Exosome drug delivery system based on calcium phosphate mediation and preparation method

Abstract

The invention belongs to the technical field of biotechnology, particularly relates to an exosome drug delivery system based on calcium phosphate mediation, and further discloses a preparation method and application of the exosome drug delivery system. According to the exosome drug delivery system based on calcium phosphate mediation, the calcium phosphate mediation exosome delivery system is constructed and formed through Ca < 2 + > and phosphate, after miRNA is transfected through calcium chloride, a nucleic acid drug can be loaded into the exosome, the content of miRNA in the exosome is remarkably increased, then the drug is transported by means of the delivery performance of the exosome, and the drug delivery efficiency is improved. The drug loading efficiency and the delivery efficiency of the drug are effectively improved, and the advantage of high loading efficiency is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and specifically relates to an exosome drug delivery system based on calcium phosphate mediation, and further discloses a preparation method and application thereof. Background Art

[0002] Exosomes (Exo) are spherical membrane vesicles secreted by cells outside the cell. They are endogenous membrane nano-vesicles. Exosomes can realize the transport of substances and information between cells. Small molecules such as proteins, lipids, nucleic acids, etc. can be transferred from donor cells to recipient cells through exosomes. Therefore, exosomes are a natural and good nano-scale drug delivery carrier, and because exosomes have the advantages of biological stability, targeting specificity, low toxicity and low immunogenicity, exosomes have great potential in drug delivery.

[0003] At present, there are two main ways to load drugs into exosomes, namely endogenous loading and exogenous loading. Endogenous loading is to directly transform the source cells, introduce the target molecules into the source cells, and then separate and purify the exosomes. Exogenous loading is to load the drugs directly into the exosomes through membrane penetration or membrane fusion techniques after the separated and purified exosomes.

[0004] Exogenous loading methods mainly include direct co-incubation, electroporation, ultrasonic treatment, extrusion, repeated freezing and thawing, and surfactant permeabilization. However, direct co-incubation and repeated freezing and thawing loading methods have the defects of low efficiency and inability to load large molecule drugs; electroporation and ultrasonic treatment loading methods are very easy to change the morphology of exosomes, easily cause exosome aggregation and require special instruments; and surfactant permeabilization loading methods make it difficult to completely remove certain surfactants, especially residual surfactants may cause cell toxicity and destroy the structure of exosomes.

[0005] Therefore, the art is looking forward to developing an exosome drug delivery system with high loading efficiency and less impact on exosome structure and cytotoxicity, which has positive significance for the development of new drugs. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide an exosome drug delivery system based on calcium phosphate mediation, which has high loading efficiency and less influence on the exosome structure and cytotoxicity;

[0007] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned calcium phosphate-mediated exosome drug delivery system, which does not require special instruments and equipment and has the advantage of being easy to operate.

[0008] In order to solve the above technical problems, the present invention provides a method for preparing a calcium phosphate-mediated exosome drug delivery system, comprising the following steps:

[0009] (1) Prepare nucleic acid, exosomes and Ca 2+ Reaction A liquid and containing PO4 2- A reaction liquid B containing phosphate ions, and mixing the reaction liquid A with the reaction liquid B to obtain a mixed liquid;

[0010] (2) subjecting the mixed solution to a first incubation treatment;

[0011] (3) Continue to add ribonuclease (RNase) and mix, and perform a second incubation treatment.

[0012] Specifically, in the method for preparing the calcium phosphate-mediated exosome drug delivery system, in step (1), the nucleic acid includes a small molecule nucleic acid drug, preferably miRNA, siRNA and / or ASO.

[0013] Specifically, the preparation method of the calcium phosphate-mediated exosome drug delivery system is as follows:

[0014] The nucleic acid drug is added in an amount of 1-100 pmol / 100 μL based on the mixed solution, preferably 10-200 pmol / 300 μL, more preferably 70 pmol / 300 μL; and / or,

[0015] The amount of exosomes added to the mixed solution is 10 9 -10 11 particles / ml, preferably 10 10 particles / ml; and / or,

[0016] The Ca 2+ Based on the added concentration of the mixed solution is 0.01-1M, preferably 0.05-0.5M, more preferably 0.1 mol / L; and / or,

[0017] The PO4 2- The added concentration based on the mixed solution is 2-8 mM, preferably 2-5 mM, and more preferably 2.5 mM.

[0018] Specifically, the preparation method of the calcium phosphate-mediated exosome drug delivery system, the Ca 2+ With PO4 2- The concentration ratio is 20-100:1.

[0019] Specifically, in the preparation method of the calcium phosphate-mediated exosome drug delivery system, the reaction solution B includes PBS buffer or HBS buffer;

[0020] Preferably, the pH of the PBS buffer or HBS buffer is 7.0-7.5.

[0021] Specifically, in the preparation method of the calcium phosphate-mediated exosome drug delivery system, the reaction liquid A and the reaction liquid B independently further contain DNase and RNase-free water.

[0022] Specifically, in the preparation method of the calcium phosphate-mediated exosome drug delivery system, in the step (2), the first incubation treatment includes: incubating the mixed solution at 0-4°C for 20-40 min, incubating at 40-45°C for 40-80 s, and incubating at 0-4°C for 3-8 min.

[0023] Specifically, in the preparation method of the calcium phosphate-mediated exosome drug delivery system, in the step (3), the temperature of the second incubation treatment is 35-40° C., and the incubation time is 20-40 min;

[0024] Preferably, in step (3), the amount of ribonuclease (RNase) added to the mixed solution is 3-8 μg / mL, preferably 5 μg / mL. The RNase can digest free miRNA, preventing large errors in detecting miRNA expression and subsequent experiments.

[0025] The present invention also discloses a calcium phosphate-mediated exosome drug delivery system prepared by the method.

[0026] The present invention also discloses the use of the calcium phosphate-mediated exosome drug delivery system for preparing nucleic acid drugs;

[0027] Optionally, the nucleic acid drug includes a nucleic acid drug for treating tumors.

[0028] The calcium phosphate-mediated exosome drug delivery system of the present invention uses Ca 2+ A calcium phosphate-mediated exosome delivery system is constructed with phosphate. After transfection of miRNA with calcium chloride, the nucleic acid drug can be loaded into the exosome. The miRNA content in the exosome is significantly increased, and the drug is then transported with the help of the delivery performance of the exosome, which effectively improves the drug loading efficiency and delivery efficiency, and has the advantage of high loading efficiency.

[0029] The calcium phosphate-mediated exosome drug delivery system of the present invention adjusts the Ca 2+ With PO4 2-The higher the ratio of the two, the better the loading efficiency, which can effectively improve the drug loading capacity and efficiency of exosomes.

[0030] The calcium phosphate-mediated exosome drug delivery system of the present invention uses calcium carbonate and calcium phosphate as one of the components in the organism, which has good biocompatibility, not only has little effect on the exosome structure and cytotoxicity, but also has good pH responsiveness, can be better released in the tumor microenvironment, and exerts a better therapeutic effect.

[0031] The calcium phosphate-mediated exosome drug delivery system of the present invention does not require special instruments and equipment and has the advantage of simple preparation method and operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 It is a schematic diagram of the miRNA transfection exosomes;

[0034] Figure 2 The results of the marker protein detection of exosomes in Experimental Example 1 (A), NTA detection results (B) and TEM detection results (C);

[0035] Figure 3 is the relative expression of miRNA in exosomes before and after transfection;

[0036] Figure 4 is the expression level of miRNA under different phosphate concentrations;

[0037] Figure 5 is the expression level of miRNA under different calcium chloride concentrations;

[0038] Figure 6 The expression levels of different miRNA concentrations were changed after transfection. DETAILED DESCRIPTION

[0039] Example 1

[0040] like Figure 1 As shown in the flow chart, 20 μL (10 10particles / mL), add 10pmol of miRNA and mix evenly, and add 30μL, 1M CaCl2 and mix (the final concentration of the system is 0.1M). The above mixture is slowly added to PBS (pH7.3) to adjust the final volume of the system to 300μL. The above mixture is placed on ice for incubation for 30 minutes, then treated in a 42℃ water bath for 60 seconds, and then incubated on ice for 5 minutes. Continue to add RNase to the above mixture at a final concentration of 5μg / mL, and continue incubation at 37℃ for 30 minutes to remove free RNA.

[0041] Example 2

[0042] like Figure 1 As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 30 μL of 1M CaCl2 to mix (the final concentration of the system is 0.1 M). Slowly add the above mixture to PBS (pH 7.3) and adjust the final volume of the system to 300 μL. Incubate the above mixture on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase to the above mixture at a final concentration of 5 μg / mL, and continue incubating at 37°C for 30 minutes to remove free RNA.

[0043] Example 3

[0044] like Figure 1 As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 120 pmol of miRNA and mix evenly, and add 30 μL of 1M CaCl2 to mix (the final concentration of the system is 0.1 M). Slowly add the above mixture to PBS (pH 7.3) and adjust the final volume of the system to 300 μL. Incubate the above mixture on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase to the above mixture at a final concentration of 5 μg / mL, and continue incubating at 37°C for 30 minutes to remove free RNA.

[0045] Example 4

[0046] like Figure 1As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 10 μL of 1M CaCl2 to mix. Slowly add the above mixture to PBS (pH 7.3) to adjust the final volume of the system to 300 μL. Incubate the above mixture on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase to the above mixture at a final concentration of 5 μg / mL, and continue incubating at 37°C for 30 minutes to remove free RNA.

[0047] Example 5

[0048] like Figure 1 As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 20 μL of 1M CaCl2 to mix. Slowly add the above mixture to PBS (pH 7.3) to adjust the final volume of the system to 300 μL. Incubate the above mixture on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase to the above mixture at a final concentration of 5 μg / mL, and continue incubating at 37°C for 30 minutes to remove free RNA.

[0049] Example 6

[0050] like Figure 1 As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 80 μL of DEPC water and 30 μL of 1M CaCl2 (the final concentration of the system is 0.1M) to obtain a mixed solution A. Take another 42 μL of PBS (pH 7.3) and 108 μL of DEPC water to mix and prepare a mixed solution B. Slowly add the mixed solution A to the mixed solution B, incubate the mixed solution on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase to the mixed solution, and continue incubating at 37°C for 30 minutes to remove free RNA.

[0051] Example 7

[0052] like Figure 1As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 40 pmol of miRNA and mix evenly, and add 30 μL of 1M CaCl2 to mix. Slowly add the above mixture to PBS (pH 7.3) to adjust the final volume of the system to 300 μL. Incubate the above mixture at 4°C for 20 minutes, then place it in a 40°C water bath for 40 seconds, and then incubate it at 4°C for 8 minutes. Continue to add RNase to the above mixture at a final concentration of 3 μg / mL, and continue incubating at 35°C for 40 minutes to remove free RNA.

[0053] Example 8

[0054] like Figure 1 As shown in the flowchart, take 20 μL of exosomes at a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 30 μL of 1M CaCl2 to mix. Slowly add the above mixture to PBS (pH 7.3) to adjust the final volume of the system to 300 μL. Incubate the above mixture at 4°C for 40 minutes, then treat it in a 45°C water bath for 20 seconds, and then incubate it at 4°C for 3 minutes. Continue to add RNase to the above mixture at a final concentration of 8 μg / mL, and continue incubating at 40°C for 20 minutes to remove free RNA.

[0055] Example 9

[0056] like Figure 1 As shown in the flowchart, take 20 μL of exosomes with a concentration of 1 μg / μL, add 70 pmol of miRNA and mix evenly, and add 100 μL of DEPC water and 30 μL of 1M CaCl2 (the final concentration of the system is 0.1M) to obtain a mixed solution A. Take another 42 μL of PBS (pH 7.3) and 108 μL of DEPC water to mix and prepare a mixed solution B. Slowly add the mixed solution A to the mixed solution B, incubate the mixed solution on ice for 30 minutes, then treat it in a 42°C water bath for 60 seconds, and then incubate it on ice for 5 minutes. Continue to add RNase with a final concentration of 5 μg / mL to the mixed solution, and continue to incubate at 37°C for 30 minutes to remove free RNA.

[0057] Example 10

[0058] The scheme described in this example is the same as that in Example 2, the only difference being that the PBS buffer is replaced by the HBS buffer.

[0059] The main component of the HBS buffer solution, phosphate ions, includes 8 g / L NaCl, 0.4 g / L KCl, 1 g / L glucose, 60 mg / L KH2PO4, and 47.5 mg / L Na2HPO4.

[0060] Comparative Example 1

[0061] The exosome delivery system was prepared with reference to Example 1 in Chinese patent CN118948800A, but the system is only suitable for the preservation of exosomes and cannot be used for the construction of exosome delivery system.

[0062] Experimental example

[0063] 1. Exosome detection

[0064] According to the method in Example 1, in order to collect cell exosomes, the cells were cultured to a stable stage, replaced with an exosome-free culture medium, and continued to be cultured until the cells were 80% to 90% confluent, and the cell supernatant was collected. The supernatant was removed by gradient centrifugation to remove large particulate matter such as residual cells and cell debris. Ultrafiltration was concentrated, and chromatography purification was performed using a multi-mode chromatography column, and exosomes were obtained by filtration and sterilization using a 0.45 μm + 0.22 μm capsule filter.

[0065] Western Blot technology was used to analyze the expression of exosome-specific markers. Figure 2 As shown in middle A, exosome-specific characteristic proteins CD9, CD81 and TSG101 are all positively expressed, while Calnexin protein expression is negative.

[0066] like Figure 2 The NTA result shown in B shows that the particle size of the exosomes is about 130.6 nm, which is in line with the normal particle size range (30-200 nm);

[0067] like Figure 2 The transmission electron microscopy results shown in center C show that the exo structure is mainly present as saucer-shaped vesicles.

[0068] It can be seen that the method of the present invention can successfully obtain the desired exosomes.

[0069] 2. Detection of relative expression of miRNA in exosomes

[0070] Take the delivery system prepared in Example 2 above (denoted as exo-miRNA), that is, the exosomes loaded with miRNA obtained by transfection.

[0071] The total RNA of the human skin fibroblasts was extracted by the Trizol method, and reverse transcription was performed using the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) reverse transcription reagent, and the corresponding miRNA quantitative detection was performed using the miRNA Universal SYBR qPCR Master Mix quantitative reagent.

[0072] At the same time, the exosomes not mediated by calcium phosphate (denoted as exo) were subjected to the same operation as a control. The exosomes not mediated by calcium phosphate were based on the scheme in Example 2, where phosphoric acid and calcium chloride were replaced with DEPC water, followed by 30 minutes on ice, 60 seconds at 42°C, 5 minutes on ice, and 30 minutes of RNase incubation at 37°C.

[0073] Figure 3 The relative expression of miRNA in exosomes before and after transfection. It can be seen that the content of miRNA in exosomes increased by more than 50 times when RNA in exosomes was directly extracted after transfection of miRNA by calcium phosphate system.

[0074] 3. Detection of miRNA expression levels at different concentrations of calcium chloride and phosphate

[0075] Referring to the preparation method in Example 2, the expression of exo-miRNA under different concentrations of calcium chloride and phosphoric acid treatment was discussed. The transfected exosomes were co-cultured with human fibroblasts, and the RNA of the cells was extracted after 24 hours, and the expression of miRNA was detected by qPCR.

[0076] Take 20ul of exosomes loaded with miRNA and add them to human fibroblasts cultured in 6-well plates (cell confluence 60%-70%), add 2ml of basal culture medium, mix well, extract RNA from cells after culturing for 24 hours, and detect the expression of miRNA by qPCR. Figure 4 The expression results of phospho-miRNA at different concentrations are shown in Figure 2. Figure 5 The expression results of miRNA under different concentrations of calcium chloride.

[0077] It can be seen that different concentrations of phosphoric acid can promote the transfer of miRNA into exosomes, but the transfection efficiency is negatively correlated with the phosphoric acid concentration, that is, high concentrations inhibit miRNA transfection, and low concentrations promote miRNA transfection. The transfer of miRNA into exosomes is positively correlated with the concentration of calcium chloride, that is, within a certain concentration range, the higher the concentration of calcium chloride, the higher the efficiency of miRNA transfection.

[0078] 4. Detection of expression levels of different exo-miRNAs

[0079] According to the preparation method in the above Example 2, the expression of exo-miRNA under different concentrations of miRNA was discussed respectively. The transfected exosomes were co-cultured with human fibroblasts, and the RNA of the cells was extracted after 24 hours, and the expression of miRNA was detected by qPCR.

[0080] Take 20ul of exosomes loaded with miRNA and add them to human fibroblasts cultured in 6-well plates (cell confluence 60%-70%), add 2ml of basal culture medium, mix well, extract RNA from cells after culturing for 24 hours, and detect the expression of miRNA by qPCR.

[0081] Attached Figure 6 The expression results of miRNA after transfection with different concentrations of miRNA are shown in Figure 2. It can be seen that the miRNA transfection efficiency is related to the concentration of added miRNA, low concentration promotes, and high concentration inhibits. In this embodiment, the optimal transfection concentration of miRNA is 70 pmol.

[0082] The calcium phosphate-mediated exosome drug delivery system of the present invention uses Ca 2+ and phosphate to form a calcium phosphate-mediated exosome delivery system, and by adjusting the Ca 2+ With PO4 2- The higher the ratio of the two, the better the loading efficiency. After transfection of miRNA with calcium chloride, the nucleic acid drug can be loaded into the exosomes. The content of miRNA in the exosomes is significantly increased, and then the drug is transported with the help of the delivery performance of the exosomes, which effectively improves the drug loading efficiency and delivery efficiency, and has the advantage of high loading efficiency.

[0083] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a calcium phosphate-mediated exosome drug delivery system, characterized in that: The steps include: (1) Prepare nucleic acid, exosomes and Ca 2+ Reaction A liquid and containing PO4 2- A reaction liquid B containing phosphate ions, and mixing the reaction liquid A with the reaction liquid B to obtain a mixed liquid; (2) subjecting the mixed solution to a first incubation treatment; (3) Continue to add ribonuclease and mix and perform a second incubation treatment to obtain the product.

2. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to claim 1, characterized in that: In step (1), the nucleic acid includes a small molecule nucleic acid drug, preferably miRNA, siRNA and / or ASO.

3. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to claim 1 or 2, characterized in that: The nucleic acid drug is added in an amount of 1-100 pmol / 100 μL based on the mixed solution, preferably 10-200 pmol / 300 μL, more preferably 70 pmol / 300 μL; and / or, The amount of exosomes added to the mixed solution is 10 9 -10 11 particles / ml, preferably 10 10 particles / ml; and / or, The Ca 2+ Based on the added concentration of the mixed solution is 0.01-1M, preferably 0.05-0.5M, more preferably 0.1 mol / L; and / or, The PO4 2- The added concentration based on the mixed solution is 2-8 mM, preferably 2-5 mM, and more preferably 2.5 mM.

4. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to any one of claims 1 to 3, characterized in that: The Ca 2+ With PO4 2- The concentration ratio is 20-100:

1.

5. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to any one of claims 1 to 4, characterized in that: The reaction solution B includes PBS buffer or HBS buffer; Preferably, the pH of the PBS buffer or HBS buffer is 7.0-7.

5.

6. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to any one of claims 1 to 5, characterized in that: The reaction liquid A and the reaction liquid B independently further include DNase and RNase-free water.

7. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to any one of claims 1 to 6, characterized in that: In the step (2), the first incubation treatment includes: incubating the mixed solution at 0-4°C for 20-40 minutes, incubating at 40-45°C for 40-80 seconds, and incubating at 0-4°C for 3-8 minutes.

8. The method for preparing the calcium phosphate-mediated exosome drug delivery system according to any one of claims 1 to 7, characterized in that: In the step (3), the temperature of the second incubation treatment is 35-40°C, and the incubation time is 20-40 minutes; Preferably, in step (3), the amount of ribonuclease added based on the mixed solution is 3-8 μg / mL, preferably 5 μg / mL.

9. A calcium phosphate-mediated exosome drug delivery system prepared by the method according to any one of claims 1 to 8.

10. Use of the calcium phosphate-mediated exosome drug delivery system according to claim 9 for preparing nucleic acid drugs.

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