Anti-tumor electrospun scaffolds and methods of making the same

Abstract

The application belongs to the technical field of medical devices, and specifically discloses an anti-tumor electrospun scaffold and a preparation method thereof. The anti-tumor electrospun scaffold comprises a drug-loaded electrospun scaffold body and tumor cell-derived drug-loaded micro-particles loaded on the surface of the scaffold body. The drug-loaded electrospun scaffold body comprises amino polymers, hydrophilic polymers and celecoxib with a mass ratio of (2-10):(2-13):1, which are prepared by using an electrospinning technology, and has a high specific surface area and a high porosity. The loading amount of the tumor cell-derived doxorubicin-loaded micro-particles on the anti-tumor electrospun scaffold is 0.6 μg / mg-6.1 μg / mg. The drug is directly delivered to a tumor resection cavity in the form of in-situ implantation, long-term drug retention, continuous drug release and complete degradation of the scaffold material are achieved. Residual tumor cells are effectively killed, the tumor immune microenvironment is reshaped, and the recurrence and metastasis of the tumor are inhibited.

Description

Technical Field

[0001] This application belongs to the field of medical device technology, and more specifically, relates to an anti-tumor electrospinning scaffold and its preparation method. Background Technology

[0002] While surgical removal of tumors has some effectiveness, residual tumor cells limit treatment options due to the risk of recurrence and metastasis. Adjuvant therapies such as chemotherapy aim to eliminate these cells, but often produce severe side effects, limiting patient survival benefits. Therefore, there is an urgent need for innovative postoperative methods to eliminate residual tumor cells, inhibit tumor recurrence and metastasis, and reduce adverse reactions.

[0003] Besides residual tumor cells, the tumor microenvironment altered by perioperative trauma also significantly impacts tumor recurrence and metastasis. Inflammation, particularly surgically induced inflammation, plays a crucial role in cancer treatment. Surgical interventions can trigger complex inflammatory responses, typically leading to the recruitment of immune cells such as polymorphonuclear neutrophils (PMNs). PMNs, as key participants in acute inflammation, exhibit a dual role in cancer treatment. On one hand, they contribute to tissue repair, fight infection, and aid in the early stages of wound healing. However, on the other hand, they can inadvertently promote tumor progression by releasing pro-tumorigenic factors, such as various cytokines, chemokines, prostaglandin E2, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs—a network of DNA, histones, and granule proteins), creating a supportive microenvironment for cancer cells. These factors released by PMNs can promote angiogenesis, tissue remodeling, and immune escape, thus promoting cancer cell survival and growth. Furthermore, NETs can trap circulating tumor cells, promoting their adhesion to distant organs and facilitating metastasis. The complex interactions between inflammation, tumor microenvironments (PMNs), and cancer highlight 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] Inflammatory responses mediated by PMNs participate in multiple signaling pathways, influencing tumor growth. Cyclooxygenase-2 (COX-2) is a key rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandins (including prostaglandin E2) in response to inflammatory signals, playing a crucial role in the regulation of tumor-associated inflammation, angiogenesis, immune escape, and metastasis. Therefore, targeting the COX-2 / prostaglandin E2 signaling pathway may offer hope for improving inflammation and cancer treatment. Celecoxib (CEL), a nonsteroidal anti-inflammatory drug, is a selective COX-2 inhibitor, belonging to type II in the Biopharmaceutics Classification System (BCS), exhibiting high permeability and near-insoluble in water. Marketed solid oral formulations (capsules) developed using dissolution-enhancing technology can improve the properties of CEL to some extent. Clinically, it is used to treat pain and inflammation associated with diseases such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. In addition, oral CEL is being used by researchers as adjuvant therapy before tumor resection. However, data from randomized controlled trials and retrospective case-control studies show that CEL has limited efficacy as an adjuvant therapy before radical surgery. This may be because CEL carries the potential risk of serious gastrointestinal and cardiovascular thrombotic events in clinical applications. For safety reasons, this method focuses on prevention and uses the minimum dose, which cannot effectively counteract the large release of prostaglandins during the perioperative period. Even with relatively small doses, the risk of serious adverse events cannot be avoided.

[0005] In existing technologies, electrospinning is typically used to mix polymeric materials such as polylactic acid and gelatin with tumor therapeutic drugs to obtain electrospun scaffolds for in vivo treatment. These scaffolds also physically isolate the tumor resection site from the peritoneum, preventing postoperative adhesions. However, using electrospun scaffolds alone faces challenges such as limited drug accumulation at the tumor site and poor deep penetration. Microparticles, vesicle-like subcellular structures ranging in size from 100 nm to 1000 nm, are released when cells undergo cytoskeleton changes during activation or apoptosis. Their good biocompatibility, homologous targeting, and low immunogenicity make them a highly promising drug delivery carrier. However, using microparticles alone faces problems such as difficulty in drug retention within the tumor resection cavity and low utilization rates. Furthermore, tumor cell-derived drug-loaded microparticles cannot be directly used as electrospinning materials and are difficult to combine with electrospun scaffolds, resulting in limited drug binding and affecting their efficacy. Chemical or biological surface modifications to electrospun scaffolds can sometimes enhance their interaction with other components, but these still face challenges such as complex preparation processes and increased toxicity. Summary of the Invention

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

[0007] To achieve the above objectives, this application provides an antitumor 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 (2-10):(2-13):1; the tumor cell-derived drug-loaded microparticles are loaded on the antitumor electrospun scaffold at a loading amount of 0.6 μg / mg to 6.1 μg / mg.

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

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

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

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

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

[0013] This application also provides a method for preparing the above-mentioned antitumor electrospinning scaffold, including the following steps:

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

[0015] S2. The electrospun scaffold body is immersed in a solution of tumor cell-derived drug-loaded microparticles at a concentration of 10 μg / mL to 200 μg / mL for 1 hour to 10 hours, so that the tumor cell-derived drug-loaded microparticles are adsorbed onto the electrospun scaffold body by electrostatic adsorption, thereby obtaining the antitumor 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 an organic acid, hexafluoroisopropanol, dimethyl sulfoxide, or dichloromethane.

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

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

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

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

[0021] In summary, compared with the prior art, the technical solutions conceived in this application have the following main technical advantages:

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

[0023] This electrospinning scaffold can be directly implanted into the tumor resection site during tumor surgery to eliminate residual tumor cells, prevent tumor recurrence, and inhibit the function of NETs released by inflammatory PMNs after tumor surgery, thereby reshaping the tumor immune microenvironment and inhibiting tumor metastasis.

[0024] Animal experiments have verified that the antitumor electrospinning scaffold of this application can maintain a high concentration of therapeutic level at the lesion site, while maintaining only a low drug level in other tissues or peripheral blood. This reduces damage to other organs, achieves in-situ local drug release, and to a certain extent reduces the occurrence of adverse drug reactions, reduces drug usage, and saves medical costs.

[0025] This application uses chemotherapy microparticles produced by tumor cells for anti-tumor treatment, which solves the problems of drug resistance after the use of chemotherapy drugs and the insensitivity of some tumor cells to chemotherapy drugs.

[0026] Verification has shown that the electrospun nanofiber scaffold used in this application has a strong anti-inflammatory effect. The drug molecule CEL encapsulated in the scaffold is inexpensive 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, eliminating the need for secondary surgery for removal. It completely degrades within 35 days after intratumoral implantation in BALB / c mice following tumor resection.

[0028] The preparation method used in this application, electrospinning technology and electrostatic adsorption method for loading chemotherapy microparticles, is applicable to a variety of polymer materials and drug-loaded microparticles, and does not require modification of the electrospinning scaffold body, with few toxic side effects and simple process. Attached Figure Description

[0029] Figure 1 A schematic diagram illustrating the effect of implanted electrospinning scaffold CEL / DOX-MPs@CP on anti-tumor recurrence and metastasis;

[0030] Figure 2 The image shows the crystal structure analysis of CEL@CP obtained in step one of the examples using powder diffraction.

[0031] Figure 3 The X-ray energy dispersive spectrum of CEL@CP obtained in step one of the embodiments is shown in the figure, with the scale bar at 400 nm.

[0032] Figure 4 The images show 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. The scale bar in the images is 2 μm.

[0033] Figure 5 The image shows a laser confocal image of Cy5.5 / DOX-MPs@CP in Comparative Example 3, with a scale bar of 10 μm.

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

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

[0036] Figure 8a The graph shows the effects of each embodiment and comparative example on the in vitro content of cyclooxygenase-2 (COX-2) (left) and inducible nitric oxide synthase (iNOS) (right) in inflammatory PMNs.

[0037] Figure 8b The graph shows the effects of each embodiment and comparative example on the in vitro content of arginase 1 (ARG1) and myeloperoxidase (MPO) activity in inflammatory PMNs (left).

[0038] Figure 8cThe graph shows the effect of DCF average fluorescence intensity on inflammatory PMNs in vitro for each embodiment and comparative example;

[0039] Figure 8d The graph 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 graph shows the effects of each embodiment and comparative example on the extracellular DNA area ratio (left) and citrullinated histone H3 (H3Cit) area ratio (right) of inflammatory PMNs in vitro.

[0041] Figure 9a The effect of in vitro intervention of inflammatory PMNs on the survival rate (left) and relative scratch width (right) of 4T1 cells in each embodiment and comparative example;

[0042] Figure 9b The diagram shows the effects of in vitro intervention of inflammatory PMNs on the migration (left) and invasion (right) of 4T1 cells in each embodiment and comparative example;

[0043] Figure 9c The effect of in vitro intervention of inflammatory PMNs on the survival rate (left) and relative scratch width (right) of 4T1 cells in each embodiment and comparative example;

[0044] Figure 9d The diagram shows the effects of in vitro intervention of inflammatory PMNs on the migration (left) and invasion (right) of 4T1 cells in each embodiment and comparative example.

[0045] Figure 10a The graphs show the retention curves of each embodiment and comparative example at the tumor resection site in mouse breast cancer; the left graph shows the total fluorescence intensity at Cy5.5, and the right graph shows the total fluorescence intensity at IR780.

[0046] Figure 10b The images show the in vitro fluorescence quantitative spectroscopy of tumors and major organs in mice on day 19 after tumor resection for each embodiment and comparative example; the left image shows the average fluorescence intensity at Cy5.5, and the right image shows the average fluorescence intensity at IR780.

[0047] Figure 11a The left image shows the tumor suppression curves after resection of breast cancer tumors in mice of each embodiment and comparative example, and the right image shows the tumor on day 19.

[0048] Figure 11b The images show the tumor suppression effects of each embodiment and comparative example on day 19 after mammary tumor resection in mice. The left image shows tumor weight, and the right image shows Ki67. + Cell proportion diagram;

[0049] Figure 12aThe left image shows the number of lung metastatic nodules and survival rate of each embodiment and comparative example on day 19 after tumor resection of breast cancer in mice.

[0050] Figure 12b Images of the lungs of each embodiment and comparative example on day 19 after resection of breast cancer tumors in mice;

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

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

[0053] Figure 13c The left image shows the area ratio (left) of vascular endothelial growth factor (VEGF) and CD8 in the tumor microenvironment of mice 19 days after tumor resection for each embodiment and comparative example. + The influence of T cell proportion (right) diagram;

[0054] Figure 13d The diagram shows the effects of each embodiment and comparative example on the proportion of myeloid-derived suppressor cells (MDSCs) (left) and regulatory T cells (Tregs) in the tumor microenvironment on day 19 after mastectomy in mice. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0056] Furthermore, throughout this specification, references to "an embodiment"; "an embodiment," "an example," or similar language indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this application. Therefore, the appearance of the phrase "in one embodiment;" throughout this specification, and similar language, may, but not necessarily, refer to the same embodiment.

[0057] Addressing the shortcomings of existing technologies and considering the problems of recurrence and metastasis after tumor surgery, as well as the adverse reactions and tumor drug resistance caused by intravenous injection of CEL and chemotherapy drugs, this application utilizes electrospinning technology to prepare an anti-tumor electrospun scaffold that co-delivers CEL and tumor cell-derived drug-loaded microparticles for direct implantation at the tumor resection site during tumor surgery. This in-situ implantation material ensures long-term retention and continuous release of drugs within the tumor resection cavity after surgery. The released tumor cell-derived drug-loaded microparticles are effectively internalized, eliminating 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 remodeling the tumor immune microenvironment. These effects are related to the disruption of the function of NETs released by inflammatory PMNs. The combination of these characteristics makes this composite material an effective strategy for inhibiting postoperative tumor recurrence and metastasis.

[0058] The preparation method of this anti-tumor electrospinning scaffold includes the following steps:

[0059] S1. An electrospun scaffold body is obtained by electrospinning, forming a three-dimensional spatial structure composed of nanofibers with a diameter of 100nm to 1000nm; the mass fraction of the electrospinning solution used in the electrospinning is 3% to 60%; its 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 amino polymers to hydrophilic polymers is 5:1 to 4:6. When the proportion of amino polymers is too high, it may lead to poor CEL release, while when the proportion of hydrophilic polymers is too high, the structure of the electrospun scaffold is not stable enough; the solvent is an organic acid, hexafluoroisopropanol, dimethyl sulfoxide, or dichloromethane; since the specific surface area of ​​nanofibers is 1000 times that of microfibers, the specific surface area of ​​the nanofiber scaffold composed of nanofibers will be several orders of magnitude larger than that of fiber scaffolds prepared by ordinary spinning technology;

[0060] S2. In an ambient environment of 4℃ to 37℃, the electrospun scaffold body is immersed in a solution of 10μg / mL to 200μg / mL of tumor cell-derived drug-loaded microparticles for 1 hour to 10 hours. Since the amino polymers in the electrospun scaffold are positively charged, the negatively charged tumor cell-derived drug-loaded microparticles can be adsorbed onto the electrospun scaffold body through electrostatic adsorption, thereby obtaining the antitumor 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 amino polymer, hydrophilic polymer and 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 fibroin, collagen, gelatin, dextran, polyamino acids, chondroitin sulfate, or heparin; the hydrophilic polymer is polyethylene oxide, polyvinylpyrrolidone, or polyvinyl alcohol.

[0064] In one embodiment, the tumor cell-derived drug-loaded microparticles in step S2 include microparticles released from the cytoskeleton by tumor cells during activation or apoptosis, and chemotherapeutic drugs encapsulated within the microparticles; wherein the tumor cells are liver cancer cells, breast cancer cells (such as mouse breast cancer cell line 4T1), or melanoma cells; and 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 (e.g., doxorubicin, hereinafter referred to as DOX), pyrimidines (e.g., 5-fluorouracil), folic acid antagonists (e.g., methotrexate), metal complexes (e.g., cisplatin), and diterpenoids (e.g., 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 soaking 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 soaking time is 4 hours, and the pH value of the solution is preferably 7.4.

[0066] The antitumor 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 antitumor electrospun scaffold is 0.6 μg / mg to 6.1 μg / mg; the main component of the electrospun scaffold is 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 aforementioned electrospun scaffold body can be stored in a light-proof glass desiccator after preparation, and the aforementioned tumor cell-derived drug-loaded microparticles can be frozen at -80°C after preparation. The antitumor electrospun scaffold obtained by electrostatic adsorption is freshly prepared before use.

[0068] Figure 1The CEL / DOX-MPs@CP (specific components can be found in Example 1 below) is an anti-tumor electrospinning scaffold in one embodiment. Due to the large specific surface area of ​​the electrospinning scaffold, it is an excellent carrier for enriching drug-loaded microparticles. Implanting this anti-tumor electrospinning scaffold can increase the concentration of local therapeutic drugs. Because CEL / DOX-MPs@CP has a long drug retention time and sustained drug release performance within the tumor resection cavity, it can effectively kill residual tumor cells after surgery. It also has a certain inhibitory effect on the proliferation, invasion, and migration of tumor cells promoted by inflammatory PMNs. Simultaneously, it reshapes the tumor immune microenvironment and increases CD8+ in the tumor microenvironment. + The proportion of T cells was reduced, as were the proportions of MDSCs and Tregs. This effectively inhibited tumor recurrence and metastasis. The CEL / DOX-MPs@CP shown in the middle figure consists of a gray nanofiber scaffold loaded with anti-inflammatory drugs and red chemotherapeutic drug microparticles attached to it. As can be seen from the figure, after tumor resection, residual tumor cells and the altered tumor microenvironment caused by surgical trauma, including enhanced aggregation of inflammatory PMNs, excessive NET formation, and the production of Tregs and MDSCs, promote tumor recurrence and metastasis. NETs, ​​in particular, can capture circulating tumor cells, promoting their adhesion to distant organs and facilitating metastasis. However, after implantation of CEL / DOX-MPs@CP, not only can it rapidly and effectively kill residual tumor cells and inhibit tumor recurrence, but it can also significantly improve the post-tumor resection microenvironment, 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 was increased, thereby inhibiting tumor metastasis.

[0069] Example 1

[0070] I. Preparation of the main body of the electrospinning scaffold

[0071] 360 mg of chitosan (average molecular weight 850,000, degree of deacetylation 95%, hereinafter referred to as C), 360 mg of polyethylene oxide (average molecular weight 600,000, hereinafter referred to as P), and 72 mg of CEL (≥99%, molecular weight 381.38 g / mol, hereinafter referred to as CEL) were dissolved in 12 mL of 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 was 1:1. This polymer solution was placed in the sample injection device of an electrospinning apparatus. The voltage was adjusted to 18 kV, the distance between the spinneret and the collecting plate was adjusted to 15 cm, the working fluid drive speed was adjusted to 1 mL / h, the receiving device rotation speed was adjusted to 30 rpm, and the ambient humidity was controlled to not exceed 30%. Electrospinning was performed to obtain CEL@CP. After electrospinning, high-performance liquid chromatography (HPLC) results 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 hours, and then stored in a light-proof glass desiccator.

[0072] II. Preparation of DOX-MPs

[0073] 4T1 cells were exposed to ultraviolet light (UVB, 300 J / m²). -2 After 1 hour, 200 μg / mL DOX was added and co-incubated with 4T1 cells for 12 hours. The supernatant was centrifuged at 1000g for 10 minutes. The supernatant was then centrifuged at 14000g for 2 minutes to remove cells and debris. The supernatant was then centrifuged at 14000g for 1 hour to obtain DOX-MPs. After washing with PBS, the DOX-MPs PBS solution was stored at -80℃. Atomic force microscopy results showed that DOX-MPs were monodisperse irregular spherical particles. Dynamic light scattering analysis showed that the average particle size of DOX-MPs was approximately 337 nm, and the zeta potential was -8.8 mV. High performance liquid chromatography results showed that the drug loading of DOX-MPs was 0.06 μg DOX per microgram of MPs protein.

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

[0075] Dry 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 DOX-MPs solution with a concentration of 100 μg / mL. At room temperature (25 °C), DOX-MPs were enriched onto the positively charged CEL@CP surface through electrostatic adsorption, yielding CEL / DOX-MPs@CP. Four hours later, the composite scaffold CEL / DOX-MPs@CP was removed for subsequent experiments. High-performance liquid chromatography (HPLC) confirmed that the DOX-MPs loading on the CEL@CP, based on the DOX content, was 6.1 μg DOX / mg CEL@CP.

[0076] Example 2-4: Control of DOX-MPs Load

[0077] Example 1 was repeated using the same steps, except that the concentration of DOX in step two and the final DOX loading on the CEL@CP were as 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 the CEL@CP can reach 6.1 μg / mg. However, when the concentration of DOX in the DOX-MPs solution before immersion is continuously increased, the increase in DOX-MPs loading on the 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 capacity (μg / mg) 0.6 2.9 6.1

[0080] Example 5

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

[0082] Comparative Example 1

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

[0084] Comparative Example 2

[0085] Example 1 was repeated using the same steps, except that in step one, CEL was replaced with a hydrophobic small molecule fluorescent dye Cy5.5 at a mass fraction of 0.0034% in the polymer solution; and in step three, the resulting composite scaffold was Cy5.5 / DOX-MPs@CP. The loading 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 one, CEL was replaced with a hydrophobic small molecule fluorescent dye Cy5.5 at a mass fraction of 0.0034% in the polymer solution; in step two, DOX-MPs were replaced with surface-labeled MPs (i.e., IR780-MPs) of the fluorescent dye IR780; and in step three, the composite scaffold obtained was Cy5.5 / IR780-MPs@CP, with a Cy5.5 loading of 0.4 μg / mg and an IR780 loading of 0.9 μg / mg on the composite scaffold.

[0088] Comparative Example 4

[0089] The same steps as described in Example 1 were repeated, except that 72 mg of CEL was not added to the polymer solution in step one. After step one, CP was obtained, and after drying, it was cut into small round pieces with a diameter of 14 mm and a mass of 2.5 mg. In step three, DOX-MPs@CP was finally obtained.

[0090] Experimental results verification

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

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

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

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

[0095] CEL / DOX-MPs@mPPPE: CEL / DOX-MPs@mPPPE obtained in step three of Comparative Example 1;

[0096] Cy5.5 / DOX-MPs@CP: Cy5.5 / DOX-MPs@CP obtained in step three of Comparative Example 2;

[0097] IR780-MPs: IR780-MPs obtained in step two of Comparative Example 3;

[0098] Cy5.5 / IR780-MPs@CP: Cy5.5 / IR780-MPs@CP obtained in step three of Comparative Example 3;

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

[0100] CP: The CP obtained in step one of Comparative Example 4;

[0101] DOX-MPs@CP: DOX-MPs@CP obtained in step three 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 validation 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 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 validation example 3): Take the 7-day release solution of CP and dilute it with PBS solution by the same multiple as the above CEL@CP release solution, DOX-MPs@CP release solution and CEL / DOX-MPs@CP release solution;

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

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

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

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

[0109] The crystal structure of CEL@CP obtained in step one of Example 1 was analyzed by X-ray powder diffraction, and the results are as follows: Figure 2As shown, the surface of CEL@CP does not have the characteristic diffraction peaks of the active pharmaceutical ingredient CEL and the polymeric materials C and P. The crystallinity of CEL@CP, C+P and CEL is calculated to be 9.15%, 36.22% and 95.29% respectively, indicating that CEL is well encapsulated in the polymeric material in an amorphous state.

[0110] Single fibers of CEL@CP and CP were characterized using a transmission electron microscope equipped with an energy-dispersive X-ray spectroscopy (EDS) instrument, with F, N, and S elements (not present in chitosan and polyethylene oxide) used as loading indicators. The scale bar is 400 nm, and Merge indicates the fused display result. From... Figure 3 It can be seen that the F, N and S elements are evenly distributed in the CEL@CP nanofiber scaffold, which further reveals the successful loading of CEL in CEL@CP, while there is no obvious distribution of the corresponding elements in the nanofiber scaffold of CP.

[0111] Following step one of Example 1, adjusting CEL, C, and P according to the parameters in Table 2 during the preparation of the electrospun scaffold body yields the same result: a stable electrospun scaffold body in which CEL is encapsulated in an amorphous state within CP, and CEL can be continuously released.

[0112] Table 2

[0113] CEL mass (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 Scanning electron microscopy and transmission electron microscopy results showed that CP and CEL@CP had smooth surfaces. In contrast, the CEL / DOX-MPs@CP obtained in step three of Example 1 showed a rough surface, with a scale bar of 2 μm. This indicates that DOX-MPs may be adsorbed on the scaffold surface and in the gaps between the CEL@CP microfibers. The average diameters of CP, CEL@CP, and CEL / DOX-MPs@CP were 412.8±83.1 nm, 438.7±86.1 nm, and 411.1±93.4 nm, respectively, with porosities of 57.8%, 58.2%, and 44.4%, respectively. The reduced porosity of CEL / DOX-MPs@CP confirms that DOX-MPs were effectively loaded into multiple gaps within the CEL@CP.

[0115] Following step three of Example 1, adjusting the soaking time between 1 hour and 10 hours also yielded the same results as described above, namely, DOX-MPs were effectively loaded onto CEL@CP and into multiple gaps of CEL@CP.

[0116] Comparative Example 2 was characterized using laser confocal imaging results, confirming the presence of Cy5.5 and DOX-MPs fluorescence in Cy5.5 / DOX-MPs@CP, as shown in the figure. Figure 5 As shown, the scale bar is 10 μm, which further demonstrates the effective loading of CP for CEL and DOX-MPs. Verification Example 2: In vivo degradation and biocompatibility of CEL / DOX-MPs@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 specified time intervals, CEL / DOX-MPs@CP was removed, washed with PBS, and vacuum-dried at 50°C for 4 hours, after which 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, skin and muscle tissue surrounding the implanted CEL / DOX-MPs@CP were subjected to H&E staining to assess its biocompatibility. Results showed that no obvious signs of inflammation or tissue necrosis were observed in the skin and muscle tissue surrounding the CEL / DOX-MPs@CP implanted in 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 of PBS solution and shaken at 37 °C and 100 rpm. The release solution was collected at specified time points, 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 determined by high-performance liquid chromatography (HPLC), and the release rates of CEL and DOX-MPs were calculated. Figure 6 (Left) As shown. In addition, 1 mL of DOX-MPs was added to a 14 kDa dialysis bag, which was then immersed in 25 mL of PBS solution and shaken at a constant speed of 100 rpm at 37°C. The concentration of DOX was detected by high-performance liquid chromatography at specified time points, and the release of DOX was calculated. The results are as follows... Figure 6 As shown on the right.

[0121] Experimental results showed that approximately 75% of DOX-MPs were released from CEL / DOX-MPs@CP within 24 hours, which helped to effectively kill residual tumor cells. Only 18% of DOX was released from DOX-MPs within 24 hours. Approximately 32.4% of CEL was released from CEL / DOX-MPs@CP in the initial 12 hours; controlled release followed over the next 7 days. The initial 12-hour burst release met the demand for high concentrations of CEL due to the excessive release of catecholamines and prostaglandins during the perioperative period; while the subsequent sustained release met the tumor microenvironment's need for continuous CEL.

[0122] However, the release effect of DOX-MPs in CEL / DOX-MPs@mPPPE is poor because CEL@mPPPE fails to effectively load DOX-MPs. The release effect of CEL is also poor, as evidenced by the maximum release rate of CEL being less than 5% after 96 hours.

[0123] Validation Example 4: In vitro cytotoxicity study of CEL / DOX-MPs@CP against 4T1 cells.

[0124] To evaluate the in vitro biological function of CEL / DOX-MPs@CP, 4T1 cells were sputtered at a concentration of 1×10⁻⁶. 3 Four T1 cells were seeded at a density of 100 cells per well in 96-well plates, with 5 replicates per group. The cells were co-incubated for 24 hours with 200 μL of CP release medium, CEL@CP release medium, DOX-MPs@CP release medium, CEL / DOX-MPs@CP release medium, CEL solution, DOX solution, and DOX-MPs solution, respectively. Then, 20 μL of CCK-8 solution was added and incubated for the appropriate time according to the manufacturer's instructions. The absorbance of each well was measured using a microplate reader. The group co-incubated with PBS was considered to have 100% survival. Figure 7 As shown, DOX-MPs exhibited stronger cytotoxicity against 4T1 cells compared to DOX, which may be due to the enhanced 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 significant cytotoxicity was detected in the CEL group and the CEL@CP group.

[0125] Validation Example 5: In vitro functional study of inflammatory PMNs by CEL / DOX-MPs@CP

[0126] While PMNs are crucial for anti-infective immune responses, they can have detrimental effects when stimulated by inflammation, enter the tumor microenvironment, and become activated, promoting tumorigenesis and development. Inhibiting the activity of inflammatory PMNs is essential to disrupting the interaction between PMNs and tumors. CEL can inhibit the function of inflammatory PMNs by targeting the COX-2 / PGE2 pathway in the inflammatory environment. To evaluate the in vitro effects of CEL / DOX-MPs@CP on inflammatory PMNs, freshly isolated PMNs (1×10⁻⁶) from the femur and tibia of BALB / c mice were used. 6 Each well was pretreated with 2000 μL of the same PBS, CEL, DOX, DOX-MPs solutions as in Validation Example 4, as well as CEL@CP, DOX-MPs@CP, and CEL / DOX-MPs@CP release solutions for 1 hour. Then, the wells were further stimulated with 200 nM phorbol ester (PMA) (a potent inflammatory stimulant) for 4 hours. The supernatant and lysate were collected, and the levels of COX-2, iNOS, and ARG1, as well as the activity of MPO, were detected using a kit. Results are as follows: Figure 8a , Figure 8b As shown. "PMA+" indicates the group treated with PMA stimulation in each example and comparative example; PBS indicates the control group treated with PBS only, without PMA; and PMA indicates the group treated with PMA only. From Figure 8a , Figure 8b It can be seen that PMA significantly increased the levels of inflammatory mediators COX-2, iNOS, and ARG1, as well as the activity of MPO in PMNs. These enzymes are expressed by PMNs and can trigger and promote tumor growth. It can also 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. Furthermore, PMNs are known to produce intracellular ROS, leading to oxidative stress, DNA damage, and activation of signaling pathways supporting tumorigenesis and development.

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

[0128] Besides inflammatory mediators and ROS, PMNs can also release NETs, ​​which can capture circulating tumor cells and help them attach to distant organs, thereby promoting tumor progression and metastasis. To determine whether CEL / DOX-MPs@CP affects NET production by inflammatory PMNs, PMNs were pretreated for 1 hour with the same PBS, CEL, DOX, and DOX-MPs solutions as in validation example 4, as well as CEL@CP, DOX-MPs@CP, and CEL / DOX-MPs@CP release solutions. They were then stimulated with 200 nM PMA for 4 hours, followed by the addition of 2 drops / mL Sytox Green (a cell-impermeable dye that fluoresces green upon binding to DNA released from PMNs). After 30 minutes, the fluorescence intensity of Sytox Green was quantitatively measured using a multi-microplate reader, and observation was performed using a laser confocal microscope. Figure 8d As shown, "PMA+" indicates the group treated with PMA in each example 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 PMA treatment significantly increased the Sytox Green fluorescence intensity of PMNs, while treatment with CEL, CEL@CP, or CEL / DOX-MPs@CP, but not with DOX, DOX-MPs, or DOX-MPs@CP, significantly suppressed PMA-triggered Sytox Green fluorescence. Figure 8e(Left) Semi-quantitative statistical analysis of the fluorescence area ratio in each group of laser confocal images further confirms the decrease in Sytox Green fluorescence intensity in the CEL / DOX-MPs@CP group. Furthermore, as... Figure 8e As shown on the right, CEL / DOX-MPs@CP significantly reduced the expression of H3Cit, a NETs-specific marker, further confirming that CEL / DOX-MPs@CP effectively inhibited the production of NETs in inflammatory PMNs.

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

[0130] The interaction between PMNs and tumors can promote tumor cell proliferation, migration, and invasion. To investigate the effect of CEL / DOX-MPs@CP-induced inhibition of inflammatory PMN 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 pretreated with CEL@CP, DOX-MPs@CP, or CEL / DOX-MPs@CP release solutions (PMNs stimulated with 200 nM PMA for 4 hours). 4T1 cell viability was then measured using the CCK-8 assay. Figure 9a As shown in the left image, "+PMA" indicates the group that received PMA stimulation in each example and comparative example, while "No PMNs" indicates the control group containing only PBS buffer. PMA-stimulated inflammatory PMNs supernatant significantly increased 4T1 cell survival. Inflammatory PMNs supernatant pretreated with DOX, DOX-MPs, or DOX-MPs@CP had no significant effect on PMN-promoted 4T1 cell proliferation. Conversely, compared to the PMNs supernatant treatment group, treatment with PMNs supernatant pretreated with CEL, CEL@CP, or CEL / DOX-MPs@CP significantly reduced 4T1 cell survival, indicating that CEL / DOX-MPs@CP effectively inhibited PMN-promoted 4T1 cell proliferation.

[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 a scratch mold. Then, 4T1 cells were co-incubated with the supernatant of inflammatory PMNs pretreated with PBS, CEL, DOX, DOX-MPs solutions, CEL@CP release solution, DOX-MPs@CP release solution, and CEL / DOX-MPs@CP release solution to conduct a wound healing experiment. Figure 9aAs shown on the right, "+PMA" indicates the group that received PMA stimulation in each example and comparative example, while "No PMNs" indicates the control group containing only PBS buffer. The supernatant of PMA-stimulated PMNs significantly promoted 4T1 cell migration, and the wounds in the PMNs+PMA treatment group closed significantly after 24 hours. Pretreatment with DOX, DOX-MPs, and DOX-MPs@CP release solutions did not significantly inhibit inflammatory PMN-induced 4T1 cell migration, while the wounds in 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 effectively inhibited inflammatory PMN-induced 4T1 cell migration. Furthermore, as... Figure 9b Transwell assays targeting invasion and migration further confirmed that CEL / DOX-MPs@CP can inhibit the migration and invasion of 4T1 cells induced by inflammatory PMNs.

[0132] NETs released from PMNs have a significant impact on tumor cell proliferation and metastasis. Since CEL / DOX-MPs@CP significantly inhibited NET release from PMNs, it was hypothesized that NETs might be involved in CEL / DOX-MPs@CP-mediated inhibition of 4T1 cell proliferation, migration, and invasion induced by inflammatory PMNs (PMNs stimulated with 200 nM PMA for 4 hours). To verify this hypothesis, PMNs pretreated with PBS or CEL / DOX-MPs@CP release solution and then stimulated with PMA for 4 hours were treated with DNase I (a recognized inhibitor of NET formation) at a concentration of 150 U / mL for 20 minutes, and NETs pretreated with PBS or CEL / DOX-MPs@CP release solution and then stimulated with PMA for 4 hours were treated with DNase I at a concentration of 150 U / mL for 20 minutes, and then applied to 4T1 cells. As expected, NETs obtained from inflammatory PMNs treated with PBS significantly promoted the proliferation, migration, and invasion of 4T1 cells. Figure 9c (Left) shows the proliferation of 4T1 cells, as... Figure 9c (right) and Figure 9d (Left) shows the migration of 4T1 cells and as shown in the figure. Figure 9d (Right) In the invasion diagram of 4T1 cells, NET ()The parentheses indicate the groups that underwent the corresponding treatment and harvested NETs. "+PMA" indicates the groups that received PMA stimulation in each example and comparative example. "+DNase I" indicates the groups that received DNase I treatment in each example and comparative example. "No PMNs" indicates the control group containing only PBS buffer. The supernatant of PMA-stimulated inflammatory PMNs significantly improved the cell viability of 4T1 cells. Similar to the supernatant obtained from PBS-treated inflammatory PMNs, this indicates that NETs play an important role in regulating 4T1 cell proliferation, migration, and invasion. Although DNase I treatment significantly reduced the proliferation, migration, and invasion of 4T1 cells promoted by PBS-treated inflammatory PMNs supernatant and NETs, ​​it did not significantly affect the aforementioned functions of the supernatant obtained from CEL / DOX-MPs@CP pretreated inflammatory PMNs or NETs-induced 4T1 cells. Figure 9c and Figure 9d As shown, CEL / DOX-MPs@CP participates in preventing the proliferation, migration, and invasion of 4T1 cells by inhibiting the generation of NETs.

[0133] Validation Example 7: Retention of CEL / DOX-MPs@CP at the surgical resection site in mice with breast cancer.

[0134] Ideal orthotopic implants require sustained drug retention at the target site. To evaluate the sustained drug retention capability of CEL / DOX-MPs@CP at the tumor resection site, it was tested using Cy5.5 / IR780-MPs@CP (Comparative Example 3). The orthotopic 4T1 mammary tumor volume in BALB / c mice reached approximately 300 mm². 3 At that time, 90% of the breast tumor was surgically removed. Subsequently, a 2.5 mg fibrous scaffold (Cy5.5 / IR780-MPs@CP) from Comparative Example 3 was placed into the surgical resection cavity, and 50 μL of an equivalent dose of Cy5.5 / IR780-MPs suspension was also placed into the surgical resection cavity. Figure 10a It can be seen that in mice treated with Cy5.5 / IR780-MPs and Cy5.5 / IR780-MPs@CP, the fluorescence intensity of Cy5.5 and IR780 decreased over time. However, compared with the Cy5.5 / IR780-MPs group, the decrease in fluorescence intensity of Cy5.5 and IR780 in the Cy5.5 / IR780-MPs@CP group was slower. Furthermore, on day 19 post-treatment, in vitro fluorescence of major organs... Figure 10bAnalysis showed significant accumulation of Cy5.5 and IR780 fluorescence in tumor tissues 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 showed a 1.8-fold increase in Cy5.5 accumulation and a 4.4-fold increase in IR780 accumulation in tumor tissues, indicating that Cy5.5 / IR780-MPs@CP has a strong retention capacity at the tumor resection site.

[0135] Example 8: Investigation of the antitumor effect of CEL / DOX-MPs@CP on mammary gland resection in mice after surgery.

[0136] Considering that CEL / DOX-MPs@CP can effectively inhibit the function of inflammatory PMNs and maintain a certain level of drug accumulation in tumor tissue, we evaluated its effect on inhibiting postoperative tumor recurrence and metastasis in a spontaneously metastatic orthotopic 4T1 breast cancer-bearing BALB / c mouse model. When the orthotopic tumor volume reached approximately 300 mm², the effect was assessed. 3 At that time, 90% of the tumor was surgically removed, and 2.5 mg of a fiber scaffold (CP, CEL@CP, DOX-MPs@CP, or CEL / DOX-MPs@CP) was implanted into the surgical cavity (the CEL loading on the scaffold was 7.2 mg / kg, and the DOX loading was 1.8 mg / kg). Simultaneously, 50 μl of a solution containing CEL, DOX, DOX-MPs, or CEL / DOX-MPs with the same CEL and DOX loading on the fiber scaffold, as well as PBS solution, was administered. Figure 11a as well as Figure 11b As shown in the left image, mice treated with PBS, CP, or CEL experienced rapid tumor recurrence after tumor resection and exhibited similar tumor growth curves. While DOX showed limited effectiveness in inhibiting tumor recurrence, DOX-MPs and CEL / DOX-MPs demonstrated some anti-recurrence ability, possibly due to DOX-MPs' more effective deep penetration into tumor cells. The stronger inhibitory effect in the DOX-MPs@CP treatment group may be attributed to the persistent drug retention at the surgical wound site promoted by CP. CEL / DOX-MPs@CP showed the strongest ability to inhibit tumor recurrence after tumor resection, reducing tumor volume by 91% and tumor weight by 84% compared to the PBS treatment group. Histopathological observation was performed using H&E staining, and semi-quantitative analysis was conducted using Ki67 immunohistochemical staining. Figure 11b (Right). The results showed that tumor cell proliferation was minimal in the CEL / DOX-MPs@CP treatment group, which confirmed that CEL / DOX-MPs@CP has a significant ability to inhibit postoperative tumor recurrence.

[0137] like Figure 12a (Left), Figure 12b As shown, 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. Furthermore, as... Figure 12a As shown on the right, CEL / DOX-MPs@CP treatment significantly prolonged the postoperative survival time of 4T1 tumor-bearing mice, with 63% of the mice still alive on day 40 postoperatively, a significantly longer survival time compared to other groups. This further confirms 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 weight changes showed that CEL / DOX-MPs@CP did not cause significant adverse reactions, indicating that CEL / DOX-MPs@CP has good biocompatibility.

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

[0139] To explore the potential mechanism by which CEL / DOX-MPs@CP inhibits postoperative tumor recurrence and metastasis, the tumor microenvironment of BALB / c mice bearing orthotopic 4T1 breast cancer was evaluated after treatment using the same method as in validation case 8. 2.5 mg of a fiber scaffold CEL@CP, DOX-MPs@CP, or CEL / DOX-MPs@CP (with a CEL loading of 7.2 mg / kg and a DOX loading of 1.8 mg / kg) was implanted into the surgical resection cavity. Simultaneously, equivalent CEL, DOX, DOX-MPs, and CEL / DOX-MPs solutions with the same CEL and DOX loading on the fiber scaffolds, as well as 50 μl of PBS solution, were administered. On postoperative day 19, the proportion of PMNs in the tumor tissue was assessed by flow cytometry. Figure 13a As shown in the left image, compared with PBS alone, treatment with DOX, DOX-MPs, or DOX-MPs@CP did not significantly alter the proportion of PMNs in the tumor tissue. Conversely, treatment with CEL@CP or CEL / DOX-MPs@CP significantly reduced the proportion of PMNs, indicating that CEL@CP or CEL / DOX-MPs@CP significantly inhibited the number of PMNs within the resected cavity. Immunofluorescence staining further confirmed the reduction in PMN numbers in the CEL / DOX-MPs@CP treatment group, as shown in the image. Figure 13aAs shown on the right, the expression level of the PMN biomarker Ly6G was low. Simultaneously, CEL / DOX-MPs@CP significantly reduced the expression of H3Cit, a key player in PMN release within NETs, ​​confirming that CEL / DOX-MPs@CP can effectively inhibit NET production. Figure 13b As shown on the left. Similarly, mice treated with CEL / DOX-MPs@CP showed significantly reduced COX-2 expression levels, such as... Figure 13b (Right) As shown. The expression of VEGF regulated by COX-2 was also significantly reduced in the CEL / DOX-MPs@CP treatment group, such as... Figure 13c As shown on the left, this suggests that implantation of CEL / DOX-MPs@CP may reduce tumor angiogenesis, thereby inhibiting tumor recurrence and metastasis.

[0140] PMNs create an immunosuppressive microenvironment, inhibiting the function of cytotoxic T cells and other immune cells, thus allowing tumor cells to evade the immune system. Flow cytometry analysis showed that CD8+ was present in tumor tissues from 4T1 tumor-bearing mice treated with DOX-MPs and DOX-MPs@CP. + The significantly upregulated percentage of T cells indicates that DOX-MPs-induced chemotherapy can trigger an anti-tumor immune response. CEL@CP also significantly increased CD8+ at the tumor site. + The percentage of T cells suggests that CEL-induced suppression of PMN function may contribute to enhanced anti-tumor immunity. Following CEL / DOX-MPs@CP treatment, CD8+ cells in the tumor microenvironment... + The proportion of T cells has further increased compared to before, such as Figure 13c (Right) As shown. Conversely, MDSCs, as Figure 13d (Left) As shown and Tregs, as Figure 13d The percentage of immunosuppressive cells, including those on the right, was significantly reduced in the CEL / DOX-MPs@CP treatment group. These data indicate that CEL / DOX-MPs@CP effectively improves the tumor immunosuppressive microenvironment and may contribute to the efficient suppression of tumor recurrence and metastasis in surgically resected 4T1 tumor-bearing mice.

[0141] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An antitumor electrospinning scaffold, characterized in that, The scaffold comprises an electrospun scaffold body and tumor cell-derived drug-loaded microparticles loaded thereon through electrostatic adsorption; the electrospun scaffold body comprises an amino polymer, a hydrophilic polymer, and celecoxib in a mass ratio of (2~10):(2~13):1; the tumor cell-derived drug-loaded microparticles are loaded onto the antitumor electrospun scaffold at a rate of 0.6 μg / mg~6.1 μg / mg. Wherein, the amino polymer is chitosan, and the hydrophilic polymer is polyethylene oxide; The tumor cell-derived drug-loaded microparticles include microparticles released from the cytoskeleton by tumor cells during activation or apoptosis, and chemotherapy drugs encapsulated within the microparticles; the tumor cells are breast cancer cells, and the chemotherapy drugs are doxorubicin.

2. The antitumor electrospinning scaffold as described in claim 1, characterized in that, The mass ratio of the amino polymer to the hydrophilic polymer is 5:1 to 4:

6.

3. The method for preparing the antitumor electrospinning scaffold as described in any one of claims 1 or 2, characterized in that, Includes the following steps: S1. Obtain the electrospun scaffold body by electrospinning; the solute in the electrospinning solution used for electrospinning includes an amino polymer, a hydrophilic polymer, and celecoxib in a mass ratio of (2~10):(2~13):

1. S2. The electrospun scaffold body is immersed in a solution of 10µg / mL to 200µg / mL of tumor cell-derived drug-loaded microparticles for 1 hour to 10 hours, so that the tumor cell-derived drug-loaded microparticles are adsorbed onto the electrospun scaffold body by electrostatic adsorption, thereby obtaining the antitumor electrospun scaffold.

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

5. The preparation method according to claim 4, characterized in that, The mass fraction of the electrospinning solution is 3% to 10%.

6. The preparation method according to claim 3, characterized in that, In step S2, the concentration of the solution of drug-loaded microparticles derived from tumor cells is 50 µg / mL to 100 µg / mL, and the soaking time is 4 to 6 hours.

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