Exosome drug delivery system and preparation method and application thereof

Abstract

The application relates to an exosome drug delivery system and a preparation method and application thereof, and relates to the technical field of biotechnology. The preparation method comprises the following steps: mixing a nucleic acid molecule and exosomes, adopting ultrasonic treatment at 40 kHz, the treatment time being 30s-240s, incubation, precipitation, and obtaining the exosome drug delivery system. The preparation method is simple and rapid, has little influence on the structure of the exosomes and little toxicity, has higher loading efficiency, and is convenient for subsequent cell treatment through the specific ultrasonic treatment process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an exosome drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Exosomes are endogenous membrane nanoscale vesicles, which are natural good carriers suitable for drug delivery, and can carry various types of molecules, including small molecule compounds, nucleic acids and proteins, etc. Compared with virus-derived carriers, exosomes have the following advantages: wide source, no risk of immune response, and can achieve repeated administration without resistance.

[0003] Currently, the most preferred drug delivery system is a nanoparticle platform based on liposomes, albumin, polymer micelles and nanoscale polymer-drug conjugates, and since exosomes are gradually developing into potential tools for drug delivery systems, it is necessary to improve nucleic acid drug delivery systems to achieve the effect of exogenous gene regulation. However, how to achieve efficient drug loading without introducing exogenous substances is still a concern. The EV-based drug delivery system has many advantages, such as high permeability, low immunogenicity and no cytotoxicity, and exosomes have been proven to be therapeutic nanocarriers for delivering various cargos, including siRNA, miRNA, proteins and drugs.

[0004] Currently, methods using exosomes as drug delivery carriers include transfection, electroporation, ultrasonic treatment and saponin permeation. However, these methods all have effects on cells or exosomes, and have the following disadvantages: (1) transfection reagents and electroporation have toxic effects on cells; (2) ordinary ultrasonic treatment and saponin permeation change the morphology of exosomes and damage their structure; (3) in previous studies, electroporation was used for loading, but the exosome aggregates formed by electroporation reduced the loading efficiency. SUMMARY

[0005] In view of the above problems, the present application provides a preparation method of an exosome drug delivery system, which is simple and fast, has little effect on the structure of exosomes and little toxicity, and has higher loading efficiency, which is convenient for subsequent cell processing.

[0006] In order to achieve the above purpose, the present application provides a preparation method of an exosome drug delivery system, comprising the following steps: mixing nucleic acid molecules and exosomes, using 40kHz ultrasonic treatment, the treatment time is 30s-240s, incubation, precipitation, to obtain an exosome drug delivery system.

[0007] The inventors found that the application of exosomes as carriers of drug delivery systems is limited for the following reasons: (1) the use of a large dose of transfection reagents will cause side effects due to the immunogenicity of lipid molecules, thus having a toxic effect on cells; (2) electroporation will neutralize the negative charge of nucleic acid probes during the discharge process, thus causing aggregation of nucleic acid molecules and reducing the loading efficiency into the exosomes, and electroporation also has a toxic effect on cells; (3) the conventional ultrasonic treatment process will change the morphology of exosomes and damage their structure; and (4) the osmotic pressure instability caused by saponin penetration will damage the structure of exosomes. Compared with the above conventional methods, ultrasonic treatment has less damage to exosomes and higher loading efficiency. Considering the defects of the conventional ultrasonic treatment process that changes the morphology of exosomes, the inventors propose to prepare an exosome drug delivery system by the above specific ultrasonic treatment process. Excessive ultrasonic treatment frequency or excessive ultrasonic treatment time will easily cause damage to exosomes and incomplete structure. Therefore, the inventors find a balance point between nucleic acid molecule loading efficiency and exosome structure integrity by using a specific ultrasonic treatment frequency and a specific ultrasonic treatment time, so that the exosome structure remains intact while effectively loading nucleic acid molecules. Compared with the conventional ultrasonic treatment process for loading nucleic acid molecules, the preparation method has a simple and fast processing flow, has less effect on the structure of exosomes and less toxicity, and has higher loading efficiency than the conventional ultrasonic treatment process, which is convenient for subsequent cell processing.

[0008] In one embodiment, the treatment time is 240 seconds.

[0009] With the above treatment time, the loading efficiency of exosomes after ultrasonic treatment is high, and the structure of exosomes remains intact.

[0010] In one embodiment, the incubation temperature is 37°C.

[0011] In one embodiment, the incubation time is 30 minutes.

[0012] After the specific ultrasonic treatment process, the structure of the exosome membrane becomes loose, generating gaps. Then, during the incubation period, nucleic acid molecules enter the exosomes through the gaps, and after the above temperature and time incubation, the gap fusion is promoted, the movement repair of the exosome membrane is accelerated, the nucleic acid overflow is prevented, the nucleic acid molecules are better retained, and the loading efficiency is maintained.

[0013] In one embodiment, the length of the nucleic acid molecule is ≤50 nt.

[0014] In one embodiment, the nucleic acid molecule is siRNA.

[0015] In one of the embodiments, the nucleic acid molecule is STAT3 siRNA, and the sequence is as follows:

[0016] Sense strand: 5'-CCGTGGAACCATACACAAATT-3' (SEQ ID NO: 1);

[0017] Antisense strand: 5'-TTTGTGTATGGTTCCACGGTT-3' (SEQ ID NO: 2).

[0018] The application further provides an exosome drug delivery system prepared by the preparation method.

[0019] The application further provides an application of the exosome drug delivery system in preparing a drug.

[0020] In one of the embodiments, the application comprises loading and delivering an anti-tumor drug by the exosome drug delivery system.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The exosome drug delivery system, the preparation method and the application thereof have the following advantages: the preparation method is simple and fast, has little influence on the structure of the exosome and little toxicity, and has higher loading efficiency, which is convenient for subsequent cell processing. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a graph of observation results of exosome morphology after different time ultrasonic treatment in the embodiments;

[0024] Figure 2 It is a flowchart of preparation of the exosome drug delivery system in the embodiments, wherein 50nt represents 50 nucleotide bases;

[0025] Figure 3 It is an electropherogram of siRNA before and after loading in the embodiments;

[0026] Figure 4 It is a graph of results of qPCR detection of STAT3 siRNA level in the embodiments;

[0027] Figure 5 It is a graph of results of WB detection of STAT3 protein expression level in the embodiments. DETAILED DESCRIPTION

[0028] For the purposes of this disclosure, reference will be made to the accompanying drawings which form a part of the disclosure. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the application. The embodiments of the application illustrated in the drawings are intended to provide a better understanding of the present application. The application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0030] Source:

[0031] HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription reagent (Novozyme), ChamQ Universal SYBR Qpcr Master Mix quantitative reagent (Novozyme), exosome (micro-nano nucleic acid biological medicine (Guangdong) Co., Ltd.), ultracentrifuge tube (Beckman, CAT#: 326823), ultracentrifuge tube (Beckman, CAT#: 344057), dialysis membrane (Millipore, CAT#: VSWP09025).

[0032] The reagents, materials and equipment used in this example are commercially available unless otherwise specified. The test methods are conventional test methods in the art unless otherwise specified.

[0033] Embodiment

[0034] An exosome drug delivery system and a preparation method thereof.

[0035] I. Extraction of exosomes

[0036] Exosomes were extracted by HEK293T cells, and the specific operation was as follows: (1) The culture supernatant of HEK293T cells was collected, and the obtained culture supernatant was centrifuged at 10,000g at 4°C for 30 min, and then the supernatant was collected. (2) The collected supernatant was transferred to an ultracentrifuge tube (Beckman, CAT#: 326823), and centrifuged at 100,000g (200uL exosome was added to the bottom) for 70 min. (3) 500uL liquid was collected at the bottom, diluted with PBS to 5.5mL, and transferred to a small ultracentrifuge tube (Beckman, CAT#: 344057), 500uL exosome was added to the bottom, and centrifuged at 100,000g for 70 min, then 250uL liquid was discarded from the bottom, and the remaining 200uL solution was collected. (4) Dialysis was performed through a dialysis membrane (Millipore, CAT#: VSWP09025) to 1xPBS buffer. About 200uL of exosome suspension was collected and stored at -80°C for standby.

[0037] II. Constructing ultrasonic treatment process

[0038] Ultrasonic treatment of exosomes and electron microscope detection: 10ug of exosomes were divided into 5 groups, 2ug in each group, diluted with 1xPBS to 50ul, and treated under ultrasonic conditions of 40kHz for 0, 30s, 60s, 120s and 240s respectively. After treatment, the exosome samples were observed by electron microscope.

[0039] The observation results are shown in Figure 1 It can be seen that the exosomes treated by ultrasonic waves with a frequency of 40kHz can maintain complete structure within different treatment time periods, provided that the treatment time does not exceed 240s.

[0040] III. Preparation of exosome drug delivery system

[0041] Ultrasonic treatment, loading of STAT3 siRNA, process as shown in Figure 2

[0042] STAT3 siRNA was designed and synthesized, and the specific sequence is shown as follows:

[0043] Sense strand 5'-CCGTGGAACCATACACAAATT-3'(SEQ ID NO: 1);

[0044] Antisense strand 5'-TTTGTGTATGGTTCCACGGTT-3'(SEQ ID NO: 2).

[0045] The above sense strand and antisense strand are RNA sequences, wherein T is the letter standard representation in WIPO Sequence table, which is uracil U.​

[0046] Take 100 μl of exosome at a concentration of 1 μg / μl, 10 μl of siRNA at a concentration of 100 μM, and a total volume of 110 μl. Ultrasonic treatment is performed at a frequency of 40 kHz for 240 s, and then incubation is performed at 37 °C for 30 min. After the exosomes are precipitated, the supernatant is detected for unloaded siRNA by 10% PAGE gel. The precipitated exosomes are resuspended in PBS to a concentration of 1 μg / μl for storage.

[0047] Detection results: After ultrasonic treatment and constant temperature water bath incubation, the residual siRNA in the supernatant is detected, and it can be observed that the content of loaded siRNA in the supernatant is very low, while siRNA bands can be detected before loading, as shown in Figure 3 .

[0048] Four, cell culture.

[0049] HCT116 cells are selected for culture, and the culture conditions are DMEM + 10% FBS + 1% double antibody, 5% CO2, and constant temperature culture at 37 °C.

[0050] Five, cell plating.

[0051] According to the cell count, the cell concentration is obtained, and the DMEM complete culture solution is diluted to 12 mL, mixed well, and 2 mL is added to each well. Incubate at 37 °C for 1-2 days. 4

[0052] Six, cell treatment and sample collection.

[0053] The exosomes loaded with STAT3 siRNA prepared in step two of this example are used, and the final concentration is 100 ng / μl. Incubate with the cells cultured in step four of this example, and incubate at 37 °C for 24 h. Collect the cells to extract RNA and protein. Detect the RNA expression and protein expression levels of STAT3. Use exosomes without siRNA loading as a control.

[0054] Seven, total RNA extraction and reverse transcription to cDNA and qPCR quantitative detection.

[0055] Use TRIzol lysis solution to lyse cells to extract total RNA. After quantification, take 1 μg of total RNA, use HiScript IIIRT SuperMix for qPCR (+gDNA wiper) reverse transcription reagent for reverse transcription, and use ChamQ Universal SYBRQpcr Master Mix quantitative reagent for quantitative detection of stat3 gene.

[0056] The detection results are as follows Figure 4 ​As shown, the obtained siRNA-loaded exosomes were used to treat cells, and the interference effect on the target gene was detected. Figure 4 As shown, the STAT3 gene expression was down-regulated, and through comparison of the RNA level, it was found that the target gene was well intervened.

[0057] Eight, protein extraction and WB detection.

[0058] The RIPA lysis solution and protease inhibitor were mixed, and the proteins of the cells in each group were extracted, and the protein concentration was determined by BCA. 20 μl of protein sample was taken, 5 μL of 5x protein loading buffer was mixed, heated at 95°C for 10 min, cooled on ice, separated by 10% SDS-PAGE protein gel, and the proteins on the separation gel were transferred to PVDF membrane, blocked with 5% skim milk for 1 h, incubated with GAPDH and STAT3 antibodies for 1 h, then incubated with horseradish peroxidase-labeled secondary antibody for 1 h, and finally reacted with ECL chemiluminescence substrate, and the fluorescence signal was detected in the Tanon gel imager.

[0059] The detection results are shown in Figure 5 As shown, the obtained siRNA-loaded exosomes were used to treat cells, and the interference effect on the target gene was detected, and through comparison of the protein expression level, it was found that the target gene was well intervened.

[0060] Nine, exosome nanoparticle size analysis.

[0061] 1 μl of exosome sample was taken, diluted 1000 times with PBS, and then slowly injected into the Nanosight instrument sample tank with 1 ml disposable syringe, and the exosome particle number and size distribution in the sample were counted and analyzed by nanosight detection and analysis software.

[0062] Ten, transmission electron microscope identification of exosome morphology.

[0063] 10 μl of exosome sample was taken, fixed with 10 μl of 2.5% glutaraldehyde, stained with 1% uranyl acetate, and then dropped onto a copper grid, and the picture was collected under a Hitachi H-7650 transmission electron microscope.

[0064] The technical features of the above-described embodiments can be combined in any way. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combination of the technical features does not exist, it should be considered as the scope of the present disclosure.

[0065] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing an exosome drug delivery system, characterized in that, Includes the following steps: Nucleic acid molecules and exosomes were mixed and treated with ultrasound at 40 kHz for 240 s, followed by incubation and precipitation to obtain an exosome drug delivery system; the incubation temperature was 37 °C and the incubation time was 30 min, and the nucleic acid molecule was siRNA.

2. The preparation method according to claim 1, characterized in that, The nucleic acid molecule is STAT3 siRNA, and its sequence is shown below: Chain of Justice: 5'-CCGTGGAACCATACACAAATT-3'(SEQ ID NO: 1); Antisense chain: 5'-TTTGTGTATGGTTCCACGGTT-3' (SEQ ID NO: 2).

3. An exosome drug delivery system obtained by the preparation method according to any one of claims 1-2.

4. The use of the exosome drug delivery system according to claim 3 in the preparation of a drug.

5. The application according to claim 4, characterized in that, The application includes: loading and delivering drugs into the exosome drug delivery system, wherein the drugs include at least one of antitumor drugs, anti-infective drugs, anti-aging drugs, and immunomodulatory drugs.

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