Anti-tumor electrospinning stent and preparation method thereof

Abstract

The invention belongs to the technical field of medical instruments, and particularly discloses an anti-tumor electrospinning stent and a preparation method thereof. The drug-loaded electrospinning stent is characterized by comprising a drug-loaded electrospinning stent main body and tumor cell-derived drug-loaded microparticles loaded on the surface of the drug-loaded electrospinning stent main body. The drug-loaded electrospinning stent main body comprises an amino polymer, a hydrophilic polymer and celecoxib which are prepared by using an electrospinning technology and have a mass ratio of (2-10): (2-13): 1, and the drug-loaded electrospinning stent main body has high specific surface area and high porosity; the loading capacity of the tumor cell source doxorubicin-loaded microparticles on the anti-tumor electrospinning bracket is 0.6 mu g / mg to 6.1 mu g / mg. The drug is directly delivered to a tumor resection cavity in an in-situ implantation mode, and long-acting drug retention, continuous drug release and complete degradation of a stent material are achieved. The tumor immune microenvironment is remodeled while residual tumor cells are effectively killed, and recurrence and metastasis of tumors are inhibited.

Description

Technical Field

[0001] The present application belongs to the field of medical device technology, and more specifically, relates to an anti-tumor electrospun scaffold and a preparation method thereof. Background Art

[0002] Although surgical resection of the tumor is effective, the treatment is limited by recurrence and metastasis due to residual tumor cells. Adjuvant therapy such as chemotherapy aims to eliminate these cells, but often produces serious side effects, limiting the survival benefit of patients. Therefore, innovative postoperative methods are urgently needed to eliminate residual tumor cells, inhibit tumor recurrence and metastasis, and reduce adverse reactions.

[0003] In addition to residual tumor cells, the tumor microenvironment altered by perioperative trauma also has an important impact on tumor recurrence and metastasis. Inflammation, especially inflammation caused by surgery, plays a key role in the treatment of cancer. Surgical intervention can trigger a complex inflammatory response, often leading to the recruitment of immune cells such as polymorphonuclear neutrophils (PMNs). As important participants in acute inflammation, PMNs exhibit a dual role in cancer treatment. On the one hand, they contribute to tissue repair, fight infection, and aid in the initial stages of wound healing. However, on the other hand, they can promote tumorigenesis by releasing pro-tumorigenic factors such as various cytokines, chemokines, and prostaglandin E. 2 PMNs release factors that inadvertently promote tumor progression by creating a supportive microenvironment for cancer cells, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs—a meshwork composed of DNA, histones, and granule proteins). These factors released by PMNs can promote angiogenesis, tissue remodeling, and immune escape, promoting the survival and growth of cancer cells. In addition, NETs can capture circulating tumor cells, promote their adhesion to distant organs, and promote metastasis. The complex interaction between inflammation, PMNs, and cancer highlights the importance of intervening in the postoperative inflammatory environment. Addressing surgery-induced inflammation can potentially mitigate its adverse effects on the tumor microenvironment, thereby limiting tumor recurrence and metastasis.

[0004] The inflammatory response mediated by PMNs participates in multiple signaling pathways and affects tumor growth. Cyclooxygenase-2 is a catalytic enzyme that converts arachidonic acid into prostaglandins (including prostaglandin E) in response to inflammatory signals. 2 ), which plays a key role in the regulation of tumor-related inflammation, angiogenesis, immune escape and metastasis. 2Signaling pathways may bring hope for improving the treatment of inflammation and cancer. The nonsteroidal anti-inflammatory drug celecoxib (CEL) is a selective cyclooxygenase-2 inhibitor, belonging to type II in the Biopharmaceutics Classification System (BCS), with high permeability and almost insoluble in water. The solid oral preparation (capsule) developed by dissolution-promoting technology can improve the properties of CEL to a certain extent. It is clinically used to treat pain and inflammation associated with diseases such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. In addition, oral CEL has also been used by researchers as an adjuvant therapy before tumor resection. However, data from randomized controlled trials and retrospective case-control studies show that the effect of CEL as an adjuvant therapy before radical surgery is limited. The reason may be that CEL has the potential risk of serious gastrointestinal and cardiovascular thrombotic events in clinical application. For safety reasons, this method focuses on prevention and uses the minimum dose, which cannot effectively counteract the large release of prostaglandins during perioperative period. Even with relatively small doses, the risk of serious adverse events cannot be avoided.

[0005] In the prior art, polylactic acid, gelatin and other polymer materials are usually mixed with tumor therapeutic drugs for electrospinning to obtain electrospun scaffolds for in vivo treatment. At the same time, the tumor resection site can be physically isolated from the peritoneum, which plays a role in postoperative adhesion prevention. However, the use of only electrospun scaffolds faces problems such as insufficient accumulation of therapeutic drugs in the tumor site and inability to penetrate deeply. Microparticles are vesicle-like subcellular structures with a size of 100nm to 1000nm that are released by cells when the cytoskeleton is changed during activation or apoptosis. Good biocompatibility, homologous targeting and low immunogenicity make it a highly potential drug delivery carrier. However, the use of only microparticles faces problems such as difficulty in drug retention in the tumor resection cavity and low utilization rate. At the same time, for tumor cell-derived drug-loaded microparticles, they cannot be used directly as electrospun materials, and are difficult to combine with electrospun scaffolds, resulting in limited drugs combined with electrospun scaffolds, affecting their effectiveness. Chemical or biological surface modification of electrospun scaffolds can sometimes enhance the interaction between electrospun scaffolds and other components, but they still face difficulties such as complex preparation processes and increased toxic and side effects. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present application is to achieve the co-delivery of CEL and tumor cell-derived drug-loaded microparticles by improving the electrospun scaffold material.

[0007] To achieve the above-mentioned purpose, the present application provides an anti-tumor electrospun scaffold, comprising: an electrospun scaffold body and tumor cell-derived drug-loaded microparticles loaded thereon; the electrospun scaffold body comprises an amino polymer, a hydrophilic polymer and CEL in a mass ratio of (2 to 10): (2 to 13): 1; the loading amount of the tumor cell-derived drug-loaded microparticles on the anti-tumor electrospun scaffold is 0.6 μg / mg to 6.1 μg / mg.

[0008] Preferably, the amino polymer is chitosan, chitin, cellulose, silk, collagen, gelatin, dextran, polyamino acid, chondroitin sulfate or heparin; the hydrophilic polymer is polyethylene oxide, polyvinyl pyrrolidone or polyvinyl alcohol.

[0009] Preferably, the tumor cell-derived drug-loaded microparticles include microparticles released from tumor cells when the cytoskeleton is changed during activation or apoptosis, and chemotherapeutic drugs encapsulated in the microparticles; the tumor cells are liver cancer cells, breast cancer cells or melanoma cells.

[0010] As further preferred, the chemotherapy drug is anthracycline, pyrimidine, folic acid antagonist, metal complex or diterpenoid drug.

[0011] Preferably, the mass ratio of the amino polymer to the hydrophilic polymer is 5:1 to 4:6.

[0012] Preferably, the electrospun support body comprises amino polymer, hydrophilic polymer and CEL in a mass ratio of 5:5:1.

[0013] The present application also provides a method for preparing the above-mentioned anti-tumor electrospun scaffold, comprising the following steps:

[0014] S1. Obtaining an electrospun stent body by electrospinning; the solute of the electrospinning solution used for electrospinning comprises an amino polymer, a hydrophilic polymer and CEL in a mass ratio of (2 to 10): (2 to 13): 1;

[0015] S2. Soak the electrospun scaffold body in a solution of 10 μg / mL to 200 μg / mL tumor cell-derived drug-loaded microparticles for 1 hour to 10 hours, so that the tumor cell-derived drug-loaded microparticles are adsorbed on the electrospun scaffold body by electrostatic adsorption, thereby obtaining the anti-tumor electrospun scaffold.

[0016] Preferably, in step S1, the mass fraction of the electrospinning solution is 3% to 60%, and the solvent of the electrospinning solution is organic acid, hexafluoroisopropanol, dimethyl sulfoxide or dichloromethane.

[0017] As further preferred, the mass fraction of the electrospinning solution is 3% to 10%.

[0018] As further preferred, the organic acid is acetic acid, formic acid, trifluoroacetic acid or acrylic acid.

[0019] Preferably, in step S2, the concentration of the solution of tumor cell-derived drug-loaded microparticles is 50 μg / mL to 100 μg / mL, the immersion time is 4 hours to 6 hours, and the pH value is 7.4.

[0020] Preferably, the ambient temperature during the electrostatic adsorption in step S2 is 4° C. to 37° C.

[0021] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art:

[0022] The present application utilizes electrospinning technology to combine amino polymers and hydrophilic polymers with CEL to prepare an anti-inflammatory nanofiber scaffold with positive charge and high specific surface area and porosity; the nanofiber scaffold can mainly load negatively charged tumor cell-derived drug-loaded microparticles through electrostatic adsorption; the electrospun nanofiber scaffold used in the present application has a large specific surface area and porosity, and is a good carrier for enriching drug-loaded microparticles, and delivers chemotherapy microparticles directly to residual tumors through in situ implantation, creating favorable conditions for their further internalization by residual tumor cells, thereby increasing the concentration of local chemotherapy drugs;

[0023] This electrospun scaffold can be directly implanted into the tumor resection site during tumor surgery to eliminate residual tumor cells and avoid tumor recurrence. At the same time, it can inhibit the function of NETs released by inflammatory PMNs after tumor surgery, reshape the tumor immune microenvironment, and inhibit tumor metastasis.

[0024] Animal experiments have shown that the anti-tumor electrospun scaffold of the present application can maintain a high concentration of therapeutic level at the lesion site, while only maintaining a low drug level in other tissues of the body or peripheral blood, which not only reduces the damage caused to other organs of the body, but also realizes the in situ local drug sustained release, and to a certain extent reduces the occurrence of adverse drug reactions, reduces the amount of drug used, and saves medical costs;

[0025] This application uses chemotherapy microparticles produced by tumor cells for anti-tumor treatment, which solves the problem of drug resistance after using chemotherapy drugs and the problem that some tumor cells are insensitive to chemotherapy drugs;

[0026] It has been verified that the electrospun nanofiber scaffold used in this application has a strong anti-inflammatory effect, and the encapsulated drug molecule CEL is cheap but can inhibit the proliferation, migration and invasion of tumor cells promoted by PMNs, and can reshape the tumor immune microenvironment;

[0027] Animal experiments have verified that the drug-loaded polymer material used in this application is a biodegradable polymer material with good biocompatibility and no need for secondary surgery to remove it. It is completely degraded within 35 days after being implanted in the tumor resection cavity of BALB / c mice;

[0028] The preparation method used in this application, the electrospinning technology and the electrostatic adsorption method of loaded chemotherapy microparticles, can be applied to a variety of polymer materials and drug-loaded microparticles, and there is no need to modify the main body of the electrospun scaffold, with small toxic and side effects and simple process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the effect of CEL / DOX-MPs@CP implanted in electrospun scaffolds to prevent tumor recurrence and metastasis;

[0030] Figure 2 This is a crystal structure analysis diagram of CEL@CP obtained in step 1 of Example using a powder diffraction method;

[0031] Figure 3 This is an X-ray energy dispersion spectrum analysis diagram of CEL@CP obtained in step 1 of the embodiment, where the scale bar is 400 nm;

[0032] Figure 4 The scanning electron microscope and transmission electron microscope results of the CP obtained in Comparative Example 4 and the CEL@CP and CEL / DOX-MPs@CP obtained in Example 1 are shown in FIG. 1 , and the scale bar in the figure is 2 μm;

[0033] Figure 5 This is the laser confocal imaging of Cy5.5 / DOX-MPs@CP of comparative example 3, where the scale bar is 10 μm;

[0034] Figure 6 This is a graph showing the in vitro release results of CEL / DOX-MPs@CP of Example 1;

[0035] Figure 7 The figure shows the cytotoxicity of each embodiment and comparative example on mouse breast cancer cell line (4T1 cell) in vitro;

[0036] Figure 8a The figures are the effects of various examples and comparative examples on the cyclooxygenase-2 (COX-2) content (left) and the inducible nitric oxide synthase (iNOS) content (right) in inflammatory PMNs in vitro;

[0037] Figure 8b The figures are the effects of various examples and comparative examples on the content of arginase 1 (ARG1) (left) and the activity of myeloperoxidase (MPO) (right) in inflammatory PMNs in vitro;

[0038] Figure 8cThe figure is the effect of each embodiment and comparative example on the DCF mean fluorescence intensity of inflammatory PMNs in vitro;

[0039] Figure 8d The figure shows the effect of each embodiment and comparative example on the average fluorescence intensity of extracellular DNA of inflammatory PMNs in vitro;

[0040] Figure 8e The figures are the effects of various embodiments and comparative examples on the extracellular DNA area ratio (left) and citrullinated histone H3 (H3Cit) area ratio (right) of inflammatory PMNs in vitro;

[0041] Figure 9a The figures are the effects of in vitro intervention of inflammatory PMNs on the survival rate (left) and relative scratch width (right) of 4T1 cells in various embodiments and comparative examples;

[0042] Figure 9b The figures are the effects of in vitro intervention of inflammatory PMNs on the migration (left) and invasion (right) of 4T1 cells in various embodiments and comparative examples;

[0043] Fig.9c The figures are the effects of in vitro intervention of inflammatory PMNs on the survival rate (left) and relative scratch width (right) of 4T1 cells in various embodiments and comparative examples;

[0044] Figure 9d The diagrams are the effects of in vitro intervention of inflammatory PMNs on the migration (left) and invasion (right) of 4T1 cells in various embodiments and comparative examples;

[0045] Fig.10a The retention curves of various embodiments and comparative examples at the resection site of mouse breast cancer tumors; the left side of the figure shows the total fluorescence intensity of Cy5.5, and the right side of the figure shows the total fluorescence intensity of IR780;

[0046] Fig.10b The in vitro fluorescence quantitative diagrams of tumors and major organs of each embodiment and comparative example on the 19th day after mouse breast cancer tumor resection are shown; the left side of the figure shows the average fluorescence intensity of Cy5.5, and the right side of the figure shows the average fluorescence intensity of IR780;

[0047] Fig.11a The figures are the tumor inhibition curves after resection of breast cancer tumors in mice of various embodiments and comparative examples (left) and the tumor photos on the 19th day (right);

[0048] Fig.11b The tumor inhibition effect diagram of each embodiment and comparative example on the 19th day after mouse breast cancer tumor resection, the left figure is the tumor weight diagram, the right figure is Ki67 + Cell ratio diagram;

[0049] Fig.12aThe figures are the number of lung metastatic nodules (left) and the survival rate of each embodiment and comparative example on the 19th day after breast cancer tumor resection in mice;

[0050] Figure 12b The lung photos of each embodiment and comparative example on the 19th day after the breast cancer tumor resection in mice;

[0051] Fig.13a The figure shows the influence of each embodiment and comparative example on the proportion of PMNs (left) and the area proportion of lymphocyte antigen 6G (Ly6G) (right) in the tumor microenvironment on the 19th day after breast cancer tumor resection in mice;

[0052] Fig.13b The figure shows the influence of each embodiment and comparative example on the area ratio of H3Cit (left) and the area ratio of COX-2 (right) in the tumor microenvironment on the 19th day after mouse breast cancer tumor resection;

[0053] Fig.13c The area ratio of vascular endothelial growth factor (VEGF) and CD8 in the tumor microenvironment of mice breast cancer tumor resection on the 19th day after each embodiment and comparative example + The proportion of T cells (right) affects the map;

[0054] Fig.13d The figure shows the influence of each embodiment and comparative example on the proportion of myeloid-derived suppressor cells (MDSCs) (left) and regulatory T cells (Tregs) (right) in the tumor microenvironment on the 19th day after breast cancer tumor resection in mice. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0056] In addition, references throughout this specification to "one embodiment"; "one embodiment", "an example" or similar language indicate that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present application. Thus, appearances of the phrase "in one embodiment"; "in one embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0057] Based on the deficiencies of the prior art, in response to the problems of recurrence and metastasis faced after tumor surgery, as well as the adverse reactions and tumor resistance caused by the intravenous injection of CEL and chemotherapy drugs, this application uses electrospinning technology to prepare an anti-tumor electrospun scaffold that co-delivers CEL and tumor cell-derived drug-loaded microparticles, which is used to be directly implanted into the tumor resection site during tumor surgery. This in situ implantation material ensures the long-term retention and continuous release of drugs in the tumor resection cavity after surgery. The released tumor cell-derived drug-loaded microparticles are effectively internalized and eliminate residual tumor cells. The released CEL can significantly inhibit the function of inflammatory PMNs after tumor surgery, thereby inhibiting the proliferation, migration and invasion of residual tumor cells induced by PMNs, and reshape the tumor immune microenvironment. These effects are related to the destruction of the function of NETs released by inflammatory PMNs. The combination of the above characteristics makes this composite material an effective strategy for inhibiting postoperative tumor recurrence and metastasis.

[0058] The preparation method of the anti-tumor electrospun scaffold comprises the following steps:

[0059] S1. Obtain the main body of the electrospun scaffold by electrospinning to form a three-dimensional spatial structure composed of nano-scale fibers with a diameter of 100nm to 1000nm; the mass fraction of the electrospinning solution used for the electrospinning is 3% to 60%; the solutes include amino polymers, hydrophilic polymers and CEL in a mass ratio of (2 to 10): (2 to 13): 1; wherein the mass ratio of the amino polymer to the hydrophilic polymer is 5:1 to 4:6, when the ratio of the amino polymer is too high, it may lead to poor release of CEL, while when the ratio of the hydrophilic polymer is too high, the structure of the electrospun scaffold is not stable enough; the solvent is organic acid, hexafluoroisopropanol, dimethyl sulfoxide or dichloromethane; since the specific surface area of ​​nano-scale fibers is 1000 times that of micron-scale fibers, the specific surface area of ​​the nanofiber scaffold composed of nano-scale fibers will be several orders of magnitude larger than that of the fiber scaffold prepared by ordinary spinning technology;

[0060] S2. In an ambient environment of 4°C to 37°C, the electrospun scaffold body is immersed in a solution of 10 μg / mL to 200 μg / mL tumor cell-derived drug-loaded microparticles for 1 hour to 10 hours. Since the amino polymers in the electrospun scaffold carry positive charges, the negatively charged tumor cell-derived drug-loaded microparticles can be adsorbed on the electrospun scaffold body by electrostatic adsorption, thereby obtaining the anti-tumor electrospun scaffold.

[0061] In one embodiment, the mass fraction of the electrospinning solution in step S1 is 3% to 10%; in another embodiment, the mass fraction of the electrospinning solution is 6.4%, wherein the mass ratio of the amino polymer, the hydrophilic polymer and the CEL is 5:5:1.

[0062] In one embodiment, the organic acid in step S1 is acetic acid, formic acid, trifluoroacetic acid or acrylic acid.

[0063] In one embodiment, the amino polymer in step S1 is chitosan, chitin, cellulose, silk, collagen, gelatin, dextran, polyamino acid, chondroitin sulfate or heparin; the hydrophilic polymer is polyethylene oxide, polyvinyl pyrrolidone or polyvinyl alcohol.

[0064] In one embodiment, the tumor cell-derived drug-loaded microparticles in step S2 include microparticles released from tumor cells when the cytoskeleton is changed during activation or apoptosis, and chemotherapeutic drugs encapsulated in the microparticles; wherein the tumor cells are liver cancer cells, breast cancer cells (such as mouse breast cancer cell line 4T1) or melanoma cells; the chemotherapeutic drugs include chemotherapeutic drugs for treating acute leukemia, lymphoma, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, liver cancer, bladder cancer, skin cancer and various other solid tumors, such as one or more of anthracyclines (such as doxorubicin, hereinafter labeled as DOX), pyrimidines (such as 5-fluorouracil), folic acid antagonists (such as methotrexate), metal complexes (such as cisplatin), and diterpenes (such as paclitaxel).

[0065] In one embodiment, in step S2, the concentration of the tumor cell-derived drug-loaded microparticle solution is 50 μg / mL to 100 μg / mL, and the immersion time is 4 hours to 6 hours; in another embodiment, in step S2, the ambient temperature is 25°C, the concentration of the tumor cell-derived drug-loaded microparticle solution is 100 μg / mL, the solvent of the tumor cell-derived drug-loaded microparticle solution is 1× phosphate buffered saline (PBS), the immersion time is 4 hours, and the pH value of the solution is preferably 7.4.

[0066] The anti-tumor electrospun scaffold prepared by the above method includes: an electrospun scaffold body and tumor cell-derived drug-loaded microparticles loaded thereon; the loading amount of the tumor cell-derived drug-loaded microparticles on the anti-tumor electrospun scaffold is 0.6 μg / mg to 6.1 μg / mg; the main components of the electrospun scaffold are consistent with the solute composition of the electrospinning solution, including amino polymers, hydrophilic polymers and CEL in a mass ratio of (2 to 10): (2 to 13): 1.

[0067] The electrospun scaffold body can be stored in a light-proof glass desiccator after preparation, and the tumor cell-derived drug-loaded microparticles can be frozen at -80°C after preparation. The anti-tumor electrospun scaffolds obtained by electrostatic adsorption are freshly prepared before use.

[0068] Figure 1The CEL / DOX-MPs@CP in the embodiment (the specific components can be found in Example 1 below) is an anti-tumor electrospun scaffold in a certain embodiment. Since the electrospun scaffold body has a large specific surface area, it is a good carrier for enriching drug-loaded microparticles. After the anti-tumor electrospun scaffold is implanted in the body, the concentration of local therapeutic drugs can be increased. Since CEL / DOX-MPs@CP has a long drug retention time and continuous drug release performance in the tumor resection cavity, it can effectively kill residual tumor cells after surgery. It has a certain inhibitory effect on the proliferation, invasion and migration of tumor cells promoted by inflammatory PMNs. At the same time, it also reshapes the tumor immune microenvironment and increases the CD8 + The proportion of T cells increased, and the proportion of MDSCs and Tregs decreased. It effectively inhibited the recurrence and metastasis of tumors. The CEL / DOX-MPs@CP in the middle figure is composed of a gray nanofiber scaffold loaded with anti-inflammatory drugs and red chemotherapeutic drug-loaded microparticles attached to it. As can be seen from the figure, after tumor surgical resection, the residual tumor cells and the tumor microenvironment changed by surgical trauma, including enhanced aggregation of inflammatory PMNs, formation of excessive NETs, ​​production of Tregs and MDSCs, etc., promote tumor recurrence and metastasis, especially NETs, ​​which can capture circulating tumor cells, promote their adhesion to distal organs and promote their metastasis. After the implantation of CEL / DOX-MPs@CP, it can not only produce rapid and effective killing of residual tumor cells, but also inhibit tumor recurrence. It can also significantly improve the microenvironment after tumor resection, which is manifested in inhibiting the widespread production of NETs, ​​reducing the proportion of immunosuppressive cells MDSCs and Tregs, and increasing cytotoxic CD8 + The proportion of T cells, thereby inhibiting tumor metastasis.

[0069] Example 1

[0070] 1. Preparation of electrospun scaffold body

[0071] 360mg chitosan (average molecular weight of 850,000, deacetylation degree of 95%, hereinafter marked as C), 360mg polyethylene oxide (average molecular weight of 600,000, hereinafter marked as P) and 72mg CEL (≥99%, molecular weight of 381.38g / mol, hereinafter marked as CEL) were dissolved in 12mL 90% acetic acid solution to prepare a polymer solution with a CEL mass fraction of 0.6%, a C mass fraction of 2.9%, and a P mass fraction of 2.9%, wherein the mass ratio of C to P is 1:1. The polymer solution was placed in the sample injection device of the electrospinning device, the voltage was adjusted to 18kv, the distance between the spinneret and the collecting plate was adjusted to 15cm, the working liquid driving speed was adjusted to 1mL / h, the speed of the receiving device was adjusted to 30rpm, and the environmental humidity was controlled not to exceed 30% for electrospinning to obtain CEL@CP. After the electrospinning was completed, the results of high performance liquid chromatography showed that the drug loading and encapsulation efficiency of CEL@CP were 9% and 96%, respectively. The CEL@CP was vacuum dried at room temperature for 24 h, and then stored in a light-proof glass desiccator.

[0072] 2. Preparation of DOX-MPs

[0073] 4T1 cells were irradiated with ultraviolet light (UVB, 300 J m -2 ) for 1 hour, and then 200μg / mL DOX was added to incubate with 4T1 cells for 12 hours. The supernatant was centrifuged at 1000g for 10 minutes. The supernatant was centrifuged at 14000g for 2 minutes to remove cells and debris. The supernatant was continued to be centrifuged at 14000g for 1 hour to obtain DOX-MPs. After washing with PBS, the PBS solution of DOX-MPs was stored at -80°C. Atomic force microscopy results showed that DOX-MPs were monodisperse irregular spherical particles. Using dynamic light scattering detection, the average particle size of DOX-MPs was about 337nm, and the zeta potential was -8.8mV; the results of high-performance liquid chromatography showed that the drug loading of DOX-MPs was 0.06μg DOX per microgram of MPs protein.

[0074] 3. Preparation of CEL / DOX-MPs@CP

[0075] The dried CEL@CP was cut into small round pieces with a diameter of 14 mm and a mass of 2.5 mg and immersed in 200 μL of a 100 μg / mL DOX-MPs solution. DOX-MPs were enriched on the positively charged surface of CEL@CP by electrostatic adsorption at room temperature (25°C) to obtain CEL / DOX-MPs@CP. After 4 hours, the composite scaffold CEL / DOX-MPs@CP was taken out for subsequent experiments. The DOX-MPs loading on CEL@CP was 6.1 μg DOX / mg CEL@CP according to the content of DOX, as verified by high performance liquid chromatography.

[0076] Example 2-4 Control of DOX-MPs loading

[0077] Example 1 was repeated with the same steps, except that the concentration of DOX in step 2 and the final DOX loading on CEL@CP were shown in Table 1. It can be seen that when the DOX concentration in the DOX-MPs solution before immersion reaches 100 μg / mL, the DOX-MPs loading on CEL@CP can reach 6.1 μg / mg, and when the DOX concentration in the DOX-MPs solution before immersion continues to increase, the increment of the DOX-MPs loading on CEL@CP is limited.

[0078] Table 1 Relationship between DOX loading and solution concentration

[0079] Example 2 Example 3 Example 4 DOX concentration (μg / mL) 12.5 50 100 Loading amount (μg / mg) 0.6 2.9 6.1

[0080] Example 5

[0081] Example 1 was repeated with the same steps, except that in step 1, C was replaced by 6% by mass of silk fibroin, and P was replaced by 8% by mass of polyvinyl alcohol in the polymer solution.

[0082] Comparative Example 1

[0083] Example 1 was repeated with the same steps, except that, in step 1, C was replaced by 4.8% by mass of methoxypolyethylene glycol-polylactic acid (hereinafter marked as mPP) in the polymer solution, and P was replaced by 4.8% by mass of polyethylene glycol (hereinafter marked as PE); the composite scaffold obtained in step 3 was CEL / DOX-MPs@mPPPE.

[0084] Comparative Example 2

[0085] Example 1 was repeated with the same steps, except that in step 1, 0.0034% of the hydrophobic small molecule fluorescent dye Cy5.5 was used to replace CEL in the polymer solution; in step 3, the composite scaffold obtained was Cy5.5 / DOX-MPs@CP. The loading amount of Cy5.5 on the composite scaffold was 0.4 μg / mg.

[0086] Comparative Example 3

[0087] Example 1 was repeated with the same steps, except that, in step 1, in the polymer solution, CEL was replaced with a hydrophobic small molecule fluorescent dye Cy5.5 with a mass fraction of 0.0034%; in step 2, DOX-MPs were replaced with fluorescent dye IR780 surface labeled MPs (i.e., IR780-MPs); and in step 3, the composite scaffold obtained was Cy5.5 / IR780-MPs@CP, and the loading amount of Cy5.5 on the composite scaffold was 0.4 μg / mg, and the loading amount of IR780 was 0.9 μg / mg.

[0088] Comparative Example 4

[0089] Example 1 was repeated with the same steps, except that 72 mg of CEL was not added to the polymer solution in step 1, CP was obtained after step 1, and the CP was dried and cut into small round pieces with a diameter of 14 mm and a mass of 2.5 mg; DOX-MPs@CP was finally obtained in step 3.

[0090] Experimental results verification

[0091] The definitions of the characters involved in the experimental results verification are as follows:

[0092] CEL@CP: CEL@CP obtained in step 1 of Example 1;

[0093] DOX-MPs: DOX-MPs obtained in step 2 of Example 1;

[0094] CEL / DOX-MPs@CP: CEL / DOX-MPs@CP obtained in step 3 of Example 1;

[0095] CEL / DOX-MPs@mPPPE: CEL / DOX-MPs@mPPPE obtained in step 3 of comparative example 1;

[0096] Cy5.5 / DOX-MPs@CP: Cy5.5 / DOX-MPs@CP obtained in step 3 of comparative example 2;

[0097] IR780-MPs: IR780-MPs obtained in step 2 of comparative example 3;

[0098] Cy5.5 / IR780-MPs@CP: Cy5.5 / IR780-MPs@CP obtained in step 3 of comparative example 3;

[0099] Cy5.5 / IR780-MPs solution: PBS solution containing Cy5.5 at an equivalent dose of 75 μg / kg and IR780-MPs at an equivalent dose of 180 μg / kg;

[0100] CP: CP obtained in step 1 of comparative example 4;

[0101] DOX-MPs@CP: DOX-MPs@CP obtained in step 3 of comparative example 4;

[0102] PBS solution: 1X PBS (pH 7.4) solution;

[0103] CEL@CP release solution, DOX-MPs@CP release solution and CEL / DOX-MPs@CP release solution (except for Verification Example 3): Take the seven-day release solutions of CEL@CP, DOX-MPs@CP and CEL / DOX-MPs@CP respectively, and dilute them with PBS solution to a solution with a CEL equivalent dose of 7.6 μg / mL and / or a DOX equivalent dose of 3.3 μg / mL;

[0104] CP release solution (except for verification example 3): take the 7-day release solution of CP and dilute it with PBS solution at the same multiple of the above CEL@CP release solution, DOX-MPs@CP release solution and CEL / DOX-MPs@CP release solution;

[0105] DOX-MPs solution: PBS solution of DOX-MPs containing an equivalent dose of 3.3 μg / mL DOX;

[0106] CEL solution: PBS solution containing CEL at an equivalent dose of 7.6 μg / mL;

[0107] DOX solution: PBS solution containing DOX at an equivalent dose of 3.3 μg / mL.

[0108] Verification Example 1 Characterization of CEL / DOX-MPs@CP

[0109] The crystal structure of CEL@CP obtained in step 1 of Example 1 was analyzed by X-ray powder diffraction. Figure 2As shown, there are no characteristic diffraction peaks of the raw material CEL and the polymer materials C and P on the surface of CEL@CP. The calculation shows that the crystallinity of CEL@CP, C+P and CEL are 9.15%, 36.22% and 95.29%, respectively, indicating that CEL is well encapsulated in the polymer material in an amorphous state.

[0110] A transmission electron microscope equipped with an energy dispersive X-ray spectrometer was used to characterize the images of single fibers of CEL@CP and CP using F, N, and S elements that are not contained in chitosan and polyethylene oxide as loading indicators. The scale bar is 400 nm, and Merge indicates the result of fusion display. Figure 3 It can be seen that the F, N, and S elements are evenly distributed in the CEL@CP nanofibrous scaffolds, further revealing the successful loading of CEL in CEL@CP, while there is no obvious distribution of the corresponding elements in the nanofibrous scaffolds in CP.

[0111] According to step 1 of Example 1, CEL, C and P in the preparation process of the electrospun scaffold body are adjusted according to the parameters in Table 2, and the same result can be obtained, that is, a stable electrospun scaffold body in which CEL is encapsulated in CP in an amorphous state can be obtained, and CEL can be continuously released.

[0112] Table 2

[0113] Mass of CEL (mg) 60 72 100 1000 800 Mass of C (mg) 120 720 1000 5000 6900 Mass of P (mg) 180 144 240 3000 10300

[0114] Figure 4 The scanning electron microscopy and transmission electron microscopy results show that CP and CEL@CP have smooth surfaces. In contrast, the CEL / DOX-MPs@CP obtained in step three of Example 1 shows a rough surface, where the scale is 2μm. This indicates that DOX-MPs may be adsorbed on the surface of the scaffold and in the gaps between CEL@CP microfibers. Among them, the average diameters of CP, CEL@CP and CEL / DOX-MPs@CP are 412.8±83.1, 438.7±86.1 and 411.1±93.4nm, respectively, and the porosities are 57.8%, 58.2% and 44.4%, respectively. The reduction in the porosity of CEL / DOX-MPs@CP confirms that DOX-MPs are effectively loaded into multiple gaps in CEL@CP.

[0115] According to step 3 of Example 1, the immersion time was adjusted between 1 hour and 10 hours, and the same results as above were detected, that is, DOX-MPs were effectively loaded onto CEL@CP and into multiple gaps of CEL@CP.

[0116] The results of laser confocal imaging were used to characterize Comparative Example 2, which confirmed the presence of Cy5.5 and DOX-MPs fluorescence in Cy5.5 / DOX-MPs@CP. Figure 5 As shown, the scale bar is 10 μm, which further proves the effective loading of CEL and DOX-MPs by CP.

[0117] To evaluate the in vivo degradation of CEL / DOX-MPs@CP, 2.5 mg of CEL / DOX-MPs@CP was surgically implanted into the tumor resection cavity of BALB / c mice. At the specified time intervals, CEL / DOX-MPs@CP was taken out, washed with PBS and vacuum dried at 50°C for 4 hours, and then its residual weight was measured. The results showed that CEL / DOX-MPs@CP was completely degraded within 35 days after implantation into the tumor resection cavity of BALB / c mice, indicating that CEL / DOX-MPs@CP is biodegradable.

[0118] At different time intervals, the skin and muscle tissues around the implanted CEL / DOX-MPs@CP were taken for H&E staining to detect its biocompatibility. The results showed that no obvious signs of inflammation and tissue necrosis were observed in the skin and muscle tissues around the CEL / DOX-MPs@CP-implanted mice, indicating good biocompatibility.

[0119] Verification Example 3 In vitro release of CEL / DOX-MPs@CP

[0120] 2.5 mg CEL / DOX-MPs@CP and 2.5 mg CEL / DOX-MPs@mPPPE were placed in 3 mL PBS solution and shaken at 37 °C and 100 rpm. The release solution was taken at the specified time point, lysed with 10% sodium dodecyl sulfate, and then centrifuged at 9600 g for 10 minutes. Finally, the concentrations of CEL and DOX-MPs in the supernatant were detected by high performance liquid chromatography, and the release of CEL and DOX-MPs was calculated, as shown in Figure 2. Figure 6 (Left) As shown. In addition, 1 mL of DOX-MPs was added to a 14 kDa dialysis bag, then immersed in 25 mL of PBS solution, and shaken at 37 °C and 100 rpm. The concentration of DOX was detected by HPLC at the specified time point, and the release of DOX was calculated. The results are shown in Figure 6 (right) shown.

[0121] The experimental results showed that about 75% of DOX-MPs were released from CEL / DOX-MPs@CP within 24 hours, which helped to effectively kill residual tumor cells. The release of DOX from DOX-MPs was only 18% within 24 hours. CEL was released from CEL / DOX-MPs@CP by burst release of about 32.4% within the first 12 hours, and then continuously released in a controlled manner within the next 7 days. The initial 12-hour burst release can meet the demand for high-concentration CEL due to the excessive release of catecholamines and prostaglandins during the perioperative period, while the subsequent sustained release of the drug during the sustained release phase meets the demand for sustained CEL in the tumor microenvironment.

[0122] However, the release of DOX-MPs in CEL / DOX-MPs@mPPPE was poor because CEL@mPPPE failed to effectively load DOX-MPs. The release of CEL was also poor, as shown by the maximum release of CEL within 96 hours being less than 5%.

[0123] Verification example 4CEL / DOX-MPs@CP in vitro cytotoxicity study on 4T1 cells

[0124] To evaluate the in vitro biological function of CEL / DOX-MPs@CP, 4T1 cells were cultured at 1×10 3 The cells were inoculated into a 96-well plate at a density of 10 cells per well, with 5 cells per group in parallel. 4T1 cells were incubated with 200 μL of CP release solution, CEL@CP release solution, DOX-MPs@CP release solution, CEL / DOX-MPs@CP release solution, CEL solution, DOX solution, and DOX-MPs solution for 24 hours. Then, 20 μL of CCK-8 solution was added and incubated for an appropriate time according to the instructions. The absorbance of each well was detected by an ELISA instrument, and the PBS co-incubation group was taken as 100% survival rate. Figure 7 As shown in the figure, DOX-MPs have a stronger cytotoxic effect on 4T1 cells than DOX, which may be due to the stronger uptake of DOX-MPs by 4T1 cells. The DOX-MPs@CP group and the CEL / DOX-MPs@CP group showed similar cell viability to the DOX-MPs group, indicating that loading CEL into CP does not affect the biological activity of DOX-MPs. No obvious cytotoxicity was detected in the CEL group and the CEL@CP group.

[0125] Verification Example 5: Study on the function of CEL / DOX-MPs@CP on inflammatory PMNs in vitro

[0126] Although PMNs are essential for immune responses against infection, they may have harmful effects and promote tumor occurrence and development when they are stimulated by inflammation and enter the tumor microenvironment and become activated. Inhibiting the activity of inflammatory PMNs is essential for cutting off the interaction between PMNs and tumors. CEL can inhibit the activity of inflammatory PMNs by targeting COX-2 / PGE 2 To evaluate the effect of CEL / DOX-MPs@CP on inflammatory PMNs in vitro, PMNs (1×10 6 The cells were pretreated with 2000 μL of PBS, CEL, DOX, DOX-MPs solution, CEL@CP, DOX-MPs@CP and CEL / DOX-MPs@CP release solution as in Verification Example 4 for 1 hour, and then stimulated with 200 nM phorbol ester (PMA) (a potent inflammatory stimulant) for 4 hours. The supernatant and lysate were collected and the contents of COX-2, iNOS, ARG1 and the activity of MPO were detected using a kit. The results are shown in Figure 8a , Figure 8b As shown. Among them, "PMA+" indicates the group after PMA stimulation in each embodiment and comparative example, PBS indicates the control group treated with PBS only without PMA, and PMA indicates the group treated with PMA only. Figure 8a , Figure 8b It can be seen that PMA significantly increased the content of inflammatory mediators COX-2, iNOS, ARG1 and the activity of MPO in PMNs, which are enzymes that can be expressed by PMNs and will trigger tumor growth and promotion. It can be seen that treatment with DOX, DOX-MPs or DOX-MPs@CP did not significantly inhibit the production of these inflammatory mediators in PMNs. In contrast, CEL, CEL@CP and CEL / DOX-MPs@CP significantly reduced the increase in inflammatory mediators induced by PMA, indicating that CEL / DOX-MPs@CP can effectively inhibit the function of inflammatory PMNs. In addition, PMNs are known to produce intracellular ROS, which leads to oxidative stress, DNA damage and activation of signaling pathways that support tumor occurrence and development.

[0127] To determine the level of ROS, PMNs freshly isolated from the femur and tibia of BALB / c mice were pretreated with the same PBS, CEL, DOX, DOX-MPs solution, CEL@CP, DOX-MPs@CP and CEL / DOX-MPs@CP release solution as in Verification Example 4 for 1 hour, and then stimulated with 200nM PMA for 30 minutes. PMNs were collected and suspended in serum-free RPMI 1640 medium containing 2.5μM DCFH-DA fluorescent probe and incubated for 10 minutes. The intracellular DCF fluorescence intensity was detected by flow cytometry, and the results are shown in Figure 2. Figure 8c As shown, "PMA+" represents the group after PMA stimulation in each embodiment and comparative example, PBS represents the control group treated with PBS only without PMA, and PMA represents the group treated with PMA only. As can be seen from the figure, CEL, CEL@CP and CEL / DOX-MPs@CP consistently and effectively eliminated the production of intracellular ROS in PMNs induced by PMA, further proving that CEL / DOX-MPs@CP can inhibit the function of inflammatory PMNs.

[0128] In addition to inflammatory mediators and ROS, PMNs can also release NETs, ​​which promote tumor progression and metastasis by capturing circulating tumor cells and helping them attach to distal organs. To determine whether CEL / DOX-MPs@CP affects the production of NETs by inflammatory PMNs, PMNs were pretreated with the same PBS, CEL, DOX, DOX-MPs solution as in Verification Example 4, as well as CEL@CP, DOX-MPs@CP and CEL / DOX-MPs@CP release solution for 1 hour, and then stimulated with 200nM PMA for 4 hours, followed by the addition of 2 drops / mL Sytox Green (a cell-impermeable dye that emits green fluorescence after binding to DNA released by PMNs). After 30 minutes, the fluorescence intensity of Sytox Green was quantitatively measured using a multifunctional microplate reader and observed using a laser confocal microscope. Figure 8d As shown, "PMA+" indicates the group after PMA stimulation in each embodiment and comparative example, PBS indicates the control group treated with PBS only without PMA, and PMA indicates the group treated with PMA only. It can be seen that the Sytox Green fluorescence intensity of PMNs treated with PMA was significantly increased, while the Sytox Green fluorescence triggered by PMA was significantly inhibited by CEL, CEL@CP or CEL / DOX-MPs@CP treatment, but not DOX, DOX-MPs and DOX-MPs@CP treatment. Figure 8e(Left) Semi-quantitative statistics of the fluorescence area ratio of each group of laser confocal images further proved the decrease in the fluorescence intensity of Sytox Green in the CEL / DOX-MPs@CP group. Figure 8e As shown in (right), CEL / DOX-MPs@CP significantly reduced the expression of NETs-specific marker H3Cit, further confirming that CEL / DOX-MPs@CP effectively inhibited the production of NETs in inflammatory PMNs.

[0129] Verification Example 6 Study on the effect of CEL / DOX-MPs@CP intervention of inflammatory PMNs on tumor cell proliferation, migration and invasion in vitro

[0130] The interaction between PMNs and tumors can promote the proliferation, migration and invasion of tumor cells. To investigate the effect of CEL / DOX-MPs@CP-induced inhibition of inflammatory PMNs function on tumor cell viability, 4T1 cells were co-incubated with the same PBS, CEL, DOX, and DOX-MPs solutions as in Verification Example 4, as well as the supernatant of inflammatory PMNs (PMNs stimulated with 200nM PMA for 4 hours) pretreated with CEL@CP, DOX-MPs@CP, or CEL / DOX-MPs@CP release solution, and then the viability of 4T1 cells was determined by the CCK-8 method. Figure 9a (Left), wherein "+PMA" indicates the group after PMA stimulation was added in each embodiment and comparative example, and No PMNs indicates the control group containing only PBS buffer. The inflammatory PMNs supernatant stimulated by PMA significantly increased the cell survival rate of 4T1. The inflammatory PMNs supernatant pretreated with DOX, DOX-MPs or DOX-MPs@CP had no significant effect on the proliferation of 4T1 cells promoted by inflammatory PMNs. On the contrary, compared with the inflammatory PMNs supernatant-treated group, the survival rate of 4T1 cells was significantly reduced after treatment with the inflammatory PMNs supernatant pretreated with CEL, CEL@CP or CEL / DOX-MPs@CP, indicating that CEL / DOX-MPs@CP can effectively inhibit the proliferation of 4T1 cells promoted by inflammatory PMNs.

[0131] To further evaluate the potential inhibitory effect of CEL / DOX-MPs@CP on the migration and invasion of inflammatory PMNs, 4T1 cells were first cultured in scratch molds, and then PBS, CEL, DOX, DOX-MPs solution, and supernatants of inflammatory PMNs pretreated with CEL@CP release solution, DOX-MPs@CP release solution, and CEL / DOX-MPs@CP release solution were co-incubated with 4T1 cells for wound healing experiments. Figure 9a(right), wherein "+PMA" indicates the group after PMA stimulation was added in each embodiment and comparative example, and No PMNs indicates the control group containing only PBS buffer. The supernatant of PMNs stimulated with PMA significantly promoted the migration of 4T1 cells, and the wounds of the PMNs+PMA treatment group were significantly closed after 24 hours. Pretreatment with DOX, DOX-MPs and DOX-MPs@CP release solution had no obvious inhibitory effect on the migration of 4T1 cells induced by inflammatory PMNs, while the wounds of the CEL, CEL@CP release solution or CEL / DOX-MPs@CP release solution treatment groups still had large gaps, indicating that CEL / DOX-MPs@CP has an effective inhibitory effect on the migration of 4T1 cells induced by inflammatory PMNs. In addition, as Figure 9b Transwell experiments on invasion and migration also confirmed that CEL / DOX-MPs@CP can inhibit the migration and invasion of 4T1 cells induced by inflammatory PMNs.

[0132] NETs released by PMNs have a significant effect on the proliferation and metastasis of tumor cells. Since CEL / DOX-MPs@CP significantly inhibited the release of NETs from PMNs, it is speculated that NETs may be involved in the CEL / DOX-MPs@CP-mediated inhibition of the proliferation, migration and invasion of 4T1 cells induced by inflammatory PMNs (PMNs stimulated with 200nM PMA for 4 hours). To verify this speculation, PMNs pretreated with PBS or CEL / DOX-MPs@CP release solution and stimulated with PMA for 4 hours were treated with 150U / mL DNase I (a recognized inhibitor of NETs formation) for 20 minutes; or NETs pretreated with PBS or CEL / DOX-MPs@CP release solution and harvested after 4 hours of PMA stimulation were treated with 150U / mL DNase I for 20 minutes, respectively, and acted on 4T1 cells. As expected, NETs obtained from inflammatory PMNs treated with PBS significantly promoted the following: Fig.9c (Left) The proliferation of 4T1 cells is shown. Fig.9c (right) and Figure 9d (Left) The migration of 4T1 cells and Figure 9d (Right) shows the invasion diagram of 4T1 cells, in which NET ()Indicates the group that underwent the corresponding treatment in the brackets and harvested NETs, ​​"+PMA" indicates the group after PMA stimulation in each embodiment and comparative example, "+DNase I" indicates the group after DNase I was added in each embodiment and comparative example, and No PMNs indicates the control group containing only PBS buffer. The supernatant of inflammatory PMNs stimulated with PMA significantly increased the cell survival rate of 4T1. Similar to the supernatant obtained by treating inflammatory PMNs with PBS, it shows that NETs play an important role in regulating the proliferation, migration and invasion of 4T1 cells. Although DNase I treatment significantly reduced the proliferation, migration and invasion of 4T1 cells promoted by the supernatant of inflammatory PMNs treated with PBS and NETs, ​​it had no significant effect on the supernatant obtained from inflammatory PMNs pretreated with CEL / DOX-MPs@CP and the above functions of 4T1 cells induced by NETs, ​​such as Fig.9c and Figure 9d As shown, it was shown that CEL / DOX-MPs@CP was involved in preventing the proliferation, migration and invasion of 4T1 cells by inhibiting the generation of NETs.

[0133] Verification Example 7 Study on the retention of CEL / DOX-MPs@CP at the site of surgical resection of breast cancer in mice

[0134] An ideal in situ implant requires that the drug be retained continuously at the target site. To evaluate the sustained drug retention ability of CEL / DOX-MPs@CP at the tumor resection site, the Cy5.5 / IR780-MPs@CP of comparative example 3 was used for testing. When the volume of the orthotopic 4T1 breast tumor in BALB / c mice reached about 300 mm 3 90% of the breast tumor was surgically removed. Then, 2.5 mg of Cy5.5 / IR780-MPs@CP fiber scaffold of Comparative Example 3 was placed into the surgical resection cavity, and 50 μL of Cy5.5 / IR780-MPs suspension with an equivalent dose was also placed into the surgical resection cavity. Fig.10a It can be seen that the fluorescence intensity of Cy5.5 and IR780 in mice treated with Cy5.5 / IR780-MPs and Cy5.5 / IR780-MPs@CP decreased over time. However, compared with the Cy5.5 / IR780-MPs group, the fluorescence intensity of Cy5.5 and IR780 in the Cy5.5 / IR780-MPs@CP group decreased more slowly. In addition, on the 19th day after treatment, the in vitro fluorescence of major organs Fig.10bThe analysis results showed that there was significant accumulation of Cy5.5 and IR780 fluorescence in the tumor tissue of the Cy5.5 / IR780-MPs@CP group, while only a small amount of fluorescence was observed in other major organs. Compared with the Cy5.5 / IR780-MPs group, the Cy5.5 / IR780-MPs@CP group mice had a significant increase of 1.8 times in the accumulation of Cy5.5 and 4.4 times in the accumulation of IR780 in the tumor tissue, indicating that Cy5.5 / IR780-MPs@CP has a strong retention ability at the tumor resection site.

[0135] Verification Example 8 Investigation of the anti-tumor effect of CEL / DOX-MPs@CP on breast cancer in mice after surgical resection

[0136] Considering that CEL / DOX-MPs@CP can effectively inhibit the function of inflammatory PMNs and maintain a certain amount of drug accumulation in tumor tissues, we evaluated its effect of inhibiting postoperative tumor recurrence and metastasis in an orthotopic 4T1 breast cancer-bearing BALB / c mouse model with spontaneous metastasis. 3 At the same time, 90% of the tumor was surgically removed, and 2.5 mg of fiber scaffolds CP, CEL@CP, DOX-MPs@CP or CEL / DOX-MPs@CP (the loading amount of CEL on the scaffold was 7.2 mg / kg, and the loading amount of DOX on the scaffold was 1.8 mg / kg) were implanted in the surgical resection cavity, and 50 μl of CEL, DOX, DOX-MPs, CEL / DOX-MPs solutions and PBS solutions with the same loading amount of CEL and DOX on the above fiber scaffolds were given at the same time. Fig.11a as well as Fig.11b As shown in (left), mice treated with PBS, CP or CEL had rapid tumor recurrence after tumor resection and showed similar tumor growth curves. Although DOX had a rather limited effect in inhibiting tumor recurrence, DOX-MPs and CEL / DOX-MPs showed a certain anti-tumor recurrence ability, which may be due to the fact that DOX-MPs can penetrate tumor cells more effectively and deeply. In the DOX-MPs@CP-treated group, the stronger inhibitory effect may be attributed to the sustained drug retention in the surgical wound promoted by CP. CEL / DOX-MPs@CP showed the strongest ability to inhibit tumor recurrence after tumor resection, with a 91% reduction in tumor volume and an 84% reduction in tumor weight compared with the PBS-treated group. Histopathological observations were performed using H&E staining and semi-quantification was performed using Ki67 immunohistochemical staining, as shown in Figure 2. Fig.11b (Right) The results showed that the proliferation of tumor cells in the CEL / DOX-MPs@CP treatment group was minimal, which confirmed that CEL / DOX-MPs@CP has the ability to significantly inhibit tumor recurrence after surgery.

[0137] like Fig.12a (Left), Figure 12b As shown in Figure 2, significantly fewer metastatic nodules were observed in the lungs of mice treated with CEL / DOX-MPs@CP, indicating that CEL / DOX-MPs@CP can effectively inhibit postoperative lung metastasis in 4T1 tumor-bearing mice. Fig.12a As shown in (right), CEL / DOX-MPs@CP treatment significantly prolonged the postoperative survival time of 4T1 tumor-bearing mice, and 63% of the mice were still alive on the 40th day after surgery, which was significantly longer than the survival time of other groups, further confirming that CEL / DOX-MPs@CP has good anti-cancer activity when implanted in situ after tumor resection. Histopathological observation of major organs, blood biochemical analysis, and body weight changes showed that CEL / DOX-MPs@CP did not cause obvious adverse reactions, indicating that CEL / DOX-MPs@CP has good biocompatibility.

[0138] Verification Example 9 Investigation of the regulatory effect of CEL / DOX-MPs@CP on the immune microenvironment after surgical resection of breast cancer in mice

[0139] To explore the potential mechanism of CEL / DOX-MPs@CP in inhibiting postoperative tumor recurrence and metastasis, the tumor microenvironment of orthotopic 4T1 breast cancer-bearing BALB / c mice was evaluated after being treated with the same method as Validation Example 8. 2.5 mg of fiber scaffolds CEL@CP, DOX-MPs@CP or CEL / DOX-MPs@CP (the loading amount of CEL on the scaffold was 7.2 mg / kg, and the loading amount of DOX was 1.8 mg / kg) were implanted in the surgical resection cavity, and 50 μl of CEL, DOX, DOX-MPs, CEL / DOX-MPs solutions and PBS solutions with the same CEL and DOX loading amounts on the above-mentioned fiber scaffolds were given at the same time. On the 19th day after surgery, the proportion of PMNs in the tumor tissue was evaluated by flow cytometry. Fig.13a As shown in (left), compared with PBS alone, treatment with DOX, DOX-MPs, or DOX-MPs@CP did not significantly change the proportion of PMNs in tumor tissue. In contrast, the proportion of PMNs was significantly reduced after treatment with CEL@CP or CEL / DOX-MPs@CP, indicating that CEL@CP or CEL / DOX-MPs@CP had a significant inhibitory effect on the number of PMNs in the resection cavity. The reduction in the number of PMNs in the CEL / DOX-MPs@CP treatment group was further confirmed by immunofluorescence staining, as shown in Figure 2. Fig.13a(Right) As shown, the expression level of PMNs marker Ly6G was low. At the same time, CEL / DOX-MPs@CP also significantly reduced the expression of H3Cit, a key player in PMNs release in NETs, ​​which confirmed that CEL / DOX-MPs@CP can effectively inhibit the generation of NETs, ​​such as Fig.13b (Left) Similarly, the expression level of COX-2 was significantly reduced in mice treated with CEL / DOX-MPs@CP, as shown in Fig.13b (right) As shown. The expression of VEGF regulated by COX-2 was also significantly reduced in the CEL / DOX-MPs@CP treatment group. Fig.13c (Left) This indicates that implantation of CEL / DOX-MPs@CP may reduce the formation of tumor angiogenesis and thus inhibit tumor recurrence and metastasis.

[0140] PMNs create an immunosuppressive microenvironment, inhibiting the function of cytotoxic T cells and other immune cells, thereby enabling tumor cells to escape the attack of the immune system. Flow cytometry analysis showed that CD8 + The percentage of T cells was significantly upregulated, indicating that DOX-MPs-induced chemotherapy can trigger anti-tumor immune response. CEL@CP can also significantly increase the CD8 + The percentage of T cells in the tumor microenvironment was significantly increased after CEL / DOX-MPs@CP treatment, indicating that the inhibition of PMNs function induced by CEL may help enhance anti-tumor immunity. + The proportion of T cells has been further improved compared with the previous period. Fig.13c (right). In contrast, MDSCs, such as Fig.13d (Left) and Tregs, as shown Fig.13d (right) The percentage of immunosuppressive cells, including 100 μg / mL, was significantly reduced in the CEL / DOX-MPs@CP treatment group. The above data indicate that CEL / DOX-MPs@CP effectively improves the tumor immunosuppressive microenvironment and may help to effectively inhibit tumor recurrence and metastasis in 4T1 tumor-bearing mice undergoing surgical resection.

[0141] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. An anti-tumor electrospun scaffold, characterized in that: It comprises an electrospun stent body and tumor cell-derived drug-loaded microparticles loaded thereon; the electrospun stent body comprises an amino polymer, a hydrophilic polymer and celecoxib in a mass ratio of (2-10):(2-13):1; the loading amount of the tumor cell-derived drug-loaded microparticles on the anti-tumor electrospun stent is 0.6 μg / mg to 6.1 μg / mg.

2. The anti-tumor electrospun scaffold according to claim 1, characterized in that: The amino polymer is chitosan, chitin, cellulose, silk, collagen, gelatin, dextran, polyamino acid, chondroitin sulfate or heparin.

3. The anti-tumor electrospun scaffold according to claim 1, characterized in that: The hydrophilic polymer is polyethylene oxide, polyvinyl pyrrolidone or polyvinyl alcohol.

4. The anti-tumor electrospun scaffold according to claim 1, characterized in that: The tumor cell-derived drug-loaded microparticles include microparticles released from tumor cells when the cytoskeleton is changed during activation or apoptosis, and chemotherapy drugs encapsulated in the microparticles; the tumor cells are liver cancer cells, breast cancer cells or melanoma cells.

5. The anti-tumor electrospun scaffold according to claim 4, characterized in that: The chemotherapy drugs are anthracyclines, pyrimidines, folic acid antagonists, metal complexes or diterpenes.

6. The anti-tumor electrospun scaffold according to claim 1, characterized in that: The mass ratio of the amino polymer to the hydrophilic polymer is 5:1 to 4:

6.

7. The method for preparing the anti-tumor electrospun scaffold according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Obtaining an electrospun stent body by electrospinning; the solute of the electrospinning solution used for the electrospinning comprises an amino polymer, a hydrophilic polymer and celecoxib in a mass ratio of (2 to 10): (2 to 13): 1; S2. Soak the electrospun scaffold body in a solution of 10 μg / mL to 200 μg / mL tumor cell-derived drug-loaded microparticles for 1 hour to 10 hours, so that the tumor cell-derived drug-loaded microparticles are adsorbed on the electrospun scaffold body by electrostatic adsorption, thereby obtaining the anti-tumor electrospun scaffold.

8. The preparation method according to claim 7, characterized in that: In the step S1, the mass fraction of the electrospinning solution is 3% to 60%, and the solvent of the electrospinning solution is organic acid, hexafluoroisopropanol, dimethyl sulfoxide or dichloromethane.

9. The preparation method according to claim 8, characterized in that: The mass fraction of the electrospinning solution is 3% to 10%.

10. The preparation method according to claim 7, characterized in that: In the step S2, the concentration of the solution of tumor cell-derived drug-loaded microparticles is 50 μg / mL to 100 μg / mL, and the immersion time is 4 hours to 6 hours.

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