Biodegradable Lung Sealant

Abstract

The present invention relates to a rapid initial setting liquid lung sealant that forms an adhesive and elastic hydrogel. For optimized setup time and extended working time, a lung sealant is provided that includes two high molecular weight (20 kDa) multi-arm PEG compositions, where the first PEG composition contains an alkaline buffer and the second PEG composition contains a weakly acidic buffer. In some embodiments, the PEG further includes a radioprotectant, such as tocopherol. In some embodiments, a colorant is added to one or both PEGs of the lung sealant to improve visualization of the lung sealant against human lung tissue.

Description

[Technical Field]

[0001] The present invention relates to a two-component biodegradable sealant for sealing lung tissue and methods of use thereof. [Background technology]

[0002] Tissue sealants are used as adjuncts to primary pulmonary pleural closure using, for example, sutures, surgical mesh, and staples. Currently, no adjunctive hemostatic sealant is adopted as standard treatment for lung sealing. PROGEL (Becton, Dickinson and Company, Franklin Lakes, New Jersey) hydrogel is the only approved lung sealant in the United States. However, PROGEL has not been widely adopted due to its high stiffness, which results in a compliance mismatch with the underlying lung tissue. The resulting shear stress at the sealant-tissue interface ultimately leads to undesirable delamination. Furthermore, PROGEL's low elongation at failure makes it impossible to apply the sealant in a fully deflated state. Therefore, PROGEL must be applied to the lung in a partially inflated state, which adds complexity to the application process. PROGEL formulations do not initially set rapidly, leading to undesirable flow and ultimately to poor coverage of the target lung surface, resulting in significant material waste. Furthermore, poor visualization due to minimal contrast of the sealant on the lung surface can lead to insufficient coverage.

[0003] In the Asia-Pacific region, NEOVEIL (Gunze Limited, Tokyo, Japan), a bioabsorbable polyglycolic acid (PGA) sheet for reinforcing sutures and staples, is utilized in conjunction with fibrin sealant to seal lung tissue. However, NEOVEIL and fibrin sealant have lower adhesive strength than synthetic sealants. The lower adhesive strength and low elongation at failure can result in undesirable detachment of the NEOVEIL mesh and fibrin sealant from the target tissue.

[0004] Pulmonary sealants are used to prevent air leaks after pulmonary surgery or injury. Pulmonary sealants are particularly important for preventing complications such as prolonged alveolar air leak (PAL) and infection after pulmonary surgery. However, sealing lung tissue is particularly difficult due to lung movement during breathing. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for a lung sealant that has high tensile strength, high elongation at break, and low modulus of elasticity that does not inhibit the natural cyclic movement of the lung. [Means for solving the problem]

[0006] The present invention addresses the limitations of currently existing biodegradable sealants. Provided herein is a synthetic, biodegradable, two-component, rapid initial setting liquid sealant composition that forms an adhesive and elastic hydrogel. The sealant provided herein has high tensile strength, high elongation at break, and low modulus, making it particularly useful as a lung sealant that does not inhibit the natural cyclic movement of the lung.

[0007] Minimizing the stiffness of the lung sealants of the present invention provides unexpectedly advantageous lung sealing performance. The lung sealants provided herein have reduced stiffness and greater elongation at failure, in contrast to sealants currently used for lung sealing. The stiffness of the lung sealants provided herein is about 2.9 times lower than PROGEL® sealant, and about 7 times higher elongation at failure.

[0008] In one embodiment of the present invention, the sealant comprises a two-component, flowable, fast-initial setting liquid composition that forms an adhesive and elastic biodegradable hydrogel.

[0009] In some embodiments, a method of making a lung sealant composition of the present disclosure comprises dissolving a first polyethylene glycol (PEG) polymer powder in an alkaline buffer to form a first component, and dissolving a second PEG polymer powder in a weakly acidic buffer to form a second component, the two components being mixed immediately before or during application to the tissue or wound.

[0010] In some embodiments, a method of making a lung sealant herein includes mixing an alkaline buffer with a 4-arm PEG-NH2-HCl containing tocopherol to form a 4-arm PEG-NH2-HCl composition; separately mixing a weakly acidic buffer with a 4-arm PEG-SG containing tocopherol to form a 4-arm PEG-SG composition; simultaneously delivering the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition onto lung tissue using an applicator device that provides adequate mixing of the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition; and allowing the lung sealant to harden on the lung tissue.

[0011] In a preferred embodiment, the lung sealant composition consists of two high molecular weight (20 kDa) four-arm PEG polymers, one PEG dissolved in an alkaline buffer and the other PEG dissolved in a weakly acidic buffer for optimized preparation time and extended working time before curing or gelling.

[0012] In some embodiments, the biodegradable lung sealant comprises a first component comprising a first multi-arm PEG and an alkaline buffer for reconstituting the first multi-arm PEG, and a second component comprising a second multi-arm PEG and a weakly acidic buffer for reconstituting the second multi-arm PEG, wherein when the first and second components are combined, they produce an adhesive and elastic biodegradable lung sealant.

[0013] In some embodiments, the flowable sealant of the present invention comprises a two-component composition, the two components being mixed immediately prior to or during application to the tissue or wound, with the first component comprising multi-arm PEG-amine (PEG-NH) and the second component comprising multi-arm PEG-succinimidyl glutarate (PEG-SG). In some preferred embodiments, the first component of the sealant composition comprises 20 kDa 4-arm PEG-amine HCl salt (4-arm PEG-NH-HCl), and the second component comprises 20 kDa 4-arm PEG-succinimidyl glutarate (4-arm PEG-SG).

[0014] In a preferred embodiment, 4-arm PEG-amine 20kDa is combined with an alkaline N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer to form the first component of the sealant composition, and 4-arm PEG-SG 20kDa is combined with a weakly acidic citrate buffer to form the second component of the sealant composition.

[0015] In some embodiments, the first and second components are delivered to the lung tissue in a device that provides adequate mixing of the components.

[0016] In some embodiments of the present disclosure, the PEG polymer includes a free radical scavenger to neutralize reactive oxygen species (ROS). In some aspects, the free radical scavenger is a radioprotector that improves the radiation robustness of the sealant composition. In aspects, the radioprotector is an antioxidant, such as tocopherol. Examples of suitable tocopherols are α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, tocotrienol, and combinations thereof. In a preferred embodiment, the radioprotector is incorporated into the PEG powder prior to adding the buffer to the PEG powder.

[0017] In some embodiments, the sealant composition further comprises a colorant to improve visualization of the sealant against human tissue and allow for more consistent coverage of the target tissue. In some preferred embodiments, one or more colorants are combined with the PEG powder. In some aspects of the invention, the colorant is FD&C Blue #1.

[0018] In some preferred embodiments, about 730 ppm to about 3000 ppm of FD&C Blue #1 is added to the PEG-amine powder, the PEG-SG powder, or both the PEG-amine powder and the PEG-SG powder.More preferably, about 1000 ppm to about 1600 ppm of FD&C Blue #1 is added to the PEG-amine powder, the PEG-SG powder, or both the PEG-amine powder and the PEG-SG powder.

[0019] In some aspects, the PEG powder for preparing a component of the lung sealant of the present invention contains both a first additive and a second additive, where the first additive is a radioprotectant such as an antioxidant and the second additive is a colorant. In one embodiment, the first additive is tocopherol and the second additive is FD&C Blue #1.

[0020] In one aspect, all of the components of the sealants and kits of the present disclosure, including the PEG powder, buffers, solutions, and containers, are sterilized starting with at least the minimum effective X-ray radiation dose, most preferably up to about 39 kiloGrays (kGy) or up to about 47 kGy.

[0021] In another embodiment of the sealant, each of the PEG-SG and / or PEG-amine is mixed with about 500 to about 4000 ppm of tocopherol. In a preferred embodiment, about 1500 to about 3000 ppm of tocopherol is added to each of the PEGs.

[0022] In some embodiments, the PEG-SG and PEG-amine each have an equal molecular weight. In a preferred embodiment, the PEG-SG and PEG-amine each have a molecular weight of about 20 kDa.

[0023] In another embodiment, the final concentration of each of PEG-SG and PEG-amine in the sealant applied to the tissue is about 39 mg / mL to about 87 mg / mL, preferably about 52 mg / mL to about 77 mg / mL, and more preferably 67 mg / mL.

[0024] In some embodiments, at least a portion of each of the multi-arm PEGs prior to dissolution is present in the form of particles having a size of about 250 micrometers or greater, hi other embodiments, the particle size range of at least one multi-arm PEG is from about 250 to about 1250 micrometers.

[0025] In another embodiment, the PEG-amine has about 20% of the particles having a size of at least 710 μm to maximize the dissolution rate of the sealant and minimize preparation time.

[0026] In another embodiment, the PEG-SG has about 40% of the particles having a size of at least 500 μm to maximize the dissolution rate of the sealant and minimize preparation time.

[0027] In another embodiment, the pKa of the reconstitution buffer for PEG-amine is preferably from about 8.0 to about 9.7 pKa, most preferably from about 8.8 to about 9.4 pKa.

[0028] In another embodiment, the pH of the reconstitution buffer for the PEG-amine is at least 0.05 pH units above the buffer pKa.

[0029] In another aspect of the sealant provided herein, the PEG-amine / CHES solution after reconstitution has a preferred pH of about 9.0 to about 10.5, more preferably about 9.20 to about 9.80. In a most preferred embodiment, the PEG-amine / CHES solution has a pH after reconstitution of about 9.35.

[0030] In another embodiment, the reconstitution buffer for PEG-amine in the pre-filled device is preferably about 190 to about 210 mM CHES buffer, having a pre-reconstitution pH of about 9.34 to about 9.80.

[0031] In another embodiment, the reconstitution buffer for the PEG-amine in the pre-filled device is about 180 mM to about 220 mM CHES buffer, having a pre-reconstitution pH of about 9.35 to about 9.80.

[0032] In another embodiment, the reconstitution buffer for PEG-amine in the pre-filled device is about 180 to about 220 mM CHES buffer, having a pre-reconstitution pH of about 9.37 to about 9.80.

[0033] In a preferred embodiment, the reconstitution buffer for the PEG-amine in the pre-filled device is 200 mM CHES buffer with a pre-reconstitution pH of 9.58.

[0034] In another embodiment, the reconstitution buffer for PEG-SG is a citrate buffer having an initial pH of 7.0 or less and a concentration of about 3.5 mM to about 15 mM, more preferably about 5.5 mM to about 12.5 mM.

[0035] In a preferred embodiment, before irradiation, the concentration of the citrate buffer is 9.04 mM and the initial pH is about 4.5.

[0036] In another embodiment, a colorant such as FD&C Blue #1 is incorporated into one or both of the dry PEG powders. In another aspect, FD&C Blue #1 is incorporated into one or both of the PEG powders at a concentration of about 730 ppm to about 3000 ppm prior to irradiation.

[0037] In another embodiment of the device, FD&C Blue #1 is increased up to 300 ppm to counter color loss caused by irradiation. Preferably, the final concentration of FD&C Blue #1 in the lung sealant of the present invention is about 50 μg / mL to about 100 μg / mL.

[0038] In a preferred embodiment, the lung sealant comprises two 4-arm PEGs, the first PEG being 4-arm PEG-NH2-HCl, the second PEG being 4-arm PEG-SG, the alkaline buffer being CHES, the weakly acidic buffer being citrate buffer, the antioxidant being tocopherol, and the colorant being FD&C Blue #1, with the final concentrations of the lung sealant composition after combining the components being 67 mg / mL 4-arm PEG-SG (20 kDa), 67 mg / mL 4-arm PEG-NH2-HCl (20 kDa), 100 mM CHES, 4.5 mM citrate buffer, 340 μg / mL tocopherol, and 88 μg / mL FD&C Blue #1.

[0039] In some embodiments of the present invention, kits are provided, each kit comprising two dry PEG powders and two buffers. In a preferred embodiment, the kit comprises dry 4-arm PEG-amine and dry 4-arm PEG-SG, an alkaline buffer, and a weakly acidic buffer.

[0040] In another embodiment, the biodegradable sealant is combined with a contrast agent for later locating the sealing site. In one aspect, the contrast agent is a non-ionic contrast agent such as iohexol for radiopacity.

[0041] In another embodiment, the biodegradable sealant is combined with a contrast agent to facilitate detection by magnetic resonance imaging.

[0042] In yet another embodiment, the imaging agent is a radioactive agent that allows localization of the seal site using radiodetection methods.

[0043] In another embodiment, the biodegradable sealant composition can be combined with a therapeutic agent to provide localized delivery of the therapeutic agent, for example, the therapeutic agent can be one or more chemotherapeutic agents for the management of cancer.

[0044] In another embodiment, a method of using the lung sealant includes adding tocopherol to a 4-arm PEG-amine to form a mixture of 4-arm PEG-amine and tocopherol, irradiating the mixture of 4-arm PEG-amine and tocopherol, adding an alkaline buffer to the mixture of 4-arm PEG-amine and tocopherol to form a first component of the lung sealant, adding tocopherol to a 4-arm PEG-NHS ester to form a mixture of 4-arm PEG-NHS ester and tocopherol, irradiating the mixture of 4-arm PEG-NHS ester and tocopherol, and adding a weakly acidic buffer to the mixture of 4-arm PEG-NHS ester and tocopherol to form a second component of the lung sealant, mixing and simultaneously applying the first and second components onto lung tissue using an applicator device to form the lung sealant, and allowing the sealant to harden on the tissue.

[0045] In some embodiments, in the methods of using the lung sealants herein, the 4-arm PEG-amine and 4-arm PEG-NHS ester are each in the form of a dry powder.

[0046] In another embodiment, a mixture of 4-arm PEG-amine and tocopherol, and a mixture of 4-arm PEG-NHS ester and tocopherol, are each irradiated with up to about 40 kilograys (kGy) of X-ray radiation.

[0047] As used herein, the terms "comprising," "including," "having," and grammatical variations thereof, are understood to identify the stated features, elements, steps, or components, but do not exclude the addition of one or more additional features, elements, steps, components, or groups thereof. These terms encompass the terms "consisting of" and "consisting essentially of." Furthermore, as used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more," unless the context clearly dictates otherwise.

[0048] These and other aspects and advantages of the present invention will become more apparent from the following description and accompanying drawings. [Brief explanation of the drawings]

[0049] [Figure 1] 1 is a graph showing the effect of various molecular weights of PEG-SG and PEG-amine on the maximum load at failure of lung sealants. [Figure 2] 1 is a graph showing the effect of various molecular weights of PEG-SG and PEG-amine on the tensile elongation at failure of lung sealants. [Figure 3] FIG. 1 is a contour plot showing the average maximum load assessment at various PEG-SG to PEG-amine ratios (r) and total polymer content (PC). [Figure 4]1 is a graph showing maximum tensile load evaluation of two PEG candidates of the present invention (Candidate #1: 62 mg / mL PEG-SG and 62 mg / mL PEG-amine, and Candidate #2: 67 mg / mL PEG-SG and 67 mg / mL PEG-amine) and a mock PROGEL formulation ("Mock PROGEL") prepared based on publicly available information for PROGEL sealant (65 mg / mL PEG-succinimidyl succinate, 150 mg / mL human serum albumin, 50 mM carbonate, pH=9.0). The maximum tensile load of the lung sealant of the present invention was 2.1-fold greater than that of Mock PROGEL. [Figure 5] 1 is a graph showing the elongation at break of two PEG candidates of the present invention (Candidate #1: 62 mg / mL PEG-SG and 62 mg / mL PEG-amine, and Candidate #2: 67 mg / mL PEG-SG and 67 mg / mL PEG-amine) and Mock ProGel (65 mg / mL PEG-succinimidyl succinate, 150 mg / mL human serum albumin, 50 mM carbonate, pH=9.0). The elongation at break of the two PEG candidates of the present invention was 7.2-fold greater than that of Mock ProGel. [Figure 6] 1 is a graph showing the stiffness of two PEG candidates of the present invention (Candidate #1: 62 mg / mL PEG-SG and 62 mg / mL PEG-amine, and Candidate #2: 67 mg / mL PEG-SG and 67 mg / mL PEG-amine) and Mock ProGel (65 mg / mL PEG-succinimidyl succinate, 150 mg / mL human serum albumin, 50 mM carbonate, pH=9.0). The stiffness of the two PEG candidates of the present invention was 2.9-fold lower than that of Mock ProGel. [Figure 7] 1 is a graph of the maximum shear modulus (Pa) of lung sealants as the concentration of PEG-SG and PEG-amine is increased in equal proportions from 40 mg / mL to 100 mg / mL. [Figure 8] 1 is a graph showing the polymerization time of lung sealant when the concentration of PEG-SG and PEG-amine is increased in equal proportions from 40 mg / mL to 100 mg / mL. [Figure 9] 1 is a graph showing the rate of polymerization of lung sealant when the concentration of PEG-SG and PEG-amine is increased in equal proportions from 40 mg / mL to 100 mg / mL. [Figure 10A] 1 is a graph showing the assessment of BHT at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of Mn (number average molecular weight). [Figure 10B] 1 is a graph showing the assessment of BHT at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of Mp (molecular weight of the highest peak). [Figure 10C] 1 is a graph showing the assessment of BHT at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of Mw (weight average molecular weight). [Figure 10D] 1 is a graph showing the assessment of BHT at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of polydispersity index (PDI). [Figure 11A] 1 is a graph showing the assessment of tocopherol at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of Mn (number average molecular weight). [Figure 11B] 1 is a graph showing the assessment of tocopherol at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of Mp (molecular weight of the highest peak). [Figure 11C] 1 is a graph showing the assessment of tocopherol at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in relation to Mw (weight average molecular weight). [Figure 11D] 1 is a graph showing the assessment of tocopherol at various concentrations in reducing the adverse effects of irradiation on 4-arm PEG-OH in terms of polydispersity index (PDI). [Figure 12A]1 is a graph showing the assessment of 2000 ppm (±)-α-tocopherol (tocopherol) on lung sealant PEG to reduce the effects of irradiation in terms of Mn (number average molecular weight). [Figure 12B] 1 is a graph showing the assessment of 2000 ppm (±)-α-tocopherol (tocopherol) to lung sealant PEG for reducing the effects of irradiation in terms of Mp (molecular weight of the highest peak). [Figure 12C] 1 is a graph showing the assessment of 2000 ppm (±)-α-tocopherol (tocopherol) to lung sealant PEG for reducing the effects of irradiation in terms of Mw (weight average molecular weight). [Figure 12D] 1 is a graph showing the assessment of 2000 ppm (±)-α-tocopherol (tocopherol) on lung sealant PEG to reduce the effects of irradiation in terms of polydispersity index (PDI). [Figure 13] 1 is a graph showing the dose-dependent effect of X-ray irradiation (kGy) on PEG-SG and PEG-amine functionality when 2000 ppm tocopherol is added to PEG-SG and PEG-amine, respectively. [Figure 14] 1 is a graph showing the effect of various X-ray irradiation (kGy) doses on aldehyde and formate formation for PEG-amine and PEG-SG. [Figure 15] 1 is a graph showing the effect of various X-ray irradiation (kGy) doses on the formation of PEG esters for PEG-amine and PEG-SG. [Figure 16] 1 is a graph showing the loss of the antioxidant tocopherol at various doses of X-ray irradiation (kGy) for PEG-amine and PEG-SG. [Figure 17] 1 is a graph showing the extent of dissolution of PEG-amine samples at various particle sizes at a controlled maximum. [Figure 18] 1 is a graph showing the extent of dissolution of PEG-amine samples at various particle sizes with controlled minimum and maximum ranges. [Figure 19]1 is a graph showing the extent of dissolution of PEG-SG samples with various particle sizes at a controlled maximum. [Figure 20] 1 is a graph showing the extent of dissolution of PEG-SG samples at various particle sizes with controlled minimum and maximum ranges. [Figure 21] 1 is a graph showing the effect of buffer pKa on polymerization time of PEG-SG and PEG-amine. [Figure 22] 1 is a graph showing the effect of pH of various CHES on the polymerization rate of PEG-amine (HCl salt). [Figure 23] 1 is a contour plot of the polymerization rate response at various pH and CHES concentrations. [Figure 24] 1 is a graph showing the results of burst pressure tests performed at various angles to demonstrate how the lung sealant of the present invention, with its improved ability to stay in place compared to Mock ProGel, affects performance. [Figure 25] 1 is an interaction plot showing the reduction of PEG-SG hydrolysis by citrate buffer. DETAILED DESCRIPTION OF THE INVENTION

[0050] The biodegradable lung sealant of the present invention is applied as a liquid that initially sets rapidly to remain at the target tissue location and provide a proper seal. The sealant has improved elasticity, tensile strength, visualization, and working time.

[0051] Furthermore, the sealants of the present invention surprisingly and unexpectedly have advantageous shear moduli, polymerization times, and polymerization rates for sealing lung tissue. In one aspect, the lung sealants provided herein comprise a combination of polyethylene glycol (PEG) polymers of equal molecular weight and an optimal buffer system. PEGs having a molecular weight of about 15 kDa to about 50 kDa are preferred for use in the lung sealants of the present invention.

[0052] The polymerization kinetics of the lung sealants provided herein can be determined as follows: (a) Maximum shear modulus, a measure of the stiffness of the sealant; (b) the polymerization time (k), defined as the time required to achieve 50% of the final sealant structure, and (c) Polymerization rate, defined as the maximum slope of the shear modulus.

[0053] Polymerization time (k) analyzes the shear modulus over time normalized to the maximum shear modulus. Polymerization time not only provides an understanding of the cure time (the time required to achieve the final structure), but also allows for the indirect determination of the initial set time, i.e., the time required for the sealant to stop flowing. Since the sealant initially cures within a few seconds, the initial set time cannot be directly quantified.

[0054] The polymerization rate provides a measure of the rate at which the shear modulus of the sealant increases over time and is sensitive to both the polymerization time and the final hydrogel structure.

[0055] The efficacy of a sealant, or its ability to provide a strong seal, is determined by the structure of the sealant, which is measured through cohesive tensile testing and shear modulus.

[0056] PEG suitable for use in the lung sealants of the present invention is a multi-arm PEG, such as a 2-, 3-, 4-, 6-, or 8-arm multi-arm PEG. In some preferred embodiments, the PEG is a 4-arm PEG. In some aspects, the multi-arm PEG is a multi-arm PEG derivative. In some embodiments, the multi-arm PEG derivative is a multi-arm PEG-amine hydrochloride (HCl) salt. For example, multi-arm PEGs are commercially available from JenKem Technology (Plano, Texas) or NOF Corporation (Tokyo, Japan). In preferred embodiments, the multi-arm PEG has a molecular weight of about 15 kDa to about 50 kDa, most preferably about 20 kDa. In some embodiments, the lung sealants provided herein comprise a multi-arm PEG having a reactive functional group at each end of the arm, such as an N-hydroxysuccinimide (NHS) ester group, which can form a stable conjugate, i.e., an amide bond, with the PEG-amine. An example of a suitable PEG for use in the sealants provided herein is 4-arm PEG-succinimidyl glutarate. glutarate (SG), 4-arm PEG-succinimidyl valerate, 4-arm PEG-succinimidyl carbonate, 4-arm PEG-succinimidyl succinate, 4-arm PEG-succinimidyl butanoate, 4-arm PEG-succinimidyl succinamide, 4-arm PEG-succinimidyl propionate, 4-arm PEG-sulfosuccinimidyl glutarate, 4-arm PEG-sulfosuccinimidyl valerate, 4-arm PEG-sulfosuccinimidyl carbonate, 4-arm PEG-succinimidyl carboxymethyl ester, 4-arm PEG-sulfosuccinimidyl succinate, 4-arm PEG-sulfosuccinimidyl butanoate, 4-arm PEG-sulfosuccinimidyl succinamide, 4-arm PEG-sulfosuccinimidyl propionate, 4-arm PEG-amine, 4-arm PEG-isocyanate, 4-arm PEG-imidoester, and 4-arm PEG-maleimide.

[0057] In another aspect, the sealants provided herein can include a thiol reactive with PEG-NHS ester and PEG-maleimide.

[0058] In some embodiments, the lung sealant contains PEG at a total final concentration in the sealant of about 39 mg / mL to about 87 mg / mL. In a preferred embodiment, the total concentration of each of the PEGs is about 67 mg / mL. Examples of preferred PEGs are 4-arm PEG-succinimidyl glutarate, MW 20 kDa (4-arm-PEG-SG-20K) (67 mg / mL), available from JenKem Technology or NOF Corporation, and 4-arm PEG-amine, MW 20 kDa (4-arm-PEG-NH2-20K) (67 mg / mL), available from JenKem Technology or NOF Corporation. In one aspect, the final concentrations of PEG-SG and PEG-amine in the sealant applied to tissue are each about 39 mg / mL to about 87 mg / mL, preferably about 52 mg / mL to about 77 mg / mL, and more preferably about 67 mg / mL.

[0059] The particle size distribution of commercially available PEG results in maximum dissolution using the method provided herein for preparing the biodegradable lung sealant of the present invention. However, particle size significantly affects the dissolution rate of PEG. Unexpectedly, larger particle size results in faster dissolution by reducing the tendency of PEG to aggregate and adhere to the wall of the glass vial.

[0060] For PEG-amine, a maximum particle size of at least 710 μm is preferred. For PEG-SG (when dissolved in a lower concentration buffer), a maximum particle size of at least 500 μm is most preferred. Lower maximum particle sizes will dissolve but require longer preparation times.

[0061] In another aspect, buffers suitable for use in preparing the lung sealants of the invention are alkaline buffers such as N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, 2-amino-2-methyl-1,3-propanediol, N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, pyridoxine buffer, and weakly acidic buffers such as citrate buffer. Preferably, the weakly acidic buffer has a pH of about 4 to about 6, or up to 7.

[0062] Carbonate buffers are not preferred because they have insufficient buffering capacity at pH 9, which causes increased fluidity. Fluidity can be decreased at higher pHs, but undesirable hydrolysis and tissue reactions can occur.

[0063] Higher molecular weight PEGs result in lung sealants with higher tensile elongation at failure. However, higher molecular weight PEGs may exhibit reduced renal clearance and undesirably longer half-lives. In a preferred embodiment, 20 kDa molecular weight PEGs provide a desirable balance between hydrogel structure and excretion / clearance time. In one aspect, the molecular weights of the two PEGs are similar or equal. In a preferred aspect, the PEGs are PEG-SG and PEG-amine, each having a molecular weight of 20 kDa.

[0064] The lung sealants provided herein have a low stiffness relative to currently commercially available lung sealants. Surprisingly and unexpectedly, the lung sealants herein exhibit greater strength, elasticity, and compliance than currently commercially available products. For example, the elongation at break of the lung sealants of the present invention is about 7 times greater than that of the Mock ProGel formulation (FIG. 5), and the stiffness is about 2.9 times lower than that of the Mock ProGel formulation (FIG. 6).

[0065] In some embodiments, a radioprotectant such as tocopherol mitigates the change in molecular weight (MW) of PEG. Irradiation of PEG is preferably carried out in a hypoxic environment.

[0066] The PEG-amine is preferably in the protonated form to prevent large irradiation-induced loss of functionality.

[0067] In some embodiments of the present invention, the lung sealant includes a colorant. In some aspects, the colorant is included in one or both of the dry PEG powders. In other aspects, the colorant is incorporated into the dry PEG-SG powder. Examples of colorants for use in the lung sealants of the present invention are FD&C Blue #1, FD&C Blue #2, FD&C Green #3, FD&C Yellow #6, and combinations thereof. In a preferred embodiment, about 1000 ppm to about 1600 ppm of the colorant FD&C Blue #1 is incorporated into the dry PEG-SG powder. Preferably, the final concentration of FD&C Blue #1 in the lung sealant of the present invention is about 50 μg / mL to about 100 μg / mL.

[0068] In one embodiment, a kit is provided that includes two dry PEG powders and two buffers. In a preferred embodiment, the kit includes dry 4-arm PEG-amine powder and dry 4-arm PEG-SG powder, CHES buffer, and citrate buffer.

[0069] The pH of the PEG-amine solution after dissolution is preferably about 9.0 to about 10.5, more preferably about 9.20 to about 9.80, and most preferably about pH 9.35.

[0070] The lung sealant of the present invention may be used to prevent prolonged air leak (PAL) after lung volume reduction (LVR), biological lung volume reduction (Bio-LVR), surgical lung volume resection (e.g., lung volume resection to treat emphysema), bullectomy, thoracotomy, sternotomy, and thoracic surgery.

[0071] Further details regarding the lung sealants of the present invention are provided in the non-limiting examples below. [Example]

[0072] Example 1 Effect of PEG polymer molecular weight on hydrogel structure. A study was conducted to understand the effect of PEG-amine and PEG-SG molecular weights on hydrogel structure at constant molar concentrations of each PEG to test tensile strength, elongation at break, and modulus. PEG-amine molecular weights were varied from 5 kDa to 20 kDa, and PEG-SG molecular weights were varied from 10 kDa to 20 kDa. A 50 mM carbonate buffer system was used for all conditions.

[0073] As shown in the graph in Figure 1, the maximum load at failure of the sealant formulations tested increased as the molecular weight of each PEG increased, with the most dramatic increase occurring when both PEG molecular weights were equal.

[0074] Interestingly, as shown in Figure 2, the tensile elongation at failure also increased with increasing molecular weight of each PEG. However, PEGs with molecular weights greater than about 50 kDa exhibit reduced renal clearance. A favorable balance between hydrogel structure and clearance time was found when each PEG had a molecular weight of 20 kDa.

[0075] Example 2 shear modulus, Effect of PEG concentration on polymerization time and polymerization rate A study was conducted to characterize a range of PEG-SG (20 kDa) and PEG-amine (20 kDa) concentrations using a 100 mM CHES (pH = 9.35), 3.75 mM citrate (pH = 5.0) buffer system. PEG-SG and PEG-amine were tested at equally increasing concentrations from 40 mg / mL to 100 mg / mL.

[0076] The polymerization kinetics of the sealant was studied via an oscillatory rheometry time sweep for 300 seconds at 1 Hz, 150 Pa. The shear modulus over time was fitted to the Hill equation to provide three outputs: (1) Maximum shear modulus, a measure of the stiffness of the compound; (2) the polymerization time (k), defined as the time to 50% of the final structure, and (3) The polymerization rate, defined as the maximum slope of the shear modulus.

[0077] To analyze the linear effect of such bulk polymerization systems, studies were designed with the total polymer content and the ratio of the two reagents as factors in a central composite design.

[0078]

number

[0079] In this study, as shown in Figure 3, the maximum tensile load was observed to increase linearly with increasing polymer content, with a quadratic effect of r being observed with a maximum value near r = 1.0, or at uniform concentrations of PEG-SG and PEG-amine.

[0080] As shown in Figure 4, the stiffness of the sealant of the present invention is very low relative to the simulated formulation of the current commercially available sealant. Two candidate sealants of the present invention were tested against Mock ProGel (65 mg / mL PEG-succinimidyl succinate, 150 mg / mL human serum albumin, 50 mM carbonate (pH = 9.0)) in a tensile evaluation. The maximum tensile load of the two candidate sealants of the present invention was observed to be 2.1 times greater than that of Mock ProGel. The intervals were calculated using the individual standard deviations.

[0081] A theoretical analysis of the ultimate tensile stress requirements for human lung sealants was performed. The lung was assumed to be a spherical balloon with an extreme worst-case (male, right lung, 99.99th percentile) volume of 5371 mL, and the sealant thickness was assumed to be 1 mm. The theoretical calculation of burst pressure was as follows:

[0082]

number

[0083] where UTS is the ultimate tensile stress of the sealant, Delta is the thickness of the sealant, and R is the radius of the lung. A safety factor of 3 was applied to the standard intrathoracic pressure of 30 cm HO, and the burst pressure was set at 90 cm HO. A required ultimate tensile strength of 47.8 kPa was calculated, corresponding to a PEG concentration of 39.5 mg / mL PEG-SG and 39.5 mg / mL PEG-amine.

[0084] Various concentrations of PEG-SG and PEG-amine were tested. The concentrations of PEG-SG and PEG-amine were increased at equal rates from 40 mg / mL to 100 mg / mL, respectively. As shown in Figure 7, there was a significant effect of PEG concentration on shear modulus (p<0.01). Interestingly, the maximum shear modulus was observed to peak between 67 and 77 mg / mL, not significantly different from 52 and 62 mg / mL. Below 52 mg / mL and above 77 mg / mL, the shear modulus decreased sharply.

[0085] As shown in Figure 8, polymerization times of less than 25 seconds were observed to correlate with acceptable polymerization structures. The lower limit determined by the mechanical requirements of 39.5 mg / mL had a calculated polymerization time of 21 seconds according to the regression model. The upper limit of 86.5 mg / mL corresponded to a polymerization time of 25 seconds.

[0086] There was a significant effect of PEG concentration on the polymerization rate (p<0.01). As shown in Figure 9, the polymerization rate exhibited a significant peak at 67 mg / mL, and a rapid polymerization rate with no significant difference was observed in the range of approximately 52 mg / mL to approximately 77 mg / mL.

[0087] Combining data from mechanical analysis and polymerization time evaluation, the final concentration of PEG in each of the lung sealants of the present invention is about 39 to about 87 mg / mL, preferably about 52 to about 77 mg / mL, and more preferably 67 mg / mL. In some embodiments, the concentration of PEG in the lung sealant of the present invention is about 39 to about 87 mg / mL, more preferably about 52 to about 77 mg / mL, and most preferably about 67 mg / mL.

[0088] Example 3 PEG irradiation Gamma and X-ray irradiation studies were conducted to evaluate the ability of the lung sealant PEG to be sterilized. Irradiation of the PEG was performed under a nitrogen purge.

[0089] PEG was administered separately under ambient conditions in glass vials and subjected to 27.9–29.5 kGy of gamma irradiation. PEG-SG was significantly degraded and did not form hydrogels when combined with non-irradiated PEG-amine. PEG-amine lost 30% of its functionality, demonstrating significant cross-linking of the PEG backbone.

[0090] Subsequent studies were performed in the same manner, except that the vials were evacuated to 100 mTorr and purged with nitrogen to 600 Torr, and the PEG-amine was irradiated in its protonated HCl salt form. Relative to ambient conditions, the loss in functionality of PEG-SG was reduced by 4%, and low molecular weight degradants were reduced by an order of magnitude, resulting in effective crosslinking when mixed with PEG-amine. The loss in functionality of PEG-amine was reduced by 18%, and chain degradation was similarly reduced.

[0091] These results suggest that irradiation of lung sealant PEG must be performed in a hypoxic environment and that the PEG-amine should be in the protonated form to prevent a significant loss of functionality.

[0092] To reduce the adverse effects of the irradiation process on the lung sealant PEG, two antioxidants, BHT and tocopherol, were added at various concentrations up to 4000 ppm.

[0093] As shown in Figures 10A-10D, BHT was added at various concentrations to test for reducing the adverse effects of irradiation on 4-arm PEG-OH with respect to Mn (number average molecular weight) (Figure 10A), Mp (molecular weight of the highest peak) (Figure 10B), Mw (weight average molecular weight) (Figure 10C), and polydispersity index (PDI) (Figure 10D).

[0094] There was no significant effect on molecular weight distribution, nor was there a relationship between viscosity and BHT concentration. Residual BHT was already present in the PEG at up to 300 ppm as an oxidation protectant. The addition of additional BHT did not reduce the effect of the irradiation process on the 4-arm PEG-OH.

[0095] As shown in Figures 11A-11D, tocopherol was added at various concentrations to test for reducing the adverse effects of irradiation on 4-arm PEG-OH with respect to Mn (number average molecular weight) (Figure 11A), Mp (molecular weight of the highest peak) (Figure 11B), Mw (weight average molecular weight) (Figure 11C), and polydispersity index (PDI) (Figure 11D).

[0096] The addition of tocopherol manifested a linear decrease in the molecular weight distribution after irradiation as the tocopherol concentration increased up to 1500 ppm and the viscosity decreased up to 2000 ppm.

[0097] Therefore, tocopherol in the range of 1500-4000 ppm added to multi-arm PEG is maximally effective in achieving the following benefits: 1) reduced loss of mechanical performance in uniaxial tension, including ultimate tensile strength and elongation at failure; 2) an increase in the maximum allowable dose of X-ray radiation; and 3) Reducing the viscosity of the resulting PEG solution to reduce pressure forces and improve the ease of spraying or instilling the lung sealant composition onto lung tissue.

[0098] To further understand the effect of tocopherol on lung sealant PEG throughout the irradiation process, the dose-dependent effects of X-ray irradiation with 2000 ppm (±)-α-tocopherol (tocopherol) were investigated. PEG was analyzed for changes in molecular weight distribution as measured via gel permeation chromatography (GPC), chemical functionality via NMR, polymerization time, and cohesive tensile testing.

[0099] As shown in Figures 12A-12D, the changes in Mn (number average molecular weight) (Figure 12A) and Mp (molecular weight of the highest peak) (Figure 12B) were relatively small. Mn had a maximum change of 6% and 13% for PEG-SG and PEG-amine, respectively. Mp demonstrated a maximum change of 13% and 4% for PEG-SG and PEG-amine, respectively. Mw (weight average molecular weight) (Figure 12C) demonstrated a more significant change of up to 18% and 34% for PEG-SG and PEG-amine, respectively. However, the change was less than 10% for each PEG up to 25 kGy and 17% at 35 kGy, suggesting that the greatest difference was observed above 35 kGy. The polydispersity index (PDI) of PEG-amine (Figure 12D) increased more than that of PEG-SG due to both a decrease in Mn and an increase in Mw.

[0100] The dose-dependent effect of X-ray irradiation (kGy) on PEG-SG and PEG-amine functionality when 2000 ppm tocopherol was added to each is shown in Figure 13. PEG was X-irradiated from 37.6 kilograys (kGy) to 46.8 kGy. There was a significant dose-dependent effect of X-ray irradiation on PEG-SG functionality (p<0.01, one-way ANOVA), but not on PEG-amine functionality (p=0.11, one-way ANOVA). Linear regression indicated that for PEG-SG, 0.14% functionality was lost for each 1 kGy increase in irradiation. The maximum loss observed at 46 kGy was 10% for PEG-SG.

[0101] Although low molecular weight PEG oxidation products were observed in the irradiated samples, no significant differences were observed (one-way ANOVA). PEG aldehyde and formate remained below 0.05% up to 37 kGy in the PEG-amine samples and at all irradiation doses in the PEG-SG samples. As shown in Figure 14, at 46 kGy, 0.01% aldehyde and formate were observed in the PEG-SG samples, and at 46 kGy, 0.01% to 0.22% aldehyde and formate were observed in the PEG-amine samples, resulting in an average of approximately 0.11% for the PEG-amine, suggesting that the response to irradiation may be more variable at this dose for the PEG-amine.

[0102] As shown in Figure 15, PEG esters remained below 0.15% on average at all irradiation doses in PEG-SG and up to 37 kGy in PEG-amine. At 46 kGy, 0.21% PEG esters were observed in PEG-amine and 0.12% PEG esters were observed in PEG-SG, suggesting that the response to irradiation may be more variable at this dose for PEG-amine.

[0103] Tocopherol concentrations were quantified via HPLC. As shown in Figure 16, PEG-amine demonstrated a linear decrease in tocopherol as the irradiation dose increased up to 37 kGy. At higher doses, tocopherol concentrations decreased to approximately 6% of the concentration in the control sample. PEG-SG demonstrated a more significant decrease in tocopherol, remaining at approximately 8% at 37 kGy.

[0104] Tocopherol concentrations were significantly affected by PEG-amine (p<0.01) and PEG-SG (p<0.01, one-way ANOVA). Given that the response was linear up to 37 kGy, regressions were performed to understand the changes in tocopherol concentrations with X-ray irradiation. 6.4 ppm and 47 ppm of tocopherol were lost per 1 kGy increase in X-ray irradiation for PEG-amine and PEG-SG, respectively.

[0105] With regard to cohesive mechanical properties, no significant differences were observed in percent elongation up to 45 kGy, and tensile strength and modulus up to 35 kGy.

[0106] There was a significant effect of irradiation dose on polymerization time and rate as assessed in the rheometric polymerization evaluation. However, all irradiation doses tested did not differ significantly between groups, except for 37 kGy, which had a polymerization time 64.2% longer than the control.

[0107] The use of about 1500 ppm to about 3000 ppm of tocopherol in the lung sealant PEG compositions provided herein is most preferred because this range minimizes the amount of tocopherol required while achieving the full benefit of tocopherol as a radioprotectant. However, concentrations of about 500 ppm to about 4000 ppm of tocopherol also have a significantly favorable effect on PEG molecular weight distribution. The maximum X-ray exposure dose is most preferably about 35 kGy to about 45 kGy.

[0108] Example 4 PEG particle size distribution The lung sealant provided herein is prepared from PEG powder and a buffer solution. The time required to dissolve the PEG powder was tested to identify the desired particle size range. PEG (JenKem) was sieved into various particle size ranges, dried under vacuum, and loaded into a lung sealant applicator device. The PEG was dissolved in the applicator device using a syringe method: three strokes (each stroke representing one cycle of pushing and pulling the syringe plunger in the device), followed by a 1-minute constant circular gyration. Dissolution was tested spectrophotometrically at 534 nm using barium chloride and potassium iodide.

[0109] Two sets of PEG-amine samples were tested: 1) particle size with a controlled maximum (Figure 17), and 2) particle size with a controlled minimum and maximum range (Figure 18). Unexpectedly, as shown in Figure 17, there was a statistically significant increase in the degree of dissolution as the maximum particle size increased up to 710 μm, after which there was no further increase in the degree. The degree of dissolution was 36%, 51%, 83%, and 97% for the 125, 250, 500, and 710 μm particle sizes, respectively, relative to the 1200 μm maximum particle size group. Small particles clumped and aggregated, slowing dissolution. A similar trend was observed when comparing particle sizes with controlled minimum and maximum ranges. As shown in the graph in Figure 18, there was a statistically significant increase in the degree of dissolution, with the range of 710 μm to 1200 μm demonstrating the greatest and most favorable dissolution. The extent of dissolution was 67%, 86%, and 87% for the 125-250 μm range, 250-500 μm range, and 500-710 μm range, respectively, for the 710-1200 μm maximum particle size group.

[0110] Two sets of PEG-SG samples were tested: 1) particle size with a controlled maximum (Figure 19), and 2) particle size with a controlled minimum and maximum range (Figure 20). In the lung sealant formulation of the present invention, PEG-SG may dissolve more readily due to the lower concentration of citrate buffer, allowing PEG-SG to solvate more readily. As with PEG-amine, there was a significant effect of particle size on the degree of dissolution. However, as shown in the graph in Figure 19, for PEG-SG, maximum particle sizes of 500 μm, 710 μm, and 1200 μm completely dissolved, significantly more than the lower maximum particle sizes (125 μm and 250 μm). Particle sizes greater than 1200 μm were also tested and similarly completely dissolved. As shown in the graph in Figure 20, comparing the particle size ranges, there was a statistically significant increase in the degree of dissolution up to 500-710 μm, after which all groups were completely dissolved. The degree of dissolution was 40% and 86% for the 125-250 μm and 250-500 μm ranges, respectively, relative to the 500-710 μm range. PEG-SG in the lower particle size range resulted in a similar phenomenon observed with PEG-amine. However, when a 500 μm particle size was present, PEG-SG was completely dissolved.

[0111] Particle size has a significant effect on the dissolution rate of PEG. Unexpectedly, larger particle sizes result in faster dissolution by reducing the tendency of PEG to aggregate and adhere to the glass vial wall. For PEG-amine, it is most preferable to have particles with a particle size of up to 710 μm or more, or up to 1200 μm, present in the particle size distribution along with smaller particles.

[0112] For PEG-SG (dissolved in a lower concentration buffer), it is most preferred that the particle size distribution have particles with particle sizes up to 500 μm or up to 1200 micrometers, distributed with smaller particles. Lower maximum particle sizes will still ultimately dissolve, but will require longer preparation times.

[0113] Example 5 PEG-amine buffer (alkaline buffer) NHS ester / amine chemistry is generally performed at pH 7.2 to pH 8.5 to yield stable amide bonds. The most common buffers utilized are phosphate, carbonate-bicarbonate, HEPES, or borate buffers. To maintain a sufficient proportion of the amine in a deprotonated state, the pH must be maintained at a slightly alkaline pH. However, as the pH increases, the rate of hydrolysis of the PEG-SG ester similarly increases. Therefore, when developing a fast-initial-cure sealant based on this chemistry, a balance between increasing the reaction rate and losing the reactivity of the PEG-SG is crucial.

[0114] Unexpectedly, carbonate buffer, a commonly used buffer for this chemistry, was found to be inappropriate for use in the method for making the lung sealant of the present invention. A 100 mM carbonate buffer system was tested with 28.5 mg / mL 4-arm PEG-amine-10K and 75 mg / mL 4-arm PEG-SG-20K systems at various pH levels using a 40-degree inclined plane migration distance assay. Migration distance was found to decrease significantly with increasing pH. Migration distances were 236 mm at pH 9.0, 140 mm at pH 10.0, and 85 mm at pH 11.0. By comparison, fibrin sealant migrated approximately 60 mm in the same assay. Considering that a pH greater than 10.5 is considered corrosive, carbonate buffer systems were found to be insufficient to maintain a pH high enough for satisfactory polymerization kinetics due to unacceptable fluidity at pH 9.

[0115] Other buffer candidates were investigated with the goal of minimizing the required initial pH of the sealant to avoid any possible corrosive effects. Several buffer candidates were tested for their ability to maintain the sealant pH at 9.0 ± 0.5 pH throughout the crosslinking reaction. A methoxy PEG-SG surrogate material was utilized in place of the 4-arm PEG-SG to avoid gelation and allow for continuous monitoring of the pH of the reaction when combined with PEG-amine.

[0116] The pH was measured over the course of 10 minutes at 100 mM (final concentration) to determine how well the buffers maintained the pH when starting at pH 9. The data demonstrated that borate, CHES, and pyridoxine buffers were acceptable in the 9-9.4 pKa range.

[0117] To understand the appropriate range for NHS ester / amine chemistry, a series of buffer pKas were investigated, ranging from 7.2 pKa to 10.3 pKa, as shown in Table 1 below. The post-reconstitution pH of all buffers was held constant at pH=9.35. Final PEG-SG and PEG-amine concentrations of 67 mg / mL were tested.

[0118] [Table 1]

[0119] As shown in Figure 21, polymerization time was significantly affected by buffer pKa (p<0.01). At pH 9.35, buffers with pKas in the range of 8.8 to 9.4 demonstrated significantly reduced polymerization times compared to those outside that range. Buffer pKas of 8.4 and 9.6 demonstrated similarly rapid polymerization rates. Quadratic regression was used to determine the pKa range that resulted in acceptable polymerization times. For polymerization times less than 25 seconds, buffers with pKas in the range of about 7.9 to about 9.7 are preferred.

[0120] This finding regarding buffers for use in making the lung sealants of the present invention is unexpected and contradicts the use of buffers commonly used in sealant formulations. Most commercially available sealant products utilizing NHS ester / amine chemistry utilize phosphate, triethanolamine, carbonate, or a combination of such buffer systems in their formulations. While there is typically little discussion of the reasons for using such buffers, the choice is likely due to the popularity of these buffers across medical devices and pharmaceutical formulations, as well as proven safety data. The lung sealant compositions of the present invention provided herein include biological buffers with a narrower pKa range, preferably from about 7.9 to about 9.7, and most preferably from about 8.8 to about 9.4.

[0121] Additionally, to select the correct buffer candidate, the set-up time of the sealant was optimized, taking into account the pH after reconstitution. In this evaluation, the polymerization time of the sealant was evaluated by migration distance evaluation on a 40-degree inclined surface. It was discovered that a significant decrease in migration distance was observed when the pH of the buffer system was increased above the system's pKa. This observation was also consistent for borate buffer (pKa = 9.0) and pyridoxine buffer (pKa = 9.4). These results suggest that the initial cure time of the sealant can be significantly decreased by increasing the pH after reconstitution by at least 0.05 pH units above the buffer's pKa.

[0122] CHES buffer (pKa = 9.3) was tested by polymerization time rheometry in a two-part experiment. In the first study, a wide pH range from 8.0 to 10.5 was tested. It was observed that the sealant did not polymerize at pH ≤ 8.5. In addition, the maximum shear modulus decreased linearly as the pH increased from 9.0 to 10.5. The polymerization time decreased significantly from 9.0 to 9.5 and did not decrease further. There was a significant effect of pH on the polymerization rate, with the fastest polymerization rate observed at pH = 9.5.

[0123] In a second experiment, a higher resolution study was conducted to assess pH from 9.05 to 9.8 in 0.15 unit increments. In this study, the maximum shear modulus was observed to decrease by approximately 3% for each 0.15 pH unit increase. However, the polymerization time decreased significantly from 9.05 to 9.35, after which no significant differences were observed. As shown in Figure 22, the polymerization rate demonstrated a maximum average value at 9.35, but did not differ significantly from 9.20 to 9.80.

[0124] Therefore, the pH of the CHES after dissolving PEG-amine is preferably about 9.0 to about 10.5, more preferably about 9.20 to about 9.80, and most preferably pH 9.35. As shown in Figure 23, there was a significant effect on the polymerization rate by both CHES concentration (p<0.01) and pH (p<0.01). There was a significant quadratic effect of pH (p<0.01). The fastest polymerization rates were observed at high CHES concentrations and intermediate pH. As the pH increased above approximately 9.55, the polymerization rate decreased.

[0125] Example 6 Adhesion evaluation The lung sealant of the present invention was compared to Mock ProGel for its ability to remain in place. In this evaluation, 4 mL of sealant was manually sprayed onto a 2 cm wide target zone at a rate of approximately 2 mL / sec on a 40 degree inclined surface. The percentage of sealant remaining within the target zone was determined gravimetrically.

[0126] A prototype lung sealant formulation of the present invention was compared with a Mock ProGel formulation. The lung sealant formulation tested was 78 mg / mL 4-arm PEG-amine HCl-20k, 63 mg / mL 4-arm PEG-SG-20k, and 100 mM CHES (pH=9.35). The Mock ProGel formulation was 65 mg / mL linear PEG-SS-4k, 150 mg / mL bovine serum albumin, and 50 mM carbonate buffer (pH=9.0).

[0127] Next, functional burst pressure testing was performed to understand how the improved ability to stay in place affected performance. In this evaluation, the formulations tested in the angled adhesion evaluation were tested here. As shown in Figure 24, burst pressure testing was performed on 0° (flat), 30°, and 45° inclined surfaces on porcine pleural tissue with a 1 mm circular defect. The sealant was sprayed onto the tissue, allowed to cure for 10 minutes, and pressure was applied to the tissue until it failed.

[0128] Qualitatively, the lung sealant of the present invention was observed to completely cover the target tissue at all angles tested: Mock ProGel completely covered the tissue at the 0° angle, but at 30° some tissue was exposed, and at 45° most of the sealant ran off, forming a hydrogel as it ran.

[0129] Quantitatively, the burst pressure of the lung sealant of the present invention did not change significantly with bevel angle, whereas that of Mock ProGel decreased significantly with increasing bevel angle, decreasing by 77% at 45° relative to flat conditions.

[0130] Example 7 PEG-SG buffer (weakly acidic buffer) NHS-activated esters such as PEG-SG are known to hydrolyze over time. Therefore, PEG-SG must be maintained in dry powder form until use. The rate of hydrolysis is dependent on the solution pH, and the half-life decreases as the pH increases. The rate of hydrolysis must be considered, as it determines the working time, defined as the time from PEG dissolution to sealant application.

[0131] As shown in Table 3 below, currently available products with NHS ester chemistry have relatively short working times. ProGel, DuraSeal, Adherus, and CoSeal include working times of 20, 60, 60, and 120 minutes in their instructions, respectively. ProGel does not use an aqueous buffer solution to dissolve the PEG ester in the sealant formulation. DuraSeal and Adherus use a phosphate buffer system to extend working time to 60 minutes, and CoSeal uses a phosphate buffer system to extend working time to 120 minutes.

[0132] [Table 2] * These values ​​are based on publicly available information.

[0133] The method for making lung sealant provided herein utilizes a weakly acidic buffer to maintain PEG-SG at a weakly acidic pH. The degree of hydrolysis of PEG-SG was monitored over time using a spectrophotometric assay. Briefly, PEG-SG was dissolved in the target buffer, and 10 μL of the sample was added to a microplate and incubated for a set time. At each time point, 100 μL of 10×PBS (pH=7.8) was added to the solution to stop further hydrolysis, and the absorbance was immediately measured at 260 nm.

[0134] Citrate buffers at pH 3.0, 4.0, 5.0, 6.0, and 7.0 were compared to water (assuming pH=7.0) by three-way ANOVA, as shown in Figure 25. Citrate buffer was found to significantly reduce the extent of PEG-SG hydrolysis by 2.5% relative to water over the course of 2 hours.

[0135] The data support that the hydrolysis rate of PEG-SG can be minimized by dissolution in a weakly acidic buffer, such as citrate buffer, at pH 4 to about 6 or up to 7, and that as the pH decreases, the hydrolysis rate also decreases. Most preferred is a pH of about 4 to less than 5. This contradicts previous teachings suggesting that a pH of 5.0 to 6.0 is optimal.

[0136] The concentration of the PEG-SG buffer is equally important. The concentration must be high enough to neutralize the acidic NHS-induced changes released by PEG-SG upon hydrolysis, yet low enough so as not to inhibit the polymerization reaction when mixed with the PEG-amine solution. Citrate buffer concentrations up to 12.5 mM do not affect the polymerization time of the lung sealant. However, citrate buffer concentrations higher than 15 mM cause an increase in polymerization time. Citrate buffer concentrations of about 3.5 mM to about 15 mM are preferred, with citrate concentrations of about 5.5 to about 12.5 mM being most preferred.

[0137] A response optimization algorithm was implemented to identify the pre-irradiation concentration and pH that would result in a post-irradiation 8 mM citrate buffer with a pH of 4.92. According to this algorithm, a pre-irradiation citrate buffer of 10.6 mM (pH = 4.09) would result in a target post-irradiation 8 mM buffer (95% confidence interval: 7.89-8.11) with a pH of 4.92 (95% confidence interval: 4.91-4.93).

[0138] Example 8 Irradiation of FD&C Blue #1 lung sealant colorant. A 20 μg / mL aqueous solution of FD&C Blue #1 was gamma-irradiated at 32.3-33.1 kGy. The absorbance at 628 nm was measured for the non-irradiated control and irradiated samples. The measured absorbances were 2.734 and -0.031, respectively. The blue color of the solution was lost as a result of irradiation. Therefore, no colorant should be added to the buffer solution.

[0139] In subsequent preclinical studies, colorants were added to vials containing dry PEG-SG and dry PEG-amine. FD&C Blue #1 was dissolved in ethanol at 1 mg / mL. 120 μL was pipetted into each vial (final concentration 20 μg / mL) and dried overnight at room temperature in a vacuum oven. The PEG was then purged with nitrogen, sealed, and irradiated at 26.9–27.4 kGy. The blue color was maintained in each of the dry PEG compositions after irradiation. Therefore, it is preferable to incorporate FD&C Blue #1 into the dry PEG compositions, since irradiating the colorant after adding it to the buffer solution results in loss of color.

[0140] Example 9 FD&C Blue#1 irradiation dose study There were significant differences in FD&C Blue #1 concentration observed with X-ray exposure dose. No differences were observed up to 15 kGy relative to the non-irradiated control. There was a moderate linear correlation between X-ray exposure dose and FD&C Blue #1 concentration, which indicated a loss of 6.6 ppm of FD&C Blue #1 for every 1 kGy increase in exposure dose. Therefore, the colorant should be overdosed by 297 ppm to counter the color loss caused by exposure to doses up to 45 kGy.

[0141] Example 10 Lung sealing efficacy in a physiological ex vivo lung chamber model Lung sealing efficacy was tested in porcine lungs in an in vitro physiological lung chamber model. A lung sealing formulation containing 28.5 mg / mL 4-arm PEG-amine-10K, 75 mg / mL 4-arm PEG-SG-20K, and 50 mM carbonate (pH = 9.0) was tested against Mock ProGel. This study focused on application with the tissue in a flat orientation to demonstrate lung sealing efficacy and avoid variability caused by the sealant's ability to stay in place. A 10 mm long, 5 mm deep linear parenchymal defect and staple line air leak were created, resulting in mild to severe air leaks. The lung sealant formulation (n = 4) and Mock ProGel (n = 3) demonstrated 100% sealing efficacy under positive ventilator pressures of up to 30 cm of HO. Under negative pressure, the lung sealant formulation demonstrated 100% success, while Mock ProGel failed in 33% of linear parenchymal defects and 66% of staple line air leaks. Mock ProGel demonstrated exclusively cohesive failure. The lung sealant formulation in this example demonstrated 100% sealing efficacy using a carbonate buffer; however, unexpectedly, as described herein, carbonate buffers are not preferred for use in the lung sealant formulations of the present invention. Further studies showed unexpectedly superior results using alkaline and weakly acidic buffers.

[0142] Example 11 Lung sealing efficacy in an in vivo model Lung sealing efficacy was tested in an acute large animal model. The presently disclosed lung sealing formulations, 62 mg / mL 4-arm PEG-amine-20K (HCl salt), 62 mg / mL 4-arm PEG-SG-20K, 100 mM CHES (pH=9.4), and the control test article Mock ProGel, were tested. A 12 mm long, 5 mm deep linear parenchymal defect and a wedge resection of the left apical lung lobe were performed using an Echelon Flex 50 mm linear stapler. An air bubble leak test was performed to characterize the severity of air leaks in 20 cm of HO. The formulations were applied using an EVICEL applicator with an airless spray accessory. Once the pre-procedure air leak assessment was completed, the visceral surface of the deflated lung was wiped with dry gauze to remove excess moisture, and then the control Mock ProGel formulation or the disclosed lung sealant formulation (delivered by the prototype device) was applied to the air leak site and staple line. After allowing the control Mock ProGel formulation or the disclosed lung sealant formulation to harden for approximately 2 minutes, the lung was fully inflated and a bubble test was performed at 20 cm and 30 cm of HO ventilator pressure to assess initial sealing efficacy. The disclosed lung sealant was 100% effective (n=4). Once initial sealing efficacy was achieved, the chest wall was temporarily closed using standard techniques. A chest drainage catheter was placed into the pleural cavity and connected to a Pleur-evac® drainage system to restore negative intrathoracic pressure. After the incision was closed, each animal was continuously maintained under anesthesia for approximately 3.5 hours, during which time mechanical ventilation was utilized. The lung sealant formulation of the present disclosure demonstrated 100% sealing efficacy under positive ventilator pressures up to 30 cm of H2O (n=4).

[0143] Additives such as chemotherapeutic agents, growth factors, cytokines, antimicrobial agents, procoagulant hemostatic agents, antifibrinolytic agents and combinations thereof may be delivered in the lung sealant of the present invention.

[0144] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0145] While various variations of the present disclosure have been shown and described, further adaptations of the methods and systems described herein may be achieved by those skilled in the art through appropriate modifications without departing from the scope of the present invention. While some such possible modifications have been described, other modifications will be apparent to those skilled in the art. For example, the embodiments, variations, geometries, materials, dimensions, proportions, steps, etc. discussed above are illustrative and not required. Accordingly, it will be understood that the scope of the present invention should be considered in terms of the following claims, and is not limited to the details of construction and operation shown and described in this specification and drawings.

[0146] [Embodiment] (1) A biodegradable lung sealant, a) a first component comprising a first multi-arm PEG and an alkaline buffer for reconstituting the first multi-arm PEG; b) a second component comprising a second multi-arm PEG and a weakly acidic buffer for reconstituting the second multi-arm PEG; A biodegradable lung sealant, wherein when components (a) and (b) are combined, they produce an adhesive and elastic biodegradable lung sealant. (2) The lung sealant of embodiment 1, wherein the multi-arm PEG of the first component and the multi-arm PEG of the second component each further comprise an antioxidant. (3) The lung sealant of embodiment 2, wherein the antioxidant is tocopherol. (4) The lung sealant of embodiment 3, wherein the antioxidant is selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, tocotrienol, and combinations thereof. (5) The lung sealant of embodiment 2, wherein the first multi-arm PEG, the second multi-arm PEG, or both the first multi-arm PEG and the second multi-arm PEG further comprise a colorant.

[0147] (6) The lung sealant of embodiment 5, wherein the colorant is selected from the group consisting of FD&C Blue #1, FD&C Blue #2, FD&C Green #3, FD&C Yellow #6, and combinations thereof. (7) The lung sealant of embodiment 1, wherein the first multi-arm PEG and the second multi-arm PEG are each four-arm PEG. (8) The lung sealant of embodiment 1, wherein the first multi-arm PEG is a PEG-amine and the second multi-arm PEG is a PEG-NHS ester. (9) The lung sealant of embodiment 8, wherein the PEG-amine is 4-arm PEG-NH2-HCl. (10) The lung sealant of embodiment 8, wherein the PEG-NHS ester is 4-arm PEG-succinimidyl glutarate (SG).

[0148] (11) The lung sealant of embodiment 8, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester in the lung sealant are each about 39 mg / mL to about 87 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa. (12) The lung sealant of embodiment 11, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each about 52 mg / mL to about 77 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa. (13) The lung sealant of embodiment 12, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each 67 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa. (14) The lung sealant of embodiment 8, wherein the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each formulated with about 1500 ppm to about 3000 ppm of an antioxidant. 15. The lung sealant of claim 14, wherein the antioxidant is tocopherol.

[0149] (16) The lung sealant of embodiment 9, wherein the pKa of the buffer for reconstituting the PEG-NH2-HCl is about 7.9 pKa to about 9.7 pKa. (17) The lung sealant of embodiment 16, wherein the pKa of the buffer for reconstituting the PEG-NH2-HCl is about 8.8 pKa to about 9.4 pKa. (18) The lung sealant of embodiment 9, wherein the pH of the buffer after reconstitution for PEG-NH2-HCl is at least 0.05 pH units above the buffer pKa. (19) The lung sealant of embodiment 18, wherein the PEG-NH2-HCl has a pH after reconstitution of about 9.0 to about 10.5. (20) The lung sealant according to embodiment 8, wherein the buffer for reconstituting the PEG-NHS ester has a concentration of about 3.5 mM to about 15 mM and a pH before reconstitution of about 4.0 to about 7.0.

[0150] (21) The lung sealant of embodiment 8, wherein the buffer for reconstituting the PEG-NHS ester has a pH before reconstitution of about 4.0 to less than 5.0. (22) The lung sealant of embodiment 8, wherein the first multi-arm PEG is PEG-NH2-HCl, the second multi-arm PEG is 4-arm PEG-SG, and the lung sealant further comprises tocopherol and FD&C Blue #1, wherein the final concentration of the lung sealant composition after combining the components comprises 67 mg / mL 4-arm PEG-SG (20 kDa), 67 mg / mL 4-arm PEG-NH2-HCl (20 kDa), 340 μg / mL tocopherol, and 80 μg / mL FD&C Blue #1. (23) The lung sealant of embodiment 1, further comprising a contrast agent. (24) A kit for preparing a biodegradable lung sealant, comprising: a first storage container containing a first multi-arm dry PEG powder; a second storage container containing an alkaline buffer solution for preparing the first multi-arm PEG composition; a third storage container containing a second multi-arm dry PEG powder; and a fourth storage container containing a weakly acidic buffer solution for preparing the second multi-arm PEG composition, wherein the biodegradable lung sealant is formed after the first multi-arm PEG composition and the second multi-arm PEG composition are combined and simultaneously applied onto lung tissue. (25) A method of using the lung sealant of embodiment 8, comprising: adding tocopherol to the 4-arm PEG-amine to form a mixture of 4-arm PEG-amine and tocopherol; irradiating the mixture of 4-arm PEG-amine and tocopherol; adding an alkaline buffer to the mixture of 4-arm PEG-amine and tocopherol to form the first component of the lung sealant; adding tocopherol to the 4-arm PEG-NHS ester to form a mixture of 4-arm PEG-NHS ester and tocopherol; irradiating the mixture of 4-arm PEG-NHS ester and tocopherol; adding a weakly acidic buffer to the mixture of 4-arm PEG-NHS ester and tocopherol to form the second component of the lung sealant; mixing and simultaneously applying the first and second components onto lung tissue using an applicator device to form the lung sealant; and allowing the sealant to harden on the tissue.

[0151] (26) The method of embodiment 25, wherein the mixture of 4-arm PEG-amine and tocopherol and the mixture of 4-arm PEG-NHS ester and tocopherol are each irradiated with up to about 40 kilograys (kGy) of X-ray radiation. (27) The method of embodiment 25, wherein each of the 4-arm PEG-amine and the 4-arm PEG-NHS ester is irradiated in a hypoxic environment, and the 4-arm PEG-amine is in a protonated form. (28) A method of making a lung sealant, comprising: mixing an alkaline buffer with a 4-arm PEG-NH2-HCl containing tocopherol to form a 4-arm PEG-NH2-HCl composition; separately mixing a weakly acidic buffer with a 4-arm PEG-SG containing tocopherol to form a 4-arm PEG-SG composition; simultaneously delivering the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition onto lung tissue using an applicator device that provides adequate mixing of the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition; and allowing the lung sealant to harden on the lung tissue. 29. The method of claim 28, wherein the 4-arm PEG-NH2-HCL containing tocopherol is irradiated before mixing with the alkaline buffer, and the 4-arm PEG-SG containing tocopherol is irradiated before mixing with the weakly acidic buffer. 30. The method of claim 28, further comprising adding 730 ppm to about 3000 ppm of FD&C Blue #1 to the 4-arm PEG-SG, wherein the 4-arm PEG-SG is in the form of a dry powder prior to combining the 4-arm PEG-SG with the weakly acidic buffer.

[0152] 31. The method of claim 28, wherein the 4-arm PEG-NH2-HCl is in the form of a dry powder, and wherein at least about 65% of the 4-arm PEG-NH2-HCl in dry powder form has a particle size of about 250 micrometers to about 1250 micrometers. 32. The method of embodiment 28, wherein the 4-arm PEG-NH2-HCl is in the form of a dry powder, and wherein at least about 20% of the 4-arm PEG-NH2-HCl in dry powder form has a particle size greater than 710 micrometers. 33. The method of embodiment 28, wherein the 4-arm PEG-SG is in the form of a dry powder, and wherein at least about 80% of the 4-arm PEG-SG in dry powder form has a particle size of about 250 micrometers to about 1250 micrometers. 34. The method of embodiment 28, wherein the 4-arm PEG-SG is in the form of a dry powder, and wherein at least about 40% of the 4-arm PEG-SG in dry powder form has a particle size greater than 500 micrometers. (35) The method of embodiment 28, wherein the pKa of the reconstitution buffer for PEG-NH2-HCl is about 7.9 pKa to about 9.7 pKa.

[0153] (36) The method of embodiment 28, wherein the pH of the buffer for PEG-NH2-HCl after reconstitution is at least 0.05 pH units above the buffer pKa. 37. The method of claim 28, wherein the PEG-NH2-HCl composition has a pH after reconstitution of about 9.0 to about 10.5. (38) The method of embodiment 28, wherein the reconstitution buffer for PEG-NH2-HCl is an alkaline buffer having a pKa of 9.3, with a concentration of about 180 mM to about 220 mM and a pre-reconstitution pH of about 9.34 to about 9.76. (39) The method of embodiment 28, wherein the reconstitution buffer for PEG-SG is a weakly acidic buffer having a concentration of about 3.5 mM to about 15 mM and a pre-reconstitution pH of about 4.0 to about 5.0.

Claims

1. 1. A biodegradable lung sealant comprising: a) a first component comprising a first multi-arm PEG and an alkaline buffer for reconstituting the first multi-arm PEG; b) a second component comprising a second multi-arm PEG and a weakly acidic buffer for reconstituting the second multi-arm PEG; A biodegradable lung sealant, wherein when components (a) and (b) are combined, they produce an adhesive and elastic biodegradable lung sealant.

2. 10. The lung sealant of claim 1, wherein the multi-arm PEG of the first component and the multi-arm PEG of the second component each further comprise an antioxidant.

3. 3. The lung sealant of claim 2, wherein the antioxidant is tocopherol.

4. 4. The lung sealant of claim 3, wherein the antioxidant is selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, tocotrienol, and combinations thereof.

5. 3. The lung sealant of claim 2, wherein the first multi-arm PEG, the second multi-arm PEG, or both the first multi-arm PEG and the second multi-arm PEG further comprise a colorant.

6. 6. The lung sealant of claim 5, wherein the coloring agent is selected from the group consisting of FD&C Blue #1, FD&C Blue #2, FD&C Green #3, FD&C Yellow #6, and combinations thereof.

7. 2. The lung sealant of claim 1, wherein the first multi-arm PEG and the second multi-arm PEG are each a four-arm PEG.

8. 2. The lung sealant of claim 1, wherein the first multi-arm PEG is a PEG-amine and the second multi-arm PEG is a PEG-NHS ester.

9. 9. The lung sealant of claim 8, wherein the PEG-amine is 4-arm PEG-NH2-HCl.

10. 9. The lung sealant of claim 8, wherein the PEG-NHS ester is 4-arm PEG-succinimidyl glutarate (SG).

11. 9. The lung sealant of claim 8, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester in the lung sealant are each from about 39 mg / mL to about 87 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa.

12. 12. The lung sealant of claim 11, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each from about 52 mg / mL to about 77 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa.

13. 13. The lung sealant of claim 12, wherein the final concentrations of the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each 67 mg / mL, and the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester have a molecular weight of 20 kDa.

14. 9. The lung sealant of claim 8, wherein the first multi-arm PEG-amine and the second multi-arm PEG-NHS ester are each formulated with about 1500 ppm to about 3000 ppm of an antioxidant.

15. 15. The lung sealant of claim 14, wherein the antioxidant is tocopherol.

16. 10. The lung sealant of claim 9, wherein the buffer for reconstituting the PEG-NH2-HCl has a pKa of about 7.9 pKa to about 9.7 pKa.

17. 17. The lung sealant of claim 16, wherein the pKa of the buffer for reconstituting the PEG-NH2-HCl is from about 8.8 pKa to about 9.4 pKa.

18. 10. The lung sealant of claim 9, wherein the pH of the buffer for PEG-NH2-HCl after reconstitution is at least 0.05 pH units above the buffer pKa.

19. 19. The lung sealant of claim 18, wherein the PEG-NH2-HCl has a pH after reconstitution of about 9.0 to about 10.

5.

20. 9. The lung sealant of claim 8, wherein the buffer for reconstituting the PEG-NHS ester has a concentration of about 3.5 mM to about 15 mM and a pre-reconstitution pH of about 4.0 to about 7.

0.

21. 9. The lung sealant of claim 8, wherein the buffer for reconstituting the PEG-NHS ester has a pre-reconstitution pH of about 4.0 to less than 5.

0.

22. 9. The lung sealant of claim 8, wherein the first multi-arm PEG is PEG-NH2-HCl, the second multi-arm PEG is 4-arm PEG-SG, the lung sealant further comprises tocopherol and FD&C Blue #1, and wherein the final concentrations of the lung sealant composition after combining the components comprise 67 mg / mL 4-arm PEG-SG (20 kDa), 67 mg / mL 4-arm PEG-NH2-HCl (20 kDa), 340 μg / mL tocopherol, and 80 μg / mL FD&C Blue #1.

23. The lung sealant of claim 1 further comprising a contrast agent.

24. 1. A kit for preparing a biodegradable lung sealant, comprising: a first storage container containing a first multi-arm dry PEG powder; a second storage container containing an alkaline buffer solution for preparing the first multi-arm PEG composition; a third storage container containing a second multi-arm dry PEG powder; and a fourth storage container containing a weakly acidic buffer solution for preparing the second multi-arm PEG composition, wherein the biodegradable lung sealant is formed after the first multi-arm PEG composition and the second multi-arm PEG composition are combined and simultaneously applied onto lung tissue.

25. 10. A method of using the lung sealant of claim 8, comprising: adding tocopherol to a 4-arm PEG-amine to form a mixture of 4-arm PEG-amine and tocopherol; irradiating the mixture of 4-arm PEG-amine and tocopherol; adding an alkaline buffer to the mixture of 4-arm PEG-amine and tocopherol to form the first component of the lung sealant; adding tocopherol to the 4-arm PEG-NHS ester to form a mixture of 4-arm PEG-NHS ester and tocopherol; irradiating the mixture of 4-arm PEG-NHS ester and tocopherol; adding a weakly acidic buffer to the mixture of 4-arm PEG-NHS ester and tocopherol to form the second component of the lung sealant; mixing and simultaneously applying the first and second components onto lung tissue using an applicator device to form the lung sealant; and allowing the sealant to harden on the tissue.

26. 26. The method of claim 25, wherein the mixture of 4-arm PEG-amine and tocopherol and the mixture of 4-arm PEG-NHS ester and tocopherol are each irradiated with up to about 40 kilograys (kGy) of X-ray radiation.

27. 26. The method of claim 25, wherein each of the 4-arm PEG-amine and the 4-arm PEG-NHS ester is irradiated in a hypoxic environment, and the 4-arm PEG-amine is in a protonated form.

28. 1. A method of making a lung sealant, the method comprising: mixing an alkaline buffer with 4-arm PEG-NH2-HCl containing tocopherol to form a 4-arm PEG-NH2-HCl composition; separately mixing a weakly acidic buffer with 4-arm PEG-SG containing tocopherol to form a 4-arm PEG-SG composition; simultaneously delivering the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition onto lung tissue using an applicator device that provides adequate mixing of the 4-arm PEG-NH2-HCl composition and the 4-arm PEG-SG composition; and allowing the lung sealant to harden on the lung tissue.

29. 29. The method of claim 28, wherein the 4-arm PEG-NH2-HCL containing tocopherol is irradiated before mixing with the alkaline buffer, and the 4-arm PEG-SG containing tocopherol is irradiated before mixing with the weakly acidic buffer.

30. 29. The method of claim 28, further comprising adding 730 ppm to about 3000 ppm of FD&C Blue #1 to the 4-arm PEG-SG, wherein the 4-arm PEG-SG is in the form of a dry powder prior to mixing the 4-arm PEG-SG with the weakly acidic buffer.

31. 29. The method of claim 28, wherein the 4-arm PEG-NH2-HCl is in the form of a dry powder, and wherein at least about 65% of the 4-arm PEG-NH2-HCl in dry powder form has a particle size of from about 250 micrometers to about 1250 micrometers.

32. 29. The method of claim 28, wherein the 4-arm PEG-NH2-HCl is in the form of a dry powder, and wherein at least about 20% of the 4-arm PEG-NH2-HCl in dry powder form has a particle size greater than 710 micrometers.

33. 29. The method of claim 28, wherein the 4-arm PEG-SG is in the form of a dry powder, and wherein at least about 80% of the 4-arm PEG-SG in dry powder form has a particle size of about 250 micrometers to about 1250 micrometers.

34. 29. The method of claim 28, wherein the 4-arm PEG-SG is in the form of a dry powder, and wherein at least about 40% of the 4-arm PEG-SG in dry powder form has a particle size greater than 500 micrometers.

35. 29. The method of claim 28, wherein the pKa of the reconstitution buffer for PEG-NH2-HCl is from about 7.9 pKa to about 9.7 pKa.

36. 29. The method of claim 28, wherein the pH of the buffer for PEG-NH2-HCl after reconstitution is at least 0.05 pH units above the buffer pKa.

37. 29. The method of claim 28, wherein the PEG-NH2-HCl composition has a pH after reconstitution of about 9.0 to about 10.

5.

38. 29. The method of claim 28, wherein the reconstitution buffer for PEG-NH2-HCl is an alkaline buffer with a pKa of 9.3, having a concentration of about 180 mM to about 220 mM and a pre-reconstitution pH of about 9.34 to about 9.

76.

39. 29. The method of claim 28, wherein the reconstitution buffer for PEG-SG is a weakly acidic buffer having a concentration of about 3.5 mM to about 15 mM and a pre-reconstitution pH of about 4.0 to about 5.0.

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