Nano vaccine based on liver cell membrane as well as preparation method and application of nano vaccine

Abstract

The invention discloses a liver cell membrane-based nano vaccine as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: extracting cell membranes of alcoholic fatty liver cells, and then mixing PLGA nanoparticles with the cell membranes to prepare the nano vaccine. The bionic vaccine prepared by the invention is small in size and high in operability in the aspect of manufacturing. According to the bionic vaccine disclosed by the invention, the generation and release of an inflammatory mediator are regulated by displaying an autoantigen, so that the inflammatory injury of the liver is relieved. The autoantigen displayed by the bionic vaccine has high individual specificity, side effects caused by non-specific immune activation can be reduced, and treatment accuracy and safety are improved at the same time.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nano vaccine based on liver cell membrane and a preparation method and application thereof. Background Art

[0002] With the improvement of living standards, the prevalence of alcohol-related liver disease (ALD) in my country has continued to rise, ultimately leading to an increase in cases of end-stage liver disease. ALD is a type of liver disease caused by alcohol dependence and long-term excessive drinking. A cross-sectional analysis of patients with liver disease worldwide showed that in clinical practice, many patients with early forms of viral hepatitis and NAFLD were referred to specialized centers, while ALD patients were rarely discovered in the early stages. There is an urgent need for a new treatment strategy that can effectively reverse ALD in the early stages, thereby avoiding the disease from progressing to a refractory and irreversible late-stage state.

[0003] Although liver toxicity caused by alcohol and its metabolites is a core factor in the pathogenesis of ALD, inflammatory immune damage plays a key role in the pathogenesis of ALD. As one of the important immune organs of the human body, the liver has a complex immune microenvironment composed of a variety of immune cells and cytokines, which jointly participate in the immune regulation of the liver. The cells in the liver have their own functions and are interrelated. The immune microenvironment not only plays an important role in maintaining liver homeostasis, but also plays an irreplaceable role in the occurrence and development of liver disease. Inflammatory immune damage to the liver plays an important role in the occurrence and development of ALD, and is also a potential target for targeted treatment of ALD.

[0004] The development of nanodelivery systems provides an effective platform for the integration of multifunctional candidate systems. Currently, a variety of nanodelivery systems have been developed, including nanoliposomes, polymers, and self-assembled nanomaterials. In recent years, biomimetic drug delivery systems, especially cell membrane-coated nanoparticles (NPs), have attracted increasing attention in the field of nanotherapeutics. By coating the natural cell membrane on the surface of the nanoparticles, the nanoparticles are able to act as decoys and absorb or bind complex pathological molecules to achieve therapeutic effects. In ALD, different types of hepatocyte death, including apoptosis, necrosis, pyroptosis, and ferroptosis, determine the severity of inflammation as well as disease progression. Damaged or stressed hepatocytes can release a large number of proinflammatory mediators, including cytokines and chemokines, which promote the recruitment of immune cells and activate the immune system, thereby promoting liver inflammation in ALD patients.

[0005] The results of searching relevant domestic and foreign literature and patents indicate that there are no reports on using polylactic-co-glycolic acid (PLGA) as a carrier and simultaneously coating AML12 liver cell membranes to prepare nanovaccines for immunotherapy of alcoholic liver disease. Summary of the invention

[0006] The purpose of the present invention is to provide a nano vaccine based on liver cell membrane and its preparation method and application to solve the problems existing in the above-mentioned prior art. The present invention develops liver cell-mimicking nanoparticles to treat ALD, and uses an alcoholic liver mouse model to evaluate the application of the above-mentioned bionic cell membrane nanoparticles in the treatment of alcoholic fatty liver disease.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing a nano vaccine based on mouse liver cell membrane, comprising the following steps:

[0009] The cell membrane of alcoholic fatty liver cells is extracted, and then PLGA nanoparticles and the cell membrane are mixed to prepare the nano vaccine.

[0010] The second technical solution of the present invention is the nano vaccine prepared by the preparation method.

[0011] The third technical solution of the present invention is the use of the nano vaccine in the preparation of drugs for preventing and treating alcoholic liver disease.

[0012] A fourth technical solution of the present invention is a drug for preventing and treating alcoholic liver disease, comprising the nano vaccine.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The bionic vaccine prepared by the present invention is small in size and has strong operability in production. The bionic vaccine of the present invention regulates the production and release of inflammatory mediators by displaying autoantigens, thereby alleviating inflammatory damage to the liver. The autoantigens displayed by the bionic vaccine of the present invention have high individual specificity, can reduce the side effects caused by nonspecific immune activation, and improve the accuracy and safety of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1The preparation and characterization of the alcoholic fatty liver cell membrane bionic nano vaccine constructed in the embodiment of the present invention. Among them, A is the cytotoxicity detection of AML12 liver cells treated with 250mmol / L alcohol and 0.25mmol / L palmitic acid for 6 hours; B is the expression of adipogenic genes and lipid-lowering genes in AML12 liver cells treated with 250mmol / L alcohol and 0.25mmol / L palmitic acid for 6 hours; C is the expression of inflammatory factors in AML12 liver cells treated with 250mmol / L alcohol and 0.25mmol / L palmitic acid for 6 hours; D is the transmission electron microscopy of the alcoholic fatty liver cell membrane bionic nano vaccine; E is the hydrated particle size and Zeta potential of the alcoholic fatty liver cell membrane bionic nano vaccine.

[0017] Figure 2 The alcoholic fatty liver cell membrane bionic nano vaccine constructed in the embodiment of the present invention can be effectively taken up by antigen presenting cell dendritic cells and its distribution in the body. Among them, A is a confocal image of bone marrow-derived dendritic cells (BMDCs) co-incubated with alcoholic fatty liver cell membrane bionic nano vaccine; B is a flow cytometry result diagram; C is an in vivo imaging image of alcoholic fatty liver cell membrane bionic nano vaccine 6, 24 and 48 hours after subcutaneous injection into the vicinity of the right inguinal lymph node of mice.

[0018] Figure 3 The in vivo therapeutic effect of the alcoholic fatty liver cell membrane biomimetic nanovaccine constructed in the embodiment of the present invention. Among them, A is the result of HE and Oil Red O staining of mouse liver; B is the result of mouse body weight, liver weight to body weight ratio, plasma alanine aminotransferase concentration, and plasma triglyceride concentration; C is the result of mouse liver alanine aminotransferase concentration, IL-10 concentration, TGF-β concentration, and IL-6 concentration. DETAILED DESCRIPTION

[0019] 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.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0021] 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.

[0022] 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 those skilled in the art. The present application description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0025] The present invention provides a method for preparing a nano vaccine based on mouse liver cell membrane, comprising the following steps:

[0026] The cell membrane of alcoholic fatty liver cells is extracted, and then PLGA nanoparticles and the cell membrane are mixed to prepare the nano vaccine.

[0027] In some specific embodiments, the method for extracting cell membranes of alcoholic fatty liver cells is: collecting alcoholic fatty liver cells, then disrupting the cells, and collecting membrane precipitates.

[0028] In some specific embodiments, the method for constructing the alcoholic fatty liver cells is: treating the liver cells with 250 mmol / L alcohol and 0.25 mmol / L palmitic acid.

[0029] In some specific embodiments, the method for constructing the alcoholic fatty liver cells is: treating AML12 hepatocytes with 250 mmol / L alcohol and 0.25 mmol / L palmitic acid for 6 hours.

[0030] In some specific embodiments, the method for disrupting cells is: ultrasonic disruption of cells under ice-water bath conditions, 35W, ultrasonic time 5min, ultrasonic 2 seconds / interval 1 second.

[0031] In some specific embodiments, the method for collecting membrane precipitate is: after cell disruption, collect the supernatant at 10000g for 15 min, transfer the supernatant to a Beckman centrifuge tube, weigh and balance it, centrifuge it at 100000g for 2 hours, discard the supernatant, and collect the membrane precipitate.

[0032] In some specific embodiments, the mass ratio of the PLGA nanoparticles to the cell membrane is 5:1.

[0033] The embodiment of the present invention also provides a nano vaccine prepared by the preparation method.

[0034] The embodiment of the present invention also provides the use of the nano vaccine in preparing a drug for preventing and treating alcoholic liver disease.

[0035] The embodiment of the present invention also provides a medicine for preventing and treating alcoholic liver disease, comprising the nano vaccine.

[0036] In some specific embodiments, the drug reduces liver damage by reducing lipid droplet accumulation, downregulating the liver weight / body weight ratio, plasma alanine aminotransferase concentration, serum triglyceride concentration, liver alanine aminotransferase concentration and IL-6 concentration, and upregulating TGF-β concentration and IL-10 concentration.

[0037] Example 1

[0038] Preparation of alcoholic fatty liver cell modeling agents: AML12 hepatocytes were treated with 250mmol / L alcohol and 0.25mmol / L palmitic acid for 6 hours, and then CCK8 was used to detect the cell status. The cell viability was good and the drug treatment did not cause obvious toxicity ( Figure 1 Middle A). The expression of cellular adipogenic genes and lipid-lowering genes was detected. The results showed that the adipogenic genes Srebp1 and ACC were significantly upregulated and the lipid-lowering genes CPT2 and AdipoQ were significantly downregulated in the alcoholic fatty liver cell model group ( Figure 1 Middle B). The expression of cellular inflammatory factors was detected, and the results showed that TNF-ɑ, IL-1β, and IL-6 were significantly upregulated in the alcoholic fatty liver cell model group ( Figure 1 Middle C).

[0039] Extraction of alcoholic fatty liver cell membrane: Wash the cells twice with 2 mL PBS, 1 min each time, then digest the adherent cells with 1 mL trypsin for 2 min, terminate the digestion with 2 mL complete medium, and make a single cell suspension, then collect the cells at 1000 rpm for 5 min. Ultrasonic disruption of cells under ice water bath conditions, 35 W, ultrasonic time 5 min, ultrasonic 2 seconds / interval 1 second, 4 ° C, 10000g, 15 min to collect the supernatant, discard the precipitate, transfer the supernatant to a Beckman centrifuge tube, weigh and balance, centrifuge at 100000g for 2 hours, discard the supernatant, and collect the membrane precipitate. BCA detects membrane protein concentration, 1 mg / tube aliquots stored in a -80 refrigerator.

[0040] Preparation of biomimetic nano-vaccine: PLGA polymer was synthesized by double emulsion method: 180 mg of PLGA was dissolved in 10 mL of dichloromethane, 1.0 mL of ultrapure water was added as the aqueous phase, and the colostrum was formed by ultrasonication for 10 min under an ultrasonic pulverizer (180 W). After adding an emulsifier (2 mL of 1% sodium cholate solution) to the colostrum, the colostrum was ultrasonicated again for 10 min under an ultrasonic pulverizer (180 W) to obtain a double emulsion. The double emulsion was taken to a round-bottom flask and suspended for 10 min, and the organic solvent was evaporated. The finished product was centrifuged at 11000g for 10 min, the supernatant was removed, and the PLGA nanoparticles were obtained by vacuum drying. Then, PLGA nanoparticles and membrane proteins were mixed in PBS at a mass ratio of 5:1, and the biomimetic nano-vaccine was prepared through a 400 nm polycarbonate membrane liposome extruder.

[0041] Transmission electron microscopy results showed that after the liver cell membrane was modified, the PLGA nanoparticles had an obvious membrane structure outside, forming a typical core-shell structure, proving that PLGA successfully modified the cell membrane ( Figure 1 D). The hydrated particle sizes of PLGA nanoparticles and biomimetic nanovaccines are both around 100 nm. After the hepatocyte membrane is modified, the hydrated particle size increases, and the potential changes from the PLGA surface potential to the membrane protein surface potential ( Figure 1 (middle D).

[0042] Nanoparticles can be effectively taken up by BMDCs. The present invention detected the internalization of synthesized membrane nanoparticles by BMDCs. Two fluorescently labeled membrane nanoparticles were prepared. Confocal results showed that three membrane nanoparticles labeled with the cell membrane red fluorescent probe DiD were internalized into BMDCs and localized in the lysosome structure ( Figure 2 Similarly, flow cytometry results showed that BMDCs took up both membrane nanoparticles within 1 hour, and the uptake efficiency of the two membrane nanoparticles was basically the same ( Figure 2Middle B). The biodistribution of the alcoholic fatty liver cell membrane biomimetic nanovaccine was then tested after subcutaneous injection into the right inguinal lymph nodes of mice. The in vivo imaging results of small animals showed that the nanoparticles were present near the injection site for at least 48 hours at 6, 24 and 48 hours after injection ( Figure 2 Middle C).

[0043] Example 2

[0044] Evaluation of the in vivo therapeutic effect of constructed biomimetic nano-vaccines of cell membranes for alcoholic fatty liver disease

[0045] The bionic nano vaccine was injected into the right groin of 6-8 week old ICR mice for a total of 3 immunizations, with an interval of 3 days between each immunization. The short-term alcoholic hepatitis model was established on the seventh day after the last immunization. The experiment was divided into a modeling group, a control group, and an alcoholic fatty liver cell membrane bionic nano vaccine group, with 5 mice in each group.

[0046] The specific implementation plan is: on the seventh day after the last immunization, the model group was given a Lieber-DeCarli control liquid feed (35% fat calories) for 5 days. On the sixth day, the model group was changed to a Lieber-DeCarli 5% (v / v) alcohol liquid feed for a total of 10 days. From 7 to 9 o'clock on the 11th day, the model group was given 5g·kg -1 Ethanol, the control group was given an equal volume of dextrin with equal calories. After 9 h, the mice were killed and blood and liver samples were collected.

[0047] The results showed that the liver damage of mice in the alcoholic fatty liver cell membrane bionic nanovaccine group was alleviated, and the accumulation of lipid droplets was reduced ( Figure 3 A), after treatment, there was no significant difference in the body weight of the three groups of mice. The liver weight / body weight ratio, plasma alanine aminotransferase concentration, and serum triglyceride concentration of the mice in the alcoholic fatty liver cell membrane bionic nanovaccine group were significantly decreased ( Figure 3 Middle B), the concentration of alanine aminotransferase and pro-inflammatory factor IL-6 in mouse liver was significantly downregulated, while the concentration of anti-inflammatory factor TGF-β and IL-10 was significantly upregulated ( Figure 3 (C) shows that the alcoholic fatty liver cell membrane bionic nanovaccine can significantly improve the liver pathology of alcoholic liver mice.

[0048] In summary, the present invention uses AML12 hepatocytes as modeling objects, constructs an alcoholic hepatocyte model by treating with 250mmol / L alcohol and 0.25mmol / L palmitic acid for 6 hours, extracts alcoholic hepatocyte membranes, and then prepares PLGA nanoparticles loaded with alcoholic hepatocyte membranes. The alcoholic liver mouse model is used to evaluate the application of the above-mentioned bionic cell membrane nanoparticles in the treatment of alcoholic fatty liver disease.

[0049] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a nano vaccine based on mouse liver cell membrane, characterized in that: The following steps are involved: The cell membrane of alcoholic fatty liver cells is extracted, and then PLGA nanoparticles and the cell membrane are mixed to prepare the nano vaccine.

2. The preparation method according to claim 1, characterized in that: The method for extracting cell membranes of alcoholic fatty liver cells comprises: collecting alcoholic fatty liver cells, then breaking the cells, and collecting membrane precipitates.

3. The preparation method according to claim 2, characterized in that: The method for constructing the alcoholic fatty liver cells is as follows: the liver cells are treated with 250 mmol / L alcohol and 0.25 mmol / L palmitic acid.

4. The preparation method according to claim 2, characterized in that: The method for disrupting cells is: ultrasonically disrupting cells under ice water bath conditions, 35W, ultrasonic time 5min, ultrasonic 2 seconds / interval 1 second.

5. The preparation method according to claim 2, characterized in that: The method for collecting the membrane precipitate is as follows: after the cells are disrupted, the supernatant is collected at 10,000 g for 15 min, the supernatant is transferred to a Beckman centrifuge tube, and after weighing and balancing, the tube is centrifuged at 100,000 g for 2 hours, the supernatant is discarded, and the membrane precipitate is collected.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the PLGA nanoparticles to the cell membrane is 5:

1.

7. The nanovaccine prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nano vaccine as claimed in claim 7 in the preparation of a medicament for preventing and treating alcoholic liver disease.

9. A drug for preventing and treating alcoholic liver disease, characterized in that: Including the nano vaccine according to claim 7.

10. The drug according to claim 9, characterized in that The drug reduces liver damage by reducing lipid droplet accumulation, downregulating the liver weight / body weight ratio, plasma alanine aminotransferase concentration, serum triglyceride concentration, liver alanine aminotransferase concentration and IL-6 concentration, and upregulating TGF-β concentration and IL-10 concentration.

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