Extracellular vesicle, preparation method, pharmaceutical composition and use

Abstract

Provided are an extracellular vesicle, a preparation method, a pharmaceutical composition, and a use. The preparation method comprises: generating an extrusion effect on an extracellular vesicle, so that an exogenous nucleic acid is loaded into the extracellular vesicle; purifying the extracellular vesicle loaded with the exogenous nucleic acid, so as to obtain a highly-expressed extracellular vesicle of the exogenous nucleic acid for diagnosis and treatment; and quantifying the exogenous nucleic acid of the highly-expressed extracellular vesicle of the exogenous nucleic acid for diagnosis and treatment. The exogenous nucleic acid having diagnosis and treatment properties is loaded into the extracellular vesicle on the basis of a high-throughput physical extrusion method. Because the structural size is similar to or slightly smaller than that of the extracellular vesicle, the extrusion effect is generated on the extracellular vesicle, a membrane of the extracellular vesicle is temporarily perforated, and the exogenous nucleic acid is loaded in the extracellular vesicle, so that the in-vitro stability of the nucleic acid is protected. In addition, by performing quantitative design on the exogenous nucleic acid of the highly-expressed extracellular vesicle of the exogenous nucleic acid for diagnosis and treatment, complicated and uncontrollable biochemical processes are avoided, and the quantitative exogenous nucleic acid enters target cells and takes an effect to accurately regulate and control protein expression, thereby increasing the utilization rate of the exogenous nucleic acid.

Description

Extracellular vesicle, preparation method, pharmaceutical composition and application Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to an extracellular vesicle, a preparation method, a pharmaceutical composition and applications. Background Art

[0002] Gene therapy uses molecular biology techniques to introduce a target gene into target cells, correcting erroneous gene expression in those cells and thereby treating diseases. It is an emerging clinical medical technology born from the integration of modern medicine and molecular biology. However, nucleic acids are unstable in vitro and easily degraded in vivo, necessitating the use of vector technology. Ideal vectors should possess stability, low immunogenicity, and tissue specificity. The enclosed interior of extracellular vesicles accommodates the loading of a variety of molecules, while the phospholipid bilayer protects exogenous nucleic acids. The specific molecular distribution ensures low immunogenicity.

[0003] The existing technology uses high-throughput physical extrusion methods to load therapeutic small molecule compounds into extracellular vesicles. However, cell transfection uses uncontrollable and unquantifiable molecular biological methods to achieve the expression of endogenous nucleic acids. In addition, exogenous nucleic acids are loaded into extracellular vesicles through electroporation, but this affects the in vitro stability of the nucleic acids and thus their functionality.

[0004] Summary of the Invention

[0005] In view of this, it is necessary to provide an extracellular vesicle, a rapid preparation method, a pharmaceutical composition and application that highly expresses exogenous nucleic acids for diagnosis and treatment, with a controllable transfection process and high in vitro stability of the nucleic acid, in order to address the defects of the existing technology.

[0006] To solve the above problems, this application adopts the following technical solutions:

[0007] One of the purposes of this application is to provide a method for preparing extracellular vesicles, comprising the following steps:

[0008] The extracellular vesicles are squeezed to load the exogenous nucleic acid into the extracellular vesicles. The concentration of the extracellular vesicles is 1×10 2 -1×10 20 particles / mL, and the extrusion action time is 30 seconds to 15 minutes;

[0009] Purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain extracellular vesicles that highly express the exogenous nucleic acid for diagnosis and treatment;

[0010] Quantify exogenous nucleic acids in extracellular vesicles that highly express exogenous nucleic acids for diagnostic and therapeutic applications.

[0011] In some embodiments, the step of squeezing the extracellular vesicles to load the exogenous nucleic acid into the extracellular vesicles specifically includes the following steps:

[0012] After the exogenous nucleic acid is mixed with the extracellular vesicles, the extracellular vesicles are squeezed by a nanoporator to temporarily open the membrane of the extracellular vesicles, thereby allowing the exogenous nucleic acid to be loaded into the extracellular vesicles.

[0013] In some embodiments, the size of the extracellular vesicles is 20 nm-12 μm, and the exogenous nucleic acid is a nucleotide sequence with a length of 4-30,000.

[0014] In some embodiments, the size of the extracellular vesicle is 30 nm-200 nm, and the exogenous nucleic acid is a nucleotide sequence with a length of 10-50.

[0015] In some embodiments, the concentration of the exogenous nucleic acid is 10 pM-10 μM, and the concentration of the extracellular vesicles is 1×10 4 -1×10 18 particles / mL, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:50-1:500.

[0016] In some embodiments, the concentration of the exogenous nucleic acid is 1 nM-1 μM, and the concentration of the extracellular vesicles is 1×10 4 -1×10 18 particles / mL, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:100-1:200.

[0017] In some embodiments, the step of purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain extracellular vesicles highly expressing the exogenous nucleic acid for diagnosis and treatment specifically includes the following steps:

[0018] The extracellular vesicles loaded with the exogenous nucleic acid are purified using ultrafiltration or RNAse method to remove free nucleic acid to obtain extracellular vesicles that highly express diagnostic and therapeutic nucleic acid.

[0019] In some embodiments, the step of quantifying the exogenous nucleic acid in extracellular vesicles containing highly expressed exogenous nucleic acid for diagnosis and treatment specifically includes the following steps:

[0020] Quantification of exogenous nucleic acids in extracellular vesicles that highly express exogenous nucleic acids for diagnostics and theranostics is performed by real-time quantitative polymerase chain reaction or droplet digital polymerase chain reaction.

[0021] In some embodiments, the extracellular vesicles include liposomes, artificial lipid vesicles, lipid nanoparticles, plant extracellular vesicles, animal extracellular vesicles, human cell-derived extracellular vesicles, bacterial outer membrane vesicles, membrane vesicles secreted by microorganisms, cell membrane recombinant nanoparticles, bacterial outer membrane recombinant nanoparticles, virus particles and virus-like particles with envelope structures, and artificial nanovesicles hybridized with cell membrane / bacterial outer membrane / viral membrane proteins; the exogenous nucleic acids used for diagnosis and treatment include plasmids, ribonucleic acids, deoxyribonucleic acids, oligonucleotides, circular RNA, miRNA, mRNA, ASO, siRNA, saRNA, SSO and EON.

[0022] The second purpose of the present application is to provide an extracellular vesicle, which is prepared by a rapid preparation method of the extracellular vesicle that highly expresses exogenous nucleic acids for diagnosis and treatment.

[0023] The third purpose of this application is to provide a pharmaceutical composition comprising the extracellular vesicles.

[0024] The fourth purpose of this application is to provide an application of the extracellular vesicles in regulating protein expression or protein replacement therapy or immune diagnosis and treatment or tissue regeneration.

[0025] This application adopts the above technical solution, and its beneficial effects are as follows:

[0026] The extracellular vesicles, preparation methods, pharmaceutical compositions and applications provided in the present application produce an extrusion effect on the extracellular vesicles so that exogenous nucleic acids are loaded into the extracellular vesicles, and the extracellular vesicles loaded with the exogenous nucleic acids are purified to obtain extracellular vesicles with highly expressed exogenous nucleic acids for diagnosis and treatment, and the exogenous nucleic acids in the extracellular vesicles with highly expressed exogenous nucleic acids for diagnosis and treatment are quantified. The present application uses a high-throughput physical extrusion method to load exogenous nucleic acids with diagnostic properties into extracellular vesicles. Because the size is similar to or slightly smaller than that of the extracellular vesicles, an extrusion effect is produced on the extracellular vesicles, causing the membrane of the extracellular vesicles to temporarily open, thereby allowing the exogenous nucleic acids to be loaded therein, thereby protecting the in vivo stability of the nucleic acids. In addition, by quantitatively designing the exogenous nucleic acids in the extracellular vesicles with highly expressed exogenous nucleic acids for diagnosis and treatment, cumbersome and uncontrollable biochemical processes are avoided, and the quantitative exogenous nucleic acids enter the target cells and function to precisely regulate protein expression, thereby improving the utilization rate of the exogenous nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0028] FIG1 is a flow chart of the steps of a method for rapidly preparing extracellular vesicles that highly express exogenous nucleic acids for diagnosis and treatment, provided in an embodiment of the present application.

[0029] FIG2 is a schematic diagram of the working principle of the nanoporator ExoNP provided in this embodiment.

[0030] FIG3 is a graph showing the relative expression levels of nucleic acids in extracellular vesicles in different treatment groups provided in this example.

[0031] FIG4 is a graph showing the results of the regulation of target genes in target cells by different treatment groups provided in this example.

[0032] FIG5 is a graph showing the relative expression levels of target genes in target cells in different treatment groups provided in this example. DETAILED DESCRIPTION

[0033] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0034] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on this application.

[0035] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0036] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0037] Please refer to Figure 1, which is a flowchart of the steps of the method for preparing extracellular vesicles provided in an embodiment of the present application, including the following steps S110 to S130. The implementation method of each step is described in detail below.

[0038] Step S110: Extruding the extracellular vesicles to load the exogenous nucleic acid into the extracellular vesicles. The concentration of the extracellular vesicles is 1×10 2 -1×10 20 particles / mL.

[0039] In this embodiment, the step of squeezing the extracellular vesicles to load the exogenous nucleic acid into the extracellular vesicles specifically includes the following steps: after mixing the exogenous nucleic acid with the extracellular vesicles, squeezing the extracellular vesicles by a nanoporator to temporarily open the membrane of the extracellular vesicles, thereby loading the exogenous nucleic acid into the extracellular vesicles.

[0040] It can be understood that the extracellular vesicles provided in this embodiment are obtained by conventional extraction steps, including cell recovery, cell culture, extracellular vesicle extraction and purification.

[0041] The extracellular vesicles include liposomes, artificial lipid vesicles, lipid nanoparticles, plant extracellular vesicles, animal extracellular vesicles, human cell-derived extracellular vesicles, bacterial outer membrane vesicles, membrane vesicles secreted by microorganisms, cell membrane recombinant nanoparticles, bacterial outer membrane recombinant nanoparticles, virus particles and virus-like particles with envelope structures, and artificial nanovesicles hybridized with cell membrane / bacterial outer membrane / viral membrane proteins; the exogenous nucleic acids used for diagnosis and treatment are plasmids, ribonucleic acids, deoxyribonucleic acids, oligonucleotides, circular RNA, miRNA, mRNA, ASO, siRNA, saRNA, SSO and EON.

[0042] Specifically, the size of the extracellular vesicles is 20 nm-12 μm, and the exogenous nucleic acid is a nucleotide sequence with a length of 4-30,000.

[0043] Specifically, the size of the extracellular vesicles is 30nm-200nm, and the exogenous nucleic acid is a nucleotide sequence with a length of 10-50.

[0044] It is understandable that extracellular vesicles smaller than 200 nm can easily cross multiple physiological and pathological barriers in the body. Currently, nucleic acid drugs with diagnostic and therapeutic effects mainly include antisense oligonucleotides (ASOs, consisting of about 15-25 nucleotides), small interfering RNA (siRNA, consisting of about 20-25 nucleotides) and micro RNA (micro RNA, consisting of about 21-23 nucleotides).

[0045] It is understood that the exogenous nucleic acid provided in this embodiment is obtained by conventional in vitro synthesis of the nucleic acid of interest.

[0046] Specifically, the concentration of the exogenous nucleic acid is 10 pM-10 μM, and the concentration of the extracellular vesicles is 1×10 4 -1×10 18 particles / mL, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:50-1:500.

[0047] Specifically, the concentration of the exogenous nucleic acid is 1 nM-1 μM, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:100-1:200.

[0048] It is understandable that large-scale purification of extracellular vesicles smaller than 200 nm presents certain challenges. Currently, the methods can achieve 1×10 6 -1×10 16 In order to achieve the minimum dose of exogenous nucleic acid for diagnosis and treatment to enter the target cells and exert its effect, a low concentration of diagnostic exogenous nucleic acid is loaded into a low concentration of extracellular vesicles to observe whether it exerts a diagnostic and therapeutic effect.

[0049] Please refer to Figure 2 for a schematic diagram of the working principle of the nanoporator ExoNP provided in this embodiment. This embodiment uses a high-throughput physical extrusion method to load therapeutic exogenous nucleic acids into extracellular vesicles. The high-throughput physical extrusion method is based on micro-nanofluidics technology. Since the size of the exosomes is similar to or slightly smaller than that of the extracellular vesicles, it exerts an extrusion effect on the extracellular vesicles, temporarily opening pores in the extracellular vesicle membrane, thereby allowing the exogenous nucleic acids to be loaded therein and protecting the in vitro stability of the exogenous nucleic acids.

[0050] It should be noted that the method provided in the above embodiment is applicable to the loading of different nucleic acid mixtures, and the time required to process samples of different concentrations is 10 seconds to 15 minutes.

[0051] Step S120: purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain extracellular vesicles that highly express the exogenous nucleic acid for diagnosis and treatment.

[0052] In this embodiment, the step of purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain extracellular vesicles highly expressing the exogenous nucleic acid for diagnosis and treatment specifically includes the following steps:

[0053] The extracellular vesicles loaded with the exogenous nucleic acid are purified using ultrafiltration or RNAse method to remove free nucleic acid to obtain extracellular vesicles that highly express diagnostic and therapeutic nucleic acid.

[0054] Step S130: quantifying the exogenous nucleic acid in the extracellular vesicles containing the highly expressed exogenous nucleic acid for diagnosis and treatment.

[0055] In this embodiment, the exogenous nucleic acids in extracellular vesicles that highly express exogenous nucleic acids for diagnosis and treatment are quantified by real-time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) methods.

[0056] It can be understood that by quantifying the exogenous nucleic acid in the extracellular vesicles, the exogenous nucleic acid can be quantitatively entered into the target cells and exert its function.

[0057] The present application also provides a pharmaceutical composition comprising the extracellular vesicles described above. The pharmaceutical composition utilizes extracellular vesicles that highly express exogenous nucleic acids for diagnosis and treatment to treat target cells, transport the target nucleic acids into the target cells, and correct the erroneous gene expression of the target cells, thereby enabling diagnosis, treatment, and integrated diagnosis and treatment operations, thereby exerting a diagnostic and therapeutic effect.

[0058] The extracellular vesicles provided in this application can be used in regulating protein expression or protein replacement therapy or immune diagnosis and treatment or tissue regeneration.

[0059] The above-mentioned embodiments of the present application use a high-throughput physical extrusion method to load therapeutic exogenous nucleic acids into extracellular vesicles. Due to the size of the extracellular vesicles being similar to or slightly smaller than that of the extracellular vesicles, an extrusion effect is exerted on the extracellular vesicles, causing the membrane of the extracellular vesicles to temporarily open pores, thereby allowing the exogenous nucleic acids to be loaded therein, thereby protecting the in vitro stability of the nucleic acids. In addition, by quantitatively designing the exogenous nucleic acids of the extracellular vesicles of highly expressed exogenous nucleic acids for diagnosis and treatment, cumbersome and uncontrollable biochemical processes are avoided, and the quantitative exogenous nucleic acids enter the target cells and function to precisely regulate protein expression, thereby improving the utilization rate of the exogenous nucleic acids. The use of the pharmaceutical composition comprising the extracellular vesicles that highly express exogenous nucleic acids for diagnosis and treatment can be used as a supplement or even a substitute for new diagnostic and therapeutic methods such as gene diagnosis and treatment and protein diagnosis and treatment.

[0060] The above technical solution is described in detail below in conjunction with specific embodiments.

[0061] The above-mentioned rapid preparation method was used, and the parameters were changed as shown in the following table.

[0062] Please refer to Figure 3, which is a graph showing the relative expression levels of nucleic acids in extracellular vesicles of the different treatment groups provided in Example 1. As can be seen from Figure 3, compared with the "extracellular vesicle" group, the extracellular vesicles treated by the physical extrusion method of the nanoporator achieved a 10-fold enrichment of diagnostic and therapeutic exogenous nucleic acids.

[0063] Please refer to Figure 4, which is a graph showing the regulation of target genes in target cells by different treatment groups provided in Example 1. As can be seen from Figure 4, compared with the "extracellular vesicle" group, the "nanoporator extracellular vesicle@exogenous nucleic acid" group has the effect of downregulating the target gene.

[0064] Please refer to Figure 5, which is a graph showing the relative expression levels of target genes in target cells in the different treatment groups provided in Example 1. As can be seen from Figure 5, compared with the "extracellular vesicle" group, the "nanoporator_extracellular vesicle@exogenous nucleic acid" group achieved a 2.5-fold downregulation of the target gene.

[0065] It can be seen from the above examples that the extracellular vesicles provided by the present application that highly express exogenous nucleic acids for diagnosis and treatment can achieve stable and process-controllable loading of exogenous nucleic acids into extracellular vesicles, and transport and release a certain amount of exogenous nucleic acids into target cells to exert their effects, thereby improving the utilization rate of exogenous nucleic acids.

[0066] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A method for preparing extracellular vesicles, characterized in that: The steps include: The extracellular vesicles are squeezed to load the exogenous nucleic acid into the extracellular vesicles. The concentration of the extracellular vesicles is 1×10 2 -1×10 20 particles / mL; Purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain extracellular vesicles highly expressing the exogenous nucleic acid for diagnosis and treatment; Quantification of exogenous nucleic acids in extracellular vesicles that highly express exogenous nucleic acids for diagnostics.

2. The method for preparing extracellular vesicles according to claim 1, characterized in that: The step of squeezing the extracellular vesicles to load the exogenous nucleic acid into the extracellular vesicles specifically comprises the following steps: After the exogenous nucleic acid is mixed with the extracellular vesicles, the extracellular vesicles are squeezed by a nanoporator to temporarily open the membrane of the extracellular vesicles, thereby allowing the exogenous nucleic acid to be loaded into the extracellular vesicles.

3. The method for preparing extracellular vesicles according to claim 1 or 2, characterized in that: The size of the extracellular vesicle is 20 nm-2 μm, and the exogenous nucleic acid is a nucleotide with a sequence length of 4-30,000.

4. The method for preparing extracellular vesicles of exogenous nucleic acid according to claim 3, characterized in that: The size of the extracellular vesicle is 30nm-200nm, and the exogenous nucleic acid is a nucleotide with a sequence length of 10-50.

5. The method for preparing extracellular vesicles according to claim 3, characterized in that: The concentration of the exogenous nucleic acid is 10 pM-10 μM, and the concentration of the extracellular vesicles is 1×10 4 -1×10 18 particles / mL, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:50-1:

500.

6. The method for preparing extracellular vesicles according to claim 5, characterized in that: The concentration of the exogenous nucleic acid is 1 nM-1 μM, and the volume ratio of the exogenous nucleic acid to the extracellular vesicles is 1:100-1:

200.

7. The method for preparing extracellular vesicles according to claim 1, characterized in that: The step of purifying the extracellular vesicles loaded with the exogenous nucleic acid to obtain the extracellular vesicles with highly expressed exogenous nucleic acid for diagnosis and treatment specifically includes the following steps: The extracellular vesicles loaded with the exogenous nucleic acid are purified using ultrafiltration or RNAse method to remove free nucleic acid, so as to obtain extracellular vesicles that highly express diagnostic and therapeutic nucleic acid.

8. The method for preparing extracellular vesicles according to claim 1, characterized in that: The step of quantifying the exogenous nucleic acid in the extracellular vesicles of the highly expressed exogenous nucleic acid for diagnosis and treatment specifically includes the following steps: The exogenous nucleic acids in extracellular vesicles that highly express exogenous nucleic acids for diagnostics and treatment are quantified by real-time quantitative polymerase chain reaction or droplet digital polymerase chain reaction.

9. The method for preparing extracellular vesicles according to claim 1, characterized in that: The extracellular vesicles include liposomes, artificial lipid vesicles, lipid nanoparticles, plant extracellular vesicles, animal extracellular vesicles, extracellular vesicles from human cells, bacterial outer membrane vesicles, membrane vesicles secreted by microorganisms, cell membrane recombinant nanoparticles, bacterial outer membrane recombinant nanoparticles, virus particles and virus-like particles with envelope structures, and artificial nanovesicles hybridized with cell membrane / bacterial outer membrane / viral membrane proteins; the exogenous nucleic acids used for diagnosis and treatment include plasmids, ribonucleic acids, deoxyribonucleic acids, oligonucleotides, circular RNA, miRNA, mRNA, ASO, siRNA, saRNA, SSO and EON.

10. An extracellular vesicle, characterized in that The extracellular vesicles are prepared by the method for preparing the extracellular vesicles according to any one of claims 1 to 8.

11. A pharmaceutical composition, characterized in that Comprising the extracellular vesicles described in claim 8.

12. Use of the extracellular vesicles according to claim 10 in regulating protein expression or protein replacement therapy or immune diagnosis and treatment or tissue regeneration.

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