A pharmaceutical composition for resisting liver fibrosis and a preparation method thereof

Abstract

The present invention discloses an anti-liver fibrosis drug, which includes exosomes and forsythiaside; the exosomes are exosomes modified with hyaluronic acid. The present invention uses hyaluronic acid to modify milk exosomes mEXO, so that the PHI loaded with the modified mEXO has liver targeting, and the efficacy of the prepared targeted nanomedicine in treating liver fibrosis is significantly improved after oral administration. Experiments have proved that the targeted oral preparation PHI-HA-mEXO prepared by the present invention not only has good stability and high safety, but also has efficient targeting ability and superior liver fibrosis effect, and has the value of practical promotion and application.

Description

Technical Field

[0001] The present invention specifically relates to a pharmaceutical composition for resisting liver fibrosis and a preparation method thereof. Background Art

[0002] Liver fibrosis is a serious problem that affects the health of millions of people worldwide. It is an ineffective reversible wound healing response to repeated liver damage caused by various acute or chronic stimuli. After liver damage caused by various reasons, hepatic stellate cells are activated by proinflammatory cytokines and reactive oxygen species secreted by apoptotic hepatocytes and Kupffer cells, and transform into myofibroblast-like cells. Activated hepatic stellate cells (aHSC) produce a large amount of extracellular matrix mainly composed of collagen. If the development of liver fibrosis is not intervened at this time, liver fibrosis will develop into more serious cirrhosis or even liver cancer.

[0003] There is currently no standard treatment for liver fibrosis. At this stage, liver fibrosis is mainly alleviated by treatments that eliminate the cause, such as anti-inflammatory, antioxidant, antiviral and immunomodulatory treatments. However, the above therapies have various side effects and cannot be used for a long time. Therefore, it is of great significance to find safe and effective anti-liver fibrosis drugs. It is reported that traditional medicine has unique advantages in anti-liver fibrosis. For example, Chinese medicine can play a liver-protecting role through various pathways such as anti-inflammatory and antioxidant. Among them, forsythiaside (PHI) has significant anti-liver fibrosis ability. It can play an anti-inflammatory role by reducing the expression of NF-κB and thus reverse liver fibrosis. In addition, it can also play an anti-liver fibrosis role by regulating intestinal microbiota and bile acid metabolism. Although PHI exhibits significant anti-liver fibrosis properties, its poor water solubility and incomplete oral absorption limit its oral bioavailability, hindering the clinical development of PHI as a potential liver fibrosis therapeutic agent.

[0004] There is a need to develop PHI into a drug with liver-targeting capabilities in order to maximize drug efficacy after oral administration. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an anti-liver fibrosis drug, which comprises exosomes and forsythiaside; the exosomes are exosomes modified with hyaluronic acid.

[0006] Furthermore, the exosomes are milk exosomes modified with hyaluronic acid.

[0007] Furthermore, the milk exosomes are mixed with hyaluronic acid and incubated to obtain milk exosomes modified with hyaluronic acid; the mass ratio of the milk exosomes to hyaluronic acid is 20 to 40:1, preferably 20:1.

[0008] Furthermore, the milk exosomes are a suspension obtained by washing and resuspending the precipitate obtained by differential ultracentrifugation of milk with a PBS solution; the protein content in the suspension is 4 to 8 mg / ml.

[0009] Furthermore, the method of differential ultracentrifugation is:

[0010] The milk was first centrifuged at 10,000×g for 30 min, the solution was centrifuged at 100,000×g for 90 min, the supernatant was collected and centrifuged at 135,000×g for 70 min, and the precipitate was collected to obtain the product.

[0011] Furthermore, when preparing the drug, the preparation ratio of exosomes to forsythiaside is 4 to 8:1.

[0012] Furthermore, when preparing the drug, the preparation ratio of exosomes to forsythiaside is 6:1.

[0013] The present invention also provides a method for preparing the aforementioned pharmaceutical composition, characterized in that it comprises the following steps:

[0014] 1) Weighing raw materials according to the ratio; mixing exosomes and hyaluronic acid, incubating, centrifuging, and taking the supernatant to obtain hyaluronic acid-modified milk exosomes;

[0015] 2) The hyaluronic acid-modified milk exosomes obtained in step 1) are mixed with forsythiaside, incubated, and ultrafiltered to obtain.

[0016] Furthermore, the incubation temperature in step 1) is 37° C. for 48 h; the centrifugation speed is 3500 g for 25 min;

[0017] Step 2) The incubation is carried out at room temperature in the dark for 3 hours; the ultrafiltration tube is 10KDa, the speed is 3500g, and the time is 10min

[0018] The present invention also provides a use of the above-mentioned medicine in preparing a medicine for treating liver fibrosis.

[0019] The present invention uses hyaluronic acid to modify milk exosome mEXO, so that the PHI loaded by the modified mEXO has liver targeting, and the efficacy of the prepared targeted nanomedicine in treating liver fibrosis is significantly improved after oral administration. Experiments have proved that the targeted oral preparation PHI-HA-mEXO prepared by the present invention not only has good stability and high safety, but also has efficient targeting ability and excellent liver fibrosis effect, and has the value of practical promotion and application.

[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0021] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Physicochemical properties of mEXO and PHI-HA-mEXO (A. TEM images of mEXO and PHI-HA-mEXO; B. Particle size measurement by NTA and surface charge measurement by zeta potential of mEXO and PHI-HA-mEXO after dilution with PBS; C. Identification of mEXO and PHI-HA-mEXO marker proteins CD9, CD81, and Calnexin)

[0023] Figure 2 Stability and dissolution of PHI-HA-mEXO (A. HPLC images of PHI and PHI-HA-mEXO; B. NTA, zeta potential and electron microscopy images of PHI-HA-mEXO after 1 month of low-temperature storage; C. Release curve of PHI-HA-mEXO in PBS at pH 7.4)

[0024] Figure 3 PHI-HA-mEXO uptake and CD44 expression assay (A. Uptake of DIR-labeled PHI-HAmEOX and PHI-mEXO in qHSCs and aHSCs; B. Uptake of DIR-labeled PHI-HAmEOX and PHI-mEXO in normal and TAA-induced liver fibrosis in zebrafish larvae; C. Representative confocal images of CD44 signals in normal and TGF-β1-activated LX2 cells; D. Representative images of CD44 signals in normal and TGF-β1-activated LX2 cells detected by flow cytometry)

[0025] Figure 4In vitro anti-fibrotic effect of PHI-HA-mEXO (A. Cell viability of LX2 cells after treatment with TGF-β1 and PHI-HA-mEXO (3.75, 7.5, 15, 30, 60 and 120 μg / mL); B. PHI-HA-mEXO significantly downregulated α-SMA and Col1α1 mRNA levels; C. Flow cytometric analysis of LX2 cell apoptosis rate; D. TGF-β1 and PHI-HA-mEX O. Cell viability of LX2 cells after treatment with mEXO, HA-mEXO, PHI, and PHI-mEXO; E. Cell viability of LX2 cells and LO2 cells after treatment with PHI-HA-mEXO (3.75, 7.5, 15, 30, 60, and 120 μg / mL). Each bar represents the mean ± SD (n = 3). #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control group, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. model group)

[0026] Figure 5 Anti-liver fibrosis effect of PHI-HA-mEXO in zebrafish model (A. Fluorescence images of zebrafish larvae exposed to TAA, TAA+PHI-HA-mEXO (25, 50, 100 μM), TAA+HAmEXO, TAA+PHI-mEXO, TAA+PHI and TAA+mEXO; B. Effects of PHI-HA-mEXO on ALT, AST and ALP levels in zebrafish larvae; C. HE staining of zebrafish liver; D. Sirius red staining of zebrafish liver; E. Quantitative results of Col1α1 and α-SMA expression in zebrafish. Each bar represents the mean ± SD (n = 6). #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. control group, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group)

[0027] Figure 6 Effects of PHI-HA-mEXO on autophagy-related genes in TAA-induced zebrafish liver fibrosis (A. Gene expression levels of Beclin1 and LC3; B. Gene expression level of P62; C. Gene expression levels of Atg5 and Atg7. Each bar represents the mean ± SD (n = 3). #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. control group, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group) DETAILED DESCRIPTION

[0028] Example 1. Preparation of milk exosomes of the present invention

[0029] Fresh milk was placed in several 50 mL centrifuge tubes, centrifuged at 10,000 × g for 30 min in a normal centrifuge, and skim milk was collected; skim milk was transferred to several 38.4 mL open ultracentrifuge tubes and balanced, and centrifuged at 100,000 × g for 90 min in an ultracentrifuge, the upper whey was collected and filtered through a 0.22 μm filter membrane to reduce possible mixed proteins; the obtained whey was centrifuged at 135,000 × g for 70 min in an ultracentrifuge, the exosome precipitate was taken, the exosome precipitate was washed three times with pre-cooled PBS solution, and the exosome precipitate was resuspended into a solution with PBS solution, and filtered through a 0.22 μm microporous filter membrane to obtain an exosome solution with a protein concentration of 6 mg / mL.

[0030] Example 2 Preparation of the Targeted Drug Preparation of the Present Invention

[0031] 1) Mix 20 mg:1 mg of exosomes (mEXO) and hyaluronic acid (DSPE-PEG2000-HA), incubate at 37°C for 48 h, centrifuge for 25 min (3500 g), collect the supernatant, and obtain hyaluronic acid-modified milk exosomes HA-mEXO;

[0032] 2) HA-mEXO obtained in step 1) was mixed with forsythiaside at a ratio of 6 mg:1 mg, incubated at room temperature in the dark for 3 h, and ultrafiltered at 3500 g for 10 min using a 10 KDa ultrafiltration tube to remove unbound components to obtain PHI-HA-mEXO.

[0033] The beneficial effects of the present invention are described below through test examples.

[0034] Experimental Example 1 Preparation of forsythiaside-targeted composite exosomes and its anti-liver fibrosis study

[0035] 1. Materials and Methods

[0036] 1. Materials

[0037] Milk was purchased from a local supermarket. Forsythiaside (Cat. No. 21080708) was obtained from Chengdu MUST Biotechnology Co., Ltd. (Chengdu, China). DSPE-PEG2000-HA (Cat. No. R-A66001-2k) was synthesized by Ruixi Biotechnology Co., Ltd. (Shaanxi). Transforming growth factor-β1 (Cat. No. CA59) was obtained from Jinan Protein Technology Co., Ltd. (Shanghai). LX-2 cells were donated by Central South University (Changsha, China). Human normal hepatocytes LO2 (iCell-h054) were purchased from iCell Biotechnology Co., Ltd. (Shanghai, China). Tricaine was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). BCA protein detection kit (Cat. No. G2026-1000T) was purchased from Lianke Biotechnology Co., Ltd. (Hangzhou, China). HA (Cat. No. CPA182Ge21; Dilution 1:1000), CD63 (Cat. No. YT5525; Dilution 1:1000), CD81 (Cat. No. YT5394; Dilution 1:1000) and Calnexin (Cat. No. YT0613; Dilution 1:1000) antibodies were purchased from Cloud-Clone Corporation (Wuhan) and Immunoway Biotechnologies (USA). Goat anti-rabbit IgG-HRP (Cat. No. 05-4030-05; Dilution 1:5000) was purchased from Lianke Biotechnology Co., Ltd. (Hangzhou, China). HPLC grade methanol (Cat. No. 216565) was purchased from Merck Chemicals (Shanghai, China). RPMI Medium 1640 basic (1x) (Cat. No. 8122651) and DMEM basic (1x) (Cat. No. 8122340) were obtained from Gibco (Australia). CD44 (Cat. No. AF6186) was from Affinity biosciences (Jiangsu). Annexin-V-FITC apoptosis detection kit (Cat. No. E-CK-A211) was from Eliorite Biotechnology Co., Ltd. (Wuhan). DIR (Cat. No. D4006) was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd. (Suzhou, China). 4% paraformaldehyde (Cat. No. G1101) was purchased from Sewell Technology Co., Ltd. (Wuhan, China). Total RNA isolation kit (Cat. No. RE-03014) was obtained from Chengdu Fuji Biotechnology Co., Ltd.ABScript III RT Master Mix for qPCR (Cat. No. 20428) and Genious 2X SYBRGreen Fast qPCR Mix (No ROX) (Cat. No. 21205) were obtained from Abotek Biotechnology Co., Ltd. (Wuhan, China). PCR primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Chengdu Branch).

[0038] 2. Methods

[0039] 2.1 Extraction and separation of exosomes

[0040] The method for extracting exosomes in this experiment was differential ultracentrifugation, and the process temperature of all centrifugation was set at 4°C. In short, fresh milk was placed in several 50mL centrifuge tubes, centrifuged at 10,000×g for 30min in a normal centrifuge to remove fat globules, casein aggregates, cell debris and other substances, and skim milk was collected. The skim milk was transferred to several 38.4mL open ultracentrifuge tubes and balanced, centrifuged at 100,000×g for 90min in an ultracentrifuge, the protein precipitate at the bottom was discarded, the upper whey was collected and filtered through a 0.22μm filter membrane to reduce possible inclusions of foreign proteins; the obtained whey was centrifuged at 135,000×g for 70min in an ultracentrifuge, and the supernatant was discarded to harvest the exosome precipitate. The exosome precipitate was washed three times with pre-cooled PBS solution, and the exosome precipitate was resuspended into a solution with PBS solution, and the exosome solution was obtained by filtering through a 0.22 μm microporous filter membrane. The protein concentration of milk exosomes was determined by the BCA protein kit and standardized to 6 mg / mL. The exosome resuspension was stored in a -80 °C refrigerator for future use.

[0041] 2.2 PHI drug loading and drug loading determination

[0042] Exosomes (mEXO) and hyaluronic acid (DSPE-PEG2000-HA) (20 mg:1 mg) were mixed, incubated at 37°C for 48 h, and centrifuged for 25 min (3500 g) to obtain HA-mEXO. Forsythiaside was mixed with HA-mEXO in a ratio of 1:2, 1:4, 1:6, and 1:8, respectively, incubated at room temperature in the dark for 3 h, and ultrafiltered at 3500 g for 10 min using a 10KDa ultrafiltration tube to remove unbound drugs to obtain PHI-HA-mEXO.

[0043] In order to evaluate the loading capacity of mEXO for PHI, the drug loading rate of PHI was determined by high performance liquid chromatography. Preparation of reference solution: Accurately weigh a certain amount of PHI reference into a 5mL volumetric flask, add methanol to make up to volume, and obtain the mother solution. Preparation of test solution: Take 10μL of PHI-HA-mEXO suspension and add 790μL of methanol to break the emulsion. Chromatographic column: Agilent ZORBAX Eclipse C18 (4.6×250mm, 5um); detection wavelength: 280nm; mobile phase: methanol-water; column temperature: 25℃; elution according to the gradient elution order in Table 1, flow rate: 1mL / min; injection volume: 10μL. The obtained sample was detected by high performance liquid chromatography instrument for peak area, and the concentration and encapsulation efficiency were calculated according to the PHI standard curve. The encapsulation efficiency calculation formula is as follows. Encapsulation efficiency = [(concentration of forsythiaside in purified sample * sample volume) / total mass of forsythiaside] × 100%.

[0044] 2.3 Characterization of mEXO and PHI-HA-mEXO

[0045] Take 10μL of exosomes or PHI-HA-mEXO extracted from milk, drop it on a 200-mesh carbon film copper grid and precipitate it for 1 minute, and remove the floating liquid with filter paper. Take 10μL of the sample and drop it on the copper grid to precipitate for 1 minute, carefully absorb the remaining liquid with filter paper, add 10μL of 1% uranyl acetate for negative staining for 5 minutes, remove the excess negative staining liquid with filter paper, let it stand at room temperature for 30 minutes to dry, and perform electron microscopy imaging at 100KV to observe the morphology of mEXO and PHI-HA-mEXO. Dilute the extracted exosomes or PHI-HA-mEXO 1000 times with ultrapure water, mix thoroughly, and detect it on the machine (nanoparticle tracking analyzer), and record the exosome particle size distribution range, exosome membrane zeta potential and exosome concentration (number of vesicles / mL). Mix mEXO or PHI-HA-mEXO with protein loading buffer at a ratio of 4:1 and treat at 100℃ for 10min; take 10μL of the treated sample for electrophoresis, transfer, washing and blocking, dilute CD9, CD81 and Calnexin primary antibodies according to the instructions and incubate the membrane in the primary antibody solution overnight. After incubation, wash the PVDF membrane with TBST solution 3 times, 10min each time. After washing, incubate with secondary antibody for 2h. After incubation, wash the PVDF membrane 3 times with TBST solution, 10min each time. Image the PVDF membrane according to the instructions in the ultrasensitive ECL chemiluminescence kit.

[0046] 2.4 Storage stability study

[0047] Since the constructed drug delivery system is mainly based on mEXO, the biofilm material is not suitable for long-term storage at 4°C. In order to explore the storage conditions of the drug delivery system to meet subsequent experiments, the PHI-HA-mEXO system was stored in a -80°C refrigerator in a solution state. After thawing for 1 month, the exosome particle size and potential changes were detected by a nanoparticle tracking analyzer to evaluate the stability of PHI-HA-mEXO.

[0048] 2.5 In vitro release

[0049] 2.5.1 Cellular uptake studies of PHI-HA-mEXO labeled with DIR dye

[0050] qHSC and aHSC cells were cultured at 5×10 4 The cells were seeded at a density of 100 μg / ml in a confocal culture dish and incubated overnight. After overnight incubation, PHI-mEXO and PHI-HA-mEXO labeled with DIR dye were added to each well at a concentration of 30 μg / ml. After incubation for 4 hours, the cells were washed with PBS and fixed with 4% paraformaldehyde solution. The cells were then observed using a laser confocal microscope.

[0051] 2.5.2 In vivo biodistribution study of PHI-HA-mEXO labeled with DIR dye

[0052] PHI-HA-mEXO labeled with DIR dye was used to treat liver fibrosis zebrafish larvae and normal zebrafish larvae, respectively. Then, the zebrafish larvae were anesthetized and placed on their side on a confocal dish, and fluorescence was observed using a confocal microscope at excitation and emission wavelengths of 750 and 779 nm.

[0053] 2.6 Study on CD44 expression

[0054] LX2 cells were cultured at 1×10 6 / well inoculated on a six-well plate, divided into a blank control group, a model group, a PHI-mEXO and a PHI-HA-mEXO group. The control group was LX2 cells cultured without drugs for 24 hours, the model group was treated with 15nM TGF-β in LX2 cells for 24 hours, and the PHI-mEXO and PHI-HA-mEXO groups were treated with PHI at a concentration of 30μg / ml in LX2 cells added with 15nM TGF-β, respectively, for 24 hours. After drug treatment, the cells were washed twice with PBS, CD44 antibody was added, incubated in the dark for 30 minutes, and analyzed by flow cytometry to calculate the level of CD44 expression in each group.

[0055] LX2 cells were seeded on a confocal dish for 24 hours, fixed with 4% paraformaldehyde at room temperature after intervention, blocked with normal non-immune serum (sheep) at room temperature, incubated with primary antibody CD44-PE (1:375) at 4°C overnight, incubated with goat anti-rabbit red fluorescent secondary antibody (1:2000) at room temperature for 30 minutes, stained with DAPI reagent and sealed, and observed and photographed using a confocal microscope.

[0056] 2.7 Cell culture, cell viability and cytotoxicity

[0057] LO2 cells and LX-2 cells were cultured in 1640 medium supplemented with 10% FBS. LO2 cells and LX-2 cells were plated at 5×10 3 The cells were inoculated into 96-well plates at a density of 10 cells and incubated overnight. Then, PHI-HA-mEXO was treated with different drug concentrations (3.75, 7.5, 15, 30, 60, and 120 μg / mL) for 24 hours. After that, the drug-containing medium was discarded and 20 μL MTT solution was added and incubated for 4 hours. Finally, the absorbance at 490 nm was measured with a microplate reader.

[0058] 2.8 Animal testing

[0059] 2.8.1 Experimental animals

[0060] TAA solution was used to induce liver fibrosis in 2dpf zebrafish larvae for 3 days. The first group was the control group in which zebrafish larvae were not given TAA and PHI-HA-mEXO. Except for the first group, all other groups of zebrafish larvae were treated with TAA. The second group was the TAA control group in which zebrafish larvae were not treated with PHI-HA-mEXO. The third, fourth, and fifth groups were treated with PHI-HA-mEXO (25, 50, and 100 μM), respectively. At the end of the experiment, all samples were fixed in 4% paraformaldehyde for paraffin embedding for histopathological examination. All procedures were in compliance with the Animal Experiment Ethics Committee of Chengdu University of Traditional Chinese Medicine.

[0061] 2.8.2 Biochemical analysis and histopathological examination of liver tissue

[0062] Liver tissue samples were fixed with 4% paraformaldehyde for 24 h, dehydrated with ethanol, transparentized, then waxed, embedded, and sliced ​​at 5 μm. The paraffin sections were dewaxed, rehydrated, stained with hematoxylin, differentiated, and stained with eosin, and then routinely stained with hematoxylin-eosin (HE) and Sirius red for histopathological examination under a microscope.

[0063] First, 30 zebrafish larvae were accurately selected and homogenized in an ice-water bath with 9 volumes of homogenate. Then, the homogenate was centrifuged at 2500r / min for 10 minutes. The supernatant was collected and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured using ALT, AST, and ALP commercial kits according to the manufacturer's instructions.

[0064] 2.8.3 Real-time quantitative PCR (RT-qPCR) analysis

[0065] Total RNA was extracted from approximately 30 zebrafish larvae using a total RNA isolation kit in 50 μL RNase-free water according to the manufacturer's instructions. The purity of the RNA was characterized by the OD260 / 280 value measured by a nucleic acid / protein analyzer. ⅢAll-in-one RT SuperMix Perfect for qPCR was used for reverse transcription to synthesize cDNA. The reaction conditions were as follows: 25°C for 5 min, 42°C for 15 min, and 85°C for 5 s. RT-qPCR was performed on a StepOnePlus Real-Time PCRSystem by adding 2× Taq Pro Universal SYBR qPCR MasterMix according to the manufacturer's protocol. The reaction conditions were set as follows: 95°C for 1 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. 2 -△△Ct The method was used to calculate the relative expression of the target gene.

[0066] 2.8.4 Statistical analysis

[0067] SPSS 26.0 software was used for statistical analysis. The experimental results were expressed as mean ± standard deviation, and one-way ANOVA was used for statistical analysis. P < 0.05 was considered statistically significant.

[0068] 3. Results

[0069] 3.1 Physical and chemical characterization of mEXO and PHI-HA-mEXO

[0070] Transmission electron microscopy results showed that the exosomes extracted by differential centrifugation had good dispersion. mEXO and PHI-HA-mEXO were mostly round or quasi-round saucer-like / cup-shaped structures with obvious membranous structures, clear boundaries, and concentrated distribution, indicating that the size uniformity of the obtained exosomes was good ( Figure 1 A).

[0071] Nanoparticle tracking analysis results showed that the overall particle size of exosomes ranged from 80 to 140 nm, with an average particle size of 114.2 nm ( Figure 1 B), the membrane surface is negatively charged, and the zeta potential is: -36.41±1.63mV( Figure 1 B), the concentration is 6.0E+10 Particles / mL. The overall particle size of PHI-HA-mEXO ranges from 80 to 130 nm, with an average particle size of 120.7 nm ( Figure 1 B), the membrane surface is negatively charged, and the zeta potential is: -36.77±1.21mV( Figure 1 B), the concentration is 2.7E+11 Particles / mL.

[0072] Western blot results showed that the obtained mEXO and PHI-HA-mEXO expressed characteristic membrane proteins CD9 and CD81, but no negative protein Calnexin was expressed ( Figure 1 C).

[0073] 3.2 Drug loading, stability and in vitro release studies of PHI-HA-mEXO

[0074] According to the injection concentration and peak area of ​​the PHI standard, the linear regression equation of the standard curve was calculated as Y = 42.457X-70.798 (R 2 =0.9997), PHI has a good linear relationship in the mass concentration range of 3.125~700mg / L. Figure 2 A shows the chromatograms of forsythiaside standard and PHI-HA-mEXO. The drug loading rate of PHI was calculated by the regression equation of the PHI standard curve. The experimental results show that the drug loading rate of mEXO on PHI is 34.10%, 40.97%, 57.06% and 48.93% when the ratio of forsythiaside to HA-mEXO is 1:2, 1:4, 1:6 and 1:8, respectively. The results show that the drug loading rate is the highest when the ratio of forsythiaside to HA-mEXO is 1:6, which is 57.06%, indicating that mEXO has a good loading efficiency on PHI, which is conducive to the subsequent pharmacological experiments.

[0075] The particle size and potential changes of PHI-HA-mEXO after being stored in a -80°C refrigerator for 1 month were measured by nanoparticle tracking analyzer. Figure 2 B Results show that after 1 month of low-temperature storage, the particle size of the PHI-HA-mEXO system was concentrated at 120.5 nm and the zeta potential was -33.21 ± 0.00 mV. Compared with the freshly prepared samples, the properties of the nanoparticles did not change significantly. These findings indicate that PHI-HA-mEXO can be stored for a long time without causing significant changes in its physicochemical properties.

[0076] The drug release kinetics of PHI-HA-mEXO were determined using a dialysis bag in a PBS buffer solution. The results showed that PHI-HA-mEXO exhibited time-dependent release. After 1, 2, 4, and 8 hours, the cumulative release of PHI was 2%, 46%, 70%, and 83%, respectively. Almost all the drug (92%) was released after 12 hours ( Figure 2 C).

[0077] 3.3. Cellular uptake, evaluation of CD44 expression on target cells, and in vivo biodistribution

[0078] The uptake of PHI-HA-mEXO by qHSC and aHSC was observed using laser confocal microscopy. Figure 3 As shown in A, aHSCs have a higher uptake of PHI-HA-mEXO than qHSCs. aHSCs overexpress CD44, while qHSCs do not upregulate the expression of this protein. In addition, we analyzed the biodistribution of PHI-HA-mEXO labeled with DIR dye in fibrotic and normal livers of zebrafish larvae ( Figure 3 B). Confocal images show that the fluorescence intensity in fibrotic liver is significantly stronger than that in normal liver. The above findings indicate the efficient targeting ability of PHI-HA-mEXO to aHSC and its potential to improve liver fibrosis. To evaluate the targeting specificity of PHI-HA-mEXO, we detected the expression of CD44 on aHSC and qHSC by flow cytometry. The results are shown in Figure 2. Figure 3 As shown in C, high levels of CD44 expression were observed in aHSCs, while low levels of CD44 expression were found in qHSCs. The expression level of CD44 in the PHI-HA-mEXO group was significantly lower than that in the model group due to the combination of CD44 and hyaluronic acid. In addition, we used confocal microscopy to measure the CD44 fluorescence on hepatic stellate cells. The results are shown in Figure 3 As shown in D, CD44 was expressed most in the PHI-mEXO group of the model group, and was downregulated in the PHI-HA-mEXO group of the model group. The reason was that the targeted drug bound to CD44, resulting in a decrease in fluorescence intensity. In the blank group, the targeted and non-targeted groups showed weaker fluorescence intensity because the hepatic stellate cells were not activated, and there was no significant difference between the two. These results indicate that PHI-HA-mEXO can enter aHSC and has a low tendency to interact with qHSC.

[0079] 3.4. Anti-fibrotic effect of PHI-HA-mEXO on aHSC

[0080] We evaluated the anti-fibrotic effect of PHI-HA-mEXO using the TGF-β1-induced LX-2 cell liver fibrosis model and compared it with free PHI. We first tested the cytotoxicity of PHI-HA-mEXO (3.75, 7.5, 15, 30, 60, and 120 μg / mL) on aHSCs. Figure 4 As shown in A, cell viability decreased with increasing concentrations of PHI-HA-mEXO and returned to normal levels at 30 μg / mL. In addition, qRT-PCR results also showed similar results, such as Figure 4 As shown in Figure B, PHI-HA-mEXO can significantly inhibit the expression levels of Col1α1 and α-SMA in LX-2 cells, while mEXO, HA-mEXO, PHI and PHI-mEXO groups cannot significantly inhibit the expression levels of Col1α1 and α-SMA in LX-2 cells. In addition, flow cytometry results also showed that PHI-HA-mEXO can promote LX-2 cell apoptosis ( Figure 4 C) In order to exclude the influence of mEXO and hyaluronic acid on the results, we conducted a parallel control experiment at a dosage concentration of 30 μg / mL. The results are as follows Figure 4 As shown in D, the exosome group, targeted exosome group, free PHI group and PHI-mEXO group showed no difference compared with the model group, while the PHI-HA-mEXO group showed significant cytotoxic effect.

[0081] To evaluate the biosafety of PHI-HA-mEXO, we investigated the cytotoxicity of PHI-HA-mEXO to normal hepatocytes and hepatic stellate cells. After incubation of PHI-HA-mEXO (3.75, 7.5, 15, 30, 60, and 120 μg / mL) with normal hepatocytes and hepatic stellate cells for 24 h, we observed no significant cytotoxicity ( Figure 4 E). This indicates that PHI-HA-mEXO is specific to aHSCs, is safe and biologically inert to other cells, and has good biocompatibility.

[0082] 3.5. Improvement of PHI-HA-mEXO on thioacetamide-induced liver fibrosis

[0083] Two-day-old zebrafish larvae were immersed in thioacetamide (TAA) for 3 days to induce liver fibrosis, and at the same time, each group was treated with PHI-HA-mEXO (25, 50, 100 μM) for 3 days. Figure 5As shown in A, the fluorescent area represents the liver tissue of zebrafish larvae. The larvae in the normal control group showed clear and strong fluorescence signals. However, the larvae treated with TAA presented relatively weak and dark fluorescence signals, and the liver area was significantly reduced, indicating that TAA exposure would definitely lead to morphological changes in the liver of the larvae. In contrast, PHI-HA-mEXO treatment significantly improved the liver changes caused by TAA excitation and dose-dependently enhanced liver fluorescence and area. In addition, zebrafish treated with PHI-HA-mEXO (25, 50, 100 μM) showed significant improvements in the liver, indicating that PHI-HA-mEXO has better anti-liver fibrosis effects than HA-mEXO, PHI-mEXO, PHI and mEXO. Samples were then collected for evaluation of biochemical indices (ALT, AST, ALP), and the results are shown in Figure 3. Figure 5 As shown in B, compared with the blank group, the ALT, AST, and ALP of zebrafish larvae in the model group were significantly increased, reflecting considerable liver damage. With the increase of PHI-HA-mEXO dose (25, 50, 100 μM), ALT, AST, and ALP were significantly reduced to the level of the blank group, showing an excellent liver protection effect.

[0084] To further explore the role of PHI-HA-mEXO in liver fibrosis, H&E and Sirius red staining were performed on the liver of the model zebrafish. Figure 5 As shown in C, the hepatocytes in the blank group showed visible nucleoli, round nuclei, and chromatin dispersed in the periphery, without cell loss and structural changes. Compared with the blank group, the model group showed obvious cell loss and structural changes. PHI-HA-mEXO restored liver damage in a dose-dependent manner. Consistent with the changes in H&E staining, significantly more collagen deposition between hepatocytes in Sirius red staining of liver sections was observed in the model group than in the blank group, while the PHI-HA-mEXO group (25, 50, 100uM) reduced the collagen deposition between hepatocytes in a dose-dependent manner ( Figure 5 D). Col1α1 and α-SMA are important components of the extracellular matrix, and their expression levels can reflect the severity of liver fibrosis. We used PCR to detect the gene expression changes of Col1α1 and α-SMA in liver tissues of different groups ( Figure 5 E). The results showed that the expression levels of Col1α1 and α-SMA in the model group were significantly increased, while PHI-HA-mEXO inhibited this change in a dose-dependent manner. The above data clearly showed that PHI-HA-mEXO treatment significantly inhibited TAA-induced liver fibrosis.

[0085] 3.6. PHI-HA-mEXO inhibits autophagy by downregulating Beclin-1 and LC3

[0086] Autophagy can aggravate liver fibrosis by degrading lipid droplets and cellular proteins to provide energy for aHSC. Beclin-1, LC3, and P62 are important markers of autophagosome formation, which is the most critical process in autophagy. In order to evaluate the level of autophagy in the liver after PHI-HA-mEXO treatment, the gene levels of these indicators were measured using qRT-PCR. The results showed that the expression levels of proteins such as Beclin-1 and LC3 were significantly increased in the model group, while the levels of Beclin-1 and LC3 in the schizotypal exosomes targeted exosomes group (25, 50, 100uM) gradually decreased in a dose-dependent manner ( Figure 6 A). P62, an autophagy-related transporter, was significantly decreased in the model group and increased in a dose-dependent manner after PHI-HA-mEXO treatment ( Figure 6 B) To further confirm our findings, we also analyzed the levels of Atg5 and Atg7, which are involved in the conversion of LC3-I to LC3-II. Figure 6 As shown in C, PHI-HA-mEXO significantly reduced the mRNA levels of Atg5 and Atg7. The above results indicate that PHI-HA-mEXO alleviates liver fibrosis at least in part by inhibiting autophagy in aHSCs.

[0087] 4. Discussion

[0088] PHI has been shown to be a drug with good anti-inflammatory and anti-fibrotic effects. However, due to its poor water solubility and incomplete oral absorption, its oral bioavailability is low. To overcome the limitations associated with oral administration, a variety of drug delivery systems have been developed for drug delivery. For example, Wang et al. developed a novel liposomal drug delivery system for the delivery of PHI; however, they have not entered clinical application due to their high cost, lack of widespread applicability, and inherent limitations including safety. Our study aims to study the oral targeted delivery of PHI by using milk-derived exosomes to overcome the problems of low bioavailability and targeted therapy of PHI. Drug loading was achieved by mixing exosomes (mEXO) and hyaluronic acid (DSPE-PEG2000-HA), incubating at 37°C for 48 h, and centrifuging for 25 min (3500 g) to obtain HA-mEXO. Take HA-mEXO, mix it with forsythiaside, incubate it at room temperature in the dark for 3 hours, and use a 10KDa ultrafiltration tube to ultrafilter at 3500g for 10 minutes to remove unbound drugs to obtain PHI-HA-mEXO. Compared with free exosomes (115.9nm), the morphological size of PHI-HA-mEXO (116nm) did not change significantly. The interaction between nanoparticles and cell membranes is significantly affected by the surface charge of the nanocomplex. In addition, positively charged nanoparticles are more likely to induce cellular oxidative stress and disrupt the antioxidant system of hepatocytes. Exosomes show obvious negative charge, which ensures the safety of the exosome drug delivery system.

[0089] When we tried to use exosomes to deliver PHI, the stability of the exosome preparation was crucial. Our stability test results showed that after storing PHI-HA-mEXO at -80°C for one month, its particle size and ζ potential values ​​did not change significantly from the freshly prepared samples, indicating that the PHI-HA-mEXO system has a certain storage stability. In vitro release is another important indicator that can be used to predict the in vivo kinetics of drugs after administration. Our in vitro release test results showed that PHI-HA-mEXO released 46% of the loaded drug within approximately 2 hours and 83% of the loaded drug within 8 hours, indicating that the PHI-HA-mEXO drug dissolution was relatively complete.

[0090] As a common hyaluronic acid receptor, CD44 is often highly expressed in cancer cells and is often used as a targeted treatment point for cancer cells. The present invention uses hyaluronic acid to target liver fibrosis cells to produce unexpected results. Cell uptake experiments show that aHSCs exhibit higher uptake compared to qHSCs, indicating that a large amount of CD44 helps PHI-HA-mEXO to enrich in the Disse cavity where hepatic stellate cells are located in the liver. In addition, in the biodistribution study, DIR-labeled PHI-HA-mEXO was clearly accumulated in the liver, showing excellent targeting.

[0091] Although hyaluronic acid ensures the targeting of PHI-HA-mEXO, off-target phenomena may occur, causing PHI to act on normal cells. The non-cytotoxicity of PHI-HA-mEXO is important for preventing apoptosis of normal hepatic stellate cells and hepatocytes during anti-fibrotic treatment. Our cytotoxicity experiments showed that after administration, the cell viability of normal hepatic stellate cells and hepatocytes was 73.54% and 83.01%, respectively, showing good safety. Therefore, hyaluronic acid-based PHI-HA-mEXO can be used in clinical trials to treat liver fibrosis.

[0092] The in vitro antiproliferative efficacy of PHI-HA-mEXO was tested on aHSCs, and the results showed that PHI-HA-mEXO had higher cytotoxicity compared with free PHI and PHI-mEXO. Based on the cytotoxicity data, we further explored the efficacy of PHI-HA-mEXO using a thioacetamide-induced zebrafish larvae liver fibrosis model. PHI-HA-mEXO was studied at three doses: 25μmol / L, 50μmol / L, and 100μmol / L. The changes in biochemical indicators ALT, AST, and ALP in zebrafish larvae with liver fibrosis given PHI-HA-mEXO indicated that liver function changed with the degree of liver fibrosis. Compared with the blank group, ALT, AST, and ALP in zebrafish larvae in the model group were significantly increased, reflecting considerable liver function damage. After treatment with PHI-HA-mEXO, all test indicators were significantly reduced, showing a good anti-fibrotic effect. In addition, liver histopathological results in the PHI-HA-mEXO-treated group showed reduced collagen fibrillation and focal necrosis in a dose-dependent manner compared with the model group. In conclusion, PHI-HA-mEXO can inhibit hepatic stellate cell activation and reduce col1α1 and α-SMA formation to prevent liver fibrosis.

[0093] Given that hepatic stellate cell activation is a typical feature of liver fibrosis, and autophagy is a highly conserved intracellular degradation pathway that mainly regulates lysosome degradation of damaged organelles and macromolecules through autophagy-related genes. It has been found that autophagy can provide energy for HSC activation by degrading lipid droplets, while inhibiting autophagy can significantly inhibit HSC activation. Our study found that PHI-HA-mEXO can target and inhibit autophagy in HSC, such as the levels of autophagy-related proteins LC3, Beclin1, P62, Atg5 and Atg7 returned to normal levels after PHI-HA-mEXO treatment. Our results indicate that PHI-HA-mEXO may partially inhibit HSC activation by downregulating autophagy.

[0094] The data from these studies showed that after oral administration, exosomes loaded with PHI significantly improved the bioavailability of PHI and enhanced the efficacy of the drug. Although exosome loading did not completely solve the problem of poor solubility of PHI, it also made PHI more likely to be used in clinical applications. As a hydrophobic drug, the efficiency of drug-loaded exosomes obtained by conventional incubation, ultrasound and other methods is not high. In order to improve the loading efficiency of exosomes for drugs, we used DMSO to dissolve the drug in advance and then incubated it with exosomes, thereby greatly improving the loading efficiency of exosomes for forsythiaside. In addition, the anti-liver fibrosis targeted drug delivery system based on milk exosomes prepared in this experiment showed good targeting in both cell and animal experiments, proving that HA functionalization is an efficient milk exosome targeted modification strategy. In order to further verify the potential of oral drug delivery of milk exosome drug delivery system, we also constructed a zebrafish liver fibrosis model induced by thioacetamide to explore the anti-liver fibrosis activity of the milk exosome targeted drug delivery system constructed by this strategy in vivo.

[0095] 5. Conclusion

[0096] In this study, we developed PHI-HA-mEXO, a natural hepatoprotectant from Forsythia suspensa, which can induce apoptosis of activated hepatic stellate cells and alleviate thioacetamide-induced liver fibrosis in zebrafish larvae. Our results showed that the particle size of PHI-HA-mEXO was about 116.3 nm. This drug system can significantly induce apoptosis of activated hepatic stellate cells without affecting normal hepatic stellate cells and hepatocytes. In addition, the therapeutic effect of PHI-HA-mEXO showed a more effective therapeutic effect compared with the same dose of free PHI. The good biosafety and significant anti-hepatic fibrosis effect indicate that PHI-HA-mEXO has great application potential in the treatment of liver fibrosis.

Claims

1. A drug for treating liver fibrosis, Features: It includes exosomes and forsythiaside; the exosomes are milk exosomes modified by hyaluronic acid; the milk exosomes are mixed and incubated with hyaluronic acid to obtain milk exosomes modified by hyaluronic acid; the mass ratio of the milk exosomes to the hyaluronic acid is 20:1; When preparing the drug, the mass ratio of exosomes to forsythiaside is 4 to 8:

1.

2. The drug according to claim 1, Features: The milk exosomes are a suspension obtained by washing and resuspending the precipitate obtained by differential ultracentrifugation of milk with a PBS solution; the protein content in the suspension is 4 to 8 mg / ml.

3. The drug according to claim 2, Features: The method of the differential ultracentrifugation is: The milk was first centrifuged at 10,000×g for 30 min, the solution was centrifuged at 100,000×g for 90 min, the supernatant was collected and centrifuged at 135,000×g for 70 min, and the precipitate was collected to obtain the product.

4. The drug according to claim 1, Features: When preparing the drug, the mass ratio of exosomes to forsythiaside is 6:

1.

5. A method for preparing the drug according to any one of claims 1 to 4, Features: It includes the following steps: 1) Weighing raw materials according to the ratio; mixing exosomes and hyaluronic acid, incubating, centrifuging, and taking the supernatant to obtain hyaluronic acid-modified milk exosomes; 2) The hyaluronic acid-modified milk exosomes obtained in step 1) are mixed with forsythiaside, incubated, and ultrafiltered to obtain.

6. The preparation method according to claim 5, Features: The incubation temperature of step 1) is 37°C for 48 hours; the centrifugation speed is 3500g for 25 minutes; the incubation of step 2) is room temperature in the dark for 3 hours; the ultrafiltration tube of the ultrafiltration is 10KDa, the speed is 3500g, and the time is 10 minutes.

7. Use of the drug according to any one of claims 1 to 4 in the preparation of a drug for treating liver fibrosis.

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