A sealing material for wet tissue and a method of preparing and using the same

Abstract

The application provides a sealing material for wet tissues, a preparation method and application thereof, and the sealing material comprises a hydrophobic oil phase and temperature-sensitive microspheres; the temperature-sensitive microspheres are dispersed in the hydrophobic oil phase to form a heterogeneous system; the temperature-sensitive microspheres have a core-shell structure, the core-shell structure comprises a core and a shell covering the core; the core comprises a temperature-sensitive cross-linked network and an ion-cross-linkable polysaccharide, and the shell is a porous shell body network and is formed by a copolymerization reaction of a light-cross-linkable natural polymer derivative and a co-monomer containing an active chemical group. The application realizes rapid hemostasis on dynamic and bleeding wet tissues through a double drainage mechanism of the hydrophobic oil phase instantaneously draining liquid and the temperature-sensitive core actively pumping, and realizes sealing on the wet tissues through the shell and tissue protein cross-linking to form stable chemical adhesion.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a sealing material for moist tissues, its preparation method, and its application. Background Technology

[0002] Ideal injectable medical adhesives should possess good biocompatibility, rapid adhesion, and stability in humid environments. Currently, the mainstream commercial medical adhesives are mainly α-cyanoacrylates and fibrin. However, these traditional adhesives perform poorly in humid environments. For example, fibrin adhesives are easily diluted or washed away by blood flow, resulting in insufficient adhesion strength; cyanoacrylates polymerize slowly on damp surfaces, and the cured material is hard and brittle, unable to integrate with soft, pulsating tissues, easily leading to secondary damage.

[0003] Currently, there has been some progress in the field of antibody-based tissue adhesives, but existing sealants still have shortcomings such as insufficient active drainage efficiency, lack of tissue regeneration promotion function, and inability to meet the needs of the entire wound healing cycle.

[0004] Therefore, there is an urgent clinical need for a novel bio-adhesive that can provide an immediate, secure, and seamless seal for dynamic, bleeding, wet tissue. Summary of the Invention

[0005] To address the problems existing in the above-mentioned technologies, the present invention provides a sealing material for moist tissues, its preparation method and application, which is suitable for injection into dynamic, bleeding surgical wounds to achieve rapid sealing and hemostasis.

[0006] This invention provides a sealing material for moist tissues, comprising a hydrophobic oil phase and temperature-sensitive microspheres; the temperature-sensitive microspheres are dispersed in the hydrophobic oil phase in a heterogeneous system; the temperature-sensitive microspheres have a core-shell structure, comprising a core and a shell covering the core; the core comprises a temperature-sensitive crosslinking network and an ionically crosslinkable polysaccharide, and the shell is a porous shell bulk network formed by a copolymerization reaction of a photocrosslinkable natural polymer derivative and a comonomer containing active chemical groups.

[0007] Preferably, the thermosensitive crosslinking network is formed by graft copolymerization of a thermosensitive polymer and a polysaccharide derivative; the thermosensitive polymer has a lower critical solution temperature between 25-35°C and includes at least one of the following copolymers: poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) (i.e., PNVCL), poly(ethylene oxide)-poly(propylene oxide) block copolymer (i.e., Pluronic); the polysaccharide derivative includes at least one of the following substances: hydroxyethyl starch, hyaluronic acid, chitosan, dextran, and carboxymethyl cellulose.

[0008] Preferably, the molar ratio of the thermosensitive polymer to the polysaccharide derivative is 1:2 to 1:4.

[0009] Preferably, the ion-crosslinkable polysaccharide includes at least one of the following substances: sodium alginate, gellan gum, and carrageenan.

[0010] Preferably, the core further includes at least one of the following drugs encapsulated therein: hemostatic drugs, repair-promoting drugs, anti-inflammatory drugs, anti-adhesion and anti-scarring drugs, and local anesthetic drugs.

[0011] Preferably, the polymer material is a photocrosslinkable natural polymer derivative including at least one of the following substances: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide alginate, and methacrylamide silk fibroin.

[0012] Preferably, the comonomer containing active chemical groups is selected from polymerizable monomers containing active groups capable of reacting with protein functional groups such as amino and thiol groups.

[0013] Preferably, the hydrophobic oil phase is a liquid medium that is hydrophobic, bioinert, immiscible with tissue fluid, and has a certain viscosity, including at least one of the following: medical-grade silicone oil, medium-chain triglycerides, mineral oil, and perfluorocarbon liquid.

[0014] Preferably, when the sealing material is applied to moist tissue, the hydrophobic oil phase displaces the liquid in the moist tissue, the temperature-sensitive crosslinking network shrinks in volume to actively draw the liquid on the surface of the moist tissue into the outer shell, the liquid drawn into the outer shell reacts with the core to form a core physical network, and at the same time the outer shell reacts with the moist tissue through its active chemical groups to form an interfacial bonding layer.

[0015] The present invention provides a method for preparing a sealing material for moist tissues as described above, comprising the following steps:

[0016] S1. Preparation of thermosensitive crosslinking solutions and polymer solutions;

[0017] S2. A core-shell structure precursor is formed by emulsion method or coaxial electrospray technology, with the temperature-sensitive crosslinking solution prepared in S1 as the core and the polymer solution prepared in S1 as the shell;

[0018] S3. The precursor is freeze-dried and low-temperature ground to obtain temperature-sensitive microspheres;

[0019] S4. The temperature-sensitive microspheres are mixed with a hydrophobic oil phase to form a sealing material for wet tissues.

[0020] Preferably, the particle size of the temperature-sensitive microspheres is 20-120 μm.

[0021] Preferably, the mass ratio of the temperature-sensitive microspheres to the hydrophobic oil phase is 1:1 to 1:3.

[0022] The present invention provides the application of a sealing material for moist tissues as described above in the preparation of medical devices for hemostasis, tissue adhesion, or promoting wound healing.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention designs a temperature-sensitive microsphere with a core-shell structure, which serves as an intelligent functional unit. In synergy with a hydrophobic oil phase, it achieves rapid hemostasis on dynamic, bleeding wet tissue surfaces through a dual drainage mechanism of instantaneous drainage from the hydrophobic oil phase and active pumping from the temperature-sensitive core. Furthermore, the outer shell forms a stable chemical bond with the tissue through cross-linking, allowing the sealing material to quickly and firmly adhere to the rapidly hemostatic wet tissue surface, thus achieving a seal on the wet tissue surface.

[0025] This invention achieves injectable properties of sealing materials by dispersing temperature-sensitive microspheres in a hydrophobic oil phase to form a heterogeneous system, making it easier to apply to wound sealing and hemostasis in surgical procedures.

[0026] This invention, by incorporating ionically cross-linkable polysaccharides within the core, enables the temperature-sensitive core to pump out calcium ions (Ca) from tissue fluid. This is achieved because the outer shell is a porous structure that allows ions and small molecules to pass freely. 2+ The inhaled Ca is drawn in and forced through the pores of the outer shell. 2+ Rapid ionic cross-linking occurs between the microspheres and the polysaccharide chains within the core, extending near the outer shell. This reaction forms a robust core physical network in situ at the interface between the temperature-sensitive microspheres and the tissue. One end of this network is anchored in the microstructure of the tissue surface, while the other end is integrated with the entire core gel network, thereby strongly binding the microspheres to the tissue and providing instantaneous high-strength adhesion.

[0027] This invention encapsulates a drug (e.g., growth factor, thrombin, etc.) within a core. As the core's physical network degrades, the drug diffuses through the outer shell and reaches the lesion at a controlled release rate, achieving sustained controlled drug release. This upgrades passive sealing materials into an active treatment system, integrating hemostasis, adhesion, and healing promotion. The core's physical network also encapsulates the drug, effectively preventing premature release during the active pumping process of the temperature-sensitive core, thus preventing it from reaching the lesion. Subsequently, as the core's physical network degrades, the drug diffuses through its channels and eventually penetrates the outer shell to reach the lesion. The drug release rate is controlled by the degradation kinetics of the core's physical network (e.g., the enzymatic / hydrolytic rate of calcium alginate and the enzymatic / hydrolytic rate of the temperature-sensitive cross-linked network) and the interaction forces between the drug and the core's physical network, thereby achieving sustained controlled drug release. Attached Figure Description

[0028] Figure 1 This is a scanning electron microscope image of the sealing material for moist tissues of the present invention before it comes into contact with the surface of the moist tissue.

[0029] Figure 2 This is a schematic diagram of the structure of the temperature-sensitive microspheres used in the sealing material for moist tissues of the present invention before pumping.

[0030] Figure 3 This is a scanning electron microscope image of the sealing material for moist tissues of the present invention after it comes into contact with the surface of moist tissues.

[0031] Figure 4 This is a schematic diagram of the structure of the temperature-sensitive microsphere pump after suction of the sealing material for moist tissues according to the present invention.

[0032] Figure 5 This is a schematic diagram of the sealing material for wet tissues of the present invention stored in a syringe.

[0033] Figure 6 This is a schematic diagram of the sealing material for moist tissue of the present invention being injected onto the surface of moist tissue using a syringe. Detailed Implementation

[0034] To make the objectives, features, and beneficial effects of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described below are merely for explaining the present invention and are not intended to limit the present invention. Furthermore, the same or similar reference numerals may be used in the drawings to refer to the same or similar elements in different embodiments, and descriptions of the same or similar elements in different embodiments, as well as descriptions of prior art elements, features, effects, etc., may be omitted.

[0035] As shown in this specification and claims, unless specifically indicated or obvious from the context, all numerical values ​​provided herein are modified with the term "about," which should be understood as being within the normal tolerance range in the art. "About" can be understood as allowing tolerances of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the numerical value.

[0036] This invention provides a sealing material for moist tissues, such as... Figure 1 As shown, it includes a hydrophobic oil phase 1 and temperature-sensitive microspheres 2, with the temperature-sensitive microspheres 2 dispersed in the hydrophobic oil phase 1 in a heterogeneous system. Figure 2 As shown, the temperature-sensitive microsphere 2 has a core-shell structure, which includes a core 21 and an outer shell 22 covering the core 21 (e.g., Figure 4 (As shown).

[0037] When the sealing material is injected onto the surface of moist tissue, such as Figure 3 As shown, utilizing the low surface energy and immiscibility of the hydrophobic oil phase matrix, the hydrophobic oil phase 1 can instantly physically displace blood and tissue fluid from the surface of moist tissue, achieving the instantaneous drainage function of the hydrophobic oil phase, thereby instantly physically sealing the surface of moist tissue and forming a temporary, relatively "dry" contact area to create the initial adhesion interface. In addition: (1) As a continuous matrix, the hydrophobic oil phase can uniformly disperse and stably suspend temperature-sensitive microspheres with core-shell structures to form a paste-like system, ensuring the injectability of the sealing material (e.g. Figure 5 and 6 As shown), it is convenient for use in surgical wound sealing and hemostasis operations, and also ensures the in-situ retention of the sealing material, making it easier to stay on the surface of moist tissue and not easy to be lost; (2) The hydrophobic environment of the hydrophobic oil phase effectively isolates the active chemical groups of the shell 22 from the water in the environment, effectively preventing the active chemical groups from prematurely hydrolyzing and failing before reaching the tissue surface, thereby ensuring that the active chemical groups carry out chemical cross-linking reaction as scheduled after contacting the surface of moist tissue, realizing "on-demand" start-up and satisfying "time-sequence synergy". Then the temperature-sensitive cross-linking network of the core 21 contacts the moist tissue, and a hydrophilic-hydrophobic phase transition occurs because the tissue temperature (e.g., 37°C) exceeds its low critical solution temperature (LCST), such as Figure 4 As shown, the core 21 undergoes a phase transition and shrinks, reducing its volume by more than 60%. The resulting powerful "pumping effect" enables the temperature-sensitive core to actively pump, drawing liquid from the surface of wet tissue through the porous outer shell 22 into the interface region of the core 21, thereby creating a relatively "dry" contact area on the tissue surface instantaneously (< 10 s).

[0038] The above process utilizes a two-stage drainage mechanism of "instantaneous drainage of hydrophobic oil phase" and "active pumping of temperature-sensitive core" to efficiently remove liquid from wet surfaces, creating an ideal interface for subsequent bonding. This enables rapid (second-level) and strong (adhesion strength > 200 kPa) adhesion in wet environments, providing a stable chemical bond.

[0039] The liquid drawn into the outer shell 22 comes into contact with the core 21, and transient ionic cross-linking (< 5 s) forms a core physical network, providing instantaneous strength. The outer shell 22 reacts with the wet tissue through its active chemical groups to form an interfacial bonding layer at the interface (> 30 s), providing a robust chemical bond.

[0040] The above process achieves phased and time-sequential synergy between "core ion crosslinking (instantaneous strength)" and "shell covalent crosslinking (long-term stability)," avoiding crosslinking interference.

[0041] Specifically, when dry temperature-sensitive microspheres come into contact with a liquid at a temperature higher than their lower critical solution temperature (LCST), two processes occur simultaneously: water absorption by the porous network structure of the outer shell and phase change shrinkage of the temperature-sensitive core material. However, the rates of these two processes differ significantly: temperature-sensitive shrinkage occurs in the microsecond to millisecond range, while water absorption and expansion take in the second to minute range. Therefore, before the microspheres have fully swelled, shrinkage has already begun and become dominant, exhibiting an overall "active absorption" behavior. "Liquid passing through the outer shell" and "active pumping by the core" are not two opposing stages, but rather an integrated process achieved through the synergy of the core-shell structure and the porous outer shell.

[0042] Specifically, when the dried microspheres come into contact with moist tissue, the porous network structure of the outer shell first absorbs the tissue fluid into the pores of the outer shell. At this time, the fluid only exists within the porous channels of the outer shell and has not yet entered the core in large quantities; the microspheres themselves do not "expand".

[0043] Simultaneously, the temperature-sensitive material in the core senses the temperature and undergoes a phase transition and shrinkage within an extremely short time (microseconds)—the molecular chains transform from a hydrophilic extended state to a hydrophobic coiled state, resulting in a sharp decrease in volume. However, due to the rigid porous structure of the outer shell, it does not shrink synchronously. Therefore, a "confined space" is formed between the core and the outer shell. After the core shrinks, the pressure within this confined space decreases, generating a negative pressure. This negative pressure is conducted outward through porous channels, "drawing" the interfacial liquid into the microsphere. The entire process macroscopically manifests as a causal relationship between "shrinkage of the temperature-sensitive network" and "liquid absorption." In this embodiment of the invention, the shrinkage rate of the temperature-sensitive microsphere 2 is much faster than its expansion rate. The phase transition shrinkage of N-isopropylacrylamide (PNIPAM) materials occurs in the microsecond to millisecond range, while the swelling of dried gels typically takes seconds to minutes. When the liquid enters the pores of the outer shell through capillary action, the core has already sensed the temperature and begun to shrink. The microsphere initiates shrinkage before it has fully swelled, resulting in an overall macroscopic manifestation of "active absorption" rather than "absorption followed by expulsion."

[0044] This invention designs a temperature-sensitive microsphere with a core-shell structure, which is used as an intelligent functional unit. In collaboration with a hydrophobic oil phase, a dual drainage mechanism of instantaneous drainage of the hydrophobic oil phase and active pumping of the temperature-sensitive core is used to achieve a three-step time-sequential synergistic mechanism of "active pumping of the temperature-sensitive core (< 10 s) → ionic cross-linking of the core (< 5 s) → covalent cross-linking of the shell (> 30 s)".

[0045] In some embodiments, the core 21 includes a temperature-sensitive crosslinking network.

[0046] In some embodiments, the thermosensitive crosslinking network of the core 21 is formed by graft copolymerization of a thermosensitive polymer and a polysaccharide derivative, wherein the low critical dissolution temperature of the thermosensitive polymer is between 25-35°C.

[0047] In some embodiments, the temperature-sensitive polymer is selected from at least one of polymers having a similar LCST (e.g., 25-35°C), such as poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) (PNVCL), and poly(ethylene oxide)-poly(propylene oxide) block copolymer (Pluronic).

[0048] In some embodiments, the polysaccharide derivative is a natural polysaccharide or a derivative thereof, selected from at least one of hydroxyethyl starch, hyaluronic acid, chitosan, dextran, and carboxymethyl cellulose.

[0049] In some embodiments, the molar ratio of the thermosensitive polymer to the polysaccharide derivative is 1:2 to 1:4.

[0050] In some embodiments, the core 21 further includes at least one of ionically crosslinkable polysaccharides such as sodium alginate, gellan gum, and carrageenan. The ionically crosslinkable polysaccharides in the core, after the temperature-sensitive core actively pumps liquid onto the surface of hygroscopic tissue, react with calcium ions (Ca) in the liquid. 2+ Rapid ionic cross-linking occurs, forming a core physical network to provide strong initial adhesion.

[0051] In some embodiments, the core 21 further includes at least one drug encapsulated therein, such as: hemostatic drugs (thrombin, aminocaproic acid); repair-promoting drugs (growth factors, cytokines); anti-inflammatory drugs (antibiotics, antimicrobial peptides, silver ions, nonsteroidal anti-inflammatory drugs); anti-adhesion and anti-scarring drugs (mitomycin C, 5-fluorouracil (5-FU), hyaluronidase, glucocorticoids); and local anesthetic drugs (lidocaine, bupivacaine).

[0052] In some embodiments, when the encapsulating material 23 of the core is a growth factor (e.g., vascular endothelial growth factor VEGF or epidermal growth factor EGF), its encapsulation amount is preferably 0.1-1.0 μg / mg microspheres; when the encapsulating material 23 is thrombin (e.g., thrombin), its encapsulation amount is preferably 10-200 IU / mg microspheres; when the encapsulating material 23 is an antibiotic or a small molecule chemical drug, its encapsulation amount can be determined by conventional experiments, for example, 0.1-10 wt% of the total weight of the microspheres.

[0053] As the core physical network degrades, the drugs encapsulated within the core 21 diffuse through the porous network of the outer shell, reaching the lesion site at a controlled release rate. This achieves sustained controlled release of the drugs, upgrading the passive sealing material into an active treatment system and integrating hemostasis, adhesion, and healing promotion. The core physical network also encapsulates the drugs, effectively preventing premature release outside the shell during the active pumping process of the temperature-sensitive core, thus preventing them from reaching the lesion site. Subsequently, as the core physical network degrades, the drugs diffuse through its pores and are ultimately released through the porous structure of the outer shell. The release rate of the drugs is jointly controlled by the degradation kinetics of the core physical network (e.g., the enzymatic / hydrolytic rate of calcium alginate and the enzymatic / hydrolytic rate of the temperature-sensitive cross-linked network) and the interaction forces between the drugs and the core physical network, thereby achieving sustained controlled release.

[0054] In some embodiments, the housing 22 has a porous structure.

[0055] In some embodiments, the outer shell 22 includes a porous shell body network formed by copolymerization of photocrosslinkable natural polymer derivatives and comonomers containing active chemical groups.

[0056] In some embodiments, the photocrosslinkable polymeric derivative is selected from at least one of photosensitive derivatives obtained by modifying natural polymeric materials, such as methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide alginate, and methacrylamide silk fibroin.

[0057] In some embodiments, the comonomer containing active chemical groups is selected from polymerizable monomers containing active groups capable of reacting with protein functional groups such as amino and thiol groups, such as N-hydroxysuccinimide acrylate (AAC-NHS ester), other derivatives of succinimide ester (such as sulfonyl-NHS), glycidyl methacrylate (GMA), etc. Furthermore, crosslinking agents such as carbodiimide (EDC) may be introduced into the shell to enhance the stability of the shell body network.

[0058] In a specific embodiment, the photocrosslinkable polymeric derivative is methacrylamide gelatin (GelMA), and the comonomer containing active chemical groups is N-hydroxysuccinimide acrylate (AAC-NHS ester). Under photoinitiation, the methacrylamide groups of GelMA and the acrylate groups of AAC-NHS ester undergo a copolymerization reaction to form a crosslinked network, while the active NHS ester groups are covalently fixed in the network.

[0059] Therefore, the outer shell 22 essentially constitutes a shell body network composed of polymer materials and active chemical groups. This network forms an interfacial bonding layer through covalent reactions between its active chemical groups and tissue proteins, which is the core basis for achieving long-term and stable chemical adhesion to tissues.

[0060] In one specific embodiment, the methacrylamide gelatin (GelMA) network of the shell 22 provides a biocompatible framework, and the N-hydroxysuccinimide (NHS) ester groups, which are in situ immobilized in the methacrylamide gelatin network via copolymerization, are the active centers for forming strong adhesion. The NHS ester groups can undergo efficient amidation reactions with primary amino groups (-NH2) in wet tissues and establish chemical linkages through two parallel pathways:

[0061] 1. Longitudinal anchoring of the outer shell of the temperature-sensitive microspheres to moist tissue:

[0062] R1-NHS + H2N-R2→ R1-CO-NH-R2

[0063] Wherein: R1: the outer shell of the temperature-sensitive microspheres, R2: wet tissue protein

[0064] 2. Lateral covalent links between multiple temperature-sensitive microspheres:

[0065] The outer shell of microsphere A -NHS + the outer shell of microsphere B -NH2 → Microsphere A - Microsphere B

[0066] The shell of the temperature-sensitive microspheres uses methacrylamide gelatin (GelMA), which is prepared by partially modifying gelatin with methacrylic anhydride. Its molecular chain retains some unreacted lysine residues, which also provide primary amino groups (-NH2). During the formation of the shell network, the active groups of N-hydroxysuccinimide ester (NHS ester) are fixed in situ within the covalently cross-linked network through the photopolymerization reaction of GelMA and AAC-NHS ester. Therefore, the shell of a single temperature-sensitive microsphere simultaneously possesses both NHS ester and primary amino groups (-NH2), allowing multiple temperature-sensitive microspheres, when tightly packed, to form lateral covalent cross-links through the amidation reaction of NHS ester and primary amino groups (-NH2), thus forming stable amide bonds.

[0067] The aforementioned longitudinal anchoring and transverse covalent linking reactions occur simultaneously, both forming covalent amide bonds, thereby constructing a pervasive three-dimensional covalent cross-linked network (i.e., interfacial bonding layer) in situ on the surface of moist tissue, adhering discrete points to form a macroscopic "surface seal" and providing long-term stability.

[0068] In some embodiments, the hydrophobic oil phase is selected from one or more of the following liquid media that are hydrophobic, bioinert, immiscible with tissue fluid, and have a certain viscosity: medical-grade silicone oil, medium-chain triglycerides, mineral oil, perfluorinated carbon liquid, etc.

[0069] Preferably, the hydrophobic oil phase is medical-grade silicone oil. The viscosity of the medical-grade silicone oil is 5-20 cSt.

[0070] This invention provides a method for preparing the sealing material for moist tissues as described above, comprising the following steps:

[0071] S1. Preparation of thermosensitive crosslinking solutions and polymer solutions;

[0072] S2. A core-shell structure precursor is formed by emulsion method or coaxial electrospray technology, with the temperature-sensitive crosslinking solution prepared in S1 as the core and the polymer solution prepared in S1 as the shell;

[0073] S3. The precursor was freeze-dried and low-temperature ground to obtain temperature-sensitive microspheres with a particle size of 20-120 μm;

[0074] S4. Thermosensitive microspheres and hydrophobic oil phase are mixed at a mass ratio of 1:1 to 1:3 to form a sealing material for wet tissues.

[0075] In some embodiments, S1 includes the following steps:

[0076] 1.1 Synthesis of temperature-sensitive crosslinking solution

[0077] Hydroxyethyl starch (HES, Mw 200kDa) and N-isopropylacrylamide (PNIPAM) monomer were copolymerized by free radical graft copolymerization to obtain a copolymer with an LCST of 32℃.

[0078] Under nitrogen protection, 5.0 g of HES was dissolved in 90 mL of deionized water and stirred at 60 °C. 3.0 g of PNIPAM monomer was added, and the mixture was stirred for 2 hours to ensure thorough mixing. 0.1 g of ammonium persulfate was added, and the reaction was carried out at 70 °C for 6 hours. After dialyzing the reaction solution (MWCO 10 kDa) for 3 days, the dialyzed solution was freeze-dried to obtain a white flocculent HES-PNIPAM graft copolymer, which was used as the thermosensitive crosslinking solution.

[0079] 1.2 Preparation of the outer shell solution and the core solution

[0080] Core phase liquid: Take 4.0g of the above copolymer, 1.0g of sodium alginate and 100μg of VEGF165, dissolve them in 15mL of deionized water at 4℃ to obtain a homogeneous viscous liquid.

[0081] Shell phase: Take 3.0 g of methacrylamide gelatin, 1.5 g of N-hydroxysuccinimide acrylate (AAC-NHS ester), and 0.15 g of α-ketoglutaric acid, dissolve them in 12 mL of deionized water at 37 °C to obtain a clear solution.

[0082] In some embodiments, S2 includes the following steps:

[0083] Coaxial electrospray preparation of temperature-sensitive microspheres:

[0084] Using a coaxial electrospray device, the core phase liquid and the outer shell phase liquid were pumped in through the inner and outer syringes respectively (flow rates of 0.8 mL / h and 1.2 mL / h respectively).

[0085] Under a high voltage of 12 kV, the droplet was sprayed into a receiver containing a 0.1 M CaCl2 solution and simultaneously irradiated with 365 nm UV light (intensity 15 mW / cm²) for 30 minutes.

[0086] This process simultaneously completes the photopolymerization crosslinking of GelMA and AAC-NHS ester in the shell and the ionic crosslinking of sodium alginate in the core, forming a temperature-sensitive microsphere with a stable structure and an outer shell covalently fixed with NHS active ester groups.

[0087] In some embodiments, S3 includes the following steps:

[0088] Post-processing and finished product preparation

[0089] Thermosensitive microspheres in the receiving solution were collected using a sieve, washed three times with pre-cooled phosphate buffer (PBS, 4°C) to remove unreacted substances, then freeze-dried, and then ground at 20 Hz for 2 minutes using a low-temperature grinder. Microspheres of 20-120 μm were obtained by sieving.

[0090] In some embodiments, S4 includes the following steps:

[0091] Finally, under sterile conditions, the microspheres and medical silicone oil were mixed evenly at a mass ratio of 1:(1.5-2). Figure 1 (As shown), dispensed into syringes, sealed, and stored at low temperature (e.g.) Figure 5 (As shown).

[0092] This invention provides the application of a sealing material for moist tissues as described above in the preparation of medical devices for hemostasis, tissue adhesion, or promoting wound healing.

[0093] The preparation method of the sealing material for moist tissues is illustrated in the following specific embodiments:

[0094] Example 1

[0095] Preparation of sealing materials based on coaxial electrospray

[0096] S1. Preparation of thermosensitive crosslinking solutions and polymer solutions

[0097] 1.1 Synthesis of temperature-sensitive HES-PNIPAM copolymer

[0098] Hydroxyethyl starch (HES, Mw 200kDa) was copolymerized with poly(N-isopropylacrylamide) monomer via free radical graft copolymerization to obtain a copolymer with an LCST of 32℃.

[0099] Under nitrogen protection, 5.0 g of HES was dissolved in 90 mL of deionized water and stirred at 60 °C. 3.0 g of NIPAM monomer was added, and the mixture was stirred for 2 hours to ensure thorough mixing. 0.1 g of ammonium persulfate was added, and the reaction was carried out at 70 °C for 6 hours. After dialyzing the reaction solution (MWCO 10 kDa) for 3 days, the dialyzed solution was freeze-dried to obtain a white flocculent HES-PNIPAM graft copolymer.

[0100] 1.2 Preparation of core-shell solution

[0101] Core phase liquid: Take 4.0g of the above copolymer, 1.0g of sodium alginate and 100μg of VEGF165, dissolve them in 15mL of deionized water at 4℃ to obtain a homogeneous viscous liquid.

[0102] Shell phase: Take 3.0 g of methacrylamide gelatin, 1.5 g of AAC-NHS ester, and 0.15 g of α-ketoglutaric acid, dissolve them in 12 mL of deionized water at 37 °C to obtain a clear solution.

[0103] S2. Coaxial electrospray preparation of core-shell microspheres

[0104] Using a coaxial electrospray device, the core phase liquid and the outer shell phase liquid were pumped in through the inner and outer syringes respectively (flow rates of 0.8 mL / h and 1.2 mL / h respectively).

[0105] Under a high voltage of 12 kV, the droplet was sprayed into a receiver containing a 0.1 M CaCl2 solution and simultaneously irradiated with 365 nm UV light (intensity 15 mW / cm²) for 30 minutes.

[0106] This process simultaneously completes the photopolymerization crosslinking of GelMA and AAC-NHS ester in the outer shell and the ionic crosslinking of sodium alginate in the core. Under these conditions, the methacryloyl groups of GelMA and the acryloyl groups of AAC-NHS ester undergo photopolymerization, forming a covalently crosslinked three-dimensional network, thereby covalently binding the active groups of NHS ester into this network. This results in a structurally stable microsphere shell. The activity of the covalently bound NHS ester is retained during brief UV irradiation, and when the microspheres are applied to tissue surfaces, they undergo a coupling reaction with the primary amine groups on the tissue surface, providing strong chemical adhesion.

[0107] S3. Post-processing and finished product preparation

[0108] The microspheres in the receiving solution were collected using a sieve, washed three times with pre-cooled phosphate-buffered saline (PBS, 4°C) to remove unreacted substances, and then freeze-dried. They were then ground at 20 Hz for 2 minutes using a cryogenic grinder and sieved to obtain microspheres of 20–120 μm.

[0109] S4. Finally, under sterile conditions, the microspheres and medical silicone oil are mixed evenly at a mass ratio of 1:(1.5-2), dispensed into syringes, sealed, and stored at low temperature.

[0110] Example 2

[0111] Alternative preparation methods based on emulsion-freeze-milling

[0112] S1. Prepare core phase and shell phase solutions according to the method of Example 1.

[0113] S2. The core phase solution is slowly added dropwise to the shell phase solution under high-speed stirring (1000 rpm) to form a water-in-oil-in-water (W / O / W) double emulsion. The emulsion is quickly poured into a mold and frozen at -20°C. Freezing and phase separation: During freezing, the core and shell components separate due to their solubility differences, forming a preliminary core-shell structure. The sample is then deep-frozen at -80°C for 12 hours to fix the phase-separated structure. Freeze-drying and grinding: The frozen block material is freeze-dried. Then, it is ground at 20 Hz for 2 minutes using a low-temperature grinder and sieved to obtain microspheres of 20-120 μm.

[0114] Steps S3-4 are the same as those in Example 1.

[0115] Example 3

[0116] Rapid gelation of temperature-sensitive core-shell microspheres

[0117] In the shell phase solution of Example 1, an additional 1.0 wt% of four-arm polyethylene glycol succinimide (4-arm PEG-NHS) was added. This crosslinking agent has a small molecular weight and diffuses rapidly, allowing it to crosslink with gelatin and tissue proteins within seconds of contact with the tissue fluid. Other steps and process parameters were consistent with those of Example 1.

[0118] Example 4

[0119] Double-layered core-shell microspheres (hemostasis + anti-adhesion)

[0120] In Example 1, a hydrophilic lubricating layer formed by photocrosslinking of polyethylene glycol diacrylate (PEGDA) was added to the outer shell. The specific preparation process is as follows:

[0121] S1. Preparation of core solution, intermediate shell solution and outer layer solution

[0122] Core phase liquid (i.e., the temperature-sensitive core of Example 1): Same as Example 1.

[0123] Intermediate shell phase liquid (i.e., the outer shell of Example 1): Same as the outer shell phase liquid of Example 1.

[0124] Outer phase liquid (i.e., hydrophilic lubricating layer): Take 2.0 g of polyethylene glycol diacrylate (PEGDA, molecular weight 1000 Da) and 0.05 g of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959, photoinitiator), dissolve them in 10 mL of deionized water, stir until completely dissolved, and obtain a clear solution. Store in the dark for later use.

[0125] S2. Triaxial electrospray preparation of core-shell-shell three-layer microspheres

[0126] A triaxial coaxial electrospray device was used to pump in the core phase (temperature-sensitive core), the intermediate shell phase (adhesive layer, i.e., the outer shell of Example 1), and the outer shell phase (i.e., the PEGDA hydrophilic lubricating layer). Specifically, they were configured as follows:

[0127] Inner needle (core phase fluid): flow rate 0.6 mL / h, 21G needle.

[0128] Intermediate shell phase liquid: flow rate 1.0 mL / h, 17G needle.

[0129] Outer needle (outer phase liquid): flow rate 0.8 mL / h, 14G needle.

[0130] Electrospray parameters: voltage 15 kV, receiving distance 15 cm, receiving liquid is 0.1 M CaCl2 solution (4℃), ambient temperature 25℃.

[0131] Droplets were sprayed into the receiving liquid, while a 365 nm UV light source (intensity 20 mW / cm²) was placed above the receiver to photocrosslink the microspheres during the spraying process. The intermediate shell underwent photopolymerization crosslinking of GelMA and AAC-NHS ester, and the outer layer underwent photohomogenization crosslinking of PEGDA, forming a hydrophilic lubricating layer. The microspheres in the receiving liquid were further immersed at 4°C for 30 minutes to allow for sufficient ionic crosslinking of the core sodium alginate with Ca²⁺. Steps S3-4 were the same as in Example 1.

[0132] Other steps and process parameters (including washing, freeze drying, grinding, sieving, mixing with medical silicone oil, etc.) are the same as in Example 1.

[0133] Comparative Example 1

[0134] No temperature-sensitive core

[0135] The HES-PNIPAM thermosensitive copolymer in the core material of Example 1 was replaced with an equal mass of unmodified ordinary hydroxyethyl starch (HES). This material does not have temperature responsiveness.

[0136] The other components, proportions, and preparation processes of the core and shell are kept exactly the same as in Example 1 to ensure that the variables are unique.

[0137] Comparative Example 2

[0138] Core-shell-free homogeneous hybrid microspheres

[0139] No separation of core-shell solutions was performed. The HES-PNIPAM, sodium alginate, and VEGF used for the core in Example 1 were mixed at once with GelMA, AAC-NHS, and photoinitiator used for the shell to prepare a homogeneous precursor solution.

[0140] The homogeneous solution was atomized and solidified using a uniaxial electrospray process to obtain homogeneous microspheres with the same chemical composition but without a core-shell structure. The dispersion matrix (hydrophobic oil phase) was the same as in Example 1.

[0141] Comparative Example 3

[0142] Microspheres without chemical cross-linking groups

[0143] When preparing the shell phase solution, AAC-NHS ester was completely omitted, and only GelMA and a photoinitiator were used. The core composition and other processes were exactly the same as in Example 1.

[0144] The microsphere shell prepared in this way only has a photocrosslinked GelMA network and lacks active groups that can covalently react with tissues.

[0145] Comparative Example 4

[0146] Homogeneous microspheres without temperature sensitivity

[0147] The HES-PNIPAM in the core was replaced with an equal amount of non-thermosensitive common hydroxyethyl starch (HES). It was then homogenized with the shell component (GelMA without AAC-NHS).

[0148] The same coaxial electrospray process as in Example 1 was used to prepare homogeneous microspheres.

[0149] Comparative Example 5

[0150] Simulate existing technology

[0151] Homogeneous microspheres were prepared by chemical crosslinking of polyacrylic acid (PAAc), NHS ester, and chitosan. These microspheres were then dispersed in medical silicone oil of the same viscosity as in Example 1 of this invention at the same proportions.

[0152] The formulations of the above comparative examples and embodiments are summarized in Table 1 below.

[0153] Table 1. Summary of Formulas

[0154]

[0155] Performance testing methods and standards

[0156] 1. Initial Adhesion Time: Refer to industry-standard methods. Adhere a 12 mm diameter patch to a moist, detached pigskin (simulating a wet tissue surface). Immediately apply a constant, slight pressure (approximately 5 kPa) to the back of the patch. Use a stopwatch to record the time (S) required from patch contact with the tissue until it can resist the initial shear force generated by its own weight without slippage or detachment. Repeat the test at least 5 times (n≥5), and take the mean ± standard deviation.

[0157] 2. Adhesion Strength Test: Following ASTM F2255 (Standard Test Method for Determining the Overlap Shear Strength of Tissue Adhesives by Tensile Load), the lap shear test was conducted. The patch was adhered between two detached pieces of pigskin and stretched at a rate of 10 mm / min. The maximum load divided by the adhesion area yielded the wet adhesion strength, expressed in kPa.

[0158] 3. Interface Liquid Removal Time Test: The surface of the test substrate is wetted to form a continuous liquid film simulating blood or tissue fluid. A measured amount of sealant is applied to the center of the liquid film, and a high-speed camera (frame rate ≥ 100 fps) is simultaneously activated to record the dynamic process. The removal time is calculated from the moment the sealant contacts the liquid surface until the surrounding liquid is completely displaced, exposing the edge of the dry substrate.

[0159] 4. Cell proliferation experiment: In accordance with GB / T 16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", L929 cells were cultured in the material extract for 24 hours and the cell viability was detected.

[0160] 5. Angiogenesis Assay: In a chicken embryo allantoic membrane model, VEGF-loaded microspheres or control samples were placed in areas with sparse CAM vessels. After incubation for 48-72 hours, the growth of blood vessels around the samples was observed under a stereomicroscope. The number of blood vessel branches or the total length of blood vessels was quantitatively analyzed using image analysis software (such as ImageJ) and compared with the blank control area.

[0161] The performance test results of the examples and control examples are shown in Table 2 below.

[0162] Table 2. Summary of Performance of Examples and Comparative Examples

[0163]

[0164] Among these, higher values ​​for overlap shear strength, cell viability, and angiogenesis enhancement are better, while shorter values ​​for initial adhesion time and interfacial drainage time are better. The test results show that Example 1 performs best in all aspects. Specifically:

[0165] 1. Compared to Example 1, the wet adhesion strength of Comparative Example 1 (without a temperature-sensitive core) decreased sharply from 208.4 kPa to 95.8 kPa, and the interfacial drainage time was significantly prolonged. This indicates that the phase change "pumping effect" of the HES-PNIPAM core can actively and efficiently remove interfacial liquid and improve wet adhesion performance.

[0166] 2. In Comparative Example 2 (without core-shell structure), the homogeneous microspheres not only showed a decrease in strength (118.6 kPa), but the failure mode was mainly cohesive failure, rather than detachment of the adhesive layer-tissue interface. This indicates that the physical separation of the core and shell ensures the temporal synergy between "instantaneous physical crosslinking of sodium alginate" and "long-term chemical crosslinking of NHS ester," avoiding crosslinking interference, thereby achieving higher adhesion strength and a more reliable adhesion interface.

[0167] 3. Comparative Example 3 (without chemical cross-linking): After short-term immersion, the adhesion strength was lost by more than 80%. This indicates that the formation of covalent bonds with the tissue surface is the decisive factor for the material to maintain long-term adhesion stability in a dynamic and humid physiological environment, and physical forces cannot replace it.

[0168] 4. Comparative Example 4 (without temperature-sensitive homogeneous spheres) served as a bivariate defect control. Its performance (72.4 kPa) was even lower than that of Comparative Examples 1 or 2, which had only a single defect, and lower than all examples and control examples with a single defect. This demonstrates that "temperature sensitivity" and "staged cross-linking of the core-shell structure" are synergistic and indispensable, playing a core role in improving the final performance.

[0169] 5. Compared with Comparative Example 5, Example 1 shows significant improvements in key indicators such as adhesion strength, initial adhesion speed, and interfacial drainage efficiency. Furthermore, by loading VEGF into the core, it adds an active healing-promoting therapeutic function, achieving an upgrade from simple adhesion to adhesion and active repair.

[0170] 6. Example 2 improves process adaptability through cryogenic grinding. By sacrificing some precision, it employs a more scalable process path, providing an option for industrialization.

[0171] 7. Example 3 (Rapid Gelization) By adding a fast-acting crosslinking agent, the initial adhesion time was shortened to 4.2 seconds, indicating that the performance of the present invention is adjustable and can be optimized in a targeted manner according to clinical needs.

[0172] 8. Example 4 (anti-adhesion double-layer microspheres) successfully endowed the invention with significant anti-adhesion function while maintaining excellent adhesion (185.6 kPa), indicating that the invention has functional scalability.

[0173] 9. The performance data from Example 1 further corroborates the core mechanism of the present invention: the interface drainage time is only 7.9 seconds, the initial adhesion time is only 8.5 seconds, and the wet adhesion strength is as high as 208.4 kPa. These data directly show that the dry temperature-sensitive microspheres can indeed achieve rapid drainage and strong adhesion after contacting moist tissue, verifying the effectiveness of the "pumping effect" from a macroscopic perspective.

[0174] Although specific embodiments have been described above, these embodiments are not intended to limit the scope of the invention, even when only a single embodiment is described with respect to a particular feature. The feature examples provided in this disclosure are intended to be illustrative and not limiting, unless otherwise stated. In practice, one or more technical features of the dependent claims may be combined with the technical features of the independent claims as needed and where technically feasible, and the technical features from the respective independent claims may be combined in any suitable manner rather than solely by the specific combinations listed in the claims.

[0175] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A sealing material for moist tissues, characterized in that, The system comprises a hydrophobic oil phase and temperature-sensitive microspheres; the temperature-sensitive microspheres are dispersed in the hydrophobic oil phase in a heterogeneous system; the temperature-sensitive microspheres have a core-shell structure, comprising a core and a shell covering the core; the core comprises a temperature-sensitive crosslinking network and an ionically crosslinkable polysaccharide, the shell is a porous shell bulk network, and is formed by copolymerization of a photocrosslinkable natural polymer derivative and a comonomer containing active chemical groups; the temperature-sensitive crosslinking network is formed by graft copolymerization of a temperature-sensitive polymer and a polysaccharide derivative; the temperature-sensitive polymer has a lower critical solution temperature between 25-35°C and includes at least one of the following copolymers: poly(N-isopropylacrylamide), poly(N-vinylcaprolactam), poly(ethylene oxide)-poly(propylene oxide) block copolymer; the polysaccharide derivative includes at least one of the following substances: hydroxyethyl starch, hyaluronic acid, chitosan, dextran, carboxymethyl cellulose; the molar ratio of the temperature-sensitive polymer to the polysaccharide derivative is 1:2-1:

4.

2. The sealing material for moist tissues according to claim 1, characterized in that, The ion-crosslinkable polysaccharide includes at least one of the following substances: sodium alginate, gellan gum, and carrageenan.

3. The sealing material for moist tissues according to claim 1, characterized in that, The core also includes at least one of the following drugs encapsulated therein: hemostatic drugs, repair-promoting drugs, anti-inflammatory drugs, anti-adhesion and anti-scarring drugs, and local anesthetic drugs.

4. The sealing material for moist tissues according to claim 1, characterized in that, The photocrosslinkable natural polymer derivatives include at least one of the following substances: methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide chitosan, methacrylamide alginate, and methacrylamide silk fibroin.

5. The sealing material for moist tissues according to claim 1, characterized in that, The comonomer containing active chemical groups is selected from polymerizable monomers containing active groups capable of reacting with protein functional groups.

6. The sealing material for moist tissues according to claim 1, characterized in that, The hydrophobic oil phase is a liquid medium that is hydrophobic, bioinert, and immiscible with tissue fluid, including at least one of the following: medical-grade silicone oil, medium-chain triglycerides, mineral oil, and perfluorocarbon liquid.

7. The sealing material for moist tissues according to claim 1, characterized in that, When the sealing material is applied to moist tissue, the hydrophobic oil phase displaces the liquid in the moist tissue, and the temperature-sensitive crosslinking network shrinks in volume to actively draw the liquid in the moist tissue into the shell. The liquid drawn into the shell reacts with the core to form a core physical network, while the shell reacts with the moist tissue through its active chemical groups to form an interfacial bonding layer.

8. A method for preparing a sealing material for moist tissues as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Preparation of thermosensitive crosslinking solutions and polymer solutions; S2. A core-shell structure precursor is formed by emulsion method or coaxial electrospray technology, with the temperature-sensitive crosslinking solution prepared in S1 as the core and the polymer solution prepared in S1 as the shell; S3. The precursor is freeze-dried and low-temperature ground to obtain temperature-sensitive microspheres; S4. The temperature-sensitive microspheres are mixed with a hydrophobic oil phase to form a sealing material for wet tissues.

9. The preparation method according to claim 8, characterized in that, The temperature-sensitive microspheres have a particle size of 20-120 μm.

10. The preparation method according to claim 8, characterized in that, The mass ratio of the temperature-sensitive microspheres to the hydrophobic oil phase is 1:1 to 1:

3.

11. The use of a sealing material for moist tissue as described in any one of claims 1-7 in the preparation of a medical device for hemostasis, tissue adhesion, or promoting wound healing.

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