CN120037408A - Pharmaceutical composition and application thereof in preparation of medicines for treating liver diseases - Google Patents

Abstract

The present invention discloses a pharmaceutical composition. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a carrier complex, the carrier complex is formed by exosomes, and lipid substances or cationic polymers through the attraction of positive and negative charges, the carrier complex also comprises an active pharmaceutical ingredient nucleic acid, and the pharmaceutical composition optionally comprises a pharmaceutically suitable carrier. The pharmaceutical composition can achieve the delivery of nucleic acids. Thereby achieving liver-targeted expression of nucleic acids and improving the survival rate of individuals with liver diseases (e.g., acute liver failure models, liver fibrosis).

Description

Pharmaceutical composition and application thereof in preparation of medicines for treating liver diseases

Technical Field

The invention belongs to the field of pharmacy, and in particular relates to a pharmaceutical composition and application thereof in preparing medicines for treating liver diseases.

Background

Gene therapy is a therapeutic strategy that delivers foreign genetic material into target cells of a patient to replace defective genes. The goal of genetic disease gene therapy is to enable sustained expression of a therapeutic gene within a cell of interest at levels sufficient to ameliorate or cure disease symptoms with minimal adverse events. Current means of gene therapy include in vitro transduction and in vivo gene delivery. In vitro transduction refers to the extraction of cells from a patient and transduction with a gene of interest, after which the cells are returned to the patient by a procedure such as hematopoietic stem cells.

In vivo transduction has high requirements on the vector. Nanoliposomes (Lipid nanoparticles, LNPs) are non-viral vectors that deliver genes that have been widely studied in recent years. Compared with viral vectors, LNP delivery can achieve transient expression, reduce off-target probability by local administration, and at the same time, is far less immunogenic than viruses. There are several problems with LNP. Firstly, it is poorly targeted and does not deliver well to the cells of interest, and secondly, the use of lipid labels containing fluorophores or radiolabels to track PEG-containing LNPs during in vitro and in vivo uptake studies is complicated. Finally, reactive lipids in LNP may also be recognized by the immune system and activate immune responses, thereby causing safety concerns and related immune activation problems have been reported in several documents. Therefore, it is an urgent need to find a highly targeted and safe nucleic acid delivery vector for gene therapy.

Exosomes are extracellular vesicles produced and secreted by somatic cells, with diameters of 30-200nm, which have a similar topology to cells and are rich in nucleic acids, proteins, lipids and metabolites. The exosomes can be used as transmitters of genetic information to transmit carried cytokines, signal molecules and genetic materials to adjacent and distant cells, thereby playing a role in regulating physiological and pathological states of receptor cells and participating in the occurrence and development processes of various diseases. Since exosomes are capable of mediating intercellular communication, donor cells can transfer proteins, mRNA, miRNA, lipids, and other exogenous substances to recipient cells through exosomes, which are used as a nanocarrier for drug delivery studies.

Disclosure of Invention

Aiming at the problem that the prior art lacks effective carriers for delivering nucleic acid medicaments for treating liver diseases, the application provides a pharmaceutical composition and application thereof in preparing medicaments for treating liver diseases. The pharmaceutical composition can realize liver targeting expression of a target gene in vivo. Compared with the traditional liposome drug delivery, the liposome drug delivery has higher liver targeting and stability, lower immune toxicity, better biocompatibility and longer body circulation time.

In order to solve the technical problems, the technical scheme is that the pharmaceutical composition comprises a carrier compound, wherein the carrier compound is formed by exosomes and lipid substances or cationic polymers through positive and negative charge attraction, the carrier compound also comprises active medicinal ingredient nucleic acid, and the pharmaceutical composition also optionally comprises a pharmaceutically applicable carrier.

In a specific embodiment of the invention, the lipid-based material comprises a liposome or a cationic lipid comprising one or more polar regions capable of binding negatively charged components, preferably DOTAP, DLin-MC3-DMA, DOTAM or DOSPA, and the cationic polymer comprises protonatable amine groups which form the carrier complex with the exosomes by means of attractive interaction of positive and negative charges, preferably polyethylenimine, polyaminoester, chitosan or diethylaminoethyl dextran.

In one embodiment of the invention, the exosomes are derived from mammalian cells including, but not limited to, 293 series cells including 293T cells, 293F cells, 293FT cells, and 293 cells, and stem cells including mesenchymal stem cells, adult stem cells, and embryonic stem cells, and the like.

As one embodiment, the 293T cells may be obtained by transfection of the 293 cells with the adenovirus E1A gene.

In a preferred embodiment of the invention, the amount of said exosomes in said carrier complex is not less than 1E 5/μl.

In a preferred embodiment of the present invention, the amount of the lipid-based substance or the cationic polymer is not less than 0.3. Mu.g/. Mu.l.

In one embodiment of the invention, the ratio of the exosomes to the lipid-based substance or cationic polymer is 1E 5: 0.3-10. Mu.g, preferably 1E5, e.g., 0.4. Mu.g, 1E5, 0.5. Mu.g, 1E5, 0.6. Mu.g, 1E5, 0.7. Mu.g, 1E5, 0.8. Mu.g, 1E5, 0.9. Mu.g, 1E5, 1.1. Mu.g, 1E5, 1.2. Mu.g, 1E5, 1.3. Mu.g, 1E5, 1.4. Mu.g, 1.5. Mu.g, 1E5, 1.6. Mu.g, 1E5, 1.7. Mu.g, 1E5, 1.8. Mu.g, 1E5, 1.9. Mu.g, 1E5, 2.1. Mu.g, 1E5, 2.2. Mu.g, 1E5, 2.3. Mu.5, 1E5, 1.4. Mu.7. Mu.g, 1E5, 1.5, 1E5, 2.6. Mu.7. Mu.g, 1E5 and 2.5.

In one embodiment of the invention, the active pharmaceutical ingredient nucleic acid is selected from the group consisting of plasmid, mRNA and small RNA.

As one embodiment, the 293T cell is obtained by transfection of an adenovirus E1A gene from a 293 cell.

In a preferred embodiment of the invention, the active pharmaceutical ingredient nucleic acid is a plasmid. Preferably, the active pharmaceutical ingredient nucleic acid is a plasmid comprising a nucleic acid encoding hIL-10 (Human Interleukin, human interleukin 10). More preferably, the active pharmaceutical ingredient nucleic acid is a nucleic acid comprising a polypeptide encoding hIL-10.

The pharmaceutically acceptable carrier is a carrier conventional in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like.

In order to solve the technical problems, the technical scheme is that the preparation method of the pharmaceutical composition is characterized by comprising the following steps of physically mixing the carrier compound in a proper buffer system, and preferably, the pH value of the buffer system is 7-8.

In order to solve the technical problems, the technical scheme provided by the invention is that the pharmaceutical composition or the 293T cell-derived exosome is applied to the preparation of medicines for treating liver diseases.

In a preferred embodiment of the invention, the liver disease is a liver disease with mutations, deletions or dysfunctions of genes. Preferably, the liver disease is liver failure or liver fibrosis. More preferably, the liver failure is exogenous drug-induced liver failure or liver fibrosis. Further preferably, the exogenous drug is canavalin a, acetaminophen or CCL4.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

The pharmaceutical composition can realize homing targeted delivery of active pharmaceutical ingredient nucleic acid. For example, the target plasmid human hIL-10 gene can be subjected to liver parenchymal targeting to realize the treatment effect on ConA-induced acute liver injury of mice, APAP-induced acute liver injury of mice and CCL 4-induced chronic liver fibrosis of mice. The 293T cell derived exosome has good liver targeting and liver protection efficacy, stability protection of expression vector and other effects, and can improve liver targeting expression of active pharmaceutical ingredient nucleic acid in cells and bodies, so that the active pharmaceutical ingredient nucleic acid can be stably expressed in bodies and the possibility of immune response is reduced.

Drawings

FIG. 1 shows the effect of expression in 293T cells of a 293T cell-derived exosome loaded with a GFP plasmid into a pharmaceutical composition together with a polymer, a lipid and a liposome, respectively.

FIG. 2 shows the effect of expression in recipient cells 293T after loading 293F and UCFT cell-derived exosomes in association with liposomes into a GFP plasmid building composition.

FIG. 3 shows the positive rate of GFP delivery into recipient cells 293T by the pharmaceutical compositions obtained by the different methods of preparation.

FIG. 4 shows the change in hIL-10 expression level over time after administration of the pharmaceutical composition.

Fig. 5 is the survival rate of mice of each group in a model of ConA-induced acute liver failure in mice treated with administration of the pharmaceutical composition.

Fig. 6 shows serum ALT levels and AST levels of mice in each group following ConA-induced acute liver failure model of mice administered with the pharmaceutical composition.

FIG. 7 shows hIL-10 mRNA levels in liver parenchymal cells of mice in each group after ConA-induced mice acute liver failure model was treated with the pharmaceutical composition.

Figure 8 is survival of groups of mice in a model of drug induced liver injury in mice treated with APAP by administration of the pharmaceutical composition.

Fig. 9 shows serum ALT levels and AST levels in each group of mice following treatment of APAP-induced mice with the pharmaceutical composition.

FIG. 10 shows hIL-10 mRNA levels in liver parenchymal cells of mice in each group after treatment of APAP-induced mice with a pharmaceutical composition.

FIG. 11 shows serum ALT levels in groups of mice following treatment of CCL 4-induced mice liver fibrosis models with administration of the pharmaceutical compositions.

Fig. 12 is the levels of serum AST from each group of mice following treatment of CCL 4-induced mice liver fibrosis model with administration of the pharmaceutical composition.

Fig. 13 is the levels of serum TBIL in mice of each group following treatment of CCL 4-induced mice liver fibrosis model with administration of the pharmaceutical composition.

FIG. 14 is H & E staining of liver histopathological sections of mice of each group following treatment of CCL4 induced mice liver fibrosis models with administration of the pharmaceutical composition.

FIG. 15 shows the mRNA levels of α -SAM in liver tissue of groups of mice following treatment of a CCL 4-induced mouse liver fibrosis model with administration of a pharmaceutical composition.

FIG. 16 is a starting plasmid map of hIL-10 used.

FIG. 17 is a starting plasmid map of GFP used.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The starting materials involved in the examples include DMSO, DOTAP from Sigma-Aldrich, DMEM (containing D-Glucose 4.5g/L, L-Glutamine, sodium Pyruvate), fetal Bovine Serum (FBS) and 10 XPBS from Gibco, 0.25% Trypsin-EDTA, phenol Red, DMEM/F12, lipofectamine liposome transfection reagent Thermo Fisher, PEI from Saint Corp. Trizol was purchased from Sigma, and reverse transcriptase, SYBR Green fluorescent dye was purchased from TAKARA. The hIL-10 assay kit in the supernatant was purchased from Biyun Tian biotechnology. 293T cells are the human embryonic kidney cell line of ATCC, accession number CRL-3216.

Laboratory instruments tissue homogenizer (Shanghai Bixiao Biotech Co.), low temperature high speed centrifuge (Eppendorf, 5415R), cell incubator (Thermo FISHER SCIENTIFIC, 371), ultra pure water preparation system (PALL CASCADA), multifunctional enzyme labeling instrument (Bio TEK SYNERGY 4), applied Bio-system 7500fast fluorescence quantitative PCR instrument (Thremo), direct and inverted integral fluorescence microscope (Echo Revolve).

Experimental animals Male C57BL/6 strain mice, weighing 22-25g, purchased from Shanghai Nannon model biotechnology Co.

Example 1 construction of 293T cell derived exosomes pharmaceutical compositions to achieve exogenous gene delivery on 293T cells

The 293T cell-derived exosomes were obtained by classical ladder differential centrifugation, specifically by collecting the supernatant of 293T cells after 72h of culture, transferring the supernatant into a new centrifuge tube and centrifuging at 2000 Xg for 10 min. Carefully remove the supernatant to a new centrifuge tube, 10000 Xg, 30min again centrifuge to remove larger vesicles. The supernatant was carefully removed to a fresh centrifuge tube and centrifuged at 110000g for 70min at 4 ℃. After centrifugation, the supernatant was removed, resuspended in pre-chilled 1 XPBS and centrifuged again at 4℃at 110000g for 70min. The supernatant was removed and resuspended with an appropriate amount of 1 XPBS to give exosomes, and the number of exosomes was detected and recorded.

293T cells were plated at a cell density of 1X10 ⁵ cells/ml (DMEM medium containing 10% FBS), 24h after cell attachment, the DMEM medium containing 10% FBS was replaced with DMEM without FBS. The specific steps of loading 1. Mu.g of GFP-expressing plasmid into 293T cell-derived exosomes include mixing ①. Mu. gGFP plasmid+lipofectamine 1. Mu.l (1 mg/ml) +1×10 ⁹ 293T cell-derived exosomes, ②. Mu. gGFP plasmid+PEI 4. Mu.l (1 mg/ml) +1×10 ⁹ 293T cell-derived exosomes, ③. Mu. gGFP plasmid+DOTAP 6. Mu.l (1 mg/ml) +1×10 ⁹ 293T cell-derived exosomes, and standing for 12h to obtain corresponding medicines, and adding into 293T cell receptor cells. The GFP plasmid map is shown in FIG. 17. Cells were added for 6h and the culture was continued with DMEM medium containing 10% fbs. After 24h of liquid exchange, the GFP expression was recorded by taking a photograph with a fluorescence microscope. The results are shown in FIG. 1, which shows that the composite vector prepared above can well realize plasmid delivery. Similar effects can be achieved for each group of cationic liposomes or cationic polymers.

The GFP coding sequence (MN 832871.1) is as follows:

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtac(SEQ ID NO:7)

Example 2 pharmaceutical compositions of 293F and UCFT cell-derived exosome constructs to achieve exogenous gene delivery on 293T cells

293T cells were plated at a cell density of 1X 10 ⁵ cells/ml, respectively, and after 24h cell attachment, the medium was changed to basal medium. 1. Mu.g of plasmid expressing GFP was loaded into UCFT cells (obtained after T transfection of fibroblasts Umbilical cord-derived fibroblasts) as source of exosomes. Specifically, 1 μg GFP plasmid+1 μl lipofectamine (1 mg/ml) +1× ⁹ exosomes derived from UCFT cells or 1× ⁹ exosomes derived from 293F cells are mixed uniformly at 4deg.C, and then left stand for 12h to obtain corresponding medicines, and added into 293T cells of recipient cells. After the solution is treated for 6 hours, the culture medium containing the serum of the receptor cells is replaced for continuous culture. GFP expression was recorded 24h after transfection by fluorescent microscopy. The results are shown in fig. 2, indicating that exosomes of different cell sources can also mediate plasmid delivery into 293T cells.

Example 3 optimization of preparation method of pharmaceutical composition for construction of 293T cell derived exosomes

293T cells were plated at a cell density of 1X 10 ⁵ cells/ml, respectively, and after 24h cell attachment, the medium was changed to basal medium. Preparation of 293T cell-derived exosomes by loading 1 μg of GFP-expressing plasmid into the 293T cell-derived exosomes the following three groups, ① μg of GFP plasmid+1 μl lipofectamine (1 mg/ml), 25℃were mixed and left for 20min, 1×10 ⁹ 293T cell-derived exosomes were added, ② μg of GFP plasmid+1×10 ⁹ 293T cell-derived exosomes were mixed and left for 20min, 1 μl lipofectamine (1 mg/ml), ③1×10⁹ 293T cell-derived exosomes+1 μl lipofectamine (1 mg/ml), 25℃were mixed and left for 20min, and 1 μg of GFP plasmid was added. All samples were then allowed to stand at 4 ℃ for 12 hours, respectively, to prepare the corresponding drug, which was added to recipient cells 293T. After the solution is treated for 6 hours, the culture medium containing the serum of the receptor cells is replaced for continuous culture. After 24h, the positive rate of GFP in the cells in each preparation mode was analyzed by flow cytometry. As a result, as shown in FIG. 3, the GFP-positive rate (19.0%) of the conventional preparation method, group ①, was lower than that of the unconventional preparation method, group ③ (56.3%), while the GFP-positive rate was not high (16.8%) in group ②, although it was also the unconventional preparation method.

EXAMPLE 4 pharmaceutical composition constructed of 293T cell-derived exosomes to achieve delivery of human hIL-10 genes on 293T cells

(1) 293T cell-derived exosomes loaded with hIL-10 transfected cells

293T cells were plated at a cell density of 1X 10 ⁵ cells/ml, respectively, and after 24h cell attachment, the medium was changed to basal medium. The hIL-10 expression plasmid (plasmid was prepared by extracting using TIANGEN endotoxin-free plasmid big extraction kit, plasmid map is shown IN FIG. 16, hIL-10 sequence is shown IN SEQ ID NO: 8) was prepared according to the preparation method of group ③ IN example 3, specifically ③1×10⁹ apoplasts derived from 293T cells +1. Mu.l lipofectamine (1 mg/ml) were 25℃mixed and left for 20min, 1. Mu.g of hIL-10 plasmid was added, and then the sample was left for 12h at 4℃to prepare a drug, which was added to the recipient cells 293T. After the solution is treated for 6 hours, the culture medium containing the serum of the receptor cells is used for continuous culture, and the transfection is completed.

The hIL-10 coding sequence (NM-000572.3) is as follows:

ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC(SEQ ID NO:8)

IL-10/hIL-10H as designed in the subsequent examples refer to the above-described nucleic acid sequences.

(2) Transfection efficiency detection

After the transfection was completed, cell culture supernatants were collected at 24h, 48h and 72h, respectively, and the levels of hIL-10 in the cell supernatants were analyzed by ELISA kit (Biyun) detection method.

(3) Data analysis

Data processing and significance analysis used GRAPHPAD PRISM 8.0.0 software. Two sets of data were compared using the two-way fastened unpaired t test, and multiple sets of univariate data were compared using one-way ANOVA (one-way ANOVA) and corrected using the Tukey test. * The/#p <0.05, ×/#p <0.01, ×/#p <0.001 represents an increasing significant difference.

The results are shown in FIG. 4, which shows that the hIL-10 can be well expressed by loading the hIL-10 plasmid into exosomes. And the expression amount increases with time.

EXAMPLE 5 delivery of pharmaceutical compositions of the 293T cell-derived exosomes for the prevention of acute liver failure in mice with the human hIL-10 Gene

(1) After C57/BL6 mice were acclimatized, 200. Mu.l of each group of the drugs prepared in example 1 was injected via tail vein. The specific preparation system is 10 mug hIL-10 plasmid (injection concentration is 0.05 mug/mul) +10 mul lipofectamine (1 mg/ml) (injection concentration is 0.05 mug/mul) +1×10 ¹⁰ exosomes derived from 293T cells (injection concentration is 0.5×10 ⁸ per mul), and PBS is supplemented to 200 mul. 16mg/kg Canavalia gladiata A (ConA, sigma) was injected 24h after the injection. Serum was collected at 0h, 24h, and 48, and sent to Qianmai medical biochemistry company for detection of levels of ALT and AST in mice by HPLC analysis. Mice were recorded for death and survival curves were drawn.

(2) The primary liver parenchymal cells of mice are isolated, and the mRNA level of hIL-10 in the liver parenchymal cells of each group of mice is detected, wherein the internal reference is beta-ACTIN. hIL-10 primer sequence F ACCCTCAGGCTGAGGCTA (SEQ ID NO: 1), R CATGGCTTTGTAGATGCC (SEQ ID NO: 2), beta-ACTIN primer sequence F TCAGCAATGCCTGGGTACAT (SEQ ID NO: 3), R ATCACTATTGGCAACGAGCG (SEQ ID NO: 4).

(3) Data processing and significance analysis used GRAPHPAD PRISM 8.0.0 software. Two sets of data were compared using the two-way fastened unpaired t test, and multiple sets of univariate data were compared using one-way ANOVA (one-way ANOVA) and corrected using the Tukey test. * The/#p <0.05, ×/#p <0.01, ×/#p <0.001 represents an increasing significant difference.

In the ConA-induced acute liver failure model of mice, after loading the 293T cell-derived exosomes of hIL-10, the survival rate of the dosed group was significantly improved over that of PBS group (P < 0.001), as shown in fig. 5. Mice in the dosing group had significantly improved 24h blood biochemical index ALT and AST compared to the PBS group (P < 0.001), as shown in fig. 6. Meanwhile, the RNA of the primary liver cells of each group of mice is extracted, and the fluorescent quantitative PCR experiment result (figure 7) shows that the exosomes loaded with hIL-10 can be well expressed in liver parenchymal cells, and have good prevention and treatment effects on ConA-induced acute liver failure of mice.

Note that since only one PBS group survived at 48h, the corresponding spot in FIG. 6 was free of error bar, and that at 72h the PBS group mice had died.

EXAMPLE 6 pharmaceutical composition of 293T cell-derived exosomes delivering intervention of human hIL-10 Gene in acute liver failure in mice

(1) After C57/BL6 mice were acclimatized, 200. Mu.l of each group of the drugs prepared in example 1 was injected via tail vein. The specific preparation system is 10 mug hIL-10 plasmid (injection concentration is 0.05 mug/mul) +10 mul lipofectamine (1 mg/ml) (injection concentration is 0.05 mug/mul) +1×10 ¹⁰ exosomes derived from 293T cells (injection concentration is 0.5×10 ⁸ per mul), and PBS is supplemented to 200 mul. 600mg/kg APAP (acetaminophen, acetaminophen, sigma) was injected into the tail vein 24h after injection. Serum was collected at 0h, 24h, and 48, and sent to Qianmai medical biochemistry company for detection of levels of ALT and AST in mice by HPLC analysis. Mice were recorded for death and survival curves were drawn.

(2) The primary hepatocytes of mice were isolated and the mRNA levels of hIL-10 in each group of mice were examined, and the primer sequences were the same as in example 5.

(3) Data processing and significance analysis used GRAPHPAD PRISM 8.0.0 software. Two sets of data were compared using the two-way fastened unpaired t test, and multiple sets of univariate data were compared using one-way ANOVA (one-way ANOVA) and corrected using the Tukey test. * The/#p <0.05, ×/#p <0.01, ×/#p <0.001 represents an increasing significant difference.

In the APAP-induced acute liver failure model of mice, the survival rate of the administered group was significantly improved (P < 0.001) compared to PBS group mice after loading the 293T cell-derived exosomes of hIL-10, as shown in FIG. 8. Mice in the dosing group had significantly improved 24h blood biochemical index ALT and AST compared to the PBS group (P < 0.001), as shown in fig. 9. Meanwhile, RNA of primary liver cells of each group of mice is extracted, and the fluorescent quantitative PCR experiment result shows that the exosomes loaded with hIL-10 can be well expressed in liver parenchymal cells, and have good prevention and treatment effects on APAP-induced acute liver failure of mice (figure 10).

EXAMPLE 7 delivery of pharmaceutical compositions of the 293T cell-derived exosomes for the treatment of chronic liver fibrosis in mice with the human hIL-10 Gene

(1) After C57/BL6 mice were adaptively cultured, a model of chronic liver fibrosis was constructed by injecting 50. Mu.l of 30% CCL4 (Guozhong) twice a week for 8 weeks. 200. Mu.l of the drug prepared according to example 1 was injected by tail vein starting at the fourth week, and the preparation system was 10. Mu.g of hIL-10 plasmid (injection concentration: 0.05. Mu.g/. Mu.l) +10. Mu.l lipofectamine (1 mg/ml) (injection concentration: 0.05. Mu.g/. Mu.l) +1X 10 ¹⁰ of 293T cell-derived exosomes (injection concentration: 0.5X10 ⁸/. Mu.l). Once a week for 4 weeks. The positive drug is obeticholic acid (OCA, erbi Shanghai biotechnology limited). After the treatment, blood was collected and serum was sent to Qianmai medical biochemistry company to detect the levels of ALT, AST and TBIL in each group of mice by HPLC analysis.

(2) And taking liver tissues of each group of mice to perform pathological staining to analyze the pathological changes of the liver.

(3) The expression level of alpha-SMA was analyzed by taking RNA extracted from liver tissue of each group of mice and performing reverse transcription, and the results of fluorescence quantitative PCR were analyzed by using beta-ACTIN as an internal reference, and the alpha-SMA primer F TCAAGGAGAAGCTGTGCTATGT (SEQ ID NO: 5), R TTCGTGGATGCCCGCTGA (SEQ ID NO: 6) and the beta-ACTIN primer were the same as in example 5.

In the CCL 4-induced acute liver failure model of mice, the mice in the dosing group after injection of the exosomes loaded with hll-10 were all lower in ALT (fig. 11), AST (fig. 12) and TBIL (fig. 13) than the PBS control group, with significant ALT differences (P < 0.05) and very significant TBIL differences (P < 0.001).

Mice in the dosing group had a lower degree of liver fibrosis than the PBS control group (fig. 14).

In the CCL 4-induced acute liver failure model of mice, the α -SMA in mice in the dosing group following injection of the exosomes loaded with hIL-10 was significantly lower than in the model group (P < 0.05) (fig. 15).

Claims (10)

1. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a carrier complex, wherein the carrier complex is formed by exosomes, and lipid substances or cationic polymers through the attraction of positive and negative charges, the carrier complex also comprises an active medicinal ingredient nucleic acid, and the pharmaceutical composition optionally comprises a pharmaceutically suitable carrier. 2. The pharmaceutical composition according to claim 1, characterized in that the lipid substance comprises liposomes or cationic lipids, the cationic lipids contain one or more polar regions, the polar regions can bind to negatively charged components, and the cationic lipids are preferably DOTAP, DLin-MC3-DMA, DOTAM or DOSPA; the cationic polymer contains protonable amine groups, and the amine groups form the carrier complex with the exosomes by attracting each other through positive and negative charges, and the cationic polymer is preferably polyethyleneimine, polyaminoester, chitosan or diethylaminoethyl dextran. 3. The pharmaceutical composition according to any one of claims 1-2, characterized in that the exosomes are derived from mammalian cells, the mammalian cells include but are not limited to 293 series cells and stem cells, the 293 series cells include 293T cells, 293F cells, 293FT and 293, and the stem cells include mesenchymal stem cells, adult stem cells and embryonic stem cells. 4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the amount of the exosomes in the carrier complex is not less than 1E5/μl. 5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the amount of the lipid substance or cationic polymer is not less than 0.3 μg/μl. 6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the active pharmaceutical ingredient nucleic acid is selected from plasmid, mRNA and small RNA. 7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that the active pharmaceutical ingredient nucleic acid is selected from a plasmid containing a nucleic acid encoding hIL-10. 8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a medicament for treating liver diseases. 9. The use according to claim 8, characterized in that the liver disease is a liver disease caused by gene mutation, deletion or abnormal function; preferably, the liver disease is liver failure or liver fibrosis; more preferably, the liver failure is exogenous drug-induced liver failure or liver fibrosis. 10. The method for preparing the pharmaceutical composition according to any one of claims 1 to 7, characterized in that the method comprises the following steps: physically mixing the carrier complex in a buffer system to obtain the carrier complex.