Multifunctional exosome for treating hepatic fibrosis and preparation method thereof
By constructing the multifunctional exosome a5HT1D-CD47-Exo-miR-29b, the existing miR-29b is easily metabolized and difficult to target during the administration process, and the efficient drug transcends ECM and targeted delivery is achieved, achieving efficient therapeutic effect of liver fibrosis.
Patent Information
- Application Number
- CN202510238499.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-03
- Publication Date
- 2025-05-30
AI Technical Summary
The existing miR-29b is easily metabolized by the liver during administration, difficult to cross ECM, easy to be engulfed by liver macrophages, and difficult to target to activated hepatic stellate cells, resulting in poor treatment effect.
By constructing the multifunctional exosome a5HT1D-CD47-Exo-miR-29b, exosomes derived from rat hepatic stellate cells were engineered, loaded miR-29b and linked 5HT1D antibodies on the surface, achieving efficient drug transcendence across ECM and targeted delivery to activated hepatic stellate cells.
The drug is effectively crossed ECM and avoided phagocytosis of liver macrophages, targeted to activated liver stellate cells, inhibited the production of collagen and activation of liver stellate cells, and achieved the efficient therapeutic effect of liver fibrosis.
Smart Images
Figure CN120060154A_ABST
Abstract
Description
Technical Field
[0001] The present invention discloses a multifunctional exosome for the treatment of liver fibrosis, and also provides a preparation process thereof, belonging to the technical field of biomedical production. Background Art
[0002] Liver fibrosis is a pathological process of excessive healing after liver injury. If not restricted, it will gradually develop into liver cirrhosis or even liver cancer, seriously threatening human health. Research shows that damaged hepatocytes will release different inflammatory mediators to induce the activation of hepatic stellate cells (HSCs), and differentiate into proliferative myofibroblasts. During this process, HSCs lose the ability to store lipid droplets and transform into an activated state that can induce the production of collagen and alpha-smooth actin (α-SMA), resulting in the excessive deposition of extracellular matrix (ECM) in the perisinusoidal space. This will promote the formation of fibrous scars, limit blood flow in the liver, and thus cause liver fibrosis.
[0003] Research shows that transforming growth factor-beta 1 (TGF-β1) is an important cytokine that induces the activation of HSCs. After the TGF-β signaling pathway is activated, downstream proteins such as α-SMA and collagen are significantly up-regulated, while the expression of matrix metalloproteinases is inhibited. This imbalance promotes the proliferation and persistence of activated HSCs (aHSCs). Therefore, inhibiting TGF-β1 can effectively inhibit the activation of HSCs and become the most effective inhibitory target for the treatment of liver fibrosis. In recent years, a new treatment strategy using microRNA (miR) to reverse liver fibrosis has attracted much attention. Among them, miR-29b inhibits the synthesis of collagen by binding to the 3'UTR of COL1A1 mRNA and has become an important treatment target. This mechanism blocks the TGF-β / Smad3 signaling pathway and simultaneously enhances the PTEN signaling cascade. Although miR-29b has good therapeutic potential, there are problems such as being easily metabolized by the liver, difficult to cross the ECM, easily phagocytosed by liver macrophages, and difficult to target aHSCs during the administration process.
[0004] Exosomes are the smallest subtype of extracellular vesicles, which can target the maternal cells that secrete exosomes and have a certain homing effect. Compared with other carriers such as liposomes and nanoparticles, exosomes have a strong ability to cross natural barriers. Moreover, they have low immunogenicity, low toxicity, and good stability as gene drug carriers. In addition, exosomes contain transmembrane and membrane-anchored proteins, which increase their engineering ability, thus avoiding being phagocytosed by liver macrophages, enhancing the endocytosis of target cells, and promoting the delivery of their contents. Therefore, exosomes as drug delivery carriers provide a new strategy for the treatment of liver fibrosis. Summary of the Invention
[0005] The present invention constructs a multifunctional exosome for the treatment of liver fibrosis, and uses exosomes derived from rat hepatic stellate cells for engineering transformation to achieve the avoidance of drugs being phagocytosed by liver macrophages and targeted delivery to activated hepatic stellate cells.
[0006] To achieve the purpose, the present invention adopts the following technical solutions: After culturing a large number of rat hepatic stellate cells, the miR-29b plasmid is loaded into CD47 protein stably transfected rat hepatic stellate cells CD47-HSC-T6 using a nanosecond pulsed microfluidic system. After culturing for 24 hours, the culture medium supernatant is centrifuged, and the supernatant is ultrafiltered and concentrated to obtain crude exosomes, which are further purified by an ultracentrifuge to obtain purified drug-loaded exosomes CD47-Exo-miR-29b; then the 5HT1D antibody is incubated with CD47-Exo-miR-29b, and the unmodified 5HT1D antibody is washed away with PBS to obtain multifunctional exosomes a5HT1D-CD47-Exo-miR-29b, that is, exosomes loaded with therapeutic drugs and modified with CD47 protein and 5HT1D antibody.
[0007] The preparation method of a multifunctional exosome for the treatment of liver fibrosis according to the present invention includes the following steps: 1) The supernatant of CD47 protein stably transfected rat hepatic stellate cells CD47-HSC-T6 is successively stimulated by a nanosecond pulsed microfluidic system, centrifuged, filtered, and ultracentrifuged to obtain purified exosomes with high expression of CD47 protein; Among them, the supernatant is used to extract exosomes by a nanosecond pulsed microfluidic system, the filtration is carried out using a 0.22 μm filter membrane, and the filtrate is centrifuged by an ultracentrifuge at 100,000 g; 2) Loading therapeutic drugs and surface modification: Load miR-29b into the purified exosomes with high expression of CD47 protein in step 1), then add 5HT1D antibody and incubate. After incubation, centrifuge the mixture with an ultrafiltration tube at 4°C, and then add PBS and centrifuge to wash the unmodified 5HT1D antibody to obtain multifunctional exosomes a5HT1D-CD47-Exo-miR-29b.
[0008] The present invention also defines that the targeted therapeutic drug loading adopts a nanosecond pulsed microfluidic system, wherein: The process of loading drugs into the nanosecond pulsed microfluidic system is as follows: Add 50 μL of miR-29b at a concentration of 100 μg / mL into the buffer channel. Under nanosecond pulses, load the miR-29b plasmid into CD47-HSC-T6 cells. After culturing for 24 hours, take the supernatant of the culture medium and centrifuge it. Concentrate the supernatant by ultrafiltration to obtain crude exosomes, and then further purify them with an ultracentrifuge to obtain drug-loaded exosomes CD47-Exo-miR-29b.
[0009] Furthermore, the present invention also defines the multifunctional exosomes obtained by the defined method. The exosome morphology is disc-shaped, contains CD63 exosome characteristic proteins, and has an average diameter of 150 nm. The multifunctional exosomes prepared by the present invention can inhibit the production of collagen and the activation of hepatic stellate cells, and inhibit the growth of liver fibrosis.
[0010] The multifunctional exosomes a5HT1D-CD47-Exo-miR-29b described in the present invention can be used to prepare drugs for treating liver fibrosis diseases.
[0011] Since the surface of activated hepatic stellate cells overexpresses 5HT1D receptors, the present invention connects 5HT1D antibodies to the surface of exosomes and loads miR-29b therein to inhibit the production of collagen and, at the same time, inhibit the activation of hepatic stellate cells, thereby achieving efficient treatment of liver fibrosis.
[0012] The positive effects of the present invention are as follows: It provides a multifunctional exosome that can efficiently treat liver fibrosis; uses exosomes derived from rat hepatic stellate cells for engineering transformation to enable the drug to efficiently cross the ECM, avoid phagocytosis by liver macrophages, and target activated hepatic stellate cells. Connect 5HT1D antibodies to its surface and load miR-29b therein to inhibit the production of collagen and, at the same time, inhibit the activation of hepatic stellate cells, thereby achieving efficient treatment of liver fibrosis. The present invention improves the therapeutic effect of exosomes on liver fibrosis through a series of engineering transformations of exosomes. Description of the Drawings
[0013] Figure 1 is the Western blot of a5HT1D-CD47-Exo-miR-29b of the present invention; Figure 2 This is the cryo-electron microscopy image of a5HT1D-CD47-Exo-miR-29b of the present invention; Figure 3 This is the flow cytometry analysis image of activated hepatic stellate cells of the present invention taking up a5HT1D-CD47-Exo-miR-29b; Figure 4 This is the confocal laser microscopy image of hepatic macrophages of the present invention taking up a5HT1D-CD47-Exo-miR-29b; Figure 5 This is the effect image of a5HT1D-CD47-Exo-miR-29b crossing the ECM of the present invention; Figure 6 This is the treatment effect image of each group of rat liver fibrosis models during treatment of the present invention. Detailed implementation manners
[0014] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art to the present invention will fall within the scope of the claims of the present invention. Example 1
[0015] After culturing CD47-HSC-T6 cells in large quantities, the cells were digested, centrifuged at 300 g for 5 minutes, and CD47-HSC-T6 cells were collected. After mixing them with 80 - 150 μg / mL miR-29b plasmid, nanosecond pulse stimulation was performed through a nanosecond pulse microfluidic system under the conditions of an applied voltage of 140 - 160 V, a pulse width of 100 - 800 ns, and a flow rate of 1 - 5 mL / h. Then, cell culture was carried out with a cell amount of 3 - 6 million cells per flask for 24 hours to increase the yield of exosomes; The supernatant was taken and subjected to gradient centrifugation at 4°C using a high-speed refrigerated centrifuge: centrifuged at 300 g for 10 min to remove dead cells, 1200 g for 10 min, and 10,000 g for 20 min to remove cell debris; the supernatant was taken and filtered through a sterile 0.22 μm filter membrane to remove larger vesicles; finally, centrifuged at 100,000 g for 70 min using an ultracentrifuge to precipitate CD47-Exo-miR-29b exosomes. The obtained CD47-Exo-miR-29b has exosome marker proteins CD63, TSG101, and Alix on its surface. See Figure 1 ; Loading of 5HT1D antibody on the surface of exosomes: CD47-Exo-miR-29b obtained by ultracentrifugation was incubated with 5HT1D antibody at 37 °C for 2 h (protein content 1 / 0 - 1 / 6). Under the condition of 4 °C, it was ultracentrifuged at 100,000 g for 2 h to remove the antibody not bound to CD47-Exo-miR-29b, and a5HT1D-CD47-Exo-miR-29b was obtained. The obtained a5HT1D-CD47-Exo-miR-29b was spherical, with a particle size of about 150 nm and good dispersibility. See Figure 2 .
[0016] The positive effects of the present invention are demonstrated by the following experimental examples:
[0017] Experimental Example 1 Take the a5HT1D-CD47-Exo-miR-29b obtained in Example 1, stain it with PKH26, and then incubate it with activated hepatic stellate cells. Flow cytometry analysis was performed on the uptake of a5HT1D-CD47-Exo-miR-29b by activated hepatic stellate cells. The specific steps are as follows: Add PKH26 dye to 0.2 - 0.6 mg / mL a5HT1D-CD47-Exo-miR-29b (100 - 150 μL), incubate at room temperature for 10 - 30 min, centrifuge at 1200 g for 20 min at 4 °C using a 100 kDa ultrafiltration tube, add 150 μL PBS, centrifuge at 1200 g for 20 min at 4 °C using a 100 kDa ultrafiltration tube, and repeat once to obtain PKH26-stained multifunctional exosomes PKH26-a5HT1D-CD47-Exo-miR-29b; Add PKH26-a5HT1D-CD47-Exo-miR-29b to activated hepatic stellate cells, incubate for 4 h, remove the supernatant, wash with PBS, digest with trypsin, centrifuge at 800 g for 5 min, remove the supernatant and add 400 μL paraformaldehyde solution for fixation. Flow cytometry analysis was performed on the uptake of PKH26-a5HT1D-CD47-Exo-miR-29b by activated hepatic stellate cells. The experimental results showed that activated hepatic stellate cells had a relatively high uptake of PKH26-a5HT1D-CD47-Exo-miR-29b. See Figure 3 .
[0018] Experimental Example 2
[0019] Take the PKH26-a5HT1D-CD47-Exo-miR-29b obtained in Experimental Example 1, and then incubate it with hepatic macrophages. Analyze the uptake of a5HT1D-CD47-Exo-miR-29b by hepatic macrophages using a flow cytometer. The specific steps are as follows: Add PKH26-a5HT1D-CD47-Exo-miR-29b to hepatic macrophages, incubate for 4 h, remove the supernatant, wash with PBS, digest with trypsin, centrifuge at 800 g for 5 min, remove the supernatant and add 400 μL of paraformaldehyde solution for fixation. Analyze the uptake of PKH26-a5HT1D-CD47-Exo-miR-29b by hepatic macrophages using a flow cytometer. The experimental results show that hepatic macrophages showed a low uptake of PKH26-a5HT1D-CD47-Exo-miR-29b; see Figure 4 .
[0020] Experimental Example 3
[0021] Take the PKH26-a5HT1D-CD47-Exo-miR-29b obtained in Experimental Example 1. Use a transwell chamber to simulate ECM. Place PKH26-a5HT1D-CD47-Exo-miR-29b in the upper layer of the chamber and examine the uptake of PKH26-a5HT1D-CD47-Exo-miR-29b by activated hepatic stellate cells in the lower chamber of the transwell. The specific steps are as follows: First, add a collagen suspension including 200 - 300 μL of type I collagen with a concentration of 5 - 8 mg / mL, 10 - 20 μL of sodium hydroxide with a concentration of 0.1 - 0.5 mol / L, 100 μL of PBS (10 mM), and 600 - 700 μL of sterile water to the upper chamber of the Transwell. Then, incubate at 37°C for 1 - 2 h to form a collagen layer. Seed activated hepatic stellate cells at a density of 5×10 4 cells / well in a 24-well plate and culture for 24 h. Then, add PKH26-a5HT1D-CD47-Exo-miR-29b to the collagen layer formed in the upper chamber of the Transwell, culture in the dark for 24 h, and use a flow cytometer to analyze the uptake of PKH26-a5HT1D-CD47-Exo-miR-29b by activated hepatic stellate cells. The experimental results show that activated hepatic stellate cells showed a high uptake of PKH26-a5HT1D-CD47-Exo-miR-29b; see Figure 5 .
[0022] Experimental Example 4
[0023] Construction of a rat liver fibrosis model: Wistar rats (male, 150 - 200 g, 6 - 8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After two weeks of adaptive cultivation, the modeling operation was carried out. The model group was intraperitoneally injected with CCl 4 olive oil mixture (1:1, V / V, 1 mL / kg); the sham operation group was intraperitoneally injected with olive oil (1 mL / kg). It was injected twice a week for 8 weeks continuously.
[0024] In vivo treatment: All liver fibrosis rats were randomly divided into 5 groups, namely the sham operation (Sham) group, the model (Model) group, the Exo-miR-29b group, the CD47-Exo-miR-29b group, and the a5HT1D-CD47-Exo-miR-29b group. Tail vein injection was carried out for drug administration starting from the 8th week, with a drug administration dose of 0.1 - 0.5 mg / kg miR-29b, lasting for 4 weeks, once every three days. After the treatment ended, the pathological conditions of the rat liver tissues were detected by H&E and Masson staining. The results are as Figure 6 shown, Conclusion: It can be proved from the Figure 6 results that a5HT1D-CD47-Exo-miR-29b has a good inhibitory effect on liver fibrosis.
[0025] The above is only a general description and implementation method of the present invention, and does not limit the patent protection scope of the present invention. Any equivalent changes made according to the specification and drawings of the present invention, or directly or indirectly using the present invention patent in other related technical fields, are regarded as within the protection scope of the present invention patent.
Claims
1. A multifunctional exosome for the treatment of liver fibrosis, characterized in that: The multifunctional exosomes a5HT1D-CD47-Exo-miR-29b are disc-shaped, contain CD63 exosome characteristic proteins, and have an average diameter of 150 nm; they can inhibit the production of collagen, inhibit the activation of hepatic stellate cells, and achieve efficient treatment of liver fibrosis.
2. The method for preparing multifunctional exosomes for treating liver fibrosis according to claim 1, comprising the following steps: 1) Loading therapeutic drugs: The CD47-HSC-T6 cell supernatant and miR-29b plasmid mixture were stimulated by a nanosecond pulse microfluidic system, centrifuged, filtered, and ultracentrifuged to obtain purified drug-loaded exosomes; The supernatant was used to extract exosomes using a nanosecond pulse microfluidic system, filtered using a 0.22 μm filter membrane, and the filtrate was centrifuged at 100,000 g using an ultracentrifuge; 2) Surface modification: 5HT1D antibody was added for incubation, and the mixture after incubation was centrifuged at 4°C using an ultrafiltration tube, and then PBS was added for centrifugation to wash the unmodified 5HT1D antibody to obtain multifunctional exosomes a5HT1D-CD47-Exo-miR-29b.
3. The method for preparing multifunctional exosomes according to claim 2, characterized in that Step 1) involves the process of loading drugs into the nanosecond pulse microfluidic system as follows: The collected CD47-HSC-T6 cells were passed through the chip channels at a density of 3-6 million / bottle, and 50 μL of miR-29b plasmid at a concentration of 100 μg / mL was added to the buffer channel. Under nanosecond pulses, the miR-29b plasmid was transfected into the CD47-HSC-T6 cells. After culturing for 24 hours, the culture supernatant was centrifuged, and the supernatant was ultrafiltrated and concentrated to obtain crude exosomes, which were further purified using an ultracentrifuge.
4. Use of the multifunctional exosomes (a5HT1D-CD47-Exo-miR-29b) according to claim 1 in the preparation of a drug for treating liver fibrosis.