Targeted hepatic stellate cell nanoparticles loaded with LATS2 nucleic acid sequence and application of targeted hepatic stellate cell nanoparticles

Abstract

The invention provides a targeted hepatic stellate cell nanoparticle loaded with an LATS2 nucleic acid sequence and application of the targeted hepatic stellate cell nanoparticle. The preparation method comprises the following steps: by taking a polylactic acid-glycolic acid copolymer (PLGA) as a nano carrier, loading an LATS2 plasmid with a GFAP specific promoter, preparing nano particles PLGA-LATS2, and then coating the surfaces of the PLGA-LATS2 nano particles with a macrophage membrane, so as to prepare the nano particles MM / PLGA-LATS2 coated with the macrophage membrane. According to the application, PLGA-LATS2 and MM / PLGA-LATS2 can be recruited to the liver fibrosis lesion site, so that accumulation of PLGA-LATS2 and MM / PLGA-LATS2 in the lesion site is increased, the gene intervention effect is enhanced, the efficient liver fibrosis lesion site targeting function is achieved, the progress of liver fibrosis can be effectively inhibited, and good biological safety is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug development, relates to the treatment of liver fibrosis, and specifically relates to a hepatic stellate cell-targeted nanoparticle loaded with a LATS2 nucleic acid sequence and a use thereof. Background Art

[0002] Liver fibrosis is the result of excessive accumulation of scar tissue due to inflammation of liver cells. Liver fibrosis can be caused by various types of chronic liver damage, such as alcoholism, hepatitis virus infection, genetic abnormalities, steatohepatitis, autoimmunity and other non-infectious diseases such as fatty liver, which can lead to liver cell damage, retention of inflammatory cells in the damaged liver and activation of collagen-producing cells, resulting in hardening of the liver and ultimately liver fibrosis.

[0003] The pathological mechanism of liver fibrosis is very complex. It is a repair response involving the entire body after liver damage. Therefore, drugs developed for a single target have not yet been successfully applied in clinical practice, so there are currently no drugs with clear anti-liver fibrosis efficacy for clinical use. Summary of the invention

[0004] Liver fibrosis is characterized by excessive deposition of ECM proteins, especially type 1 collagen, which is mainly produced by hepatic stellate cells (HSCs). LATS2 is a negative regulator of YAP1 in the Hippo signaling pathway and plays a key role in organ size control and tumor suppression by limiting proliferation and promoting apoptosis. Elevated LATS2 increases phosphorylation of the downstream transcription factor YAP, inhibits YAP nuclear translocation, and reduces the expression of pro-fibrotic genes. If LATS2 can be promoted to express in HSCs and inhibit the proliferation and activation of HSCs, it may be possible to treat liver fibrosis.

[0005] In order to solve the problems in the prior art, the present invention provides a targeted hepatic stellate cell nanoparticle loaded with LATS2 nucleic acid sequence, using polylactic acid-co-glycolic acid copolymer PLGA as a nanocarrier, loading LATS2 plasmid with GFAP specific promoter, preparing nanoparticle PLGA-LATS2, and then coating the surface of PLGA-LATS2 nanoparticle with macrophage membrane to prepare macrophage membrane coated nanoparticle MM / PLGA-LATS2. The present invention can recruit PLGA-LATS2 and MM / PLGA-LATS2 to the lesion site of liver fibrosis, thereby increasing the accumulation of PLGA-LATS2 and MM / PLGA-LATS2 in the lesion site, enhancing the gene intervention effect, and realizing the efficient targeting function of the lesion site of liver fibrosis, which can not only effectively inhibit the progression of liver fibrosis, but also has good biosafety.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] A hepatic stellate cell-targeted nanoparticle loaded with a LATS2 nucleic acid sequence, characterized in that polylactic acid-glycolic acid copolymer PLGA is used as a nanocarrier, and a LATS2 plasmid with a GFAP-specific promoter is loaded to prepare the nanoparticle PLGA-LATS2.

[0008] The macrophage membrane-coated LATS2 nucleic acid sequence loaded with GFAP-specific promoter and targeted hepatic stellate cell nanoparticles (MM / PLGA-LATS2) are characterized in that the macrophage membrane is coated on the surface of PLGA-LATS2 nanoparticles to prepare the nanoparticles.

[0009] The sequence of the specific promoter GFAP in the plasmid GFAP-mLATS2 is shown in SEQ ID NO.1:.

[0010] According to an embodiment of the present invention, the LATS2 sequence is a mouse LATS2 nucleic acid sequence, and the mouse LATS2 nucleic acid sequence is shown as SEQ ID NO.2:

[0011] ATGAGGCCAAAGACTTTTCCTGCCACAACTTACTCTGGAAATAGCCGGCAGCGAT

[0012] TGCAAGAGATTCGAGAGGGGCTGAAGCAGCCATCCAAGGCTTCCACCCAGGGGC

[0013] TGCTGGTGGGACCAAACAGTGACACTTCCCTGGATGCCAAAGTCCTGGGGAGCA

[0014] AAGATGCCTCCAGGCAGCAGCAAATGAGAGCCACCCCGAAGTTTGGACCTTATCA

[0015] AAAAGCTCTCAGGGAAATCCGATATTCCCTCCTGCCTTTTGCCAACGAGTCAGGC

[0016] ACTTCGGCAGCTGCAGAGGTGAACCGGCAGATGCTTCAGGAGTTGGTGAATGCG

[0017] GGATGTGACCAGGAGATGGCTGGCAGAGCGCTCAAGCAGACGGGCAGTAGGAGT

[0018] ATCGAAGCTGCCTTGGAGTACATCAGTAAGATGGGCTACCTGGACCCCAGGAATG

[0019] AGCAGATTGTGCGAGTCATCAAGCAGACCTCCCCAGGAAAGGGCCTGGCGCCCA

[0020] CCCCGGTGACTCGGCGGCCCAGTTTCGAGGGCACAGGGGAAGCACTCCCATCCTA

[0021] CCACCAGCTGGGTGGTGCAAACTACGAGGGCCCCGCCGCACTGGAGGAGATGCC

[0022] GCGGCAATATTTAGACTTTCTCTTCCCTGGAGCCGGAGCCGGCACCCACGGTGCCC

[0023] AGGCTCACCAGCATCCTCCCAAAGGGTACAGCACAGCAGTAGAGCCAAGTGCGC

[0024] ACTTTCCGGGCACACACTATGGTCGTGGTCATCTACTATCGGAGCAGCCTGGGTAT

[0025] GGGGTGCAGCGCAGTTCCTCCTTCCAGAACAAGACGCCACCAGATGCCTATTCCA

[0026] GCATGGCCAAGGCCCAGGGTGGCCCTCCCGCCAGCCTCACCTTTCCTGCCCATGC

[0027] TGGGCTGTACACTGCCTCGCACCACAAGCCGGCGGCTACCCCACCTGGGGCCCAC

[0028] CCATTACATGTGTTGGGCACCCGGGGTCCCACGTTTACTGGCGAAAGCTCTGCAC

[0029] AGGCTGTGCTGGCACCGTCCAGGAACAGCCTCAATGCTGACTTGTACGAGCTGGG

[0030] CTCCACGGTGCCCTGGTCTGCAGCTCCACTGGCACGCCGCGACTCGCTGCAGAAG

[0031] CAGGGTCTAGAAGCCTCGCGGCCGCATGTGGCTTTTCGGGCTGGCCCCAGCAGGA

[0032] CCAACTCCTTCAACAACCCACAACCTGAGCCCTCACTGCCCGCCCCCAACACGGT

[0033] CACCGCCGTGACGGCCGCACACATCCTTCACCCTGTGAAGAGCGTGCGTGTGCTG

[0034] CGGCCCGAGCCCCAGACAGCCGTGGGGCCCTCGCACCCCGCCTGGGTGGCTGCG

[0035] CCCACAGCACCTGCCACTGAGAGCCTGGAGACGAAGGAGGGCAGCGCAGGCCCA

[0036] CACCCGCTGGATGTGGACTATGGCGGCTCCGAGCGCAGGTGCCCACCGCCTCCGT

[0037] ACCCAAAGCACTTGCTGCTGCCCAGTAAGTCTGAGCAGTACAGCGTGGACCTGGA

[0038] CAGCCTGTGCACCAGTGTGCAGCAGAGTCTGCGAGGGGGCACTGAGCAAGACAG

[0039] GAGTGACAAGAGCCACAAAGGTGCGAAGGGAGACAAAGCTGGCAGAGACAAAA

[0040] AGCAGATTCAGACCTCCCCGGTGCCTGTCCGCAAGAATAGCAGAGATGAAGAGA

[0041] AGAGAGAGTCTCGCATCAAGAGTTACTCCCCTTATGCCTTCAAATTCTTCATGGAG

[0042] CAACACGTGGAGAATGTCATCAAAACCTACCAGCAGAAGGTCAGCCGGAGGCTA

[0043] CAGCTGGAGCAGGAAATGGCCAAAGCTGGGCTCTGTGAGGCCGAGCAGGAGCAG

[0044] ATGAGGAAGATCCTCTACCAGAAGGAGTCTAACTACAACCGGCTGAAGAGGGCC

[0045] AAGATGGACAAGTCCATGTTTGTGAAAATCAAGACTCTAGGCATCGGTGCCTTTG

[0046] GGGAAGTGTGCCTCGCTTGTAAGCTGGACACTCACGCTCTGTACGCCATGAAGAC

[0047] TCTCAGGAAGAAGGATGTCCTGAACCGGAATCAAGTGGCCCATGTCAAGGCTGAG

[0048] AGGGACATCCTGGCTGAAGCAGACAATGAGTGGGTGGTCAAACTCTACTACTCCT

[0049] TCCAGGACAAGGACAGCCTGTACTTTGTGATGGACTACATACCAGGCGGGGATAT

[0050] GATGAGCCTGCTGATCAGGATGGAGGTCTTCCCTGAGCACCTGGCCCGCTTCTACA

[0051] TTGCAGAGTTGACCCTGGCCATTGAAAGTGTCCACAAGATGGGCTTTATCCACCG

[0052] GGACATCAAGCCTGACAACATACTCATCGACCTGGATGGTCATATTAAGCTGACAG

[0053] ATTTTGGCCTCTGCACTGGATTCAGGTGGACTCACAATTCCAAGTACTACCAGAAA

[0054] GGGAACCACATGAGACAGGACAGCATGGAGCCCGGTGACCTCTGGGACGATGTT

[0055] TCCAACTGTCGCTGTGGAGACAGGTTAAAGACCCTGGAGCAGAGGGCGCAGAAG

[0056] CAGCACCAGAGGTGCCTGGCACATTCTCTTGTCGGGACACCAAATTACATCGCTC

[0057] CGGAGGTGCTTCTCCGCAAAGGGTACACGCAGCTCTGTGACTGGTGGAGCGTCG

[0058] GTGTGATTCTCTTTGAGATGCTGGTTGGGCAGCCGCCTTTCTTGGCCCCCACCCCC

[0059] ACAGAGACGCAGCTGAAGGTGATCAACTGGGAGAGCACGCTGCATATCCCTACGC

[0060] AGGTGAGGCTCAGCGCTGAGGCCCGAGACCTCATCACGAAGCTGTGCTGCGCGG

[0061] CTGACTGCCGCCTGGGCAGGGATGGGGCAGATGACCTCAAGGCACACCCGTTCTT

[0062] CAACACCATCGACTTTTCCCGTGACATCCGAAAGCAGCCTGCACCCTACGTCCCC

[0063] ACCATCAGCCACCCCATGGACACCTCCAATTTTGACCCGGTGGATGAAGAAAGCC

[0064] CCTGGCACGAGGCCAGCGGAGAGAGCGCCAAGGCCTGGGACACGCTGGCCTCCC

[0065] CCAGCAGCAAGCATCCAGAGCACGCCTTCTATGAGTTCACCTTCCGCAGGTTCTTC

[0066] GATGACAACGGCTATCCCTTCCGGTGCCCGAAGCCCTCAGAGCCCGCAGAGAGTG

[0067] CAGACCCAGGGGATGCGGACTTGGAAGGTGCGGCCGAGGGCTGCCAGCCGGTGTACGTGTAA。

[0068] According to an embodiment of the present invention, the preparation method of the nanoparticle PLGA-LATS2 is as follows:

[0069] PLGA was dissolved in dichloromethane, and LATS2 plasmid and MC3 cationic lipid were added, wherein the mass ratio of LATS2 plasmid to MC3 cationic lipid was 1:6000-7000; the mixture was then added into polyvinyl alcohol (PVA) aqueous solution, ultrasonicated in an ice bath for 4-9 minutes, stirred with a magnetic stirrer to volatilize the organic solvent for 2-6 hours, and then dialyzed in ultrapure water for 8-16 hours, and passed through a 200 nm polycarbonate membrane to obtain PLGA-LATS2 nanoparticles.

[0070] According to one embodiment of the present invention, the preparation method of the nanoparticles MM / PLGA-LATS2 is as follows: PLGA-LATS2 nanoparticles and extracted macrophage membranes are fully mixed in a ratio of 1:7-13, and water bath sonicated for 7-16 minutes; a micro membrane extruder is used to first pass through a 400nm polycarbonate membrane and then through a 200nm polycarbonate membrane to obtain MM / PLGA-LATS2 nanoparticles.

[0071] According to one embodiment of the present invention, the method for extracting the macrophage membrane is as follows: macrophages are subjected to a hypotonic solution gradient to obtain a cell lysate, which is obtained by differential centrifugation and probe ultrasonic extraction; the hypotonic solution gradient is specifically a cell suspension in which a 0.25X buffer solution containing a protease inhibitor is added, and the cells are lysed on ice for 1.5 to 2.5 hours and then probe ultrasonicated for 4 to 6 minutes to fully lyse the cells, and the cell lysate is collected, centrifuged at 800 to 1200 rpm and 3 to 5°C for 4 to 7 minutes, and the supernatant is carefully collected; then centrifuged at 12000 to 15000 rpm and 3 to 5°C for 8 to 12 minutes to sequentially remove cell debris and organelles, and collect the precipitate to obtain the macrophage membrane; the obtained macrophage membrane is resuspended in ultrapure water, and then passed through a 400nm polycarbonate membrane using a micro membrane extruder, and repeatedly extruded 10-20 times to obtain the macrophage membrane.

[0072] According to one embodiment of the present invention, PLGA-LATS2 nanoparticles are used in the preparation of drugs for preventing or treating liver fibrosis.

[0073] According to one embodiment of the present invention, MM / PLGA-LATS2 nanoparticles are used in the preparation of drugs for preventing or treating liver fibrosis.

[0074] Beneficial effects:

[0075] The present invention provides a hepatic stellate cell-targeted nanoparticle loaded with a LATS2 nucleic acid sequence, using polylactic acid-glycolic acid copolymer PLGA as a nanocarrier, adding cationic lipid MC3 at the same time, loading a LATS2 plasmid with a GFAP-specific promoter, preparing nanoparticles PLGA-LATS2, and then coating the surface of the PLGA-LATS2 nanoparticles with macrophage membranes to prepare macrophage membrane-coated nanoparticles MM / PLGA-LATS2. The present invention can recruit PLGA-LATS2 and MM / PLGA-LATS2 to liver fibrosis lesions, thereby increasing the accumulation of PLGA-LATS2 and MM / PLGA-LATS2 in the lesions, enhancing the gene intervention effect in specific cells, inhibiting the proliferation of hepatic stellate cells, and realizing efficient liver fibrosis lesion targeting function, which can not only effectively inhibit the progression of liver fibrosis, but also has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Plasmid GFAP-LATS2 expression profile.

[0077] Figure 2 Related characterization diagrams of PLGA-LATS2 and MM / PLGA-LATS2 nanoparticle preparation; Figure 2 A is the particle size of PLGA-LATS2 and MM / PLGA-LATS2; Figure 2 B is the potential of PLGA-LATS2 and MM / PLGA-LATS2; Figure 2 C is the electron microscopy images of PLGA-LATS2 and MM / PLGA-LATS2; Figure 2 D is the Coomassie blueprint of PLGA-LATS2, macrophages, macrophage membrane, and MM / PLGA-LATS2.

[0078] Figure 3 PLGA-LATS2 and MM / PLGA-LATS2 nanoparticle stability test diagram; Figure 3 A is the stability of PLGA-LATS2 and MM / PLGA-LATS2; Figure 3 B is the particle size of PLGA-LATS2 and MM / PLGA-LATS2 at 7 days; Figure 3 C is PLGA-LATS2, MM / PLGA-LATS27 days PDI.

[0079] Figure 4 PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles cellular uptake experiments.

[0080] Figure 5PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles were labeled with Cy5 to evaluate their liver targeting ability.

[0081] Figure 6 Detection of relevant biochemical indicators in mouse serum; Figure 6 A is the serum ALT of mice in the three groups of CCL4, PLGA-LATS2 and MM / PLGA-LATS2; Figure 6 B is the serum AST of mice in the CCL4, PLGA-LATS2 and MM / PLGA-LATS2 groups.

[0082] Figure 7 HE staining of mouse liver.

[0083] Figure 8 Masson staining of mouse liver.

[0084] Fig. 9 Sirius Red staining of mouse liver. DETAILED DESCRIPTION

[0085] The present invention is described in detail below by specific examples. It is pointed out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above invention content. Unless otherwise specified, the parts described in the present invention are all parts by weight, and the percentages are all mass percentages.

[0086] The raw materials and reagents used in the present invention are all commercially available products, and some of the reagents are as follows:

[0087]

[0088] The specific embodiments of the present invention are further described in detail as follows:

[0089] Example 1: Construction of plasmid GFAP-mLATS2.

[0090] The sequence of the specific promoter GFAP in plasmid GFAP-mLATS2 is shown in SEQ ID NO.1: AACATATCCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGC。

[0091] The nucleic acid sequence of mouse LATS2 is shown in SEQ ID NO.2:

[0092] ATGAGGCCAAAGACTTTTCCTGCCACAACTTACTCTGGAAATAGCCGGCAGCGAT

[0093] TGCAAGAGATTCGAGAGGGGCTGAAGCAGCCATCCAAGGCTTCCACCCAGGGGC

[0094] TGCTGGTGGGACCAAACAGTGACACTTCCCTGGATGCCAAAGTCCTGGGGAGCA

[0095] AAGATGCCTCCAGGCAGCAGCAAATGAGAGCCACCCCGAAGTTTGGACCTTATCA

[0096] AAAAGCTCTCAGGGAAATCCGATATTCCCTCCTGCCTTTTGCCAACGAGTCAGGC

[0097] ACTTCGGCAGCTGCAGAGGTGAACCGGCAGATGCTTCAGGAGTTGGTGAATGCG

[0098] GGATGTGACCAGGAGATGGCTGGCAGAGCGCTCAAGCAGACGGGCAGTAGGAGT

[0099] ATCGAAGCTGCCTTGGAGTACATCAGTAAGATGGGCTACCTGGACCCCAGGAATG

[0100] AGCAGATTGTGCGAGTCATCAAGCAGACCTCCCCAGGAAAGGGCCTGGCGCCCA

[0101] CCCCGGTGACTCGGCGGCCCAGTTTCGAGGGCACAGGGGAAGCACTCCCATCCTA

[0102] CCACCAGCTGGGTGGTGCAAACTACGAGGGCCCCGCCGCACTGGAGGAGATGCC

[0103] GCGGCAATATTTAGACTTTCTCTTCCCTGGAGCCGGAGCCGGCACCCACGGTGCCC

[0104] AGGCTCACCAGCATCCTCCCAAAGGGTACAGCACAGCAGTAGAGCCAAGTGCGC

[0105] ACTTTCCGGGCACACACTATGGTCGTGGTCATCTACTATCGGAGCAGCCTGGGTAT

[0106] GGGGTGCAGCGCAGTTCCTCCTTCCAGAACAAGACGCCACCAGATGCCTATTCCA

[0107] GCATGGCCAAGGCCCAGGGTGGCCCTCCCGCCAGCCTCACCTTTCCTGCCCATGC

[0108] TGGGCTGTACACTGCCTCGCACCACAAGCCGGCGGCTACCCCACCTGGGGCCCAC

[0109] CCATTACATGTGTTGGGCACCCGGGGTCCCACGTTTACTGGCGAAAGCTCTGCAC

[0110] AGGCTGTGCTGGCACCGTCCAGGAACAGCCTCAATGCTGACTTGTACGAGCTGGG

[0111] CTCCACGGTGCCCTGGTCTGCAGCTCCACTGGCACGCCGCGACTCGCTGCAGAAG

[0112] CAGGGTCTAGAAGCCTCGCGGCCGCATGTGGCTTTTCGGGCTGGCCCCAGCAGGA

[0113] CCAACTCCTTCAACAACCCACAACCTGAGCCCTCACTGCCCGCCCCCAACACGGT

[0114] CACCGCCGTGACGGCCGCACACATCCTTCACCCTGTGAAGAGCGTGCGTGTGCTG

[0115] CGGCCCGAGCCCCAGACAGCCGTGGGGCCCTCGCACCCCGCCTGGGTGGCTGCG

[0116] CCCACAGCACCTGCCACTGAGAGCCTGGAGACGAAGGAGGGCAGCGCAGGCCCA

[0117] CACCCGCTGGATGTGGACTATGGCGGCTCCGAGCGCAGGTGCCCACCGCCTCCGT

[0118] ACCCAAAGCACTTGCTGCTGCCCAGTAAGTCTGAGCAGTACAGCGTGGACCTGGA

[0119] CAGCCTGTGCACCAGTGTGCAGCAGAGTCTGCGAGGGGGCACTGAGCAAGACAG

[0120] GAGTGACAAGAGCCACAAAGGTGCGAAGGGAGACAAAGCTGGCAGAGACAAAA

[0121] AGCAGATTCAGACCTCCCCGGTGCCTGTCCGCAAGAATAGCAGAGATGAAGAGA

[0122] AGAGAGAGTCTCGCATCAAGAGTTACTCCCCTTATGCCTTCAAATTCTTCATGGAG

[0123] CAACACGTGGAGAATGTCATCAAAACCTACCAGCAGAAGGTCAGCCGGAGGCTA

[0124] CAGCTGGAGCAGGAAATGGCCAAAGCTGGGCTCTGTGAGGCCGAGCAGGAGCAG

[0125] ATGAGGAAGATCCTCTACCAGAAGGAGTCTAACTACAACCGGCTGAAGAGGGCC

[0126] AAGATGGACAAGTCCATGTTTGTGAAAATCAAGACTCTAGGCATCGGTGCCTTTG

[0127] GGGAAGTGTGCCTCGCTTGTAAGCTGGACACTCACGCTCTGTACGCCATGAAGAC

[0128] TCTCAGGAAGAAGGATGTCCTGAACCGGAATCAAGTGGCCCATGTCAAGGCTGAG

[0129] AGGGACATCCTGGCTGAAGCAGACAATGAGTGGGTGGTCAAACTCTACTACTCCT

[0130] TCCAGGACAAGGACAGCCTGTACTTTGTGATGGACTACATACCAGGCGGGGATAT

[0131] GATGAGCCTGCTGATCAGGATGGAGGTCTTCCCTGAGCACCTGGCCCGCTTCTACA

[0132] TTGCAGAGTTGACCCTGGCCATTGAAAGTGTCCACAAGATGGGCTTTATCCACCG

[0133] GGACATCAAGCCTGACAACATACTCATCGACCTGGATGGTCATATTAAGCTGACAG

[0134] ATTTTGGCCTCTGCACTGGATTCAGGTGGACTCACAATTCCAAGTACTACCAGAAA

[0135] GGGAACCACATGAGACAGGACAGCATGGAGCCCGGTGACCTCTGGGACGATGTT

[0136] TCCAACTGTCGCTGTGGAGACAGGTTAAAGACCCTGGAGCAGAGGGCGCAGAAG

[0137] CAGCACCAGAGGTGCCTGGCACATTCTCTTGTCGGGACACCAAATTACATCGCTC

[0138] CGGAGGTGCTTCTCCGCAAAGGGTACACGCAGCTCTGTGACTGGTGGAGCGTCG

[0139] GTGTGATTCTCTTTGAGATGCTGGTTGGGCAGCCGCCTTTCTTGGCCCCCACCCCC

[0140] ACAGAGACGCAGCTGAAGGTGATCAACTGGGAGAGCACGCTGCATATCCCTACGC

[0141] AGGTGAGGCTCAGCGCTGAGGCCCGAGACCTCATCACGAAGCTGTGCTGCGCGG

[0142] CTGACTGCCGCCTGGGCAGGGATGGGGCAGATGACCTCAAGGCACACCCGTTCTT

[0143] CAACACCATCGACTTTTCCCGTGACATCCGAAAGCAGCCTGCACCCTACGTCCCC

[0144] ACCATCAGCCACCCCATGGACACCTCCAATTTTGACCCGGTGGATGAAGAAAGCC

[0145] CCTGGCACGAGGCCAGCGGAGAGAGCGCCAAGGCCTGGGACACGCTGGCCTCCC

[0146] CCAGCAGCAAGCATCCAGAGCACGCCTTCTATGAGTTCACCTTCCGCAGGTTCTTC

[0147] GATGACAACGGCTATCCCTTCCGGTGCCCGAAGCCCTCAGAGCCCGCAGAGAGTG

[0148] CAGACCCAGGGGATGCGGACTTGGAAGGTGCGGCCGAGGGCTGCCAGCCGGTGTACGTGTAA.

[0149] Example 2: Characterization of PLGA-LATS2 and MM / PLGA-LATS2 nanoparticle preparation.

[0150] (1) Extraction of macrophages: Macrophages were disrupted by gradient of hypotonic solution to obtain cell lysate, which was obtained by differential centrifugation and ultrasonic extraction; the hypotonic solution gradient was specifically a cell suspension with 0.25X buffer solution containing protease inhibitors added, and the cells were lysed on ice for 2 hours and then ultrasonicated for 5 minutes to fully lyse the cells, and the cell lysate was collected, centrifuged at 1000 rpm and 4°C for 5 minutes, and the supernatant was carefully collected; the cell debris and organelles were removed by centrifugation at 14000 rpm and 4°C for 10 minutes, and the precipitate was collected to obtain macrophage membrane; the obtained macrophage membrane was resuspended in ultrapure water, and then passed through a 400 nm polycarbonate membrane using a micro membrane extruder, and extruded repeatedly 10-20 times to obtain macrophage membrane vesicles for subsequent coating of PLGA nanoparticles.

[0151] (2) Preparation of PLGA-LATS2 nanoparticles: Weigh 10 mg of PLGA, add 1 ml of dichloromethane, sonicate for 60 seconds to fully dissolve PLGA, then add 1 mg of LATS2 plasmid and 6.4 g of MC3 cationic lipid and mix well. Slowly add the above solvent dropwise to 3 ml of 1% polyvinyl alcohol (PVA) aqueous solution, sonicate for 5 minutes in an ice bath, with an interval of 5 seconds. Stir the volatile organic solvent with a magnetic stirrer for 4 hours, then dialyze in ultrapure water for 12 hours, and filter through a 200 nm polycarbonate membrane to obtain PLGA-LATS2 nanoparticles.

[0152] (3) The prepared PLGA-LATS2 nanoparticles were fully mixed with the extracted macrophage membranes at a ratio of 1:10 and subjected to water bath sonication for 10 min. The nanoparticles were first passed through a 400 nm polycarbonate membrane and then through a 200 nm polycarbonate membrane using a micro membrane extruder to obtain MM / PLGA-LATS2 nanoparticles.

[0153] (4) PLGA-LATS2 nanoparticles and MM / PLGA-LATS2 nanoparticles were diluted 20 times with PBS and their particle sizes were measured using a Malvern laser particle size analyzer; they were diluted 200 times with ddH2O and their zeta potential was measured using a Malvern laser particle size analyzer. Figure 2 .As shown in A and B: The particle size of the nanoparticles is about 200 nm, and the potential of MM / PLGA-LATS2 is lower than that of PLGA-LATS2 nanoparticles.

[0154] (5) PLGA-LATS2 nanoparticles and MM / PLGA-LATS2 nanoparticles were diluted 10 times with PBS and their microscopic morphology was observed using a transmission electron microscope. Figure 2 .C shows: MM / PLGA-LATS2 nanoparticles have an obvious "core-shell" structure.

[0155] (6) PLGA-LATS2, macrophages, macrophage membranes, MM / PLGA-LATS2 nanoparticles, and PLGA-LATS2 nanoparticles were extracted with protein lysate and subjected to SDS-PAGE gel electrophoresis. The integrity of the membrane protein was characterized by the classic Coomassie brilliant blue staining method. The results are as follows: Figure 2 .D.

[0156] Example 3: Stability test of PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles.

[0157] Images of MM / PLGA-LATS2 nanoparticles and PLGA-LATS2 nanoparticles were taken for 7 consecutive days and their particle size and zeta potential were measured. Figure 3As shown: MM / PLGA-LATS2 nanoparticles and PLGA-LATS2 nanoparticles are relatively stable, the particle size and PDI values ​​fluctuate within a small range, and there is no precipitation.

[0158] Example 4: Cellular uptake experiment of PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles.

[0159] (1) JS-1 cells were plated and divided into three groups (Blank group, PLGA-LATS2 group, and MM / PLGA-LATS2 group), and intervention was performed after they adhered to the cell wall.

[0160] (2) After the cells adhered, 100 μL of PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles were added at 1 h, 3 h, and 6 h.

[0161] (3) After the intervention time point, the cells were digested with trypsin, then resuspended with PBS and collected. After filtering impurities with a cell strainer, the cells were tested on a flow cytometer. Figure 4 As shown: The uptake rate of MM / PLGA-LATS2 nanoparticles in JS-1 cells is higher than that of PLGA-LATS2, and the uptake rate of nanoparticles increases over time.

[0162] Example 5: Cy5-labeled PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles to evaluate their liver targeting properties. (1) Cy5-labeled PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles.

[0163] (2) Wild-type mice (WT mice) were induced with CCL4 for 8 weeks and divided into two groups (cy5-PLGA-LATS2 group: mice were injected with 200 μL of cy5-PLGA-LATS2 via tail vein; cy5-MM / PLGA-LATS2 group: mice were injected with 200 μL of cy5-MM / PLGA-LATS2 via tail vein).

[0164] (3) 24 hours after the mice were injected with cy5-PLGA-LATS2 and cy5-MM / PLGA-LATS2 via the tail vein, the mice were killed, and their hearts, livers, spleens, lungs, and kidneys were removed by dissection and fluorescent photographs were taken. Figure 5 As shown: the injected nanoparticles were mainly enriched in the liver, and the enrichment amount of cy5-MM / PLGA-LATS2 nanoparticles was higher than that of cy5-PLGA-LATS2.

[0165] Example 6: Detection of relevant biochemical indicators in mouse serum

[0166] (1) Experimental groups: CCL4 group: WT mice were injected with CCL4 intraperitoneally for 8 weeks, 3 times a week; CCL4+PLGA-LATS2 group: WT mice were injected with CCL4 intraperitoneally for 8 weeks, 3 times a week, and PLGA-LATS2 nanoparticles were injected into the tail vein starting at the 4th week for 4 consecutive weeks; CCL4+MM / PLGA-LATS2 group: WT mice were injected with CCL4 intraperitoneally for 8 weeks, 3 times a week, and MM / PLGA-LATS2 nanoparticles were injected into the tail vein starting at the 4th week for 4 consecutive weeks.

[0167] (2) After the mouse model was established, the mice were anesthetized and blood was collected from the eyeballs before they were killed. The ALT and AST levels in the mouse serum were then measured using the kit provided by Nanjing Jiancheng. Figure 6 As shown: The serum ALT and AST levels of mice injected intraperitoneally with CCL4 were significantly increased, but after treatment with PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles, the ALT and AST levels of mice decreased, and the serum ALT and AST levels of mice in the CCL4+MM / PLGA-LATS2 group decreased more.

[0168] Example 7: HE staining of mouse liver.

[0169] (1) Liver tissues of mice (experimental grouping is the same as in Example 6) were collected, fixed with 4% paraformaldehyde for 2 days, dehydrated with gradient alcohol, embedded in paraffin, and paraffin sections were prepared.

[0170] (2) After the paraffin sections are dewaxed in xylene, they are gradiently hydrated through alcohol solutions of different concentrations.

[0171] (3) Stain the cell nuclei with hematoxylin for 5 min, place them in saturated lithium carbonate solution to blue for 30 s, and then rinse with double distilled water for 5 min.

[0172] (4) Stain with eosin for 4 min and rinse off excess dye with double distilled water.

[0173] (5) Dehydrate with ethanol solution in a gradient manner, and then place in xylene for 30 minutes to make it transparent; after sealing with neutral resin, observe the liver tissue morphology under a microscope and evaluate liver tissue damage. Figure 7 As shown: CCL4 induced a large amount of hepatocyte necrosis, inflammatory cell infiltration and fibrogenesis in the liver tissue of mice, but after intervention with PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles, the above pathological characteristics in the mouse liver were significantly reduced.

[0174] Example 8: Masson staining of mouse liver

[0175] (1) Dewaxing of paraffin sections: xylene I for 5 min, xylene II for 5 min, xylene III for 5 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and washing with distilled water for 5 min;

[0176] (2) Chromization or mercury removal precipitation (this step can be omitted for formaldehyde-fixed tissues);

[0177] (3) Wash with tap water and distilled water in turn;

[0178] (4) Stain the nuclei with Regaud hematoxylin stain or Weigert iron hematoxylin stain for 5–10 min.

[0179] (5) Differentiate with 1% hydrochloric acid and alcohol for 5 to 15 seconds, and then wash thoroughly with water;

[0180] (6) Add 0.1-1% lithium carbonate for 5 minutes to increase the degree of blueing, and wash with water;

[0181] (7) Use Masson Ponceau acid red solution for 5 to 10 minutes;

[0182] (8) Wash with 2% glacial acetic acid solution for 1 min;

[0183] (9) Differentiation with 1% phosphomolybdic acid aqueous solution for 3-5 min, and washing with 2% glacial acetic acid aqueous solution for 1 min;

[0184] (10) Dye with aniline blue aqueous solution or 1% light green aqueous solution for 1 to 2 minutes without washing; wash with 0.2% glacial acetic acid aqueous solution for 1 minute;

[0185] (11) Dehydrate with 95% ethanol for 2-3 seconds, dehydrate with anhydrous ethanol for 5 seconds three times, and transparentize with xylene for 1 minute three times. After sealing with neutral resin, observe the liver tissue morphology under a microscope and evaluate liver tissue fibrosis. Figure 8 As shown: CCL4 induced a large amount of fibrogenesis in mouse liver tissue, but after intervention with PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles, the collagen fibers in the mouse liver were significantly reduced.

[0186] Example 9: Sirius Red staining of mouse liver

[0187] (1) Dewaxing and hydration: xylene I for 5 min, xylene II for 5 min, xylene III for 5 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and washing with distilled water for 5 min.

[0188] (2) Add hematoxylin staining solution for 5-10 minutes, and then wash with distilled water for 10-20 seconds to remove excess staining solution.

[0189] (3) Wash with tap water for 5 minutes.

[0190] (4) Dye with Sirius Red solution for 5-10 minutes.

[0191] (5) Rinse lightly with running water to remove the dye on the surface of the slice.

[0192] (6) Dehydration and transparency: 1 minute in anhydrous ethanol, 10 minutes in xylene, and sealing with neutral resin. Observe the liver tissue morphology under a microscope and evaluate liver tissue damage. Fig. 9 As shown: CCL4 induced a large amount of fibrogenesis in mouse liver tissue, but after intervention with PLGA-LATS2 and MM / PLGA-LATS2 nanoparticles, the collagen fibers in the mouse liver were significantly reduced.

Claims

1. A targeted hepatic stellate cell nanoparticles loading the LATS2 nucleic acid sequence, Its characteristics are: The nanoparticle PLGA-LATS2 was prepared by loading the LATS2 plasmid with GFAP-specific promoter using polylactic acid-glycolic acid copolymer PLGA-LATS2; The sequence of the specific promoter GFAP in the plasmid GFAP-mLATS2 is shown in SEQ ID NO.1: AACATATCCTGGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGCCTGAGCTGGCTCTGTGAGCTGGGAGGAGGAGGAGACAGCCAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACT CACCTTGGCACAGACACAATGTTCGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCCCCTCAAATGCCTTCCGAGAAGCCCCATTGAGCAGGGGCTTGCATTGCACCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCCCCCCCCTAGGGGCTGC CCTTGCTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGAGAGAGGAGGAGGGCTGTCTGCTTCCC CCATAGCTGGGCTGCGGCCCAACCCCCCCCCCTCAGGCTATGCCAGGGGTGTTGCCAGGGCACCCGGCATCGCCAGTCTAGCCACTCCTTCATCATAAAGCCTCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGC.

2. The targeted hepatic stellate cell nanoparticles as claimed in claim 1, It is characterized in that The LATS2 sequence is a mouse LATS2 nucleic acid sequence, and the mouse LATS2 nucleic acid sequence is shown in SEQ ID NO.2: ATGAGGCCAAAGACTTTTCCTGCCACAACTTACTCTGGAAATAGCCGCAGCGATTGCAAGAGATTCGAGAGGGCTGAAGCAGCCATCCAAGGCTTCCACCCAGGGCTGCTGGTGGACCAAACAGTGACACTTCCCTGGATGCCAAAGTCCTGGGGAGCAAAGATGCCTCCAGCAGCAAATGAGAGCC ACCCCGAAGTTTGGACCTTATCAAAAAGCTCTCAGGGAAATCCGATATTCCCTCCTGCCTTTTGCCAACGAGTCAGGCACTTCGGCAGCTGCAGAGGTGAACCGGCAGATGCTTCAGGAGTTGGTGAATGCGGATGTGACCAGGAGATGGCTGGCAGCTCAAGCAGACGGGCAGTAGGAGTATCGAAGCTG CCTTGGAGTACATCAGTAAGATGGGCTACCTGGACCCCCAGGAATGAGCAGATTGTGCGAGTCATCAAGCAGACCTCCCCAGGAAAGGGCCTGGCGCCACCCCCGGTGACTCGGCGCCCAGTTTCGAGGGCACAGGGGAAGCACTCCCCATCCTACCACCAGCTGGGTGCAAACTACGAGGGCCCCGCCGCAC TGGAGGAGATGCCGCGGCAATATTTAGACTTTCTCTTCCCTGGAGCCGGAGCCGGCACCACCCACGGTGCCCAGGCTCACCAGCATCCTCCCAAAGGGTACAGCAGCAGTAGAGCCAAGTGCGCACTTCCGGCACACTATGGTCGTGGTCATCTACTATCGGAGCAGCCTGGTATGGGTGCAGCGCAGTTC CTCCTTCCAGAACAAGACGCCACCAGATGCCTATTCCAGCATGGCCAAGGCCCAGGGTGGCCCTCCCCGCCAGCCTCACCTTTCCTGCCATGCTGGGCTGTACACTGCCTCGCACCACCAAGCCGGCGCTACCCCCACCTGGGGCCACCCATTACATGTGTTGGGCACCCGGGTCCCACGTTTACTGGCGAAAG CTCTGCACAGGCTGTGCTGGCACCGTCCAGGAACAGCCTCAATGCTGACTTGTACGAGCTGGGCTCCACGGTGCCCTGGTCTGCAGCTCCACTGGCACGCCGCGACTCGCTGCAGAAGCAGGGTCTAGAAGCCTCGCGCCGCATGTGGCTTTCGGCTGGCCCAGCAGGACCAACTCCTTCAACAA CCTGAGCCCTCACTGCCCGCCCCCAACACACGGTCACCGCCGTGACGCCGCACACATCCTTCACCCTGTGAAGAGCGTGCGTGTGCTGCGCCGAGCCCAGACAGCCGTGGGGCCTCGCACCCCGCCTGGGTGGCTGCGCCCACAGCACCTGCCACTGAGAGCCTGGAGACGAAGGAGGGCAGCGCAGCCACAC ACCCGCTGGATGTGGACTATGGCGGCTCCGAGCGCAGGTGCCCACCGCCTCCGTACCCAAAGCACTTGCTGCTGCCCAGTAAGTCTGAGCAGTACAGTACAGCGTGGACCTGGACAGCCTGTGCACCAGTCTGCAGTGCAGAGTCTGCGAGGGGCACTGAGCAAGACAGGAGGAGTGACAAGAGGAGGAAGCCACAAAGGTGCGAAGGGAGA CAAAGCTGGCAGAGACAAAAAAGCAGATTCAGACCTCCCGGTGCCTGTCCGCAAGAATAGCAGAGATGAAGAGAGAGAGTCTCGCATCAAGAGTTACTCCCCCTTATGCCTCAAATTCTTCATGGAGCAACACGTGGAGAATGTCATCAAAACCTACCAGCAGAAGGTCAGCCGGAGCTACAGCTGGAGCA GGAAATGGCCAAAGCTGGGCTCTGTGAGGCCGAGCAGGAGCAGATGAGGAAGATCCTCTACCCAGAAGGAGTCTAACTACAACCGGCTGAAGAGGGCCAAGATGGACAAGTCCATGTTTGTGAAATCAAGACTCTAGGCATCGGTGCCTTTGGGAAGTGCCTCGCTTGTAAGCTGGACACTCACGCTCTGTAC GCCATGAAGACTCTCAGGAAGAAGGATGTCCTGAACCGGAATCAAGTGGCCATGTCAAGGCTGAGAGGGACATCCTGGCTGAAGCAGACAATGAGTGGGTGGTCAAACTCTACTACTCCTTCCAGGACAAGGACAGCCTGTACTTTGTGATGGACTACATACCAGGCGGGATATGATGAGCCTGCTGATCAGG ATGGAGGTCTTCCCTGAGCACCTGGCCCGCTTCTACATTGCAGAGTTGACCCTGGCCATTGAAAGTGTCCACAAGATGGGCTTTATCCACCGGGACATCAAGCCTGACAACATACTCGACCTGGATGGTCATTAAGCTGACAGATTTGGCCTCTGCACTGGATTCAGGTGGACTCACAATTCCAAGTACT ACCAGAAAAGGGAACCACATGAGACAGCAGCATGGAGCCCGGTGACCTCTGGGACGATGTTTCCAACTGTCGCTGGAGACAGGTTAAAGACCCTGGAGCAGAGGGCGCAGAAGCAGCAGCACCAGAGGTGCCTGGCACATTCTCTTGTCGGGACACCAAATTACATCGCTCCGGAGGTGCTTCTCCGCAAAGGGT ACACGCAGCTCTGTGACTGGTGGAGCGTCGGTGTGATTCTCTTTGAGATGCTGGTGGCAGCCGCCTTTCTTGGCCCCACCCCCCACAGAGACGCAGCTGAAGGTGATCAACTGGGAGAGCACGCTGCATATCCCTACGCAGGTGAGGCTCAGCGCTCAGCGCTGAGGCCCCGAGACCTCATCACGAAGCTGTGCTGCGCGCGC TGACTGCCGCCTGGGCAGGGATGGGGCAGATGACCTCAAGGCACACCCGTTCTTCAACACCATCGACTTTTCCCGTGACATCCGAAAGCAGCCTGCACCCTACGTCCCCACCATCAGCCCCATGGACACCTCCAATTTTGACCCGGTGGATGAAGAAAGCCCTGGCACGAGCGGAGAGAGCGCCAAG GCCTGGGACACGCTGGCCTCCCCCCAGCAGCAAGCATCCAGAGCACGCCTTCTATGAGTTCACCTTCCGCAGGTTCTTCGATGACAACGGCTATCCCTTCCGGTGCCGAAGCCCTCAGAGCCGCAGAGAGTGCAGACCCAGGGGATGCGGACTTGGAAGGTGCGCCGAGGGCTGCCAGCCGGTGTAA.

3. The targeted hepatic stellate cell nanoparticles as claimed in claim 1 or 2, Its characteristics are: The preparation method of the nanoparticle PLGA-LATS2 is: dissolve PLGA with dichloromethane, add LATS2 plasmid and MC3 cationic lipid, wherein the mass ratio of LATS2 plasmid and MC3 cationic lipid is 1:6000-7000; then the mixture is added to the aqueous polyvinyl alcohol PVA solution, sonicate the ice bath for 4-9 minutes, stir the volatile organic solvent by a magnetic stirrer for 2-6 hours, and then dialyze in ultrapure water for 8-16 hours, and pass through a 200nm polycarbonate film to obtain PLGA-LATS2 nanoparticles.

4. The macrophage membrane is coated with the LATS2 nucleic acid sequence loaded with GFAP-specific promoter to target liver stellate cell nanoparticles (MM / PLGA-LATS2). Its characteristics are: The macrophage membrane is coated on the surface of the PLGA-LATS2 nanoparticle according to any one of claims 1-3.

5. The nanoparticle of claim 4, Its characteristics are: The preparation method of the nanoparticle MM / PLGA-LATS2 is: the PLGA-LATS2 nanoparticles are fully mixed with the extracted macrophage membrane in a ratio of 1:7-13, and the water bath is ultrasonic for 7-16 minutes; a micro-film extruder is used to pass through the 400nm polycarbonate membrane and then pass through the 200nm polycarbonate membrane to obtain MM / PLGA-LATS2 nanoparticles.

6. The nanoparticle of claim 4 or 5, It is characterized in that The extraction method of the macrophage membrane is: the macrophages are broken through a gradient of hypotonic solution to obtain cell lysate, and the method of differential centrifugation and ultrasonic extraction of probes is obtained; the gradient of the hypotonic solution is specifically the cell suspension, the 0.25X buffer solution containing protease inhibitor is added to the cell suspension, and the probe is lysed for 1.5 to 2.5 hours, and the probe is ultrasonic for 4 to 6 minutes to fully lyse the cells, and collects the cell lysate. Centrifuge at 800-1200 rpm, centrifuge at 3-5°C for 4-7 minutes, and carefully collect the supernatant; then centrifuge at 12000-15000 rpm, centrifuge at 3-5°C for 8-12 minutes, and collect precipitation to obtain macrophage membrane; the obtained macrophage membrane is resuspended with ultrapure water, and then a micro-membrane extruder is used to pass through a 400nm polycarbonate membrane, and repeatedly extrude 10-20 times to obtain macrophage membrane.

7. The use of PLGA-LATS2 nanoparticles according to any one of claims 1-3 in the preparation of drugs for preventing or treating liver fibrosis.

8. Use of MM / PLGA-LATS2 nanoparticles as claimed in any one of claims 4-6 in the preparation of drugs for preventing or treating liver fibrosis.

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