Reactive Hydrogel-Forming Formulations and Related Methods

Abstract

Compositions and methods related to hydrogel tissue sealants are generally described. In certain embodiments, the hydrogel-forming composition is provided in dry form (e.g., as one or more powder mixtures) and includes at least an electrophilic polymeric crosslinker and a nucleophilic polymer, such as a protein, capable of crosslinking with the crosslinker. One or more solvents capable of dissolving the crosslinker and the protein are provided and can be used to dissolve the hydrogel-forming composition and promote crosslinking.

Description

[Technical Field]

[0001] Technical Field Compositions and methods relating to hydrogel tissue sealants are generally described. [Background technology]

[0002] background Pneumothorax is a problematic complication of lung biopsy procedures, in which air enters the pleural space as a result of puncture of the parietal and visceral pleura. Pneumothorax causes significant concern for clinicians performing percutaneous lung biopsies and for patients undergoing percutaneous lung biopsies. The incidence of pneumothorax in patients undergoing percutaneous lung biopsies has been reported to range from approximately 9% to approximately 54% of patients, averaging approximately 15%. Furthermore, on average, approximately 7% of all percutaneous lung biopsies result in pneumothorax, requiring the placement of a chest tube in the patient, which subsequently results in an average hospital stay of approximately three days. Factors that increase the risk of pneumothorax include increasing patient age, obstructive pulmonary disease, increasing lesion depth, passage of multiple pleural layers, increased needle traversal time, and traversing a fissure. Pneumothorax can occur during or immediately after the lung biopsy procedure. Other complications of percutaneous lung biopsy include hemoptysis, hemothorax, infection, and air embolism. The development of novel hydrogel tissue sealants and related methods that have the ability to adhere to and / or seal against tissue (eg, the pleura) to address pneumothorax and other surgical applications would be beneficial. Summary of the Invention [Means for solving the problem]

[0003] overview Compositions and methods for forming hydrogel tissue sealants are generally described. The subject matter of the present invention includes, in some cases, interrelated products, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

[0004] In some embodiments, a hydrogel-forming composition for forming a hydrogel tissue sealant is described. In certain embodiments, the hydrogel-forming composition includes a first component comprising a crosslinker, wherein the crosslinker has the formula: G-LM-PEG-LM-G is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate diradical of formula -C(O)-, b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl. In certain embodiments, the hydrogel-forming composition includes a second component comprising a protein capable of crosslinking with the crosslinker. In some embodiments, the hydrogel-forming composition includes one or more solvents capable of dissolving the first component and the second component, and a surfactant. In certain embodiments, when the first component, the second component, and the surfactant are all dissolved in one or more solvents, crosslinking of the crosslinker and the protein occurs, forming the hydrogel tissue sealant.

[0005] In some embodiments, the hydrogel-forming composition for forming the hydrogel tissue sealant comprises a crosslinker, wherein the crosslinker has the formula: G-LM-PEG-LM-G is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d-OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and (Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In certain embodiments, the hydrogel-forming composition comprises a protein capable of crosslinking with a crosslinker, one or more solvents capable of dissolving the first and second components, and a surfactant, such that when the crosslinker, protein, and surfactant are all dissolved in the one or more solvents, crosslinking of the crosslinker and protein occurs to form a hydrogel tissue sealant.

[0006] According to certain embodiments, a hydrogel-forming composition for forming a hydrogel tissue sealant includes a first component comprising a crosslinker, wherein the crosslinker has the formula: G-LM-PEG-LM-G is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CHd an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and (Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In some embodiments, the hydrogel-forming composition comprises a second component comprising a protein capable of being crosslinked with a crosslinker, and one or more solvents capable of dissolving the first and second components, wherein when the first and second components are dissolved in the one or more solvents, upon mixing of the first and second components dissolved in the one or more solvents, crosslinking of the crosslinker and the protein occurs with a gelation time of 20 seconds or less to form a hydrogel tissue sealant.

[0007] According to some embodiments, a hydrogel-forming composition for forming a hydrogel tissue sealant includes a first component comprising a crosslinker, wherein the crosslinker has the formula: G-LM-PEG-LM-G is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl. In certain embodiments, the hydrogel-forming composition comprises a second component comprising a protein capable of being crosslinked with a crosslinker, a first solvent capable of dissolving the first component, and a second solvent capable of dissolving the second component, wherein when the second component is dissolved in the second solvent, the pH of the solution of the second component in the second solvent is 10.2 or more and 10.6 or less, and when the first component is dissolved in the first solvent and combined with the solution of the second component in the second solvent, a crosslinked solution of the first component and the second component is formed.

[0008] In certain embodiments, a hydrogel-forming composition for forming a hydrogel tissue sealant comprises a first component comprising a crosslinker, wherein the crosslinker has the formula: G-LM-PEG-LM-G is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d-OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) According to certain embodiments, the hydrogel-forming composition comprises a second component comprising a protein capable of being crosslinked with a crosslinker, and one or more solvents capable of dissolving the first component and the second component such that, when the first component and the second component are mixed separately with the one or more solvents, at least the second component is capable of having a dissolution time of 30 seconds or less at 25°C.

[0009] In certain embodiments, methods of forming a hydrogel tissue sealant are described. In some embodiments, the methods include dissolving a first component in a first solvent, wherein the first component has the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; da divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oxiimidazolyl, and tresyl.) In certain embodiments, the method includes dissolving a second component comprising a protein capable of cross-linking with a cross-linking agent in a second solvent, and combining the dissolved first component with the dissolved second component to form a hydrogel-forming composition comprising the cross-linking agent, the protein, and a surfactant to initiate cross-linking of the cross-linking agent and the protein, thereby forming the hydrogel tissue sealant.

[0010] According to certain embodiments, a method of forming a hydrogel tissue sealant includes dissolving a first component in a first solvent, wherein the first component has the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In some embodiments, the method includes dissolving a second component comprising a protein capable of crosslinking with a crosslinker in a second solvent, and combining the dissolved first component with the dissolved second component to form a hydrogel-forming composition comprising the crosslinker and the protein, thereby initiating crosslinking of the crosslinker with the protein such that the crosslinking is characterized by a gelation time of 20 seconds or less to form the hydrogel tissue sealant.

[0011] In some embodiments, a method of forming a hydrogel tissue sealant comprises dissolving a first component in a first solvent to form a solution of the first component, wherein the first component has the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d-OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oxiimidazolyl, and tresyl.) In certain embodiments, the method includes dissolving a second part comprising a protein capable of crosslinking with a crosslinker in a second solvent to form a solution of the second part having a pH of 10.2 or greater and 10.6 or less, and combining the solutions of the first and second parts to form a hydrogel-forming composition comprising the crosslinker and the protein, thereby initiating crosslinking of the crosslinker and the protein to form the hydrogel tissue sealant.

[0012] According to certain embodiments, a method of forming a hydrogel tissue sealant includes dissolving a first component in a first solvent, wherein the first component has the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; da divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In some embodiments, the method includes dissolving a second component comprising a protein capable of crosslinking with a crosslinker in a second solvent, wherein the dissolution time of the second component in the second solvent at 25° C. is 30 seconds or less, and combining the dissolved first component with the dissolved second component to form a hydrogel-forming composition comprising the crosslinker and the protein, thereby initiating crosslinking of the crosslinker and the protein and forming the hydrogel tissue sealant.

[0013] In some embodiments, the method of forming a hydrogel tissue sealant comprises providing a hydrogel tissue sealant having the formula: G-LM-PEG-LM-G forming a hydrogel-forming composition comprising a crosslinker, the crosslinker being a difunctionalized polyalkylene oxide-based component of the formula: (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In some embodiments, the solution comprises a protein capable of cross-linking with a cross-linking agent and a surfactant, wherein the hydrogel-forming composition, upon formation, results in the initiation of cross-linking of the cross-linking agent with the protein, thereby forming a hydrogel tissue sealant.

[0014] In some embodiments, methods of sealing tissue are described. In certain embodiments, the methods include delivering a hydrogel-forming composition to a tissue site, wherein the hydrogel-forming composition comprises: formula: G-LM-PEG-LM-G a first component comprising a crosslinker, the crosslinker being a difunctionalized polyalkylene oxide-based component of (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2)c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl; and a second component comprising a protein capable of being cross-linked with the cross-linking agent; In certain embodiments, the hydrogel-forming composition further comprises a surfactant.

[0015] According to certain embodiments, a method of sealing tissue includes delivering a hydrogel-forming composition to a tissue site, wherein the hydrogel-forming composition comprises: formula: G-LM-PEG-LM-G a solution of a first component comprising a crosslinker, the crosslinker being a difunctionalized polyalkylene oxide-based component of (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d- (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl; and A second component solution containing a protein capable of being cross-linked with a cross-linking agent, the solution having a pH of 10.2 or more and 10.6 or less. is the reaction product of

[0016] According to some embodiments, a method of sealing tissue includes delivering a hydrogel-forming composition to a tissue site, wherein the hydrogel composition comprises: formula: G-LM-PEG-LM-G a first component comprising a crosslinker, the crosslinker being a difunctionalized polyalkylene oxide-based component of (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated;d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl; and a second component comprising a protein capable of being cross-linked with the cross-linking agent; In certain embodiments, the method includes forming a hydrogel tissue sealant at the tissue site by a crosslinking reaction characterized by a gelation time of 20 seconds or less.

[0017] In certain embodiments, a kit for forming a hydrogel tissue sealant is described, wherein the kit includes a first component contained within a first container, the first component having the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In certain embodiments, the kit includes a second component housed with a second container, wherein the second component comprises a protein capable of cross-linking with the cross-linking agent and a surfactant.

[0018] According to some embodiments, a kit for forming a hydrogel tissue sealant includes a first component in powder form contained within a first container, wherein the first component has the formula: G-LM-PEG-LM-G a crosslinker which is a difunctionalized polyalkylene oxide-based element; (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and (Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In some embodiments, the kit includes a second component in powder form contained in a second container, the second component including a protein capable of being crosslinked with a crosslinking agent, a first aqueous hydration solution contained in a third container and capable of dissolving the first component, and a second aqueous hydration solution contained in a fourth container and capable of dissolving the second component.

[0019] In certain embodiments, a kit for forming a hydrogel tissue sealant includes one or more syringes collectively containing at least three separate containers, wherein a first container contains a first component in powder form, a second container contains a second component in powder form, and at least a third container contains one or more solvents, wherein the one or more syringes are configured to facilitate mixing of the first component with the one or more solvents to form a solution of the first component and to facilitate mixing of the second component with the one or more solvents to form a solution of the second component, and wherein the one or more syringes are further configured to mix the solution of the first component with the solution of the second component to form a crosslinked solution of the first component and the second component capable of forming a hydrogel tissue sealant, wherein the first component comprises an electrophilic biodegradable polymer and the second component comprises a nucleophilic biodegradable polymer capable of crosslinking with the electrophilic biodegradable polymer.

[0020] According to some embodiments, the hydrogel-forming composition for forming the hydrogel tissue sealant has the formula: G-LM-PEG-LM-G a first component comprising a crosslinker that is a difunctionalized polyalkylene oxide-based component of the formula: (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) bMonoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c -C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl.) In certain embodiments, the hydrogel-forming composition comprises a second component comprising a protein capable of being crosslinked with a crosslinker and one or more solvents, wherein the first component and the second component are dissolved in the one or more solvents.

[0021] In certain embodiments of the hydrogel-forming composition, the difunctionalized polyalkylene oxide-based entity has the formula G-LM-(OCH2CH2) nO-LM-G, where n is an integer of 10 to 500, preferably 50 to 200.

[0022] In certain embodiments of the hydrogel-forming composition, the leaving group G in the difunctionalized polyalkylene oxide-based component is N-oxysuccinimidyl.

[0023] In certain embodiments of the hydrogel-forming composition, the bifunctional linking moiety L in the bifunctionalized polyalkylene oxide-based element is -(CH) b -C(O)- and -C(O)-(CH2) c -C(O)-, where b and c are both integers from 1 to 10.

[0024] In certain embodiments of the hydrogel-forming composition, the difunctionalized polyalkylene oxide-based component is [ka] In both formulas, n is an integer of 10 to 500, preferably 50 to 200.

[0025] In certain embodiments of the hydrogel-forming composition, the protein is selected from the group consisting of human serum albumin, recombinant human serum albumin, and animal-derived albumin.

[0026] In certain embodiments of the hydrogel-forming composition, the protein is recombinant human serum albumin.

[0027] In certain embodiments of the hydrogel-forming composition, the composition further comprises a surfactant dissolved in one or more solvents.

[0028] In certain embodiments of the hydrogel-forming composition, the surfactant is selected from non-functionalized PEG, dextran sulfate, poloxamer, polysorbate, oil, siloxane, stearate, and / or glycol, preferably having a weight average molecular weight of 1000 g / mol to 40000 g / mol.

[0029] In certain embodiments of the hydrogel-forming composition, the one or more solvents comprise water in an amount of 50% to 100% by weight, preferably 90% to 100% by weight, based on the total amount of solvent.

[0030] In certain embodiments of the hydrogel-forming composition, the difunctionalized polyalkylene oxide-based component is [ka] (wherein n is an integer of 10 to 500, preferably 50 to 200); The protein is recombinant human serum albumin; The surfactant is a non-functionalized PEG; and Water comprises 90% or more by weight of the total amount of the one or more solvents.

[0031] In certain embodiments of the hydrogel-forming composition, the composition further comprises a cross-linking initiator, an antioxidant, and / or a radio-opacifying agent.

[0032] In certain embodiments of the hydrogel-forming composition, the composition comprises a base or a basic buffer, preferably a carbonate and / or bicarbonate.

[0033] In certain embodiments of the hydrogel-forming composition, the composition comprises an antioxidant, preferably butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate d-alpha tocopheryl polyethylene glycol-1000 succinate, or sodium metabisulfite, and / or mixtures thereof.

[0034] In certain embodiments of the hydrogel-forming composition, the composition comprises a radiopaque agent, preferably gold, silver, iodine, potassium chloride, barium sulfate, iohexol, or diatrizoate, and / or mixtures thereof.

[0035] In certain embodiments of the hydrogel-forming composition, the first component of the composition is dissolved in a first solvent.

[0036] In certain embodiments of the hydrogel-forming composition, the second component of the composition is dissolved in a second solvent.

[0037] In certain embodiments of the hydrogel-forming composition, the second component dissolved in the second solvent has a pH of 10.2 to 10.6.

[0038] In certain embodiments, a first container containing a first component comprising a cross-linking agent as defined herein; a second container containing a second component comprising a protein, preferably a protein selected from the group consisting of human serum albumin, recombinant human serum albumin, and animal-derived albumin; and, optionally, one or more additional containers containing one or more solvents, preferably water, for dissolving the first and second components. 10. A kit for forming a hydrogel tissue sealant, comprising:

[0039] In certain embodiments, the kit comprises a first container containing a first component; a second container containing a second component; and a third container containing a solvent, preferably water, for dissolving the first and second components.

[0040] In certain embodiments, the kit includes two syringes, wherein a first syringe includes a first container and a second container; a second syringe includes a third container; the first and second components contained in the syringes are in powder form; the first and second syringes are configured to be fluidically connectable to each other such that the first and second containers are disposed in fluid communication with the third container to facilitate mixing of the first and second components with a solvent to form a solution of the first component in the first container and a solution of the second component in the second container, and the first syringe is further configured to mix the solution of the first component with the solution of the second component to form a hydrogel-forming composition for forming a hydrogel tissue sealant. In certain embodiments, such a kit comprises a first container housing a first component; a second container housing a second component; a third container housing a solvent, preferably water, for dissolving the first component; and a fourth container housing a solvent, preferably water, for dissolving the second component, and may further comprise two syringes, the first syringe comprising the first and second containers; the second syringe comprising the third and fourth containers; the first and second components being in powder form; the first and second syringes configured to be fluidly connectable to each other such that the first and second containers are disposed in fluid communication with the third and fourth containers, respectively, to facilitate mixing of the first component with the solvent in the third container to form a solution of the first component in the first container and to facilitate mixing of the second component with the solvent in the fourth container to form a solution of the second component in the second container, and the first syringe is further configured to mix the solution of the first component with the solution of the second component to form a hydrogel-forming composition for forming a hydrogel tissue sealant.

[0041] In certain embodiments, any hydrogel-forming composition described herein and / or prepared using any kit described herein is suitable for use in a method of surgical treatment. In certain embodiments, such a method of surgical treatment includes delivering the hydrogel-forming composition to a tissue site and forming a hydrogel tissue sealant at the tissue site. In certain embodiments, the surgical treatment is a lung biopsy procedure, and the composition is used to prevent or reduce the risk of pneumothorax during or after the lung biopsy procedure, which can be a procedure in which any hydrogel-forming composition described herein is delivered to a patient's pleural cavity to form a hydrogel tissue sealant through which a biopsy sample is taken.

[0042] In certain embodiments, a kit for forming a hydrogel tissue sealant includes a first container containing a first component comprising a crosslinker, a second container containing a second component comprising a protein, preferably a protein selected from the group consisting of human serum albumin, recombinant human serum albumin, and animal-derived albumin, and, optionally, one or more additional containers containing one or more solvents, preferably water, for dissolving the first and second components.

[0043] According to certain embodiments, the hydrogel-forming compositions described above, or prepared using the kits described above, can be used in methods of surgical treatment.

[0044] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention are described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical element depicted is typically represented by a single numeral. For clarity, not every element is labeled in every figure, and not every element of every embodiment of the present invention is shown, unless explanation is necessary for those skilled in the art to understand the invention. [Brief explanation of the drawings]

[0046] [Figure 1] 1 illustrates steps in an exemplary method for forming a hydrogel tissue sealant, according to certain embodiments. [Figure 2A] 1 shows a schematic diagram of a syringe device configured to store and / or mix one or more components of a hydrogel-forming composition, according to certain embodiments. [Figure 2B] 1 shows a schematic diagram of a syringe device configured to deliver a hydrogel-forming composition to a tissue site, according to certain embodiments. [Figure 3A] 2B shows a cross-sectional schematic view of the syringe device shown in FIG. 2A, according to certain embodiments. [Figure 3B] 2C illustrates a cross-sectional schematic view of the syringe device shown in FIG. 2B, according to certain embodiments. [Figure 4] 1 illustrates steps in an exemplary method for hydrating and delivering a hydrogel-forming composition, according to certain embodiments. [Figure 5A] 1 shows an x-ray image of a pig lung model, according to certain embodiments. [Figure 5B] 1 shows an x-ray image of a post-biopsy pig lung model, according to certain embodiments. [Figure 6A] 1 shows an x-ray image of the coaxial insertion of a syringe needle to deliver hydrogel tissue sealant into a porcine lung model, according to certain embodiments. [Figure 6B] 1 shows an x-ray image of a porcine lung model having a hydrogel tissue sealant, according to certain embodiments. [Figure 7A] 1 shows an image of a hydrogel tissue sealant adhered to the parietal pleura of a porcine lung model, according to certain embodiments. [Figure 7B] 1 shows an image of a hydrogel tissue sealant adhered to the parietal and visceral pleura of a porcine lung model, according to certain embodiments. [Figure 8A] FIG. 2C shows a schematic diagram of the syringe device depicted in FIG. 2B with the coaxial cannula of the syringe device inserted into the pleural cavity of a subject being treated, prior to deployment of the hydrogel-forming composition contained within the syringe, according to certain embodiments. [Figure 8B] FIG. 8B shows a schematic diagram of the syringe device of FIG. 8A with the plunger depressed to deposit a hydrogel-forming composition to deliver and form a hydrogel lung sealant in the pleural cavity of a subject being treated, according to certain embodiments. [Figure 8C] 8C shows a schematic diagram of a biopsy needle inserted through the coaxial cannula of the syringe device of FIG. 8B, according to certain embodiments. [Figure 9A] 1 shows a CT scan of a subject (Subject 5) three days after hydrogel placement and subsequent lung biopsy procedure, according to certain embodiments. [Figure 9B] 1 shows a CT scan of a control subject (Control Subject 9) demonstrating air embolism immediately following a lung biopsy procedure, according to certain embodiments. [Figure 9C] 1 shows a CT scan of a control subject (Control Subject 10) exhibiting pneumothorax immediately following a lung biopsy procedure, according to certain embodiments. [Figure 9D] 1 shows a CT scan of a control subject (Control Subject 6) exhibiting pneumothorax two days after a lung biopsy procedure, according to certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description Compositions and methods related to hydrogel tissue sealants are generally described. In certain embodiments, the hydrogel-forming composition is provided in dry form (e.g., as one or more powder mixtures) and includes at least a crosslinker and a protein capable of crosslinking with the crosslinker. A solvent (i.e., one or more solvents) capable of dissolving the crosslinker and protein may be provided and used to dissolve the hydrogel-forming composition and promote crosslinking. Surfactants that can stabilize the hydrogel-forming composition, increase the rate at which the protein dissolves in one or more solvents, and / or prevent protein aggregation may also be added to either one or more components of the powder mixture or one or more solvents. While in certain embodiments, all of the components of the composition may be part of a single dry mixture (e.g., a powder mixture), in other embodiments that may result in further stability and improved shelf life, the composition may be separated into two or more reactive components (e.g., two or more dry powder mixtures), with at least a first and a second component containing a component that reacts with one or more components of another of the components. Preferably, in such embodiments, the components in each of the components are substantially unreactive with the other components in such components, so that reaction may be prevented until the components are dissolved (e.g., hydrated) in one or more suitable solvents and combined prior to or during use, thereby allowing them to react to form a hydrogel. When the components are separated into components that are substantially unreactive with the other components in such components, the components may be formulated and stored in a hydrated, flowable form rather than a dry, non-hydrated form. In many of the following discussions and examples, the compositions are provided as two dry powder components prior to hydration and mixing of the components to form a crosslinked hydrogel tissue sealant, although, as indicated above, other dry and flowable formulations are possible.

[0048] As an example, crosslinking to form a hydrogel tissue sealant can be initiated by combining a first component containing a crosslinker with a second component containing a protein. In certain embodiments, a surfactant can be part of the second component. The first component can further include, for example, an antioxidant (e.g., a first antioxidant), which can be selected to increase the stability of the crosslinker. The second component can further include a second antioxidant, which can be selected to increase the stability of the protein. As a result, the hydrogel-forming composition can have both an increased shelf life and an increased storage capacity compared to other conventional hydrogel-forming compositions. For example, in some embodiments, the hydrogel-forming compositions described herein can be stored at room temperature for extended periods of time (e.g., 3 years or more) without the need for refrigeration. Other advantages of the hydrogel-forming compositions can include shorter, more tunable gelation times and increased pot life, both of which are further described in more detail below.

[0049] In certain embodiments, multi-component (e.g., two-component, three-component, four-component) composition formulations can be used. In some embodiments, the first component comprises a bifunctionalized polyalkylene oxide crosslinker, and the second component comprises a protein (e.g., lyophilized albumin) capable of crosslinking with the bifunctionalized polyalkylene oxide. In certain embodiments, the second component can also comprise a crosslinking initiator (e.g., a base such as sodium carbonate or a basic buffer) that initiates crosslinking between the crosslinker and the protein. In certain embodiments, as noted above, both the first and second components can be provided and stored as a powdered mixture. The powdered mixtures can be hydrated separately or simultaneously (e.g., with a solvent such as water, a biocompatible organic solvent, or an aqueous solution) and then combined (if hydrated separately) to form the hydrogel tissue sealant. In certain embodiments, the hydration solution that hydrates the first component (and / or the second component) can further comprise a radiopaque agent that renders the hydrogel tissue sealant visible, for example, spectroscopically. The hydration solution in which the second component (and / or the first component) is hydrated may include an anti-foaming additive, such as a poloxamer. In certain embodiments, the anti-foaming additive may assist in protein refolding upon hydration.

[0050] The hydrogel-forming compositions can be used to bond or seal tissues in vivo. In certain non-limiting embodiments, for example, the hydrogel-forming compositions may be particularly useful as pleural lung sealants to seal off air or fluid from entering the pleural cavity. In some such embodiments, the hydrogel lung sealant may advantageously reduce the incidence of complications, such as pneumothorax, during and / or after a lung biopsy procedure. In certain embodiments, in addition to or instead of use as a hydrogel tissue sealant, the compositions and methods described herein may be useful in various other medical applications, such as post-surgical adhesion barriers or wound dressings.

[0051] As used herein, the term "crosslink" refers to a chemical reaction between two or more similar or dissimilar polymers, copolymers, oligomers, or macromers that links the two or more similar or dissimilar polymers, copolymers, oligomers, or macromers by the formation of at least one covalent and / or ionic bond, or a chain extension between one or more polymers, copolymers, oligomers, and / or macromers that provides a longer chain of one or more polymers, copolymers, oligomers, and / or macromers by the formation of at least one covalent and / or ionic bond.

[0052] Electrophilic Crosslinkers According to certain embodiments, the hydrogel-forming composition comprises an electrophilic biodegradable polymer. In certain embodiments, the electrophilic biodegradable polymer can be a synthetic or naturally occurring polymer that contains, or has been functionalized to contain, one or more, preferably two or more, reactive electrophilic groups. Many suitable electrophilic biodegradable polymers are known to those skilled in the art. In some embodiments, for example, a particularly advantageous and preferred crosslinker of the hydrogel-forming composition comprises a difunctionalized polyalkylene oxide. In certain embodiments, the difunctionalized polyalkylene oxide has the formula: G-LM-PEG-LM-G having a composition described by: (In the formula: PEG is polyethylene glycol; Each LM is a carbonate divalent radical of formula -C(O)-, a divalent radical of formula -(CH) b Monoester divalent groups of the formula -C(O)- (wherein b is an integer from 1 to 10), c diester groups of the formula -C(O)-, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; d a divalent dicarbonate group of the formula -OC(O)- (wherein d is an integer of 1 to 10), a divalent group of the formula -N(H)-C(O)-(CH d an amide-containing divalent group of the formula -C(O)- (wherein d is an integer from 1 to 10), c-C(O)-N(H)-(CH2) d - (wherein c is an integer of 1 to 10 and d is an integer of 1 to 10), and amide-containing divalent groups of the formula -RC(O)-, -RC(O)-(CH2) c -C(O)-, -RC(O)-O-(CH2) d -OC(O)-, -RN(H)-C(O)-(CH2) d -C(O)- or -R-(CH2) c -C(O)-N(H)-(CH2) d - (wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments); and Each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oxiimidazolyl, and tresyl).

[0053] According to certain embodiments, the crosslinker has the formula: G-LM-PEG-LM-G a difunctionalized polyalkylene oxide of the formula: (In the formula: PEG is polyethylene glycol; Each LM is the same and has the formula -C(O)-, -(CH b -C(O)- (wherein b is an integer of 1 to 5), -C(O)-(CH2) c -C(O)- (where c is an integer from 2 to 10, and the aliphatic portion of the group can be saturated or unsaturated), -C(O)-O-(CH2) d -OC(O)- (wherein d is an integer of 2 to 10), and the formulas -RC(O)-, -RC(O)-(CH2) c -C(O)- or -RC(O)-O-(CH2) da difunctional linking moiety represented by an oligomeric divalent group represented by -OC(O)-, where c is an integer from 2 to 10, d is an integer from 2 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments; and Each G is the same and is a leaving group selected from the group N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl).

[0054] According to some embodiments, the hydrogel-forming composition comprises any of a variety of suitable crosslinkers (e.g., difunctionalized polyalkylene oxides). In some embodiments, the crosslinker is form: [ka] The compound is or comprises 2-arm PEG disuccinimidyl succinate (PEG(SS)2).

[0055] In certain embodiments, the cross-linking agent is form: [ka] is or comprises a 2-arm PEG carboxymethyl ester of

[0056] According to certain embodiments, the crosslinker (e.g., a bifunctionalized polyalkylene oxide) of the formula G-LM-PEG-LM-G can have any of a variety of suitable weight-average molecular weights. For example, in certain embodiments, the degree of ethoxylation in PEG (and the value of n in the above formula) is such that the crosslinker can have a weight-average molecular weight of 1 kDa or more, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 10 kDa or more, or 15 kDa or more. In certain embodiments, the crosslinker can have a weight-average molecular weight of 20 kDa or less, 15 kDa or less, 10 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, or 2 kDa or less. Combinations of the above-listed ranges are also possible (e.g., the crosslinker can have a weight-average molecular weight of 1 kDa or more and 20 kDa or less, and the crosslinker can have a weight-average molecular weight of 3 kDa or more and 5 kDa or less). Other ranges are also possible. In certain embodiments, with respect to the formulas for 2-arm PEG disuccinimidyl succinate and 2-arm PEG carboxymethyl ester shown above, n ranges from 10 to 500, more preferably from 50 to 200. In some embodiments, the weight average molecular weight of the crosslinker is measured using size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS).

[0057] According to certain embodiments, bifunctionalized polyalkylene oxide crosslinkers, which can be described by the formula G-LM-PEG-LM-G, such as but not limited to the examples above, can be prepared by any of a variety of suitable synthetic methods known to those skilled in the art, see, for example, U.S. Pat. No. 6,576,263, U.S. Re. Pat. No. RE38,827, and U.S. Re. Pat. No. RE38,158, each of which is incorporated herein by reference in its entirety.

[0058] In some embodiments, a difunctionalized polyalkylene oxide that can be described by the formula G-LM-PEG-LM-G can be prepared using known processes, procedures, or synthetic methods, such as those reported in U.S. Pat. No. 4,101,380 or U.S. Pat. No. 4,839,345, each of which is incorporated herein by reference in its entirety, or in International Application Publication No. PCT / US90 / 02133, filed April 19, 1990, or by Abuchowski et al., Cancer Biochem. Biophys., 7:175-186 (1984). Briefly, in certain embodiments, a polyalkylene oxide compound (e.g., polyethylene glycol, discussed below as illustrative) and a suitable acid anhydride are dissolved in a suitable polar organic solvent in the presence of a base and refluxed for a period of time sufficient to form a polyethylene glycol diester diacid. The diester diacid is then reacted with a leaving group such as an N-hydroxyimide compound in the presence of dicyclohexylcarbodiimide or another condensing agent in a suitable polar organic solvent and stirred at room temperature to form the desired bifunctional crosslinker.

[0059] All or part of the bifunctionalized polyalkylene oxide-based compounds that can be described by the formula G-LM-PEG-LM-G can be purchased commercially from commercial sources, including, but not limited to, NOF America Corporation, Laysan Bio, Inc., Sigma-Aldrich, and / or JenKem Technology USA. The bifunctionalized polyalkylene oxide-based compounds can also be readily synthesized by those skilled in the art of chemical synthesis in light of the teachings and exemplary methods described herein with respect to exemplary compositions, published literature, and the level of ordinary skill and knowledge of those of ordinary skill in the art.

[0060] In certain non-limiting embodiments, PEG(SS)2 can be synthesized by obtaining a linear PEG having an average weight-average molecular weight of 3,350 Da, corresponding to 75.7 oxyethylene repeating units. Linear PEG can be obtained, for example, from Dow Chemical Company. In some embodiments, linear PEG can be converted to PEG(SS)2 by a two-step synthesis. For example, in some instances, the first step can involve reacting the linear PEG with two equivalents of succinic anhydride to form an ester. The second step can involve reacting the ester with two equivalents of N-hydroxysuccinimide (NHS) to produce the crosslinker PEG(SS)2, resulting in a white solid two-arm crosslinker with two succinimidyl groups per molecule.

[0061] In certain embodiments, the bifunctionalized polyalkylene oxide-based compound of formula G-LM-PEG-LM-G comprises a leaving group G (e.g., N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oximidazolyl, and tresyl). In such embodiments, the leaving group G is an electrophilic leaving group capable of reacting with a nucleophilic group, such as an amine group of a protein. According to certain embodiments, the leaving group G reacts with an amine group of a nucleophile (e.g., a protein) to produce a crosslinked composition with simultaneous release of the leaving group G by formation of an amide bond. Such reactivity is further described in U.S. Pat. No. 6,458,147, which is incorporated herein by reference in its entirety.

[0062] According to certain embodiments, the purity of a difunctionalized polyalkylene oxide crosslinker can be measured by its percent difunctionality. A higher percentage of difunctionality can advantageously result in a higher degree and / or rate of crosslinking, providing hydrogels formed from the hydrogel-forming compositions with increased performance characteristics, such as faster gelation times, longer pot lives, and / or longer shelf lives, and / or improved mechanical properties or resorption times, each of which is described in more detail below. In some embodiments, the difunctionalized polyalkylene oxide crosslinker has a percent difunctionality of 75% by weight or greater, 80% by weight or greater, 85% by weight or greater, 90% by weight or greater, 95% by weight or greater, or 99% by weight or greater. In certain embodiments, the difunctionalized polyalkylene oxide crosslinker has a percent difunctionality of 70% by weight to 99.9% by weight, or 90% by weight to 95% by weight. Other ranges are possible. As used herein, the percent difunctionality of the difunctionalized polyalkylene oxide crosslinkers is determined by high performance liquid chromatography (HPLC).

[0063] In powdered form, the difunctionalized polyalkylene oxide crosslinker can have a relatively low weight percent water content. A low weight percent water content of the powdered difunctionalized polyalkylene oxide crosslinker can advantageously provide a hydrogel-forming composition with improved shelf life because of reduced hydrolysis of the difunctionalized polyalkylene oxide, thus maintaining the reactivity of the crosslinker over storage time. In certain embodiments, for example, the weight percent water content of the powdered difunctionalized polyalkylene oxide crosslinker can be 10 wt% or less, 9 wt% or less, 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4 wt% or less, 3 wt%, or 2 wt% or less, based on the total weight of the powdered difunctionalized polyalkylene oxide crosslinker. In certain embodiments, the weight percent water content of the powdered crosslinker can be 1% to 10% by weight, based on the total weight of the powdered difunctionalized polyalkylene oxide crosslinker, or 4% to 6% by weight, based on the total weight of the powdered difunctionalized polyalkylene oxide crosslinker. Other ranges are possible. The weight percent water content stated herein is measured using a moisture meter and / or Karl Fischer titration.

[0064] According to certain embodiments, the hydrogel-forming composition includes a powdered crosslinking agent in any of a variety of suitable amounts, expressed as a weight percent by mass (wt%) relative to the total amount of the powdered hydrogel-forming composition. For example, in some embodiments, the hydrogel-forming composition includes the crosslinking agent in an amount, on a powdered basis, of 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more, 45 wt% or more, 50 wt% or more, or 55 wt% or more of the total weight of the powdered hydrogel-forming composition. In certain embodiments, the hydrogel-forming composition includes the crosslinking agent in an amount, on a powdered basis, of 60 wt% or less, 55 wt% or less, 50 wt% or less, 45 wt% or less, 40 wt% or less, 35 wt% or less, 30 wt% or less, 25 wt% or less, 20 wt% or less, or 15 wt% or less of the total weight of the powdered hydrogel-forming composition. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes the crosslinker in an amount, on a powdered basis, of 10% to 60% by weight of the total weight of the powdered hydrogel-forming composition; the hydrogel-forming composition includes the crosslinker in an amount, on a powdered basis, of 25% to 30% by weight of the total weight of the powdered hydrogel-forming composition). Other ranges are also possible.

[0065] Nucleophilic biodegradable polymers such as proteins In certain embodiments, the hydrogel-forming composition comprises a nucleophilic biodegradable polymer capable of crosslinking with the electrophilic biodegradable polymer. In certain embodiments, the nucleophilic biodegradable polymer can be a synthetic or naturally occurring polymer that contains or has been functionalized to contain one or more, preferably two or more, reactive nucleophilic groups. Many suitable nucleophilic biodegradable polymers are known to those skilled in the art. In some embodiments, for example, a particularly advantageous and preferred nucleophilic biodegradable polymer is a protein. According to some embodiments, for example, the hydrogel-forming composition comprises a protein capable of crosslinking with the above-mentioned electrophilic crosslinkers (e.g., PEG(SS)2). In certain embodiments, the protein comprises serum albumin (SA). In some embodiments, the serum albumin can be human serum albumin (HSA) derived from donor blood, recombinant human serum albumin (rHSA) expressed in yeast and / or rice, and / or an animal-derived albumin, such as bovine serum albumin (BSA). According to certain embodiments, the protein (e.g., rHSA) can be lyophilized. Lyophilization of proteins may advantageously, according to some embodiments, prevent protein degradation and improve shelf life and / or dissolution time when dissolved in aqueous solvents, as described below.

[0066] In certain non-limiting embodiments, the protein can be Cohn analog culture grade BSA obtained from Proliant Biologicals. In some embodiments, the recombinant human serum albumin can be Cellastim recombinant human serum albumin, Healthgen recombinant human serum albumin, Optibumin recombinant human serum albumin, InVitria human serum albumin, or Albumedix human serum albumin.

[0067] In certain embodiments, the protein may be or include collagen or gelatin. Other proteins are also possible.

[0068] According to certain embodiments, the purity and / or amount of protein aggregates can be determined by the percentage of the amount of protein monomer in the protein source. In certain embodiments, for example, the protein may comprise 60% or more by weight of protein monomer, 65% or more by weight of protein monomer, 70% or more by weight of protein monomer, 75% or more by weight of protein monomer, 80% or more by weight of protein monomer, 85% or more by weight of protein monomer, 90% or more by weight of protein monomer, or 95% or more by weight of protein monomer. In certain embodiments, the protein comprises 99% or less by weight of protein monomer, 95% or less by weight of protein monomer, 90% or less by weight of protein monomer, 85% or less by weight of protein monomer, 80% or less by weight of protein monomer, 75% or less by weight of protein monomer, 70% or less by weight of protein monomer, 70% or less by weight of protein monomer, or 65% or less by weight of protein monomer. Combinations of the above-listed ranges are also possible (e.g., the protein comprises 60% or more by weight of protein monomer and 99% or less by weight of protein monomer, and the protein comprises 90% or more by weight of protein monomer and 95% or less by weight of protein monomer). Other ranges are also possible. As explained in more detail below, certain components of the hydrogel-forming composition (e.g., surfactants and / or antifoaming agents) can act to prevent aggregation and / or the formation of protein dimers or higher order multimeric structures.

[0069] The powdered protein may have a relatively low weight percent moisture content. In certain embodiments, for example, the weight percent moisture content of the powdered protein components may be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more (greater than or equal to), 8% or more, or 9% or more, based on the total weight of the powdered protein components. In some embodiments, the weight percent moisture content of the powdered protein may be 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less, based on the total weight of the powdered protein components. Combinations of the above-listed ranges are also possible (e.g., the weight percent moisture content of the powdered protein may be between 1% or more and 10% or less, based on the total weight of the powdered protein components, and the weight percent moisture content of the powdered protein may be between 4% or more and 6% or less, based on the total weight of the powdered protein components). Other ranges are also possible. As described herein, the weight percent water content is measured using a moisture meter and / or Karl Fischer titration.

[0070] According to certain embodiments, the overall powdered hydrogel-forming composition comprises a protein (e.g., albumin) in any of a variety of suitable weight percent by mass (wt%) amounts, based on the total weight of the powdered hydrogel-forming composition (i.e., based on the combined weight of both the protein-containing powdered component and the electrophilic polymer-containing crosslinker powdered component). For example, in certain embodiments, the hydrogel-forming composition comprises the protein in an amount, on a powdered basis, of 40 wt% or more, 45 wt% or more, 50 wt% or more, 55 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, or 75 wt% or more of the total weight of the powdered hydrogel-forming composition. In certain embodiments, the hydrogel-forming composition comprises the protein in an amount, on a powdered basis, of 80 wt% or less, 75 wt% or less, 70 wt% or less, 65 wt% or less, 60 wt% or less, 55 wt% or less, 50 wt% or less, or 45 wt% or less of the total weight of the powdered hydrogel-forming composition. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes the protein in an amount, on a powdered basis, of 40% to 80% by weight of the total weight of the powdered hydrogel-forming composition; the hydrogel-forming composition includes the protein in an amount, on a powdered basis, of 55% to 65% by weight of the total weight of the powdered hydrogel-forming composition). Other ranges are also possible.

[0071] The ratio of the number of leaving groups G (e.g., NHS) to the number of amine groups (e.g., of a protein) in the bifunctionalized polyalkylene oxide-based compound of the formula G-LM-PEG-LM-G can be any suitable ratio. In certain embodiments, for example, the ratio of leaving groups G to amine groups is 0.5:1 or more, 1:1 or more, 1.5:1 or more, 2:1 or more, or 2.5:1 or more. In some embodiments, the ratio of leaving groups G to amine groups is 3:1 or less, 2.5:1 or less, 2:1 or less, 1.5:1 or less, or 1:1 or less. Combinations of the above-listed ranges are also possible (e.g., the ratio of leaving groups G to amine groups is 0.5:1 or more and 3:1 or less, and the ratio of leaving groups G to amine groups is 2:1 or more and 2.5:1 or less). Other ranges are also possible.

[0072] In certain non-limiting embodiments, the crosslinker is PEG(SS)2, the protein is rHSA, and the ratio of NHS groups on PEG(SS)2 to amine groups on rHSA is 2.21: 1. In other non-limiting embodiments, the crosslinker is PEG(SS)2, the protein is rHSA, and the ratio of NHS groups on PEG(SS)2 to amine groups on rHSA is 2.65:1.

[0073] Crosslinking initiator In some embodiments, the cross-linking reaction occurring between an electrophilic cross-linker and a nucleophile (e.g., a protein) is pH-sensitive. In certain such embodiments, for example, the cross-linking reaction is inhibited at acidic pH and can be initiated and sustained by increasing the pH to neutral or basic values. In some embodiments, the hydrogel-forming composition includes a cross-linking initiator that initiates cross-linking between the cross-linker and the nucleophile (e.g., a protein). In certain embodiments, the cross-linking initiator can be combined with the protein as a powder mixture. In some such embodiments, the cross-linking initiator can be lyophilized along with the protein.

[0074] In certain embodiments, the crosslinking initiator may be a base or a basic buffer. In certain embodiments, for example, the crosslinking initiator comprises a base and / or a basic buffer that promotes the reaction between the leaving group G in a bifunctionalized polyalkylene oxide-based compound of the formula G-LM-PEG-LM-G and an amine group of a protein. Any of a variety of suitable bases or basic buffers may be utilized. In certain embodiments, where the nucleophilic compound comprises an amine group that reacts with the crosslinker, the basic crosslinking initiator is a base and / or a basic buffer that does not contain an amine functionality. In some embodiments, the base comprises a carbonate and / or bicarbonate (e.g., carbonate and / or bicarbonate). For example, in certain embodiments, the base or basic buffer comprises sodium carbonate. In some embodiments, the base or basic buffer comprises sodium bicarbonate. Other bases or basic buffers are possible.

[0075] The crosslinking reaction between the leaving group G and the amine group of the nucleophile (e.g., a protein) can occur at any of a variety of suitable pH values. In some embodiments, the crosslinking reaction is favored at high pH values. In certain embodiments, for example, the crosslinking reaction between the leaving group G and the amine group of the nucleophile (e.g., a protein) is initiated and occurs at a pH of 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher. In certain embodiments, the crosslinking reaction between the leaving group G and the amine group of the nucleophile is initiated and occurs at a pH of 12 or lower, 11 or lower, 10 or lower, or 9 or lower. Combinations of the above-listed ranges are also possible (e.g., the crosslinking reaction between the leaving group G and the amine group of the nucleophile is initiated and occurs at a pH between 7 and 11, inclusive; the crosslinking reaction between the leaving group G and the amine group of the nucleophile is initiated and occurs at a pH between 8 and 11, inclusive; the crosslinking reaction between the leaving group G and the amine group of the nucleophile is initiated and occurs at a pH between 9 and 11, inclusive; or the crosslinking reaction between the leaving group G and the amine group of the nucleophile is initiated and occurs at a pH between 10 and 11, inclusive). Other ranges are also possible.

[0076] In certain non-limiting embodiments, the crosslinking reaction between the leaving group G and the amine group of a nucleophile (e.g., a protein) can be initiated at a suitable pH to promote the reaction by combining a solution of the crosslinker with a solution of the nucleophile having a pH between 10.2 or more and 10.6 or less.

[0077] The hydrogel-forming composition can include a powdered cross-linking initiator (e.g., a base or basic buffering agent) in any of a variety of suitable amounts, in weight percent by mass (wt%), based on the total weight of the powdered hydrogel-forming composition. The amount of base or basic buffering agent can affect the reactivity of the hydrogel-forming composition, such as gelation time (described below) or other measures of the time it takes for the cross-linking agent to cross-link with a nucleophile (e.g., a protein). Thus, in certain embodiments, it can be advantageous to select the type and / or amount of base or basic buffering agent to promote the rate and / or extent of cross-linking so that the hydrogel can cross-link and form to effectively seal the tissue before or upon delivery of the hydrogel-forming composition to a tissue site.

[0078] In certain embodiments, the hydrogel-forming composition comprises a crosslinking initiator in an amount, on a powdered basis, of 0.1 wt.% or more, 0.2 wt.% or more, 0.5 wt.% or more, 1 wt.% or more, 1.5 wt.% or more, 2 wt.% or more, 2.5 wt.% or more, 3 wt.% or more, 4 wt.% or more, 5 wt.% or more, 6 wt.% or more, 7 wt.% or more, 8 wt.% or more, or 9 wt.% or more of the total weight of the powdered hydrogel-forming composition. In certain embodiments, the hydrogel-forming composition comprises a crosslinking initiator in an amount, on a powdered basis, of 10 wt.% or less, 9 wt.% or less, 8 wt.% or less, 6 wt.% or less, 5 wt.% or less, 4 wt.% or less, 3 wt.% or less, 2 wt.% or less, 1.5 wt.% or less, 1 wt.% or less, 0.5 wt.% or less, 0.2 wt.% or less, or 5 wt.% or less of the total weight of the powdered hydrogel-forming composition. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes a cross-linking initiator in an amount, on a powdered basis, of greater than or equal to 0.1 wt % and less than or equal to 10 wt % of the total weight of the powdered hydrogel-forming composition, or greater than or equal to 0.1 wt % and less than or equal to 5 wt % of the total weight of the powdered hydrogel-forming composition, and the hydrogel-forming composition includes a cross-linking initiator in an amount, on a powdered basis, of greater than or equal to 4 wt % and less than or equal to 8 wt % of the total weight of the composition). Other ranges are also possible.

[0079] According to certain embodiments, the cross-linking initiator may be compartmentalized to be part of the second component. For example, in some embodiments, the cross-linking initiator may be combined with the protein (e.g., albumin) as a powder mixture. In some such embodiments, the cross-linking initiator and protein may be lyophilized. In some alternative embodiments, the cross-linking initiator may be dissolved in a solvent (e.g., water) and used as a hydration solution to hydrate the second component (e.g., protein).

[0080] surfactants In certain embodiments, the hydrogel-forming composition includes a surfactant. In some embodiments, the surfactant can stabilize one or more components (e.g., the first component, the second component) of the hydrogel-forming composition. In certain embodiments, the surfactant can increase the rate of dissolution of a protein (e.g., albumin) in one or more solvents used to dissolve the protein. In some embodiments, the surfactant can be selected to prevent aggregation (e.g., clumping) of the protein (e.g., albumin).

[0081] Any of a variety of suitable surfactants may be utilized. In some embodiments, for example, the surfactant comprises non-functionalized polyethylene glycol (PEG). Any of a variety of non-functionalized PEGs may be utilized. In certain embodiments, the non-functionalized PEG will be solid at room temperature. In certain embodiments, the non-functionalized PEG will have, for example, a weight average molecular weight of 100 g / mol or more and 40,000 g / mol or less. In certain embodiments, the non-functionalized PEG is PEG 8000 (e.g., PEG having a molecular weight of 8000 g / mol). In certain embodiments, the surfactant comprises dextran sulfate. In some embodiments, the surfactant may comprise a poloxamer, a polysorbate (e.g., TWEEN®), or may include a purely lipophilic material such as an oil (e.g., mineral oil, vegetable oil), a siloxane, a stearate, a glycol, and / or a mixture thereof. In certain embodiments, for example, the poloxamer is Pluronic® L61. Other Pluronic® poloxamers may also be utilized.

[0082] In some embodiments, the surfactant may further function as an anti-foaming additive (although not all anti-foaming additives need be surfactants). An anti-foaming additive may advantageously prevent foaming and / or the formation of air bubbles when the hydrogel-forming composition is hydrated, thereby promoting cross-linking. In some embodiments, for example, the anti-foaming additive prevents the formation of air bubbles that would otherwise be present in the hydrated hydrogel-forming composition in the absence of the anti-foaming additive. Such air bubbles can interfere with cross-linking and weaken the resulting hydrogel network of the tissue sealant. In certain non-limiting embodiments, a poloxamer (e.g., Pluronic® L61) is an anti-foaming additive.

[0083] According to certain embodiments, the hydrogel-forming composition includes a powdered or liquid surfactant in any of various amounts, expressed as a weight percent by mass (wt%), based on the total weight of the powdered or aqueous solution of the powdered hydrogel-forming composition. In some embodiments, for example, the hydrogel-forming composition includes the surfactant in an amount, on a powdered or liquid basis, of 0.01 wt% or more, 0.1 wt% or more, 0.5 wt% or more, 1 wt% or more, 5 wt% or more, 10 wt% or more, or 15 wt% or more of the total weight of the powdered or aqueous hydrogel-forming composition. In certain embodiments, the hydrogel-forming composition includes the surfactant in an amount, on a powdered or liquid basis, of 20 wt% or less, 15 wt% or less, 10 wt% or less, 5 wt% or less, 1 wt% or less, 0.5 wt% or less, 0.1 wt% or less, or 0.01 wt% or less of the total weight of the powdered or aqueous hydrogel-forming composition. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes the surfactant in an amount, on a powdered or liquid basis, of 1% to 30% by weight of the total weight of the powdered or aqueous hydrogel-forming composition; the hydrogel-forming composition includes the surfactant in an amount, on a powdered or liquid basis, of 10% to 20% by weight of the total weight of the powdered or aqueous solution of the powdered hydrogel-forming composition). Other ranges are also possible.

[0084] In some embodiments, a surfactant can be part of the second component. For example, in certain embodiments, the surfactant can be combined with the protein (and cross-linking initiator, in some embodiments) as a powder mixture. In some such embodiments, the protein, cross-linking initiator, and surfactant can be lyophilized (e.g., prior to dissolution in a solvent). Without wishing to be bound by theory, in some embodiments in which a liquid surfactant is utilized (e.g., Pluronic® L61), the liquid surfactant can hydrogen bond with one or more powder components (e.g., proteins) of the second component. In some embodiments, the surfactant can be dissolved and / or mixed in a solvent (e.g., water) used as a hydration solution to hydrate the second component and form a hydrated solution that, when mixed with a solution of the first component, is capable of cross-linking with the first component. In certain embodiments, for example, the surfactant can be dispersed and / or suspended in the solvent used to hydrate the second component.

[0085] According to certain embodiments, the hydrogel-forming composition may include two or more surfactants (e.g., two surfactants, three surfactants, etc.), with each individual surfactant, or the cumulative amount of all surfactants combined, falling within any of the weight percent ranges listed above.

[0086] antioxidants According to certain embodiments, the hydrogel-forming composition may include at least one antioxidant. The antioxidant may advantageously increase the storage stability of one or more components of the hydrogel-forming composition. For example, the use of one or more antioxidants may increase the shelf life and / or storage capacity of the hydrogel-forming composition. Because the one or more antioxidants are more susceptible to oxidation than the cross-linking reagents used to form the hydrogel (e.g., due to a lower oxidation potential), the antioxidants may be oxidized during storage before the cross-linking reagents, resulting in a hydrogel-forming composition with a longer shelf life than an otherwise equivalent hydrogel-forming composition without the one or more antioxidants.

[0087] Any of a variety of suitable antioxidants may be utilized. In certain embodiments, for example, the composition includes butylated hydroxytoluene (BHT). In some such embodiments, BHT prevents free radical-mediated oxidation. In certain embodiments, BHT may be utilized to prevent oxidation of a bifunctionalized polyalkylene oxide crosslinker. In some embodiments, the composition includes N-acetyl-DL-tryptophan. In some such embodiments, N-acetyl-DL-tryptophan prevents oxidation of one or more amino acids and / or other residues of a protein. In certain embodiments, the antioxidant is or includes butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate d-alpha tocopheryl polyethylene glycol-1000 succinate, sodium metabisulfite, and / or mixtures thereof.

[0088] In some embodiments, the hydrogel-forming composition comprises at least two antioxidants. For example, in certain embodiments, the hydrogel-forming composition may comprise a first antioxidant (e.g., BHT) that prevents oxidation of the difunctionalized polyalkylene oxide crosslinker and a second antioxidant (e.g., N-acetyl-DL-tryptophan) that prevents oxidation of proteins.

[0089] In addition to preventing oxidation of one or more components of the hydrogel-forming composition, one or more antioxidants may, in some cases, stabilize one or more components of the hydrogel-forming composition (e.g., crosslinkers, proteins, etc.) to allow for sterilization with lethal doses of radiation (e.g., electron beam or gamma radiation). In certain embodiments, for example, one or more components of the hydrogel-forming composition (e.g., the first component and / or the second component) may be sterilized using electron beam irradiation. In some such embodiments, one or more components of the hydrogel may be exposed to one or more doses of electron beam irradiation, with a cumulative dose of between 25 kGy and 30 kGy.

[0090] According to certain embodiments, the hydrogel-forming composition includes each antioxidant (e.g., first antioxidant, second antioxidant) in any of a variety of suitable amounts, expressed as weight percent by mass (wt%), based on the total weight of the powdered hydrogel-forming composition. In some embodiments, for example, the hydrogel-forming composition includes each antioxidant in an amount, on a powdered basis, of 0.1 wt% or more, 1 wt% or more, 2 wt% or more, 5 wt% or more, 10 wt% or more, or 15 wt% or more of the total weight of the powdered hydrogel-forming composition. In certain embodiments, the hydrogel-forming composition includes each antioxidant in an amount, on a powdered basis, of 20 wt% or less, 15 wt% or less, 10 wt% or less, 5 wt% or less, 2 wt% or less, 1 wt% or less, or 0.1 wt% or less of the total weight of the powdered hydrogel-forming composition. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes each antioxidant in an amount, on a powdered basis, of greater than or equal to 0.1% and less than or equal to 20% by weight of the total weight of the powdered hydrogel-forming composition; the hydrogel-forming composition includes each antioxidant in an amount, on a powdered basis, of greater than or equal to 1% and less than or equal to 5% by weight of the total weight of the powdered hydrogel-forming composition). Other ranges are also possible.

[0091] According to certain embodiments, the antioxidant can be part of the first and / or second component. In some embodiments, for example, at least one antioxidant can be combined with the crosslinker and / or protein as a powder mixture. In some such embodiments, the antioxidant can be lyophilized along with the protein (and / or crosslinker, in some cases). In certain alternative embodiments, the antioxidant can be dissolved (e.g., in a solvent such as water) used as a hydration solution to hydrate the first and / or second component.

[0092] Radiopaque agents In some embodiments, the hydrogel-forming composition includes a radiopaque agent. The use of a radiopaque agent can impart the resulting hydrogel with the ability to be imaged spectroscopically, for example, by X-ray or computed tomography (CT) imaging. Any of a variety of suitable radiopaque agents can be added. In some embodiments, the radiopaque agent includes gold (e.g., gold nanoparticles), silver (e.g., silver nanoparticles), or iodine. In certain embodiments, the radiopaque agent is potassium chloride (KCl), barium sulfate, iohexol, or diatrizoate.

[0093] According to certain embodiments, the hydrogel-forming composition includes a radiopaque agent in any of various amounts, expressed as a weight percent by mass (wt%), based on the total weight of the powdered hydrogel-forming composition. In some embodiments, for example, the hydrogel-forming composition includes the radiopaque agent in an amount of 0.1 wt% or more, 0.5 wt% or more, 1 wt% or more, 5 wt% or more, 10 wt% or more, or 15 wt% or more of the total weight of the powdered hydrogel-forming composition, on a powdered basis. In certain embodiments, the hydrogel-forming composition includes the radiopaque agent in an amount of 20 wt% or less, 15 wt% or less, 10 wt% or less, 5 wt% or less, 1 wt% or less, or 0.5 wt% or less of the total weight of the powdered hydrogel-forming composition, on a powdered basis. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition includes the radiopaque agent in an amount, on a powdered basis, of greater than or equal to 0.1% and less than or equal to 20% by weight of the total weight of the powdered hydrogel-forming composition; the hydrogel-forming composition includes the radiopaque agent in an amount, on a powdered basis, of greater than or equal to 1% and less than or equal to 10% by weight of the total weight of the powdered hydrogel-forming composition). Other ranges are also possible.

[0094] Other agents In any of the above-described embodiments, the hydrogel-forming composition can include other active agents or components for a variety of purposes, such as any of a variety of suitable active agents, such as antimicrobial agents, anti-inflammatory agents, hemostatic agents, etc.

[0095] According to some embodiments, the hydrogel-forming composition is in the form of one or more powders (e.g., during storage of the hydrogel-forming composition). In certain embodiments, one or more powders of the hydrogel-forming composition can be hydrated with water or one or more aqueous solutions to form an aqueous solution of the hydrogel-forming composition comprising the crosslinker, protein, optional surfactant, optional antioxidant, and / or optional crosslinking initiator. In certain embodiments, forming the hydrogel-forming composition in the form of an aqueous solution comprising the crosslinker and protein initiates crosslinking of the crosslinker and protein, thereby forming the hydrogel tissue sealant.

[0096] As noted above, in certain embodiments, the hydrogel-forming composition may be stored and / or provided as a multi-component formulation in which some of the components are separated from others in different powdered or hydrated components. In some embodiments, for example, the hydrogel-forming composition comprises at least a first component, a second component, a solvent capable of dissolving the first and second components, and an optional surfactant. In some such embodiments, the first component comprises a crosslinker and an optional antioxidant, and the second component comprises a protein. The surfactant, in certain embodiments, may be part of the second component (e.g., a powder mix with the protein) or may be dissolved and / or otherwise mixed in the solvent used to hydrate the first and / or second components. In some embodiments, the hydrogel-forming composition comprises a crosslinking initiator, which may be part of the second component (e.g., a powder mix with the protein) or may be dissolved in the solvent used to hydrate one or more of the components (e.g., the second component). In certain embodiments, for example, the second component is a mixture (e.g., a lyophilized powder mixture) comprising a protein, a cross-linking initiator, and a surfactant. The hydrogel-forming composition may also include at least one antioxidant, which in some embodiments may be part of the first and / or second component or may be dissolved in the solvent used to hydrate the first and / or second component. In certain embodiments, the hydrogel-forming composition may also include a radiopaque agent, which in some embodiments may be part of the first and / or second component or may be dissolved in the solvent used to hydrate the first and / or second component.

[0097] In certain embodiments, it may be advantageous to store the first component (e.g., comprising the crosslinking agent (and optional antioxidant)) and the second component (e.g., comprising the protein (and optionally a crosslinking initiator and / or surfactant)) separately to avoid crosslinking of the crosslinking agent and protein during storage and / or to delay crosslinking until the hydrogel-forming composition is delivered to a tissue site. In certain embodiments, at least the second component comprising the protein, crosslinking initiator, and surfactant may be lyophilized, or at least the protein of such component is lyophilized.

[0098] According to some embodiments, the first component can be in the form of a first powder mixture, and the second component can be in the form of a second powder mixture (e.g., during storage of the first component and / or the second component). In certain embodiments, the first component and / or the second component, which are powder mixtures, can be separately solvated or hydrated with water or one or more solvents (e.g., water, a biocompatible organic solvent such as DMSO) or an aqueous solution (in the following description, "hydrated" is used for brevity, but it should be understood that "solvated" should substitute for "hydrated" in embodiments in which a non-aqueous solvent is used), thereby providing the first component in the form of a first solution (e.g., a first aqueous solution) and the second component in the form of a second solution (e.g., a second aqueous solution). In certain embodiments, it can be advantageous to dissolve the first component (e.g., a crosslinker) in a biocompatible organic solvent such as DMSO to extend the pot life of the hydrogel-forming composition. In some embodiments, the hydrated first component and the hydrated second component may be combined to initiate cross-linking of the protein with a cross-linking agent, thereby forming a hydrogel tissue sealant.

[0099] In certain embodiments, a first solution (e.g., for hydrating a first component containing a cross-linking agent) can include a radiopaque agent and / or a first antioxidant. In some embodiments, a second solution (e.g., for hydrating a second component containing a protein) can include a surfactant, a cross-linking initiator, and / or a second antioxidant.

[0100] When a protein is included as the nucleophilic polymer, the second component can be hydrated so that the concentration of the protein in the resulting hydrated solution is any of a variety of suitable amounts. In some embodiments, for example, the concentration of the protein in the hydrated second component is 10% by weight by volume or more, 15% by weight by volume or more, 20% by weight by volume or more, 25% by weight by volume or more, or 30% by weight by volume or more. In certain embodiments, the concentration of the protein in the hydrated second component is 35% by weight by volume or less, 30% by weight by volume or less, 25% by weight by volume or less, 20% by weight by volume or less, or 15% by weight by volume or less. Combinations of the above-listed ranges are also possible (e.g., the concentration of the protein in the hydrated second component is 10% by weight by volume or more and 35% by weight by volume or less, and the concentration of the protein in the hydrated second component is 20% by weight by volume or more and 25% by weight by volume or less). Other ranges are also possible.

[0101] According to certain embodiments, the lyophilized second component containing a protein may have a relatively fast dissolution time. As used herein, the term "dissolution time" is given its ordinary meaning in the art and generally refers to the time it takes for the second component containing a protein to completely dissolve when hydrated (or solvated) by mixing or agitation. A relatively fast dissolution time may advantageously reduce the time it takes to form a hydrogel tissue sealant. The dissolution time is calculated by starting a timer, hydrating the second component by mixing the second component with the hydration solution, and stopping the timer when the second component is completely dissolved. In some embodiments, the dissolution time of the second component at 25°C may be 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, or 35 seconds or more. In certain embodiments, the dissolution time of the second component at 25°C is 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, or 15 seconds or less. Combinations of the listed ranges are possible (e.g., the dissolution time of the second component at 25° C. is between 10 and 40 seconds, inclusive, and the dissolution time of the second component at 25° C. is between 20 and 30 seconds, inclusive). Other ranges are also possible.

[0102] In certain embodiments, the dissolution time of the second component may depend on the amount of protein in the second component upon hydration, i.e., its mass relative to the volume of the solution. For example, in some embodiments, the dissolution time of the second component is directly proportional to the amount of protein in the second component. In some non-limiting embodiments, assuming the final volumes of the second component containing a lower amount of protein and the second component containing a higher amount of protein are the same, for example, a second component containing a relatively lower amount of protein (e.g., 10% by mass to volume in the resulting hydration solution) will have a shorter dissolution time than an otherwise equivalent second component having a relatively higher amount of protein (e.g., 30% by mass to volume in the resulting hydration solution).

[0103] The solution of the lyophilized second component (e.g., comprising a protein and a cross-linking initiator) may have a relatively high pH upon dissolution. In certain embodiments, for example, the solution of the lyophilized second component has a pH of 9 or greater, 9.5 or greater, 10 or greater, or 10.5 or greater upon dissolution. In some embodiments, the solution of the lyophilized second component has a pH of 11 or less, 10.5 or less, 10 or less, or 9.5 or less upon dissolution. Combinations of the above-listed ranges are also possible (e.g., the solution of the lyophilized second component has a pH of 9 or greater and 11 or less upon dissolution, and the solution of the lyophilized second component has a pH of 10 or greater and 10.5 or less upon dissolution). Other ranges are also possible.

[0104] It should be understood that the amount of protein added need not be specified, as long as there is an amount of protein that, when hydrated as described herein, produces a pH within the range of the resulting solution. That said, in some embodiments, dissolving even a relatively small amount of the second component (e.g., 10% by weight to volume) can produce a solution having a pH within the range. For example, in some embodiments, the second component, when hydrated to form the resulting solution of protein, results in a solution having a pH of 9 or greater, 9.5 or greater, 10 or greater, or 10.5 or greater.

[0105] In certain embodiments, as described herein, the first and second components can be dissolved in one or more solvents and then combined / mixed together to form a crosslinked hydrogel-forming composition comprising a solution of the first and second components. In some embodiments, for example, the first component can be dissolved in a first solvent, the second component can be dissolved in a second solvent, and then they can be combined to form a hydrogel-forming composition solution. The hydrogel-forming composition solution of the first and second components can have a pH less than or substantially similar to the pH of the lyophilized solution of the second component. In certain embodiments, for example, the crosslinked solution of the first and second components has a pH of 7 or greater, 8 or greater, 9 or greater, 9.5 or greater, 10 or greater, or 10.5 or greater. In some embodiments, the crosslinked solution of the first and second components has a pH of 11 or less, 10.5 or less, 10 or less, or 9.5 or less. Combinations of the above-listed ranges are also possible (e.g., the cross-linking solutions of the first and second parts have a pH of 9 or greater and 11 or less, and the cross-linking solutions of the first and second parts have a pH of 10 or greater and 10.5 or less). Other ranges are also possible.

[0106] In certain non-limiting but advantageous embodiments, the first and second part hydrogel-forming composition solutions are formed by combining an unbuffered solution of the first part with a solution of the second part having a pH of 10.2 or greater and 10.6 or less.

[0107] The time it takes for a hydrated hydrogel-forming composition to crosslink and form a gel can determine how quickly the composition can act as a tissue sealant when the hydrogel / hydrogel-forming composition is delivered to a tissue site. When the hydrogel-forming composition is applied to a tissue site, it can be beneficial for the hydrogel-forming composition to crosslink within a short enough time frame to allow the applied composition to quickly seal the tissue puncture site or other wound surface. As used herein, "measured gelation time" refers to the time measured by dispensing a hydrated first component and a hydrated second component into a vial containing a stir bar on a stir plate adjusted to 300 RPM, recording the initial time (T0) at which the components of the composition are dispensed, followed by the end time (T) at which the stir bar stops rotating due to gelation. F ) The measured crosslinking time is the time the timer is stopped minus the initial time. In certain embodiments, rheometry may be utilized to determine the measured gel time.

[0108] The hydrogel-forming composition can have any of a variety of measured gel times suitable for a particular application and method of application. In some embodiments, for example, the hydrogel-forming composition can have a measured gel time of 0.1 seconds or more, 0.5 seconds or more, 1 second or more, 2 seconds or more, 3 seconds or more, 4 seconds or more, 5 seconds or more, 10 seconds or more, or 15 seconds or more. In certain embodiments, the hydrogel-forming composition has a measured gel time of 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, 2 seconds or less, 1 second or less, or 0.5 seconds or less. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition can have a measured gel time of 0.1 seconds or more and 20 seconds or less, and the hydrogel-forming composition can have a measured crosslinking time of 1 second or more and 3 seconds or less). Other ranges are also possible.

[0109] According to certain embodiments, the measured gelation time can be advantageously shortened by adjusting the amount of cross-linking initiator. In some embodiments, for example, sufficient cross-linking initiator is added to provide a suitable pH value, as described herein, to initiate the cross-linking reaction between the cross-linker and the protein in a given surgical environment. In certain embodiments, the cross-linking initiator provides a pH value of 10 or greater (e.g., 10.2-10.6, 10.3-10.4), which facilitates a faster cross-linking reaction, as the reaction is generally favored at higher pH values. In certain embodiments, the gelation time can be adjusted depending on the amount of base or basic buffer in the hydrogel-forming composition.

[0110] In some cases, it may be advantageous for the hydrogel-forming composition to have a sufficiently long measured pot life. As used herein, the term "measured pot life" refers to the duration during which the hydrated first and second components of the hydrogel-forming composition remain usable after hydration of one or more of the powdered reactive components (e.g., the first component and / or the second component) but before combining solutions of the first and second components to form a crosslinked hydrogel-forming composition solution. A sufficiently long pot life may, in some embodiments, advantageously allow the hydrated first and second components of the hydrogel-forming composition to remain usable after a user (e.g., a physician) hydrates the first and second components until the user is ready to deliver one or more components to a site of administration to form a hydrogel tissue sealant. As used herein, "measured pot life" is determined by measuring a particular performance indicator, such as, for example, gel time and / or burst strength (both of which are described in more detail herein), of a crosslinked hydrogel-forming composition or formed hydrogel and comparing it to an equivalent hydrogel-forming composition or formed hydrogel formed from freshly hydrated first and second components of the hydrogel-forming composition that has not been stored or used after delayed hydration. The measured pot life is the time it takes for the performance indicator of a crosslinked hydrogel composition formed from one or more hydrated components stored for a particular period of time to differ by a defined percentage (e.g., ±10%) from the performance indicator of a crosslinked hydrogel composition formed from one or more freshly hydrated components, based on clinically-based minimum values ​​for each performance indicator that ensure the hydrogel tissue sealant can safely perform its function (e.g., sealing tissue).

[0111] The hydrogel-forming components of the composition can have any of a variety of suitable pot-life times (defined as a performance index that varies by ±10% or less). In some embodiments, for example, the hydrogel-forming composition has a pot-life of 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 5 hours or more, or 10 hours or more. In certain embodiments, the hydrogel-forming composition has a pot-life of 24 hours or less, 10 hours or less, 5 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, or 20 minutes or less. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition has a pot-life of 10 minutes or more and 24 hours or less, and the hydrogel-forming composition has a pot-life of 1 hour or more and 2 hours or less). Other ranges are also possible.

[0112] According to certain non-limiting embodiments, the pot life of either or both of the reagent components of the hydrogel-forming composition may be increased, particularly in embodiments in which the components are provided in solvated form by dissolving such components in a biocompatible nonpolar organic solvent, such as DMSO, to form the solvated components of the hydrogel-forming composition. The use of such a solvent may, in certain cases, significantly increase the pot life, for example, to one week or more, one month or more, six months or more, one year or more, or two years or more.

[0113] According to some embodiments, it may be advantageous for a hydrogel-forming composition to have a sufficiently long measured shelf life. As used herein, the term "measured shelf life" refers to the duration during which one or more of the powdered components of a hydrogel-forming composition remain suitably usable following storage of the one or more powdered components. A sufficiently long shelf life may, in some embodiments, advantageously allow a hydrogel-forming composition to remain usable following extended storage of the hydrogel-forming composition. As used herein, "measured shelf life" is determined by, for example, measuring a particular performance indicator, such as gel time, dissolution time, and / or burst strength (described in more detail herein), of a crosslinked hydrogel-forming composition solution and / or a prepared crosslinked hydrogel composition formed therefrom that has been subjected to a storage period, relative to the corresponding measured indicator resulting from such one or more powdered components (e.g., the first and / or second components) of the hydrogel-forming composition that have not been subjected to storage prior to hydration. The measured shelf life is the time it takes for the performance index of a crosslinked hydrogel composition derived from one or more powdered components that has been subjected to storage for a period of time before hydration to differ from the same index measured for the fresh components by a specified percentage (e.g., ±10%), based on clinically based minimum values ​​for each performance index that ensure the hydrogel tissue sealant can safely perform its function (e.g., sealing tissue).

[0114] The first and second components of the hydrogel-forming composition can have any of a variety of suitable shelf-life times. In some embodiments, for example, the hydrogel-forming composition has a shelf-life (defined as ±10% difference from fresh components) of 1 week or more, 1 month or more, 6 months or more, 1 year or more, 2 years or more, 3 years or more, or 4 years or more. In certain embodiments, the hydrogel-forming composition has a shelf-life of 5 years or less, 4 years or less, 3 years or less, 2 years or less, 1 year or less, 6 months or less, or 1 month or less. Combinations of the above-listed ranges are also possible (e.g., the hydrogel-forming composition has a shelf-life of 1 week or more and 5 years or less, and the hydrogel-forming composition has a shelf-life of 1 year or more and 2 years or less). Other ranges are also possible.

[0115] Methods for forming a hydrogel tissue sealant are provided. In some embodiments, the methods include forming a crosslinking solution comprising at least a crosslinker and a nucleophilic biodegradable polymer (e.g., a protein), where the formation of the crosslinking solution initiates crosslinking of the crosslinker and the nucleophilic biodegradable polymer (e.g., a protein), thereby forming the hydrogel tissue sealant. In certain embodiments, the crosslinking solution comprises a crosslinking initiator, a surfactant, and / or an antioxidant, as described herein.

[0116] According to certain embodiments, a method of forming a hydrogel tissue sealant includes dissolving a first powdered component comprising a crosslinker (e.g., an electrophilic biodegradable polymer) and a second powdered component comprising a nucleophilic biodegradable polymer (e.g., a protein) in one or more solvents. In some embodiments, for example, the first component is dissolved in a first solvent (e.g., water or an aqueous solution) and the second component is dissolved in a second solvent (e.g., water or an aqueous solution). In some such embodiments, the dissolved component and the dissolved second component are combined to form a crosslinked hydrogel-forming composition in the form of a solution comprising the crosslinker and the protein, thereby initiating crosslinking of the crosslinker and the nucleophilic biodegradable polymer (e.g., a protein) to form the hydrogel tissue sealant.

[0117] In certain embodiments, a method for forming a hydrogel tissue sealant includes hydrating a first powdered component including a crosslinker and at least one antioxidant, hydrating a second powdered component including a protein, a crosslinking initiator, and a surfactant, and combining the hydrated first component with the hydrated second component to initiate crosslinking between the crosslinker and the protein. FIG. 1 illustrates steps in such an exemplary method for forming a hydrogel tissue sealant. Method 150 includes, in step 152, hydrating a first powdered component including, for example, a crosslinker. In some embodiments, the first powdered component optionally includes an antioxidant. In certain embodiments, the first powdered component is hydrated with a first solvent including water or a first aqueous solution. The first solvent, in some embodiments, can include a radiopaque agent. Step 154 ​​includes hydrating a second powdered component including, for example, a protein. In certain embodiments, the second powdered component includes a crosslinking initiator and / or a surfactant. In some embodiments, the second powdered component is hydrated with a second solvent comprising water or a second aqueous solution. In certain embodiments, the second solvent comprises an antifoaming agent. According to certain embodiments, steps 152 and 154 can occur simultaneously (but in separate containers). Step 156 comprises combining the hydrated first component with the hydrated second component to initiate crosslinking of the crosslinker with the protein to form the hydrogel tissue sealant.

[0118] Also disclosed herein are methods related to sealing tissue with a formed hydrogel composition. In some embodiments, for example, such methods include delivering to a tissue site a hydrogel-forming composition comprising a first component and a second component, or delivering to a tissue site a partially or fully crosslinked hydrogel composition, wherein the hydrogel composition comprises a reaction product of the first component reagent and the second component reagent described above.

[0119] The hydrogel composition can be delivered to the tissue site by any of a variety of suitable methods. In some embodiments, the first part of the hydrogel-forming composition and the second part of the hydrogel-forming composition can be at least partially combined and crosslinking initiated prior to delivery of the hydrogel composition to the tissue site. In certain embodiments, the first part of the hydrogel-forming composition and the second part of the hydrogel-forming composition are fully combined prior to delivery of the hydrogel composition to the tissue site. According to certain embodiments, the hydrated first part of the hydrogel-forming composition and the hydrated second part of the hydrogel-forming composition can crosslink simultaneously as the hydrogel composition is delivered to the tissue site. In some embodiments, for example, the hydrogel tissue sealant is at least partially formed prior to or during delivery to the tissue site. In certain embodiments, the tissue site is a pleural site, such as the parietal and / or visceral pleura.

[0120] In certain embodiments, the hydrogel-forming composition can be delivered to a tissue site using one or more syringes, sprayers, or other applicators. In certain embodiments, for example, applicators that can be used to deliver the hydrogel-forming composition are described in U.S. Patent Application No. 62 / 822,490, entitled "LUNG BIOPSY FLOWABLE SEALANT DELIVERY SYSTEM," or PCT / US2020 / 023772, entitled "SEALANT DELIVERY APPARATUS, AND SYSTEM AND METHOD FOR PREPARING SAME, FOR USE IN A LUNG PROCEDURE," both of which are incorporated herein by reference in their entireties. The following application, filed on the same date as this application, is also incorporated by reference in its entirety: International Application Publication No. PCT / US21 / 23171, filed March 19, 2021, entitled "MULTI-COMPONENT SEALANT DELIVERY SYSTEMS INCORPORATING QUARTER-TURN CONNECTORS." Further details regarding hydrogel-forming composition delivery devices are provided below.

[0121] According to some embodiments, a hydrogel tissue sealant can be formulated to adhere to a tissue site. In certain embodiments, the adhesion of a hydrogel tissue sealant at a tissue site can be determined using a liquid burst pressure strength model based on ASTM F2392-04 (the Standard Test Method for Burst Strength of Surgical Sealants). According to some embodiments, the test is designed to measure the pressure required to burst a sealant patch over a simulated liquid leak, thereby indirectly measuring the sealant's adhesion to the simulated tissue. In certain embodiments, the hydrogel tissue sealant can have any of a variety of suitable burst pressure strengths (e.g., liquid burst pressure strengths). For example, in some embodiments, the burst pressure strength of the hydrogel tissue sealant measured by such a test is 10 mmHg or greater, 50 mmHg or greater, 100 mmHg or greater, 150 mmHg or greater, 200 mmHg or greater, or 250 mmHg or greater. In certain embodiments, the burst pressure strength of the hydrogel tissue sealant as measured by such a test is 300 mmHg or less, 250 mmHg or less, 200 mmHg or less, 150 mmHg or less, 100 mmHg or less, or 50 mmHg or less. Combinations of these ranges are also possible (e.g., the burst pressure strength of the hydrogel tissue sealant is 10 mmHg or more and 300 mmHg or less, and the burst pressure strength of the hydrogel tissue sealant is 100 mmHg or more and 150 mmHg or less). Other ranges are also possible.

[0122] In some embodiments, the crosslinked hydrogel tissue sealant can swell (e.g., with water) after delivery to the tissue site. In some embodiments, the hydrogel tissue sealant can advantageously have a relatively high swelling rate and / or degree (characterized by mass gain after a defined swelling period). A hydrogel tissue sealant with a relatively high swelling rate can be advantageous because the hydrogel tissue sealant swells to conform to the tissue delivery site, improving sealing. In certain embodiments, and as described in more detail below, the hydrogel composition can be delivered to the tissue site via a coaxial cannula. In certain embodiments, the coaxial cannula becomes surrounded by the hydrogel composition during and / or after delivery to perform a biopsy procedure (e.g., a lung biopsy). The coaxial cannula, in some embodiments, can be removed through the bulk of the hydrogel after the biopsy procedure, creating a puncture, void, or hole in the hydrogel tissue sealant. In some such embodiments, the hydrogel tissue sealant may swell (e.g., with water) after removal of the coaxial cannula, thus substantially closing and / or filling the puncture, void, and / or hole caused by the coaxial cannula. The swelling ratio of the hydrogel tissue sealant may be measured by forming a crosslinked hydrogel sealant as described herein, recording the weight of the hydrogel composition, incubating the hydrogel composition in a phosphate buffered saline (PBS) solution at 37°C, removing the hydrogel composition from the PBS solution after 2 hours, and recording the weight of the hydrogel composition, where the percentage is calculated as the percent weight gain.

[0123] According to certain embodiments, the hydrogel tissue sealant has a swelling mass increase of 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more. In some embodiments, the hydrogel tissue sealant has a swelling mass increase of 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, or 35% or less. Combinations of the above-listed ranges are also possible (e.g., the hydrogel tissue sealant has a swelling mass increase of 30% or more and 70% or less, and the hydrogel tissue sealant has a swelling ratio of 40% or more and 50% or less). Other ranges are also possible.

[0124] According to certain embodiments, the hydrogel tissue sealant may be utilized as a pulmonary sealant for the pleura, sealing off air and / or fluid from entering the pleural cavity. In some embodiments, the hydrogel tissue sealant may be utilized to seal the pleura as a whole.

[0125] The hydrogel tissue sealant may degrade over time in a subject. In certain embodiments, the degradation time of a hydrogel may be measured using a degradation model based on ASTM F1635 (the Standard Test Method for in Vitro Degradation). In some embodiments, the test is designed to measure the degradation rate (i.e., rate of mass loss) and / or changes in material or structural properties of materials used in surgical implants. The hydrogel tissue sealant may have any suitable degradation time. In some embodiments, for example, the hydrogel tissue sealant has a degradation time of 1 day or more, 5 days or more, 7 days or more, 10 days or more, or 15 days or more. In certain embodiments, the hydrogel tissue sealant has a degradation time of 20 days or less, 15 days or less, 10 days or less, 7 days or less, or 5 days or less. Combinations of the above-listed ranges are also possible (e.g., the hydrogel tissue sealant has a degradation time of 1 day or more and 20 days or less, and the hydrogel tissue sealant has a degradation time of 10 days or more and 15 days or less). Other ranges are also possible.

[0126] According to certain embodiments, kits are provided. The kits may include one or more devices, such as a container or a syringe (e.g., a syringe barrel) containing a container, capable of storing one or more components of the hydrogel-forming composition, mixing one or more components of the hydrogel-forming composition, and / or delivering the hydrogel-forming composition (or one or more components thereof) to a tissue site. For example, FIG. 2A shows a schematic diagram, and FIG. 3A shows a cross-section, of a dual-syringe, four-compartment / container device 200 capable of storing and / or mixing one or more components of the hydrogel-forming composition with one or more solvents, according to certain embodiments. FIG. 2B shows a schematic diagram, and FIG. 3B shows a cross-section, of the device of FIG. 2A, in which a second syringe element containing hydrated / dissolved first and second components of the hydrogel-forming composition is coupled with a coaxial cannula to deliver the hydrogel-forming composition to a tissue site, according to some embodiments.

[0127] The kit may include any of the above-described first components contained within a first container, which may conveniently be the barrel of a syringe device, as shown in Figures 2A-3B. With reference to Figure 3A, for example, syringe device 200 (e.g., having two interconnected syringe components of a device configured to mix the stored components with one or more solvents) may include a first component containing a cross-linking agent contained within first barrel / container 102, e.g., during storage. In certain embodiments, the first component may further include an antioxidant (e.g., BHT), a surfactant, and / or a radiopaque agent. The first component may be in the form of a first dry powder.

[0128] The kit may further include a second container housing any of the second components described above. With reference to FIG. 3A , for example, syringe device 200 may house a second component in second barrel / container 104, e.g., during storage. In some embodiments, the second component may include a protein capable of cross-linking with a cross-linking agent. The second component may further include a cross-linking initiator and / or surfactant (e.g., PEG 8000) that initiates cross-linking of the protein with the cross-linking agent. In some embodiments, the second component may further include a radiopaque agent. The second component may be in the form of a second dry powder.

[0129] In some embodiments, the kit further includes a third component, e.g., a solvent, contained within a third container. For example, referring to FIG. 3A, the apparatus 200 may contain such a third component within the third barrel / container 106. According to certain embodiments, the third component includes a first solvent (e.g., water, DMSO) or solution (e.g., an aqueous solution) for dissolving and / or hydrating the first powdered component. In some embodiments, the first solvent or solution includes an antioxidant (e.g., BHT), a radiopaque agent (e.g., KCl), and / or a surfactant.

[0130] In certain embodiments, the kit further includes a fourth component, e.g., a solvent, contained in a fourth container. For example, referring to FIG. 3A, the apparatus 200 may contain such a fourth component in the fourth barrel container 108. According to some embodiments, the fourth compartment includes a second solvent (e.g., water, DMSO) or solution (e.g., an aqueous solution) for dissolving and / or hydrating the second powder component. In certain embodiments, the second solvent or solution includes a cross-linking initiator (e.g., a base such as sodium carbonate or a basic buffer). In some embodiments, the second solvent or solution includes an antioxidant (e.g., N-acetly-DL-tryptophan), a radiopaque agent, or a surfactant (e.g., Pluronic® L61).

[0131] According to some embodiments, and as shown, one or more of the first, second, third, and fourth containers are compartments (barrels) of a syringe or applicator. For example, with reference to Figures 2A and 3A, the first barrel / container 102 and the second barrel / container 104 can be the double barrels of a first syringe 100 (e.g., in disclosed embodiments, also used as an applicator syringe—see Figure 2B), and the third barrel / container 106 and the fourth barrel / container 108 are the double barrels of a second syringe 120 (e.g., containing a mixing or hydration solvent).

[0132] According to certain embodiments, a kit for forming a hydrogel tissue sealant includes one or more syringes that collectively provide at least three separate containers. In some embodiments, for example, the kit includes a first container (e.g., first container 102 in first syringe 100) containing a first component (e.g., an electrophilic biodegradable polymer) in powder form, a second container (e.g., second container 104 in first syringe 100) containing a second component (e.g., a nucleophilic biodegradable polymer) in powder form, and at least a third container (e.g., third container 106 and fourth container 108 in second syringe 120) containing one or more solvents.

[0133] The kit may include one or more syringes (e.g., one syringe, two syringes, three syringes, four syringes). The one or more syringes may have any of a variety of suitable configurations. In certain embodiments, for example, the kit includes two syringes (e.g., a first syringe 100 and a second syringe 120). Each syringe of the one or more syringes may be a double-barrel syringe in certain embodiments. The first syringe 100 (e.g., an applicator syringe) may, in some embodiments, include a first container 102 containing a first component in powder form and a second container 104 containing a second component in powder form. According to certain embodiments, the second syringe 120 (e.g., a mixing or hydration syringe) includes a third container 106 containing a first solvent capable of dissolving the first component and a fourth container 108 containing a second solvent capable of dissolving the second component.

[0134] In some embodiments, one or more syringes (e.g., first syringe 100 and second syringe 120) are configured such that first container 102 and second container 104 can be placed in fluid communication with at least a third container 106 containing one or more solvents. Configuring the kit in this manner facilitates mixing of the first component with the one or more solvents to form a solution of the first component, and facilitates mixing of the second component with the one or more solvents to form a solution of the second component. For example, in certain embodiments, the first syringe 100 (e.g., an applicator syringe) and the second syringe 120 (e.g., a mixing or hydration syringe) are configured to be fluidly connectable to each other such that the first container 102 and the second container 104 are separately placed in fluid communication with the third container 106 and the fourth container 108, respectively, and are capable of facilitating mixing of a first component with a first solvent to form a solution of the first component in the first container 102 and of facilitating mixing of a second component with a second solvent to form a solution of the second component in the second container 104.

[0135] The kit may include one or more devices capable of delivering the hydrogel-forming composition (or one or more components thereof) to a tissue site. For example, FIG. 2B shows a schematic diagram of a device capable of delivering a hydrogel-forming composition to a tissue site, according to some embodiments, and FIG. 3B shows a cross-section thereof. As shown in FIG. 2B , device 250 (e.g., a delivery device) includes a first syringe 100 and a needle assembly. The needle assembly, in some embodiments, includes a coaxial cannula 130 and a needle 132. In some embodiments, first syringe 100 is configured to mix and contain a dissolved first component (e.g., a solution of the first component) and a dissolved second component (e.g., a solution of the second component) to form a cross-linked solution of the first and second components that can form a hydrogel tissue sealant upon delivery to the tissue site by the needle assembly.

[0136] 4 shows an overview of the steps of an exemplary method for hydrating and delivering a hydrogel-forming composition using the apparatus depicted in FIGS. 2A-3B, according to certain embodiments. The method 400 for hydrating and delivering a hydrogel-forming composition includes, in some embodiments, step 402, which includes mechanically and fluidically interconnecting an applicator syringe (e.g., first syringe 100 in FIGS. 2A and 3A) and a mixing syringe (e.g., second syringe 120 in FIGS. 2A and 3A). In some such embodiments, the applicator syringe (e.g., first syringe 100 in FIGS. 2A and 3A) includes a first element housed in a first container (e.g., first container 102 in FIGS. 2A and 3A) and a second element housed in a second container (e.g., second container 104 in FIGS. 2A and 3A), and the mixing syringe (e.g., second syringe 120 in FIGS. 2A and 3A) includes a third element housed in a third container (e.g., third container 106 in FIGS. 2A and 3A) and a fourth element housed in a fourth container (e.g., fourth container 108 in FIGS. 2A and 3A).

[0137] In certain embodiments, method 400 includes step 404, which includes sequentially depressing the plungers of an applicator syringe (e.g., first syringe 100 in FIGS. 2A and 3A) and a mixing syringe (e.g., second syringe 120 in FIGS. 2A and 3A) to hydrate a first component contained in a first container (e.g., first container 102 in FIGS. 2A and 3A) and a second component contained in a second container (e.g., second container 104 in FIGS. 2A and 3A). Step 406 of method 400 includes assessing whether the first component and / or second component are fully hydrated (e.g., fully dissolved). If the first component and / or second component are not fully hydrated, step 404 is repeated. If the first component and / or second component are fully hydrated, the user can proceed to step 408.

[0138] According to some embodiments, step 408 of method 400 includes separating a mixing syringe (e.g., second syringe 120 in FIGS. 2A and 3A) from an applicator syringe (e.g., first syringe 100 in FIGS. 2A and 3A) when the hydrated first component and the hydrated second component are contained in the applicator syringe. Step 410 includes coupling a needle assembly (e.g., coaxial cannula 130 and needle 132 in FIG. 2B) to the applicator syringe (e.g., first syringe 100 in FIGS. 2B and 3B).

[0139] According to some embodiments, method 400 includes step 420, which includes disposing a material (e.g., a solution of a first component and a solution of a second component) at a tissue site from an applicator syringe (e.g., first syringe 100 in FIGS. 2B and 3B). In certain embodiments, disposing the material includes mixing the solution of the first component and the solution of the second component to form a cross-linked solution of the first component and the second component (e.g., at one or more mixing points within the needle assembly, proximal to the needle assembly, and / or at the distal end of the needle assembly) simultaneously as the material is delivered to the tissue site.

[0140] According to certain embodiments, a needle assembly including a coaxial cannula is used to deliver a hydrogel composition and perform a biopsy procedure (e.g., a lung biopsy). In some embodiments, a device 250 (e.g., a delivery device) is used to insert the coaxial cannula into a subject's pleural cavity so that the hydrogel-forming composition can be deposited at a tissue site. See, for example, FIG. 8A , which illustrates a schematic diagram of a hydrogel delivery device having a coaxial cannula inserted into a subject's pleural cavity, according to certain embodiments. The hydrated hydrogel-forming composition is deposited at the tissue site. See, for example, FIG. 8B , which illustrates a schematic diagram of a hydrogel delivery device having a hydrogel-forming composition deposited through a coaxial cannula to provide a hydrogel tissue sealant at a tissue site, according to certain embodiments. In some embodiments, the hydrogel-forming composition is deposited while or after a user advances the cannula through the tissue site.

[0141] Following delivery of the hydrogel composition, in some embodiments, a biopsy is performed. For example, according to certain embodiments, the syringe element 100 of the device 250 is removed from the needle assembly including the coaxial cannula, and a biopsy device including a standard biopsy needle is inserted through the coaxial cannula. A biopsy (e.g., lung biopsy) procedure is then performed. See, for example, FIG. 8C, which shows a schematic diagram of a biopsy needle inserted through the coaxial cannula to perform the biopsy procedure, according to certain embodiments.

[0142] In certain embodiments, after the hydrogel composition is placed at the tissue site to provide the hydrogel tissue sealant and the biopsy procedure is performed, the needle assembly including the biopsy device and coaxial cannula is removed from the administration site. According to some embodiments, the hydrogel tissue sealant may swell (e.g., with water) as described herein to seal any punctures, voids, and / or holes in the hydrogel tissue sealant caused by removal of the needle assembly.

[0143] To prevent increased moisture or oxygen uptake by the powdered ingredients during storage, one or more of the containers / syringes containing one or more of the powdered components (particularly the powdered first component (e.g., including the crosslinker)) may be placed in a sealed pouch (e.g., a sealed foil pouch), optionally flushed with an inert gas such as nitrogen, and optionally containing a desiccant material (e.g., a desiccant or molecular sieve material such as a desiccant packet containing either PharmaKeep® (Mitsubishi Gas Chemical America, Inc.) or 4A molecular sieves (Multisorb Filtration Group)) within the pouch. [Example]

[0144] Example 1 The following example describes the use of a hydrogel tissue sealant in a porcine lung model. An X-ray image of the porcine lung model was obtained, as shown in Figure 5A. A biopsy was performed on the porcine lung model. Figure 5B shows the normal airway pressure of the post-biopsy image of the porcine lung model. A hydrogel-forming composition containing the reaction product of PEG(SS)2 and albumin, along with iohexol as a radiopaque material, was prepared. As shown in Figure 6A, the hydrogel-forming composition was delivered to the porcine lung model pre-biopsy via a needle (circled) at the time of initial puncture of the lung. The hydrogel tissue sealant is visible through the soft tissue and pleural cavity. The applicator's coaxial element was left in place, and the sealant application needle was removed. A biopsy needle was inserted through the coaxial element into the target tissue, a biopsy sample was obtained, and the coaxial element and biopsy needle were removed. As shown in Figure 6B, the hydrogel tissue sealant (circled) remained in place and was easily visualized after the procedure, where it closed the puncture site. The hydrogel tissue sealant allowed for normal ventilation. Figure 5B shows an X-ray image of the porcine lung model after biopsy. As shown in Figure 7A, the hydrogel tissue sealant (circled) adheres to the parietal pleura of the porcine lung model. Furthermore, as shown in Figure 7B, the hydrogel tissue sealant (circled) protrudes from the inside of the lung and adheres to the parietal and visceral pleura of the porcine lung model, thus sealing the biopsy tract.

[0145] Example 2 The following example illustrates the evaluation of the stability of hydrogel-forming compositions in accelerated aging tests.

[0146] Samples of hydrogel-forming compositions containing a PEG(SS)2-containing component and a recombinant human serum albumin (rHSA)-containing component were produced according to Table 1. PEG(SS)2 was obtained from Sigma (samples 1, 3, and 4) or Laysan Bio (sample 2) and handled under a nitrogen atmosphere. The PEG(SS)2-containing component contained either PEG(SS)2 or PEG(SS)2 with added BHT. In certain cases, desiccant or molecular sieve material was also enclosed in the pouch—desiccant packets containing either PharmaKeep® (Mitsubishi Gas Chemical America, Inc.) or 4A molecular sieves (Multisorb Filtration Group). The PEG(SS)2-containing component was aliquoted into a first syringe and stored in a sealed foil pouch under a nitrogen atmosphere. The rHSA-containing component solution used to prepare the rHSA-containing component used to form the hydrogel by lyophilization contained rHSA obtained from InVitria (Junction City, KS) combined with reverse osmosis (RO) water to provide a 30% rHSA concentration by weight to volume, along with sodium carbonate and PEG 8000. The rHSA-containing component solution in RO water was lyophilized, ground into a powder, and aliquoted into a second syringe and sealed in a foil pouch under a nitrogen atmosphere.

[0147] A hydration kit was prepared using deionized (DI) water in the third syringe (to hydrate the PEG(SS)2-containing component) and DI water with Pluronic® L61 in the fourth syringe (to hydrate the lyophilized rHSA-containing component). The hydration kit was sealed in a foil pouch with two Leur-Lock® connectors for connection to each syringe, allowing the powder mixture to be hydrated at the time of use. Samples were e-beam sterilized twice. All samples were stored and conditioned at 40°C to mimic advanced degradation compared to room temperature storage.

[0148] [Table 1]

[0149] Samples were aged according to Table 2, withdrawn at the indicated time points, and evaluated as described in more detail below.

[0150] [Table 2]

[0151] The gelation times of the four hydrogel-forming composition samples shown in Table 1 were assessed at each stability time point by (i) hydrating the PEG(SS)2-containing component and the rHSA-containing component, (ii) waiting a period of 2, 30, or 60 minutes after hydration, (iii) dispensing the hydrated components into vials containing stir bars on a stir plate adjusted to 300 RPM, (iv) recording the initial time upon dispensing the components, and (v) recording the final time when the stir bar stopped rotating due to gelation. The results are shown in Table 3.

[0152] [Table 3]

[0153] The dissolution times of the four rHSA-containing component samples shown in Table 1 were assessed at each stability time point by (i) connecting the syringe containing the rHSA-containing component to the syringe containing DI water and Pluronic® L61, (ii) starting a timer, (iii) pushing fluid back and forth into the powder syringe, and (iv) stopping the timer when the rHSA-containing component was completely dissolved. The results are shown in Table 4.

[0154] [Table 4]

[0155] The pH of the four rHSA-containing component samples shown in Table 1 was assessed at each stability time point by (i) dissolving the powder mixture with a syringe containing DI water and Pluronic® L61 as described above for dissolution time measurements, and (ii) measuring the pH of the solution using a calibrated Mettler Toledo FiveEasy pH Meter. The results are shown in Table 5.

[0156] [Table 5]

[0157] The swelling ratios of the hydrogel compositions formed from the four samples shown in Table 1 were evaluated at each stability time point by (i) forming the hydrogel composition by hydrating the PEG(SS)2-containing component and the rHSA-containing component, dispensing the components using a mixing tip, and allowing them to gel; (ii) recording the weight of the hydrogel composition at time zero; (iii) incubating the hydrogel composition in a phosphate-buffered saline (PBS) solution at 37°C; (iv) removing the hydrogel composition from the PBS solution after 2 hours; and (v) recording the weight of the hydrogel composition. The swelling percentage was calculated based on the percentage weight gain. The results are shown in Table 6.

[0158] [Table 6]

[0159] Example 3 The following examples illustrate the liquid burst pressure strength of hydrogel compositions according to certain embodiments.

[0160] Hydrogel compositions containing PEG(SS)2 and rHSA were produced according to Table 7. The rHSA-containing component included lyophilized rHSA with Pluronic® L61 and antioxidants. 45 samples of three different hydrogel-forming compositions were investigated. The mass-to-volume percentage of rHSA varied from 10 to 30% among the compositions, and the amount of PEG(SS)2 was also varied so that the NHS ester:amine ratio was kept constant at 2.21 for all three compositions.

[0161] [Table 7]

[0162] The powdered PEG(SS)2 and rHSA-containing components were dispensed into their own syringes, and each was hydrated using a separate syringe containing 1 mL of water. The components were then dispensed using a mixing tip and allowed to gel. The adhesion of the hydrogel compositions was measured using a liquid burst pressure model based on ASTM F2392-04 (the Standard Test Method for Surgical Sealants). The results are shown in Table 8.

[0163] [Table 8]

[0164] Example 4 The following example illustrates the evaluation of a hydrogel composition according to the present invention as a sealant for use during a lung biopsy procedure in a porcine model to prevent pneumothorax complications.

[0165] Five hydrogel compositions were produced. PEG(SS)2 was handled under a nitrogen atmosphere. PEG(SS)2 was dispensed into a first syringe and stored in a sealed pouch under a nitrogen atmosphere. The rHSA-containing component solution used to prepare the rHSA-containing component used to form the hydrogel by lyophilization contained rHSA combined with RO water, along with sodium carbonate, PEG 8000, and Pluronic® L61. The rHSA-containing component solution in RO water was lyophilized, ground into a powder, dispensed into a second syringe, and sealed in a foil pouch under a nitrogen atmosphere.

[0166] A hydration kit (e.g., a double-barrel syringe) was prepared using DI water in the first compartment of the double-barrel syringe (to hydrate the PEG(SS)2-containing component) and DI water in the second compartment of the double-barrel syringe (to hydrate the lyophilized rHSA-containing component). The hydration kit was sealed in a foil pouch with connections to each syringe containing the PEG(SS)2- and rHSA-containing component so that the powder mixture could be hydrated with their respective solutions at the time of use.

[0167] A total of 10 pig subjects were evaluated. Five of the ten pigs were designated as test subjects, and the hydrogel composition was implanted into the left lower lobe of the lung. To deliver the hydrogel composition, a coaxial technique was utilized with computed tomography (CT) guidance. A delivery device was inserted through the soft tissue until it was in close proximity to the lung and pleural cavity (see FIG. 8A). The hydrogel composition was hydrated and passed through the ported needle system of the delivery device, and placed in the subcutaneous tissue, pleural cavity, and immediately adjacent lung parenchyma (see FIG. 8B). CT imaging was utilized to confirm hydrogel placement.

[0168] After successful implantation of the hydrogel composition, a lung biopsy was obtained within 5 minutes of implantation using a coaxial cannula. Briefly, the needle was adjusted as needed, advanced to the biopsy site, and the ported needle delivery system was removed. A standard biopsy needle was inserted through the coaxial system, and a standard lung biopsy procedure was performed using a 16G Bard Mission Biopsy Needle, continuing to use CT guidance (see Figure 8C).

[0169] Follow-up evaluations of two of the five study subjects were performed 72 (±8) hours after hydrogel implantation, and evaluations of the other three pigs were performed 144 (±8) hours after implantation. During the follow-up evaluations, CT scans were completed to assess the presence of the hydrogel composition and the presence (or absence) of pneumothorax. After completing the CT scans, the animals were euthanized, a comprehensive necropsy was performed, and target organs (i.e., lungs) were removed for gross pathology. The inner chest wall (e.g., parietal pleura) was also examined.

[0170] Five control pigs underwent lung biopsy procedures as described above, but without implantation of the hydrogel composition. Follow-up evaluations (including CT scans to assess the presence (or absence) of pneumothorax) were performed 48 (±8) hours after lung biopsy. Animals were then euthanized after their CT scans (unless otherwise noted), and comprehensive necropsies were performed, with target organs removed for gross pathology.

[0171] An overview of the study design is shown in Table 9.

[0172] [Table 9]

[0173] Each of the five subjects successfully implanted the hydrogel composition prior to the lung biopsy procedure. Placement of the hydrogel composition was successful and caused no immediate problems or concerns for the physician performing the procedure. No pneumothorax complications occurred during the biopsy procedure or during the 20-30 minute monitoring period following the procedure. All five subjects survived to their scheduled follow-up visits on days 3 or 6. Furthermore, none of the five subjects showed signs of postoperative or delayed pneumothorax on their postoperative CT scans (see representative subject 5 in Figure 9A, where the arrow indicates the site of the hydrogel). Autopsy revealed retained hydrogel material on days 3 or 6, as expected. The hydrogel compositions in the day 6 subjects (i.e., samples 1, 3, and 4) displayed decreased hydrogel firmness, indicating resorption. All five subjects experienced slight irritation of the parietal pleura around the needle insertion site, but nothing of major concern.

[0174] Five control subjects underwent lung biopsies without the addition of the hydrogel composition. Two of the five control subjects (i.e., subjects 9 and 10) experienced pneumothorax during the procedure and subsequent air embolism, as evidenced by CT scans. Figure 9B shows an example of air embolism (circled) in subject 9, and Figure 9C shows an example of pneumothorax (circled) in subject 10. Due to the severity of these complications, two test subjects were sacrificed after completing the biopsies. The remaining three control subjects tolerated the lung biopsies, and follow-up CT scans revealed the presence of a large pneumothorax in one pig (i.e., subject 6; see Figure 9D, where the circle indicates the pneumothorax). The other two pigs (i.e., subjects 7 and 8) were completely free of complications. Necropsies revealed no notable findings in any of the control subjects.

[0175] A summary of the study results is shown in Table 10. The results showed improved outcomes of lung biopsy procedures in test subjects (0% pneumothorax rate) compared to control subjects (60% pneumothorax rate).

[0176] [Table 10]

[0177] While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are utilized. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the above-described embodiments are presented by way of example only, and that, within the scope of the appended claims and equivalents thereof, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0178] The indefinite articles "a" and "an," as used herein in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0179] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and present unconjunctically in other cases. Unless clearly indicated to the contrary, other elements, whether related or unrelated to the elements specifically identified, may optionally be present in addition to the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so forth.

[0180] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., the inclusion of at least one of more than one of the elements of the list, and optionally the inclusion of additional items not listed. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of the elements of the list. In general, the term "or" as used herein shall be construed to indicate exclusive alternatives (i.e., "either / or, but not both") only when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0181] As used herein in the specification and claims, the phrase "at least one," in connection with a list of one or more elements, should be understood to mean at least one element selected from one or more elements in the list of elements, and not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any and all combinations of elements in the list of elements. This definition allows for elements, whether related or unrelated to the specifically identified elements, that may optionally be present in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers. Thus, as a non-limiting example, "at least one of A and B" (or, similarly, "at least one of A or B," or, similarly, "at least one of A and / or B") can refer in one embodiment to at least one A (and optionally including more than one element other than B), with no B present; in another embodiment to at least one B (and optionally including more than one element other than A), with no A present; in yet another embodiment to at least one A, optionally including more than one element, and at least one B (and optionally including other elements), with optionally more than one element; and so forth.

[0182] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," etc., shall be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. 1. A hydrogel-forming composition for forming a hydrogel tissue sealant, comprising: formula: G-LM-PEG-LM-G a first component comprising a crosslinker which is a difunctionalized polyalkylene oxide-based component of formula (I); (In the formula: PEG is polyethylene glycol; Each LM is a divalent carbonate group of the formula -C(O)-, a divalent carboxylate group of the formula -(CH ２ ) ｂ Monoester divalent groups of the formula —C(O)— (wherein b is an integer from 1 to 10), ２ ) ｃ diester groups of the formula —C(O)—, where c is an integer from 1 to 10, and the aliphatic portion of the group can be saturated or unsaturated; ２ ) ｄ dicarbonate divalent radicals of the formula —O—C(O)— (wherein d is an integer of 1 to 10), ２ ) ｄ an amide-containing divalent radical of the formula —C(O)—, where d is an integer from 1 to 10; ２ ) ｃ -C(O)-N(H)-(CH ２ ) ｄ - (wherein c is an integer from 1 to 10 and d is an integer from 1 to 10), and amide-containing divalent radicals of the formula -R-C(O)-, -R-C(O)-(CH ２ ) ｃ -C(O)-, -R-C(O)-O-(CH ２ ) ｄ -O-C(O)-, -RN(H)-C(O)-(CH ２ ) ｄ -C(O)- or -R-(CH ２ ) ｃ -C(O)-N(H)-(CH ２ ) ｄ -wherein c is an integer from 1 to 10, d is an integer from 1 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments; and each G is a leaving group independently selected from the group consisting of N-oxysuccinimidyl, N-oxymaleimidyl, N-oxyphthalimidyl, nitrophenoxyl, N-oxiimidazolyl, and tresyl; a second component comprising a protein capable of cross-linking with said cross-linking agent; a crosslinking initiator in an amount selected to provide the desired gel time; one or more solvents capable of dissolving the first component, the second component, and the crosslinking initiator; and surfactants; Including, When the first component, the second component, the cross-linking initiator, and the surfactant are all dissolved in the one or more solvents, cross-linking of the cross-linking agent and the protein occurs simultaneously with mixing of the first component, the second component, the cross-linking initiator, and the surfactant dissolved in the one or more solvents, with the selected amount of cross-linking initiator providing a gelation time of 5 seconds or less, thereby forming the hydrogel tissue sealant, which is capable of preventing or reducing the risk of pneumothorax during or after a lung biopsy procedure.

2. 10. The composition of claim 1, wherein the surfactant is capable of stabilizing, increasing the rate at which the protein dissolves, and / or preventing aggregation of the protein.

3. The composition of claim 1 or 2, wherein the crosslinking initiator is part of the second component.

4. The composition of any one of claims 1 to 3, wherein the surfactant is part of the second component.

5. 5. The composition of claim 4, wherein the second component is a lyophilized powder containing both the protein and the surfactant prior to dissolution in the one or more solvents.

6. The composition according to any one of claims 1 to 5, wherein at least one of the one or more solvents is water or an aqueous solution.

7. The composition of any one of claims 1 to 6, further comprising a first antioxidant.

8. The composition described in claim 7, wherein the first antioxidant is part of the first element.

9. The composition of claim 7 or 8, wherein the first antioxidant is butylated hydroxytoluene.

10. Each LM is the same and has the formula —C(O)—, —(CH ２ ) ｂ -C(O)- (wherein b is an integer of 1 to 5), -C(O)-(CH ２ ) ｃ -C(O)- (where c is an integer from 2 to 10, and the aliphatic portion of the group can be saturated or unsaturated), -C(O)-O-(CH ２ ) ｄ -O-C(O)- (wherein d is an integer of 2 to 10), and compounds of the formula -R-C(O)-, -R-C(O)-(CH ２ ) ｃ -C(O)-, or -R-C(O)-O-(CH ２ ) ｄ a difunctional linking moiety represented by an oligomeric divalent group represented by -O-C(O)-, where c is an integer from 2 to 10, d is an integer from 2 to 10, and R is a polymer or copolymer having 1 to 10 monomeric lactide, glycolide, trimethylene carbonate, caprolactone, or p-dioxanone fragments; and Each G is the same, and may be N-oxysuccinimidyl, N-oxymaleimidyl, N-oxy The composition of any one of claims 1 to 9, wherein the leaving group is selected from the group consisting of thiophenoxyl, N-oximidazolyl, nitrophenoxyl, N-oximidazolyl, and tresyl.

11. The crosslinking agent has the formula: 【Chemistry 1】 or 【Chemistry 2】 a difunctionalized polyalkylene oxide-based element of the formula:

11. The composition of any one of claims 1 to 10, wherein the value of n in the formula is such that the crosslinker has a weight average molecular weight of from 1 kDa to 20 kDa.

12. The composition of any one of claims 1 to 11, wherein the protein is selected from the group consisting of human serum albumin, recombinant human serum albumin, and animal-derived albumin.

13. The composition of any one of claims 1 to 12, wherein the protein is recombinant human serum albumin.

14. The composition of any one of claims 1 to 13, wherein the protein is lyophilized.

15. The composition of any one of claims 1 to 14, wherein the crosslinking initiator comprises a base and / or a basic buffer.

16. 16. The composition of claim 15, wherein the base and / or basic buffer comprises a carbonate and / or bicarbonate.

17. The composition of claim 15, wherein the base and / or basic buffer comprises sodium bicarbonate.

18. 16. The composition of claim 15, wherein the base and / or basic buffer comprises sodium carbonate.

19. The composition of any one of claims 1 to 18, wherein the surfactant is a non-functionalized PEG.

20. 20. The composition of claim 19, wherein the non-functionalized PEG has a weight average molecular weight of 1000 g / mol or more and 40000 g / mol or less.

21. The composition of claim 20, wherein the weight average molecular weight of the non-functionalized PEG is 8000 g / mol.

22. The composition of any one of claims 1 to 18, wherein the surfactant is dextran sulfate, poloxamer, polysorbate, oil, siloxane, stearate, and glycol.

23. 23. The composition of claim 22, wherein the poloxamer is Pluronic® L61 or the oil is a mineral oil or a vegetable oil.

24. 24. The composition of claim 23, wherein the surfactant is an antifoam additive.

25. The composition of any one of claims 7 to 24, further comprising a secondary antioxidant.

26. 26. The composition of any one of claims 7 to 25, wherein the first antioxidant is N-acetyl-DL-tryptophan.

27. 27. The composition of any one of claims 1 to 26, wherein the first component is in the form of a first powder or powder mixture and / or the second component is in the form of a second powder or powder mixture.

28. 27. The composition of any one of claims 1 to 26, wherein the first part is in the form of a first aqueous solution and / or the second part is in the form of a second aqueous solution.

29. A composition according to any one of the preceding claims, comprising one or more powders or powder mixtures, or one or more aqueous solutions.

30. The composition of any one of claims 1 to 29, comprising a radiopaque agent.

31. The composition of claim 30, wherein the radiopaque agent comprises gold, silver, or iodine.

32. The composition of claim 30 or 31, wherein the radiopaque agent is selected from the group consisting of KCl, barium sulfate, iohexol, and diatrizoate.

33. The composition of any one of claims 1 to 32, wherein the selected amount of the crosslinking initiator provides a gel time of 0.5 seconds or less.

34. 34. The composition of any one of claims 1 to 33, wherein when the first component, the second component, the crosslinking initiator, and the surfactant are all dissolved in the one or more solvents, the resulting hydrogel-forming composition has a measured pot life of not less than 10 minutes and not more than 24 hours.

35. 1. A kit for forming a hydrogel tissue sealant, comprising: a first container containing a first element comprising the cross-linking agent defined in claim 1; and a second container containing a second component comprising a protein; Kit including:

36. The protein is selected from the group consisting of human serum albumin, recombinant human serum albumin, and animal serum albumin.

36. The kit of claim 35, wherein the albumin is selected from the group consisting of albumin derived from a plant.

37. A kit as described in claim 35 or 36, comprising one or more additional containers containing one or more solvents for dissolving the first element and the second element.

38. The kit described in claim 37, wherein the one or more solvents are water.

39. the first container containing the first element; the second container containing the second component; and a third container containing a solvent for dissolving the first component and the second component; 39. The kit of claim 37 or 38, comprising:

40. The kit of claim 39, wherein the solvent is water.

41. A kit comprising two syringes, a first syringe containing the first container and the second container; and a second syringe containing the third container; the first component and the second component are in powder form; the first syringe and the second syringe are configured to be fluidly connectable to one another such that the first container and the second container are disposed in fluid communication with the third container to facilitate mixing of the first component and the second component with the solvent to form a solution of the first component in the first container and a solution of the second component in the second container; and 41. The kit of claim 39 or 40, wherein the first syringe is further configured to mix the solution of the first part with the solution of the second part to form a hydrogel-forming composition for forming a hydrogel tissue sealant.

42. the first container containing the first element; the second container containing the second component; a third container containing a solvent for dissolving the first component; and a fourth container containing a solvent for dissolving the second component; 39. The kit of claim 37 or 38, comprising:

43. The kit of claim 42, wherein the solvent in the third container is water.

44. A kit as described in claim 42 or 43, wherein the solvent in the fourth container is water.

45. A kit comprising two syringes, a first syringe containing the first container and the second container; and a second syringe containing the third container and the fourth container; the first component and the second component are in powder form; the first syringe and the second syringe are configured to be fluidly connectable to each other such that the first container and the second container are disposed in fluid communication with the third container and the fourth container, respectively, and mixing of the first component and the solvent in the third container can be promoted to form a solution of the first component in the first container, and mixing of the second component and the solvent in the fourth container can be promoted to form a solution of the second component in the second container; and 45. The kit of any one of claims 42-44, wherein the first syringe is further configured to mix the solution of the first part with the solution of the second part to form a hydrogel-forming composition for forming a hydrogel tissue sealant.

46. 46. ​​The kit of any one of claims 35 to 45, wherein the first component further comprises an antioxidant.

47. The kit of any one of claims 35 to 46, wherein the second component further comprises a surfactant and / or a cross-linking initiator.

48. 46. ​​The kit of claim 41 or 45, wherein mixing the solution of the first part with the solution of the second part provides the hydrogel-forming composition of any one of claims 1 to 34.

49. 35. The hydrogel-forming composition of any one of claims 1 to 34 for use in a method of surgical treatment.

50. 50. The hydrogel-forming composition for use according to claim 49, wherein the method of surgical treatment comprises delivering the hydrogel-forming composition to a tissue site and forming a hydrogel tissue sealant at the tissue site.

51. 51. The hydrogel-forming composition for use according to claim 49 or 50, wherein the surgical treatment is a lung biopsy procedure, and the composition is used to prevent or reduce the risk of pneumothorax during or after the lung biopsy procedure.

52. 52. The hydrogel-forming composition for use according to any one of claims 49 to 51, wherein the composition is used to prevent or reduce the risk of pneumothorax in a lung biopsy procedure in a patient, wherein the hydrogel-forming composition is delivered to the pleural cavity of the patient to form a hydrogel tissue sealant through which a biopsy sample is taken.

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