Preparation and application of quadruple bioactive molecule engineered fusion extracellular vesicle

Abstract

The invention discloses preparation and application of quadruple bioactive molecule engineered fusion extracellular vesicles, and relates to the technical field of biology. The preparation method of the quadruple bioactive molecule engineered fusion extracellular vesicles comprises the step of fusing human umbilical cord mesenchymal stem cell extracellular vesicles and M2 type macrophage extracellular vesicles by adopting a polyethylene glycol induction method to prepare the quadruple bioactive molecule engineered fusion extracellular vesicles. Meanwhile, the quadruple bioactive molecule engineered fusion extracellular vesicle can be used as a nano-carrier, and functional molecules are loaded through an electrotransduction and co-incubation method, so that the effects of dual targeting on damaged liver, promotion of hepatocyte regeneration, anti-inflammation and anti-inflammation and / or real-time tracing imaging are achieved, and the curative effect of the quadruple bioactive molecule engineered fusion extracellular vesicle is improved. Therefore, the dual self-targeting acute hepatic failure can be synergistically and efficiently treated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the preparation and application of an engineered fusion extracellular vesicle of quadruple bioactive molecules. Background Art

[0002] Extracellular vesicles (EVs) have shown great application prospects in the efficient treatment of acute liver failure (ALF) due to their nano-size effect, low immunogenicity, and good biocompatibility. However, the weak targeting, limited function and therapeutic effect of single EVs have seriously hindered the application and development of EVs in disease treatment.

[0003] Hybrid membrane nanovesicles (HMNVs) are vesicles formed by the fusion of homologous or heterologous membrane substances that are naturally secreted or artificially synthesized. They have various preparation methods. While avoiding some inherent problems of natural vesicles, they also have the multifunctional characteristics of modified vesicles. They have gradually become a substitute for natural vesicles and have been widely studied and developed. HMNVs have significant advantages: (1) They can be prepared on a large scale. Compared with the low yield and strong restrictions of traditional EVs, fusion nanovesicles can be prepared through a variety of preparation methods to obtain sufficient yield to meet experimental needs. (2) Strong targeting. Vesicle fusion "inherits" the characteristics of both parental cells and strengthens the dual targeting effect. (3) High biosafety. The preparation ingredients are clear, and most of them are mainly composed of biological drug-carrying ingredients, which reduces the safety risk of pure nanomedicines. (4) Multifunctionality. Breaking the limitation of the single function of natural EVs, HMNVs are modified by multiple methods, providing more application possibilities. At this stage, the application of HMNVs is becoming more and more extensive, which provides a new development idea for the biomedical application of EVs.

[0004] The present invention intends to develop a quadruple bioactive molecule engineered fusion extracellular vesicle to achieve efficient synergistic treatment of dual self-targeted acute liver failure. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation and application of a quadruple bioactive molecule engineered fusion extracellular vesicle to solve the problems of the above-mentioned prior art. The quadruple bioactive molecule engineered fusion extracellular vesicle can achieve efficient synergistic treatment of dual self-targeted acute liver failure.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing quadruple bioactive molecule engineered fusion extracellular vesicles for treating acute liver failure, comprising the step of fusing human umbilical cord mesenchymal stem cell extracellular vesicles and M2 macrophage extracellular vesicles by a polyethylene glycol induction method to prepare the quadruple bioactive molecule engineered fusion extracellular vesicles.

[0008] Furthermore, the particle number ratio of the human umbilical cord mesenchymal stem cell extracellular vesicles and the M2 macrophage extracellular vesicles is 1:1.

[0009] Furthermore, the polyethylene glycol is PEG4000.

[0010] The present invention also provides a quadruple bioactive molecule engineered fusion extracellular vesicle prepared according to the above preparation method.

[0011] The present invention also provides a method for preparing drug-loaded fusion vesicles, comprising the steps of uniformly mixing the above-mentioned quadruple bioactive molecule engineered fusion extracellular vesicles with a solution of functional molecules, and then performing electro-conversion and co-incubation treatment to prepare drug-loaded fusion vesicles loaded with the functional molecules.

[0012] Furthermore, the functional molecules include hepatocyte growth factor (HGF) and polyene phosphatidyl choline (PPC).

[0013] Furthermore, the functional molecules also include fluorescent imaging tracer molecules. The fluorescent imaging tracer molecules may be indocyanine green (ICG).

[0014] Furthermore, the parameters of the electrical conversion are: voltage 100V, resistance 200Ω, capacitance 100μF.

[0015] The present invention also provides a drug-loaded fusion vesicle prepared according to the above preparation method.

[0016] The present invention also provides the use of the above-mentioned quadruple bioactive molecule engineered fusion extracellular vesicles or drug-loaded fusion vesicles in the preparation of drugs for treating acute liver failure.

[0017] The present invention also provides a drug for treating acute liver failure, wherein the active ingredients include the above-mentioned quadruple bioactive molecule engineered fusion extracellular vesicles or drug-loaded fusion vesicles.

[0018] The present invention discloses the following technical effects:

[0019] The present invention successfully and innovatively prepares a molecularly engineered fusion extracellular vesicle for efficient synergistic treatment of dual self-targeted acute liver failure. The fusion extracellular vesicle is prepared by fusion of human umbilical cord mesenchymal stem cell extracellular vesicles (MSC-EVs) with homing targeting and pro-regeneration effects and interleukin-4 polarization-induced M2 macrophage extracellular vesicles (M2-EVs) with inflammatory targeting and anti-inflammatory and anti-inflammatory effects.

[0020] At the same time, the fusion vesicles can be used as nanocarriers, and liver growth factor (HGF), liver protection drug polyene phosphatidylcholine (PPC) and real-time tracer molecule indocyanine green (ICG) can be loaded into the fusion vesicles through electrotransduction and co-incubation methods, thereby playing the role of dual targeting of damaged liver, promoting liver cell regeneration, anti-inflammatory and real-time tracer imaging, thereby achieving synergistic and efficient treatment of dual self-targeted acute liver failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Fluorescence microscopic observations of the mixed group MIX and the fusion group FuEVs;

[0023] Figure 2 TEM morphology of FuEVs;

[0024] Figure 3 This is the detection result diagram of the particle size and Zeta potential of FuEVs;

[0025] Figure 4 It is a statistical graph of cell activity under different PPC concentrations;

[0026] Figure 5 It is a statistical graph of cell activity under different HGF concentrations;

[0027] Figure 6 Characterization diagram of drug-loaded fusion vesicles HP@FuEVs; a: TEM morphology of HP@FuEVs; b: particle size and potential of HP@FuEVs; c: seven-day stability results of HP@FuEVs; d: UV absorption spectrum of HGF; e: UV absorption spectrum of PPC;

[0028] Figure 7Characterization diagram of drug-loaded fusion vesicles HIP@FuEVs; a: TEM morphology of HIP@FuEVs; b: particle size and potential of HIP@FuEVs; c: UV absorption spectrum of ICG; d: standard curve of ICG;

[0029] Figure 8 The tracing images of free ICG and HIP@FuEVs in mice at different times;

[0030] Fig. 9 The tracing images of free ICG and HIP@FuEVs in the main organs of mice (a) and the statistical graph of fluorescence intensity (b);

[0031] Fig.10 This is a graph evaluating the therapeutic effect of HP@FuEVs on mice with acute liver failure after surgical resection; a: weight change of mice in different treatment groups after 14 days; bf: serum biochemical indexes ALT, AST, BUN, Crea and TBiL of mice in different treatment groups after 14 days of treatment; gh: serum inflammatory factors IL-6 and TNF-α of mice in different treatment groups after 14 days of treatment; i: statistical graph of liver growth index of mice in different treatment groups after 14 days;

[0032] Fig.11 H&E staining for the biosafety assessment of HP@FuEVs in mice with acute liver failure model established by surgical liver resection. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The present invention provides a molecularly engineered fusion extracellular vesicle for efficient synergistic treatment of dual self-targeted acute liver failure. First, extracellular vesicles (MSC-EVs) derived from human umbilical cord mesenchymal stem cells and extracellular vesicles (M2-EVs) derived from interleukin-4 polarized M2 macrophages were extracted respectively by differential centrifugation. Subsequently, the two EVs from different sources were fused by a polyethylene glycol (PEG)-induced method to obtain fusion vesicles (FuEVs). The FuEVs not only inherit the ability of mesenchymal stem cells to target inflammation and promote regeneration, but also have the inflammatory targeting and anti-inflammatory functions of M2 macrophages. In the treatment of ALF, dual self-targeting effects and synergistic therapeutic effects can be achieved for damaged livers. In order to further improve the therapeutic effect of FuEVs and monitor its distribution and metabolism in the body in real time, the present invention loaded three functional molecules in FuEVs by electroporation and co-incubation, namely, hepatocyte growth factor (HGF), hepatoprotective drug polyene phosphatidyl choline (PPC) and fluorescent imaging tracer molecule indocyanine green (ICG), and prepared two molecularly engineered fusion EVs, HP@FuEVs and HIP@FuEVs. Finally, the regenerative and anti-inflammatory effects of HP@FuEVs at the cellular and animal levels were explored, and the targeting ability and tracer imaging ability of HIP@FuEVs on damaged livers were evaluated, and the safety and effectiveness of the drug-carrying system in the treatment of ALF were comprehensively evaluated.

[0039] Example 1

[0040] 1. Construction of FuEVs

[0041] (1) MSC-EVs and M2-EVs were extracted by differential centrifugation. The detailed steps are as follows: Two types of extracellular vesicles (EVs) were extracted by differential ultracentrifugation. Taking the extraction of MSC-EVs as an example, the culture medium after 48 hours of cell culture was first collected. Then, the culture medium was centrifuged at 300g for 15 minutes (4°C) to remove residual cells. Next, the supernatant was taken and centrifuged at 2000g for 15 minutes (4°C) to remove cell debris. After discarding the precipitate, continue to centrifuge at 10,000g for 30 minutes (4°C) to remove apoptotic bodies and larger vesicles. At this time, the supernatant was filtered through a 0.22μm filter membrane to remove potential bacterial contamination. Then, an ultracentrifuge was used to centrifuge at 100,000g for 70 minutes (4°C) and then at 120,000g for 70 minutes (4°C), and the supernatant was discarded after each centrifugation. Finally, the pellet was resuspended in an appropriate amount of sterile PBS (200 μL) and gently mixed to obtain MSC-EVs suspension. Afterwards, MSC-EVs were stored at -80 °C or in liquid nitrogen to avoid repeated freezing and thawing. For the extraction of M2-EVs, the same steps were followed, but differential ultracentrifugation was performed using the conditioned medium after 24 h of cell polarization.

[0042] (2) The particle numbers of MSC-EVs and M2-EVs were measured using NTA, and the measured particle numbers and concentrations were similar. Therefore, 200 μL of EVs were added to each at a volume ratio of 1:1, and the mixture was placed in a 50-mL centrifuge tube. Then, 400 μL of PEG4000 solution with the same volume as the mixed liquid was slowly added dropwise to the centrifuge tube. After gently shaking, the mixture was allowed to stand at room temperature and the reaction was timed for 10 min.

[0043] (3) Prepare a serum-free pure culture medium in advance. After the reaction in step (2) is completed, first slowly add 4 mL of pure culture medium (5 times the total volume of the solution in the centrifuge tube) to the centrifuge tube, and control the addition time within 2 minutes. Then continue to add 12 mL to make the added pure culture medium 20 times the total volume. Then transfer the liquid in the centrifuge tube to a culture dish and incubate at 37°C for 40 minutes.

[0044] (4) After the incubation, the liquid was transferred to a 100 KD ultrafiltration tube and centrifuged at 8500 rpm and 4°C for 10 min. The liquid remaining in the ultrafiltration tube was collected as FuEVs.

[0045] 2. Fusion Verification of FuEVs

[0046] Laser confocal microscopy and flow cytometry were used to verify the fusion of FuEVs. The detailed steps are as follows:

[0047] (1) Similar to the confocal laser characterization of single extracellular vesicles, the verification of FuEVs requires the simultaneous addition of two fluorescent-labeled antibodies for marker proteins, FITC-anti-human CD105 for MSC-EVs and APC-anti-mouse CD206 for M2-EVs. If two overlapping fluorescences are observed at the same time under 100× confocal microscope, it proves that the vesicles have successfully fused.

[0048] (2) Flow cytometry also uses two fluorescently labeled antibodies to simultaneously label FuEVs, which can not only verify fusion but also quantify the fusion rate.

[0049] The prepared FuEVs were observed by laser confocal microscopy using fluorescent labeling dyes of two EVs marker proteins CD105 and CD206 antibodies. Figure 1 Compared with the MIX group in which MSC-EVs and M2-EVs were simply mixed, FuEVs showed a yellow color after the fusion of the two EVs, proving that the fusion vesicles were successfully prepared.

[0050] Fusion was verified by using surface marker proteins and other properties of FuEVs. FuEVs still need to be structurally characterized. TEM was used to characterize the morphology of FuEVs, NTA was used to measure the particle size, and the Malvern particle sizer was used to measure the Zeta potential to complete the overall characterization of FuEVs. The results are shown in Figure 2-Figure 3 The results showed that the vesicles in FuEVs had obvious fusion phenomenon, and their particle size was about 240.9nm and the potential was about -13.28mV.

[0051] Example 2

[0052] After the fusion vesicles FuEVs are successfully prepared, the liver protection drugs polyene phosphatidylcholine (PPC), hepatocyte growth factor (HGF) and real-time tracer molecule indocyanine green (ICG) need to be loaded into the fusion vesicles according to the needs. Among them, the fusion vesicles FuEVs loaded only with PPC and HGF are named HP@FuEVs, and the fusion vesicles FuEVs loaded with PPC, HGF and ICG are named HIP@FuEVs.

[0053] 1. Screening of drug concentration

[0054] When using electroporation to deliver drugs, it is necessary to consider the toxic side effects of excessively high drug concentrations on cells, so the drug concentration must be screened first.

[0055] (1) PPC concentration screening: PPC was dissolved in a mixture of methanol and water at a ratio of 1:9, and then the whole culture medium was used to prepare PPC solutions with concentrations of 0, 2.5, 5, 10, 20, 50, 100, 200, and 500 μM. After the solution was prepared, the MTT method was used to determine the toxicity of PPC to cells. The experimental steps are as follows:

[0056] ① Mouse hepatocytes AML12 were cultured at 10 4 The cells were seeded at a density of 1.54 × 10 / well in a 96-well plate and cultured in a carbon dioxide incubator at 37°C for 24 h.

[0057] ② Use a pipette to discard the old culture medium, and add the prepared culture medium containing different concentrations of PPC to the 96-well plate in appropriate amounts. The corresponding blank control group only adds the special culture medium. Be careful to pipette and add gently. After adding the sample, place the cells in the incubator and incubate for 24 hours.

[0058] ③ After the incubation, the culture medium in each well was discarded, and a 10% volume concentration of MTT solution (the solution was prepared by diluting the culture medium) was added thereto, and incubated in the dark for 4 hours.

[0059] ④After the incubation, immediately add 110 μL of DMSO to each well to dissolve the crystalline formazan produced in the living cells to facilitate the subsequent determination of the absorbance value.

[0060] ⑤ Set the parameters of the microplate reader, incubate the 96-well plate in the microplate reader with shaking for 15 minutes, and measure its absorbance at 490nm. The value represents the strength of cell activity, which is ultimately used to screen the appropriate PPC concentration.

[0061] result( Figure 4 ) showed that when the concentration of PPC was 100 μM, it would not cause damage to normal liver cells. Therefore, the concentration of PPC was selected to be 100 μM when preparing fully active nanomedicines.

[0062] (2) Optimization of HGF concentration: Dissolve the lyophilized HGF standard in pure culture medium to prepare gradient solutions with concentrations of 0, 20, 50, 100, 200, and 400 ng / mL. The preparation steps are as follows:

[0063] First, place the lyophilized cytokine standard at room temperature for 20 minutes. Then centrifuge at 11000rpm, 4℃ for 1 minute; and 3500rpm, 4℃ for 5 minutes. After centrifugation, add an appropriate amount of sterile water or culture medium to the lyophilized standard tube in a sterile operating table to prepare an HGF stock solution of appropriate concentration. Subsequently, dilute the solution to different concentrations with PBS containing 5% seaweed protein and store it in a -20℃ freezer or use it directly. As described in the operation of screening the appropriate PPC solution concentration, mix different concentrations of HGF with cells and incubate them, measure the relevant MTT value, measure the effect of HGF on cell viability, and finally determine the appropriate HGF concentration.

[0064] result( Figure 5 ) showed that when the concentration of HGF was 200ng / mL, it would not cause damage to normal liver cells. Combining literature review and experience accumulated within the research group, the concentration of HGF selected for the preparation of fully active nanomedicines was 200ng / mL.

[0065] (3) Determination of ICG concentration: In the present invention, ICG mainly plays a role in observing the ability of cells to take up materials and observing the real-time tracing process of materials in liver failure model mice. Therefore, based on the principles of saving materials, simple operation and ensuring safety, the present invention uses HP@FuEVs as the main research object in all other stages. Therefore, through a large number of article reviews and experience accumulation within the research group, the present invention directly determined the appropriate ICG concentration, i.e. 100 μg / mL, and the prepared HIP@FuEVs will play a role at the appropriate time.

[0066] 2. Preparation and characterization of drug-loaded fusogenic vesicles HP@FuEVs

[0067] 2.1 Preparation of drug-loaded fusogenic vesicles HP@FuEVs

[0068] The PPC, HGF and FuEVs with appropriate concentrations obtained by screening were mixed in a certain ratio, and the drugs were introduced into FuEVs by electroporation. After a series of operations such as incubation and ultrafiltration, the HP@FuEVs drug delivery system was obtained. The specific construction process is as follows:

[0069] ① Mix the prepared HGF solution (200 ng / mL, 135 μL), PPC solution (100 μM, 135 μL) and FuEVs (1.2 mg / mL, 30 μL), transfer the mixture to an electroporation cup pre-cooled on ice, set the parameters of the electroporator to voltage 100 V, resistance 200 Ω, capacitance 100 μF, and perform electroporation according to the instructions.

[0070] ② After electroporation, transfer the sample in the electroporation cup to a sterile culture dish, mark it and place it in a carbon dioxide incubator for incubation for 1 hour to promote membrane repair.

[0071] ③After the incubation period, transfer the liquid in the culture dish to a 100KD ultrafiltration tube and centrifuge at 8500rpm and 4°C for 10 minutes to remove various free drugs. ④Collect the sample retained in the ultrafiltration tube, which is the drug-loaded fusion vesicle HP@FuEVs.

[0072] 2.2 Characterization of drug-loaded fusogenic vesicles HP@FuEVs

[0073] 2.2.1 TEM morphology, particle size and Zeta potential characterization

[0074] The TEM morphology, particle size and Zeta potential of HP@FuEVs were detected. Figure 6 a and b.

[0075] At the same time, the seven-day stability of HP@FuEVs was measured. The stability measurement operation is as follows: first, the frozen drug-loaded fusion vesicles HP@FuEVs were thawed on ice, and then the changes in the particle size and potential of the drug-loaded fusion vesicles HP@FuEVs in sterile PBS were measured at the same time point for 7 consecutive days. Finally, the dispersion of the material was presented in the form of a change curve (the smaller the change, the more stable the material). The results showed that the particle size and potential of HP@FuEVs did not change significantly, and it had excellent stability ( Figure 6 (c).

[0076] 2.2.2 Determination of drug loading

[0077] After preparing the HP@FuEVs drug delivery system, it is necessary to verify the success of drug loading and determine the loading amount of various drugs. The experiment can directly prove the success of drug loading of fusion vesicles while determining the drug loading amount by ultraviolet absorption method.

[0078] (1) Determination of HGF drug loading:

[0079] The HGF loading was determined by ELISA enzyme-linked immunosorbent assay. According to the instructions of the human HGF ELISA kit, the washing solution and working solution were first prepared, and the standard sample and HP@FuEVs sample were diluted with the standard diluent. Then, the samples were incubated and reacted for 90, 60, 30, and 15 minutes. Finally, the absorbance of the sample and the standard sample at 450nm was measured in an ELISA instrument, and the HGF loading in the HP@FuEVs sample was calculated based on the HGF concentration standard curve.

[0080] The UV absorption spectrum of HGF is shown in Figure 6 The drug loading and encapsulation efficiency are shown in Table 1.

[0081] (2) Determination of drug loading of PPC:

[0082] ① Use a mixed solution of methanol and water to prepare PPC solutions with gradient concentrations of 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 μM.

[0083] ② Transfer the PPC solutions of different concentrations to the cuvettes for UV spectrophotometer, calibrate the measurement baseline of UV spectrophotometer, put them into the cuvettes, and measure the absorbance of samples of different concentrations. At the same time, dilute HP@FuEVs according to appropriate multiples and measure their absorbance under the same baseline. In order to prevent methanol from volatilizing during the experimental operation, the measurement error can be minimized by preparing and measuring immediately.

[0084] ③ Calculate the standard absorption curve according to the absorbance value of the PPC solution, and calculate and obtain the drug loading amount of PPC in the HP@FuEVs drug delivery system.

[0085] The UV absorption spectrum of PPC is shown in Figure 6 In e, drug loading and encapsulation efficiency are shown in Table 1.

[0086] Table 1 Drug loading and encapsulation efficiency of HGF and PPC in HP@FuEVs

[0087] project Encapsulation efficiency Drug loading HGF 82.5±7.4％ 12.1±1％ PPC 61.3±6.8％ 3.9±0.4％

[0088] 3. Preparation and Characterization of Drug-Loaded Fusogenic Vesicles HIP@FuEVs

[0089] 3.1 Preparation of drug-loaded fusogenic vesicles HIP@FuEVs

[0090] The PPC, HGF, ICG and FuEVs with appropriate concentrations obtained by screening were mixed in a certain ratio, and the drugs were introduced into FuEVs by electroporation. After a series of operations such as incubation and ultrafiltration, the HIP@FuEVs drug delivery system was obtained. The specific construction process is as follows:

[0091] ① Mix the prepared HGF solution (200 ng / mL, 140 μL), PPC solution (100 μM, 140 μL), ICG solution (100 μg / mL, 80 μL) and FuEVs (1.2 mg / mL, 40 μL), transfer the mixture to an electroporation cup pre-cooled on ice, set the parameters of the electroporator to 100 V, 200 Ω, 100 μF, and perform electroporation according to the instructions.

[0092] ② After electroporation, transfer the sample in the electroporation cup to a sterile culture dish, mark it and place it in a carbon dioxide incubator for incubation for 1 hour to promote membrane repair.

[0093] ③ After the insulation is completed, the liquid in the culture dish is transferred to a 100KD ultrafiltration tube and centrifuged at 8500rpm and 4℃ for 10 minutes to remove various free drugs.

[0094] ④ Collect the samples retained in the ultrafiltration tube, which are the drug-loaded fusion vesicles HIP@FuEVs.

[0095] 3.2 Characterization of drug-loaded fusogenic vesicles HIP@FuEVs

[0096] The concentration of ICG was determined by ultraviolet absorption method. Figure 7 d) and absorption spectra ( Figure 7 c), verifying the successful drug loading of ICG and analyzing its TEM morphology ( Figure 7 a), particle size and potential ( Figure 7 b), which laid the foundation for the subsequent use of HIP@FuEVs in in vivo experiments.

[0097] 3.3 Evaluation of the targeting and tracing imaging capabilities of drug-loaded fusogenic vesicles HIP@FuEVs on damaged liver

[0098] Acute liver failure model mice were established by surgical resection, and the model mice were randomly divided into two groups. Free ICG solution and HIP@FuEVs solution (ICG concentration in the two solutions was the same) were injected through the tail vein, respectively. The imaging tracing of free ICG and HIP@FuEVs in mice was observed at the time after injection (0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h). The results are shown in Figure 8 The results showed that compared with free ICG, HIP@FuEVs could quickly target and accumulate in the damaged liver 2 hours after intravenous injection, and the content was significantly higher than that of free ICG. In order to further verify the targeted aggregation ability of HIP@FuEVs in the damaged liver, the mice were euthanized and dissected 2 hours after tail vein injection, and the fluorescence intensity of free ICG and HIP@FuEVs in the main organs of the mice (heart, liver, spleen, lung, and kidney) was observed and compared. The results are shown in Fig. 9 The results showed that the fluorescence intensity of HIP@FuEVs in the damaged liver was much higher than that of free ICG, demonstrating the good targeting ability of the nanomedicine to the damaged liver in vivo.

[0099] Example 3

[0100] In order to further evaluate the therapeutic effect of HP@FuEVs in vivo, mice with acute liver failure model established by surgical resection were randomly divided into 6 groups, namely healthy group (WT group), model group (PBS group), HGF group, PPC group, FuEVs group and HP@FuEVs group. The 6 groups were injected with corresponding materials, weighed every two days, and the treatment records were kept for 14 days, and the weight change map was drawn (see Fig.10 In (a), it was observed that there was no significant change in the body weight of mice in each group.

[0101] At the same time, the serum of mice in each group was taken to measure the biochemical indexes and inflammatory factor indexes: AST, ALT, Crera, BUN, T-Bil, IL-6, TNF-α. The results are shown in Fig.10 Compared with the PBS group, the serum biochemical indexes and inflammatory factors of the other groups, especially the HP@FuEVs group, were close to normal levels, proving that the HP@FuEVs material can eliminate liver inflammation to a certain extent and promote the repair of the liver and kidneys.

[0102] At the same time, the mice in each group were dissected after euthanasia, the liver weight was weighed, and the liver growth index after 14 days of treatment was calculated. The results are shown in Fig.10 The experiment found that compared with the PBS group, the HP@FuEVs group had a higher liver growth index, which could promote the regeneration and repair of damaged liver cells to a certain extent.

[0103] In order to investigate the biosafety of HP@FuEVs in mice with acute liver failure after surgical resection, the HP@FuEVs group mice were euthanized after 14 days of treatment, and their hearts, spleens, lungs, kidneys and intestines were removed after dissection. H&E stained sections of each organ were then prepared and compared with those of the healthy group of mice. The sections showed that the mice's organs did not show any damage or lesions ( Fig.11 ), indicating that the material HP@FuEVs has good biosafety and can be used for the treatment of acute liver failure.

[0104] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing quadruple bioactive molecule engineered fusion extracellular vesicles for treating acute liver failure, characterized in that: The method comprises the steps of fusing human umbilical cord mesenchymal stem cell extracellular vesicles and M2 macrophage extracellular vesicles by a polyethylene glycol induction method to prepare the quadruple bioactive molecule engineered fusion extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: The particle number ratio of the human umbilical cord mesenchymal stem cell extracellular vesicles to the M2 macrophage extracellular vesicles is 1:

1.

3. The preparation method according to claim 1, characterized in that: The polyethylene glycol is PEG4000.

4. An engineered extracellular vesicle containing quadruple bioactive molecules prepared according to the preparation method according to any one of claims 1 to 3.

5. A method for preparing drug-loaded fusion vesicles, characterized in that: The method comprises the steps of uniformly mixing the quadruple bioactive molecule engineered fusion extracellular vesicles described in claim 4 with a solution of functional molecules, and then performing electro-conversion and co-incubation treatment to prepare drug-loaded fusion vesicles loaded with the functional molecules.

6. The preparation method according to claim 5, characterized in that: The functional molecules include hepatocyte growth factor and polyene phosphatidylcholine.

7. The preparation method according to claim 6, characterized in that: The functional molecules also include fluorescent imaging tracer molecules.

8. A drug-loaded fusion vesicle prepared according to the preparation method according to any one of claims 5 to 7.

9. Use of the quadruple bioactive molecule engineered fusion extracellular vesicles as claimed in claim 4 or the drug-loaded fusion vesicles as claimed in claim 8 in the preparation of a drug for treating acute liver failure.

10. A drug for treating acute liver failure, characterized in that: The active ingredient includes the quadruple bioactive molecule engineered fusion extracellular vesicles as described in claim 4 or the drug-loaded fusion vesicles as described in claim 8.

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