Liposome nano drug-loaded particle modified by stem cell membrane and preparation method of liposome nano drug-loaded particle

Abstract

The invention discloses a stem cell membrane modified liposome nano drug-loaded particle and a preparation method thereof, belongs to the technical field of nano drug loading, and particularly relates to a core layer, 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 comprises lecithin choline and cholesterol; the membrane layer is a stem cell membrane, and the core layer is coated with the stem cell membrane; an outer layer, wherein the outer layer comprises a chitosan-based derivative; the core layer and the membrane layer form the stem cell membrane coated liposome nano drug-loaded particle, and the amount of the chitosan-based derivative is 400-600wt% of the stem cell membrane coated liposome nano drug-loaded particle. The invention provides a stem cell membrane modified liposome nano drug-loaded particle and a preparation method thereof. The stem cell membrane modified liposome nano drug-loaded particle is good in stability, good in drug release effect in an acidic tumor microenvironment, good in targeting, good in immunocompatibility, good in biological safety and good in biocompatibility.

Description

Technical Field

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

[0002] The treatment of breast cancer has undergone profound changes from single surgery to comprehensive treatment, including various means such as surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy. However, traditional chemotherapy drugs often face problems such as poor water solubility, large side effects, and poor targeting in application, which affect the treatment effect and the quality of life of patients. In recent years, with the rapid development of nano-science and technology, nano-drug delivery technology has been widely used in the field of breast cancer drug research and development and treatment. Although traditional nano-drug delivery particles such as liposomes can improve the bioavailability and targeting of drugs, they still face many challenges in crossing biological barriers, evading immune surveillance, and achieving precise delivery. In addition, the toxicity and safety of nano-drug delivery systems also need to be fully studied and evaluated.

[0003] Stem cell membranes are rich in various functional proteins and carbohydrate molecules, which play important roles in cell recognition, signal transduction, and immune escape. In recent years, combining stem cell membranes with nano-drug delivery systems to prepare nano-drug delivery particles with stem cell membrane characteristics, which can mimic the behavior of stem cells, such as targeted migration, immune escape, and cell-cell interaction, etc., also provides new ideas for the treatment of breast cancer.

[0004] In the research and development process of stem cell membrane biomimetic nano-drug delivery systems, although this field shows great potential, its construction is still in the primary stage, facing a series of complex technical problems such as the stability of material preparation, biological safety, drug targeting, and the performance optimization of nano-materials themselves. Therefore, we urgently need to develop a new type of stem cell membrane-modified nano-drug delivery system, continuously explore a nano-drug delivery system that matches the stem cell membrane, and conduct sufficient toxicity and safety evaluations 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 nano-drug delivery particle with good stability, good drug release effect under acidic tumor microenvironment, good targeting, good immunocompatibility, good biological safety, and good biocompatibility, and a preparation method thereof.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows: A stem cell membrane-modified liposome nano-drug delivery particle, comprising: A core layer, the core layer being a carrier and a drug, the drug being loaded on the carrier, the carrier being a lipid carrier, and the lipid carrier including phosphatidylcholine and cholesterol; A membrane layer, wherein the membrane layer is a stem cell membrane, and the stem cell membrane is coated outside the core layer; an outer layer, the 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-based 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 nanoparticles loaded with dual drugs to solve the problems existing in the existing nanoparticle system in clinical applications. The nanoparticles include drug-loaded albumin paclitaxel nab-PTX and doxorubicin DXR, liposome nanocarrier lipo, stem cell membrane MSCm, and externally modified chitosan-based derivatives from the inside to the outside structure; wherein the water-soluble drug is wrapped in the central aqueous phase inside the liposome nanocarrier, the outside of the liposome nanocarrier is coated with the stem cell membrane, and the outer surface of the stem cell membrane is further modified by chitosan, and the chitosan-based derivatives @ stem cell membrane-lipid carrier-drug nanoparticles are assembled together. The above materials of the present invention all have good biosafety, greatly reduce the potential toxicity risk that may be caused by the accumulation of the nanoparticle system in the organism, and provide a safer and more reliable basis for the development of drug delivery systems. In addition, when chitosan derivatives are used to modify stem cell membranes, they can not only improve the stability of the stem cell membranes and protect stem cells from damage by the external environment, but also enhance the immunomodulatory ability of the stem cell membranes to nano-drug-carrying particles, which will help reduce immune responses and rejection reactions during treatment. Chitosan derivatives can use carboxymethyl chitosan.

[0007] Preferably, the amount of cholesterol is 10-20wt% of 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 0.05-5wt% of egg phosphatidylcholine.

[0008] Preferably, the chitosan-based derivatives include at least one of carboxymethyl chitosan, chitosan amino acid derivatives and chitosan polyenamine derivatives. When chitosan-based derivatives are used 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 polyenamine derivatives alone cannot enhance the immunomodulatory ability of the prepared stem cell membrane-modified liposome nanoparticles, and cannot effectively reduce the immune response and rejection during the treatment process.

[0009] More preferably, the chitosan amino acid derivative is obtained by reacting chitosan with S-lactylglutathione.

[0010] More preferably, the chitosan polyenamine derivative is obtained by reacting chitosan with N-methacryloylglycine.

[0011] Preferably, the chitosan-based derivatives include carboxymethyl chitosan, chitosan amino acid derivatives, and chitosan polyamine 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 polyamine derivatives is 5 - 200:2 - 200.

[0012] Preferably, in the preparation of chitosan amino acid derivatives, chitosan is added to deionized water, then HOBt is added, and the mixture is stirred and dispersed evenly. Subsequently, S-lactoylglutathione and EDC·HCl are added, and the pH of the solution is controlled to be 4.5 - 5.5. The reaction is carried out under dark conditions with stirring at 20 - 40 °C for 1 - 8 h. After the reaction is completed, dialysis is carried out in a dialysis bag, and after dialysis is completed, freeze-drying is carried out to obtain chitosan derivatives.

[0013] More preferably, in the preparation of chitosan amino acid derivatives, the usage amount of chitosan is 0.5 - 10 wt% of deionized water.

[0014] More preferably, in the preparation of chitosan amino acid derivatives, the usage amount of HOBt is 60 - 90 wt% of chitosan.

[0015] More preferably, in the preparation of chitosan amino acid derivatives, the usage amount of S-lactoylglutathione is 300 - 500 wt% of chitosan.

[0016] More preferably, in the preparation of chitosan amino acid derivatives, the usage amount of EDC·HCl is 50 - 80 wt% of chitosan.

[0017] Preferably, in the preparation of chitosan polyamine derivatives, chitosan is added to an acetic acid solution, and the mixture is stirred and mixed at 20 - 40 °C. In an inert gas atmosphere, an initiator and N-methylacryloylglycine are added, and then the reaction is carried out with stirring at 50 - 80 °C for 1 - 4 h. After the reaction is completed, the pH is adjusted to 10 - 12, and the product is washed successively with deionized water and methanol, and then freeze-dried to obtain chitosan derivatives.

[0018] More preferably, in the preparation of chitosan polyamine derivatives, the acetic acid solution contains 1 - 3 wt% of acetic acid, and the usage amount of chitosan is 2 - 10 wt% of the acetic acid solution.

[0019] More preferably, in the preparation of chitosan polyamine derivatives, the initiator is benzoyl peroxide, and the usage amount of the initiator is 1 - 10 wt% of chitosan.

[0020] More preferably, in the preparation of chitosan polyamine derivatives, the usage amount of N-methylacryloylglycine is 20 - 60 wt% of chitosan.

[0021] The present invention discloses a preparation method of liposome nano-drug-loaded particles modified with a stem cell membrane, including: S1. Add the drug into deionized water to prepare a drug solution, add the lipid carrier raw materials into a solvent to prepare a lipid carrier raw material solution, and mix the drug solution with the lipid carrier raw material solution to prepare liposome nano-drug-loaded particles, namely the core layer; the lipid carrier raw materials include lecithin and cholesterol. S2. Extract the stem cell membrane, and mix the stem cell membrane with the liposome nano-drug-loaded particles to prepare stem cell membrane-coated liposome nano-drug-loaded particles. S3. Add the chitosan-based derivative into deionized water, and then mix it with an isopropanol solution containing stem cell membrane-coated liposome nano-drug-loaded particles to prepare chitosan-based-coated liposome nano-drug-loaded particles, namely stem cell membrane-modified liposome nano-drug-loaded particles; the amount of the chitosan-based derivative is 400 - 600 wt% of the stem cell membrane-coated liposome nano-drug-loaded particles.

[0022] Preferably, the stem cell membrane is derived from mesenchymal stem cells.

[0023] Preferably, the dosage of the drug in the drug solution is 0.01 - 0.6 wt% of the deionized water, and the drug includes nab-PTX and / or DXR.

[0024] Preferably, the dosage of lecithin is 2 - 20 wt% of the solvent.

[0025] Preferably, in the isopropanol solution containing stem cell membrane-coated liposome nano-drug-loaded particles, the amount of the stem cell membrane-coated liposome nano-drug-loaded particles is 0.005 - 0.2 wt% of the isopropanol.

[0026] Preferably, adding the chitosan-based derivative into deionized water to obtain a chitosan-based solution, and the chitosan-based solution contains 0.05 - 2 wt% of carboxymethyl chitosan.

[0027] The present invention discloses the use of the above-mentioned stem cell membrane-modified liposome nano-drug-loaded particles in the preparation of anti-tumor drugs.

[0028] Preferably, in the preparation of single-drug-loaded liposome nano-drug-loaded particles, add the drug into deionized water, stir at 50 - 70 °C for 10 - 60 min to obtain a single drug solution; add lecithin and cholesterol into chloroform, spin-coat at 20 - 40 °C and 60 - 100 rpm / min to form a film, then add the single drug solution, perform water bath sonication for 1 - 20 min, and then dialyze in deionized water for 12 - 48 h to remove the unencapsulated drug, and extrude through a membrane to obtain single-drug-loaded liposome nano-drug-loaded particles.

[0029] More preferably, in the preparation of single-drug-loaded liposome nano-drug-loaded particles, the drug is nab-PTX or DXR, and the dosage of the drug is 0.01 - 0.6 wt% of the deionized water.

[0030] More preferably, in the preparation of the single-drug-loaded liposome nano-drug-loaded particles, the usage amount of phosphatidylcholine is 2-20 wt% of chloroform.

[0031] More preferably, in the preparation of the single-drug-loaded liposome nano-drug-loaded particles, the usage amount of cholesterol is 10-20 wt% of phosphatidylcholine.

[0032] More preferably, in the preparation of the single-drug-loaded liposome nano-drug-loaded particles, taking the initial usage amount of the drug in the single drug solution, the usage amount of the drug is 0.05-5 wt% of phosphatidylcholine.

[0033] Preferably, in the preparation of the dual-drug-loaded liposome nano-drug-loaded particles, the drug is added to deionized water and stirred at 50-70 °C for 10-60 min to obtain a dual drug solution; phosphatidylcholine and cholesterol are added to chloroform and spin-coated into a film at 20-40 °C and 60-100 rpm / min, then the dual drug solution is added, sonicated in a water bath for 1-20 min, and then dialyzed in deionized water for 12-48 h to remove the unencapsulated drug, and extruded through a membrane to obtain the single-drug-loaded liposome nano-drug-loaded particles.

[0034] More preferably, in the preparation of the dual-drug-loaded liposome nano-drug-loaded particles, the drugs are nab-PTX and DXR, and nab-PTX and DXR in the drug are compounded and used in a mass ratio of 1:0.3-0.8, and the usage amount of the drug is 0.01-0.6 wt% of deionized water.

[0035] More preferably, in the preparation of the dual-drug-loaded liposome nano-drug-loaded particles, the usage amount of phosphatidylcholine is 2-20 wt% of chloroform.

[0036] More preferably, in the preparation of the dual-drug-loaded liposome nano-drug-loaded particles, the usage amount of cholesterol is 10-20 wt% of phosphatidylcholine.

[0037] More preferably, in the preparation of the dual-drug-loaded liposome nano-drug-loaded particles, taking the initial usage amount of the drug in the dual drug solution, the usage amount of the drug is 0.05-5 wt% of phosphatidylcholine.

[0038] Preferably, in the preparation of the stem cell membrane-coated liposome nano-drug-loaded particles, mesenchymal stem cells are cultured, mesenchymal stem cells in the logarithmic growth phase are collected, centrifuged after digestion, resuspended with PBS solution, and then the cells are lysed by the repeated freeze-thaw method, and the stem cell membrane fragments of mesenchymal stem cells are extracted with a cell membrane protein extraction kit; then the stem cell membrane fragments are repeatedly extruded through a polycarbonate porous membrane to prepare vesicles, and then the vesicles are mixed with liposome nano-drug-loaded particles and sonicated in a water bath at 0-10 °C in the dark for 10-35 min to obtain the stem cell membrane-coated liposome nano-drug-loaded particles.

[0039] More preferably, in the preparation of the stem cell membrane-coated liposome nano drug-loaded particles, the vesicles and the liposome nano drug-loaded particles are mixed at a mass ratio of 1:0.05 - 2.

[0040] Preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, the stem cell membrane-coated liposome nano drug-loaded particles are added to isopropanol, and then the isopropanol solution containing the stem cell membrane-coated liposome nano drug-loaded particles is dropped into the chitosan-based solution, followed by ultrasonic oscillation for 10 - 60 min, and then dialysis in deionized water for 12 - 48 h to obtain the chitosan-based coated liposome nano drug-loaded particles.

[0041] More preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, the usage amount of the stem cell membrane-coated liposome nano drug-loaded particles is 0.005 - 0.2 wt% of the isopropanol.

[0042] More preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, the chitosan-based solution is formed by mixing carboxymethyl chitosan and deionized water, and the chitosan-based solution contains 0.05 - 2 wt% of carboxymethyl chitosan.

[0043] More preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, the isopropanol solution containing the stem cell membrane-coated liposome nano drug-loaded particles is measured by the stem cell membrane-coated liposome nano drug-loaded particles, the chitosan-based solution is measured by carboxymethyl chitosan, and the usage amount of carboxymethyl chitosan is 400 - 600 wt% of the stem cell membrane-coated liposome nano drug-loaded particles.

[0044] More preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, in addition to carboxymethyl chitosan, the chitosan-based solution also contains chitosan amino acid derivatives and chitosan polyamine derivatives. The chitosan-based solution contains 0.05 - 0.5 wt% of chitosan amino acid derivatives, and the chitosan-based solution contains 0.02 - 0.2 wt% of chitosan polyamine derivatives.

[0045] More preferably, in the preparation of the chitosan-based coated liposome nano drug-loaded particles, the chitosan-based solution also contains polyethylene glycol glutarate. The chitosan-based solution contains 0.02 - 0.5 wt% of polyethylene glycol glutarate. After using carboxymethyl chitosan, chitosan amino acid derivatives and chitosan polyamine derivatives in the present invention, and further adding polyethylene glycol glutarate, the immunomodulatory ability of the obtained chitosan-based coated liposome nano drug-loaded particles is improved, while the single use of polyethylene glycol glutarate has no effect. Polyethylene glycol glutarate can enhance the effect of the use of carboxymethyl chitosan, chitosan amino acid derivatives and chitosan polyamine derivatives, and can reduce the immune response and rejection reaction during the treatment process.

[0046] Since the present invention uses lecithin acylcholine and cholesterol as lipid carriers to load drugs and prepare a core layer, a stem cell membrane is coated on the core layer, and finally a chitosan-based derivative is coated to form an outer layer, obtaining liposome nanoparticles modified with a stem cell membrane; the drugs include nab-PTX and / or DXR, thus having the following beneficial effects: The liposome nanoparticles prepared by the present invention, the liposome nanoparticles coated with a stem cell membrane, and the liposome nanoparticles coated with a chitosan-based derivative 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 liposome nanoparticle modified with a stem cell membrane and its preparation method, which has good stability, good drug release effect in an acidic tumor microenvironment, good targeting, good immunocompatibility, good biosafety, and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a morphological characterization diagram.

[0048] Figure 2 It is a particle size test result diagram.

[0049] Figure 3 It is an in vitro drug release result diagram.

[0050] Figure 4 It is a targeting test result diagram.

[0051] Figure 5 It is an immune escape test result diagram.

[0052] Figure 6 It is a comparative result diagram of the immune escape test.

[0053] Figure 7 It is a biosafety test result diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0055] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and the features in the embodiments of the present application can be combined with each other. Next, the present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0056] Example 1: A preparation method of liposome nano-drug-loaded particles Preparation of single-drug-loaded liposome nano-drug-loaded particles: Add the drug into deionized water and stir at 60 °C for 30 min to obtain a single-drug solution; Add lecithin and cholesterol into chloroform, spin-coat into a film at 30 °C and 80 rpm / min, then add the single-drug solution, perform water bath sonication for 10 min, then dialyze in deionized water for 24 h to remove the unencapsulated drug, and extrude through a membrane to obtain single-drug-loaded liposome nano-drug-loaded particles. The drug is nab-PTX, and the dosage of the drug is 0.1 wt% of deionized water; The dosage of lecithin is 2.52 wt% of chloroform, the dosage of cholesterol is 16.67 wt% of lecithin, and the dosage of the single-drug solution is based on the initial dosage of the drug therein, and the dosage of the drug is 0.1 wt% of lecithin.

[0057] Example 2: A preparation method of liposome nano-drug-loaded particles Preparation of single-drug-loaded liposome nano-drug-loaded particles: Add the drug into deionized water and stir at 60 °C for 30 min to obtain a single-drug solution; Add lecithin and cholesterol into chloroform, spin-coat into a film at 30 °C and 80 rpm / min, then add the single-drug solution, perform water bath sonication for 10 min, then dialyze in deionized water for 24 h to remove the unencapsulated drug, and extrude through a membrane to obtain single-drug-loaded liposome nano-drug-loaded particles. The drug is DXR, and the dosage of the drug is 0.1 wt% of deionized water; The dosage of lecithin is 2.52 wt% of chloroform, the dosage of cholesterol is 16.7 wt% of lecithin, and the dosage of the single-drug solution is based on the initial dosage of the drug therein, and the dosage of the drug is 0.1 wt% of lecithin.

[0058] Example 3: A preparation method of liposome nano-drug-loaded particles Preparation of dual-drug-loaded liposome nano-drug-loaded particles: Add the drugs into deionized water and stir at 60 °C for 30 min to obtain a dual-drug solution; Add lecithin and cholesterol into chloroform, spin-coat into a film at 30 °C and 80 rpm / min, then add the dual-drug solution, perform water bath sonication for 10 min, then dialyze in deionized water for 24 h to remove the unencapsulated drug, and extrude through a membrane to obtain single-drug-loaded liposome nano-drug-loaded particles. The drugs are nab-PTX and DXR, and nab-PTX and DXR in the drugs are compounded and used in a mass ratio of 1:0.5, and the dosage of the drugs is 0.1 wt% of deionized water; The dosage of lecithin is 2.52 wt% of chloroform, the dosage of cholesterol is 16.7 wt% of lecithin, and the dosage of the dual-drug solution is based on the initial dosage of the drugs therein, and the dosage of the drugs is 0.1 wt% of lecithin.

[0059] Example 4: Preparation method of a stem cell membrane-modified liposome nanocarrier particle Preparation of stem cell membrane-coated liposome nanocarrier particles: Culture mesenchymal stem cells, collect mesenchymal stem cells in the logarithmic growth phase, centrifuge after digestion, resuspend the cells with PBS solution, then break the cells by the repeated freeze-thaw method, and extract the stem cell membrane fragments of mesenchymal stem cells with a cell membrane protein extraction kit; then use a polycarbonate porous membrane to repeatedly extrude the stem cell membrane fragments to prepare vesicles, and then mix the vesicles with liposome nanocarrier particles, and perform water bath sonication at 4 °C in the dark for 20 min to obtain stem cell membrane-coated liposome nanocarrier particles. The vesicles and liposome nanocarrier particles are mixed at a mass ratio of 1:0.5. The liposome nanocarrier particles are the single-drug-loaded liposome nanocarrier particles prepared in Example 1.

[0060] Example 5: Preparation method of a stem cell membrane-modified liposome nanocarrier particle This example is different from Example 4 in that the liposome nanocarrier particles are the single-drug-loaded liposome nanocarrier particles prepared in Example 2.

[0061] Example 6: Preparation method of a stem cell membrane-modified liposome nanocarrier particle This example is different from Example 4 in that the liposome nanocarrier particles are the double-drug-loaded liposome nanocarrier particles prepared in Example 3.

[0062] Example 7: Preparation method of a stem cell membrane-modified liposome nanocarrier particle Preparation of chitosan-based coated liposome nanocarrier particles: Add the stem cell membrane-coated liposome nanocarrier particles to isopropanol, and then drop the isopropanol solution containing the stem cell membrane-coated liposome nanocarrier particles into the chitosan-based solution, ultrasonically oscillate for 20 min, and then dialyze in deionized water for 24 h to obtain chitosan-based coated liposome nanocarrier particles. The usage amount of the stem cell membrane-coated liposome nanocarrier particles is 0.02 wt% of the isopropanol solution. The chitosan-based solution is prepared by mixing carboxymethyl chitosan and deionized water. The chitosan-based solution contains 0.1 wt% of carboxymethyl chitosan. The isopropanol solution containing the stem cell membrane-coated liposome nanocarrier particles is measured by the stem cell membrane-coated liposome nanocarrier particles, and the chitosan-based solution is measured by carboxymethyl chitosan. The usage amount of carboxymethyl chitosan is 500 wt% of the stem cell membrane-coated liposome nanocarrier particles. The stem cell membrane-coated liposome nanocarrier particles are prepared from Example 4.

[0063] Example 8: Preparation method of a stem cell membrane-modified liposome nanocarrier particle This example is different from Example 7 in that the stem cell membrane-coated liposome nanoparticles are prepared from Example 5.

[0064] Example 9: A method for preparing stem cell membrane-modified liposome nanoparticles This example is different from Example 7 in that the stem cell membrane-coated liposome nanoparticles are prepared from Example 6.

[0065] Example 10: A method for preparing stem cell membrane-modified liposome nanoparticles This example is different from Example 7 in that the chitosan-based solution in the preparation of chitosan-based coated liposome nanoparticles further contains chitosan amino acid derivatives and chitosan polyamine derivatives. The chitosan-based solution contains 0.2 wt% of chitosan amino acid derivatives, and the chitosan-based solution contains 0.12 wt% of chitosan polyamine derivatives.

[0066] Preparation of chitosan amino acid derivatives: Chitosan is added to deionized water, then HOBt is added, and the mixture is stirred and dispersed evenly. Subsequently, S-lactoylglutathione and EDC·HCl are added, and the pH of the solution is controlled at 5. The reaction is carried out under dark stirring at 30 °C for 5 h. After the reaction is completed, dialysis is carried out in a dialysis bag. After dialysis is completed, freeze-drying is carried out to obtain chitosan derivatives. The usage amount of chitosan is 2 wt% of deionized water, the usage amount of HOBt is 80 wt% of chitosan, the usage amount of S-lactoylglutathione is 400 wt% of chitosan, and the usage amount of EDC·HCl is 60 wt% of chitosan.

[0067] Preparation of chitosan polyamine derivatives: Chitosan is added to an acetic acid solution, and the mixture is stirred and mixed at 30 °C. Under an inert gas atmosphere, an initiator and N-methacryloylglycine are added, and then the reaction is carried out under stirring at 60 °C for 2 h. After the reaction is completed, the pH is adjusted to 11, and the product is washed successively with deionized water and methanol, and then freeze-dried to obtain chitosan derivatives. The acetic acid solution contains 2 wt% of acetic acid, the usage amount of chitosan is 6 wt% of the acetic acid solution, the initiator is benzoyl peroxide, the usage amount of the initiator is 5 wt% of chitosan, and the usage amount of N-methacryloylglycine is 40 wt% of chitosan.

[0068] Example 11: A method for preparing stem cell membrane-modified liposome nanoparticles This example is different from Example 10 in that in the preparation of chitosan-based coated liposome nanoparticles, the stem cell membrane-coated liposome nanoparticles are prepared from Example 5.

[0069] Example 12: A method for preparing stem cell membrane-modified liposome nanoparticles This example is different from Example 10 in that in the preparation of chitosan-based coated liposome nano drug-loaded particles, the stem cell membrane-coated liposome nano drug-loaded particles are prepared according to Example 6.

[0070] Example 13: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This example is different from Example 10 in that the chitosan-based solution in the preparation of chitosan-based coated liposome nano drug-loaded particles further contains polyethylene glycol glutarate. The chitosan-based solution contains 0.15 wt% of polyethylene glycol glutarate.

[0071] Example 14: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This example is different from Example 13 in that in the preparation of chitosan-based coated liposome nano drug-loaded particles, the stem cell membrane-coated liposome nano drug-loaded particles are prepared according to Example 5.

[0072] Example 15: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This example is different from Example 13 in that in the preparation of chitosan-based coated liposome nano drug-loaded particles, the stem cell membrane-coated liposome nano drug-loaded particles are prepared according to Example 6.

[0073] Comparative Example 1: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This comparative example is different from Example 9 in that the chitosan in the chitosan-based solution is replaced by carboxymethyl chitosan.

[0074] Comparative Example 2: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This comparative example is different from Example 9 in that the chitosan in the chitosan-based solution is replaced by the chitosan amino acid derivative prepared in Example 10.

[0075] Comparative Example 3: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This comparative example is different from Example 9 in that the chitosan in the chitosan-based solution is replaced by the chitosan polyeneamine derivative prepared in Example 10.

[0076] Comparative Example 4: A method for preparing stem cell membrane-modified liposome nano drug-loaded particles This comparative example is different from Example 9 in that the chitosan in the chitosan-based solution is replaced by polyethylene glycol glutarate.

[0077] Test Example: The present invention performs morphological characterization on the liposome nano-drug-loaded particles and stem cell membrane-coated liposome nano-drug-loaded particles prepared in the examples. Taking the dual-drug-loaded liposome nano-drug-loaded particles prepared in Example 3 and the stem cell membrane-coated liposome nano-drug-loaded particles prepared in Example 6 as examples, the morphological characterization results are as follows Figure 1 shown, where Figure 1 (a) is the morphology of the dual-drug-loaded liposome nano-drug-loaded particles, which is a spherical structure as a whole, with a size of about 100 nm; Figure 1 (b) is the morphology of the stem cell membrane-coated liposome nano-drug-loaded particles. Its overall shape and structure have not changed significantly, and the boundary of the membrane coating can be clearly observed, indicating that the stem cell membrane-coated liposome nano-drug-loaded particles have been successfully synthesized.

[0078] The present invention conducts stability tests on the liposome nano-drug-loaded particles and stem cell membrane-coated liposome nano-drug-loaded particles prepared in the examples. The particle size tests of the dual-drug-loaded liposome nano-drug-loaded particles prepared in Example 3, the single-drug-loaded liposome nano-drug-loaded particles prepared in Example 1, the stem cell membrane-coated liposome nano-drug-loaded particles prepared in Example 6, and the chitosan-based coated liposome nano-drug-loaded particles prepared in Example 9 are carried out in PBS solution for 18 days. The results are as follows Figure 2 shown, where lip-nab-PTX-DXR is the dual-drug-loaded liposome nano-drug-loaded particles prepared in Example 3, MSCm-lip-nab-PTX-DXR is the stem cell membrane-coated liposome nano-drug-loaded particles prepared in Example 6, lip-nab-PTX is the single-drug-loaded liposome nano-drug-loaded particles prepared in Example 1, and CMCTS@MSCm-lip-nab-PTX-DXR is the chitosan-based coated liposome nano-drug-loaded particles prepared in Example 9. With the increase of time, the particle size of the liposome nano-drug-loaded particles not coated with the stem cell membrane increases significantly. However, the stem cell membrane-coated liposome nano-drug-loaded particles and the chitosan-based coated liposome nano-drug-loaded particles show relatively stable particle size information during the test, and the chitosan-based coated liposome nano-drug-loaded particles modified with carboxymethyl chitosan show the best stability, indicating that carboxymethyl chitosan can further enhance the environmental stability of the stem cell membrane.

[0079] The present invention conducts in vitro drug release tests on the chitosan-based coated liposome nano-drug-loaded particles prepared in Example 9, simulating the tumor microenvironment with a pH of 6.8 and the normal cell environment with a pH of 7.4 for drug release tests. The results are as follows Figure 3As shown in the figure, as time goes by, the drug is continuously released, and the chitosan-based coated liposome nanoparticles have a faster drug release rate and a larger drug release amount in the pH = 6.8 environment than in the pH = 7.4 environment. This indicates that the liposome nanoparticles modified with stem cell membranes of the present invention are very suitable for applications in the acidic tumor microenvironment.

[0080] The present invention conducts targeting tests on the single-drug-loaded liposome nanoparticles prepared in Example 3 and the stem cell membrane-coated liposome nanoparticles prepared in Example 6. 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, the control group 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 are inoculated in a confocal cell culture dish. After culturing with DMEM containing 10% fetal bovine serum for 24 h, the culture medium is discarded. Take 100 μL of FITC-MSCm-lipo-nab-PTX-DXR, FITC-lipo-nab-PTX-DXR, and FITC-MSCm-lipo-nab-PTX-DXR and mix them evenly with 900 μL of the culture medium containing 10% fetal bovine serum. The total volume is 1 mL and the final concentration of the nanoparticles is 1 mg / mL. Incubate with 4T1 and EMT6 for 4 hours. Discard the incubation solution, wash 3 times with PBS solution, and then use confocal microscopy. Then, breast cancer cells 4T1 are co-incubated with the above samples respectively, and observed and imaged with CLSM. The experimental results are as Figure 4 shown. The FITC-MSCm-lipo-nab-PTX-DXR + 4T1 cells show stronger fluorescence intensity than the FITC-lipo-nab-PTX-DXR + 4T1 cells and the FITC-MSCm-lipo-nab-PTX-DXR + EMT6 cells. This indicates that the stem cell-coated 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.

[0081] The present invention conducts immune escape tests on the dual-drug-loaded liposome nanocarrier particles prepared in Example 3, the stem cell membrane-coated liposome nanocarrier particles prepared in Example 6, and the chitosan-based coated liposome nanocarrier particles prepared in Example 9. Macrophages were inoculated in confocal cell culture dishes. After 24 hours of culture, the culture medium was discarded. The above three samples were respectively mixed evenly with the culture medium containing 10% fetal bovine serum at a volume ratio of 1:9, incubated with macrophages for 4 hours, washed with PBS, and then subjected to confocal imaging. The results are as Figure 5 shown. Among them, lipo-nab-PTX-DXR is the dual-drug-loaded liposome nanocarrier particles of Example 3, MSCm-lipo-nab-PTX-DXR is the stem cell membrane-coated liposome nanocarrier particles of Example 6, and CMCTS@MSCm-lipo-nab-PTX-DX is the chitosan-based coated liposome nanocarrier particles of Example 9. Compared with the stem cell membrane-coated liposome nanocarrier particles and the chitosan-based coated liposome nanocarrier particles, the dual-drug-loaded liposome nanocarrier particles not coated with the stem cell membrane showed the characteristic of being largely taken up by macrophages, indicating that the stem cell membrane-coated liposome nanocarrier particles have higher immunocompatibility. In addition, it was also found that the chitosan-based coated liposome nanocarrier particles modified with carboxymethyl chitosan were less likely to be taken up by macrophages than the stem cell membrane-coated liposome nanocarrier particles, which also indirectly demonstrated the protective effect of carboxymethyl chitosan on the stem cell membrane and its potential immunostimulatory ability.

[0082] The present invention conducts immune escape tests on the chitosan-based coated liposome nanocarrier particles prepared in Example 9, 12, 15 and Comparative Examples 1-4 according to the above method. Among them, the sample was mixed evenly with the 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 as Figure 6As shown, after the core layer is coated with the stem cell membrane in the present invention, the immune escape ability of the stem cell membrane-coated liposome nanomedicine particles 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, a chitosan-based derivative is added for coating treatment in the present invention. Among them, the single use of carboxymethyl chitosan can further improve the immune escape ability of the chitosan-based coated liposome nanomedicine particles. If carboxymethyl chitosan is replaced with chitosan, chitosan can hardly improve the immune escape ability of the obtained chitosan-based coated liposome nanomedicine particles. If carboxymethyl chitosan is further replaced with the chitosan amino acid derivative or chitosan polyenamine derivative prepared in the present invention, the immune escape ability of the obtained chitosan-based coated liposome nanomedicine particles can also hardly be improved. The chitosan amino acid derivative in the present invention is prepared by reacting chitosan with S-lactoylglutathione, and the chitosan polyenamine derivative in the present invention is prepared by reacting chitosan with N-methacryloylglycine. The present invention finds that when carboxymethyl chitosan, chitosan amino acid derivative, and chitosan polyenamine derivative are used together, the immune escape ability of the obtained chitosan-based coated liposome nanomedicine particles can be enhanced. When carboxymethyl chitosan, chitosan amino acid derivative, and chitosan polyenamine derivative are used, the present invention finds that if polyethylene glycol glutarate is further added, the outer layer formed by the co-use of carboxymethyl chitosan, chitosan amino acid derivative, chitosan polyenamine derivative, and polyethylene glycol glutarate has better immune escape ability.

[0083] The present invention conducts cell safety tests on the dual-loaded liposome nanomedicine particles prepared in Example 3, the stem cell membrane-coated liposome nanomedicine particles prepared in Example 6, and the chitosan-based coated liposome nanomedicine particles prepared in Examples 7-9, and evaluates the killing effect of the nanomedicine particles of the present invention on breast cancer 4T1 cells and the biosafety on normal breast EMT6 cells based on the CCK8 method. The above test samples are selected, the specific culture time is set to 24 h, the sample concentration is set to 10 μg / mL, and after incubation for 3 h, the optical density (OD) value of each well is measured at 450 nm with an enzyme-labeled instrument and the cell survival rate is calculated. The experimental results are as Figure 7As shown in the figure, 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 obvious inhibitory effects on breast cancer 4T1 cells. Among them, the chitosan-based coated liposome nanoparticles of Example 9 show better proliferation inhibition rates compared to the two samples loaded with single drugs 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 have similar inhibitory effects 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 of the stem cell membrane and the modification of carboxymethyl chitosan do not hinder the killing of breast cancer cells. On the other hand, the above samples generally have good biocompatibility with normal breast EMT6 cells. Among them, the chitosan-based coated liposome nanoparticles prepared in Examples 7-9 have similar cell activities; 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 show effects that are nearly 20% better than the above samples respectively, which can be attributed to the fact that the liposome is coated with the stem cell membrane and the stem cell membrane is modified with carboxymethyl chitosan, and it also further illustrates the protective effect of carboxymethyl chitosan on the stem cell membrane.

[0084] The present invention conducted a biosafety test on the chitosan-based coated liposome nanoparticles prepared in Examples 9, 12, and 15. Using normal breast EMT6 cells, the cell survival rates of breast EMT6 cells in the chitosan-based coated liposome nanoparticles of Examples 12 and 15 are not much different from those of Example 9, indicating that the chitosan-based coated liposome nanoparticles of Examples 12 and 15 in the present invention also have excellent biosafety.

[0085] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0086] In this text, specific examples are used to elaborate on the principles and implementation modes of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation mode of the present application. It should be noted that due to the limited nature of literal expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A stem cell membrane-modified liposome nano-drug-loading particle, comprising: A core layer, where 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 lecithin acylcholine and cholesterol; A membrane layer, where the membrane layer is a stem cell membrane, and the stem cell membrane coats the outside of the core layer; An outer layer, where the outer layer includes a chitosan-based derivative; The core layer and the membrane layer form a stem cell membrane-coated liposome nano-drug-loading particle, and the amount of the chitosan-based derivative is 400-600 wt% of the stem cell membrane-coated liposome nano-drug-loading particle.

2. The lipidosome nano-drug-loaded particle modified by a stem cell membrane according to claim 1, characterized in that: The amount of the cholesterol is 10-20 wt% of the lecithin acylcholine; 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 lecithin acylcholine.

3. A liposome nano-drug-loaded particle modified with a stem cell membrane according to claim 1, characterized in that: The chitosan-based derivative includes at least one of carboxymethyl chitosan, chitosan amino acid derivative, and chitosan polyamine derivative.

4. A liposome nano-drug-loaded particle modified with a stem cell membrane according to claim 1, characterized in that: The chitosan-based derivative includes carboxymethyl chitosan, chitosan amino acid derivative, and chitosan polyamine derivative, and the mass ratio of carboxymethyl chitosan to chitosan amino acid derivative is 5-200:5-50, and the mass ratio of carboxymethyl chitosan to chitosan polyamine derivative is 5-200:2-200.

5. A preparation method of a stem cell membrane-modified liposome nano-drug-loading particle, comprising: S1, adding a drug into deionized water to make a drug solution, adding lipid carrier raw materials into a solvent to make a lipid carrier raw material solution, and mixing the drug solution and the lipid carrier raw material solution to make a liposome nano-drug-loading particle, that is, the core layer; the lipid carrier raw materials include lecithin acylcholine and cholesterol; S2, extracting the stem cell membrane, and mixing the stem cell membrane with the liposome nano-drug-loading particle to make a stem cell membrane-coated liposome nano-drug-loading particle; S3, adding the chitosan-based derivative into deionized water, and then mixing it with an isopropanol solution containing the stem cell membrane-coated liposome nano-drug-loading particle to prepare a chitosan-based-coated liposome nano-drug-loading particle, that is, a stem cell membrane-modified liposome nano-drug-loading particle; the amount of the chitosan-based derivative is 400-600 wt% of the stem cell membrane-coated liposome nano-drug-loading particle.

6. The preparation method of a liposome nano-drug-loaded particle modified with a stem cell membrane according to claim 5, characterized in that: The stem cell membrane is derived from mesenchymal stem cells.

7. The preparation method of a liposome nano-drug-loaded particle modified with a stem cell membrane according to claim 5, characterized in that: The dosage of the drug in the drug solution is 0.01-0.6 wt% of the deionized water, and the drug includes nab-PTX and / or DXR.

8. The preparation method of a liposome nano-drug-loaded particle modified by a stem cell membrane according to claim 5, characterized in that: The dosage of the lecithin acylcholine is 2-20 wt% of the solvent.

9. The preparation method of a liposome nano-drug-loaded particle modified by a stem cell membrane according to claim 5, characterized in that: In the isopropanol solution containing the stem cell membrane-coated liposome nano-drug-loading particle, the amount of the stem cell membrane-coated liposome nano-drug-loading particle is 0.005-0.2 wt% of the isopropanol.

10. Use of the stem cell membrane-modified liposome nano-drug-loading particle according to any one of claims 1-4 in the preparation of anti-tumor drugs.

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