KR20220128568A - A pharmaceutical composition for preventing or treating liver fibrosis containing exosomes surface-modified with vitamin A - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis, wherein the pharmaceutical composition contains exosomes surface-modified with vitamin A as active ingredients. The exosomes surface-modified with vitamin A, which are active ingredients of the present invention, are purified ingredients which contain genes, proteins, growth factors, and the like related to the cell proliferation, differentiation, and regeneration of stem cells, and do not contain harmful factors of antibiotics, serum, or culture media. The exosomes have the characteristic of specifically targeting hepatic stellate cells, and therefore have remarkable effects as compared with existing exosomes.

Description

A pharmaceutical composition for preventing or treating liver fibrosis containing exosomes surface-modified with vitamin A

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis.

Liver fibrosis is caused by excessive deposition of extracellular matrix due to chronic inflammation, and if this chronic liver disease persists, it eventually progresses to cirrhosis due to the transformation of the structure of the liver and a decrease in the number of hepatocytes. Such chronic liver disease can be viewed as a continuous disease that progresses from chronic hepatitis to cirrhosis through fibrosis rather than an independent disease. Currently, drug treatment or liver transplantation is being performed for various hepatitis and liver cirrhosis, but the need for a new treatment is emerging due to drug resistance or lack of liver transplant donors.

On the other hand, liver regeneration therapy using stem cells is a treatment that has recently received a lot of attention, and it is known that the injected stem cells differentiate into functional hepatocytes in the damaged liver to show therapeutic effects. Functional hepatocyte differentiation using stem cells in vitro is possible, but there is a problem that the efficiency of hepatocyte differentiation is low with stem cells transplanted from animal models and humans. In addition, the biggest problem of the conventional treatment using stem cells is that the cell engraftment rate and survival rate are not constant depending on the patient. In addition, allogenic transplantation has limitations due to the immune response by stem cells.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are hereby incorporated by reference in their entirety to more clearly set forth the content of the present invention and the level of the art to which the present invention pertains.

Republic of Korea Patent Publication No. 10-2018-0057546

As a result of research efforts to develop a technology that can specifically target hepatic stellate cells, which are the main causative cells of liver fibrosis, in order to maximize the treatment efficiency of liver fibrosis, the present inventors made efforts to convert stem cells into normal cells while alleviating liver fibrosis. By extracting exosomes containing various growth factors and effective protein components for and surface modification of them with vitamin A, selectively targeting hepatic stellate cells and remarkably increasing the treatment efficiency of liver fibrosis, the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis, comprising, as an active ingredient, an adipose stem cell-derived exosome surface-modified with vitamin A.

Another object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating liver fibrosis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis comprising, as an active ingredient, an adipose stem cell-derived exosome surface-modified with vitamin A.

The exosome surface-modified with vitamin A, the active ingredient of the present invention, contains genes, proteins, and growth factors related to cell proliferation, differentiation, and regeneration of stem cells, and does not contain antibiotics, serum, or harmful factors of culture medium. It has a remarkable effect compared to the exosomes used previously because it has the characteristic of specifically targeting hepatic stellate cells. An example of such a basic concept of the present invention is shown in FIG. 1 .

Exosomes, which are active ingredients of the present invention, may be surface-modified by a vitamin A ligand comprising a covalently linked vitamin A moiety and a phospholipid moiety.

The vitamin A may be at least one selected from the group consisting of retinol, retinal, provitamin A carotenoids, and salts thereof.

The phospholipid refers to a compound in which a fatty acid and a phosphate group are bonded to glycerol. The head of phospholipids is polar, and the tail with fatty acids is hydrophobic because it does not mix well with water and is easily miscible with non-polar substances, and the region with phosphate groups is hydrophilic because it is easily miscible with water molecules or other polar molecules. These phospholipids are important because they are a major component of biological membranes, and are contained in large amounts in the myelin sheaths of nerves and tissues.

The phospholipids include, for example, phosphorylethanolamine, Distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), Ceramide phosphorylethanolamine, Phosphosphingolipids, and phosphorylcholine ( phosphorylcholine), sphingomyelin, phosphoryllipid, ceramide phosphoryllipid, diacylglyceride, glycerophospholipid, phosphatidate, Phosphatidylethanolamine, phosphatidylcholine, lecithin, phosphatidylserine, phosphoinositide, Phosphatidyl inositol, Phosphatidylinositol, Phosphatidylinositol (Phosphatidylinositol bisphosphate), phosphatidylinositol trisphosphate, etc. may be used, but is not limited thereto.

Since the vitamin A ligand of the present invention includes a covalently linked vitamin A moiety and a phospholipid moiety, it may exhibit a particularly remarkable effect in selectively targeting hepatic stellate cells.

In one embodiment, when evaluating the targeting rate based on the number of exosome particles accumulated in hepatic stellate cells activated with TGF-β, adipose stem cell-derived exosomes surface-modified with the vitamin A ligand of the present invention are phospholipids Compared to adipose stem cell-derived exosomes surface-modified with only vitamin A without and without vitamin A, it exhibits an intracellular targeting rate that is at least 1.5 times, at least 1.8 times, at least 2 times, and at least 3 times higher.

The vitamin A ligand may further include a covalently linked polyethylene glycol (PEG) moiety. Pegylation of phospholipids can further improve blood circulation time and stability of surface-modified exosomes. In a preferred embodiment, the vitamin A ligand may have a phospholipid-PEG-vitamin A structure.

As used herein, the term “prevention” refers to any action of suppressing or delaying the onset of liver fibrosis by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms due to liver fibrosis are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “stem cell” refers to a cell that is the basis of cells or tissues constituting an individual, and its characteristics are that it can self-renew by dividing repeatedly, and has a specific function depending on the environment. It refers to a cell having a multidifferentiation ability capable of differentiating into a cell. It occurs in all tissues during the development of the fetus and is found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissue, even in adulthood. Stem cells are totipotent stem cells, which are formed when a fertilized egg begins dividing, and pluripotent stem cells in the blastocyst, which are formed by continuing division of these cells, depending on the type of differentiated cell. And they are classified as multipotent stem cells that exist in mature tissues and organs. At this time, the pluripotent stem cells are cells that can differentiate only into cells specific to the tissues and organs that contain these cells. It is involved in maintenance and inducing regeneration in the event of tissue damage. These tissue-specific pluripotent cells are collectively referred to as adult stem cells.

Mesenchymal stem cells, classified as adult stem cells, are cells that are attracting attention as materials for regenerative medicine. They can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord. It has the ability to differentiate into cells constituting various human tissues such as cells, osteocytes, chondrocytes, nerve cells, and cardiomyocytes.

In the present invention, stem cells isolated from human adipose tissue were used, but the present invention is not limited thereto. In addition, as the adipose stem cells, those derived from autologous adipose tissue of a patient to which the pharmaceutical composition is administered may be used.

As used herein, the term “exosome” refers to cell-derived vesicles present in almost all eukaryotic fluids including blood, urine, and cell culture fluids. Because exosomes have been reported to perform important specific functions in processes such as aggregation, intracellular signal transduction, and cellular waste management, exosomes are secreted by fusion to the plasma membrane or directly from the plasma membrane. In general, it is attracting attention as a biomarker for specific diseases, diagnosis, and treatment. In the present invention, the exosomes may be naturally secreted from the cells by culturing human adipose stem cells.

The pharmaceutical composition according to the present invention contains adipose stem cell-derived exosomes as an active ingredient, and may include a pharmaceutically acceptable carrier.

Adipose stem cell-derived exosomes are a composition generated in the process of promoting the secretion of various factors for the treatment of liver fibrosis, and contain genetic information, proteins, and growth factors for the treatment of liver fibrosis. Absorption is also excellent. Therefore, it is possible to solve problems that can be caused by the currently used therapeutic drugs, or the limitations of stem cell cancer cellization or allogenic transplantation that can occur in treatment using stem cells that are being researched recently.

The adipose stem cell-derived exosome of the present invention may have a diameter of 30 to 400 nm, and in a narrower range, 100 to 300 nm.

On the other hand, the pharmaceutically acceptable carrier is commonly used in formulation, and includes saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. The present invention is not limited thereto, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection, inhalant, external preparation for skin, or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, applied to the skin, nasal passages, and airways) according to a desired method, preferably intravenously. The dosage varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple.

In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, is administered daily or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

Another aspect of the present invention comprises the steps of (a) activating the carboxyl group of vitamin A; (b) synthesizing a vitamin A ligand by coupling the phospholipid compound with the carboxyl group of the activated vitamin A; And (c) to provide a method for preparing the pharmaceutical composition of the present invention, comprising the step of modifying the surface of the adipose stem cell-derived exosomes with the synthesized vitamin A ligand.

In one embodiment, step (a) may be performed by converting the carboxyl group of vitamin A into an amine-reactive ester.

The pharmaceutical composition of the present invention prepared as described above can effectively inhibit the expression of α-SMA (α-smooth muscle actin) in hepatic stellate cells, and can inhibit the deposition of extracellular matrix in liver tissue. have.

In addition, the pharmaceutical composition of the present invention comprises blood aspartate aminotransferase (AST), blood alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (Total bilirubin (TP), or LW/BW (Ratio of liver weight to body weight) levels.

On the other hand, adipose stem cell-derived exosomes surface-modified with vitamin A of the present invention are a major cause of hepatic fibrosis, while general adipose stem cell-derived exosomes have a behavior of indiscriminate accumulation throughout the liver tissue, thus limiting efficient liver fibrosis treatment. It can specifically target hepatic stellate cells, which are causative cells.

1 shows an example of the basic concept of the present invention in which exosomes extracted from adipose stem cells are surface-modified with vitamin A and used as an active ingredient of a fibrosis therapeutic composition targeting hepatic stellate cells.
Figure 2 shows (a) the synthesis process of vitamin A ligand for hepatic stellate cell target and (b) 1H-NMR results of retinoic acid, DSPE-PEG2000-amine, DSPE-PEG2000-Vit A.
Figure 3 shows (a) the size change by the surface modification of the exosome and (b) the shape change by the surface modification of the exosome.
4 shows (a) biomarker evaluation of exosomes using a confocal microscope and (b) results of exosome marker evaluation using flow cytometry.
Figure 5 shows (a) adipose stem cell-derived exosomes and cellular uptake evaluation of exosomes surface-modified with vitamin A ligand and (b) fluorescence quantitative results of (a).
Figure 6 shows (a) adipose stem cell-derived exosomes and fibrosis factor expression inhibition evaluation of exosomes surface-modified with vitamin A ligand and (b) fluorescence quantitative results of (a).
7 shows (a) evaluation of biodistribution behavior of adipose stem cell-derived exosomes and exosomes surface-modified with vitamin A ligand, and (b) real-time fluorescence change results in liver tissue of (a).
Figure 8 shows (a) the distribution evaluation of adipose stem cell-derived exosomes and exosomes surface-modified with vitamin A ligand in liver tissue, and (b) co-localization evaluation results of exosomes and α-SMA.
9 shows an animal experimental plan for evaluating the therapeutic efficacy of exosomes surface-modified with vitamin A ligand.
10 shows the results of aspartate aminotransferase (AST) blood analysis in an animal model of liver fibrosis.
Figure 11 shows the results of evaluation of (a) histopathological analysis of liver tissue and (b) liver tissue fibrous area distribution.
12 shows the evaluation results of collagen I distribution in liver tissue.
13 shows the evaluation results of hydroxyproline assay of liver tissue.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example

1. Extraction of exosomes effective for the treatment of liver fibrosis from human adipose stem cells

Human adipose stem cell-derived exosomes were extracted during culturing of human adipose stem cells. Specifically, human adipose stem cells were cultured in a general culture medium (HyClone ^™ , Cat#: SH30919) and replaced with a serum-free medium (HyClone ^™ , Cat#: SH30243) 24 hours before exosome extraction and cultured for 24 hours. After that, the cell culture supernatant was recovered. The recovered cell culture supernatant was first centrifuged at 3,000 x g, 4° C. for 20 minutes, and secondarily, 0.22 μm filtered to remove cell debris and wastes. Thereafter, the recovered supernatant was separated and purified using a tangential flow filtration (TFF) system using a 300 kDa filter to separate and purify the exosomes. After recovering the supernatant, normal culture medium was added to human adipose stem cells and cultured, and this process was repeated until passage 7. In this way, the exosomes extracted in the process of proliferation until passage 7 were used as a freeze-dried formulation composition.

2. Preparation of adipose stem cell-derived exosomes surface-modified with vitamin A according to the previous method and evaluation of hepatic stellate cell targeting ability

The surface of the lyophilized exosome obtained in Example 1 was modified with vitamin A, and in order to evaluate the targeting ability of hepatic stellate cells, the exosomes were first fluorescently labeled before modification (Flamma 675, red).

Then, according to a previously known method (Sato et al, NATURE BIOTECHNOLOGY , VOLUME 26 NUMBER 4 APRIL 2008, pp. 431-442), the surface of the fluorescently labeled exosomes was modified with vitamin A. Specifically, the fluorescence-labeled exosomes were diluted to a concentration of 1.5x10 ⁹ cells/ml in Diluent C (sigma aldrich) as a reaction buffer. To prepare VA-coupled exosomes, vitamin A (retinol, Sigma) was mixed with the exosome suspension. Free vitamin A not attached to the exosomes was separated from the exosome preparations using a micro-partition system (VIVASPIN 2 concentrator 30,000 MWCO PES, VIVASCIENCE). Finally, through Amicon® (Sigma aldrich, Ultra-4 Centrifugal Filter Unit), centrifuged at 2600 g at 4°C for 20 min to obtain exosomes surface-modified with Vitamin A.

Then, using the surface-modified exosomes and non-modified exosomes with the Vitamin A was evaluated for intracellular accumulation (cellular uptake). Specifically, exosomes derived from adipose stem cells or exosomes surface-modified with vitamin A, which are fluorescently labeled (Flamma 675, red) in hepatic stellate cells activated with TGF-β1, were treated at a concentration of 1×10 ⁷ particles/dish for 12 hours. treated, and fluorescence was quantified.

As a result of the experiment, in the case of adipose stem cell-derived exosomes, a fluorescence intensity of about 2000 a.u. was obtained in activated hepatocytes, and in the case of exosomes surface-modified with vitamin A, a fluorescence intensity of about 2200 a.u. in activated hepatocytes was obtained. It was confirmed that exosomes were accumulated in hepatic stellate cells.

3. Surface modification of exosomes using vitamin A ligand (FIGS. 2 and 3)

As a novel method different from those previously known, the exosome surface was modified with a vitamin A ligand.

First, as shown in Fig. 2(a), a vitamin A ligand for a hepatic stellate cell target was synthesized. Specifically, after completely dissolving 100.0 mg of retinoic acid (RA) in 1 ml DMSO, 113.7 mg of EDC·HCl and 76.7 mg of NHS were added and reacted at 40° C. for 48 hours to activate the carboxyl group of RA. Then, 836.2 mg of DSPE-PEG2000-amine was added and reacted at 40° C. for 36 hours (see FIG. 2(a)). After completion of the reaction, it was put in a dialysis tube (MWCO = 1 kDa) and dialyzed in methanol: ultrapure water = 1:1 solution for two days. Thereafter, dialysis was further performed in ultrapure water for two days. 1H-NMR results of the used or prepared Retinol, DSPE-PEG2000-amine, and DSPE-PEG2000-Vit A are shown in FIG. 2(b).

The surface of human adipose stem cell exosomes was modified by a hydrophobic insertion method using the modified moiety (DSPE-PEG-Vitamin A) synthesized above. Specifically, first, the concentration of exosomes was measured using Nano Tracking Ananysis (NTA) and diluted to a concentration of 1.5x10 ⁹ cells/ml in Diluent C (sigma aldrich) as a reaction buffer. Similarly, the modified moiety, DSPE-PEG-Vitamin A stock (anhydrous ethanol, 1 mM), was diluted in 1 ml of Diluent C to a final concentration of 400 nM. The diluted exosomes and the modified moiety were mixed and stirred at 4°C for 15 minutes. Thereafter, unreacted modified moiety and Diluent C were removed through PD-10. Finally, through Amicon® (Sigma aldrich, Ultra-4 Centrifugal Filter Unit), centrifuged at 2600 g at 4°C for 20 min to obtain vitamin A-modified exosomes.

Figure 3 shows the change in the size and shape of the exosome according to the modification of the surface of the adipose stem cell-derived exosome with a vitamin A ligand in the novel method in 3 above. As a result of confirming the size of the exosomes before and after modification with nanoparticle tracking analysis (NTA) equipment, it was confirmed that the average size of the exosomes increased by about 10 nm by the surface modification (FIG. 3a). In addition, as a result of observation by TEM, it was confirmed that there was no change in the shape of the exosomes due to surface modification (FIG. 3b).

4. Evaluation of biomarkers of exosomes (Fig. 4)

In order to evaluate the biomarkers of the adipose stem cell-derived exosomes and the exosomes surface-modified with vitamin A in step 3, CD63-capture magnetic beads (6000 pieces) and 100ul of adipose stem cell-derived exosomes or vitamin A ligands were used to evaluate the biomarkers. The modified exosomes (5×10 ⁹ pieces/ml) were mixed and reacted at room temperature for 12 hours. Then, anti-CD9 antibody (1ug) or anti-CD63 (1ug) antibody was added and reacted at 4°C for 1 hour. Then, it was labeled with a secondary antibody of Alexa Fluor 488 and measured by confocal microscopy and flow cytometry. The results of evaluation of the markers of the exosomes using a confocal microscope are shown in FIG. 4(a), and the results of evaluation of the markers of the exosomes using flow cytometry are shown in FIG. 4(b).

5. Evaluation of hepatic stellate cell targeting ability of adipose stem cell-derived exosomes and exosomes surface-modified with vitamin A ligand (FIG. 5)

Human umbilical vein endothelial cells (HUVEC), hepatic stellate cells (LX-2), adipose stem cell-derived exosomes fluorescently labeled with activated hepatic stellate cells (Flamma 675, red) or vitamin A ligand according to the novel method in 3 above. The surface-modified exosomes were treated with a concentration of 1×10 ⁷ particles/dish for 12 hours. The cells used in this experiment were used after a 60-hour stabilization process, and the activated hepatic stellate cells were activated for 48 hours with TGF-β1 (10ng/ml) for the last 48 hours. The results of quantification of fluorescence are shown in FIG. 5 .

As a result of the experiment, it was confirmed that the exosomes surface-modified with vitamin A ligand were effectively accumulated in the activated hepatic stellate cells compared to the adipose stem cell-derived exosomes. Specifically, in the case of activated hepatic stellate cells, a fluorescence intensity of about 2000 a.u. was obtained for adipose stem cell-derived exosomes, and in the case of exosomes modified with vitamin A ligand in 3 above, about 3500 a.u. The fluorescence intensity was obtained, and it was confirmed that the exosomes surface-modified with vitamin A ligand were effectively accumulated in the activated hepatic stellate cells compared to the adipose stem cell-derived exosomes (FIG. 5). Therefore, in all subsequent experiments, exosomes surface-modified with vitamin A ligand were used according to the new method.

6. Evaluation of fibrosis factor expression inhibition and fluorescence quantification of adipose stem cell-derived exosomes and exosomes surface-modified with vitamin A ligand (Fig. 6)

α-smooth muscle actin (α-SMA) is utilized as a representative liver fibrosis indicator and is expressed by hepatic stellate cells. To the hepatic stellate cells or activated hepatic stellate cells prepared as described above, adipose stem cell-derived exosomes or exosomes surface-modified with vitamin A ligand at a concentration of 1×10 ⁷ particles/dish or 1×10 ⁸ particles/dish 24 hours treatment. Thereafter, α-SMA was fluorescently labeled (Alexa 594) and measured with a confocal microscope, and the results are shown in FIG. 6 . As a result of the experiment, it was confirmed that the expression of α-SMA (red) was effectively suppressed in the group treated with the exosomes surface-modified with a vitamin A ligand compared to the adipose stem cell-derived exosomes ( FIG. 6 ).

7. Evaluation of biodistribution behavior of adipose stem cell-derived exosomes and exosomes surface-modified with vitamin A ligand (Fig. 7)

The biodistribution behavior of two types of exosomes labeled with fluorescence (Flamma 675) was evaluated in real time using small animal optical imaging equipment. Exosomes were administered intravenously through the tail in an amount of 1×10 ⁹ particles/200ul/head to the liver fibrosis animal model to evaluate the residence time in the body. In the animal model used in this experiment, liver fibrosis was induced by intraperitoneal injection of thioacetamide, a liver toxin, at a concentration of 250 mg/kg 3 times a week for 12 weeks. After measuring real-time fluorescence change in liver tissue and setting the liver of an animal model as a region of interest (ROI), the fluorescence intensity of the corresponding region was quantified, and the results are shown in FIG. 7 . As a result of the experiment, it was confirmed that the biodistribution behavior was not significantly changed by the vitamin A ligand surface modification of the exosomes ( FIG. 7 ).

8. Evaluation of hepatic stellate cell targeting ability of exosomes surface-modified with vitamin A ligand (FIG. 8)

Fluorescently stained exosomes were administered to an animal model inducing liver fibrosis, and immunohistochemistry was performed on liver tissue sections to evaluate their targeting ability to hepatic stellate cells. Specifically, the exosomes before modification were mixed with Cy5.5-NHS in a weight ratio of 200:1, stirred at 4° C. for 4 hours, and after staining, the modification step was performed as above. Unmodified exosomes were also fluorescently stained in the same way. To evaluate whole body biodistribution, the amount of fluorescence was measured using IVIS equipment for each time after treatment. After the last measurement, the main organs were removed by euthanasia, fixed with 4% neutral buffered formalin solution, and embedded in paraffin to prepare tissue sections with a thickness of 5 μm. After deparaffination and protein retrieval, the paraffin sections were treated with an antibody targeting α-Smooth Muscle Actin (α-SMA), a hepatic stellate cell marker, and stained with fluorescence. The stained sections were observed with a confocal fluorescence microscope to compare and evaluate the targeting ability of the modified exosomes.

In addition, exosomes were administered to the animal model and sacrificed 24 hours later to extract liver tissue. Liver tissue was fixed in 4% paraformaldehye solution and then made of paraffin blocks and slides with a thickness of 5 μm. Then, the liver tissue was labeled with α-SMA (Alexa 594, green) through immunohistochemistry, and the fluorescence of exosomes (Flamma 675, red) accumulated in the liver tissue was measured with a confocal microscope, and the results were analyzed. 8 shows. As a result of evaluating the co-localization of exosomes and α-SMA, adipose stem cell-derived exosomes show indiscriminate accumulation in liver tissue, whereas exosomes surface-modified with vitamin A ligand selectively accumulate in hepatic stellate cells. was confirmed (FIG. 8).

9. Evaluation of the therapeutic efficacy of exosome-based liver fibrosis surface-modified with vitamin A ligand using an animal model of disease (FIG. 9)

As shown in FIG. 9, an animal experiment plan was established for evaluating the therapeutic efficacy of the exosomes surface-modified with vitamin A ligand. Adipose stem cell-derived exosomes or exosomes surface-modified with vitamin A ligands were intravenously administered 1, 2, 3 times at two-day intervals from the day after completing the production of the established animal model of liver fibrosis. After 24 hours of exosome administration, blood was collected and sacrificed, and blood and histological analysis were performed.

The production of the animal model was performed as follows. To create a liver fibrosis model, TAA was used among drugs that cause liver fibrosis (carbon tetrachloride (CCL ₄ ), Thioacetamine (TAA), dimethylnitrosamine (DMN)). Specifically, a disease model was created by intraperitoneal administration of TAA to C57BL/6 mice for 12 weeks, and the therapeutic effect of liver fibrosis was confirmed by intravenous injection of a composition containing exosomes surface-modified with vitamin A ligand. To check the therapeutic effect of exosomes surface-modified with vitamin A ligand, blood was collected two days after treatment with exosomes, centrifuged at 4° C. at 12000 rpm for 15 minutes, and the supernatant, serum, was collected for liver function in Chemon Co., Ltd. inspection was ordered. Using an automatic blood analyzer (ADVIA2120, SIEMENS, USA), biochemical indicators such as total protein, alkaline phosphatase (ALP), and glutamic pyruvate transminase (GPT) were measured to determine the liver function improvement effect. In addition, the liver was removed for visual and histopathological examination, fixed in 10% neutral buffered formalin solution, embedded in paraffin, and 4 μm thick tissue sections were prepared and stained with Hematoxilin-Eosin for comparison of histological findings. , Masson's trichrome staining was performed and observed with an optical microscope. The therapeutic effect of liver fibrosis was confirmed by observing whether the liver cells were regenerated and the fibrous septum was formed through the macroscopic observation and the Trichrome staining result indicating the degree of fibrosis.

10. Aspartate aminotransferase (AST) hematological analysis in an animal model of liver fibrosis (Fig. 10)

Among the animal models prepared above, the blood of the exosome-administered group was coagulated at room temperature, and then serum was obtained and analyzed by centrifugation, and the results are shown in FIG. 10 . AST is an enzyme secreted when liver damage occurs and decreases as liver function returns to normal. As a result of the experiment, it was confirmed that the group administered with the exosomes surface-modified with a vitamin A ligand showed improved liver function values within a short period of time compared to the adipose stem cell-derived exosomes (FIG. 10).

11. Histopathological analysis of liver tissue (Fig. 11)

The exosomes were administered to the animal model prepared in 9 above, and the liver tissue was removed by sacrificing 24 hours later. Liver tissue was fixed in 4% paraformaldehye solution and then made of paraffin blocks and slides with a thickness of 5 μm. The extracellular matrix of liver tissue was stained by Sirius red staining. It was evaluated based on the distribution of extracellular matrix around blood vessels, septa formation, and bridging between blood vessels, and the results are shown in FIG. 11 . As a result of the experiment, a decrease in septa and bridging was observed in exosomes surface-modified with vitamin A ligand compared to exosomes derived from adipose stem cells (FIG. 11a).

In addition, in order to evaluate the distribution of the fibrous area in the liver tissue, the ratio of the stained part (red) in the liver tissue of the exosome administration group 3 times among the animal models prepared in step 9 was quantified using the Image J program. As a result of the experiment, a decrease in the extracellular matrix was clearly observed in the group administered with the exosomes surface-modified with vitamin A ligand compared to the adipose stem cell-derived exosomes (FIG. 11b).

12. Evaluation of Collagen I (colⅠ) Distribution in Liver Tissue (FIG. 12)

Among the animal models prepared in 9 above, the distribution of collagen I in the liver tissue of the exosome-administered group three times was evaluated by immunohistochemistry (Alexa 594, red). As a result of the experiment, a decrease in col I was clearly observed in the group administered with the exosomes surface-modified with vitamin A ligand compared to the adipose stem cell-derived exosomes (FIG. 12),

13. Evaluation of hydroxyproline assay in liver tissue (FIG. 13)

Among the animal models prepared in 9 above, hydroxyproline contained in the liver tissue of the exosome administration group 3 times was quantified. By quantifying hydroxyproline, an essential amino acid constituting collagen, it is possible to show the collagen content in liver tissue. As a result of the experiment, it was confirmed that a decrease in hydroxyproline was observed in the group administered with the exosomes surface-modified with vitamin A ligand compared to the adipose stem cell-derived exosomes (FIG. 13).

Claims (13)

A pharmaceutical composition for preventing or treating liver fibrosis, comprising, as an active ingredient, an adipose stem cell-derived exosome surface-modified with vitamin A. The pharmaceutical composition according to claim 1, wherein the exosome is surface-modified with a vitamin A ligand comprising a covalently linked vitamin A moiety and a phospholipid moiety. The pharmaceutical composition according to claim 1, wherein the exosomes surface-modified with vitamin A selectively target hepatic stellate cells. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits the expression of α-SMA (α-smooth muscle actin) in hepatic stellate cells. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits the deposition of extracellular matrix in liver tissue. The pharmaceutical composition according to claim 1, wherein the adipose stem cells are human adipose stem cells. The pharmaceutical composition according to claim 1, wherein the adipose stem cells are derived from autologous adipose tissue of a patient to which the pharmaceutical composition is administered. The pharmaceutical composition according to claim 1, wherein the composition is for intravenous administration. The pharmaceutical composition according to claim 2, wherein the vitamin A ligand further comprises a covalently linked polyethylene glycol (PEG) moiety. The pharmaceutical composition according to claim 9, wherein the vitamin A ligand has a phospholipid-PEG-vitamin A structure. The pharmaceutical composition according to claim 1, wherein the vitamin A is at least one selected from the group consisting of retinol, retinal, provitamin A carotenoids and salts thereof. (a) activating the carboxyl group of vitamin A;
(b) synthesizing a vitamin A ligand by coupling the phospholipid compound with the carboxyl group of the activated vitamin A; and
(c) modifying the surface of the adipose stem cell-derived exosome with the synthesized vitamin A ligand
A method for preparing a pharmaceutical composition according to claim 1, comprising a. 13. The method according to claim 12, wherein step (a) is carried out by converting the carboxyl group of vitamin A into an amine-reactive ester.

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