Electronic injection self-healing porous microsphere composite material as well as preparation method and application thereof

Abstract

The invention relates to an electronic injection self-healing porous microsphere composite material and a preparation method and application thereof.Porous microspheres are prepared through an electronic injection method, indocyanine green ICG is encapsulated through the heating self-healing effect, and then a Fe < 3 + >-polyphenol network and an FN modification layer are sequentially constructed. The preparation process is simple and efficient, and products are easy to purify and separate. The obtained FN functionalized microspheres have good dispersibility and slow release characteristics, and can significantly improve tumor targeting and cellular uptake efficiency. The system integrates the photo-thermal effect of ICG, the photo-thermal enhancement effect of Fe < 3 + >-polyphenol and the dual functions of chemical kinetics treatment and the tumor targeting specificity of FN, chemical kinetics-photo-thermal synergistic treatment can be achieved through peritumoral injection, and a new thought is provided for tumor treatment.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to an electrospray self-healing porous microsphere composite material and a preparation method and application thereof. Background Art

[0002] Nasopharyngeal carcinoma is a highly invasive malignant tumor of the head and neck. It has a special anatomical location (adjacent to the skull base and important neurovascular structures) and is difficult to resect surgically. It mainly relies on radiotherapy and chemotherapy. However, traditional radiotherapy can easily damage surrounding normal tissues, and the systemic toxicity and tumor resistance of chemotherapy drugs (such as cisplatin and 5-fluorouracil) severely limit the therapeutic effect. Therefore, the development of new targeted drug delivery systems to increase drug enrichment at the tumor site, reduce toxic side effects and enhance therapeutic effects has become a key challenge in the treatment of nasopharyngeal carcinoma. In recent years, local delivery strategies based on nanomaterials have provided new ideas for the treatment of nasopharyngeal carcinoma. However, traditional nanodelivery systems have problems such as poor stability, sudden drug release and difficulty in staying at the tumor site for a long time, which limits their clinical application. Among them, metal polyphenol nanostructures (such as Fe 3+ -Tannic acid, Cu 2+ Epigallocatechin-3-(-epigallocatechin-3-ol) has attracted considerable attention for its excellent chemodynamic therapeutic (CDT) properties. It catalyzes H₂O₂ in the tumor microenvironment through Fenton / Fenton-like reactions to generate highly toxic hydroxyl radicals (·OH), which specifically kill tumor cells (Wang et al. Adv. Mater. 2022, 34, 2107009). While metal polyphenol nanostructures possess CDT properties, they suffer from limitations such as in vivo stability, biocompatibility, safety, and tumor microenvironment responsiveness, necessitating innovative technological improvements.

[0003] As a drug carrier, PLGA exhibits significant advantages in controlled and targeted drug delivery, primarily due to its excellent biocompatibility, degradability, low immunogenicity, and low toxicity. These properties make it suitable for long-term drug release or targeted therapy. However, the degradation and release rates of PLGA are influenced by its molecular weight and copolymer ratio, making regulation complex and requiring precise control. Furthermore, drug delivery effectiveness depends on the design and optimization of specific diseases and drugs. Our group has previously demonstrated promising results in improving drug loading and bioavailability using electrospinning fiber microspheres (Xiao et al. Nano Today 2021, 38, 101-123; Zhang et al. Adv. Fiber Mater. 2022, 4, 807–819; Huang et al. J. Colloid Interface Sci. 2025, 686, 498-508). However, these microspheres still face numerous drawbacks, including poor mechanical properties, unclear biodegradability, limited drug loading, uncontrolled release, potential immune responses, unknown long-term toxicity, complex preparation processes, and limited application areas, requiring further innovation and improvement. Indocyanine Green (ICG), due to its remarkable photothermal conversion and fluorescence properties, enables high-resolution, real-time tissue imaging and precise tumor-targeted therapy. It holds broad promise for medical applications, but its rapid metabolism (half-life of only 2-4 minutes) and susceptibility to plasma protein binding can lead to decreased fluorescence signal stability, limiting its application in long-term monitoring or repeated dosing scenarios.

[0004] Single-treatment approaches have significant limitations in anti-tumor treatment, primarily in terms of drug resistance in tumor cells, individual differences in efficacy, and damage to normal tissues. While traditional chemotherapy or radiotherapy can inhibit tumor growth to a certain extent, their effectiveness is often affected by the adaptation and escape mechanisms of tumor cells, leading to a gradual weakening of the therapeutic effect. Furthermore, single-treatment approaches often struggle to account for tumor heterogeneity, potentially preventing some patients from receiving effective treatment due to individual differences. These factors collectively limit the overall effectiveness of tumor treatment, necessitating the urgent need for new treatment strategies.

[0005] A search of relevant domestic and foreign literature and patents has not yet found any reports on the use of electrosprayed microspheres as a carrier for the co-delivery of ICG, metal polyphenols, and FN for the combined photothermal and chemodynamic treatment of solid tumors. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an electrosprayed self-healing porous microsphere composite material, its preparation method, and application. Specifically, it relates to a preparation method and application of an electrosprayed self-healing porous microsphere composite for delivering metal polyphenols / indocyanine green / fibronectin. This composite material can achieve responsive drug release, reduce toxic side effects, and achieve efficient tumor treatment by combining chemodynamic therapy with photothermal therapy. Furthermore, the fibronectin delivered by this platform imparts tumor-targeting specificity to the drug carrier, enhancing its recognition and binding capabilities for tumor cells.

[0007] The present invention provides a drug-loaded microsphere composite material. The drug-loaded microsphere composite material uses polymer microspheres as carriers, the polymer microspheres are loaded with drugs, and the surfaces of the microspheres are sequentially coated with a functional layer and a modified protein.

[0008] The polymer microspheres include PLGA microspheres; the drug includes indocyanine green ICG; the functional layer includes metal-polyphenol coordination complex Fe 3+ A functional layer is formed; the protein is fibronectin FN.

[0009] The drug-loaded microsphere composite material is an electrosprayed self-healing porous microsphere composite material for delivering metal polyphenols / indocyanine green / fibronectin.

[0010] By precisely controlling the solvent evaporation dynamics during the electrospraying process of poly(lactic acid-co-glycolic acid) (PLGA), the difference in the evaporation rate of different solvents was used to induce the formation of a porous structure on the surface of the microspheres. In the subsequent heating self-healing stage, the photothermal conversion agent indocyanine green (ICG) was efficiently encapsulated inside the microspheres, and Fe was complexed on the surface of the microspheres through metal-polyphenol coordination. 3+ A functional coating, namely tannic acid / iron complex (TAF), was formed, and ultimately the bioactivity was optimized by surface modification of fibronectin (FN).

[0011] The present invention provides a method for preparing a drug-loaded microsphere composite material, comprising:

[0012] (1) mixing PLGA porous microspheres and drug solution, heating and stirring to obtain self-healing drug-loaded microspheres;

[0013] (2) mixing the self-healing drug-loaded microspheres, tannic acid TA solution, and an iron source, and stirring the mixture to react to obtain drug-loaded microspheres coated with tannic acid iron;

[0014] (3) The drug-loaded microspheres coated with tannic acid iron and the protein solution are mixed and extruded to obtain a drug-loaded microsphere composite material.

[0015] The drug delivery platform uses electrosprayed PLGA porous microspheres. By adjusting the ratio of organic solvents used to dissolve the PLGA and controlling the rate of solvent evaporation, pores are formed on the surface of the droplets as they solidify under the force of the electric field.

[0016] Preferably, the preparation of the PLGA porous microspheres includes: dissolving PLGA in a mixed organic solvent, performing electrospraying, adjusting the voltage and receiving distance, and using polyvinyl alcohol (PVA) solution as a receiving liquid to collect the microspheres to obtain the PLGA porous microspheres.

[0017] Preferably, the molecular weight of the PLGA is Mw=20-40 kDa, the viscosity is 1.2-1.5, and the mass concentration of PLGA is 4-6%;

[0018] Preferably, the mixed organic solvent is a mixture of hexafluoroisopropanol (HFIP) and dichloromethane (DCM).

[0019] Preferably, the molar ratio of HFIP to DCM is 3:1 to 5:1.

[0020] Preferably, the polyvinyl alcohol (PVA) solution is prepared by dissolving PVA in water to a mass fraction of 1-3%, heating the solution in a water bath to 80-86° C., stirring the solution for 2-4 hours, and placing the solution in a receiving portion of an electrospray device after the solid powder is completely dissolved and cooled; wherein the molecular weight of the PVA is Mw = 20-30 kDa.

[0021] Preferably, the process parameters of the electrospray are as follows: adjusting the high voltage power supply to a range of 4 to 10 kV; adjusting the distance between the electrospray needle tip and the receiving device to 10 to 15 cm; and adjusting the flow rate of the propulsion pump to 1 to 2 mL / h.

[0022] After the microspheres are collected, they are post-processed, wherein the post-processing comprises first curing at 4-8°C for 8-12 hours, then differential centrifugation, further curing at 4-8°C for 8-12 hours, and freeze-drying; wherein the differential centrifugation comprises first centrifuging at 2000-4000 rpm for 3-10 minutes, discarding the precipitate and taking the supernatant; then centrifuging at 8000-12000 rpm for 10-30 minutes, discarding the supernatant and taking the precipitate.

[0023] Preferably, the concentration of the drug solution in step (1) is 0.5-2 mg / mL; the heating and stirring is heating to 40-45° C., stirring at a speed of 700-1000 rpm, and stirring for 2-4 hours; and the solvent of the drug solution is water.

[0024] Preferably, the drug in step (1) comprises indocyanine green ICG.

[0025] After the heating and stirring in step (1) is completed, the microspheres are collected and then post-processed, wherein the post-processing is first solidified at 4-8°C for 8-12 hours, then differential centrifuged, further solidified at 4-8°C for 8-12 hours, and freeze-dried; wherein the differential centrifugation is first centrifuged at 2000-4000 rpm for 3-10 minutes, discarding the precipitate and taking the supernatant; then centrifuged at 8000-12000 rpm for 10-30 minutes, discarding the supernatant and taking the precipitate.

[0026] In the step (2), tannic acid TA solution and iron source are sequentially added to the self-healing drug-loaded microspheres.

[0027] Furthermore, the self-healing drug-loaded microspheres in step (2) are mixed with the tannic acid solution, and iron salt is added under stirring to react, thereby obtaining a drug-loaded microsphere material of the tannic acid / iron complex loaded with ICG.

[0028] Preferably, the molar ratio of TA, iron source and self-healing drug-loaded microspheres in step (2) is (24:22:1) to (33.52:49.2:1); the iron source is an iron salt; wherein the iron salt is ferric chloride FeCl3.

[0029] The solvent of the TA solution in step (2) is anhydrous ethanol.

[0030] In the step (2), the reaction is stirred for 8-24 hours.

[0031] After the reaction in step (2) is stirred, the mixture is centrifuged.

[0032] Preferably, the solvent of the protein solution in step (3) is deionized water; the concentration of the protein solution is 2 to 4 mg / mL; the mass ratio of the drug-loaded microspheres encapsulating ferric tannate to the protein is 1:2 to 1:6;

[0033] Preferably, the protein in step (3) is fibronectin FN;

[0034] Furthermore, the protein in step (3) is a purified protein.

[0035] Preferably, the extrusion in step (3) is repeated 10 to 20 times through a film extruder.

[0036] Furthermore, in step (3), after repeated extrusion and contacting by a film extruder, the precipitate is collected by centrifugation, solidified at 4° C. for 8-12 hours, and freeze-dried.

[0037] The present invention provides a drug-loaded microsphere composite material, or use of the drug-loaded microsphere composite material prepared by any of the methods described in preparing contrast agents, and / or preparing drugs for combined chemokinetic / dual photothermal therapy of tumors, such as drugs for treating nasopharyngeal carcinoma.

[0038] The present invention adopts electrospraying method to prepare porous microspheres, and after encapsulating indocyanine green (ICG) through heating self-healing effect, Fe 3+ -polyphenol network and FN modified layer. The preparation process is simple and efficient, and the product is easy to purify and separate. The obtained FN functionalized microspheres have good dispersibility and sustained release properties, which can significantly improve tumor targeting and cell uptake efficiency. The system integrates the photothermal effect of ICG, Fe 3+ -The dual functions of the photothermal enhancement effect and chemodynamic therapy of polyphenols and the tumor targeting specificity of FN can achieve chemodynamic-photothermal synergistic therapy through peritumoral injection, providing a new idea for tumor treatment.

[0039] The present invention provides a self-healing porous microsphere-metal polyphenol targeted drug composite for delivering ICG. The composite is prepared using an electrospray method, and then heated with ICG to the glass transition temperature of PLGA. The composite utilizes self-healing pores to achieve efficient drug loading, forming PI. Subsequently, a tannic acid iron metal polyphenol network is coated on the surface of the PI microspheres using a one-pot method to form PI-TAF. Finally, the composite is encapsulated with fibronectin (FN) through physical extrusion and hydrogen bonding to form PI-TAF@FN.

[0040] The physical and chemical properties of the prepared PI-TAF@FN were characterized using methods such as zeta potential and dynamic light scattering analysis (DLS), ultraviolet-visible spectroscopy (UV-vis), inductively coupled plasma atomic emission spectroscopy (ICP-OES), and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The drug release rate of the material in solutions with different pH values ​​was determined, and its pH-responsive drug release performance was measured. The cytotoxicity of PI-TAF@FN was then evaluated using the CCK-8 assay. Flow cytometry was used to examine cellular phagocytosis of the material and the apoptosis efficiency of tumor cells after treatment with the material and light. The effect of the material on intracellular GSH levels was detected using GSH and GSSG detection kits. Laser confocal microscopy was used to evaluate the material's effects on intracellular ROS and LPO levels. Finally, a nude mouse subcutaneous tumor model was established for anti-tumor experiments.

[0041] The present invention provides a self-healing porous microsphere-metal polyphenol composite drug for delivering ICG, metal polyphenol and FN, which can reduce the toxic side effects of ICG when used alone, and at the same time, the Fe 3+ The resulting metal polyphenol network not only enables chemodynamic therapy but also further enhances photothermal effects, further inhibiting tumor growth. Fibronectin (FN) coating the microspheres allows for targeted delivery to tumor sites, enhancing the accumulation of the drug delivery system at the tumor site, thus simplifying the procedure and improving anti-tumor efficacy.

[0042] Beneficial effects

[0043] (1) The process of the present invention is simple, the cost is low, the product is easy to operate and separate, and it has good development prospects;

[0044] (2) The microsphere drug prepared by the present invention has good dispersibility and biocompatibility, and can release ICG in a pH-responsive manner in the tumor microenvironment, providing a new idea for constructing a safe, intelligent, and efficient photothermal drug carrier;

[0045] (3) The composite prepared by the present invention can be complexed with Fe 3+ It triggers a "Fenton-like reaction" to implement chemodynamic therapy. At the same time, the metal polyphenol network formed by the complexation of iron ions and polyphenols further enhances the photothermal therapeutic effect of ICG by improving light absorption capacity, photothermal conversion efficiency and leveraging its structural stability advantages.

[0046] (4) The complex prepared by the present invention showed significant anti-tumor effect after being injected into mice through peritumoral injection, achieving dual photothermal / chemodynamic combined therapy and having potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the synthesis and application of PI-TAF@FN prepared in the present invention.

[0048] Figure 2 is the differential scanning calorimetry (DSC) spectrum of the PLGA porous microspheres prepared in the present invention;

[0049] Figure 3 The hydrodynamic diameter (a) and surface potential results (b) of PLGA, PI, PI-TAF and PI-TAF@FN prepared in the present invention;

[0050] Figure 4 The hydrodynamic diameter changes of PI-TAF@FN prepared by the present invention in water, PBS, and 1640 cell culture medium over seven days;

[0051] Figure 5 Schematic diagram (a) and scanning electron microscope (SEM) image (b) of the self-healing changes of the self-healing porous microspheres PI prepared by the present invention upon heating;

[0052] Figure 6 SEM image (a) and particle size distribution histogram (b) of PI-TAF@FN microspheres prepared in the present invention;

[0053] Figure 7 SDS-PAGE images of PI-TAF and PI-TAF@FN microspheres prepared in the present invention;

[0054] Figure 8The linear relationship between 1 / T1 and Fe concentration of PI-TAF@FN prepared in the present invention at pH 6.5 and 7.4 respectively;

[0055] Figure 9 Cumulative release curves of ICG (a) and iron ions (b) from PI-TAF and PI-TAF@FN prepared in the present invention at pH 6.5 and 7.4;

[0056] Figure 10 The temperature rise curves of PI and PI-TAF@FN prepared in the present invention (a), the temperature rise curves of PI-TAF@FN with different concentrations (b), a temperature rise and fall cycle curve of PI-TAF (c), the relationship between cooling time and -lnθ (d), and the temperature curves of PI-TAF@FN in five temperature rise and fall cycles (e);

[0057] Figure 11 Cell viability graphs of nasopharyngeal carcinoma cells (CNE-2) after incubation with PBS, ICG, PI, PI-TAF, or PI-TAF@FN for 24 h with (a) and without (b) laser irradiation.

[0058] Figure 12 Flow cytometry histogram (a) and quantitative analysis bar graph (b) after cells were co-incubated with PBS, ICG, PI, PI-TAF or PI-TAF@FN for 6 h;

[0059] Figure 13 is the percentage of GSH consumption in CNE-2 cells after 24 h of treatment with PBS, ICG, PI, PI-TAF, or PI-TAF@FN; Figure 14 CLSM results (a) and flow cytometry analysis results (bc) of the changes in intracellular ROS levels after CNE-2 cells were co-incubated with PBS, ICG, PI, PI-TAF, or PI-TAF@FN for 6 h.

[0060] Figure 15 CLSM results of changes in intracellular LPO levels after CNE-2 cells were co-incubated with PBS, PI-TAF@FN, PI+L, PI-TAF+L, or PI-TAF@FN+L for 24 h;

[0061] Figure 16 Flow cytometry detection results (a) and quantitative analysis (bc) of cell apoptosis after PBS, ICG, PI, PI-TAF or PI-TAF@FN were co-incubated with CNE-2 cells for 12 h;

[0062] Figure 17Western blot results (a) and quantitative analysis (bc) of GPX4 and Caspase-3 expression levels after PBS, PI-TAF@FN, PI+L, PI-TAF+L, and PI-TAF@FN+L were co-incubated with CNE-2 cells for 24 h.

[0063] Figure 18 The distribution of PI-TAF (a) and PI-TAF@FN (b) in various organs and tumor sites in mice after peritumoral injection at different time points;

[0064] Figure 19 Tumor volume change curves (ae) recorded over 14 days in tumor-bearing mice treated with peritumoral injection of PBS, PI-TAF@FN, PI+Laser, PI-TAF+Laser, or PI-TAF@FN+Laser.

[0065] Figure 20 Figure 3 is the body weight change curve of tumor-bearing mice during the 14-day treatment period. DETAILED DESCRIPTION

[0066] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0067] Unless otherwise specified, all chemical reagents are commercially available and used directly without further purification. PLGA was purchased from Jinan Daigang Bioengineering Co., Ltd. ICG was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Fibronectin was from Shanghai Xianlian Biotechnology Co., Ltd. CNE-2 cells (human nasopharyngeal carcinoma cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. 1640 culture medium, fetal bovine serum, penicillin-streptomycin antibodies, and trypsin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Qihai Biotechnology Co., Ltd. GSH and GSSH detection kits and ROS detection kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Apoptosis detection kits were purchased from Jiangsu Keyi Biotechnology Co., Ltd. Nude mice were purchased from Shanghai Slake Laboratory Animal Center. ELISA kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd. Water with a resistivity higher than 18.2 MΩ.cm used in all experiments was purified by a laboratory water purification system (Cascada I, PALL, Beijing).

[0068] Example 1

[0069] (1) 5.3 mg of PLGA (Mw = 20 kDa, viscosity 1.5) was dissolved in a mixed organic solvent of hexafluoroisopropanol and dichloromethane (molar ratio 3:1) to a 4% PLGA concentration. After stirring for 2 h, the solution was transferred to a 1 mL syringe as the precursor solution for the electrospray device. Separately, 100 mg of PVA (Mw = 20 kDa) was dissolved in 10 mL of ultrapure water to a 1% PVA concentration. The solution was heated and stirred in an 85°C water bath for 2 h to dissolve the PVA, and then cooled to room temperature.

[0070] (2) Place the PVA solution prepared in (1) below as the receiving solution. Adjust the parameters of the electrospray equipment: high-voltage power supply 6kV, receiving distance 15cm, injection pump propulsion speed 1.5mL / h, and place a magnetic stirring device under the receiving device to stir at a constant speed of 800rpm to prevent microsphere aggregation. After collecting the obtained microspheres, solidify them at 4℃ for 12h, and then perform differential centrifugation: first centrifuge at 3000rpm for 10min, discard the precipitate and take the supernatant; then centrifuge at 10000rpm for 30min, discard the supernatant and take the precipitate, and continue to solidify at 4℃ for 12h. Finally, freeze-drying treatment is performed to obtain porous PLGA microspheres with uniform particle size.

[0071] (3) Then, 4 mg of ICG was weighed and dissolved in 5 mL of ultrapure water and stirred evenly. The porous PLGA microspheres prepared in step (2) were immersed in the prepared ICG solution and heated and stirred at 42°C for 2 h. The obtained microspheres were collected and solidified at 4°C for 12 h, followed by differential centrifugation: first centrifuged at 3000 rpm for 10 min, discarded the precipitate and took the supernatant; then centrifuged at 10000 rpm for 30 min, discarded the supernatant and took the precipitate, and continued to solidify at 4°C for 12 h. Finally, after freeze-drying, self-healing drug-loaded microspheres PI with uniform particle size were obtained.

[0072] (4) 3.5 mg / mL PI microspheres were dispersed in an anhydrous ethanol solution containing 2.6 mg / mL tannic acid (TA). 0.74 mg / mL FeCl3 aqueous solution was slowly added during continuous stirring. The mixed solution was stirred for 12 hours overnight. After the ethanol was completely evaporated, it was centrifuged at 13,000 rpm at 4°C for 30 minutes to obtain ICG-loaded tannic acid-iron complex composite microsphere material PI-TAF, in which the molar ratio of TA, FeCl3, and PI was 24:26:1.

[0073] (5) Take 15 mL of fibronectin stock solution and place it in a 10,000 Da dialysis bag for 24 hours, changing the water twice in the middle. The dialyzed stock solution is placed in a -80°C freezer for more than 12 hours and freeze-dried to obtain purified fibronectin FN.

[0074] (6) 4 mg of the purified FN from step (5) was dissolved in 1 mL of ultrapure water to obtain an FN solution. The solution was then mixed with 1 mg of PI-TAF microspheres. The solution was extruded 12 times using an Avanti microextruder, centrifuged at 10,000 rpm for 6 min, and the supernatant was discarded. The precipitate was then solidified at 4°C for 12 h. Finally, the solution was freeze-dried to obtain PI-TAF@FN.

[0075] Example 2

[0076] 5 mg of the porous PLGA microspheres prepared in Example 1 were placed in a crucible and tested in a DSC instrument. The temperature range was set at 0-400°C, the heating rate was 10°C / min, and the protective gas (nitrogen or argon) flow rate was 20-100 mL / min. The heat flow change was monitored in real time, and parameters such as the glass transition temperature and phase change enthalpy were measured. Figure 2 As shown in Figure 2, thermal performance parameters were obtained by analyzing the characteristic peak position, peak area, and baseline change of the heat flow curve. The test results showed that the glass transition temperature of the porous PLGA microspheres was 41.56°C.

[0077] Example 3

[0078] The PLGA, PI, PI-TAF, and PI-TAF@FN microspheres prepared in Example (1) were dispersed in 1 mL of ultrapure water to measure the surface potential, hydrodynamic diameter, and dispersion coefficient. Figure 3 As shown in Figure a, the hydrodynamic diameter of PLGA microspheres is 1021.2 nm. After ICG loading (PI), the diameter increases to 1266.1 nm. After TAF encapsulation (PI-TAF), the diameter further increases to 2299 nm. Finally, the diameter of FN-coated PI-TAF@FN microspheres reaches 2509 nm. The surface potential test results are shown in Figure 3. Figure 3 As shown in b, the voltage of PLGA is -22.98 mV, PI increases to -12.4 mV, PI-TAF decreases to -24.62 mV, and PI-TAF@FN further decreases to -27.28 mV. These data confirm the successful loading of ICG and the layer-by-layer coating of TAF and FN.

[0079] Example 4

[0080] To evaluate the stability of the microsphere complex, the PI-TAF@FN microspheres prepared in Example 1 were dispersed in three media: water, PBS, and RPMI-1640 medium. Three parallel samples were set up for each group of experiments, and each sample was measured three times and the average value was taken. Dynamic light scattering (DLS) technology was used to monitor the changes in its hydrodynamic diameter over time. The experimental results showed that the hydrodynamic diameter of PI-TAF@FN remained stable during the one-week observation period ( Figure 4), indicating that the composite microspheres have excellent colloidal stability.

[0081] Example 5

[0082] The PLGA porous microspheres prepared in Example 1, the PLGA porous microspheres heated and stirred in the ICG aqueous solution, and the PI microspheres that completed self-healing after heating for 2 hours were dispersed in 1 mL of deionized water. 10 μL of the dispersion was evenly added dropwise to the surface of a clean silicon wafer and allowed to air-dry at room temperature. The dried silicon wafer was fixed to a sample stage coated with a conductive adhesive. After 60 seconds of gold (Au) ion sputtering treatment, the surface morphology of the three microspheres was characterized using a Hitachi S-4800 field emission scanning electron microscope (accelerating voltage 5 kV) ( Figure 5 The experimental results show that heating the PLGA porous microspheres to above their glass transition temperature (Tg) while loading ICG can effectively promote the self-healing of the porous microsphere surface and form a dense and uniform PI microsphere structure.

[0083] Example 6

[0084] The PI-TAF@FN microspheres prepared in Example 1 were dispersed in 1 mL of deionized water, and 10 μL was added dropwise to the surface of a clean silicon wafer and allowed to air dry at room temperature. The SEM sample was processed according to the method of Example 5, and the surface morphology was characterized using a Hitachi S-4800 field emission scanning electron microscope (accelerating voltage 5 kV). Figure 6 a). ImageJ software (Version 1.53) was used to analyze SEM images, and the diameters of more than 60 microspheres were counted and a size distribution histogram was drawn ( Figure 6 b). The results show that the PI-TAF@FN microspheres have uniform morphology and an average diameter of 1.91±0.06μm.

[0085] Example 7

[0086] Take the PI-TAF, FN, and PI-TAF@FN prepared in Example 1 and perform SDS-PAGE gel electrophoresis to verify whether the microspheres have successfully coated FN. Prepare a material solution with a protein concentration of 2 mg / mL, take 20 μL and mix with 5 μL of loading buffer, boil in a boiling water bath for 5 minutes, add PI-TAF, FN, PI-TAF@FN and standard protein Marker (5 μL) to the corresponding channels of the gel in turn, and then electrophorese at 120V for 30 minutes. After the gel run is completed, take out the gel and place it in a container filled with Coomassie Brilliant Blue for staining for 30 minutes, then replace the staining solution with the same volume of decolorizing solution and decolorize on a decolorizing shaker for 2 hours. Finally, use a gel scanner to record the protein bands on the precast gel. The results are as follows Figure 7As shown in the figure, PI-TAF did not show obvious protein bands, while PI-TAF@FN showed the same bands as FN, indicating that FN was successfully coated on the surface of the microspheres.

[0087] Example 8

[0088] In order to determine whether PI-TAF@FN has pH responsiveness in the tumor microenvironment, the material PI-TAF@FN was diluted into solutions with different concentration gradients (Fe element concentrations were 0.06, 0.11, 0.22, 0.43, 0.85, and 1.7 mM, respectively) using buffer solutions with different pH values ​​(pH = 6.5 and 7.4), and 2 mL was taken for testing. The T1 relaxation time of PI-TAF@FN under different pH conditions (pH = 6.5 and 7.4) was measured using a nuclear magnetic resonance analyzer, and the reciprocal of the relaxation time was linearly fitted with the Fe concentration, and the slope was the relaxation rate r1 (e.g. Figure 8 ). At pH 6.5, the r1 of PI-TAF@FN was 9.27 mM -1 s -1 At pH 7.4, the r1 of PI-TAF@FN was 5.46 mM -1 s -1 The above results further demonstrate that PI-TAF@FN has good T1 MR imaging performance under weakly acidic conditions and can be used as an MR T1 contrast agent.

[0089] Example 9

[0090] Disodium hydrogen phosphate and sodium dihydrogen phosphate were used to prepare buffer solutions of pH = 7.4 and pH = 6.5, respectively. The prepared PI-TAF and PI-TAF@FN were dissolved in 1 mL of the two buffer solutions of different pH, respectively, to prepare a 1 mg / mL solution, which was then placed in a dialysis bag. The dialysis bag was then placed in 9 mL of the corresponding pH buffer solution and shaken in a constant temperature shaker at 37°C. At different time points (0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, 48h and 72h), 1 mL of the liquid outside the dialysis bag was aspirated, and then 1 mL of fresh buffer solution of the corresponding pH was added. After the sustained release was completed, the absorbance at 795 nm was tested by ultraviolet light, and the drug release curves of PI-TAF and PI-TAF@FN under different pH conditions were drawn, as shown in FIG. Figure 9As shown in Figure a, the 48-hour drug release rate of PI-TAF at pH = 7.4 is 34.6%, while the 48-hour drug release rate at pH = 6.5 is 62.8%, indicating that PI-TAF has good pH-responsive drug release performance and is more conducive to drug release under acidic conditions. The drug release rate of PI-TAF@FN is lower than that of PI-TAF under the same conditions, indicating that the drug release rate of the material is reduced after protein coating.

[0091] The buffer solution was prepared in the above manner, and the prepared PI-TAF@FN was prepared into a 1 mg / mL solution, which was then placed in a dialysis bag. The dialysis bag was then placed in 9 mL of the corresponding pH buffer solution and oscillated in a constant temperature shaker at 37°C. At different time points (0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, 48 h and 72 h), 1 mL of the liquid outside the dialysis bag was drawn, and then 1 mL of fresh buffer solution of the corresponding pH was added. After the sustained release was completed, the sample liquid was digested with aqua regia, and the characteristic spectral line intensity of the iron element at each time point was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and the cumulative sustained release curve of iron ions was drawn, as shown in FIG. Figure 9 As shown in (b), the 48-h drug release rate of PI-TAF@FN at pH = 6.5 was 40.26%, while at pH = 7.4, the cumulative release of iron ions was 25.5%, indicating that PI-TAF has good pH response performance and is more conducive to the release of iron ions under acidic conditions.

[0092] Example 10

[0093] The PI and PI-TAF@FN prepared in Example 1 were dispersed in 1 mL of deionized water (wherein the ICG concentration was 50 μg / mL), and their temperature rise under 808 nm laser irradiation (10 min) was investigated. Figure 10 As shown in a, at a certain power density (1W / cm 2 ), the heating rate of PI-TAF@FN is greater than that of PI. After 10 minutes of irradiation, the temperature of PI-TAF@FN solution exceeds 50℃. Figure 10 b. Take the PI-TAF@FN prepared in Example (1) and investigate the temperature rise of solutions with different concentrations (0, 50, 100, 200, 300 μg / mL) under 808 nm laser irradiation (10 min). 2), the temperature-raising effect of PI-TAF@FN gradually increased with the increase of concentration. At a concentration of 900 μg / mL, after 10 minutes of irradiation, the temperature of PI-TAF@FN reached 51.2°C, while the temperature of ultrapure water remained basically unchanged. Through a single cycle of heating and cooling curves, the photothermal conversion efficiency of PI-TAF@FN was calculated to be 51.4% ( Figure 10 cd), indicating that PI-TAF@FN has good photothermal conversion performance. Figure 10 e. The photothermal stability of PI-TAF@FN was tested by performing five cycles of heating and cooling. One heating cycle was completed by irradiating the sample with an 808nm laser for 10 minutes, followed by cooling to the starting temperature with the laser turned off, and then another cooling cycle. The figure shows no significant difference in temperature change between the five cycles, demonstrating the excellent photothermal stability of the prepared PI-TAF@FN.

[0094] Example 11

[0095] CNE-2 cells were used as model cells to study the in vitro anti-tumor activity of PI, PI-TAF and PI-TAF@FN prepared in Example 1. CNE-2 cells in the logarithmic growth phase were collected and plated at 1×10 4 The cells were seeded at a density of 100 cells per well in two 96-well plates. The culture medium used was 1640 complete medium supplemented with 100 U / mL penicillin-streptomycin and 10% fetal bovine serum, and incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and complete culture medium containing different concentrations of ICG, PI, PI-TAF and PI-TAF@FN (the ICG concentrations of the materials used were 5, 10, 20, 40, 60, 80, 100, and 120 μg / mL) was added to each well plate. One of the 96-well plates was irradiated with laser for 10 minutes (808 nm, power 1 W / cm 2 After the illumination, the original culture medium was discarded from both plates, and the plates were washed three times with PBS. The culture medium was replaced with serum-free culture medium containing 10% CCK-8 solution. After incubation at 37°C for 2 hours, the absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated based on this value. Cells treated with PBS were used as blank controls, and the cell viability was recorded as 100%. The results are shown in Figure 2. Figure 11As shown in a, within the experimental concentration range, with the increase of ICG concentration, the cytotoxicity of the corresponding PI-TAF and PI-TAF@FN material groups gradually increased. This shows that TAF, as a chemodynamic therapy material, can effectively kill cancer cells and the therapeutic effect is enhanced in a concentration-dependent manner. Compared with the PI-TAF group, the PI-TAF@FN group showed a stronger cell killing effect, which is related to the targeting of FN to tumor cells. The loading of FN enhances the recognition and binding ability of cancer cells and composite materials, thereby improving the killing effect on cancer cells. The cell viability of the laser irradiation group is shown in Figure 1. Figure 11 As shown in b, the cell viability of the PI, PI-TAF, and PI-TAF@FN groups was significantly reduced after laser irradiation. When the ICG concentration was 40 μg / mL, the cell viability was approximately 55.72%, 49.76%, and 45.43%, respectively, indicating that the prepared microsphere drug carrier can mediate photothermal therapy to effectively inhibit the growth of cancer cells.

[0096] Example 12

[0097] CNE-2 cells were cultured at a rate of 2 × 10 5 Cells were seeded at a high density in a 6-well cell culture plate overnight and adhered to the wall. Then, different material groups of ICG, PI, PI-TAF, and PI-TAF@FN (ICG concentration was 40 μg / mL) were cultured in a 37°C incubator under complete medium conditions for 12 hours. After that, all cells in the well plate were washed three times with PBS, digested, centrifuged, and collected. The fluorescence intensity of the samples was detected by flow cytometry ( Figure 12 ab). The experimental results showed that the fluorescence intensity of the PI group was higher than that of the ICG group, indicating that the drug carrier microspheres can effectively deliver drugs to cells. More importantly, the PI-TAF@FN group showed the strongest fluorescence signal, with an intensity significantly higher than that of the PI and PI-TAF groups. This result fully demonstrates that FN coating can significantly enhance the carrier's targeting of tumor cells, thereby significantly improving drug delivery efficiency.

[0098] Example 13

[0099] CNE-2 cells were used as a cell model to evaluate the effects of different materials on intracellular glutathione (GSH) levels. 5The cells were seeded into two 6-well plates at a density of 100 cells / mL. The culture medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% fetal bovine serum. The plates were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and the cells were incubated at 5% CO2 and 37°C for 6 hours with culture medium containing 10% PBS or culture medium containing ICG, PI, PI-TAF, or PI-TAF@FN (ICG concentration was 40 μg / mL). One of the plates was irradiated with laser for 10 minutes (808 nm, power 1 W / cm 2 ). The cells in the two 6-well plates were then digested, centrifuged, and repeatedly frozen and thawed under liquid nitrogen and 37°C. The GSH and GSSG detection kits were then used to detect the intracellular GSH content. The results are shown in the figure. Figure 13 As shown in the figure, both PI-TAF and PI-TAF@FN treated groups can significantly deplete intracellular GSH, and the mechanism is mainly dependent on the redox cycle of iron-tannic acid. It is worth noting that light treatment further enhances the depletion effect of GSH, which is attributed to the triple mechanism of photocatalytic ROS generation, accelerated iron cycle and photothermal synergy. In particular, under laser irradiation conditions, TAF-loaded PI-TAF exhibits a stronger GSH consumption ability than PI. However, under the same concentration conditions, the GSH level of the PI-TAF group is lower than that of the PI-TAF@FN group. This phenomenon can be explained by the fact that the FN coated on the surface of the material specifically targets the α- v β3 integrin significantly enhances the phagocytosis of cells on materials, thereby more effectively consuming intracellular GSH.

[0100] Example 14

[0101] CNE-2 cells were used as a cell model to evaluate the effects of different materials on intracellular ROS levels. 5 The cells were seeded into confocal dishes at a density of 100 cells / mL. The culture medium used was 1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing 10% PBS or culture medium containing ICG, PI, PI-TAF, and PI-TAF@FN (the ICG concentration of the materials used was the corresponding concentration when 40 μg / mL) was added to each well and incubated with the cells at 5% CO2 and 37°C for 12 hours. At the same time, the corresponding light treatment group (808 nm, power 1 W / cm 2The original culture medium was discarded and the cells were incubated with DCFH-DA probe diluted in serum-free culture medium for 30 minutes in the dark. The culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde solution for 15 minutes, washed three times with PBS, and stained with the nuclear dye DAPI for 10 minutes. After washing three times with PBS, the experimental results were observed using a laser confocal microscope. The DCFH-DA probe can be oxidized by ROS in cells to DCF, which displays green fluorescence. The results are shown in Figure 2. Figure 14 As shown in (a), intracellular ROS levels remained unchanged in the ICG and PI groups, whereas they were significantly increased in cells treated with PI-TAF and PI-TAF@FN. This is likely due to the TAF-mediated Fenton-like reaction, which generates a redox cycle and catalyzes ROS production in these treated cells. The PI-TAF@FN group exhibited the highest green fluorescence, demonstrating the targeted production of ROS by FN-loading. Specifically, under laser irradiation, PI, PI-TAF, and PI-TAF@FN exhibited significantly higher ROS levels. This is primarily attributed to the synergistic effects of the photosensitizer's photothermal effect, the photothermally enhanced Fenton reaction of the iron-tannic acid complex, and FN-mediated targeted cancer cell endocytosis, which collectively promote the explosive generation of reactive oxygen species.

[0102] To quantitatively analyze the level of ROS generated in cells, CNE-2 cells in the logarithmic growth phase were collected and plated at 2×10 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate and cultured for 24 h. The original culture medium was discarded and culture medium containing 10% PBS or culture medium containing ICG, PI, PI-TAF and PI-TAF@FN (the ICG concentration of the materials used was the corresponding concentration when 40 μg / mL) was added to each well and incubated with the cells at 5% CO2 and 37°C for 12 h. At the same time, the corresponding light treatment group (808 nm, power 1 W / cm 2 The original culture medium was discarded, the cells were washed with PBS three times, and the cells were collected by trypsin digestion. They were incubated with DCFH-DA probe diluted in serum-free medium for 30 minutes in the dark. After washing with PBS three times, the cells were resuspended in 300 μL PBS and the fluorescence intensity was measured by flow cytometry. Figure 14As shown in Figures (b-c), similar to the results from laser confocal microscopy, ROS levels in cancer cells treated with TAF-containing microspheres increased significantly compared to the PBS group. Laser irradiation also significantly increased ROS levels in cells, indicating that TAF loading endows the microsphere drug carrier with the ability to stimulate cellular ROS production. After loading with FN, ROS levels in cells treated with PI-TAF@FN were even more significantly enhanced, indicating that FN loading promoted the targeted uptake of PI-TAF@FN within cancer cells, enhancing the therapeutic effect.

[0103] Example 15

[0104] CNE-2 cells were used as a cell model to evaluate the lipid peroxidation accumulation effect of different materials prepared in Example 1 on cancer cells. CNE-2 cells in the logarithmic growth phase were collected and plated at 2×10 5 The cells were seeded into confocal dishes at a density of 100 cells / mL. The culture medium used was 1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing 10% PBS or culture medium containing PI, PI-TAF and PI-TAF@FN (the corresponding concentration when the ICG concentration of the materials used was 40 μg / mL) was added to each well and incubated with the cells at 5% CO2 and 37°C for 12 hours. The corresponding light treatment group (808 nm, power 1 W / cm 2 Discard the original culture medium and add 1 μL LPO probe and 500 μL 1640 culture medium to each well in the dark. Incubate in an incubator for 20 minutes. After incubation, wash three times with PBS and fix with 4% paraformaldehyde for 15 minutes. After fixation, stain with DAPI for 5 minutes and observe the red, green and blue fluorescence signals of the cells under a 63x oil microscope. Figure 15 As shown in the figure, compared with the PBS control group, the other groups showed enhanced green fluorescence signals (oxidized C11-BODIPY581 / 591) and correspondingly weakened red fluorescence signals (non-oxidized C11-BODIPY581 / 591). Secondly, the lipid peroxidation level of cancer cells increased significantly after being treated with TAF-containing microsphere materials (PI-TAF, PI-TAF@FN), which was attributed to the Fe 3+ Compared with the single treatment group, the red fluorescence signal intensity of the cells in the PI-TAF@FN+Laser group was further reduced, while the green fluorescence signal intensity was correspondingly increased, indicating that photothermal combined with chemodynamic therapy can significantly promote the accumulation of intracellular LPO.

[0105] Example 16

[0106] CNE-2 cells were used as a cell model to evaluate the killing effects of different materials prepared in Example 1 on cancer cells. CNE-2 cells in the logarithmic growth phase were collected and plated at 2×10 5 The cells were seeded into 6-well plates at a density of 100 cells / mL. The culture medium used was 1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The cells were incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing 10% PBS or culture medium containing ICG, PI, PI-TAF, and PI-TAF@FN (the concentrations of the materials used were the corresponding concentrations when the concentration of ICG was 40 μg / mL) was added to each well and incubated with the cells at 5% CO2 and 37°C for 12 hours. At the same time, the corresponding light treatment group (808 nm, power 1 W / cm 2 , 10 min). Subsequently, all cells in the well plate were digested, centrifuged, and collected. They were incubated with Annexin V-FITC and PI, a cell apoptosis detection reagents diluted 100 times with PBS, for 10 min. Cell apoptosis and necrosis were detected by flow cytometry. Figure 16 As shown, the PI group showed no significant apoptosis, indicating that the material has good cytocompatibility. All experimental groups containing TAF exhibited significant cell killing effects, indicating a significant increase in cell apoptosis caused by the iron ion Fenton reaction, which is due to cell damage caused by chemodynamic treatment. Subsequently, after 808nm laser irradiation, the PI and PI-TAF groups showed enhanced tumor killing ability. After loading with FN, the percentage of cell apoptosis in the PI-TAF@FN group further increased, indicating that the loading of FN enhanced the recognition and specific binding of cancer cells to the composite microspheres, thereby improving the efficiency of cancer cell treatment.

[0107] Example 17

[0108] In order to verify the apoptosis pathway of cancer cells after treatment with the prepared microsphere platform, CNE-2 cells were used as a model to evaluate the effects of different materials in Example 1 on the expression levels of related proteins (GPX4 and Caspase-3). 5Cells were seeded at a density of 100 μg / mL in 6-well plates using RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The cells were incubated at 37°C with 5% CO2 for 24 h. The original medium was discarded, and culture medium containing 10% PBS, PI-TAF@FN, PI+Laser, PI-TAF+Laser, and PI-TAF@FN+Laser ([Fe] = 20 μM) were added and incubated with the cells at 37°C with 5% CO2 for 24 h. After the incubation, the original culture medium was discarded and the cells were washed three times with PBS. Then, the cells in all wells were digested, centrifuged, and collected. The cells were lysed on ice and centrifuged at 4°C, 12000 rpm for 5 minutes. The supernatant protein solution was collected and the protein concentration was determined. Subsequently, SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The content of GPX4 and Caspase-3 in the cells was studied. β-actin was used as an internal reference. The results are shown in Figure 2. Figure 17 As shown in (ac). In the PBS control group, GPX4 and Caspase-3 expression remained stable; in the PI-TAF@FN group, GPX4 decreased to 70%, while Caspase-3 increased by 1.3 times, indicating that ferric tannate alone can induce ferroptosis and apoptosis. In the PI-TAF+L group, GPX4 was significantly downregulated to 40%, while Caspase-3 increased by 2.1 times, confirming that laser treatment synergistically enhances the dual photothermal effect of ferric tannate and ICG, promoting tumor cell apoptosis while simultaneously accelerating ferroptosis. This gradient change reveals the key synergistic mechanism of laser treatment. The introduction of the FN carrier significantly enhanced the therapeutic effect of the PI-TAF complex, as demonstrated by a further decrease in GPX4 protein levels and a significant increase in Caspase-3 activity. This phenomenon indicates that FN modification effectively promotes the specific recognition and uptake of the composite microspheres by cancer cells, thereby more effectively activating the dual cell death pathways of ferroptosis and apoptosis, significantly enhancing the anti-tumor effect.

[0109] Example 18

[0110] All animal experiments were conducted in strict accordance with the standards of the Animal Care Association. Six-week-old female BALB / c nude mice were purchased from the Shanghai Slake Laboratory Animal Center. To investigate the distribution and metabolism of the microspheres in various organs and tissues, the two prepared microspheres (PI-TAF and PI-TAF@FN) were prepared in PBS with an Fe concentration of 50 μg / mL. 2×10 6 CNE-2 cells were inoculated into the inner thigh of black mice and the tumor volume reached 200 mm. 3Around 1 hour, each group of materials (200 μL) were injected peritumorally. Tumor-bearing mice were sacrificed and dissected at different time points (0.5, 1, 3, 6, 12, 24, and 48 hours). Tumor-bearing mice injected with PBS (200 μL) via the tail vein served as blank controls. The heart, liver, spleen, lung, kidney, tumor, and blood were removed and weighed, and then digested with aqua regia for 3-5 days. Finally, the iron content of each sample was determined by ICP-OES, and the iron content of each organ and tumor was calculated ( Figure 18 ).from Figure 18 As can be seen from Figures AB, 24 hours after the peritumoral injection of each group of materials, the iron content of each organ (heart, liver, spleen, lung, and kidney) returned to the level before injection. These results prove that the prepared materials (PI-TA, PI-TAF@FN) can be metabolized and cleared normally in mice. Figure 18 It can be seen that 1 hour after the tail vein injection of each group of materials (PI-TAF, PI-TAF@FN), the relative Fe content in the tumor site increased significantly and reached a peak around 3 hours, which may be due to the retention of materials caused by high permeability and retention effect in the tumor; and after 6 hours, the relative Fe content began to decrease, which may be due to the dissociation of the materials and their metabolism and transport to other organs and tissues of the mice through the blood circulation.

[0111] Example 19

[0112] A CNE-2 subcutaneous transplant tumor model was established in nude mice. 6 CNE-2 cell suspension was inoculated subcutaneously in the right hind limb of mice. 3 The tumor-bearing mice were randomly divided into 6 groups (n=5), as follows: control group (PBS, 200 μL); experimental groups including PI-TAF@FN group, PI+Laser group, PI-TAF+Laser group and PI-TAF@FN+Laser group (ICG dose of 10 mg kg -1 , 200 μL), all administered by peritumoral injection. The treatment start day was defined as day 1, and treatment was performed every 3 days for a total of 4 interventions. The weight changes and tumor volume of the mice were monitored and recorded every 2 days until day 14. The length of the mouse tumor was recorded as L and the width as W. The tumor volume was (L×W 2 ) / 2. After the treatment, the mice in each group were euthanized on the 14th day. Figure 19As shown in (ae), compared with the PBS control group, each treatment group showed different degrees of tumor growth inhibition. Among them, the tumor volume of the PI-TAF@FN group was significantly smaller than that of the control group. This result confirms that the targeting function of FN can effectively promote the enrichment of the microsphere carrier at the tumor site, and the loaded TAF can achieve chemodynamic therapy. It is worth noting that the PI-TAF+Laser group showed a more significant therapeutic effect than the PI+Laser group, which may be attributed to the dual photothermal effect of ICG and TAF in PI-TAF and the synergistic effect with TAF-mediated chemodynamic therapy. More importantly, the PI-TAF@FN+Laser group showed the strongest tumor inhibition effect, which fully demonstrated the multiple synergistic effects of FN-mediated targeted delivery, dual photothermal therapy of ICG and TAF, and chemodynamic therapy.

[0113] Figure 20 The weight change curves of mice in each group during treatment are shown. During the experiment, the weight of mice was measured every 2 days using a precision electronic balance (accuracy 0.1g) and performed at the same time point (9:00-11:00 am) to reduce the impact of diurnal fluctuations. The data showed that the weight of mice in all treatment groups showed a similar growth trend, and no significant weight loss or behavioral abnormalities were observed. This result indicates that the PI-TAF@FN microsphere material and its combined treatment strategy (dual photothermal / chemodynamic therapy) did not cause obvious systemic toxicity reactions and had good in vivo biocompatibility.

Claims

1. A drug-loaded microsphere composite material, characterized in that: The drug-loaded microsphere composite material uses polymer microspheres as carriers, the interior of the polymer microspheres is loaded with drugs, and the surface of the microspheres is coated with a functional layer and a modified protein in sequence.

2. A method for preparing a drug-loaded microsphere composite material, comprising: (1) mixing PLGA porous microspheres and drug solution, heating and stirring to obtain self-healing drug-loaded microspheres; (2) mixing the self-healing drug-loaded microspheres, tannic acid TA solution, and an iron source, and stirring the mixture to react to obtain drug-loaded microspheres coated with tannic acid iron; (3) The drug-loaded microspheres coated with tannic acid iron and the protein solution are mixed and extruded to obtain a drug-loaded microsphere composite material.

3. The preparation method according to claim 2, characterized in that: The preparation of the PLGA porous microspheres comprises: dissolving PLGA in a mixed organic solvent, performing electrospraying, and using a polyvinyl alcohol (PVA) solution as a receiving liquid to collect the microspheres to obtain the PLGA porous microspheres.

4. The preparation method according to claim 3, characterized in that The molecular weight of the PLGA is Mw=20-40 kDa, the viscosity is 1.2-1.5, and the mass concentration of the PLGA is 4-6%; The mixed organic solvent is a mixture of hexafluoroisopropanol (HFIP) and dichloromethane (DCM); The molar ratio of HFIP to DCM is 3:1 to 5:

1.

5. The preparation method according to claim 3, characterized in that: The polyvinyl alcohol (PVA) solution is prepared by dissolving PVA in water to a mass fraction of 1-3%, heating the solution in a water bath to 80-86° C., stirring the solution for 2-4 hours, and placing the solution in a receiving portion of an electrospray device after the solution is completely dissolved and cooled. The molecular weight of the PVA is 20-30 kDa.

6. The preparation method according to claim 3, characterized in that: The process parameters of the electrospray are as follows: adjusting the high voltage power supply to a range of 4 to 10 kV; adjusting the distance between the electrospray needle tip and the receiving device to 10 to 15 cm; and adjusting the flow rate of the propulsion pump to 1 to 2 mL / h.

7. The preparation method according to claim 2, characterized in that: The concentration of the drug solution in step (1) is 0.5-2 mg / mL; the heating and stirring is heating to 40-45° C. and stirring for 2-4 hours; The drug in step (1) includes indocyanine green ICG.

8. The preparation method according to claim 2, characterized in that: The molar ratio of TA, iron source and self-healing drug-loaded microspheres in step (2) is (24:22:1) to (33.52:49.2:1); the iron source is an iron salt; and the solvent of the TA solution is anhydrous ethanol.

9. The preparation method according to claim 2, characterized in that: The solvent of the protein solution in step (3) is deionized water; the concentration of the protein solution is 2 to 4 mg / mL; the mass ratio of the drug-loaded microspheres encapsulating ferric tannate to the protein is 1:2 to 1:6; the protein in step (3) is fibronectin FN; The extrusion in step (3) is repeated 10 to 20 times through a film extruder.

10. Use of the drug-loaded microsphere composite material according to claim 1 or the drug-loaded microsphere composite material prepared by the method according to any one of claims 2 to 9 in preparing contrast agents and / or preparing drugs for chemokinetic / photothermal therapy of tumors.

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