Bionic nanoparticle MDI-CCM as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of pharmaceutical preparations, and provides bionic nanoparticles MDI-CCM as well as a preparation method and application thereof. Bionic nanoparticles MDI-CCM are constructed, based on mesoporous silica nanoparticles loaded with doxorubicin (DOX) and indocyanine green (ICG), acid-sensitive controlled-release column [5] arene (WP [5] as a nano valve) is constructed on the surface, a tumor cell membrane is coated to realize homologous targeting of tumor cells, the nano valve triggers controllable release of drugs in a tumor microenvironment, and the drug delivery effect is improved. DOX chemotherapy and ICG near-infrared laser photo-thermal effect form synergy, and the treatment effect on 4T1 breast cancer tumors is enhanced. Experimental results show that the MDI-CCM can significantly inhibit tumor growth, has the characteristics of low toxicity, high drug loading capacity, active targeting and intelligent controlled release, can be applied to homologous targeting and chemotherapy-photothermal synergistic treatment of cancers such as breast cancer, and provides new insight and basis for related treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a biomimetic nanoparticle MDI-CCM and a preparation method and application thereof. Background Art

[0002] Breast cancer (BC) is a malignant tumor caused by genetic mutations in mammary epithelial cells under the influence of multiple oncogenic factors, leading to uncontrolled cell proliferation. Chemotherapy is a common treatment modality used throughout the early, middle, and late stages of cancer. However, drug bioavailability in the body is low, and its cytotoxic effects affect both cancer cells and normal cells, leading to increased cytotoxicity in normal cells and a higher incidence of drug resistance. Photothermal therapy (PTT) uses photothermal agents to convert near-infrared laser (NIR) light energy into thermal effects, ablating tumor cells. Unlike surgery and chemotherapy, PTT is non-invasive and has minimal toxic side effects. However, PTT alone has limited tumor-killing effects and is limited by its monotherapy nature.

[0003] In recent years, nanoparticle drug delivery systems have attracted considerable attention in the field of targeted tumor therapy. They can enhance and maintain the clinical efficacy of chemotherapeutic drugs with minimal side effects. However, they still suffer from drawbacks such as removal by the reticuloendothelial system (RES) and inefficient tumor accumulation. Research has shown that nanoparticle surfaces can be modified with membranes derived from different cell types or biological vesicles to enhance biocompatibility and targeting efficiency. Tumor cell membranes (CCMs) contain specific proteins and receptors that can evade immune surveillance while enhancing homologous targeting of tumor cells. Consequently, coating drug carriers with CCMs to prepare biomimetic nanoparticles has become an effective approach for achieving active targeting and enhancing therapeutic efficacy.

[0004] Doxorubicin (DOX) is a common chemotherapy drug for breast cancer, but its application is limited due to problems such as the easy development of drug resistance. Indocyanine green (ICG) can convert NIR light energy into heat energy, causing local temperature rise in the tumor site and damaging tumor cells, thereby achieving the purpose of tumor inhibition. Mesoporous silica nanoparticles (MSNs) are good drug loading carriers due to their porous structure, large specific surface area and easy surface functionalization. Carboxylate column[5]arene can be combined with pyridine-modified MSNs (MSN-Py) through electrostatic interaction. In slightly acidic tumor sites, the interaction between the two is weakened due to the protonation of column[5]arene (WP[5]), causing it to fall off the surface of MSNs, achieving the purpose of drug release.

[0005] In view of the shortcomings of existing breast cancer treatment technologies and the potential of tumor cell membrane modification in nanomedicine targeted therapy, the present invention proposes a biomimetic nanoparticle MDI-CCM and its preparation method and application. Summary of the Invention

[0006] The purpose of the present invention is to provide a biomimetic nanoparticle MDI-CCM and a preparation method and application thereof, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: A biomimetic nanoparticle MDI-CCM, comprising: mesoporous silica nanoparticles loaded with doxorubicin and indocyanine green; A surface-constructed acid-sensitive controlled-release nanovalve, wherein the nanovalve is pillar[5]arene, i.e., WP[5]; Encapsulated tumor cell membranes; Among them, the tumor cell membrane achieves homologous targeting, the nanovalve triggers controlled drug release under acidic or warm conditions, doxorubicin kills tumor cells through chemotherapy, and indocyanine green produces photothermal effect under near-infrared laser irradiation, forming a chemotherapy-photothermal synergistic treatment effect.

[0008] Furthermore, the loading ratio of doxorubicin and indocyanine green is 1:2.

[0009] Furthermore, the tumor cell membrane is a 4T1 cell membrane, and its membrane protein is completely retained on the surface of the nanoparticles by an extrusion method.

[0010] A method for preparing the biomimetic nanoparticle MDI-CCM according to the above-mentioned method comprises the following steps: Mesoporous silica nanoparticles (MSN-OH) bearing hydroxyl groups were prepared by hexadecyltrimethylammonium bromide template method. MSN-OH was modified with chlorine groups by (3-chloropropyl)trimethoxysilane and then modified with pyridine to obtain pyridine-modified mesoporous silica nanoparticles MSN-Py; Doxorubicin and indocyanine green were loaded on MSN-Py in a ratio of 1:2, and then WP[5] was added to obtain nanovalve-modified drug-loaded mesoporous silica nanoparticles MDI-WP[5]; 4T1 cell membranes were extracted and coated on MDI-WP[5] by extrusion method to obtain biomimetic nanoparticles MDI-CCM.

[0011] Furthermore, the ratio of the 4T1 cell membrane to MDI-WP[5] was 1×10 7 4T1 cell membranes correspond to 1 mgMDI-WP[5].

[0012] A use of the biomimetic nanoparticle MDI-CCM described above in the preparation of a drug for treating breast cancer.

[0013] A pharmaceutical composition comprises the above-mentioned biomimetic nanoparticle MDI-CCM and pharmaceutically acceptable excipients.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a biomimetic nanoparticle, MDI-CCM, based on mesoporous silica nanoparticles loaded with doxorubicin (DOX) and indocyanine green (ICG). It features an acid-sensitive controlled-release nanovalve on its surface and is coated with tumor cell membranes. This coating achieves homologous targeting of tumor cells, triggering controlled drug release in the acidic, warm tumor microenvironment via the nanovalve. DOX kills tumor cells through chemotherapy, while ICG generates photothermal activity under near-infrared laser irradiation, leading to a local temperature increase. This accelerates DOX release while simultaneously killing the tumor, enhancing the therapeutic effect against 4T1 breast cancer and creating a synergistic chemotherapy-photothermal effect. Experimental evaluations have demonstrated that MDI-CCM exhibits excellent dispersibility, morphological integrity, and biocompatibility. The synergistic chemotherapy-photothermal therapy it mediates significantly inhibits tumor growth. The MDI-CCM exhibits low toxicity, high drug loading, active tumor cell targeting, and intelligent controlled release properties. This reduces systemic drug side effects while enhancing therapeutic efficacy. This biomimetic nanodelivery system is suitable for homologous targeting and synergistic chemotherapy-photothermal therapy for breast cancer and other cancers. This study provides new insights into chemotherapy-photothermal synergistic therapy mediated by tumor cell membrane biomimetic carriers, and also provides a basis for developing new drug delivery systems with targeted delivery, intelligent controlled release characteristics and reduced systemic toxicity to improve the anti-tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of scanning electron microscopy are shown in Figure 2. A is the SEM image of MSN-OH; B is the SEM image of MSN-OH based on the results of scanning electron microscopy. Figure 1 A is the size distribution histogram of MSN-OH; C is the SEM image of MDI-CCM; D is the size distribution histogram of MSN-OH based on Figure 1 Size distribution histogram of MDI-CCM in Figure C (scale bar: 200 nm).

[0016] Figure 2 Characterization of physical and chemical properties; A is the TEM image of MDI-CCM; B is the hydrodynamic diameter distribution of MDI-CCM determined by DLS; C is the Zeta potential value of MSN-OH, MSN-Py, MSN-WP[5] and MDI-CCM nanoparticles; D is the FT-IR spectrum of MSN-OH, MSN-Py, MSN-WP[5] and MDI-CCM.

[0017] Figure 3 Elemental mapping images of MDI-CCM (scale: 100 nm); A is the grayscale morphology of MDI-CCM; B is the C element mapping of MDI-CCM; C is the N element mapping of MDI-CCM; D is the O element mapping of MDI-CCM; E is the Si element mapping of MDI-CCM; F is the P element mapping of MDI-CCM; G is the S element mapping of MDI-CCM; H is the Cl element mapping of MDI-CCM.

[0018] Figure 4 The results of membrane protein analysis using Coomassie Brilliant Blue are shown in Figure 1. I: 4T1 cell membrane protein; II: MDI-WP[5]; III: MDI-CCM.

[0019] Figure 5 Figure 2 is the effect of different pH and temperature on the in vitro release of MDI-CCM; A is the effect of different temperatures on the in vitro release of MDI-CCM; B is the effect of different pH on the in vitro release of MDI-CCM.

[0020] Figure 6 The cytotoxicity of different concentrations of MSN-WP[5]@CCM on 4T1, B16 and BV2 cells was investigated; A shows the cytotoxicity of different concentrations of MSN-WP[5]@CCM on 4T1 cells; B shows the cytotoxicity of different concentrations of MSN-WP[5]@CCM on B16 cells; C shows the cytotoxicity of different concentrations of MSN-WP[5]@CCM on BV2 cells.

[0021] Figure 7Results of cytotoxicity experiments; A is the determination of the killing ability of free ICG on 4T1 cells under light-shielded conditions; B is the determination of the killing ability of free DOX, MDI-CCM and MDI-CCM+NIR under a power density of 1.0 W / cm 2 The killing ability of 4T1 cells was determined after 808 nm laser irradiation for 10 min; C is the IC value of the drug in B 50 Fitting curve; D is the investigation of the cell killing ability of MDI-CCM+NIR on BV2, B16, and 4T1 cells (****P<0.0001).

[0022] Figure 8 Fluorescence images of 4T1 cells incubated with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h. Cell nuclei were stained with Hoechst 33342 in blue, DOX in red, and ICG in green (scale bar: 50 μm).

[0023] Figure 9 Fluorescence microscopic images of B16 and 4T1 cells incubated with MDI-CCM for 4 h, respectively. The cell nuclei were stained with Hoechst 33342 in blue, DOX in red, and ICG in green (scale bar: 50 μm).

[0024] Figure 10 Figure 3 Flow cytometric analysis of the uptake of DOX and ICG by 4T1 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h, and the mean fluorescence intensities of DOX and ICG. A shows the uptake of DOX by 4T1 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; B shows the mean fluorescence intensity of DOX by 4T1 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; C shows the uptake of ICG by 4T1 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; D shows the mean fluorescence intensity of ICG by 4T1 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h.

[0025] Figure 11Figure 3 Flow cytometric analysis of the uptake of DOX and ICG by B16 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h, and the mean fluorescence intensities of DOX and ICG. A shows the uptake of DOX by B16 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; B shows the mean fluorescence intensity of DOX by B16 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; C shows the uptake of ICG by B16 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h; D shows the mean fluorescence intensity of ICG by B16 cells after incubation with MDI-CCM for 0 h, 1 h, 2 h, 4 h, and 6 h.

[0026] Figure 12 The photothermal ablation effects of different treatment groups on cells (scale bar: 100 μm).

[0027] Figure 13 The in vivo distribution of MDI-WP[5] and MDI-CCM at different time points after intravenous injection in BALB / C mice bearing 4T1 tumors (n=3).

[0028] Figure 14 The mean fluorescence intensity of major organs and tumor sites in BALB / C mice bearing 4T1 tumors 24 h after intravenous injection of MDI-WP[5] and MDI-CCM (n=3, **P<0.01, ***P<0.001).

[0029] Figure 15 The power density was 1 W / cm after intravenous injection of MDI-WP[5] and MDI-CCM. 2 Infrared thermal imaging results of nude mice bearing 4T1 tumors after irradiation with 808 nm NIR for 10 min.

[0030] Figure 16 Figure 3 is the curve of changes in mouse body weight and tumor volume during treatment; A is the curve of changes in mouse tumor volume (n=5); B is the curve of changes in mouse body weight (n=5).

[0031] Figure 17 These are H&E-stained photographs of the main organs of mice after treatment (scale bar: 100 μm). DETAILED DESCRIPTION

[0032] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0033] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0034] Example 1: Preparation and characterization of MDI-CCM; Preparation of hydroxyl-bearing mesoporous silica nanoparticles (MSN-OH): 0.5 g of hexadecyltrimethylammonium bromide, 250 mL of ultrapure water, and 1.8 mL of 2 M aqueous NaOH solution were mixed and placed in a 500 mL three-necked flask. The mixture was heated to 80°C in an oil bath and stirred for 30 minutes. Subsequently, 2.5 mL of ethyl orthosilicate was added, and the reaction was continued at 80°C with stirring for 2 hours. After the reaction, the mixture was filtered while hot and washed alternately with deionized water and methanol three times (20 mL each). The mixture was dried in a vacuum oven at room temperature overnight to obtain silica nanoparticles (MSNs). Subsequently, 200 mg of MSNs were dispersed in 20 mL of methanol containing 1% hydrochloric acid and stirred at 65°C for 6 hours. After the reaction, the mixture was centrifuged at 10,000 rpm for 15 min. The resulting white precipitate was washed alternately with methanol and ultrapure water three times (20 mL each time), and then dried in a vacuum drying oven at room temperature overnight to obtain mesoporous silica nanoparticles MSN-OH with hydroxyl groups.

[0035] Preparation of pyridine (Py)-modified mesoporous silica nanoparticles (MSN-Py): 100 mg of MSN-OH was dispersed in 10 mL of toluene and sonicated for 30 minutes. Subsequently, 0.2 mL of (3-chloropropyl)trimethoxysilane was quickly added under nitrogen, and the mixture was stirred and refluxed at 120°C for 24 hours. After the reaction, the reaction mixture was centrifuged at 10,000 rpm for 15 minutes. The supernatant was discarded and the resulting mixture was dispersed in a mixture of 10 mL of toluene and 1 mL of pyridine. The mixture was stirred and refluxed at 120°C for 24 hours under nitrogen. The resulting product was washed alternately with anhydrous ethanol and ultrapure water three times (20 mL each time) and then dried in a vacuum oven at room temperature to obtain pyridine-modified mesoporous silica nanoparticles (MSN-Py).

[0036] Preparation of drug-loaded mesoporous silica nanoparticles MDI-WP[5] modified with nanovalve: drug loading was performed according to the mass ratio of DOX:ICG=1:2. That is, 5 mg of MSN-Py was dissolved in 3 mL of ultrapure water, and after sonication to fully dissolve it, 1.5 mg of DOX and 3 mg of ICG were added and stirred for 24 h in the dark. Subsequently, 9 mg of column[5] aromatic hydrocarbon (WP[5], as a nanovalve) was added and stirred for 24 h in the dark. After the reaction was completed, centrifugation was performed at 10,000 rpm for 15 min, washed with ultrapure water 3 times, and the precipitate was collected and placed in a vacuum drying oven for drying at room temperature to obtain drug-loaded mesoporous silica nanoparticles MDI-WP[5] modified with nanovalve. At the same time, the supernatant after centrifugation was collected for the determination of the drug loading amount of DOX and ICG.

[0037] In addition, the preparation method of nanovalve-modified mesoporous silica nanoparticles (MSN-WP[5], MSN-ICG) without DOX and ICG or without DOX was the same as above, except that DOX and ICG or without DOX was added.

[0038] Preparation of biomimetic nanoparticles MDI-CCM: 4T1 cells (about 1×10 7 After digestion with trypsin, the cell fluid was collected and centrifuged at 4°C and 600 g for 5 min, and the supernatant was retained. Subsequently, 500 μL of membrane protein extraction solution A containing 2 μL of PMSF was added to the centrifuge tube. After ice bathing for 15 min, it was placed in a 37°C water bath for 30 s, and repeatedly frozen and thawed 3 times, each time for 30 s. Subsequently, it was centrifuged at 4°C and 700 g for 10 min, and the supernatant was aspirated. The supernatant was then centrifuged at 4°C and 14,000 g for 30 min to precipitate the cell membrane fragments. Finally, the precipitate was retained and the cell membrane fragments were suspended in 0.5 mL of sterile water for later use. 1×10 7 The cell membrane of MDI-WP[5] was coated on the surface of MDI-WP[5] at a ratio of 4T1 cell membranes to 1 mg of MDI-WP[5]. 1 mg of MDI-WP[5] was ultrasonically suspended in 0.5 mL of sterile water and slowly added dropwise to the cell membrane suspension obtained above. The mixture was passed through 400 nm and 200 nm polycarbonate membranes in an extruder in sequence. The extrusion operation was repeated 15 times for each pore size membrane to finally obtain biomimetic drug-loaded nanoparticles MDI-CCM.

[0039] In addition, the preparation method of biomimetic nanoparticles without DOX or without ICG and DOX (MSN-ICG@CCM, MSN-WP[5]@CCM) was the same as above, except that MDI-WP[5] was replaced by the corresponding MSN-ICG or MSN-WP[5].

[0040] Experimental results: like Figure 1 As shown in Figures AD, MSN-OH and MDI-CCM are both approximately spherical ( Figure 1 A and C), the average particle size of MSN-OH is about 137.4 nm ( Figure 1 Middle B), the average particle size of MDI-CCM after coating is about 150 nm ( Figure 1 Middle D).

[0041] like Figure 2 As shown in Figures AD, the morphology and size of MDI-CCM were further characterized by TEM and DLS. TEM results showed that MDI-CCM had a clear core-shell structure ( Figure 2 (A) The cell membrane thickness is approximately 8 nm. Figure 2 The DLS results of B were similar to those of Figure 1 The average particle size of the coated MDI-CCM is about 150 nm, which is consistent with the data shown in D, indicating that the prepared MDI-CCM has good uniformity. Then, the Zeta potential of the sample was tested using a Malvern laser particle size analyzer. Figure 2 As shown in Figure C, the surface potential of MSN-OH is -33 mV, while MSN-Py presents a positive charge (+12.5 mV). After the modification of the nanovalve, the potential of MSN-WP[5] turns negative (-5.5 mV), indicating the successful modification of the nanovalve. After drug loading and cell membrane coating, the potential of the nanocarrier further decreases (-27.3 mV), which is caused by the modification of the negatively charged cell membrane, thus proving the successful coating of the cell membrane. At the same time, the Fourier transform infrared spectroscopy analysis results are shown in Figure 4. Figure 2 As shown in D, at 1523 cm -1 The appearance of the characteristic peak at 1279 cm is due to the stretching vibration of the benzene ring, which directly proves the successful modification of Py on the surface of MSN-OH; and the peaks of MSN-WP[5] and MDI-CCM at 1279 cm -1 There is an absorption peak at , which corresponds to the CH stretching mode of the aromatic ring, thus confirming the successful modification of WP[5] on the MSN-Py surface.

[0042] Depend on Figure 3 As can be seen from Figures AH, the chlorine (Cl) element is evenly distributed on the particle surface as a modification intermediate. At the same time, the evenly distributed area of ​​common elements in tumor cell membranes (such as C, N, O, P, S, etc.) is slightly larger than that of silicon (Si), which is consistent with the coating characteristics of tumor cell membranes. The results further indicate the successful coating of tumor cell membranes on nanocarriers.

[0043] MDI-CCM biomimetic nanoparticles mainly rely on surface proteins of tumor cell membranes to achieve homologous targeting. Therefore, it is necessary to explore whether MDI-CCM prepared by extrusion can completely retain the proteins on the 4T1 cell membrane. The proteins in 4T1 cell membrane, MDI-WP[5] and MDI-CCM were analyzed by SDS-PAGE. Figure 4 As shown in the figure, the protein bands in the MDI-CCM pores are clear and essentially identical to those in the 4T1 cell membrane pores, indicating that the tumor cell membrane proteins encapsulated in the MDI-CCM are well preserved, demonstrating that the MDI-CCM preparation process does not result in membrane protein loss. These results demonstrate the complete encapsulation of the tumor cell membrane, ensuring the active targeting effect of MDI-CCM.

[0044] Example 2: In vitro evaluation of MDI-CCM; 2.1 Investigation of in vitro drug release performance of MDI-CCM; The in vitro drug release performance of MDI-CCM was tested using dynamic dialysis, which was divided into two dimensions: pH response and temperature response. Considering the generally acidic pH at the tumor site, to investigate the release of DOX from MDI-CCM at different pH values, 1 mL of 500 μg / mL MDI-CCM was transferred to a dialysis bag with a molecular weight cutoff of 14 kDa. The bag was then placed in 4 mL of PBS buffer (release medium) with different pH values ​​(5.7, 6.4, and 7.4). Under magnetic stirring, the absorbance of DOX was measured at 482 nm at different time points, and the DOX release amount was calculated and the release curve was plotted.

[0045] To investigate the release of DOX from MDI-CCM at different temperatures, 1 mL of 500 μg / mL MDI-CCM was transferred to a 14 kDa dialysis bag and placed in 4 mL of PBS buffer (release medium) at different temperatures (37°C, 45°C, and 60°C). Under magnetic stirring, the absorbance of DOX was measured at 482 nm at different time points to calculate the release amount and plot the release curve.

[0046] like Figure 5 As shown in Figures A and B. Under physiological conditions (pH = 7.4), the release rate of DOX from MDI-CCM was slow, while under pathological conditions (pH 5.7 and pH 6.4), the release rate of DOX gradually accelerated with the increase of acidity, but no severe burst release occurred ( Figure 5 In addition, the release rate of DOX also accelerated with increasing temperature ( Figure 5(A in the middle). This is due to the varying release behavior of DOX under different conditions, fundamentally stemming from the varying binding strengths between Py and the nanovalve. Under physiological conditions, the nanovalve binds more strongly to Py, inhibiting premature drug release. However, in the acidic tumor microenvironment or under simulated photothermal conditions, the nanovalve is triggered and "opens," enabling controlled drug release. This mechanism demonstrates that MDI-CCM can precisely control the release of loaded drugs through the nanovalve, minimizing drug loss during delivery. Furthermore, based on the absorbance of the supernatant collected after centrifugation during preparation, the DOX loading in the MDI-CCM was calculated to be 127 mg / g.

[0047] 2.2 Cytotoxicity study; In order to study the cytotoxicity of MSN-WP[5]@CCM, the MTT method was used to study the cell viability after incubation with different cells. The specific operation was as follows: 4T1 cells, B16 cells and BV2 cells in the logarithmic growth phase were collected and incubated with 1×10 4 The cells were seeded at a density of 100 μg / well in a 96-well plate and incubated overnight at 37°C with 5% CO2. Different concentrations of MSN-WP[5]@CCM were added to the corresponding wells and incubated at 37°C for 24 h and 48 h, respectively. After the incubation, MTT reagent was added to each well and incubated for another 4 h. The formazan crystals were dissolved in DMSO. The absorbance was measured at 490 nm using a microplate reader and the cytotoxicity of MSN-WP[5]@CCM was calculated using the following formula: ; Among them, A e is the absorbance of the experimental group, A0 is the absorbance of the control group, A C is the absorbance of the blank group.

[0048] The results are as follows Figure 6 As shown, 4T1 cells ( Figure 6 Middle A), B16 cells ( Figure 6 Middle B) and BV2 cells ( Figure 6 (C) After incubation with different concentrations of MDI-CCM for 24 h and 48 h, the cell viability was higher than 80%, indicating that the biomimetic nanoparticles MSN-WP[5]@CCM have good biosafety.

[0049] In order to study the killing ability of free DOX, free ICG and MDI-CCM on tumor cells under the condition of light and verify the synergistic chemotherapy-photothermal therapy ability of MDI-CCM, the inhibitory effect of different drug groups on 4T1 cells was detected by MTT assay. The specific operation is as follows: 4T1 cells in the logarithmic growth phase were collected and seeded into 96-well cell culture plates. The culture plates were placed in a cell culture incubator and incubated overnight. The next day, the culture medium was discarded, and 100 μL of different concentrations of drugs were added to each group. At the same time, each well of the light group was illuminated at 808 nm and 1 W / cm 2 After 10 min of near-infrared laser irradiation, the cells were placed in an incubator overnight. MTT reagent was added and the cells were incubated for another 4 h. The culture medium was then removed with a syringe, DMSO was added, the cells were shaken, and the cells were incubated at 37°C for 10 min. The cell absorbance was measured at 490 nm using a microplate reader, and the cell survival rate and cell half inhibition rate (IC) were calculated using GraphPad Prism 9. 50 To further verify the targeting ability of MDI-CCM, the MTT assay was used to examine the inhibitory effects of MDI-CCM combined with light on the growth of 4T1 cells, B16 cells, and BV2 cells, and to investigate its targeting ability. The specific procedures were the same as above.

[0050] The results are as follows Figure 7 As shown in Figure AD. First, the cell-killing ability of free ICG in the dark was studied. Figure 7 The results in Figure A show that the viability of cells treated with free ICG in the concentration range of 0-800 μg / mL was above 80%, indicating that free ICG had no obvious killing ability on cells under light-proof conditions. Figure 7 Figure B is the study of the cell killing ability of MDI-CCM with or without light at different DOX concentrations, and the drug IC is plotted based on the results. 50 The fitting curve, that is Figure 7 C. The results showed that as the DOX concentration increased, cell viability decreased and cell killing ability increased. Compared with the free DOX treatment group, the cell viability of the MDI-CCM treatment group with the same free DOX drug concentration was significantly reduced. This was due to the controlled release of DOX by MDI-CCM and the active targeting effect on the 4T1 tumor cell membrane. In addition, compared with other groups, the cell killing ability of the MDI-CCM+NIR group was stronger, proving that chemotherapy and photothermal therapy have synergistic advantages. Figure 7 As shown in Figure D, compared with B16 and BV2, MDI-CCM had a more significant killing effect on 4T1 cells under the same conditions, further verifying the homologous targeting effect of MDI-CCM.

[0051] 2.3 Investigation of the cell uptake ability of MDI-CCM; In order to study the uptake capacity of 4T1 cells for MDI-CCM, the fluorescence distribution of DOX and ICG in cells was observed by confocal laser scanning microscope (CLSM). The specific operation was as follows: cells in logarithmic growth phase were collected and 4T1 cells were plated at 2×10 5 Cells were seeded at a density of 100 cells / well onto a 12-well plate with a slide mounted on the bottom. The plates were cultured in an incubator for 12 hours, and the supernatant medium was discarded. After careful washing with sterile PBS and evaporation, MDI-CCM solutions were prepared at a concentration of 0.15 μg / mL DOX and incubated with the cells for varying times. Following incubation, the cells were washed twice with sterile PBS to remove any residual MDI-CCM. Each well was fixed with 4% paraformaldehyde for 15 minutes at room temperature, and the supernatant was discarded. After washing with PBS, the slides were stained with Hoechst 33342 anti-quenching mounting medium. The slides were then inverted onto the mounting medium. Laser confocal microscopy was used to observe cell nuclei using the Hoechst 33342 channel, DOX using the Cy3 channel, and ICG using the FITC channel to detect ICG and DOX cellular uptake and intracellular colocalization.

[0052] The results are as follows Figure 8 As shown in the figure, after incubation with MDI-CCM for 1 h, 2 h, 4 h, and 6 h, the red fluorescence intensity of DOX and the green fluorescence intensity of ICG in 4T1 cells increased with the extension of incubation time, indicating that the cellular uptake of MDI-CCM increased over time. In addition, the red fluorescence of DOX and the green fluorescence of ICG overlapped with the blue fluorescence of the cell nucleus, indicating that DOX and ICG had been released from MDI-CCM and internalized into the 4T1 cell nucleus. To further verify the targeting effect of MDI-CCM, MDI-CCM was incubated with 4T1 cells and B16 cells for 4 h, respectively. The results are shown in the figure. Figure 9 As shown in the figure, after 4 h of co-incubation with MDI-CCM, the fluorescence intensity of 4T1 cells was significantly higher than that of B16 cells, indicating that 4T1 cells took up more MDI-CCM than B16 cells, which indicates that MDI-CCM can homologously target the same type of cells.

[0053] Flow cytometry was used to quantitatively investigate the uptake of MDI-CCM by 4T1 cells. The main procedures were as follows: 4T1 cells were plated at 3×10 5Cells were plated at a density of 100 cells / well in a 12-well plate and cultured in an incubator for 12 hours. The original culture medium was discarded, and the cells were washed twice with PBS (pH 7.4). Depending on the time point, medium containing MDI-CCM (DOX concentration of 0.15 μg / mL) was added and incubated for different time points. The medium was discarded, and the cells were washed twice with PBS (pH 7.4). The cells were harvested and resuspended in sterile PBS. After washing twice, each well was fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then analyzed by flow cytometry, with three replicates injected per group. DOX fluorescence was analyzed in the PE channel and ICG fluorescence in the APC-A750 channel. The data were quantified using FlowJo software.

[0054] The flow cytometry results were as follows Figure 10 AD and Figure 11 As shown in Figures A to D, the fluorescence shift in both 4T1 and B16 cells increased significantly with increasing incubation time, indicating a positive correlation between cellular uptake efficiency and time. This result is consistent with the qualitative uptake analysis of 4T1 cells by confocal laser scanning. Furthermore, after 6 hours of incubation with MDI-CCM, the mean fluorescence intensities of DOX and ICG in 4T1 cells were approximately 1.9 times those in B16 cells. This is due to the fact that the MDI-CCM coated with the 4T1 cell membrane is internalized through self-recognition of the tumor cell membrane and selectively "homing" to the homologous tumor in vivo, achieving homologous targeting and further enhancing the uptake efficiency of the homologous cancer cells.

[0055] 2.4 Photothermal ablation capability assessment; To evaluate the photothermal ablation ability of different drug administration groups, live / dead cells were stained and recorded by fluorescence microscopy. 4T1 cells were collected and plated at 4 × 10 5 The cells were incubated overnight in a 6-well plate at a density of 100 cells / well. The cells were divided into 6 groups, including PBS (Control group), NIR, ICG, MSN-ICG@CCM, ICG+NIR, and MSN-ICG@CCM+NIR. After the corresponding treatment, they were incubated for 4 h and then heated with 1.0 W / cm 2 The NIR, ICG+NIR, and MSN-ICG@CCM+NIR groups were irradiated with 808 nm near-infrared lasers for 10 minutes, respectively. The culture medium was removed, and the cells were washed three times with PBS. 0.5 mL of Calcein-AM / PI staining solution was added to each well and incubated in the dark for 45 minutes. The cells were then washed and observed under a fluorescence microscope.

[0056] The results are as follows Figure 12As shown, compared with the control group, cells in the NIR, ICG, and MSN-ICG@CCM groups appeared green (Calcein-AM staining for live cells), indicating that 808 nm laser irradiation alone or with a photothermal agent but without NIR irradiation had no significant cell killing effect, demonstrating the safety of NIR therapy and the low cytotoxicity of the nanodrug delivery system. Compared with the aforementioned treatment groups, some cells in the ICG+NIR group appeared red (PI staining for dead cells), while cells in the MSN-ICG@CCM+NIR group were almost entirely red, demonstrating the excellent photothermal ablation capability of MSN-ICG@CCM+NIR.

[0057] Example 3: In vivo evaluation of MDI-CCM; 3.1 In vivo distribution experiments; To investigate the targeting ability of MDI-CCM in mice and its accumulation at the tumor site, in vivo imaging experiments were performed on mice. ICG is a common contrast agent in clinical practice and can be used as a fluorescent probe. 7 The tumor-bearing mouse model was established by inoculating 4T1 tumor cells at a concentration of 100 μL / mouse, diluted in 3 mg / mL Matrigel. When the tumor volume reached 150 mm 3 Around 24 hours after injection, 4T1 model mice were randomly divided into three groups and injected intravenously with PBS, MDI-WP[5], and MDI-CCM, respectively. The fluorescence of the tumor site was observed at 2, 4, 8, and 24 hours using an in vivo imaging system. Mice were killed 24 hours after injection, and the tumor and major organs including heart, liver, spleen, lung, and kidney were taken for ex vivo fluorescence imaging. The fluorescence intensity of the tumor and major organs was quantified using software.

[0058] The results are as follows Figure 13 As shown in the figure, compared with the MDI-WP[5] group, the MDI-CCM group had obvious fluorescence signals at the tumor site, and the fluorescence intensity increased with the extension of injection time, almost reaching the peak at 8 hours, and gradually weakened after 24 hours. The quantitative results of in vitro fluorescence imaging showed that ( Figure 14 ), the mean fluorescence intensity of the tumor in the MDI-CCM group (5.735×10 7 ) is the mean fluorescence intensity of the tumor in the MDI-WP[5] group (3.431×10 7 This is likely due to its homologous targeting ability to the tumor cell membrane. These results demonstrate that MDI-CCM possesses excellent targeting capabilities and is expected to be further applied in in vivo targeted drug delivery.

[0059] 3.2 Investigation of photothermal effects in vivo; In order to investigate the photothermal effect in vivo, a near-infrared thermal imaging experiment was conducted: tumors with a volume of 150 mm were selected.3 The corresponding drugs were injected into the tail vein of mice. 4 h later, the PBS+NIR group, MDI-WP[5]+NIR group and MDI-CCM+NIR group were treated with a power density of 1.0 W / cm 2 The tumors were irradiated with 808 nm laser for 10 min, and the temperature changes at the tumor site were recorded using a near-infrared thermal imager.

[0060] The results are as follows Figure 15 As shown, the body temperature of mice in the PBS+NIR group did not change significantly, while the temperature of the tumor site of mice in the MDI-WP[5] group rose to 45.6°C, indicating that the ICG photothermal conversion efficiency was excellent. The temperature of the tumor site in the MDI-CCM+NIR group was higher than that in the MDI-WP[5]+NIR group, reaching 50.2°C, indicating that the targeting ability of the tumor cell membrane enabled MDI-CCM to accumulate at the tumor site, increasing the local ICG concentration in the tumor, thereby further increasing the temperature of the tumor site.

[0061] 3.3 In vivo anti-tumor efficacy and biosafety assessment; By subcutaneously injecting 6 × 10 7 cell / mL of 4T1 tumor cells (100 μL / mouse) were used to establish a tumor-bearing mouse model. When the tumor volume reached 100 mm 3 Around 24 hours after injection, mice were randomly divided into PBS, PBS+NIR, free DOX (5 mg / kg), free ICG+NIR, DOX+ICG+NIR, MSN-WP[5]@CCM, MDI-WP[5]+NIR, MDI-CCM and MDI-CCM+NIR (equivalent to 5 mg / kg free DOX) groups, with 5 mice in each group. On days 0, 3, 6, 9 and 12, mice were treated with 5 mg / kg DOX via tail vein injection. At the same time, 4 hours after injection, the PBS+NIR group, DOX+ICG+NIR group, MDI-WP[5]+NIR group and MDI-CCM+NIR group were injected with a power density of 1.0 W / cm 2 The tumor site was irradiated with an 808 nm laser for 10 minutes. The body weight and tumor volume of the mice were measured and recorded every three days, and time-weight and time-tumor volume curves were plotted. After treatment, the mice were euthanized, and their major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining to assess potential side effects of the prepared nanodrug delivery system on these organs.

[0062] The results are as follows Figure 16 and 17As shown, the tumor volume of the PBS group, PBS+NIR group and MSN-WP[5]@CCM group increased rapidly during the experiment, and the tumor inhibition effect of the ICG+NIR treatment group was slight. The therapeutic effect of the free DOX group was better than that of the ICG+NIR group, but lower than that of the DOX+ICG+NIR group, indicating that DOX chemotherapy has an inhibitory effect on tumors, and that the chemotherapy-photothermal synergistic treatment effect is better than that of DOX alone. The therapeutic effect of the MDI-CCM group was stronger than that of the DOX group but lower than that of the DOX+ICG+NIR group, indicating that the cell membrane-coated nanocarriers can enhance the killing effect of DOX on tumors, but because there is no laser irradiation, its therapeutic effect is weaker than the synergistic treatment effect of DOX+ICG+NIR. The therapeutic effect of the MDI-WP[5]+NIR group was better than that of the DOX+ICG+NIR group, which also proved that nanocarriers can enhance the therapeutic effect. The results showed that the MDI-CCM+NIR group had the best therapeutic effect on tumors, which effectively proved that tumor cell membrane-coated nanoparticles can improve the therapeutic effect of tumors through homologous targeting ability ( Figure 16 At the same time, the weight change curve of mice is as follows Figure 16 As shown in Figure B, there was no significant change in the body weight of mice in each treatment group during the treatment period, indicating that the drugs in each group had no significant side effects on the mice. In addition, no obvious inflammatory damage or tissue damage was observed in all major organs collected after treatment in different treatment groups ( Figure 17 ), which indicates that the prepared nanodrug delivery system can be used as a potential drug delivery platform with good biocompatibility for synergistic treatment of breast cancer.

[0063] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A biomimetic nanoparticle MDI-CCM, characterized in that: include: mesoporous silica nanoparticles loaded with doxorubicin and indocyanine green; A surface-constructed acid-sensitive controlled-release nanovalve, wherein the nanovalve is pillar[5]arene, i.e., WP[5]; Encapsulated tumor cell membranes; Among them, the tumor cell membrane achieves homologous targeting, the nanovalve triggers controlled drug release under acidic or warm conditions, doxorubicin kills tumor cells through chemotherapy, and indocyanine green produces photothermal effect under near-infrared laser irradiation, forming a chemotherapy-photothermal synergistic treatment effect.

2. The biomimetic nanoparticle MDI-CCM according to claim 1, characterized in that The loading ratio of doxorubicin and indocyanine green is 1:

2.

3. The biomimetic nanoparticle MDI-CCM according to claim 1, characterized in that: The tumor cell membrane is a 4T1 cell membrane, and its membrane protein is completely retained on the surface of the nanoparticles through an extrusion method.

4. A method for preparing the biomimetic nanoparticle MDI-CCM according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mesoporous silica nanoparticles (MSN-OH) bearing hydroxyl groups were prepared by hexadecyltrimethylammonium bromide template method. MSN-OH was modified with chlorine groups by (3-chloropropyl)trimethoxysilane and then modified with pyridine to obtain pyridine-modified mesoporous silica nanoparticles MSN-Py; Doxorubicin and indocyanine green were loaded on MSN-Py in a ratio of 1:2, and then WP[5] was added to obtain nanovalve-modified drug-loaded mesoporous silica nanoparticles MDI-WP[5]; 4T1 cell membranes were extracted and coated on MDI-WP[5] by extrusion method to obtain biomimetic nanoparticles MDI-CCM.

5. The preparation method according to claim 4, characterized in that The ratio of 4T1 cell membrane to MDI-WP[5] was 1×10 7 4T1 cell membranes correspond to 1 mg of MDI-WP[5].

6. Use of the biomimetic nanoparticle MDI-CCM according to any one of claims 1 to 3 in preparing a drug for treating breast cancer.

7. A pharmaceutical composition, characterized in that The biomimetic nanoparticle MDI-CCM comprises the biomimetic nanoparticle MDI-CCM according to any one of claims 1 to 3 and pharmaceutically acceptable excipients.

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