Gel Compositions, Systems, and Methods

Abstract

Methods of forming gels and related methods of treating a subject with such gels are described. The methods may include preparing a composition by combining a first polyethylene glycol (PEG)-based polymer, poly(ethyleneimine)-based polymer, or poly(1,2-glycerol) carbonate-based polymer comprising a macromer that comprises at least one first functional moiety, a crosslinker comprising a second PEG-based polymer comprising at least one second functional moiety, and a photoinitiator, and forming the gel by activating the photoinitiator via a light source.

Description

[Technical field]

[0001] The present disclosure relates generally to therapeutic gels useful in medical procedures, including endoscopic surgery. For example, the present disclosure includes gels, as well as compositions and systems formulated to form gels (e.g., for application to body tissues, such as the gastrointestinal tract). [Background technology]

[0002] Endoscopic procedures, such as endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), and anastomosis, as well as health conditions such as intentional or disease-induced fistula formation, inflammatory bowel disease (IBD), and diseases associated with IBD, can result in and / or contribute to damage to the tissues of the gastrointestinal (GI) tract. Colorectal cancer is one of the leading causes of cancer death in developed countries. Standard preventive care for patients over 50 years of age includes biopsy of polyps (known as polypectomy) via colonoscopy to evaluate for colorectal cancer. In practice, a physician inserts an endoscope into the patient's colon under anesthesia and removes the polyps after inspecting the colon. After removal, the wound is left open to the colon's internal environment or is thermally closed using electrocoagulation. Post-popectomy or other endoscopic procedures wounds in the GI tract can result in bleeding and sepsis. Electrocoagulation can result in other complications such as irrigation or post-polypectomy coagulation syndrome.

[0003] These types of medical procedures and health conditions can leave a relatively thin layer of tissue in the GI tract wall. Currently, physicians often rely on time or surgical procedures, including clipping or endoscopic suturing, to allow the GI tract wall to heal. However, these practices may be inappropriate in certain cases, such as large defects and / or fragile or fibrous tissue. Potential complications include perforation, infection, and sepsis. Summary of the Invention

[0004] Methods of forming gels useful in medical procedures are disclosed. The disclosure includes, for example, a method of forming a gel, comprising preparing a composition by combining a macromer comprising a first polyethylene glycol (PEG)-based polymer, a poly(ethyleneimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer comprising at least one first functional moiety, a crosslinker comprising a second PEG-based polymer comprising at least one second functional moiety, and a photoinitiator, and forming a gel by activating the photoinitiator via a light source. The gel may be biocompatible and / or biodegradable. The at least one first functional moiety may comprise, for example, a thiol group, a vinyl group, an allyl group, an acrylate group, or a norbornene group, and / or the at least one second functional moiety may comprise a thiol group, a vinyl group, an allyl group, an acrylate group, or a norbornene group, the at least one first functional moiety being different from the at least one second functional moiety. In at least one example, the at least one first functional moiety or the at least one second functional moiety may comprise a vinyl group, an allyl group, an acrylate group, or a norbornene group, and the other of the at least one first functional moiety or the at least one second functional moiety may comprise a thiol group. According to some examples herein, the macromer, the crosslinker, and the photoinitiator may collectively comprise 10-25% by weight of the composition, based on the total weight of the composition. Optionally, the molar ratio of the at least one first functional moiety to the at least one second functional moiety may range from 1:1 to 2:1. Additionally or alternatively, the macromer may collectively comprise 5-15% by weight of the composition, based on the total weight of the composition. The crosslinker may collectively comprise 5-10% by weight of the composition, based on the total weight of the composition. The concentration of the photoinitiator in the composition may range from about 0.1 mM to about 100 mM. In some examples, the crosslinker comprises an N-hydroxysuccinimide group and / or a maleimide group. Additionally or alternatively, the macromer may comprise a hyperbranched polymer.

[0005] According to some aspects of the present specification, the composition may further include a physiological buffer. The light source may emit UV light or visible light. For example, when illuminated with UV light, the gel may form within 5 seconds. In some examples, when the photoinitiator is activated with visible light, the gel may form within 10 seconds. Optionally, the composition may further include an additive, including a tyrosine derivative, to accelerate the gelation time of the composition. In some aspects, the composition may include up to 10 mM of the additive. The tyrosine derivative may include, for example, tyrosine methyl ester or tyrosine ethyl ester.

[0006] The gels described above and elsewhere herein can be used to treat tissue in a subject, such as a human subject. For example, the gels can be used to treat tissue in the gastrointestinal tract of a subject.

[0007] The present disclosure also includes a method of forming a gel, comprising: preparing a first solution by combining a macromer comprising a polyethylene glycol (PEG)-based polymer, a poly(ethyleneimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer comprising at least one first functional group, with a first buffer; preparing a second solution by combining a crosslinker comprising a second (PEG)-based polymer comprising a plurality of second functional groups, with a second buffer having a lower pH than the first buffer; and mixing the first and second solutions to form a gel. The gel may be biocompatible and / or biodegradable. The at least one first functional group may comprise, for example, a thiol group or an amine group, and / or the plurality of second functional groups may comprise an N-hydroxysuccinimide group or a maleimide group. In some examples, the molecular weight of the macromer may be about 2,000 Da. Additionally or alternatively, the molecular weight of the crosslinker may be about 3,400 Da. In some examples, the molar ratio of crosslinker to macromer may range from 3:2 to 7:3.

[0008] As described above, the gels disclosed herein can be used to treat tissues of a subject. For example, a method of forming a gel can include treating a subject by forming a gel on tissues of the gastrointestinal tract of the subject. In at least one example, the method includes applying to the tissue a first solution comprising a macromer comprising a polyethylene glycol (PEG)-based polymer, a poly(ethyleneimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer comprising at least one first functional group and a first buffer; and applying to the tissue a second solution comprising a crosslinker comprising a second (PEG)-based polymer comprising a plurality of second functional groups and a second buffer having a lower pH than the first buffer, whereby the first solution contacts the second solution to form a gel on the tissue. The first solution can be applied to the tissue before, after, or simultaneously with the second solution.

[0009] The disclosure also includes compositions comprising macromers comprising polyethylene glycol (PEG)-based polymers, poly(ethyleneimine)-based polymers, or poly(1,2-glycerol) carbonate-based polymers, the macromers comprising at least one thiol or amine group; the crosslinker comprises a PEG-based polymer comprising an N-hydroxysuccinimide functional group, a maleimide functional group, or both, and the composition is formulated as a hydrogel. The hydrogel has a gel strength of at least 2,000 Pa and / or a viscosity of 0.03-0.90 N / cm when adhered to a body lumen. 2 Additionally or alternatively, the hydrogel may be formulated to withstand a burst pressure of up to about 15,000 Pa (about 150 mbar) when adhered to colon tissue to fill an opening in the tissue of about 1 mm by about 5 mm. [Brief description of the drawings]

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments. [Figure 1A]1A and 1B show exemplary macromer structures according to some embodiments of the present disclosure. [Figure 1B] Same as above. [Figure 2A] 2A-2G show exemplary macromers according to some embodiments of the present disclosure. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 3A] 3A and 3B show exemplary crosslinker structures according to some embodiments of the present disclosure. [Figure 3B] Same as above. [Figure 4A] 4A-4D show exemplary cross-linking agents according to some embodiments of the present disclosure. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Diagram 5] FIG. 1 is a schematic diagram of the formation of a gel according to some aspects of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram of the dissolution of a gel according to some aspects of the present disclosure. [Figure 7] 1 shows the mechanism of gel dissolution in the presence of cysteine. [Figure 8] 1 shows the dissolution mechanism of the gel in the presence of water. [Figure 9] 1 illustrates possible reactions with exemplary crosslinkers according to some aspects of the present disclosure. [Figure 10] 1 is a gel strength chart as described in Example 1. [Figure 11A] 11A and 11B are charts of gel strength and gel swell ratio as described in Example 2. [Figure 11B] Same as above. [Figure 12A]12A-12E are charts of gel strength and gel time as described in Example 3. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 13] 1 shows the synthesis of an exemplary crosslinker as described in Example 4. [Figure 14] 1 shows the crosslinker and macromer used in forming the gel described in Example 6. [Figure 15] 4 shows the results of gelation measurements as described in Example 6. [Figure 16] 16 and 17 show the rheological measurements of the hydrogels described in Example 6. [Figure 17] Same as above. [Figure 18] The amidation reaction kinetics at the NHS ester and internal ester linkages described in Example 6 are reported. [Figure 19] 1H NMR data for monitoring the hydrolysis described in Example 6. [Figure 20] 1 shows storage modulus data for hydrogels at different temperatures as described in Example 6. [Figure 21] The swelling of the hydrogel described in Example 6 is reported. [Figure 22] 1 shows the results of adhesion measurements of the hydrogels described in Example 6. [Figure 23] Cytotoxicity results of the hydrogels described in Example 6 are reported. [Figure 24] 24 and 25 report bacterial translocation studies of the hydrogels described in Example 6. [Diagram 25] Same as above. [Figure 26] 1 is an SEM image of the hydrogel described in Example 6. [Figure 27] 27 and 28 show the agar plate assay results for the hydrogels described in Example 6. [Figure 28] Same as above. [Figure 29]1 shows the synthesis of several exemplary crosslinkers described in Example 7. [Diagram 30] 1 shows the properties measured for various hydrogels described in Example 8. [Diagram 31] 1 shows SEM images of various hydrogels described in Example 8. [Diagram 32] 1 shows 1H NMR spectra of the crosslinker before and after reaction with the macromer described in Example 8. [Diagram 33] 1 shows 1H NMR spectra investigating the NHS hydrolysis of the crosslinker described in Example 8. [Diagram 34] Kinetic studies of various hydrogels are reported in Example 8. [Diagram 35] The strain and frequency sweeps of the hydrogel described in Example 8 are reported. [Diagram 36] 36 and 37 report the storage modulus of the hydrogels described in Example 8. [Figure 37] Same as above. [Figure 38] The swelling of the hydrogel described in Example 8 is reported. [Figure 39] The dissolution of the hydrogel described in Example 8 is reported. [Diagram 40] 40 and 41 report the rheological measurements of the hydrogels described in Example 8. [Diagram 41] Same as above. [Diagram 42] Dissolution results for the hydrogel described in Example 8 are reported. [Diagram 43] Cell viability of the hydrogels described in Example 8 is reported. [Figure 44-1] FIG. 44 shows the in vivo study design described in Example 8. [Figure 44-2] Same as above. [Diagram 45] 45-49 show H&E staining of various tissue samples described in Example 8. [Figure 46] Same as above. [Figure 47] Same as above. [Figure 48] Same as above. [Figure 49] Same as above. [Figure 50] FIG. 1 is a schematic representation of the dissolution of a hydrogel used as a wound dressing as described in Example 8. [Figure 51] 1 shows the synthesis of several exemplary crosslinkers described in Example 9. [Figure 52] 1 shows the synthesis of an exemplary macromer as described in Example 10. [Diagram 53] 1 shows the results of a TNBS assay detecting primary amines on PEI and PEI-SH molecules as described in Example 10. [Figure 54] 1 shows the results of rheological measurements of the hydrogel described in Example 11. [Figure 55] 1 shows the 1H NMR spectrum of the crosslinker described in Example 11. [Figure 56] 1 shows the system used to measure burst pressure as described in Example 11. [Figure 57] Burst pressure data for the hydrogels described in Example 11 are reported. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the features set forth in the claims. As used herein, the terms "comprises," "having," "including," or other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements does not include only those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. In this disclosure, relative terms such as "about," "substantially," "generally," and "approximately" are used to indicate a possible variation of ±10% in a stated value or characteristic. All ranges are understood to be inclusive of the upper and lower limits, for example, a macromer content of 5% to 10% by weight includes 5%, 10%, and all values ​​therebetween.

[0012] The embodiments of the present disclosure may address one or more limitations in the art. However, the scope of the present disclosure is defined by the appended claims, not by the ability to solve a particular problem. The present disclosure includes compositions and systems formulated to form gels, e.g., hydrogels, as well as compositions in gel / hydrogel form useful for application to tissues, e.g., of the gastrointestinal tract. The hydrogels herein may function as temporary, minimally invasive, in situ hydrogel dressings applied immediately after a medical procedure, such as a polypectomy. The hydrogels can prevent or reduce potential complications by covering and protecting the wound. From a biomaterial design perspective, the dressing can achieve one or more of the following: 1) form rapidly in situ; 2) adhere to colonic tissue; 3) be non-cytotoxic; 4) dissolve spontaneously in 3-5 days; 5) swell up to 200% to absorb wound exudate; 6) prevent bacterial spread or migration; and / or 7) conform to the malleable shape of the colonic lumen. The gels herein can be formulated to provide desired properties, such as gelation rate, adhesive strength, swelling, cytotoxicity, and / or degradation, depending on the hydrogel composition. The compositions herein can be delivered to a subject by a suitable medical device, such as a catheter inserted through an endoscope. For example, a dual lumen catheter may be used. The barrier properties of the hydrogel can help prevent bacterial translocation.

[0013] The compositions, systems, and methods herein may provide a set of properties, including inherent cohesion and adhesion to tissue, among others. Such properties allow the gels herein to function as a protective barrier for thin, damaged, and / or otherwise damaged tissues in a body lumen, such as the GI tract. For example, exemplary compositions, such as formulations or systems for forming gels, may be applied to a target site along the GI tract, and the composition may crosslink to form a gel, thereby providing barrier protection / treatment to the target site. Components of the compositions and gel systems herein may provide desired properties that are advantageous for tissue protection, for example, before, during, and / or after a medical procedure. The compositions and systems herein may be delivered to the target site by any suitable method or technique. Properties of the composition, such as viscosity, may facilitate the deliverability of the gel-forming formulation to the target site via a suitable medical device, such as, for example, single / multi-lumen catheters, including endoscopes, and syringes, among other devices useful for medical procedures. For example, the compositions herein in gel form may have a viscosity ranging from about 0.010 Pa·s, such as about 0.015 Pa·s, such as about 0.013 Pa·s, at room temperature (about 22-25° C.). The components of the composition or gel system may be crosslinked to form a gel, which may include activating one or more components at or in the presence of a stimulus, such as pH value or light. The hydrogels herein may be hydrophilic three-dimensional polymer networks formed from macromers and a crosslinker. The gels herein, such as hydrogels, may be formed by combining the macromers and the crosslinker under appropriate pH conditions or exposure to light to initiate crosslinking.

[0014] Exemplary macromers useful in the present disclosure include polyethylene glycol (PEG)-based polymers, poly(1,2-glycerol) carbonate (PGC)-based polymers, and poly(ethyleneimine)-based polymers. The macromers may have multiple functional groups, such as amine, alkene, and / or thiol functional groups, available for reaction with a crosslinker. Figures 1A and 1B show exemplary macromer structures depicting a branched poly(ethyleneimine) with amine functional groups (Figure 1A) and a branched poly(ethyleneimine) with thiol functional groups (Figure 1B). Further examples of macromers that can be used herein are shown in FIG. 2A (poly(ethyleneimine)), FIG. 2B (4-arm PEG-NH2), FIG. 2C (PEG-based macromer with alkene functionality), FIG. 2D (poly(1,2-glycerol) carbonate-based macromer with alkene functionality, where m and n are integers representing the amount of each unit that add up to 100%, and "ran" refers to a random copolymer), FIG. 2E (PEG-based macromer containing norbornene moieties with alkene functionality), FIG. 2F (poly(1,2-glycerol) carbonate-based macromer with alkene functionality), FIG. (Poly(1,2-glycerol carbonate)-based macromers containing norbornene moieties with alkene functional groups, where l, m, and n are integers equal to or greater than 1), and FIG. 2G (hyperbranched poly(ethyleneimine)-thiol) (see also Example 7). In at least one example, the macromer comprises a poly(1,2-glycerol) carbonate-based polymer having at least one norborene group, where the norborene group constitutes between 1% and 90% of the macromer.

[0015] Examples of crosslinkers useful in the present disclosure include PEG-based polymers containing one or more N-hydroxysuccinimide or maleimide functional groups. Figures 3A and 3B show exemplary crosslinker structures representing N-hydroxysuccinimide-PEG polymers (Figure 3A) and maleimide-PEG polymers (Figure 3B). Further examples of crosslinkers that can be used herein are shown in Figures 4A-4D. Figures 4A and 4B are two different types of N-hydroxysuccinimide functionalized PEG crosslinkers. The structures are the same except that the one shown in Figure 4A contains an internal hydrolyzable ester bond. Figure 4C shows another exemplary structure of an N-hydroxysuccinimide functionalized PEG crosslinker, where m is an integer equal to or greater than 1, e.g., m=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (see also Example 5). FIG. 4D shows an exemplary structure of a maleimide-functionalized PEG crosslinker, where n is an integer equal to or greater than 1, e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (see also Example 6).

[0016] As mentioned above, the gels herein can form a three-dimensional polymer network that can form a barrier over a wound or other site of interest in the body, such as the colon or another portion of the GI tract. FIG. 5 is a simplified diagram of crosslinking between a macromer and a crosslinker, where a functional group of the macromer reacts with a functional group of the crosslinker. Depending on the strength of the bond between the macromer and the crosslinker, the polymer network of the gel can be disrupted, for example, dissolving the gel. FIG. 6 is a simplified diagram of dissolution of a gel. Dissolution can occur, for example, by hydrolysis. FIG. 7 shows the hydrolysis of a hydrogel having thiol groups. Dissolution can also occur via thiol-thioester exchange, such as by reaction with cysteine ​​methyl ester, as described in some examples below (FIG. 8). In the latter case, it is believed that the primary amine of the cysteine ​​methyl ester rearranges to form an irreversible amide bond, preventing reformation of the gel after the polymer network has collapsed.

[0017] The structure of the crosslinker can help control the dissolution rate. Figure 9 shows possible reactions at different sites of an exemplary N-hydroxysuccinimide functionalized PEG crosslinker: (A) reaction with NHS ester, (B) reaction with internal thioester, and (C) reaction with internal ester. Reactions of these sites with macromer poly(ethyleneimine), cysteine ​​methyl ester, and water are shown, with the darker shaded areas corresponding to the more reactive moieties. Thus, the NHS ester (A) is most reactive with the macromer poly(ethyleneimine), and the internal thioester (B) is most reactive with cysteine ​​methyl ester. The internal esters have equal reactivity with the macromer, cysteine ​​methyl ester, and water. Without wishing to be bound by theory, it is believed that the stability of the gel is at least partially derived from the length of the hydrophobic methylene chain that protects adjacent thioesters from hydrolysis or thiol-thioester exchange.

[0018] The gels herein can be formed on a target tissue of a subject, such as tissue of the GI tract (e.g., intestinal tissue, colonic tissue, etc.). For example, the crosslinker and macromer can be delivered separately to the target tissue site such that the two components do not contact each other until they reach the target tissue site. In some examples, a dual lumen catheter can be used, for example, where the crosslinker and macromer are delivered in separate lumens to the target tissue site. The two components can contact each other at the target tissue site, and gelation occurs at the target tissue site in the presence of a photoinitiator activated by an appropriate pH (e.g., components formulated to crosslink at the physiological pH of the GI tract) or by UV or visible light. For example, a photoinitiator can be applied to the target tissue site before, after, or simultaneously with the crosslinker and / or macromer, and then light can be applied to activate the photoinitiator to initiate crosslinking and form a gel. Gelation can be initiated in a time greater than 0 seconds and less than 30 seconds, less than 25 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds, for example, greater than 1 second and less than 15 seconds. The crosslinker and macromer (and photoinitiator, if present) may be selected to provide a relatively fast gelation rate to form a gel when exposed to the attractive force of gravity in a tortuous environment such as the GI tract.

[0019] The gel, when formed in situ on the tissue, can form a barrier with sufficient strength to remain intact for a desired period of time. For example, the gel can remain on the tissue for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 5 days, at least 7 days, at least 14 days, at least 21 days, or at least 30 days. According to some embodiments of the present disclosure, the gel can form a barrier on the tissue that lasts for a period ranging from about 1 hour to about 60 days, from about 1 hour to about 30 days, from about 1 hour to about 14 days, from about 1 hour to about 24 hours, from about 12 hours to about 48 hours, or from about 2 days to about 21 days, from about 5 days to about 14 days.

[0020] The crosslinker and macromer may be selected to provide sufficient strength for the gel to meet the desired time period for forming a barrier on tissue. As discussed in the Examples below, a higher crosslink density (which depends at least in part on the number and type of functional groups on the crosslinker and macromer) and / or relative hydrophobicity is expected to provide a stronger gel with a longer residence time when applied to tissue. The gels herein may be biocompatible and / or biodegradable. For example, the gel may dissolve over time (e.g., by hydrolysis and / or in the presence of exogenous thioesters such as thiol-thioester exchange, as described in the Examples below) depending on the crosslink density and strength of the gel.

[0021] Exemplary compositions and systems useful in medical procedures, including endoscopic procedures, are further described below, e.g., compositions or systems that include or are formulated to form a gel. The compositions can be applied to a subject for therapeutic purposes, and the compositions can be activated via a variety of mechanisms (e.g., to form a gel in situ).

[0022] pH Activated Gel System In some aspects of the present disclosure, the composition or system may be formulated to crosslink and form a cohesive gel at physiological pH, e.g., about 7 to about 7.5, e.g., about 7.35 to 7.45. Thus, such compositions and systems may be pH-activated such that the gel, e.g., hydrogel, selectively crosslinks at neutral to basic pH (e.g., little or no crosslinking at acidic pH) and the reaction rate increases as the pH increases. According to some aspects of the present disclosure, the composition or system may be pH-activated to form a gel, e.g., hydrogel. For example, the composition may include at least two components, e.g., a first component (e.g., a first partial solution) and a second component (e.g., a second partial solution), that crosslink at physiological pH, e.g., a pH within the range of about 7 to about 7.5. Thus, for example, the first and second partial solutions may have different pH values ​​and may be mixed together to provide a physiological pH to form a gel.

[0023] The first component of the exemplary system may include a macromer. The macromer may be a multifunctional polyethylene glycol (PEG)-based or poly(ethyleneimine)-based polymer. For example, the PEG-based or poly(ethyleneimine)-based polymer may have a molecular weight of at least 1500 Da (g / mol), such as about 1800 Da to about 2200 Da, such as about 2000 Da. For example, the macromer may have a molecular weight in the range of about 1500 Da to about 2500 Da, about 1500 Da to about 2000 Da, or about 1800 Da to about 2200 Da. The poly(ethyleneimine) may be linear or branched. In some examples, the macromer may be a multifunctional PEG-based or poly(ethyleneimine)-based polymer having multiple functional groups. The multiple functional groups may react with a crosslinker of the system (examples of crosslinkers are discussed in more detail below). Such functional groups may be, for example, amine or thiol functional groups. According to some embodiments, the multifunctional PEG- or poly(ethyleneimine)-based polymer may include a plurality of 2-20 functional groups, e.g., 4, 6, 8, or 15 functional groups. Exemplary structures depicting branched poly(ethyleneimine) with amine functional groups (FIG. 1A) and branched poly(ethyleneimine) with thiol functional groups (FIG. 1B) may be used in the present disclosure. In some embodiments, the macromer is dissolved in a buffer. Exemplary buffers in which the macromer can be dissolved include, but are not limited to, borate buffer. For example, the borate buffer may have a pH of about 8.5-9.0.

[0024] The second component of the system may include a crosslinker. Examples of crosslinkers include, but are not limited to, PEG-based polymers. For example, the PEG-based polymers used as crosslinkers may have a molecular weight greater than 3000 Da, such as about 3200 Da to about 3500 Da, such as about 3400 Da. According to some embodiments of the present disclosure, the crosslinker has a molecular weight ranging from about 3000 Da to about 3800 Da, about 3200 Da to about 3500 Da, or about 3400 Da to about 3800 Da. In some examples, the crosslinker may be a PEG-based polymer that includes one or more N-hydroxysuccinimide or maleimide functional groups. The N-hydroxysuccinimide or maleimide groups may react with the macromer, as described in more detail below. Exemplary structures representing N-hydroxysuccinimide-PEG polymers and maleimide-PEG polymers are shown in Figures 3A and 3B, respectively. However, it should be noted that suitable crosslinkers, such as PEG-based polymers, are not limited to N-hydroxysuccinimide or maleimide functional groups. PEG-based polymers suitable for the present disclosure may include other functional groups that can react with the macromers of the first component of the system to form a gel.

[0025] In some embodiments, the crosslinker may be provided in a solution that includes a buffer, e.g., the crosslinker is dissolved in a buffer. For example, the buffer may be a phosphate buffer, such as phosphate buffered saline (PBS). The buffer in which the crosslinker is provided, e.g., dissolved, may have a lower pH than the buffer in which the macromer is dissolved. For example, the crosslinker may be provided, e.g., dissolved, in a PBS solution having a pH of about 6.0-6.5. Thus, a system for forming a gel according to the present disclosure may include at least two buffers that may be pH activated, one having a higher pH than the other.

[0026] The crosslinker and macromer may be present in a molar ratio of about 3:2 to 7:3, such as a molar functional ratio of 2:1, respectively (e.g., N-hydroxysuccinimide:amine, N-hydroxysuccinimide:thiol, maleimide:amine, maleimide:thiol, etc.). The gel may be an aqueous composition having a combined content of crosslinker and macromer of at least 15% by weight based on the total weight of the composition. For example, the content of crosslinker may be about 10 to 20% by weight based on the total weight of the composition, such as in the range of about 10% to about 15% by weight, about 12% to about 18% by weight, or about 15% to about 20% by weight. Additionally or alternatively, the content of macromer may be about 5 to 10% by weight, such as in the range of about 5% to about 8% by weight, or about 7% to about 9% by weight based on the total weight of the composition. As mentioned above, the first and second partial solutions may comprise at least two different buffers, for example, a first buffer suitable for the crosslinker and a second buffer suitable for the macromer. According to some embodiments, the first buffer comprises a phosphate buffer and the second buffer comprises a borate buffer. The aqueous composition may comprise any suitable salt as a buffering agent. It should be noted that the mechanical properties of the gel, e.g., hydrogel, formed by the compositions herein may be determined at least in part by the amount of macromer and / or crosslinker. For example, gel strength may increase as the content of macromer and crosslinker in the aqueous composition increases. Thus, for example, a composition comprising about 20% or about 25% by weight of a combination of macromer and crosslinker relative to the total weight of the aqueous gel system may form a gel having a higher gel strength than a gel formed from a composition comprising about 15% by weight of a combination of macromer and crosslinker.

[0027] The components of the composition or system (eg, macromer, crosslinker, and their respective buffers) can be mixed together. Under physiological pH, the functional groups of the crosslinker and the macromer react with each other through chemical bonds, thereby allowing instant gelation. For example, when the composition is at a pH of about 7 to about 7.5, the macromer and the crosslinker may react to form a gel. In some embodiments, the gel may be formed within 20 seconds, 15 seconds, 10 seconds, or about 5 seconds upon mixing a first component comprising the macromer with a first buffer and a second component comprising the crosslinker with a second buffer. For example, the gel may be formed in a time range of about 1 second to about 15 seconds, about 3 seconds to about 8 seconds, about 5 seconds to about 10 seconds, or about 2 seconds to about 5 seconds. The resulting gel, e.g., a hydrogel, may be dissolvable either passively over time, e.g., in a physiological environment, or on demand, e.g., by application of an agent capable of disrupting the hydrogel network. For example, the gel may dissolve within about 10 to 30 minutes. Solubility may be measured in a laboratory environment, e.g., by measuring the rheology of the gel when immersed in an aqueous solution.

[0028] Gels formed from the macromers and crosslinkers can exhibit desirable properties. For example, the storage modulus (as a measure of gel strength) of the resulting gel, e.g., hydrogel, can range from about 2.0 to 10.5 kPa, e.g., from about 2.5 kPa to about 10 kPa, from about 5 kPa to about 8 kPa, or from about 3.5 kPa to about 7.5 kPa. Additionally or alternatively, the gel can retain a gel strength (also referred to herein as storage modulus G') in the range of about 2000 to 10,000 Pa for a desired period of time, e.g., up to 30 days or more, at about room temperature settings. Further, for example, the gel, e.g., hydrogel, when adhered to tissue, e.g., tissue of a body lumen, e.g., colonic tissue of the GI tract, can have a gel strength of about 0.03 to 0.90 N / cm 2 , for example, about 0.1 to 0.6 N / cm 2 , for example, about 0.05 N / cm 2 ~about 0.4N / cm 2 , about 0.5N / cm 2 ~about 0.9N / cm 2 , or about 0.75 N / cm 2 ~about 0.9N / cm 2 The shear force may range from 0.1 to 0.5.

[0029] Additionally or alternatively, the gel may be formulated to withstand a burst pressure (corresponding to the pressure at which the gel will tear or break when adhered to tissue) of up to about 200 mbar, such as up to 150 mbar, for example, greater than 1 mbar and less than or equal to 200 mbar (1 mbar=100 Pa). It should be noted that the burst pressure of the gel can be measured by a catheter and pressure transducer (including, for example, a Millar catheter equipped with a pressure sensor), which can be utilized to measure the baseline pressure and the pressure just before bursting. To measure the burst pressure, the gel can be formed in situ in an opening in a tissue sample and exposed to fluids of increasing pressure until the gel's cohesion and / or adhesion to the tissue is broken and the fluid passes through the opening in the tissue. The pressure corresponding to the maximum pressure of the fluid just before the gel breaks is the burst pressure.

[0030] The burst pressure of a gel applied to tissue of the GI tract, such as colon tissue, can be measured as follows: First, an opening with dimensions of approximately 1 mm by 5 mm width and length is cut into the tissue (the depth of the opening corresponds to the thickness of the tissue, approximately 5 mm for colon tissue). The tissue sample is fixed onto the open end of a container such that an area of ​​approximately 51 mm (approximately 2 inches) in diameter is placed as an unobstructed window on the container. Saline is introduced into the container and allowed to flow through the opening, and the pressure sensor is calibrated to a baseline pressure. A gel is then allowed to form in situ and the opening is closed. Saline is then introduced into the container and the increasing fluid pressure is measured until the solution cannot pass through the opening in the gel and exit the container. The maximum pressure just before the saline breaks through the gel and exits the opening is the burst pressure. In some examples herein, the gel can be formulated to withstand a burst pressure of at least 50 mbar, at least 100 mbar, or at least 120 mbar (1 mbar = 100 Pa) when adhered to colon tissue. For example, the gels herein can be formulated to withstand a burst pressure of up to about 150 mbar when adhered to colon tissue, e.g., a burst pressure in the range of about 50 mbar to about 150 mbar, about 100 mbar to about 150 mbar, or about 125 mbar to about 150 mbar. Burst pressure can be measured on colon tissue using a 1 mm x 5 mm aperture size as described above.

[0031] The burst pressure of gels used as arterial or other vascular occlusion devices can be measured by forming the gel in situ to close a blood vessel 4-6 mm in diameter. A syringe pump and pressure transducer can be used (see FIG. 56 and Example 11) and DO can be pumped through the syringe pump at a rate of 1 mL / min until leakage in the gel sample is observed. The peak pressure detected from the pressure transducer is recorded as the burst pressure (units of pressure, 1 mmHg = 133.322 Pa).

[0032] Light-activated gel system The present disclosure also includes compositions and systems formulated to form a gel when activated by light as a stimulus. In some examples, the composition may be formulated to crosslink and form a cohesive gel when exposed to light (e.g., UV light or visible light). Such compositions and systems may therefore be described as being light activated. Such compositions and systems may include, for example, a macromer, a crosslinker, a photoinitiator, and a buffer. The buffer may be any suitable buffer near or slightly above physiological pH, depending on the buffer used. For example, a phosphate buffer may be in the pH range of 7.0 to 8.0.

[0033] The macromer can be a multifunctional PEG-based polymer that includes at least one functional group. The PEG-based polymer can be linear or branched. The at least one functional group of the macromer can include, for example, a thiol group, or an alkene group, such as a vinyl group, an allyl group, an acrylate group, or a norbornene group, among other alkene groups. The functional group of the macromer can be selected based on the desired properties of the gel, including, for example, gel time. The number of functional groups of the macromer can be between 4 and 100, for example, between 10 and 50, between 25 and 65, or between 45 and 85.

[0034] In some examples, the crosslinker can be a PEG-based polymer that includes at least one functional group. The functional group of the crosslinker can be complementary to the functional group of the macromer to crosslink the macromer and the crosslinker. For example, the at least one functional group of the crosslinker can include a thiol group, or an alkene group, such as a vinyl group, an allyl group, an acrylate group, a norbornene group, or other types of alkene groups. The functional group of the crosslinker can be selected based on the desired degradation characteristics of the gel. In some examples herein, the number of functional groups of the crosslinker can be between 2 and 4, e.g., 2, 3, or 4 different functional groups.

[0035] In some examples, the macromer can include a thiol group and the crosslinker can include an alkene group, or vice versa. For example, the crosslinker can include a PEG-based polymer that includes a thiol group and the macromer includes an alkene group, such as an acrylate group. In another example, the macromer can include a PEG-based polymer that includes a thiol group and the crosslinker can include a PEG-based polymer that includes an alkene group, such as an allyl ether group.

[0036] As discussed above, the composition may include a photoinitiator, for example, to initiate gelation. Thus, for example, the photoinitiator may be a compound that absorbs light at a given wavelength of light. According to aspects of the present disclosure, the photoinitiator may absorb UV light (e.g., wavelengths between about 100-390 nm) or visible light (e.g., wavelengths between about 390-800 nm). Examples of photoinitiators suitable for the compositions herein that are activated by UV light include, but are not limited to, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) and lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP). UV light-activated gelation may occur immediately, for example, within about 5 seconds of UV light exposure. Examples of photoinitiators suitable for the compositions herein that are activated by visible light include, but are not limited to, Eosin Y. Visible light activated gelation can occur shortly or immediately after visible light exposure, for example, within about 10 seconds of visible light exposure. In some examples, the photoinitiator absorbs white light, for example, at wavelengths of about 390 nm to about 700 nm. Thus, when the composition is irradiated with UV light or visible light (e.g., white light), depending on the photoinitiator used, the composition may crosslink to form a gel. The intensity of the UV light or visible light is about 1 mW / cm 2 to about 150 mW / cm 2 For example, UV light (365 nm) intensity can range from about 4 mW / cm 2 to about 120 mW / cm 2 and the white light intensity may be in the range of about 10 mW / cm 2 (e.g., at the maximum absorption of the photoinitiator) to about 45 mW / cm 2up to 42.9 W / cm 2 may be also possible.

[0037] In some examples, the composition may include additives to facilitate or enhance the photopolymerization gelation reaction rate. Exemplary additives include small molecule additives such as, for example, tyrosine derivatives, e.g., tyrosine methyl ester or tyrosine ethyl ester. Such compositions may include photoinitiators that absorb visible light, e.g., white light.

[0038] The aforementioned components of macromer, crosslinker, and photoinitiator may be present in a combined concentration of about 10-25% by weight based on the total weight of the composition. At higher amounts (e.g., about 20-25% by weight), the composition may have a relatively short gelation time and may result in a gel with relatively high elasticity. The content of the crosslinker may be 5-10% by weight based on the total weight of the composition. Additionally or alternatively, the content of the macromer may be 5-15% by weight based on the total weight of the composition. The molar ratio between the functional groups of the macromer and the functional groups of the crosslinker may range from 1:1 to 2:1 in the composition. In some examples, the molar ratio is about 1:1. It is noted that a 1:1 ratio of the aforementioned moieties may result in an increase in elasticity, i.e., gel modulus, compared to the aforementioned 2:1 molar ratio. In some examples, the composition or system may include a macromer that includes more than one functional group per crosslinker of the composition or system (e.g., a 1:1 ratio of macromer to crosslinker, where the macromer includes at least two functional groups, or a 1:2 ratio of macromer to crosslinker, where the macromer includes at least four functional groups). Additionally, it is noted that different stoichiometries of functional moieties, e.g., the number of functional or reactive groups per macromer, may affect both the mechanical properties and swelling ratio of the resulting gel, e.g., hydrogel. For example, increasing the number of functional groups in the macromer of a photoactivated gel system may result in a stiffer (e.g., more viscous) gel with a lower swelling ratio.

[0039] The composition may include about 0.1 mM to about 100 mM of photoinitiator. Higher photoinitiator concentrations, for example, about 90 to 100 mM, may provide a relatively faster gelation rate compared to lower concentrations, for example, about 0.1 to 1 mM. When the composition includes an additive, the composition may include up to 10 mM of the additive, for example, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, or about 0.5 mM to about 1 mM.

[0040] The resulting light-activated gels, e.g., hydrogels, may exhibit many desirable properties that are beneficial for application to tissue before, during, and / or after a medical procedure. For example, the gels, e.g., hydrogels, may exhibit gel strengths (also referred to as storage modulus G') ranging from 500 to 2500 Pa, e.g., from about 500 Pa to about 1500 Pa, from about 1000 Pa to about 2000 Pa, from about 750 Pa to about 1250 Pa, from about 1750 Pa to about 2500 Pa. The gel strength G' may depend on the concentrations, macromers and crosslinkers in the composition, and / or the ratio of the components to each other. The gels, e.g., hydrogels, may exhibit swelling ratios mf / mi (the fold change in weight of the gel due to water absorption, i.e., mf is the weight of the gel at a particular time point after immersion of the gel in a buffer solution, and mi is the initial weight of the gel before immersion in the buffer solution) ranging from about 1.8 to about 1.9 times the initial mass, or from about 2.3 to about 2.4 times the initial mass. The resulting gel may exhibit a relatively low level of cytotoxicity, for example, the gel may exhibit at least greater than 97% viability after exposure to a cell line such as NIH3T3 fibroblasts for 24 hours.

[0041] The following examples are intended to illustrate the present disclosure, but are not limiting in nature. It is understood that the present disclosure encompasses additional embodiments in line with the preceding description and the following examples. The present disclosure is not limited to the examples further described below, but encompasses additional conditions without departing from the scope of the present disclosure.

[0042] Working Example Example 1 Exemplary pH-activated compositions (gel systems) were prepared ex vivo at room temperature in a humid environment according to Table 1. A first partial solution was prepared by mixing amine-terminated PEG-based macromers or poly(ethyleneimine) macromers with borate buffer at pH 8.5. Separately, a second partial solution was prepared comprising N-hydroxysuccinimide crosslinker dissolved in phosphate buffer at pH 6.5.

[0043] [Table 1]

[0044] The first and second partial solutions were mixed together to form an aqueous solution which then formed a gel. The composition was left for 1 hour to fully gel before evaluating the resulting properties.

[0045] The gel strength (storage modulus G') of the gels was measured at room temperature (approximately 22-25°C) using a TA instruments DHR-2 rheometer and assessed using a 1-100% strain sweep at a frequency of 1 Hz. The linear viscoelastic region was determined as the strain rate increased until the slope of the gel strength curve decreased by 10%. A frequency sweep from 1-10 Hz was then performed within the linear viscoelastic region at 3% strain. The gels were immersed in 50 mM PBS until the gel dissolved, after which frequency sweeps were performed at t=0, 4 h, 24 h, 48 h, 7 days, and 30 days.

[0046] FIG. 10 shows the gel strength of the gel over the above time periods. As shown, the gel exhibited a gel strength of at least 1,000 Pa for at least 30 days. The gel exhibited an initial gel strength of about 4,000 Pa at t=0 and a peak gel strength of about 8,000 Pa at t=24 hours. These properties indicate that the pH-activated gel can function as a sustained protective barrier for tissue for at least 30 days.

[0047] Gel times were determined using the inverted tube test. Gel was determined immediately after mixing the first and second part solutions when the gel no longer ran down the side of the vial when inverted. Gel times were measured in less than 1 second.

[0048] The swelling ratio was calculated as a weight percent of the hydrogel after immersion in 50 mM PBS according to the following formula:

[0049]

number

[0050] where mf is the weight of the gel at a particular time point after the gel is immersed in the buffer, and mi is the initial weight of the gel before immersion in the buffer.

[0051] [Table 2]

[0052] Adhesion measurements were determined using ex vivo porcine colon tissue by lap shear testing with an Instron® machine. The tissue was cut into pieces measuring approximately 51 mm x 25 mm (approximately 2 in x 1 in). The gel was placed between the two pieces of colon tissue and left in a humid chamber for 1 hour to fully gel. Note that in this example, gelation was delayed until approximately 5-10 minutes, which allows for better processing of the ex vivo tissue / adhesion measurements. Thus, to ensure complete gelation, the tissue sample was left in the chamber for 1 hour. The gel-tissue construct was then mounted in the Instron® and the two strips of colon tissue were pulled in opposite directions to each other at a rate of 10 mm / min, with the force continuously measured until cohesive failure of the hydrogel was observed. Adhesion measurements ranged from 0.03 to 0.85 N / cm 2 The range was.

[0053] Example 2 Two UV-activated compositions (gel compositions 1 and 2) were prepared ex vivo at room temperature by mixing an alkene-containing PEG-based macromer, a thiol-containing PEG-based crosslinker, and the photoinitiator LAP in PBS at pH 7.4 according to Table 3. Gel composition 1 was prepared using the PGC-based macromer shown in Figure 2D, and gel composition 2 was prepared using the PEG-based macromer shown in Figure 2C. The crosslinkers were a PEG dithiol crosslinker or a 4-arm-PEG-thiol crosslinker.

[0054] [Table 3]

[0055] The composition was gelled using a handheld 4 W lamp of 365 nm UV light. The gel strength before and after swelling the gel in buffer was measured with a TA Instruments DHR-2 rheometer using 8 mm parallel plates at room temperature (approximately 22-25 °C). A frequency sweep was performed at 1% strain and the gel strength G' was determined from the linear viscoelastic portion of the sweep.

[0056] FIG. 11A shows the gel strength of the gels before and after swelling. The gel obtained from composition 1 exhibited a gel strength of about 1,800 Pa before swelling and a gel strength of about 1,500 Pa after swelling. The gel obtained from composition 2 exhibited a gel strength of about 700 Pa before swelling and a gel strength of about 1000 Pa after swelling. It is noted that composition 1 contains macromers with more reactive moieties per molecule compared to composition 2, which has macromers with four reactive moieties per molecule. Thus, composition 1 exhibited a higher crosslink density than composition 2, resulting in a stronger gel strength.

[0057] The swelling ratio was determined according to the above formula 1 by obtaining the initial gel mass (mi), then swelling in buffer for 24 hours, and obtaining the gel mass (mf) after wiping off the excess buffer.

[0058] FIG. 11B shows the gel swelling ratios of both gels (expressed in FIG. 11B as a ratio of mf divided by mi, rather than a percentage). The gel obtained from composition 1 showed a ratio of about 1.9, while the gel obtained from composition 2 showed a ratio of about 2.4. The difference in swelling ratio between the two compositions may also be a result of the difference in the macromers used, which results in different crosslink densities. It can be seen that a higher crosslink density results in a lower swelling ratio.

[0059] Example 3 An exemplary visible light-activated gel system (Composition 3) was prepared ex vivo at room temperature by mixing an alkene-containing PEG-based macromer with a thiol-containing PEG-based crosslinker, photoinitiator Eosin Y, additive tyrosine ethyl ester, and pH 7.0 phosphate buffer according to Table 4.

[0060] [Table 4]

[0061] The system was gelled using an AmScope 150W halogen lamp equipped with a dual gooseneck fiber optic illuminator with broad spectrum white light (400-700 nm).

[0062] Gel strength was measured at room temperature (about 22 to 25° C.) at t=0, 4 hours, 24 hours, 48 ​​hours, and 7 days in the same manner as in Example 2, as shown in FIG. 12A. The gels exhibited an initial gel strength of approximately 600 Pa at t=0, which increased in gel strength over the next 7 days with a peak gel strength of approximately 2,100 Pa at t=24 hours. The gels exhibited a gel strength of at least 600 Pa for at least 7 days. Thus, these results indicate that the gels can function as a sustained protective barrier for tissues for at least 7 days.

[0063] Gel times were measured on a DHR-2 rheometer at room temperature (approximately 22-25 °C) using 20 mm parallel plates with two goosenecks of a halogen lamp aimed at the solution and then exposed between the two parallel plates. Time sweeps were performed at a frequency of 1 Hz. The lamp was turned on at 30 seconds and the time after which gelation occurred was recorded. The gel times are shown in Figure 12B. As shown, the gelation rate increases as the concentration of tyrosine ethyl ester is increased (from 0 to 10 mM).

[0064] The viscosity of the gel precursor was measured from flow sweeps on a DHR-2 rheometer using a 50 mm 1.008° cone plate at 37° C. The results, shown in FIG. 12C, show that prior to photoinitiation, the gel precursor solution exhibited Newtonian properties and sufficiently low viscosity in the shear rate range above 1 / s to allow the solution to be applied through long catheters. Furthermore, the solution did not exhibit significant shear thinning or thickening.

[0065] The in vitro cytotoxicity of the photoinitiators and gels was tested using NIH3T3 fibroblast cells cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded in either 96-well or 12-well plates at a density of 2,500 or 25,000 cells / well, respectively, and allowed to adhere overnight. Photoinitiator and gel solutions were sterile filtered using a 0.22 μm filter before being tested in vitro. The gel solutions were then gelled in a biosafety cabinet using a 150 W halogen lamp and co-cultured with the cells using 3 μm pore size Transwell inserts. Cells were incubated with treatment for 24 hours and then viability was measured using the MTS (3-(4,5-dimethyltriazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tratozolium, inner salt) assay (CellTiter96™ AQ ueous One, Promega). Cell viability was normalized to control untreated cells.

[0066] 12D-12E show the results of the cytotoxicity tests described above. 50 The IC was approximately 0.223 mM, which exceeds the concentration used in the gel formulations. As seen in FIG. 12E, which demonstrates greater than 97% cell viability for all three weight percent gel formulations, 50 By using less than 100% photoinitiator, no cytotoxicity issues were anticipated in the gels. Furthermore, no differences were observed between the viability of the three gel formulations, indicating that the other components of the gels do not have any significant toxicity associated with them, even when their concentrations in solution are increased.

[0067] Example 4 The crosslinker shown in FIG. 4A ("SA crosslinker") was synthesized as follows, as shown in FIG.

[0068] First, SA-PEG-SA was synthesized as follows: Poly(ethylene glycol) (PEG; average Mn 3000 g / mol; Sigma Aldrich) (5 g, 1.6 mmol) was dissolved in a three-neck round-bottom flask with stirring at 120 °C. Once dissolved, the flask was placed under vacuum and the temperature was reduced to 80 °C and stirred for 30 min. The flask was purged with nitrogen three times. Succinic anhydride (SA) (99%; Aldrich) (0.75 g, 7.5 mmol) was added to the flask. The reaction was stirred under nitrogen for 18 h. The contents were then dissolved in a minimum amount of anhydrous methylene chloride (DCM; 99%; anhydrous; Sigma Aldrich) and precipitated in diethyl ether. Finally, the product was filtered and dried under vacuum for 1 day (white solid, 99% yield). Proton and carbon nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) spectra were obtained in CDCl3 on an Agilent 500 MHz spectrometer. The NMR spectrum of the SA-PEG-SA product was as follows: 1 H NMR (500 MHz), CDCl3: δ 2.62 (m, 8H), 3.64 (overlap, 288H), 4.24 (m, J = 4.6 Hz, 4H); 13C NMR (500 MHz), CDCl3: δ 174.0, 172.1, 70.5, 63.8, 29.3, 28.3 ppm. The SA-PEG-SA intermediate was obtained in a reaction yield of 99%.

[0069] SA-PEG-SA (4 g, 1.3 mmol) was then added to a dry round-bottom flask and dissolved in 15 mL of dry DCM. N-hydroxysulfonimide (NHS; 99%, Sigma Aldrich) (0.4 g, 3.8 mmol) and dicyclohexylcarbodiimide (DCC; 99%, Sigma Aldrich) (0.8 g, 3.8 mmol) were added and the flask was purged with argon. The mixture was stirred at room temperature for 18 h. Dicyclohexylurea was filtered and the solution was concentrated and precipitated in diethyl ether. The resulting product, SA crosslinker (white water-soluble powder), was collected by filtration and dried under vacuum overnight (white solid, 98% yield). The structure was: 1 H NMR, 13 The NMR spectrum of the SA crosslinker measured as above was as follows: 1 H NMR (500MHz), CDC13: δ2.70(t,J=1.0Hz,4H),2.77(t,J=1.0Hz,8H),2.89(t,J=1.0Hz,4H),3.57(overlap,296H),4.20(t,J=1.0Hz,4H)ppm. 13 C NMR (500MHz), CDC13: δ170.9, 168.9, 167.6, 70.7, 64.1, 28.6, 26.2, 25.5ppm.

[0070] Molecular weight and polymer distribution were measured using gel permeation chromatography (GPC) at a flow rate of 1.0 mL / min in tetrahydrofuran (THF) as the mobile phase. For the SA crosslinker, M w PDI: 2949 g / mol; PDI: 1.02. Two identical Jordi Gel DVB columns (Jordi Labs, 250 mm × 10 mm, pore size 10 5GPC analysis was performed on an OptiLab DSP interference refractometer (Wyatt Technology) equipped with a 1000 Å (1000 Å) for SA crosslinker. n : 2893 g / mol. Matrix-assisted laser desorption / ionization (MALDI-TOF) was performed on a Bruker autoflex Speed ​​spectrometer equipped with a SMART-beam II and a flash detector. For the SA crosslinker, MALDI-TOF(pos):M w : 3600 m / z. Differential scanning calorimeter (DSC) spectra were obtained on a Q100TA Instruments calorimeter and used to determine melting points (mp). For SA crosslinker, Mp(DSC): 43.5°C.

[0071] Example 5 The crosslinker shown in FIG. 4B ("SVA crosslinker") (average Mn 3400) was obtained from Laysan Bio, Inc. and stored in a glove box. The NMR spectrum measured as described above was as follows: 1 H NMR(500MHz), CDC13:δ1.69(tt,J=7.3,7.4 4H), 1.83(tt,J=6.1,7.3,4H),2.64(t,J=7.3,4H),2.83(b,8H),3.49(t,J=6.1,4H),3.63(m,300H)ppm. 13 C NMR (500 MHz), CDCl3: δ 169.1, 168.6, 70.4, 30.6, 28.4, 25.5, 21.4 ppm. The following were measured for the SVA crosslinker and were measured as described in Example 4. MALDI-TOF (pos): w :3700m / z;GPC:M n :4635g / mol;M w :4812g / mol;Polydispersity index PDI:1.03;Mp(DSC):47.6℃.

[0072] Example 6 Hydrogels were prepared as shown in FIG. 14 by mixing the crosslinker shown in FIG. 4A ("SA crosslinker") and the crosslinker shown in FIG. 4B ("SVA crosslinker") with hyperbranched polyethyleneimine (PEI) (average Mn 2000 g / mol, manufactured by Polysciences) or 4-arm PEG-NH2 HCl salt (4-arm PEG-NH2) (star polymer, Mn 5000 g / mol, manufactured by JenKem). Briefly, each PEG crosslinker was dissolved in 0.1 M phosphate buffer, pH 6.5. Each of PEI and 4-arm PEG-NH2 was dissolved in 0.3 M borate buffer, pH 8.6. The pH obtained after mixing the crosslinker and macromer solutions was adjusted to pH 8.5. The molar ratio of amine:NHS was 1:15, and hydrogels were prepared at 10, 15, or 20 weight percent (wt%). The ratio of amine groups to NHS groups of the macromer in the crosslinks was kept constant and the weight percentage was increased to increase the amount in solution. The properties of the hydrogels were measured and analyzed as described in the next section.

[0073] Data were analyzed with Graph Pad Prism8. For hydrogel characterization studies, error bars represent the standard deviation of results from ≥3 replicates. For bacterial translocation studies, error bars represent the standard deviation of results from 3 biological replicates, each performed with ≥3 technical replicates. Results were compared and significance was assessed using Student's T-test. *p<0.05 is significant.

[0074] Gelation measurement A relatively fast gelation time (e.g., less than 3 seconds) may be useful for forming gels in situ, for example, during polypectomy procedures or other internal wound coverage. For gelation measurements, the crosslinker and amine-terminated macromer solutions were mixed and placed in a 2 mL glass vial. Gelation was tested using an inverted tube test setup. The tube was inverted every 10 seconds. Gelation was defined by the time the solution remained at the bottom when the vial was inverted. All gelation studies were performed at room temperature, 25°C.

[0075] Figure 15 shows gelation measurements. Panel A) Gel time of hydrogels at various weight percentages and various formulations, and Panel B) Gel time of SA crosslinker + PEI hydrogels, 15 wt%, at increasing pH. *p<0.05. SA crosslinker + PEI hydrogels gelled faster as the weight percentage increased from 10 wt% to 20 wt%, as shown in Figure 15, Panel A) (all hydrogels in Panel A were formed at pH 8.5). The increase in gelation time is attributed to a higher concentration of reactive groups, thus promoting more rapid gelation. Next, gelation times between SA crosslinker and either PEI or 4-arm PEG-NH2 macromer at 15 wt% were compared. Gel times were similar at about 90 seconds (1.5 minutes), suggesting that gelation is independent of the amine macromer.

[0076] The SVA crosslinker + PEI hydrogel was found to gel at a similar rate to the SA crosslinker + 4-arm PEG-NH2 and SA crosslinker + PEI hydrogels. However, the SVA crosslinker + 4-arm PEG-NH2 hydrogel was observed to gel faster than the SA crosslinker + 4-arm PEG-NH2. This increase in gelation time is believed to be due to two factors. The 4-arm PEG-NH2 macromer contains four long amine-terminated arms and has a molecular weight of 5 kDa. PEI is a more condensed branched macromer with a molecular weight of 2.0 kDa. The longer PEG arms of the 4-arm PEG-NH2 are believed to favor the faster gelation time of the SVA crosslinker + 4-arm PEG-NH2 hydrogel, as they provide increased steric freedom compared to PEI, a branched polymer with shorter arms containing terminal amines and a smaller molecular weight. This steric hindrance observed in the smaller branched PEI structure is believed to reduce its ability to readily react with NHS reactive groups in the hydrogel network compared to the higher molecular weight and longer armed star-shaped 4-arm PEG-NH2 structure.

[0077] Regarding defects in the hydrogel network, the terminal amines facilitate conjugation with NHS esters, but the SA crosslinker contains internal esters that are also susceptible to macromer amidation and hydrolysis. The preferred site for amidation in the SA crosslinker is the NHS ester, which is consistent with the model system. 1 H NMR analysis, t 1 / 2 =0.60min -1 FIG. 19 shows the NMR spectral changes of the reaction, and FIG. 18 shows the monitoring changes in the signals in the NMR of the NHS ester and the inner ester as a function of time. Although unlikely, it is possible that amidation occurs at the inner ester, leading to defects in the hydrogel network (t 1 / 2 =1.8min -1 ) (see Figure 18). Furthermore, the half-life for hydrolysis of the ester bond is t 1 / 2 = <5 min, which further leads to defects in the hydrogel network. Hydrolysis and amidation of the internal esters are competing reactions, which may result in slower gelation times compared to the SVA crosslinker + 4-arm PEG-NH2 hydrogel.

[0078] The effect of pH on gelation time was also evaluated for 15 wt % SA crosslinker+PEI hydrogels at pH 8.5, pH 9.5, and pH 10.5, as summarized in FIG. 15, panel B). The gelation rate increased with increasing pH of the PEI buffer solution: 100 s at pH 8.6, 60 s at pH 9.5, and 5 s at pH 10.5. For comparison, see FIG. 15, panel A), at pH 8.5, SA crosslinker+4-arm PEG-NH2 hydrogels gelled in 80 s, SVA crosslinker+PEI hydrogels gelled in 90 s, and SVA crosslinker+4-arm PEG-NH2 hydrogels gelled in 45 s. Increasing the pH of the PEI solution resulted in faster gelation.

[0079] Rheological measurements Rheological measurements were obtained using a TA Instruments DHR-2 rheometer. Rheological measurements were performed at 22 °C using 8 mm parallel plates. Oscillatory strain sweeps were performed from 0.1 to 10% strain at a frequency of 0.1 Hz. From the strain sweeps, the linear viscoelastic region was determined as the percentage strain below which G' deviates 10° from horizontal. Frequency sweeps were then performed at all time points over 30 days. Strain was set to fall within the linear viscoelastic region of 3%, and frequencies were performed ranging from 0.1 Hz to 10 Hz according to previously published protocols. Data are expressed as mean ± standard deviation (n ≥ 3).

[0080] The storage modulus (G') of the hydrogels was measured at 0, 4, 24, 48 hours, 7 days, and 30 days after swelling in 100 mM PBS, pH 7.4 (3% strain applied). The swelling ratio and storage modulus (G') of each hydrogel were measured over 30 days or until the hydrogel was dissolved in 100 μM PBS, pH 7.4, as an indication of strength and swelling of the hydrogel. Figure 16 shows the strength (G') measured for the following hydrogels: Panel A) SA crosslinker + PEI at various weight percent; Panel B) 15 wt% SA crosslinker + PEI or SA crosslinker + 4-arm PEG-NH2; Panel C) 15 wt% SA crosslinker + 4-arm PEG-NH2 and SVA + 4-arm PEG-NH2; Panel D) 15 wt% SA crosslinker + PEI and SVA crosslinker + PEI. All rheometry was recorded over time after swelling. *p<0.05.

[0081] The 10 wt%, 15 wt%, and 20 wt% SA crosslinker + PEI hydrogels exhibited average G' of 638 Pa, 992 Pa, and 2930 Pa, respectively, upon gelation. The 15 wt% and 20 wt% hydrogels maintained mechanical integrity (G'>300 Pa) up to 48 h, whereas the 10 wt% SA crosslinker + PEI hydrogel dissolved after 4 h (G'<300 Pa). To evaluate the effect of degradable ester bonds in the crosslinker on mechanical strength, hydrogels were prepared using SA or SVA crosslinkers and 4-arm PEG-NH2 (panels B) and D). In comparison, the 15 wt% SA crosslinker + 4-arm PEG-NH2 hydrogel exhibited a G' of 3814 Pa at time t=0 and maintained integrity over 7 days of swelling, with minimal change in G' over 24 h, and reduced mechanical strength at 48 h and 7 days. The 15 wt% SVA crosslinker + PEI and SVA crosslinker + 4-arm PEG-NH2 hydrogels exhibited G' of 1683 Pa and 7739 Pa, respectively. The G' of all hydrogels initially increased upon swelling (Figure 16). The SA crosslinker + 4-arm PEG-NH2 hydrogel was found to maintain its integrity with unchanged G' (3591 Pa) over 48 hours and maintain its hydrogel morphology for 7 days of swelling. On the other hand, the SVA crosslinker + 4-arm PEG-NH2 hydrogel maintained its mechanical strength (13766 Pa) over 30 days of swelling. A similar trend was observed for hydrogels prepared using PEI, while the G' remained similar over 48 hours for the SA crosslinker + PEI hydrogel (1380 Pa).

[0082] As shown for the SA crosslinker + PEI hydrogels, increasing the hydrogel weight percentage resulted in a larger G' and longer-lasting mechanical strength (see Panel A). Additionally, G' decreased over time for each SA crosslinker + PEI hydrogen, likely due to hydrolysis at the internal ester bonds. On the other hand, G' remained unchanged for the SVA crosslinker + PEI hydrogels over 30 days of swelling, likely due to the lack of degradable bonds in the SVA crosslinker structure (see Panel C).

[0083] The increased degradation rate of the SA crosslinker+PEI hydrogel compared to the SA crosslinker+4-arm PEG-NH2 hydrogel is believed to be due to the local basic pH within the hydrogel network as a result of PEI. The effect of pH was evaluated by swelling the SA crosslinker+PEI hydrogel in dH2O, pH 5.0, and the SA crosslinker+4-arm PEG-NH2 in aqueous TEA (tertiary amine, [M] equivalent to that present in PEI-based hydrogels) at pH 8.0. Figure 17 shows the storage modulus of SA crosslinker+PEI hydrogels swollen at pH 7.4 and pH 5.0 in panel A) and the storage modulus of SA crosslinker+4-arm PEG-NH2 hydrogels swollen at pH 7.4 and pH 8.0 in panel B). *p<0.05. The SA crosslinker+PEI hydrogel swelled at pH 5.0 and hydrolyzed in 48 hours, similar to swelling in PBS at pH 7.4 (see panel A). The similar degradation rates are likely due to the strongly basic nature of PEI and its inability to buffer the local pH. Swelling the SA crosslinker + 4-arm PEG-NH2 hydrogel in a solution containing TEA accelerated hydrolysis of the hydrogel, with the hydrogel degrading in 24 h compared to 7 days in PBS at pH 7.4. This increased hydrolysis of the SA crosslinker + 4-arm PEG-NH2 hydrogel is consistent with the local basic pH of PEI promoting hydrolysis of internal esters within the hydrogel network (see Panel B).

[0084] Rheological measurements were performed on the SA crosslinker + PEI hydrogel in dH2O, pH 5.0, and on the SA crosslinker + 4-arm PEG-NH2 at pH 8.0. The SA crosslinker + PEI hydrogel exhibited a G' of 1362 Pa at time t=0 and maintained similar mechanical integrity over 48 hours until significant hydrolysis occurred (loss of hydrogel integrity is defined as G'<300 Pa since the hydrogel cannot retain its mechanical structure during rheological measurements if it exhibits a storage modulus below 300 Pa). This was regardless of the pH of the water in which the gel was swelled (see Figure 17). The G' of the SA crosslinker + 4-arm PEG-NH2 hydrogel formulation was 2239 Pa at time t=0 and was soluble in pH 8.0 aqueous solution by 48 hours (G'<300 Pa). On the other hand, the G' of the SA crosslinker + 4-arm PEG-NH2 hydrogel swollen at pH 7.4 decreased over time but maintained its mechanical integrity (G' > 300 Pa) through 7 days (see Figure 17).

[0085] Hydrolysis of the internal ester bond was investigated in a model system. 1 The hydrolysis of the SA crosslinker's internal ester (top) shifted upward in pH 8.0 DO compared to the unhydrolyzed internal ester of the SA crosslinker (bottom) in unbuffered DO based on the change in NMR signal. The half-life (t 1 / 2 ) in the presence of TEA (again, at [M] similar to that in the PEI-based hydrogels) in DO at pH 8.0 for ester linkage was 19.8 min, whereas in the absence of TEA (pH 5 or 6), over 24 h 1 H NMR showed no evidence of hydrolysis of the ester bond.

[0086] Hydrolysis of SA crosslinker + PEI hydrogels was evaluated at 37°C to mimic the colonic environment. Figure 20 shows storage modulus G' values ​​at room temperature compared to 37°C for panel A) SA crosslinker + PEI hydrogels and panel B) SA crosslinker + 4-arm PEG-NH2 hydrogels. SA crosslinker + PEI hydrogels degraded within 24 hours at 37°C, whereas SA crosslinker + 4-arm PEG-NH2 hydrogels degraded at the same rate regardless of temperature (room temperature (RT) or 37°C) and existed for 7 hours. It is believed that the increase in temperature further accelerated the PEI-catalyzed hydrolysis of the hydrogels. SA crosslinker + PEI hydrogels showed a larger G' value (3579 Pa) at 37°C compared to hydrogels maintained at room temperature, whereas SA + 4-arm PEG-NH2 hydrogels were stable during 7 days of swelling regardless of temperature.

[0087] Regardless of the hydrogel composition, immersion of the hydrogels in 100 mM PBS resulted in an initial increase in storage modulus and swelling after the first 4 hours. Figure 21 reports the swelling of the hydrogels over a 24 hour period. The 10 wt% SA crosslinker + PEI hydrogel hydrolyzed at 24 hours, so swelling data was not available. The swelling percentage of the hydrogels was reported after 24 hours of swelling. The hydrogels swelled until they reached equilibrium or degraded at 24 hours. All hydrogels swelled to at least 200% of their initial weight in the buffer. Increasing the weight percentage of the hydrogels (SA crosslinker + 10, 15, and 20 wt% PEI) swelled by 153%, 259%, and 411%, respectively. The SA crosslinker + 4-arm PEG-NH2 hydrogel swelled by 396%. The SVA crosslinker + PEI and SVA crosslinker + 4-arm PEG-NH2 hydrogels swelled by 274% and 376%, respectively. This absorptive property may be useful for covering tissue, such as for use as a wound dressing in polypectomy procedures and other medical procedures.

[0088] Adhesion Adhesion of hydrogels to ex vivo porcine colon tissue was performed with an Instron 5944 Micro-tester. Hydrogels were mixed and placed between two colon tissue pieces. Colon tissue was divided into 25 mm x 25 mm (1 inch x 1 inch) pieces and hydrogel was allowed to gel directly on the tissue. When applying hydrogel to colon tissue, an additional piece of tissue was placed on top in a "sandwich" fashion (tissue-hydrogel-tissue). In each sample, the tissue adhering to the hydrogel (obtained by scraping the colon tissue with a scalpel) was either the mucosal layer or the submucosal layer of the colon tissue. After 1 hour of gelation in a humid chamber, lap shear testing was performed according to the ASTM D3165 protocol for adhesion of hydrogels on colon tissue. Tissue pieces were pulled apart at a speed of 5 mm / min at room temperature until failure of adhesion was detected. Data are presented as mean ± standard deviation (n=3).

[0089] The adhesive strength of 15 wt% SA crosslinker+PEI, SVA crosslinker+PEI, and SA crosslinker+4-arm PEG-NH2 hydrogels was measured at 25°C on colon tissue with and without the presence of a mucosal layer. These hydrogels were chosen to determine whether the presence of PEI or 4-arm PEG-NH2 in the hydrogel alters the adhesive force and whether a hydrolyzable SA crosslinker or a non-hydrolyzable SVA crosslinker affects the adhesive force. In some samples, the mucosal layer on the colon tissue was removed with a scalpel after polypectomy to expose the submucosal layer to a more suitable model tissue. As shown in Figure 22, SA crosslinker+PEI, SVA crosslinker+PEI, and SA crosslinker+4-arm PEG-NH2 hydrogels each had an adhesive strength of 0.18 N / cm with an intact mucosal layer. 2 , 0.36N / cm 2 , and 0.03N / cm 2 and 0.31 N / cm without intact mucosal layer, respectively. 2 , 0.29N / cm 2 , and 0.64 N / cm 2The mean adhesion strength of 1 mm on colon tissue with intact mucosa layer (left data shown in black) and without mucosa layer (right data shown in dark grey) is shown in Figure 22. *p<0.05.

[0090] The SA crosslinker + PEI and SVA crosslinker + PEI hydrogels adhered best to tissue with an intact mucosal layer, with a resistance of 0.18 N / cm, respectively. 2 and 0.36N / cm 2 The SA crosslinker + 4-arm PEG-NH2 hydrogel showed the strongest adhesion to the tissue without a mucosal layer (0.64 N / cm 2 ) (Figure 22). This difference in adhesion is believed to be due to hydrogen bonding and charge-charge interactions between cationic PEI and the mucus layer compared to neutral PEG. Mucus is an anionic, hydrophobic, and viscoelastic network containing glycoproteins that are available for hydrogen bonding and electrostatic interactions with molecules such as PEI. PEG, on the other hand, is uncharged, hydrophilic, and non-adhesive; all properties understood to retard adhesion to mucus. SA crosslinker + 4-arm PEG-NH2 hydrogels adhered most strongly to colon tissue without a mucus layer, likely due to the lack of electrostatic interactions with the tissue matrix. A force of at least 0.3 N / cm was required to maintain adhesion to colon tissue. 2 is considered to be sufficient.

[0091] Cytotoxicity research The cytotoxicity of 15 wt% hydrogels was evaluated against NIH3T3 fibroblasts. Crosslinker and PEI solutions were passed through a 0.22 μm PVDF filter before mixing and gelling under sterile conditions. 50 mg, 25 mg, and 10 mg (±2.5 mg) portions of hydrogels were placed in permeable cell culture inserts (PES, 3 μm pores) (Cell Treat, 230637). Permeable cell culture inserts containing hydrogel samples were incubated in sterile deionized water for 16 h at 4 °C to allow swelling. NIH3T3 (ATCC, CRL-1658) were cultured in DMEM + 10% BCS + 1% PS at 37 °C, 5% CO2 and 95% humidified air. In the experiments, all cells were at passages 4–8. Cells were plated in 24-well plates at 1.25 × 10 4 cells / cm 2 Cells were seeded with 0.01% CO and allowed to attach for 16 hours. The medium was changed and the cell culture inserts containing the swollen hydrogels were transferred to the wells containing the attached cells. The hydrogel samples were briefly equilibrated to 37 °C before transfer. The hydrogels were incubated in the presence of cells for 24 hours. The cell culture inserts were removed and MTS reagent (Promega, G5421) diluted 1:9 in medium was added to each well. The absorbance (490 nm) was measured after 4 hours. Relative cell viability was determined by normalizing the absorbance with cells exposed to hydrogels versus unexposed controls. All experiments were completed in triplicate and error bars represent one standard deviation from the mean. All hydrogels were found to be minimally cytotoxic (>88% cell viability) (Figure 23).

[0092] Bacterial migration Bacterial translocation studies were performed using isolates of Escherichia coli and Bacteroides fragilis, as both are commonly found in the gut and are known to cause infections. E. coli is highly mobile and may potentially cross hydrogels. B. fragilis isolates are known to exhibit multidrug resistance and cause sepsis. These two common gut bacteria with pathogenic potential were evaluated for their ability to cross SA crosslinker + PEI and SA crosslinker + 4-arm PEG-NH2 hydrogels.

[0093] In vitro tests on agar plates and microscopy studies were performed. The advantage of the agar-based assay is that individual bacteria can grow for approximately 24 h until they become visible colonies, so that it is possible to detect whether even a small number of bacterial cells have invaded the hydrogel. Clinical isolates E. coli (ADR129Q-SMC9096) and B. fragilis (CFPLTA004_1B-SMC9107) were obtained from children with cystic fibrosis. Prior to inoculation of the hydrogels, the E. coli isolate was cultured aerobically in LB (lysogeny broth) overnight, and the B. fragilis isolate was cultured anaerobically on blood agar (TSA + 5% sheep blood) for 48 h using a GasPak system. Hydrogel discs (8 mm diameter × 2.5 mm height) were placed on LB agar (for E. coli) or TSA + 5% sheep blood agar (for B. fragilis), and 5 μL of bacteria or PBS was added to the top of each hydrogel. Plates were then incubated aerobically (E. coli) or anaerobically (B. fragilis) at 37°C for 24 hours. After 24 hours, the hydrogel was removed and the agar plates were incubated for an additional 24 hours under conditions appropriate for each microorganism to test bacterial growth under the hydrogel as a measure of whether the microorganism was able to pass through the hydrogel. After growth, B. fragilis isolates were scraped into 1 mL of PBS and homogenized. One mL each of B. fragilis and E. coli was centrifuged at 16,000 x g for 30 seconds and resuspended in PBS. Each isolate was then normalized to an OD600 of 1.0 in PBS for the agar plate experiments and an OD600 of 1.0 in minimal medium for the microscopy experiments as reported (BioProject Accession No. PRJNA557692). Wells treated with medium alone were used to determine background fluorescence and were subtracted from each sample before analysis.

[0094] Microscopy was performed on a Nikon Eclipse Ti inverted microscope equipped with a Hamamatsu ORCA-Flash 4.0 camera running on a Nikon Elements AR. Images were acquired through a Plan Fluor 40x DIC M N2 objective using fast scan mode and 2X2 binning. Images were processed with ImageJ, background subtracted, and signal intensity was quantified by measuring the average signal intensity per pixel through the Integrated Density (IntDen) function. For microscopy studies, 300 μL of SA crosslinker + 4-arm PEG-NH2 was inoculated into each well of an 8-well plate (Cellvis, Cat. No. C8-1.5HN). To visualize the bacteria, Syto9 was added to each culture prior to hydrogel inoculation. Bacterial cultures were inoculated either on top of the hydrogel or underneath the hydrogel where a hole had first been punctured with a pipette tip. Plates were imaged both before and after incubation to determine whether bacteria inoculated on the top were able to pass through the hydrogel. The results show that these microorganisms do not cross the SA+4-arm PEG-NH2 hydrogel, indicating the potential for sepsis prevention in vivo. Figure 24 shows bacterial mitigation by SA crosslinker+4-arm PEG-NH2 hydrogel. The presence of E. coli (left) and B. fragilis (right) was assessed in porous hydrogels where bacteria were inoculated at the bottom of the well (top two panels) and in nonporous hydrogels where bacteria were placed on the surface (bottom two panels). Three independent experiments were performed, with three technical replicates for each. Representative images are shown combining bright field and Syto9 staining. The thickness of the hydrogels was approximately 1 mm. E. coli and B. fragilis added to the bottom of the porous hydrogels were observed under a microscope (Figure 24, top left and top right, respectively), but were absent when added to the top of the nonporous hydrogels (Figure 24, bottom panel).

[0095] To quantify the effect of hydrogels on bacterial mitigation, Syto9 signal intensity was assessed at the bottom of the hydrogels after subtracting background fluorescence from the media-only control. Figure 25 reports the measured surface area of ​​Syto9 stained bacteria 24 hours after inoculation with E. coli (top graph A) and B. fragilis (bottom graph B) on SA crosslinker + 4-arm PEG-NH2 hydrogels. Bacterial presence was measured in three independent experiments. The surface area occupied by bacteria was compared between porous hydrogels inoculated with bacteria at the bottom of the well and non-porous hydrogels inoculated with bacteria on the surface. Error bars represent standard deviation, and *, **, and **** indicate significant differences in bacterial surface area at P values ​​less than 0.05, 0.01, and 0.0001, respectively. Consistent with the images in Figure 24, bacteria inoculated at the bottom of the porous hydrogels show elevated Syto9 staining compared to the control inoculated at the top (Figure 25). The SA crosslinker + 4-arm PEG-NH2 hydrogel was found to be an effective barrier against E. coli and B. fragilis for at least 24 h. In contrast, the SA crosslinker + PEI hydrogel hydrolyzed under laboratory conditions at 37 °C and therefore could not be studied.

[0096] The lack of bacterial migration through the hydrogels could be a result of the hydrogel pore size relative to the size of the bacteria. The pore sizes of the hydrogels ranged from less than 1 μm to 20 μm, and the pores were not connected to each other, forming a mesh-like network, as shown by scanning electron microscopy for the SA crosslinker + 4-arm-PEG-NH2 hydrogel (Figure 26). The length of E. coli and B. fragilis is approximately 1.0-4.5 μm. Thus, the pore size and porosity created a tortuous path for the bacteria to move through, inhibiting bacterial migration. The SA crosslinker + PEI hydrogel was not suitable for this study because it hydrolyzed under laboratory conditions at 37 °C.

[0097] The results of the agar plate assay are shown in Figures 27-28. Figure 27 tests whether B. fragilis can pass through SA crosslinker + 4-arm PEG-NH2 hydrogels by placing the hydrogels on TSA + 5% sheep blood agar, applying bacteria to the surface of the hydrogel, and then evaluating the growth of B. fragilis on the agar after total incubation times of 24 h and 48 h. Three independent experiments were performed with n=3 controls and n=4-5 Bacteroides-inoculated hydrogel discs per experiment. For each plate, bacteria were spotted directly on the plate as a positive control (large arrow, top left). One representative plate per independent replicate is shown. The apical side of each hydrogel was filled with 10 μL of sterile PBS or 1OD 600 Each plate was inoculated with either 10 μL of B. fragilis culture in 100 / mL PBS. Plates were incubated anaerobically at 37 °C for 24 h (top row). After 24 h, the hydrogel was removed (middle row) and the plates were incubated under the same conditions for an additional 24 h (bottom row). After 24 h, B. fragilis growth was evident on the apical side of the hydrogel but not on the agar. This indicates that B. fragilis had not passed through the hydrogel in significant quantities. After 48 h, contamination was confirmed in 11 of 14 total technical replicates. Of these, 10 / 11 were most likely edge contaminations that occurred when removing the hydrogel from the plate (black arrows). In experiment 1, hydrogels were flipped over on plates after 24 h to confirm viability of B. fragilis on the apical side of the hydrogel. Growth originating from the apical side of the hydrogel at 48 h indicates that B. fragilis was still viable (small white arrow, bottom left).

[0098] Figure 28 shows E. coli results for SA crosslinker + 4-arm PEG-NH2 hydrogels by placing the hydrogels on LB agar plates, applying E. coli to the surface of the hydrogels, and evaluating E. coli growth on the agar plates after 24 and 48 h total incubation times. Three independent experiments were performed with n=3 control and n=4-5 E. coli inoculated hydrogel discs per experiment. One representative plate per independent replicate is displayed. For each plate, bacteria were spotted directly on the plate as a positive control (large white arrow, top left). The apical side of each hydrogel was filled with 10 μL of sterile PBS or 1OD 600 Each plate was inoculated with either 10 μL of E. coli culture in 100 / mL PBS. The plates were incubated aerobically at 37°C for 24 h (top row). After 24 h, the hydrogels were removed (middle row) and the plates were incubated under the same conditions for another 24 h (bottom row). After 24 h, E. coli growth was evident on the apical side of the hydrogels, but not on the agar of E. coli-inoculated hydrogels that remained intact (n=11 / 15). This indicates that E. coli did not penetrate the agar in significant quantities. At 48 h, n=8 / 15 disks showed contamination of the plate (black arrows). All of the 24 h and most of the 48 h contamination occurred during experiment 2. These hydrogels were slightly thinner than the other experiments, and some had melted by 24 h. This is the most likely cause of contamination. In experiment 1, the hydrogels were flipped over on the plates after 24 h to check for E. coli viability on the apical side of the hydrogels. Growth from the apical side at 48 h shows that E. coli was still viable (small white arrow, bottom left).

[0099] The application and handling of the hydrogels was investigated by administering the crosslinker and macromer components through a dual-lumen catheter for subsequent hydrogel formation at the exit site at the target site on colon tissue samples. A dual-lumen catheter was used. This catheter can be inserted into the colon through an endoscope in vivo, eliminating the need for a separate device. Air pressure can be applied through the dual-lumen catheter to spray the hydrogel precursor components onto the wound and gel in situ. The two-part hydrogel system was delivered to colon tissue ex vivo. All 12 hydrogel formulations were injected through the dual-lumen catheter and subsequently gelled and adhered to the colon tissue both with and against gravity.

[0100] Example 7 Additional crosslinkers (crosslinkers 5, 6, and 7) were added to PEG (M w Briefly, the synthesis was carried out starting from PEG (M 3000). w 3000) was reacted with the appropriate anhydride to form PEG diacid, which was then activated with NHS ester to give crosslinker 1. Crosslinker 1 was reacted with 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) and thiol-terminated carboxylic acids of 1, 5, and 10 methylene to give intermediates 2, 3, and 4, respectively. NHS-activated crosslinkers were then prepared by dicyclohexylcarbodiimide (DCC) coupling chemistry with NHS, and the products were purified by precipitation in diethyl ether. Yields were 85-98% for all reactions. The crosslinker structures are shown in Table 1. 1 H NMR, 13 Characterization was performed as described in Example 6. The data were as follows:

[0101] PEG diacid: The synthesis of PEG diacid compounds was based on previously reported protocols. 1H NMR(500MHz), CDCl3:δ1.93(q,J=7.21Hz,4H),2.4(tt,J=7.21,8H),3.62(m,292H),4.22(tt,J=4.73Hz,4H)ppm; 13 C NMR (500MHz), CDCl3:175.3,172.8,70.6,68.9,63.4,33.1,32.6,19.9ppm.

[0102] Crosslinker 1. The synthesis of the starting material was based on previously reported protocols. 1 H NMR(500MHz), CDCl3:δ4.15(tt,J=3.3,1.5,4H),3.54(m,296H),2.8(b,8H),2.6(t,J=7.3,4H),2.4(t,J=7.3,4H),2.0(q,J=7.3,4H)ppm; 13 C NMR (500MHz), CDCl3:172.3,169.0,168.0,70.5,69.0,63.6,32.4,29.9,25.5,19.7ppm.

[0103] Intermediate 2. Synthesis was based on previously reported protocols. 1 H NMR(500MHz), CDCl3:δ4.21(m,J=4.6,4.9,4H),3.62(m,296H),2.68(t,J=7.3,4H),2.40(t,J=7.2,4H),1.98(t,J=7.2,4H)ppm; 13 C NMR (500MHz), CDCl3:196.8,172.6,169.8,70.6,69.0,63.6,42.3,32.8,31.0,20.5ppm.

[0104] Intermediate 3. In a flame-dried flask, 1,8-diazabicyclo(5.4.0)undec-7-ene (265 μL) and 6-mercaptohexanoic acid (122 μL) were added to a solution of crosslinker 1 (1 g) in anhydrous DMF (5 mL). The solution was stirred at room temperature for 16 h. The organic phase was extracted with 1 M HCl solution, water, and brine. The organic phase was dried over sodium sulfate, filtered, and precipitated in diethyl ether. The precipitate was filtered and dried under vacuum to give intermediate 3 as a white solid (96% yield). 1 H NMR(500MHz), CDCl3:δ4.22(t,J=4.8,4H),3.63(m,308H),2.86(t,J=7.2,4H),2.61(t,J=7.3,4 H),2.38(t,J=7.4,4H),2.30(t,J=7.4,4H),1.97(t,J=7.3,4H),1.60(m,8H),1.39(m,4H),ppm; 13 C NMR (500MHz), CDCl3:198.6,176.1,172.7,70.7,69.0,42.8,33.5,32.9,29.2,28.5,28.1,24.2,20.6ppm.

[0105] Intermediate 4. Synthesis was carried out according to the procedure described above using 11-mercaptoundecanoic acid (0.190 g) as the thiol source (yield 92%). 1 H NMR(500MHz), CDCl3:δ4.22(t,J=4.9,4H),2.85(t,J=7.4,7.3,4H),2.60(t=7.3,4H),2. 38(t,J=7.3,4H),2.30(t,J=7.5,4H),1.97(t,J=7.3,4H),1.60(m,8H),1.39(m,24H)ppm; 13 C NMR (500MHz), CDCl3:198.7,176.5,172.7,70.5,69.0,63.5,33.8,32.9,29.4,29.3,29.2,29.1,29.0,28.95,28.8,28.7,24.7,20.6ppm.

[0106] Crosslinkers 5, 6, and 7. The synthesis of crosslinkers 5, 6, and 7 was based on previously reported protocols (yields 96–98%). Crosslinking agent 5. 1 H NMR(500MHz),CDCl3:δ4.16(t,J=4.3,4H),3.92(s,4H),3.57(m,257H),2.78(b,8H),2.67(t,J=7.3,4H),2.34(t,J=7.3,4H),1.95(q,J=7.3,4H)ppm; 13 C NMR(500MHz),CDCl3:δ ppm;MALDI-TOF(pos):M w :3763m / z;GPC:M n :5077;M w :5312;PDI:1.05;Mp(DSC):46.06℃。

[0107] Crosslinking agent 6. 1 H NMR(500MHz),CDCl3:δ4.21(tt,J=1.5,3.4,4H),3.63(m,290H),2.86(t,J=7.3,4H),2.81(b,8H),2.60(tt,J=2.5,4.9,8H),2.37(t,J=7.3,4H),1.96(q,J=7.3,7.4,4H),1.74(q,J=7.4,7.7,4H),1.59(m,4H),1.46(m,4H)ppm; 13 C NMR(500MHz),CDCl3:δ198.6,172.7,169.1,168.4,70.5,69.1,63.6,42.9,33.0,29.1,28.4,27.8,25.6,24.1,20.6ppm;MALDI-TOF(pos):M w :3807m / z;GPC:M n :4999;M w :5196;PDI:1.04;Mp(DSC):45.80℃。

[0108] Crosslinking agent 7. 1H NMR(500MHz), CDCl3:δ4.22(m,4H),3.62(m,278H),2.85(m,8H),2.70(t,J=7.2,7.3,2H),2.60(tt,J=7.3,4H)),2.45(t,J =7.2,7.4,4H),2.37(t,J=7.2,7.3,4H),2.04(q,J=7.2,7.4,4H),1.95(m,4H),1.71(m,2H),1.52(m,4H),1.25(m,10H)ppm; 13 C NMR(500MHz), CDCl3:δ198.8,172.7,169.2,168.6,70.5,69.0,63.5,42.8,32.9, 30.9,29.5,29.3,29.2,29.0,28.8,28.7,25.6,24.5,20.6ppm;MALDI-TOF(pos):M w :4210m / z;GPC:M n :6038;M w :6313;PDI:1.05;Mp(DSC):47.42℃.

[0109] Example 8 The crosslinkers of Example 7 (i.e., crosslinkers 5, 6, and 7) dissolved in 0.1 M phosphate buffer (pH 6.5) were mixed with branched polyethyleneimine (PEI; wHydrogels of 10 wt%, 15 wt%, and 20 wt% were prepared by mixing with PEI (1800). The solubility of crosslinker 7 in buffer was observed to be minimal, which is believed to be due to the hydrophobicity of the methylene chains in its structure. To overcome the low solubility, crosslinker 7 was dissolved in 0.1 M phosphate buffer (pH 6.5) containing 50% ethanol before mixing with the PEI solution. The ratio of NHS:NH2 was 2:1 to ensure amidation of PEI and the respective crosslinker. No significant difference was observed in the mechanical properties of the hydrogels with NHS:NH2 ratios of 2:1 or 1:1. Clear solid hydrogels were formed within 5 minutes for all compositions (Hydrogels 5, 6, and 7, respectively) as measured by the inverted tube gelation test (see discussion in Example 6). The gelation time of the hydrogels was found to be positively correlated with increasing hydrophobic chain length. As shown in Figure 30, hydrogels prepared with crosslinkers 5, 6, and 7 gelled in less than 5 seconds, less than 90 seconds, and less than 3-5 minutes, respectively. Figure 30 reports: Panel A) gelation time for 10 wt%, 15 wt%, and 20 wt% hydrogels; Panel B) storage modulus for 10 wt%, 15 wt%, and 20 wt% hydrogel 7; Panel C) storage modulus for 15 wt% hydrogels 5, 6, and 7; Panel D) swelling over time of 15 wt% hydrogel. Gelation time was also found to be positively correlated with weight percent; i.e., the higher the weight percent, the longer the gelation time.

[0110] Next, the morphology of the hydrogels was characterized using scanning electron microscopy (SEM). All hydrogels had various pore sizes ranging from 5 μm to 100 μm and had a honeycomb-like structure. Hydrogel 7 showed a more layered structure, unlike the other hydrogels. Figure 31 shows SEM images of hydrogels 5 (top), 6 (middle), and 7 (bottom). Due to this observed secondary structure, the critical aggregation concentration (CAC) of crosslinker 7 was evaluated using a pyrene assay. A CAC of 0.050 mM was observed, which was a lower concentration than the hydrogel crosslinker concentration (0.053 mM). This indicates that a self-assembled structure was formed within the hydrogel itself, resulting in the layered structure seen in SEM. From the standpoint of chemical reactivity, the terminal amines of PEI can react with the terminal NHS ester or the internal thioester to form amide bonds. The preferred attack site of the amines can be determined by: 1 The amidation reaction was determined by H NMR. Specifically, N-butylamine was used as a model for the primary terminal amine of PEI and was added to an aqueous solution containing crosslinker 6. 1 The reactivity was followed by H NMR. Selective reactivity was observed between PEI and the NHS ester of the crosslinker, but not with the internal thiol ester (>99% at NHS sites in 20 min). Amidation of the NHS ester was confirmed by an upfield shift from bound NHS ester at 2.82 ppm to free NHS at 2.49 ppm on crosslinker 6, while the methylene peak at 2.6 ppm of the thioester does not shift. Figure 32 shows a representative image of crosslinker 6 before (bold line) and after (thin line) reaction with the PEI mimetic 4-butylamine. 1 1 H NMR spectra are shown. Upon reaction with 4-butylamine, a shift of the NHS peak attached to crosslinker 6 was observed from 2.78 ppm (bold line) to 2.49 ppm (thin line) after the NHS ester was cleaved from crosslinker 6. Figure 33 shows a representative peak of intact crosslinker 6 (bottom) (2.78 ppm NHS) and NHS hydrolyzed (2.54 ppm) crosslinker 6 (top) in 0.3 M sodium bicarbonate buffer, pH 8.0. 1 The H NMR spectrum is shown.

[0111] The attack of the terminal amine on the NHS ester occurred quickly, less than 10 seconds, but in hydrogels this reaction is likely slower. The reason is that if one of the amines attacks the NHS ester, entanglement and solidification would occur, which would result in increased steric hindrance. Thus, gelation times would be longer. In addition, competitive hydrolysis reactions occurred in the NHS ester. Figure 34 shows panel A) the rate sequence of thioester hydrolysis for crosslinker 5 in 0.3 M borate buffer, pH 8.0; panel B) the rate sequence of thioester hydrolysis for crosslinker 6 in 0.3 M borate buffer, pH 8.0; and panel C) the stability of NHS esters in 0.1 M phosphate buffer, pH 6.5. However, hydrolysis of the NHS esters was negligible over 20 minutes at pH 6.5, which is more than enough time to prepare hydrogels (see panel C). This selectivity of amidation in NHS esters ensures that the internal thioester bonds are preserved, allowing dissolution by cysteine ​​methyl ester (CME).

[0112] Regarding mechanical properties, strain and frequency sweeps were performed at various time points before and after swelling in 50 mM PBS. First, a strain sweep was used to determine the linear viscoelastic region (Figure 35 (left)). A frequency sweep was performed for all hydrogels at 3% strain from 1 to 10 Hz (Figure 35 (right)). These hydrogels exhibited viscoelastic solid-like behavior with storage modulus (G') > loss modulus (G").

[0113] FIG. 36 reports the storage modulus of hydrogels 5, 6, and 7 prepared with 10 wt% (left) and 20 wt% (right) of crosslinkers 5, 6, and 7, respectively. Also, FIG. 37 reports the storage modulus of hydrogels prepared with 10 wt%, 15 wt%, and 20 wt% of crosslinkers 5 (left), 6 (center), and 7 (right) until 30 days of swelling or dissolution. Over the 30 days of swelling, the lowest storage modulus was observed for hydrogel 5, which maintained a G' below 10 kPa for a period of time after swelling. The storage modulus of hydrogels prepared with crosslinkers 6 and 7 were both higher, with peak storage moduli at 15 wt% of approximately 12 kPa and 20 kPa, respectively. This increase in storage modulus for each hydrogel is believed to be due to the hydrophobicity of the methylene, with the longer methylene chain length resulting in greater hydrophobic interactions and stronger hydrogels. This observation also holds true for the weight percent dependence: the higher the weight percent, the greater the storage modulus.

[0114] Figure 38 reports the swelling of 20 wt% hydrogels. Figure 39 reports the dissolution of hydrogels 5, 6, and 7 prepared with 10 wt% (left) and 20 wt% (right) crosslinkers 5, 6, and 7, respectively, upon immersion in 0.3 M CME solution, pH 8.6. Figure 40 reports the rheological measurements of hydrogels prepared from crosslinker 6 at 2:1 (black) or 1:1 (grey) NHS:NH2 molar ratios. Figure 41 reports the rheological measurements of hydrogels made from crosslinker 6 with and without EtOH.

[0115] To confirm that the storage modulus of hydrogels prepared with crosslinker 7 was not increased by the presence of ethanol, rheological measurements were performed on hydrogels prepared with crosslinker 6 under the same conditions as the hydrogels used for crosslinker 7. No significant differences in storage modulus were observed between hydrogels prepared with or without EtOH, indicating that the buffer conditions did not alter the mechanical properties of the hydrogels (Figure 41).

[0116] During the 30-day swelling period, the hydrogels swelled from 150 to 350%, depending on the weight percent and hydrophobicity of the hydrogel formulation (Figure 38). Swelling of all hydrogels reached equilibrium after 48 hours. Hydrogels prepared using crosslinker 7 swelled the least, likely as a result of hydrophobicity within the long methylene chain length, while hydrogels prepared using crosslinker 5 swelled the most.

[0117] All hydrogels underwent hydrolysis over 30 days of swelling, as indicated by a loss of overall structure and a decrease in storage modulus over time. Hydrogel 5 showed an immediate loss of storage modulus and overall structure, whereas hydrogels 6 and 7 increased in strength as they swelled. However, a decrease in storage modulus was observed in hydrogels 6 and 7 by 30 days after swelling. This loss of structure and mechanical properties is believed to be due to hydrolysis of the crosslinker. To further characterize the hydrolysis, the rate of crosslinker hydrolysis was measured in 0.1 M sodium bicarbonate buffer, pH 8.0, at 20°C. 1 The hydrolysis was measured by H NMR with k = 0.055 min for crosslinkers 5 and 6, respectively. -1 and k=0.003min -1It was observed that dissolution of the thioester bond occurred preferentially at the thioester bond, at a rate of 0.01 to 0.01% (Figure 34). This is in contrast to the ester bond between the glutaric acid of the crosslinker and PEG. Hydrogel 7 was stable for over 7 days. The stability of the thioester bond in crosslinker 7 was attributed to the length of the hydrophobic methylene chain that protects the adjacent thioester from hydrolysis (see Figure 9). Apart from hydrolysis, the thioester promoted the dissolution of the hydrogel through thiol-thioester exchange in the presence of cysteine ​​methyl ester (CME). When the hydrogel was exposed to a 0.3 M CME solution at pH 8.6, the thiol of the cysteine ​​methyl ester would attack and displace the internal thioester of the crosslinker. The amine of the internal cysteine ​​methyl ester would then rearrange to form an amide bond by displacing the thioester (see Figure 29). This amide bond would prevent the re-attack of the original internal thiol. This dissolution process would fragment the hydrogel network, leading to the degradation of the hydrogel over time. The storage modulus of the hydrogels in CME solution was evaluated as a function of time at pH 8.6. Complete dissolution, defined as G'<300 Pa, was found to occur in less than 10 minutes to more than 90 minutes depending on the hydrogel formulation and weight percent, with higher weight percents resulting in longer methylene chain lengths and longer times to complete dissolution. Figure 42 shows panel A) dissolution of 15 wt% hydrogels in 0.3M CME solution; panel B) adhesion of hydrogels to human breast tissue using lap shear testing; and panel C) adhesion of 15 wt% hydrogel 6 to burned and unburned human abdominal tissue. See also Figure 39. Specifically, at 15 wt%, hydrogel 5 dissolved within 10 minutes, hydrogel 6 dissolved within 30 minutes, and hydrogel 7 dissolved within 80 minutes. This trend continued across all hydrogels, regardless of weight percent. This slower dissolution of hydrogel 7 compared to hydrogels 5 and 6 is believed to be due to the additional hydrophobic methylenes near the thioester reducing the local hydrophilicity.Dissolution rates using CME in sodium bicarbonate buffer pH 8.0 with crosslinker 6 due to the competitive reaction at the thioester between water hydrolysis and thiol-thioester exchange. 1 The thiol-thioester exchange rate was determined by monitoring the decrease in the methylene protons adjacent to the thioester, and was found to be k = 0.084 min -1 This rate was determined to be faster than the rate of hydrolysis and was therefore interpreted as indicating that thiol-thioester exchange was the preferred mode of dissolution under 0.3 M CME solution conditions.

[0118] The adhesive properties of the hydrogels were studied on human skin. Lap shear tests were performed to measure the adhesive strength on human breast and abdominal tissue in vitro. All hydrogels had a strength of approximately 0.5 N / cm 2 The hydrogels adhered to the tissues similarly with values ​​of 0.01 and showed cohesive failure at the hydrogel-skin interface (Figure 42). Furthermore, the hydrogels adhered similarly to burned skin as well as healthy skin. This adhesive strength is believed to be due to physical entanglement between the hydrogel and human skin.

[0119] Prior to the in vivo study, cytotoxicity was evaluated using NIH3T3 fibroblasts. Figure 43 reports the cell viability of hydrogels prepared with crosslinkers 5, 6, 7, and PEI on NIH3T3 fibroblasts. Hydrogels 6 and 7 showed >85% viability, while hydrogel 5 showed very low viability. This may be due to the increase in local acidity due to the rapid release and dissolution of glutaric acid.

[0120] Based on these summary results, 15 wt% Hydrogel 6 was selected for in vivo testing. Hydrogel 6 was non-toxic, exhibited a storage modulus comparable to human skin, maintained mechanical strength and structure over 7 days, adhered to skin, swelled, and dissolved in 30 minutes. In the in vivo model, second degree burns were induced in four pigs by heating a brass cylinder to 80°C and placing it on the pigs' backs for 20 seconds. As shown in Figure 44, the dressings were changed once or twice and treatment groups were evaluated at days 7 and 14 to observe the difference in healing between groups. Hydrogel 6 was compared to gauze sponge dressings, Mepilex®, and xeroform. Triple antibiotic ointment was applied to each burn before dressing. After necropsy, tissues were dissected and stained with hematoxalin & eosin (H&E). FIG. 44 shows Panel A) a schematic of the experiment; Panel B) representative photographs and histology of the burn site; Panel C) histological scores of necrosis and angiogenesis.

[0121] Figures 45-49 and Tables 5-9 report data obtained from the samples. Figure 45 shows the H&E of Group 1 for gauze (left), no dressing (center), and hydrogel dressing (right) (see Table 5). Figure 46 shows the H&E of Group 1 for gauze (left), no dressing (center), and hydrogel dressing (right) (see Table 6). Figure 47 shows the H&E of Group 2 for gauze (left), no dressing (center), and hydrogel dressing (right) (see Table 7). Figure 48 shows the H&E of Group 4 for gauze (left), no dressing (center), and hydrogel dressing (right) (see Table 8). Figure 49 shows the H&E of Group 5 for gauze (left), no dressing (center), and hydrogel dressing (right) (see Table 9). Tables 5-9 show the mean ± SD, median, and incidence of inflammation and inflammatory cell types.

[0122] [Table 5]

[0123] [Table 6]

[0124] [Table 7]

[0125] [Table 8]

[0126] [Table 9]

[0127] Overall, mild / moderate necrosis, epidermal ulceration, inflammation, and angiogenesis were observed in all treatment groups. However, Hydrogel 6 showed less necrosis, epidermal ulceration, and inflammation than the other treatment groups, and showed similar angiogenesis, burn depth (mm), and epidermal dermal thickness (mm) as all treatment groups by day 14 (Figure 44, Panel C). Additionally, all hydrogels showed some degree of re-epithelialization by day 14, with Hydrogel 6 showing complete re-epithelialization in two burns and partial re-epithelialization in one burn after two dressing changes (N=3). This is the only dressing that showed complete re-epithelialization in multiple burns. The only treatment groups that showed complete re-epithelialization in burns included Hydrogel 6 at day 14 (one dressing change) and the sterile gauze dressing at day 14 (two dressing changes). Although the differences between groups were not statistically significant (P>0.05), Hydrogel 6 tended to perform better than conventional gauze, Mepilex®, and xeroform dressings. Our hydrogel spray application and removal process allows for easy application and debridement during dressing, eliminating the need for mechanical debridement or disruption of newly formed tissue. Figure 50 shows a schematic of the dissolution of Hydrogel 6 used as a burn dressing. Gauze was soaked in 0.3 M CME solution and placed on the Hydrogel 6 burn dressing for 10 minutes to induce dissolution of the dressing. Subsequently, the burn area was wiped with gauze soaked in HO, and a new hydrogel dressing was prepared on the wound.

[0128] Example 9 Additional hydrogels according to the present disclosure are prepared. The macromer is either a PEG-based macromer or a poly(1,2-glycerol carbonate) (PGC)-based macromer featuring an alkene functional moiety. The crosslinker is a PEG-based crosslinker featuring a thiol moiety (see Example 9). These components are dissolved in a phosphate buffer ranging from pH 7 to 8 at a total polymer concentration ranging from 10% to 25% by weight. As the weight percentage of the gel solution increases, the gelation time decreases and the elastic modulus of the gel increases. The molar ratio between the alkene moiety and the thiol moiety can range between 1:1 and 2:1 in the gel formulation. A 1:1 ratio results in a slight increase in the elastic modulus of the gel compared to a 2:1 ratio. The alkene functional moiety encompasses many different structures, such as, but not limited to, alkyl ethers as shown in Figures 2C and 2D, or norbornene as shown in Figures 2E and 2F. The choice of the moiety affects the gelation time, with norbornene exhibiting a faster reaction rate than alkyl ethers. Macromers may feature 4-100 alkene moieties per molecule. Macromers featuring higher amounts of alkene moieties result in stiffer gels and lower swelling ratios. Crosslinkers may contain 2-4 thiols per molecule. The gel precursor solution does not solidify until irradiated with 365 nm UV light or white light, depending on the photoinitiator used. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or eosin Y. The photoinitiator concentration ranges from 0.1 mM to 100 mM depending on the photoinitiator and the gelation rate, with higher photoinitiator concentrations resulting in faster rates. At very high concentrations, the gel macrostructure may be destroyed and there may be a risk of cytotoxicity. For visible light systems, up to 10 mM tyrosine ethyl ester is included to increase the gelation rate. These gels can be gelled at 4-120 mW / cm. 2 Wide range of UV (365nm) intensity and 10mW / cm 2 (at maximum absorption of the photoinitiator) to 42.9 W / cm 2 Formed under full spectrum white light.

[0129] For UV-activated hydrogels, the storage modulus can be tuned between 500 Pa and 2,000 Pa by altering the concentration and ratio of macromer and crosslinker. These formulations are single solutions and exhibit suitable viscosities for application along the length of an endoscope through a single-lumen catheter. These formulations utilize stimuli-responsive gelation with fast kinetics in response to long-wave UV light, forming a gel within 5 seconds after irradiation. The gel adheres to porcine colon tissue and exhibits strong burst pressures when used to seal small defects. In vivo studies are performed to apply the components using an endoscopic catheter. The resulting gel is still present 2.5 hours after application. The resulting gel has low cytotoxicity and shows over 97% viability over 24 hours in NIH 3T3 fibroblasts. This photoinitiator has been shown to induce IC 50 Although used at concentrations below 10 mM, when combined with up to 10 mM tyrosine ethyl ester, it exhibits fast (<10 seconds) gelation kinetics in response to a wide range of white light sources such as bike lights, lamps, and endoscopes.

[0130] Example 9 As shown in Figure 51, an additional crosslinker bearing a thiol moiety was added to PEG(M wThe crosslinkers were synthesized starting from PEG-3000 (Figure 3). These crosslinkers were prepared for studies on the formation of hydrogels formed in situ via a Michael addition reaction between branched PEI-thiols and bifunctional maleimide-activated PEG crosslinkers, as further described in Examples 10 and 11. There is an internal thioester bond within this crosslinker that facilitates dissolution by thiol-thioester exchange with a cysteine ​​methyl ester (CME) solution. After thiol-thioester exchange, the primary amines of the CME rearrange to form irreversible amide bonds, which are believed to prevent reformation of the hydrogel after the polymer network collapses (see Figure 8). The PEG crosslinkers were prepared with methylene chain lengths of 2, 3, or 4. The dependence of the methylene chain length on the mechanical properties, swelling, dissolution time, and burst pressure of the hydrogels was determined by varying the length of the methylene chain. The hydrogels contained internal thioesters for dissolution by thiol-thioester exchange and maleimide end groups for conjugation with hyperbranched poly(ethyleneimine)-thiols ("PEI-SH").

[0131] As summarized in Figure 51, PEG-diols were reacted with the respective anhydrides (succinic anhydride, glutaric anhydride, or adipic anhydride) to give the corresponding PEG diacids. The PEG diacids were then functionalized with N-hydroxysuccinimide (NHS) end groups via DCC coupling to produce crosslinkers 1, 2, and 3. Crosslinker 1, 2, or 3 (1 g) was dissolved in dimethylformamide (DMF) in a flame-dried round-bottom flask equipped with a magnetic stir bar. Thioglycolic acid (68.8 μL) and diisopropylethylamine (DIPEA) (279 μL) were added in that order. Thioglycolic acid was chosen due to its hydrophilic nature adjacent to the thioester and fast dissolution time. The reaction was stirred overnight at room temperature. The organic phase was extracted with 1N HCl solution, water, and then brine. The organic phase was dried over sodium sulfate, filtered through filter paper, and precipitated in diethyl ether to give a white powder (98% yield).

[0132] Following the previous step, intermediates 1, 2, and 3 were functionalized with maleimide reactive end groups by peptide coupling method using maleimidotrifluoroacetic acid, PyBOP, DIPEA in dry DCM to give the final crosslinkers, crosslinkers 4, 5, and 6. The methylene chain lengths are 2, 3, and 4, respectively. Intermediate 1, 2, or 3 was dissolved in dry methylene chloride in a flame-dried round-bottom flask equipped with a magnetic stir bar. Maleimido-ethylamine trifluoroacetic acid, DIPEA, HOBt, and EDC were added to the reaction. The solution was stirred at room temperature overnight. The organic phase was extracted using saturated citric acid solution, water, and brine. The organic phase was then dried over sodium sulfate, filtered through filter paper, and precipitated in diethyl ether to give an off-white solid. The solid was dried under vacuum overnight. The solid was then dissolved in water, filtered through a 0.22 μm syringe filter, and lyophilized to give an off-white solid (80-90% yield). The yields of all the above reactions were above 80%.

[0133] 1 H NMR, 13 Characterization data by C NMR, GPC, and DSC are as follows: PEG diacid. This polymer was prepared according to a previously published protocol (see also Example 7).

[0134] Crosslinkers 1, 2, 3. The synthesis of crosslinkers 1, 2, and 3 was based on previously published protocols (see also Example 7). Intermediates 1, 2, 3. Synthesis was carried out as described above. 11H NMR (500 MHz), characterization in CDCl3: Intermediate 1 - δ 4.22 (tt, J = 4.7 Hz, 4H), 3.62 (m, 310H), 2.93 (t, J = 6.8 Hz, 4H), 2.68 (t, J = 6.8 Hz, 4H) ppm; Intermediate 2 - δ 4.22 (tt, J = 4.8 Hz, 4H), 3.63 (m, 308H), 2.86 (t, J = 7.2 Hz, 4H), 2.61 (t, J = 7.3 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.4 Hz, 4H), 1.97 (t, J = 7.3 Hz, 4H), 1.60 (m, 8H), 1.39 (m, 4H) ppm; Intermediate 3 - δ 4.21 (tt, J = 4.4, 4.9 Hz, 4H), 3.63 (m, 277H), 2.62 (t, J = 6.7, 7.2 Hz, 4H), 2.34 (t, J = 6.7, 7.2 Hz, 4H), 1.69 (m, 8H) ppm. 13 13C NMR (500 MHz), characterization in CDCl3: Intermediate 1 - 195.9, 171.5, 70.5, 64.1, 30.9, 29.1 ppm; Intermediate 2 - 198.6, 172.7, 70.7, 69.0, 33.5, 32.9, 20.6 ppm; Intermediate 3 - 197.0, 173.0, 70.5, 63.5, 33.7, 31.0, 24.7, 24.0 ppm.

[0135] Crosslinking agents 4, 5, and 6. Synthesis was carried out as described above. Crosslinking agent 4. 1 1H NMR: δ 6.71 (s, 2H), 6.55 (b, 1H), 4.23 (tt, J = 4.2, 4.9 Hz, 4H), 3.62 (m, 322H), 2.96 (t, J = 6.8 Hz, 4H), 2.74 (t, J = 6.8 Hz, 4H) ppm; 13 13C NMR: 197.5, 171.9, 134.2, 70.5, 64.0, 32.3, 29.1 ppm; M w (GPC, THF): 2868 Da; M n (GPC, THF): 2801 Da; PDI (GPC, THF): 1.02; Melting point (DSC): 41.78 °C; Crystallization point (DSC): 39.9 °C.

[0136] Crosslinking agent 5. 1H NMR: δ6.72(s,2H),6.51(b,1H),4.23(tt,J=4.8Hz,4H),3.63(m,297H),2.73(t,J=7.3Hz,4H),2.42(t,J=7.2Hz,4H),2.01(m,J=7.2,7.3Hz,4H)ppm; 13 C NMR:198.2,172.6,134.2,70.4,63.6,32.8,32.3,20.2ppm;M w (GPC, THF): 3028 Da;M n (GPC,THF): 2955Da; PDI(GPC,THF): 1.02; Melting point (DSC): 40.22°C; Crystallization point (DSC): 21.3°C.

[0137] Crosslinker 6.1H NMR: δ6.71(s,2H),6.50(b,1H),4.21(tt,J=,4H),2.67(t,3H),2.36(t,J=,4H),1.67(m,8H)ppm; 13 C NMR:198.6,173.1,134.2,70.5,63.5,33.5,32.4,24.6,24.0ppm;M w (GPC,THF):3351Da;M n (GPC,THF): 3162Da; PDI(GPC,THF): 1.06; Melting point (DSC): 45.04°C; Crystallization point (DSC): 33.5°C.

[0138] Example 10 Thiol-terminated polyethyleneimine (PEI-SH) hyperbranched macromers (Figure 2G) were synthesized by reacting with the maleimide-terminated PEG crosslinker of Example 9 as summarized in Figure 51. The synthesis of PEI-SH involved reacting pentafluorophenyl-functionalized 3-(tritylthio)propionic acid with PEI overnight to obtain a trityl-protected PEI-thiol hyperbranched polymer (hereafter "PEI-STr") (yield = 68%). The trityl groups were then deprotected using TFA and Et3Si to obtain the final PEI-SH hyperbranched polymer (yield = 96%). 1 H NMR, 13 Characterization data including C NMR, GPC, and DSC are as follows:

[0139] In the initial PEI-SH synthesis, PEI was fully thiolated by reacting 15 equivalents of thiol per PEI molecule. However, due to the high concentration of thiol per polymer, intra- and intermolecular disulfide bonds were formed as visually observed via a pink solution of PEI-SH in borate buffer (pH 8.6). This minimized the number of free thiols available for Michael addition reaction with the maleimide-functionalized crosslinker of Example 9. Therefore, the equivalents of thiol reacted with PEI were reduced to minimize the number of intermolecular and intramolecular disulfide bonds. The number of free amines was measured by a colorimetric TNBS assay as shown in Figure 53. This assay was performed by reacting a 0.01% (w / v) solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in 0.1 M sodium bicarbonate buffer, pH 8.5, with PEI-SH. After incubating the solution at 37°C for 2 hours, the resulting yellow solution was diluted with 10% SDS and 1 N HCl to stop the reaction. The absorbance, which correlates with the number of primary amines present in solution, was read at 335 nm. A standard curve was constructed based on various concentrations of PEI and fully thiolated PEI-SH. The slope of the RFU vs. concentration (μg / mL) graph correlates with the number of free amines on a particular molecule. PEI (MW 1800) has an average of 15 free amines, with a slope of 0.007 for the TNBS assay. Fully thiolated PEI-SH exhibits a slope of 0.000 as expected, indicating the absence of primary amines on the molecule. The slope of the line representing PEI-SH prepared with 4 equivalents of tritylthiopropionic acid was evaluated. The slope of that line is 0.002, one-third the slope of unfunctionalized PEI. From these data, it was confirmed that approximately 2 / 3 of the PEI polymer is thiolated, meaning that there are 5-6 primary amines remaining. This partial functionalization of PEI is expected to minimize intra- and intermolecular disulfide bonds and facilitate hydrogel formation with maleimide-functionalized crosslinkers.

[0140] PEI-STr. PEI (3 g) was dissolved in DMF. 3-(tritylthio)propionic acid pentofluorophenol (3.4 g), HOBt (3.2 g), and DIPEA (4.7 mL) were added. The reaction was stirred at room temperature overnight. The reaction was dissolved in methylene chloride and the organic phase was extracted with sodium bicarbonate, water, and brine. The organic solution was dried over sodium sulfate, filtered through paper, and concentrated. The organic solution was precipitated in diethyl ether and dried under vacuum to give a pale yellow solid (68% yield). 1 H NMR: δ8.00(s,1H),7.49-7.10(m,48H),3.65-2.01(m,60H)ppm; 13 C NMR:162.5,144.6,129.5,127.9,126.7,36.5,35.1,27.7ppm.

[0141] PEI-SH. In a round bottom flask equipped with a magnetic stir bar, PEI-STr (2 g) was dissolved in a minimum amount of methylene chloride. Trifluoroacetic acid (TFA) (12.3 mL) and triethylsilane (2.7 mL) were added dropwise simultaneously to the stirred solution. The reaction was stirred at room temperature for 3 hours. The methylene chloride and TFA were removed under vacuum and redissolved in a minimum amount of methylene chloride. The solution was precipitated in diethyl ether and the product was dried under vacuum overnight. The product was dissolved in 1N HCl, filtered through a 0.22 μm syringe filter, and lyophilized to give a pale yellow solid (96% yield). 1 H NMR:7.9(s,1H),3.61-2.49(m,217.13H)ppm; 13 C NMR:163.1,162.8,117.6,115.3,39.5,22.7ppm;M w (GPC, aqueous):5660Da;M n (GPC, aqueous): 6994 Da; PDI (GPC, aqueous): 1.12; Mp (DSC): 15.6°C.

[0142] Example 11 Hydrogels were prepared by combining the crosslinker of Example 9 with the macromer of Example 10. The hydrogels were prepared at a ratio of 2:1 crosslinker:PEI(SH)4. The crosslinker and PEI-SH were dissolved in 0.1 M phosphate buffer pH 6.5 and 0.3 M borate buffer pH 8.6, respectively. Each solution was loaded into a dual lumen syringe equipped with a mixing tip and injected into a cylindrical mold to form a solid hydrogel.

[0143] The gelation kinetics was investigated by mixing the maleimide crosslinker with the PEI-SH mimic mercaptopropionic acid. 1 The gelation rate was assessed by following the disappearance of the maleimide alkene peak at 6.70 ppm in H NMR. After injection of 2 equivalents of mercaptopropionic acid, used as a PEI-SH mimic in situ, NMR spectra were recorded every 0.4 seconds for approximately 20 seconds. No alkene peak was observed at 6.70 ppm immediately after injection of the PEI-SH mimic, indicating a gelation rate faster than 0.4 seconds.

[0144] After gelation, the storage modulus of the hydrogels was measured by strain and frequency sweeps to determine the linear viscoelastic region (LVER) as an assessment of mechanical strength. The LVER exists up to 10% strain, which is the maximum strain that can be applied to these hydrogels before plastic deformation occurs. Frequency sweeps were performed at 3% strain and 0.1-10 Hz within the LVER. The initial storage modulus of our hydrogels was 2000-5000 Pa. Upon swelling of the hydrogels in 50 mM PBS, crosslinkers 4, 5, and 6 showed a decrease in storage modulus. Figure 54 shows panel A) rheological measurements of the hydrogels; panel B) swelling in 50 mM PBS; and panel C) dissolution of the hydrogels in 0.3 M CME solution. The decrease in G' over time is attributed to degradation of the crosslinkers due to hydrolysis.

[0145] To estimate the rate of decomposition and to confirm the location of hydrolysis at the internal thioester rather than the ester, 1H NMR crosslinker spectra were monitored for 20 min in 0.3 M sodium bicarbonate buffer, pH 8.0. The methylene adjacent to the internal thiol shifted from 3.41 ppm to 3.17 ppm, whereas the methylene peak at 4.15 ppm adjacent to the ester bond, corresponding to the other terminal methylene of the diacid bond (succinic, glutaric, adipic) in the crosslinker, did not shift during base-catalyzed hydrolysis. 1 The selective hydrolysis of the thioester was confirmed by H NMR shifts. Figure 55 shows the hydrolysis of the thioester as observed by the shift of the methylene adjacent to the thiol from 3.41 ppm (bond) to 3.17 ppm (hydrolysis). 1 The H NMR spectrum is shown.

[0146] The degradation rates of hydrogels prepared using crosslinkers 4, 5, and 6 varied from over 4 hours, over 24 hours, and over 7 days, and increased corresponding to the hydrophobic methylene chain length of the internal diacid linkages that protect the internal thioesters of the crosslinks from hydrolysis. Crosslinker 4 contains an internal succinic acid linkage with two methylenes, while crosslinkers 5 and 6 contain glutaric and adipic acid linkages with three and four methylenes, respectively. The longer and more hydrophobic methylene chain length in the crosslinker is believed to make the thioester more stable against hydrolytic cleavage, resulting in a slower degradation rate. The variation in degradation rate versus methylene chain length within crosslinkers 4, 5, and 6 allows the mechanical properties of the hydrogel to be tuned through the crosslinker structure in order to maintain mechanical integrity.

[0147] Swelling between 200-400% was observed (Figure 54, Panel B). The swelling reached a maximum after 24 hours of immersion in 50 mM PBS. Swelling in aqueous solutions is believed to be advantageous for hydrogels, as they have the ability to expand in size upon absorbing aqueous liquids from the surrounding environment.

[0148] The on-demand dissolution time of hydrogels via thiol-thioester exchange was evaluated when immersed in 0.3 M cysteine ​​methyl ester (CME) solution (Figure 8). Frequency sweeps were performed at 10 min intervals to allow sufficient CME exposure until the hydrogel network completely collapsed or degraded (G' < 300 Pa). Thiol-thioester exchange caused the hydrogel network to collapse and the amines of the CME to rearrange to form irreversible amide bonds, preventing the reformation of the hydrogel. Dissolution occurred within 10 min for all three hydrogel formulations (Figure 54, panel C). While acid bonds (succinic, glutaric, adipic) retard hydrolysis under neutral conditions, the basic conditions of the CME solution catalyze the thiol-thioester exchange reaction. The rapid dissolution of the hydrogel network via thiol-thioester exchange may be useful in medical settings, such as minimizing the anesthesia time of the patient.

[0149] Prior to the ex vivo studies, the cytotoxicity of the hydrogels towards NIH3T3 fibroblasts was assessed over a 24-hour exposure. Hydrogels 4 and 5 showed an average cell viability of 60%, while hydrogel 6 showed an average cell viability of 98%. The lower cell viability of hydrogels 4 and 5 is likely due to the rapid release of succinic and glutaric acids due to the breakdown of the hydrogel network and the increased local acidity in the closed environment of the transwell plate.

[0150] Hydrogel burst pressure was measured by injecting the macromer into one end of an ex vivo 2 cm porcine carotid artery in a total volume of 1 mL to form a hydrogel. The hydrogel filled the vessel and remained in place. After storing the occluded artery in a humid environment for 30 minutes (e.g., mimicking the time during an exemplary surgical procedure), the vessel was mounted on a proprietary burst pressure system equipped with a pressure transducer and syringe pump connected to a computer (Figure 56). Deionized HO was pumped into the vessel at 1 mL / min until leakage was observed, and pressure was recorded until failure. The burst pressures of hydrogels 4, 5, and 6 prepared with crosslinkers 4, 5, and 6 were 382 mmHg, 440 mmHg, and 231 mmHg, respectively, up to 4 times greater than the arterial pressure (120 / 60) (Figure 57). A burst pressure of 200-600 mmHg is considered sufficient for a hydrogel occlusion device.

[0151] It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A method for forming a gel, the method comprising: a macromer comprising a first polyethylene glycol (PEG)-based polymer, a poly(ethyleneimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer comprising at least one first functional moiety; a crosslinking agent comprising a second PEG-based polymer comprising at least one second functional moiety; and a photoinitiator preparing a composition by combining them; and forming a biocompatible gel by activating the photoinitiator via a light source. A method comprising the above.

2. The method according to claim 1, wherein the at least one first functional moiety comprises a thiol group, a vinyl group, an allyl group, an acrylate group, or a norbornene group, the at least one second functional moiety comprises a thiol group, a vinyl group, an allyl group, an acrylate group, or a norbornene group, and the at least one first functional moiety is different from the at least one second functional moiety.

3. The method according to claim 1, wherein one of the at least one first functional moiety or the at least one second functional moiety comprises a vinyl group, an allyl group, an acrylate group, or a norbornene group, and the other of the at least one first functional moiety or the at least one second functional moiety comprises a thiol group.

4. The method according to claim 1, wherein the macromer, the crosslinking agent, and the photoinitiator together account for about 10-25% by weight of the composition based on the total weight of the composition.

5. The method according to claim 1, wherein the molar ratio of the at least one first functional moiety to the at least one second functional moiety is in the range of 1:1 to 2:

1.

6. The method according to claim 1, wherein the macromer accounts for about 5-15% by weight of the composition based on the total weight of the composition, and / or the crosslinking agent accounts for 5-10% by weight of the composition based on the total weight of the composition.

7. The method according to claim 1, wherein the crosslinking agent comprises an N-hydroxysuccinimide group and / or a maleimide group.

8. The method according to claim 1, wherein the concentration of the photoinitiator in the composition is in the range of about 0.1 mM to about 100 mM.

9. The method according to claim 1, wherein the composition further comprises a physiological buffer.

10. The method according to claim 1, wherein the light source emits UV light or visible light.

11. The method according to claim 10, wherein when irradiated with UV light, the gel is formed within 5 seconds, or when the photoinitiator is activated with visible light, the gel is formed within 10 seconds.

12. The method according to claim 1, wherein the macromer is a hyperbranched polymer.

13. The method according to claim 1, wherein the composition further comprises an additive comprising a tyrosine derivative to accelerate the gelation time of the composition, and optionally, the tyrosine derivative comprises tyrosine methyl ester or tyrosine ethyl ester.

14. The method according to claim 13, wherein the composition comprises up to 10 mM, for example about 0.1 mM to about 5 mM, of the additive.

15. The method according to any one of claims 1 to 14, wherein the gel is formulated for treating human gastrointestinal tissue.

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