Bionic drug-loaded nanoparticle with expressed CXCR4, preparation method therefor, and use thereof

Abstract

Provided are a bionic drug-loaded nanoparticle with expressed CXCR4, a method for preparing same, and use thereof. The method comprises: mixing a lactic acid-glycolic acid copolymer and an immunosuppressant in a certain ratio, and preparing the mixture into a drug-loaded nanoparticle using a microfluidic technology; preparing an MSC cell stably expressing membrane protein CXCR4 and lysing same to extract the cell membrane, so as to give an engineered stem cell membrane; and fusing the engineered stem cell membrane with the drug-loaded nanoparticle to give a bionic drug-loaded nanoparticle with expressed CXCR4. The bionic drug-loaded nanoparticle with expressed CXCR4 is a bionic cell membrane drug-loaded nanoparticle with chemotaxis functionality. By means of genetic engineering, the engineered stem cell membrane is modified to encapsulate a therapeutic agent, and directionally migrates to the lesions in the body and targets specific cells, thereby improving the drug distribution in vivo and reducing adverse effects of immunosuppressants.

Description

A biomimetic drug-loaded nanoparticle expressing CXCR4 and its preparation method and application Technical Field

[0001] The present invention relates to the field of bionic cells, and in particular to a bionic drug-loaded nanoparticle expressing CXCR4, and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have been widely used in the clinical treatment of various diseases due to their low immunogenicity and broad spectrum of immunomodulatory functions. MSCs possess inherently low immunogenicity due to the absence or low expression of major histocompatibility complex class I and class II molecules and co-stimulatory molecules on their cell membranes. This allows them to evade clearance by immune cells upon entry into the body and prolong their circulation. In recent years, research on stem cell-based "carrier stem cells" has gained increasing attention. MSCs express numerous receptors and cell adhesion molecules that facilitate their migration and homing to target tissues. The CXCR4 / SDF-1 signaling axis, consisting of the membrane chemokine receptor CXCR4 and the high concentration of stromal-derived factor-1 (SDF-1) at sites of inflammatory injury, is one of the most important mechanisms for MSC chemotaxis and homing. This underpins the use of stem cell-delivered nanoparticles for targeted therapy of tumors and inflammatory diseases. Dysregulation of the SDF-1 / CXCR4 axis plays a key role in the differentiation, migration, recruitment, and engraftment, as well as the survival and proliferation, of bone marrow mesenchymal stem cells.

[0003] While stem cell drug delivery systems can improve the environmental adaptability of nanomedicines, the microscopic size of MSCs limits their precise homing to target tissues and organs. Multiple studies have shown that intravenously infused MSCs are primarily distributed in the lungs, liver, and spleen. Furthermore, MSC migration and homing to damaged tissues are influenced by numerous factors, including cell age and passage number, culture conditions, and delivery methods. These factors limit the application of MSC delivery vehicles in biomedical applications.

[0004] Currently, immunosuppressants are primarily used for organ transplant rejection, autoimmune diseases, and malignant tumors. However, patients experience significant toxic side effects from high-dose immunosuppressants, while low-dose treatments severely limit their efficacy. Nanodrug delivery systems can solubilize drugs, increase their half-life, improve their distribution in the body, enhance their targeting, and reduce their toxic side effects, offering significant potential for application in disease diagnosis and treatment. Cells proliferate within the capillaries at the site of injury, while the lymphatic system is obstructed, making it easy for nanodrugs to accumulate in this area. Most studies suggest that a major reason for the poor efficacy of nanoparticles is their lack of targeting for arthritis, but their safety profile is relatively low. Summary of the Invention

[0005] In order to solve the problems of low homing rate, large toxic and side effects, and unstable therapeutic effects of traditional nanomedicines for the treatment of autoimmune diseases, the present invention provides a biomimetic drug-loaded nanoparticle expressing CXCR4 and its preparation method and application. The nanomedicine is prepared by using a biodegradable nanomaterial with excellent biocompatibility to load an immunosuppressant. The mesenchymal stem cell membrane overexpressing CXCR4 is coated on the surface of the nanomedicine, further synergizing the immune escape and inflammatory chemotaxis functions of the stem cells to prepare a cell membrane-coated biomimetic nanomedicine, improving the drug distribution in the body and reducing the toxic and side effects of immunosuppressive drugs, thereby realizing an effective strategy for efficient drug delivery and targeted lesion sites.

[0006] A method for preparing biomimetic drug-loaded nanoparticles expressing CXCR4, comprising:

[0007] Lactic acid-glycolic acid copolymer and immunosuppressant were mixed in a certain proportion and prepared into drug-loaded nanoparticles using microfluidic technology;

[0008] Prepare MSC cells stably expressing CXCR4 membrane protein, lyse them and extract the cell membrane to obtain engineered stem cell membrane;

[0009] fusing the engineered stem cell membrane with drug-loaded nanoparticles to obtain biomimetic drug-loaded nanoparticles expressing CXCR4, wherein the biomimetic drug-loaded nanoparticles expressing CXCR4 are biomimetic cell membrane drug-loaded nanoparticles with chemotactic properties;

[0010] Among them, the method for fusing the engineered stem cell membrane with the drug-loaded nanoparticles is: mixing the engineered stem cell membrane and the drug-loaded nanoparticles at a mass ratio of 0.8~1: 0.8~1, and fusing them by ultrasound, wherein the ultrasound treatment frequency is 10~30 kHz, the time is 30~60 s, and the temperature is 5~10°C.

[0011] To optimize the above technical solutions, specific measures / limitations adopted also include:

[0012] The immunosuppressant is selected from at least one of cyclosporine, methotrexate and cyclophosphamide.

[0013] Lactic acid-glycolic acid copolymer and immunosuppressant are mixed at a mass ratio of 5-10:1.

[0014] The specific method for preparing drug-loaded nanoparticles using microfluidic technology is as follows: lactic acid-glycolic acid copolymer and immunosuppressant are mixed and dissolved in dimethyl sulfoxide to form an aqueous phase mixture, the mass / volume ratio of lactic acid-glycolic acid copolymer and immunosuppressant to dimethyl sulfoxide is 5-15:1 mg / mL, the aqueous phase mixture is dripped dropwise into ultrapure water, the volume ratio of ultrapure water to aqueous phase mixture is 6~10:1, and stirred at room temperature for 0.5~3 hours.

[0015] Drug-Loaded Nanoparticle Purification: The prepared drug-loaded nanoparticles were transferred to a dialysis bag to remove excess organic phase and reagents. Water was removed by distillation and dialyzed against water for 30 h. The suspension was then freeze-dried to obtain purified nanoparticles. The molecular weight cutoff of the filter membrane was 3–7 kDa.

[0016] The MSC cells are selected from at least one of human umbilical cord MSC cells, human adipose MSC cells or human bone marrow MSC cells.

[0017] The preparation method of MSC cells stably expressing CXCR4 membrane protein is as follows: P3 MSC cells are cultured until the confluence reaches 80-90%, the MSC cells are transfected with a culture medium containing Lv-CXCR4 overexpression lentivirus, and the cells are cultured at 37°C for 12-24 hours. The culture medium containing the Lv-CXCR4 overexpression lentivirus is replaced with DMEM medium and cultured for a further 36-48 hours. Resistance screening is then performed to obtain MSC cells stably expressing CXCR4 membrane protein.

[0018] The method for lysing and extracting the cell membrane to obtain the engineered stem cell membrane is as follows: after washing the cells two to three times with pre-chilled phosphate buffer solution, resuspending them in hypotonic lysis buffer, and repeatedly freezing and thawing them in liquid nitrogen 4 to 5 times to fully lyse the cells. The cell membrane pellet is then centrifuged and resuspended in phosphate buffer solution. The pellet is then repeatedly extruded through a polycarbonate membrane 8 to 13 times to obtain the engineered stem cell membrane. The polycarbonate membrane has a pore size of 400 nm, 200 nm, or 100 nm, preferably 200 nm.

[0019] The hypotonic lysis buffer includes Tris-HCl buffer, protease inhibitors, and phosphatase inhibitors.

[0020] The present invention also protects biomimetic drug-loaded nanoparticles expressing CXCR4 prepared by the method.

[0021] The present invention also protects the use of the bionic drug-loaded nanoparticles in the preparation of immunosuppressant drugs.

[0022] Furthermore, the application is the application of biomimetic drug-loaded nanoparticles in the preparation of rheumatoid arthritis delivery drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention transforms in vitro 2D cultured stem cells with weakened chemotaxis so that they can "home" to inflammatory damage or tumor microenvironment. The isolated CXCR4 stem cell membrane is used to encapsulate clinical therapeutic drugs. The resulting biomimetic cell membrane-loaded drug nanoparticles can migrate directionally to damaged areas in the body and target specific cells.

[0025] The present invention first analyzes the characteristics of MSC cell membranes, combines the properties of biomaterials, and constructs stem cells that overexpress CXCR4 through genetic engineering technology. PLGA drug-loaded nanoparticles are prepared by nanoprecipitation method, which wrap the CXCR4 stem cell membrane to form bionic nanomedicines. The bionic membrane-wrapped nanomedicines are then systemically delivered to the injured area to alleviate the course of the disease. The method is simple and easy to operate.

[0026] The method of fusing the engineered stem cell membrane with drug-loaded nanoparticles and preparing drug-loaded nanoparticles using microfluidic technology is critical to the successful acquisition of the product CMPNs and the impact on product performance. The process method of the present invention can achieve relatively ideal experimental results.

[0027] Further research found that the engineered MSCs of the present invention highly expressed CXCR4, which enabled the cells to acquire stronger migration ability and reduced the proportion of co-cultured inflammatory cells. Therefore, when nanomedicines wrapped in engineered MSCs membranes are injected during the progression of the disease, the bionic nanoparticles can migrate directionaly toward damaged organs and enhance their immune regulation ability, which is conducive to further improving the disease treatment effect of the MSC biomimetic membrane.

[0028] The present invention also provides related medical and pharmaceutical applications, such as organ transplantation anti-rejection, autoimmune diseases and malignant tumor treatment, alleviating the problem of systemic toxicity of nanomedicines, while ensuring that the carried therapeutic drugs survive for a long time at the site of injury and stably exert their biological functions, so that immunosuppressive drugs can reach the minimum therapeutic dose in the body.

[0029] The present invention has good application prospects in the treatment research of bionic nanomedicines, such as the development of bionic intravenous preparations, which are safe, feasible and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a transmission electron micrograph of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to the present invention.

[0031] FIG2 is a schematic diagram of the preparation and identification of biomimetic drug-loaded nanoparticles expressing CXCR4 according to the present invention.

[0032] FIG3 is a hemolytic experiment of the biomimetic drug-loaded nanoparticles expressing CXCR4 of the present invention.

[0033] FIG4 is a chemotaxis verification of the biomimetic drug-loaded nanoparticles expressing CXCR4 of the present invention.

[0034] FIG5 is a photograph of joints treated with RA using the biomimetic drug-loaded nanoparticles expressing CXCR4 of the present invention.

[0035] FIG6 is an H&E pathological picture of RA treated by the biomimetic drug-loaded nanoparticles expressing CXCR4 according to the present invention. DETAILED DESCRIPTION

[0036] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0038] Example 1

[0039] A method for preparing biomimetic drug-loaded nanoparticles expressing CXCR4 comprises the following steps:

[0040] 1. Preparation of drug-loaded nanoparticles using microfluidic technology: 10 mg of PLGA (lactic acid-co-glycolic acid copolymer) was dissolved in 1 mL of DMSO (dimethyl sulfoxide), and 1 mg of MTX (methotrexate) was added to obtain an aqueous mixture; the obtained aqueous mixture was dropped into 8 ml of ultrapure water and stirred at 25°C for 1 hour; the resulting product was transferred to a dialysis tube (MW: 3500 Da) to remove excess organic phase and reagents; after dialysis with distilled deionized water for 24 hours, the suspension was freeze-dried to obtain purified drug-loaded nanoparticles.

[0041] 2. Preparation of MSC cells stably expressing CXCR4 membrane protein: Culture P3 MSC cells until confluency reaches 80%, then transfect the MSC cells with medium containing Lv-CXCR4 overexpression lentivirus and culture at 37°C for 12-24 hours. Replace the medium containing Lv-CXCR4 overexpression lentivirus with DMEM medium and continue culturing for 36-48 hours. Subsequently, use medium containing 1-3 mg / ml puromycin for resistance screening to obtain cells stably transfected with CXCR4 membrane protein.

[0042] 3. Characterization of CXCR4-engineered stem cell membranes: Flow cytometry (FCM) was used to detect cell growth cycles and stem cell surface markers. The human CXCR4 gene was aligned using BLAST software to obtain the gene sequence, construct the pDsRed-CXCR4 plasmid, and package it into lentivirus. MSCs were transfected with the gene, and CXCR4 receptor expression was detected using laser scanning confocal microscopy (LSCM), reverse transcription PCR (RT-PCR), Western blot (WB), immunofluorescence, and FCM. Transwell assays were used to evaluate changes in the in vitro migration ability of transfected MSCs.

[0043] 4. Preparation of CXCR4 engineered stem cell membrane: The MSC cells stably expressing CXCR4 membrane protein prepared in step 2 were rinsed two to three times with pre-chilled phosphate buffer solution, resuspended in hypotonic lysis buffer, and repeatedly frozen and thawed in liquid nitrogen five times. After the cells were fully lysed, they were centrifuged at 1850 g for 10 min at 4°C to extract the cell membrane pellet. Finally, the extracted cell membrane pellet was resuspended in phosphate buffer solution and the cell membrane suspension was squeezed back and forth 11 times through a 200 nm polycarbonate membrane using a micro-extruder to obtain the CXCR4 engineered stem cell membrane;

[0044] 5. Preparation of Chemotactic Biomimetic Cell Membrane Drug-Loaded Nanoparticles (CMPNs): The engineered cell membrane prepared in Step 3 was ultrasonically treated and then blended with the drug-loaded nanoparticles prepared in Step 1 at a 1:1 mass ratio. Ultrasonic fusion was performed at a frequency of 20 kHz, a duration of 30 s, and a temperature of 8°C to obtain biomimetic cell membrane drug-loaded nanoparticles with chemotactic properties. (See Figure 1.) The results showed that the biomimetic cell membrane drug-loaded nanoparticles exhibited a distinct core-shell structure, were round and uniform, with a particle size of approximately 121.4 nm and a charge of approximately -20 mV. The CMPNs had a drug loading of 4.3% and an encapsulation efficiency of 93.2%, achieving effective methotrexate loading.

[0045] Example 2

[0046] In vitro characterization of biomimetic drug-loaded nanoparticles expressing CXCR4.

[0047] To test the long-term stability of the prepared CXCR4-expressing biomimetic drug-loaded nanoparticles, the nanoparticles were dispersed in PBS buffer to a final concentration of 1 mg / mL, and their particle size was observed using dynamic light scattering. The particle size remained largely unchanged over the course of a week, demonstrating the good solution stability of the CXCR4-expressing biomimetic drug-loaded nanoparticles.

[0048] In vitro drug release experiments were conducted to study the drug release capacity of biomimetic drug-loaded nanoparticles expressing CXCR4. Red blood cell hemolysis experiments were used to evaluate the blood compatibility of biomimetic drug-loaded nanoparticles expressing CXCR4. The results showed that biomimetic drug-loaded nanoparticles expressing CXCR4 had good blood compatibility, as shown in Figure 3.

[0049] Example 3

[0050] Application of biomimetic drug-loaded nanoparticles expressing CXCR4 in the targeted treatment of RA joints. To evaluate the in vivo therapeutic effect of biomimetic drug-loaded nanoparticles expressing CXCR4, a collagen adjuvant-induced rheumatoid arthritis mouse model was used in the experiment. The model group, MTX group, MPNs group, and CMPNs group were set up. Among them, the model group did not receive any treatment, the MTX group was directly injected with MTX solution (methotrexate injection), the MPNs group was injected with MPNs (stem cell membrane-wrapped PLGA & MTX), and the CMPNs group was injected with CMPNs (CXCR4-modified stem cell membrane-wrapped PLGA & MTX). The concentration of each group was 100 ul, 2.5 mg MTX / kg; injected twice through the tail vein on day 28 and day 35, respectively, and the mice were killed on day 42 to observe the therapeutic effect. Peripheral blood, synovium and joint tissues were collected at the same time. Figure 5 shows the joint tissue diagram, from which it can be found that the joint redness and swelling of CIA mice were significantly inhibited after treatment with stem cell membrane nanoparticles, and the swelling of the mouse paws was significantly relieved. Pathological analysis of the knee joint (HE staining) showed that the inflammation and bone destruction in the joint were significantly alleviated after nanoparticle treatment, as shown in Figure 6.

[0051] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A preparation method of a biomimetic drug-loaded nanoparticle expressing CXCR4, characterized in that, Including: Mixing poly (lactic-co-glycolic acid) and an immunosuppressant in a certain proportion, and using microfluidic technology to prepare drug-loaded nanoparticles; Preparing MSC cells stably expressing CXCR4 membrane protein, lysing them to extract cell membranes, and obtaining engineered stem cell membranes; Fusing the engineered stem cell membranes with the drug-loaded nanoparticles to obtain biomimetic drug-loaded nanoparticles expressing CXCR4, and the biomimetic drug-loaded nanoparticles expressing CXCR4 are biomimetic cell membrane drug-loaded nanoparticles with chemotactic properties; Among them, the method for fusing the engineered stem cell membranes with the drug-loaded nanoparticles is: mixing the engineered stem cell membranes and the drug-loaded nanoparticles at a mass ratio of 0.8~1:0.8~1, and performing ultrasonic fusion, where the ultrasonic treatment frequency is 10~30 kHz, the time is 30~60 s, and the temperature is 5~10 °C.

2. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, wherein: The immunosuppressant is selected from at least one of cyclosporine, methotrexate, and cyclophosphamide.

3. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, wherein: Poly (lactic-co-glycolic acid) and the immunosuppressant are mixed at a mass ratio of 5~10:

1.

4. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, characterized in that: The specific method for preparing drug-loaded nanoparticles using microfluidic technology is: mixing poly (lactic-co-glycolic acid) and the immunosuppressant and dissolving them in dimethyl sulfoxide to form an aqueous phase mixture, and the mass / volume ratio of poly (lactic-co-glycolic acid), the immunosuppressant, and dimethyl sulfoxide is 5-15:1 mg / mL. Drop the aqueous phase mixture into ultrapure water drop by drop, and the volume ratio of ultrapure water to the aqueous phase mixture is 6~10:1, and stir at room temperature for 0.5~3 h.

5. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, characterized in that: The MSC cells are selected from at least one of human umbilical cord MSC cells, human adipose MSC cells, or human bone marrow MSC cells.

6. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, characterized in that: The method for preparing MSC cells stably expressing CXCR4 membrane protein is: culturing P3-generation MSC cells until they reach a confluence of 80~90%, transfecting the MSC cells with a medium containing Lv-CXCR4 overexpression lentivirus, then replacing the medium containing Lv-CXCR4 overexpression lentivirus with DMEM medium and continuing to culture, and then performing resistance screening to obtain MSC cells stably expressing CXCR4 membrane protein.

7. The preparation method of the biomimetic drug-loaded nanoparticles expressing CXCR4 according to claim 1, characterized in that: The method for lysing them to extract cell membranes and obtaining engineered stem cell membranes is: rinsing the cells with pre-cooled phosphate buffer solution, resuspending them with a hypotonic lysis solution, placing them in liquid nitrogen and freezing and thawing repeatedly to fully lyse the cells, centrifuging to extract the cell membrane precipitate, resuspending it with phosphate buffered saline solution, and extruding repeatedly to obtain engineered stem cell membranes.

8. The biomimetic drug-loaded nanoparticles expressing CXCR4 prepared by the method according to any one of claims 1-7.

9. The application of the biomimetic drug-loaded nanoparticles according to claim 8 in the preparation of immunosuppressant drugs.

10. The application according to claim 9, wherein: The application of the biomimetic drug-loaded nanoparticles in the preparation of drugs for delivering to rheumatoid arthritis.

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