Stem cell membrane-modified liposome nanoparticles and preparation method thereof

Abstract

The present invention discloses a stem cell membrane-modified liposome nanoparticle and a preparation method thereof, belonging to the field of nano-drug delivery technology, and specifically relates to: a core layer, wherein the core layer is a carrier and a drug, the drug is loaded on the carrier, the carrier is a lipid carrier, and the lipid carrier includes egg phosphatidylcholine and cholesterol; a membrane layer, wherein the membrane layer is a stem cell membrane, and the stem cell membrane is coated on the outside of the core layer; an outer layer, wherein the outer layer includes a chitosan derivative; the core layer and the membrane layer constitute the stem cell membrane-coated liposome nanoparticle, and the amount of the chitosan derivative is 400-600wt% of the stem cell membrane-coated liposome nanoparticle. The present invention provides a stem cell membrane-modified liposome nanoparticle with good stability, good drug release effect in an acidic tumor microenvironment, good targeting, good immunocompatibility, good biosafety, and good biocompatibility, and a preparation method thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano drug delivery, and in particular relates to a stem cell membrane-modified liposome nano drug delivery particle and a preparation method thereof. Background Art

[0002] Breast cancer treatment has undergone a profound transformation from single surgery to comprehensive treatment, encompassing multiple approaches including surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy. However, traditional chemotherapy drugs often face challenges such as poor water solubility, significant side effects, and poor targeting, which compromise therapeutic efficacy and patients' quality of life. In recent years, with the rapid development of nanoscience and technology, nanodrug delivery technology has been widely used in the development and treatment of breast cancer drugs. Although traditional nanoparticles, such as liposomes, can improve drug bioavailability and targeting, they still face numerous challenges in crossing biological barriers, evading immune surveillance, and achieving precise delivery. Furthermore, the toxicity and safety of nanodrug delivery systems require thorough research and evaluation.

[0003] Stem cell membranes are rich in a variety of functional proteins and carbohydrate molecules, which play an important role in cell recognition, signal transduction, and immune evasion. In recent years, stem cell membranes have been combined with nanoparticle drug delivery systems to create nanoparticles with stem cell membrane properties. These particles can mimic stem cell behaviors, such as targeted migration, immune evasion, and intercellular interactions, providing new insights into breast cancer treatment.

[0004] While the research and development of stem cell membrane-inspired nano-drug delivery systems has shown tremendous potential, their development remains in its early stages, facing a series of complex technical challenges, including material stability, biosafety, drug targeting, and optimizing the performance of the nanomaterials themselves. Therefore, we urgently need to develop novel stem cell membrane-modified nano-drug delivery systems, continuously explore nano-drug delivery systems that are compatible with stem cell membranes, and conduct thorough toxicity and safety assessments to achieve efficient and safe drug delivery in vivo. Summary of the Invention

[0005] The purpose of the present invention is to provide a stem cell membrane-modified liposome nanoparticle with good stability, good drug release effect in acidic tumor microenvironment, good targeting, good immunocompatibility, good biosafety and good biocompatibility, and a preparation method thereof.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0007] A stem cell membrane-modified liposome nanoparticle containing drugs, comprising:

[0008] The core layer comprises a carrier and a drug, the drug is loaded on the carrier, the carrier is a lipid carrier, and the lipid carrier comprises egg phosphatidylcholine and cholesterol;

[0009] The membrane layer is a stem cell membrane, and the stem cell membrane is coated on the outside of the core layer;

[0010] an outer layer comprising a chitosan-based derivative;

[0011] The core layer and the membrane layer constitute the stem cell membrane-coated liposome nanoparticles, and the amount of the chitosan derivative is 400-600wt% of the stem cell membrane-coated liposome nanoparticles. The object of the present invention is to develop a stem cell membrane-modified liposome nanoparticle loaded with dual drugs to solve the problems existing in the clinical application of the existing nano drug delivery system. The nanoparticles include, from the inside to the outside, the drug-loaded drugs albumin paclitaxel nab-PTX and doxorubicin DXR, liposome nanocarrier lipo, stem cell membrane MSCm, and the outer modified chitosan derivative; wherein the liposome nanocarrier wraps the water-soluble drug in the central aqueous phase, the liposome nanocarrier is coated with the stem cell membrane outside, and the stem cell membrane outer surface is further modified by chitosan, and together they are assembled into chitosan derivative @ stem cell membrane-lipid carrier-drug nanoparticles. The above materials of the present invention all have good biosafety, greatly reducing the potential toxicity risk that may be caused by the accumulation of the nano drug delivery system in the organism, and providing a safer and more reliable basis for the development of drug delivery systems. Furthermore, when chitosan derivatives are used to modify stem cell membranes, they not only improve the membrane's stability and protect the stem cells from damage by the external environment, but also enhance the membrane's ability to immunomodulate drug-loaded nanoparticles, which helps reduce immune responses and rejection during treatment. Chitosan derivatives can include carboxymethyl chitosan.

[0012] Preferably, the amount of cholesterol is 10-20 wt% of the egg phosphatidylcholine; or, the mass ratio of the stem cell membrane to the core layer is 1:0.05-2; or, the amount of the drug is 0.05-5 wt% of the egg phosphatidylcholine.

[0013] Preferably, the chitosan-based derivative includes at least one of carboxymethyl chitosan, chitosan amino acid derivatives, and chitosan polyalkylene amino derivatives. When using chitosan-based derivatives to modify stem cell membranes, not all chitosan or its derivatives can be used. In the present invention, the use of chitosan alone, chitosan amino acid derivatives alone, or chitosan polyalkylene amino derivatives alone cannot enhance the immunomodulatory ability of the prepared stem cell membrane-modified liposome nanoparticles, nor can it effectively reduce immune responses and rejection reactions during treatment.

[0014] More preferably, the chitosan amino acid derivative is obtained by reacting chitosan with S-lactylglutathione.

[0015] More preferably, the chitosan polyalkylene amino derivative is obtained by reacting chitosan with N-methacryloylglycine.

[0016] Preferably, the chitosan-based derivatives include carboxymethyl chitosan, chitosan amino acid derivatives and chitosan polyalkylene amino derivatives, the mass ratio of carboxymethyl chitosan to chitosan amino acid derivatives is 5-200:5-50, and the mass ratio of carboxymethyl chitosan to chitosan polyalkylene amino derivatives is 5-200:2-200.

[0017] Preferably, in the preparation of chitosan amino acid derivatives, chitosan is added to deionized water, and then HOBt is added, stirred and dispersed evenly, and then S-lactylglutathione and EDC·HCl are added, the pH of the solution is controlled to 4.5-5.5, and the reaction is stirred in the dark at 20-40° C. for 1-8 hours. After the reaction is completed, dialyzed in a dialysis bag, and freeze-dried to obtain the chitosan derivative.

[0018] More preferably, in the preparation of chitosan amino acid derivatives, the amount of chitosan used is 0.5-10 wt % of deionized water.

[0019] More preferably, in the preparation of chitosan amino acid derivatives, the amount of HOBt used is 60-90 wt % of chitosan.

[0020] More preferably, in the preparation of chitosan amino acid derivatives, the amount of S-lactylglutathione used is 300-500 wt % of chitosan.

[0021] More preferably, in the preparation of chitosan amino acid derivatives, the amount of EDC·HCl used is 50-80 wt % of chitosan.

[0022] Preferably, in the preparation of chitosan polyalkyleneamine derivatives, chitosan is added to an acetic acid solution, stirred and mixed at 20-40° C., an initiator and N-methylacryloylglycine are added under an inert gas atmosphere, and then stirred and reacted at 50-80° C. for 1-4 hours. After the reaction is completed, the pH is adjusted to 10-12, and the solution is washed with deionized water and methanol in sequence, and freeze-dried to obtain a chitosan derivative.

[0023] More preferably, in the preparation of the chitosan polyalkyleneamine derivative, the acetic acid solution contains 1-3 wt % of acetic acid, and the amount of chitosan used is 2-10 wt % of the acetic acid solution.

[0024] More preferably, in the preparation of the chitosan polyalkyleneamine derivative, the initiator is dibenzoyl peroxide, and the amount of the initiator used is 1-10 wt % of the chitosan.

[0025] More preferably, in the preparation of chitosan polyalkyleneamine derivatives, the amount of N-methacryloylglycine used is 20-60 wt % of chitosan.

[0026] The present invention discloses a method for preparing stem cell membrane-modified liposome nanoparticles loaded with drugs, comprising:

[0027] S1, adding the drug to deionized water to prepare a drug solution, adding a lipid carrier raw material to a solvent to prepare a lipid carrier raw material liquid, and mixing the drug solution and the lipid carrier raw material liquid to prepare liposome nanoparticles loaded with drugs, i.e., the core layer; the lipid carrier raw materials include egg phosphatidylcholine and cholesterol;

[0028] S2, extracting the stem cell membrane, and mixing the stem cell membrane with liposome nanoparticles to prepare stem cell membrane-coated liposome nanoparticles;

[0029] S3, adding a chitosan-based derivative to deionized water, and then mixing it with an isopropanol solution containing stem cell membrane-coated liposome nanoparticles to prepare chitosan-based liposome nanoparticles, i.e., stem cell membrane-modified liposome nanoparticles; the amount of the chitosan-based derivative is 400-600 wt % of the stem cell membrane-coated liposome nanoparticles.

[0030] Preferably, the stem cell membrane is derived from mesenchymal stem cells.

[0031] Preferably, the amount of the drug in the drug solution is 0.01-0.6 wt % of deionized water, and the drug includes nab-PTX and / or DXR.

[0032] Preferably, the amount of egg phosphatidylcholine used is 2-20 wt % of the solvent.

[0033] Preferably, in the isopropanol solution containing the stem cell membrane-coated liposome nanoparticles, the amount of the stem cell membrane-coated liposome nanoparticles is 0.005-0.2 wt % of the isopropanol.

[0034] Preferably, the chitosan-based derivative is added to deionized water to obtain a chitosan-based solution, wherein the chitosan-based solution contains 0.05-2 wt % of carboxymethyl chitosan.

[0035] The present invention discloses the use of the stem cell membrane-modified liposome nanometer drug-carrying particles in the preparation of tumor drugs.

[0036] Preferably, in the preparation of single-drug-loaded liposome nanoparticles, the drug is added to deionized water and stirred at 50-70°C for 10-60 minutes to obtain a single drug solution; egg phosphatidylcholine and cholesterol are added to chloroform, and rotated at 20-40°C and 60-100 rpm / min to form a film, and then the single drug solution is added, and the mixture is ultrasonicated in a water bath for 1-20 minutes, and then dialyzed in deionized water for 12-48 hours to remove unentrapped drugs, and squeezed through the membrane to obtain single-drug-loaded liposome nanoparticles.

[0037] More preferably, in the preparation of single drug-loaded liposome nanoparticles, the drug is nab-PTX or DXR, and the amount of the drug used is 0.01-0.6 wt % of deionized water.

[0038] More preferably, in the preparation of single drug-loaded liposome nanoparticles, the amount of egg phosphatidylcholine used is 2-20 wt % of chloroform.

[0039] More preferably, in the preparation of single drug-loaded liposome nanoparticles, the amount of cholesterol used is 10-20 wt % of egg phosphatidylcholine.

[0040] More preferably, in the preparation of single drug-loaded liposome nanoparticles, the amount of the single drug solution used is calculated based on the initial amount of the drug used therein, and the amount of the drug used is 0.05-5wt% of the egg phosphatidylcholine.

[0041] Preferably, in the preparation of dual-drug-loaded liposome nanoparticles, the drug is added to deionized water and stirred at 50-70°C for 10-60 minutes to obtain a dual-drug solution; egg phosphatidylcholine and cholesterol are added to chloroform, and rotated at 20-40°C and 60-100 rpm / min to form a film, and then the dual-drug solution is added, and water bath ultrasound is performed for 1-20 minutes, and then dialyzed in deionized water for 12-48 hours to remove unentrapped drugs, and squeezed through the membrane to obtain single-drug-loaded liposome nanoparticles.

[0042] More preferably, in the preparation of dual-drug-loaded liposome nanoparticles, the drugs are nab-PTX and DXR, and nab-PTX and DXR in the drugs are compounded in a mass ratio of 1:0.3-0.8, and the amount of the drugs used is 0.01-0.6wt% of deionized water.

[0043] More preferably, in the preparation of dual-drug-loaded liposome nanoparticles, the amount of egg phosphatidylcholine used is 2-20 wt % of chloroform.

[0044] More preferably, in the preparation of dual-drug-loaded liposome nanoparticles, the amount of cholesterol used is 10-20 wt % of egg phosphatidylcholine.

[0045] More preferably, in the preparation of dual-drug-loaded liposome nanoparticles, the amount of the dual-drug solution used is based on the initial amount of the drug used, and the amount of the drug used is 0.05-5wt% of the egg phosphatidylcholine.

[0046] Preferably, in the preparation of stem cell membrane-coated liposome nanoparticles, mesenchymal stem cells are cultured, mesenchymal stem cells in the logarithmic growth phase are collected, digested and centrifuged, the cells are resuspended with PBS solution, and then the cells are broken by repeated freezing and thawing, and the stem cell membrane fragments of the mesenchymal stem cells are extracted using a cell membrane protein extraction kit; then, the stem cell membrane fragments are repeatedly squeezed using a polycarbonate porous filter membrane to prepare vesicles, and then the vesicles are mixed with the liposome nanoparticles, and ultrasonicated in a water bath at 0-10°C in the dark for 10-35 min to obtain stem cell membrane-coated liposome nanoparticles.

[0047] More preferably, in the preparation of the stem cell membrane-coated liposome nanoparticles, the vesicles and the liposome nanoparticles are mixed at a mass ratio of 1:0.05-2.

[0048] Preferably, in the preparation of chitosan-coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are added to isopropanol, and then the isopropanol solution containing the stem cell membrane-coated liposome nanoparticles is added dropwise to the chitosan-based solution, ultrasonically shaken for 10-60 minutes, and then dialyzed in deionized water for 12-48 hours to obtain chitosan-coated liposome nanoparticles.

[0049] More preferably, in the preparation of chitosan-based coated liposome nanoparticles, the amount of stem cell membrane-coated liposome nanoparticles is 0.005-0.2 wt % of isopropanol.

[0050] More preferably, in the preparation of chitosan-coated liposome nanoparticles loaded with drugs, the chitosan-based solution is prepared by mixing carboxymethyl chitosan with deionized water, and the chitosan-based solution contains 0.05-2 wt % of carboxymethyl chitosan.

[0051] More preferably, in the preparation of chitosan-coated liposome nanoparticles, the isopropanol solution containing stem cell membrane-coated liposome nanoparticles is measured in terms of stem cell membrane-coated liposome nanoparticles, and the chitosan-based solution is measured in terms of carboxymethyl chitosan, and the amount of carboxymethyl chitosan used is 400-600 wt % of the stem cell membrane-coated liposome nanoparticles.

[0052] More preferably, in the preparation of chitosan-coated liposome nanoparticles, the chitosan-based solution contains, in addition to carboxymethyl chitosan, a chitosan amino acid derivative and a chitosan polyalkylene amino derivative. The chitosan-based solution contains 0.05-0.5 wt% of the chitosan amino acid derivative and 0.02-0.2 wt% of the chitosan polyalkylene amino derivative.

[0053] More preferably, in the preparation of chitosan-coated liposome nanoparticles, the chitosan-based solution further contains polyethylene glycol glutarate. The chitosan-based solution contains 0.02-0.5wt% polyethylene glycol glutarate. In the present invention, after using carboxymethyl chitosan, chitosan amino acid derivatives, and chitosan polyenamino derivatives, and then further adding polyethylene glycol glutarate, the resulting chitosan-coated liposome nanoparticles have an immunomodulatory ability, while the use of polyethylene glycol glutarate alone has no effect. The polyethylene glycol glutarate can enhance the effects of the carboxymethyl chitosan, chitosan amino acid derivatives, and chitosan polyenamino derivatives, and can reduce immune responses and rejection reactions during treatment.

[0054] The present invention uses egg phosphatidylcholine and cholesterol as lipid carriers to load drugs, prepares a core layer, coats the core layer with a stem cell membrane, and finally coats it with a chitosan-based derivative to form an outer layer, thereby obtaining stem cell membrane-modified liposome nanoparticles; the drugs include nab-PTX and / or DXR, thereby having the following beneficial effects: the liposome nanoparticles, stem cell membrane-coated liposome nanoparticles, and chitosan-based coated liposome nanoparticles prepared by the present invention have good stability, good drug release effect in an acidic tumor microenvironment, good targeting, good immunocompatibility, good biosafety, and good biocompatibility. Therefore, the present invention is a stem cell membrane-modified liposome nanoparticle with good stability, good drug release effect in an acidic tumor microenvironment, good targeting, good immunocompatibility, good biosafety, and good biocompatibility, and a preparation method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 A morphology representation diagram.

[0056] Figure 2 This is the particle size test result diagram.

[0057] Figure 3 The figure shows the in vitro drug release results.

[0058] Figure 4 This is the result of the targeted test.

[0059] Figure 5 This is the result of immune escape test.

[0060] Figure 6 This is a comparison chart of immune escape test results.

[0061] Figure 7 This is a diagram showing the biosafety test results. DETAILED DESCRIPTION

[0062] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0063] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0064] Example 1: A method for preparing liposome nanoparticles

[0065] Preparation of single-drug liposomal nanoparticles: The drug was added to deionized water and stirred at 60°C for 30 minutes to obtain a single-drug solution. Egg phosphatidylcholine and cholesterol were added to chloroform and rotated at 30°C and 80 rpm to form a film. The single-drug solution was then added and the mixture was sonicated in a water bath for 10 minutes. The mixture was then dialyzed in deionized water for 24 hours to remove unencapsulated drug. The particles were then extruded through the membrane to obtain single-drug liposomal nanoparticles. The drug was nab-PTX, and the amount of drug used was 0.1wt% of the deionized water. The amount of egg phosphatidylcholine used was 2.52wt% of the chloroform, and the amount of cholesterol used was 16.67wt% of the egg phosphatidylcholine. The amount of the single-drug solution used was based on the initial amount of drug used, which was 0.1wt% of the egg phosphatidylcholine.

[0066] Example 2: A method for preparing liposome nanoparticles

[0067] Preparation of single-drug liposome nanoparticles: The drug was added to deionized water and stirred at 60°C for 30 minutes to obtain a single-drug solution. Egg phosphatidylcholine and cholesterol were added to chloroform and rotated at 30°C and 80 rpm to form a film. The single-drug solution was then added and the mixture was sonicated in a water bath for 10 minutes. The mixture was then dialyzed in deionized water for 24 hours to remove unencapsulated drug. The particles were then extruded through the membrane to obtain single-drug liposome nanoparticles. The drug was DXR, and the amount of drug used was 0.1wt% of the deionized water. The amount of egg phosphatidylcholine used was 2.52wt% of the chloroform, and the amount of cholesterol used was 16.7wt% of the egg phosphatidylcholine. The amount of the single-drug solution used was based on the initial amount of drug used, which was 0.1wt% of the egg phosphatidylcholine.

[0068] Example 3: A method for preparing liposome nanoparticles

[0069] Preparation of dual-drug liposome nanoparticles: The drug was added to deionized water and stirred at 60°C for 30 minutes to obtain a dual-drug solution. Egg phosphatidylcholine and cholesterol were added to chloroform and rotated at 30°C and 80 rpm to form a film. The dual-drug solution was then added and the mixture was sonicated in a water bath for 10 minutes. The mixture was then dialyzed in deionized water for 24 hours to remove unencapsulated drug and extruded through the membrane to obtain single-drug liposome nanoparticles. The drugs were nab-PTX and DXR, mixed at a mass ratio of 1:0.5. The drug dosage was 0.1 wt% of the deionized water. The egg phosphatidylcholine dosage was 2.52 wt% of the chloroform dosage, and the cholesterol dosage was 16.7 wt% of the egg phosphatidylcholine dosage. The dosage of the dual-drug solution was based on the initial drug dosage, resulting in a drug dosage of 0.1 wt% of the egg phosphatidylcholine dosage.

[0070] Example 4: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0071] Preparation of stem cell membrane-coated liposomal nanoparticles: Mesenchymal stem cells were cultured and collected during the logarithmic growth phase. After digestion and centrifugation, the cells were resuspended in PBS and disrupted by repeated freeze-thaw cycles. Mesenchymal stem cell membrane fragments were extracted using a cell membrane protein extraction kit. Vesicles were then repeatedly extruded using a polycarbonate porous filter membrane to prepare vesicles. The vesicles were then mixed with liposomal nanoparticles and sonicated in a water bath at 4°C in the dark for 20 minutes to obtain stem cell membrane-coated liposomal nanoparticles. The vesicles and liposomal nanoparticles were mixed at a mass ratio of 1:0.5. The liposomal nanoparticles were the single-drug-loaded liposomal nanoparticles prepared in Example 1.

[0072] Example 5: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0073] The difference between this embodiment and embodiment 4 is that the liposome nanoparticles are the single drug-loaded liposome nanoparticles prepared in embodiment 2.

[0074] Example 6: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0075] The difference between this embodiment and embodiment 4 is that the liposome nanoparticles are the dual-drug-loaded liposome nanoparticles prepared in embodiment 3.

[0076] Example 7: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0077] Preparation of chitosan-based coated liposome nanoparticles: stem cell membrane coated liposome nanoparticles are added to isopropanol, and then the isopropanol solution containing stem cell membrane coated liposome nanoparticles is added dropwise to the chitosan-based solution, ultrasonically shaken for 20 minutes, and then dialyzed in deionized water for 24 hours to obtain chitosan-based coated liposome nanoparticles. The usage amount of stem cell membrane coated liposome nanoparticles is 0.02wt% of isopropanol solution, and the chitosan-based solution is mixed with deionized water by carboxymethyl chitosan. The chitosan-based solution contains 0.1wt% of carboxymethyl chitosan. The isopropanol solution containing stem cell membrane coated liposome nanoparticles is measured with stem cell membrane coated liposome nanoparticles, and the chitosan-based solution is measured with carboxymethyl chitosan. The usage amount of carboxymethyl chitosan is 500wt% of stem cell membrane coated liposome nanoparticles. Stem cell membrane coated liposome nanoparticles is prepared by Example 4.

[0078] Example 8: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0079] The difference between this embodiment and embodiment 7 is that the stem cell membrane-coated liposome nanoparticles are prepared according to embodiment 5.

[0080] Example 9: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0081] The difference between this embodiment and embodiment 7 is that the stem cell membrane-coated liposome nanoparticles are prepared according to embodiment 6.

[0082] Example 10: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0083] This embodiment differs from embodiment 7 in that the chitosan-based solution used in the preparation of chitosan-coated liposome nanoparticles further contains a chitosan amino acid derivative and a chitosan polyalkylene amino derivative. The chitosan-based solution contains 0.2 wt% of the chitosan amino acid derivative, and the chitosan-based solution contains 0.12 wt% of the chitosan polyalkylene amino derivative.

[0084] Preparation of chitosan amino acid derivatives: Chitosan was added to deionized water, followed by HOBt, and stirred to disperse uniformly. S-lactylglutathione and EDC·HCl were then added. The pH of the solution was controlled at 5, and the mixture was stirred at 30°C in the dark for 5 hours. After completion of the reaction, the mixture was dialyzed in a dialysis bag and freeze-dried to obtain a chitosan derivative. The chitosan was used in an amount of 2 wt% of the deionized water, 80 wt% of the HOBt, 400 wt% of the S-lactylglutathione, and 60 wt% of the EDC·HCl.

[0085] Preparation of chitosan polyeneamine derivatives: Chitosan was added to an acetic acid solution and stirred at 30°C. An initiator and N-methacryloylglycine were added under an inert gas atmosphere, and the mixture was stirred at 60°C for 2 hours. After completion of the reaction, the pH was adjusted to 11, and the mixture was washed with deionized water and then methanol, followed by freeze-drying to obtain a chitosan derivative. The acetic acid solution contained 2 wt% acetic acid, and the amount of chitosan used was 6 wt% of the acetic acid solution. The initiator was dibenzoyl peroxide, which was used at 5 wt% of the chitosan. The amount of N-methacryloylglycine used was 40 wt% of the chitosan.

[0086] Example 11: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0087] The difference between this embodiment and embodiment 10 is that, in the preparation of chitosan-based coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are prepared by embodiment 5.

[0088] Example 12: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0089] The difference between this embodiment and embodiment 10 is that, in the preparation of chitosan-based coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are prepared by embodiment 6.

[0090] Example 13: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0091] The difference between this embodiment and embodiment 10 is that the chitosan-based solution in the preparation of the chitosan-coated liposome nanoparticles further contains polyethylene glycol glutarate, wherein the chitosan-based solution contains 0.15 wt% polyethylene glycol glutarate.

[0092] Example 14: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0093] The difference between this embodiment and embodiment 13 is that, in the preparation of chitosan-based coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are prepared by embodiment 5.

[0094] Example 15: Preparation of Stem Cell Membrane-Modified Liposome Nanoparticles

[0095] The difference between this embodiment and embodiment 13 is that, in the preparation of chitosan-based coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are prepared by embodiment 6.

[0096] Comparative Example 1: Preparation method of stem cell membrane-modified liposome nanoparticles

[0097] The difference between this comparative example and Example 9 is that the carboxymethyl chitosan in the chitosan-based solution is replaced by chitosan.

[0098] Comparative Example 2: Preparation method of stem cell membrane-modified liposome nanoparticles

[0099] This comparative example is different from Example 9 in that the carboxymethyl chitosan in the chitosan-based solution is replaced by the chitosan amino acid derivative prepared in Example 10.

[0100] Comparative Example 3: Preparation method of stem cell membrane-modified liposome nanoparticles

[0101] This comparative example is different from Example 9 in that the carboxymethyl chitosan in the chitosan-based solution is replaced by the chitosan polyalkylene amino derivative prepared in Example 10.

[0102] Comparative Example 4: Preparation method of stem cell membrane-modified liposome nanoparticles

[0103] This comparative example is different from Example 9 in that the carboxymethyl chitosan in the chitosan-based solution is replaced by polyethylene glycol glutarate.

[0104] Test example:

[0105] The present invention performs morphological characterization on the liposome nanoparticles and stem cell membrane-coated liposome nanoparticles prepared in the examples. Taking the dual-drug-loaded liposome nanoparticles prepared in Example 3 and the stem cell membrane-coated liposome nanoparticles prepared in Example 6 as examples, the morphological characterization results are shown in FIG. Figure 1 As shown, Figure 1 a is the morphology of dual-drug-loaded liposome nanoparticles, which are spherical in structure and about 100 nm in size; Figure 1 b is the morphology of stem cell membrane-coated liposome nanoparticles. Its overall shape and structure have not changed significantly, and the boundaries of the membrane coating can be clearly observed, which indicates that the stem cell membrane-coated liposome nanoparticles have been successfully synthesized.

[0106] The present invention conducted a stability test on the liposome nanoparticles and stem cell membrane-coated liposome nanoparticles prepared in the examples. The dual-drug-loaded liposome nanoparticles prepared in Example 3, the single-drug-loaded liposome nanoparticles prepared in Example 1, the stem cell membrane-coated liposome nanoparticles prepared in Example 6, and the chitosan-based coated liposome nanoparticles prepared in Example 9 were tested for particle size in PBS solution for 18 days. The results are as follows: Figure 2 As shown, lip-nab-PTX-DXR is the dual-drug-loaded liposome nanoparticles prepared in Example 3, MSCm-lip-nab-PTX-DXR is the stem cell membrane-coated liposome nanoparticles prepared in Example 6, lip-nab-PTX is the single-drug-loaded liposome nanoparticles prepared in Example 1, and CMCTS@MSCm-lip-nab-PTX-DXR is the chitosan-coated liposome nanoparticles prepared in Example 9. As time goes by, the particle size of the liposome nanoparticles not coated by the stem cell membrane increases significantly. However, the stem cell membrane-coated liposome nanoparticles and the chitosan-coated liposome nanoparticles showed relatively stable particle size information during the test, and the chitosan-coated liposome nanoparticles modified with carboxymethyl chitosan showed the best stability, indicating that carboxymethyl chitosan can further enhance the environmental stability of the stem cell membrane.

[0107] The present invention conducted an in vitro drug release test on the chitosan-coated liposome nanoparticles prepared in Example 9, simulating a tumor microenvironment with a pH of 6.8 and a normal cell environment with a pH of 7.4. The results are as follows: Figure 3As shown in the figure, the drug is continuously released as time goes by, and the chitosan-coated liposome nanoparticles release the drug faster and in a larger amount in a pH = 6.8 environment than in a pH = 7.4 environment, which indicates that the stem cell membrane-modified liposome nanoparticles of the present invention are very suitable for application in an acidic tumor microenvironment.

[0108] The present invention performs a targeting test on the single drug-loaded liposome nanoparticles prepared in Example 3 and the stem cell membrane-coated liposome nanoparticles prepared in Example 6. The samples are respectively selected and co-incubated with breast cancer cells 4T1 and normal breast cells EMT6. MSCm-lipo-nab-PTX-DXR is the stem cell membrane-coated liposome nanoparticles of Example 6, and lipo-nab-PTX-DXR is the single drug-loaded liposome nanoparticles of Example 3. MSCm-lipo-nab-PTX-DXR+4T1 cells and control groups lipo-nab-PTX-DXR+4T1 cells and MSCm-lipo-nab-PTX-DXR+EMT6 cells are selected and loaded with fluorescein FITC. The specific test method is as follows: 4T1 and EMT6 were inoculated in a confocal cell culture dish, cultured with DMEM containing 10% fetal bovine serum for 24 hours, and then the culture medium was discarded. 100 μL of FITC-MSCm-lipo-nab-PTX-DXR, FITC-lipo-nab-PTX-DXR, and FITC-MSCm-lipo-nab-PTX-DXR were taken and mixed evenly with 900 μL of culture medium containing 10% fetal bovine serum, totaling 1 mL, and the final concentration of the nanoparticles was 1 mg / mL, and incubated with 4T1 and EMT6 for 4 hours. The incubation solution was discarded, washed 3 times with PBS solution, and then the breast cancer cells 4T1 were co-incubated with the above samples using confocal microscopy, and observed and imaged using CLSM. The experimental results are as follows. Figure 4 As shown in the data, FITC-MSCm-lipo-nab-PTX-DXR+4T1 cells showed stronger fluorescence intensity than FITC-lipo-nab-PTX-DXR+4T1 cells and FITC-MSCm-lipo-nab-PTX-DXR+EMT6 cells, indicating that stem cells coated with liposome nanoparticles have the best targeting effect on breast cancer cells and will not cause excessive interference to normal cells, which will be beneficial for subsequent treatment.

[0109] The present invention conducted immune escape tests on the dual-drug-loaded liposome nanoparticles prepared in Example 3, the stem cell membrane-coated liposome nanoparticles prepared in Example 6, and the chitosan-coated liposome nanoparticles prepared in Example 9. Macrophages were inoculated in a confocal cell culture dish and cultured for 24 hours. The culture medium was discarded and the three samples were mixed with culture medium containing 10% fetal bovine serum at a volume ratio of 1:9. The samples were incubated with macrophages for 4 hours, washed with PBS, and then confocal imaging was performed. The results are shown in Figure 2. Figure 5 As shown, lipo-nab-PTX-DXR is the dual-drug-loaded liposome nanoparticles of Example 3, MSCm-lipo-nab-PTX-DXR is the stem cell membrane-coated liposome nanoparticles of Example 6, and CMCTS@MSCm-lipo-nab-PTX-DX is the chitosan-based coated liposome nanoparticles of Example 9. Compared with the stem cell membrane-coated liposome nanoparticles and the chitosan-based coated liposome nanoparticles, the dual-drug-loaded liposome nanoparticles that are not coated with the stem cell membrane show the characteristics of being taken up in large quantities by macrophages, which shows that the stem cell membrane-coated liposome nanoparticles have higher immunocompatibility. In addition, it was found that the chitosan-based coated liposome nanoparticles modified with carboxymethyl chitosan are less likely to be taken up by macrophages than the stem cell membrane-coated liposome nanoparticles, which also indirectly illustrates the protective effect of carboxymethyl chitosan on the stem cell membrane and its potential immune-promoting ability.

[0110] The chitosan-coated liposome nanoparticles prepared in Examples 9, 12, 15 and Comparative Examples 1-4 were subjected to immune escape testing according to the above method, wherein the samples were mixed evenly with a culture medium containing 10% fetal bovine serum at a volume ratio of 1:10, incubated with macrophages for 4 hours, washed with PBS, and then subjected to confocal imaging. The test results are shown in FIG. Figure 6As shown in the figure, after the core layer is coated with the stem cell membrane, the immune escape ability of the obtained stem cell membrane-coated liposome nanoparticles is improved, and the immune escape ability comes from the effect of the stem cell membrane. In order to further improve the immune escape ability, the present invention adds a chitosan derivative for coating treatment. Among them, the single use of carboxymethyl chitosan can further improve the immune escape ability of the chitosan-coated liposome nanoparticles. If carboxymethyl chitosan is replaced by chitosan, chitosan can hardly improve the immune escape ability of the obtained chitosan-coated liposome nanoparticles. If Replacing carboxymethyl chitosan with chitosan amino acid derivatives or chitosan polyenamino derivatives prepared in the present invention in one step also barely improves the immune escape ability of the resulting chitosan-coated liposome nanoparticles. The chitosan amino acid derivatives in the present invention are prepared by reacting chitosan with S-lactylglutathione, and the chitosan polyenamino derivatives in the present invention are prepared by reacting chitosan with N-methylacryloylglycine. The present invention has found that the immune escape ability of the resulting chitosan-coated liposome nanoparticles can be enhanced when carboxymethyl chitosan, chitosan amino acid derivatives, or chitosan polyenamino derivatives are used together. When carboxymethyl chitosan, chitosan amino acid derivatives, or chitosan polyenamino derivatives are used, the present invention has found that if polyethylene glycol glutarate is further added, the outer layer formed by the combined use of carboxymethyl chitosan, chitosan amino acid derivatives, chitosan polyenamino derivatives, or polyethylene glycol glutarate has better immune escape ability.

[0111] The present invention conducted cell safety tests on the dual-drug-loaded liposome nanoparticles prepared in Example 3, the stem cell membrane-coated liposome nanoparticles prepared in Example 6, and the chitosan-coated liposome nanoparticles prepared in Examples 7-9, and evaluated the killing effect of the nanoparticles of the present invention on breast cancer 4T1 cells and the biosafety on normal breast EMT6 cells based on the CCK8 method. The above-mentioned test samples were selected, the specific culture time was set to 24 h, the sample concentration was set to 10 μg / mL, and after incubation for 3 h, the optical density (OD) value of each well was measured at 450 nm on a microplate reader and the cell survival rate was calculated. The experimental results are as follows: Figure 7As shown, lipo-nab-PTX-DXR is the dual-drug-loaded liposome nanoparticles of Example 3, MSCm-lipo-nab-PTX-DXR is the stem cell membrane-coated liposome nanoparticles of Example 6, CMCTS@MSCm-lipo-nab-PTX is the chitosan-based coated liposome nanoparticles of Example 7, CMCTS@MSCm-lipo-DXR is the chitosan-based coated liposome nanoparticles of Example 8, and CMCTS@MSCm-lipo-nab-PTX-DX is the chitosan-based coated liposome nanoparticles of Example 9. On the one hand, the above samples generally have a significant inhibitory effect on breast cancer 4T1 cells, among which the chitosan-based coated liposome nanoparticles of Example 9 show a better proliferation inhibition rate than the two samples loaded with a single drug in the same system, which can be attributed to the synergistic effect between the two drugs. In addition, the chitosan-based coated liposome nanoparticles of Example 9 had a similar inhibitory effect on 4T1 cells as the dual-drug-loaded liposome nanoparticles of Example 3 and the stem cell membrane-coated liposome nanoparticles of Example 6, indicating that the coating with the stem cell membrane and the modification with carboxymethyl chitosan did not hinder the killing of breast cancer cells. On the other hand, the above samples generally had good biocompatibility with normal breast EMT6 cells, among which the chitosan-based coated liposome nanoparticles prepared in Examples 7-9 had similar cell activity; however, compared with the dual-drug-loaded liposome nanoparticles of Example 3 and the stem cell membrane-coated liposome nanoparticles of Example 6, the chitosan-based coated liposome nanoparticles of Example 9 showed an effect that was nearly 20% better than the above samples, which can be attributed to the coating of the liposomes with the stem cell membrane and the modification of the stem cell membrane with carboxymethyl chitosan, which further illustrates the protective effect of carboxymethyl chitosan on the stem cell membrane.

[0112] The present invention conducted a biosafety test on the chitosan-coated liposome nanoparticles prepared in Examples 9, 12, and 15. Normal breast EMT6 cells were used. The cell survival rate of breast EMT6 cells in the chitosan-coated liposome nanoparticles in Examples 12 and 15 was not much different from that in Example 9, indicating that the chitosan-coated liposome nanoparticles in Examples 12 and 15 of the present invention also have excellent biosafety.

[0113] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0114] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A stem cell membrane-modified liposome nanoparticle drug-loaded particle comprising: The core layer comprises a carrier and a drug, the drug is loaded on the carrier, the carrier is a lipid carrier, and the lipid carrier comprises egg phosphatidylcholine and cholesterol; The membrane layer is a stem cell membrane, and the stem cell membrane is coated on the outside of the core layer; an outer layer comprising a chitosan-based derivative; The core layer and the membrane layer constitute the stem cell membrane-coated liposome nanoparticles, and the amount of the chitosan derivative is 400-600 wt % of the stem cell membrane-coated liposome nanoparticles; The chitosan-based derivative is a combination of carboxymethyl chitosan, chitosan amino acid derivatives and chitosan polyenamino derivatives; the chitosan amino acid derivative is obtained by reacting chitosan with S-lactyl glutathione, and the amount of S-lactyl glutathione used is 300-500wt% of the chitosan; the chitosan polyenamino derivative is obtained by reacting chitosan with N-methacryloyl glycine, and the amount of N-methacryloyl glycine used is 20-60wt% of the chitosan.

2. The stem cell membrane-modified liposome nanoparticles according to claim 1, characterized in that: The amount of cholesterol is 10-20 wt% of the egg phosphatidylcholine; or, the mass ratio of the stem cell membrane to the core layer is 1:0.05-2; or, the amount of the drug is 0.05-5 wt% of the egg phosphatidylcholine.

3. The stem cell membrane-modified liposome nanoparticles according to claim 1, characterized in that: The mass ratio of the carboxymethyl chitosan to the chitosan amino acid derivative is 5-200:5-50, and the mass ratio of the carboxymethyl chitosan to the chitosan polyalkylene amino derivative is 5-200:2-200.

4. The method for preparing stem cell membrane-modified liposome nanoparticles according to claim 1, comprising: S1, adding the drug to deionized water to prepare a drug solution, adding a lipid carrier raw material to a solvent to prepare a lipid carrier raw material liquid, and mixing the drug solution and the lipid carrier raw material liquid to prepare liposome nanoparticles loaded with drugs, i.e., the core layer; the lipid carrier raw materials include egg phosphatidylcholine and cholesterol; S2, extracting the stem cell membrane, and mixing the stem cell membrane with liposome nanoparticles to prepare stem cell membrane-coated liposome nanoparticles; S3, adding a chitosan-based derivative to deionized water, and then mixing it with an isopropanol solution containing stem cell membrane-coated liposome nanoparticles to prepare chitosan-based liposome nanoparticles, i.e., stem cell membrane-modified liposome nanoparticles; the amount of the chitosan-based derivative is 400-600 wt % of the stem cell membrane-coated liposome nanoparticles.

5. The method for preparing stem cell membrane-modified liposome nanoparticles according to claim 4, characterized in that: The stem cell membrane is derived from mesenchymal stem cells.

6. The method for preparing stem cell membrane-modified liposome nanoparticles according to claim 4, characterized in that: The dosage of the drug in the drug solution is 0.01-0.6 wt % of deionized water, and the drug includes nab-PTX and / or DXR.

7. The method for preparing stem cell membrane-modified liposome nanoparticles according to claim 4, characterized in that: The usage amount of the egg phosphatidylcholine is 2-20wt% of the solvent.

8. The method for preparing stem cell membrane-modified liposome nanoparticles according to claim 4, characterized in that: In the isopropanol solution containing the stem cell membrane-coated liposome nanoparticles, the amount of the stem cell membrane-coated liposome nanoparticles is 0.005-0.2 wt % of the isopropanol.

9. Use of the stem cell membrane-modified liposome nanoparticles according to any one of claims 1 to 3 in the preparation of a drug for treating breast cancer.

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