Preparation method and application of double-membrane fusion targeted nano-drug delivery system

Abstract

The application provides a preparation method and application of a double-membrane fusion targeted nano drug delivery system, a targeted material Cholesterol-PEG2000-RT modified liposome loaded with hydroxychloroquine is prepared, then the similarity of the liposome and the membrane component of the exosome is utilized, and the fusion of the two is realized through an extrusion method, so that the double-membrane fusion targeted drug delivery system for activating hepatic stellate cells is prepared. The double-membrane fusion targeted nano drug delivery system can not only exert the synergistic anti-hepatic fibrosis treatment effect of hydroxychloroquine and the exosome, but also can realize the specific targeted autophagy inhibition of hydroxychloroquine on activated hepatic stellate cells, so that the further proliferation and differentiation of the activated hepatic stellate cells are prevented, the generation of fibrosis-related proteins is reduced, the anti-hepatic fibrosis effect is exerted, and in addition, the genes and active factors carried by the exosome derived from bone marrow mesenchymal stem cells can exert the liver protection and synergistic anti-hepatic fibrosis effect, so that a new thought and method are provided for the clinical treatment of hepatic fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanodrug delivery systems, in particular, a preparation method and application thereof, and relates to a preparation method of a hydroxychloroquine-loaded liposome with targeted modification and a double-membrane fusion targeted nanodrug delivery system fused with an exosome. The nanodrug delivery system can target activated hepatic stellate cells and is used for comprehensive treatment of liver fibrosis. BACKGROUND

[0002] Liver fibrosis is a chronic liver injury characterized by excessive deposition of extracellular matrix and collagen, and its occurrence and development are a complex process involving multiple factors, multiple genes and multiple stages. So far, no effective drug has been approved for the treatment of liver fibrosis [Acs Nano.2021, 15(4):7547-7562]. Traditional drugs can exacerbate fibrosis due to their toxic side effects and secondary damage to the liver during treatment, so it is urgent to develop new treatment methods for liver fibrosis.

[0003] A large number of studies have confirmed that hepatic stellate cells are the central hub of the occurrence and development of liver fibrosis, and autophagy plays a crucial role in the process of occurrence and development of liver fibrosis. However, the effects of autophagy on different types of cells in the liver are completely opposite. Autophagy of hepatic stellate cells can induce a fibrosis cascade and exacerbate fibrosis [Journal of Hepatology.2011, 55(6):1176-1177.], and the treatment of liver fibrosis can be achieved by inhibiting autophagy of hepatic stellate cells. However, autophagy of hepatocytes, macrophages and endothelial cells can inhibit liver cell damage, maintain endothelial cell homeostasis and reduce inflammatory cytokines produced by macrophages and endothelial cells, and inhibition of autophagy may lead to the occurrence of liver fibrosis. Therefore, it is particularly important to target and inhibit the autophagy level of activated hepatic stellate cells in the treatment of liver fibrosis. In addition, existing studies have clearly pointed out that activated hepatic stellate cells are the key cells for the excessive deposition of extracellular matrix and collagen and the generation of fibrous tissue, so based on comprehensive consideration, specifically targeting and inhibiting autophagy of activated hepatic stellate cells is the best choice for treating liver fibrosis.

[0004] Hydroxychloroquine is a drug that has been widely used in clinical treatment of diseases such as malaria, and as a clear autophagy inhibitor, it has also entered phase IV clinical trials. However, the traditional preparation of the drug has poor targeting, poor water solubility, large dosage, and easy distribution in red blood cells and around the eyeball in the body, which can cause blurred vision and aplastic anemia in patients after long-term use, and cannot achieve specific autophagy inhibition of activated hepatic stellate cells.

[0005] Therefore, it is necessary to improve the above-mentioned shortcomings by means of new preparation, reduce the damage to other tissues and organs and achieve specific autophagy inhibition. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the application provides a preparation method of a double-membrane fusion targeted nano drug delivery system and application thereof. The purpose of the application is to provide a preparation method of a double-membrane fusion targeted nano drug delivery system. The drug delivery system utilizes the easy modification characteristics of the surface of liposomes, first prepares RT modified liposomes loaded with hydroxychloroquine, and then realizes the fusion of the liposomes and the membrane components of the exosomes by extrusion, thereby preparing a double-membrane fusion targeted activated hepatic stellate cell drug delivery system. The double-membrane fusion targeted nano drug delivery system can not only exert the synergistic anti-liver fibrosis treatment effect of hydroxychloroquine and exosomes, but also can realize the specific autophagy inhibition of hydroxychloroquine on activated hepatic stellate cells, reduce the generation of fibrosis-related proteins by preventing further proliferation and differentiation, and play an anti-liver fibrosis role. In addition, the genes and active factors carried by the exosomes derived from bone marrow mesenchymal stem cells can play a liver protection and synergistic anti-liver fibrosis role, thereby providing a new idea and method for the clinical treatment of liver fibrosis.

[0007] The technical solution adopted by the application to solve the technical problems comprises the following steps:

[0008] A preparation method of a double-membrane fusion targeted nano drug delivery system, wherein Cholesterol-PEG 2000 -NH2 and retinol treated by acylation reaction are used as raw materials, and the two are subjected to amidation reaction to obtain a targeted material Cholesterol-PEG 2000 -RT, the obtained targeted material Cholesterol-PEG 2000 -RT is prepared by using cholesterol, hydrogenated lecithin, and is loaded with hydroxychloroquine, and finally is subjected to membrane extrusion with exosomes to obtain a double-membrane fusion targeted activated hepatic stellate cell nano drug delivery system.

[0009] The specific steps of the preparation method of the double-membrane fusion targeted nano drug delivery system are as follows:

[0010] Step (1): Retinol (RT) was stirred at room temperature until completely dissolved in chloroform to obtain a chloroform solution. After adding succinic anhydride and triethylamine to the chloroform solution, it was stirred at room temperature for 48 h. The chloroform organic layer was washed with water three times, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were added. After stirring at room temperature for 1 h, reaction solution A was obtained for standby; Cholesterol-PEG 2000 -NH2 and triethylamine were added to reaction solution A, which was stirred at the reaction temperature for a reaction time T to obtain reaction solution B. Reaction solution B was washed with water three times and dried over anhydrous sodium sulfate. Then the organic phase was added to ice ethyl ether, the precipitate was collected, and vacuum drying obtained the carrier material Cholesterol-PEG 2000 -RT;

[0011] Step (2): Cholesterol-PEG 2000 -RT, cholesterol and hydrogenated lecithin were dissolved in chloroform, and the organic solvent was removed by rotary evaporation under reduced pressure. After adjusting the pH value to x by adding 0.1 mol / L sodium citrate, the hydration time t1 was obtained. The obtained liposome suspension after hydration was collected in a centrifuge tube, and then the probe was used for ultrasonic treatment at 0°C for a time t2. Then hydroxychloroquine sulfate was added to the liposome suspension to dissolve, and then 0.2 mol / L Na2PHO4 was added until the pH was adjusted to 7. The mixture was incubated at 37°C on a constant temperature shaker for a time t3. After high-speed centrifugation, the precipitate was resuspended with water, and the resuspension was filtered through 0.8 μm and 0.22 μm microporous filters, respectively, to obtain the hydroxychloroquine-loaded targeting liposomes;

[0012] Step (3): The hydroxychloroquine-loaded targeting liposomes were uniformly mixed with the exosomes, and the mixture was extruded N times with a 400 nm and 200 nm particle size liposome extruder to obtain the double-membrane fusion targeting activated hepatic stellate cell nanodrug delivery system.

[0013] In step (1), the molar ratio of retinol, triethylamine and succinic anhydride was 1:1:1-1:10:15; the molar ratio of retinol, EDC and NHS was 1:0.5:0.5-1:20:30; when Cholesterol-PEG 2000 -NH2 was added to reaction solution A, the molar ratio of Cholesterol-PEG 2000The molar ratio of -NH2 to retinol is 1:4 to 1:20, and triethylamine and Cholesterol-PEG are also present. 2000 The molar ratio of -NH2 is 1:2 to 20:1.

[0014] In step (1), the reaction temperature is 0℃~100℃ and the reaction time T is 0.5~50h.

[0015] In step (2), Cholesterol-PEG 2000 -RT, cholesterol, and hydrogenated lecithin were in a mass ratio of 1:1:5 to 1:1:10; hydroxychloroquine sulfate and liposome suspension (Cholesterol-PEG) 2000 The feed ratio of total RT, cholesterol and hydrogenated lecithin is 1:1 to 1:20.

[0016] In step (2), the pH value x ranges from 1 to 6.5; the hydration time t1 is 10 to 180 min; the probe ultrasound time t2 is 1 to 60 min; and the constant temperature oscillation incubation time on a 37℃ shaker is 0.5 to 4 h.

[0017] In step (3), the mass ratio of the targeted liposomes loaded with hydroxychloroquine to the exosomes (based on total protein content) is 1:1 to 50:1.

[0018] In step (3), the number of extrusions N is 1 to 20 times.

[0019] The dual-membrane fusion targeted nanodelivery system prepared by the method described herein can be used in anti-liver fibrosis drugs.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention is the first to use Cholesterol-PEG 2000 Using -NH2 and acylated retinol as raw materials, the two are subjected to an amidation reaction to obtain the targeting material Cholesterol-PEG. 2000 -RT. Utilizing the targeted material Cholesterol-PEG. 2000 -RT was used to prepare liposomes loaded with hydroxychloroquine, which were then fused with exosomes to obtain a nano-drug delivery system that specifically targets and activates hepatic stellate cells. This invention not only enables active targeted drug delivery to activated hepatic stellate cells, enhancing the efficacy of hydroxychloroquine while reducing toxic side effects on other liver cells, but also synergistically exerts the hepatoprotective function of exosomes.

[0022] 2, the activity evaluation experiment results of the obtained double-membrane fusion targeted activated hepatic stellate cell nano drug delivery system also show that the drug delivery system can realize the targeted drug delivery of activated hepatic stellate cells, inhibit the autophagy level of hepatic stellate cells at the same time, and can play an anti-liver fibrosis role with exosomes, and show high in-vivo and in-vitro anti-liver fibrosis treatment effect. Moreover, no damage is caused to other main organs during treatment, and it has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 In the present application, Figure 1 (a) is a transmission electron micrograph of Lip (100 nm), Figure 1 (b) is a transmission electron micrograph of Exo (100 nm), Figure 1 (c) is a transmission electron micrograph of HCQ@RT-Lip-Exo (100 nm), Figure 1 (d) is a particle size distribution graph of HCQ@RT-Lip-Exo.

[0024] Figure 2 is a comparison graph of RT competitive uptake inhibition experiment of HCQ@RT-Lip-Exo.

[0025] Figure 3 is the expression of a-SMA and Collagen1 protein of aHSCs cells in different treatment groups.

[0026] Figure 4 is the photo, HE, Masson and Sirius red staining graph of liver of mice in different treatment groups.

[0027] Figure 5 is the HE staining graph of main organs of liver fibrosis model mice after different treatments. DETAILED DESCRIPTION

[0028] The present application will be further described below in combination with the drawings and examples.

[0029] The present application takes hepatic stellate cells as a specific targeting intervention target, utilizes the high expression characteristic of retinol binding protein receptor on the surface of activated hepatic stellate cells (aHSCs), takes RT as a targeting group, and prepares a double-membrane fusion targeted activated hepatic stellate cell nano drug delivery system by taking a liposome (Lip) and an exosome (Exo) fusion as a drug delivery carrier. The drug delivery system can specifically target aHSCs, plays an anti-fibrosis role through the autophagy inhibition effect thereof, at the same time, plays a synergistic anti-fibrosis role with exosomes rich in RNA, proteins and active cytokines and other related anti-fibrosis substances acting on aHSCs, together with hydroxychloroquine, so as to realize precise targeting and accumulation, and achieve a synergistic anti-liver fibrosis treatment effect.

[0030] The present application takes Cholesterol-PEG 2000 -NH2 and retinol treated by acylation reaction as raw materials, and the two are obtained by amidation reaction to obtain the targeting material Cholesterol-PEG 2000 -RT. The obtained targeting material is combined with cholesterol and hydrogenated lecithin to obtain a targeting modified liposome by a thin film dispersion method, and then a hydroxychloroquine-loaded liposome is obtained by a pH gradient method, and finally a double-membrane fusion targeting activated hepatic stellate cell nanodrug delivery system is obtained by a membrane extrusion method, and a high-efficiency anti-liver fibrosis drug is obtained by in vivo and in vitro activity evaluation.

[0031] Example 1

[0032] Preparation and characterization of double-membrane fusion nanodrug delivery system HCQ@RT-Lip-Exo

[0033] Step (1): weigh 500.0 mg (1.75 mmol) of retinol into 5 mL of chloroform, and stir the retinol at room temperature in the dark until it is completely dissolved in chloroform. After adding 354.2 mg (3.5 mmol) of triethylamine and 350.3 mg (3.5 mmol) of succinic anhydride, stir at room temperature in the dark for 48 h, wash with water three times, dry with anhydrous sodium sulfate, then add 296.9 mg (3 mmol) of EDC and 345.3 mg (3 mmol) of NHS, continue to stir at room temperature for 1 h, and reserve; add 750.0 mg (0.5 mmol) of Cholesterol-PEG 2000 -NH2 and 354.2 mg (3.5 mmol) of triethylamine to the reaction solution, stir at 45°C for 5 h, wash the reaction solution with water three times, and dry with anhydrous sodium sulfate, then add to 50 mL of ice ethanol, collect the precipitate, and vacuum dry to obtain the targeting material Cholesterol-PEG 2000 -RT.

[0034] Cholesterol-PEG 2000 -RT of 1 The H NMR analysis results are as follows: 1 H-NMR (400MHz, CDCl3) δ H : 1 H-NMR (400MHz, CDCl3): δ H0.66 (3H, s, CH3), 0.83-0.86 (6H, m, CH3), 0.90 (3H, d, J = 4 Hz), 0.99 (3H, s, CH3), 1.01 (3H, s, CH3), 1.38 (3H, s, CH3), 1.40 (6H, s, CH3), 1.42 (3H, s, CH3), 1.69-2.04 (6H, m, CH), 2.34 (4H, s, CH2), 3.06-3.11 (10H, m, CH2), 3.59-3.81 (194H, m, CH2 and CH2 in PEG), 4.06-4.12 (1H, m, CH=), 5.20-5.36 (2H, m, CH=), 5.71 (1H, s, CH=), 6.08-6.25 (3H, m, CH=), 6.86-6.92 (1H, m, CONH).

[0035] Step (2): 10 mg Cholesterol-PEG 2000 - RT, 10 mg Cholesterol and 80.0 mg Hydrogenated lecithin were dissolved in 5 mL chloroform, ultrasonic at room temperature for 5 min until completely dissolved, remove the organic phase slowly by rotary evaporation under reduced pressure, add 3 mL 0.1 M citric acid and hydrate for 30 min. The liposome suspension after hydration was collected in an EP tube, and ultrasonic with a probe for 5 min under ice bath conditions. Then 35 mg hydroxychloroquine sulfate was added to the above suspension to dissolve, and then an appropriate amount of 0.2 M Na2PHO4 was added to adjust the pH to neutral, and incubated at 37°C on a constant temperature shaking incubator for 1 h, and the precipitate was obtained by high speed centrifugation at 26000 rpm for 30 min, and then resuspended in water and filtered through 0.8 μm and 0.22 μm microporous filter membranes to obtain hydroxychloroquine-loaded targeting liposomes.

[0036] Step (3): Take 2.0 mg of exosome suspension and add 60 mg of HCQ@RT-Lip, mix well and then extrude 15 times through 400 nm and 200 nm polycarbonate membranes with a liposome extruder to obtain the double-membrane fusion targeted activated hepatic stellate cell nanodrug delivery system HCQ@RT-Lip-Exo. The drug loading of HCQ was determined by HPLC.

[0037] The particle size, PDI and zeta potential of the HCQ@RT-Lip-Exo nanodrug delivery system were determined as shown in Table 1. Figure 1 The particle size of the nanodrug delivery system was 152.6 ± 0.77 nm, the distribution coefficient PDI was 0.119 ± 0.013, the particle size distribution range was narrow (PDI < 0.3), and a uniformly dispersed nanodrug delivery system was obtained, and the drug loading of HCQ was 5.49%.

[0038] Example 2

[0039] Evaluation of the targeting of the double-membrane fusion targeted nanodelivery system HCQ@RT-Lip-Exo

[0040] The targeting was evaluated by RT competitive uptake inhibition experiment. DMEM complete culture medium containing 0 mg / mL and 3 mg / mL of RT was incubated with aHSCs cells for 2 h, and then the cells were incubated with NBD fluorescently labeled HCQ@Lip-Exo-NBD and HCQ@RT-Lip-Exo-NBD (HCQ concentration 50 μM) for 2 h, washed the cell surface with PBS for 2 times, fixed with 4% paraformaldehyde for 20 min, incubated with 1 μg / mL of DAPI for 15 min, washed the cell surface with PBS for 2 times, and observed the cell uptake by laser confocal microscopy.

[0041] The results of the RT competitive uptake experiment show that Figure 2 ), after the addition of free RT, the competitive binding of RT and retinol binding protein receptor on the surface of aHSCs cells, the cell uptake of HCQ@RT-Lip-Exo-NBD nanoparticles was significantly reduced, but the cell uptake of HCQ@Lip-Exo-NBD nanoparticles without RT target head was not affected. It can be seen that RT can produce competitive uptake inhibition of HCQ@RT-Lip-Exo-NBD nanoparticles, which verifies that the constructed nanodelivery system can achieve active targeted delivery and treatment of aHSCs.

[0042] Example 3

[0043] In vitro anti-hepatic fibrosis activity study of the double-membrane fusion targeted nanodelivery system HCQ@RT-Lip-Exo

[0044] HSCs cells were inoculated in a 24-well cell culture plate at 1 × 10 5 After adhering, except for the control group, the rest of the groups were activated with 10 ng / mL of TGF-β1 for 24 h. HCQ, Exo, HCQ@Lip-Exo and HCQ@RT-Lip-Exo were added for treatment (HCQ dose was 50 μM) and incubated for 24 h. Washed with PBS for 3 times, fixed with 4% paraformaldehyde for 20 min, washed with PBS for 3 times, incubated with 0.1% Triton-X-100 at room temperature for 10 min. After washing with PBS for 3 times, add 5% serum at room temperature for blocking for 30 min, primary antibody (α-SMA and Collagen I antibody) was blocked at 4°C overnight, and then fluorescent secondary antibody was blocked at room temperature for 1 h. The expression of α-SMA and Collagen I protein was observed by laser confocal microscopy.

[0045] As Figure 3As shown, the in vitro anti-hepatic fibrosis experiment results show that the expression amounts of fibrosis related proteins a-SMA and Collagen I are reduced after treatment by the drugs of each group, and the double-membrane fusion targeted nano drug delivery system HCQ@RT-Lip-Exo group shows the best in vitro anti-hepatic fibrosis effect.

[0046] Example 4

[0047] In vivo anti-hepatic fibrosis activity research of the double-membrane fusion targeted nano drug delivery system HCQ@RT-Lip-Exo

[0048] BALB / c mice of 6-8 weeks old were selected to construct a hepatic fibrosis mouse model by intraperitoneal injection of thioacetamide for 5 weeks (100 mg / kg in the first week, 200 mg / kg in the 2nd-5th week), and were randomly divided into 6 groups (6 mice in each group). From the second week of modeling, the mice were treated by intravenous injection of saline, HCQ, Exo, HCQ@Lip-Exo and HCQ@RT-Lip-Exo (the dose of HCQ was 20 mg / kg) every other day. After 35 days, the in vivo anti-hepatic fibrosis activity of each treatment group was evaluated. 24 hours after the last administration, the mice were sacrificed to collect heart, liver, spleen, lung and kidney tissues. The heart, liver, spleen, lung and kidney tissues of the mice were fixed in 4% paraformaldehyde, paraffin-embedded, and processed into 4 mm thick sections. The heart, spleen, lung and kidney paraffin sections were stained with HE, and the liver was stained with HE, Sirius red and Masson, respectively. The stained sections were observed by upright microscope.

[0049] The in vivo anti-hepatic fibrosis activity evaluation experiment shows that after treatment by the HCQ@RT-Lip-Exo nanoparticles, the liver fibrosis deposition is significantly reduced, the liver lobule structure is complete, and no obvious fibrous septum is found, which shows the best anti-hepatic fibrosis effect compared with other treatment groups Figure 4 ). At the same time, no obvious organ damage was found from the HE staining results of the heart, spleen, lung and kidney, as Figure 5 shown. Therefore, the HCQ@RT-Lip-Exo nano drug delivery system constructed in the application not only has excellent anti-hepatic fibrosis effect, but also has good treatment safety, which has important clinical guiding significance for the research of hepatic fibrosis treatment drugs.

Claims

1. A method for preparing a double-membrane fusion targeted nano-delivery system, characterized by The method comprises the following steps: Cholesterol-PEG 2000 -NH2 and retinol treated by acylation reaction, and the two are obtained by amidation reaction. Cholesterol-PEG 2000 -RT, the obtained targeted material Cholesterol-PEG 2000 -RT and cholesterol, hydrogenated lecithin to prepare the targeted modified liposome, and load hydroxychloroquine, and finally get double membrane fusion targeted activated hepatic stellate cell nano drug delivery system by membrane extrusion.

2. The preparation method of the dual-membrane fusion targeted nanodrug delivery system according to claim 1, characterized in that... The specific steps of the preparation method of the double-membrane fusion targeted nanodelivery system are as follows: Step (1): Retinol was stirred at room temperature until it was completely dissolved in chloroform to obtain a chloroform solution. Succinic anhydride and triethylamine were added to the chloroform solution, which was stirred at room temperature for 48 h. The chloroform organic layer was washed with water three times, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide were added. The reaction solution A was obtained after stirring at room temperature for 1 h and was ready for use. Cholesterol-PEG 2000 -NH2 and triethylamine were added to the reaction solution A, which was stirred at the reaction temperature for 0.5-50 h to obtain reaction solution B. The reaction solution B was washed with water three times and dried over anhydrous sodium sulfate. Then the organic phase was added to ice ethyl ether, the precipitate was collected, and vacuum drying was performed to obtain the carrier material Cholesterol-PEG 2000 -RT; Step (2): Cholesterol-PEG 2000 -RT, cholesterol and hydrogenated lecithin were dissolved in chloroform, the organic solvent was removed by rotary evaporation under reduced pressure, 0.1 mol / L sodium citrate was added to adjust the pH value to 1-6.5, and the hydration time was 10-180 min; the obtained liposome suspension after hydration was collected in a centrifuge tube, and the probe was used for ultrasonic treatment at 0°C for 1-60 min, then hydroxychloroquine sulfate was added to the liposome suspension for dissolution, 0.2 mol / L Na2HPO4 was added until the pH was adjusted to 7, and the mixture was incubated at 37°C on a constant temperature shaker for 0.5-4 h; the precipitate was obtained by high-speed centrifugation, resuspended with water, and then filtered through 0.8 μm and 0.22 μm microporous filters, respectively, to obtain hydroxychloroquine-loaded targeted liposomes; Step (3): uniformly mixing the hydroxychloroquine-loaded targeted liposome with the exosome, and extruding the mixture 1-20 times by using a 400 nm and 200 nm particle size liposome extruder respectively to obtain the double-membrane fusion targeted activated hepatic stellate cell nanodelivery system.

3. The method according to claim 1, wherein the method comprises the following steps: In the step (1), the molar ratio of retinol, triethylamine and succinic anhydride is 1:1:1-1:10:15; the molar ratio of retinol, EDC and NHS is 1:0.5:0.5-1:20:30; the reaction solution A is added with Cholesterol-PEG 2000 -NH2 when the molar ratio of Cholesterol-PEG 2000 -NH2 and retinol is 1:4-1:20, the molar ratio of triethylamine and Cholesterol-PEG 2000 -NH2 is 1:2-20:

1.

4. The method according to claim 1, wherein the method comprises the following steps: In step (1), the reaction temperature is 0-100 DEG C.

5. The method according to claim 1, wherein the method comprises the following steps: In the step (2), Cholesterol-PEG 2000 - The mass ratio of RT, cholesterol and hydrogenated lecithin is 1:1:5-1:1:10; the mass ratio of hydroxychloroquine sulfate and the liposome suspension is 1:1-1:

20.

6. The method according to claim 1, wherein the method comprises the following steps: In step (3), the mass ratio of the hydroxychloroquine-loaded targeted liposome to the exosome is 1:1-50:1.

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