2-D conjugate: The study using FESEM

[0187] To understand the hydrogel network structures formed from Cn fatty acid- AA1-AA2-D conjugates, we performed the FESEM study of the representative one, namely C8-ILD-NH2(2) hydrogel. FESEM showed the upper layer of fibrous networks under * 10,000 magnification (Fig. 6 panel a). These networks were polygonal. The SEM image of the hydrogel under *35,000 magnification revealed the networks comprising fibrils with lengths > 1 pm comparable to those reported by other researchers (Fig. 6b) [42,43], There is a high propensity for C8-ILD-NH2(2) molecules to form fibrils through intermolecular hydrogen bonds between N-H (hydrogen bonding donor) and C=O (hydrogen bonding acceptor) groups, as it contains four N-H groups and five C=O groups to form hydrogen bonds along its backbone (Fig. 2a). Of note, the nonpolar hydrocarbon side chains of I (isoleucine) and L (leucine) as well as the hydrocarbon tail of C8 acid in (2) can interact with the counterpart groups of another (2) through hydrophobic force, which also contributes to the formation of a hydrogel network

[0066] ,

[0188] 3.4. The hydrogel formed from a selected Cn fatty acid-AAl-AA2-D conjugate accelerated the healing of third-degree burn wounds

[0189] C8-ILD-NH2(2) was selected as the initial candidate for burn-wound treatment from the 13 new Cn fatty acid-AAl-AA2-D gelable conjugates synthesized in this study (Table 1) because it formed a hydrogel faster and with greater elasticity than the other 13 conjugates. This selection was further supported by our finding that the hydrogel formed from (2) had injectable, shear-thinning, and rapid re-gelling properties in saline at a broad pH range (pH 2-8) compatible with wound treatment. The C8-ILD-NH2(2) hydrogel (3% in PBS and 30 pL / wound) was injected to fill necrotic skin excised by third-degree bum wounds in 18- month-old C57BL / 6J mice at day 2 dpb. The wound sizes were measured during the course of healing. The wound closure at 7 dpb, at 23.4% in the C8-ILD-NH2(2) gel -treated group, was significantly higher than the 5.2% closure for the control group (p < 0.05) (Fig. 7a and 7b). At 14 dpb, the difference in wound closure was more significant (p < 0.01), as the wounds were 94.7% closed in the hydrogel-treated group compared to 52.9% in the control. Wound closure is a crucial step in the early healing phase, as it forms a barrier against infection and water loss while participating in and supporting other healing processes. The Tegaderm film dressing can markedly reduce the skin contraction as demonstrated by prior reports [50-52], This forces the wound closure more by re-epitheliazation. Our method of film application is likely to render the contraction control more effective and durable than those in references [50-52], The promotion of wound closure by the C8-ILD-NH2(2) gel demonstrated its prohealing properties, and should act on wound re-epitheliazation besides contraction.

[0190] The acceleration of wound healing by the C8-ILD-NH2(2) hydrogel was confirmed with a histological analysis of the wound re-epithelialization (Fig. 7 panel b). Effective wound healing is characterized by prompt regrowth of neoepithelium and regeneration of connective tissue by fibroblasts in the wound region

[0067] , Fig. 7 panel b shows regeneration of epithelia from the wound edges, which resulted in a wound area with a higher degree of re-epithelialization in the C8-ILD-NH2(2) gel-treated wounds than in the nongel control wounds (411.4 vs. 2175.9 pm, p < 0.01). The control wounds also formed a scab on the wound surface (Fig. 7 panel b, left). Thus, C8-ILD-NH2(2) hydrogel significantly promoted re-epithelialization of third-degree bum wounds in C57 / BL6j mice.

[0191] In summary, C8-ILD-NH2(2) hydrogel can accelerate wound healing. No differences were found between the hydrogel -treated group and the PBS -treated control group in terms of mouse behavior, including drinking, foraging, grooming, and eating, or in terms of bodyweight or the size and weight of the liver, kidneys, and spleen. This hydrogel did not show systematic or local toxicity during the wound healing test. Our in vivo observations suggestthat the application of hydrogel to wounds did not elicit any toxic or adverse effects on mouse health. Our study of the C8-ILD-NH2(2) hydrogel for wound treatment has provided preclinical data for the further development of Cn fatty acid-AAl-AA2-D conjugate-based biomaterials for treating burn wounds in humans.

[0192] Notably, at 3 dpb, the wound sizes in both groups were actually larger than the initial size (6 mm in diameter) of the burn wounds created at 0 dpb, whereas the closure (%) at 3 dpb was negative (Fig. 7 panel a). The 6 mm heated rod used for burning cauterized / destroyed the 6-mm-diameter full-thickness skin underneath it; however, it also injured the tissue around its perimeter, causing the burn-wound margin to expand at 3 dpb, even though the necrotic tissue created by burning had already been debrided by conducting the 6-mm-diameter fullthickness excision at 2 dpb. However, the promotion of wound closure and re-epithelialization by the hydrogel (Fig. 7) may reflect its action in promoting growth from surrounding noninjured tissues and cells and / or its ability to partially rescue heat-injured tissue / cells [30,31], The hydrogel can also affect wound vascularization, inflammation, and late-phase remodeling [30,31], The hydrogels described herein can also have effects on bacteria and other microbes [4].

[0193] This report demonstrated the promising potential of new gelable analogs formed by conjugating fatty acids and AA1-AA2-D tripeptides for wound treatment. We are screening other new analogs in which D is replaced by other amino acids, such as glutamic acid, lysine, arginine, or histidine, with electrically charged side chains, and in which the Cn fatty acid is replaced with other fatty acids and their derivatives to generate biomaterials that can be used for better therapeutics. Without wishing to be bound by theory, the Cn fatty acid-AAl-AA2- D analogs likely self-assembled into fibrous structures that further interacted with each other to form the hydrogel network. This can be tested in transmission electron microscopy and circular dichroism studies in the future.

[0194] 4. Conclusions

[0195] We conducted a rational design and organic synthesis of molecular structures capable of forming prohealing hydrogels using two two Fmoc / tBu-based SPPS routes, and we identified 13 new conjugates of saturated fatty acid-aminoacid l-amonoacid2-asparatic acid that can form hydrogels under wound-compatible conditions. The compounds were purified, and analyzed using LCMS and 'H and13C NMR spectroscopic techniques. Among them, C8 acid-ILD-NH2(2) was the best in terms of hydrogelation. Our results further revealed that C8 acid-ILD-NH2(2), in media resembling the pathophysiological wound condition, has desirableinjectability, shear-thinning, and re-gelation features, as well as wound-compatible mechanical properties, that make this compound valuable in wound treatment. The C8 acid-ILD-NH2(2) hydrogel can markedly accelerate the healing of third-degree bum wounds. These results indicate the ability of the Cn fatty acid-AAl-AA2-D molecular template to form hydrogels capable of effectively promoting wound healing.

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[0267] Molecular Modification of Queen Bee Acid and 10-Hydroxy decanoic Acid with Specific Tripeptides: Rational Design, Organic Synthesis, and Assessment for Prohealing and Antimicrobial Properties

[0268] To overcome the limitation of MGH / RJ, we developed new amphiphiles by the modification of their reparative component queen bee acid (hda) and 10-hydroxyl decanoic acid (hdaa) with specific tripeptides via peptide bonding through a rational design and organic synthesis. Our design of the molecular sequences incorporated the amphiphile targets to be biocompatible in wound healing, biodegradable, non-toxic, hydrogelable in wound niche, prohealing, and antimicrobial. Consequently, we designed a molecular template hfa-aal-aa2-aa3with rational selection criteria for each moiety in this short sequence. Following this template, we have then successfully synthesized many amphiphiles by Fmoc / tBu-based SPPS and verified their structures by NMR and LC / MS / MS. Among these new compounds, we tested several hda, hdaa, ILD-NH2, ILK-NH2 -containing amphiphiles, and found that they are suitable to promote wounding as they can form hydrogels with pertinent and stable elastic moduli compatible to wounds for cell and tissue repair and regeneration, injectable, and shearthinning. Our assay revealed that hdaa-ILK-NFF markedly inhibited S aureus. Overall, oOur molecular modification of hda and hdaa with specific tripeptide via peptide bonding is capable in amplifying the prohealing and antimicrobial properties of these active components of MGH / RJ, and having potential to overcome the limitation of MGH / RJ in medical treatments.

[0269] Introduction

[0270] Queen bee acid (10-hydroxy-2-decenoic acid or hda) and 10-hydroxy decanoic acid (hdaa) are the bioactive components of royal jelly and medical grade honey1-6. Medical grade honey (MGH) and royal jelly (RJ) are used to heal various wounds, especially infected wounds, pressure ulcers (bedsores), diabetic ulcers, and / or venous or arterial ulcers1, 7'16. Mitogen- Activated Protein Kinase (MAPK) signaling can be activated by hda5. Hda reduces inflammatory cytokine and signaling, exerts bactericidal activity against infections in gastrointestinal tract5, promotes smooth muscle cell survival against hydroxyl radical-induced injury17, prevents photoaging damage by enhancing collagen production in human dermal fibroblasts18, promotes neurogenesis by neural stem cells19. The hda alleviated blood-brain barrier damage via activating the AMPK / PI3K / AKT pathway20, increases longevity and TOR signaling in C. elegans, and fortifies immunity21. The mechanism for hda actions also involves reducing IKB-(^ expression22, inhibiting the TNF-U / NF-KB axis and NLRP3 inflammasome-IL- ip pathway, increasing FOXO1 -activation of autophagy21, and down-regulating MMPs23. The hda and hdaa contribute to the key pharmacological effects of royal jolly, including antiinflammatory, wound healing, anti-oxidant, anti-bacterial, and insulin-like properties1, 5’24'26. However, the effectiveness of hda, hdaa, MGH, or RJ are still too insufficient to meet the clinical demand in healing infected or chronic woundslj 7'16. Additionally the efficacies of MGH or RJ vary because their compositions rely on honeybees and flowers, which can vary widely and are not realistic to rigorously quality-control and regulate1, posing a high risk to patients. Because they are products of flowers and bees, MGH / RJ can trigger allergic reactions27, 28. Allergy could be avoided if the allergens are not in the modality. Gels and otherwound dressings1, 29, 30and nanomaterials1, 31, 32were used to provide slow release of MGH / RJ, but still do not resolve these weaknesses.

[0271] Wound healing is a crucial physiological process that allows the body to repair tissue damage from injuries or surgeries, preventing infection, maintaining tissue integrity, and enabling recovery from various medical procedures. Impeded wound healing potentially results in serious complications like chronic ulcers, infections, and tissue loss, significantly impairing a patient's quality of life and overall health33'36. The field of regenerative hydrogels is emerging and heavily explored for ideal treatment of wounds. Hydrogels (gels) can hold large amounts of biological fluid, mimic the 3D structure of tissue extracellular matrix to scaffold tissue repair and regeneration37-43and barricade wounds from the external environment. They are semi- permeable to H2O vapor and O2 and CO2 exchange. Gel flexibility and ability to conform to wound contours offer optimal tissue contact, increasing healing. They can be engineered to release the therapeutic at the optimal rate and concentration or in response to a niche or pathological change. Gels can also disrupt microbial biofilms, prevent biofilm re-formation, and render bacteria more susceptible to the treatment and host immune system44. Gel dressing can be removed without the pain caused by changing non-gel dressings45, such as gauzes, plasters, and bandages. Therefore, we incorporate gel functions into our therapeutic development.

[0272] Staphylococcus (S) aureus, a major bacterium in wound infections, often develops antimicrobial resistance (AMR), leading to life-threatening conditions46. Methicillin- resistant S. aureus (MRSA) causes worse clinical outcomes. S. aureus in biofilms exhibits increased virulence and AMR, making antimicrobials less effective and resulting in infected wounds47'49. There is a lack of effective therapies to the AMR of S. aureus for non-healing infected wounds50-52, particularly among older adults, underscoring the need for innovative therapeutics. Moreover, S. aureus can cause a broad range of diseases including pneumonia, endocarditis, osteomyelitis, mastitis, toxic shock syndrome, cellulitis, and impetigo53, 54. In the United States alone, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) results in an annual burden of $478 million to 2.2 billion on third-party payers and $1.4-13.8 billion on society55.

[0273] Described herein are approaches to develop therapeutic molecules that can enhance the medicinal functions of MGH / RJ and their reparative component hda and hdaa and overcome the limitations of these natural products. We performed the molecular modification of hda and hdaa with specific tripeptides through rational design, and solid phase organicsynthesis in order to create pro-healing antimicrobial amphiphiles. We can engineer these new amphiphiles to be biocompatible in wound healing, biodegradable, non-toxic, hydrogelable in wound niche, pro-healing, and antimicrobial. Additionally, we generated new molecules by replacing hda or hdaa in these amphiphiles with representative long linear carbon carboxylic acids including 3 -hydroxyl-decanoic acid (a minor RJ component), 12-hydroxyl dodecanoic acid, and 12-hydroxyl octadecanoic acid for comparison. We then assessed the self-assembling hydrogelability and anti-5 aureus property of selected molecules that we engineered.

[0274] 2. Results and Discussion

[0275] 2.1. Rational Design and Solid Phase Organic Synthesis of New AmphiphilesCovalently Modified from Queen Bee Acid, 10-Hydroxy decanoic Acid, or Other Hydroxy Fatty Acids with Specific Tripeptides

[0276] Amphiphilic molecular structures were targeted for our covalent modification of queen bee acid and 10-hydroxy decanoic acid using specific peptides. Appropriately designed amphiphiles can form pro-healing gels under wound-compatible conditions56'63. Amphiphiles possess a hydrophilic head and a hydrophobic tail. Their ability to form hydrogels depends on the balance between these two regions, which facilitates self-assembly into complex structures, such as fibrils or networks, through non-covalent interactions like hydrogen bonding, electrostatic forces, and van der Waals interactions. This self-assembly results in a gel-like network that can retain large amounts of water. Factors such as the length and structure of the hydrophobic tail, as well as the polarity of the hydrophilic head, play a crucial role in this process64'67. Certain amphiphiles target bacterial cell membranes, disrupting or compromising their integrity while sparing mammalian cell membranes. This includes cationic amphiphiles inspired by natural antimicrobial peptides68-7°. Bacterial membranes are generally more negatively charged, which allows positively charged amphiphiles to bind strongly and disrupt them. In contrast, mammalian membranes have a more neutral charge, reducing interaction and potential damage71-73. When an amphiphile interacts with a bacterial membrane, its hydrophobic region inserts into the lipid bilayer, while the hydrophilic region interacts with the aqueous environment, causing disruption of the membrane structure and leading to cell leakage and death. Depending on the structure of amphiphiles, it can also form pores in the bacterial membrane, allowing the leakage of essential cellular components74. The amphiphile structure dependent balance between the hydrophobic and hydrophilic regions of an amphiphile significantly affects its selectivity75. The arrangement of positive charges on the amphiphile molecule can also influence its interaction with different membranes68.

[0277] We linked hda or hdaa via peptide bond by solid phase organic synthesis to a rationally designed tripeptide to create the gelable Janus amphiphile that is prohealing and antimicrobial76. This amphiphile has hda or hdaa moiety acting as the suitable hydrophobic tail and the tripeptide domain providing the appropriate hydrophilic head and aqueous solubility, and linkage to hda / hdaa, consequently is able to self-assemble into a prohealing and antimicrobial hydrogel. Another advantage to use specific tripeptides for the modification is that they are biocompatible and biodegradable as they can be broken down by tissue enzymes to individual nutrient amino acids77.

[0278] Based on above rationale, we designed an amphiphilic molecular template that links a hydroxyl fatty acid (hfa) and a specific tripeptide via a covalent bondfor example hfa- amino acidl-amino acid2-amino acid3 or hfa-aal-aa2-aa3. The hfa includes hda, hdaa, 3 -hydroxyl-decanoic acid (a minor RJ component), 12-hydroxyl dodecanoic acid, 12-hydroxyl octadecanoic acid, or other hydroxylated fatty acid. The aal includes a hydrophobic amino acid that can enhance the hydrophobicity of our targeted amphiphile, such as alanine (Ala or A), valine (Vai or V), isoleucine (I or He), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), tyrosine (Tyr or Y), or tryptophan (Trp or W). The aa2 can be used to facilitate the hydrophoilicity or act as a linker, thereby it can be hydrophobic including Ala, Vai, He, Leu, Met, Phe, Tyr, or Trp; polar uncharged, such as serine (Ser or S), threonine (Thr or T), asparagine (Asn or N), glutamine (Gin or Q); or with other case side chains, such as glycine (gly or G), cysteine (Cys or C), proline (Pro or P), or selenocysteine (Sec or U). The aa2 can also be used to facilitate the hydrophilicity or provide the linkage, thus it can be polar uncharged (such as Ser, Thr, Asn, or Gin), with other case side chains (such as gly, Cys, Pro, or Sec), or charged (such as Arg, Lys, Asp, or Glu). The aa3 at the C-terminus of the tripeptide is the amino acid that needs to provide the appropriate hydrophilic head and aqueous solubility of our targeted amphiphile, including an amino acid with electrically charged side-chain that can function as the hydrophilic head, such as arginine (Arg or R), lysine (Lys or K), aspartic acid (Asp or D), and glutamic acid (Glu or E). At pathophysiological pH (~7.4), R or K is positively charged due to its side chain containing a guanidinium (pKa -12.5) or alkyl amino group (pKa - 10.7), respectively78; R or K side chain remains protonated and carries a positive charge while its terminus carboxyl is amidized and a-amino group is used in the peptide bond linking to aa2, providing the pertinent hydrophilic head and aqueous solubility for gelation; in contrast, D or E contains a net negative charge because its carboxyl containing side chain loses its proton (with pKa -3.9 for D or -4.1 for E) while its terminus carboxyl is free or amidizedand a-amino group is used in the peptide bond linked to aa2, providing the pertinent hydrophilic head and aqueous solubility for gelation78. Histidine (His or H) can be also used as aa3 due to its imidazole side chain (pKa 6.0) that is protonated and hydrophilic at pH < 5, a wound condition during treatment79, 80, while its terminus carboxyl is amidized and a-amino group is used in the peptide bond linking to aa2, providing the pertinent hydrophilic head and aqueous solubility for gelation.

[0279] This hfa-aal-aa2-aa3 template is modified from our amphiphilic template fatty acid Cn -aal-aa2-D tripeptide, where the carbon chain length is n for the fatty acid Cn81, 82. The hydroxyl of hfa reduces its hydrophobicity compared to the Cn with the same carbon chain length, whereas the hydroxyl can donate hydrogen for hydrogen bonding that could facilitate hydrogelation.

[0280] The synthesis of these hfa-aal-aa2-aa3 amphiphiles was carried out through standard Fmoc-based solid phase peptide synthesis (SPPS) using Rink-amide-resin (Scheme 1). The representative new amphiphiles that we generated from the modification of hda, hdaa, and several other hfa by this approach are presented in Table 1. More of these new amphiphiles are presented in Supplemental Materials. The NMR spectra for compounds 1-4 and 6 provided chemical shifts 5 and coupling constants J that verify their designed structures (Table 1). The LC-MS / MS analysis of these amphiphiles validated their molecular masses and structures (Table 1).

[0281] Compound 1 (hdaa-ILD-NHi). The structure of hdaa-ILD (M = 528.5 Daltons) was identified by the LC-MS / MS diagnostic fragmentation ions m / z 527.3 {the ion of deprotonated molecule: [M - H+]’ = (528 -1 = 527)}, 509 [527 - H2O]’, 412, 394 [412- H2O]’, 242, 224 [242- H2O]’, 207, 165, 131, and 114 (Figure 37D-F, Table 1) and by our NMR data as follows:

[0282] ’H NMR (400 MHz, DMSO-d6): 5 12.23 (brs, 1H), 7.96 (t, J = 8.4 Hz, 2H), 7.87 (d, J = 10.4 Hz, 2H), 7.09 (d, J = 6.1 Hz, 2H), 4.41 (t, J = 7.1 Hz, 1H), 4.37 (t, J = 8.4 Hz, 1H), 4.24 (q, J = 10.0 Hz, 1H), 4.10 (t, J = 10.0 Hz, 1H), 2.65 (q, J = 6.1 Hz, 1H), 2.54 (d, J = 6.1 Hz, 1H), 2.14-2.10 (m, 2H), 1.70-1.66 (m, 2H), 1.59-1.51 (m, 2H), 1.47-1.39 (m, 5H), 1.29-1.24 (m, 12H), 0.87 (d, J = 8.2 Hz, 3H), 0.81 (s, J = 8.2 Hz, 9H);13C NMR (100 MHz, DMSO-de): 5 172.4, 172.2, 171.8, 171.6, 171.4, 68.5, 63.6, 60.7, 57.0, 51.2, 49.4, 40.2, 35.8, 35.6, 33.5, 32.5, 29.0, 28.8, 28.7, 28.6, 28.5, 28.4, 28.3, 28.1, 27.4, 25.5, 25.3, 24.9, 23.0, 21.4, 15.4, 10.7.

[0283] Compound 2 (3-hydroxyl decanoic acid-ILD-NH2PH167). 'll NMR (400 MHz, DMSO-d6): 5 12.18 (brs, 1H), 8.13-8.07 (m, 1H), 8.01-7.88 (m, 2H), 7.10-7.03 (m, 2H), 5.44-5.34 (m, 1H), 4.66 (m, 1H), 4.44-4.39 (m, 1H), 4.28-4.10 (m, 2H), 3.76 (brs 1H), 2.69- 2.55 (m, 2H), 2.28-2.19 (m, 1H), 2.09 (s, 2H), 1.66-1.56 (m, 2H), 1.46-1.42 (m, 4H), 2.09 (s, 1H), 1.66-1.56 (m, 2H), 1.46-1.42 (m, 4H), 1.28 (brs, 10H), 0.88 (brs, 3H), 0.82 (m, 9H). Its LC-MS / MS fragmentation ions verified the molecular mass M 528 Daltons and structure (m / z 527 [M-H+], 509, 399, 284, and 266) (Table 1).

[0284] Compound 3 (hda-ILD-NH2PH166). 'II NMR (400 MHz, DMSO-d6): 5 12.18 (brs, 1H), 8.06 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 14.0 Hz, 1H), 6.67-6.60 (m, 1H), 6.08 (d, J = 16.0 Hz, 1H), 4.43-4.36 (m, 2H), 4.25-4.17 (m, 2H), 3.38 (t, J = 13.0 Hz, 1H), 2.68-2.63 (m, 1H), 2.55 (d, J = 7.5 Hz, 1H), 2.15-2.10 (m, 2H), 1.76-1.67 (m, 1H), 1.62-1.52 (m, 1H), 1.48-1.39 (m, 6H), 1.31-1.26 (m, 8H), 0.87 (d, J = 8.5 Hz, 3H), and 0.81 (d, J = 8.5 Hz, 9H);13C NMR (100 MHz, DMSO-d6): 5 172.3, 171.8, 171.7, 171.3, 165.2, 143.0, 124.2, 68.5, 60.6, 57.2, 51.3, 49.5, 36.2, 35.8, 32.5, 31.2, 28.7, 28.6, 28.4, 28.2, 27.7, 27.6, 27.3, 25.4, 24.9, 24.5, 24.0, 23.0, 21.4, 15.3, 10.9. Its LC-MS / MS fragmentation ions verified the molecular mass M 526 Daltons and structure (m / z 525 [M - H+], 507 [525 - H2O]’ , 410, 392 [410 - H2O]’, 357, 242, 224, 207, 165, 131, 114) (Figure 37 panel A, Table 1).

[0285] Table 1. Representative new amphiphiles generated by hydroxy fatty acid- tripeptide conjugates reported in this work: a brief of their chemical structures, MS / MS ions, hydrogelation features, and gelation time.

[0286] Notes: (a) C-terminus, amidated; N-terminus, hydroxyl fatty acid acylated; hda, queen bee acid (10-hydroxy-2-decenoic acid); hdaa, 10-hydroxy decanoic acid; letters I, L, D, and K denote amino acids: I - isoleucine, L - leucine, D- aspartic acid, K - lysine; NH2- C- terminus amide, (b) MS / MS ion acquired by LC-MS / MS, single charged (z = 1, m / z = Daltons); (c) Compound concentration, 1.5% w / v; pH in PBS saline, 7.0-7.5; (d) gelation time in minutes.

[0287] Compound 4 (12-hydroxyl dodecanoic acid-ILD-NHi PH165). 'II NMR (400 MHz, DMSO-d6): 5 12.26 (brs, 1H), 7.96 (t, J = 8.4 Hz, 2H), 7.88 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 5.1 Hz, 2H), 4.42-4.36 (m, 2H), 4.24 (q, J = 7.2 Hz, 1H), 3.99 (t, J = 8.3 Hz, 1H), 3.34 (brs, 2H), 2.67-2.61 (m, 1H), 2.55 (d, J = 8.0 Hz, 1H), 2.26 (t, J = 6.5 Hz, 1H), 2.13 (d, J = 5.8 Hz, 1H), 1.70-1.66 (m, 2H), 1.59-1.46 (m, 9H), 1.24 (brs, 20H), 0.87 (d, J = 7.2 Hz, 3H), and 0.82 (d, J = 6.5 Hz, 9H);13C NMR (100 MHz, DMSO-d6): 5 172.2, 172.1, 171.8, 171.7, 171.4, 68.5, 63.5, 60.7, 56.9, 51.1, 49.3, 40.3, 35.9, 35.8, 35.0, 33.5, 32.5, 29.0, 28.9, 28.8, 28.6, 28.5, 28.4, 28.1, 27.4, 25.4, 25.3, 24.9, 24.4, 24.0, 23.0, 21.4, 15.3, 10.7. Its LC-MS / MS fragmentation ions verified the molecular mass M 556.5 Daltons and structure (m / z 555 [M- H+], 537, 438, 423, 327, 282) (Table 1).

[0288] Compound 5 (12-hydroxyl octadecanoic acid-ILD-NHi PH168). Its LC- MS / MS fragmentation ions verified the molecular mass M 640.5 Daltons and structure (m / z 639 [M - H+], 621, 524, 506, 394, 298) (Table 1).

[0289] Compound 6 (hdaa-ILK-NH2PH165). 'II NMR (600 MHz, DMSO-d6); 7 97(d, J = 7.5 Hz, 1H), 7.87 (d, J = 9.5 Hz, 6H), 7.75 (brs, 4H), 7.26 (brs, 1H), 7.03 (brs, 1H), 4.37 (t, J = 11.8 Hz, 1H), 4.29-4.25 (m, 1H), 4.16-4.11 (m, 2H), 3.98 (t, J = 5.5 Hz, 1H), 3.98 (t, J = 5.5 Hz, 1H), 3.36 (t, J = 6.5 Hz, 1H), 2.76-2.74 (m, 2H), 2.27-2.24 (m, 1H), 2.17-2.08 (m, 2H), 1.68-1.66 (m, 4H), 1.53-1.45 (m, 12H), 1.23 (s, 18H), 0.87 (d, J = 6.8 Hz, 2H), 0.81 (t, J= 11.8 Hz, 3H);13C NMR (150 MHz, DMSO-ck); 173.2, 172.3, 171.5, 171.3, 68.5, 63.5, 60.7,56.9, 52.0, 51.1, 40.3, 38.6, 36.0, 35.1, 33.5, 32.5, 31.4, 29.0, 28.9, 28.7, 27.4, 26.6, 25.5, 25.4,24.9, 24.4, 24.0, 23.0, 22.1, 21.4, 15.3, 10.7. Its LC-MS / MS fragmentation ions verified the molecular mass M 640.5 Daltons and structure (m / z 542.5 [M + H+], 524, 496, 414, 395, 297) (Table 1).

[0290] 2.2. Prohealing Relevant Hydrogelability of Amphiphiles Covalently Modified from hda, hdaa, or Other hfa with Specific Tripeptides

[0291] The hydrogelation of these representative new amphiphiles (1-6, Table 1) was undertaken in wound compatible PBS buffer at 1.5% w / v concentration (0.75 mg in 0.5 mL PBS). We confirmed every gel formation by widely used vial inversion test81, 82. These compounds were visually insoluble in PBS but attained solubility by adjusting the pH using 0.1 M NaOH and / or 0.1M HC1. The pH was adjusted again to 7.4. The white precipitate (suspension) was formed after the addition, the solution was sonicated for a minute, and incubated at ambient conditions for gel formation. All the compounds except compound 5, formed hydrogels within 30 min, the duration termed gelation time (GT) (Table 1). At 1.5% w / v and pH 7-7.5, the GT is shorter for compound 1 (12 mins) with hydroxyl at CIO of the decanoyl than compound 2 (30 mins) with hydroxyl at C3 of the decanoyl moiety, suggesting ClO-hydroxyl is more favorable than C3-hydroxyl for that hydrogelation (Table 1). The double-bond of 10-hydroxyl dec-2-enoyl moiety of compound 3 corresponded to shorter GT (5 mins) compared to compound 1 (12 mins) where this double-bond did not exist (Table 1), which reflects the effect of C2 double-bond on hydrogelation. The 7i-electrons of the C2 double bond conjugate with the 7t-electrons of the Cl carbonyl double bond, delocalizing the electron density across the conjugated system, making the electron donor O atom of carbonyl in a hydrogen bond more electron-rich and thus increasing the strength of the hydrogen bond interaction; essentially, the conjugated system can stabilize the partial positive charge on the hydrogen bond donor, consequently contributing to hydrogelation. The GT is shorter for compound 1 (12 mins) with a 10-hydroxyl decanoyl moiety than for compound 4 (20 mins) with a 12-hydroxyl-dodecanoyl moiety, suggesting 10-hydroxyl decanoyl is better for hydrogelation than 12-hydroxyl-dodecanoyl moiety when other molecular structural features are the same. The 12-hydroxyl dodecanoyl with longer hydrophobic carbon-chain should reduce hydrophobicity and aqueous solubility of the hfa-ILD-NH2 amphiphile more than 10- hydroxyl-decanoyl, which could contribute to the longer GT of compound 4. Hydrophoblic domain with pertinent hydrophobicity is essential for an amphiphile to self-assemble intohydrogel. Too much or too little hydrophobicity can tip off the balance between the hydrophobicity and hydrophilicity of an amphiphile, resulting in the failure in hydrogelation. This could explain why in the same hfa-ILD-NH2 template compound 5 with 12-hydroxyl octadecanoyl moiety failed to hydrogelate whereas compound 4 with a 12-hydroxyl- dodecanoyl moiety is gelable (Table 1).

[0292] Furthermore, we observed that the GT for a particular compound is inversely proportional to the concentration. For example, the GT for a 0.5% w / v compound is longer as compared to the 1% w / v concentration. It was also observed that the hydrogel formed at 0.5% w / v concentration was clear whereas hydrogels formed at higher concentrations were opaque.

[0293] We also examined the stability of these hydrogels at various pH levels, which encompass the pH range of human wounds under different treatments83, 84These hydrogels remained gelated from pH 2 to pH 8, indicating their ability to maintain stability within a pathophysiologically relevant pH range. The hydrogel of compound 1 at 1.5% w / v changed from opaque to more translucent when pH was increased from 2 to 10 (Figure 39 panel A), and became liquid solution at pH 11.5. In comparison, the hydrogel of compound 3 changed similarly when pH increased from 2 to 8 (Figure 39 panel B), and dissolved at pH 10. The double-bond in 10-hydroxyl dece-2-noyl moiety of compound 3 reduced the tolerance from pH 10 to 8 in hydrogel stability although it can reduce the gelation time (Table 1). The conjugation of the C2 double bond with Cl carbonyl double bond could be more sensitive to maximal pH although it can enhance pro-gelling hydrogen bonding.

[0294] 2.3. Prohealing and Antimicrobial Related Rheological and InjectableProperties of New Hydrogels Self-assembled from Selected Amphiphiles Covalently Modified from hda and hdaa with Specific Tripeptides

[0295] We determined prohealing and antimicrobial related rheological and injectable properties of new hydrogels that were self-assembled from selected amphiphiles covalently modified from hda and hdaa with ILD tripeptide. ILD was selected because we recently found that caprylic acid-ILD-NH2 amphiphile is able to self-assemble to prohealing hydrogel that is wound-compatible, shear-thinning, injectable, non-toxic, and biodegradable81, 82. The gel injectability represents its ability to precisely fill 3D surfaces and cavities as a liquid and then solidify onsite as an elastic matrix although this gel can be applied to wounds by pipetting, swapping, or spraying other than injection. The hydrogel should comply with wound healing physiochemically and mechanically for the repair and regeneration of cells and tissue.

[0296] The mechanical properties of our new hda-ILD-NFE (3) gel and hdaa-ILD-NFE (1) gel prepared from compounds 3 and 1 respectively (Table 1) were assessed using an Anton- Paar MCR 092 rheometer, and presented in Figure 40. The amplitude / shear strain sweep test of these hydrogels showed that storage modulus (G’) was higher than loss modulus (G”) in the limit of the linear viscoelastic region (LVE) (Figure 3 panels A and E), indicating the presence of an elastic hydrogel structure. When the shear strain was increased from 0.01% to 100%, the storage modulus (G’) and loss modulus (G”) slowly decreased until 1% strain, followed by a sharp decrease, and reached a point where G’ and G” curves crossed over each other (crossover point G’ = G”). Further increase in strain leads to shear thinning where G’ < G”, indicating a transition from the gel state to a viscous liquid state. The hda-ILD-NFE (3) gel showed the highest value (G’ = 100000 Pa) for storage modulus (G’) and loss modulus (G”). The frequency sweep experiment (Figure 40 panels B and F) was carried out at a constant 1% strain (within the LVE range) with a frequency range from 100-0.1 rad / s; both hydrogels showed gel status with constant values of storage modulus and loss modulus, where G’ is more than G” and independent of the frequency, reflecting the high stability of these hydrogels; and both hydrogels had storage G’ moduli between ~lk and ~10k Pa, compatible to skin tissue85, 86. The elastic nature of these gels further supports their suitability for wound treatment.

[0297] The shear-thinning properties of hda-ILD-NFE (3) gel and hdaa-ILD-NFE (1) gel were examined using the time-dependent shear thinning thixotropic tests (Figure 40 panels C and G). In this experiment, the shear strain was suddenly increased from 0.1% to 200% for 4 minutes and then reduced back to 0.1%. The G’ and G” values remained constant when 0.1% strain was applied for the initial 2 mins, then the G’ and G” values dropped abruptly when 200% strain was imposed. It was worth noting that G” was more than G’, where the fluid turned into solution and remained as a solution until the 200% strain was removed. These changes revealed the rapid shear-thinning properties of both gels, as the gels were liquefied by the increased shear strain. The G’ and G” values completely switched back to their original values when the 0.1% strain was restored. The shear-thinning and regenerative properties of both hydrogels provided mechanistic insights into their injectability. To directly determine the gel injectability, we liquefied each gel by shaking, drew the liquid into a syringe through a 25- gauge needle, then injected the liquid into the target site. We observed spontaneous re-gelation, as demonstrated in the photo of this process (Figure 40 panel D lower panel).

[0298] 2.4 Determination of Gelation-related fibrous Structures Using TransmissionElectronic Microscopy

[0299] To determine the hydrogelation-related fibrous structures formed from hfa- ILD-NH2, we conducted transmission electronic microscopy (TEM) study of the representative one, i.e. hda-ILD-NFE (3). The TEM study revealed the nano-fibrous rod-like structures formed from hda-ILD-NEk (3) at *60000 magnification (Figure. 4). There is a high propensity for hda-ILD-NFE (3) molecules to form fibrils through intermolecular hydrogen bonds between N-H (hydrogen bonding donor) and C=O (hydrogen bonding acceptor) groups as it contains four N-H groups, one hydroxy group, and 5 C=O groups to form hydrogen bonds along its backbone (Table 1). Additionally, the nonpolar side chains of I and L, along with the hydrocarbon tail of hda in hda-ILD-NH2 (3), can interact with the corresponding groups of another (3) through hydrophobic interactions, thereby promoting fibril formation87

[0300] 2.5. The assessment of hdaa-ILK-NEE (6) inhibition of S. aureus

[0301] We explored the antimicrobial activity of hdaa-ILK-NEE (6) described in Table 1 against S. aureus using the standard Kirby-Bauer disc diffusion method. We found that hdaa- ILK-NH2 (6) markedly inhibited S. aureus in the culture dish with 25 mm diameter of the inhibition zone (Figure 5). This observation indicates that the amphiphile hdaa-ILK-NEE (6) modified from hdaa with tripeptide ILK-NH2 by peptide bonding is anti-X aureus. It is likely to be also antimicrobial against other bacteria as it is a unique amphiphile that tends to interrupt the bacteria membrane based on its structure. Additional amphiphiles that we designed and synthesized by the modification of hda, hdaa, and other hydroxyl fatty acids with specific tripeptides could also have the antimicrobial activities. These warrant the investigation in the future.

[0302] 3. Materials and Methods

[0303] 3.1. Materials

[0304] Fmoc-protected amino acids namely Fmoc-Ile-OH (CAS No. 71989-23-6, 98%), Fmoc-Leu-OH (CAS No. 35661-60-0, 99.78%), Fmoc-Asp(OtBu)-OH (CAS No. 71989-14-5, 99.92%) and fatty acids, namely 10-hydroxy decanoic acid (CAS No. 1679-53-4, 99.77%), 3-hydroxydecanoic acid (CAS No. 14292-26-3, 99.25%), (E)- 10-hydroxy dec-2- enoic acid (CAS No. 14113-05-4, 98.0%), 12-hydroxydodecanoic acid (CAS No. 505-95-3, 97%), were purchased from BLD Pharmatech Co. Ltd. (Cincinnati, USA) and used without further purification. 12-Hydroxystearic acid (CAS No. 106-14-9, 97%), was purchased from Chemsavers Inc. (Bluefield, VA, USA). Fmoc rink amide resin (0.57 mmol / g, 100-200 mesh), the Kaiser test kit (Catalog no. KGZ001), and O -benzotri azol e-N, N, N’ and N’- tetramethyluronium-hexafluoro-phosphate (CAS No. 94790-37-1) were purchased fromAapptec, LLC (Louisville, KY, USA). Hydroxybenzotriazole (HOBt, CAS No. 2592-95-2, 98.75%) was purchased from Apexbio (Houston, TX, USA). Trifluoroacetic acid (TFA; CAS No. 76-05-1, 99%) was purchased from Honeywell Research Chemicals (Muskegon, MI, USA). Piperidine (CAS No. 110-89-4, 99%) was purchased from Sigma-Aldrich Co., LLC (St. Louis, MI, USA). N, N-diisopropylethylamine (CAS No. 7087-68-5, 99%) was purchased from TCI America (Portland, OR, USA). N, N-dimethylformamide (DMF; CAS No. 68-12-2), diethyl ether (DE; CAS No. 60-29-7), and dichloromethane (DCM; CAS No. 75-09-2) were obtained from Thermo-Scientific (Ward Hill, MA, USA).

[0305] 3.2. Synthesis and structural analysis of fatty acid-conjugated tripeptides

[0306] The hydroxy fatty acid-conjugated peptides were synthesized using Fmoc- based solid-phase peptide synthesis (SPPS) strategies manually using PolyPrep columns obtained from Bio-Rad Laboratories (Hercules, CA, USA). The synthesis was carried out on a 0.1 mmol scale on the Fmoc-Rink amide resin.

[0307] 3.2.1. General procedure for synthesis of fatty acid-conjugated compounds 1-5.

[0308] The Fmoc-Rink amide resin (175 mg) was swollen in di chloromethane (DCM)(5.0 mL) in a Biorad column for 30 min. The solvent was then pushed out and 20% piperidine in dimethylformamide (DMF) (5.0 mL) was added, and the mixture was shaken well for 20 min. The solvent was removed, and the resin was washed with DMF (3 x 5 mL) and DCM (3 x 5 mL). The Fmoc group removal was confirmed by a positive Kaiser test. The Fmoc- Asp(tBu)-OH (164 mg) was placed in a scintillation vial and dissolved in DMF (5.0 mL) together with HBTU (152 mg), HOBt (54 mg), and DIPEA (0.2 mL). The mixture was sonicated for 1 min and then added to a resin column, which was shaken on a vortex mixer for 6 h. The solvent was removed from the column and the column was washed with DMF (3 x 5 mL) and DCM (3 x 5 mL). The amino acid coupling was confirmed by a negative Kaiser test. The resin was end-capped by adding 5 mL acetic anhydride: pyridine solution (3:2, v / v) and rocking the resin for 1 h, followed by washing with DMF (3 x 5 mL) and DCM (3 x 5 mL). The Fmoc group was removed by the addition of 20% piperidine in DMF (5.0 mL) and shaking for 20 min. The solvent was removed, and the column was washed with DMF (3 x 5 mL) and DCM (3 x 5 mL). The second, and third amino acid coupling and fatty acid coupling were carried out by using the same protocol of Fmoc group removal and next amino acid / fatty acid coupling. The cleavage of the fatty acid-peptide conjugate from the resin was carried out by adding a mixture of TFA: H2O: TIPS (5.0 mL; 95:2.5:2.5 v / v) to the resin, and the mixture was stirred at room temperature for 2 h. The resin was filtered and washed by DCM (5 mL). Thefiltrate was evaporated on a rotary evaporator to remove excess TFA. Ice-cold diethyl ether was added to the resulting crude product to yield a white solid precipitate. The white precipitate was filtered through the sintered funnel, and the precipitate was washed three times with cold diethyl ether. The trifluoroacetic acid counter-anion was replaced with HC1 by dissolving the white precipitate obtained after ether trituration in 5.0 mL of 0.1 M HC1 solution, stirring for 15 min, and then adding 5 mL acetonitrile. The soluble mixture was then dried in a dry ice bath and lyophilized overnight in a freeze-dryer (Thermo Savant, Holbrook, NY, USA), to get white powder. The structure of the fatty acid-peptide conjugates was confirmed by LC-MS / MS analysis.

[0309] 3.2.2. Structural analysis. The reagents and the fatty acid-amino acid / peptide conjugates were analyzed using an LC-MS / MS system consisting of an Agilent 1100 LC system (HPLC-DAD-autosampler, Agilent Technologies, Inc., Santa Clara, CA, USA) and a QTRAP 6500+quadruple-linear trap mass spectrophotometer with electrospray ionization (Sciex.com, Framingham, MA, USA). The NMR analysis was carried out using a Brucker 400 MHz NMR instrument, DMSO-de (CAS No. 2206-27-1, 99.9 atom% D, Thermo-scientific, Fair Lawn, NJ, USA) as a solvent, and Topspin 4.3.0 version software. The 'H NMR (400 MHz), 13C NMR (100 MHz), and 13C DEPT data were acquired using a purified compound (20 mg) dissolved in 0.7 ml DMSO-de in a 5 mm diameter NMR tube. DEPT-135 was used to determine the multiplicity of carbon atoms, CH2 groups showed inverted signals whereas, CH and CH3 groups were upright. The quaternary carbon (C) did not show any signal.

[0310] 3.3. Hydrogel formation

[0311] 3.3.1. Hydrogel preparation. Lyophilized hydroxy fatty acid peptide conjugates were dissolved in phosphate-buffered saline (PBS) at a final concentration of 0.5% (5.0 mg in 1 mL PBS), 1.5% (15.0 mg in 1 mL PBS), and 3% (30.0 mg in 1 mL PBS). The pH of the peptide solutions was increased to 9.0 by adding 0.1 M NaOH to dissolve the compounds and readjusted to pH 7.4 by drop-wise addition of 0.1 N HC1, followed by sonication. Compounds 1-4 immediately formed suspension after sonication; then the solution was kept at room temperature for gel formation. The gel formation was confirmed by the vial inversion method, and photographs were taken.

[0312] 3.3.2. Hydrogel sterilization. PBS, 0.1 N HC1, 0.1 M NaOH, pipette tips, andEppendorf tubes were autoclaved (Steris, AMSCO 250 LS) at 130°C for 45 min. The other procedures for hydrogel formation were performed under pathogen-free conditions inside a BSL-2 hood.

[0313] 3.4. Rheological studies

[0314] Tests were performed on 50 pL hydrogel samples using an Anton-Paar MCR 092 rheometer (Anton Paar USA, Inc., Houston, TX, USA) with a 20 mm cone plate at a measuring gap of 39 pm. The effects of concentration on gel strength and viscoelastic behavior were assessed by conducting amplitude / strain sweep experiments for all gels using oscillatory shearing strain. Storage (G') and loss (G") moduli were measured as a function of strain (ranging from 0.01 to 100%) at a constant frequency of 10 rad / s. The mechanical stability of the peptide hydrogels was tested by running frequency sweep experiments at angular frequencies ranging from 1 to 100 rad / s at a constant 1% strain, which was under the limit of the linear viscoelastic region obtained from the amplitude sweep test. The hydrogel structure survived the tests under this strain. We studied the thixotropic properties to understand the time-dependent shear-thinning of gels under 4 min of constant high shear strain (200%) that liquefied the gel, and we then followed the re-gelation after the shear strain returned to the low shear strain of 0.1% used for the initial 2 min test. The G' and G" values provide the elastic gel-like and viscous liquid behaviors of our gels, respectively88.

[0315] 3.5. Procedures for Transmission Electronic Microscopy. Hydrogels dissolved with water was drop-casted (1 pL) onto carbon-coated 300 mesh carbon grids (Ted Pella Inc., Redding, CA, USA) and then air-dried for 10 mins. The carbon film was washed 3 times with deionized water drops and the grids were negatively stained with 2% uranyl acetate (VWR LLC, Philadelphia, PA, USA) in water and air-dried. Stained grids were examined under a JEOL 2010 transmission electron microscope (operated at 200 keV) and TEM images were acquired.

[0316] 3.6. Antibacterial Activity Assessment by Kirby-Bauer Disc Diffusion Method

[0317] The antibacterial activity of hdaa-ILK-NEE (6) against S. aureus was assayed by the agar disc diffusion method, the Kirby-Bauer disc diffusion method, following the published procedures89. Nutrient Agar (NA) medium was used to cultivate S. aureus (stain: Zen 29). S. aureus suspension was diluted and adjusted to the equivalent of the 0.5 McFarland standard (~1 x 108CFU / mL) and then inoculated to the plates (100 pl / plate). A gel puncture was use to generate 6 mm diameter wells. The hdaa-ILK-NEE (6) (5 mg) was added to the well. The plates were incubated at 37 °C for 24 h. The inhibition of S. aureus was assessed visually based on the size of clear zone surrounding the well and recorded as the diameter in millimeters.

[0318] 4. Conclusion

[0319] To overcome the limitation of MGH / RJ, we developed new amphiphiles by the modification of their reparative component hda and hdaa with specific tripeptides via peptide bonding through a rational design and organic synthesis. Our design of the molecular sequences incorporated the amphiphile targets to be biocompatible in wound healing, biodegradable, nontoxic, hydrogelable in wound niche, pro-healing, and antimicrobial. Consequently, we designed a molecular template hfa-aal-aa2-aa3 with rational selection criteria for each moiety in this short sequence. Following this template, we have then successfully synthesized many amphiphiles by Fmoc / tBu-based SPPS and verified their structures by NMR and LC / MS / MS. Among these amphiphiles, we tested several hda, hdaa, ILD-NH2, ILK-NH2 -containing ones, and found that they are suitable to promote wounding as they can form hydrogels with pertinent and stable elastic moduli compatible to wounds for cell and tissue repair and regeneration, injectable, and shear-thinning. Our assay indicated that hdaa-ILK-NFB markedly inhibited S aureus.

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[0410] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMSWhat is claimed is:

1. A compound according to Formula (I) or Formula (II):O aa2ORA\N^RI(Formula I); whereinRi is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C,P, U, W, D, E, L, H, or R; aas is selected from the group consisting of D, E, L, H, or R; andR4 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic group, or a hydroxylated derivative thereof;(Formula II); whereinRi is -NH2 or -OH; aa2 is selected from the group consisting of A, V, I, L, M, F, Y, W, S, T, N, Q, G, C, P, U, W, D, E, L, H, or R; aas is selected from the group consisting of D, E, L, H, or R; aai is selected from the group consisting of A, V, I, L, M, F, Y, or W; andR2 is a C4-C40 saturated aliphatic group, a C4-C40 unsaturated aliphatic, or a hydroxylated derivative thereof.

2. The compound of claim 1, wherein the R2-C(O)- group or R4-C(0)- group are selected from the group consisting of 10-Hydroxydecanoyl, 3 -Hydroxy decanoyl, 10- Hydroxydec-2-enoyl, 12-Hydroxydodecanoyl, 12-Hydroxy octadecenoyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, decadecanoyl, docosanoicoyl, tetracosanoyl, hexacosanoyl, docosahexaenoyl, docosahexaenoyl, docosapentaenoyl, prostaglandin El, eicosapentaenoyl, arachidonoyl, linol enoyl, linoleicoyl, octadecenoyl, hexacosanoyl, a C4-40 saturated fatty carbonyl, or a hydroxylated derivative thereof.

3. The compound of claim 1, wherein the compound is:CI2-ILD-NH2;C16-ILD-0H;C8-VLD-NH2;-hydroxyl dodecanoic acid-LD-NH2;-hydroxyl octadecanoic acid-ID-NH2;-hydroxyl octadecanoic acid-LD-NH2;-hydroxyl octadecanoic acid-IK-NH2;4. A hydrogel comprising one or more compounds of claim 1.

5. The hydrogel of claim 4, further comprising a buffer or cell culture media.

6. The hydrogel of claim 5, wherein the buffer is selected from the group consisting of a saline buffer, a phosphate buffer, PBS (Phosphate Buffered Saline), DPBS (Dulbecco's Phosphate Buffered Saline), HBSS (Hank's Balanced Salt Solution), or any combination thereof.

7. The hydrogel of claim 4, wherein the cell culture media is selected from the group consisting of MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI-1640, IMDM (Iscove's Modified Dulbecco's Medium), or any combination thereof.

8. The hydrogel of claim 5, wherein the saline buffer comprises a phosphate-buffered saline solution.

9. The hydrogel of claim 4, wherein the gel has a viscoelasticity of about 0.1-15 kPa.

10. The hydrogel of claim 4, wherein the gel comprises about 0.05 % w / v to about 10% w / v of one or more compounds of claim 1.

11. The hydrogel of claim 4, wherein the hydrogel comprises a pH of about 2 to about 10.5.

12. The hydrogel of claim 4, wherein the hydrogel comprises a swelling ratio of about 100% to about 400%.

13. The hydrogel of claim 4, further comprising an additional active agent.

14. The hydrogel of claim 13, wherein the additional active agent comprises an antiviral agent, an antimicrobial agent, an analgesic agent, an anti-inflammatory agent, or any combination thereof.

15. A method of promoting wound healing and / or tissue regeneration, the method comprising administering the hydrogel of claim 4 to a wound.

16. The method of claim 1, wherein the administration comprises topical administration or parenteral administration.

17. The method of claim 15, wherein the wound comprises a bum wound, an infected wound, a pressure ulcer, a diabetic ulcer, a venous ulcer, an arterial ulcer, or any combination thereof.

18. The method of claim 15, wherein the gel increases the healing rate of the wound by about 1.5 times to about 20 times.

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