Microspheres containing calcium and gallic acid and their application in reducing postoperative tissue adhesion

Abstract

A microsphere, which is a porous microsphere containing calcium and polyphenolic substances, is a metal polyphenol network composed of calcium ions and gallic acid. It has been verified that the microspheres of the present invention combine the therapeutic effects of Ca<supgt;2+< / supgt; and gallic acid, and significantly reduce the formation of abdominal wall adhesions in a cecal-abdominal wall adhesion injury model. The optimization of the therapeutic effect is achieved in the following aspects: (i) CaPNs with a porous structure and good biocompatibility are synthesized by a simple method, (ii) the CaPNs produced by the coordination complexation of Ca<supgt;2+< / supgt> and gallic acid exhibit synergistic hemostatic and antibacterial effects, and (iii) the sustained release of gallic acid in CaPNs makes full use of its anti-inflammatory and antioxidant properties.

Description

Technical Field

[0001] The invention relates to a biomaterial, in particular to a metal polyphenol network particle containing calcium ions and gallic acid, which can not only shorten the hemostasis time but also effectively reduce the degree of tissue adhesion after surgery. Background Art

[0002] Abdominal adhesions are one of the most common complications of abdominal and pelvic surgery, with an incidence rate of up to 90%. Postoperative adhesions often lead to a variety of clinical complications, including intestinal obstruction, female infertility, and chronic abdominal pain, which bring burdens to patients and society. Factors that affect abdominal adhesions, such as inevitable peritoneal injury, foreign body retention, bleeding, and postoperative infection during abdominal surgery, can easily cause adhesions between damaged surfaces and form adhesion bands. Residual foreign bodies can cause abdominal inflammation and increase the degree of postoperative adhesions. Most foreign bodies come from residual blood in the body (especially continuous bleeding from wounds after surgery). After abdominal surgery, blood exuded from the injured area coagulates to form clots and insoluble fibrin, stimulating fibroblast proliferation and exacerbating adhesion formation and development. In addition, unintentional abdominal infection during surgery can change the abdominal microenvironment, leading to fibrin exudation, fibrin formation, and exacerbating the development of adhesions. In general, these factors can induce inflammatory responses and lead to excessive fibrosis. Although the formation mechanism and treatment of postoperative abdominal adhesions have been widely studied, the current anti-adhesion strategies are not ideal.

[0003] Adhesion lysis is commonly used in clinical practice to relieve tissue adhesions. However, approximately one-quarter of abdominal re-exploration surgeries result in intestinal injury, leading to new adhesions. Therefore, the most effective clinical adhesion management strategy is to avoid the formation of adhesions. This can be achieved by adopting good surgical techniques, minimizing damage to intra-abdominal organs (e.g., laparoscopic surgery), and optimizing and improving external barrier materials and drugs to prevent abdominal adhesions. Barrier materials are widely used in clinical practice as anti-adhesion materials due to their convenience and effectiveness. However, commercially available membrane barrier products (e.g., Seprafilm and Interceed) are limited by their limitations in processing difficulties and low efficiency. Drugs help reduce fibrin exudation, promote fibrin decomposition, and avoid adhesions by reducing abdominal infections. However, topical or systemic use of anti-inflammatory drugs and antibiotics (e.g., aspirin, dexamethasone, penicillin, and metronidazole, etc.) has the disadvantages of short retention time and rapid leakage in the body, which significantly impairs the effectiveness of these drugs in preventing adhesions.

[0004] Combined strategies of physical barriers and drug therapy, such as injectable hydrogels, have excellent wound coverage properties and the potential to inhibit abdominal adhesions. Despite their established efficiency, currently available hydrogel systems still have the following limitations: (1) rapid decomposition and limited retention time; (2) the presence of hazardous residues in the cross-linking agent; (3) lack of self-healing ability during injection; (4) slow hydrogel formation; and (5) inability to reduce local oxidative stress and inflammatory responses. In addition, most hydrogels mainly act as physical barriers to isolate injured tissues and avoid postoperative adhesions. These approaches or strategies generally provide only limited benefits because they cannot reduce blood extravasation at the site of injury or achieve adequate hemostasis. Therefore, the rational design of anti-adhesion materials with hemostatic, antibacterial, anti-inflammatory, and antioxidant properties requires further research.

[0005] Polyphenolic compounds have been extensively studied due to their inherent hemostatic, antimicrobial, anti-inflammatory, antioxidant, and disease-treating properties. Our previous studies have shown that polyphenols composed of gallic acid and Mg 2+ The synthesized magnesium metal-organic framework (Mg-MOF) can be gradually released into the acidic microenvironment. The released Mg 2+ It can promote angiogenesis and regulate inflammation. At the same time, the released gallic acid can eliminate excessive reactive oxygen species (ROS) in cells and reduce macrophage inflammation caused by ROS. Gallic acid is a low molecular weight triphenol compound with good anti-inflammatory and antioxidant activities. In addition, gallic acid also has pharmacological effects such as hemostasis and antibacterial. It has been shown that gallic acid can inhibit the inflammatory response by inhibiting the activation of the p65-NF-κB and IL-6 / STAT3 pathways, thereby reducing postoperative abdominal adhesions. Despite this, Mg-MOF does not solve the problem of tissue bleeding.

[0006] Ca 2+ It is often used in hemostasis research to promote the conversion of prothrombin into thrombin and accelerate thrombosis. 2+ The material can accelerate hemostasis and reduce bleeding from damaged tissue to a certain extent. Gallic acid or Ca 2+ It is easy to cause sudden release and loss, resulting in poor therapeutic effect.

[0007] Metal polyphenol networks (MPNs) are supramolecular network structures mainly formed by the coordination of metal ions with polyphenol structures. MPNs have shown great potential in biomedical applications because they provide a rapid and simple method to construct multifunctional nanoplatforms while exhibiting excellent physicochemical properties and good biocompatibility. As a new type of nanomaterial self-assembled from metal ions and polyphenols, MPNs exhibit a variety of beneficial properties, such as bioadhesion, controllable composition, tunable pore size, pH responsiveness, selective permeability, and thermal stability. 3+The MPNs coating formed by chelation was assembled on medical gauze as a hemostatic dressing, which solved the potential problems of tannic acid absorption and poisoning in large-area wounds and exhibited excellent hemostatic properties. Summary of the invention

[0008] One object of the present invention is to provide a microsphere having a metal polyphenol network structure, which can achieve rapid hemostasis after acting on bleeding tissue.

[0009] Another object of the present invention is to provide a microsphere having a metal polyphenol network structure, which can not only shorten the hemostasis time but also effectively improve the anti-inflammatory and antioxidant capabilities.

[0010] Another object of the present invention is to provide a microsphere having a metal polyphenol network structure for alleviating tissue adhesions, such as abdominal adhesions.

[0011] Another object of the present invention is to provide a microsphere as a medical device for use in abdominal and pelvic surgery to relieve tissue adhesion.

[0012] A fifth object of the present invention is to provide a medical device comprising microspheres having a metal polyphenol network structure for use in abdominal and pelvic surgery to relieve tissue adhesions.

[0013] The invention discloses a microsphere, which is a porous microsphere containing calcium and polyphenol substances and a metal polyphenol network composed of calcium ions and gallic acid.

[0014] Another type of microsphere is a porous microsphere containing calcium and polyphenols. It is a metal polyphenol network composed of calcium ions and gallic acid, including micropores (<2nm), mesopores (2nm-50nm) and macropores (>50nm), among which macropores are the main pores, accounting for more than 50%.

[0015] Another type of microsphere is a porous microsphere containing calcium and polyphenols. It is a metal polyphenol network composed of calcium ions and gallic acid, with a specific surface area of ​​1.8504m 2 / g.

[0016] Another type of microsphere is a porous microsphere containing calcium and polyphenols, which is a metal polyphenol network composed of calcium ions and gallic acid. The ratio of calcium ions to gallic acid is 1:3.8 by weight.

[0017] The particle size distribution range of the microspheres of the present invention, namely, the CaPNs microspheres, is 0 to 250 μm, and the evaluated particle size is micron-level.

[0018] After the microspheres of the present invention act on bleeding tissue, they have the ability to quickly stop bleeding and can effectively reduce the occurrence of tissue adhesion, or reduce the degree of tissue adhesion in wound tissue.

[0019] The microspheres of the present invention act on the wound surface alone, or are added to scaffold materials, such as PGA, PLA, PLGA and PLL, to form medical devices such as anti-adhesion scaffolds with a certain shape, which are used to reduce the degree of tissue adhesion during tissue repair.

[0020] The present invention also provides a method for preparing microspheres having a metal polyphenol network structure, comprising:

[0021] Dissolve calcium ions (such as CaCl2) and gallic acid in water, mix well, adjust the pH of the solution to 8.0±0.2, heat to 120°C, centrifuge (10000rpm, 15min, 4°C), and obtain the precipitate, which is recorded as CaPNs.

[0022] Ca 2+ It plays an important role in blood coagulation, and the released gallic acid has anti-inflammatory, antioxidant, antibacterial and anti-fibrinogenic properties. 2+ The therapeutic effects of gallic acid and cecal PNs were shown to be superior to those of cecal PNs, and significantly reduced the formation of abdominal wall adhesions in the cecal-abdominal wall adhesion injury model. The results show that the therapeutic effects of cecal PNs were optimized in the following aspects: (i) CaPNs with porous structure and good biocompatibility were synthesized using a simple method; (ii) Ca 2+ The CaPNs generated by complexation with gallic acid exhibited synergistic hemostatic and antibacterial effects, and (iii) the sustained release of gallic acid in CaPNs fully utilized its anti-inflammatory and antioxidant properties.

[0023] In vitro and in vivo studies have shown that CaPNs have multiple functions such as hemostasis, antibacterial, anti-inflammatory, antioxidant, and anti-adhesion. This type of CaPNs with multiple therapeutic functions has potential clinical application prospects in preventing postoperative adhesions.

[0024] Compared with the commercially available standard anti-adhesion solid film barrier materials that cannot completely cover the wound surface, the CaPNs provided by the present invention can be easily applied by spraying onto irregularly shaped surfaces. Secondly, unlike the hydrogel anti-adhesion barrier materials in the literature that require toxic photocrosslinkers or complex chemical reactions, the preparation process of CaPNs microspheres is simple, does not require toxic reagents or complex chemical reactions, and is suitable for clinical applications. In addition, the CaPNs provided by the present invention solves the problem that current commercial products and most preclinically evaluated biomaterial-based abdominal anti-adhesion materials cannot simultaneously exhibit outstanding hemostatic, antioxidant, anti-inflammatory and antibacterial properties, which is conducive to the application of anti-adhesion after abdominal surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Figure 1 is a diagram showing the synthesis and characterization results of CaPNs microspheres; wherein: a is a schematic diagram showing the synthesis of CaPNs microspheres, b is an SEM image of CaPNs microspheres, c is an SEM local magnified image of the CaPNs microspheres in Figure b, d is an SEM image of a single CaPNs microsphere, e is a C element distribution diagram of the CaPNs microspheres, f is a Ca element distribution diagram of the CaPNs microspheres, g is an O element distribution diagram of the CaPNs microspheres, h is an X-ray diffraction (XRD) spectrum of the CaPNs microspheres, i is a diagram showing the specific surface area and cumulative pore volume of the CaPNs microspheres, and j is a diagram showing the viability detection results of L929 cells after incubation with CaPNs for 48 hours;

[0026] Figure 2 Figures 1 and 2 are the blood compatibility and in vitro coagulation performance results of CaPNs, wherein: a is a photo of blood after incubation with PBS, Celox, CaPNs and 1% Triton X-100, b is a hemolysis rate result diagram of the sample (n=3), c is a SEM image of red blood cell adhesion and aggregation on the surface of Celox and CaPNs, d is a SEM image of platelet adhesion and aggregation on the surface of Celox and CaPNs (where the arrows point to in the figure), e is a statistical diagram of BCI results of different materials, n=3 for each group in the figure, f is a statistical diagram of HGB content of red blood cells adhered to the control group (Control) and Celox and CaPNs hemostatic materials after incubation with diluted whole blood, n=3 for each group in the figure, g is a statistical diagram of the number of adhered platelets after treatment with the control group (Control) and Celox and CaPNs hemostatic materials, n=3 for each group in the figure, h is a statistical diagram of in vitro coagulation time of the control group (Control) and Celox and CaPNs hemostatic materials, n=5 for each group in the figure;

[0027] Figure 3 Evaluation of the in vivo hemostatic ability of CaPNs, wherein: a is a photograph of the hemostatic results of the control group (Control) and Celox and CaPNs hemostatic materials on liver incision, b is a photograph of the hemostatic results of the control group (Control) and Celox and CaPNs hemostatic materials on tail amputation, c is a statistical graph of blood loss in each group of rat liver injury model and rat tail amputation model, n=5 in the figure, d is a statistical graph of hemostasis time in each group of rat liver injury model and rat tail amputation model, n=5;

[0028] Figure 4Figure 2 is the result of the in vitro anti-inflammatory, antibacterial and antioxidant abilities of CaPNs, where: a is the SEM image of cell polarization after Raw264.7 cells were treated with LPS or LPS+CaPNs for 24 h, b is the statistical graph of the mRNA expression of IL-6 and iNOS after Raw264.7 cells were incubated with LPS or LPS+CaPNs for 24 h, c is the representative photo of each bacterial strain treated with Celox or CaPNs, d is the statistical graph of the resistance evaluation results of Celox and CaPNs to Staphylococcus aureus, e is the statistical graph of the resistance evaluation results of Celox and CaPNs to Escherichia coli, and f is 50 μg mL -1 CaPNs(1), 100 μg mL -1 CaPNs(2), 150 μg mL - 1 Representative fluorescence images of ROS in mouse fibroblasts treated with CaPNs (3), ROS was stained by DCFA-DA, g is the statistical result of DCFA-DA fluorescence intensity in figure f, n = 3 for each group in the figure;

[0029] Figure 5 Figure 2 is the result of the postoperative anti-adhesion, oxidative stress and inflammation effects of CaPNs in the rat lateral wall defect-cecal abrasion model, wherein: a is a representative image of the adhesion formation between the abdominal wall and the cecal abdominal wall in the Sham group (sham operation group), Model group (model group), Celox group and CaPNs group at the beginning of modeling (day 0) and on the 7th day after surgery, and the arrows show the adhesion sites; b is the Nair score on the 7th day after surgery in each group, and n=6 in each group in the figure; c is the Zuhlke score on the 7th day after surgery in each group, and n=6 in each group in the figure; d is the statistical graph of the expression results of IL-1β, IL-6 and TNF-α in the serum of rats in each group, and n=3 in each group in the figure; e is the statistical graph of the expression results of IL-6, NF-κB and STAT3 genes in each group, and n=3 in each group in the figure; f is the result of Western blot detection of NF-κB and STAT3 activity in each group;

[0030] Figure 6 Figure 7 shows the histopathological results on the 7th day after surgery, where: a is the H&E staining image of the tissue sections of the animals in each group, b is the MT staining image of the tissue sections of each group, and c is the representative immunohistochemical staining result of α-SMA in abdominal wall adhesions. In the figure, "CE" represents the cecum; "AW" represents the abdominal wall, and the position indicated by the arrow is the adhesion site;

[0031] Figure 7 The results of pathological section staining of liver injury model in rats of control group (Control), Celox group and CaPNs group at 1 week (1W) and 2 weeks (2W) after surgery;

[0032] Figure 8 Schematic diagram of the anti-adhesion mechanism of the CaPNs microspheres of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0034] Figure 8 Schematic diagram of the following embodiments of the present invention based on CaPNs and its anti-adhesion mechanism. Figure 8 As shown, the CaPNs microspheres applied to the wound surface released gallic acid and calcium ions after fluid absorption, exhibiting outstanding hemostatic, antioxidant, anti-inflammatory and antibacterial properties, and significantly reduced the formation of abdominal wall adhesions in the cecum-abdominal wall adhesion injury model.

[0035] Peritoneal adhesions are mainly caused by unavoidable peritoneal injury, bleeding, inflammatory response and potential infection risk during surgery. Therefore, the best drug for preventing postoperative abdominal adhesions must meet the above criteria. According to the results of this embodiment, the effect of CaPNs in preventing adhesion is consistent with the above considerations.

[0036] The sources of the main materials used in the following embodiments of the present invention are as follows:

[0037] Gallic acid was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0038] Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0039] Antibodies were obtained from Abcam (Cambridge, MA, USA).

[0040] Other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Nanjing, China) and used as received without further purification.

[0041] In an incubator (37° C., 5% CO 2 ), complete culture medium (DMEM cell culture medium supplemented with 10% fetal bovine serum (FBS)) was used.

[0042] Sprague-Dawley (SD) rats, weighing 220–250 g, were purchased from JESJ Laboratory Animal Co., Ltd. (Shanghai, China). One week before the experiment, the animals had free access to water and food and were housed in a room with a temperature of 20–24 °C and a relative humidity of 55–75%. The experiments were performed in accordance with European Communities Council Directive 86 / 609 / EEC of November 24, 1986. All experimental procedures were performed in accordance with the Institutional Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Animal Care and Use Committee of Shanghai Ninth People's Hospital.

[0043] The various test methods used in the following examples of the present invention are specifically described as follows:

[0044] 1) Preparation of CaPNs microspheres:

[0045] First, 10 g of CaCl2, 38 g of gallic acid and 500 mL of ultrapure water (18.2 MΩ, Millipore Co., Burlington, MA, USA) were added to a round-bottom flask and mixed with a magnetic stirrer for 10 minutes. Next, 10 M KOH aqueous solution was added to adjust the pH to 8, and the mixture was heated in a muffle furnace at 140 ° C for 24 h (Sxi-8-10, Xinhan Instrument Equipment Co., Ltd., Zhengzhou, China), centrifuged (10000 rpm, 15 min, 4 ° C) to separate the gray-yellow solid, and then washed twice with ultrapure water.

[0046] 2) Cytocompatibility analysis

[0047] Mouse fibroblasts (L929) were cultured at 1×10 5 mL -1 L929 cells were inoculated into 96-well plates at different cell densities. -1 ) for 48 hours, with 3 replicates per group. The control group did not receive CaPNs treatment. Cell viability was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA). To stain live cells, 10% CCK-8 solution and culture medium were added to each well after 48 hours. The cells were maintained in an incubator (5% CO2, 37°C) for 2 hours. The 96-well plate was examined using a microplate reader (SpectraMAX iD3, Molecular Devices, LLC, San Jose, CA, USA), and the data were statistically analyzed and plotted.

[0048] 3) In vitro hemolysis and hemostasis assessment

[0049] For in vitro hemolysis and hemostasis tests, fresh whole blood was drawn from SD rats and immediately collected in anticoagulant tubes containing sodium heparin. Commercially available chitosan hemostatic particles were used. as a control. Blood was diluted with normal saline and incubated with various materials, and blood compatibility was assessed using a microplate reader. In vitro hemostasis time and coagulation index (BCI) tests were performed using a modified protocol described previously. Absorbance assay based on lactate dehydrogenase (LDH) activity was used to quantitatively analyze the platelet adhesion capacity of Celox and CaPNs. The number of platelets adhered to the surfaces of different samples was measured by detecting LDH activity in adhered platelet lysates and comparing it with the relevant calibration curve. The modified method was used to quantitatively evaluate the erythrocyte adhesion properties of Celox and CaPNs in diluted whole blood.

[0050] To characterize the morphology of platelets and red blood cells on the hemostatic material, 10 mg of samples (Celox and CaPNs) were compressed into thin sheets and placed in 24-well plates. Whole blood was centrifuged at 2000 rpm for 15 minutes to obtain platelet-rich plasma (PRP). Whole blood or PRP (100 μL) was dropped onto the sample surface and placed at 37°C for 30 minutes. Subsequently, all samples were carefully washed with PBS to eliminate non-adherent platelets and blood cells. The samples were fixed at 4°C for 4 hours in electron microscopy fixative (2.5% glutaraldehyde) and gradually dehydrated by immersion in 60%, 70%, 80%, 90% and 100% ethanol solutions to characterize the morphology of the samples. Red blood cells and platelets. After drying, the samples were sprayed with gold and examined using a scanning electron microscope (SEM).

[0051] 4) In vitro macrophage polarization analysis

[0052] For SEM analysis, 5 × 10 4 Raw264.7 cells were seeded into one well of a 24-well plate containing a sterilized silicon wafer. -1 LPS, LPS+100 μg mL -1After 24 h of co-culture with CaPNs or normal cell culture medium, the silicon wafer containing Raw264.7 cells was removed from the plate and fixed with 2.5% glutaraldehyde for 30 min. Dehydration was performed using different alcohol gradients (30%, 5 min; 50%, 5 min; 70%, 10 min; 80%, 10 min; 95%, 15 min and 100%, 15 min). Each set of images was captured using SEM (Nikon ECLIPSEE 100; Nikon Corporation, Tokyo, Japan). Raw264.7 cells were treated using the aforementioned method to evaluate cytokine expression. Total RNA from RAW264.7 cells treated with LPS or LPS+CaPNs or normal cell culture medium was extracted using TRIzol reagent. RT-qPCR was used to detect the expression levels of IL-6 and iNOS. GAPDH was used as a housekeeping gene. The primer sequences used in RT-qPCR are shown in Table 1.

[0053] Table 1

[0054]

[0055] 5) In vitro antioxidant test

[0056] Using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, the reduction of ROS in CaPNs solution can be observed. Mouse fibroblasts (L929) were incubated in 6-well plates with different concentrations of CaPNs (50 μg mL -1 , 100 μg mL -1 or 150 μg mL -1 ) for 24 hours and reacted with 0.3 mM H2O2 for 30 minutes. After discarding the supernatant, 1 mL DCFH-DA (10×10 -6 M, dissolved in DMEM medium) and incubated for 20 min (5% CO2, 37°C). Cell fluorescence was observed using an inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). Untreated cells were used as controls. Cells that received only CaPNs or H2O2 produced the same results as above.

[0057] 6) Biological antibacterial activity analysis

[0058] The Escherichia coli and Staphylococcus aureus strains were activated in a 37°C constant temperature incubator for 18 h and diluted to a viable bacterial concentration of about 1.0 × 10 5 CFU mL -1. Sterilize the prepared LB solid culture medium with high pressure and high temperature. When cooled to about 50°C, pour the culture medium into a sterilized culture dish. After the culture medium cools and solidifies, evenly apply 200μL of diluted bacterial solution on the surface of the solid culture medium. Use sterile tweezers to soak a sterilized circular filter paper with a diameter of 6mm in 3mg Celox powder and CaPNs powder; then, stick the filter paper on the surface disk area. Place the watch dish in a 37°C constant temperature incubator for 24h, and use a blank filter paper as a control group. Next, place 3mg Celox and CaPNs powder in a 1.5mL sterile centrifuge tube, and add 400μL of a concentration of approximately 1.0×10 5 CFU mL -1 The solution was incubated for 24 hours and the absorbance at 600 nm was recorded. The pure bacteria solution was used as a control.

[0059] 7) In vivo hemostasis study

[0060] To evaluate the in vivo hemostatic potential of the powders (Celox and CaPNs), a rat model of liver incision and tail amputation was created. Celox was used as a commercial control. Briefly, female SD rats (6-8 weeks) were anesthetized and immobilized. To calculate the amount of blood loss, a weighed gauze was placed under the liver, and a 5 mm (depth) and 10 mm (length) incision was made with a sterile scalpel blade to injure the liver. In the experimental groups, the bleeding liver was randomly treated with drugs coated with Celox or CaPNs (100 mg / kg, n=5). The control group was not treated after liver hemorrhage, and the amount of bleeding and the time of hemostasis were recorded throughout the hemostatic process. For hematoxylin and eosin (H&E) and Masson's trichrome (MT) staining, liver tissues were fixed at the incision site, dehydrated with alcohol, immersed in xylene, and embedded in paraffin. The paraffin-embedded samples were cut into 5 μm sections and dewaxed with xylene, and then hydrated with different gradients of alcohol. After staining with H&E and MT, sections were examined under an optical microscope. Tail amputation model was processed with sterile cutting of 50% of the length, placed in air for 5 s, and then treated with Celox or CaPNs (100 mg / kg, n=5). The control group received no treatment (n=5). Hemostasis time and total blood loss were recorded.

[0061] 8) Evaluation of anti-adhesion in vivo in rat model with cecal abdominal wall defect

[0062] SD rats were randomly divided into sham operation group, model group, Celox group and CaPNs group, with 6 rats in each group. As described above, SD rats were sedated with 50 mg / kg body weight of sodium pentobarbital, their abdominal hair was removed and cleaned, and then disinfected using three alternating cycles of iodone and 75% ethanol. A 2 cm long incision was made along the midline of the abdominal wall using surgical scissors. The cecum was separated and gently wiped with sterile surgical gauze to cause surface damage until punctate bleeding was observed. The abdominal wall was scraped with a knife to produce a 1×1 cm 2 Lesions, the worn cecum was placed opposite to the injured abdominal wall. The rats in the model group were rinsed with 1 mL of sterile saline to flush the damaged cecum and abdomen. Celox and CaPNs powders were sprayed on the injured cecum and abdominal wall of the rats in the Celox and CaPNs groups using a powder spray bottle (150 mg / kg, n=6). Finally, the abdominal wall and skin were sutured layer by layer with 5-0 and 6-0 sutures. All surgeries were performed under sterile conditions.

[0063] The rats in each group were killed 7 days after the heart blood was collected. After the heart blood supernatant was collected by centrifugation, the inflammatory-related indicators in the rat serum were detected by enzyme-linked immunosorbent assay (ELISA) kit. The cecal and abdominal wall adhesions were photographed and graded using a conventional scoring system. The cecal and abdominal wall tissues associated with injury and adhesion were then collected and stained with H&E and MT. RT-qPCR and western blotting were used to analyze the gene expression of the p65-NF-κB and IL-6 / STAT3 signaling pathways and the expression of phosphorylation-related proteins. The primer sequences used in RT-qPCR are shown in Table 2. The primer sequences were provided by BioTNT Biotechnology Co., Ltd. (Shanghai, China).

[0064] Table 2

[0065]

[0066] 9) Immunohistochemical staining of rat cecum and abdominal wall injury sites

[0067] Paraffin sections were dewaxed with water and environmentally friendly dewaxing agent (10 minutes each in three cylinders), gradient alcohol (anhydrous, 95%, 75%) for 5 minutes each cylinder, and rinsed 3 times with Tris buffered saline (TBS) for 3 minutes each time. Cells were repaired with citrate antigen retrieval buffer (pH 6.0) and washed with TBS (5 minutes × 3 times) and soaked. After cooling (rewarming), the sections were taken out and soaked in TBS for 3 minutes, and soaked in 3% H2O2 at 25°C for 30 minutes. The serum was discarded and the primary antibody was used with 0.1% 20 μL detergent (TBST, pH 7.4) was diluted in Tris-buffered saline, and 50 μL primary antibody (Abcam, #ab7817, 1:6000) was added dropwise to each section and incubated overnight at 4°C.

[0068] The next day, the slides were removed from the refrigerator and incubated at room temperature for 15 minutes. The slides were washed three times with TBST and then rinsed three times with immersion solution for 3 minutes each. The TBST was discarded and goat anti-mouse secondary antibody (Abcam, #ab205719, 1:2000) was added to each slide. The slides were incubated at 37°C for 45 minutes and rinsed three times with TBST for 3 minutes each. The TBST was removed and 50 μL of freshly prepared 3,3'-diaminobenzidine (DAB) was added dropwise to each section and observed under a microscope, timed, and the reaction was terminated with tap water. The nuclei were stained with hematoxylin for 1 minute, washed, differentiated with hydrochloric acid alcohol for 1-2 seconds, re-washed, and washed with blue reflux solution for a few seconds. The nuclei were stained under a microscope. The slides were sealed with environmental sealant, dried, and examined under a microscope. The samples were then stored in a dry, cool room at room temperature. Images were captured using a white light microscope or scanner.

[0069] 10) Statistical analysis

[0070] All experiments were repeated 3 times, and the quantitative data were expressed as mean ± standard deviation. GraphPad Prism 8.0 (GraphPad Software, Boston, MA, USA) was used to analyze the data. When the p value was *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, the test was considered statistically significant.

[0071] Example 1 Synthesis and Characterization of CaPNs

[0072] Gallic acid is a polyphenol with anti-inflammatory, antioxidant, antimicrobial, antidiabetic and hemostatic properties. It optimizes hemostasis by increasing its porosity. It protects the various biological properties of gallic acid and achieves Ca 2+ and the gradual release of gallic acid is necessary. 2+ and gallic acid to assemble into bioporous microspheres ( Figure 1 a). CaPNs microspheres appear as brown powder in optical images. SEM ( Figure 1 b) shows that CaPNs are typical universal spheres with a rough surface. The particle size distribution of CaPNs microspheres was evaluated using a particle size analyzer, and the results showed that the particle size distribution was narrow (0-250 μm). Particles of this specification may reduce the migration of microspheres into the systemic circulation, thereby minimizing the difficulties caused by possible distal thrombosis. CaPNs microspheres have a suitable size and a rough surface, which promotes the attachment and aggregation of platelets and red blood cells. SEM image of the microsphere surface ( Figure 1c) shows that the surface of the material is uneven and there are quite obvious gaps between the rectangular crystals, indicating that the material has an excellent porous structure and improves the water absorption rate of CaPNs microspheres.

[0073] The swelling behavior of CaPNs microspheres can be divided into three stages: rapid absorption, slow absorption, and stability. The microsphere expansion rate increased significantly within the first 2 minutes, reaching 182%. After 6 minutes of water addition, CaPNs reached saturation, reaching 241%, and the water absorption rate remained almost unchanged in the following time. The swelling experiment showed that CaPNs exhibited excellent water absorption speed and water absorption. The element distribution of CaPNs by bright field scanning SEM ( Figure 1 d. Figure 1 e. Figure 1 f and Figure 1 g) shows that C, Ca, and O are evenly distributed throughout the CaPNs microspheres. This result indicates that the microspheres combined with organic molecules and metal ions were successfully synthesized. The crystal structure of CaPNs was further confirmed by X-ray diffraction (XRD). Figure 1 h), the results are consistent with previous studies.

[0074] In order to study the void structure of CaPNs microspheres, we tested the specific surface area of ​​CaPNs microspheres. The specific surface area and cumulative pore volume of CaPNs were analyzed using N2 adsorption-desorption isotherms ( Figure 1 i). Brunauer-Emmett Teller (BET) calculations show that the specific surface area of ​​CaPNs is 1.8504 m 2 / g. The pore size distribution of CaPNs was observed using the DFT method, and the results revealed the pore size of CaPNs, including micropores, mesopores, and macropores, among which macropores are the main pores. Mesoporous materials have higher specific surface areas than macroporous materials. However, macropores can better adsorb high-viscosity liquids (e.g., blood). The subsequent verification that CaPNs have good hemostatic properties can be attributed to the porosity of the particles.

[0075] The gallic acid release curve of CaPNs in PBS release medium (pH 7.4) was also tested. Six hours after CaPNs entered the release medium, the release rate of gallic acid reached 19.28%, and the cumulative release rate of CaPNs was 63.57% at 168h, with about 36% of gallic acid not released. Therefore, CaPNs were confirmed to have good sustained-release function.

[0076] Cytocompatibility is a prerequisite for in vivo application. This example determines the cytotoxicity of CaPNs in a mouse fibroblast cell line (L929). Figure 1 As shown in j, when the CaPNs concentration reached 200 μg mL -1There was no obvious cytotoxicity to mouse fibroblasts.

[0077] Example 2 Hemolysis and in vitro hemostasis evaluation

[0078] The blood compatibility of biomaterials is a prerequisite for their in vivo application. The blood compatibility of CaPNs was tested using fresh anticoagulated blood, and 1% Triton X-100 was used as a positive control. Hemolysis in the control group with 1% Triton X-100 was expected; however, no hemolysis was observed in the PBS, Celox, or CaPNs-treated groups ( Figure 2 a). Quantitative results showed that the hemolysis rates of different concentrations of CaPNs were all lower than the safe range (5%) ( Figure 2 b), indicating that the synthesized CaPNs have good blood compatibility.

[0079] Gallic acid adsorbs and aggregates substances necessary for the hemostatic mechanism. Adhesion of CaPNs to blood cells was observed using SEM, indicating that CaPNs inherit a hemostatic mechanism similar to gallic acid ( Figure 2 c). In addition, the SEM images showed that the red blood cells in the CaPNs group were intact and distributed in a round pie shape. At the same time, the absorbance of free hemoglobin (HGB) was measured after incubation to quantify the number of free red blood cells. A lower absorbance value indicated that more red blood cells adhered to the surface of the material ( Figure 2 g). CaPNs and Celox have the same blood cell adhesion ability, and there is no statistical difference in their relative HGB absorbance, which is consistent with the SEM observation results, indicating that CaPNs have excellent hemostatic ability. When effective hemostatic substances come into contact with blood, they stimulate platelet adhesion and aggregation, triggering the activation of coagulation events. SEM is used to study the interaction between PRP and various materials. Figure 2 As shown in d, synthetic CaPNs stimulate platelet adhesion and aggregation on their surface, similar to Celox. LDH assay was used to quantify platelet adhesion and calculate platelet number based on the absorbance of LDH ( Figure 2 f). After 60 min of incubation, the number of platelets adhered to CaPNs or Celox was significantly higher than that adhered to gauze. The platelet counts of Celox and CaPNs were statistically significant compared with those of gauze.

[0080] As a quantitative indicator for evaluating the coagulation ability of various materials, the larger the BCI value, the slower the coagulation speed. In addition, the coagulation ability of different materials was quantified by measuring the absorbance at 540nm and calculating the BCI. Figure 2As shown in Figure 5, the relative BCI of the Celox group was about 43.6%, and the relative BCI of CaPNs was about 25.7%. From the perspective of BCI, CaPNs have better hemostatic ability than commercially available Celox. The in vitro hemostatic properties of the materials were further evaluated by measuring the clotting time after mixing with recalcified blood ( Figure 2 h). The coagulation time of the control group was 6.348±0.41 minutes, that of the Celox group was 3.14±0.111 minutes, and that of the CaPNs group was 1.336±0.086 minutes. The coagulation time was shorter, and the hemostatic ability was the strongest among all the groups.

[0081] Therefore, the good hemostatic ability of CaPNs can be attributed to two aspects: 1) The high specific surface area and high porosity of CaPNs can concentrate the coagulation factors in the blood, and the Ca in CaPNs can 2+ As a hemostatic factor, it activates the coagulation mechanism and accelerates blood coagulation; 2) Gallic acid in CaPNs promotes the adhesion and aggregation of coagulated red blood cells and platelets.

[0082] Example 3 In vivo hemostasis study

[0083] Tail amputation and liver bleeding experiments were performed on rats to evaluate the in vivo hemostatic effects of different hemostatic materials. The results showed that CaPNs rapidly stopped bleeding and formed blood clots in the microparticles and wounds. The amount of blood loss in both injury models was significantly reduced ( Figure 3 a and Figure 3 b) In this example, the coagulation time and blood loss of rats were also quantified. Figure 3 c, Blood loss in the control group, Celox group, and CaPNs group in the liver bleeding and tail amputation hemostasis experiments. In the liver bleeding model, the blood loss in the CaPNs group (0.564±0.154g) was significantly lower than that in the control group (1.828±0.43g) and the Celox group (1.03±0.133g). The blood loss in the tail amputation model in the control group, Celox group, and CaPNs group showed that the blood loss in the CaPNs group was the least (0.394g±0.245g) compared with the control group (3.812±0.639g) and the Celox group (1.326±0.158g).

[0084] Figure 3 d shows the hemostasis time of the control group, Celox, and CaPNs groups in the liver bleeding and tail amputation hemostasis experiments. In the liver bleeding model, the mean bleeding time of the CaPNs group (0.372 minutes) was significantly shorter than that of the control group (2.692 minutes) and the Celox group (1.65 minutes). In the tail amputation model, the mean hemostasis time after CaPNs treatment was 14.89 minutes faster than that of the untreated group and 1.292 minutes faster than that of the Celox group.

[0085] In conclusion, based on the significant reduction in blood loss and bleeding time in the liver bleeding model and tail amputation paradigm, CaPNs exhibited excellent hemostatic properties in vivo and were suitable for complex bleeding sites.

[0086] Pathological sections of the rat liver injury model were analyzed 1 week and 2 weeks after surgery. H&E staining was used to observe the distribution of inflammatory cells. MT staining was used to observe the distribution of collagen fibers in the wound surface. Figure 7 As shown in the figure, at 1 and 2 weeks after surgery, the wound surface of the control group and Celox group showed a greater inflammatory reaction, with an increase in inflammatory cells, and hepatocyte degeneration and local necrosis were observed in the local incision. In addition, MT staining showed that compared with the control group and Celox group, the incision of the CaPNs group had less collagen fiber deposition and less fibrosis. Two weeks after surgery, the incision in the CaPNs group healed, and the hepatocytes around the incision were neatly arranged. These results indicate that Celox is more toxic to hepatocytes than CaPNs, and that CaPNs promote liver wound healing.

[0087] Example 4 In vitro anti-inflammatory, antibacterial and antioxidant activities of CaPNs

[0088] SEM images show that LPS100ng mL -1 The treated macrophages developed an inflammatory phenotype, M1 polarization, which was different from that of control (untreated) Raw264.7 cells. However, CaPNs 100 μg mL -1 The addition of LPS promoted the repolarization of pro-inflammatory M1 macrophages to pro-healing M2 macrophages ( Figure 4 a). RT-qPCR was performed on Raw264.7 cells that had been exposed to LPS or LPS+CaPNs for 24 hours, with untreated cells as controls. M1 macrophage markers (IL-6 and iNOS) were measured using RT-qPCR ( Figure 4 b). The results showed that the expression of IL-6 and iNOS in macrophages increased significantly after LPS stimulation, and CaPNs inhibited the LPS-induced expression of IL-6 and iNOS, indicating that CaPNs had an inhibitory effect on M1 polarization. In conclusion, CaPNs inhibited the expression of inflammatory genes and promoted the repolarization of macrophages from M1 to M2, thus exerting a positive effect on wound healing and preventing abdominal adhesions.

[0089] Infection is a clinical challenge that leads to delayed or non-healing wounds. Therefore, biomaterials must have inherent antibacterial properties to meet the clinical requirements for long-lasting antimicrobial agents. In this example, the antibacterial properties of Celox and CaPNs were studied in vitro using Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli). Figure 4As shown in c, an obvious inhibition ring was observed in the CaPNs group, and the inhibition ring in the Celox group was weaker than that in the CaPNs group, indicating that the prepared CaPNs had a strong inhibitory effect on both bacteria. The 24-hour culture results showed that the OD values ​​of Staphylococcus aureus and Escherichia coli in the CaPNs treatment group were 600 The values ​​were 0.078±0.010 and 0.110±0.004, respectively. The OD values ​​of Staphylococcus aureus and Escherichia coli in the control group 600 The values ​​were 0.373±0.021 and 0.428±0.004, respectively. The values ​​of E. coli in the Celox group were 0.21±0.004 and 0.247±0.008 ( Figure 4 d and Figure 4 e). Therefore, CaPNs exhibited excellent antibacterial ability.

[0090] Intra-abdominal adhesions can be prevented and treated by using an antioxidant system or maintaining low reactive oxygen levels. Although many antioxidants have been tried, they consume oxidants and cannot be used for a long time. In contrast, synthetic CaPNs with a microsphere structure can control the release of gallic acid and prevent abdominal adhesions due to the antioxidant properties of gallic acid. Using DCFH-DA as a ROS probe, the ROS level during incubation was evaluated by green fluorescence intensity ( Figure 4 f) When medium containing 3 mM H2O2 was added, mouse fibroblasts produced strong green fluorescence. -1 Treatment of cells with CaPNs and then stimulation with the same dose of H2O2 resulted in a significant decrease in fluorescence intensity. In addition, by increasing the concentration of CaPNs to 100 μg mL -1 or 150 μg mL -1 , the fluorescence further decreased. As the concentration of CaPNs increased, the fluorescence intensity gradually decreased ( Figure 4 g). These results suggest that CaPNs can effectively scavenge cytotoxic ROS.

[0091] Example 5 CaPNs exhibit anti-adhesion efficacy in vivo

[0092] A rat cecal abdominal wall adhesion model was used to evaluate the effectiveness of CaPNs in preventing postoperative cecal abdominal wall adhesions and compare them with commercially available Celox hemostatic powder and untreated rats. After inducing cecal and abdominal wall injuries, Celox or CaPNs were sprayed onto the wound surface. Rats given normal saline served as a negative control group (referred to as the model group). Rats with exposed cecum and abdominal wall but not injured served as the sham operation group (Sham group). On the 7th day after surgery, the peritoneum of the rats was opened, and the degree of adhesion was assessed and scored. The model group developed severe, non-removable adhesions ( Figure 5a), similar to the literature. Before separation of adhesions to harvest cecal and abdominal wall tissue, images were captured and sent to a blinded assessor for review and scoring on a standard scoring system of 0 to 4 based on the presence and severity of adhesions.

[0093] We used two alternative assessment methods to evaluate postoperative abdominal adhesions in rats: Nair grading and Zuhlke grading. Nair grading is mainly related to the amount of adhesions, and the grading criteria are as follows:

[0094] 0: no adhesion;

[0095] 1: single band between viscera or between viscera and wall;

[0096] 2: Two bands between viscera or between visceral walls;

[0097] 3: Two or more intestinal bands or a piece of intestine;

[0098] 4: The internal organs are directly attached to the wall.

[0099] The Zuhlke classification focuses on the severity of adhesions and the degree of damage to the damaged wound surface. The classification criteria are as follows:

[0100] 0: no adhesion;

[0101] 1: Thin film (gentle blunt peeling);

[0102] 2: Mild (aggressive blunt dissection);

[0103] 3: mild (sharp dissection);

[0104] 4: Severe (indivisible without damage).

[0105] The Nair and Zuhlke grading scores were obtained in each group on the 7th day after surgery.

[0106] The results are as follows Figure 5 b and Figure 5 As shown in Figure c, the average Nair grading score of the model group was 3.7, and the average Zuhlke grading score was 3.7. The model group rats had obvious adhesions on the abdominal wall and cecum. Due to the severe adhesions, sharp dissection was required to cut the thick fibrous tissue surrounding the cecum and abdominal wall. In the Celox group, the average Nair grading score was 1.8, and the average Zuhlke grading score was 1.7, indicating that Celox was able to minimize postoperative adhesions. The average Nair grading score of the CaPNs group was 0.7, and the average Zuhlke grading score was 0.7, with almost no adhesion tissue, while the average score of the sham group was 0, indicating that the CaPNs group successfully inhibited adhesions.

[0107] Example 6 Anti-inflammatory and antioxidant properties of CaPNs in vivo

[0108] RT-qPCR, ELISA, and Western blotting were used to investigate the anti-inflammatory and antioxidant activities of CaPNs. Serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 levels were detected by ELISA. The results showed that the levels of TNF-α, IL-1β, and IL-6 in the serum of rats in the CaPNs-treated group were significantly lower than those in the model group. However, there was no significant difference between the CaPNs-treated group and the Celox group ( Figure 5 d).

[0109] Studies have shown that gallic acid has anti-inflammatory, antioxidant, anti-cancer, anti-diabetic and anti-fibrin properties. As an anti-inflammatory mechanism, gallic acid can inhibit the inflammatory response through the p65-NF-κB and IL-6 / STAT3 pathways, which are also key factors in the formation of intra-abdominal adhesions. Therefore, this example evaluated the effects of CaPNs on the expression of genes and proteins in the p65-NF-κB and IL-6 / STAT3 signaling pathways in damaged tissues. RT-qPCR results showed that the expression levels of IL-6, NF-κB and STAT3 in the Celox group were significantly lower than those in the model group; the CaPNs group had the lowest expression of IL-6, NF-κB and STAT3; the expression of NF-κB and STAT3 was significantly different from that of the other two groups ( Figure 5 e). Western blotting was used to detect the protein levels of phosphorylated NF-κB, non-phosphorylated NF-κB, phosphorylated STAT3, and non-phosphorylated STAT3 in each group. Compared with the control group, the expression levels of phosphorylated NF-κB and phosphorylated STAT3 in the CaPNs group were significantly decreased ( Figure 5 f). It can be seen that CaPNs also have anti-inflammatory and antioxidant properties in vivo.

[0110] Example 7 Histopathological analysis of specimens from different groups

[0111] Use H&E ( Figure 6 a) and MT staining ( Figure 6 b) Histological observation of tissue adhesion and healing at the injury site. Severe adhesion was observed between the abdominal muscle layer and the cecum in the model group (indicated by the arrow) ( Figure 6a). This group showed high connective tissue accumulation and a large number of inflammatory cells, indicating that the development of adhesions is directly related to inflammation. The Celox group had moderate adhesions with moderate bridging between the cecum and the surrounding abdominal wall. In contrast, there was no obvious adhesion between the abdominal wall and the cecum after CaPNs treatment. MT staining showed abundant collagen deposition in the model group. Despite the protective effect of Celox, collagen deposition was still visible, resulting in mild to moderate adhesions. Consistent with the H&E staining data, almost no collagen deposition was detected in the CaPNs-treated group, indicating good anti-adhesion efficacy. α-Smooth muscle actin (α-SMA) is an important marker for mesenchymal cells and fibroblasts in abdominal adhesions. α-SMA expression was significantly increased at the site of abdominal wall adhesions with fibrotic scars between the abdominal wall and the visceral layer. The results of immunohistochemical staining of α-SMA are shown in Figure 6 c. In the model group, there was significant expression of α-SMA at the adhesion site, and after treatment with Celox or CaPNs, the expression of α-SMA decreased, especially in the CaPNs group. This suggests that CaPNs reduced the fibrosis of intra-abdominal adhesions and inhibited intra-abdominal adhesions.

Claims

1. A use of microspheres in the preparation of a medical device for reducing abdominal wall tissue adhesion, characterized in that The microspheres are composed of a metal polyphenol network composed of calcium ions and gallic acid, and the ratio of calcium ions to gallic acid is 1:3.8 by weight; the microspheres include micropores, mesopores and macropores, the micropores are <2nm, the mesopores are 2nm-50nm, the macropores are >50nm, and the macropores are the main pores, accounting for more than 50%, and are prepared as follows: Dissolve calcium ions and gallic acid in water, mix well, adjust the pH of the solution to 8.0±0.2, heat to 120°C, centrifuge at 4°C, 10000 rpm, for 15 minutes, and take the precipitate.

2. The use according to claim 1, characterized in that The specific surface area is 1.8504m 2 / g.

3. The use according to claim 1, characterized in that The particle size distribution range of the microspheres is 0 to 250 μm.

4. The use according to claim 1, characterized in that The heating is carried out in a muffle furnace.

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