CN114831961B - Liver-targeted bionic cell membrane drug-loaded nano-particle and preparation method and application thereof - Google Patents

Abstract

The invention provides a liver-targeted bionic cell membrane drug-loaded nanoparticle, a preparation method and application thereof, wherein the outside of the drug-loaded nanoparticle is coated by an engineering cell membrane, and an anti-liver fibrosis drug is encapsulated in the drug-loaded nanoparticle for treating liver fibrosis. The engineering cell membrane selects LX2 cells (human hepatic stellate cells) as source cells, and specifically expresses tumor necrosis factor related apoptosis-inducing ligand (TRAIL) protein through Lv-TRAIL-zsgreen over-expression lentivirus transfection. The drug-loaded nano-particles consist of lactic acid-glycolic acid copolymer (PLGA) and all-trans retinoic acid (ATRA), and the ATRA and TRAIL can exert synergistic effect to induce hepatic fibroblast apoptosis. The bionic cell membrane nano particles are mainly concentrated in liver tissues after entering the body, and have remarkable treatment effect in anti-hepatic fibrosis treatment, realize targeted delivery and efficient synergistic effect, and have wide application prospects.

Description

Liver-targeted biomimetic cell membrane drug-loaded nanoparticles and its preparation method and application

technical field

The field of pharmaceutical preparations of the present invention specifically relates to a liver-targeted biomimetic cell membrane drug-loaded nanoparticle and its preparation method and application.

Background technique

The activation of hepatic stellate cells (HSC) is an effector of liver fibrosis and its continuous progression leading to liver cirrhosis and the development of hepatocellular carcinoma. As the most important mesenchymal cells in the liver, HSCs are in a quiescent state under normal liver conditions and store vitamin A in the form of retinol esters. After liver injury, HSC will be activated to produce α-smooth muscle actin (α-smooth muscle actin, α-SMA) and type I, III collagen fibers and other extracellular matrix. The increase in the number of fibroblasts and the excessive deposition of extracellular matrix proteins disrupt the normal structure of the liver and further lead to liver fibrosis. While the loss of retinoic acid may play a potential role in promoting cell activation, related derivatives of vitamin A may help prevent liver fibrosis and carcinogenesis. As one of the activation products of vitamin A, ATRA can inhibit the activation of HSC by inhibiting the expression of thioredoxin interacting protein, thereby reducing its oxidative stress level and playing a therapeutic effect.

In recent years, the bionic cell membrane nano-drug delivery system has gradually realized innovative applications in many fields such as vaccine preparation and targeted disease therapy. The genetically engineered cell membrane has high biocompatibility and biosafety, which can reduce the series of immune reactions caused by exogenous substances entering the body, such as opsonizing proteins and complement activation, and effectively prevent the mononuclear macrophage system from being affected by drugs. capture. In addition, the engineered cells use LX2 cells, which can express specific proteins on the cell membrane, so that the biomimetic nanocarriers can activate the downstream caspase cascade reaction through the combination of TRAIL protein and death receptor DR5 while possessing heterologous targeting properties , NF-κB and JNK/AP signaling pathways induce the corresponding apoptosis of fibroblasts.

The biomimetic cell membrane drug-loaded nanoparticles described in the present invention are different from the widely used biomimetic nanomaterials coated with red blood cell membranes. In addition to the selection of cell types, functional therapeutic proteins are expressed on the cell membrane through genetic engineering design to further realize biomimetic Extended applications of nanosystems. Through the dual effects of engineered cells expressing TRAIL protein to promote the apoptosis of activated fibroblasts and drug ATRA to inhibit fibroblast activation and return to resting state, liver-targeted biomimetic cell membrane drug-loaded nanoparticles will be an effective strategy for the treatment of liver fibrosis , to provide a new idea for the treatment of tumors.

Contents of the invention

The object of the present invention is to provide a liver-targeted biomimetic cell membrane drug-loaded nanoparticle and its preparation method and application.

The object of the present invention is achieved through the following technical solutions:

In a first aspect, the present invention provides a method for preparing liver-targeted biomimetic cell membrane drug-loaded nanoparticles, comprising the following steps:

(1) Preparation of drug-loaded nanoparticles: lactic acid-glycolic acid copolymer and all-trans retinoic acid are dissolved in anhydrous dichloromethane, and the mass ratio of the lactic acid-glycolic acid copolymer and all-trans retinoic acid is 8- 10:1, the water phase uses polyvinyl alcohol, the water phase is prepared by dissolving polyvinyl alcohol in deionized water, adding 4g of polyvinyl alcohol per 100ml of deionized water; then use the probe ultrasonic to emulsify on ice, ultrasonic The condition is that the output power is 260-325W, and the duration is 5-15min; the white emulsion formed by ultrasonic emulsification is slowly added dropwise to 12-14mL single distilled water, and stirred at room temperature for 4-5h to volatilize the dichloromethane and solidify the drug-loaded nano Particles; finally, the drug-loaded nanoparticles were collected after ultrafiltration and centrifugation at 3000 rpm for 30 minutes;

(2) Preparation of LX2 cell line stably expressing TRAIL protein: culture LX2 cells in the logarithmic growth phase until the cells adhere to the wall, and then use the medium containing Lv-trail-zsgreen overexpression lentivirus to transfect the LX2 cells , cultured at 37°C for 24 hours, replaced the medium containing Lv-trail-zsgreen overexpressed lentivirus with DMEM medium and continued to cultivate for 48 hours, and then used G418 medium containing 200 μmol/L for resistance screening to obtain stable transfection and stable LX2 cell line expressing TRAIL protein;

(3) Preparation of engineered cell membrane: The LX2 cell line stably expressing TRAIL protein prepared in step (1) was washed three times with phosphate buffered saline at 4°C, resuspended with hypotonic cell lysate and lysed on a shaker at 4°C After 4-6 hours, the output power is 260-325W, and the probe is ultrasonicated for 1-3 minutes; then differential centrifugation is performed to remove the cell membrane pellet; finally, the proposed cell membrane pellet is resuspended in phosphate buffered saline solution, and the cell membrane is suspended by a micro extruder The liquid is squeezed back and forth 9-13 times from the polycarbonate membrane to obtain the engineered cell membrane;

(4) Preparation of liver-targeted biomimetic cell membrane drug-loaded nanoparticles: After the engineered cell membrane prepared in step (3) was ultrasonically treated, it was mixed with the drug-loaded nanoparticles prepared in step (1) at a mass ratio of 1:1. The mixture was mixed, and the blend was extruded back and forth 9-12 times from a polycarbonate membrane with a pore size of 200 nm through a micro-extruder to obtain liver-targeted biomimetic cell membrane drug-loaded nanoparticles.

Further, the differential centrifugation in step (3) includes: first centrifuging at 1,000-1500g centrifugal force and 4°C for 10-15min, and taking the supernatant; Under centrifugal conditions, centrifuge for 30-40min; finally, under centrifugal conditions of 900000-100000g centrifugal force and 4°C, centrifuge for 1-3h.

Further, the hypotonic cell lysate includes 0.25× phosphate buffered saline solution, protease inhibitors and phosphatase inhibitors.

Further, the pore size of the polycarbonate membrane in step (3) is 50nm, 100nm, 200nm or 400nm.

Further, the polycarbonate membrane preferably has a pore size of 200 nm.

In a second aspect, the present invention provides a liver-targeted biomimetic cell membrane drug-loaded nanoparticle prepared by the above method.

In a third aspect, the present invention provides an application of a liver-targeted biomimetic cell membrane drug-loaded nanoparticle in the preparation of a drug for treating liver fibrosis.

Beneficial effects of the present invention: 1. The present invention provides a biomimetic cell membrane drug-loaded nanoparticle that can exert a synergistic targeting effect to treat liver fibrosis. Liver targeting can be achieved by using LX2 cells as source cells. The therapeutic protein TRAIL expressed on the cell membrane can promote the apoptosis of activated liver fibroblasts, combined with the drug ATRA to inhibit the activation of fibroblasts, can effectively reduce the progression of liver fibrosis. At the same time, the biomimetic cell membrane drug-loaded nanoparticles are transported with the help of a stable membrane structure, which can prolong their residence time in the blood circulation and smoothly reach the target cells to achieve effective drug penetration and treatment;

2. The polymer material used in the engineered cell membrane nanoparticles of the present invention is PLGA, which is a medical polymer material approved by the U.S. Food and Drug Administration (FDA) to ensure the safety of the drug delivery system, and the coated cell membrane is strengthened. Its biological applicability has high clinical application value.

Description of drawings

Figure 1 is a schematic diagram of the preparation and structure of a liver-targeted biomimetic cell membrane drug-loaded nanoparticle;

Figure 2 is a transmission electron microscope image of a liver-targeted biomimetic cell membrane drug-loaded nanoparticle;

Fig. 3 is western blotting detection figure;

Figure 4 is a diagram of the effect of detecting TM-ATRA/NP and NP, M-NP, ATRA/NP, M-ATRA/NP, TM-NP on apoptosis induced by crystal violet;

Figure 5 is a graph showing the effect of TM-ATRA/NP, TM-NP and ATRA/NP on inducing apoptosis by classical collagen fiber staining method;

Figure 6 is a graph showing the effect of TM-ATRA/NP, TM-NP and ATRA/NP on apoptosis induced by Sirius red staining;

Figure 7 is a graph showing the effect of TM-ATRA/NP, TM-NP and ATRA/NP on inducing apoptosis by hematoxylin-eosin staining;

Figure 8 is a schematic diagram of evaluating the safety of treatment by HE staining of tissue sections of different organs.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, rather than all Example. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts are within the protection scope of the present invention.

Fig. 1 is a schematic diagram of the preparation and structure of a liver-targeted biomimetic cell membrane drug-loaded nanoparticle (TM-ATRA/NP) of the present invention.

The present invention will be further described below in conjunction with the accompanying drawings. The content described in the embodiments of this specification is only an enumeration of the implementation forms of the inventive concept. The protection scope of the present invention should not be regarded as limited to the specific forms stated in the embodiments. The protection scope of the invention also includes equivalent technical means that those skilled in the art can think of based on the concept of the invention.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

Example Preparation of a liver-targeted biomimetic cell membrane drug-loaded nanoparticle (TM-ATRA/NP)

(1) Preparation of drug-loaded nanoparticles (ATRA/NP)

10 mg of all-trans retinoic acid (ATRA) and 100 mg of lactic acid-glycolic acid copolymer (PLGA) were blended in 1 mL of anhydrous dichloromethane, and the concentration of the aqueous phase was 4% polyvinyl alcohol (PVA). Prepared by dissolving polyvinyl alcohol in deionized water, adding 4g of polyvinyl alcohol per 100ml of deionized water; then emulsifying on ice using a probe ultrasonic, the ultrasonic conditions are output power 325W, power 720W, duration 15min; The white emulsion formed by ultrasonic emulsification was slowly added dropwise to 14 mL of single distilled water, and stirred at room temperature for 5 h to volatilize the methylene chloride and solidify the drug-loaded nanoparticles (ATRA/NP); finally, the drug-loaded nanoparticles were collected by ultrafiltration and centrifugation at 3000 rpm for 30 min. Nanoparticles (ATRA-loaded nanoparticles, ATRA/NP);

(2) Preparation of LX2 cell line (LX2-trail-zsgreen) stably expressing TRAIL protein

The LX2 cells in the logarithmic growth phase were cultured until the cells adhered to the wall, and then the LX2 cells were transfected with the medium containing Lv-trail-zsgreen overexpression lentivirus, cultured at 37°C for 24 hours, and replaced with fresh medium containing The Lv-trail-zsgreen overexpressed lentivirus culture medium continued to be cultured for 48 hours. At this time, the degree of cell confluence in each well reached about 90%, and then the G418 medium containing 200 μmol/L was used for resistance screening, and stable transfection was obtained. LX2 cell line expressing TRAIL protein (LX2-trail-zsgreen);

Described fresh medium is DMEM medium;

The Lv-trail-zsgreen overexpression lentivirus is from Shanghai Jikai Gene Medical Technology Co., Ltd., which contains a gene fragment that can express TRAIL protein and a gene fragment that can express zsgreen fluorescent protein.

(3) Preparation of engineered cell membrane (LX2-zsgreen-trail cell membrane)

The LX2 cell line (LX2-trail-zsgreen) stably expressing TRAIL protein prepared in step (1) was washed three times with phosphate buffered saline at 4°C, resuspended with hypotonic cell lysate and lysed on a shaker at 4°C for 4h , and then use the output power of 260W, and ultrasonic probe for 1min; then carry out differential centrifugation to remove the cell membrane precipitation; the differential centrifugation includes: first centrifuging at 1000g centrifugal force and 4°C for 10min, and taking the supernatant; The supernatant was centrifuged for 30 min at 10,000g centrifugal force and 4°C; finally, centrifuged for 1 hour at 100,000g centrifugal force and 4°C to obtain a transparent cell membrane pellet; finally, the proposed cell membrane pellet was resuspended in phosphate buffered saline solution, passed The cell membrane suspension was extruded 11 times back and forth from the polycarbonate membrane with a pore size of 200 nm by a micro-extruder to obtain an engineered cell membrane (LX2-zsgreen-trail cell membrane).

The hypotonic cell lysate includes 0.25×phosphate buffered saline solution, protease inhibitors and phosphatase inhibitors.

(4) Preparation of liver-targeted biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP)

After sonicating 10 mg of the engineered cell membrane (LX2-zsgreen-trail cell membrane) prepared in step (3), it was blended with 10 mg of the drug-loaded nanoparticles (ATRA/NP) prepared in step (1), and the micro-extrusion The blended solution was squeezed back and forth 11 times from the polycarbonate membrane with a pore size of 200 nm to obtain liver-targeted biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP).

Fig. 2 is the transmission electron micrograph of the biomimetic cell membrane drug-loaded nanoparticle (TM-ATRA/NP) of liver targeting; from Fig. 2, the biomimetic cell membrane drug-loaded nanoparticle of liver target prepared by the present invention is a spherical particle, and Has a classic core-shell structure.

Figure 3 is the detection diagram of western blot method. BCA kits were used to measure LX2 cell lysate (LX2 celllysate, CL), LX2 cell line lysate stably expressing TRAIL protein (LX2-trail-zsgreen CL), LX2 cell membrane (LX2cell membrane) and engineered cell membrane (LX2-zsgreen -trail cell membrane), the measurement results are shown in Figure 3, it can be seen that the LX2 cell line lysate (LX2-trail-zsgreen CL) stably expressing TRAIL protein and the engineered cell membrane (LX2-zsgreen-trail cell membrane) can express TRAIL protein.

Application example 1

(1) Cytotoxicity test

Crystal violet staining detection process: Huh7 cells were inoculated into 12-well plates according to the number of 5×10^4 cells/well, and the cells were allowed to adhere to the wall after culturing for 12 hours; Nanoparticles (nanoparticles, NP) cell membrane-coated empty nanoparticles (nanoparticles coated with fibroblast membrane, M-NP), expressing TRAIL protein cell membrane coated empty nanoparticles (nanoparticles coated with TRAIL-expressing fibroblast membrane, TM -NP), drug-loaded nanoparticles (ATRA-encapsulated nanoparticles, ATRA/NP), cell membrane-coated ATRA-loaded nanoparticles (ATRA-encapsulated nanoparticles coated with fibroblast membrane, M-ATRA/NP), and liver-targeted biomimetic Cell membrane drug-loaded nanoparticles (ATRA-encapsulated nanoparticles coated with TRAIL-expressing fibroblast membrane, TM-ATRA/NP) were treated with medium for 24h as the experimental group, and Huh7 cells were treated with fresh medium for 24h as the control group; Discard the medium containing NP, M-NP, TM-NP, ATRA/NP, M-ATRA/NP or TM-ATRA/NP, wash twice with phosphate buffered saline solution, and place the plate upside down on the filter paper On the top, control the water; each of the orifice plates of 6 experimental groups (NP, M-NP, TM-NP, ATRA/NP, M-ATRA/NP or TM-ATRA/NP) and a control group (control) Add 50 μL of crystal violet staining solution to the wells, and incubate on a shaker at room temperature for 20 min at a frequency of 20 vibrations per minute. Discard the crystal violet staining solution in the plate, wash it four times with phosphate buffered saline, put the plate upside down on the filter paper, drain the water, and observe the cells under a microscope; the test results are shown in Figure 4.

Fig. 4 is a graph showing the effects of TM-ATRA/NP and NP, M-NP, TM-NP, ATRA/NP, M-ATRA/NP on apoptosis induced by crystal violet staining solution. It can be seen from Figure 4 that the cell membrane of TRAIL itself has the effect of apoptosis. TM-ATRA/NP had the highest cytotoxicity, which indicated that ATRA could synergistically enhance the cell-killing effect of TRAIL.

(2) Liver fibrosis experiment

A mouse model of liver fibrosis was constructed. CCl4 was dissolved in corn oil at a ratio of 1:4, and the corresponding dose was given according to the body weight of the mice according to the ratio of the volume of CCl4 to 0.5 μL/g of the mouse body weight, but for practical reasons (the range of the syringe) the mouse was injected with a constant volume of 50 μL and make up the remaining volume with corn oil. The mice were injected intraperitoneally every 7 days and weighed in time. The mice were treated with phosphate-buffered saline (PBS), TM-NP, ATRA/NP, and TM-ATRA/NP respectively, and then the mice in each group were weighed to get the average value.

Fig. 5 shows the effects of staining TM-ATRA/NP and control vectors TM-NP and ATRA/NP on apoptosis induced by classical collagen fiber staining method (Masson) on the mouse liver fibrosis model. It can be seen from the experimental results that the control group, that is, the healthy mice that did not receive treatment, had no obvious blue collagen fibers, indicating that their livers were relatively healthy, while the PBS group had a large number of blue collagen fibers deposited, extending outward from the portal area. The thicker fiber cords and darker staining indicate that there are more collagen fibers and pseudolobules have been formed, indicating that the liver fibrosis is more serious and there is no therapeutic effect. In the TM-NP group, there were some blue collagen fibers deposited, which extended outward from around the portal area, but compared with the PBS group, there was a certain therapeutic effect. The deposition of blue collagen fibers in the ATRA/NP group was significant, but the blue collagen fibers decreased as time progressed, indicating that there was a certain therapeutic effect. The collagen fibers in the TM-ATRA/NP group were far less than those in the other groups, indicating that the liver-targeted biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP) had the most significant effect on reducing collagen fiber deposition.

Figure 6 shows the effect of TM-ATRA/NP and control vectors TM-NP and ATRA/NP on apoptosis induced by Sirius red staining on the mouse liver fibrosis model. Sirius red staining showed that various types of collagen fibers expanded outward from the portal area. Under a polarizing microscope, collagen deposition and expansion had formed pseudolobules, including a large number of red type I collagen fibers, a small amount of sparse reticular type II collagen fibers, and light yellow type IV collagen fibers. Collagen fibers. It can be seen from the experimental results that the healthy mice in the control group, that is, not receiving treatment, have no yellow collagen fiber deposition, less red collagen fiber, and lighter staining, indicating that their liver is healthy and has no liver fibrosis. In the PBS group, a large number of red and light yellow collagen fibers were deposited, extending outward from the portal area. The fiber cords were thicker and the staining was darker, indicating that there were more collagen fibers, indicating that the liver fibrosis was more serious, and there was no therapeutic effect. The TM-NP group had some red collagen fiber deposition, but compared with the control group, there was still some fibrosis, and compared with the PBS group, it had a certain therapeutic effect. The ATRA/NP group was similar to the TM-NP group, and the deposition of red collagen fibers was less than that of the PBS group, indicating that there was a certain therapeutic effect. Collagen fibers in TM-ATRA/NP group were far less than those in other groups, indicating that liver-targeted biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP) had a significant therapeutic effect in the process of liver fibrosis, and collagen fiber deposition was significantly reduced.

Fig. 7 is the effect of TM-ATRA/NP and control vectors TM-NP and ATRA/NP in inducing cell apoptosis detected by Hematoxylin-Eosin staining on the mouse liver fibrosis model. It was found that the control group, that is, the healthy mice that did not receive treatment, had no obvious tubular structure, indicating that there was no liver fibrosis. The histological structure of the liver slices in the PBS group showed that the structure of the hepatic lobule and the portal area was not obvious, and a large number of collagen fibers were formed, which were interconnected and surrounded the hepatic cell group, forming a ring-shaped pseudo-lobular structure with different shapes; in the pseudo-lobular structure The hepatocytes were scattered, the cord-like arrangement was not obvious, and the gaps were large. The liver section showed a large area of pseudolobular tissue structure, and obvious circular vacuoles could be seen in the hepatocytes. The TM-NP group had some collagen fiber deposition, compared with the PBS group, it had a certain therapeutic effect but the effect was not obvious. The deposition of red collagen fibers in the ATRA/NP group was less than that in the PBS group, indicating a certain therapeutic effect. TM-ATRA/NP still exhibited normal liver tissue structure, which indicated that liver-targeted biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP) had a significant effect on liver fibrosis.

Organ distribution experiment of nanocarriers: The mice with liver fibrosis model were killed by necking. The heart (Heart), liver (Liver), spleen (Spleen), lung (Lung) and kidney (Kidney) were dissected, fixed in 10% neutral formalin for 4 hours, dehydrated with conventional ethanol step by step, transparent in xylene, Embedded in paraffin; after serial sectioning, routine xylene dewaxing, ethanol at various levels to wash with water. HE staining was performed, and the staining results are shown in Figure 8. Figure 8 is a schematic diagram of evaluating the safety of treatment by HE staining of tissue sections of different organs. It can be seen from Figure 8 that, except for the significant difference in liver section staining, other organs were found to be in a physiologically normal state in each group. The biosafety and liver targeting of the liver-targeting biomimetic cell membrane drug-loaded nanoparticles (TM-ATRA/NP) prepared in the present invention are proved.

The liver-targeted biomimetic cell membrane drug-loaded nanoparticle prepared by the preparation method of the liver-targeted biomimetic cell membrane drug-loaded nanoparticle can be used to prepare a drug for treating liver fibrosis.

Claims (6)

1. A method for preparing liver-targeted biomimetic cell membrane drug-loaded nanoparticles, comprising the following steps: (1) Preparation of drug-loaded nanoparticles: lactic acid-glycolic acid copolymer and all-trans retinoic acid are dissolved in anhydrous dichloromethane, and the mass ratio of the lactic acid-glycolic acid copolymer and all-trans retinoic acid is 8- 10:1, the water phase uses polyvinyl alcohol, the water phase is prepared by dissolving polyvinyl alcohol in deionized water, adding 4g of polyvinyl alcohol per 100ml of deionized water; then use the probe ultrasonic to emulsify on ice, ultrasonic The condition is that the output power is 260-325W, and the duration is 5-15min; the white emulsion formed by ultrasonic emulsification is slowly added dropwise to 12-14mL single distilled water, and stirred at room temperature for 4-5h to volatilize the dichloromethane and solidify the drug-loaded nano Particles; finally, the drug-loaded nanoparticles were collected after ultrafiltration and centrifugation at 3000 rpm for 30 minutes; (2) Preparation of LX2 cell line stably expressing TRAIL protein: culture LX2 cells in the logarithmic growth phase until the cells adhere to the wall, and then use the medium containing Lv-trail-zsgreen overexpression lentivirus to transfect the LX2 cells , cultured at 37°C for 24 hours, replaced the medium containing Lv-trail-zsgreen overexpressed lentivirus with DMEM medium and continued to cultivate for 48 hours, and then used G418 medium containing 200 μmol/L for resistance screening to obtain stable transfection and stable LX2 cell line expressing TRAIL protein; (3) Preparation of engineered cell membrane: The LX2 cell line stably expressing TRAIL protein prepared in step (2) was washed three times with phosphate buffered saline at 4°C, resuspended with hypotonic cell lysate and lysed on a shaker at 4°C After 4-6 hours, the output power is 260-325W, and the probe is ultrasonicated for 1-3 minutes; then differential centrifugation is performed to remove the cell membrane pellet; finally, the proposed cell membrane pellet is resuspended in phosphate buffered saline solution, and the cell membrane is suspended by a micro extruder The liquid is squeezed back and forth 9-13 times from the polycarbonate membrane to obtain the engineered cell membrane; The differential centrifugation described in step (3) includes: first, centrifuge for 10-15min under the centrifugal condition of 1,000-1500g centrifugal force and 4°C, and take the supernatant; , centrifuge for 30-40min; finally, centrifuge for 1-3h under the centrifugal force of 900000-100000g and 4℃; (4) Preparation of liver-targeted biomimetic cell membrane drug-loaded nanoparticles: After the engineered cell membrane prepared in step (3) was ultrasonically treated, it was mixed with the drug-loaded nanoparticles prepared in step (1) at a mass ratio of 1:1. The mixture was mixed, and the blend was extruded back and forth 9-12 times from a polycarbonate membrane with a pore size of 200 nm through a micro-extruder to obtain liver-targeted biomimetic cell membrane drug-loaded nanoparticles. 2. A method for preparing liver-targeted biomimetic cell membrane drug-loaded nanoparticles according to claim 1, wherein the hypotonic cell lysate comprises 0.25× phosphate buffered saline, protease inhibitors and phosphoric acid Enzyme inhibitors. 3. A method for preparing liver-targeted biomimetic cell membrane drug-loaded nanoparticles according to claim 1, wherein the polycarbonate membrane pore size in step (3) is 50nm, 100nm, 200nm or 400nm. 4 . The method for preparing liver-targeted biomimetic cell membrane drug-loaded nanoparticles according to claim 3 , wherein the pore size of the polycarbonate membrane is preferably 200 nm. 5. A liver-targeted biomimetic cell membrane drug-loaded nanoparticle prepared by any one of claims 1-4. 6. The application of the liver-targeted biomimetic cell membrane drug-loaded nanoparticle according to claim 5 in the preparation of a medicament for treating liver fibrosis.