Sprayable hydrogel for tumor postoperative adhesion prevention and immunotherapy as well as preparation method and application of sprayable hydrogel

Abstract

The invention discloses sprayable hydrogel for tumor postoperative adhesion prevention and immunotherapy as well as a preparation method and application of the sprayable hydrogel, and belongs to the technical field of biomedical materials. The sprayable hydrogel comprises a hydrogel matrix material and a nano material dispersed in the hydrogel matrix material; wherein the nano material is an ovalbumin liposome; the hydrogel matrix material is prepared from oxidized konjac glucomannan and methacrylated gelatin. According to the sprayable hydrogel, on a biological tissue interface, one side has stable tissue adhesion capacity, and the other side keeps free from surrounding tissues; due to the asymmetric adhesion design, the unexpected adhesion problem caused by indifference adhesion of traditional hydrogel can be reduced; in addition, drug release is doubly regulated and controlled through liposome and a gel network, antigen burst release is avoided, immune cell activation is remarkably promoted, and then the tumor recurrence rate is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a sprayable hydrogel for post-tumor adhesion prevention and immunotherapy, as well as a preparation method and application thereof. Background Art

[0002] Colorectal cancer is a common malignant tumor of the digestive system. Surgical resection remains the first-line treatment option in clinical practice. However, this therapy is often accompanied by thorny issues such as residual tumor cells and tissue defects, which can easily lead to tumor recurrence and the formation of abdominal adhesions. To address these challenges, current strategies mainly include: (1) Eliminate residual tumor cells and inhibit tumor recurrence through systemic chemotherapy; (2) Using laparoscopic surgery instead of traditional abdominal and pelvic open surgery to reduce the incidence of adhesions; (3) Mechanically remove the formed adhesion tissue through a secondary operation.

[0003] However, chemotherapy is often associated with severe side effects, laparoscopic surgery is very limited in reducing adhesions, and mechanical dissection of adhesions also has a very high recurrence rate. Therefore, developing effective adjuvant treatment strategies after colorectal cancer surgery to simultaneously prevent abdominal adhesions and tumor recurrence is of great clinical value.

[0004] Immunotherapy, as an emerging cancer treatment, not only eliminates residual tumor cells at the resection site through local immune activation but also targets circulating tumor cells that have metastasized to distant sites by inducing a systemic immune response. Furthermore, these types of intraperitoneal tumors present another challenging challenge: postoperative adhesions. Currently, the most effective method for preventing postoperative tissue adhesions in clinical practice is to physically isolate the damaged tissue by implanting a barrier material, thereby reducing its contact with adjacent tissues. Various polymer solutions and anti-adhesion films have been widely used to prevent postoperative adhesions. However, traditional materials (such as Seprafilm®) degrade slowly, potentially leading to secondary injury or foreign body reactions. Most hydrogels (such as chitosan-based materials) exhibit good biocompatibility but exhibit weak tissue adhesion, making them difficult to adapt to the dynamic and humid in vivo environment. Even though hydrogels with underwater adhesion have been developed, the potential adverse consequences of indiscriminate adhesion are a significant concern. Furthermore, simple physical barriers only effectively isolate the damaged tissue from surrounding tissues and lack anti-inflammatory or immunomodulatory properties, failing to fundamentally suppress excessive inflammation and fibrosis. Furthermore, these materials present challenges such as inconvenient handling, short residence time, and instability. Asymmetric adhesive hydrogels with anti-inflammatory properties may address these needs and serve as localized drug release reservoirs to extend drug release time and enhance efficacy. However, reports on asymmetric adhesive hydrogels suitable for minimally invasive surgery and capable of simultaneously preventing postoperative tissue adhesion and tumor recurrence are limited. Summary of the Invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the present invention aims to provide a sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors and a preparation method thereof.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing a sprayable hydrogel for anti-adhesion and immunotherapy after tumor surgery, including a hydrogel matrix material and a nanomaterial dispersed in the hydrogel matrix material; wherein the nanomaterial is ovalbumin liposome (OVA@Lipo); the hydrogel matrix material includes oxidized konjac glucomannan (OKGM) and methacryloylated gelatin (GelMA).

[0007] Furthermore, the concentration of the ovalbumin liposomes in the sprayable hydrogel is 40-60 mg / mL; preferably, the concentration of the ovalbumin liposomes is 50 mg / mL.

[0008] Furthermore, ovalbumin liposomes were prepared by a reverse evaporation method.

[0009] Furthermore, an organic solution containing lipids is mixed with an ovalbumin solution to construct a W / O type emulsion, and then the organic solvent is removed by evaporation, followed by freeze-drying to prepare ovalbumin liposomes.

[0010] Furthermore, the lipid is at least one of lecithin, hydrogenated lecithin and cholesterol; preferably, the mass ratio of lecithin, hydrogenated lecithin and cholesterol is 2-4:0-2:1.

[0011] Furthermore, the mass ratio of oxidized konjac glucomannan to methacryloylated gelatin is 1:1-2; preferably, the mass ratio of oxidized konjac glucomannan to methacryloylated gelatin is 1:1.2-1.5; more preferably, the mass ratio of oxidized konjac glucomannan to methacryloylated gelatin is 1:1.5.

[0012] Furthermore, the sprayable hydrogel further comprises an immune adjuvant, and the concentration of the immune adjuvant in the sprayable hydrogel is 5-20 μg / mL; preferably, the concentration of the immune adjuvant is 10-15 μg / mL; more preferably, the concentration of the immune adjuvant is 10 μg / mL.

[0013] Furthermore, the immune adjuvant is a cytokine adjuvant, a Toll-like receptor agonist or a polysaccharide adjuvant; wherein the cytokine adjuvant is granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2) or interferon-γ (IFN-γ).

[0014] The method for preparing the sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors comprises the following steps: (1) Blending an oxidized konjac glucomannan solution, a methacrylated gelatin solution, and a photoinitiator to form a hydrogel precursor solution; (2) Encapsulating the ovalbumin liposome lyophilized powder in a hydrogel precursor liquid to prepare a sprayable hydrogel; or encapsulating the ovalbumin liposome and an immune adjuvant together in a hydrogel precursor liquid to prepare a sprayable hydrogel.

[0015] Furthermore, the specific process of step (1) is as follows: dissolving methacrylated gelatin in a photoinitiator solution at 60-70°C, dissolving oxidized konjac glucomannan in deionized water at 70-80°C, mixing the two solutions, adding ovalbumin liposome freeze-dried powder and immune adjuvant, and mixing to obtain the product.

[0016] Furthermore, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP); and the concentration of the photoinitiator solution is 0.25% (w / v).

[0017] The sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors can be used for postoperative anti-adhesion and immunotherapy of tumors.

[0018] The present invention has the following beneficial effects: (1) The present invention co-loads ovalbumin-loaded liposomes and granulocyte-macrophage colony-stimulating factor into an oxidized konjac glucomannan / methacrylated gelatin double-network hydrogel to form a sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors. The sprayable hydrogel achieves initial adhesion through the Schiff base reaction between the aldehyde groups of oxidized konjac glucomannan (OKGM) and the amino groups on the tissue surface; at the same time, methacrylated gelatin (GelMA) as the second gel component can not only cross-link with OKGM through Schiff base reaction, but also limit the mobility of the aldehyde groups after the photocross-linking network is formed, thereby making the outer surface of the hydrogel exhibit non-adhesive properties, that is, the hydrogel can achieve stable tissue adhesion on one side at the biological tissue interface while the other side remains free from the surrounding tissue. This asymmetric adhesion design can reduce the unexpected adhesion problem caused by indiscriminate adhesion of traditional hydrogels, and provide a new solution for the application of hydrogels in the biomedical field.

[0019] (2) The present invention uses liposomes as antigen carriers. Liposomes have a shear-induced "self-renewal" hydrated lubricating layer. Its combination with the gel network further effectively reduces the surface friction coefficient and prevents postoperative adhesion.

[0020] (3) The sprayable hydrogel prepared by the present invention can dually regulate the release of drugs (OVA and GM-CSF) through liposomes and gel networks, avoid sudden release of antigens, significantly promote the activation of immune cells, and thus significantly reduce the recurrence rate of tumors.

[0021] (4) The mechanism of action of the sprayable hydrogel prepared by the present invention is explained as follows: After surgical resection of the tumor, the wound surface is sprayed with hydrogel precursor fluid and rapidly forms a gel under 405 nm ultraviolet light. On the one hand, the aldehyde groups of the oxidized polysaccharide can achieve stable adhesion to the tissue through Schiff base reaction, and the outward side is smooth and non-sticky due to the encapsulation of the aldehyde groups by the photocrosslinking network; on the other hand, the liposomes further reduce the friction coefficient of the gel surface through shear responsive rearrangement, thereby effectively preventing the formation of postoperative adhesions. In addition, GM-CSF loaded inside the gel can induce immature DCs to proliferate and migrate to the tumor resection bed. Liposomes loaded with the model antigen OVA can induce DCs to mature after being slowly released from the gel and continuously stimulate T cell activation and the generation of immune memory effects to inhibit tumor recurrence and metastasis.

[0022] (5) The sprayable hydrogel prepared by the present invention has both sprayable operability and light-controlled asymmetric adhesion properties, and is particularly suitable for minimally invasive scenarios such as laparoscopy, providing an innovative treatment strategy for patients with various abdominal tumors such as colorectal cancer after surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of the synthesis and action mechanism of Lipo@GO Gel; Figure 2 The morphological structure characterization diagram of OVA@Lipo; Figure 3 To test the stability of OVA@Lipo; Figure 4 Fourier transform infrared spectrum characterization diagram of the gel matrix material in Lipo@GO Gel; Figure 5 This is the H NMR spectrum of the gel matrix material in Lipo@GO Gel; Figure 6 This is the self-healing performance evaluation diagram of Lipo@GO Gel; Figure 7 Asymmetric adhesion evaluation diagram of Lipo@GO Gel; Figure 8 This is the lubrication performance evaluation diagram of Lipo@GO Gel; Figure 9 The drug controlled release of Lipo@GO Gel; Figure 10 This is the biosafety assessment diagram of Lipo@GO Gel; Figure 11 This is the effect of Lipo@GO Gel in preventing tissue adhesion after surgery in rats; Figure 12 This is the effect of Lipo@GO Gel in preventing tumor recurrence after subtotal resection. DETAILED DESCRIPTION

[0024] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0025] Example 1 A sprayable hydrogel (Lipo@GO Gel) for postoperative anti-adhesion and immunotherapy of tumors includes a hydrogel matrix material and a nanomaterial uniformly dispersed in the hydrogel matrix material; wherein the nanomaterial is ovalbumin liposome (OVA@Lipo); the hydrogel matrix material includes oxidized konjac glucomannan (OKGM) and methacryloylated gelatin (GelMA).

[0026] The synthesis and mechanism of action of the sprayable hydrogel for anti-adhesion and immunotherapy after tumor surgery are shown in the following figure. Figure 1 , and its specific preparation method comprises the following steps: (1) Preparation of ovalbumin liposomes Lecithin, hydrogenated lecithin, and cholesterol were dissolved in 20 mL of chloroform at a mass ratio of 2:2:1. Then, 5 mL of OVA (4 mg / mL) solution was slowly added dropwise. The mixed solution was ultrasonically treated and then evaporated under reduced pressure. 5 mL of deionized water was then added again for hydration, and the mixture was evaporated under reduced pressure for 20 min. The resulting suspension was ultrasonically treated for 6 min using a 20% power probe with a program set to work for 2 s and rest for 1 s. After filtration, 7.5% (w / v) trehalose was added as a lyoprotectant and the mixture was freeze-dried to obtain ovalbumin liposomes.

[0027] (2) Preparation of oxidized konjac glucomannan and methacrylated gelatin 2.4 g of konjac glucomannan was dissolved in 300 mL of deionized water, and then 9 mM sodium periodate was added to the system and stirred continuously for 12 h in a dark environment at 30°C. Subsequently, ethylene glycol was added to the system and reacted for 2 h to stop oxidation; then, the system was dialyzed with deionized water for 3 days and freeze-dried to obtain oxidized konjac glucomannan.

[0028] 5 g of gelatin was dissolved in 50 mL of Dulbecco's phosphate-buffered saline (DPBS), and then 1 mL of methacrylic anhydride was added dropwise to the dissolved gelatin solution. The mixture was then stirred at 50 °C for 3 h, and then 5 times the volume of DPBS was added and stirred for 10 min to terminate the reaction. The mixture was then dialyzed against deionized water for 7 days and freeze-dried to obtain methacrylated gelatin.

[0029] (3) Preparation of nano-hydrogel composite system 0.45 g of GelMA was dissolved in 3 mL of 0.25% (w / v) photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP)) standard solution at 60°C, and 0.3 g of OKGM was dissolved in 10 mL of deionized water at 70°C. The two prepolymer solutions, GelMA and OKGM, were mixed in a volume ratio of 3:1, and 50 mg / mL ovalbumin liposome lyophilized powder and 10 μg / mL GM-CSF (granulocyte-macrophage colony-stimulating factor) were added and mixed to obtain a nanohydrogel composite system (Lipo@GO Gel).

[0030] Example 2 A sprayable hydrogel (Lipo@GO Gel) for postoperative anti-adhesion and immunotherapy of tumors includes a hydrogel matrix material and a nanomaterial uniformly dispersed in the hydrogel matrix material; wherein the nanomaterial is ovalbumin liposome (OVA@Lipo); the hydrogel matrix material includes oxidized konjac glucomannan (OKGM) and methacryloylated gelatin (GelMA).

[0031] The method for preparing the sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors comprises the following steps: (1) Preparation of ovalbumin liposomes Lecithin, hydrogenated lecithin, and cholesterol were dissolved in 18 mL of chloroform at a mass ratio of 3:1:1. Then, 5 mL of OVA (4 mg / mL) solution was slowly added dropwise. The mixed solution was ultrasonically treated and then evaporated under reduced pressure. 5 mL of deionized water was then added again for hydration, and the mixture was evaporated under reduced pressure for 30 min. The resulting suspension was ultrasonically treated for 6 min using a probe at 25% power, with a program set to operate for 2 s and rest for 1 s. After filtration, 5% (w / v) trehalose was added as a lyoprotectant and the mixture was freeze-dried to obtain ovalbumin liposomes.

[0032] (2) Preparation of oxidized konjac glucomannan and methacrylated gelatin 3 g of konjac glucomannan was dissolved in 300 mL of deionized water, and then 12 mM sodium periodate was added to the system and stirred continuously at 45°C in the dark for 15 h. Ethylene glycol was then added to the system for 2 h to stop oxidation; the system was then dialyzed with deionized water for 3 days and freeze-dried to obtain oxidized konjac glucomannan.

[0033] 5 g of gelatin was dissolved in 50 mL of DPBS, and then 0.8 mL of methacrylic anhydride was added dropwise to the dissolved gelatin solution. The mixture was then stirred at 50 °C for 3 h, and then 2 times the volume of DPBS was added and stirred for 10 min to terminate the reaction. The mixture was then dialyzed against deionized water for 7 days and freeze-dried to obtain methacrylated gelatin.

[0034] (3) Preparation of nano-hydrogel composite system 0.6 g of GelMA was dissolved in 3 mL of 0.25% (w / v) photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP)) standard solution at 65°C, and 0.5 g of OKGM was dissolved in 10 mL of deionized water at 75°C. The two prepolymer solutions, GelMA and OKGM, were mixed in a volume ratio of 3:2, and 50 mg / mL ovalbumin liposome lyophilized powder and 10 μg / mL GM-CSF (granulocyte-macrophage colony-stimulating factor) were added and mixed to obtain a nanohydrogel composite system (Lipo@GO Gel).

[0035] Example 3 A sprayable hydrogel (Lipo@GO Gel) for postoperative anti-adhesion and immunotherapy of tumors includes a hydrogel matrix material and a nanomaterial uniformly dispersed in the hydrogel matrix material; wherein the nanomaterial is ovalbumin liposome (OVA@Lipo); the hydrogel matrix material includes oxidized konjac glucomannan (OKGM) and methacryloylated gelatin (GelMA).

[0036] The method for preparing the sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors comprises the following steps: (1) Preparation of ovalbumin liposomes Lecithin, hydrogenated lecithin, and cholesterol were dissolved in 15 mL of chloroform at a mass ratio of 4:0:1. Then, 5 mL of OVA (4 mg / mL) solution was slowly added dropwise. The mixed solution was ultrasonically treated and then evaporated under reduced pressure. 5 mL of deionized water was then added again for hydration, and the mixture was evaporated under reduced pressure for 40 min. The resulting suspension was ultrasonically treated for 8 min using a 30% power probe with a program set to work for 2 s and rest for 1 s. After filtration, 7.5% (w / v) trehalose was added as a lyoprotectant and the mixture was freeze-dried to obtain ovalbumin liposomes.

[0037] (2) Preparation of oxidized konjac glucomannan and methacrylated gelatin 2.4 g of konjac glucomannan was dissolved in 300 mL of deionized water, and then 9 mM sodium periodate was added to the system and stirred continuously at 30°C in the dark for 18 h. Ethylene glycol was then added to the system for 2 h to stop oxidation; the system was then dialyzed with deionized water for 3 days and freeze-dried to obtain oxidized konjac glucomannan.

[0038] 5 g of gelatin was dissolved in 50 mL of DPBS, and then 1 mL of methacrylic anhydride was added dropwise to the dissolved gelatin solution. The mixture was then stirred at 50 °C for 3 h, and then 5 times the volume of DPBS was added and stirred for 10 min to terminate the reaction. The mixture was then dialyzed with deionized water for 7 days and freeze-dried to obtain methacrylated gelatin.

[0039] (3) Preparation of nano-hydrogel composite system 0.45 g of GelMA was dissolved in 3 mL of 0.25% (w / v) photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP)) standard solution at 70°C, and 0.3 g of OKGM was dissolved in 10 mL of deionized water at 80°C. The two prepolymer solutions were mixed in a 1:1 volume ratio and 50 mg / mL ovalbumin liposome lyophilized powder and 15 μg / mL GM-CSF (granulocyte-macrophage colony-stimulating factor) were added and mixed to obtain a nanohydrogel composite system (Lipo@GO Gel).

[0040] Experimental example The sprayable hydrogels for postoperative anti-adhesion and immunotherapy of tumors prepared in the three embodiments of the present invention have similar performances. Taking Example 1 as an example, the performance of the relevant products is described as follows: 1. Characteristics of intermediate products (1) The ovalbumin liposomes prepared by the present invention were observed under transmission electron microscope and scanning electron microscope. Figure 2 .

[0041] Transmission electron microscopy results showed that the ovalbumin liposomes prepared by the present invention were spherical in shape, well dispersed, and had a particle size of 100-200 nm (see Figure 2 (a)). Scanning electron microscopy was used to test the morphology of the freeze-dried liposome powder. Figure 2 (b) shows that the nanoliposomes after freeze-drying also have a spherical structure and the particle size has not changed significantly, laying the foundation for subsequent loading into the hydrogel matrix as a freeze-dried powder.

[0042] (2) The stability of the ovalbumin liposomes prepared by the present invention was tested. Figure 3 As shown in the figure, the hydrodynamic diameter and dispersibility index of OVA@Lipo did not change significantly within 10 days, indicating that it has good stability.

[0043] (3) The hydrogel matrix materials oxidized konjac glucomannan (OKGM) and methacrylated gelatin (GelMA) prepared in the present invention were subjected to infrared analysis. Figure 4 As shown in the figure, compared with the original KGM, the infrared spectrum of OKGM after oxidation modification is at 1730 cm -1 and 890 cm -1 Two new characteristic peaks appeared at the position: corresponding to the symmetrical vibration of the aldehyde group and the hemiacetal structure formed by the aldehyde group and its adjacent hydroxyl group. In addition, the nuclear magnetic resonance spectroscopy revealed that (see Figure 5), GelMA showed two characteristic peaks at chemical shifts δ = 5.3 ppm and 5.5 ppm. The signal peaks were attributed to the two equivalent protons of the carbon-carbon double bond (C=C) in the methacrylamide group, indicating that methacrylic anhydride was successfully grafted to the amino group of the side chain of the gelatin molecule.

[0044] 2. Self-healing properties of nano-hydrogel composite system The crystal violet stained gel and the unstained gel were cut and assembled together, and incubated at 37°C to observe their self-healing phenomenon. Figure 6 As shown in (a), after the cut crystal violet-labeled gel was brought into contact with the unstained gel at 37°C, the dye molecules were observed to diffuse between the gels. Moreover, under the action of external force, the healing gel still maintained its intact structure, suggesting that the gel may undergo self-healing behavior through dynamic covalent bonding.

[0045] Furthermore, the Lipo@GO Gel was subjected to a step strain scan at 1 Hz to test the gel structure recovery. The specific conditions of the structure recovery test were: (1) first section strain = 1%; first period: 120 s; (2) second section strain = 400%; second period: 120 s; three cycles. Figure 6 As shown in (b), under low strain, the G' of the composite gel system is higher than G'', and the system exhibits typical solid-like behavior. Under high strain, it has liquid-like properties, as reflected by G'' being higher than G'. This solid-liquid transition behavior showed good repeatability over three consecutive cycles of testing, confirming the excellent self-healing ability of Lipo@GO Gel.

[0046] 3. Asymmetric Adhesion Properties of Nanohydrogel Composite Systems In the "sol-adhesion" experiment, the hydrogel precursor liquid dyed with fluorescein isothiocyanate (FITC) was injected into a homemade mold. After the precursor liquid was slightly solidified, the Cy5.5-dyed fibrinogen thrombin solution was slowly dripped onto the surface of the precursor liquid, and then immediately irradiated with ultraviolet light for cross-linking. The gel was then placed in a 37°C incubator for 1 hour. Finally, the double-layer gel was rinsed with flowing deionized water for 3 minutes. In the "gel-non-adhesion" experiment, the FITC-dyed gel precursor liquid was first photocrosslinked in the mold to form a gel. The Cy5.5-dyed fibrinogen thrombin solution was then dripped onto the surface of the gel and placed in a 37°C incubator for 1 hour. Finally, it was detected by fluorescence microscopy. Figure 7As shown in the "gel-non-adhesion" test, fibrin did not adsorb to the gel surface, while in the "sol-adhesion" test, fibrin clearly adsorbed to the gel surface. This indicates that the adhesion ability of the hydrogel is significantly reduced after UV curing. It is speculated that this is because the photocrosslinked network restricts the free flow of aldehyde groups, thus providing the necessary guarantee for effectively inhibiting adhesion.

[0047] 4. Lubrication properties of nano-hydrogel composite system Dil-stained nanoliposomes were added to the hydrogel matrix, and a 10 mm PE ball was repeatedly rolled on the gel surface for 10 minutes. The gel was then immediately transferred to an inverted fluorescence microscope to observe the exudation of nanoliposomes on the gel surface before and after friction. Finally, a scanning electron microscope was used to observe the distribution of liposomes on the freeze-dried gel surface before and after friction. The results showed (see Figure 8 ), friction causes more nanoliposomes to be distributed on the gel surface, and the relative fluorescence intensity of nanoliposomes on the hydrogel surface after friction testing is significantly improved, laying the foundation for the boundary lubrication function of the composite system based on nanoliposomes.

[0048] 5. Drug controlled release characteristics of nano-hydrogel composite system The OVA and GM-CSF contents in the release medium were determined using BCA protein assay kit and ELISA assay kit, respectively. Figure 9 As shown in (a), in the initial stage of release (0-24 h), the OVA@Lipo group showed a significant burst release effect. In contrast, the composite gel system only released about 40% of the antigen, indicating that the gel network effectively inhibited the rapid diffusion of the drug. Considering that an ideal drug delivery system should also have environmental responsiveness to achieve controlled release, the release behavior of the system in a simulated tumor microenvironment (pH 5.5) was further investigated. It was found that the cumulative release amount and release rate of OVA under acidic conditions were higher than those under physiological pH conditions. This may be because the reversible dissociation of Schiff base bonds in an acidic environment leads to an increase in the swelling of the gel network, thereby accelerating drug diffusion. As shown in Figure 9 As shown in (b), the release rate and cumulative amount of GM-CSF at pH 5.5 were both higher than those at pH 7.4. This pH-dependent release pattern exhibits similar response characteristics to the Lipo@GO gel system. Compared to Lipo@GO gel, a significant burst of GM-CSF occurred at the initial release stage, suggesting that the presence of nanoliposomes can effectively slow the rate of antigen release, and this sequential release pattern can, to a certain extent, synergistically enhance the immune response.

[0049] VI. Biosafety of Nanohydrogel Composite Systems The CCK-8 detection kit was used to test the cell viability of mouse colon cancer cells (CT26) and mouse embryonic fibroblasts (NIH-3T3) after treatment with different concentrations of hydrogel extracts. Figure 10 As shown in (a), CT26 and NIH-3T3 cells treated with different concentrations and types of gel extracts showed a survival rate of more than 80% after 24 hours. In addition, the results of Calcein-AM / PI live-dead cell staining showed (see Figure 10 (b) Compared with the untreated group, there was no significant difference in NIH-3T3 cell proliferation or cell morphology after treatment with the gel extract. These results confirm that the composite system has good biosafety.

[0050] VII. Effect of Nano-hydrogel Composite System in Preventing Postoperative Tissue Adhesion A rat cecum-abdominal wall defect wear model was used to simulate the postoperative adhesion formation process. First, the rats were anesthetized and fixed, and then their abdomens were depilated and disinfected. A scalpel was used to make an incision in the midline of the abdomen to expose the abdominal cavity and cecum. A scalpel blade was used to scrape the surface of the left abdominal wall and cecum to form a corresponding 1 cm × 1 cm defect, including the peritoneum and part of the muscle, accompanied by bleeding. The cecum wound and the abdominal wall defect were then connected with surgical sutures to ensure that adhesions of the peritoneum and abdominal wall could be formed. The control group was sprayed with 500 μL of normal saline, and the experimental group was covered with 150 μL of GelMA, GO Gel, and Lipo@GO Gel, respectively, to cover the damaged abdominal wall and cecum. Finally, the abdomen was sutured layer by layer, and the adhesions in the abdominal cavity were evaluated on the 7th and 14th days. Figure 11 As shown in the figure, severe dense adhesions formed between the cecum and the abdominal wall of the control rats; similar thick film adhesions also appeared in the abdominal cavity of the rats treated with GelMA. In contrast, GO Gel treatment had a significant inhibitory effect on the formation of adhesions at the injury site, and only some rats showed thin film adhesions. The Lipo@GOGel group containing nanoliposomes showed the best anti-adhesion effect, and no obvious adhesions were observed in all experimental individuals, indicating that the introduction of nanoliposomes further enhanced the anti-adhesion efficiency of the material.

[0051] 8. Effect of Nano-hydrogel Composite System in Preventing Postoperative Tumors CT26 cells in good growth condition were digested and centrifuged, resuspended in pre-cooled physiological saline and counted; 100 μL CT26 cells (3.0×10 7cells / mL) to form subcutaneous primary CT26 tumors; after 14 days of growth, the CT26 tumor-bearing mice were randomly divided into four groups, and then underwent subtotal resection. The tumor resection site was locally sprayed with 150 μL of different preparations, specifically normal saline, blank gel GO Gel, free Lipo (OVA@Lipo=50 mg / mL), and Lipo@GO Gel ([GM-CSF]=10 μg / mL; [OVA@Lipo]=50 mg / mL).

[0052] like Figure 12 As shown, the control group (normal saline) showed rapid tumor recurrence and a growth rate that exceeded that of the initial tumor. Compared with the control group, the blank gel GO Gel group showed a slight inhibitory effect on tumor growth, presumably because the gel physically blocked the vascular system necessary for tumor growth. In contrast, the free Lipo group showed a certain tumor suppression effect, but all mice in this group still experienced tumor recurrence, which may be due to the rapid release of the cytokine GM-CSF and antigens, which failed to continuously activate the mouse immune system. Treatment with the Lipo@GO Gel group significantly inhibited tumor progression, with only one mouse experiencing tumor recurrence. This indicates that the nanohydrogel composite system, as a local drug and antigen reservoir, can prolong the release time of antigens and immunomodulatory cytokines, successfully reducing the tumor recurrence rate.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors, characterized in that: The invention comprises a hydrogel matrix material and a nano material dispersed in the hydrogel matrix material; wherein the nano material is an ovalbumin liposome; and the hydrogel matrix material comprises oxidized konjac glucomannan and methacryloylated gelatin.

2. The sprayable hydrogel for anti-adhesion and immunotherapy after tumor surgery according to claim 1, characterized in that: The concentration of ovalbumin liposomes in the sprayable hydrogel is 40-60 mg / mL.

3. The sprayable hydrogel for anti-adhesion and immunotherapy after tumor surgery according to claim 1 or 2, characterized in that: Ovalbumin liposomes were prepared by the reverse evaporation method.

4. The sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors according to claim 3, characterized in that: An organic solution containing lipids is mixed with an ovalbumin solution to construct a W / O emulsion, and then the organic solvent is removed by evaporation, followed by freeze-drying to prepare ovalbumin liposomes.

5. The sprayable hydrogel for anti-adhesion and immunotherapy after tumor surgery according to claim 1, characterized in that: The mass ratio of oxidized konjac glucomannan to methacryloyl gelatin is 1:1-2.

6. The sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors according to any one of claims 1 to 5, characterized in that: The sprayable hydrogel further includes an immune adjuvant. The concentration of the immune adjuvant in the sprayable hydrogel is 5-20 μg / mL.

7. The sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors according to claim 6, characterized in that: The immune adjuvant is a cytokine adjuvant, a Toll-like receptor agonist or a polysaccharide adjuvant; granulocyte-macrophage colony-stimulating factor, interleukin-2 or interferon-γ.

8. The method for preparing the sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Blending an oxidized konjac glucomannan solution, a methacrylated gelatin solution, and a photoinitiator to form a hydrogel precursor solution; (2) Encapsulating the ovalbumin liposome lyophilized powder in a hydrogel precursor liquid to prepare a sprayable hydrogel; or encapsulating the ovalbumin liposome and an immune adjuvant together in a hydrogel precursor liquid to prepare a sprayable hydrogel.

9. The method for preparing a sprayable hydrogel for postoperative anti-adhesion and immunotherapy of tumors according to claim 8, characterized in that: The specific process of step (1) is as follows: dissolving methacrylated gelatin in a photoinitiator solution at 60-70° C., dissolving oxidized konjac glucomannan in deionized water at 70-80° C., mixing the two solutions, adding ovalbumin liposome freeze-dried powder and immune adjuvant, and mixing well to obtain the product.

10. Use of the sprayable hydrogel for postoperative tumor adhesion prevention and immunotherapy according to any one of claims 1 to 7 in the preparation of postoperative tumor adhesion prevention and immunotherapy drugs.

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