Hybrid biomimetic rBCM / Zn-MOF nanodrug delivery carrier and preparation method and application thereof

Abstract

The application discloses a hybrid biomimetic RBCM / Zn-MOF nano drug delivery carrier and a preparation method and application thereof, relates to the technical field of biomimetic nano drug delivery, and the nano drug delivery carrier is FA-RBCM-CuS@ZIF-8. The process comprises the following steps: synthesizing PVP-CuS NPs, mixing 2-methyl imidazole with the PVP-CuS NPs, adding zinc nitrate hexahydrate, and preparing CuS@ZIF-8; connecting FA to the surface of RBCM to obtain FA-RBCM, and mixing the FA-RBCM with CuS@ZIF-8, and extruding to obtain the FA-RBCM-CuS@ZIF-8 nano drug delivery carrier. The obtained sample has uniform particle size distribution and stable physical and chemical properties. The carrier has high encapsulation rate and loading capacity for DOX, has responsive drug release capacity in a simulated tumor microenvironment, and has good biological safety.

Description

Technical Field

[0001] This invention relates to the field of biomimetic nanomedicine delivery technology, and more specifically to a hybrid biomimetic RBCM / Zn-MOF nanomedicine delivery carrier, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common cancer among women worldwide and a leading cause of death. Currently, clinical treatments for breast cancer primarily include surgery, radiotherapy, and chemotherapy. However, breast cancer treatment still faces a series of serious challenges, such as damage to normal tissues, high recurrence rates, and the development of drug resistance. To overcome the shortcomings of traditional treatments, synergistic multi-treatment approaches have been explored and proven to be an effective innovative anti-cancer strategy. Synergistic multi-strategy approaches can achieve complementary advantages among various therapies, inhibiting tumor growth and proliferation through different mechanisms and methods, greatly improving anti-tumor efficacy. Functionalized nanoparticles can serve as carriers for chemical drugs and other therapeutic agents, enabling their efficient delivery. Compared with traditional chemical drugs, nanoparticle drug delivery systems have advantages such as high in vivo bioavailability, good targeting, and controllable drug release.

[0003] Phototherapy is an emerging cancer treatment modality that utilizes light radiation of different wavelengths to induce photochemical or photothermal changes in target tissues. The most common phototherapies include photodynamic therapy (PDT) and photothermal therapy (PTT), both of which utilize light and exogenous or endogenous absorbers to generate cytotoxic reactive oxygen species (ROS) or induce local temperature increases, respectively. At the cellular level, both PDT and PTT have been shown to effectively combat drug resistance and compensate for related signaling pathways. In the tumor microenvironment, these two strategies can modulate tumor perfusion, angiogenesis, and extracellular matrix homeostasis to enhance the efficiency of targeted intratumoral drug delivery. Furthermore, compared to traditional chemotherapy, PDT and PTT offer greater and more precise spatiotemporal control, reducing off-target toxicity. Compared to radiotherapy, phototherapy uses non-ionizing radiation, reducing the risk of secondary cancer development. In conclusion, due to its unique mechanism of action and significant advantages, phototherapy can serve as an adjunct to highly effective cancer treatment, used in conjunction with traditional therapies.

[0004] ROS-based therapies can target cancer cells or specific organelles, reverse drug resistance, and exhibit strong oxidative lethality. Chemodynamic therapy (CDT) is a novel ROS-related cancer treatment method. This method utilizes a Fenton or Fenton-like reaction to react with excess intracellular hydrogen peroxide (H₂O₂) in tumor tissue, generating hydroxyl radicals (·OH). Compared to other types of ROS (such as E(·OH / H₂O) = 2.17V, E(H₂O₂ / H₂O) = 1.78V), ·OH (E(·OH / H₂O) = 2.80V), as a type of ROS, exhibits stronger oxidative capacity and more potent tumor cell-killing activity. In particular, the generation of ·OH does not require O₂; instead, it specifically utilizes the overexpressed H₂O₂ in tumor cells. Solid tumors possess a unique microenvironment characterized by slight acidity, hypoxia, and high H₂O₂, leading to faster proliferation and metastasis rates compared to normal tissues. Therefore, tumor cells overexpressing H₂O₂ can specifically trigger CDT, reducing potential damage to normal tissues.

[0005] However, selecting appropriate media or platforms to integrate various therapies remains a key challenge in implementing multi-strategy synergistic treatment. Currently, metal-organic frameworks (MOFs) composed of organic ligands and their coordinating metal ions / clusters have become a major research hotspot in the biomedical field. Zeolite imidazolium acid framework-8 (ZIF-8) is one of the most commonly used MOFs; it is a framework composed of zinc ions (Zn... 2+ ZIF-8, a biocompatible MOF composed of 2-methylimidazole, is particularly suitable as a drug carrier for tumor therapy due to its high porosity, tunable pore size, ease of preparation, effective encapsulation / loading of "cargo" from small molecules to biomacromolecules, and responsive biodegradability. These advantages also endow ZIF-8 with excellent performance in mediating various tumor therapies, including chemotherapy, PDT, PTT, CDT, sonodynamic therapy (SDT), starvation therapy, and immunotherapy. ZIF-8 exhibits good stability under physiological conditions, and due to its low extracellular pH, it disintegrates at the tumor site, achieving responsive drug release, making it a high-performance drug carrier. Previous studies have combined ZIF-8 with other nanoparticles to create core-shell structured composite carrier materials for multimodal synergistic therapy.

[0006] Red blood cell membranes (RBCMs) are natural long-cycle drug carriers obtained through hemolysis and have become a hot topic in the design and research of artificial biomimetic materials and delivery systems. Studies have shown that the CD47 protein on the surface of RBCMs can flexibly transmit signals to macrophages, preventing macrophage phagocytosis. Therefore, coating other nanocarriers with RBCMs can reduce reticuloendothelial system (RES) uptake, decrease immune recognition, and prolong in vivo circulation time. Simultaneously, the RBCM coating can prevent drug leakage, increase drug concentration at the lesion site, and improve treatment efficiency. Furthermore, RBCMs are widely available, have low development costs, and possess significant application potential.

[0007] CuS NPs, as a near-infrared II (NIR-II) photothermal converter, exhibit higher photothermal conversion efficiency compared to NIR-I photothermal agents. Under NIR-II light irradiation, they can rapidly raise the temperature of tumor tissue within a short time. This high temperature induces protein denaturation in tumor cells, inhibits their division and post-damage repair processes, ultimately killing them. The warming effect also promotes vasodilation, increasing the amount of oxygen reaching distal hypoxic regions. 2 It significantly reduces hypoxia at the tumor site, increases the ROS production area, and provides sufficient O2 to broken DNA to form stable oxygen compounds, thus preventing the automatic repair of damaged DNA. Furthermore, Cu... 2+ It can also catalyze the production of highly cytotoxic ·OH from endogenous H2O2, causing damage to tumor cells and possessing strong CDT capabilities.

[0008] Doxorubicin (DOX) is one of the most commonly used chemotherapy drugs in clinical practice, exhibiting excellent inhibitory effects against various solid tumors. Its main mechanism of action is to insert into DNA, disrupting topoisomerase II-mediated DNA repair and generating free radicals that cause cell damage, thereby achieving a tumor-suppressing effect. However, long-term use of this drug can lead to serious adverse consequences, including cardiotoxicity and bone marrow suppression. These toxic side effects significantly limit its frequency and scope of use as an anticancer drug. Furthermore, because DOX does not specifically target tumor cells, it is expelled from the cell membrane by ABC transporters after entering the cell, leading to drug resistance. Targeted nanocarriers, as a cutting-edge strategy and technology for improving cancer chemotherapy, can enhance the delivery efficiency of drugs such as DOX, reduce toxic side effects, reverse multidrug resistance, and improve the efficacy of chemotherapy.

[0009] Folic acid (FA), also known as vitamin B9, participates in many important biochemical processes in the body, such as DNA replication and methylation. More importantly, elevated FA receptor levels are a key biochemical characteristic of various solid tumors. FA receptor overexpression has been found on the surface of epithelial tumor cells in various organs, including the breast, colon, lung, prostate, ovary, and brain. Therefore, drugs or NPs bound to FA can be rapidly internalized and enter cancer cells via receptor-mediated endocytosis. Furthermore, using FA as a targeting modifier is believed to bypass the drug efflux pump of cancer cells, alleviating multidrug resistance. Combined with the non-immunogenic properties of FA, it offers advantages over monoclonal antibodies. Therefore, FA-based targeted therapy is a promising cancer treatment approach.

[0010] Therefore, how to provide a hybrid biomimetic nanodrug delivery carrier based on the above materials is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention provides a hybrid biomimetic RBCM / Zn-MOF nanodrug delivery carrier, its preparation method and application.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] One embodiment of the present invention provides a hybrid biomimetic RBCM / Zn-MOF nanomedicine delivery carrier, wherein the nanomedicine delivery carrier is FA-RBCM-CuS@ZIF-8.

[0014] Preferably, the process includes: (1) synthesizing PVP-CuS NPs;

[0015] (2) Synthesis of CuS@ZIF-8: 2-methylimidazole was mixed with PVP-CuS NPs, and zinc nitrate hexahydrate was added and mixed again to prepare a pH-responsive nanocarrier CuS@ZIF-8;

[0016] The specific preparation process is as follows: 8 mg of the prepared CuS NPs were dissolved in 2 mL of methanol and sonicated until homogeneous. 16 mg of the prepared ZIF-8 was dissolved in 6 mL of methanol and sonicated until homogeneous. The prepared CuS NPs solution and ZIF-8 solution were mixed, and 6 mL of methanol was added and sonicated until homogeneous. The mixture was stirred at 400 rpm for 5 min at room temperature and then allowed to stand for 2.5 h. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 20 min using a high-speed centrifuge. The precipitate was collected and frozen at -80℃ for 12 h. Lyophilization yielded the pH-responsive nanocarrier CuS@ZIF-8.

[0017] (3) Connect FA to the surface of RBCM to obtain FA-RBCM, and mix it with CuS@ZIF-8 prepared in step (2), and extrude it to obtain FA-RBCM-CuS@ZIF-8 nanomedicine delivery carrier.

[0018] Preferably, in step (1), the specific process for synthesizing PVP-CuS NPs is as follows:

[0019] Copper chloride dihydrate and polyvinylpyrrolidone were weighed at a mass ratio of 1:5, dispersed in ultrapure water, stirred at room temperature for 1 h, sodium sulfide nonahydrate was added, stirred for 5 min, transferred to an oil bath, heated at 90 °C for 40 min, and after the reaction was completed, centrifuged, the precipitate was collected and freeze-dried to synthesize PVP-CuS NPs; wherein the volume-to-mass ratio of sodium sulfide nonahydrate to polyvinylpyrrolidone was 1 ml: 20 mg.

[0020] Preferably, the particle size of the prepared PVP-CuS NPs is 45-50 nm.

[0021] Preferably, in step (2), the mass ratio of 2-methylimidazole to PVP-CuS NPs is 6:1, and the synthesized CuS@ZI F-8 has a particle size of about 180 nm.

[0022] The second aspect of this invention provides the application of the prepared nanomedicine delivery carrier in the preparation of nanomedicine delivery systems and in the preparation of drugs for treating breast cancer.

[0023] A third aspect of the present invention provides a drug for treating breast cancer, the drug being FA-RBCM-CuS / DOX@ZIF-8.

[0024] The fourth aspect of this invention provides a method for preparing a drug, the process of which is as follows: 20 mg CuS@ZIF-8 is dispersed in 2 mL PBS solution, sonicated for 0.5 h, then 2 mL (5 mg / mL) DOX solution is added, and the mixture is stirred at 37 °C for 24 h. After the reaction is completed, 1 mL FA-RBCM is added and the mixture is stirred for another 24 h. Finally, the mixture is centrifuged at 12000 rpm for 10 min, washed three times with 10 mL PBS, the precipitate is collected and freeze-dried to obtain FA-RBCM-CuS / DOX@ZIF-8.

[0025] As can be seen from the above technical solution, compared with the prior art, this invention uses Zn-based MOF-ZIF-8 as the base material and coats it with FA-functionalized RBCM to develop a green biomimetic hybrid nanocarrier for the co-delivery of DOX and CuS NPs. Its preparation process is simple, and the resulting sample has a uniform particle size distribution and stable physicochemical properties. The carrier exhibits high encapsulation efficiency and loading capacity for DOX, demonstrates responsive drug release capability in a simulated tumor microenvironment, and shows good biosafety. The presence of FA-RBCM can increase the uptake efficiency of MCF-7 cells. This carrier can carry DOX and CuS NPs into tumor cells, exerting PTT and CDT effects under NIR irradiation, inducing the production of a large amount of ROS, and achieving synergistic anti-tumor effects. Therefore, FR-CuS / DOX@ZIF-8 is a highly efficient drug delivery system with significant development and application value. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The attached figure shows the particle size distribution of different nanosamples.

[0028] Figure 2 The attached figure shows the potential diagrams of different nanosamples.

[0029] Figure 3 The attached figures are transmission electron microscope (TEM) images of different samples. A: CuS NPs, B: ZIF-8, C: CuS@ZIF-8, D: RBCM-CuS / ZIF-8.

[0030] Figure 4 The attached figures are scanning electron microscope images of different samples. A: ZIF-8, B: CuS@ZIF-8, C: RBCM-CuS / ZIF-8.

[0031] Figure 5 The attached figure shows the ultraviolet absorption spectra of different nano-samples.

[0032] Figure 6 The attached figures show the infrared absorption spectra of different nanomaterials. A: Infrared spectrum of nanomaterials before drug loading; B: Infrared spectrum of each nanomaterial after drug loading.

[0033] Figure 7 The attached figure shows the N2 adsorption curves of different nanoparticles.

[0034] Figure 8The attached figure shows XPS curves of different nanosamples.

[0035] Figure 9 The attached figure shows the XRD patterns of different nanosamples.

[0036] Figure 10 The attached figure shows the DSC curves of different nanosamples.

[0037] Figure 11 The attached image shows the fluorescence pattern of FA-RBCM.

[0038] Figure 12 The attached figure shows the in vitro release curve of FA-RBCM-CuS / DOX@ZIF-8 in simulated photothermal and tumor environments.

[0039] Figure 13 The attached figure shows the results of the cytotoxicity (MCF-7) assay for each nanomaterial. A: 24h, B: 48h.

[0040] Figure 14 The attached figure shows the cytotoxicity (L-02) results of various nanomaterials. A: 24h, B: 48h.

[0041] Figure 15 The attached figure shows the cytotoxicity (MCF-7) assay results for each drug delivery system. A: 24h, B: 48h, C: 24h + NIR, D: 48h + NIR.

[0042] Figure 16 The attached image shows the photothermal verification diagram of the carrier.

[0043] Figure 17 The attached figure shows the relationship between the photothermal conversion efficiency of the carrier and time.

[0044] Figure 18 The attached figure shows the CDT effect verification results of the nanosamples. A: Catalytic efficiency of samples at different concentrations; B: Catalytic efficiency of samples at different pH values; C: Full wavelength scan.

[0045] Figure 19 The attached figures show cellular uptake images (fluorescence photographs) of each nanosample. A: 4h; B: 24h.

[0046] Figure 20 The attached image shows the detection results of reactive oxygen species (ROS) (fluorescence photograph). Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Sample names and abbreviations: CuS@ZIF-8(CZ), DOX@ZIF-8(ZD), CuS / DOX@ZIF-8(CZD), FA-RBCM-CuS / DOX@ZIF-8(FRCZD).

[0049] Example 1: Preparation of a hybrid biomimetic nanodrug delivery carrier FA-RBCM-CuS@ZIF-8

[0050] Step 1: Preparation of CuS NPs: Weigh 20 mg of copper chloride dihydrate (CuCl2·2H2O) and 100 mg of polyvinylpyrrolidone, disperse them separately in 5 mL of ultrapure water, and add them to a round-bottom flask containing 90 mL of ultrapure water. Stir at 400 rpm for 1 h at room temperature. Add 1 mL of sodium sulfide nonahydrate (Na2S·9H2O) dissolved in ultrapure water, stir for 5 min, transfer to an oil bath, and heat at 90 °C for 40 min (the solution turns dark green). After the reaction is complete, centrifuge at 12000 rpm for 20 min using a high-speed centrifuge. Collect the precipitate and freeze it in a -80 °C freezer for 12 h. After freeze-drying, the precipitate is obtained.

[0051] Step 2, Preparation of ZIF-8: Weigh 48 mg of 2-methylimidazole and dissolve it in 5 mL of methanol. Dissolve 4 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) in 5 mL of methanol. After ultrasonic mixing, slowly add the methanol solution of Zn(NO3)2·6H2O to the 2-methylimidazole solution. Stir at 400 rpm for 1 h at room temperature (the solution turns milky white). After the reaction is complete, centrifuge at 12000 rpm for 20 min using a high-speed centrifuge. Collect the precipitate and freeze it at -80℃ for 12 h. After lyophilization, the product is obtained.

[0052] Step 3: Preparation of CuS@ZIF-8: Weigh 8 mg of CuS NPs prepared in Step 1 and dissolve in 2 mL of methanol, then sonicate until homogeneous. Weigh 16 mg of ZIF-8 prepared in Step 2 and dissolve in 6 mL of methanol, then sonicate until homogeneous. Mix the prepared CuS NPs solution with the ZIF-8 solution, add 6 mL of methanol, and sonicate until homogeneous. Stir at 400 rpm for 5 min at room temperature, then let stand for 2.5 h. After the reaction is complete, centrifuge at 12000 rpm for 20 min using a high-speed centrifuge. Collect the precipitate and freeze it at -80℃ for 12 h. After lyophilization, the product is obtained.

[0053] Step 4: Preparation and Storage of Red Blood Cell Regulators (RBCMs): First, whole blood was obtained from mouse sinuses. Fresh blood was collected in heparin-coated blood collection tubes and centrifuged at 1500 rpm for 10 min at 4°C to separate plasma. The blood was washed repeatedly with PBS / physiological saline until the supernatant was colorless, yielding red blood cells (RBCs). The RBCs were then dissolved in 50 mL of ultrapure water and placed in a -80°C freezer, undergoing repeated freeze-thaw cycles to ensure full rupture. Subsequently, the RBCs were centrifuged at 15000 rpm for 5 min to remove hemoglobin. The RBCs were washed several times with ultrapure water and filtered sequentially using 0.45 μm and 0.2 μm filters until the supernatant was colorless and the precipitate was pale pink. The collected red blood cell ghost images were then sonicated for 5 min at 12% power using an ultrasonic cell disruptor (3 s on, 2 s off). The resulting RBCs were dispersed in ultrapure water containing a small amount of protease inhibitor and stored at -80°C.

[0054] Step 5, Preparation and storage of FA-RBCM: Weigh 10 mg of DSPE-PEG2000-FA, dissolve it by sonication with dimethyl sulfoxide, mix it with 500 μL of RBCM prepared in step (4), shake it in a shaker at 37°C in the dark for 3.5 h, and place it at 4°C for 48 h to obtain the product.

[0055] Step 6: Preparation of FA-RBCM-CuS@ZIF-8: Weigh 20mg of CuS@ZIF-8 obtained in step (3), dissolve it in 2mL of ultrapure water, mix it with FA-RBCM in step (5), and then extrude it 10 times with 200nm and 100nm polycarbonate films through an Avanti micro extruder (Avanti PolarLipids) to obtain hybrid nanoparticles.

[0056] Example 2: DOX loaded onto the carrier prepared in Example 1

[0057] Preparation of DOX@ZIF-8: Take 20 mg of ZIF-8 prepared in Example 1 and disperse it in 2 mL of PBS solution. After sonication for 0.5 h, add 2 mL (5 mg / mL) of DOX solution and stir at 37 °C for 24 h. After the reaction is completed, centrifuge at 12000 rpm for 10 min and wash three times with 10 mL of PBS. Collect the precipitate and freeze dry to obtain DOX@ZIF-8.

[0058] Preparation of CuS / DOX@ZIF-8: 20 mg of CuS@ZIF-8 prepared in Example 1 was dispersed in 2 mL of PBS solution and sonicated for 0.5 h. Then, 2 mL (5 mg / mL) of DOX solution was added and stirred at 37 °C for 24 h. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 min and washed three times with 10 mL of PBS. The precipitate was collected and freeze-dried to obtain CuS / DOX@ZIF-8.

[0059] Preparation of FA-RBCM-CuS / DOX@ZIF-8: 20 mg of CuS@ZIF-8 prepared in Example 1 was dispersed in 2 mL of PBS solution and sonicated for 0.5 h. Then, 2 mL of (5 mg / mL) DOX solution was added and stirred at 37 °C for 24 h. After the reaction was completed, 1 mL of FA-RBCM was added and stirring was continued for 24 h. Finally, the mixture was centrifuged at 12000 rpm for 10 min, washed three times with 10 mL of PBS, and the precipitate was collected and freeze-dried to obtain FA-RBCM-CuS / DOX@ZIF-8.

[0060] The loading efficiency of DOX-loaded nanosamples—DOX@ZIF-8, CuS / DOX@ZIF-8, and FA-RBC M-CuS / DOX@ZIF-8—was determined using the provided information. Each nanoformulation was centrifuged, and the supernatant volume was measured. HPLC analysis was performed using a mobile phase prepared with methanol, acetonitrile, and phosphoric acid in a specific ratio. Since DOX exhibits strong UV absorption at 480 nm, this wavelength was chosen as the detection wavelength. Standard concentrations of DOX solutions were prepared for establishing standard curves. The peak area A was then substituted into the standard curve to determine the DOX content in the supernatant. The encapsulation efficiency and drug loading of DOX were calculated using the following formula:

[0061] Drug loading capacity = Drug amount (encapsulation) / Nanocarrier mass

[0062] Encapsulation efficiency = Drug load / Total drug load × 100%

[0063] The drug loading and encapsulation efficiency of each nano-formulation are shown in Table 1.

[0064] Table 1. Results of drug loading and encapsulation efficiency determination for different nano-formulations

[0065] Drug loading (ug / mg) Encapsulation efficiency (%) DOX@ZIF-8 326.8 98 CuS / DOX@ZIF-8 282.7 84.8 FR-CuS / DOX@ZIF-8 266.1 79.8

[0066] The drug loading (μg / mg) of ZIF-8, CuS@ZIF-8, and FR-CuS@ZIF-8 were 326.8, 282.7, and 266.1, respectively, and the encapsulation efficiencies were 98%, 84.8%, and 79.8%, respectively. The constructed ZIF-8 nanosystem did not show a significant decrease in drug loading or encapsulation efficiency due to the increase in modifying components. Therefore, FR-CuS@ZIF-8 in this invention has high drug loading and encapsulation efficiency.

[0067] For each carrier—ZIF-8, CuS NPs, CuS@ZIF-8, CuS / DOX@ZIF-8, and FR-CuS / DOX@ZIF-8—using deionized water as the dispersion medium, the particle size and zeta potential of the samples were determined using a laser particle size analyzer. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0068] Figure 1 This is a particle size distribution diagram of different nanoparticles, from Figure 1 It is known that the synthesized ZIF-8 has excellent dispersibility and small particle size. The particle size of the drug-loaded nanoparticles is stably maintained between 250-280 nm, and the particle size of the nanosystem coated with FA-RBCM is about 300 nm. This particle size makes it easy for the nanocarrier to be taken up by tumor cells, thereby exerting therapeutic effects.

[0069] Figure 2 These are Zeta potential diagrams for different nanoparticles, derived from... Figure 2 It can be seen that the charge of ZIF-8 is +23.1mV. After encapsulating CuS NPs, the potential drops to +11.9mV. DOX is positively charged. The potential of CuS / DOX@ZIF-8 loaded with DOX rises to +15.9mV. FA and RBCM are both negatively charged. After coating with FA-RBCM, the potential of the hybrid nano-drug delivery system remains at around +2.4mV and has relatively high stability.

[0070] Example 3 Sample Characterization

[0071] The samples provided in Examples 1 and 2—ZIF-8, CuS NPs, CuS@ZIF-8, CuS / DOX@ZIF-8, FR-CuS / DOX@ZIF-8, FA-RBCM, etc.—were characterized by FT-IR, UV-Vis, TEM, SEM, XPS, etc.

[0072] Transmission electron microscopy (TEM) images of CuS NPs, ZIF-8, CuS@ZIF-8, and RBCM-CuS@ZIF-8 were analyzed, see [link to TEM images]. Figure 3 , Figure 3As shown in A, CuS NPs appear as relatively uniform small particles with an approximately spherical shape and a particle size of 50-60 nm. Figure 3 As shown in B, ZIF-8 exhibits excellent dispersibility and stability, with a size stable at 160-200 nm. It presents a uniform hexagonal structure with a large internal space for loading DOX. Figure 3 As shown in C, the CuS@ZIF-8 has a size of 160-200 nm, and it is clear that it contains small CuS NPs, indicating that the CuS NPs are successfully anchored inside ZIF-8. Figure 3 As shown in D, the RBCM-CuS@ZIF-8 has a size of 280-300nm and a somewhat blurry film on its surface, indicating that RBCM was successfully coated on the ZIF-8 surface.

[0073] Scanning electron microscopy analysis was performed on ZIF-8, CuS@ZIF-8, and RBCM-CuS@ZIF-8, as shown in the figure. Figure 4 , Figure 4 As shown in A, the ZIF-8 surface is relatively smooth and the shape is relatively regular. Figure 4 As shown in B, some small particles are attached to the surface of CuS@ZIF-8. During the preparation process, CuS NPs are not only embedded inside, but also adsorbed on the ZIF-8 surface. Moreover, due to the use of PVP, CuS@ZIF-8 mostly exists in the form of several aggregates. Figure 4 As shown in C, RBCM-CuS@ZIF-8 becomes smoother and more transparent due to the thin layer of red blood cell membrane covering its surface.

[0074] UV absorption spectra of CuS NPs, CuS@ZIF-8, and CuS / DOX@ZIF-8 were analyzed, see [link to data]. Figure 5 The CuS NPs exhibit UV absorption in the 1000-1100 nm range, and CuS@ZIF-8 also shows UV absorption in the same region, indicating that CuS NPs have been successfully doped both inside and on the surface of ZIF-8. The CuS / DOX@ZIF-8 shows an absorption peak at 540 nm, which is due to the interaction of DOX with metal ions (Cu). 2+ The combination of ) caused a redshift, proving the successful loading of DOX.

[0075] Infrared absorption spectroscopy analysis was performed on CuS@ZIF-8, ZIF-8, FA-RBCM, CuS / DOX@ZIF-8, and FR-CuS / DOX@ZIF-8, see [link to data]. Figure 6 , Figure 6 In the image, A represents the infrared absorption spectra of CuS@ZIF-8 and ZIF-8, with a scan wavenumber range of 500-4000 cm⁻¹. -1The X-axis represents transmittance (%), and the Y-axis represents wavenumber, both in cm. -1 As shown in the figure, the peak shapes of the two are basically the same, indicating that the structure of the parent ZIF-8 was not destroyed during further synthesis, highlighting the stability of ZIF-8. CuS@ZIF-8 peaked at 1671 cm⁻¹. -1 The presence of an absorption peak at this location is due to the stretching vibration of the ortho-hydroxyl group on the benzene ring of PVP-CuS, indicating that CuS NPs are successfully anchored inside ZIF-8. Figure 6 In the figure, B represents the infrared absorption spectra of FA-RBCM, CuS / DOX@ZIF-8, and FR-CuS / DOX@ZIF-8. As shown in the figure, CuS / DOX@ZIF-8 exhibits absorption spectra at 2924 cm⁻¹. -1 There is a -CH2- stretching vibration peak at 1730 cm⁻¹. -1 There is a C=O vibration peak at 991 cm⁻¹. -1 The presence of a CH vibration peak at 2850 cm⁻¹ indicates successful DOX loading. FR-CuS / DOX@ZIF-8 at 2850 cm⁻¹ -1 The appearance of antisymmetric stretching vibration peaks indicates successful coating of FA-RBCM.

[0076] The adsorption of the nanoparticles prepared in Example 1 before and after drug loading was determined by nitrogen adsorption method, see [reference needed]. Figure 7 Therefore, the specific surface area of ​​ZIF-8 is 1091.3 m². 2 / g, with a relatively large internal surface area; after internal anchoring of CuS NPs, the specific surface area becomes 484.7m². 2 / g indicates that a considerable portion of CuS NPs are embedded internally; the specific surface area of ​​FR-CuS / DOX@ZIF-8 is 93.4m². 2 / g, the internal surface area is further reduced, which proves that a considerable portion of DOX was successfully loaded inside ZIF-8, and also indicates the successful coating of RBCM, proving that ZIF-8 nanoparticles are a carrier with excellent drug loading performance.

[0077] XPS Survey spectral analysis was performed on different nanosamples, see [link / reference]. Figure 8 The main constituent elements of ZIF-8 are known to be C, N, and O. All samples showed C1s, N1s, and O1s peaks, confirming the successful synthesis of ZIF-8. Furthermore, CuS@ZIF-8 and FR-CuS@ZIF-8 exhibited a significant Cu2p peak at a binding energy of 930-940 eV, demonstrating the successful embedding of CuS NPs into ZIF-8 and the successful construction of the composite nanocarrier.

[0078] XRD pattern analysis of different nano samples is shown in the figure. Figure 9The diffraction peaks of ZIF-8 and CuS@ZIF-8 are largely consistent, indicating that the internal embedding of CuS NPs did not affect the crystal form of ZIF-8. The characteristic diffraction peaks of CuS@ZIF-8 at 47.5° and 59.4° indicate that CuS NPs are embedded inside ZIF-8.

[0079] DSC spectral analysis of different nanosamples is shown in the figure. Figure 10 As shown in the figure, the glass transition temperatures of all nanosamples are relatively low, indicating their high stability. Using ZIF-8 as the drug carrier, the embedding of CuS NPs and the coating with RBCM do not affect the stability.

[0080] Fluorescence image analysis of different nanosamples using FA-RBCM is shown in the figure. Figure 11 RBCM was labeled with DiD, and FA was labeled with FITC. After treatment at 37℃ and 4℃, images were taken using a fluorescence microscope. As shown in the figures, RBCM exhibits red light, FA exhibits green light, and the fused color is light yellow, indicating that FA coats the surface of RBCM.

[0081] Based on the above characterization results, this invention successfully prepared a ZIF-8 nanocarrier capable of loading CuS NPs and DOX. After coating with RBCM, it was functionalized with FA. The final nanosystem exhibits good physical stability and biomimetic properties, as well as good dispersibility, and has the potential to be used as an excellent delivery carrier for antitumor drugs.

[0082] Example 4: DOX release from FR-CuS / DOX@ZIF-8 under simulated in vivo environment and photothermal action.

[0083] 3 mg FR-CuS / DOX@ZIF-8 was dispersed in 3 ml of PBS buffer solution with corresponding conditions (pH 7.4, pH 5). The resulting solutions were then placed in dialysis bags with a molecular weight cutoff of 3500 Da, and then in 30 ml of PBS buffer solution. The bags were continuously shaken in a constant temperature shaker at 37°C. The NIR group was irradiated with a 1064 nm laser for 5 min at regular intervals. At 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, and 96 h, 2 ml of the release solution was aspirated into 5 ml centrifuge tubes, and 2 ml of PBS buffer solution with the corresponding conditions was added. The peak area of ​​the release solution at different time points was determined by high-performance liquid chromatography (HPLC). The cumulative release rate of DOX was calculated based on the DOX standard curve. A graph was plotted with time on the x-axis and cumulative release rate on the y-axis. Figure 12The results show that FR-CuS / DOX@ZIF-8 exhibits the fastest release rate under NIR conditions and pH 5.0, reaching 67.7% release rate at 24 hours. The release rate then steadily increases, reaching 81% at 96 hours. Conversely, under conditions without NIR and pH 7.4, the release rate is the slowest, stabilizing at 12 hours and reaching only 17.3% at 96 hours. These results indicate that photothermal effects and the slightly acidic environment of the tumor can promote drug release from the nano-drug delivery system.

[0084] Example 5

[0085] The MTT assay was used to investigate the cytotoxicity of each nanocarrier (material) to MCF-7 and L-02 cell lines, as well as the cytotoxicity of each drug delivery system to MCF-7 cells. A plot was created with concentration on the x-axis and cell viability on the y-axis. The cell viability of each carrier on MCF-7 cells is shown in the figure. Figure 13 The survival rates of each vector on L-02 cells are shown in the figure. Figure 14 Survival rates of MCF-7 cells under different drug delivery systems are shown in the figure. Figure 15 .

[0086] contrast Figure 13 A and Figure 13 As shown in B, ZIF-8, CuS NPs, CuS@ZIF-8, FR-CuS@ZIF-8 and other materials have cell survival rates of over 80% at concentrations of 0-100 μg / mL for 24h and 48h, indicating that the nanocarriers have high biocompatibility.

[0087] Depend on Figure 14 It can be seen that, compared with MCF-7 cells, L-02 cells had a higher survival rate after treatment with different concentrations of nanomaterials for 24 h or 48 h, indicating that the nanomaterials basically do not affect the growth of normal cells and have good biocompatibility. The hybrid nanocarrier coated with RBCM has better biocompatibility.

[0088] Depend on Figure 15 A and Figure 15 As shown in B, the cytotoxicity of each nano-drug delivery system increases sequentially with increasing concentration. The survival rate of the free DOX group was 73%, and the survival rate of the FR-CuS / DOX@ZIF-8 group was 58%. At 48 hours, the survival rate of the FR-CuS / DOX@ZIF-8 group decreased to 53%. Figure 15 C and Figure 15As shown in D, under NIR irradiation, the cell survival rate further decreased. In particular, after 48 hours of drug administration and under NIR irradiation, the cell survival rate of the FR-CuS / DOX@ZIF-8 group was only 36%, while the survival rate of the free DOX group was still close to 70%. This indicates that the constructed nano-drug delivery system FR-CuS / DOX@ZIF-8 has a good killing effect on MCF-7 cells.

[0089] Example 6

[0090] Verification of the photothermal effect: A temperature change curve of the carrier was plotted with time on the x-axis and temperature on the y-axis. Based on the determined concentrations, ZIF-8, CuS NPs, CuS@ZIF-8, FR-CuS@ZIF-8, and water were prepared (see [link to relevant documentation]). Figure 16 As shown in the figure, the highest temperature of water stabilized at around 27℃ within 5 minutes, while the highest temperature of ZIF-8 stabilized at around 30℃ within 5 minutes. The temperature of CuSNPs increased gradually with the extension of light irradiation time, reaching around 50℃ within 30 seconds, indicating good photothermal conversion performance. CuS@ZIF-8 and FR-CuS@ZIF-8 also approached 50℃ within 5 minutes and gradually stabilized. The results indicate that CuS NPs have a good photothermal effect, and the loading of CuS on ZIF-8 and the surface coating with RBCM do not affect its photothermal effect.

[0091] Verification of the relationship between the photothermal conversion efficiency of the carrier and time: ZIF-8, CuS NPs, CuS@ZIF-8, and FR-CuS@ZIF-8 were prepared into solutions of 30 μg / mL, and the photothermal conversion efficiency was measured at 2 W / cm². 2 Under NIR irradiation conditions, images were taken every 30 seconds using an infrared thermal imager. See Figure 17 The results showed that, at a constant power, the photothermal conversion effect of the nano-sample increased with time, and the temperature also increased accordingly, confirming that the nanocarrier has a good photothermal conversion effect and is time-dependent.

[0092] Photodynamic chemistry (CDT) verification diagram of FR-CuS@ZIF-8: Samples of different concentrations were reacted with MB, H2O2, and PBS solutions, and the absorbance at 652 nm was recorded every 10 min. Since the nanomaterial can catalyze the generation of ·OH from excess H2O2, thereby decolorizing MB, this verifies the CDT effect of the nanocarrier. See Figure 18 ,Depend on Figure 18 As shown in A, the degradation rate of MB increases with increasing concentration; when the concentration exceeds 100 μg / mL, the degradation rate of MB reaches over 30%. Figure 18As shown in B, pH affects the degradation rate of MB by FR-CuS@ZIF-8. At pH 7.4, 6.5, and 5 and a catalytic time of 60 min, the degradation rates of MB reached 87%, 61%, and 49%, respectively, indicating that pH 5 is the optimal pH condition for FR-CuS@ZIF-8 to catalyze the degradation of MB. Figure 18 C in the figure represents the full-wavelength scan under different pH conditions for 60 min of catalysis. The figure shows that as the pH decreases, the absorption peak of MB also decreases, indicating an increase in MB degradation rate. These results demonstrate that the CDT effect of this nanocarrier is time-, concentration-, and pH-dependent.

[0093] Example 7

[0094] The uptake of various nanosamples by MCF-7 cells was analyzed using fluorescence localization. Groups were set up: DOX (Control), DOX@ZIF-8, CuS / DOX@ZIF-8, FR-CuS / DOX@ZIF-8, and FR-CuS / DOX@ZIF-8 + NIR. Each sample was prepared at a concentration of 5 μg / mL. MCF-7 cells were incubated at this concentration in six-well plates for 4 h and 24 h. After incubation, the culture medium was discarded, and the cells were washed twice with 1 mL of PBS. 1 mL of nuclear staining agent (Hoechst 33342) was added for staining for 15 min. After staining, the staining solution was discarded, and the cells were washed twice with 1 mL of PBS. 1 mL of PBS was added to keep the cells moist, and cell uptake was observed under a fluorescence microscope. Cell uptake fluorescence is shown in the figure. Figure 19 The results showed that the fluorescence intensity induced by DOX delivery using nanocarriers was significantly higher than that of the control group (free DOX). As the surface modification degree of the carrier increased, the red fluorescence of DOX gradually increased, and the overlap with the blue fluorescence of the cell nucleus increased, indicating that the nanocarriers can significantly improve the cellular uptake of chemotherapeutic drugs and have excellent delivery performance. NIR irradiation can enhance the uptake of hybrid carriers.

[0095] Verification of ROS generation induced by nanosamples in cells:

[0096] The effect of various nanosamples on inducing ROS production in MCF-7 cells was analyzed using fluorescence localization. Groups were set up: Control, DOX, DOX@ZIF-8, CuS / DOX@ZIF-8, FR-CuS / DOX@ZIF-8, and FR-CuS / DOX@ZIF-8+NI R. Each sample was prepared at a concentration of 5 μg / mL. At this concentration, MCF-7 cells were incubated in six-well plates for 4 h and 24 h. After incubation, the culture medium was discarded, and the cells were washed twice with 1 mL PBS. 1 mL of ROS fluorescent probe was added for staining for 15 min. After staining, the staining solution was discarded, and the cells were washed twice with 1 mL PBS. 1 mL of PBS was added to keep the cells moist, and intracellular ROS production was observed under a fluorescence microscope. Results are shown below. Figure 20 As shown in the figure, compared with free DOX, the drug delivery system can induce ROS production more efficiently. The hybrid drug delivery system produces a higher amount of intracellular ROS. After NIR irradiation, the intracellular ROS level increases to a certain extent. This result indicates that FR-CuS / DOX@ZIF-8 can effectively deliver drugs into cells and induce a large amount of ROS production, achieving synergistic therapy.

[0097] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0098] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A drug for treating breast cancer, characterized in that, The drug is FA-RBCM-CuS / DOX@ZIF-8. The preparation process of FA-RBCM-CuS / DOX@ZIF-8 is as follows: 20 mg CuS@ZIF-8 is dispersed in 2 mL PBS solution, sonicated for 0.5 h, then 2 mL of 5 mg / mL DOX solution is added, and the mixture is stirred at 37℃ for 24 h. After the reaction is completed, 1 mL of FA-RBCM is added and the mixture is stirred for another 24 h. Finally, the mixture is centrifuged at 12000 rpm for 10 min, washed three times with 10 mL PBS, the precipitate is collected and freeze-dried to obtain FA-RBCM-CuS / DOX@ZIF-8. The preparation process of CuS@ZIF-8 includes: (1) Synthesis of PVP-CuS NPs: Copper chloride dihydrate and polyvinylpyrrolidone were weighed at a mass ratio of 1:5, dispersed in ultrapure water, stirred at room temperature for 1 h, sodium sulfide nonahydrate was added, stirred for 5 min, transferred to an oil bath, heated at 90℃ for 40 min, centrifuged after the reaction was completed, the precipitate was collected and freeze-dried to synthesize PVP-CuS NPs; wherein, the volume mass ratio of sodium sulfide nonahydrate to polyvinylpyrrolidone was 1 ml: 20 mg; (2) Synthesis of CuS@ZIF-8: 2-methylimidazole was mixed with PVP-CuS NPs, and zinc nitrate hexahydrate was added and mixed again to prepare a pH-responsive nanocarrier CuS@ZIF-8; wherein the mass ratio of 2-methylimidazole to PVP-CuS NPs was 6:

1. The preparation process of the FA-RBCM is as follows: Weigh 10 mg of DSPE-PEG2000-FA, dissolve it by sonication with dimethyl sulfoxide, mix it with 500 μL of RBCM, shake it in a shaker at 37°C in the dark for 3.5 h, and then place it at 4°C for 48 h to obtain the final product.

2. The drug according to claim 1, characterized in that, The particle size of the prepared PVP-CuS NPs was 45-50 nm.

3. The drug according to claim 1, characterized in that, The synthesized CuS@ZIF-8 particles had a size of 180 nm.

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