A membrane-coated manganese dioxide nanocarrier modified by a cell-penetrating peptide, and a preparation method and application thereof

Abstract

The application discloses a preparation method and application of a hollow manganese dioxide nanocarrier coated with a human umbilical cord mesenchymal stem cell membrane and a transmembrane peptide TAT, and is characterized in that: hollow mesoporous manganese dioxide is synthesized through a selective etching technology, and a layer of human umbilical cord mesenchymal stem cell membrane is coated outside the manganese dioxide through a repeated extrusion method. Since the cell membrane has special tumor homing characteristics, and lacks major histocompatibility complex-II and only expresses major histocompatibility complex-I in a trace amount, the key scientific problems and difficulties such as low biological safety, strong immunogenicity and easy elimination by the body of artificially synthesized nanomaterials can be effectively solved. Finally, the cell membrane surface is modified with the transmembrane peptide TAT through a phospholipid insertion method, precise subcellular localization-nucleus targeting is realized, and tumor cells can be more effectively killed.

Description

Technical Field

[0001] This invention relates to medicine, and in particular to a membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier, its preparation method, and its application. Background Technology

[0002] Lung cancer poses a serious threat to human life, with both its incidence and mortality rates steadily increasing worldwide, resulting in over 600,000 deaths annually. For most patients with non-small cell lung cancer, paclitaxel (PTX)-based chemotherapy remains a first-line treatment; however, the side effects of PTX cannot be ignored. One of the focuses of this invention is how to retain the anti-tumor advantages of PTX, reduce systemic toxicity and side effects, and enhance its concentration in the tumor microenvironment.

[0003] The rapid development of nanomedicine has brought new opportunities for the efficient diagnosis and treatment of tumors. Compared with traditional drugs, nano-drug delivery systems have advantages such as good stability, long retention time in the body, low systemic side effects, and high local concentration within tumors. However, there are still some key scientific problems in the artificial synthesis of nanomaterials, such as low biosafety, strong immunogenicity, and easy clearance by the body. Manganese dioxide (MnO2) has attracted special attention as a unique tumor microenvironment-responsive nanocarrier. The research group of Liu Zhuang at Soochow University prepared a hollow mesoporous MnO2 nanolayer encapsulated in polyethylene glycol (PEG). The ultrasensitive pH-responsive MnO2 nanoparticles can achieve tumor-specific magnetic resonance imaging and acid-responsive controlled drug release. Although modifying the MnO2 surface with PEG can improve the biodistribution of the nanocarrier, it cannot completely avoid immune clearance, and PEG can activate the human complement system.

[0004] Currently, publicly available cell membrane-coated nanomaterials, both domestically and internationally, mainly consist of cancer cell membranes, erythrocyte membranes, leukocyte membranes, macrophage membranes, and bone marrow mesenchymal stem cell membranes. However, they have several drawbacks. For example, the injection of cancer cell membrane-coated nanomaterials into the human body remains controversial; erythrocyte membranes lack relevant protein receptors and therefore lack targeting ability; leukocyte and macrophage membranes are immunogenic and can easily activate the human complement system; and bone marrow mesenchymal stem cells can transform into tumor-associated cells after several generations of culture. Therefore, there is an urgent need to find a cell membrane with natural targeting ability and low immunogenicity as a transport carrier for PTX.

[0005] Umbilical cord mesenchymal stem cells (UC-MSCs) are derived from Wharton's jelly, the gel-like connective tissue of the umbilical cord. They possess unique tumor homing characteristics and lack major histocompatibility complex (MHC)-II, expressing only trace amounts of MHC-I. Therefore, allogeneic MSCs injected into the human body are immune-immune. Human umbilical cord mesenchymal stem cells (HU-MSCs) are easier to isolate and culture than mesenchymal stem cells from other sources, have fewer ethical controversies in clinical application, and do not transform into tumor-associated fibroblasts after several generations of cell culture, unlike bone marrow-derived mesenchymal stem cells. Therefore, HU-MSC membrane-encapsulated nanoparticles have broader clinical application prospects, but no publicly reported applications have been found to date.

[0006] The key to controlling tumor cell DNA synthesis is the delivery of chemotherapeutic drugs to the cell nucleus. Currently, most nanomedicine delivery vehicles reach the cytoplasm, rarely targeting the nucleus, and are unable to reach deep tumor tissues far from blood vessels. The transmembrane peptide TAT, a short-chain polypeptide rich in basic amino acids (YGRKKRRQRRR), possesses strong cell membrane penetration capabilities. When linked to nanocarriers, it can be successfully delivered to the cell nucleus to exert its effects. The research group of Jianlin Shi at the Shanghai Institute of Ceramics, Chinese Academy of Sciences, designed TAT-modified mesoporous silica nanoparticles capable of reaching the tumor cell nucleus to release drugs, thereby disrupting tumor cell DNA. It is well known that the proteins on the surface of HU-MSC membranes are fundamental to their specific functions; therefore, the integrity of these surface proteins must be ensured from the preparation of cell membrane nanovesicles to the modification of the cell membrane surface with TAT. Currently, there are no reports on TAT-modified nanomedicine delivery vehicles for HU-MSC membranes. Summary of the Invention

[0007] The purpose of this invention is to provide a transmembrane peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier, its preparation method, and its application. The core of this drug delivery system consists of hollow mesoporous manganese dioxide nanoparticles, coated with a human umbilical cord mesenchymal stem cell membrane. The transmembrane peptide TAT is modified on the outer surface of the human umbilical cord mesenchymal stem cell membrane. The preparation process is described below. Figure 1 .

[0008] The first objective of this invention is to provide a membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier, the carrier comprising a drug, hollow mesoporous manganese dioxide nanoparticles, human umbilical cord mesenchymal stem cell membrane nanovesicles, and a membrane-penetrating peptide; wherein the hollow mesoporous manganese dioxide nanoparticles are loaded with the drug, the human umbilical cord mesenchymal stem cell membrane nanovesicles coat the hollow mesoporous manganese dioxide nanoparticles, and the membrane-penetrating peptide is modified on the outer surface of the human umbilical cord mesenchymal stem cell membrane nanovesicles.

[0009] Furthermore, the drug is a chemotherapy drug; preferably, the chemotherapy drug is paclitaxel, imiquimod, or doxorubicin.

[0010] Furthermore, the amino acid sequence of the transmembrane peptide is YGRKKRRQRRR (SEQ ID NO.1).

[0011] Furthermore, the particle size of the hollow manganese dioxide nanocarrier coated with the membrane-penetrating peptide TAT modified human umbilical cord mesenchymal stem cell membrane is 100-110 nm.

[0012] A second objective of this invention is to provide a method for preparing a membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarriers, the method comprising the following steps:

[0013] (1) Preparation of hollow mesoporous manganese dioxide nanoparticles; preferably, the hollow mesoporous manganese dioxide nanoparticles have a particle size of 80-90 nm and a pore size of 9 nm.

[0014] (2) Preparation of drug-loaded hollow mesoporous manganese dioxide nanoparticles: The drug is loaded into the hollow mesoporous manganese dioxide nanoparticles prepared in (1) to obtain drug-loaded hollow mesoporous manganese dioxide nanoparticles.

[0015] (3) Preparation of human umbilical cord mesenchymal stem cell membrane nanovesicles: human umbilical cord mesenchymal stem cells were taken, and the human umbilical cord mesenchymal stem cell membrane was retained to obtain human umbilical cord mesenchymal stem cell membrane nanovesicles.

[0016] (4) The human umbilical cord mesenchymal stem cell membrane nanovesicles prepared in (3) are coated with drug-loaded hollow mesoporous manganese dioxide nanoparticles prepared in (2) to obtain human umbilical cord mesenchymal stem cell membrane nanovesicles coated with drug-loaded hollow mesoporous manganese dioxide nanocarriers.

[0017] (5) The outer surface of the human umbilical cord mesenchymal stem cell membrane nanovesicles prepared in (4) is coated with a membrane-penetrating peptide to obtain the membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarrier.

[0018] Furthermore, (1) hollow mesoporous manganese dioxide nanoparticles were prepared using selective etching technology;

[0019] Preferably, the specific steps of the selective etching technique are as follows: Anhydrous ethanol, ultrapure water, and ammonia are mixed at room temperature, with a molar ratio of 9-10:1:1-2. Orthosilicate is added dropwise, with a molar ratio of 1:10 between the orthosilicate and ultrapure water. The mixture is stirred at room temperature for 8-14 hours, centrifuged to collect the precipitate, and then potassium permanganate solution is added dropwise, with a molar ratio of 1.9-2.8:1 between potassium permanganate and orthosilicate. The mixture is sonicated for 6 hours, centrifuged to collect the precipitate, washed with water, and finally sodium carbonate solution is added, with a molar ratio of 2.2-3.3:1 between sodium carbonate and orthosilicate. The mixture is dissolved at 60°C for 12 hours, centrifuged to retain the precipitate, and the precipitate is vacuum dried to obtain hollow mesoporous manganese dioxide nanoparticles.

[0020] Furthermore, (2) the loading process involves mixing a drug solution with porous manganese dioxide nanoparticles and then drying them.

[0021] Furthermore, (3) the particle size of the human umbilical cord mesenchymal stem cell membrane nanovesicles is 150nm~200nm.

[0022] Furthermore, (4) the coating is to mix human umbilical cord mesenchymal stem cell membrane nanovesicles with drug-loaded hollow mesoporous manganese dioxide nanoparticles in PBS to obtain a mixed solution, and to repeatedly squeeze the mixed solution with a filter membrane with a particle size of 300nm to 400nm.

[0023] Further, (5) the specific operation is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and the membrane-penetrating peptide are dissolved in PBS, and phospholipid-polyethylene glycol-maleimide (Mal-PEG-DSPE) is added to form phospholipid-polyethylene glycol-membrane-penetrating peptide. The phospholipid-polyethylene glycol-membrane-penetrating peptide is added to the PBS solution of human umbilical cord mesenchymal stem cell membrane nanovesicle-coated drug-loaded hollow mesoporous manganese dioxide nanocarrier, stirred, centrifuged, and the supernatant is discarded to obtain the membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier; preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and the membrane-penetrating peptide is 12-15:18-20:1-1.5, and the concentration of the membrane-penetrating peptide in PBS is 0.1-0.15 mg / mL.

[0024] A third objective of this invention is to provide the application of the aforementioned membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier in the preparation of drugs that kill tumor cells.

[0025] Compared with existing technologies, it has the following unique advantages:

[0026] (1) This invention is the first to combine manganese dioxide nanocarriers with human umbilical cord mesenchymal stem cell membranes, which not only has a specific targeting effect, promotes drug accumulation at the tumor site and improves delivery efficiency, but also solves key scientific problems and difficulties such as low biosafety, strong immunogenicity and easy clearance by the body of artificially synthesized nanomaterials.

[0027] (2) Since the human umbilical cord mesenchymal stem cell membrane has special tumor homing characteristics and lacks major histocompatibility complex-II and only expresses major histocompatibility complex-I in trace amounts, it can effectively solve key scientific problems and difficulties such as low biosafety, strong immunogenicity and easy clearance by the body of artificially synthesized nanomaterials.

[0028] (3) For the first time, the transmembrane peptide TAT was modified by phospholipid insertion on the surface of human umbilical cord mesenchymal stem cell membrane to achieve precise subcellular localization—nuclear targeting, thereby more effectively killing tumor cells.

[0029] (4) This system specifically targets tumor tissue. Manganese dioxide dissolves in the tumor microenvironment to generate oxygen, which relieves hypoxia in the tumor site and opens up a new route for anti-tumor drugs, with significant economic and social benefits.

[0030] (5) The preparation method of this invention is simple, easy to produce, and low in cost. Its product can effectively deliver drugs. In particular, the drug that kills tumor cells has been used in the treatment of non-small cell lung cancer, and relevant experiments have achieved very satisfactory and beneficial technical results. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the preparation of a membrane-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarriers.

[0032] Figure 2 Physicochemical characterization of hollow manganese dioxide nanocarriers coated with the transmembrane peptide TAT-modified human umbilical cord mesenchymal stem cell membrane. (a) Transmission electron microscopy image of HMnO2-MSC-TAT, scale bar 100 nm; (b) UV-Vis-NIR spectra of TAT alone, HMnO2, and HMnO2-MSC-TAT; (c) Zeta potential diagram of HMnO2-MSC-TAT; (d) SDS-PAGE protein analysis of HMnO2-MSC-TAT, HMnO2, and MSC (MSC: human umbilical cord mesenchymal stem cell membrane nanovesicles); (e) Particle size changes of HMnO2-MSC-TAT after 2 weeks in H2O, PBS, and cell culture medium.

[0033] Figure 3 To assess the ability of hollow mesoporous manganese dioxide nanocarriers to respond to the tumor microenvironment, the degradation behavior of HMnO2 at different pH values ​​(7.4, 5.5) was determined using ultraviolet-visible spectroscopy.

[0034] Figure 4 The loading and release of drugs from HMnO2-MSC-TAT@PTX nanocarriers are shown. (a) UV-Vis spectra of PTX, HMnO2, and HMnO2@PTX alone; (b) the loading amount of PTX in cell membrane-coated nanomaterial HMnO2-MSC-TAT@PTX and non-cell membrane-coated nanomaterial HMnO2-TAT@PTX at different drug-to-HMnO2 ratios; and (c) the percentage of PTX released from HMnO2-MSC-TAT@PTX and HMnO2-TAT@PTX carriers at different pH values ​​(7.4, 5.5).

[0035] Figure 5 The relative cell viability of liver LO2 cells after culturing with different concentrations of HMnO2-MSC-TAT for 24 hours.

[0036] Figure 6 Study on apoptosis-induced in A549 cells by HMnO2-MSC-TAT@PTX nanocarrier.

[0037] Figure 7 Investigation of the nucleation performance of HMnO2-MSC-TAT@PTX nanoparticles.

[0038] Figure 8 This study investigates the targeting of cell membrane-coated nanocarrier HMnO2-MSC-TAT@PTX and non-cell membrane-coated nanocarrier HMnO2-TAT@PTX in a mouse subcutaneous tumor model. Detailed Implementation

[0039] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. The following embodiments are further illustrations of the present invention and do not limit the scope of the invention.

[0040] Clinical-grade HU-MSCs were obtained from Jiangsu Cell Technology Medical Research Institute and Jiangsu Cell Technology Biotechnology Co., Ltd. Non-small cell lung cancer A549 cells were purchased from the American Type Culture Collection (ATCC).

[0041] The present invention

[0042] TAT refers to a membrane-penetrating peptide, and the amino acid sequence of the membrane-penetrating peptide TAT is YGRKKRRQRRR.

[0043] MSC or HU-MSC refers to human umbilical cord mesenchymal stem cells.

[0044] MnO2 refers to manganese dioxide.

[0045] PTX stands for paclitaxel.

[0046] EDC refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0047] NHS stands for N-hydroxysuccinimide.

[0048] PAH refers to polyallylamine.

[0049] PAA refers to: polyacrylic acid

[0050] NH2-PEG-NH2 refers to: amino-polyethylene glycol-amino

[0051] Mal-PEG-DSPE refers to: phospholipid-polyethylene glycol-maleimide.

[0052] DSPE-PEG-TAT refers to: Phospholipid-Polyethylene Glycol-Penetrating Peptide TAT

[0053] H-MnO2 or HMnO2 refers to hollow mesoporous manganese dioxide nanoparticles.

[0054] HMnO2@PTX refers to hollow mesoporous manganese dioxide nanoparticles loaded with paclitaxel.

[0055] HMnO2-MSC@PTX refers to a hollow mesoporous manganese dioxide nanocarrier loaded with paclitaxel, coated with human umbilical cord mesenchymal stem cell membrane nanovesicles.

[0056] HMnO2-MSC-TAT@PTX refers to a membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarriers.

[0057] HMnO2-TAT@PTX refers to: membrane-penetrating peptide-modified manganese dioxide nanocarriers.

[0058] Example 1

[0059] Preparation of hollow mesoporous manganese dioxide nanoparticles (HMnO2 nanoparticles): 1700 mmol anhydrous ethanol, 180 mmol ultrapure water, and 200 mmol ammonia were mixed and stirred at 400 rpm for 10 min at room temperature. 18 mmol orthosilicate was added dropwise, and the mixture was stirred at room temperature for 12 h. The mixture was centrifuged at 12000g for 10 min to collect the precipitate. Then, 36 mmol potassium permanganate solution was added dropwise, and the mixture was sonicated for 6 h. The mixture was centrifuged at 14800 rpm for 15 min to collect the precipitate, washed three times with water, and finally dissolved in a sodium carbonate solution containing 54 mmol sodium carbonate at 60℃ for 12 h. The precipitate was centrifuged at 14800 rpm for 10 min and the precipitate was retained. The precipitate was dried in a vacuum drying oven to obtain HMnO2 nanoparticles. The hollow mesoporous manganese dioxide nanoparticles had a particle size of 80-90 nm and a pore size of 9 nm.

[0060] Example 2

[0061] Preparation of drug-loaded hollow mesoporous manganese dioxide nanoparticles: Take 1 ml of 3 mg / ml paclitaxel solution, add 1 ml of 1 mg / ml HMnO2 nanoparticle PBS solution, mix well, sonicate for 24 h, centrifuge at 12000 r / min for 10 min, and dry in a vacuum drying oven to obtain PTX-loaded hollow mesoporous manganese dioxide nanoparticles HMnO2@PTX.

[0062] Example 3

[0063] Preparation of human umbilical cord mesenchymal stem cell membrane nanovesicles (HU-MSC membrane nanovesicles): Clinical-grade HU-MSCs were obtained from Jiangsu Cell Technology Medical Research Institute and Jiangsu Cell Technology Biotechnology Co., Ltd., China. Umbilical cord mesenchymal stem cells (5 × 10^6 cells) in cell culture flasks were washed with PBS buffer. 2 ml of 0.25% trypsin solution was added to the cell culture flasks, and the cells were digested at 37°C. After 1 min, digestion was terminated with cell culture medium, and the cell culture medium was removed by centrifugation. The stem cells in the centrifuge tubes were washed three times with PBS buffer. The washed stem cells were redispersed in hypotonic cell lysis buffer and lysed overnight at 4°C. The lysed cells were lysed on ice for 2 min using a cell disruptor, then centrifuged at 3500g for 5 min at 4°C, and the supernatant was retained. The supernatant was then centrifuged at 15000g for 30 min at 4°C and the supernatant was discarded to obtain a human umbilical cord mesenchymal stem cell membrane. A polycarbonate filter membrane with a pore size of 200 nm was selected, and the stem cell membrane was extruded using a mini extruder to prepare HU-MSC membrane nanovesicles with a particle size of 150 nm to 200 nm.

[0064] Example 4

[0065] Preparation of drug-loaded hollow mesoporous manganese dioxide nanocarriers coated with human umbilical cord mesenchymal stem cell membrane nanovesicles (H-MnO2-MSC@PTX): 2 mg / ml of HMnO2@PTX in PBS solution was thoroughly mixed with 2 mg / ml of HU-MSC membrane nanovesicles in PBS solution to obtain a mixed solution. The mixed solution was loaded into a mini squeezer, and a polycarbonate filter membrane with a pore size of 200 nm was used to squeeze the mixed solution. The squeezing was repeated for 21 cycles. Then, the mixture was centrifuged at 12000 r / min for 10 min at 4 °C to remove the residual HU-MSC membrane nanovesicles in the supernatant. The obtained H-MnO2-MSC@PTX was placed back into PBS buffer and stored at 4 °C.

[0066] Example 5

[0067] Preparation of membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarriers (HMnO2-MSC-TAT@PTX): First, 12.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 18 mg of N-hydroxysuccinimide (NHS), and 1 mg of TAT were dissolved in 10 mL of PBS and stirred vigorously at room temperature for 1 h. Then, under sonication, 50 mg of Mal-PEG-DSPE was added to form DSPE-PEG-TAT. Finally, 10 μL of 10 mg / mL DSPE-PEG-TAT was added to 1 mL of 0.5 mg / mL HMnO2-MSC@PTX PBS solution, stirred at 4 °C for 1 h, centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was discarded, and the obtained H-MnO2-MSC-TAT@PTX was resuspended in PBS and stored at 4 °C. The membrane-penetrating peptide TAT-modified human umbilical cord mesenchymal stem cell membrane-coated hollow manganese dioxide nanocarrier has a particle size of 100-110 nm.

[0068] Comparative Example 6

[0069] Preparation of membrane-penetrating peptide-modified manganese dioxide nanocarriers (HMnO2-TAT@PTX)

[0070] Hollow mesoporous manganese dioxide nanoparticles were prepared according to the method in Example 1. 10 mg of HMnO2 aqueous solution (2 mg / mL) was added to 50 mg of PAH aqueous solution (5 mg / mL), and after sonication for 2 h, centrifugation and washing were performed to obtain HMnO2 / PAH. Under sonication conditions, the HMnO2 / PAH aqueous solution was added to 50 mg of PAA solution (5 mg / mL), and after sonication for 2 h, centrifugation and washing were performed to obtain HMnO2 / PAH / PAA. Subsequently, under sonication conditions, 50 mg of NH2-PEG-NH2 was added, and centrifugation was performed to obtain aminated manganese dioxide nanoparticles (HMnO2-NH2). HMnO2-TAT was obtained by amide reaction of the carboxyl group of the transmembrane peptide with the amino group of HMnO2-NH2. EDC, NHS, and the transmembrane peptide were dissolved in PBS (EDC 12.5 mg, NHS 18 mg, and TAT 1 mg), respectively. After 30 minutes, HMnO2-NH2 was added and stirred for 12 hours. The mixture was then centrifuged and washed to obtain HMnO2-TAT. For paclitaxel loading, the HMnO2-TAT aqueous solution could be mixed with different concentrations of paclitaxel and reacted for 12 hours. The particle size of the transmembrane peptide-modified manganese dioxide nanocarrier was 100–110 nm.

[0071] The preparation method of this invention is simple, easy to produce, and low in cost. Its product can be effectively used in the treatment of non-small cell lung cancer, and relevant experiments have yielded very satisfactory and beneficial technical results. The relevant experimental details are as follows:

[0072] Experimental Example 1: Physicochemical Characterization of HMnO2-MSC-TAT@PTX

[0073] Take 100 μL of the PBS solution of HMnO2-MSC-TAT@PTX prepared in Example 5 above, dilute it 10 times with deionized water, and drop 20 μL onto a carbon film copper grid. After the sample solution is allowed to air dry, observe the particle morphology and size under a transmission electron microscope. The results show that a stem cell membrane is clearly observed on the surface of the HMnO2-MSC-TAT@PTX nanoparticles, exhibiting a distinct core-shell structure, with a particle size of approximately 108 nm. Figure 2 (a).

[0074] The modification of TAT peptides in the PBS solution, TAT-FITC solution, and HMnO2 prepared in Example 5 was determined by UV-Vis spectroscopy. The results showed that the PBS solution of HMnO2-MSC-TAT@PTX exhibited a clear UV absorption peak at 495 nm, belonging to TAT-FITC, indicating that TAT was successfully modified on the surface of MSCs. Figure 2 (b)

[0075] 100 μL of PBS solutions of HMnO2 nanoparticles from Example 1, HU-MSC membrane nanovesicles from Example 3, H-MnO2-MSC@PTX from Example 4, HMnO2-MSC-TAT@PTX prepared in Example 5, and PBS suspension of umbilical cord mesenchymal stem cell membrane were taken, diluted 10 times with deionized water, and added to the sample cell. The Zeta potential of the nanoparticles was measured using a nanoparticle size potentiostat. The results showed that the Zeta potential of HMnO2 nanoparticles was -30.63 mV, the Zeta potential of HU-MSC membrane nanovesicles was -17.47 mV, and the Zeta potential of H-MnO2-MSC@PTX was -17.83 mV. Comparison of surface potentials revealed that the Zeta potential of H-MnO2-MSC@PTX was close to that of HU-MSC membrane nanovesicles, indicating the successful preparation of H-MnO2-MSC@PTX nanoparticles. The Zeta potential of the TAT-modified HMnO2-MSC-TAT nanoparticles was -11.70 mV. Figure 2 (c)

[0076] To evaluate the cell membrane surface proteins of the outer layer of HMnO2-MSC-TAT@PTX, polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the HMnO2-MSC-TAT@PTX cell membrane surface proteins. The umbilical cord mesenchymal stem cell membrane vesicle solution prepared in Example 3, the HMnO2-TAT@PTX solution prepared in Example 6, and the HMnO2-MSC-TAT@PTX solution prepared in Example 5 were boiled separately. After 10 min, heating was stopped, the samples were cooled to room temperature, centrifuged, and set aside. 25 μL of sample solution was added to each channel of the electrophoresis system. After electrophoresis, the protein bands were stained with Coomassie Brilliant Blue solution and destained overnight with glacial acetic acid. The results showed that HMnO2-MSC-TAT@PTX maintained the integrity of the MSC membrane surface proteins well, realizing the transfer of MSC membrane proteins from mesenchymal stem cells to HMnO2 nanoparticles, laying the foundation for subsequent nanoparticle targeting of tumor tissues. Figure 2 (d)

[0077] To investigate the stability of the particles, the diluted HMnO2-MSC-TAT@PTX solution was placed at 4°C and dispersed in different solvents (PBS, H2O, and cell culture medium) to a final concentration of 1 mg / ml. After incubation for 2 weeks, the particle size of HMnO2-MSC-TAT@PTX was observed using dynamic light scattering. The results showed that the particle size of the HMnO2-MSC-TAT@PTX nanoparticles did not change significantly within two weeks, indicating that the nanoparticles possess good solution stability. Figure 2 (e).

[0078] Experiment 2: The ability of H-MnO2-MSC-TAT@PTX nanocarriers to respond to the tumor microenvironment

[0079] Manganese dioxide is stable at neutral and alkaline pH levels, but it can decompose into Mn under acidic conditions. 2+ Therefore, HMnO2-MSC-TAT@PTX was incubated with PBS at various pH values ​​(7.4, 6.5, and 5.5) at different time points (0.5, 2, 4, and 8 h). The dissolution of the nanocarrier was measured by UV-Vis spectroscopy and observed by transmission electron microscopy. The results showed that the UV absorption peak of MnO2 did not change much at pH 7.4, but decreased rapidly at pH 6.5 and 5.5. This in-situ verification of the ultrasensitive pH response of HMnO2-MSC-TAT@PTX nanoparticles indicates that the nanocarrier is acid-sensitive and that manganese dioxide can dissolve in the tumor microenvironment to form MnO2. 2+ This releases the internal drug ( Figure 3 ).

[0080] Experiment 3: The ability of HMnO2-MSC-TAT@PTX nanocarriers to load paclitaxel

[0081] Drug loading and encapsulation efficiency are two important parameters for evaluating the drug loading capacity of nanoparticles. To investigate the ability of HMnO2-MSC-TAT@PTX nanoparticles to load the anticancer drug PTX, the characteristic absorption peak of PTX at 227 nm was measured by UV-Vis spectroscopy using the standard curve method.

[0082] The specific steps are as follows: First, 1 mg of HMnO2 was dissolved in 1 ml of pure water, and a methanol solution containing PTX (5 mg / ml) was added (the feed ratio was drug:MnO2 = 1:2, 1:1, 2:1, 3:1). After soaking for 12 h, the free PTX in the solution was removed by centrifugation and the nanoparticles were washed with water to obtain drug-loaded H-MnO2@PTX nanoparticles. Then, the drug delivery carrier HMnO2-MSC-TAT@PTX, which is loaded with PTX and modified with a transmembrane peptide, was prepared by repeated extrusion.

[0083] The characteristic absorption peak of PTX was determined by ultraviolet-visible spectroscopy. The presence of an absorption peak at a wavelength of 227 nm indicates successful loading. Figure 4 a). The results showed that when the mass ratio of drug PTX to nanocarrier was 3:1, the drug loading was 80% (a). Figure 4 b).

[0084] Experiment 4: Drug release capacity of HMnO2-MSC-TAT@PTX nanocarriers

[0085] The prepared HMnO2-MSC-TAT@PTX and HMnO2-TAT@PTX were dispersed in 1 ml of PBS buffer and added to a 3500 Da dialysis bag. Both ends were tied tightly, and the dialysis bag was then completely immersed in 900 ml of release medium at pH 5.5. The bag was placed in a 37°C water bath and stirred at a constant speed of 100 rpm / min. Samples (3 ml) were taken at specific time points, and an equal volume of fresh release medium was added promptly. The samples were filtered through a 0.22 μm microporous filter and analyzed by HPLC under the following chromatographic conditions (column: Inertsil ODS-3C18 column (150 × 4.6 mm, pore size 5 μm, Agilent, USA), mobile phase: acetonitrile: pure water = 50:50, flow rate: 1.0 ml / min, column temperature: 30°C, detection wavelength: 227 nm, injection volume: 20 μl). The cumulative release was calculated, and a time-cumulative release curve was plotted. The results showed that the nanocarrier exhibited good environmentally responsive sustained-release effect for anticancer drugs, releasing 20% ​​of PTX at pH 7.4; and a higher release rate at pH 5.5, with only about 40% of the drug released in the first 4 hours, after which the release rate slowed down, with a cumulative release of 60% at 12 hours and 67% at 24 hours. Figure 4 c).

[0086] Experiment 5: Biocompatibility Evaluation of Unloaded HMnO2-MSC-TAT Nanoparticles

[0087] Good biocompatibility is a necessary prerequisite for the design and fabrication of micro / nano drug delivery carriers. Therefore, the biocompatibility of unloaded HMnO2-MSC-TAT nanoparticles was evaluated in this study. The HMnO2-MSC-TAT nanoparticles were prepared sequentially using the methods described in Examples 1, 3, 4, and 5. In the cytotoxicity assay of the HMnO2-MSC-TAT nanoparticles, normal liver cells LO2 (source: ATCC) were used as the research subject, and the cytocompatibility of the HMnO2-MSC-TAT nanoparticles was evaluated using the standard MTT assay. The specific steps are as follows: LO2 cells were seeded in 96-well plates at a density of 5 × 10^4 / ml, with a cell suspension volume of 100 μL per well. After culturing in a cell culture incubator for 24 h, the cell culture medium was aspirated, and different concentrations (3.13, 6.25, 12.5, 25, 50, 100, 200 μg / mL) of cell membrane-coated HMnO2-MSC-TAT nanoparticle solution were added to the cells. After culturing in an incubator for 24 h, the 96-well plates were removed, and 20 μL of MTT was added to each well. After incubation for 4 h, the culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken, and the absorbance (OD) at a wavelength of 490 nm was measured using a microplate reader. Each group had 6 replicates. The absorbance was recorded, and the cell viability was calculated as: Cell viability = (OD value of experimental group / OD value of control group) × 100%. The results showed that after 24 hours of incubation of LO2 with different concentrations of HMnO2-MSC-TAT nanoparticles, HMnO2-MSC-TAT exhibited almost no toxicity to LO2, with cell viability reaching nearly 80%. This indicates that our nanomedicine delivery system possesses high biocompatibility, providing a solid foundation for future clinical translation. It also demonstrates that this nanocarrier has no significant impact on the activity of human liver cells. Figure 5 ).

[0088] Experiment 6: Investigation of the nucleation performance of HMnO2-MSC-TAT@PTX nanoparticles

[0089] To investigate the time-dependent entry of nanoparticles into the cell nucleus, human non-small cell lung cancer A549 cells were co-cultured for 4 h, 12 h, and 24 h. The concentrations of Mn in the cells and nucleus were then measured by ICP-OES. The specific steps were as follows: In a 10 cm culture dish, nanoparticles were injected with 5 × 10⁻⁶ nanoparticles... 6A549 cells were seeded at a density suitable for incubation at 37°C and 5% CO2 for 24 hours. Then, unloaded HMnO2-MSC-TAT (HMnO2-MSC-TAT nanoparticles were prepared sequentially according to Examples 1, 3, 4, and 5), HMnO2-MSC (HMnO2-MSC nanoparticles were prepared sequentially according to Examples 1, 3, and 4), and HMnO2-TAT nanoparticles (HMnO2-TAT nanoparticles were prepared according to the method in Example 6) were added at a concentration of 100 μg / mL. After co-culturing for 24 hours, the cell culture medium was aspirated, and the cells were washed three times with PBS. The cells were then digested with 0.25% trypsin for 2 minutes and collected by centrifugation. Cell lysis buffer was added according to the instructions, and the cells were lysed under sonication. Finally, the Mn content in the cells was determined by ICP-OES.

[0090] To determine the Mn content in the cell nucleus, cells were cultured according to the above procedure, digested with trypsin, and collected by centrifugation. Then, the cells were... 6 A concentration of Mn / mL was added to the nucleus separation buffer. The cells were incubated on ice for 10 min, then centrifuged at 800 rpm for 15 min to obtain A549 cell nuclei. Cell lysis buffer was then added, and the cells were lysed under sonication. The Mn content in the nuclei was then determined by ICP-OES.

[0091] The results showed that after co-culturing HMnO2-MSC-TAT, HMnO2-MSC, and HMnO2-TAT with A549 cells for different times (4h, 12h, and 24h), the modification with TAT peptide enhanced the nuclear targeting effect, and the Mn content in the cell nucleus increased significantly with the extension of culture time. After 24h, the Mn content in the cell nucleus of HMnO2-MSC-TAT was 10-fold higher than that of unmodified HMnO2-MSC, demonstrating its significant nuclear targeting performance. Figure 7 ).

[0092] Experiment 7: Study on apoptosis induced by H-MnO2-MSC-TAT@PTX nanocarrier in human non-small cell lung cancer A549 cells

[0093] To evaluate the killing effect of drug-loaded stem cell membrane-masked nanomedicine delivery vehicles on cancer cells, HMnO2-MSC-TAT@PTX nanoparticles loaded with anticancer drugs were co-cultured with A549 cells for 24 hours, and the activity of cancer cells in each experimental group was analyzed.

[0094] The specific steps are as follows: A549 cells were seeded in 96-well plates at a density of 5×10^4 / ml, with a cell suspension volume of 100ul per well. After culturing in a cell culture incubator for 24 hours, the cell culture medium was aspirated, and the same concentration of free PTX and HMnO2-MSC-TAT@PTX nanoparticle solution was added to the cells. After culturing in an incubator for 24 hours, the apoptosis of cancer cells was detected by flow cytometry.

[0095] The results showed that, compared with free PTX, the nanomedicine delivery carrier could increase the tumor cell apoptosis rate; compared with the non-cell membrane-coated nanocarrier HMnO2-TAT@PTX group, the cell membrane-coated nanocarrier HMnO2-MSC-TAT@PTX group induced more tumor cell apoptosis. Figure 6 ).

[0096] Experiment 8: Targeting study of HMnO2-MSC-TAT@PTX nanocarriers in a mouse subcutaneous tumor model

[0097] To evaluate the in vivo tumor-targeting ability of the HMnO2-MSC-TAT@PTX nanocarrier, the distribution of HMnO2-MSC-TAT@PTX in mice after tail vein injection was studied using a small animal in vivo imaging system.

[0098] The specific steps are as follows: Healthy C57BL / 6 mice weighing approximately 15g were selected, and 5×10^5 Lewis lung cancer cells (source: ATCC) were subcutaneously injected into the right axilla. The tumor volume was allowed to reach 500mm². 3 Subsequently, mice were injected with 200 μL of HMnO2-MSC-TAT@PTX labeled with the near-infrared dye ICG at a concentration of 4 mg / ml. The distribution of the nanoparticles in mice at 1, 2, 6, 8, 12, and 24 hours after tail vein injection was observed using the near-infrared fluorescence of ICG. Before scanning, mice were anesthetized with chloral hydrate via intraperitoneal injection, and then placed in a live imaging system for whole-body scanning. Images were obtained using Living... The software performs the analysis.

[0099] The results showed that 2 hours after tail vein injection, the nanoparticles were clearly distributed in mice; 4 hours later, the fluorescence intensity at the tumor site was significantly enhanced, indicating that more HMnO2-MSC-TAT@PTX nanocarriers accumulated at the tumor site. Compared with the non-cell membrane-coated HMnO2-TAT@PTX group, less HMnO2-MSC-TAT@PTX was taken up by organs such as the spleen and liver, indicating that cell membrane-coated nanomaterials reduce the phagocytosis of nanoparticles by organs. Figure 8 ).

[0100] Experiments show that the method is stable and reliable, and has the following unique advantages compared with existing technologies:

[0101] This invention is the first to combine MnO2 nanocarriers with HU-MSCs membranes, which not only has a specific targeting effect, promoting drug aggregation at the tumor site and improving delivery efficiency, but also solves key scientific problems and difficulties such as low biosafety, strong immunogenicity, and easy clearance by the body of artificially synthesized nanomaterials.

[0102] For the first time, TAT was modified on the surface of HU-MSCs membrane to achieve precise subcellular localization—nuclear targeting—thereby more effectively killing tumor cells.

[0103] This system specifically targets tumor tissue. Manganese dioxide dissolves in the tumor microenvironment to generate oxygen, relieving hypoxia in the tumor site and opening up a new avenue for anti-tumor drugs, with significant economic and social benefits. sequence list <110> The Second Affiliated Hospital of Nanjing Medical University <120> A membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarrier, its preparation method and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <400> 1 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10

Claims

1. A membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier, characterized in that, The carrier comprises a drug, hollow mesoporous manganese dioxide nanoparticles, human umbilical cord mesenchymal stem cell membrane nanovesicles, and a membrane-penetrating peptide; the hollow mesoporous manganese dioxide nanoparticles are loaded with the drug, the human umbilical cord mesenchymal stem cell membrane nanovesicles encapsulate the drug-loaded hollow mesoporous manganese dioxide nanoparticles, and the membrane-penetrating peptide is modified on the outer surface of the human umbilical cord mesenchymal stem cell membrane nanovesicles, the amino acid sequence of the membrane-penetrating peptide being YGRKKRRQRRR; the drug is a chemotherapy drug or imiquimod.

2. The membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier according to claim 1, characterized in that, The chemotherapy drugs mentioned are paclitaxel and doxorubicin.

3. A method for preparing a membrane-transfer peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarrier, characterized in that, The method includes the following steps: (1) Preparation of hollow mesoporous manganese dioxide nanoparticles; (2) Preparation of drug-loaded hollow mesoporous manganese dioxide nanoparticles: The drug is loaded into the hollow mesoporous manganese dioxide nanoparticles prepared in (1) to obtain drug-loaded hollow mesoporous manganese dioxide nanoparticles. (3) Preparation of human umbilical cord mesenchymal stem cell membrane nanovesicles: human umbilical cord mesenchymal stem cells were taken, and the human umbilical cord mesenchymal stem cell membrane was retained to obtain human umbilical cord mesenchymal stem cell membrane nanovesicles. (4) The human umbilical cord mesenchymal stem cell membrane nanovesicles prepared in (3) are coated with drug-loaded hollow mesoporous manganese dioxide nanoparticles prepared in (2) to obtain human umbilical cord mesenchymal stem cell membrane nanovesicles coated with drug-loaded hollow mesoporous manganese dioxide nanocarriers. (5) The outer surface of the human umbilical cord mesenchymal stem cell membrane nanovesicles prepared in (4) is coated with a transmembrane peptide to obtain the transmembrane peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarrier. The amino acid sequence of the membrane-penetrating peptide is YGRKKRRQRRR; the drug is a chemotherapy drug or imiquimod.

4. The preparation method according to claim 3, characterized in that, (1) Hollow mesoporous manganese dioxide nanoparticles were prepared by selective etching technology.

5. The preparation method according to claim 4, characterized in that, The specific steps of the selective etching technique are as follows: Anhydrous ethanol, ultrapure water, and ammonia are mixed at room temperature, with a molar ratio of 9-10:1:1-2. Orthosilicate is added dropwise, with a molar ratio of 1:10 between orthosilicate and ultrapure water. The mixture is stirred at room temperature for 8-14 hours, centrifuged to collect the precipitate, and then potassium permanganate solution is added dropwise, with a molar ratio of 1.9-2.8:1 between potassium permanganate and orthosilicate. The mixture is sonicated for 6 hours, centrifuged to collect the precipitate, washed with water, and finally sodium carbonate solution is added, with a molar ratio of 2.2-3.3:1 between sodium carbonate and orthosilicate. The mixture is dissolved at 60°C for 12 hours, centrifuged to retain the precipitate, and the precipitate is vacuum dried to obtain hollow mesoporous manganese dioxide nanoparticles.

6. The preparation method according to claim 3, characterized in that, (2) The loading process involves mixing a drug solution with hollow mesoporous manganese dioxide nanoparticles and then drying them.

7. The preparation method according to claim 3, characterized in that, (3) The particle size of the human umbilical cord mesenchymal stem cell membrane nanovesicles is 150 nm to 200 nm.

8. The preparation method according to claim 3, characterized in that, (4) The coating is to mix human umbilical cord mesenchymal stem cell membrane nanovesicles with drug-loaded hollow mesoporous manganese dioxide nanoparticles in PBS to obtain a mixed solution, and to repeatedly squeeze the mixed solution with a filter membrane with a particle size of 300 nm to 400 nm.

9. The preparation method according to claim 3, characterized in that, (5) The specific operation is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and membrane-penetrating peptide are dissolved in PBS, and phospholipid-polyethylene glycol-maleimide is added to form phospholipid-polyethylene glycol-membrane-penetrating peptide. The phospholipid-polyethylene glycol-membrane-penetrating peptide is added to the PBS solution of human umbilical cord mesenchymal stem cell membrane nanovesicles coated with drug-loaded hollow mesoporous manganese dioxide nanocarriers, stirred, centrifuged, and the supernatant is discarded to obtain the membrane-penetrating peptide-modified human umbilical cord mesenchymal stem cell membrane coated with manganese dioxide nanocarriers.

10. The preparation method according to claim 9, characterized in that, The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and the membrane-penetrating peptide is 12~15:18~20:1~1.5, and the concentration of the membrane-penetrating peptide in PBS is 0.1~0.15 mg / mL.

11. The use of the membrane-penetrating peptide modified human umbilical cord mesenchymal stem cell membrane-coated manganese dioxide nanocarrier according to claim 1 in the preparation of a drug for killing tumor cells, wherein the tumor is non-small cell lung cancer.

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