Non-cationic nucleic acid nanoparticle and preparation method thereof

Abstract

The invention discloses a non-cationic nucleic acid nanoparticle and a preparation method and application thereof, the non-cationic nucleic acid nanoparticle comprises metal ions, polyphenol and a nucleic acid drug, the metal ions and the polyphenol are coordinated and complexed to form a nano network structure, and the nucleic acid drug is entrapped in the nano network structure to form the nanoparticle. The non-cationic nucleic acid nanoparticle can be used as a novel nucleic acid delivery carrier, the preparation process of the nano delivery carrier is simple and controllable, and the economic cost is low; nucleic acid drugs can be effectively protected from being degraded by nuclease, and the stability is improved; the non-cationic delivery carrier can effectively avoid the problem of cytotoxicity of a cationic carrier, and has good in-vivo and in-vitro safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a non-cationic nucleic acid nanoparticle and a preparation method and application thereof. Background Art

[0002] Nucleic acid drugs are drugs with specific base sequences, including oligonucleotide drugs, nucleic acid aptamer drugs and nucleic acid vaccines. Currently, nucleic acid products have been widely used in basic research and clinical diagnosis and treatment of diseases such as tumors, infectious diseases, blood diseases and neurodegenerative diseases. Compared with traditional drugs, nucleic acid drugs have the advantages of high therapeutic efficiency, low drug toxicity and strong specificity, and are expected to become the third largest category of drugs after small molecule drugs and antibody drugs. For example, mRNA nucleic acid vaccines represented by mRNA technology have achieved great success in the prevention of the new coronavirus.

[0003] Due to their inherent large molecular weight and negative charge, free nucleic acid drugs are difficult to enter cells and are easily degraded by RNase. Therefore, nucleic acid drugs require suitable carriers to deliver them into cells to exert their therapeutic effects. A large number of studies have shown that, unlike microparticles, nanoscale particles are easily endocytosed by cells, and therefore have the potential to solve the problem of nucleic acid drug entry into cells. For example, the most advanced non-viral gene delivery system in clinical practice: lipid nanoparticle (LNP) technology. However, cationic lipids will inevitably cause an increase in host cytokine levels, leading to high inflammation and cytotoxicity. In addition, cationic carriers also have problems such as adsorbing protein coronas and being easily cleared, and difficulty in releasing nucleic acid drugs into cells. Therefore, safer and more efficient non-cationic delivery systems still need to be developed.

[0004] Nanoparticles formed by self-assembly of metal ions and organic ligands through coordination have been proven to be able to load drugs through direct coordination, physical embedding or covalent binding, effectively improving the stability of drugs in the body and prolonging the circulation time in the body, and have broad application prospects in nucleic acid drug delivery. In addition, this type of nanoparticle based on metal coordination can integrate the various characteristics of metal ions, organic ligands and loaded drugs, and combine metal immunotherapy and gene therapy. Summary of the invention

[0005] The object of the present invention is to provide a non-cationic nucleic acid nanoparticle, comprising metal ions, polyphenols and nucleic acid drugs, wherein the metal ions and polyphenols are coordinated and complexed to form a nano-network structure, and the nucleic acid drugs are contained in the nano-network structure to form nanoparticles; The molar ratio of the metal ions to the polyphenols is 1:1 to 20:1.

[0006] Furthermore, the metal ion is Al 3+ , Fe 3+ 、Zn2+ , Mn 2+ or Mg 2+ One or a mixture of several of them.

[0007] Furthermore, the polyphenol is one or a mixture of tannic acid, epigallocatechin gallate, gallic acid, and quercetin.

[0008] Furthermore, the nucleic acid drug is one or a mixture of DNA, siRNA, and mRNA.

[0009] The method for preparing the non-cationic nucleic acid nanoparticles comprises the following steps: Step 1, adding a polyphenol solution and a nucleic acid drug into a buffer solution and mixing; Step 2, adding the metal ion solution to the mixed solution of step 1, stirring and reacting; Step 3, after centrifuging the reaction solution of step 2, collecting the precipitate, resuspending it and then dispersing it by ultrasonication to obtain non-cationic nucleic acid nanoparticles.

[0010] Furthermore, the metal ion solution is a metal salt solution.

[0011] Furthermore, the buffer is a HEPES buffer with a pH range of 5.0 to 9.0.

[0012] Preferably, the concentration of the HEPES buffer is 10-100 mM, the pH is 7.4-9.0; the reaction temperature is room temperature, and the reaction time is 5-120 min.

[0013] In a specific embodiment of the present invention, the polyphenol is tannic acid, the metal salt solution is AlCl3 solution, and the molar ratio of metal ions to polyphenol is 1:1 to 8:1, preferably 4:1 to 8:1.

[0014] The third object of the present invention is to provide the use of the above non-cationic nucleic acid nanoparticles in the preparation of tumor prevention and treatment drugs.

[0015] The present invention designs a non-cationic nucleic acid nanoparticle based on metal coordination. The nanoparticle is formed by metal ions, polyphenols and nucleic acid drugs. The metal ions form a nano-network structure by coordination and complexation with the ortho-diphenol hydroxyl groups on the polyphenols. The nucleic acid drugs are encapsulated by the coordination with the metal ions and the hydrogen bonding of the polyphenols during the formation of the nano-network structure. The preparation method of the non-cationic nucleic acid nanoparticle is simple and controllable, and can effectively improve the stability and safety of the nucleic acid drugs. At the same time, the coordination bond is broken under the action of ATP in the cell, which is conducive to the release of the nucleic acid drugs and metal ions, and realizes the combination of gene therapy and metal immunotherapy.

[0016] The non-cationic nucleic acid nanoparticles in the present invention can release metal ions in cells and be used in combination with metal immunotherapy, which is scalable. For example, aluminum ions can activate and enhance the body's immune response through NLRP3 inflammasomes, improving the efficacy of vaccines; manganese ions activate the cGAS-STING pathway to induce anti-tumor immune response; magnesium ion supplementation can enhance natural killer cells and CD8 + T cell cytotoxicity.

[0017] The non-cationic nucleic acid nanoparticles of the present invention can be used as a novel nucleic acid delivery carrier, and the coordination complex between metal ions and polyphenols is used to form a nano skeleton, and the loaded nucleic acid is efficiently compressed through the coordination effect between the nucleic acid drug and the metal ions and the hydrogen bonding effect between the nucleic acid drug and the polyphenols; the preparation process of the nano delivery carrier is simple and controllable, and the economic cost is low; the nucleic acid drug can be effectively protected from being degraded by nucleases, and the stability is improved; the non-cationic delivery carrier can effectively avoid the cytotoxicity problem of the cationic carrier, and has good in vitro and in vivo safety; the coordination bond is responsively broken under the action of ATP in the cell, thereby facilitating the release of the nucleic acid drug and exerting a therapeutic effect, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For different molar ratios of Al 3+ Particle size results of non-cationic nucleic acid nanoparticles prepared with TA.

[0019] Figure 2 For different molar ratios of Al 3+ The mRNA encapsulation results of non-cationic nucleic acid nanoparticles prepared with TA.

[0020] Figure 3 This is an investigation of the mRNA encapsulation capacity of the non-cationic nucleic acid nanoparticles in Comparative Example 1.

[0021] Figure 4 The particle size distribution and zeta potential results of non-cationic nucleic acid nanoparticles.

[0022] Figure 5 This is a transmission electron microscopy image of non-cationic nucleic acid nanoparticles. The scale bar is 200 nm.

[0023] Figure 6 These are the results of the colloidal stability study of non-cationic nucleic acid nanoparticles.

[0024] Figure 7 These are the results of the RNase stability study of non-cationic nucleic acid nanoparticles.

[0025] Figure 8 This is the in vitro ATP-responsive release curve of non-cationic nucleic acid nanoparticles.

[0026] Fig. 9 The cytotoxicity results of non-cationic nucleic acid nanoparticles.

[0027] Fig.10 The results show the uptake of non-cationic nucleic acid nanoparticles by DC2.4 cells. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] In the following examples, the nucleic acid drug mRNA is a model mRNA, which is obtained by in vitro transcription using an eGFP plasmid (Addgene, Plasmid #129020); the nucleic acid drug siRNA is a negative control siRNA (Gene Gene). Example 1

[0032] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution and 68, 27.2, 13.6, 6.8, 3.4, 1.36, and 0 µg of nucleic acid drug mRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 10 µL of 4.2 mg / mL AlCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ATNs@mRNA). Example 2

[0033] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution and 68, 27.2, 13.6, 6.8, 3.4, 1.36, and 0 µg of nucleic acid drug mRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 5 µL of 4.2 mg / mL AlCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in HEPES buffer (pH 7.4, 10 mM) and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ATNs@mRNA). Example 3

[0034] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution and 68, 27.2, 13.6, 6.8, 3.4, 1.36, and 0 µg of nucleic acid drug mRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 2.5 µL of 4.2 mg / mL AlCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ATNs@mRNA).

[0035] Figure 1 For different molar ratios of Al 3+ The particle size results of non-cationic nucleic acid nanoparticles prepared with tannic acid (TA). 3+ Non-cationic nucleic acid nanoparticles were prepared with a molar ratio of TA to 2, 4, and 8, and the particle size was measured. The results showed that the particle size was smaller when the molar ratio was 4 and 8.

[0036] Figure 2 For different molar ratios of Al 3+ The nucleic acid encapsulation capacity of non-cationic nucleic acid nanoparticles prepared by using tannic acid (TA) was investigated. 3+A non-cationic nucleic acid nano-delivery system with a molar ratio of 2, 4, and 8 to TA was prepared by adding different amounts of mRNA to obtain ATNs@mRNA nanoparticles with a mass ratio of ATNs to mRNA of 0, 1, 2.5, 5, 10, 20, and 50. Agarose gel electrophoresis was performed to observe whether mRNA was successfully encapsulated. 3+ The higher the molar ratio of TA to TA, the stronger the nucleic acid encapsulation capacity of the non-cationic nucleic acid delivery system. Example 4

[0037] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL epigallocatechin gallate solution and 68, 27.2, 13.6, 6.8, 3.4, 1.36, and 0 µg of nucleic acid drug siRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 10 µL of 15 mg / mL FeCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (FENs@siRNA).

[0038] After testing, when the dosage of nucleic acid drug siRNA is 3.4 µg, the particle size of the prepared FENs@siRNA nanoparticles is about 120.56 nm. When the mass ratio of FENs to siRNA is greater than or equal to 7.5, siRNA can be completely encapsulated. Example 5

[0039] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution and 68, 27.2, 13.6, 6.8, 3.4, 1.36, and 0 µg of nucleic acid drug siRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 5 µL of 4.3 mg / mL ZnCl2 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ZTNs@siRNA).

[0040] After testing, when the dosage of nucleic acid drug siRNA is 3.4 µg, the particle size of the prepared ZTNs@siRNA nanoparticles is about 147.25nm. When the mass ratio of ZTNs to siRNA is greater than or equal to 10, siRNA can be completely encapsulated. Comparative Example 1

[0041] The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly. (2) Add 10 µL of 4.2 mg / mL AlCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) Add 68, 27.2, 13.6, 6.8, 3.4, and 0 µg of nucleic acid drug mRNA to the reaction solution in step (2) and mix thoroughly; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ATNs@mRNA).

[0042] Figure 3 The mRNA encapsulation capacity of the non-cationic nucleic acid nanoparticles of this example is shown in Figure 1. According to the preparation method of this example, metal ions and polyphenols are first prepared to form nanoparticles, which are then used to load nucleic acid drugs. The results show that the nanoparticles obtained by this preparation method cannot achieve effective mRNA encapsulation.

[0043] The non-cationic nucleic acid nanoparticles are characterized and investigated below. The preparation process of the non-cationic nucleic acid nanoparticles is as follows: The preparation of non-cationic nucleic acid nanoparticles comprises the following steps: (1) Add 10 µL of 6.8 mg / mL tannic acid solution and 6.8 µg of nucleic acid drug mRNA to 100 µL of pH 7.4 10 mM HEPES buffer and mix thoroughly; (2) Add 10 µL of 4.2 mg / mL AlCl3 solution to the solution in step (1) and react at room temperature for 1 h; (3) The reaction solution was centrifuged at 9000 g for 5 min, and the precipitate was collected. It was resuspended in pH 7.4 10 mM HEPES buffer and then dispersed by ultrasonication (power 15%, 1 s on, 2 s off, 2 min) to obtain non-cationic nucleic acid nanoparticles (ATNs@mRNA).

[0044] Figure 4 The particle size distribution and zeta potential results of non-cationic nucleic acid nanoparticles. It can be seen from the figure that the particle size of the prepared ATNs@mRNA nanoparticles is about 163.78 nm, and the average zeta potential is -28.63 mV.

[0045] Figure 5 This is a transmission electron micrograph of non-cationic nucleic acid nanoparticles. Take the nanoparticle preparation and drop it on a 300-mesh copper grid covered with a carbon film. After it dries, place it under a transmission electron microscope for observation. The results show that the ATNs@mRNA nanoparticles have a uniform particle size distribution and are rod-shaped.

[0046] Figure 6 To investigate the colloidal stability of non-cationic nucleic acid nanoparticles. ATNs@mRNA nanoparticles were diluted to a concentration of 80 µg / mL in water and DMEM medium containing 10% FBS, and the changes in particle size over time were investigated. The results showed that the non-cationic nucleic acid nanoparticles had good stability in water and DMEM medium containing 10% FBS within 7 days.

[0047] Figure 7 To investigate the RNase stability of non-cationic nucleic acid nanoparticles. Free mRNA and ATNs@mRNA nanoparticles (mRNA concentration was 20 µg / mL) were treated with 5 µg / mL RNase solution at 37°C for different time periods (0, 2, 4, 8, 12, 24h), and then mRNA was displaced by adding adenosine triphosphate (ATP). Agarose gel electrophoresis was used to detect whether mRNA was degraded by RNase. The results showed that non-cationic nano nucleic acid delivery vectors can effectively protect mRNA from being degraded by RNase and have RNase stability.

[0048] Figure 8 The figure is a graph of the in vitro ATP-responsive release curve of non-cationic nucleic acid nanoparticles. After the preparation (with mRNA concentration of 20 µg / mL) was incubated in the presence or absence of 2mM ATP solution for different times (0, 1, 2, 4, 8, 12, 24h), the released free mRNA content was measured by agarose gel electrophoresis. From the figure, it can be seen that the release of mRNA is ATP-sensitive. In the ATP environment, the cumulative release in 24h can reach 63%, while only 12% of the nucleic acid drug can be released without ATP.

[0049] Fig. 9The present invention is a cytotoxicity analysis of non-cationic nucleic acid nanoparticles. ATNs nanoparticles were prepared according to Example 1, and DC2.4 cells were inoculated in a 96-well plate. After the cells adhered to the wall, 0, 20, 40, 80, 160, and 320 μg / mL of the preparations were added and incubated for 24 hours; then the culture medium was discarded, and MTT solution was added. After 4 hours, the MTT solution was discarded, and DMSO was added to dissolve the generated formazan. The absorbance at 490 nm was measured using an ELISA instrument to evaluate the toxic effect of the nanoparticles on cells. The results showed that the cell survival rate was high when the concentration of the non-cationic nano nucleic acid delivery carrier was in the range of 0~80 μg / ml, and it had good safety.

[0050] Fig.10 The results of DC2.4 cell uptake of non-cationic nucleic acid nanoparticles. Nanoparticles were prepared using FAM-labeled RNA (provided by Jima Gene), and DC2.4 cells were seeded in 24-well plates. After the cells adhered to the wall, they were treated with culture media containing PBS, FAM-RNA, and ATNs@FAM-RNA, respectively, and the FAM-RNA administration concentration was maintained at 2 µg / ml. After 4 hours, the drug-containing culture medium was discarded, and the cells were washed with PBS and digested with trypsin and collected in EP tubes, and the fluorescence intensity of intracellular FAM was detected by flow cytometry. The results showed that compared with free FAM-RNA, the non-cationic nanodelivery system ATNs can significantly improve the uptake level of nucleic acid drugs by DC2.4 cells.

Claims

1. A non-cationic nucleic acid nanoparticle, characterized in that: It comprises metal ions, polyphenols and nucleic acid drugs, wherein the metal ions and the polyphenols are coordinated and complexed to form a nano-network structure, and the nucleic acid drugs are contained in the nano-network structure to form nanoparticles; The molar ratio of the metal ions to the polyphenols is 1:1 to 20:

1.

2. The non-cationic nucleic acid nanoparticle according to claim 1, characterized in that: The metal ion is Al 3+ , Fe 3+ 、Zn 2+ , Mn 2+ or Mg 2+ One or a mixture of several of them.

3. The non-cationic nucleic acid nanoparticle according to claim 1, characterized in that: The polyphenol is one or a mixture of tannic acid, epigallocatechin gallate, gallic acid and quercetin.

4. The non-cationic nucleic acid nanoparticle according to claim 1, characterized in that: The nucleic acid drug is one or a mixture of DNA, siRNA, and mRNA.

5. The method for preparing the non-cationic nucleic acid nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1, adding a polyphenol solution and a nucleic acid drug into a buffer solution and mixing; Step 2, adding the metal ion solution to the mixed solution of step 1, stirring and reacting; Step 3, after centrifuging the reaction solution of step 2, collecting the precipitate, resuspending it and then dispersing it by ultrasonication to obtain non-cationic nucleic acid nanoparticles.

6. The preparation method according to claim 5, characterized in that: The metal ion solution is a metal salt solution.

7. The preparation method according to claim 5, characterized in that: The buffer is HEPES buffer with a pH of 5.0-9.

0.

8. Use of the non-cationic nucleic acid nanoparticles according to any one of claims 1 to 4 in the preparation of tumor prevention and treatment drugs.

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