A nanosystem targeting tumor-associated fibroblasts, preparation method and application

Abstract

This paper proposes a tumor-associated fibroblast-targeted nanosystem, consisting of PLGA nanoparticles co-encapsulating anti-fibrotic and anti-aging drugs as the core, and a 4T1 cell membrane expressing a specific FAP chimeric antigen receptor as the biomimetic shell. The present invention constructs FAP-CAR-4T1 cells by embedding FAP scFv onto 4T1 cells. FAP-CAR-CM@PLGA NPs co-encapsulate anti-fibrotic and anti-aging drugs, enabling FAP-CAR-CM@PLGA-AB NPs to target CAFs and SC CAFs, improving radiotherapy resistance and achieving a dual clearance effect while simultaneously relieving the tumor's immunosuppressive microenvironment, effectively reducing breast cancer recurrence after radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically relates to a tumor-associated fibroblast-targeted nanosystem, a preparation method, and an application thereof. Background Art

[0002] Cancer-associated fibroblasts (CAFs) are crucial components of the tumor microenvironment (TME). Located within and around tumor tissue, they serve as crucial stromal cells that mediate the tumor microenvironment and contribute to tumor progression and metastasis. Radiotherapy induces senescence of stromal cells within tumor tissue. Senescent stromal cells, through a senescence-associated secretory phenotype, can produce large quantities of cytokines (such as TGF-β, IL-6, and IL-10), inducing stem-like transformation in surrounding tumor cells, further exacerbating radioresistance. Currently, there is a lack of strategies for simultaneously targeting both CAFs and senescent CAFs (SCCAFs) to improve radioresistance in breast cancer.

[0003] Nanoscale drugs can reduce drug toxicity in vivo while improving therapeutic efficacy, but they suffer from drawbacks such as low drug loading (<10%) and excipient degradation and metabolism. Studies on nanoscale drug delivery systems that simultaneously target and eliminate CAFs and senescent CAFs to improve tumor radioresistance have yet to be reported. Summary of the Invention

[0004] The present invention discloses a nanosystem targeting tumor-associated fibroblasts. The FAP-CAR-4T1 cells are constructed by embedding FAP scFv on 4T1 cells. FAP-CAR-CM@PLGA NPs co-encapsulate anti-fibrosis drugs and anti-aging drugs, so that FAP-CAR-CM@PLGA-AB NPs can achieve a targeted effect on CAFs and SC CAFs, improve radiotherapy resistance, achieve a dual clearance effect, and simultaneously eliminate the tumor immunosuppressive microenvironment, effectively reducing tumor recurrence after radiotherapy.

[0005] The present invention is achieved through the following technical solutions:

[0006] A tumor-associated fibroblast-targeted nanosystem is composed of PLGA nanoparticles co-encapsulating anti-fibrosis drugs and anti-aging drugs as the core, and 4T1 cell membrane expressing specific FAP chimeric antigen receptors as the bionic shell.

[0007] In a preferred embodiment, the anti-fibrosis drug is nintedanib.

[0008] In a preferred embodiment, the anti-aging drug is ABT-263.

[0009] In another aspect of the present invention, a method for preparing a tumor-associated fibroblast-targeting nanosystem is provided, comprising the following steps:

[0010] PLGA-AB NPs were prepared by mixing PLGA, anti-fibrotic drugs, and anti-aging drugs;

[0011] FAP-scfv was embedded in 4T1 cells to construct FAP-CAR-4T1 cells. FAP-CAR-4T1 cells were cultured, cell debris was removed, centrifuged, and precipitated to obtain FAP-CAR-CM.

[0012] PLGA-AB NPs and FAP-CAR-CM were mixed and co-extruded to obtain FAP-CAR-CM@PLGA-AB NPs, which is a tumor-associated fibroblast-targeted nanosystem.

[0013] In a preferred embodiment, the steps of constructing FAP-CAR-4T1 cells include: using FAP scfv as the target protein, obtaining a lentiviral solution by the three-plasmid calcium phosphate method to infect 4T1 cells; obtaining a stably transfected cell line by G418 screening and cloning selection, screening the integration and expression of the target gene in the cell line by PCR detection, and detecting the binding efficiency of FAP-CAR-4T1 cells and fluorescently labeled FAP protein by fluorescence microscopy.

[0014] In a preferred embodiment, PLGA-AB NPs are prepared by an emulsification dispersion method, comprising the following steps:

[0015] S11, dispersing poly(lactic-co-glycolic acid) PLGA, an anti-fibrotic drug, and an anti-aging drug in a volatile solvent to form an oil phase;

[0016] S12, adding PVA to deionized water, heating and stirring, then stopping heating, continuing stirring overnight, and filtering the solution through a membrane to obtain a PVA aqueous solution for preparing nanoparticles;

[0017] S13, adding the oil phase dropwise to the stirred PVA aqueous solution;

[0018] S14, ultrasonically emulsifying the crude emulsion using an ultrasonic disruptor;

[0019] S15, using deionized water to diffuse the volatile solvent into the aqueous phase;

[0020] S16, stirring overnight to allow the volatile solvent in the emulsion to fully evaporate.

[0021] In a preferred embodiment, the volatile solvent is ethyl acetate.

[0022] In a preferred embodiment, FAP-CAR-CM is prepared by ultraviolet irradiation, comprising the following steps:

[0023] S21, FAP-CAR-4T1 cells were cultured in a culture dish. When the cells were fully grown, the dish was opened and placed under ultraviolet light in a clean bench for irradiation;

[0024] S22, after cell irradiation, the culture dish is covered and the cells are placed back into the incubator for further culture;

[0025] S23, the cells were blown off with a pipette tip, and the cell debris was removed by centrifugation. The supernatant was further centrifuged to obtain the precipitate, which was FAP-CAR-CM.

[0026] In a preferred embodiment, the co-extrusion process to obtain FAP-CAR-CM@PLGA-AB NPs specifically includes the following steps:

[0027] S31, PLGA-AB NPs and FAP-CAR-CM resuspended in PBS were mixed; the PLGA concentration of PLGA-AB NPs was 2 mg / mL, and the membrane protein concentration of FAP-CAR-CM was 0.5 mg / mL;

[0028] S32, the two were co-extruded through a 1 μm filter membrane using a liposome extruder, with the extrusion times being 7 times, and the product was FAP-CAR-CM@PLGA-AB NPs.

[0029] In another aspect of the present invention, a nanosystem targeting tumor-associated fibroblasts is provided for use in inhibiting tumor cells, or in preparing / serving as a drug for inhibiting tumors.

[0030] In a preferred embodiment, the tumor is breast cancer.

[0031] The beneficial effects produced by the present invention are:

[0032] (1) Cleverly designing a CAFs-targeted, highly biomimetic nanosystem to achieve the combined delivery of the anti-aging drug ABT-263 and the anti-fibrotic drug nintedanib while effectively reducing the side effects of both.

[0033] (2) Innovatively combining targeted dual clearance of CAFs, especially senescent CAFs, with remodeling of the tumor immune microenvironment, to provide a new strategy for reducing tumor recurrence after radiotherapy;

[0034] (3) The biomimetic nanosystem constructed by genetically engineered tumor cells can effectively avoid off-target phenomena during delivery and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 A is a graph showing GFP fluorescence expression in transfected 4T1 cells and untreated 4T1 cells during the construction of FAP-CAR-4T1 cells in this example.

[0037] Figure 1 B is the morphology of the nanoparticles, where Figure 1 B(a) is the morphology of FAP-CAR-CM. Figure 1 B(b) is the morphology of PLGA-ABNPs. Figure 1 B(c) is the morphology of FAP-CAR-CM@PLGA-AB NPs.

[0038] Figure 1 C is the electrophoresis diagram of FAP-CAR-CM, PLGA-AB NPs, and FAP-CAR-CM@PLGA-AB NPs.

[0039] Figure 1 D is the particle size diagram of FAP-CAR-CM, PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs.

[0040] Figure 1 E is the potential diagram of FAP-CAR-CM, PLGA-AB NPs, and FAP-CAR-CM@PLGA-AB NPs.

[0041] Figure 1 F: PLGA-AB NPs were dispersed in PBS and FBS, respectively, and stored in a refrigerator at 4 °C. The hydrated particle size was measured for 5 consecutive days.

[0042] Figure 1 G: FAP-CAR-CM@PLGA-AB NPs were dispersed in PBS and FBS, respectively, and stored in a refrigerator at 4 °C. The hydrated particle size was measured for 5 consecutive days.

[0043] Figure 1 H is a comparison of the encapsulation efficiency of ABT-263 and nintedanib in PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs.

[0044] Figure 1J is the cumulative release curve of ABT-263 from PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs.

[0045] Figure 1 K is the cumulative release curve of nintedanib from PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs.

[0046] Figure 2 Comparison of the viability of Free AB, CM@PLGA-A NPs, CM@PLGA-B NPs, FAP-CAR-CM@PLGA-A NPs, FAP-CAR-CM@PLGA-B NPs and FAP-CAR-CM@PLGA-AB NPs on CAFs cells at different concentrations of ABT-263 and nintedanib.

[0047] Figure 3 Comparison of cell viability of Free AB, CM@PLGA-A NPs, CM@PLGA-BNPs, FAP-CAR-CM@PLGA-A NPs, FAP-CAR-CM@PLGA-B NPs and FAP-CAR-CM@PLGA-AB NPs after 24 hours of treatment with ABT-263 and nintedanib at different concentrations.

[0048] Figure 4 Comparison of the cytotoxicity of Free AB, CM@PLGA-A NPs, CM@PLGA-B NPs, FAP-CAR-CM@PLGA-A NPs, FAP-CAR-CM@PLGA-B NPs and FAP-CAR-CM@PLGA-AB NPs against 4T1 cells at different concentrations of ABT-263 and nintedanib.

[0049] Figure 5 A is the growth curve of the tumor in the mice of different experimental groups after tumor implantation.

[0050] Figure 5 B is the weight change curve of mice in different experimental groups after tumor implantation.

[0051] Figure 5 C: The tumor volume of the PBS group reached 1500 mm 3 After the above, the mice were sacrificed and the morphology of tumor tissues in different experimental groups was taken out.

[0052] Figure 5 D is Figure 5 C. Tumor weights recorded in different experimental groups.

[0053] Figure 5 E is Figure 5 In C, the tumor inhibition rate of treated mice was calculated based on the tumor tissue weights recorded in different experimental groups. DETAILED DESCRIPTION

[0054] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0055] Nintedanib is a small molecule tyrosine kinase receptor inhibitor primarily used clinically for idiopathic pulmonary fibrosis. Studies have shown that nintedanib targets CAFs. ABT-263 is a novel small molecule inhibitor of anti-apoptotic proteins with high affinity for Bcl-xL, Bcl-w, and Bcl-2 proteins. It can target these key proteins for senescent cell survival and selectively eliminate them.

[0056] Fibroblast activation protein (FAP) is an important molecular marker on the surface of CAFs. It is highly specifically expressed only in activated fibroblasts, and more than 90% of breast cancer fibroblasts highly express FAP.

[0057] To achieve precise delivery of the anti-fibrotic drug nintedanib and the anti-aging drug ABT-263, this example constructs a nanocarrier with long-circulating function and specific targeting to CAFs. Using the membrane of 4T1 cells expressing the specific FAP chimeric antigen receptor as a biomimetic shell, nintedanib and ABT-263 are co-encapsulated within PLGA nanoparticles to form the core, creating a highly biomimetic nanodelivery system targeting CAFs, namely FAP-CAR-CM@PLGA-AB NPs. Leveraging the long-circulating and homing effects of highly biomimetic nanodelivery, nintedanib accumulates in the tumor microenvironment. Through FAP scFv-mediated delivery, active targeting of breast cancer CAFs and senescent CAFs is achieved. Nintedanib efficiently kills CAFs, while ABT-263 effectively eliminates senescent CAFs, suppressing their SASP and cancer stem cell phenotype, thereby improving radiotherapy resistance. This dual-clearing effect simultaneously relieves the tumor's immunosuppressive microenvironment and effectively reduces breast cancer recurrence after radiotherapy.

[0058] 1. Construction of FAP-CAR-4T1 cells

[0059] Using FAP scFv as the target protein, a lentiviral solution was generated using the three-plasmid calcium phosphate method to infect 4T1 cells. Stable transfected cell lines were obtained through G418 screening and cloning selection. PCR was used to screen the integration and expression of the target gene in the cell lines. Fluorescence microscopy was used to examine the binding efficiency of FAP-CAR-4T1 cells and fluorescently labeled FAP protein. Immunofluorescence observation showed that the transfected 4T1 cells highly expressed GFP fluorescence, while untreated 4T1 cells did not express green GFP ( Figure 1 A) Since GFP is fused with FAP scFv, it indirectly proves that the transfected 4T1 cells express FAP scFv, and our FAP-CAR-4T1 cells were successfully constructed.

[0060] 2. Preparation of the highly biomimetic nanosystem FAP-CAR-CM@PLGA-AB NPs

[0061] 2.1 Preparation of PLGA-AB NPs by emulsification dispersion method. The process is as follows:

[0062] (1) Disperse 20 mg of poly(lactic-co-glycolic acid) copolymer (PLGA) (weight average molecular weight Mw 24,000-38,000), 0.1 mg of nintedanib, and 0.1 mg of ABT-263 in 1 mL of ethyl acetate to form an oil phase;

[0063] (2) Add 2 g of PVA to 100 mL of deionized water, stir at 60-70 °C for 6-7 h, then stop heating and continue stirring overnight. Pass the solution through a 0.22 μm filter membrane to obtain a 2% PVA aqueous solution for preparing nanoparticles.

[0064] (3) Add the oil phase dropwise to the stirred water phase and continue stirring for 1 h;

[0065] (4) Ultrasonic emulsification of the crude emulsion was performed using an ultrasonic disruptor with an ultrasonic power of 500 J and an ultrasonic time of 30 s.

[0066] (5) Use 6 mL of deionized water to diffuse ethyl acetate into the aqueous phase;

[0067] (6) Stir overnight to allow the ethyl acetate in the emulsion to evaporate completely.

[0068] 2.2 Preparation of FAP-CAR-CM by UV irradiation:

[0069] (1) FAP-CAR-4T1 cells were cultured in a 10 cm culture dish. When the cells were fully grown, the dish was opened and placed under ultraviolet light in a clean bench for 1 h.

[0070] (2) After 1 hour of cell irradiation, close the lid of the culture dish and place the cells back into the incubator for a further 16 hours.

[0071] (3) Blow off the cells with a pipette tip and centrifuge at 3000 g for 15 min to remove cell debris. Centrifuge the supernatant at 10000 g for another 1 h. The resulting precipitate is FAP-CAR-CM.

[0072] 2.3 Preparation of FAP-CAR-CM@PLGA-AB NPs by co-extrusion method:

[0073] (1) PLGA-AB NPs (PLGA concentration of 2 mg / mL) and FAP-CAR-CM (membrane protein concentration of 0.5 mg / mL) resuspended in PBS were mixed;

[0074] (2) The two were co-extruded through a 1 μm filter membrane using a liposome extruder for 7 times. The product was FAP-CAR-CM@PLGA-AB NPs, which was stored at 4 °C.

[0075] 3. Characterization of FAP-CAR-CM@PLGA-AB NPs

[0076] The morphology, particle size and potential of the nanoparticles were analyzed by transmission electron microscopy and dynamic light scattering particle size analyzer. Figure 1 As shown in Figure B(a), the particle size distribution of FAP-CAR-4T1 cell-derived membrane vesicles (FAP-CAR-CM) ranges from 50 to 500 nm, with a significant concentration between 300 and 400 nm. Further DLS analysis reveals that the particle size distribution is primarily between 600 and 700 nm. This is due to the presence of a hydration layer, which slightly increases the hydrated particle size compared to that observed under transmission electron microscopy. DLS also measures the surface zeta charge of -8.0 ± 0.4 mV, further confirming that FAP-CAR-CM is derived from cell-derived membrane vesicles.

[0077] like Figure 1 As shown in Figure 1(b), the prepared PLGA-AB NPs were observed by TEM and found to be relatively uniform in size, with a smooth spherical morphology and no obvious aggregation. Their hydrated particle size was around 180 nm (Figure 1D) and the zeta potential was -14.6 ±1.0 mV. This was because one end of the PLGA polymer chain had a free carbonic acid and was negatively charged.

[0078] like Figure 1As shown in Figure B(c), FAP-CAR-CM@PLGA-AB NPs were obtained by co-extrusion of the prepared FAP-CAR-CM and PLGA-AB NPs. TEM observation revealed a distinct core-shell structure. DLS analysis revealed a hydrated particle size of approximately 200 nm, approximately 20 nm larger than that of PLGA-AB NPs. The zeta potential was -10.8 ± 0.17 mV, significantly higher than that of PLGA-AB NPs. These results demonstrate the successful preparation of FAP-CAR-CM@PLGA-AB NPs, comprising FAP-CAR-CM encapsulated with PLGA-AB NPs.

[0079] The membrane proteins of FAP-CAR-4T1, FAP-CAR-CM and FAP-CAR-CM@PLGA-AB NPs were analyzed by gel electrophoresis. Figure 1 As shown in (C), the protein profiles of FAP-CAR-CM and FAP-CAR-CM@PLGA-AB NPs were consistent with those of FAP-CAR-4T1 cells, indicating that the membrane proteins were well preserved after coextrusion.

[0080] PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs were dispersed in PBS and FBS, respectively, and stored in a refrigerator at 4°C. The hydrated particle size was measured for 5 consecutive days to investigate their storage stability. The results showed that the hydrated particle size of PLGA-AB NPs in PBS and FBS was 177.4 ± 1.10 nm and 194.2 ± 2.18 nm on the first day, and 181.7 ± 4.17 nm and 196.6 ± 4.47 nm on the fifth day (e.g. Figure 1 F), the hydrated particle sizes of FAP-CAR-CM@PLGA-AB NPs in PBS and FBS were 196.6 ± 4.47 and 209.3 ± 14.8 nm on the first day, and 199.2 ± 0.40 and 238.0 ± 13.0 nm on the fifth day (e.g. Figure 1 G), the changes were all within the normal range and were negligible, indicating that both NPs had good storage stability.

[0081] The encapsulation efficiency of ABT-263 and nintedanib in PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs was detected by high performance liquid chromatography. Figure 1As shown in Figure 3, when the dosage of ABT-263 and nintedanib was 0.1 mg, the encapsulation efficiencies of the two drugs in PLGA-AB NPs were 53.7 ± 10.05% and 36.1 ± 5.7%, respectively. The encapsulation efficiencies of the two drugs in FAP-CAR-CM@PLGA-AB NPs were 36.4 ± 8.08% and 18.2 ± 2.8%, respectively. This may be due to drug loss caused by membrane retention during the co-extrusion of PLGA-AB NPs and FAP-CAR-CM.

[0082] The cumulative release curves of ABT-263 and nintedanib from PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs in PBS at pH = 7.4, which simulates the normal physiological environment, were investigated. The results showed that after 24 h of in vitro release, the cumulative release rates of ABT-263 from PLGA-AB NPs and FAP-CAR-CM@PLGA-AB NPs reached 50.57 ± 1.10% and 41.51 ± 0.8%, respectively (Fig. Figure 1 J), nintedanib was 60.12 ± 2.53% and 48.99 ± 2.19% (e.g. Figure 1 K). The cumulative release rates of ABT-263 and nintedanib from FAP-CAR-CM@PLGA-AB NPs were both reduced, which may be due to the fact that the FAP-CAR-CM coated on the periphery of PLGA-AB NPs affected the release of the two drugs, which further confirmed the successful construction of FAP-CAR-CM@PLGA-AB NPs.

[0083] 4. In vitro antitumor activity of FAP-CAR-CM@PLGA-AB NPs

[0084] To evaluate the cytotoxicity of FAP-CAR-CM@PLGA-AB NPs on CAFs, we applied Free AB, CM@PLGA-A NPs, CM@PLGA-B NPs, FAP-CAR-CM@PLGA-A NPs, FAP-CAR-CM@PLGA-B NPs, and FAP-CAR-CM@PLGA-AB NPs within a certain dose range to CAFs.

[0085] MTT assay results Figure 2For example, at a 3 μM concentration of both ABT-263 and nintedanib, the cell viability of CAFs co-incubated with FAP-CAR-CM@PLGA-AB NPs was 35.99 ± 9.66%. The cell viabilities of CAFs co-incubated with Free AB, CM@PLGA-A NPs, CM@PLGA-B NPs, FAP-CAR-CM@PLGA-A NPs, and FAP-CAR-CM@PLGA-B NPs were 80.98 ± 6.59%, 56.36 ± 8.65%, 65.78 ± 8.65%, 41.87 ± 1.97%, and 53.80 ± 5.77%, respectively. The cytotoxicity of FAP-CAR-CM@PLGA-AB NPs to CAFs was consistently much stronger than that of Free AB, likely due to the enhanced uptake of CAFs by the FAP-CAR-CM modification. When administered at the same ABT-263 concentration, FAP-CAR-CM@PLGA-A NPs exhibited a generally stronger cytotoxic effect on CAFs than CM@PLGA-A NPs. Similarly, when administered at the same nintedanib concentration, FAP-CAR-CM@PLGA-B NPs exhibited a generally stronger cytotoxic effect on CAFs than CM@PLGA-B NPs. These results further demonstrate that the FAP scFv modified on FAP-CAR-CM specifically binds to FAP on CAFs, ultimately enabling the FAP-CAR-CM-coated nanoparticles to achieve targeted cytotoxicity against CAFs. Furthermore, the cytotoxic effect of FAP-CAR-CM@PLGA-AB NPs on CAFs was consistently stronger than that of FAP-CAR-CM@PLGA-A NPs and FAP-CAR-CM@PLGA-B NPs, indicating a synergistic effect between ABT-263 and nintedanib on CAF cytotoxicity. In summary, all the results showed that FAP-CAR-CM@PLGA-ABNPs had a good killing effect on CAFs.

[0086] To investigate the in vitro toxicity of FAP-CAR-CM@PLGA-AB NPs on CAFs, FreeAB, CM@PLGA-A NPs, CM@PLGA-B NPs, FAP-CAR-CM@PLGA-A NPs, FAP-CAR-CM@PLGA-B NPs, and FAP-CAR-CM@PLGA-AB NPs within a certain concentration range were treated with SC CAFs for 24 h, and the cell viability of SC CAFs was determined by CCK-8. The results are shown in Figure 2. Figure 3As shown, when both ABT-263 and nintedanib concentrations were 1 μM, the cell viabilities of SC CAFs co-incubated with CM@PLGA-A NPs and FAP-CAR-CM@PLGA-A NPs were 43.54 ± 3.67% and 34.43 ± 3.87%, respectively. The cell viabilities of SC CAFs co-incubated with CM@PLGA-B NPs and FAP-CAR-CM@PLGA-B NPs were 91.88 ± 5.56% and 72.27 ± 11.06%, respectively. This is because ABT-263 itself is a potent anti-aging drug that can effectively kill senescent cells. Nintedanib, an anti-fibrotic drug, has a strong cytotoxic effect on CAFs, but no studies have shown its potent killing effect on senescent cells. Furthermore, the results also showed that FAP-CAR-CM@PLGA-A NPs and FAP-CAR-CM@PLGA-B NPs had stronger cytotoxicity against SC CAFs than CM@PLGA-A NPs and CM@PLGA-B NPs. This suggests that the modification of FAPscfv enables FAP-CAR-CM to specifically bind to FAP on SC CAFs, enhancing the uptake of NPs by SC CAFs and thus achieving a targeted killing effect on SC CAFs. Furthermore, the killing effect of FAP-CAR-CM@PLGA-AB NPs was consistently stronger than that of FAP-CAR-CM@PLGA-A NPs and FAP-CAR-CM@PLGA-B NPs, indicating that ABT-263 and nintedanib have a synergistic effect on the cytotoxicity of SC CAFs. In summary, FAP-CAR-CM@PLGA-AB NPs have a good targeted killing effect on SC CAFs.

[0087] Finally, the cytotoxicity of FAP-CAR-CM@PLGA-AB NPs to 4T1 cells was investigated. Figure 4As shown. After 24 hours of co-incubation with each drug, the cell viability of 4T1 cells decreased, indicating that each drug group had a killing effect on 4T1 cells. The killing effect of each drug group on 4T1 cells was dose-dependent within the dose range of 0.1-3 μM. When the concentration of ABT-263 and nintedanib was 1 μM, the cell viability of 4T1 cells in the CM@PLGA-A NPs group was 57.34 ± 3.62%, in the CM@PLGA-B NPs group was 69.69 ± 5.05%, in the FAP-CAR-CM@PLGA-A NPs group was 39.17 ± 2.65%, in the FAP-CAR-CM@PLGA-B NPs group was 52.73 ± 4.38%, in the FAP-CAR-CM@PLGA-AB NPs group was 30.43 ± 2.60%, and in the Free AB group was 35.43 ± 4.89%. The results show that both ABT-263 and nintedanib exhibit cytotoxicity against 4T1 cells. The FAP-CAR-CM@PLGA-AB NPs group exhibited stronger cytotoxicity against 4T1 cells than Free AB. This is because the nanoscale size of FAP-CAR-CM@PLGA-AB NPs facilitated NP uptake by 4T1 cells, and the FAP-CAR-CM modification enabled membrane fusion between the NPs and 4T1 cells. The 4T1 cell viability in the FAP-CAR-CM@PLGA-AB NPs group was lower than that in the FAP-CAR-CM@PLGA-A NPs and FAP-CAR-CM@PLGA-B NPs groups, indicating that ABT-263 and nintedanib have a synergistic effect on 4T1 cell killing. In summary, FAP-CAR@PLGA-AB NPs exhibited a strong cytotoxic effect against 4T1 cells.

[0088] 5. In vivo antitumor effect of FAP-CAR-CM@PLGA-AB NPs

[0089] To investigate the in vivo antitumor effects of FAP-CAR-CM@PLGA-AB NPs, Babl / c mice bearing breast cancer were randomly divided into seven groups: PBS, PBS + IR, Free AB, Free AB + IR, PLGA-AB NPs, FAP-CAR-CM@PLGA-AB NPs, and FAP-CAR-CM@PLGA-AB NPs + IR. 4T1 cells (1×10^5) were co-injected with SC CAFs (2×10^5) into the second mammary pad of Babl / c mice. Drugs were injected via the tail vein on days 6, 9, 12, and 16 after tumor implantation. On day 17 after tumor implantation, mice were irradiated with a dose of 8 Gy. Body weight and tumor volume were measured every two days in each group. Figure 5 A shows the tumor growth curve. First, it can be seen that the tumor growth in the FAP-CAR-CM@PLGA-AB NPs + IR group was the slowest and the inhibitory effect was the best, followed by FAP-CAR-CM@PLGA-AB NPs, then PLGA-AB NPs, Free AB + IR, PBS + IR, and Free AB.

[0090] In addition, the weight change curve Figure 5 B shows that there was no significant change in the body weight of mice in the FAP-CAR-CM@PLGA-AB NPs+IR group, indicating that FAP-CAR-CM@PLGA-AB NPs had excellent biosafety. 3 After the above, the mice were sacrificed, the tumor tissues were removed, and photos were taken (e.g. Figure 5 C) and record the tumor weight (eg Figure 5 D), and then the tumor inhibition rate of treated mice was calculated. The results are shown in Figure 5 As shown in Figure E, the tumor inhibition rates of FAP-CAR-CM@PLGA-AB NPs+IR and FAP-CAR-CM@PLGA-AB NPs were as high as 86.69 ± 3.63% and 82.29 ± 5.90%, respectively. The tumor inhibition rate of PLGA-AB NPs was 63.59 ± 9.66%, and the tumor inhibition rates of Free AB+IR and PBS+IR were 50.17 ± 15.07%, 42.58 ± 25.53%, and 39.49 ± 17.54%, respectively. This result is consistent with the tumor growth curve, indicating that FAP-CAR-CM@PLGA-AB NPs have a strong tumor inhibitory effect.

[0091] In summary, FAP-CAR-CM@PLGA-AB NPs are simple to prepare, have high drug loading capacity, good stability, high safety and good antitumor effect, and are a very potential antitumor drug.

Claims

1. A nanosystem targeting tumor-associated fibroblasts, characterized in that: The biomimetic shell is formed by PLGA nanoparticles that co-encapsulate anti-fibrosis drugs and anti-aging drugs as the core and 4T1 cell membranes that express specific FAP chimeric antigen receptors as the biomimetic shell. The anti-fibrosis drug is nintedanib, and the anti-aging drug is ABT-263.

2. A method for preparing the tumor-associated fibroblast-targeting nanosystem according to claim 1, characterized in that: The following steps are involved: PLGA-AB NPs were prepared by mixing PLGA, anti-fibrotic drugs, and anti-aging drugs; FAP-scfv was embedded in 4T1 cells to construct FAP-CAR-4T1 cells. FAP-CAR-4T1 cells were cultured, cell debris was removed, centrifuged, and precipitated to obtain FAP-CAR-CM. PLGA-AB NPs and FAP-CAR-CM were mixed and co-extruded to obtain FAP-CAR-CM@PLGA-AB NPs, which is a tumor-associated fibroblast-targeted nanosystem.

3. The method for preparing a tumor-associated fibroblast-targeting nanosystem according to claim 2, wherein: The steps for constructing FAP-CAR-4T1 cells include: using FAP scfv as the target protein, obtaining a lentiviral solution using the three-plasmid calcium phosphate method to infect 4T1 cells; obtaining a stably transfected cell line through G418 screening and cloning selection, screening the integration and expression of the target gene in the cell line by PCR, and detecting the binding efficiency of FAP-CAR-4T1 cells and fluorescently labeled FAP scfv protein using a fluorescence microscope.

4. The method for preparing a tumor-associated fibroblast-targeting nanosystem according to claim 2, wherein: PLGA-AB NPs were prepared by emulsification dispersion method, which includes the following steps: S11, dispersing poly(lactic-co-glycolic acid) PLGA, an anti-fibrotic drug, and an anti-aging drug in a volatile solvent to form an oil phase; S12, adding PVA to deionized water, heating and stirring, then stopping heating, continuing stirring overnight, and filtering the solution through a membrane to obtain a PVA aqueous solution for preparing nanoparticles; S13, adding the oil phase dropwise to the stirred PVA aqueous solution; S14, ultrasonically emulsifying the crude emulsion using an ultrasonic disruptor; S15, using deionized water to diffuse the volatile solvent into the aqueous phase; S16, stirring overnight to allow the volatile solvent in the emulsion to fully evaporate.

5. The method for preparing a tumor-associated fibroblast-targeting nanosystem according to claim 4, wherein: The volatile solvent is ethyl acetate.

6. The method for preparing a tumor-associated fibroblast-targeting nanosystem according to claim 2, wherein: The FAP-CAR-CM was prepared by UV irradiation, which included the following steps: S21, FAP-CAR-4T1 cells were cultured in a culture dish. When the cells were fully grown, the dish was opened and placed under ultraviolet light in a clean bench for irradiation; S22, after cell irradiation, the culture dish is covered and the cells are placed back into the incubator for further culture; S23, the cells were blown off with a pipette tip, and the cell debris was removed by centrifugation. The supernatant was further centrifuged to obtain the precipitate, which was FAP-CAR-CM.

7. The method for preparing a tumor-associated fibroblast-targeting nanosystem according to claim 2, wherein: Specific properties of FAP-CAR-CM@PLGA-AB NPs obtained by co-extrusion The following steps are involved: S31, PLGA-AB NPs and FAP-CAR-CM resuspended in PBS were mixed; the PLGA concentration of PLGA-AB NPs was 2 mg / mL, and the membrane protein concentration of FAP-CAR-CM was 0.5 mg / mL; S32, the two were co-extruded through a 1 μm filter membrane using a liposome extruder, with the extrusion times being 7 times, and the product was FAP-CAR-CM@PLGA-AB NPs.

8. Use of the tumor-associated fibroblast-targeting nanosystem according to claim 1 in the preparation of a drug for inhibiting breast cancer.

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