Tumor-targeted photo-thermal chemotherapy drug delivery system and preparation method and application thereof

Abstract

The invention belongs to the technical field of biology and medicine, and particularly relates to a tumor targeted photo-thermal chemotherapy drug delivery system and a preparation method and application thereof. According to the tumor targeted photo-thermal chemotherapy drug delivery system provided by the invention, a core carrier adopts a black phosphorus nanosheet (BPNS) and ferroferric oxide (Fe3O4) composite structure, BPNS generates heat under irradiation of near-infrared light to realize photo-thermal therapy, and Fe3O4 endows a magnetic targeting function; the medicine part is a ferroptosis inducer RSL3, glutathione (GSH) is reduced and reactive oxygen species (ROS) is increased through an inhibition system Xc-, and tumor cell ferroptosis is induced; the targeting modification layer is liver cancer cell membrane protein (Pro), and liver cancer cell surface receptors are specifically recognized through a homologous targeting mechanism, so that the enrichment efficiency of drugs in tumor tissues is improved. The system can significantly improve the treatment effect and reduce the toxicity to normal tissues, and provides a new solution for liver cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medicine, and particularly relates to a tumor-targeted photothermal chemotherapy drug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors globally, with high morbidity and mortality rates over a long period. Although current clinical treatment methods have covered various options such as surgical resection, chemotherapy, radiotherapy, interventional therapy, targeted therapy, and immunotherapy, due to problems such as tumor heterogeneity, metastasis and recurrence risks, and drug resistance, the overall treatment effect is still difficult to break through the bottleneck.

[0003] In the direction of precision treatment of malignant tumors, there are still obvious technical shortcomings in existing targeting strategies: Although magnetic targeting technology can guide drug enrichment to the tumor area through an external magnetic field, it is difficult to achieve precise cell-level positioning due to the influence of the complex tumor microenvironment; Although molecular targeting technology is based on the specific recognition mechanism of tumor surface markers, due to the high heterogeneity of liver cancer cells and the dynamic change characteristics of targets, problems such as off-target effects and acquired drug resistance often occur.

[0004] In recent years, the development of nanobiotechnology has provided new ideas for tumor diagnosis and treatment integration. Researchers have integrated molecular imaging and treatment functions by developing multifunctional nanomaterials, and among them, image-guided photothermal therapy (PTT) has shown significant advantages. This technology uses the local heat effect (42 - 45 °C) generated by irradiating photosensitizers with near-infrared light to selectively kill tumor cells. Among many photothermal materials, two-dimensional black phosphorus (BP) nanosheets have become one of the most promising candidate materials due to their high photothermal conversion efficiency, degradable characteristics, and good biocompatibility. Multiple in vitro and in vivo studies have confirmed their photothermal treatment effect.

[0005] However, the clinical translation of PTT still faces key technical obstacles: First, traditional nanodrug delivery systems generally have problems such as poor biocompatibility and poor controllability of drug release. Conventional nanoparticles are easily cleared quickly by the mononuclear phagocytic system, resulting in a shortened effective circulation time; Second, the dense extracellular matrix in tumor tissues forms a physical barrier, resulting in insufficient penetration depth of nanoparticles; Third, non-specific photothermal effects are likely to cause irreversible damage to surrounding normal tissues. This off-target thermal damage and the thermal tolerance of tumor cells together lead to a significant narrowing of the treatment window. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides a tumor-targeted photothermal chemotherapy drug delivery system, a preparation method thereof, and an application thereof.

[0007] The object of the present invention is to provide a preparation method and application of a tumor-targeted photothermal chemotherapy nano-drug delivery system. Through the dual strategies of molecular targeting and magnetic targeting, this system realizes precise treatment of tumor cells, increases the enrichment amount of drugs in tumor tissues, enhances the combined treatment effect of targeted photothermal chemotherapy, and reduces the toxic and side effects on normal tissues.

[0008] In the first aspect of the present invention, a tumor-targeted photothermal chemotherapy drug delivery system is provided, which includes a composite carrier formed by black phosphorus nanosheets and iron oxide nanoparticles, the ferroptosis inducer RSL3 loaded on the composite carrier, and an outer layer of liver cancer cell membrane protein modification layer; the magnetic responsiveness of the iron oxide nanoparticles in the composite carrier endows the system with the ability of directional enrichment under an external magnetic field, and the black phosphorus nanosheets have near-infrared light responsiveness; the liver cancer cell membrane protein modification layer enables the system to have homologous targeting and immune escape characteristics; the ferroptosis inducer RSL3 induces ferroptosis programmed cell death by regulating the glutathione metabolic pathway.

[0009] Among them, the black phosphorus nanosheets and the iron oxide nanoparticles form a composite carrier through electrostatic adsorption or chemical bonding, and the iron oxide nanoparticles are distributed on the surface of the black phosphorus nanosheets.

[0010] Among them, the ferroptosis inducer RSL3 is loaded on the surface of the composite carrier through physical adsorption or chemical bonding.

[0011] The acidic microenvironment can induce the release of RSL3. When the system is under the condition of pH 5.5, the cumulative release rate of the ferroptosis inducer RSL3 is ≥60%.

[0012] Preferably, the liver cancer cell membrane protein is wrapped on the surface of the composite carrier by physical adsorption or chemical bonding to form the liver cancer cell membrane protein modification layer.

[0013] In the second aspect of the present invention, a preparation method of a tumor-targeted photothermal chemotherapy drug delivery system is provided, which includes the following steps: Step 1: Prepare black phosphorus nanosheets by a liquid-phase exfoliation method. Disperse black phosphorus powder in deionized water, and after ultrasonic treatment and centrifugal separation, obtain a black phosphorus nanosheet dispersion; Step 2: Mix the black phosphorus nanosheet dispersion with iron oxide nanoparticles, and make the iron oxide nanoparticles distributed on the surface of the black phosphorus nanosheets through electrostatic adsorption or chemical bonding to obtain a composite carrier of black phosphorus nanosheets and iron oxide nanoparticles; Step 3: Dissolve the small molecule anti-cancer drug RSL3 in a solvent, mix it with the composite carrier of black phosphorus nanosheets and iron oxide nanoparticles, and load RSL3 onto the surface of the composite carrier through physical adsorption or chemical bonding; Step 4: Mix the liver cancer cell membrane protein with the complex prepared in Step 3, and wrap the liver cancer cell membrane protein on the outer layer of the complex by physical adsorption or chemical bonding to obtain a tumor-targeted photothermal chemotherapy drug delivery system.

[0014] As a further optimized scheme of the above preparation method, in Step 3, the composite carrier of black phosphorus nanosheets and iron oxide nanoparticles is mixed with RSL3 in water at a mass ratio of 5:1, and the obtained solution is vigorously stirred in the dark, and the composite carrier and the complex of RSL3 are collected by washing with water and centrifugation.

[0015] As a further optimized scheme of the above preparation method, in Step 4, an equal volume of the complex solution of the composite carrier and RSL3 and the protein solution are mixed, ultrasonically treated in an ice bath, filtered with a sterile syringe equipped with microporous membranes with pore sizes of 0.22 μm and 0.45 μm, and extruded at a constant rate using a three-channel syringe, repeating 10 - 20 times; after centrifuging to remove the excess protein, the tumor-targeted photothermal chemotherapy drug delivery system is collected.

[0016] The third aspect of the present invention is to provide the application of the above tumor-targeted photothermal chemotherapy drug delivery system, especially for the combined photothermal chemotherapy treatment of liver cancer tumors.

[0017] Under the guidance of an external magnetic field, the tumor-targeted photothermal chemotherapy drug delivery system utilizes the magnetic responsiveness of iron oxide nanoparticles to achieve directional enrichment at the tumor site; the photothermal effect of black phosphorus nanosheets is triggered by near-infrared light irradiation, locally heating up to 42 - 45 °C to destroy the tumor cell structure; under the acidic conditions of the tumor microenvironment, the RSL3 drug is rapidly released from the surface of the composite carrier, inducing ferroptosis by inhibiting the activity of glutathione peroxidase 4; the liver cancer cell membrane protein modification layer mediates homologous target recognition, enhancing tumor cell uptake and avoiding immune clearance; the dual mechanisms of combined photothermal ablation and ferroptosis induction synergistically inhibit tumor growth and metastasis.

[0018] Beneficial effects Compared with the prior art, the present invention provides a new tumor-targeted photothermal chemotherapy nano-drug delivery system. By constructing a nano-platform integrating dual-targeted precise delivery, tumor microenvironment-responsive drug release, photothermal-chemotherapy synergistic effect and ferroptosis mechanism, while improving the precision of tumor treatment and enhancing the curative effect, it reduces the toxic and side effects on normal tissues, showing a promising application prospect.

[0019] Dual-targeted precise uptake: The developed Pro@Fe3O4 / BPNS-RSL3 nanoplatform integrates molecular targeting (hepatocellular carcinoma cell membrane protein Pro) and magnetic targeting (Fe3O4) to achieve selective uptake of Huh7 hepatocellular carcinoma cells, with the uptake amount 2.7 times higher than that of LO2 normal cells, enhancing drug tumor enrichment and reducing normal tissue damage.

[0020] Acid microenvironment-responsive drug release: It has pH-responsive drug release characteristics, and the RSL3 drug can be precisely released at the tumor site in a simulated tumor acidic microenvironment (pH 5.5), enhancing the treatment targeting.

[0021] Chemotherapy-photothermal synergistic therapy: Combining pH-responsive RSL3 release and black phosphorus-mediated photothermal conversion (photothermal conversion efficiency η = 32.1%), the survival rate of Huh7 cells is less than 15% under the synergistic effect, showing a powerful chemotherapeutic-photothermal combined anti-tumor effect.

[0022] Ferroptosis mechanism: It is confirmed by Western blotting that the expressions of GPX4, FACL4, ferritin, etc. are down-regulated, verifying that ferroptosis is the key treatment pathway. Description of the drawings

[0023] Figure 1 : Schematic diagram of the preparation of the Pro@Fe3O4 / BPNS-RSL3 nanodrug delivery system and the mechanism of synergistic anti-tumor by photothermal chemotherapy.

[0024] Figure 2 : Characterization of Pro@Fe3O4 / BPNS. (a) TEM image of Pro@Fe3O4 / BPNS; (b) EDS spectrum of Fe3O4 / BPNS; (c) Magnetic hysteresis loop.

[0025] Figure 3 : Particle size and zeta potential distribution of Pro@Fe3O4 / BPNS. (a) Particle size and Zeta potential distribution of Fe3O4 / BPNS and (b) Pro@Fe3O4 / BPNS.

[0026] Figure 4 : Photothermal performance and stability of Pro@Fe3O4 / BPNS. (a) Temperature change curves of Fe3O4 / BP solutions with different concentrations. (b) Photothermal heating curves of Fe3O4 / BPNS under laser irradiation with different powers. (c) Photothermal heating curves of water-dispersed Fe3O4 / BPNS-based nanoparticles.

[0027] Figure 5:Cumulative release curves of RSL3 under different pH conditions. (a) Encapsulation efficiency and drug loading rate of Pro@Fe3O4 / BPNS-RSL3 nanocomposites; (b) RSL3 release curves of Pro@Fe3O4 / BPNS-RSL3 at different pH values.

[0028] Figure 6 :Cell uptake behavior. (a) Images of Huh7 and (b) LO2 cells after treatment with BPNS, Fe3O4 / BPNS, and Pro@Fe3O4 / BPNS-RSL3 for 24 h; (c) Average green fluorescence intensity.

[0029] Figure 7 :Changes in ferroptosis-related protein expression. (a) Western blotting results of the protein expressions of Ferrtin, Lactoferrin, FACL4, and GPX4 after co-culturing Fe3O4 / BPNS-based nanoparticles with Huh7 cells for 48 h. i, ii, iii, and iv represent the control group, Fe3O4 / BPNS, Pro@Fe3O4 / BPNS, and Pro@Fe3O4 / BPNS-RSL3, respectively. (b) Relative expression levels (using β-actin as a reference). Detailed implementation manners

[0030] The tumor-targeted photothermal chemotherapy drug delivery system of the present invention mainly consists of the following three parts: Core carrier part: A composite of black phosphorus nanosheets (BPNS) and iron oxide (Fe3O4). BPNS, as the basic material for drug carriers and photothermal therapy, can generate heat under near-infrared light irradiation for photothermal therapy; Fe3O4 provides magnetism, endowing the system with magnetic targeting function, and can make the drug more accurately enriched in the tumor site under the guidance of an external magnetic field.

[0031] Drug part: The small molecule anti-cancer drug RSL3, a ferroptosis inducer. RSL3 inhibits system Xc⁻, reduces the content of intracellular glutathione (GSH), increases the level of intracellular reactive oxygen species (ROS), and ultimately leads to the death of tumor cells. It is the key component for exerting anti-cancer effects.

[0032] Targeting modification part: Hepatocellular carcinoma cell membrane protein (Pro). Pro is wrapped on the outer layer of the whole composite, enabling the system to have homologous targeting advantages, specifically recognizing and binding to the corresponding receptors on the surface of hepatocellular carcinoma cells, thereby achieving precise targeting of hepatocellular carcinoma cells, increasing the enrichment amount of drugs in tumor tissues, and reducing the toxic and side effects on normal tissues.

[0033] The preparation method of the tumor-targeted photothermal chemotherapy drug delivery system includes the following steps: Step 1: Preparation of BPNS. Black phosphorus nanosheets (BPNS) were prepared by liquid-phase exfoliation method. Black phosphorus powder was dispersed in deionized water, and after ultrasonic treatment and centrifugal separation, a BPNS dispersion was obtained.

[0034] Step 2: Preparation of Fe3O4 / BPNS composite. The BPNS dispersion was mixed with Fe3O4 nanoparticles, and the Fe3O4 nanoparticles were evenly distributed on the surface of BPNS by electrostatic adsorption or chemical bonding to obtain the Fe3O4 / BPNS composite.

[0035] Step 3: Loading of RSL3 drug. The small molecule anticancer drug RSL3 was dissolved in an appropriate solvent and mixed with the Fe3O4 / BPNS composite. RSL3 was loaded onto the surface of the composite by physical adsorption or chemical bonding to obtain the Fe3O4 / BPNS-RSL3 composite.

[0036] Step 4: Targeted modification. The liver cancer cell membrane protein (Pro) was mixed with the Fe3O4 / BPNS-RSL3 composite, and Pro was wrapped on the outer layer of the composite by physical adsorption or chemical bonding to obtain the final tumor-targeted photothermal chemotherapy nanodrug delivery system Pro@Fe3O4 / BPNS-RSL3.

[0037] The above-mentioned tumor-targeted photothermal chemotherapy nanodrug delivery system includes a core carrier part, a drug part, and a targeted modification part. Among them, BPNS is the basic material for drug carriers and photothermal therapy, which can generate heat under near-infrared light irradiation for photothermal therapy. Fe3O4 provides magnetism and endows the system with magnetic targeting function. Under the guidance of an external magnetic field, the drug can be more accurately enriched in the tumor site. The drug part is triggered by the slightly acidic environment of the tumor to rapidly and accurately release the drug. The drug part is the small molecule anticancer drug RSL3, an iron death inducer, which can induce iron death in tumor cells. By inhibiting system Xc-, it reduces the content of intracellular glutathione (GSH), increases the level of intracellular reactive oxygen species (ROS), and ultimately leads to the death of tumor cells. It is the key component for exerting the anticancer effect. The targeted modification part is the liver cancer cell membrane protein (Pro), which is wrapped on the outer layer of the whole composite, making the system have the advantage of homologous targeting. Compared with traditional targeting strategies, it can specifically recognize and bind to the corresponding receptors on the surface of liver cancer cells, thereby achieving precise targeting of liver cancer cells, increasing the enrichment amount of the drug in tumor tissues, and reducing the toxic and side effects on normal tissues. The above-mentioned multiple effects cooperate with each other, enabling the Pro@Fe3O4 / BPNS-RSL3 composite nanosystem to possess dual targeting capabilities of molecular targeting and magnetic targeting, as well as the combined treatment function of chemotherapy and photothermal therapy, and can more effectively kill tumor cells.

[0038] The present invention will be further illustrated by specific embodiments below. These embodiments are exemplary and are intended to illustrate the problem and explain the present invention, rather than a limitation.

[0039] As Figure 1 shown, a magnetic black phosphorus nanosheet composite system modified with liver cancer cell membrane protein (Pro@Fe3O4 / BPNS-RSL3) was constructed. Through the dual precise positioning of Fe3O4 magnetic targeting and cell membrane protein molecular targeting, combined with the synergistic effect of the near-infrared photothermal effect of BPNS and the ferroptosis inducer RSL3, efficient enrichment of liver cancer cells, acidic microenvironment-responsive drug release, and combined photothermal chemotherapy were achieved, significantly improving the tumor killing effect and reducing normal tissue damage.

[0040] Preparation of Pro@Fe3O4 / BPNS-RSL3 Step (1): Weigh 0.1 g of black phosphorus powder and add it to 50 mL of deionized water. Place the mixture in an ultrasonic instrument and ultrasonically treat it at a power of 200 W for 2 hours. After ultrasonic treatment, centrifuge the mixture and take the supernatant to obtain the BPNS dispersion.

[0041] Step (2): Add 10 mg of BPNS to 1 mL of deionized water and ultrasonicate for 10 min. Add 1 mg of Fe3O4 to the BPNS dispersion, briefly oscillate for premixing, and then ultrasonically treat it in an ice bath for 1 h. Obtain the Fe3O4 / BPNS complex by centrifugation.

[0042] Step (3): Mix 10 mg of Fe3O4 / BPNS with 2 mg of RSL3 in water, and vigorously stir the resulting solution in the dark for 24 h. Wash with water and centrifuge to collect the Fe3O4 / BPNS-RSL3 complex. Measure the absorbance peak at 224 nm with a UV-visible spectrophotometer to quantify the RSL3 loading amount.

[0043] Step (4): Mix an equal volume of the Fe3O4 / BPNS-RSL3 solution and the protein solution, and then ultrasonically treat it in an ice bath for 10 min. Next, filter the mixture using a sterile disposable syringe equipped with microporous membranes with pore sizes of 0.22 μm and 0.45 μm. Perform an extrusion operation at a constant rate using a three-channel syringe, repeating 10 - 20 times. After centrifuging to remove the excess protein, collect the Pro@Fe3O4 / BPNS-RSL3 complex and store it at -80°C.

[0044] Characterization of Materials The morphology and size of Pro@Fe3O4 / BPNS were characterized by high-resolution transmission electron microscopy (HRTEM), its magnetic properties were analyzed by vibrating sample magnetometer (VSM), its composition was detected by Fourier transform infrared spectroscopy (FT-IR), and the particle size distribution and surface zeta potential of Pro@Fe3O4 / BPNS were determined by dynamic light scattering (DLS).

[0045] Experimental results: Figure 2 (a)TEM showed the morphology of Fe3O4 / BPNS, revealing a thin film on the surface, indicating successful functionalization. Figure 2 (b)EDS confirmed the presence of elements such as P, Fe, C, N, and O in Pro@Fe3O4 / BPNS. Figure 2 (c)The hysteresis loop of Pro@Fe3O4 / BPNS at room temperature was shown. The saturation magnetization of Fe3O4 nanocubes was 48.14 emu / g, slightly higher than that of Fe3O4 / BPNS at 46.78 emu / g. The saturation magnetization of Pro@Fe3O4 / BPNS was 38.58 emu / g because the encapsulated protein changed the magnetism of Fe3O4. Nevertheless, Pro@Fe3O4 / BPNS still retained sufficient magnetic responsiveness and could be effectively attracted by an external magnet, which ensured its applicability in magnetic targeting applications. Figure 3 The particle size and zeta potential data measured by dynamic light scattering showed that the average particle size of BPNS was 162.06 nm, that of Fe3O4 was 138.29 nm, that of Fe3O4 / BPNS was 261.84 nm, and the particle size distribution range was narrow. After modification, the particle size of Pro@Fe3O4 / BPNS was 282.12 nm, and the particle size increased slightly due to the loading of Fe3O4. The zeta potential of Pro@Fe3O4 / BPNS was -10.57 mV, indicating the success of the modification.

[0046] In vitro photothermal performance and stability analysis BPNS, Fe3O4 / BPNS, Pro@Fe3O4 / BPNS, and Pro@Fe3O4 / BPNS-RSL3 were irradiated with an 808 nm laser at 1 W / cm 2 , for 10 min. A near-infrared camera recorded the temperature and infrared images of the solution.

[0047] Experimental results: Figure 4 a For Fe3O4 / BPNS with concentrations of 50, 100, 200, and 500 μg / mL, the temperature increases were 6.8 °C, 14 °C, 21.4 °C, and 32.1 °C, respectively. Figure 4b shows the temperature response of the Fe3O4 / BPNS solution to near-infrared lasers with different power densities of 0.5 W / cm², 1.0 W / cm², 1.5 W / cm², and 2.0 W / cm². The recorded temperature increases were 11.2 °C, 14 °C, 19.2 °C, and 23.6 °C, respectively. Figure 4 c shows that Fe3O4 / BPNS still retains excellent photothermal performance even after being encapsulated with proteins. Fe3O4 / BPNS has excellent photothermal performance, with concentration-dependent and power-dependent photothermal effects.

[0048] Drug release characteristics of Pro@Fe3O4 / BPNS-RSL3 In vitro drug release experiments were carried out under the conditions of pH 7.4 (simulating normal physiological environment) and pH 5.5 (simulating tumor microenvironment) to evaluate the release characteristics of RSL3 from the Pro@Fe3O4 / BPNS-RSL3 complex. Two 1 mg samples of the nanodrug system were taken and dissolved in 1 mL of phosphate buffered saline (PBS) with the corresponding pH value. The samples were placed in a dialysis bag and immersed in 30 mL of PBS with the same pH value. These samples were incubated in a thermostatic shaking water bath at 37 °C with an oscillation speed of 120 revolutions per minute. At predetermined time intervals, 3 mL of the release medium was taken out, and the fluorescence intensity at 224 nm was measured using a spectrophotometer to quantitatively determine the amount of released RSL3.

[0049] Experimental results: Calculate the drug loading and encapsulation efficiency of the material according to the standard curve, as Figure 5 shown in (a). The encapsulation rate and drug loading of the Pro@Fe3O4 / BPNS-RSL3 nanocomposite were 62.5% and 11.25%, respectively. Figure 4 (b) shows the release curve of RSL3 from Pro@Fe3O4 / BPNS-RSL3 at different pH values. The drug release ability of Pro@Fe3O4 / BPNS-RSL3 is enhanced under acidic conditions, highlighting its potential for effective drug delivery in the weakly acidic tumor microenvironment.

[0050] In vitro cellular uptake experiment of Pro@Fe3O4 / BPNS-RSL3 The in vitro targeting ability of Pro@Fe3O4 / BPNS-RSL3 was evaluated by the uptake of Huh7 (hepatocarcinoma cells) and LO2 (normal hepatocytes). Huh7 and LO2 cells were seeded at 6×10 4Cells were seeded at a density of [number of cells] per well in a 24-well plate and cultured overnight. After 12 h, the medium was replaced with 200 μL of DMEM containing FITC-labeled BPNS, Fe3O4 / BPNS, and Pro@Fe3O4 / BPNS-RSL3. After incubation for 12 and 24 h, the old medium was discarded, and the cells were washed with PBS, fixed with 4% paraformaldehyde at room temperature, washed again with PBS, permeabilized with 0.2% Triton X-100 for 10 min, and then washed with PBS. The nuclei were stained with DAPI, washed with PBS, and imaged using a fluorescence inverted microscope.

[0051] Experimental results: Figure 6 (a)Green fluorescence of Pro@Fe3O4 / BPNS-RSL3 aggregated around the blue-stained nuclei. In contrast, cells treated with Fe3O4 / BPNS showed weaker green fluorescence around the nuclei, while cells treated with BPNS showed no visible green fluorescence around the nuclei, indicating that Huh7 cells had a stronger uptake of Fe3O4 / BPNS. Figure 6 (b)Showed that LO2 cells had a relatively weak uptake of BPNS, Fe3O4 / BPNS, and Pro@Fe3O4 / BPNS-RSL3. Figure 6 (c)Quantitative analysis further confirmed that Huh7 cells had a significantly higher uptake of Pro@Fe3O4 / BPNS-RSL3 compared with Fe3O4 / BPNS and BPNS, and was much higher than the uptake of all three nanocomposites by LO2 cells. These results clearly demonstrated that Pro@Fe3O4 / BPNS-RSL3 had superior hepatocellular carcinoma cell targeting ability, could effectively deliver drugs to cancer cells, thereby inhibiting the growth of cancer cells. In addition, this selective targeting ability helped to reduce toxic side effects, making Pro@Fe3O4 / BPNS-RSL3 a platform for targeted cancer therapy.

[0052] Verification of ferroptosis in vitro Huh7 cells were seeded at 6×10 per well 5Cells were seeded in 6-well plates at a density of. After 24 h, the medium was replaced with medium containing different treatments, and a total of four groups were set up: group i: control group (without adding nanoparticles); group ii: Fe3O4 / BPNS treatment group; group iii: Pro@Fe3O4 / BPNS treatment group; group iv: Pro@Fe3O4 / BPNS-RSL3 treatment group, and then incubated for another 48 h. After incubation, the cells were rinsed with PBS, digested with trypsin, and an appropriate amount of cell lysis buffer was added to dissociate the cells. Total protein was collected by centrifugation at 4 °C. The protein concentration in each sample was quantified using a BCA protein assay kit. After adding the loading buffer, 10% SDS-PAGE was used to separate the sample proteins for 20 min within the voltage range of 80 V - 120 V. Then the separated proteins were transferred to PVDF, and the membrane was transferred wet for 60 min. Subsequently, the PVDF membrane was incubated in 5% BSA at 37 °C for 1 h. The membrane was incubated with the primary antibody in 3% BSA-TBST (4 °C, overnight), washed three times with TBST, and incubated with the appropriate enzyme-labeled secondary antibody (1:2000) in 1% BSA-TBST for 2 h at 37 °C. After washing three times with TBST, enhanced chemiluminescence (ECL) was used to visualize the bands. Band image analysis was performed using an automatic chemiluminescence / fluorescence image analysis system. Finally, semi-quantitative analysis of the samples was performed using ImageJ software. At least three independent experiments were conducted.

[0053] Experimental results: After 2 days of co-culture, Western blot analysis showed the effect of the composite particles on the expression of apoptosis-related proteins in Huh7 cells. Figure 7a shows the original band images of ferroptosis-related proteins (FACL4, Ferritin, Lactoferrin, GPX4) and the internal reference β-actin in the four groups (i control group, ii Fe3O4 / BPNS group, iii Pro@Fe3O4 / BPNS group, iv Pro@Fe3O4 / BPNS-RSL3 group) in the Western blot experiment; Figure 7b is the semi-quantitative analysis result based on Figure 7a, presenting the relative expression levels of the proteins in each treatment group through a bar chart, and quantitatively comparing the differences in the inhibitory degrees of different nanoparticle treatments on the expression of ferroptosis-related proteins. Compared with the control group, in the experimental groups treated with Fe3O4 / BPNS and Pro@Fe3O4 / BPNS, the expression of ferritin, lactoferrin, FACL4, and GPX4 in Huh7 cells was only moderately inhibited, and the inhibitory effect was relatively slight. In the Pro@Fe3O4 / BPNS-RSL3 treatment group, the expression of these proteins showed a more significant downward trend, which was consistent with the role of RSL3 as a ferroptosis inducer.

[0054] Summary In the present invention, Fe3O4 / BPNS-RSL3 was encapsulated into Pro nanovesicles by extrusion method to obtain Pro@Fe3O4 / BPNS-RSL3. The Pro@Fe3O4 / BPNS-RSL3 nanodrug delivery system has a strong photothermal effect and is expected to be used as a photothermal agent for tumor treatment. The drug release study showed that it has a high release efficiency in the acidic tumor environment, indicating its potential in the combined photothermal-chemical treatment of tumors. Fluorescence microscopy tracking showed that Pro@Fe3O4 / BPNS-RSL3 targeted and entered tumor cells more effectively than Pro@BPNS-RSL3 through endocytosis. Cell experiments confirmed that Pro@Fe3O4 / BPNS-RSL3 has good biocompatibility and excellent synergistic antitumor effects. Generally speaking, Pro@Fe3O4 / BPNS-RSL3 is expected to become a pH-responsive drug release system for targeted combined photothermal-chemical treatment of tumors.

[0055] The above embodiments are exemplary, and the purpose is to illustrate the technical concept and characteristics of the present invention, so that those skilled in this field can understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A tumor-targeted photothermal chemotherapy drug delivery system, characterized in that: It includes a composite carrier formed by black phosphorus nanosheets and ferroferric oxide nanoparticles, an iron death inducer RSL3 loaded on the composite carrier, and an outer wrapped liver cancer cell membrane protein modification layer; the magnetic responsiveness of the ferroferric oxide nanoparticles in the composite carrier gives the system a directional enrichment ability under an external magnetic field; the black phosphorus nanosheets have near-infrared light responsiveness; the liver cancer cell membrane protein modification layer enables the system to have homologous targeting and immune escape characteristics; the iron death inducer RSL3 induces ferroptosis programmed cell death by regulating the glutathione metabolic pathway.

2. The tumor-targeted photothermal chemotherapy drug delivery system according to claim 1, characterized in that: The black phosphorus nanosheets and ferroferric oxide nanoparticles are combined by electrostatic adsorption or chemical bonding to form the composite carrier, and the ferroferric oxide nanoparticles are distributed on the surface of the black phosphorus nanosheets.

3. The tumor-targeted photothermal chemotherapy drug delivery system according to claim 2, characterized in that: The ferroptosis inducer RSL3 is loaded on the surface of the composite carrier by physical adsorption or chemical bonding.

4. The tumor-targeted photothermal chemotherapy drug delivery system according to claim 3, characterized in that: When the system is at pH 5.5, the cumulative release rate of ferroptosis inducer RSL3 is ≥ 60%.

5. The tumor-targeted photothermal chemotherapy drug delivery system according to claim 3, characterized in that: The liver cancer cell membrane protein is wrapped on the surface of the composite carrier by physical adsorption or chemical bonding to form the liver cancer cell membrane protein modification layer.

6. A method for preparing a tumor-targeted photothermal chemotherapy drug delivery system, characterized in that: The following steps are involved: Step 1: Prepare black phosphorus nanosheets by liquid phase exfoliation method, disperse black phosphorus powder in deionized water, and obtain black phosphorus nanosheet dispersion by ultrasonic treatment and centrifugal separation; Step 2: mixing the black phosphorus nanosheet dispersion with ferroferric oxide nanoparticles, distributing the ferroferric oxide nanoparticles on the surface of the black phosphorus nanosheets by electrostatic adsorption or chemical bonding, and obtaining a composite carrier of the black phosphorus nanosheets and ferroferric oxide nanoparticles; Step 3: dissolving the small molecule anticancer drug RSL3 in a solvent, mixing it with a composite carrier of black phosphorus nanosheets and ferroferric oxide nanoparticles, and loading RSL3 onto the surface of the composite carrier by physical adsorption or chemical bonding; Step 4: Mix the liver cancer cell membrane protein with the complex prepared in step 3, and wrap the liver cancer cell membrane protein in the outer layer of the complex by physical adsorption or chemical bonding to obtain the tumor-targeted photothermal chemotherapy drug delivery system.

7. The method for preparing the tumor-targeted photothermal chemotherapy drug delivery system according to claim 6, characterized in that: In step 3, the composite carrier of black phosphorus nanosheets and ferrosoferric oxide nanoparticles is mixed with RSL3 in water at a mass ratio of 5:

1. The obtained solution is vigorously stirred in the dark, washed with water, and centrifuged to obtain a complex of the composite carrier and RSL3.

8. The method for preparing the tumor-targeted photothermal chemotherapy drug delivery system according to claim 7, characterized in that: In step 4, equal volumes of the composite carrier were mixed with the complex solution of RSL3 and the protein solution, sonicated in an ice bath, and the mixture was filtered using a sterile syringe equipped with a microporous membrane with a pore size of 0.22 μm and 0.45 μm, and extruded at a constant rate using a three-channel syringe, which was repeated 10-20 times; After removing excess protein by centrifugation, the tumor-targeted photothermal chemotherapy drug delivery system is collected.

9. The use of the tumor-targeted photothermal chemotherapy drug delivery system according to any one of claims 1 to 5, characterized in that: Targeted photothermal-chemotherapy combined with chemotherapy for liver cancer tumors.

10. The use of the tumor-targeted photothermal chemotherapy drug delivery system according to claim 9, characterized in that: Under the guidance of an external magnetic field, the tumor-targeted photothermal chemotherapy drug delivery system uses the magnetic responsiveness of ferroferric oxide nanoparticles to achieve directional enrichment in the tumor site; the photothermal effect of black phosphorus nanosheets is triggered by near-infrared light irradiation, and the local temperature rises to 42-45°C to destroy the tumor cell structure; under the acidic conditions of the tumor microenvironment, the RSL3 drug is rapidly released from the surface of the composite carrier, inducing ferroptosis by inhibiting the activity of glutathione peroxidase 4; the liver cancer cell membrane protein modification layer mediates homologous targeted recognition, enhances tumor cell uptake and evades immune clearance; the dual mechanism of combined photothermal ablation and ferroptosis induction synergistically inhibits tumor growth and metastasis.

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