Patent Application
20230039987
This disclosure relates generally to therapeutic gels useful for medical procedures, including endoscopic procedures. For example, the disclosure includes gels, and compositions and systems formulated to form a gel, e.g., for application to bodily tissue, such as, e.g., the gastrointestinal tract.
Endoscopic procedures, such as endomucosal resection (EMR), endosubmucosal dissection (ESD), and anastomosis, and health conditions such as intentional or disease-originated creation of a fistula, inflammatory bowel disease (IBD), and IBD subsidiary diseases, may result in and/or contribute to damage to tissues of the gastrointestinal (GI) tract. Colorectal cancer is among the leading causes of cancer death in the developed countries. Standard preventative care for patients over 50 years old involves a colonoscopy to biopsy polyps, known as a polypectomy, to assess for colorectal cancer. Practically, a physician inserts an endoscope into the patient's colon while under anesthesia, examines the colon, and then removes the polyps. After removal, the wound is either left open to the internal colon environment or thermally sealed using electrocoagulation. Open wounds after a polypectomy or other endoscopic procedures in the GI tract can result in bleeding, hemorrhaging, and sepsis. Electrocoagulation can result in other complications such as perfusion or post polypectomy coagulation syndrome.
These types of medical procedures and health conditions may leave relatively thin tissue layers of the GI tract wall. Currently, physicians often rely on time or surgical procedures, including clipping or endoscopic suturing to allow healing of the GI tract wall. However, these practices may be unsuitable in certain cases, such as large defects, and/or friable or fibrotic tissue. Complications that may arise include perforation, infection, and sepsis.
Methods of forming gels useful in medical procedures are disclosed. The present disclosure includes, for example, a method for forming a gel comprising preparing a composition by combining a macromer comprising a first polyethylene glycol (PEG)-based polymer, a poly(ethylenimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer including at least one first functional moiety; a crosslinking agent comprising a second PEG-based polymer that includes at least one second functional moiety; and a photoinitiator; and activating the photoinitiator via a light source to form the gel. 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 group, an allyl group, an acrylate group, or a norbornene group, and the other of the at least one first functional moiety or the at least one second functional moiety may comprise a thiol group. According to some examples herein, the macromer, the crosslinking agent, and the photoinitiator may represent a total of 10-25 wt % of the composition, in relation to a 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 may range from 1:1 to 2:1. Additionally or alternatively, the macromer may represent a total of 5-15 wt % of the composition, in relation to a total weight of the composition. The crosslinking agent may represent a total of 5-10 wt % of the composition, in relation to a total weight of the composition. The concentration of the photoinitiator within the composition may range from about 0.1 mM to about 100 mM. In some examples, the crosslinking agent comprises N-hydroxysuccinimide groups and/or maleimide groups. 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 UV light or visible light. For example, the gel may be formed within five seconds when illuminated with UV light. In some examples, the gel may be formed within ten seconds when the photoinitiator is activated with visible light. Optionally, the composition may further comprise an additive to expedite a gelation time of the composition, the additive comprising a tyrosine derivative. In some aspects, the composition may comprise up to 10 mM of the additive. The tyrosine derivative may comprise, for example, tyrosine methyl ester or tyrosine ethyl ester.
The gels described above and elsewhere herein may be used to treat tissue of a subject, e.g., a human subject. For example, the gels may be used to treat tissue of a gastrointestinal tract of a subject.
The present disclosure also includes a method forming a gel comprising preparing a first solution by combining a macromer comprising a polyethylene glycol (PEG)-based polymer, a poly(ethylenimine)-based polymer, or a poly(1,2-glycerol) carbonate-based polymer, the macromer comprising at least one first functional group, and a first buffer; preparing a second solution by combining: a crosslinking agent comprising a second (PEG)-based polymer that comprises 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 the gel. The gel may be biocompatible and/or biodegradable. The at least one first functional group may comprise a thiol group or an amine group, for example, and/or the plurality of second functional groups may comprise N-hydroxysuccinimide groups or maleimide groups. In some examples, the molecular weight of the macromer may be approximately 2,000 Da. Additionally or alternatively, the molecular weight of the crosslinking agent may be approximately 3,400 Da. In some examples, the molar ratio of the crosslinking agent to the macromer may range from 3:2 to 7:3.
As mentioned above, the gels disclosed herein may be used to treat tissue of a subject. For example, the method of forming a gel may include treating a subject by forming a gel on tissue of a gastrointestinal tract of the subject. In at least one example, the method comprises applying to the tissue a first solution comprising a macromer comprising a polyethylene glycol (PEG)-based polymer, a poly(ethylenimine)-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 crosslinking agent comprising a second (PEG)-based polymer that comprises 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 contacts the second solution to form the gel on the tissue. The first solution may be applied to the tissue before, after, or at the same time as the second solution.
The present disclosure also includes a composition comprising a macromer comprising a polyethylene glycol (PEG)-based polymer, a poly(ethylenimine)-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 crosslinking agent comprising a PEG-based polymer that includes a N-hydroxysuccinimide functional group, a maleimide functional group, or both; wherein the composition is formulated as a hydrogel. The hydrogel may have a gel strength of at least 2,000 Pa and/or a shear force between 0.03-0.90 N/cm2 when adhered to a bodily lumen. Additionally or alternatively, the hydrogel may be formulated to withstand a burst pressure of up to approximately 150 mbar when the hydrogel is adhered to colon tissue to fill an aperture in the tissue of about 1 mm by about 5 mm.
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.
Both 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,” “having,” “including,” 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 a process, method, article, or apparatus. In this disclosure, relative terms, such as, for example, “about,” “substantially,” “generally,” and “approximately” are used to indicate a possible variation of ±10% in a stated value or characteristic. All ranges are understood to include endpoints, e.g., a macromer content between 5 wt % and 10 wt % includes 5 wt %, 10 wt %, and all values between.
Embodiments of this disclosure may address one or more limitations in the art. The scope of the disclosure, however, is defined by the attached claims and not the ability to solve a specific problem. The disclosure includes compositions and systems formulated to form a gel, e.g., hydrogel, and compositions in gel/hydrogel form, e.g., useful for application to tissues of the gastrointestinal tract. The hydrogels herein may serve as a temporary, minimally invasive, in situ hydrogel dressing, applied immediately after the time of a medical procedure such as a polypectomy. The hydrogel may prevent or reduce the likelihood of complications by covering and protecting a wound. From a biomaterials design perspective, the dressing may achieve one or more of the following: 1) form rapidly in situ; 2) adhere to colon tissue; 3) be non-cytotoxic; 4) naturally dissolve over 3-5 days; 5) swell up to 200% to absorb wound exudate; 6) prevent the spread or migration of bacteria; and/or 7) conform to the malleable shape of a colon lumen. The gels herein may be formulated with desired characteristics such as gelation rate, adhesion strength, swelling, cytotoxicity, and/or degradation, as a function of hydrogel composition. The compositions herein may be delivered to a subject by a suitable medical device such as a catheter inserted through an endoscope. For example, a dual lumen catheter may be used. Barrier properties of the hydrogel may help to prevent bacterial migration.
The compositions, systems, and methods herein may offer a range of properties, including among others, inherent cohesion and adhesion to tissue. With such properties, the gels herein may function as a protective barrier to thin, damaged, and/or otherwise compromised tissue of a bodily lumen, e.g., the GI tract. For example, an exemplary composition, e.g., a formulation or system for forming a gel, may be applied to a targeted site along the GI tract and the composition may crosslink to form the gel, which may provide barrier protection/therapy to the targeted site. Components of the compositions and gel systems herein may provide desired properties advantageous for tissue protection, e.g., before, during, and/or after a medical procedure. The compositions and systems herein may be delivered to a targeted site by a suitable method or technique. The properties of the compositions such as, e.g., viscosity, may facilitate the deliverability of the gel-forming formulations to targeted sites via suitable medical devices such as, e.g., single/multi lumen catheters, including endoscopes, and syringes, among other devices useful for medical procedures. For example, the compositions herein in gel form may have a viscosity ranging from about 0.010 Pa*s, e.g. to about 0.015 Pa*s, e.g., approximately 0.013 Pa*s at room temperature (about 22-25° C.). Components of the composition or gel system may crosslink to form the gel, which may include activating one or more components with or in the presence of a stimulus, such as pH value or light. The hydrogels herein may be hydrophilic, three-dimensional polymeric networks formed from a macromer and a crosslinking agent (alternatively referred to herein as a crosslinker). The gels, e.g., hydrogels, herein may be formed by combining a macromer with a crosslinking agent under suitable pH conditions or light exposure to initiate crosslinking.
Exemplary macromers useful for the present disclosure include polyethylene glycol (PEG)-based polymers, poly(1,2-glycerol) carbonate (PGC)-based polymers, and poly(ethylene imine)-based polymers. The macromer may have a plurality of functional groups such as amine, alkene, and/or thiol functional groups, available to react with the crosslinking agent.
Exemplary crosslinking agents useful for the present disclosure include PEG-based polymers that comprise one or more N-hydroxysuccinimide or maleimide functional groups.
As mentioned above, the gels herein may form three-dimensional polymeric networks capable of forming a barrier over a wound or other site of interest in the body, e.g., in the colon or another portion of the GI tract.
The structure of the crosslinking agents may help to control the rate of dissolution.
The gels herein may be formed on target tissue of a subject, such as tissue of the GI tract (e.g., intestinal tissue, colon tissue, etc.). For example, a crosslinking agent and a macromer may be delivered separately to a target tissue site, such that the two components do not contact each other until they reach the target tissue site. In some examples, a dual lumen catheter may be used, e.g., the crosslinking agent and the macromer being delivered to the target tissue site in separate lumens. The two components may come into contact with each other at the target tissue site, wherein gelation occurs due to a suitable pH (e.g., the components being formulated to crosslink at physiological pH of the GI tract) or in the presence of a photoinitiator activated by UV or visible light at the target tissue site. For example, a photoinitiator may be applied to the target tissue site before, after, or at the same time as the crosslinking agent and/or macromer, and light applied thereafter to activate the photoinitiator and initiate crosslinking to form the gel. Gelation may be initiated in a time 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, e.g., a period of time greater than 1 second and less than 15 seconds. The crosslinking agent and macromer (and photoinitiator, when present) may be selected to provide relatively fast gelation kinetics to form a gel in tortuous environments such as the GI tracts and when subjected to the pull of gravity.
Once formed on tissue in situ, the gel may form a barrier with sufficient strength to remain intact for a 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 tissue that remains for a period of time ranging from about 1 hour to about 60 days, about 1 hour to about 30 days, about 1 hour to about 14 days, about 1 hour to about 24 hours, about 12 hours to about 48 hours, or about 2 days to about 21 days, about 5 days to about 14 days.
The crosslinking agent and the macromer may be selected to provide sufficient strength to suit the period of time desired for the gel to form a barrier on tissue. As discussed in the examples below, greater crosslinking density (at least partially dependent on the number and type of functional groups of the crosslinking agent and the macromer) and/or relative hydrophobicity is expected to provide for stronger gels with longer residence times when applied to tissue. The gels herein may be biocompatible and/or biodegradable. For example, the gels may dissolve over time (e.g., by hydrolysis and/or in the presence of an external thioester such as by thiol-thioester exchange as discussed in the examples below), depending on crosslinking density and strength of the gel.
Below is further discussion of exemplary compositions and systems useful for medical procedures including endoscopic procedures, e.g., the compositions or systems comprising a gel or being formulated to form a gel. The compositions may be applied to a subject for treatment purposes, and the composition may be activated (e.g., to form a gel on-site) via different mechanisms.
pH-Activated Gel Systems
In some aspects of the present disclosure, the composition or system may be formulated to crosslink and form an adhesive, cohesive gel under physiological pH, e.g., a pH of about 7 to about 7.5, such as about 7.35-7.45. Thus, such compositions and systems may be pH-activated such that the gel, e.g., hydrogel, selectively crosslinks at a neutral to basic pH (e.g., little to no crosslinking at acidic pH), with reaction kinetics increasing as the pH increases. According to some aspects of the present disclosure, the composition or system may be pH-activated in order to from a gel, e.g., a hydrogel. For example, the composition may comprise at least two components, e.g., a first component (e.g., a first part solution) and a second component (e.g., a second part solution), that crosslink at a physiological pH, e.g., within a range of about 7 to about 7.5. Thus, for example, the first and second part solutions may have different pH values and may form a gel when mixed together so as to provide for the physiological pH.
A first component of an exemplary system may include a macromer. The macromer may be a multi-functional polyethylene glycol (PEG)-based or poly(ethylene imine)-based polymer. For example, the PEG-based polymer or poly(ethylenimine)-based polymer may have a molecular weight of at least 1500 Da (g/mol), such as about 1800 Da to about 2200 Da, e.g., about 2000 Da. For example, the macromer may have a molecular weight ranging from about 1500 Da to about 2500 Da, from about 1500 Da to about 2000 Da, or from about 1800 Da to about 2200 Da. The poly(ethylenimine) may be linear or branched. In some examples, the macromer may be a multi-functional PEG-based or poly(ethylenimine)-based polymer having a plurality of functional groups. The plurality of functional groups may react with a crosslinking agent of the system (examples of crosslinking agents discussed in further detail below). Such functional groups may be, for example, amine or thiol functional groups. According to some aspects, the multi-functional PEG-based or poly(ethylenimine)-based polymer may comprise a plurality of 2-20 functional groups, e.g., 4, 6, 8, or 15 functional groups. Exemplary structures representing a branched poly(ethylenimine) having amine functional groups (
A second component of the system may include a crosslinking agent. Exemplary crosslinking agents include, but are not limited to, PEG-based polymers. For example, the PEG-based polymer used as the crosslinking agent may have a molecular weight greater than 3000 Da, such as about 3200 Da to about 3500 Da, e.g., approximately 3400 Da. According to some aspects of the present disclosure, the crosslinking agent have a molecular weight ranging from about 3000 Da to about 3800 Da, from about 3200 Da to about 3500 Da, or from about 3400 Da to about 3800 Da. In some examples, the crosslinking agent may be a PEG-based polymer that comprises one or more N-hydroxysuccinimide or maleimide functional groups. The N-hydroxysuccinimide or maleimide group may react with a macromer as discussed in further detail below. Exemplary structures representing a N-hydroxysuccinimide-PEG polymer and a maleimide-PEG polymer are shown in
In some aspects, the crosslinking agent may be provided in solution with a buffer, e.g., the crosslinking agent being dissolved in a buffer. For example, the buffer may be a phosphate buffer, e.g., phosphate-buffered saline (PBS). The buffer in which the crosslinking agent is provided, e.g., dissolved, may have a pH that is lower than the buffer in which the macromer is dissolved. For example, the crosslinking agent may be provided, e.g., dissolved, in a solution of PBS having a pH of approximately 6.0-6.5. Thus, the 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 crosslinking agent and the macromer may be present with a functional group ratio (e.g., N-hydroxysuccinimide:amine, N-hydroxysuccinimide:thiol, maleimide:amine, maleimide:thiol, etc.) of approximately a 3:2-7:3 molar ratio, for example a 2:1 molar ratio, respectively. The gel may be an aqueous composition in which a combined content of the crosslinking agent and the macromer is at least 15% by weight, with respect to the total weight of the composition. For example, the content of the crosslinking agent may be between approximately 10-20 wt %, in relation to the total weight of the composition, e.g., ranging 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 %. Additionally or alternatively, the content of the macromer may be between approximately 5-10 wt %, in relation to the total weight of the composition, e.g., ranging from about 5 wt % to about 8 wt %, or from about 7 wt % to about 9 wt %. As discussed above, the first and second part solutions may comprise at least two different buffers, e.g., a first buffer suitable for the crosslinking 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 include any suitable salts for the buffers. It is noted that the mechanical properties of the gels, e.g., hydrogels, formed by the compositions herein may be at least partially determined by the amounts of macromer and/or crosslinking agent. For example, the gel strength may increase as the content of the macromer and crosslinking agent in the aqueous composition increases. Thus, for example, a composition comprising about 20 wt % or about 25 wt % of combined macromer and crosslinking agent, in relation to the total weight of the aqueous gel system, may form a gel that has a higher gel strength than a gel formed from a composition comprising about 15 wt % of combined macromer and crosslinking agent.
The components of the composition or system (e.g., the macromer, the crosslinking agent, and respective buffers) may be mixed together.
When under physiological pH, the functional groups of the crosslinking agent and the functional groups of the macromer may react with one another via chemical conjugation, thereby allowing for immediate gelation. For example, when the composition is at a pH of about 7 to about 7.5, the macromer and crosslinking agent may react to form a gel. In some aspects, the gel may form within 20 seconds, within 15 seconds, within 10 seconds or within approximately 5 seconds, upon mixing a first component comprising the macromer with a first buffer with a second component comprising the crosslinking agent with a second buffer. For example, the gel may form in a time ranging from about 1 second to about 15 seconds, from about 3 second to about 8 seconds, from about 5 seconds to about 10 seconds, or from about 2 seconds to about 5 seconds. The resulting gel, e.g., hydrogel, may be dissolvable, e.g., passively within the physiological environment over time, or on demand, such as by application of an agent capable of disrupting the hydrogel network. For example, the gel may dissolve within a time period of about 10-30 minutes. Dissolution may be measured within a laboratory environment, for example, by measuring rheology when submerging the gel in an aqueous solution.
Gels formed from the macromer and crosslinking agent may exhibit desired properties. For example, storage moduli of the resulting gel, e.g., hydrogel (as a measure of gel strength), may range from about 2.0-10.5 kPa, such as from about 2.5 kPa to about 10 kPa, from about 5 kPa to about 8 kPa, or from about 3.5 kPa to about 7.5 kPa. Additionally or alternatively, the gels may retain a gel strength (also referred to herein as storage modulus G′) ranging from about 2000-10,000 Pa, in approximately room temperature settings, for a desired duration of time, such as, e.g., up to 30 days or longer. Further, for example, the gel, e.g., hydrogel, may have a shear force between about 0.03-0.90 N/cm2, e.g., between about 0.1-0.6 N/cm2, such as ranging from about 0.05 N/cm2 to about 0.4 N/cm2, from about 0.5 N/cm2 to about 0.9 N/cm2, or from about 0.75 N/cm2 to about 0.9 N/cm2, when adhered to tissue, such as tissue of a bodily lumen, e.g., colon tissue of the GI tract.
The gel may additionally or alternatively be formulated to withstand a burst pressure (corresponding to the pressure at which the gel when adhered to tissue will rip or fail) of up to approximately 200 mbar of pressure, e.g., 150 mbar of pressure, for example a burst pressure greater than 1 mbar and less than or equal to 200 mbar (1 mbar=100 Pa). It is noted that the burst pressure of the gel may be measured by a catheter and pressure transducer (including, e.g., Millar catheters equipped with pressure sensors), which may be utilized to measure the baseline pressure and the pressure just before bursting. To measure burst pressure, a gel may be formed in situ in an aperture of a tissue sample and exposed to fluid of increasing pressure up to the point the cohesion of the gel and/or adhesion of the gel to the tissue breaks down to allow fluid to pass through the aperture of the tissue. The pressure corresponding to the maximum pressure of the fluid just before the gel fails is the burst pressure.
Burst pressure of a gel applied to tissue of the GI tract, such as colon tissue, may be measured as follows. First, an aperture having approximate width and length dimensions of 1 mm×5 mm is cut within the tissue (the depth of the aperture corresponding to the thickness of the tissue, approximately 5 mm in the case of colon tissue). The tissue sample is secured over the open end of a container such that an area approximately 2 inches in diameter is arranged as an unencumbered window over the container. Saline solution is introduced into the container and allowed to flow through the aperture to calibrate the pressure sensor to a baseline pressure. The gel is then formed in situ to close the aperture. The saline solution is then introduced into the container and the increasing fluid pressure measured until the gel fails to permit the solution to pass through the aperture to exit the container. The maximum pressure just prior to the saline solution breaking through the gel to exit through the aperture is the burst pressure. In some examples herein, the gel may be formulated to withstand a burst pressure of at least 50 mbar, at least 100 mbar, or at least 120 mbar when adhered to colon tissue. For example, the gels herein may be formulated to withstand a burst pressure of up to approximately 150 mbar when adhered to colon tissue, such as a burst pressure ranging from about 50 mbar to about 150 mbar, from about 100 mbar to about 150 mbar, or from about 125 mbar to about 150 mbar. The burst pressure may be measured against colon tissue as described above, using an aperture size of 1 mm×5 mm.
Burst pressure of a gel used as an artery or other vessel occlusion device may be measured by forming the gel in situ to close the vessel, wherein the vessel has an approximate diameter of 4-6 mm. A syringe pump and pressure transducer may be used (see
Light-Activated Gel Systems
The disclosure also includes compositions and systems formulated to form a gel upon activation by light as a stimulus. In some examples, the composition may be formulated to crosslink and form a cohesive gel when exposed to light, e.g., UV light or visible light. Thus, such compositions and systems may be described as being light-activated. Such compositions and systems may comprise, for example, a macromer, a cross-linking agent, a photoinitiator, and a buffer. The buffer may be any suitable buffer at about or slightly beyond physiological pH, depending on the buffer used. For example, phosphate buffers may be in the pH range of 7.0-8.0.
The macromer may be a multi-functional PEG-based polymer that includes 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 alkene group such as a vinyl group, an allyl group, an acrylate group, or a norbornene group, among other alkene groups. The functional group of the macromer may be selected based on the desired properties of the gel, including, e.g., gelation time. The number of functional groups of the macromer may be between 4-100, e.g., between 10-50, between 25-65, or between 45-85.
In some examples, the crosslinking agent may be a PEG-based polymer that includes at least one functional group. The functional group of the crosslinking agent may be complementary to the functional group of the macromer so as to crosslink the macromer and the crosslinking agent. For example, the at least one functional group of the crosslinking agent may comprise a thiol group or an alkene group, such as a vinyl group, an allyl group, an acrylate group, a norbornene group, or other type of alkene group. The functional group of the crosslinking agent may be selected based on the desired degradation properties of the gel. In some examples herein, the number of functional groups of the crosslinking agent may be between 2-4, e.g., 2, 3, or 4 different functional groups.
In some examples, the macromer comprises a thiol group and the crosslinking agent comprises an alkene group, or vice versa. For example, the crosslinking agent may comprise a PEG-based polymer that comprises a thiol group, and the macromer comprises an alkene group, e.g., an acrylate group. In another example, the macromer may comprise a PEG-based polymer comprising a thiol group, and the crosslinking agent may comprise a PEG-based polymer comprising an alkene group, such as an allyl ether group.
As mentioned above, the composition may comprise a photoinitiator, e.g., to initiate gelation. Thus, for example, the photoinitiator may be a compound that absorbs light of a given wavelength of light. According to aspects of the present disclosure, the photoinitiator may absorb UV light (e.g., wavelength between about 100-390 nm) or visible light (e.g., wavelength between about 390-800 nm). Examples of photoinitiators suitable for the compositions herein that are activated by UV light include, but are not limited to, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Gelation activated by UV light may take place immediately, e.g., within approximately 5 seconds of UV light exposure. Examples of photoinitiators suitable for the compositions herein that are activated by visible light include, but are not limited to, Eosin Y. Gelation activated by visible light may take place briefly or immediately after visible light exposure, e.g., within approximately 10 seconds of exposure to visible light. In some examples, the photoinitiator absorbs white light, e.g., a wavelength of about 390 nm to about 700 nm. Thus, when the composition is illuminated with UV light or visible light (e.g., white light), depending on the photoinitiator used, the composition may crosslink to form a gel. The intensity of UV light or visible light may range from about 1 mW/cm2 to about 150 mW/cm2. For example, the UV light (365 nm) intensity may range from about 4 mW/cm2 to about 120 mW/cm2, and white light intensity may range from about 10 mW/cm2 (e.g., at the maximal absorption of the photoinitiator) to about 45 mW/cm2, such as 42.9 W/cm2.
In some examples, the composition may comprise an additive to facilitate or enhance photopolymerization gelation kinetics. Exemplary additives include, for example small-molecule additives such as a tyrosine derivative, e.g., tyrosine methyl ester or tyrosine ethyl ester. Such compositions may comprise photoinitiators that absorb visible light, e.g., white light.
The aforementioned components of the macromer, the crosslinking agent, and the photoinitiator, may be present at a combined concentration of about 10-25 wt %, in relation 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 5-10 wt %, in relation to the total weight of the composition. Additionally or alternatively, the content of the macromer may be between 5-15 wt %, in relation to the total weight of the composition. The molar ratio between the functional groups of the macromer and the functional groups of the crosslinking agent may range between 1:1 to 2:1 in the composition. In some examples, the molar ratio is about 1:1. It is noted that the aforementioned moiety ratio of 1:1 may result in an increase in elasticity, i.e., the gel elastic modulus, compared to the aforementioned molar ratio of 2:1. In some examples, the composition or system may include a macromer that comprises two or more functional groups for every crosslinking agent of the composition or system (e.g., a macromer and crosslinking agent in a 1:1 ratio wherein the macromer comprises at least two functional groups; or a macromer and a crosslinking agent in a 1:2 ratio wherein the macromer comprises at least four functional groups). It is further noted that different stoichiometric amounts of a functional moiety, e.g., the number of functional or reactive groups per macromer, may affect both the mechanical properties and swelling ratios of the resulting gel, e.g., hydrogel. For example, an increase in the number of functional groups on a macromer of a light-activated gel system may result in a stiffer (e.g., more viscous) gel with a lower swelling ratio.
The composition may comprise from about 0.1 mM to about 100 mM of the photoinitiator. Higher photoinitiator concentrations, e.g., about 90-100 mM, may provide for relatively faster gelation kinetics as compared to lower concentrations, e.g., about 0.1-1 mM. In cases in which the composition comprises an additive, the composition may comprise up to 10 mM of the additive, such as, e.g., about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, or about 0.5 mM to about 1 mM.
The resulting light-activated gel, e.g., hydrogel, may exhibit a number of desired properties beneficial for application to tissue before, during, and/or after a medical procedure. For example, the gel, e.g., hydrogel, may exhibit a gel strength (also referred to as storage modulus G′) between 500-2500 Pa, such as ranging from about 500 Pa to about 1500 Pa, from about 1000 Pa to about 2000 Pa, from about 750 Pa to about 1250 Pa, from about 1750 Pa to about 2500 Pa. The gel strength G′ may be dependent on the concentration and macromer and crosslinking agent in the composition and/or ratios of the components relative to each other. The gel, e.g., hydrogel, may exhibit a swelling ratio mf/mi (the fold change in the weight of the gel due to water absorption, that is, mf being the weight of the gel at a particular time point after submerging the gel in the buffer, and mi being the initial weight of the gel before submerging it in the buffer) ranging from about 1.8 to about 1.9 times the initial mass or from about 2.3 to about 2.4 times the initial mass. The resultant gels may also exhibit relatively low levels of cytotoxicity. For example, the gel may exhibit greater than at least 97% viability over a 24 hour exposure to cell lines such as NIH3T3 fibroblasts.
The following examples are intended to illustrate the present disclosure without, however, being limiting in nature. It is understood that the present disclosure encompasses additional embodiments consistent with the foregoing description and following examples. The present disclosure is not limited to the examples further described below and encompasses additional conditions without departing from the scope of the present disclosure.
An exemplary pH-activated composition (gel system) was prepared ex vivo at room temperature according to Table 1 in a humid environment. A first part solution was prepared by combining an amine terminated PEG-based or poly(ethylenimine) macromer with a borate buffer having a pH of 8.5. Separately, a second part solution including a N-hydroxysuccinimide crosslinking agent dissolved in phosphate buffer having a pH of 6.5 was prepared.
The first part solution and second part solution were mixed together to form an aqueous solution that subsequently formed a gel. The compositions were left to complete gelling for one hour before assessing the resulting properties.
Gel strength (storage modulus G′) of the gel was measured at room temperature (about 22-25° C.) using a TA instruments DHR-2 rheometer and assessed using a strain sweep from 1-100%, at a 1 Hz frequency. The linear viscoelastic region was determined as the strain percentage increased, until the curve exhibited a 10% decline in the slope of the gel strength. A frequency sweep was then performed from 1-10 Hz within the linear viscoelastic region at 3% strain. Frequency sweeps were performed at times t=0, 4 hours, 24 hours, 48 hours, 7 days, and 30 days after submerging the gel in 50 mM PBS until the gel dissolved.
Gelation time was determined using the inverted tube test, wherein gelation was determined as the time at which the gel no longer runs down the side of the vial when inverted, just after mixing the first and second part solutions. The gelation time was measured at under 1 second.
The swelling ratio was determined as a percentage by weight of the hydrogel after submerging in 50 mM PBS following the equation:
with mf being the weight of the gel at that particular time point after submerging the gel in the buffer, and mi being the initial weight of the gel before submerging it in the buffer.
Adhesion measurements were determined by a lap shear test via an Instron® machine, using ex vivo porcine colon tissue. This tissue was dissected into pieces of approximately 2″×1″. The gel was placed between two pieces of colon tissue, and left in a humid chamber for 1 hour to allow for complete gelation. It is noted 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 was then mounted on the Instron® and force was continuously measured as the two strips of colon tissue were pulled in opposite directions away from each other at a rate of 10 mm/min until cohesive failure of the hydrogel was observed. Adhesion measurements ranged from 0.03-0.85 N/cm2.
Two UV-activated compositions (gel compositions 1 and 2) were prepared ex vivo at room temperature according to Table 3 by combining an alkene containing PEG-based macromer, a thiol containing PEG-based crosslinking agent, and the photoinitiator LAP in PBS having a pH of 7.4. Gel composition 1 was prepared using the PGC-based macromer shown in
The compositions were gelled using a handheld 4 W lamp at 365 nm UV light.
Gel strength before and after swelling of the gel in the buffer was measured on a TA instruments DHR-2 rheometer using 8 mm parallel plates at room temperature (about 22-25° C.). A frequency sweep was done with 1% strain and the gel strength G′ identified from the linear viscoelastic portion of the sweep.
Swelling ratio was determined by taking the mass of the initial gel (mi) and then the mass of the gel after swelling in buffer for 24 hours (mf), after blotting off excess buffer according to Equation 1 above.
An exemplary visible light-activated gel system (composition 3) was prepared ex vivo at room temperature according to Table 4 by combining an alkene containing PEG-based macromer with a thiol containing PEG-based crosslinking agent, the photoinitiator Eosin Y, additive tyrosine ethyl ester, and a phosphate buffer having a pH of 7.0.
The system was gelled using an AmScope 150 W halogen lamp with dual gooseneck fiber-optic illuminators with broad spectrum white light (400-700 nm).
The gel strength was measured at room temperature (about 22-25° C.) as in Example 2 at times t=0, 4 hours, 24 hours, 48 hours, and 7 days, as shown in
The gel exhibited an initial gel strength of approximately 600 Pa at t=0 and an increased gel strength over the next 7 days, with a peak gel strength of approximately 2,100 Pa at t=24 hours. The gel exhibited a gel strength of at least 600 Pa for at least 7 days. Thus, these results indicate that the gel may serve as a lasting protective barrier for tissue for at least a 7-day period.
Gelation time was measured on the DHR-2 rheometer at room temperature (about 22-25° C.) with 20 mm parallel plates with the two goosenecks of the halogen lamp directed at the solution before light exposure between the two parallel plates. A time sweep was performed with 1 Hz frequency. The lamp was turned on at 30 seconds, and the time at which gelation occurred thereafter was recorded. Gelation time is shown in
Gel precursor viscosity was determined from a flow sweep on the DHR-2 rheometer at 37° C. using a 50 mm 1.008° cone plate. Results shown in
In vitro cytotoxicity of the photoinitiator and the gel were tested using NIH 3T3 fibroblasts cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded in either 96 well plates or 12 well plates at a density of 2,500 cells/well or 25,000 cells/well, respectively, and allowed to adhere overnight. Photoinitiator solutions and the gel solution were sterile filtered using a 0.22 um filter prior to testing in vitro. The gel solution was then gelled within the biosafety cabinet using the 150 W halogen lamp and co-cultured with the cells utilizing a 3 μm pore size transwell insert. Cells were incubated with treatments for 24 hours and then viability was measured using an MTS (3-(4,5-dimethyltriazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tratrazolium, inner salt) assay (CellTiter 96® AQueous One, Promega). Cell viabilities were normalized to control untreated cells.
The crosslinking agent shown in
SA-PEG-SA was first synthesized as follows. Poly(ethylene glycol)(PEG; average Mn 3000 g/mol; Sigma Aldrich) (5 g, 1.6 mmol) was melted in a tri-neck round bottom flask at 120° C. while stirring. Once melted, the flask was put under vacuum and the temperature was then decreased to 80° C. and allowed to stir for 30 minutes. The flask was purged with nitrogen three times. Succinic anhydride (SA) (99%; Aldrich) (0.75 g, 7.5 mmol) was added to the flask. The reaction was stirred under nitrogen for 18 hours. The contents were then dissolved in minimal anhydrous methylene chloride (DCM; 99%; anhydrous; Sigma Aldrich), and precipitated in diethyl ether. Finally, the product was filtered and dried under vacuum for 1 day (white solid, 99% yield). Proton and carbon nuclear magnetic resonance (1H-NMR, 13C-NMR) spectra were obtained on an Agilent 500 MHz spectrometer in CDCl3 The NMR spectra for the SA-PEG-SA product were as follows: 1H NMR (500 MHz), CDCl3: δ 2.62 (m, 8H), 3.64 (overlap, 288H), 4.24 (m, J=4.6 Hz, 4H); 13C NMR (500 MHz), CDCl3: δ 174.0, 172.1, 70.5, 63.8, 29.3, 28.3 ppm. The SA-PEG-SA intermediate was obtained with a reaction yield of 99%.
Next, SA-PEG-SA (4 g, 1.3 mmol) was added to a dry, round bottom flask and dissolved in 15 mL 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 for 18 hours at room temperature. Dicyclohexylurea was filtered, the solution was concentrated, and precipitated in diethyl ether. The resulting product SA crosslinker, a white, water-soluble powder, was collected through filtration and dried on vacuum overnight (white solid, 98% yield). The structure was confirmed via 1H NMR, 13C NMR, DSC, and GPC. NMR spectra for the SA crosslinker measured as described above were as follows: 1H NMR (500 MHz), CDCl3: δ 2.70 (t, J=1.0 Hz, 4H), 2.77 (t, J=1.0 Hz, 8H), 2.89 (t, J=1.0 Hz, 4H), 3.57 (overlap, 296H), 4.20 (t, J=1.0 Hz, 4H) ppm. 13C NMR (500 MHz), CDCl3: ≡ 170.9, 168.9, 167.6, 70.7, 64.1, 28.6, 26.2, 25.5 ppm.
The molecular weight and polymeric distribution were determined using gel permeation chromatography (GPC) in tetrahydrofuran (THF) as the mobile phase with flow rate of 1.0 mL/min. For the SA crosslinker Mw: 2949 g/mol; PDI: 1.02. GPC analyses were performed on an OptiLab DSP Interferometric Refractometer (Wyatt Technology) fitted with two identical Jordi Gel DVB columns (Jordi Labs, 250 mm×10 mm, 105 Å pore size). For the SA crosslinker GPC: Mn: 2893 g/mol. Matrix-assisted laser desorption/ionization (MALDI-TOF) was performed on a Bruker autoflex Speed spectrometer equipped with a SMART-beam II and a flash detector. For the SA crosslinker MALDI-TOF (pos): Mw: 3600 m/z. Differential scanning calorimeter (DSC) spectra was taken on Q100 TA instrument calorimeter and used to determine melting point (mp). For the SA crosslinker Mp (DSC): 43.5° C.
The crosslinking agent shown in
Hydrogels were prepared by combining the crosslinking agents shown in
Data was analyzed with Graph Pad Prism 8. For the hydrogel characterization studies, error bars represent standard deviation of the results from three or more replicates. For the bacterial migration studies, error bars represent standard deviation of the results from three biological replicates each performed with three technical replicates or more. A student's T-test was used to compare results and to assess significance. A *p<0.05 is significant.
Gelation Measurements
A relatively fast gelation time (e.g., <3 seconds) may be useful for in situ forming gel, e.g., during polypectomy procedures or other internal wound dressings. For gelation measurements, the crosslinking agent and amine-terminal macromer solutions were mixed and put in a 2 mL glass vial. Gelation was tested using the inverted tube test mechanism. Every 10 seconds the tube was inverted. Gelation was defined by the time at which the solution remained at the bottom of the vial when inverted. All gelation studies were performed at room temperature, 25° C.
The SVA crosslinker+PEI hydrogels were found to gel at a similar rate to the SA crosslinker+4-arm PEG-NH2 and SA crosslinker+PEI hydrogels. However, the SVA crosslinker+4-arm PEG-NH2 hydrogel was observed to gel faster than the SA crosslinker+4-arm PEG-NH2. This increase in gelation time was attributed to two factors. The 4-arm PEG-NH2 macromer contains four, long, amine-terminal arms and has a molecular weight of 5 kDa. The PEI is a more condensed branched macromer with a molecular weight of 2.0 kDa. The long PEG arms of the 4-arm PEG-NH2 are believed to favor a faster gelation time for the SVA crosslinker+4-arm PEG-NH2 hydrogel due to increased steric freedom relative to PEI, a branched polymer with shorter arms containing terminal amines and a smaller molecular weight. The steric hindrance observed in the, smaller, branched PEI structure is believed to reduce the ability to readily react with NHS reactive groups in the hydrogel network relative to the higher molecular weight, longer armed, star 4-arm PEG-NH2 structure.
With regards to defects in the hydrogel network, terminal amines favor conjugation at the NHS-ester, however the SA crosslinker contains an internal ester that is also susceptible to macromer amidation and hydrolysis. The preferred site for amidation in the SA crosslinker is at the NHS ester as confirmed via 1H NMR analysis of a model system, t1/2=0.60 min−1.
The effect of pH on gelation time was also evaluated for SA crosslinker+PEI hydrogels at 15 wt %, for pH 8.5, pH 9.5, and pH 10.5, as summarized in panel B) of
Rheological Measurements
Rheological measurements were obtained using a TA instruments DHR-2 Rheometer. Rheological 8 mm parallel plates were used to perform rheological measurements at 22° C. Oscillatory strain sweeps were performed at a frequency of 0.1 Hz from 0.1-10% strain. The linear viscoelastic region was determined from the strain sweep as the strain percent at below which G′ deviates 10° from horizontal. Frequency sweeps were subsequently performed at all time points over 30 days. Strain was set to be within the linear viscoelastic region at 3%, and the frequency ran from 0.1 Hz to 10 Hz following a previously published protocol. Data are expressed as mean±standard deviation (n 3).
The storage modulus (G′) of the hydrogels was measured at 0 hour, 4 hours, 24 hours, 48 hours, 7 days, and 30 days after swelling in 100 mM PBS, pH 7.4 (applied strain of 3%). The swelling percent and storage modulus (G′) of each hydrogel was measured as an indicator for strength as well as hydrogel swelling over 30 days or until the hydrogel dissolved in 100 uM PBS (pH 7.4).
The SA crosslinker+PEI hydrogels of 10 wt %, 15 wt %, and 20 wt % exhibited average G′ of 638 Pa, 992 Pa, and 2930 Pa, respectively, upon gelation. The hydrogels of 15 wt % and 20 wt % maintained mechanical integrity (G′>300 Pa) through 48 hours, while the 10 wt % SA crosslinker+PEI hydrogels dissolved after 4 hours (G′<300 Pa). To assess the effects of a degradable ester linkage in the crosslinking agent relative to mechanical strength, hydrogels were prepared using the SA or SVA crosslinker and a 4-arm PEG-NH2 (see panels B) and D)). In comparison, the 15 wt % SA crosslinker+4-arm PEG-NH2 hydrogels exhibited a G′ of 3814 Pa at time t=0, and maintained integrity through 7 days of swelling with minimal change in G′ through 24 hours and a decrease in mechanical strength at 48 hours and 7 days. The SVA crosslinker+PEI and SVA crosslinker+4-arm PEG-NH2 hydrogels at 15 wt % exhibited a G′ of 1683 Pa and 7739 Pa, respectively. The G′ of all of the hydrogels initially increased upon swelling (
Increased hydrogel weight percent afforded greater G′ and longer sustained mechanical strength as shown for the SA crosslinker+PEI hydrogels (see panel A)). Additionally, the G′ decreased over time for each SA crosslinker+PEI hydrogen, believed to be due to hydrolysis at the internal ester linkage, while G′ remained unchanged for the SVA crosslinker+PEI hydrogels over 30 days of swelling, believed to be due to a lack of degradable linkage within the SVA crosslinker structure (see panel C)).
The increased degradation rate in the SA crosslinker+PEI hydrogels relative to the SA crosslinker+4-arm PEG-NH2 hydrogels was attributed to a local basic pH within the hydrogel network as a consequence of the PEI. The effect of pH was evaluated by swelling the SA crosslinker+PEI hydrogels in dH2O, pH 5.0, and the SA crosslinker+4-arm PEG-NH2 in an aqueous TEA solution (a tertiary amine; comparable [M] to that present in the PEI-based hydrogels) of pH 8.0.
Rheological measurements were performed on the SA crosslinker+PEI hydrogels in dH2O, pH 5.0, and SA crosslinker+4-arm PEG-NH2 at pH 8.0. The SA crosslinker+PEI hydrogel exhibited a G′ of 1362 Pa at time t=0 and maintained similar mechanical integrity through 48 hours until significant hydrolysis (loss of hydrogel integrity defined as G′<300 Pa, due to the hydrogel's inability to retain its mechanical structure during rheological measurements when presenting a storage moduli below 300 Pa) regardless of the pH of the water in which the gels were swollen (see
Hydrolysis at the internal ester linkage was confirmed by following a shift in the methylene peak adjacent to the ester from 4.19 ppm to 4.15 ppm on 1H NMR in a model system. The SA crosslinker was dissolved in a 0.3 M sodium bicarbonate buffered solution in D2O, pH 8.0.
Hydrolysis of the SA crosslinker+PEI hydrogel was evaluated at 37° C. to mimic a colon environment.
Regardless of the hydrogel composition, an initial increase in storage moduli and swelling occurred after the first 4 hours when the hydrogels were immersed in 100 mM PBS.
Adhesion
Adhesion of hydrogels to ex vivo porcine colon tissue was performed on an Instron 5944 Micro-tester. Hydrogels were mixed and placed between two pieces of colon tissue. The colon tissue was divided into 1″×1″ pieces, and the hydrogels gelled directly on the tissue. Upon applying the hydrogel to colon tissue, an additional piece of tissue was placed on top “sandwich style” (tissue-hydrogel-tissue). Tissue adhering to the hydrogel was either the mucosa layer of the colon tissue, or the submucosa layer, obtained by scraping the colon tissue with a scalpel, on each sample. After allowing gelation for one hour in a humid chamber, a lap shear test following ASTM D3165 protocol for adhesion of the hydrogels on colon tissue was performed. Tissue pieces were pulled apart at a rate of 5 mm/min at room temperature until failure in adhesion was detected. Data is expressed as mean±standard deviation (n=3).
Adhesive strength of the SA crosslinker+PEI, SVA crosslinker+PEI, and SA crosslinker+4-arm PEG-NH2 hydrogels at 15 wt % was measured on colon tissue with and without the mucosa layer present at 25° C. These hydrogels were chosen to determine whether the presence of the PEI vs 4-arm PEG-NH2 in the hydrogel alters the adhesion, and if the hydrolyzable SA crosslinker vs non-hydrolyzable SVA crosslinker affects the adhesion. For some samples, the mucosa layer on the colon tissue was removed with a scalpel to expose the submucosa to better model tissue after a polypectomy. The SA crosslinker+PEI, SVA crosslinker+PEI, and SA crosslinker+4-arm PEG-NH2 hydrogels exhibited mean adhesive strengths of 0.18 N/cm2, 0.36 N/cm2, and 0.03 N/cm2 with the mucosa layer intact, and 0.31 N/cm2, 0.29 N/cm2, and 0.64 N/cm2 without the mucosa layer intact, respectively, as shown in
The SA crosslinker+PEI and SVA crosslinker+PEI hydrogels adhered the greatest to the tissue with an intact mucosa layer with an adhesivity value of 0.18 N/cm2 and 0.36 N/cm2, respectively. The SA crosslinker+4-arm PEG-NH2 hydrogel adhered the strongest to the tissue without the mucosa layer (0.64 N/cm2) (
Cytotoxicity Studies
The cell cytotoxicity of hydrogels, at 15 wt % was evaluated against NIH3T3 fibroblasts. Crosslinking agent and PEI solutions were passed through a 0.22 μm PVDF filter prior to mixing and gelation under aseptic conditions. Portions 50 mg, 25 mg, and 10 mg (±2.5 mg) of hydrogel were placed into permeable cell culture inserts (PES, 3 μm pore) (Cell Treat, 230637). Permeable cell culture inserts containing hydrogel samples were incubated in sterile deionized water at 4° C. for 16 hours to allow for swelling. NIH3T3 (ATCC, CRL-1658) were cultured in DMEM+10% BCS+1% PS at 37° C. in 5% CO2 and 95% humidified air. All cells were passage 4-8 for the experiments. Cells were seeded at 1.25×104 cells/cm2 in 24 well plates and allowed to adhere for 16 hours. Media was exchanged and cell culture inserts with swelled hydrogel were transferred into the wells containing adhered cells. Hydrogel samples were briefly equilibrated to 37° C. prior to transfer. Hydrogels were incubated for 24 hours in the presence of cells. Cell culture inserts were removed and a 1:9 dilution of MTS reagent (Promega, G5421) in media was added to each well. Absorbance (490 nm) was measured after 4 hours. Relative cell viability was determined by normalizing absorbance of cells exposed to hydrogel vs a non-exposed control. All experiments were completed in triplicate and error bars represent 1 standard deviation from the mean. All hydrogels were found to be minimally cytotoxic (>88% cell viability) (
Bacterial Migration
Bacterial migration studies were conducted with isolates of Escherichia coli and Bacteroides fragilis because these microbes are both commonly found in the intestine and are known to cause infections. E. coli is highly mobile and considered to have potential to cross the hydrogel. B. fragilis isolates are known to display multi-drug resistance and cause sepsis. These two common intestinal microbes with pathogenic potential were assessed for the ability to cross the SA crosslinker+PEI and SA crosslinker+4-arm PEG-NH2 hydrogels.
In vitro testing on agar plates and microscopy studies were performed. An advantage of the agar-based assay is that it detects whether even a few bacterial cells penetrate the hydrogel, because the individual bacteria are allowed to grow for ˜24 hours into a visible colony. Clinical isolates E. coli (ADR129Q-SMC9096) and B. fragilis (CFPLTA004_1B-SMC9107) were obtained from children with cystic fibrosis. Prior to inoculation of hydrogels, E. coli isolates were cultured aerobically overnight in LB (lysogeny broth) and B. fragilis isolates were cultured anaerobically for 48 hours on blood agar (TSA+5% sheep's blood) using the GasPak system. Hydrogel discs (8 mm diameter×2.5 mm height) were placed onto LB agar (for E. coli) or TSA+5% sheep's blood agar (for B. fragilis), and 5 uL of bacteria or PBS was added to the top of each hydrogel. Plates were then incubated at 37° C. for 24 hours aerobically (E. coli) or anaerobically (B. fragilis). After 24 hours, hydrogels were removed, and the agar plates were incubated for an additional 24 hours under the appropriate conditions for each organism to test for bacterial growth below the hydrogel as a measure as to whether the microbes could transit through the hydrogel. After growth, the B. fragilis isolate was scraped into 1 mL PBS and homogenized. 1 mL each of B. fragilis and E. coli were centrifuged for 30 seconds at 16,000×g and resuspended in PBS. Each isolate was then normalized to OD600 of 1.0 in PBS for agar plate experiments and OD600 of 0.1 in minimal medium, as reported (bioproject accession number PRJNA557692), for microscopy experiments. Wells treated with medium-only served to determine background fluorescence, which was subtracted from each sample before analysis.
Microscopy was conducted on a Nikon Eclipse Ti inverted microscope equipped with a Hamamatsu ORCA-Flash 4.0 camera running on Nikon Elements AR. Fast scan mode and 2×2 binning was used and images were acquired through a Plan Fluor 40×DIC M N2 objective. Images were processed in ImageJ in which background was subtracted and signal strength quantified by measuring mean signal intensity/pixel through the Integrated Density (IntDen) function. For microscopy studies, 300 uL of SA crosslinker+4-arm PEG-NH2 was inoculated into each well of an 8-well plate (Cellvis, catalog #C8-1.5H-N). To visualize bacteria, Syto9 was added to each culture prior to hydrogel inoculation. Bacterial cultures were inoculated either on top of the hydrogel, or below hydrogels that had first been perforated with a pipette tip. Plates were imaged both before and after incubation to determine whether top-inoculated bacteria were able to cross the hydrogel. Results show that these microbes do not transverse across the SA+4-arm PEG-NH2 hydrogel, indicating its potential for preventing sepsis in vivo.
To quantify the impact on bacterial mitigation by the hydrogels, the Syto9 signal intensity was assessed at the bottom of the hydrogels after subtracting background fluorescence from a media-only control.
The lack of bacterial migration through the hydrogel may be a result of hydrogel pore size relative to the bacteria size. The pore sizes of the hydrogels ranged from <1 μm to 20 μm and the pores were not connected giving a mesh-like network as shown by scanning electron microscope for a SA crosslinker+4-arm-PEG-NH2 hydrogel (
Agar plate assay results are shown in
The application and handleability of the hydrogels was investigated by administering the crosslinking agent and macromer components through a dual lumen catheter for subsequent hydrogel formation upon exit at a target site on a sample of colon tissue. A dual lumen catheter was used; the catheter is capable of being inserted through an endoscope into the colon in vivo, eliminating the need for a separate device. Air pressure can be applied through the dual lumen catheter to spray the hydrogel precursor components onto a wound to gel in situ. The two-part hydrogel system was delivered on ex vivo colon tissue. All 12 hydrogel formulations were injected through the dual lumen catheter and subsequently gelled and adhered to colon tissue both with and against gravity.
Additional crosslinking agents (crosslinkers 5, 6, and 7) were synthesized starting from PEG (Mw 3000) as shown in
PEG Diacid: Synthesis of the PEG diacid compound was based on a previously reported protocol. 1H NMR (500 MHz), CDCl3: δ 1.93 (q, J=7.21 Hz, 4H), 2.4 (tt, J=7.21, 8H), 3.62 (m, 292H), 4.22 (tt, J=4.73 Hz, 4H) ppm; 13C NMR (500 MHz), CDCl3: 175.3, 172.8, 70.6, 68.9, 63.4, 33.1, 32.6, 19.9 ppm.
Crosslinker 1. The synthesis of the starting material was based on a previously reported protocol. 1H NMR (500 MHz), CDCl3: δ 4.15 (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 (500 MHz), CDCl3: 172.3, 169.0, 168.0, 70.5, 69.0, 63.6, 32.4, 29.9, 25.5, 19.7 ppm.
Intermediate 2. Synthesis was based off of a previously reported protocol. 1H NMR (500 MHz), CDCl3: δ 4.21 (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 (500 MHz), CDCl3: 196.8, 172.6, 169.8, 70.6, 69.0, 63.6, 42.3, 32.8, 31.0, 20.5 ppm.
Intermediate 3. In a flame dried flask, 1,8-diazabicyclo(5.4.0)undec-7-ene (265 μL) and 6-mercaptohexanoic acid (122 μL) were added to a solution of crosslinker 1 (1 g) in anhydrous DMF (5 mL). The solution was stirred at room temperature for 16 hours. The organic phase was extracted with a 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 afford intermediate 3 as a white solid (96% yield). 1H NMR (500 MHz), CDCl3: δ 4.22 (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 (500 MHz), 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, 20.6 ppm.
Intermediate 4. Synthesis followed the above procedure using 11-mercaptoundecanoic acid (0.190 g) as the thiol source (92% yield). 1H NMR (500 MHz), CDCl3: δ 4.22 (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 (500 MHz), 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, 20.6 ppm.
Crosslinkers 5, 6 and 7. The synthesis of crosslinkers 5, 6 and 7 was based off of a previously reported protocol (yield 96-98%).
Crosslinker 5. 1H NMR (500 MHz), CDCl3: δ 4.16 (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 (500 MHz), CDCl3: δ ppm; MALDI-TOF (pos): Mw: 3763 m/z; GPC: Mn: 5077; Mw: 5312; PDI: 1.05; Mp (DSC): 46.06° C.
Crosslinker 6. 1H NMR (500 MHz), CDCl3: δ 4.21 (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 (500 MHz), CDCl3: δ 198.6, 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, 20.6 ppm; MALDI-TOF (pos): Mw: 3807 m/z; GPC: Mn: 4999; Mw: 5196; PDI: 1.04; Mp (DSC): 45.80° C.
Crosslinker 7. 1H NMR (500 MHz), CDCl3: δ 4.22 (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 (500 MHz), CDCl3: δ 198.8, 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, 20.6 ppm; MALDI-TOF (pos): Mw: 4210 m/z; GPC: Mn: 6038; Mw: 6313; PDI: 1.05; Mp (DSC): 47.42° C.
Hydrogels at 10 wt %, 15 wt %, and 20 wt % were prepared by mixing the crosslinking agents of Example 7 (i.e., crosslinkers 5, 6, and 7), dissolved in 0.1 M phosphate buffer pH 6.5, with branched polyethyleneimine (PEI; Mw 1800) in 0.3 M borate buffer, pH 8.5. Minimal solubility of crosslinker 7 in buffer was observed and believed to be due to the hydrophobicity of the methylene chains in its structure. In order to overcome the low solubility, crosslinker 7 was dissolved in 0.1 M phosphate buffer pH 6.5 with 50% ethanol prior to mixing it with the PEI solution. The ratio of NHS:NH2 was 2:1 to ensure amidation of PEI and the respective crosslinking agent. No major difference in hydrogel mechanical properties was observed with a 2:1 or 1:1 NHS:NH2 ratio. A transparent, solid hydrogel formed within 5 minutes for all compositions (respective hydrogels 5, 6, and 7) as determined by the inverted tube gelation test (see discussion in Example 6). Hydrogel gelation time was found to positively correlate with increasing hydrophobic chain lengths. As shown in
Next, the morphology of the hydrogels was characterized using scanning electron microscopy (SEM). All of the hydrogels possessed pore sizes varying from 5 μm to 100 μm with a honeycomb-like structure. Hydrogel 7, unlike the other hydrogels, exhibited a more lamellar-like structure.
The attack of the terminal amine to the NHS-ester occurred quickly, under 10 seconds, however in hydrogels this reaction is likely slower because once one of the amines attacks the NHS-ester, entanglement and solidification is believed to occur with a resulting increase in steric hindrance. Hence the lengthier gelation times. Additionally, a competitive hydrolysis reaction occurred at the NHS ester.
With regards to mechanical properties, strain and frequency sweeps were performed at various time points before and after swelling in 50 mM PBS. First, the linear viscoelastic region was determined using the strain sweep (
To ensure that the presence of ethanol did not increase the storage modulus for hydrogels prepared with crosslinker 7, rheological measurements were taken for hydrogels prepared with crosslinker 6 under the same conditions as those hydrogels used for crosslinker 7. No significant difference in storage modulus was observed between hydrogels prepared with or without EtOH, indicating that the buffer conditions did not alter mechanical properties of the hydrogels (
During the 30 days of swelling, the hydrogels swelled between 150-350% depending on weight percent and hydrophobicity of the hydrogel formulation (
All of the hydrogels underwent hydrolysis over 30 days of swelling as indicated by a loss of gross structure and a reduction in storage modulus over time. Hydrogel 5 exhibited an immediate loss in storage modulus and gross structure while hydrogels 6 and 7 increased in strength as they swelled. However, a reduction in storage modulus was observed in hydrogels 6 and 7 by 30 days post swelling. This loss in structure and mechanical properties was attributed to hydrolysis of the crosslinker. To further characterize the hydrolysis, the rate of crosslinker hydrolysis was measured in 0.1 M sodium bicarbonate buffer, pH 8.0, via 1H NMR. It was observed that hydrolysis preferentially occurred at the thioester linkage with a rate of k=0.055 min−1 and k=0.003 min−1 for crosslinkers 5 and 6, respectively (
Adhesive properties of the hydrogels was studied against human skin. A lap shear test was conducted to determine adhesion strength on ex vivo human breast and abdominal tissue. All the hydrogels adhered similarly to tissue with values of approximately 0.5 N/cm2 and display cohesive failure at the hydrogel-skin interface (
Prior to the in vivo studies, cytotoxicity was assessed using NIH3T3 fibroblasts.
Based on the sum of these results, hydrogel 6 at 15 wt % was selected for in vivo testing. Hydrogel 6 exhibited non-toxicity, storage modulus on the same order as that of human skin, maintenance of mechanical strength and structure over 7 days' time, adhered to skin, swelling, and dissolution in 30 minutes. For the in vivo model, second-degree burns were induced on four pigs by heating a brass cylinder to 80° C. and placing it on the back of the pig for 20 seconds. The treatment groups were assessed at days 7 and 14, with one or two dressing changes as depicted in
Generally, all treatment groups showed mild/moderate necrosis, epidermal ulceration, inflammation, and neovascularization. Hydrogel 6, however, exhibited less necrosis, epidermal ulceration, and inflammation than other treatment groups, with similar neovascularization, and burn depth (mm) and epidermal dermal thickness (mm) to all treatment groups by day 14 (
Additional hydrogels according to the present disclosure are prepared. The macromer is either a PEG-based macromer or a poly(1,2-glycerol carbonate) (PGC) based macromer, featuring an alkene functional moiety. The crosslinking agent is a PEG-based crosslinking agent featuring thiol moieties (see Example 9). These components are dissolved in a phosphate buffer solution in the pH range of 7-8 at total polymer concentrations ranging from 10 wt % to 25 wt %. As the weight percentage of the gel solution increases, the gelation time decreases and the gel elastic modulus increases. The molar ratio between alkene and thiol moieties may range between 1:1 to 2:1 in a gel formulation. A ratio of 1:1 results in a small increase in gel elastic modulus compared to a ratio of 2:1. The alkene functional moiety encompasses many different structures, such as, but not limited to, the alkyl ether shown in
For the UV activated hydrogels, the concentrations and ratios of macromer and crosslinking agent can be modified to modulate the storage modulus between 500 Pa and 2,000 Pa. These formulations are single solutions and display a viscosity amenable to application via a single lumen catheter down the length of an endoscope. These formulations utilize stimuli-responsive gelation in response to long wave UV light with fast kinetics, forming a gel within 5 seconds of illumination. The gels adhere to porcine colon tissue and exhibit strong burst pressure when used to seal a small defect. In vivo studies are performed to apply the components using an endoscopic catheter; the resultant gels are still present 2.5 hours after application. The resultant gels have low cytotoxicity, showing greater than 97% viability in NIH 3T3 fibroblasts over 24 hrs. The photoinitiator is used at concentrations below the IC50 in NIH 3T3 fibroblasts and still exhibits fast (<10 seconds) gelation kinetics in response to a broad range of white light sources, such as bike lights, lamps, or endoscopes, when combined with tyrosine ethyl ester up to 10 mM.
Additional crosslinking agents with thiol moieties were synthesized starting from PEG (Mw 3000) as shown in
As summarized in
Following the previous step, 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 crosslinking agents, crosslinkers 4, 5, and 6 with methylene chain lengths of 2, 3, and 4, respectively. In a flame-dried, round bottom flask with a magnetic stir bar, intermediate 1, 2, or 3, was dissolved in dry methylene chloride. 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 using a saturated citric acid solution, water, and brine. The organic phase was then dried with sodium sulfate, filtered through filter paper, and precipitated in diethyl ether to obtain 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 obtain an off-white solid (80-90% yield). All yields for the above reactions were above 80%.
Characterization data by 1H NMR, 13C NMR, GPC, and DSC was as follows:
PEG Diacid. This polymer was prepared from a previously published protocol (see also Example 7).
Crosslinkers 1, 2, 3. The synthesis of crosslinkers 1, 2, and 3 were based on a previously published protocol (see also Example 7).
Intermediates 1, 2, 3. Synthesis was conducted as described above. Characterization by 1H NMR (500 MHz), CDCl3: Intermediate 1-δ 4.22 (tt, J=4.7 Hz, 4H), 3.62 (m, 310H), 2.93 (t, J=6.8 Hz, 4H), 2.68 (t, J=6.8 Hz, 4H) ppm; Intermediate 2-δ 4.22 (tt, J=4.8 Hz, 4H), 3.63 (m, 308H), 2.86 (t, J=7.2 Hz, 4H), 2.61 (t, J=7.3 Hz, 4H), 2.38 (t, J=7.4 Hz, 4H), 2.30 (t, J=7.4 Hz, 4H), 1.97 (t, J=7.3 Hz, 4H), 1.60 (m, 8H), 1.39 (m, 4H) ppm; Intermediate 3-δ 4.21 (tt, J=4.4, 4.9 Hz, 4H), 3.63 (m, 277H), 2.62 (t, J=6.7, 7.2 Hz, 4H), 2.34 (t, J=6.7, 7.2 Hz, 4H), 1.69 (m, 8H) ppm. Characterization by 13C NMR (500 MHz), CDCl3: Intermediate 1—195.9, 171.5, 70.5, 64.1, 30.9, 29.1 ppm; Intermediate 2-198.6, 172.7, 70.7, 69.0, 33.5, 32.9, 20.6 ppm; Intermediate 3-197.0, 173.0, 70.5, 63.5, 33.7, 31.0, 24.7, 24.0 ppm.
Crosslinkers 4, 5, 6. Synthesis was conducted as described above.
Crosslinker 4. 1H NMR: δ 6.71 (s, 2H), 6.55 (b, 1H), 4.23 (tt, J=4.2, 4.9 Hz, 4H), 3.62 (m, 322H), 2.96 (t, J=6.8 Hz, 4H), 2.74 (t, J=6.8 Hz, 4H) ppm; 13C NMR: 197.5, 171.9, 134.2, 70.5, 64.0, 32.3, 29.1 ppm; Mw (GPC, THF): 2868 Da; Mn (GPC, THF): 2801 Da; PDI (GPC, THF): 1.02; Melting point (DSC): 41.78° C.; Crystallization point (DSC): 39.9° C.
Crosslinker 5. 1H NMR: δ 6.72 (s, 2H), 6.51 (b, 1H), 4.23 (tt, J=4.8 Hz, 4H), 3.63 (m, 297H), 2.73 (t, J=7.3 Hz, 4H), 2.42 (t, J=7.2 Hz, 4H), 2.01 (m, J=7.2, 7.3 Hz, 4H) ppm; 13C NMR: 198.2, 172.6, 134.2, 70.4, 63.6, 32.8, 32.3, 20.2 ppm; MW (GPC, THF): 3028 Da; Mn (GPC, THF): 2955 Da; PDI (GPC, THF): 1.02; Melting point (DSC): 40.22° C.; Crystallization point (DSC): 21.3° C.
Crosslinker 6. 1H NMR: δ 6.71 (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, 24.0 ppm; Mw (GPC, THF): 3351 Da; Mn (GPC, THF): 3162 Da; PDI (GPC, THF): 1.06; Melting point (DSC): 45.04° C.; crystallization point (DSC): 33.5° C.
A thiol-terminated polyethyleneimine (PEI-SH) hyperbranched macromer (
Initially in the PEI-SH synthesis, 15 equivalents of thiol were reacted per PEI molecule to fully thiolate PEI. However, due to the high concentration of thiols per polymer, intra- as well as inter-molecular disulfide bonds formed, as observed visually via a pink solution of PEI-SH in borate buffer, pH 8.6. This minimized the number of available free thiols for a Michael addition reaction with the maleimide-functionalized crosslinking agents of Example 9. The equivalents of thiol reacted with PEI were therefore reduced to minimize the number of inter- and intra-molecular disulfide bonds. The number of free amines was determined via a colorimetric TNBS assay as shown in
PEI-STr. PEI (3 g) was dissolved in DMF. 3-(tritylthio)propionic-pentofluorophenol (3.4 g), HOBt (3.2 g), and DIPEA (4.7 mL) were added. The reaction was stirred at room temperature, overnight. The reaction was dissolved in methylene chloride, and the organic phase was extracted 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 obtain a light yellow, solid (68% yield). 1H NMR: δ 8.00 (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, 27.7 ppm.
PEI-SH. In a round bottom flask with a magnetic stir bar, PEI-STr (2 g) was solubilized in a minimal amount of methylene chloride. Trifluoroacetic acid (TFA) (12.3 mL) and triethylsilane (2.7 mL) were added to the stirring solution dropwise, simultaneously. The reaction was stirred for 3 hours at room temperature. Methylene chloride and TFA were removed under vacuum, and redissolved in a minimal amount of methylene chloride. The solution was precipitated in diethyl ether and the product was dried under vacuum overnight. The product was dissolved in 1N HCl, filtered through a 0.22 μm syringe filter, and lyophilized to afford a light-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, 22.7 ppm; Mw (GPC, Aqueous): 5660 Da; Mn (GPC, Aqueous): 6994 Da; PDI (GPC, Aqueous): 1.12; Mp (DSC): 15.6° C.
Hydrogels were prepared by combining the crosslinking agents of Example 9 and the macromer of Example 10. The hydrogels were prepared at a ratio of 2:1, crosslinking agent:PEI(SH)4. The crosslinking agents 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-lumen syringe with a mixing tip and injected into a cylindrical mold to form a solid hydrogel.
Gelation kinetics were assessed by following the disappearance of the maleimide alkene peak in 1H NMR upon mixing the maleimide crosslinking agents with mercaptopropionic acid, a PEI-SH mimetic, at 6.70 ppm. An NMR spectrum was recorded every 0.4 s for approximately 20 seconds after injecting 2 equivalents of mercaptopropionic acid, used as a PEI-SH mimetic in situ. No alkene peak was observed at 6.70 ppm immediately following injection of PEI-SH mimetic, exhibiting gelation kinetics faster than 0.4 s.
After gelation, storage modulus of the hydrogel was determined as an assessment of mechanical strength by strain and frequency sweeps to determine the linear viscoelastic region (LVER). The LVER exists to 10 strain %, and is the maximum strain that can be applied to these hydrogels before plastic deformation occurs. A frequency sweep was performed within the LVER at 3% strain, from 0.1-10 Hz. The initial storage moduli of our hydrogels were between 2000-5000 Pa. Upon hydrogel swelling in 50 mM PBS, crosslinkers 4, 5, and 6 exhibited decreasing storage moduli.
To estimate the rates of degradation and confirm the location of hydrolysis at the internal thioester instead of the ester, the 1H NMR crosslinking agent spectrum was monitored over 20 minutes in 0.3M sodium bicarbonate buffer, pH 8.0. The methylene adjacent to the internal thiol shifted from 3.41 ppm to 3.17 ppm, while the methylene peak adjacent to the ester linkage at 4.15 ppm, corresponding to the other terminal methylene on the diacid linkage in the crosslinking agent (succinic acid, glutaric acid, adipic acid), did not shift during base-catalyzed hydrolysis. This 1H NMR shift confirmed selective hydrolysis of the thioester.
Varying degradation rates, of >4 hours, >24 hours, and >7 days, for hydrogels prepared with crosslinkers 4, 5, and 6, increased relative to the hydrophobic methylene chain lengths of the internal diacid linkage protecting the internal thioesters of the crosslinking agent from hydrolysis. Crosslinker 4 contains an internal succinic acid linkage with two methylenes, while crosslinkers 5 and 6 contain glutaric acid and adipic acid linkages of three and four methylenes, respectively. The longer and more hydrophobic methylene chain length in the crosslinking agent, the more stable the thioester is believed to be against hydrolytic cleavage, resulting in slower degradation rates. The varying degradation rates, relative to the methylene chain length within crosslinkers 4, 5, and 6, allows for tuning the hydrogel mechanical properties through crosslinking agent structure in order to maintain mechanical integrity.
Swelling was observed between 200-400% (
On-demand dissolution time of the hydrogels via thiol-thioester exchange was assessed when submerged in a 0.3M cysteine methyl ester (CME) solution (
Prior to ex vivo studies, the cytotoxicity of the hydrogels against NIH3T3 fibroblasts was assessed over 24 hours of exposure. Hydrogels 4 and 5 exhibited a mean cell viability of 60%, while hydrogel 6 showed a mean cell viability of 98%. Low cell viability in hydrogels 4 and 5 was attributed to rapid release of succinic and glutaric acid, due to disassembly of the hydrogel network, and increased local acidity in the confined environment of a trans-well plate.
The hydrogel burst pressure was determined by injecting the macromers into one end of an ex vivo 2 cm porcine carotid artery at a total volume of 1 mL to form the hydrogel. The hydrogel filled the vessels and remained in place. After storing the plugged artery in a humid environment for 30 minutes (e.g., mirroring the time during an exemplary surgical procedure) the vessel was attached to a custom in-house burst pressure system with a pressure transducer connected to a computer, and a syringe pump (
It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims priority to U.S. Provisional Application No. 63/223,808 filed on Jul. 20, 2021, and to U.S. Provisional Application No. 63/260,113 filed on Aug. 10, 2021, both of which are incorporated by reference herein in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63223808 | Jul 2021 | US | |
| 63260113 | Aug 2021 | US |
