CN118055782A

CN118055782A - Gel compositions, systems, and methods

Info

Publication number: CN118055782A
Application number: CN202280062765.0A
Authority: CN
Inventors: 劳伦·S·莱德克; 杰拉尔德·弗雷德里克森; 萨曼莎·贝瑞; 凯瑟琳·A·库克; 马克·格林斯塔夫
Original assignee: Boston University; Boston Scientific Scimed Inc
Current assignee: Boston University; Boston Scientific Scimed Inc
Priority date: 2021-07-20
Filing date: 2022-07-19
Publication date: 2024-05-17

Abstract

Methods of forming a gel and related methods of treating a subject with such a gel are described. The method may include: preparing a composition by combining a macromer, a crosslinker comprising a second PEG-based polymer comprising at least one second functional moiety, and a photoinitiator; and activating the photoinitiator via the light source to form a gel. The macromer comprises 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.

Description

Gel compositions, systems, and methods

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application 63/223,808 filed on 7.20 of 2021 and U.S. application 63/260,113 filed on 8.10 of 2021, both of which are incorporated herein by reference in their entireties.

Technical Field

The present disclosure relates generally to therapeutic gels for use in medical procedures, including endoscopic procedures. For example, the present disclosure includes gels, as well as compositions and systems formulated to form gels, e.g., for application to body tissue (e.g., the gastrointestinal tract).

Background

Endoscopic procedures (e.g., endomucosal resection (EMR), endomucosal dissection (ESD) and anastomosis) and health conditions (e.g., intentional or disease-induced fistula formation, inflammatory Bowel Disease (IBD) and IBD accessory diseases) may result in and/or contribute to damage to tissue of the Gastrointestinal (GI) tract. Colorectal cancer is one of the leading causes of cancer death in developed countries. Standard preventative care for patients over 50 years old includes colonoscopy of polyp biopsies (known as polypectomy) to assess colorectal cancer. In practice, the physician inserts the endoscope into the patient's colon under anesthesia, examines the colon, and then resects the polyp. After excision, the wound is either open to the internal environment of the colon or heat sealed with electrocoagulation. Polypectomy or other post-endoscopic open wounds in the GI tract may lead to bleeding, blood loss, and sepsis. Electrocoagulation can lead to other complications such as perfusion, or post-polypectomy coagulation syndrome.

These types of medical procedures and health conditions may leave a relatively thin layer of tissue of the wall of the GI tract. Currently, doctors often rely on time or surgery (including clipping or endoscopic suturing) to heal the GI tract walls. However, these practices may be unsuitable in certain situations, such as large defects and/or fragile or fibrotic tissue. Complications that may occur include perforation, infection, and sepsis.

Disclosure of Invention

Methods for forming gels useful in medical procedures are disclosed. For example, the present disclosure includes a method for forming a gel, the method comprising: preparing a composition by combining a macromer, a crosslinker comprising a second PEG-based polymer comprising at least one second functional moiety, and a photoinitiator; and activating the photoinitiator via a light source to form a gel, the 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. The gel may be biocompatible and/or biodegradable. The at least one first functional moiety may comprise a thiol group, a vinyl group, an allyl group, an acrylate group, or a norbornene group, for example, 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, allyl, acrylate, or 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 together represent from 10 to 25 weight percent of the composition, relative to the total weight of the composition. Optionally, the molar ratio between the at least one first functional moiety and the at least one second functional moiety ranges from 1:1 to 2:1. Additionally or alternatively, the macromers may represent in total 5 to 15 weight percent of the composition, relative to the total weight of the composition. The cross-linking agents may total 5-10% by weight of the composition, relative to the total weight of the composition. The concentration of the photoinitiator in the composition may be in the range of about 0.1mM 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.

According to some aspects herein, the composition may further comprise a physiological buffer. The light source may emit ultraviolet light or visible light. For example, a gel may form in five seconds when irradiated with ultraviolet light. In some examples, the gel may form within ten seconds when the photoinitiator is activated with visible light. Optionally, the composition may further comprise an additive that accelerates the gelation time of the composition, the additive comprising a tyrosine derivative. In some aspects, the composition may comprise up to 10mM of the additive. The tyrosine derivative may comprise, for example, methyl tyrosine or ethyl tyrosine.

The gels described above and elsewhere herein can be used to treat tissue of a subject (e.g., a human subject). For example, the gel may be used to treat gastrointestinal tissue of a subject.

The present disclosure also includes a method of forming a gel comprising preparing a first solvent 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, and a first buffer, the macromer comprising at least one first functional group; preparing a second solution by combining a cross-linking agent comprising a second (PEG) based polymer comprising a plurality of second functional groups and a second buffer, the second buffer having a lower pH than the first buffer; and mixing the first solution with the second solution to form a gel. The gel may be biocompatible and/or biodegradable. For example, the at least one first functional group may comprise 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 2000Da. Additionally or alternatively, the molecular weight of the crosslinker may be about 3400Da. In some examples, the molar ratio of crosslinker to macromer may be in the range of 3:2 to 7:3.

As described above, the gels disclosed herein can be used to treat tissue of a subject. For example, a method of forming a gel may comprise treating a subject by forming a gel on gastrointestinal tissue of the subject. In at least one example, the method includes applying to the tissue a first solvent 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 cross-linking agent comprising a second (PEG) based polymer comprising a plurality of second functional groups and a second buffer, the second buffer having a lower pH than the first buffer; wherein the first solution is contacted with the second solution to form a gel on the tissue. The first solution may be applied to the tissue before, after, or simultaneously with the second solution.

The present disclosure also includes a composition comprising a macromer comprising a polyethylene glycol (PEG) based polymer, a poly (ethyleneimine) based polymer, or a poly (1, 2-glycerol) carbonate based polymer, wherein the macromer comprises at least one thiol group or amine group, and a crosslinker comprising a PEG based polymer comprising an N-hydroxysuccinimide functionality, a maleimide functionality, or both; wherein the composition is formulated as a hydrogel. When adhered to a body cavity, the hydrogel may have a gel strength of at least 2000Pa and/or a shear force between 0.03 and 0.90N/cm ². Additionally or alternatively, when the hydrogel is adhered to colon tissue to fill a hole of about 1mm by about 5mm in the tissue, the hydrogel may be formulated to withstand a burst pressure (pressure) of up to about 150 mbar.

Drawings

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.

Fig. 1A and 1B illustrate exemplary macromer structures according to some aspects of the present disclosure.

Fig. 2A-2G are exemplary macromers according to some aspects of the present disclosure.

Fig. 3A and 3B illustrate exemplary crosslinker structures according to some aspects of the present disclosure.

Fig. 4A-4D illustrate exemplary cross-linking agents according to some aspects of the present disclosure.

Fig. 5 is a schematic illustration of forming a gel according to some aspects of the present disclosure.

Fig. 6 is a schematic dissolution diagram of a gel according to some aspects of the present disclosure.

Figure 7 depicts the mechanism of gel dissolution in the presence of cysteine.

Fig. 8 depicts the mechanism of gel dissolution in the presence of water.

Fig. 9 depicts a possible reaction of an exemplary cross-linking agent according to some aspects of the present disclosure.

Fig. 10 is a graph of gel strength discussed in example 1.

Fig. 11A and 11B are graphs of gel strength and gel swelling ratio discussed in example 2.

FIGS. 12A-12E are graphs of gel strength and gelation time discussed in example 3.

FIG. 13 depicts the synthesis of an exemplary cross-linking agent discussed in example 4.

Figure 14 shows the cross-linking agent and macromer discussed in example 6 for forming a gel.

FIG. 15 is a gel measurement as discussed in example 6.

Fig. 16 and 17 show the results of rheological measurements of the hydrogels discussed in example 6.

Figure 18 reports the amidation kinetics at the NHS ester and internal ester linkages discussed in example 6.

Figure 19 shows ¹ H NMR data to monitor the hydrolysis discussed in example 6.

FIG. 20 shows storage modulus data for hydrogels discussed in example 6 at different temperatures.

Fig. 21 reports the swelling of the hydrogels discussed in example 6.

Fig. 22 shows the adhesion measurement results of the hydrogels discussed in example 6.

Figure 23 reports the cytotoxicity results of the hydrogels discussed in example 6.

Fig. 24 and 25 report bacterial migration studies of the hydrogels discussed in example 6.

Fig. 26 is an SEM image of the hydrogel discussed in example 6.

FIGS. 27 and 28 show the agar plate test results of the hydrogels discussed in example 6.

FIG. 29 depicts the synthesis of several exemplary cross-linking agents discussed in example 7.

Figure 30 shows the characteristics of various hydrogel measurements discussed in example 8.

Fig. 31 shows SEM images of various hydrogels discussed in example 8.

FIG. 32 shows ¹ H NMR spectra of the cross-linking agent before and after reaction with the macromer discussed in example 8.

FIG. 33 shows ¹ H NMR spectra of the studied NHS hydrolysis of the crosslinker discussed in example 8.

Figure 34 reports the kinetic studies of the various hydrogels discussed in example 8.

Fig. 35 reports the strain sweep and frequency sweep of the hydrogels discussed in example 8.

Fig. 36 and 37 report the storage modulus of the hydrogels discussed in example 8.

Fig. 38 reports the swelling of the hydrogels discussed in example 8.

FIG. 39 reports the dissolution of the hydrogels discussed in example 8.

Figures 40 and 41 report the results of rheological measurements on the hydrogels discussed in example 8.

FIG. 42 reports the dissolution results of the hydrogels discussed in example 8.

FIG. 43 reports the cell viability of the hydrogels discussed in example 8.

Fig. 44 shows the in vivo study design discussed in example 8.

FIGS. 45-49 show H & E staining of various tissue samples discussed in example 8.

Fig. 50 is a schematic dissolution diagram of a hydrogel for use as a wound dressing discussed in example 8.

FIG. 51 depicts the synthesis of several exemplary cross-linking agents discussed in example 9.

FIG. 52 depicts the synthesis of an exemplary macromer discussed in example 10.

FIG. 53 shows the results of the TNBS assay discussed in example 10 for detecting primary amines on PEI and PEI-SH molecules.

Fig. 54 shows the rheological measurements of the hydrogels discussed in example 11.

FIG. 55 shows ¹ H NMR spectra of the crosslinker discussed in example 11.

Fig. 56 shows a system for measuring burst pressure as discussed in example 11.

Fig. 57 reports the burst pressure data discussed in example 11.

Detailed Description

The foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the features as claimed. As used herein, the terms "comprises," "comprising," "includes," "including," "having," or other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises 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 that the stated value or characteristic may vary by ±10%. All ranges are understood to include endpoints, for example, macromer content between 5% and 10% by weight includes 5% by weight, 10% by weight, and all values in between.

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 rather than 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, such as compositions useful for application to gastrointestinal tissue. The hydrogels herein may function as temporary, minimally invasive in situ hydrogel dressings for immediate application following a medical procedure (e.g., polypectomy). Hydrogels can prevent or reduce the likelihood of complications by covering and protecting wounds. From a biomaterial design perspective, the dressing may achieve one or more of the following: 1) Rapid in situ formation; 2) Adhere to colonic tissue; 3) No cytotoxicity; 4) Naturally dissolving for 3-5 days; 5) Swelling up to 200% to absorb wound exudate; 6) Preventing bacteria from diffusing or migrating; and/or 7) conform to the malleable shape of the colon lumen. Depending on the hydrogel composition, the gels herein may be formulated to have desired properties (e.g., gelation rate, adhesion strength, swelling, cytotoxicity, and/or degradation). The compositions herein may be delivered to a subject by a suitable medical device (e.g., a catheter inserted through an endoscope). For example, a dual lumen catheter may be used. The barrier properties of the hydrogels may help to prevent bacterial migration.

The compositions, systems, and methods herein can provide a range of properties including inherent cohesion and adhesion to tissue. By virtue of such properties, the gels herein can act as protective barriers for thin, damaged and/or otherwise damaged tissues of a body lumen (e.g., the gastrointestinal tract). For example, an exemplary composition (e.g., formulation) or system for forming a gel may be applied to a target site along the gastrointestinal tract, and the composition may be crosslinked to form a gel, which may provide barrier protection/therapy to the target site. The components of the gel systems and compositions herein may provide desirable properties that facilitate tissue protection, for example, before, during, and/or after medical procedures. The compositions and systems herein may be delivered to a target site by suitable methods or techniques. The nature (e.g., viscosity) of the composition may facilitate delivery of the gel-forming formulation to the target site via suitable medical devices (e.g., single/multi-lumen catheters, including endoscopes and syringes) as well as other devices used in medical procedures. For example, a composition in gel form herein may have a viscosity of about 0.010pa-s to, for example, about 0.015 pa-s, for example, about 0.013 pa-s, at room temperature (about 22-25 ℃). The components of the composition or gel system may be crosslinked to form a gel, which may include activating one or more components in the presence of a stimulus (e.g., pH or light). The hydrogels herein may be hydrophilic three-dimensional polymer networks formed from macromers and crosslinking agents (also referred to herein as crosslinkers). The gels (e.g., hydrogels) herein may be formed by combining a macromer with a crosslinker under suitable pH conditions or light exposure to initiate crosslinking.

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 macromer may have a plurality of functional groups, such as amine, alkene, and/or thiol functional groups, that are available for reaction with the crosslinker. FIGS. 1A and 1B illustrate exemplary macromer structures representing branched poly (ethyleneimine) with amine functionality (FIG. 1A) and branched poly (ethyleneimine) with thiol functionality (FIG. 1B). Further examples of macromers that may be used herein are shown in fig. 2A (poly (ethyleneimine)), fig. 2B (4-arm PEG-NH ₂), fig. 2C (PEG-based macromer with olefinic functionality), fig. 2D (poly (1, 2-glycerol) carbonate-based macromer with olefinic functionality, where m and n are integers representing an amount of each unit up to 100% in total, and "ran" refers to a random copolymer), fig. 2E (PEG-based macromer comprising a norbornene moiety with olefinic functionality), fig. 2F (poly (1, 2-glycerol) carbonate-based macromer comprising a norbornene moiety with olefinic functionality, where l, m and n are integers of 1 or more), 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 norbornene group, wherein the norbornene group comprises from 1% to 90% of the macromer.

Exemplary cross-linking agents useful in the present disclosure include PEG-based polymers comprising one or more N-hydroxysuccinimide or maleimide functional groups. Fig. 3A and 3B illustrate exemplary crosslinker structures representing N-hydroxysuccinimide-PEG polymer (fig. 3A) and maleimide-PEG polymer (fig. 3B). Further examples of cross-linking agents that may be used herein are shown in fig. 4A-4D. FIGS. 4A and 4B are two different types of N-hydroxysuccinimide functionalized PEG cross-linkers. The structures are similar except that the structure shown in fig. 4A contains a hydrolyzable internal ester linkage. Fig. 4C shows another exemplary structure of an N-hydroxysuccinimide functionalized PEG cross-linker, wherein m is an integer of 1 or greater, 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 of 1 or greater, 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).

As described above, the gels herein may form a three-dimensional polymeric network capable of forming a barrier over a wound or other target site in the body (e.g., in the colon or another portion of the gastrointestinal tract). FIG. 5 is a simplified illustration of crosslinking between a macromer and a crosslinker, wherein the functional groups of the macromer react with the functional groups of the crosslinker. The polymeric network of the gel may be disrupted, for example, to dissolve the gel, depending on the strength of the bond between the macromer and the crosslinker. Fig. 6 is a simplified illustration of gel dissolution. Dissolution may occur, for example, by hydrolysis. Fig. 7 depicts the hydrolysis of hydrogels with thiol groups. Dissolution may also occur via thiol-transesterification, for example by reaction with methyl cysteine (fig. 8), as discussed in several examples below. In the latter case, primary amines on the cysteine methyl esters are believed to rearrange to form irreversible amide bonds, preventing the reformation of the gel after decomposition of the polymeric network.

The structure of the cross-linking agent helps control the dissolution rate. FIG. 9 shows the possible reactions of an exemplary N-hydroxysuccinimide functionalized PEG cross-linker at different sites: (A) reaction of NHS esters, (B) reaction of internal thioesters, and (C) reaction of internal esters. The reaction of these sites with the macromer poly (ethyleneimine), methyl cysteine and water is shown, with darker shaded areas corresponding to greater reactivity. Thus, NHS ester (A) reacts most strongly with the macromer poly (ethyleneimine) and internal thioester (B) reacts most strongly with cysteine methyl ester. The internal esters are quite reactive towards macromers, methyl cysteine and water. Without being limited by theory, it is believed that the stability of the gel may be derived, at least in part, from the hydrophobic methylene chain length that protects adjacent thioesters from hydrolysis or thiol-transesterification.

The gel herein may be formed on a target tissue of a subject, such as gastrointestinal tract tissue (e.g., intestinal tissue, colon tissue, etc.). For example, the cross-linking agent and the macromer may be delivered separately to the target tissue site such that the two components do not contact each other until the target tissue site is reached. In some examples, a dual lumen catheter may be used, for example, the cross-linking agent and macromer are delivered to the target tissue site in separate lumens. The two components may be in contact with each other at the target tissue site, where gelation occurs due to a suitable pH (e.g., the components are formulated to crosslink at physiological pH of the gastrointestinal tract) or in the presence of a photoinitiator activated by ultraviolet or visible light at the target tissue site. For example, the photoinitiator may be applied to the target tissue site before, after, or simultaneously with the crosslinking agent and/or macromer, and then light applied to activate the photoinitiator and initiate crosslinking to form the gel. Gelation may begin in a time period of greater than 0 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, a time period of greater than 1 second and less than 15 seconds. The cross-linking agent and macromer (and photoinitiator, when present) may be selected to provide relatively rapid gelation kinetics to form a gel in a tortuous environment (e.g., the gastrointestinal tract) and when subjected to gravitational pull.

Once formed in situ on the tissue, the gel may form a barrier of sufficient strength to remain intact for the desired period of time. For example, the gel may 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 aspects of the present disclosure, the gel may form a barrier on the tissue that remains 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.

The cross-linking agent and macromer may be selected to provide sufficient strength to accommodate the time desired for the gel to form a barrier on the tissue. As discussed in the examples below, greater crosslink density (depending at least in part on the number and type of functional groups of the crosslinker and macromer) and/or relative hydrophobicity is expected to provide a stronger gel with 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 an external thioester, such as by thiol-transesterification, as discussed in the examples below), depending on the crosslink density and strength of the gel.

Exemplary compositions and systems useful in medical procedures, including endoscopic procedures, are further discussed below, e.g., compositions or systems comprising a gel or formulated to form a gel. The composition may be used for therapeutic purposes, and the composition may be activated by different mechanisms (e.g., gel formation in situ).

PH activated gel system

In some aspects of the disclosure, the compositions or systems may be formulated to crosslink and form a viscous, coagulated gel at physiological pH (e.g., pH of about 7 to about 7.5, e.g., about 7.35-7.45). Thus, such compositions and systems may be pH-activated such that the gel (e.g., hydrogel) is selectively crosslinked at neutral to alkaline pH (e.g., little or no crosslinking at acidic pH), with reaction kinetics increasing with increasing pH. According to some aspects of the present disclosure, the composition or system may be pH-activated so as to form a gel (e.g., a hydrogel). For example, the composition may comprise at least two components, such as a first component (e.g., a first portion of solution) and a second component (e.g., a second portion of solution), that crosslink at physiological pH (e.g., in the range of about 7 to about 7.5). Thus, for example, the first and second portions of solution may have different pH values and may form a gel when mixed together to provide a physiological pH.

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

The second component of the system may include a cross-linking agent. Exemplary cross-linking agents include, but are not limited to, PEG-based polymers. For example, the PEG-based polymer used as the cross-linking agent may have a molecular weight of greater than 3000Da, such as about 3200Da to about 3500Da, such as about 3400Da. According to some aspects of the present disclosure, the crosslinking agent may have a molecular weight of about 3000Da to about 3800Da, about 3200Da to about 3500Da, or about 3400Da to about 3800 Da. In some examples, the crosslinker may be a PEG-based polymer comprising one or more N-hydroxysuccinimide or maleimide functional groups. As discussed in further detail below, the N-hydroxysuccinimide or maleimide groups can be reacted with macromers. Exemplary structures representing N-hydroxysuccinimide-PEG polymer and maleimide-PEG polymer are shown in FIGS. 3A and 3B, respectively. It should be noted, however, that suitable cross-linking agents (e.g., PEG-based polymers) are not limited to N-hydroxysuccinimide or maleimide functional groups. PEG-based polymers suitable for use in 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.

In some aspects, the cross-linking agent may be provided in the form of a solution with a buffer, e.g., the cross-linking agent is dissolved in the buffer. For example, the buffer may be a phosphate buffer, such as Phosphate Buffered Saline (PBS). The buffer in which the crosslinking agent (e.g., a dissolution crosslinking agent) is provided may have a pH lower 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 be pH-activated and may comprise at least two buffers, one having a higher pH than the other.

The crosslinker and macromer may be present in a molar ratio of 3:2 to 7:3 (e.g., molar ratio 2:1), respectively (e.g., N-hydroxysuccinimide: amine, N-hydroxysuccinimide: thiol, maleimide: amine, maleimide: thiol, etc.). The gel may be an aqueous composition wherein the combined amount of crosslinker and macromer is at least 15 weight percent, relative to the total weight of the composition. For example, the crosslinker may be present in an amount ranging from about 10 to 20 wt%, for example, from about 10 wt% to about 15 wt%, from about 12 wt% to about 18 wt%, or from about 15 wt% to about 20 wt%, relative to the total weight of the composition. Additionally or alternatively, the macromer may be present in an amount of between about 5 to 10 weight percent, for example, in the range of about 5 weight percent to about 8 weight percent, or about 7 weight percent to about 9 weight percent, relative to the total weight of the composition. As described above, the first and second partial solutions may comprise at least two different buffers, for example, a first buffer suitable for the cross-linking agent and a second buffer suitable for the macromer. According to some aspects, the first buffer comprises a phosphate buffer and the second buffer comprises a borate buffer. The aqueous composition may comprise any suitable salt for a buffer. Note that the mechanical properties of a gel (e.g., hydrogel) formed from the compositions herein may be determined, at least in part, by the amount of macromer and/or cross-linking agent. For example, gel strength may increase with increasing amounts of macromer and crosslinker in the aqueous composition. Thus, for example, a composition comprising about 20 wt% or about 25 wt% of the composition and a cross-linking agent may form a gel having a higher gel strength than a gel formed from a composition comprising about 15 wt% of the composition and a cross-linking agent, relative to the total weight of the aqueous gel system.

The components of the composition or system (e.g., macromer, cross-linker, and corresponding buffers) may be mixed together.

When at physiological pH, the functional groups of the crosslinker and the functional groups of the macromer can react with each other via chemical bonding (conjugation), allowing for immediate gelation. For example, the macromer and the crosslinker may react to form a gel when the pH of the composition is from about 7 to about 7.5. In some aspects, the gel may form within 20 seconds, 15 seconds, 10 seconds, or about 5 seconds when the first component comprising the macromer and the first buffer is mixed with the second component comprising the crosslinker and the second buffer. For example, the gel may form 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., hydrogel) may be dissolvable, e.g., passively in a physiological environment over time, or dissolvable as desired, e.g., by employing an agent capable of disrupting the hydrogel network. For example, the gel may dissolve in about 10-30 minutes. Dissolution can be measured in a laboratory environment, for example, by measuring the flow change when the gel is immersed in an aqueous solution.

The gel formed from the macromer and the crosslinker may exhibit desirable properties. For example, the storage modulus (as a measure of gel strength) of the resulting gel (e.g., hydrogel) may be in the range of about 2.0-10.5kPa, such as about 2.5kPa to about 10kPa, about 5kPa to about 8kPa, or about 3.5kPa to about 7.5kPa. Additionally or alternatively, the gel may maintain a gel strength (also referred to herein as storage modulus G') of about 2000-10000Pa at about room temperature for a desired period of time, for example, up to 30 days or more. Further, when adhered to tissue (e.g., tissue of a body cavity, such as colon tissue of the gastrointestinal tract), for example, the gel (e.g., hydrogel) may have a shear force of between about 0.03-0.90N/cm ², such as between about 0.1-0.6N/cm ², such as ranging from about 0.05N/cm ² to about 0.4N/cm ², about 0.5N/cm ² to about 0.9N/cm ², or from about 0.75N/cm ² to about 0.9N/cm ².

The gel may additionally or alternatively be formulated to withstand pressures up to about 200 mbar, for example 150 mbar, such as a burst pressure of greater than 1 mbar and less than or equal to 200 mbar (1 mbar = 100 pa) (corresponding to a pressure at which the gel will tear or fail when adhered to tissue). Note that the burst pressure of the gel may be measured by a catheter and a pressure sensor (including, for example, a milli catheter equipped with a pressure sensor), which may be used to measure the baseline pressure and the pressure immediately prior to burst. To measure burst pressure, a gel may be formed at Kong Zhongyuan bits of the tissue sample and exposed to a fluid of increased pressure that increases the pressure up to the point where the cohesion of the gel and/or adhesion of the gel to the tissue breaks down to allow the fluid to pass through the pores of the tissue. The pressure corresponding to the maximum pressure of the fluid immediately before the gel fails is the burst pressure.

The burst pressure of a gel applied to gastrointestinal tissue (e.g., colon tissue) can be measured as follows. First, a hole having width and length dimensions of about 1mm×5mm (the depth of the hole corresponds to the thickness of the tissue, about 5mm in the case of colon tissue) is cut in the tissue. The tissue sample was secured over the open end of the container such that an area of approximately 2 inches in diameter was disposed as an unobstructed window over the container. A saline solution is introduced into the vessel and allowed to flow through the orifice to calibrate the pressure sensor to the baseline pressure. A gel is then formed in situ to close the pores. The saline solution is then introduced into the container and the increasing fluid pressure is measured until the gel does not allow the solution to leave the container through the orifice. The maximum pressure immediately before the saline solution breaks through the gel and is expelled through the pores is the break-up pressure. In some examples herein, the gel may be formulated to withstand a burst pressure of at least 50 millibars, at least 100 millibars, or at least 120 millibars when adhered to colon tissue. For example, the gels herein may be formulated to withstand a burst pressure of up to about 150 millibar, such as from about 50 millibar to about 150 millibar, from about 100 millibar to about 150 millibar, or from about 125 millibar to about 150 millibar, when adhered to colon tissue. As described above, using a pore size of 1mm by 5mm, the burst pressure can be measured for colon tissue.

The burst pressure of a gel used as an arterial or other vascular occlusion device can be measured by forming the gel in situ to occlude a blood vessel, wherein the blood vessel has a diameter of about 4-6 mm. A syringe pump and pressure sensor may be used (see fig. 56 and example 11), wherein D ₂ O may 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 sensor was recorded as burst pressure (unit of pressure, 1 mmhg=133.322 Pa).

Photoactivated gel system

The present disclosure also includes compositions and systems formulated to form gels when activated by light as a stimulus. In some examples, the composition may be formulated to crosslink and form a viscous gel when exposed to light (e.g., ultraviolet light or visible light). Thus, such compositions and systems can be described as photoactivated. Such compositions and systems may include, for example, macromers, cross-linking agents, photoinitiators, and buffers. The buffer may be any suitable buffer at about physiological pH or slightly above physiological pH, depending on the buffer used. For example, the phosphate buffer may be in the pH range of 7.0-8.0.

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

In some examples, the crosslinker can include a PEG-based polymer of at least one functional group. The functional groups of the crosslinking agent may be complementary to the functional groups of the macromer, thereby crosslinking the macromer and the crosslinking agent. For example, the at least one functional group of the crosslinker may include a thiol group or an alkenyl group, such as a vinyl group, an allyl group, an acrylate group, a norbornene group, or other type of olefinic group. The functional groups of the macromer may be selected based on the desired degradation properties of the gel. In some examples herein, the number of functional groups of the crosslinker may be between 2 and 4, such as 2, 3, or 4 different functional groups.

In some examples, the macromer comprises thiol groups, and the crosslinker comprises alkenyl groups, or vice versa. For example, the crosslinker may comprise a PEG-based polymer comprising thiol groups and the macromer comprises alkenyl groups, such as acrylate groups. In another example, the macromer may comprise a PEG-based polymer comprising thiol groups, and the crosslinker may comprise a PEG-based polymer comprising alkenyl groups (e.g., allyl ether groups).

As described above, the composition may comprise a photoinitiator, for example for initiating gelation. Thus, for example, a photoinitiator may be a compound that absorbs light of a given wavelength. In accordance with aspects of the present disclosure, the photoinitiator may absorb ultraviolet light (e.g., wavelengths between about 100-390 nm) or visible light (e.g., wavelengths between about 390-800 nm). Examples of photoinitiators activated by ultraviolet light suitable for use in the compositions herein include, but are not limited to, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (Irgacure 2959) and lithium phenyl-2, 4, 6-trimethylbenzoyl phosphonate (LAP). Gelation activated by ultraviolet light may occur immediately, for example, within about 5 seconds of ultraviolet light exposure. Examples of visible light activated photoinitiators suitable for use in the compositions herein include, but are not limited to Eosin Y. Gelation activated by visible light may occur shortly or immediately after exposure to visible light, for example, within about 10 seconds of exposure to visible light. In some examples, the photoinitiator absorbs white light, for example white light having a wavelength of about 390 nanometers to about 700 nanometers. Thus, when the composition is irradiated with ultraviolet 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 ultraviolet light or visible light may be from about 1mW/cm ² to about 150mW/cm ². For example, the ultraviolet light (365 nm) intensity may range from about 4mW/cm ² to about 120mW/cm ², and the white light intensity may range from about 10mW/cm ² (e.g., at the maximum absorption of the photoinitiator) to about 45mW/cm ², such as 42.9W/cm ².

In some examples, the composition may include an additive that promotes or enhances the kinetics of photopolymerization gelation. Exemplary additives include, for example, small molecule additives such as tyrosine derivatives, e.g., methyl tyrosine or ethyl tyrosine. Such compositions may include photoinitiators that absorb visible light (e.g., white light).

The foregoing macromer, crosslinker, and photoinitiator components may be present in combined concentrations ranging from about 10 to 25 weight percent, relative to the total weight of the composition. At higher amounts (e.g., about 20-25 wt.%) the composition may have a relatively shorter gelation time and result in a gel with relatively higher elasticity. The content of the crosslinking agent may be between 5 and 10% by weight relative to the total weight of the composition. Additionally or alternatively, the content of macromer may be between 5 and 15% by weight relative to the total weight of the composition. In the composition, the molar ratio between the functional groups of the macromer and the functional groups of the crosslinker may be between 1:1 and 2:1. In some examples, the molar ratio is about 1:1. Note that the aforementioned 1:1 ratio of functional groups can result in an increase in elasticity, i.e., gel elastic modulus, compared to the aforementioned 2:1 molar ratio. In some examples, the composition or system may include a macromer comprising two or more functional groups for each crosslinker in the composition or system (e.g., a macromer to crosslinker ratio of 1:1, where the macromer comprises at least two functional groups, or a macromer to crosslinker ratio of 1:2, where the macromer comprises at least four functional groups). It should also be noted that different stoichiometric amounts of functional moieties (e.g., the number of functional groups or reactive groups per macromer) can affect the mechanical properties and swelling ratio of the resulting gel (e.g., hydrogel). For example, an increase in the number of functional groups on the macromer of the photoactivated gel system may result in a harder (e.g., more viscous) gel with less swelling.

The composition may comprise from about 0.1mM to about 100mM of photoinitiator. Higher photoinitiator concentrations (e.g., about 90-100 mM) may provide relatively faster gelation kinetics compared to lower concentrations (e.g., about 0.1-1 mM). Where the composition comprises an additive, the composition may comprise up to 10mM of the additive, for example from about 0.1mM to about 10mM, from about 0.1mM to about 5mM, from about 1mM to about 5mM, or from about 0.5mM to about 1mM.

The resulting photoactivated gels (e.g., hydrogels) can exhibit a number of desirable properties that are advantageous for application to tissue before, during, and/or after medical procedures. For example, a gel (e.g., hydrogel) may exhibit a gel strength (also referred to as storage modulus G') of between 500-2500Pa, e.g., ranging from about 500Pa to about 1500Pa, from about 1000Pa to about 2000Pa, from about 750Pa to about 1250Pa, from about 1750Pa to about 2500Pa. The gel strength G' may depend on the concentration in the composition, the macromer and the crosslinker and/or the ratio of the components relative to each other. The gel (e.g., hydrogel) may exhibit a swelling ratio mf/mi (a fold change in gel weight due to water absorption, i.e., mf is the weight of the gel at a particular point in time after immersing the gel in the buffer, mi is the initial weight of the gel before immersing the gel in the buffer) in the range of about 1.8 to about 1.9 times the initial mass or about 2.3 to about 2.4 times the initial mass. The resulting gel may also exhibit relatively low levels of cytotoxicity. For example, the gel may exhibit greater than at least 97% viability over the entire 24 hours of exposure to a cell line (e.g., NIH3T3 fibroblasts).

The following examples are intended to illustrate the disclosure, but are not limiting in nature. It is to be understood that the present disclosure includes additional embodiments consistent with the foregoing description and the examples below. The present disclosure is not limited to the examples described further below, and additional conditions are included without departing from the scope of the present disclosure.

Examples

Example 1

Exemplary pH-activated compositions (gel systems) were prepared ex-vivo at room temperature in a humid environment according to table 1. The first part solution was prepared by combining amine-terminated PEG groups or poly (ethyleneimine) macromers with borate buffer at pH 8.5. Separately, a second portion of solution comprising an N-hydroxysuccinimide crosslinker dissolved in a phosphate buffer at pH 6.5 was prepared.

TABLE 1

	First part of solution	Second part of the solution
			Macromer (mg)	1.4	Is not suitable for
Borate buffer (mu L)	100	Is not suitable for
			Crosslinking agent (mg)	Is not suitable for	36
Phosphate buffer (mu L)	Is not suitable for	100

The first and second portions of solution are mixed together to form an aqueous solution, which then forms a gel. The composition was left for one hour to complete gelation before evaluating the resulting properties.

Gel strength (storage modulus G') of the gel was measured using a TA instruments DHR-2 rheometer at room temperature (about 22-25 ℃) and evaluated at 1Hz frequency using a strain sweep of 1-100%. As the percent strain increases, the linear viscoelastic region is determined until the curve shows a 10% decrease in the slope of the gel strength. Frequency sweeps were then performed from 1-10Hz in the linear viscoelastic region at 3% strain. Frequency scans were performed at times t=0, 4 hours, 24 hours, 48 hours, 7 days and 30 days of immersing the gel in 50mM PBS until the gel was dissolved.

Figure 10 shows the gel strength of the gel during the above time. As shown, the gel exhibited a gel strength of at least 1000Pa for at least 30 days. The gel exhibited an initial gel strength of about 4000Pa at t=0 and a peak gel strength of about 8000Pa at t=24 hours. These properties indicate that the pH activated gel can act as a durable protective barrier for tissue for at least 30 days.

Gelation time was measured using an inverted tube test, wherein gelation was measured after mixing the first and second portions of solution, as the time when the gel was no longer flowing down the sides of the vial when inverted. The gelation time was measured at 1 second or less.

The swelling ratio was determined as the weight percent of hydrogel after immersion in 50mM PBS according to the following formula:

Where mf is the weight of the gel at that particular point in time after immersing the gel in the buffer, and mi is the initial weight of the gel before immersing the gel in the buffer.

TABLE 2

	Swelling ratio (%)
		15 Wt% composition	220-300
20% By weight of composition	186-234
		25% By weight of composition	125-162

Using isolated pig colon tissue viaLap shear test of the machine to determine adhesion measurements. The tissue was dissected into approximately 2 "x 1" pieces. The gel was placed between two pieces of colon tissue and left in the moist chamber for 1 hour to completely gel. Note that in this example, gelation was slowed to about 5-10 minutes to better handle ex vivo tissue/adhesion measurements. Thus, the tissue sample was left in the chamber for 1 hour to ensure complete gelation. The gel-tissue construct is then mounted at/>The force was measured continuously while pulling the two pieces of colon tissue away from each other in opposite directions at a rate of 10mm/min until cohesive failure of the hydrogel was observed. The adhesion measurement range was 0.03-0.85N/cm ².

Example 2

According to table 3, two UV-activated compositions (gel compositions 1 and 2) were prepared ex-vivo at room temperature by combining an alkene-containing PEG-based macromer, a thiol-containing PEG-based crosslinker, and a photoinitiator LAP in PBS at pH 7.4. Gel composition 1 was prepared using the PGC-based macromer shown in fig. 2D, and gel composition 2 was prepared using the PEG-based macromer shown in fig. 2C. The cross-linking agent is PEG dithiol cross-linking agent or 4-arm-PEG mercaptan cross-linking agent.

TABLE 3 Table 3

	Composition 1	Composition 2
			Macromer (mg)	71.5	62.5
Crosslinking agent (mg)	53.5	62.5
			Photoinitiator (mg)	10	10
Buffer solution (mL)	0.5	0.5

The composition was gelled under 365nm ultraviolet light using a hand-held 4W lamp.

Gel strength of the gel before and after swelling in buffer was measured on a TA instruments DHR-2 rheometer using 8mm parallel plates at room temperature (about 22-25 ℃). Frequency sweep was performed with 1% strain, and gel strength G' was identified from the linear viscoelastic portion of the sweep.

Fig. 11A shows the gel strength of the gel before and after swelling. The gel obtained from composition 1 exhibited a gel strength of about 1800Pa before swelling and about 1500Pa after swelling. The gel obtained from composition 2 exhibited a gel strength of about 700Pa before swelling and about 1000Pa after swelling. Note that composition 1 includes a macromer having more reactive moieties per molecule than composition 2, and composition 2 has a macromer having 4 reactive moieties per molecule. Thus, composition 1 exhibits a higher crosslink density than composition 2, which results in a stronger gel strength.

The swelling ratio was determined by taking the mass (mi) of the initial gel and then swelling in buffer for 24 hours after absorbing the excess buffer according to equation 1 above.

Fig. 11B shows the gel swelling ratio (expressed in fig. 11B as the ratio of mf divided by mi, instead of the percentage) of the two gels. 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 is also believed to be a result of the different macromers used, resulting in different crosslink densities. Notably, higher crosslink density results in lower swelling ratio.

Example 3

According to table 4, an exemplary visible light activated gel system (composition 3) was prepared ex-vivo at room temperature by combining an olefin-containing PEG-based macromer with a thiol-containing PEG-based crosslinker, photoinitiator Eosin Y, additive ethyl tyrosine and phosphate buffer at pH 7.0.

TABLE 4 Table 4

	Composition 3
		Macromer (mg)	278
Crosslinking agent (mg)	222
		Photoinitiator (mg)	0.129
Additive (mg)	0-4
		Buffer solution (mL)	2

The system was gelled using AmScope W halogen lamp and a double gooseneck fiber illuminator with broad spectrum white light (400-700 nm).

Gel strength was measured at t=0, 4 hours, 24 hours, 48 hours and 7 days at room temperature (about 22-25 ℃) as in example 2, as shown in fig. 12A.

The gel exhibited an initial gel strength of about 600Pa at t=0, and the gel strength increased over the next 7 days, exhibiting a peak gel strength of about 2100Pa at t=24 hours. The gel exhibits a gel strength of at least 600Pa for at least 7 days. Thus, these results indicate that the gel can act as a permanent protective barrier for tissue for at least 7 days.

Gelation time was measured on a DHR-2 rheometer with 20mm parallel plates at room temperature (about 22-25 ℃) and two goosenecks of a halogen lamp were aligned with the solution prior to exposure between the two parallel plates. The time sweep was performed with a frequency of 1 Hz. The lamp was turned on at 30 seconds and the time after which gelation occurred was recorded. The gelation time is shown in fig. 12B, and as shown, gelation kinetics increase with increasing concentration of ethyl tyrosine (from 0 to 10 mM).

The gel precursor viscosity was determined by flow scanning on a DHR-2 rheometer using a 50mm 1.008℃conical plate at 37 ℃. The results shown in fig. 12C demonstrate that the gel precursor solution exhibited newtonian properties and a viscosity sufficiently low in the shear rate range above 1/s prior to photoinitiation to allow application of the solution through a long catheter. In addition, the solution did not exhibit significant shear thinning or thickening.

Photoinitiators and gels were tested for in vitro cytotoxicity using NIH 3T3 fibroblasts cultured in darbeck Modified Eagle Medium (DMEM, dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded in 96-well or 12-well plates at a density of 2500 cells/well or 25000 cells/well, respectively, and allowed to adhere overnight. The photoinitiator solution and the gel solution were sterile filtered using a 0.22um filter prior to in vitro testing. The gel solution was then gelled in a biosafety cabinet using a 150W halogen lamp and co-cultured with cells using a 3 μm pore size transwell insert. Cells were incubated for 24 hours and then tested (CellTiter) using MTS (3- (4, 5-dimethyltriazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-triazole, inner salt)AQ _ueous One, promega) and viability was measured. Cell viability was normalized to control untreated cells.

Figures 12D-12E demonstrate the results of the cytotoxicity assays discussed above. The photoinitiator had an IC ₅₀ of about 0.223mM, which was higher than the concentration used in the gel formulation. As shown in fig. 12E, by using photoinitiators below IC ₅₀, the gel was not expected to present cytotoxicity problems, indicating that the cell viability was higher than 97% for all three weight percent gel formulations. Furthermore, there was no observable difference between the viability of the three gel formulations, indicating that as the concentration of the other components of the gel in the solution increased, they were not significantly toxic.

Example 4

As shown in fig. 13 below, the crosslinking agent ("SA crosslinking agent") shown in fig. 4A was synthesized.

SA-PEG-SA was first synthesized as follows. Poly (ethylene glycol) (PEG; average Mn 3000g/mol; SIGMA ALDRICH) (5 g,1.6 mmol) was melted in a three-necked round bottom flask with stirring at 120 ℃. Once melted, the flask was placed under vacuum, then the temperature was reduced to 80 ℃ and stirred for 30 minutes. The flask was purged three times with nitrogen. Succinic Anhydride (SA) (99%; aldrich) (0.75 g,7.5 mmol) was added to the flask. The reaction was stirred under nitrogen for 18 hours. The contents were then dissolved in a very small amount of anhydrous dichloromethane (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). Spectral proton and carbon nuclear magnetic resonance (¹H-NMR,¹³ C-NMR) spectra were obtained in CDCl ₃ on an Agilent 500MHz spectrometer. The NMR spectrum of the SA-PEG-SA product is as follows: ¹H NMR(500MHz),CDCl₃: δ2.62 (m, 8H), 3.64 (overlap, 288H), 4.24 (m, j=4.6 hz, 4H); ¹³C NMR(500MHz),CDCl₃: delta 174.0, 172.1, 70.5, 63.8, 29.3, 28.3ppm. The SA-PEG-SA intermediate is obtained with a reaction yield of 99%.

Next, SA-PEG-SA (4 g,1.3 mmol) was added to the dried round bottom flask and dissolved in 15mL of dry DCM. N-hydroxysuccinimide (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 hours. The dicyclohexylurea was filtered, the solution was concentrated and precipitated in diethyl ether. The resulting product, SA crosslinker, was collected by filtration and dried under vacuum overnight, as a white water-soluble powder (white solid, 98% yield). The structure was confirmed by ¹H NMR、¹³ C NMR, DSC and GPC. The NMR spectra of SA crosslinkers measured as described above overlap as follows ：¹H NMR(500MHz),CDCl₃：δ2.70(t,J＝1.0Hz,4H),2.77(t,J＝1.0Hz,8H),2.89(t,J＝1.0Hz,4H),3.57( ,296H),4.20(t,J＝1.0Hz,4H)ppm.¹³C NMR(500MHz),CDCl₃：δ170.9,168.9,167.6,70.7,64.1,28.6,26.2,25.5ppm.

Molecular weight and polymerization distribution were determined by Gel Permeation Chromatography (GPC) with Tetrahydrofuran (THF) as the mobile phase at a flow rate of 1.0 mL/min. For SA crosslinkers, M _w: 2949g/mol; PDI:1.02.GPC analysis was performed on a OptiLab DSP interferometric refractometer (Wyatt Technology) equipped with two identical Jordi gel DVB columns (Jordi Labs,250mm x 10mm, Pore size). For SA crosslinkers, GPC: m _n: 2893g/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 SA crosslinker, MALDI-TOF (pos): m _w: 3600m/z. Differential Scanning Calorimeter (DSC) spectra were collected on a Q100 TA instrument calorimeter for determination of melting point (mp). For SA crosslinker, mp (DSC): 43.5 ℃.

Example 5

The cross-linker ("SVA cross-linker") (average Mn 3400) shown in fig. 4B was obtained from Laysan Bio, inc. And stored in a glove box. The NMR spectra measured as described above were measured as follows ：¹H NMR(500MHz),CDCl₃：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.¹³C NMR(500MHz),CDCl₃：δ169.1,168.6,70.4,30.6,28.4,25.5,21.4ppm. for SVA cross-linker as described in example 4: MALDI-TOF (pos): m _w：3700m/z;GPC：M_n：4635g/mol;M_w: 4812g/mol; polydispersity index PDI:1.03; mp (DSC): 47.6 ℃.

Example 6

Hydrogels were prepared by combining the crosslinker shown in FIG. 4A ("SA crosslinker") and the crosslinker shown in FIG. 4B ("SVA crosslinker") with hyperbranched Polyethyleneimine (PEI) (average Mn 2000g/mol; manufacturer Polysciences) or 4-arm PEG-NH ₂ HCl salt (4-arm PEG-NH ₂) (star polymer; mn 5000g/mol; manufacturer JenKem) as shown in FIG. 14. Briefly, each PEG cross-linker was dissolved in 0.1M phosphate buffer pH 6.5. Each of PEI and 4-arm PEG-NH ₂ was dissolved in 0.3M borate buffer at pH 8.6. The pH obtained after mixing the crosslinker and the macromer solution was adjusted to pH 8.5. The molar ratio of amine to NHS was 1:15 and hydrogels were prepared at 10, 15 or 20 weight percent (wt%). The ratio of amine groups in the macromer to NHS groups in the crosslinker was kept constant, increasing the weight% to increase the amount in solution. The properties of the hydrogels were measured and analyzed as discussed in the following sections.

Data were analyzed with GRAPH PAD PRISM a. For hydrogel characterization studies, error bars represent standard deviations of three or more replicates. For bacterial migration studies, the error bars represent the standard deviation of the results of three biological replicates, repeated with three or more technical replicates at a time. Results were compared and evaluated for significance using Student's T-test. A p <0.05 has significance.

Gelation measurement

Relatively rapid gelation times (e.g., < 3 seconds) may be used to form gels in situ, such as during a polypectomy or other internal wound dressing. For gelation measurements, the crosslinker and amine-terminated macromer solution were mixed and placed in a 2mL glass bottle. Gelation was tested using an inverted tube test member. The tube was inverted every 10 seconds. Gelation is defined by the time the solution stays at the bottom of the bottle when inverted. All gelation studies were performed at room temperature (25 ℃).

Fig. 15 shows gelation measurement results; gelation time of hydrogels at different weight percentages and different formulations in panel a) and gelation time of SA crosslinker+pei hydrogels at increasing pH in panel B) with 15 weight%. * p <0.05. The SA crosslinker+pei hydrogel gelled faster as the weight percent increased from 10 wt% to 20 wt%, as all the hydrogels in fig. 15, panel a), were formed at pH 8.5. The increase in gelation time is due to the higher concentration of reactive groups and thus favors faster gelation. Next, the gelation time between SA crosslinker and PEI or 4-arm PEG-NH ₂ macromer was compared at 15 wt.%. The gelation time was similar, about 90 seconds (1.5 minutes), indicating that gelation was independent of the amine macromer.

The gel rate of the SVA crosslinker+PEI hydrogel is similar to the gel rate of the SA crosslinker+4-arm PEG-NH ₂ and SA crosslinker+PEI hydrogel. However, the SVA crosslinker +4-arm PEG-NH ₂ hydrogel gels faster than the SA crosslinker +4-arm PEG-NH ₂. The increase in gelation time is due to two factors. The 4-arm PEG-NH ₂ macromer contained four long amine terminated arms and had a molecular weight of 5kDa. PEI is a more concentrated branched macromer with a molecular weight of 2.0 kDa. The long PEG arm of 4-arm PEG-NH ₂ is believed to facilitate faster gelation time of the SVA crosslinker + 4-arm PEG-NH ₂ hydrogel due to the increased spatial freedom relative to PEI, a branched polymer with shorter arms containing terminal amine and smaller molecular weight. The steric hindrance observed in the smaller branched PEI structure relative to the higher molecular weight, longer arm, star-shaped 4-arm PEG-NH ₂ structure is believed to reduce the ability to readily react with NHs reactive groups in the hydrogel network.

Regarding defects in the hydrogel network, terminal amines facilitate binding on NHS-esters, whereas SA crosslinkers contain internal esters, which are also susceptible to amidation and hydrolysis of macromers. The preferred site for amidation in the SA crosslinker is at the NHS ester, t _1/2＝0.60min^-1 as confirmed by nuclear magnetic resonance by ¹ H NMR analysis of the model system. FIG. 19 shows the variation of the NMR spectrum of the reaction, FIG. 18 shows the variation as a function of time, monitoring the variation of the NMR signals of the NHS esters and internal esters. Although considered unlikely, amidation can occur on the internal ester, resulting in defects in the hydrogel network, t _1/2＝1.8min^-1 (see fig. 18). Furthermore, the half-life of hydrolysis at the ester linkage at pH 8.0 is t _1/2 = <5 minutes, further providing a defect in the hydrogel network. Hydrolysis and amidation of the internal esters are competing reactions and thus may result in slower gelation times relative to the SVA crosslinker+4-arm PEG-NH ₂ hydrogels.

As summarized in fig. 15, panel B), the effect of pH on gelation time was also assessed for 15 wt% SA crosslinker+pei hydrogel for pH 8.5, pH 9.5 and pH 10.5. The gelation rate increases with higher pH of the PEI buffer solution: 100 seconds at pH 8.6, 60 seconds at pH 9.5, and 5 seconds at pH 10.5. For comparison, refer to panel a of fig. 15), at pH 8.5, the SA crosslinker+4-arm PEG-NH ₂ hydrogel gelled in 80 seconds, the SVA crosslinker+pei hydrogel gelled in 90 seconds, and the SVA crosslinker+4-arm PEG-NH ₂ hydrogel gelled in 45 seconds. Increasing the pH of the PEI solution provides faster gelation.

Rheometry measurement

Rheological measurements were obtained using a TA instruments DHR-2 rheometer. Rheo 8mm parallel plates were used for rheo metry at 22 ℃. The oscillatory strain sweep is performed from 0.1-10% strain at a frequency of 0.1 Hz. The linear viscoelastic region is determined by the strain sweep as the percentage of strain below which G' deviates from the horizontal by 10 °. Frequency scans were then performed at all time points throughout 30 days. The strain is set in the 3% linear viscoelastic region and the frequency is from 0.1Hz to 10Hz according to the previously published protocols. Data are expressed as mean.+ -. Standard deviation (n.gtoreq.3).

The storage modulus (G') of the hydrogels was measured 0 hours, 4 hours, 24 hours, 48 hours, 7 days and 30 days after swelling in 100mM PBS pH 7.4 (applied strain of 3%). The percent swelling and storage modulus (G') of each hydrogel were measured as an indicator of strength, as well as hydrogel swelling throughout 30 days or until the hydrogel was dissolved in 100uM PBS (pH 7.4). Fig. 16 shows the strength (G') measured for the following hydrogels: panel a) different weight percentages of SA crosslinker+pei; panel B) 15 wt% SA crosslinker+PEI or SA crosslinker+4-arm PEG-NH ₂; panel C) 15 wt% SA crosslinker+4-arm PEG-NH ₂ and SVA+4-arm PEG-NH ₂; panel D) 15 wt% SA crosslinker+PEI and SVA crosslinker+PEI. All rheologies were recorded over time after swelling, p <0.05.

10 Wt%, 15 wt% and 20 wt% SA crosslinker+pei hydrogels showed average G' at gelation of 638Pa, 992Pa and 2930Pa, respectively. 15 wt% and 20 wt% hydrogels maintained mechanical integrity (G '>300 Pa) over 48 hours, while 10 wt% SA crosslinker+pei hydrogel dissolved after 4 hours (G' <300 Pa). To evaluate the effect of degradable ester linkages in the crosslinker on mechanical strength, hydrogels were prepared using SA or SVA crosslinkers and 4-arm PEG-NH ₂ (see B) and D)). In contrast, a 15 wt% SA crosslinker+4-arm PEG-NH ₂ hydrogel exhibited G 'of 3814Pa at time t=0 and remained intact in 7 days of swelling with minimal G' change over 24 hours, reduced mechanical strength at 48 hours and 7 days. 15% by weight of the SVA crosslinker+PEI and SVA crosslinker+4-arm PEG-NH ₂ hydrogel had G's of 1683Pa and 7739Pa, respectively. The G' of all hydrogels initially increased upon swelling (fig. 16). It was found that the SA crosslinker +4-arm PEG-NH ₂ hydrogel remained intact, G' unchanged (3591 Pa) over 48 hours, and maintained the hydrogel morphology throughout the 7 day swelling period, while the SVA crosslinker +4-arm PEG-NH ₂ hydrogel remained mechanically strong (13766 Pa) throughout the 30 day swelling period. A similar trend was observed for hydrogels prepared with PEI, however for SA crosslinker+pei hydrogel, G' remained similar (1380 Pa) throughout 48 hours.

The increase in weight percent of hydrogel provides greater G' and longer sustained mechanical strength as shown by the SA crosslinker + PEI hydrogel (see panel a)). Furthermore, for each SA crosslinker+pei hydrogel, G 'decreased over time, which is believed to be due to hydrolysis at the internal ester linkages, while for the SVA crosslinker+pei hydrogel, G' remained unchanged throughout the 30 day swelling period, which is believed to be due to the lack of degradable linkages within the structure of the SVA crosslinker (see figure C)).

The increase in the degradation rate of the SA crosslinker + PEI hydrogel relative to the SA crosslinker + 4-arm PEG-NH ₂ hydrogel is due to the local alkaline pH within the hydrogel network caused by PEI. The effect of pH was evaluated by swelling SA crosslinker+PEI hydrogel in dH ₂ O, pH 5.0.0 and SA crosslinker+4-arm PEG-NH ₂ hydrogel in TEA aqueous solution at pH 8.0 (tertiary amine; equivalent to [ M ] present in PEI-based hydrogels). FIG. 17 shows in FIG. A) the storage modulus of SA crosslinker+PEI hydrogels swollen in pH 7.4 and pH 5.0, and in FIG. B) the storage modulus of SA crosslinker+4-arm PEG-NH ₂ hydrogels swollen in pH 7.4 and pH 8.0; * p <0.05. The SA crosslinker+PEI hydrogel swells at pH 5.0 and hydrolyzes within 48 hours, similar to swelling in PBS at pH 7.4 (see FIG. A)). Similar degradation rates are due to the strong basicity of PEI and the inability to buffer local pH. By swelling the SA crosslinker +4-arm PEG-NH ₂ hydrogel in TEA-containing solution, hydrolysis of the hydrogel was accelerated, allowing the hydrogel to degrade within 24 hours, and 7 days in PBS at pH 7.4. This increase in hydrolysis of the SA crosslinker +4-arm PEG-NH ₂ hydrogel is consistent with the local alkaline pH of PEI, accelerating the hydrolysis of the internal esters in the hydrogel network (see panel B)).

Rheology tests were performed on SA crosslinker+PEI hydrogel in dH ₂ O at pH 5.0, and on SA crosslinker+4-arm PEG-NH ₂ at pH 8.0. SA crosslinker+pei hydrogels exhibited G 'of 1362Pa at time t=0 and maintained similar mechanical integrity throughout 48 hours until significant hydrolysis (loss of hydrogel integrity was defined as G' <300Pa, since the hydrogel was unable to maintain its mechanical structure during rheological measurements when the gel exhibited a storage modulus below 300 Pa) regardless of the pH of the water in which the gel swelled (see fig. 17). The G 'of the SA crosslinker+4-arm PEG-NH ₂ hydrogel formulation was 2239Pa at time t=0 and it was dissolved in aqueous solution at pH 8.0 for 48 hours (G' <300 Pa), whereas the G 'of the SA crosslinker+4-arm PEG-NH ₂ hydrogel swollen at pH 7.4 decreased over time but maintained its mechanical integrity (G' > 300 Pa) for 7 days (see fig. 17).

Hydrolysis at internal ester linkages was confirmed by following a shift in the ¹ H NMR from 4.19ppm to 4.15ppm of the methylene peak of the adjacent ester in the model system. The SA crosslinker was dissolved in 0.3M sodium bicarbonate buffer at pH 8.0 in D ₂ O. Fig. 19 shows hydrolysis of the internal ester of SA crosslinker moving upward (top) in D ₂ O at pH 8.0 relative to the unhydrolyzed internal ester of SA crosslinker in unbuffered D ₂ O based on the change in NMR signal. Via ¹ H NMR throughout 24 hours, the half-life of the ester linkage (t _1/2) was 19.8 minutes at pH 8.0 in D ₂ O in the presence of TEA (again, comparable to [ M ] present in PEI based hydrogels), whereas no ester linkage hydrolysis occurred when TEA was not present (at pH 5 or 6).

Hydrolysis of SA crosslinker+PEI hydrogel was evaluated at 37℃to simulate the colonic environment. FIG. 20 shows the storage modulus G' values at room temperature compared to 37℃for the SA crosslinker+PEI hydrogel in FIG. A) and the SA crosslinker+4-arm PEG-NH ₂ hydrogel in FIG. B). The SA crosslinker+PEI hydrogel degraded within 24 hours at 37℃regardless of the temperature (room temperature (RT) or 37 ℃), and the SA crosslinker+4-arm PEG-NH ₂ hydrogel degraded at the same rate and was present for 7 days. It is believed that the increase in temperature further accelerates the hydrolysis of the hydrogel catalyzed by the PEI. The SA crosslinker+PEI hydrogel at 37℃showed a larger G' value (3579 Pa) than the hydrogel held at room temperature, whereas the SA+4-arm PEG-NH ₂ hydrogel was stable after the entire 7 day swelling, regardless of temperature.

Regardless of the composition of the hydrogel, an initial increase in storage modulus and swelling occurred after the first 4 hours when the hydrogel was immersed in 100mM PBS. FIG. 21 reports swelling of hydrogels over 24 hours; 10 wt% of SA crosslinker+PEI hydrogel hydrolyzed at 24 hours, so there was no swelling data. The percent swelling of the hydrogels was reported after 24 hours of swelling. Hydrogels swelled until they reached equilibrium or degraded at 24 hours. All hydrogels swelled in buffer to at least 200% of their original weight. As the weight percent increases (SA crosslinker+pei at 10, 15, and 20 wt%), the hydrogels swell 153%, 259%, and 411%, respectively. SA crosslinker+4-arm PEG-NH ₂ hydrogel swelled 396%. The SVA crosslinker+PEI and SVA crosslinker+4-arm PEG-NH ₂ hydrogels swelled 274% and 376%, respectively. This absorbent property is believed to be useful for covering tissue, for example, as a wound dressing in a polypectomy or other medical procedure.

Adhesion to

Adhesion of hydrogels to isolated porcine colon tissue was performed on an Instron 5944 microassay. The hydrogel was mixed and placed between two pieces of colon tissue. Colon tissue was divided into 1 inch x 1 inch pieces and the hydrogel gelled directly onto the tissue. In applying the hydrogel to colon tissue, another piece of tissue is placed on top of the "sandwich" (tissue-hydrogel-tissue). The tissue adhering to the hydrogel is a mucosal layer or submucosa of colon tissue, which is obtained by scraping the colon tissue on each sample with a surgical knife. After one hour of gelation in the moist chamber, the adhesion of the hydrogel to the colon tissue was subjected to a lap shear test according to ASTM D3165 protocol. The tissue pieces were pulled apart at room temperature at a rate of 5mm/min until adhesion failure was detected. Data are expressed as mean ± standard deviation (n=3).

15 Wt% of the adhesive strength of SA crosslinker+PEI, SVA crosslinker+PEI and SA crosslinker+4-arm PEG-NH ₂ hydrogels were measured on colon tissue with and without mucosal layer at 25 ℃. These hydrogels were selected to determine if the presence of PEI or 4-arm PEG-NH ₂ in the hydrogel altered adhesion and whether the hydrolyzable SA crosslinker affected adhesion as compared to the non-hydrolyzable SVA crosslinker. For some samples, after polypectomy, the mucosal layer on the colon tissue was removed with a scalpel to expose the submucosa to better model tissue. SA crosslinker+pei, SVA crosslinker+pei and SA crosslinker+4-arm PEG-NH ₂ hydrogels showed average adhesive strength of 0.18N/cm ²、0.36N/cm² and 0.03N/cm ² in the case of intact mucosal layers and 0.31N/cm ²、0.29N/cm² and 0.64N/cm ² in the case of incomplete mucosal layers, respectively, as shown in fig. 22, where the adhesion of hydrogels with a thickness of 1mm to colon tissue with intact mucosal layers (data on the left are shown in black) and to colon tissue without mucosal layers (data on the right are shown in dark grey). * p <0.05.

In the case of intact mucosa, the adhesion of SA crosslinker+PEI and SVA crosslinker+PEI hydrogel to the mucosa is maximal with adhesion values of 0.18N/cm ² and 0.36N/cm ², respectively. The SA crosslinker+4-arm PEG-NH ₂ hydrogel showed the strongest adhesion to the submucosa free tissue (0.64N/cm ²) (FIG. 22). This difference in adhesion compared to neutral PEG is due to charge interactions and hydrogen bonding between the mucosal layer and the cationic PEI. Mucus is an anionic, hydrophobic and viscoelastic network whose glycoproteins are available for electrostatic interactions and hydrogen bonding with molecules such as PEI. PEG, on the other hand, is uncharged, hydrophilic, non-fouling; all of these features are believed to retard adhesion to mucus. The SA crosslinker+4-arm PEG-NH ₂ hydrogel adhered most strongly to colon tissue without mucosal layer, probably due to the lack of electrostatic interactions with the tissue matrix. It is believed that a force of at least 0.3N/cm ² may be sufficient to maintain adhesion to colon tissue.

Cytotoxicity study

The cytotoxicity of 15 wt% hydrogels against NIH3T3 fibroblasts was assessed. The crosslinker and PEI solution were passed through a 0.22 μmPVDF filter, then mixed and gelled under sterile conditions. Portions of 50mg, 25mg and 10mg (+ -2.5 mg) of hydrogel were placed into permeable cell culture inserts (PES, 3 μm wells) (CELL TREAT, 230637). The permeable cell culture insert containing the hydrogel sample was incubated at 4℃for 16 hours in sterile deionized water to allow it to swell. NIH3T3 (ATCC, CRL-1658) was cultured in DMEM+10% BCS+1% Ps at 37℃in 5% CO ₂ and 95% humidified air. All cells were 4-8 substitutes for the experiment. Cells were seeded in 24-well plates at 1.25X10 ⁴ cells/cm ² and allowed to adhere for 16 hours. The medium was exchanged and the cell culture inserts with swollen hydrogels were transferred to wells containing adherent cells. The hydrogel samples were equilibrated briefly to 37 ℃ prior to transfer. The hydrogel was 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. Absorbance (490 nm) was measured after 4 hours. Relative cell viability was determined by normalizing the absorbance of hydrogel-exposed cells relative to the unexposed control. All experiments were done in triplicate, error bars representing 1 standard deviation from the mean. All hydrogels were found to have minimal cytotoxicity (> 88% cell viability) (fig. 23).

Bacterial migration

Bacterial migration studies were performed on isolates of E.coli and Bacteroides fragilis, as both of these microorganisms are commonly found in the intestinal tract and are known to cause infection. Coli is highly mobile and is believed to have the potential to pass through hydrogels. Bacteroides fragilis isolates are known to exhibit multi-drug resistance and to cause sepsis. The ability of these two common intestinal microorganisms with pathogenic potential to cross SA crosslinker+pei and SA crosslinker+4-arm PEG-NH ₂ hydrogels was assessed.

In vitro testing and microscopy studies were performed on agar plates. One advantage of the agar-based assay is that it can detect if even a few bacterial cells penetrate the hydrogel, as a single bacterium can grow in a visible colony for about 24 hours. The clinical isolates E.coli (ADR 129Q-SMC 9096) and Bacteroides fragilis (CFPLTA _1B-SMC 9107) were obtained from children with cystic fibrosis. Coli isolates were aerobically cultured overnight in LB (lysogenic broth) and bacteroides fragilis isolates were anaerobically cultured on blood agar (tsa+5% sheep blood) for 48 hours using GasPak system prior to inoculating the hydrogel. Hydrogel discs (diameter 8mm x height 2.5 mm) were placed on LB agar (for E.coli) or TSA+5% sheep blood agar (for Bacteroides fragilis) and 5uL of bacteria or PBS was added to the top of each hydrogel. Plates were then incubated aerobically (E.coli) or anaerobically (Bacteroides fragilis) for 24 hours at 37 ℃. After 24 hours, the hydrogel was removed and the agar plates were incubated under appropriate conditions for each organism for an additional 24 hours to test bacterial growth under the hydrogel as a measure of whether microorganisms could pass through the hydrogel. After growth, the bacteroides fragilis isolate was scraped into 1mL PBS and homogenized. Each 1mL of Bacteroides fragilis and Escherichia coli was centrifuged at 16000x g for 30 seconds and resuspended in PBS. Then, for agar plate experiments, each isolate was normalized to an OD600 of 1.0 in PBS, and for microscopic experiments, as reported (bioproject accession No. PRJNA 557692), to an OD600 of 0.1 in the lowest medium. Wells treated with medium alone were used to determine background fluorescence, which was subtracted from each sample prior to analysis.

Microscopy was performed on a Nikon ECLIPSE TI inverted microscope equipped with a Hamamatsu ORCA-Flash 4.0 camera operating on Nikon ELEMENTS AR. A fast scan mode and 2X2 pixel binning (binning) were used and images were acquired through a Plan Fluor 40X DIC M N2 objective. The image was processed in ImageJ, where the background was subtracted and the signal intensity was quantified by measuring the average signal intensity per pixel as a function of integrated density (IntDen). For microscopic studies, 300uL SA crosslinker+4-arm PEG-NH ₂ was seeded into each well of an 8-well plate (Cellvis, catalog #C8-1.5H-N). To observe bacteria, syto9 was added to each culture prior to hydrogel inoculation. Bacterial cultures were inoculated on top of the hydrogel or under the hydrogel that was first perforated with pipette tips. Plates were imaged before and after incubation to determine if top inoculated bacteria were able to pass through the hydrogel. The results indicate that these microorganisms did not cross the sa+4-arm PEG-NH ₂ hydrogel, indicating their potential to prevent sepsis in vivo. Figure 24 shows bacterial remission through SA crosslinker+4-arm PEG-NH ₂ hydrogel. Coli (left) and bacteroides fragilis (right) were evaluated in perforated hydrogel, in which bacteria were inoculated into the bottom of the well (top two plates), and in unperforated hydrogel, in which bacteria were placed onto the surface (bottom two plates). Three independent experiments were performed, each with three technical replicates. Representative images combining bright field and Syto9 staining are shown. The thickness of the hydrogel was about 1 mm. Coli and bacteroides fragilis added to the bottom of the perforated hydrogel were observed under the microscope (fig. 24, top left and top right, respectively), but were absent when added to the top of the unperforated hydrogel (fig. 24, bottom panel).

To quantify the effect of the hydrogel on bacterial remission, the Syto9 signal intensity was assessed at the bottom of the hydrogel after subtraction of background fluorescence from the medium-only control. FIG. 25 reports Syto 9-stained bacterial surface area measured 24 hours after inoculating E.coli (upper panel A) and Bacteroides fragilis (lower panel B) on SA crosslinker +4-arm PEG-NH ₂ hydrogel. The presence of bacteria was measured in three independent experiments. The surface area occupied by bacteria was compared between perforated hydrogels where bacteria were inoculated into the bottom of the wells and unperforated hydrogels where bacteria were inoculated onto the surface. Error bars represent standard deviations, and represent significant differences in bacterial surface area at P values less than 0.05, 0.01 and 0.0001, respectively. Consistent with the imaging in fig. 24, bacteria inoculated into the bottom of the perforated hydrogel showed elevated Syto9 staining compared to the top-inoculated control (fig. 25). SA crosslinker+4-arm PEG-NH ₂ hydrogel was found to be an effective barrier to E.coli and Bacteroides fragilis for at least 24 hours. Whereas SA crosslinker+pei hydrogel was hydrolyzed at 37 ℃ under laboratory conditions, and therefore was not studied.

The lack of bacterial migration by hydrogels may be a result of hydrogel pore size relative to bacterial size. The pore size of the hydrogels ranged from <1 μm to 20 μm and the pores were not connected to create a network of cells as shown by scanning electron microscopy of the SA crosslinker+4-arm-PEG-NH ₂ hydrogels (fig. 26). Coli and bacteroides fragilis are about 1.0-4.5 μm in length. Thus, pore size and porosity create a tortuous path for bacterial migration and inhibit migration. SA crosslinker+pei hydrogel was hydrolyzed under laboratory conditions at 37 ℃ and thus was not available for this study.

The agar plate test results are shown in FIGS. 27 and 28. FIG. 27 test whether Bacteroides fragilis can pass through SA crosslinker +4-arm PEG-NH ₂ hydrogel by placing the hydrogel on TSA +5% sheep blood agar, applying bacteria to the surface of the hydrogel and assessing subsequent Bacteroides fragilis growth on the agar after a total incubation time of 24 hours and 48 hours. Three independent experiments were performed, each with an n=3 control group and an n=4-5 bacteroides-inoculated hydrogel disc. For each plate, bacteria were found directly on the plate as a positive control (large arrow, upper left corner). For each independent repeat, a representative plate is shown. The top end of each hydrogel was inoculated with 10. Mu.L of sterile PBS or 10. Mu.L of Bacteroides fragilis culture at 1OD ₆₀₀/mL in PBS. Plates were incubated anaerobically for 24 hours at 37 ℃ (top row). After 24 hours, the hydrogel was removed (middle row) and the plates were incubated under the same conditions for an additional 24 hours (bottom row). After 24 hours, bacteroides fragilis grew evident on the top side of the hydrogel but not on the agar, indicating that bacteroides fragilis did not pass through the hydrogel in high abundance. After 48 hours, contamination was visible in the total technical repeat of 11/14. Of these, 10/11 is most likely the edge contamination (black arrow) that occurs when the hydrogel is removed from the plate. In experiment 1, the hydrogel was turned over onto a plate after 24 hours to confirm the viability of bacteroides fragilis on the tip side of the hydrogel. Growth derived from the apical side of the hydrogel at 48 hours indicated that bacteroides fragilis was still viable (small white arrow, lower left).

FIG. 28 shows the results of subsequent E.coli growth on an LB agar plate, SA crosslinker of E.coli +4-arm PEG-NH ₂ hydrogel by placing the hydrogel on the agar plate, applying bacteria to the surface of the hydrogel and evaluating the total incubation time of 24 and 48 hours. Three independent experiments were performed, each with an n=3 control group and an n=4-5 e.coli inoculated hydrogel disc. For each independent repeat, a representative plate is shown. For each plate, bacteria were found directly on the plate as a positive control (large white arrow, upper left corner). The apical side of each hydrogel was inoculated with 10. Mu.L of sterile PBS or 10. Mu.L of E.coli culture at 1OD ₆₀₀/mL in PBS. Plates were incubated aerobically for 24 hours at 37 ℃ (top row). After 24 hours, the hydrogel was removed (middle row) and the plates were incubated under the same conditions for an additional 24 hours (bottom row). After 24 hours, for those hydrogels inoculated with E.coli that remained intact (n=11/15), E.coli growth was evident on the apical side of the hydrogel and not on agar, indicating that E.coli did not pass through the hydrogel in high abundance. At 48 hours, plate contamination was visible for n=8/15 plates (black arrow). Most of the contamination occurred during experiment 2 for all 24 hours and 48 hours. These hydrogels were slightly thinner than those in other experiments and some had melted at 24 hours, which was the most likely cause of contamination. In experiment 1, the hydrogel was turned over onto a plate after 24 hours to confirm the viability of E.coli on the tip side of the hydrogel. Growth from the apical side at 48 hours indicated that E.coli was still viable (small white arrow, bottom left).

The application and handleability of hydrogels were studied by administering a crosslinking agent and a macromer component via a dual lumen catheter for subsequent hydrogel formation at the exit of a target site on a colon tissue sample. A dual lumen catheter is used; the catheter is capable of being inserted into the colon through the endoscope body, eliminating the need for a separate device. Air pressure may be applied through the dual lumen catheter to spray the hydrogel precursor components onto the wound to gel in situ. The two-part hydrogel system is delivered in ex vivo colon tissue. All 12 hydrogel formulations were injected through a double lumen catheter and subsequently gelled and adhered to the colon tissue under and against gravity.

Example 7

Additional crosslinkers (crosslinkers 5, 6 and 7) were synthesized starting from PEG (M _w 3000) as shown in fig. 29. Briefly, PEG (M _w 3000) was reacted with the appropriate anhydride to form PEG diacid, followed by activation with NHS ester to give crosslinker 1. Crosslinker 1 is reacted with 1, 8-diazabicyclo (5.4.0) undec-7-ene (DBU) and the corresponding thiol-terminated carboxylic acids of 1, 5 and 10 methylene groups to afford intermediates 2,3 and 4, respectively. Next, NHS-activated crosslinkers were prepared via Dicyclohexylcarbodiimide (DCC) coupling chemistry with the product purified by precipitation in diethyl ether and NHS. The yield of all reactions was 85-98%. The structure of the crosslinker was determined by ¹H NMR、¹³ C NMR, GPC, MALDI and DSC; characterization measurements were performed as discussed in example 6. The data are as follows:

PEG diacid: the synthesis of PEG diacid compounds is 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;¹³C NMR(500MHz),CDCl₃：175.3,172.8,70.6,68.9,63.4,33.1,32.6,19.9ppm.

Crosslinker 1. Synthesis of starting Material based on previously reported schemes .¹H 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;¹³C NMR(500MHz),CDCl₃：172.3,169.0,168.0,70.5,69.0,63.6,32.4,29.9,25.5,19.7ppm.

Intermediate 2. Synthesis of protocols based on previous reports .¹H 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;¹³C NMR(500MHz),CDCl₃：196.8,172.6,169.8,70.6,69.0,63.6,42.3,32.8,31.0,20.5ppm.

Intermediate 3. In a flame dried flask, 1, 8-diazabicyclo (5.4.0) undec-7-ene (265. Mu.L) and 6-mercaptohexanoic acid (122. Mu.L) were added to a solution of crosslinker 1 (1 g) in anhydrous DMF (5 mL). The reaction was stirred at room temperature for 16 hours. The organic phase was extracted with 1M 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 (96% yield) as a white solid ).¹H 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＝7.3,4H),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;¹³C 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.

Intermediate 4. Synthesis by the procedure described above using 11-mercaptoundecanoic acid (0.190 g) as the thiol source (92% yield) .¹H NMR(500MHz),CDCl₃：δ4.22(t,J＝4.9,4H),2.85(t,J＝7.4,7.3,4H),2.60(t,J＝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;¹³C 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.

Crosslinking agents 5, 6 and 7. The synthesis of crosslinking agents 5, 6 and 7 was based on previously reported schemes (96-98% yield).

Crosslinking agent 5.¹H 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;¹³C NMR(500MHz),CDCl₃：δppm;MALDI-TOF(pos)：M_w：3763m/z;GPC：M_n：5077;M_w：5312;PDI：1.05;Mp(DSC)：46.06℃.

Crosslinking agent 6.¹H 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;¹³C 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℃.

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;¹³C 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℃.

Example 8

10, 15 And 20 wt% hydrogels were prepared by mixing the cross-linker of example 7 (i.e., cross-linkers 5, 6 and 7) dissolved in 0.1M phosphate buffer pH 6.5 with branched polyethylenimine (PEI; M _w 1800) in 0.3M borate buffer pH 8.5. The minimum solubility of crosslinker 7 in the buffer was observed and is believed to be due to the hydrophobicity of the methylene chains in its structure. To overcome the low solubility, the crosslinker 7 was dissolved in 0.1M phosphate buffer with pH 6.5 of 50% ethanol before mixing the crosslinker 7 with the PEI solution. NHS: NH ₂ ratio of 2:1 to ensure amidation of PEI and the corresponding crosslinker. No significant difference in mechanical properties of the hydrogels was observed with a NHS:NH ₂ ratio of 2:1 or 1:1. All compositions (corresponding hydrogels 5, 6 and 7) formed clear solid hydrogels within 5 minutes as determined by the inverted tube gelation test (see discussion in example 6). The hydrogel gelation time was found to be positively correlated with an increase in hydrophobic chain length. As shown in FIG. 30, hydrogels prepared with crosslinkers 5, 6 and 7 gelled in less than 5 seconds, 90 seconds and 3-5 minutes, respectively. Fig. 30 reports the following: panel A) gelation time of hydrogels at 10 wt%, 15 wt% and 20 wt%; panel B) storage modulus of hydrogel 7 at 10 wt%, 15 wt% and 20 wt%; panel C) storage modulus of hydrogels 5, 6 and 7 at 15 wt%; panel D) swelling of 15 wt% hydrogels over time. It was also found that the gelation time was positively correlated with the weight percent, which means that the higher the weight percent, the longer the gelation time.

Next, the morphology of the hydrogels was characterized using Scanning Electron Microscopy (SEM). All hydrogels had a cellular structure with pore sizes from 5 μm to 100 μm. Unlike other hydrogels, hydrogel 7 exhibits a more lamellar structure. Fig. 31 shows SEM images of hydrogels 5 (upper), 6 (middle) and 7 (lower). Due to this secondary structure observed, the Critical Aggregation Concentration (CAC) of crosslinker 7 was evaluated using the pyrene assay. CAC was observed at 0.050mM, at a concentration lower than the hydrogel crosslinker concentration (0.053 mM), indicating that a self-assembled structure was formed within the hydrogel itself, yielding a layered structure seen under SEM. From a chemical reactivity point of view, the terminal amine of PEI may react with terminal NHS ester or internal thioester to form an amide bond. The preferential attack site of the amine was determined via ¹ H NMR. Specifically, n-butylamine was used as a model for the terminal primary amine on PEI and added to the aqueous solution containing crosslinker 6. Amidation was performed by ¹ H NMR. A selective reaction between PEI and NHS ester was observed on the crosslinker, instead of internal thiol ester (> 99% at the NHS site over 20 minutes). Amidation on NHS ester was confirmed by a forefield shift on crosslinker 6 from bound NHS ester at 2.82ppm to free NHS at 2.49ppm, whereas the methylene peak of thioester at 2.6ppm was not shifted. FIG. 32 shows representative ¹ H NMR spectra of crosslinker 6 before (bold line) and after (narrow line) reaction with PEI mimetic (N-butylamine). Upon reaction with N-butylamine, a shift in the NHS peak bound to crosslinker 6 at 2.78ppm (bold line) to 2.49ppm (narrow line) was observed after cleavage of the NHS ester from crosslinker 6. FIG. 33 shows representative ¹ H NMR spectra of intact crosslinker 6 (bottom panel) (NHS at 2.78 ppm) and NHS hydrolyzed (2.54 ppm) crosslinker 6 in 0.3M sodium bicarbonate buffer (pH 8.0) (top panel).

Attack of the terminal amine on the NHS-ester occurs rapidly, less than 10 seconds, however in hydrogels this reaction may be slower because entanglement and curing is believed to occur once one of the amines attacks the NHS-ester, resulting in increased steric hindrance. Thus the gelation time is longer. In addition, competing hydrolysis reactions occur on NHS esters. FIG. 34 shows the reaction progression of the hydrolysis of thioester in crosslinker 5 of FIG. A) in 0.3M borate buffer at pH 8.0; panel B) reaction progression of thioester hydrolysis in crosslinker 6 in 0.3M borate buffer at pH 8.0; and panel C) NHS ester stability in 0.1M phosphate buffer pH 6.5. However, the hydrolysis of NHS ester was negligible at pH 6.5 for the entire 20 minutes, which was longer than sufficient to prepare the hydrogel. (see panel C)). This selectivity of NHS ester amidation ensures retention of internal thioester linkages, allowing dissolution by Cysteine Methyl Ester (CME).

Regarding mechanical properties, strain and frequency scans were performed at different time points before and after swelling with 50mM PBS. First, a linear viscoelastic region is determined using strain sweep (fig. 35 (left)). All hydrogels were scanned in frequency at 3% strain in the range of 1 to 10Hz (fig. 35 (right)). These hydrogels exhibit viscoelastic, solids-like behavior, storage modulus (G') > loss modulus (G ").

FIG. 36 shows the storage modulus of hydrogels 5, 6 and 7 prepared with crosslinking agents 5, 6 and 7 at 10 wt% (left) and 20 wt% (right), respectively; FIG. 37 reports the storage modulus of hydrogels prepared with crosslinkers 5 (left), 6 (middle) and 7 (right) at 10 wt%, 15 wt% and 20 wt% swelled for 30 days or until dissolved. After 30 days of swelling, the lowest storage modulus of hydrogel 5 was observed, remaining below 10kPa G' for the duration after swelling. Hydrogels prepared with crosslinkers 6 and 7 have a relatively high storage modulus at 15 wt% with peak storage moduli of about 12kPa and 20kPa, respectively. The increase in storage modulus in each hydrogel is due to the hydrophobicity of the methylene groups, so the longer the methylene chain length, the greater the hydrophobic interactions, the stronger the hydrogel. This observation applies to the weight percent dependence; the higher the weight percent, the greater the storage modulus.

FIG. 38 reports swelling of hydrogels at 20 wt%. FIG. 39 reports the dissolution of hydrogels 5, 6 and 7 prepared with crosslinkers 5, 6 and 7, respectively, when immersed in 10 wt% (left) and 20 wt% (right) 0.3M CME solutions at pH 8.6. FIG. 40 reports the results of rheological measurements of hydrogels prepared with crosslinker 6 at a 2:1 (black) or 1:1 (gray) NHS:NH ₂ molar ratio. Fig. 41 reports the results of rheological measurements of EtOH-containing and EtOH-free hydrogels made from crosslinker 6.

In order to ensure that the presence of ethanol does not increase the storage modulus of the hydrogels prepared with crosslinker 7, the hydrogels prepared with crosslinker 6 were rheologically measured under the same conditions as those used for crosslinker 7. No significant difference in storage modulus was observed between hydrogels prepared with or without ethanol, indicating that the buffer conditions did not change the mechanical properties of the hydrogels (fig. 41).

During 30 days of swelling, the hydrogels swelled between 150-350%, depending on the weight percent and hydrophobicity of the hydrogel formulation (fig. 38). All swelling of the hydrogels reached equilibrium after 48 hours. The hydrogel prepared with crosslinker 7 swells least, probably due to hydrophobicity within the long methylene chain length, while the hydrogel prepared with crosslinker 5 swells most.

All hydrogels underwent hydrolysis during 30 days of swelling, as indicated by loss of overall structure and decrease in storage modulus over time. Hydrogel 5 exhibited immediate loss of storage modulus and overall structure, while hydrogels 6 and 7 increased in strength as they swelled. However, a decrease in storage modulus of 30 days after swelling was observed in hydrogels 6 and 7. This loss of structure and mechanical properties is due to hydrolysis of the crosslinker. To further characterize the hydrolysis, the rate of crosslinker hydrolysis was measured via ¹ H NMR in 0.1M sodium bicarbonate buffer (ph 8.0). Hydrolysis was observed to occur preferentially at the thioester linkages, at rates k=0.055 min ^-1 and k=0.003 min ^-1 for crosslinkers 5 and 6, respectively (fig. 34), as opposed to the ester linkage between glutaric acid and PEG on the crosslinker. Hydrogel 7 was stable for more than 7 days. The stability of the thioester linkages in crosslinker 7 is due to the hydrophobic methylene chain length that protects the adjacent thioesters from hydrolysis (see fig. 9). In addition to hydrolysis, thioesters promote hydrogel dissolution by thiol-transesterification in the presence of Cysteine Methyl Ester (CME). Upon exposure of the hydrogel to a 0.3M CME solution at pH 8.6, it is believed that the thiol on the methyl cysteine ester attacks and displaces the internal thioester in the crosslinker. The amine on the internal cysteine methyl ester is thought to subsequently rearrange to form an amide bond by substitution of the thioester (see figure 29). Such amide linkages are believed to prevent re-attack of the original internal thiols. This dissolution process breaks down the hydrogel network, degrading the hydrogel over time. The storage modulus of the hydrogels in CME solution was evaluated as a function of time at pH 8.6. Based on the hydrogel formulation and weight percentages, it was found that complete dissolution (as defined by G' <300 Pa) occurred in less than 10 minutes to more than 90 minutes, with higher weight percentages and longer methylene chain lengths resulting in increased time to complete dissolution. Fig. 42 shows: dissolution of 15 wt% of the hydrogel in 0.3M CME solution in panel a); adhesion of hydrogels to human breast tissue using lap shear test in panel B); and 15% by weight of hydrogel 6 in panel C) on burned and non-burned human abdominal tissue. See also fig. 39. Specifically, at 15 wt%, hydrogel 5 dissolved within 10 minutes, whereas hydrogel 6 dissolved within 30 minutes, and hydrogel 7 dissolved within 80 minutes. This trend persists in all hydrogels regardless of weight percent. This slower dissolution of hydrogel 7 is due to the reduced local hydrophilicity due to the additional hydrophobic methylene groups near the thioesters compared to hydrogels 5 and 6. Since hydrolysis and thiol-transesterification reacted competitively on the thioester, the dissolution rate in sodium bicarbonate buffer at pH8.0 using CME was studied via ¹ H NMR using crosslinker 6. The reduction of methylene protons near the thioester was monitored and the thiol-transesterification rate was determined to be k=0.084 min ^-1. This rate is faster than the hydrolysis rate and therefore is interpreted as a thiol-thioester exchange being the preferred dissolution mode under 0.3M CME solution conditions.

The adhesion properties of hydrogels to human skin were studied. Lap shear tests were performed to determine the adhesive strength on isolated human breast and abdominal tissues. All hydrogels adhered to tissue with an adhesion value of about 0.5N/cm ² and showed cohesive failure at the hydrogel-skin interface (fig. 42). In addition, hydrogels similarly adhere to burned skin and healthy skin. The adhesive strength is due to the physical entanglement between the hydrogel and the human skin.

In vivo studies, NIH3T3 fibroblasts were used to assess cytotoxicity. FIG. 43 reports the cell viability of hydrogels prepared with crosslinkers 5, 6 and 7 and PEI on NIH3T3 fibroblasts. Hydrogels 6 and 7 exhibited >85% viability, while hydrogel 5 exhibited very low viability, which is believed to be due to the rapid release of glutaric acid and the increase in local acidity caused by dissolution.

Based on the sum of these results, 15 wt% of hydrogel 6 was selected for in vivo testing. Hydrogel 6 exhibited no toxicity, a storage modulus on the same order of magnitude as human skin, remained mechanically strong and structurally strong throughout 7 days, adhered to the skin, swelled, and dissolved within 30 minutes. For in vivo models, second degree burns were induced on 4 pigs by heating the brass cylinders to 80 ℃ and placing them on the backs of the pigs for 20 seconds. Treatment groups were evaluated on days 7 and 14, with one or two dressing changes as shown in fig. 44, to observe any differences in healing between groups. Hydrogel 6 was compared to gauze sponge dressings Mepilex ^TM and xeroform. Triple antibiotic ointments were applied to each burn site prior to dressing. Following necropsy, the tissues were dissected and stained with sappan Sha Linhe Eosin (H & E, hematoxalin & Eosin). Fig. 44 shows: schematic of the experiment in figure a); representative photographs and histology of burn sites in panel B); and histological scoring of necrosis and neovascularization in panel C).

Figures 45-49 and tables 5-9 report the data obtained from the samples. Fig. 45 shows H & E of group 1 (see table 5) of gauze (left), no dressing (middle) and hydrogel dressing (right); fig. 46 shows H & E of group 1 (see table 6) of gauze (left), no dressing (middle) and hydrogel dressing (right); fig. 47 shows H & E of group 2 of gauze (left), no dressing (middle) and hydrogel dressing (right) (see table 7); fig. 48 shows H & E of group 4 of gauze (left), no dressing (middle) and hydrogel dressing (right) (see table 8); fig. 49 shows H & E (see table 9) for gauze (left), no dressing (middle) and hydrogel dressing (right) of group 5. Tables 5-9 show the mean ± standard deviation, median and incidence of inflammatory cell types and inflammation.

Table 5: day 3, group 1, no dressing change.

Table 6: day 7, group 3, without dressing change

Table 7: day 7, group 2, 1 dressing change

Table 8: day 14, group 4, 1 dressing change

Table 9: day 14, group 4, 2 dressing changes

In general, all treatment groups showed mild/moderate necrosis, epidermal ulcers, inflammation and neovascularization. By day 14, however, hydrogel 6 exhibited less necrosis, epidermal ulcers and inflammation than the other treatment groups, with neovascularization, burn depth (mm) and epidermal dermis thickness (mm) similar to all treatment groups (fig. 44, panel C)). Furthermore, by day 14, all hydrogels showed some re-epithelialization (re-epithelialization), hydrogel 6 showed complete re-epithelialization on the two burns, and partial re-epithelialization on the one burn after dressing change twice (n=3); only one dressing has more than one complete re-epithelialization of the burn. The only treatment group for complete re-epithelialization on burns included hydrogel 6 on day 14 of one dressing change and sterile gauze dressing on day 14 of two dressing changes. Although the difference between the two groups was not statistically significant (P > 0.05), hydrogel 6 tended to have better performance than conventional gauze Mepilex ^TM and xeroform dressings. Our spray application and removal process of hydrogels eases application in dressing and debridement procedures, eliminating the need for mechanical debridement and destruction of newly formed tissue. Fig. 50 shows a schematic dissolution of hydrogel 6 for use as a burn wound dressing. Gauze was soaked in 0.3M CME solution and placed on hydrogel 6 burn wound dressing for 10 minutes to induce dissolution of the dressing. Subsequently, the burn wound was rubbed with gauze immersed in H ₂ O, and a new hydrogel dressing was prepared on top of the wound.

Example 9

Additional hydrogels according to the present disclosure were prepared. The macromer is a PEG-based macromer or a poly (1, 2-glycerol carbonate) (PGC) -based macromer, characterized by an olefinic functional moiety. The crosslinker is a PEG-based crosslinker featuring thiol moieties (see example 9). These components were dissolved in phosphate buffer solutions having a pH in the range of 7-8, with a total polymer concentration of 10 to 25% by weight. As the weight percent of the gel solution increases, the gelation time shortens and the gel elastic modulus increases. In gel formulations, the molar ratio between the olefin and thiol moieties may be between 1:1 and 2:1. The 1:1 ratio resulted in a small increase in gel elastic modulus compared to the 2:1 ratio. The olefin functionality comprises a number of different structures such as, but not limited to, alkyl ethers as shown in fig. 2C and 2D or norbornene as shown in fig. 2E and 2F. The selection of the moiety affects the gelation time and norbornene has faster reaction kinetics than alkyl ethers. The macromer may have 4 to 100 olefin moieties per molecule. Macromers with higher number of olefinic moieties result in stiffer gels and lower swelling ratios. The crosslinker may contain 2-4 thiols per molecule. The gel precursor solution does not solidify before irradiation with 365nm uv light or white light, depending on the photoinitiator used. The photoinitiator is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate (LAP) or Eosin Y. The photoinitiator concentration ranges from 0.1mM to 100mM, depending on the photoinitiator and the gelation kinetics, higher concentrations of photoinitiator result in faster kinetics. Very high concentrations are believed to be likely to disrupt the macrostructure of the gel and risk cytotoxicity. For visible light systems, up to 10mM ethyl tyrosine was included to increase gelation kinetics. These gels were formed over a broad range of ultraviolet (365 nm) intensities of 4 to 120mW/cm ² and white light of 10mW/cm ² to full spectrum white light of 42.9W/cm ² (at maximum absorption of photoinitiator).

For ultraviolet activated hydrogels, the concentration and ratio of macromer and crosslinker can be varied to adjust the storage modulus between 500Pa and 2000 Pa. These formulations are single solutions and exhibit viscosities suitable for application through single lumen catheters down the length of an endoscope. These formulations utilize stimulus-responsive gelation to rapidly and dynamically respond to long-wave ultraviolet light to form gels within 5 seconds of illumination. The gel adheres to porcine colon tissue and exhibits a strong burst pressure when used to seal small defects. In vivo studies were performed to apply the components using an endoscopic catheter; the resulting gel was still present after 2.5 hours of administration. The resulting gel has low cytotoxicity, exhibiting greater than 97% viability in NIH 3T3 fibroblasts throughout 24 hours. Photoinitiators were used in NIH 3T3 fibroblasts at concentrations below IC ₅₀ and still exhibit rapid (< 10 seconds) gelation kinetics when combined with up to 10mM of ethyl tyrosine, in response to a broad range of white light sources such as bicycle lights, lights (lamps) or endoscopes.

Example 9

From PEG (M _w 3000,3000), a cross-linker with thiol moieties was synthesized, as shown in fig. 51. These crosslinkers were prepared for investigation of hydrogel formation in situ via a Michael addition reaction between branched PEI-thiol and a difunctional maleimide activated PEG crosslinker, discussed further in examples 10 and 11. In this crosslinker there is an internal thioester linkage that is readily soluble via thiol-thioester exchange with a Cysteine Methyl Ester (CME) solution. It is believed that after thiol-transesterification, primary amines on the CME rearrange to form irreversible amide bonds, preventing the re-formation of hydrogels after decomposition of the polymer network (see fig. 8). PEG crosslinkers having methylene chain lengths of 2, 3 or 4 were prepared, where the methylene chain lengths were varied to determine the dependence on hydrogel mechanical properties, swelling, dissolution time and burst pressure. The hydrogels contain internal thioesters for dissolution via thiol-transesterification, and maleimide end groups for binding to hyperbranched poly (ethyleneimine) -thiols ("PEI-SH").

As summarized in fig. 51, PEG-diol was reacted with the corresponding anhydride (succinic anhydride, glutaric anhydride, or adipic acid) to give the corresponding PEG diacid. Next, PEG-diacid was functionalized with N-hydroxysuccinimide (NHS) end groups via DCC coupling to give crosslinkers 1,2, and 3. In a flame-dried round-bottomed flask with a magnetic stirrer bar, crosslinker 1,2 or 3 (1 g) was dissolved in Dimethylformamide (DMF). Thioglycollic acid (68.8 μl) and Diisopropylethylamine (DIPEA) (279 μl) were added in this order. Thioglycollic acid is selected because of its hydrophilicity adjacent to thioesters, allowing for rapid dissolution times. The reaction was stirred at room temperature overnight. The organic phase was extracted with 1N HCl solution, water, 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).

After the preceding steps, intermediates 1, 2 and 3 were functionalized with maleimide reactive end groups via a peptide coupling method using maleimide trifluoroacetic acid, pyBOP, DIPEA in dry DCM to obtain the final crosslinkers, i.e. crosslinkers 4, 5 and 6 with methylene chain lengths of 2, 3 and 4, respectively. In a flame-dried round-bottomed flask with a magnetic stirring bar, intermediate 1, 2 or 3 was dissolved in dry dichloromethane. Maleimide-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 with 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 the above reactions were all greater than 80%.

Characterization data by ¹H NMR、¹³ C NMR, GPC and DSC are as follows:

the polymer was prepared according to the previously published protocol (see also example 7).

Crosslinking agents 1,2, 3. The synthesis of crosslinking agents 1,2 and 3 is based on previously reported protocols (see also example 7).

Intermediates 1,2, 3. Were synthesized as described above. Characterization is performed by ¹H NMR(500MHz),CDCl₃: intermediate 1- δ4.22 (tt, j=4.7hz, 4H), 3.62 (m, 310H), 2.93 (t, j=6.8hz, 4H), 2.68 (t, j=6.8hz, 4H) ppm; intermediate 2-δ4.22(tt,J＝4.8Hz,4H),3.63(m,308H),2.86(t,J＝7.2Hz,4H),2.61(t,J＝7.3Hz,4H),2.38(t,J＝7.4Hz,4H),2.30(t,J＝7.4Hz,4H),1.97(t,J＝7.3Hz,4H),1.60(m,8H),1.39(m,4H)ppm; intermediate 3–δ4.21(tt,J＝4.4,4.9Hz,4H),3.63(m,277H),2.62(t,J＝6.7,7.2Hz,4H),2.34(t,J＝6.7,7.2Hz,4H),1.69(m,8H)ppm. was characterized by ¹³C NMR(500MHz),CDCl₃: intermediate 1-195.9,171.5,70.5,64.1,30.9,29.1ppm; intermediate 2-198.6,172.7,70.7,69.0,33.5,32.9,20.6ppm; intermediate 3-197.0,173.0,70.5,63.5,33.7,31.0,24.7,24.0ppm.

Crosslinking agents 4, 5, 6. The synthesis is performed as described above.

Crosslinking agent 4.¹H NMR：δ6.71(s,2H),6.55(b,1H),4.23(tt,J＝4.2,4.9Hz,4H),3.62(m,322H),2.96(t,J＝6.8Hz,4H),2.74(t,J＝6.8Hz,4H)ppm;¹³C NMR：197.5,171.9,134.2,70.5,64.0,32.3,29.1ppm;M_w(GPC,THF)：2868Da;M_n(GPC,THF)：2801Da;PDI(GPC,THF)：1.02; melting point (DSC): 41.78 deg.C; crystallization point (DSC): 39.9 ℃.

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;¹³C NMR：198.2,172.6,134.2,70.4,63.6,32.8,32.3,20.2ppm;M_w(GPC,THF)：3028Da;M_n(GPC,THF)：2955Da;PDI(GPC,THF)：1.02; melting point (DSC): 40.22 ℃; crystallization point (DSC): 21.3 ℃.

Crosslinking agent 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;¹³C 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 ℃; crystallization point (DSC): 33.5 ℃.

Example 10

As outlined in fig. 51, thiol-terminated polyethylenimine (PEI-SH) hyperbranched macromer (fig. 2G) was synthesized to react with the maleimide-terminated PEG crosslinker of example 9. Synthesis of PEI-SH involves reacting pentafluorophenyl functionalized 3- (trityl mercapto) propionic acid with PEI overnight to obtain trityl protected PEI-thiol hyperbranched polymer (hereinafter "PEI-STr") (yield = 68%). The trityl group was then deprotected with TFA and Et ₃ Si to give the final PEI-SH hyperbranched polymer (yield = 96%). Characterization data including ¹H NMR、¹³ C NMR, GPC, and DSC are as follows.

Initially in the PEI-SH synthesis, 15 equivalents of thiol per PEI molecule was reacted to fully thiolate the PEI. However, due to the high concentration of thiols in each polymer, intramolecular and intermolecular disulfide bonds are formed, as visually observed via a pink solution of PEI-SH in borate buffer at pH 8.6. This minimizes the amount of free thiol available for Michael addition reaction with the maleimide-functionalized crosslinker of example 9. Thus, the equivalent weight of thiol reacted with PEI is reduced to minimize the number of intermolecular and intramolecular disulfide bonds. As shown in FIG. 53, the amount of free amine was determined by the colorimetric TNBS assay. The test was performed by reacting a 0.01% (w/v) solution of 2,4, 6-trinitrobenzenesulfonic acid (TNBS) with PEI-SH in 0.1M sodium bicarbonate buffer at pH 8.5. After incubation of the solution for 2 hours at 37 ℃, the resulting yellow solution was diluted with 10% sds and 1N HCl to stop the reaction. Absorbance was read at 335nm, which correlates to the amount of primary amine present on the solution. Standard curves were prepared based on different concentrations of PEI and fully thiolated PEI-SH, where the slope of the RFU versus concentration (ug/mL) plot correlates with the amount of free amine on a particular molecule. PEI (molecular weight 1800) contains on average 15 free amines with a TNBS test slope of 0.007. As expected, fully thiolated PEI-SH exhibited a slope of 0.000, indicating the absence of primary amines on the molecule. The slope of a line representing PEI-SH prepared with 4 equivalents of trityl mercaptopropionic acid was evaluated. The slope of this line was 0.002, one third of the slope of unfunctionalized PEI. These data confirm about 2/3 of the thiolation of PEI polymer, which means that there are still 5-6 primary amines. This partial functionalization of PEI is expected to minimize intramolecular and intermolecular disulfide bonds and promote hydrogel formation with maleimide-functionalized crosslinkers.

PEI-STr. PEI (3 g) was dissolved in DMF. 3- (tritylthio) propionic acid-pentafluorophenol (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 dichloromethane and the organic phase was extracted/extracted from sodium bicarbonate, water and brine. The organic solution was dried over sodium sulfate, filtered through filter paper and concentrated. The organic solution was precipitated in diethyl ether and dried under vacuum to give a pale yellow solid (68% yield) ).¹H NMR：δ8.00(s,1H),7.49-7.10(m,48H),3.65-2.01(m,60H)ppm;¹³C NMR：162.5,144.6,129.5,127.9,126.7,36.5,35.1,27.7ppm.

PEI-SH. PEI-STr (2 g) was dissolved in a minimum amount of methylene chloride in a round bottom flask with a magnetic stirrer bar. 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. Dichloromethane and TFA were removed under vacuum and redissolved in a minimum amount of dichloromethane. 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 ).¹H NMR：7.9(s,1H),3.61-2.49(m,217.13H)ppm;¹³C NMR：163.1,162.8,117.6,115.3,39.5,22.7ppm;M_w(GPC, aqueous): 5660Da; m _n (GPC, aqueous): 6994Da; PDI (GPC, aqueous): 1.12; mp (DSC): 15.6 ℃.

Example 11

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

Gelation kinetics were assessed by tracking the disappearance of maleimide olefin peaks in ¹ H NMR after mixing the maleimide crosslinker with mercaptopropionic acid (a PEI-SH mimetic) at 6.70 ppm. After injection of 2 equivalents of mercaptopropionic acid (used as in situ PEI-SH mimetic), NMR spectra were recorded every 0.4 seconds for about 20 seconds. No olefin peak was observed at 6.70ppm immediately after injection of the PEI-SH mimetic, showing faster gelation kinetics than 0.4 s.

After gelation, the storage modulus of the hydrogel was determined as an assessment of mechanical strength by strain and frequency sweep to determine the linear viscoelastic region (LVER). LVER is present at 10 strain and is the maximum strain that can be applied to these hydrogels before plastic deformation occurs. Frequency sweeps were performed in LVER at 3% strain of 0.1-10 Hz. Our hydrogels have an initial storage modulus between 2000 and 5000 Pa. Crosslinking agents 4, 5 and 6 exhibited reduced storage modulus when the hydrogels were swollen in 50mM PBS. Fig. 54 shows: rheological measurements of the hydrogel in panel a; panel B) swelling in 50mM PBS; and, panel C) dissolution of the hydrogel in 0.3M CME solution. The decrease in G' over time is due to degradation of the crosslinker via hydrolysis.

To estimate the degradation rate and determine the position of hydrolysis was internal to the thioester rather than on the ester, ¹ H NMR crosslinker spectra were monitored in 0.3M sodium bicarbonate buffer at ph8.0 for the entire 20 minutes. In the base-catalyzed hydrolysis, the methylene group near the internal thiol shifts from 3.41ppm to 3.17ppm, while the methylene peak near the ester linkage is at 4.15ppm, corresponding to no shift in the other terminal methylene group on the diacid linkage in the crosslinker (succinic, glutaric, adipic). This ¹ H NMR shift confirms the selective hydrolysis of thioesters. FIG. 55 shows ¹ H NMR spectra demonstrating hydrolysis of thioesters, as observed by the movement of methylene groups near the thiol from 3.41ppm (bound) to 3.17ppm (hydrolyzed).

For hydrogels prepared with crosslinkers 4, 5 and 6, the different degradation rates (> 4 hours, >24 hours and >7 days) are increased relative to the hydrophobic methylene chain length of the internal diacid linkages that protect the internal thioesters of the crosslinkers from hydrolysis. Crosslinking agent 4 contains an internal succinic linkage with two methylene groups, while crosslinking agents 5 and 6 contain three and four methylene glutaric and adipic linkages, respectively. The longer and more hydrophobic the methylene chain in the crosslinker, the more stable the thioester is believed to be to hydrolytic cleavage, resulting in a slower degradation rate. The varying degradation rate allows the mechanical properties of the hydrogels to be tuned by the crosslinker structure relative to the methylene chain lengths within crosslinkers 4, 5, and 6 to maintain mechanical integrity.

Swelling was observed to be between 200-400% (fig. 54, panel B)). After 24 hours immersion in 50mM PBS, swelling reached a maximum. Swelling in aqueous solutions is believed to be advantageous for hydrogels because it is capable of expanding in size upon absorption of aqueous fluids from the surrounding environment.

The on-demand dissolution time of hydrogels via thiol-transesterification was assessed when immersed in 0.3M Cysteine Methyl Ester (CME) solution (fig. 8). Frequency sweeps were performed every 10 minutes to allow adequate CME exposure until the hydrogel network was completely decomposed or degraded (G' <300 Pa). Upon thiol-transesterification, the hydrogel network disintegrates and the amines on the CME rearrange to form irreversible amide bonds, preventing the re-formation of the hydrogel. Dissolution of all three hydrogel formulations occurred in 10 minutes or less (fig. 54, panel C)). The acidic linkages (succinic, glutaric and adipic) retard hydrolysis under neutral conditions, whereas the alkaline conditions of the CME solution catalyze the thiol-transesterification reaction. Rapid dissolution of hydrogel networks via thiol-transesterification may be useful in medical environments, for example, to help minimize anesthesia time for patients.

Prior to ex vivo studies, hydrogels were evaluated for cytotoxicity on NIH3T3 fibroblasts over 24 hours of exposure. Hydrogels 4 and 5 exhibited 60% of the average cell viability, while hydrogel 6 exhibited 98% of the average cell viability. The low cell viability in hydrogels 4 and 5 is due to the rapid release of succinic and glutaric acids due to the breakdown of the hydrogel network and the increase in local acidity in the restricted environment across the well plate.

The hydrogel burst pressure was determined by injecting macromer into one end of an isolated 2cm pig carotid artery in a total volume of 1mL to form a hydrogel. The hydrogel fills the blood vessel and remains in place. After storing the blocked artery in a humid environment for 30 minutes (e.g., reflecting time during an exemplary surgical procedure), the vessel was connected to a custom internal (in-house) burst pressure system with a pressure sensor and syringe pump connected to a computer (fig. 56). Deionized H ₂ O was pumped into the vessel at 1mL/min until leakage was observed and the pressure was recorded until failure. The burst pressures of hydrogels 4, 5,6 prepared with the crosslinking agents 4, 5,6 were 382mmHg, 440mmHg, and 231mmHg, respectively, up to 4 times higher than the arterial pressure (120/60) (FIG. 57). Burst pressures of 200-600mmHg are considered sufficient for hydrogel occluding devices.

It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosure without departing from the scope of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the practice and specification 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 of forming a gel, the method comprising:

The composition was prepared 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, said macromer comprising at least one first functional moiety;

A cross-linking agent comprising a second PEG-based polymer comprising at least one second functional moiety; and

A photoinitiator; and

Activating the photoinitiator via a light source to form a gel, wherein the gel is biocompatible.

2. The method of 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, and wherein the at least one second functional moiety comprises 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.

3. The method of claim 1 or 2, wherein one of the at least one first functional moiety or the at least one second functional moiety comprises a vinyl, allyl, acrylate, or 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 of any of the preceding claims, wherein the macromer, the crosslinker, and the photoinitiator together comprise from about 10 to 25 weight percent of the composition, relative to the total weight of the composition.

5. The method of any one of the preceding claims, wherein the molar ratio between the at least one first functional moiety and the at least one second functional moiety ranges from 1:1 to 2:1.

6. The method of any of the preceding claims, wherein the macromers together comprise from about 5 to 15 weight percent of the composition, relative to the total weight of the composition, and/or wherein the cross-linking agents together comprise from about 5 to 10 weight percent of the composition, relative to the total weight of the composition.

7. The method of any of the preceding claims, wherein the crosslinker comprises an N-hydroxysuccinimide group and/or a maleimide group.

8. The method of any one of the preceding claims, wherein the concentration of the photoinitiator within the composition ranges from about 0.1mM to about 100mM.

9. The method of any one of the preceding claims, wherein the composition further comprises a physiological buffer.

10. The method of any one of the preceding claims, wherein the light source emits ultraviolet or visible light.

11. The method of claim 10, wherein the gel forms within 5 seconds when irradiated with ultraviolet light, or wherein the gel forms within 10 seconds when the photoinitiator is activated with visible light.

12. The method of any one of the preceding claims, wherein the macromer is a hyperbranched polymer.

13. The method of any one of the preceding claims, wherein the composition further comprises an additive that accelerates the gelation time of the composition, the additive comprising a tyrosine derivative, optionally wherein the tyrosine derivative comprises methyl tyrosine or ethyl tyrosine.

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

15. Use of a gel formed according to the method of any one of the preceding claims for treating human gastrointestinal tissue.