Preparation method and application of anti-adhesion nanocomposite drug delivery system

Abstract

The application discloses a preparation method and application of an anti-adhesion nano composite drug delivery system, relates to the technical field of adhesives, and discloses the following technical scheme: polyacrylamide and aldehyde functionalized starch with a preset mass ratio are taken, different amounts of zeolite imidazolate framework materials are added after mixing, and then the mixture after mixing is ground and sieved to obtain the anti-adhesion nano composite drug delivery system. The application can be used as a peritoneal tumor local drug delivery system, has high drug encapsulation efficiency and drug loading rate, can release antitumor drugs in tumor tissues specifically, continuously and stably through a tumor acid microenvironment, prolongs the exposure time of the drugs, improves the curative effect of the drugs, reduces the side effects of chemotherapy, can reshape an immune microenvironment, has a good killing effect on tumor cells, and has no systemic toxicity; meanwhile, the gel powder forms a physical barrier on a surgical wound after being changed into a gel by water, so that abdominal cavity adhesion caused by surgical trauma and chemotherapy can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of adhesive preparation, more particularly, it relates to a preparation method and application of an anti-adhesion nano-composite drug delivery system. BACKGROUND

[0002] Hydrogel is a highly water-absorbing material, usually composed of water molecules and one or more water-soluble polymers. These polymers can form a combination with water molecules and absorb a large amount of water to form a cross-linked three-dimensional network gel with a three-dimensional structure.

[0003] This three-dimensional network structure can make MOFs uniformly and stably distributed in the hydrogel. The MOFs-based hydrogel not only solves the shortcomings of MOFs instability and aggregation, but also simultaneously plays the advantages of hydrogel and MOFs material, can target the tumor acid microenvironment, can specifically and continuously release antitumor drugs in tumor tissue, prolongs the exposure time of the drug, improves the efficacy of the drug, and reduces the side effects of chemotherapy.

[0004] In addition, abdominal adhesion is one of the most common complications of abdominal surgery and postoperative abdominal chemotherapy, which often causes chronic abdominal pain, intestinal obstruction and other serious problems, and affects the quality of life of patients. Therefore, finding an ideal method to prevent abdominal adhesion is still a problem to be solved in current clinical practice. The tissue adhesion and anti-adhesion of nano-biomaterials are two contradictory properties. The currently developed tissue-adhesive hydrogel can achieve anti-adhesion effect through the construction of a dense material barrier, a super-hydrophobic surface, a negatively charged surface, etc. On the other hand, the anti-tissue-adhesive hydrogel promotes tissue adhesion by changing the surface properties of the material to form a neutral hydrophilic surface and a positively charged surface.

[0005] Therefore, the present application aims to provide an anti-adhesion nano-composite drug delivery system that takes into account the two completely opposite material properties of tissue adhesion and anti-adhesion, in order to solve the above-mentioned related problems. SUMMARY

[0006] The purpose of the present application is to provide a preparation method and application of an anti-adhesion nano-composite drug delivery system to meet the demand of taking into account the two completely opposite material properties of tissue adhesion and anti-adhesion.

[0007] The above technical purpose of the present application is achieved by the following technical solution: a preparation method of an anti-adhesion nano-composite drug delivery system, the preparation method comprising the following steps:

[0008] S1: preparing aldehyde-functionalized starch;

[0009] S2: preparing zeolite imidazolate framework material:

[0010] S3: taking polyacrylamide and aldehyde group functionalized starch with a preset mass ratio, mixing, then adding different mass of zeolite imidazolate framework material, grinding and sieving the mixture, to obtain an anti-blocking nano composite drug delivery system;

[0011] The anti-blocking nano composite drug delivery system is a PAM / DAS / ZIF-8 hydrogel adhesive.

[0012] The application further provides that the preparation of the aldehyde group functionalized starch in step S1 comprises the following steps:

[0013] S101: taking 10 g of branched amylopectin, and preparing a suspension with a concentration of 10%;

[0014] S102: placing the suspension in a 35 DEG C water bath, and adding 100 mL of 10% sodium periodate solution drop by drop, and avoiding light for 12 h;

[0015] S103: centrifuging the suspension after reaction, and taking the white precipitate at the bottom of the centrifugate;

[0016] S104: washing the white precipitate with deionized water for multiple times until the pH of the deionized water after washing is 7;

[0017] S105: adding anhydrous ethanol into the white precipitate after washing to prevent caking;

[0018] S106: placing the white precipitate into a freeze dryer for freeze drying for 3 days, and fully grinding the white precipitate after freeze drying, to obtain the aldehyde group functionalized starch.

[0019] The application further provides that the preparation of the zeolite imidazolate framework material in step S2 comprises the following steps:

[0020] S201: dissolving zinc nitrate hexahydrate with a preset concentration in deionized water to obtain solution A;

[0021] S202: dissolving 2-methylimidazole with a preset concentration in methanol to obtain solution B;

[0022] S203: mixing solutions A and B to obtain solution C and stirring for 45 min;

[0023] S204: centrifuging solution C according to a preset program, and discarding the supernatant to obtain a precipitate;

[0024] S205: dispersing the precipitate in a 6 mg / mL PVP aqueous solution and stirring for 20 min, then centrifuging according to a preset program, and discarding the supernatant to obtain the zeolite imidazolate framework material.

[0025] The application is further provided with: the preset mass ratio in step S3 is 2:1.

[0026] The application is further provided with: the straight-chain starch in step S101 is from corn.

[0027] The application is further provided with: the preset concentration in step S201 is 0.05 mM.

[0028] The application is further provided with: the preset concentration in step S202 is 0.4 mM.

[0029] The application is further provided with: the preset procedures in steps S204 and S205 are both centrifugal speed of 10,000 rpm and centrifugal time of 15 min.

[0030] The application also provides an application of the anti-adhesion nano-composite drug delivery system, and the application of the anti-adhesion nano-composite drug delivery system in peritoneal drug treatment of peritoneal cancer.

[0031] The application also provides an anti-adhesion nano-composite drug delivery system.

[0032] In summary, the application has the following beneficial effects: the application can be used as a peritoneal tumor local drug delivery system, has high drug encapsulation rate and drug loading rate, can specifically and continuously release anti-tumor drugs in tumor tissues through targeting tumor acidic microenvironment, prolongs the exposure time of the drugs, improves the curative effect of the drugs, reduces the side effects of chemotherapy, can reshape the immune microenvironment, has a good killing effect on tumor cells, and has no systemic toxicity. Meanwhile, the gel powder forms a physical barrier on the surgical wound after forming a gel when meeting water, which can reduce the abdominal cavity adhesion caused by surgical trauma and chemotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a preparation method flow chart of an anti-adhesion nano-composite drug delivery system in embodiment 1 of the application;

[0034] Figure 2 is a PAM / DAS@ZIF-8 synthesis and drug loading schematic diagram of an anti-adhesion nano-composite drug delivery system in embodiment 2 of the application;

[0035] Figure 3 is a gel mechanism and morphology analysis of PAM / DAS@ZIF-8 in embodiment 2 of the application;

[0036] Figure 4 is a microstructure of PAM / DAS@ZIF-8 gels with different ZIF-8 contents in embodiment 2 of the application;

[0037] Figure 5is the microstructure of PAM / DAS@ZIF-8 gel with different ZIF-8 contents in embodiment 2 of the present application;

[0038] Figure 6 is the frequency scanning test simulation diagram of PAM / DAS@ZIF-8 hydrogel with different ZIF-8 contents in embodiment 2 of the present application;

[0039] Figure 7 is the statistical analysis diagram of the storage modulus (G') of PAM / DAS@ZIF-8 hydrogel with different ZIF-8 contents in embodiment 2 of the present application, wherein *P<0.05, **P<0.01, ***P<0.001, n=3;

[0040] Figure 8 is the adhesion schematic diagram of PAM / DAS@ZIF-8 gel on fresh pig skin in embodiment 2 of the present application;

[0041] Figure 9 is the lap shear adhesion curve diagram of PAM / DAS@ZIF-8 hydrogel with different ZIF-8 contents in embodiment 2 of the present application;

[0042] Figure 10 is the maximum adhesion strength of PAM / DAS@ZIF-8 hydrogel with different ZIF-8 contents in embodiment 2 of the present application;

[0043] Figure 11 is the blasting experiment schematic diagram of PAM / DAS@ZIF-8 hydrogel in embodiment 2 of the present application;

[0044] Figure 12 is the maximum blasting pressure schematic diagram of PAM / DAS@ZIF-8 hydrogel with different ZIF-8 contents in embodiment 2 of the present application, wherein "ns" represents no significant difference between the two groups;

[0045] Figure 13 is the macroscopic schematic diagram of the self-healing performance of PAM / DAS@ZIF-8 hydrogel in embodiment 2 of the present application;

[0046] Figure 14 is the strain scanning experiment simulation diagram of PAM / DAS@ZIF-8 hydrogel at a fixed frequency of 1 Hz in embodiment 2 of the present application;

[0047] Figure 15 is the continuous cyclic strain scanning experiment (1%→500%→1%) of PAM / DAS@ZIF-8 hydrogel in embodiment 2 of the present application to demonstrate its self-healing performance;

[0048] Figure 16is a schematic diagram of blood compatibility of PAM / DAS@ZIF-8 gel in Example 2 of the present application;

[0049] Figure 17 is a schematic diagram of cytotoxicity of PAM / DAS@ZIF-8 gel in Example 2 of the present application;

[0050] Figure 18 is a detection diagram of blood-related indexes of two groups of mice in Example 2 of the present application;

[0051] Figure 19 is a schematic diagram of HE sections of liver, heart, kidney, lung, spleen and intestine of two groups of mice after being placed in the hydrogel for 1w and 2w in Example 2 of the present application;

[0052] Figure 20 is a degradation diagram of the hydrogel under different pH environments in Example 2 of the present application;

[0053] Figure 21 is a diagram of monitoring the size change of the hydrogel by a small animal live imaging instrument in Example 2 of the present application;

[0054] Figure 22 is a diagram of monitoring the size change of the hydrogel by a small animal live imaging instrument in Example 2 of the present application;

[0055] Figure 23 is a physical diagram of degradation of the hydrogel in Example 2 of the present application;

[0056] Figure 24 is a representative picture of construction of a rat abdominal wall defect-cecum abrasion model in Example 2 of the present application;

[0057] Figure 25 is a schematic diagram of peritoneal adhesion of the control group, the Film group and the hydrogel treatment group at day 7 and day 14 after modeling in Example 2 of the present application;

[0058] Figure 26 is an adhesion score diagram of the control group, the Film group and the hydrogel treatment group at day 7 and day 14 in Example 2 of the present application;

[0059] Figure 27 is a HE staining and Masson staining result diagram of each group of specimens at day 7 and day 14 after operation in Example 2 of the present application, wherein blue represents adhesion fibrin;

[0060] Figure 28 is a standard curve diagram of HPLC determination of GSK126 and SRF in Example 2 of the present application;

[0061] Figure 29 is a release diagram of GSK126 and sorafenib in PAM / DAS@ZIF-8 at different pH values in Example 2 of the present application;

[0062] Figure 30 is a graph of the results of live imaging of tumor-bearing mice in different treatment groups at different times in Example 2 of the present application;

[0063] Figure 31 is a graph of the results of live imaging of tumor-bearing mice in different treatment groups at different times in Example 2 of the present application;

[0064] Figure 32 is a graph of the results of live imaging of tumor-bearing mice in different treatment groups at different times in Example 2 of the present application;

[0065] Figure 33 is a graph of the results of live imaging of tumor-bearing mice in different treatment groups at different times in Example 2 of the present application; DETAILED DESCRIPTION

[0066] The following will be described in detail with reference to the accompanying drawings. Figures 1-33 The present application will be further described in detail.

[0067] Example 1: A preparation method of an anti-adhesion nano-composite drug delivery system, as shown in the following scheme, the preparation method comprises the following steps: Figure 1

[0068] S1: preparing aldehyde-functionalized starch (DAS);

[0069] S101: taking 10 g of amylopectin from corn and preparing a suspension with a concentration of 10% (w / v);

[0070] S102: placing the suspension in a 35°C water bath, adding 100 mL of 10% (w / v) sodium periodate solution drop by drop, and avoiding light for 12 h;

[0071] S103: centrifuging the above reaction suspension, and taking the white precipitate at the bottom;

[0072] S104: washing the white precipitate with deionized water multiple times until the pH of the deionized water after washing is 7; it should be noted that the deionized water is a cleaning solution;

[0073] S105: adding anhydrous ethanol to the above washed white precipitate to prevent caking;

[0074] S106: placing the above white precipitate in a freeze dryer and freeze-drying for 3 days, and fully grinding the freeze-dried white precipitate (oxidized starch) to obtain aldehyde-functionalized starch (DAS).

[0075] ​S2: Preparation of zeolite imidazolium ester framework material (ZIF-8):

[0076] S201: Dissolve 0.05 mM zinc nitrate hexahydrate in deionized water to obtain solution A;

[0077] S202: Dissolve 0.4 mM 2-methylimidazole in methanol to obtain solution B;

[0078] S203: Mix solutions A and B to obtain solution C and stir for 45 minutes;

[0079] S204: Centrifuge solution C at 10,000 rpm for 15 min and discard the supernatant to obtain the precipitate;

[0080] S205: Disperse the above precipitate in a 6 mg / mL PVP aqueous solution and stir for 20 min. Then centrifuge at 10,000 rpm for 15 min and discard the supernatant to obtain the zeolite imidazole ester framework material, also known as ZIF-8 nanoparticles.

[0081] S3: Mix polyacrylamide (PAM) and aldehyde-functionalized starch (DAS) in a mass ratio of 2:1. After mixing, add different masses of zeolite imidazole ester framework material (ZIF-8). Then grind and sieve the mixture (60 mesh) to obtain an anti-adhesion nanocomposite drug delivery system.

[0082] The concentrations of ZIF-8 contained were 0%, 1%, 3%, and 5% (w / w), and were named PD, PDZ-1, PDZ-3, and PDZ-5, respectively.

[0083] The anti-adhesion nanocomposite drug delivery system is a PAM / DAS / ZIF-8 hydrogel adhesive or a PAM / DAS@ZIF-8 nanocomposite material.

[0084] Example 2

[0085] The microstructure of the zeolite imidazole ester framework material (ZIF-8) was observed using scanning electron microscopy (SEM). Figure 3 As shown, ZIF-8 exhibits a regular truncated rhombic dodecahedral morphology with a particle size of approximately 180 nm. Furthermore, elemental analysis of ZIF-8 using energy-dispersive spectroscopy (EDS) confirmed the uniform distribution of carbon (C), nitrogen (N), oxygen (O), and zinc (Zn) within the ZIF-8.

[0086] The microstructure of the PAM / DAS@ZIF-8 hydrogel was observed by scanning electron microscopy (SEM). The PAM / DAS@ZIF-8 hydrogel has a three-dimensional porous network and a large specific surface area. The porous structure of the PAM / DAS@ZIF-8 hydrogel is not destroyed by the incorporation of ZIF-8, but the incorporation of ZIF-8 makes the pore wall of the hydrogel rough. As the amount of ZIF-8 incorporated increases, the pore size of the PAM / DAS@ZIF-8 hydrogel becomes larger, which increases the spacing between the polymer chains, thereby facilitating cell ingrowth Figure 4 ). Further, the carbon (C), nitrogen (N), oxygen (O), and zinc (Zn) elements in the PAM / DAS@ZIF-8 hydrogel were analyzed by EDS. As shown in Figure 5 , ZIF-8 is successfully incorporated into the PAM / DAS@ZIF-8 hydrogel and uniformly distributed in the hydrogel network.

[0087] As a biological implant material, it must have mechanical properties that match the tissue. In order to evaluate the effect of different amounts of ZIF-8 incorporation on the mechanical properties of the hydrogel, the viscoelasticity of the composite hydrogel was evaluated by a rotational rheometer. As shown in Figure 6 , within the entire scanning range, the storage modulus (G’) of the PAM / DAS@ZIF-8 hydrogel is always greater than the loss modulus (G”), indicating that the PAM / DAS@ZIF-8 hydrogel always exhibits stable viscoelastic solid. Subsequently, the storage modulus (G’) of each sample was statistically analyzed, as shown in Figure 7 , when the frequency is 1 Hz, the storage modulus of PD, PDZ-1, PDZ-3 and PDZ-5 is 1636.67 ± 423.95 Pa, 2426.67 ± 378.99 Pa, 4550 ± 245.15 Pa and 4993.33 ± 778.80 Pa, respectively. It can be found that after the incorporation of ZIF-8, the storage modulus (G’) of the PAM / DAS@ZIF-8 hydrogel increases significantly, and the storage modulus (G’) of the PAM / DAS@ZIF-8 hydrogel increases with the increase of the amount of ZIF-8 incorporated. This is because ZIF-8 can act as a linker in the hydrogel network, thereby increasing the crosslinking density of the hydrogel and enhancing the mechanical strength of the hydrogel.

[0088] Commonly used anti-adhesion materials can be used as a physical barrier to isolate damaged tissue to prevent postoperative adhesion. However, these materials tend to be easily peeled off from the damaged tissue because of their low adhesion to soft tissue under wet conditions. Therefore, an ideal anti-adhesion material needs to have adhesion to the damaged tissue and anti-adhesion to other tissues. The aldehyde groups in the PAM / DAS@ZIF-8 hydrogel can react with the amino groups on the surface of the tissue to form a Schiff base, resulting in a tight bond on the interface, so that the hydrogel has good adhesion to the tissue, thereby avoiding the random movement of the hydrogel with the tissue activity. As shown in Figure 8 PAM / DAS@ZIF-8 mixed powder was sprinkled on fresh pigskin, and after injecting deionized water to form a gel, the PAM / DAS@ZIF-8 hydrogel immediately gelled and could be tightly adhered to the fresh pigskin. Even if the pigskin is subjected to arbitrary torsional stress, the hydrogel can still adhere to the pigskin and maintain the shape intact. More importantly, the PAM / DAS@ZIF-8 hydrogel adhered to the fresh pigskin is treated with water, and the hydrogel can also adhere to the pigskin without falling off, while the surface of the hydrogel has no adhesion. The Schiff base and hydrogen bond between the PAM / DAS@ZIF-8 hydrogel during gelling and the tissue, and the stable hydrogel system after gelling, fully consume the free groups on the surface, so that it no longer has adhesion. It is shown that the PAM / DAS@ZIF-8 hydrogel can be used as a barrier material and has anti-adhesion ability, thereby preventing postoperative adhesion and avoiding inflammation.

[0089] Further, the adhesion capacity of the PAM / DAS@ZIF-8 hydrogel was quantitatively analyzed by lap shear test with fresh pigskin, and whether the incorporation amount of ZIF-8 would affect the adhesion effect between the hydrogel and the tissue was studied and analyzed. Figure 9 and Figure 10 are the mechanical curves of the adhesion force between the AM / DAS / ZIF-8 hydrogel and fresh pigskin and the corresponding maximum adhesion strength, respectively. As shown in Figure 10 After adding ZIF-8, the adhesion capacity of the PAM / DAS@ZIF-8 hydrogel is significantly enhanced, and the maximum adhesion strength of the PD, PDZ-1, PDZ-3 and PDZ-5 hydrogels to fresh pigskin is 21.63±3.42 kPa, 29.38±1.22 kPa, 53.65±4.20 kPa and 47.82±0.73 kPa, respectively. Among them, the adhesion strength of the PDZ-3 hydrogel is the highest, about 2.5 times of the PD hydrogel without incorporating ZIF-8.

[0090] As a biomaterial, the burst strength of hydrogel is a key parameter to evaluate its sealing ability. It is related to the mechanical properties of the material, the bonding strength between the material and the tissue, and the application conditions of the material (e.g. temperature and wound size). The adhesion ability of PAM / DAS@ZIF-8 hydrogel to tissue was further evaluated by the burst test. As shown in Figure 11 , the PAM / DAS@ZIF-8 mixed powder was covered on the fresh pigskin with holes, and after the injection of deionized water to form a gel, PBS was pumped into the test device by a syringe pump until the PAM / DAS@ZIF-8 hydrogel could not withstand and burst. As shown in Figure 12 , the maximum burst pressure that the PD, PDZ-1, PDZ-3 and PDZ-5 hydrogels could withstand were 27.33±0.32 kPa, 30.33±0.32 kPa, 34.27±4.12 kPa and 39.13±3.84 kPa, respectively. Compared with PAM / DAS gel, the maximum burst pressure that the hydrogel could withstand was significantly increased after the addition of ZIF-8, which also confirmed that ZIF-8 participated in crosslinking in the network of PAM / DAS@ZIF-8 hydrogel, which significantly improved the cohesion of the hydrogel. The highest burst pressure was achieved when the content of ZIF-8 was 5%, and the maximum burst pressure of PDZ hydrogel was significantly higher than the normal human blood pressure (10-16 kPa). This indicates that PAM / DAS@ZIF-8 hydrogel has good sealing ability and can effectively prevent the leakage of blood vessels and airways.

[0091] The self-healing properties of PAM / DAS@ZIF-8 hydrogel were further studied by a rotational rheometer. First, strain sweep tests were performed on PAM / DAS@ZIF-8 hydrogel at physiological temperature 37℃, and the strain range was 0.01%~1000%. The experimental results are shown in Figure 14 , when the strain value was less than 200%, the storage modulus (G’) of PAM / DAS@ZIF-8 hydrogel was always greater than the loss modulus (G”), and as the strain increased to 200%, the storage modulus suddenly decreased and intersected with the loss modulus, proving that the network structure of the hydrogel was destroyed at this time.

[0092] Next, under constant frequency (1 Hz), an alternating shear strain sweep test was performed. As shown in Figure 15As shown, in the state of low strain (1% strain), the storage modulus (G’) of PAM / DAS@ZIF-8 hydrogel is always greater than the loss modulus (G”), indicating that PAM / DAS@ZIF-8 has a stable hydrogel network at this time. When in the state of high strain (500% strain) beyond the critical strain, the storage modulus and the loss modulus decrease sharply, the hydrogel network is broken, and the storage modulus (G’) is significantly lower than the loss modulus (G”) at this time. However, once the strain returns to 1%, the storage modulus (G’) and the loss modulus (G”) can return to the original state. During this test, the reversible sol-gel transition of PAM / DAS@ZIF-8 hydrogel proves its excellent self-healing properties.

[0093] Biological materials need to have good blood compatibility to be safely applied in vivo. In this embodiment, the hemolysis experiment of red blood cells was used to detect the blood compatibility of PAM / DAS@ZIF-8. PBS, 1% Triton X-100 and PAM / DAS@ZIF-8 self-gel were added to fresh whole blood, respectively, and the results are shown in Figure 16 As shown, there is basically no hemolysis of red blood cells in the PBS group, and a large number of red blood cells are deposited at the bottom of the centrifuge tube. In the PAM / DAS@ZIF-8 gel group, a small amount of hemoglobin is released from the red blood cells in the solution, and there is a large amount of red blood cell sediment at the bottom of the centrifuge tube, indicating that PAM / DAS@ZIF-8 hydrogel has slight hemolytic activity, which has no statistical difference with the PBS group, while the red blood cells incubated with 1% Triton X-100 have severe hemolysis and release a large amount of hemoglobin. At the same time, we quantitatively calculated the hemolysis rate by measuring the absorbance of the supernatant at 540 nm, and the results showed that the hemolysis rate of PAM / DAS@ZIF-8 hydrogel was 3.37% ± 0.44, which had no statistical difference with the control group, indicating that the gel has good blood compatibility.

[0094] Low cytotoxicity is one of the main indicators for evaluating the biocompatibility of biomaterials. In this embodiment, human gastric cancer AGS cells were used as the research object, and the LDH release method was used to detect the cytotoxicity of PAM / DAS@ZIF-8 gel. First, AGS cells were seeded in a 96-well plate at a density of 3000 cells / well, and after they were completely adhered, the gel / culture medium was added, and then incubated in a cell incubator for 24 h, 48 h and 72 h. After incubation, the LDH release amount of each group of cells was determined by LDH release method to judge the cell death. As shown in Figure 17 As shown in A, the LDH release amount of the gel group is comparable to that of the blank control group, indicating that PAM / DAS@ZIF-8 hydrogel has good cell compatibility. The toxicity of the hydrogel to AGS was detected by MTT colorimetric method. The results showed that PAM / DAS@ZIF-8 gel had no inhibitory effect on AGSFigure 17 B). The results of subsequent cell live / dead staining experiments were consistent with the results of the MTT colorimetric assay Figure 17 C).

[0095] To further evaluate the safety of PAM / DAS@ZIF-8 self-gel powder in vivo application, we opened the mouse abdominal cavity, sprayed PAM / DAS@ZIF-8 self-gel powder in the mouse abdominal cavity, and PBS as a negative control, observed whether the mice had acute poisoning reaction, and whether the diet, defecation, mental state was normal, and on the 5th, 10th, 15th day, the mouse serum was obtained, and the indexes of glutamic oxalacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), alkaline phosphatase (ALP), urea nitrogen (BUN), white blood cells (WBC), red blood cells (RBC), red blood cell distribution width (RDW), platelets (PLT) and other indicators were detected, and the results showed that compared with the negative control, the mice in the PAM / DAS@ZIF-8 self-gel powder group had no obvious poisoning reaction, and the blood routine and liver and kidney function of the mice did not change Figure 18 ). Subsequently, we sacrificed the mice at 1w and 2w time points, obtained the mouse heart, liver, kidney, lung, spleen and intestine and performed HE staining, as shown in Figure 19 compared with the control group, the hydrogel remained in the mouse for 1 week and 2 weeks, and no damage to each organ was observed, which indicated that the self-gel powder had no obvious toxicity to normal tissues, which was consistent with the cell experiment.

[0096] To study the in vivo degradation performance of the gel, we implanted Cy5.5-labeled gel into the subcutis of mice and monitored the fluorescence changes by in vivo imaging system to analyze the degradation of the gel in vivo. We observed and recorded the residual gel after 1 day, 7 days, 14 days and 21 days after the gel was implanted subcutaneously. As shown in Figures 21-23 , the amount of subcutaneous gel gradually decreased; after 21 days, basically no residual gel was detected, which indicated that the gel was basically degraded after 21 days of implantation. The chemotherapy cycle of gastrointestinal tumors is usually 2-3 weeks, so PAM / DAS@ZIF-8 hydrogel has good biodegradation performance as a DDs for gastrointestinal tumors.

[0097] We evaluated the in vivo anti-adhesion efficacy of the self-gel by using a rat abdominal wall defect-cecum abrasion abdominal adhesion model. The construction of the rat abdominal wall defect-cecum abrasion abdominal adhesion model is shown in Figure 24 , first, the damage to the cecum of the rat and the abdominal wall surface at the corresponding position was caused, then PAM / DAS@ZIF-8 self-gel powder / polylactic acid anti-adhesion film(Film) / normal saline were used for treatment respectively, and then the cecum and the defect abdominal wall were fixed, and the adhesion of the cecum and the abdominal wall of the rats in each group was observed on day 7 and day 14 after treatment. As shown in Figures 25-26As shown, in the saline group, severe and firm adhesion was formed between the cecum and abdominal wall at day 7 and day 14, with the median adhesion scores of 4 and 3, respectively, indicating that the peritoneal adhesion model was successfully constructed in this experiment; in the Film treatment group, mild adhesion was observed between the cecum and abdominal wall at day 7 and day 14, and the median adhesion scores of the Film group at day 7 and day 14 were 2 and 1, respectively, which significantly weakened the adhesion between the cecum and abdominal wall compared with the saline group; in the self-gelling powder treatment group, no adhesion band was observed between the cecum and abdominal wall, and the median adhesion scores at day 7 and day 14 were both 0, which were significantly lower than those of the control group (P<0.05), showing a very good anti-adhesion effect.

[0098] At the same time, we collected the cecum and abdominal wall tissues at the injury site of each group for HE and Masson staining to evaluate the formation of adhesion. As shown in Figure 27 , in the saline group, dense adhesion bands were formed between the cecum and abdominal wall at day 7 and day 14, and there were a large number of inflammatory cells and collagen fibers in the adhesion bands; in the Film treatment group, mild adhesion was observed between the cecum and abdominal wall at day 7 and day 14, which was significantly reduced compared with the saline group; while in the gel group, no adhesion band was observed, and the epithelial tissues of the abdominal wall and cecum had also completed complete healing. The results of HE and Masson staining were consistent with the scoring results, and we believe that the self-gelling powder can effectively prevent peritoneal adhesion.

[0099] According to the results of high performance liquid chromatography (HPLC), the peak area at different GSK126 concentrations (4ug / mL, 8ug / mL, 12ug / mL, 16ug / mL, 20ug / mL, 24ug / mL, 28ug / mL) was calculated and the standard curve of GSK126 was drawn, the results showed that GSK126 had a good linear relationship between the concentration and the peak area in the concentration range of 4-28ug / mL Figure 28 A). The regression equation was: Y=7767*X-660.1 (R2=0.9993). In the same way, according to the HPLC results, the standard curve of SRF was drawn, and linear regression analysis was performed, the regression equation was: Y=10531*X+4686 (R2=0.9997), the results showed that the concentration of SRF had a good linear relationship with the peak area in the concentration range of 2-16ug / mL Figure 28 B). Therefore, the conditions for measuring the concentration of GSK126 and SRF by HPLC are ideal.

[0100] To further explore the encapsulation efficiency and drug loading of PAM / DAS@ZIF-8 self-gel simultaneously loaded with GSK126 and SRF, we loaded PAM / DAS@ZIF-8 hydrogel according to the pre-experiment results, following the ratio of GSK126:SRF as 1:1, and evaluated the encapsulation efficiency and drug loading of GSK126 / SRF:PAM / DAS@ZIF-8 after mixing at different ratios (as shown in the table below). The results showed that with the increase of the ratio of GSK126 / SRF:PAM / DAS@ZIF-8, the encapsulation efficiency and drug loading both showed a trend of first increasing and then tending to be stable. When the ratio of GSK126 / SRF:Gel (w / w) was 10%, the encapsulation efficiency was 29.4±0.34% and the drug loading was 12.6±0.08%. When the ratio of GSK126 / SRF:Gel (w / w) increased to 30%, the encapsulation efficiency increased to 75.4±0.24% and the drug loading also increased to 29.5±0.04%. When the ratio of GSK126 / SRF:Gel (w / w) continued to increase to 40%, the encapsulation efficiency and drug loading both tended to be stable, which were 75.3±0.1% and 28.1±0.01% respectively. The experimental results showed that the nanomaterials we synthesized had ideal drug loading and encapsulation efficiency, and finally we selected GSK126 / SRF:PAM / DAS@ZIF-8 as 30% as the drug loading ratio for subsequent experiments.

[0101] Encapsulation efficiency and drug loading of different GSK126:SRF ratios

[0102]

[0103] The release of GSK126 and SRF encapsulated by DDs in PBS buffer with pH=6.5 and pH=7.4 was studied by dialysis bag method. High performance liquid chromatography and ultraviolet spectrophotometer were used to determine the cumulative release content of drugs. GSK126 and SRF encapsulated in PAM / DAS / ZIF-8 hydrogel were slowly released in PBS with pH=6.5 and pH=7.4, and the time exceeded 21 days (as shown in the figures below). Figure 29). Among them, the release rate of GSK126 and SRF from PAM / DAS / ZIF-8 hydrogel in PBS with pH = 6.5 was faster than that in PBS buffer with pH = 7.4. On the 21st day, the cumulative release amount of GSK126 and SRF in PBS with pH = 7.4 was 28.7%, 31.7% respectively, while in PBS with pH = 6.5 was 90.5%, 92.7% respectively. There was no significant difference in the release rate of the two drugs from PAM / DAS / ZIF-8 hydrogel, which released nearly 50% within 72h, and then stably and continuously released, reaching 90% at 21 days. These data show that PAM / DAS / ZIF-8 hydrogel can maintain the release of GSK126 and SRF and has pH sensitivity.

[0104] The results show that PAM / DAS@ZIF-8 self-gel can be rapidly degraded in an acidic environment and reach equilibrium in 72 hours, continuously and stably releasing drugs, thereby achieving the purposes of drug loading, slow release, targeting, and controlled release, and is expected to become an ideal nanomedicine carrier for peritoneal cavity treatment, especially for various drugs, especially hydrophobic drugs.

[0105] Evaluation of in vivo anti-tumor effect: The in vivo anti-tumor experiment was carried out using the successfully constructed MFC-luc gastric cancer peritoneal metastasis tumor model. After the model was successfully constructed, the tumor-bearing mice were randomly divided into 7 groups: (1) Blank group, (2) NK cell adoptive therapy group (NK group), (3) NK cell adoptive therapy group + PAM / DAS@ZIF-8@ (NK+M@), (4) NK cell adoptive therapy group + PAM / DAS@ZIF-8@GSK126 group (NK+M@GSK126), (5) NK cell adoptive therapy group + PAM / DAS@ZIF-8@SRF group (NK+M@SRF), (6) NK cell adoptive therapy group + GSK126 + SRF (NK+GSK126 / SRF), (7) NK cell adoptive therapy group + PAM / DAS@ZIF-8@GSK126 / SRF (NK+M@GSK126 / SRF) group, 12 in each group, and tumor reduction surgery and corresponding experimental treatment were performed, wherein the hydrogel group was evenly sprayed with hydrogel powder in the abdominal cavity after peritoneal tumor reduction surgery, forming a covering film, and NK cell adoptive therapy (1x106 per mouse) was performed. Considering that the half-life of NK cells is about 1 week, we performed intraperitoneal injection of 1x106 NK cells every other day after surgery.

[0106] As Figures 30-31As shown in FIG. 9, the intraperitoneal tumor growth (in vivo imaging) of each treatment group at different time points (0th day, 1st day, 7th day, 14th day and 21st day after surgery) was shown. In the PBS control group, all tumors recurred (recurrence rate = 100%) and grew rapidly, and the mice began to die after 2 weeks of treatment. The NK group and the NK combined material group had a certain effect on peritoneal metastasis of gastric cancer, although all tumors in the group recurred, but the overall survival time was longer than the control group. The efficacy of GSK126 and sorafenib loaded in the hydrogel was better than that of simple NK cell treatment. GSK126 combined with sorafenib had better effect than single drug, and under the assistance of nanomaterials, it could exert its effect to a greater extent. The recurrence rate of the mice in this group was 50%, which was significantly better than that in other groups. Figure 31

[0107] Through flow cytometry analysis, it was found that compared with the NK and M@ groups, the NK cell activity in the M@GSK126, M@SRF and GSK126+SRF groups was significantly enhanced, among which the M@GSK126 / SRF group had the most obvious effect, and the proportion of M2 type tumor-associated macrophages was also significantly reduced (as shown in FIG. 6). Figure 32

[0108] GSK126 was labeled with cy5.5 dye, and three groups of experiments were carried out: the first group was injected with GSK126 through the tail vein (GSK126-I.V group), the second group was injected with GSK126 through the abdominal cavity (GSK126-I.P group) and PAM / DAS@ZIF-8 hydrogel loaded with GSK126 was uniformly sprayed into the abdominal cavity of the mice (M@GSK126-I.P group). The mice were sacrificed 48 hours after administration, and the organs of the mice were taken out, and the distribution of the drug in the body was observed by the in vivo imaging instrument. The results are shown in FIG. 8. Figure 33 As shown in FIG. 8, 48 hours after administration, the drug concentration in the abdominal cavity of the GSK126-I.V group and the GSK126-I.P group was much lower than that of the M@GSK126-I.P group. In addition, the liver is one of the common metastatic sites of gastrointestinal tumors, and most of the intraperitoneal perfusion chemotherapy can be absorbed into the liver through the portal vein, which has a certain killing effect on systemic tumors. The M@GSK126-I.P group can improve the drug concentration in the liver and improve the treatment effect of gastric cancer. At the same time, it does not enhance the drug concentration in other organs such as the heart, lungs and spleen, thereby reducing the systemic toxic side effects. We used the same method to detect the distribution of SRF in the mouse body after different administration methods, and the results were consistent with those of GSK126. Therefore, we believe that the synergistic targeting effect of PAM / DAS@ZIF-8 hydrogel can form a long-acting and high-concentration drug effect in the abdominal cavity, thereby exerting better in vivo anti-tumor effect.

[0109] ​​The embodiments are only used for explaining the present application, and are not used for limiting the present application, and the person skilled in the art can make the modification of the embodiments without the creative contribution according to the need after reading the description, and as long as the modification is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for preparing an anti-adhesion nanocomposite drug delivery system, characterized in that, The preparation method comprises the following steps: S1: preparing aldehyde group functionalized starch; S2: preparing zeolite imidazolate framework material: S3: taking polyacrylamide and aldehyde group functionalized starch with a preset mass ratio of 2:1, mixing, and then adding different amounts of zeolite imidazolate framework material, the concentration of the zeolite imidazolate framework material being 1%, 3% or 5%, grinding and sieving the mixture after mixing, and obtaining an anti-adhesion nano composite drug delivery system; The anti-adhesion nano composite drug delivery system is a PAM / DAS / ZIF-8 hydrogel adhesive.

2. The method for preparing an anti-adhesion nanocomposite drug delivery system according to claim 1, characterized in that, The preparation of the aldehyde group functionalized starch in step S1 comprises the following steps: S101: taking 10 g of branched starch, and preparing a suspension with a concentration of 10%; S102: placing the suspension in a 35℃ water bath, adding 100 mL of 10% sodium periodate solution drop by drop, and avoiding light for 12 h; S103: centrifuging the suspension after reaction, and taking the white precipitate at the bottom; S104: washing the white precipitate with deionized water multiple times until the pH of the deionized water after washing is 7; S105: adding anhydrous ethanol to the white precipitate after washing to prevent caking; S106: placing the white precipitate in a freeze dryer and freeze-drying for 3 days, and fully grinding the white precipitate after freeze-drying to obtain aldehyde group functionalized starch.

3. The method of claim 1, wherein the anti-adhesion nanocomposite drug delivery system is prepared by the steps of: The preparation of the zeolite imidazolate framework material in step S2 comprises the following steps: S201: dissolving zinc nitrate hexahydrate with a preset concentration in deionized water to obtain solution A; S202: dissolving 2-methylimidazole with a preset concentration in methanol to obtain solution B; S203: mixing solutions A and B to obtain solution C and stirring for 45 min; S204: centrifuging solution C according to a preset program, and discarding the supernatant to obtain a precipitate; S205: dispersing the precipitate in a 6 mg / mL PVP aqueous solution and stirring for 20 min, then centrifuging according to a preset program, and discarding the supernatant to obtain a zeolite imidazolate framework material.

4. The method of claim 2, wherein the anti-adhesion nanocomposite drug delivery system is prepared by the steps of: The branched starch in step S101 is derived from corn.

5. The method for preparing an anti-adhesion nanocomposite drug delivery system according to claim 3, characterized in that, The preset concentration in step S201 is 0.05 mM.

6. The method of claim 3, wherein the anti-adhesion nanocomposite drug delivery system is prepared by the steps of: The preset concentration in step S202 is 0.4 mM.

7. The method for preparing an anti-adhesion nanocomposite drug delivery system according to claim 3, characterized in that, The preset programs in steps S204 and S205 are both centrifugation at a speed of 10,000 rpm for 15 min.

8. Use of the anti-adhesion nano composite drug delivery system according to any one of claims 1 to 7 in the preparation of a product for peritoneal cancer intraperitoneal drugs.

9. An anti-adhesion nano composite drug delivery system prepared by the method of any one of claims 1 to 7.

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