Injectable adhesive

Abstract

The present disclosure is directed to injectable adhesives designed for muscle regeneration that are self-healing, highly stretchable, and biodegradable, along with providing strong tissue adhesion. Applied to muscle injuries, they improved healing, with treated mice showing greater muscle weight and strength than controls, advancing medical adhesive technology.

Description

[0001] INJECTABLE ADHESIVE

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 553,452, filed on February 14, 2024. The entire contents of the foregoing application are expressly incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under DE013349 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

[0006] BACKGROUND

[0007] Traumatic muscle injuries, including those from volumetric muscle loss (VML), are clinically challenging due to their profound impact on patients' mobility, overall well-being, and quality of life1'4|. Currently, the leading treatments primarily involve scar tissue debridement, the positioning of muscle flaps at the defect site, and surgical intervention15,61. However, these strategies generally yield only sub-optimal outcomes in both aesthetics and function13,71. With the increasing prevalence of muscle-related injuries, there is a growing need for more effective treatment strategies18 101. In recent years, biomaterials placed in muscle defects have been demonstrated to enhance tissue regeneration1" '7|. They provide a supportive matrix at the injured site for cellular infiltration, and can serve as a depot for therapeutics to facilitate tissue integration and growth1'8 221. Thus, biomaterial -based strategies could yield new therapeutic options for patients suffering from debilitating muscle injuries, provide alternative solutions to traditional surgical procedures, and enhance understanding of the complex biological processes involved in muscle regeneration.

[0008] To promote muscle regeneration, biomaterials likely need to fill tissue voids and remain in defects over the time course of healing, and tissue adhering biomaterials are attractive for these purposes123,241. Compared to the use of sutures and staples to affix biomaterials in a defect, tissue adhesives can reduce the risk of infection, decrease inflammation, and simplify surgical procedures125 27Injectable adhesives are particularly attractive as their minimally invasive delivery can shorten recovery time and reduce the risk of post-operative complications, while adapting their shape to match irregular defects and ensure complete filling and better integration with the surrounding tissue128 311. A key challenge though, relates to the dynamic mechanical loads intrinsic to muscle during a healing process. Muscles facilitate force generation and body movement through the continuous cycles of contraction and relaxation132’331, and defect-filling adhesives need to withstand these deformations without compromising their structural, mechanical, and adhesive properties. In addition, adhesives should degrade over time after being applied to muscle to allow regenerating tissue to replace the biomaterial[34,35]. However, most existing adhesives fail to meet all these criteria.

[0009] SUMMARY

[0010] The present invention discloses a new class of injectable adhesives to promote muscle regeneration by leveraging dynamic covalent Schiff base chemistry for hydrogel crosslinking and tissue adhesion. These adhesives are viscoelastic and highly deformable, and can be easily injectable through needles (Example 2). They rapidly self-heal after fracture, with a return to the initial mechanical properties. More importantly, these adhesives demonstrated strong adhesion to various tissues and tunable biodegradability (Example 3). When tested on VML injuries, these adhesives remained securely in place during the healing process, and treated mice exhibited increased muscle weight and generated higher forces compared to those in the control groups (Example 4).

[0011] Accordingly, in one aspect, the present invention provides an injectable adhesive comprising: i) a polymer containing a carboxylic acid and a side chain represented by formula (I): -C(=O)-NH-L-NHC(O)-R (I);

[0012] R is optionally substituted benzaldehyde;

[0013] L is Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene; wherein one or two carbon atoms of Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene may optionally be replaced with O, N, S, S(O) or S(O)2, and / or two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or more R1; wherein said Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene represented by L is optionally substituted with one or more R1; wherein

[0014] R1, in each occurrence, is independently halogen, CN, Ci-ealkyl, -ORla, oxo (=0, as appropriate) or -NRlaRlb; wherein Rlaand Rlbare independently selected from the group consisting of hydrogen, Ci-ealkyl, Ci-ehaloalkyl, 3-6 membered carbocyclyl, and 4-6 membered heterocyclyl; and ii) a primary or secondary amine polymer.

[0015] In some embodiments, in the injectable adhesive disclosed herein, the polymer is poly(acrylic acid), poly(methacrylic acid), poly(crotonic acid), poly(isocrotonic acid), poly(2-ethylpropenoic acid); poly(maleic acid), poly(fumaric acid), poly(itaconic acid), poly(citraconic acid), or co-polymers of carboxylic acids, each of which contains the side chain of formula (I), wherein the co-polymers of carboxylic acids contains acrylic acid, methacrylic acid, crotonic acid, isocrotonic acid, 2-ethylpropenoic acid, maleic acid, fumaric acid, itaconic acid, or citraconic acid.

[0016] In some embodiments, the co-polymers used in the present application can be any co-polymer comprising carboxylic acid. In one specific embodiment, the co-polymers of carboxylic acids is poly(m ethyl methacrylate / methacrylic acid), poly(butadiene / maleic acid).

[0017] In some embodiments, in the injectable adhesive disclosed herein, the polymer is poly(acrylic acid) or poly(methacrylic acid), each of which contains the side chain represented by formula (I). In another embodiment, the polymer is poly(acrylic acid) which contains the side chain represented by formula (I).

[0018] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 5:95 to 50:50.

[0019] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 5:95 to 40:60.

[0020] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 5:95 to 35:65.

[0021] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 5:95 to 10:90.

[0022] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 10:90 to 40:60.

[0023] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 20:80 to 40:60.

[0024] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 25:75 to 35:65.

[0025] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 30:70 to 40:60. In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 30:70 to 35:65.

[0026] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 8(±2):92(±2).

[0027] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 30(±2):70(±2).

[0028] In some embodiments, the molar ratio between the side chain and the carboxylic acid contained in the polymer is 35(±2):65(±2).

[0029] In some embodiments, in the side chain represented by formula (I), L is Ci-ealkylene optionally substituted with one or more R1, and two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or two R1.

[0030] In some embodiments, in the side chain represented by formula (I), L is Ci-ealkylene optionally substituted with one or two R1, and two adjacent carbon atoms are replaced to form 5-6 membered heteroaryl optionally substituted with one or two R1.

[0031] In some embodiments, in the side chain represented by formula (I), R1, in each occurrence, is independently halogen, CN, OH, Ci-4alkyl, Ci-4alkoxy, or -NRlaRlb; wherein Rlaand Rlbare independently selected from the group consisting of hydrogen and Ci-4alkyl.

[0032] In some embodiments, in the side chain represented by formula (I), the side chain is represented by -C(=O)-NH-CH2-triazole-(CH2)2-NH(CO)-R.

[0033] In some embodiments, in the side chain represented by formula (I), R is benzaldehyde optionally substituted with one to three halogen, CN, Ci-ealkyl, -ORla, or -NRlaRlb(other than the -C(=O)-NH-L-NHC(O)- moiety). In one embodiment, R is benzaldehyde optionally substituted with one or two F, CN, Ci-4alkyl, -OH, or -NH2 (other than the -C(=O)-NH-L- NHC(O)- moiety). In another embodiment, R is unsubstituted benzaldehyde (other than the -C(=O)-NH-L-NHC(O)- moiety).

[0034] In some embodiments, in the injectable adhesive disclosed herein, the primary or secondary amine polymer is gelatin, chitosan, glycol chitosan, collagen, polyallylamine, polylysine, polyethylamine, or polyethyleneimine. In one embodiment, the primary or secondary amine polymer is gelatin, chitosan, glycol chitosan, polyallylamine, or polylysine. In another embodiment, the primary or secondary amine polymer is gelatin.

[0035] In some embodiments, the injectable adhesive disclosed herein further comprises a crosslinker for the primary or secondary amine polymer. In one embodiment, the crosslinker is transglutaminase. In another embodiment, the crosslinker Factor Xllla. In some embodiments, the injectable adhesive disclosed herein is degradable in vivo. In one embodiments, the injectable adhesive described herein degrades in vivo in 12 to 24 hours, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, 1-6 weeks, 1-7 weeks, 1-8 weeks, or 1 to 12 weeks after injection. In an exemplary embodiment, the injectable adhesive disclosed herein degrades in 20-30 days after injection. In another exemplary embodiment, the injectable adhesive disclosed herein degrades by 50% in 7 to 10 days after injection. In another exemplary embodiment, the injectable adhesive disclosed herein degrades by 50% in 8 days after injection.

[0036] The present invention is illustrated by the following drawings and detailed description, which do not limit the scope of the invention described in the claims.

[0037] BRIEF DESCRIPTION THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] Figure 1 A is a schematic view showing the application of an injectable adhesive of the present invention to injured tissue. The adhesive is primarily composed of polyacrylic acid (PAA) functionalized with benzaldehyde (BLA) and gelatin. Figure IB is a representative image of the injectable adhesive of the present application applied to porcine skin. Figure 1C shows the chemical structure of PAA modified with BLA. BLA forms dynamic crosslinks with the primary amines presented in both gelatin and the tissue surface.

[0040] Figure 2A shows representative photographic images depicting an adhesive undergoing mechanical stretching. Figure 2B is stress-stretch curve for PAA-BLA adhesives. Figure 2C is a plot of quantification of maximum stretch, and Figure 2D is a plot of quantification of tensile modulus of adhesives of the present invention, evaluated through 4 - 5 individual samples with varying degrees of modification. Figure 2E shows force versus displacement for the adhesives of the present invention, showing the fracture toughness of the adhesive matrix. The red curve (the bottom curve) depicts the tensile profile for a notched sample, while the black curve (the top curve) represents that of an unnotched sample. Figure 2F shows a stress relaxation profile that demonstrates the viscoelastic behavior of the adhesives of the present invention. Figure 2G is degradation profiles of adhesives of the present invention. Adhesives were either immersed in a medium with or without collagenase, n = 5 hydrogels per group. Figure 2H is visual demonstration of the self-healing capacity of adhesives of the present invention. The adhesives were cut in half, repositioned for one minute, and then mechanically stretched. Data are presented as means ± standard deviation.

[0041] Figure 3 A is photographic representation of adhesives of the present invention applied between porcine skin tissues. The residual adhesives observed on both sides of the skin after testing are indicative of cohesive failure. Figure 3B is a plot showing force versus displacement for adhesives with varying formulations; unmodified PAA mixed with gelatin (PAA / Gel), PAA-BLA / Gel, and PAA-BLA mixed with gelatin and transglutaminase (PAA-BLA / Gel-TG). Figure 3C is a plot of interfacial toughness measurements for various adhesives, n = 3 - 6 individual adhesives per group. Figure 3D is a plot of the impact of the degree of substitution of BLA on interfacial toughness (n = 3 - 4). Figure 3E is a plot of interfacial toughness following adhesion of PAA-BLA / Gel to various tissues (n = 3 - 4). Figure 3F is a plot of alteration in interfacial toughness throughout multiple adhesion cycles (n = 4). Figure 3G is a plot of evaluation of the recovery of interfacial toughness over time (n = 3 - 4). Data are presented as means ± standard deviation. Figure 3H illustrates comparative analysis of the injectable adhesives of the present invention with commercial injectable adhesives.

[0042] Figure 4Ais schematic representation illustrating the experimental setup in animal studies. VML was induced in mice, followed by the application of materials to the created void. Figure 4B is sequential photographs capturing the tibial anterior muscle post- VML surgery (left), and after the adhesive application (right). Figure 4C and Figure 4D are ultrasound imaging of injured muscles treated with gelatin and PAA-BLA / Gel at day 4, respectively. Single plane view (left) and 3D reconstruction (right). Figure 4E is histological assessment by H&E staining of muscle tissues with the indicated treatments. Specific areas of interest are indicated by dotted rectangles. Scale bar, 1 mm. Figure 4F is a series of enlarged images showing a detailed view of the regions highlighted in Figure 4E. Scale bar, 100 pm. Figure 4G is a plot of quantification of inflammation score observed in the treated muscles (n = 5). Figure 4H is a plot of the number of central nuclei, normalized to the tissue area (n = 5). Figure 41 is a plot of fluorescence mean intensity of CD31. Figure 4J is a plot of fluorescence mean intensity of desmin (n = 5). Data are presented as means ± standard deviation. In Figures 4H, 41, and 4J, an ordinary one-way ANO VA with post hoc Tukey’s multiple comparisons test was used; *P < 0.05, ***p < 0.001. n.s. not significant.

[0043] Figure 5 A is a series of photos of tibial anterior muscles from untreated mice (control), and mice treated with gelatin and PAA-BLA / Gel. Figure 5B is a plot of muscle weight and Figure 5C is a plot of body weight of mice treated with the indicated treatments (n = 5 - 10). Figure 5D is a plot of muscle force profiles as a function of time, measured 21 days after injury. Figure 5E is a plot of quantification of muscle force over the weeks of recovery (n = 10). The red arrow indicates the muscle force level of normal, uninjured mice. Data are presented as means ± standard deviation. In Figures 5B and 5E, an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons test was used; *P < 0.05.

[0044] Figure 6 A is a release profile of different sizes of FITC-dextran as a model drug. 10, 70, 250 and 2000 kDa fluorophore molecules were encapsulated in a gelatin-PAA gel. Figure 6B is a release profile of FITC functionalized bovine serum albumine (BSA) as a protein based model drug of 69 kDa size compared to a FITC-dextran model drug of 70 kDa.

[0045] Figure 7Ais a stress-stretch curve for adhesives with and without transglutaminase. Figure 7B is a plot of quantification of maximum stretch, and Figure 7C is a plot of quantification of tensile modulus of adhesives of the present invention, evaluated through 4 - 5 individual samples.

[0046] Figure 8 A is a plot of swelling ratio measured after 24 hours by the ratio of the change in length, normalized to initial length (n=3). Figure 8B is a plot of swelling ratio measured after 24 hours by the ratio of the change in weight, normalized to initial weight (n=3).

[0047] Figure 9 is a plot of quantification of cell viability from live and dead cell staining for cells cultured with a conditioned medium with PAA-BLA / Gel and untreated cells.

[0048] Figure 10 is a plot of quantification of hydrogel volume over time (n = 3). The volume was normalized to day 1. Data is presented as means ± standard deviation.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] Wounds often necessitate the use of instructive biomaterials to facilitate effective healing. Yet, consistently filling the wound and retaining the material in place presents notable challenges. Disclosed herein are a new class of injectable tissue adhesives by leveraging the dynamic crosslinking chemistry of Schiff base reactions. These adhesives demonstrate outstanding mechanical properties, especially in regard to stretchability and self- healing capacity, and biodegradability. Furthermore, they also form robust adhesion to biological tissues. Their therapeutic potential was evaluated in a rodent model of volumetric muscle loss (VML). Ultrasound imaging confirmed that the adhesives remained within the wound during the regeneration process, and effectively filled the void. Histological analysis indicated that the adhesives promoted activation and differentiation of muscle cells and enhanced the formation of blood vessels. Notably, the injured muscles of mice treated with the adhesives displayed increased weight and higher force generation than the control groups.

[0051] Accordingly, the present disclosure describes dynamically crosslinked injectable adhesives with strong tissue adhesion and rapid self-healing for muscle regeneration.

[0052] I. Definitions

[0053] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this invention.

[0054] In the following description, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one having ordinary skill in the art that the invention may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, reference in the specification to phrases such as "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of phrases such as "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0055] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0056] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0057] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0058] By “treatment”, “prevention” or “amelioration” of a disease or disorder is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or stopping the progression, aggravation or deterioration, the progression or severity of a condition associated with such a disease or disorder, e.g., a wound or a heart defect. In one embodiment, the symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0059] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Mammals other than humans can be advantageously used as subjects that represent animal models of tissue or organ injuries, or other related pathologies. A subject can be male or female. The subject can be an adult, an adolescent or a child. A subject can be one who has been previously diagnosed with or identified as suffering from or having a risk for developing a tissue injury, disease or condition associated with tissue injury, or requires a device to be attached within or onto the body of the subject.

[0060] II. Injectable Adhesives of the Invention

[0061] The present invention provides a pre-formed injectable adhesive comprising: i) a polymer containing a carboxylic acid and a side chain represented by formula (I): -C(=O)-NH-L-NHC(O)-R (I); and ii) a primary or secondary amine polymer. The definitions of variables L and R are disclosed herein.

[0062] The polymer used in the injectable adhesive of the present invention includes one or more carboxylic acid groups which will improve the solubility of the polymer, and is modified by benzaldehyde. Specifically, one or more carboxylic acid groups contained in the polymer are linked with benzaldehyde through a linker. As such, it forms a side chain represented by formula (I) as described above.

[0063] Thus, the polymer modified by benzaldehyde can be any polymer including a copolymer containing carboxylic acid groups or the corresponding salts, in particular the sodium, potassium or ammonium salts. In some embodiments, the polymer containing one or more carboxylic acid groups is prepared by monomers selected from the group consisting of ethylenically unsaturated Cj-Cs-monocarboxyhc acids (such as acrylic acid, methacrylic acid, crotonic acid, isocrotonic acid, 2-ethylpropenoic acid); ethylenically unsaturated Ch-Cs-di carboxylic acids (such as maleic acid, fumaric acid, itaconic acid, citraconic acid), and also the salts of the monocarboxylic and dicarboxyiic acids mentioned, in particular the sodium, potassium or ammonium salts.

[0064] In some embodiments, the polymer modified by benzaldehyde is poly(acrylic acid) or poly(methacrylic acid), and also their sodium, potassium and ammonium salts.

[0065] The polymer modified by benzaldehyde can also be a co-polymer which contains carboxylic acid groups or the corresponding salts, in particular the sodium, potassium or ammonium salts. Representative co-polymers include, but are not limited to poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid).

[0066] The weight average molecular weight of the polymer modified by benzaldehyde is in the range from 1,000 to 500,000 Dalton, preferably in the range from 5,000 to 450,000 Dalton, in particular in the range from 35,000 to 400,000 Dalton and very particularly preferably in the range from 50,000 to 350,000 Dalton.

[0067] When a polymer is modified with benzaldehyde, it may lead to reduced polymer solubility in water. Yet, the carboxylic acid group(s) contained in the polymer are not only amenable to chemical modifications but also enhance water solubility after functionalization. The benzaldehyde modification degree on the polymer is adjustable. In some embodiments, the molar ratio between the side chain (z.e., a carboxylic acid group modified with benzaldehyde) and the free carboxylic acid (or the corresponding salts) contained in the polymer is 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, or 50:50, or in a range between the values recited herein.

[0068] The phrase “degree of modification” or “modification degree” as used herein refers to the percentage of carboxylic acid in a polymer (e.g. PAA) that has been modified with the side chain of Formula (I) which contains benzaldehyde. The terms degree of modification / modification degree and molar ratio between the side chain and the free carboxylic acid are used interchangeably herein.

[0069] The side chain connected with the polymer and benzaldehyde is represented by formula (I):

[0070] -C(=O)-NH-L-NHC(O)-R (I).

[0071] Specifically, both the carboxylic acid (or the corresponding salt) group from the polymer and benzaldehyde form an amide bond with a linker L. L is Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene; wherein one or two carbon atoms of Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene may optionally be replaced with O, N, S, S(O) or S(O)2, and / or two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or more R1; wherein said Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene represented by L is optionally substituted with one or more R1; wherein R1, in each occurrence, is independently halogen, CN, Ci-ealkyl, -ORla, oxo (=0, as appropriate) or -NRlaRlb; wherein Rlaand Rlbare independently selected from the group consisting of hydrogen, Ci-ealkyl, Ci-ehaloalkyl, 3-6 membered carbocyclyl, and 4-6 membered heterocyclyl.

[0072] In an exemplary embodiment, L is Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene; wherein one carbon atom of Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene may optionally be replaced with O, N, S, S(0) or S(0)2, and / or two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or more R1; wherein said Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene represented by L is optionally substituted with one or more R1; wherein R1, in each occurrence, is independently halogen, CN, Ci-ealkyl, -0Rla, oxo (=0, as appropriate) or -NRlaRlb; wherein Rlaand Rlbare independently selected from the group consisting of hydrogen, Ci-ealkyl, Ci-ehaloalkyl, 3-6 membered carbocyclyl, and 4-6 membered heterocyclyl.

[0073] Benzaldehyde R is optionally substituted (other than the -C(=0)-NH-L-NHC(0)- moiety). Optional substituents include but are not limited to one to three halogen, CN, Ci-ealkyl, -0Rla, or -NRlaRlb. In one embodiment, benzaldehyde R is optionally substituted with one or two F, CN, Ci-4alkyl, -OH, or -NH2. In one embodiment, benzaldehyde R is unsubstituted.

[0074] As used herein, the primary or secondary amine polymer refers to a polymer that contains one or more primary or secondary amine groups. In some embodiments, the primary or secondary amine polymer is gelatin, chitosan, glycol chitosan, collagen, polyallylamine, polylysine, polyethylamine, or polyethyleneimine.

[0075] In particular, polyallylamine (PolyNH2) is represented by the following structural formula: In particular, chitosan is represented by the following structural formula: . In particular, polyethylenimine (PEI) is represented by the following structural formula: . In particular, polylysine is represented by the following structural formula: . Collagen and / or gelatin include approximately -10% amino acid with primary amine (e.g., Arg, Lysine).

[0076] In one embodiment, the primary or secondary amine polymer is gelatin.

[0077] The ratio between the polymer containing a carboxylic acid and a side chain represented by formula (I) and the primary or secondary amine polymer can be from 1 : 10 to 10: 1 by weight. In some embodiments, the ratio between the polymer containing a carboxylic acid and a side chain represented by formula (I) and the primary or secondary amine polymer is from 1 :8 to 8: 1 by weight, from 1 :5 to 5: 1 by weight, from 1 :3 to 3: 1 by weight, or from 2: 1 to 2: 1 by weight. In an exemplary embodiment, the ratio between the polymer containing the carboxylic acid and a side chain represented by formula (I) and the primary or secondary amine polymer is 1 : 1 by weight. In another embodiment, the injectable adhesive of the present invention has a polymer containing a carboxylic acid, e.g. PAA-BLA, and a primary or secondary amine polymer, e.g. gelatin, that are mixed in a ratio of from 1 : 10 to 10: 1 by weight. In an exemplary embodiment, the PAA-BLA and the gelatin are mixed in a ratio of from 1 : 1 to 4: 1 by volume such that the carboxylic acid and the primary or secondary amine polymer has a ratio of 1 :2 to 2: 1 by weight. In a further exemplary embodiment, the PAA- BLA and gelatin are mixed in a ratio of 2: 1 by volume or 1 : 1 by weight.

[0078] The injectable adhesive of the present invention can be exemplified by benzaldehyde- modified poly(acrylic acid) (PAA-BLA) combined with gelatin, a biological component that provides a supportive matrix for cells. The resulting hydrogel matrix would be crosslinked by reversible imine bonds (Figures 1 A-1C), and their dynamic nature was expected to impart needle-inj ectability to the hydrogels. Additionally, the benzaldehyde groups were expected to enable the formation of dynamic covalent bonds with primary amines present on the tissue surface, ensuring effective tissue adhesion[23,36’37].

[0079] PAA-BLA was first synthesized with defined molecular weight and degree of modification. Given the low equilibrium constant of the reversible imine chemistry, a significant degree of benzaldehyde modification on polymers is essential for stable hydrogel formation. However, the innate hydrophobicity of benzaldehyde compounds typically leads to reduced polymer solubility in water after modification. To address these conflicting features, PAA was chosen as a backbone polymer due to its pronounced polyelectrolyte nature. The abundant carboxylate groups present on PAA are amenable to chemical modifications and enhance water solubility after functionalization. Forty eight percent of the PAA carboxylate groups were modified with alkyne through carbodiimide coupling. Subsequently, azide-functionalized benzaldehyde was conjugated using copper-catalyzed click chemistry.

[0080] Scheme 1 Scheme 1 above demonstrates a representative route to prepare PAA-BLA. A-(2-azidoethyl)-4-formylbenzamide (SI) was obtained through published methods (Lou et al, Biomaterials 154, 213-222 (2018)). PAA was first modified with alkyne through carbodiimide coupling followed by a copper-catalyzed click reaction to conjugate BLA functional moieties (e.g., SI).

[0081] Notably, the benzaldehyde modification degree on a polymer (e.g. PAA) was adjustable up to 40% modification; beyond this threshold, the polymer became insoluble in water.

[0082] The present invention also provides a method of preparing an injectable adhesive. The method comprises mixing a solution of a polymer containing a carboxylic acid and a side chain represented by formula (I) described herein (i.e., a benzaldehyde modified polymer) with a solution of primary or secondary amine polymer to form the adhesive.

[0083] III. Methods Of the Invention

[0084] The present invention also provides a method of adhering an injectable adhesive to a surface, the method including the steps of a) mixing a solution of a polymer containing a carboxylic acid and a side chain represented by formula (I) described herein (i.e., a benzaldehyde modified polymer) with a solution of primary or secondary amine polymer to form an injectable adhesive, and b) applying the obtained adhesive on the surface. In some embodiments, the surface is a tissue. The system can be applied to any tissue, including, but not limited to, heart tissue, skin tissue, blood vessel tissue, bowel tissue, liver tissue, kidney tissue, pancreatic tissue, lung tissue, trachea tissue, eye tissue, cartilage tissue, tendon tissue. Alternatively, the surface is a medical device. The system can be applied to any medical device, including, but not limited to, the group consisting of a defibrillator, a pacemaker, a stent, a catheter, a tissue implant, a screw, a pin, a plate, a rod, an artificial joint, a elastomerbased (e.g., PDMS, PTU) device, a hydrogel -based device (e.g., scaffolds for drug or cell delivery or sensors), and sensors for measuring, for example, temperature, pH, and local tissue strains.

[0085] The present invention also includes methods to encapsulate a medical device, or to coat a surface of a device. In particular, the benzaldehyde modified polymer disclosed herein and the primary or secondary amine polymer are mixed and applied to the surface of the device.

[0086] The present invention also includes methods to close a wound or injury and promote wound healing. In particular, the benzaldehyde modified polymer disclosed herein and the primary or secondary amine polymer are mixed to obtain an injectable adhesive and the obtained injectable adhesive is applied to the location of the wound or injury. In a particular embodiment, the obtained injectable adhesive is applied to the heart in order to repair a heart defect.

[0087] The present invention also includes methods of delivering a therapeutically active agent to a subject, the method including a) mixing a solution of benzaldehyde modified polymer disclosed herein and a solution of a primary or secondary amine polymer to obtain an injectable adhesive; and b) placing the obtained an injectable adhesive on the surface of a subject; and wherein at least one therapeutically active agent is encapsulated in, or attached to the injectable adhesive, thereby delivering a therapeutically active agent to the subject.

[0088] The methods of the present invention include contacting the surface with an injectable adhesive. The surface can be contacted with the composition by any known routes in the art. As used herein, the term “delivery” refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that a desired effect is produced.

[0089] Exemplary modes of delivery include, but are not limited to, injection, insertion, implantation, or delivery within a scaffold that encapsulates the composition of the invention at the target surface, e.g., a tissue or organ. When the compositions of the invention are dissolved in a solution, they can be injected into the surface by a syringe.

[0090] The methods of the present invention are suitable for medical purposes, e.g. , wound closure, delivery of a therapeutic agent, or attachment of a medical device, in a subject, wherein the subject is a mammal. In some embodiments, a mammal is a primate, e.g., a human or an animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, a subject is selected from the group consisting of a human, a dog, a pig, a cow, a rabbit, a horse, a cat, a mouse and a rat. In preferred embodiments, the subject is a human.

[0091] Exemplary modes of delivery include, but are not limited to, injection, insertion, implantation, or delivery within a scaffold that encapsulates the composition of the invention at the target tissue. In some embodiments, the composition is delivered to a natural or artificial cavity or chamber of a tooth of a subject by injection. When the compositions of the invention are dissolved in a solution, they can be injected into the tissue by a syringe.

[0092] In another aspect, the present invention provides a method of adhering an injectable adhesive to a surface (e.g., tissue or device), the method including the steps of a) mixing a solution of benzaldehyde modified polymer disclosed herein and a solution of a primary or secondary amine polymer to obtain an injectable adhesive, and b) placing the obtained an injectable adhesive on the surface.

[0093] In another aspect, the present invention provides a method of treating volumetric muscle loss (VML). In some embodiments, VML is caused by a surgical resection. In some embodiments, the surgical resection is a result of the removal of tumors, infections, or other health complications. In some embodiments, VML can result from bum injuries. When burns are severe, not only the skin, but also the underlying muscle can be destroyed leading to VML. In some embodiments, VML is caused by compartment syndrome.

[0094] IV. Kits

[0095] The present invention also provides kits. Such kits can include a pre-formed injectable adhesive described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the injectable adhesive system can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to, a benzaldehyde modified polymer disclosed herein, a solution containing the primary or secondary amine component. In a particular embodiment, the present invention is directed to a two component system including a solution of benzaldehyde modified polymer; and a solution of a primary or secondary amine polymer. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0096] In certain embodiments, kits can be supplied with instructional materials which describe performance of the methods of the invention. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0097] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated herein by reference.

[0098] EXAMPLES

[0099] Example 1. Synthesis of Injectable Adhesive

[0100] Chemical synthesis of PAA-BLA:

[0101] PAA was first dissolved in DI water at a concentration of 20 mg / mL, neutralized by adjusting solution pH to 7.4 using JSfeCCh, dialyzed against DI water for 3 d, and lyophilized to give a white powder. PAA was then functionalized with alkyne via carbodiimide coupling

[0061] . Neutralized PAA was dissolved in MES buffer (0.2 M, pH 6.5) at a concentration of 10 mg / mL followed by the addition of A-hydroxysuccinimide (0.61 g per gram of neutralized PAA, 0.5 eq. to acrylic acid unit), l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (1.53 g per gram neutralized PAA, 0.75 eq.) and propargyl amine (510 uL per gram neutralized PAA, 0.75 eq.) successively. After adjusting pH to 6, the mixture was stirred at room temperature for 4 h. The solution was then dialyzed against DI water for 3 d and lyophilized to give a white powder of PAA-alkyne. The degree of modification was quantified by proton nuclear magnetic resonance (XH NMR) spectroscopy, indicating that 48% carboxylate group on polymer was modified with alkyne groups.

[0102] PAA-alkyne was next modified with BLA using copper-catalyzed click chemistry

[0061] . PAA-alkyne was dissolved in phosphate buffered saline (PBS, pH 7.4) at a concentration of 10 mg / mL. Azido-benzaldehyde (SI) was dissolved in a minimal amount of DMSO and added to the PAA solution. The amount of azido-benzaldehyde added varied from 0.05 - 0.4 eq. to PAA. The mixture was purged with N2 for 30 min followed by the addition of prepurged copper (II) sulfate pentahydrate (0.01 eq.) and sodium ascorbate (0.05 eq.) solutions in DI water. After stirring at room temperature for 1 d, the mixture was filtered to remove insoluble solids, dialyzed against DI water for 3 d, and lyophilized to get PAA-BLA. In exemplary embodiments of the invention, PAA-alkyne was mixed with 0.08 equivalents, 0.30 equivalents, and 0.35 equivalents of azidobenzaldehyde (SI) to afford PAA-BLA with 8 ± 2, 30 ± 2, and 35 ± 2 percent modification.

[0103] Characterization of functionalized PAA:

[0104] The molecular weight and molecular weight distribution of PAA were determined by gel permeation chromatography (GPC) using Agilent 1260 Infinity II GPC / SEC system and a TSKgel G5000PWxl column (Tosoh Bioscience). Sodium sulfate solution (0.05 M in DI water) was used as eluent at 0.6 mL / min. Poly(ethylene glycol) (PEG) was used as the standards to generate the calibration curve.

[0105] The degree of alkyne modification on PAA was characterized byJH NMR spectroscopy (D2O, 500 MHz) using an internal standard method

[0062] . Potassium hydrogen phthalate (KHP) was used as the internal standard (5 at 7.50 ppm) and prepared at a constant concentration of 0.04 wt%. A standard calibration curve of the integration ratio of 'H NMR peak area between KHP (5 at 7.50 ppm) and methylene units on propargyl amine (5 at 3.90 ppm) versus the concentration of propargyl amine (varied from 0.1 wt% to 0.5 wt%) was first plotted. PAA-alkyne was then dissolved in D2O containing 0.04 wt % of KHP. The concentration of alkyne groups in the sample was analyzed by correlating the integration ratio of1H NMR peak area between KHP and propargyl amine modified on PAA to the standard curve. The degree of alkyne modification was calculated according to the mass fraction and molecular weight of propargyl amine and PAA. On average, 48% of the carboxylate group on PAA was conjugated with alkyne groups.

[0106] The degree of benzaldehyde modification on PAA was measured by the absorption at 258 nm via UV-vis spectroscopy (Agilent Cary 300 UV-vis spectrophotometer). Difunctional benzaldehyde-modified PEG (PEG-diBLA) was used as a model compound to measure the extinction coefficient of the benzaldehyde structure. PEG-diBLA was dissolved at a benzaldehyde concentration of 0.0155, 0.0464, 0.0774 mM in DI water. The absorbance at 258 nm was plotted versus the concentration to obtain the standard calibration curve.

[0107] Adhesive preparation:

[0108] Firstly, PAA-BLA was dissolved in Hank's Balanced Salt Solution without calcium and phenol red (HBSS) to achieve a final concentration of 10% (w / w). The solution was supplemented with 0.04% (w / w) phenol red to indicate the pH of the solution. Gelatin was dissolved to a final concentration of 20% (w / w) in HBSS, also without calcium and phenol red. To prepare the adhesive, PAA-BLA and gelatin were warmed up at 40 °C, loaded into separate Luer lock syringes, and mixed homogeneously using a female-female Luer lock coupler (Value-plastics). The ratio of PAA-BLA to gelatin was 2: 1 by volume, yielding a final concentration of 6.67% (w / w) for both. The mixed adhesive was then quickly injected into either a mold or tissue samples for experiments.

[0109] For adhesive formulations containing TG, transglutaminase (Ajimoto) was dissolved to a final concentration of 10% (w / w) in HBSS, without calcium and phenol red. TG was included in the PAA-BLA syringe for mixing with gelatin. The ratio of PAA-BLA, gelatin, and TG was set at 2: 1 :0.2.

[0110] Example 2. The Mechanical Properties of Adhesives

[0111] Characterization of mechanical properties:

[0112] Uniaxial tensile tests were conducted using an Instron instrument (3342, equipped with a 50 N load cell) to determine the elastic modulus of the adhesives. The adhesives were formed in a mold and secured by the grippers, with sandpaper attached to prevent slippage during testing. The samples were stretched at a rate of 50 mm / min, and the elastic modulus was calculated as the slope of the stress-strain curve between 0 and 100% strain.

[0113] For the rheological analysis, a Discovery Hybrid Rheometer HR-3 (TA Instruments) was utilized. The adhesive was deposited between two plates of the rheometer immediately following mixing, forming an 8-mm-diameter disk with a thickness of 1.5 mm. Mineral oil (Sigma) was applied to the exposed hydrogel surfaces to prevent dehydration

[0063] . The storage modulus and the loss modulus were monitored over time. Once the hydrogels were fully gelled — indicated by the equilibrium of the storage and loss modulus — frequency sweep and stress relaxation tests were conducted. During the frequency sweep, the storage and loss modulus were recorded across a range of 0.01 to 100 Hz at 1% stain. In the stress relaxation test, a constant strain of 15% was maintained while measuring stress over time

[0056] .

[0114] To evaluate the swelling behavior, adhesives were fully immersed in HBSS buffer overnight. The swelling ratio was determined either by the ratio of the change in length compared to the initial length or by the ratio of the change in weight compared to the initial weight.

[0115] To determine whether these adhesives could form a robust and stretchable hydrogel matrix after injection, they were fabricated in a mold with and without the addition of transglutaminase (TG), a crosslinker for gelatin, and subjected to mechanical testing. The adhesives exhibited high fracture toughness, stretching up to three times their initial length (Figures 2B-2E). The tensile modulus was approximately 6 kPa when modified with about 30% BLA, a stiffness comparable to that of many soft tissues, including muscle[38,39](Figure 2D). Furthermore, rheological analysis revealed the viscoelastic nature of the adhesives, with a substantial degree of stress relaxation observed under deformation (Figure 2F). This can be attributed to the rapid bond dissociation of dynamic imine crosslinking between PAA-BLA and gelatin. The adhesives displayed a swelling ratio of 40% in length and 160% in weight. While TG enhanced the tensile modulus of the adhesives, it reduced the overall stretchability (Figure 7A-C). Additionally, introduction of TG led to a decrease in the swelling properties, yet the rheological behavior remained unaltered (Figure 8A-B). The degradability of the adhesives was next analyzed in the presence and absence of collagenase. Collagenase expedited the degradation process, but the inclusion of TG effectively decelerated the process (Figure 2G).

[0116] To investigate whether the dynamic bonds in the PAA-BLA / Gel hydrogels could allow self-healing, pre-formed adhesives were cut in half, and the two pieces were then placed next to each other. Within only one minute, the cut pieces spontaneously rejoined, presumably via the formation of new bonds, and the interface was able to withstand stretching forces (Figure 2H). Similarly, when pieces were placed on top of each other, they also formed new bonds within a minute, successfully resisting subsequently applied forces (Figure 21).

[0117] Example s. Adhesion Performance

[0118] Adhesion tests'.

[0119] To quantify the adhesion performance, we measured the interfacial toughness, representing the adhesion energy necessary to propagate an interfacial crack over a unit area, by T-peeling tests

[0038] . For sample preparation, we injected the adhesive between two layers of tissue, utilizing porcine skin, heart, and muscle as the tissue sources. We attached the backside of these tissues to a rigid, non-stretchable film (e.g., polyethylene terephthalate (PET)) using Krazy glue. This prevented any stretching along the peeling direction and ensured that all the work done by the mechanical tester contributed to the energy dissipated at the crack tip. The free ends of the non-stretching film were then secured to the grips of the testing machine. Using an Instron instrument (3342, with a 50 N load cell), unidirectional tension was applied, during which both the force and extension were recorded. We maintained a constant loading rate of 50 mm / min. The adhesion energy was calculated as twice the plateau force, divided by the width of the sample.

[0120] To assess repeatable adhesion performance, tissue samples with adhesives were fully separated during adhesion tests, then repositioned and left for a designated duration before undergoing subsequent adhesion tests. To prevent dehydration during extended periods, samples were housed in a wet chamber. The adhesion performance of the adhesives was next quantified. The adhesives were prepared and subsequently first injected between two pieces of porcine skin tissue (Figure 3 A). Adhesion tests revealed an interfacial toughness of -150 J / m2(Figure 3B, 3C), which outperforms other injectable adhesives available in the market (Figure 3H).

[0121] Following the test, residual adhesives were observed on both skin surfaces, indicating cohesive failure at the interface (Figure 3A). Strong adhesion (-100 J / m2) was subsequently confirmed on fully hydrated tissue surfaces. To elucidate the role of BLAin tissue adhesion, tests were conducted using an adhesive formulated with unmodified PAA and gelatin. This led to a marked decrease in interfacial toughness to 10 J / m2(Figure 3B, 3C). Interestingly, the addition of TG did not enhance the interfacial toughness (Figure 3C). The degree of modification of BLA and the molecular weight of the PAA backbone were next varied, and adhesion again quantified. Increasing the degree of modification enhanced the interfacial toughness, with an optimal value achieved at 30% of modification (Figure 3D). A higher molecular weight of PAA (5kDa to 345kDa) correspondingly improved adhesion strength. Beyond skin, the adhesives successfully adhered to other tissues, including heart and skeletal muscle (Figure 3E).

[0122] To test whether the adhesives could re-form adhesions after fracture, adhesion tests were conducted using displaced tissue samples with residual adhesives. After the initial test, these samples were repositioned and left for one minute before undergoing another adhesion test. Remarkably, the adhesives recovered approximately 60% of their initial strength within just one minute (Figure 3F). Furthermore, they retained about 50% of the adhesive strength over five cycles (Figure 3F). The degree of recovery increased with extended healing time, and given 24 hours to recover, the adhesives regained the initial adhesion strength (Figure 3G).

[0123] The biological compatibility of the adhesives was initially analyzed by co-culturing with C2C12 myoblasts. No significant decrease in cell viability was observed upon exposure to the adhesives. Following this in vitro assessment, the adhesives were implanted into mice. Histological evaluations revealed only minimal to mild inflammation, further demonstrating biocompatibility.

[0124] Example 4. The Therapeutic Effects of Adhesives in the VML Model Biodegradability:

[0125] An enzymatic biodegradation medium was prepared by adding 3.5 mg of collagenase to 100 ml of HBSS

[0064] . The adhesives were injected into a mold and allowed to gel. Subsequently, they were cut into small samples using a biopsy punch, each 5 mm in diameter and 2 mm in height, and then weighed. Each sample was immersed in 1 ml of the enzymatic medium and incubated at 37°C. Approximately 0.01% (w / v) sodium azide was added to the enzymatic medium to prevent any bacterial contamination. At each time interval, the samples were removed, lyophilized, and weighed. The weight loss was calculated as the percentage ratio of the mass of the lyophilized sample at each time interval, normalized by the dry mass of sample at day 0.

[0126] Self-healing experiment:

[0127] For the self-healing experiment, the adhesive was injected into a mold, allowed to gel, and subsequently cut in half using a razor blade. The two resultant pieces were then placed adjacent to each other, and after one minute, were manually stretched using forceps. In a similar fashion, additional adhesive pieces were positioned atop each other and, following a minute, were manually separated with forceps. Video recordings were captured throughout the cutting, stretching, and separating processes.

[0128] Biocompatibility tests:

[0129] For cytotoxicity tests, C2C12 cells (CRL-1772, ATCC) were cultured in the growth medium, DMEM containing 10% FBS and 1% Pen strep. When the confluency of cells reached 60 - 70%, cells were cultured in the presence of PAA-BLA. Pristine culture medium was used as a control. After one day, cytotoxicity was determined using a live / dead viability kit (Thermo Fisher Scientific) by adding 4 pM calcein and ethidium homodimer- 1 into the medium. Fluorescence images were taken through GFP and RFP channels in EVOS-FL (AMG).

[0130] No significant decrease in cell viability was observed upon exposure to the adhesives (Figure 9).

[0131] In vivo biocompatibility of the adhesive was assessed through subcutaneous implantation. Following a period of 10 days post-implantation, the tissues surrounding the adhesive were excised and subsequently collected for histological analysis.

[0132] Animal surgery:

[0133] All animal procedures were performed in accordance with the Harvard University Faculty of Arts and Sciences Institutional Animal Care and Use Committee guidelines. Female C57BL6 / J mice, aged 8-12 weeks (Jackson Laboratory), were used for the experiments. Mice were housed under a 12-hour light / 12-hour dark cycle, at a temperature of 20-24°C and a humidity level of 35-65%. First, a lateral incision was made through the skin lengthwise along the lateral aspect of the tibialis anterior muscle of the left hind limb

[0065] .

[0134] After reflecting the skin and fascia from the anterior surface, a sterile metal plate was inserted between the tibialis anterior muscle and underlying extensor digitorum longus muscle. A 3mm biopsy punch was used to excise a full thickness defect from the belly of the tibialis anterior muscle against the previously inserted metal plate. Wounds were either left untreated or treated with biomaterials directly to the defect region. The incision was closed with several sutures. The animals were allowed to recover from anesthesia and returned to their cages. Analgesics were administered to control pain, and the animals were monitored for pain levels, incision healing, and general health for at least 96 hours post-surgery, regardless of whether postoperative analgesia administration had ceased. The non-operated limb served to allow the animal to remain ambulatory if the operated limb became disabled.

[0135] Histology and immunofluorescence analysis:

[0136] For the histological analysis, isolated muscles were fixed in 4% paraformaldehyde, washed, paraffin-embedded and stained with H&E or Masson’s Tri chrome. The sections were imaged with a Nikon E800 or ZEISS Axio Scan.Zl Slide Scanner in bright-field mode. Histological assessment was performed by a blinded pathologist and representative images of each group were shown in the corresponding figures.

[0137] Immunohistochemistry was performed for both paraffin sections. Paraffin sections were first deparaffinized and then processed with antigen retrieval treatments using Dako Target Retrieval Solution (Agilent), prior to standard immunostaining procedures. The sectioned samples were stained using standard immunohistochemistry protocols. The samples were permeabilized with phosphate buffered saline (PBS) containing 0.1% Tween 20 or Triton X (PBST), and blocked with PBST containing 10% bovine serum albumin and 10% goat serum. The following antibodies and reagents were used for immunohistochemistry: desmin (1 : 100, abl5200, Abeam), CD31 (1 : 100, abl24432, Abeam), Prolong Gold antifade reagent (Invitrogen) and DAPI (Invitrogen). Imaged was used for post-image analysis.

[0138] Ultrasound imaging and analysis:

[0139] For ultrasound imaging, animals were anaesthetized and placed on a heating pad, and the hind limbs were fixed to prevent movement. Muscles were analyzed with high-frequency ultrasound imaging using a Vevo 3100 scanner with a 50 MHz transducer and a 3D motor (VisualSonics; axial resolution, 30 m; lateral resolution, 140 pm). The scanner was placed directly above where the injury site was located. Ultrasound images were acquired every 0.1 mm in the axial plane throughout the muscle. After imaging, animals were subsequently allowed to recover to their normal behavior. 3D volume reconstruction of the gel, defect area, and muscle was performed using the multi-slice method in Vevolab software (Vevolab 5.7.1). Each volume is created by segmenting a series of contours from a 2D slice of the ultrasound images and rendering them into a 3D image.

[0140] Muscle function analysis:

[0141] Skeletal muscle contractile function was assessed at various intervals over a 4-week period following VML surgery using the Dynamic Muscle Data Acquisition and Analysis System (Aurora Scientific, Aurora, ON). For these evaluations, the animals were anesthetized. Each animal's foot was firmly secured to a footplate connected to a dual-mode muscle lever system, with both the knee and ankle positioned at right angles. Needle electrodes were placed just beneath the skin, ensuring they weren't inserted too deeply into the muscle to prevent activation of the antagonist compartment. Muscles were stimulated at a frequency of 100 Hz, employing a pulse- width of 2 ms over 500 ms at 10 V. During the measurements, the body temperature was consistently maintained at 37°C. Muscle force data were filtered using a low-pass filter set with a cut-off frequency of 3333 Hz, a filter order of 4, and a block size of 10.

[0142] Statistical Analysis:

[0143] Data are presented as means ± standard deviation. For comparisons between two groups, Student’s t-tests were employed. When comparing more than two groups, a one-way analysis of variance followed by Tukey’s multiple comparison test was used, employing GraphPad software. Ap-value of < 0.05 was considered statistically significant.

[0144] Finally, the potential of the adhesives of the present invention to facilitate muscle regeneration were explored. Volumetric loss was created in the tibial anterior muscles of mice (Figure 4A, 4B), and mice were subsequently divided into three groups for treatment: one with gelatin alone, one with the PAA-BLA / gel adhesive, and a control group that received no treatment. Using real-time high-resolution ultrasound imaging, all of the adhesives remained at the defect site at day 4 were observed, whereas some of the mice treated with gelatin showed displacement of the gel from the defects (Figures 4C and 4D). Subsequently, the long term stability of the adhesives of the present invention was evaluated. Ultrasound imaging and 3D reconstruction confirmed that the adhesives remained well-maintained within the defect throughout the two-week duration. Further, by analyzing changes in size, we assessed the in vivo degradation profile. The adhesives’ volume noticeably decreased over time, with only about 40 % of the original volume left after 11 days (Figure 10). By the end of the second week, the adhesives had almost completely degraded.

[0145] Histological analysis of muscle tissues at day 28 demonstrated that muscles treated with the adhesives showed minimal to mild inflammation, comparable to both untreated muscles and those treated with gelatin (Figures 4E-4G). Moreover, Masson’s tri chrome staining indicated that the fibrosis levels in muscles treated with adhesives were similar to the control and gelatin groups. However, an increased number of centrally located nuclei in muscle fibers, indicative of regenerating fibers1401, was observed in muscles treated with the adhesives (Figure 4H). Immunohistochemical staining for CD31, a marker for blood vessel formation1411, and desmin, indicative of muscle satellite cell activation and differentiation1421, showed that muscles treated with the adhesive had elevated levels of both markers compared to untreated muscles (Figures 41 and 4J).

[0146] The functional impact of treating VML with the adhesives was then analyzed. The gross size and weight of the muscles were observed to be greater in mice treated with the adhesives than the control groups (Figures 5 A and 5B). All sets of mice maintained similar body weights after two weeks (Figure 5C). Importantly, longitudinal muscle function analyses conducted over a 4-week period showed that muscles in the adhesive-treated group generated substantially higher tetanic forces, as compared to both the untreated group and those treated with gelatin (Figures 5D and 5E).

[0147] Drug-Release Analysis:

[0148] For drug-release experiments, PAA-BLA was dissolved in HBSS without calcium and phenol red at a final concentration of 10% (w / w). Gelatin was dissolved to a final concentration of 20% (w / w) in HBSS, also without calcium and phenol red in the presence of 6 mg / mL drug.

[0149] To prepare the adhesive, PAA-BLA and gelatin / drug were warmed up at 40 °C, loaded into separate Luer lock syringes, and mixed homogeneously using a female-female Luer lock coupler (Value-plastics). The ratio of PAA-BLA to gelatin was 2: 1, yielding a final concentration of 6.67% (w / w) for both and 2mg / mL for the encapsulated drug. The mixed adhesive was then quickly injected into a mold 2 mm thick and 4 mm in diameter. These gels were left to crosslink in the dark for at least 1 hour, removed from the mold, and placed at the bottom of a 1.5 mLEppendorf tube and covered with 250 pL of 0.1M PBS solution at 37 °C in the dark. In set intervals of time, 2.5 pl of supernatant solution sample was taken from the tube and replaced with the same PBS buffer used for starting the release to maintain the volume of supernatant. The samples were diluted in 97.5 pl of DI water to measure their fluorescence. The release of cargo was measured by their fluorescence using a multi-mode reader (BioTek SYNERGY neo2) (ex 488 nm, em 515 nm, bandwidth 10 nm). The concentration of the cargo was determined according to the standard curve for each fluor ophore.

[0150] To investigate the drug release capacity of the PAA-BLA / gelatin hydrogel, a series of experiments to quantify the release of fluorescent model drugs have been carried out. Different FITC-dextran compounds or FITC-BSA of known molecular weight were encapsulated in the gel. After mixing, the gel was injected into a mold 2 mm thick and 4 mm in diameter. These were placed at the bottom of a 1.5 mL Eppendorf tube and covered with 250 pL of 0. IM PBS solution at 37 °C. The release of cargo was measured by their fluorescence (ex 488 nm, em 515 nm).

[0151] FITC-dextran of 10, 70, 250, and 2000 kDa were utilized as model drug molecules to determine the effect of molecular weight on release rate. Lower molecular weight FITC- dextran demonstrated an increased rate of release while high molecular weight analogues release more slowly (Figure 6A). Importantly, all analogues provided a slow release over 5- 15 days. Release of FITC functionalized bovine serum alubumine (BSA) as a protein-based model drug of 69 kDa was also performed. FITC functionalized BSA demonstrated a marginally faster release rate than the 70 kDa FITC-Dextran (Figure 6B). Similarly, both the FITC functionalized BSA and FITC-dextran were released over a period of 5-10 days.

[0152] Conclusion

[0153] A novel class of injectable tissue adhesives that exhibit both high stretchability and rapid self-healing capabilities are disclosed herein. Previous studies have explored dynamically crosslinked injectable hydrogels through various mechanisms, including guesthost interactions, Schiff base reactions, hydrazone linkages, weak molecular interactions, and thiol-alkynone|23‘2°6'43 4S|. However, these biomaterials often exhibit poor mechanical properties, with low stretchability and toughness. In contrast, injectable adhesives of the present invention can stretch up to three times their initial length and possess a rapid self- healing ability; if damaged, about 60% of the adhesive strength can be restored within just a minute. Such characteristics are not typically observed in other injectable adhesives128 31'49'501. Additionally, injectable adhesives of the present invention displayed significantly higher adhesion energy compared to commercially available adhesives. These properties may be attributed to the long polymer chains of poly(acrylic acid), combined with a high degree of modification of benzaldehyde, and the abundant primary amines of gelatin.

[0154] The dynamic crosslinking of adhesives through Schiff base reactions is pH dependent151]. At acidic conditions, these bonds tend to dissociate, resulting in a weakened adhesive matrix and compromised tissue adhesion. Conversely, under alkaline conditions, the formation of strong covalent bonds makes the adhesive matrix and tissue adhesion more brittle. At a neutral pH, an optimal balance of bonding is achieved. Recent studies have shown that during muscle injury, the pH remains relatively stable within the neutral range

[0052] . Thus, injectable adhesives of the present invention, when applied to muscle injuries, are expected to retain their dynamic properties.

[0155] It is noted that tissue adhesion is a crucial role in the development of biomaterials for muscle-related applications, while previous studies investigating biomaterials for muscle treatments often overlooked this feature[11-15]. As the dynamic movements of muscles can possibly displace these materials from their intended site, strategies to securely anchor these biomaterials are likely essential. Indeed, ultrasound imaging demonstrated that gelatin, when used alone, shifted away from the wound. In contrast, when combined with PAA-BLA, it remained in place at the injury site.

[0156] The therapeutic potential of adhesives was demonstrated in a VML model. These adhesives were found to promote muscle regeneration, as indicated by the increased number of centrally located nuclei in muscle fibers, elevated expression levels of CD31 and Desmin, and improvements in both muscle mass and force generation. These findings may relate to the improved residence of the gelatin in the wound when formulated into the adhesive. Alternatively or in addition, the adhesives' viscoelastic properties might play a role in this regenerative process, as a large body of recent work has revealed that matrix viscoelasticity can influence a wide range of cellular activities, including cell proliferation, migration, and differentiation154-60] .

[0157] While this work focused primarily on skeletal muscle application, the versatility of the adhesive likely will make it useful with other tissues. The injectable adhesives of the present invention can strongly adhere to diverse tissues, such as skin and heart, indicating that they are broadly effective in dynamic and mechanically challenging environments, including the lungs and heart.

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Claims

CLAIMS1. An injectable adhesive comprising: i) a polymer containing a carboxylic acid and a side chain represented by formula (I):-C(=O)-NH-L-NHC(O)-R (I);R is optionally substituted benzaldehyde;L is Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene; wherein one or two carbon atoms of Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene may optionally be replaced with O, N, S, S(O) or S(O)2, and / or two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or more R1; wherein said Ci-ealkylene, C2-ealkenylene, or C2-ealkynylene represented by L is optionally substituted with one or more R1; whereinR1, in each occurrence, is independently halogen, CN, Ci-ealkyl, -ORla, oxo (=0, as appropriate) or -NRlaRlb; whereinRlaand Rlbare independently selected from the group consisting of hydrogen, Ci-ealkyl, Ci-ehaloalkyl, 3-6 membered carbocyclyl, and 4-6 membered heterocyclyl; and ii) a primary or secondary amine polymer.

2. The injectable adhesive of claim 1, wherein the polymer is poly(acrylic acid), poly(methacrylic acid), poly(crotonic acid), poly(isocrotonic acid), poly(2-ethylpropenoic acid); poly(maleic acid), poly(fumaric acid), poly(itaconic acid), poly(citraconic acid), or co-polymers of carboxylic acids, each of which contains the side chain of formula (I), wherein the co-polymers of carboxylic acids contains acrylic acid, methacrylic acid, crotonic acid, isocrotonic acid, 2-ethylpropenoic acid, maleic acid, fumaric acid, itaconic acid, or citraconic acid.

3. The injectable adhesive of claim 2, wherein the co-polymers of carboxylic acids is poly(methyl methacrylate / methacrylic acid) or poly(butadiene / maleic acid).

4. The injectable adhesive of any one of claims 1 to 3, wherein the molar ratio between the side chain and the carboxylic acid contained in the polymer is 5:95 to 50:50.

5. The injectable adhesive of claim 4, wherein the molar ratio between the side chain and the carboxylic acid contained in the polymer is 10:90 to 40:60.

6. The injectable adhesive of any one of claims 1 to 5, wherein L is Ci-ealkylene optionally substituted with one or more R1, and two adjacent carbon atoms may optionally be replaced to form 4-6 membered carbocyclyl, 4-6 membered heterocyclyl, phenyl, or 5-6 membered heteroaryl, each of which is optionally substituted with one or two R1.

7. The injectable adhesive of claim 6, wherein L is Ci-ealkylene optionally substituted with one or two R1, and two adjacent carbon atoms are replaced to form 5-6 membered heteroaryl optionally substituted with one or two R1.

8. The injectable adhesive of any one of claims 1 to 7, wherein R1, in each occurrence, is independently halogen, CN, OH, Ci-4alkyl, Ci-4alkoxy, or -NRlaRlb; wherein Rlaand Rlbare independently selected from the group consisting of hydrogen and Ci-4alkyl.

9. The injectable adhesive of any one of claims 1 to 8, wherein the side chain is represented by -C(=O)-NH-CH2-triazole-(CH2)2-NHC(O)-R.

10. The injectable adhesive of any one of claims 1 to 9, wherein R is benzaldehyde optionally substituted with one to three halogen, CN, Ci-ealkyl, -ORla, or -NRlaRlb.

11. The injectable adhesive of claim 10, wherein R is benzaldehyde optionally substituted with one or two F, CN, Ci-4alkyl, -OH, or -NH2.

12. The injectable adhesive of claim 10, wherein R is unsubstituted benzaldehyde.

13. The injectable adhesive of any one of claims 1 to 12, wherein the primary or secondary amine polymer is gelatin, chitosan, glycol chitosan, collagen, polyallylamine, polylysine, polyethylamine, or polyethyleneimine.

14. The injectable adhesive of claim 13, wherein the primary or secondary amine polymer is gelatin, chitosan, glycol chitosan, polyallylamine, or polylysine.

15. The injectable adhesive of any one of claims 1 to 14, wherein the polymer is poly(acrylic acid) or poly(methacrylic acid), each of which contains the side chain represented by formula (I).

16. The injectable adhesive of claim 15, wherein the polymer is poly(acrylic acid) which contains the side chain represented by formula (I).

17. The injectable adhesive of any one of claims 1 to 16, wherein the primary or secondary amine polymer is gelatin.

18. The injectable adhesive of any one of claims 1 to 17, wherein the injectable adhesive further comprises a crosslinker for the primary or secondary amine polymer.

19. The injectable adhesive of claim 18, wherein the crosslinker is transglutaminase or Factor Xllla.

20. The injectable adhesive of any one of claims 1 to 17, wherein the injectable adhesive degrades in vivo in 1 to 12 weeks after injection.

Images