Super-oxidation protection preparation for stably storing siRNA (small interfering ribonucleic acid) nanoparticles and application of super-oxidation protection preparation

Abstract

The invention relates to the field of biology, in particular to a superoxide protection preparation for stable storage of siRNA nanoparticles and application of the superoxide protection preparation. The invention provides a novel superoxide protection preparation for stable storage of siRNA nanoparticles, and the superoxide protection preparation is helpful for further prolonging the gene silencing function of functional siRNA nanoparticles, and can ensure the quality and stability after the superoxide protection preparation and the siRNA nanoparticles are mixed. Meanwhile, proliferation of harmful microorganisms can be inhibited in a short period, and the effect of prolonging siRNA targeted silence related genes in the middle and later periods is achieved, so that the purposes of enhancing the curative effect and prolonging the action time are achieved, and the superoxide protection preparation provided by the invention can meet the current application requirements. In conclusion, the superoxide protection provided by the invention not only has strong sterilization performance, but also has the characteristic of being harmless to a human body, and meanwhile, the RNA interference capability can be maintained not to be influenced by strong oxidizability.

Description

Technical Field

[0001] The present invention relates to the biological field, and particularly to a superoxide protection preparation for stable storage of siRNA nanoparticles and its application. Background Art

[0002] In recent years, the importance of non-coding RNAs (such as microRNA, lncRNA, etc.) in biological research has become increasingly prominent. Researchers of siRNA nanoparticles with similar structures to them have found that these functional RNA products also change during storage and may become markers of storage damage. Therefore, the storage and protection of functional RNAs have also become a new research direction. The storage of RNA products is a key link in biological research. Traditional storage methods for RNA products include: quick-freezing method and improved storage method.

[0003] Quick-freezing method: After fresh tissues or cells are sampled using a sampling bag or sampling tube, they are immediately frozen with liquid nitrogen and then transferred to an environment of -80°C for long-term storage. However, this method generally does not exceed two years because low temperature only reduces the activity of RNase rather than completely inactivating it, and partial RNA degradation may occur during storage and thawing for extraction.

[0004] Improved storage method: On the basis of the quick-freezing method, before the target product is transferred to -80°C for storage, RNAKeeper-ICE Tissue Transition Buffer or other stabilizers are added before extraction, and stored at ultra-low temperature for at least 12 hours to ensure that the buffer completely penetrates into the tissue, thereby softening the tissue and inhibiting the activity of RNase, facilitating subsequent RNA extraction.

[0005] In addition to traditional low-temperature storage, researchers are also exploring other storage conditions, such as using specific pH values, salt concentrations, or additives to stabilize RNA. However, under normal conditions, the half-life of RNA is extremely short and is greatly affected by environmental factors, making it difficult to maintain biological functions in the natural environment for a long time. There is currently no report on a preparation that can be used for both sterilization and RNAi stabilization. Summary of the Invention

[0006] The object of the present invention is to provide a superoxide protection preparation for stable storage of siRNA nanoparticles and its application to solve the problems existing in the above-mentioned prior art. The preparation provided by the present invention can fill the current application needs, ensure the strong sterilization performance of the superoxide preparation, ensure its harmlessness to the human body, and at the same time maintain the RNAi interference ability from being affected by strong oxidizing properties.

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

[0008] The present invention provides a superoxide protection preparation for stable storage of siRNA nanoparticles, comprising a superoxide protectant and a superoxide protection solution;

[0009] The superoxide protectant includes a metal chelator, mannitol, a protein freeze-thaw stabilizer, a disintegrant, povidone, tubulin, and an oxidation-reducing agent; the metal chelator includes ethylenediaminetetraacetic acid and / or disodium ethylenediaminetetraacetate; the protein freeze-thaw stabilizer includes trehalose and / or sucrose; the disintegrant includes one or more of sodium carboxymethyl starch, effervescent disintegrant, sodium carboxymethyl cellulose, and polyvinylpyrrolidone; the effervescent disintegrant includes citric acid and NaHCO3; the oxidation-reducing agent includes chlorine dioxide;

[0010] The superoxide protection solution includes a metal chelator, a pH buffer, a film-forming agent, an oxidation-maintaining agent, chitosan, sodium chloride, and a polypeptide; the metal chelator includes ethylenediaminetetraacetic acid and / or disodium ethylenediaminetetraacetate; the pH buffer includes one or more of acetic acid, α-hydroxy acid, tretinoin, disodium hydrogen phosphate, potassium dihydrogen phosphate, and salicylic acid; the film-forming agent includes one or more of urea, ceramide, and glycerol; the oxidation-maintaining agent includes hydrogen peroxide and / or sodium hypochlorite; the polypeptide includes cecropin and / or bacitracin.

[0011] Preferably, the concentration of the metal chelator in the superoxide protectant is 10-40 mg / g; the concentration of mannitol is 100-400 mg / g; the concentration of the protein freeze-thaw stabilizer is 200 mg / g; the mass percentage of the disintegrant is 2-8%; the mass percentage of povidone is 0.5-2%; the mass percentage of tubulin is 1-4%; the chlorine dioxide is 0.03 g - 0.12 g / g.

[0012] Preferably, when the pH buffer in the superoxide protection solution is acetic acid, disodium hydrogen phosphate, and potassium dihydrogen phosphate, the film-forming agent is urea, and the oxidation-maintaining agent is hydrogen peroxide, the concentration of the metal chelator in the superoxide protection solution is 5-20 mM, the concentration of acetic acid is 50-200 mM, the concentration of disodium hydrogen phosphate is 1.44 g / L, the concentration of potassium dihydrogen phosphate is 0.24 g / L, the concentration of the film-forming agent is 1.8-7.2 mM, the mass percentage of the oxidation-maintaining agent is 1-4%, the concentration of chitosan is 50-200 mg / L, the mass percentage of sodium chloride is 0.9%, and the mass percentage of the polypeptide is 0.05-0.2%;

[0013] When three of α-hydroxy acid, tretinoin, disodium hydrogen phosphate, potassium dihydrogen phosphate and salicylic acid are used as the pH buffer in the superoxide protection solution, ceramide or glycerol is used as the film-forming agent, and sodium hypochlorite is used as the oxidation maintainer, the concentration of the metal chelator in the superoxide protection solution is 5-20 mM, the mass percentage of α-hydroxy acid, tretinoin or salicylic acid is 1-4%, the concentration of disodium hydrogen phosphate is 1.44 g / L, the concentration of potassium dihydrogen phosphate is 0.24 g / L, the mass percentage of the film-forming agent is 2.5-10%, the concentration of the oxidation maintainer is 50-200 ppm, the concentration of chitosan is 50-200 mg / L, the mass percentage of sodium chloride is 0.9%; the mass percentage of the polypeptide is 0.05-0.2%.

[0014] Preferably, the mass-to-volume ratio of the superoxide protector to the superoxide protection solution is 1 g:(0.25 L - 1 L).

[0015] The present invention provides the application of the above-mentioned superoxide protection preparation in maintaining the properties of siRNA.

[0016] Preferably, the properties include one or more of stability, interference, antioxidant property and antifungal property.

[0017] The present invention provides the application of the above-mentioned superoxide protection preparation in the preparation of siRNA nanoparticle preparations.

[0018] The present invention provides an siRNA nanoparticle preparation, which includes the above-mentioned superoxide protection preparation and siRNA nanoparticles.

[0019] Preferably, the mass-to-volume ratio of the superoxide protector, the superoxide protection solution and the siRNA particles is 1 g:(0.25 L - 1 L):1 g.

[0020] The present invention provides a preservation bottle for siRNA nanoparticle preparations, which includes a bottle cap, a barrel wall, a drug-loading groove and a bottle body; the drug-loading groove includes an A groove for loading the superoxide protector in the above-mentioned superoxide protection preparation and a B groove for loading siRNA nanoparticles; the bottle body loads the superoxide protection solution in the above-mentioned superoxide protection preparation.

[0021] The preservation bottle for siRNA nanoparticle preparations of the present invention has two states, namely a closed state and a mixed state, as Figure 1 and Figure 2 shown. Figure 1It is the closed state of the preservation bottle of the siRNA nanoparticle preparation. The top cover is not pressed down, the drug-loading groove is in close contact with the cylinder wall, maintaining a closed state. The superoxide protection solution in the siRNA nanoparticle preparation preservation bottle is not in contact with the superoxide protectant and the RNA nanoparticles, and the superoxide protectant and the RNA nanoparticles remain independent; Figure 2 It is the mixed state of the preservation bottle of the siRNA nanoparticle preparation. The top cover is pressed down, the drug-loading groove moves downward, and it can be mixed with the superoxide protection solution in the bottle. After inversion, the solution is evenly mixed and can be used.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a superoxide protection preparation for the stable storage of novel siRNA nanoparticles. This superoxide protection preparation helps to further extend the gene silencing function of functional siRNA nanoparticles, can ensure the quality and stability after mixing the superoxide protection preparation and siRNA nanoparticles, and can also inhibit the proliferation of harmful microorganisms in the short term and play a role in prolonging the siRNA-targeted silencing of related genes in the middle and late stages, so as to achieve the purpose of enhancing the curative effect and prolonging the action time. Therefore, the superoxide protection preparation provided by the present invention can fill the current application needs. In summary, the superoxide protection provided by the present invention has both strong sterilization performance and the characteristics of being harmless to the human body, and can also maintain the RNA interference ability without being affected by strong oxidability, which is specifically reflected in the following aspects:

[0024] 1. When ensuring that the oxidation-reduction potential (ORP) is greater than 1000 mV, it will not cause oxidative damage to siRNA nanoparticles within 96 hours of storage, and will not affect the transmission of RNA sequence genetic information and the RNA interference effect;

[0025] 2. The siRNA nanoparticle preparation containing the superoxide protection preparation provided by the present invention has a super-stable structure, can resist oxidation, prolong the degradation time, and can maintain the integrity of siRNA, preventing siRNA from being attacked by proteases and nucleases;

[0026] 3. The bactericidal action time of the novel superoxide protection preparation is long. Within a certain period of time, the bactericidal ability of this superoxide protection preparation remains unchanged, and it ensures the lowest cytotoxicity and the lowest sensitization to human skin and mucous membranes;

[0027] 4. After applying the superoxide protection preparation provided by the present invention, the siRNA nanoparticles have antioxidant ability and can remain relatively stable in the natural environment for 7 to 14 days;

[0028] 5. After mixing siRNA nanoparticles with a superoxide protectant and coating them onto a culture medium, it can cause the death of pathogenic bacteria, with a bactericidal performance of 99.9%. The inhibition rate against the active mycelia of fungi can reach over 99.0%.

[0029] 6. Recover the siRNA nanoparticles preserved with the superoxide protection preparation provided by the present invention and conduct an RNA interference experiment. The siRNA still maintains a high degree of integrity, proving that the superoxide protection preparation provided by the present invention can extend the biological function of RNA interference in the later stage of antibacterial.

[0030] 7. After combining the superoxide protection preparation provided by the present invention with siRNA nanoparticles, it can maintain a highly efficient bactericidal effect in the initial stage, and can extend the gene silencing function of siRNA in the middle and later stages, inhibit the expression of related genes, thereby achieving the ability to inhibit the growth of pathogenic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is the closed state of the preservation bottle for the siRNA nanoparticle preparation; wherein, 1 is the bottle cap, 2 is the barrel wall, 3 is the drug-loading groove, 4 is the bottle body (brown for light protection), A is the drug-loading groove for placing the superoxide protectant, and B is the drug-loading groove for placing the RNA nanoparticles;

[0033] Figure 2 It is the mixed state of the preservation bottle for the siRNA nanoparticle preparation; wherein, 1 is the bottle cap, 2 is the barrel wall, 3 is the drug-loading groove, 4 is the bottle body (brown for light protection), A is the drug-loading groove for placing the superoxide protectant, and B is the drug-loading groove for placing the RNA nanoparticles;

[0034] Figure 3 It is the experimental result of the bactericidal ability against Staphylococcus aureus; wherein, Ctr is the positive control group, 25U is the 25U working solution, 50U is the 50U working solution, 100U is the 100U working solution, and 200U is the 200U working solution;

[0035] Figure 4Figure showing the stability experiment results of siRNA nanoparticles after 96 hours; where M is the molecular weight marker, B is the cryopreserved control of siRNA nanoparticles, 50 is the siRNA nanoparticles recovered after treatment with a 50 ppm working solution, 100 is the siRNA nanoparticles recovered after treatment with a 100 ppm working solution, 200 is the siRNA nanoparticles recovered after treatment with a 200 ppm working solution, and C is the treatment with the uncoated working solution. Detailed implementation manners

[0036] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0040] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0041] Unless otherwise specified, "%" in the present invention means mass percentage content; the substances used in the present invention are all routinely purchased by those skilled in the art.

[0042] Example 1

[0043] A superoxide protection preparation for stable storage of siRNA nanoparticles, which consists of a superoxide protectant (1 g) and a superoxide protection solution. Among them, the components and concentrations of the superoxide protectant are shown in Table 1, and the components and concentrations of the superoxide protection solution are shown in Table 2.

[0044] Table 1 Composition table of the superoxide protectant

[0045]

[0046] Table 2 Composition table of the superoxide protection solution (pH 6.5 - 7.5)

[0047]

[0048]

[0049] The superoxide protectant and the superoxide protection solution prepared respectively according to Table 1 and Table 2 are filled in a preservation bottle for siRNA nanoparticle preparation. The structure of the preservation bottle for siRNA nanoparticle preparation is as shown in Figure 1 and Figure 2 The preservation bottle for siRNA nanoparticle preparation consists of the following parts: a bottle cap, a barrel wall, a drug-loading groove and a bottle body. Among them, part A of the drug-loading groove loads the superoxide protectant (loading amount 1 g), part B loads RNA nanoparticles (loading amount 1 g, containing 50 mg / g of RNA nanoparticle mixture), and the bottle body loads the superoxide protection solution (loading amount 1 L); the mass-volume ratio of siRNA nanoparticles (from the laboratory of Zhejiang Sci-Tech University, whose sequence is publicly disclosed in the Chinese patent "An RNA Bacteriostatic Agent siR2 and a Crop Pathogen Inhibitor", that is, the sequence of siR2 in this patent), the superoxide protectant and the superoxide protection solution is 1 g:1 g:(0.25 L - 1 L). Figure 1 is the closed state of the preservation bottle for siRNA nanoparticle preparation. The top cover is not pressed down, and the drug-loading groove is in close contact with the barrel wall, maintaining a closed state. The superoxide protection solution in the preservation bottle for siRNA nanoparticle preparation does not contact the superoxide protectant and siRNA nanoparticles, and the superoxide protectant and siRNA nanoparticles remain independent; Figure 2 is the mixed state of the preservation bottle for siRNA nanoparticle preparation. The top cover is pressed down, the drug-loading groove moves downward, and it can be mixed with the superoxide protection solution in the bottle. After inversion, the solution is evenly mixed and can be used. After the bottle cap is pressed down and the bottle body is inverted several times, the superoxide protection solution meets the superoxide protectant, making the superoxide protectant dissolve quickly. Its ORP > 1000 mv within 1 h. At this time, a working solution with a concentration of 100 mg / L is obtained, and its antibacterial ability reaches 99.9% when contacting Staphylococcus aureus within 10 min.

[0050] Example 2 Concentration range of the new preparation

[0051] Prepare the superoxide protectant and the superoxide protection solution according to the components and concentrations in Table 1 and Table 2 to obtain the superoxide protectant and the superoxide protection solution in Table 3 and Table 4. Then, mix the siRNA nanoparticles and the superoxide protectant in a mass ratio of 1:1, add 250 mL of 4% hydrogen peroxide diluted with double-distilled water to prepare a superoxide protection solution with a total concentration of 400 ppm, and then dilute it with double-distilled water to 200 ppm (200 U), 100 ppm (100 U), 50 ppm (50 U), 25 ppm (25 U) and 0 ppm (Ctr) respectively as the working solution. Add the Staphylococcus aureus suspension (1.0×10 5 CFU / mL, much higher than the diagnostic order of magnitude of burn wound sepsis) according to a volume ratio of 1:1, mix it with the suspension and let it stand for 10 min. Take 100 μL and coat it on the bacterial culture medium plate. After 24 h, investigate its bactericidal effect on Staphylococcus aureus. The results are shown in Table 5 and Figure 3 as follows.

[0052] Table 3 Components and Concentrations of Superoxide Protectant

[0053] Serial number Inclusion name Content range per gram of solution 1 Ethylenediaminetetraacetic acid 40 mg / g 2 Mannitol 400 mg / g 3 Trehalose 200 mg / g 4 Sodium carboxymethyl starch 8 wt.% 5 Polyvinylpyrrolidone (PVP) 2 wt.% 6 Tubulin 4 wt.% 7 Chlorine dioxide 0.12 g / g

[0054] Table 4 Configuration Table of Superoxide Protection Solution

[0055] Serial number Inclusion name Final content range per liter of solution (final concentration) 1 Ethylenediaminetetraacetic acid 20 mM 2 Acetic acid 200 mM 3 Urea 6.5 mM 4 Hydrogen peroxide 2 wt.% 5 Chitosan 200 mg / L 6 Sodium chloride 0.9 wt.% 7 Monosodium hydrogen phosphate 1.44 g / L 8 Potassium dihydrogen phosphate 0.24 g / L 9 Cecropin 0.5 wt.% 10 Double distilled water /

[0056] Table 5 Bactericidal Effects of Different Concentrations on Staphylococcus aureus

[0057]

[0058] The experimental results recorded in Table 5 show that: within 50 - 200 ppm, the bactericidal efficiency is greater than 99.9%.

[0059] Example 3 Screening of Action Time at the Effective Concentration of the New Preparation

[0060] Prepare the superoxide protectant and the superoxide protection solution according to the components and concentrations in Table 1 and Table 2 to obtain the superoxide protectant and the superoxide protection solution in Table 3 and Table 4. Then, mix the siRNA nanoparticles and the superoxide protectant in a mass ratio of 1:1, add 250 mL of 4% hydrogen peroxide diluted with double-distilled water, and add water to 1 L to prepare a working solution with a final concentration of 100 ppm. Place it for 0 h, 2 h, 12 h, 24 h, 36 h, 48 h respectively. Then, mix it with the Staphylococcus aureus suspension (1.0×10 5 CFU / mL) at a ratio of 1:1 and let it stand for 10 min (the final concentration of the liquid medicine is 50 ppm), coat it on the bacterial culture medium plate. After 24 h, investigate its bactericidal effect on Staphylococcus aureus. The results are shown in Table 6.

[0061] Table 6 Bactericidal effects of different standing times on Staphylococcus aureus after configuration

[0062]

[0063] As can be seen from Table 6, the superoxide protectant and superoxide protection solution provided by the present invention have obvious bactericidal effects on Staphylococcus aureus.

[0064] Example 4 In vitro cytotoxicity test

[0065] Optimization of the lowest cytotoxic preparation concentration of the superoxide protection solution According to GB / T 16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", the superoxide protectant and superoxide protection solution prepared according to Tables 3 and 4 were used. Then, siRNA nanoparticles and the superoxide protectant were mixed in a ratio of 1:1 by mass, and hydrogen peroxide diluted with double-distilled water at 4% was added to prepare a superoxide protection solution with a total concentration of 400 ppm. Then, it was diluted to 200 ppm, 100 ppm, 50 ppm, 25 ppm, and 12.5 ppm with MEM medium to obtain 5 sample solutions.

[0066] Cytotoxicity test method (MTT method): Inoculate 1×10 5 / ml mouse fibroblast suspension into a 96-well plate, 100 μL per well; after culturing in a 5% CO2 incubator at 37 °C for 24 h, discard the original culture medium; add sample solution, blank control solution (clear water), negative control solution (MEM medium), and positive control solution (6% H2O2), 100 μL per well, and culture in a CO2 incubator at 37 °C for 24 h; remove the original culture medium, add 50 μL MTT solution to each well; continue to culture for 2 h, then remove the MTT solution in the wells, add 100 μL isopropanol, shake for 10 min, and measure the absorbance (OD value) at double wavelengths of 570 nm and 650 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the survival rate according to the following formula:

[0067] Survival rate (%) = (100 × OD 570e ) / OD 570b ; where OD 570e is the average absorbance of the experimental sample group (negative and positive control groups), and OD 570b is the average absorbance of the blank control group. The results are shown in Table 7;

[0068] Table 7 Effects of different concentrations of the superoxide protection solution on cell viability

[0069]

[0070] As can be seen from the results in Table 7, the cytotoxicity is the lowest at a concentration below 50 ppm. Combining with Table 5, it can be obtained that 50 ppm can achieve the best bactericidal effect and the lowest cytotoxicity.

[0071] Example 5 Guinea Pig Skin Sensitization Test

[0072] Guinea pig skin hypersensitivity test: Thirty guinea pigs were randomly divided into 3 groups according to body weight, namely, a vehicle group (common vehicles include olive oil, lanolin, petrolatum, etc., and petrolatum was used in this example) and a positive control group (2,4-dinitrofluorobenzene), with 10 guinea pigs in each group, and 20 guinea pigs in the sample solution (50 ppm) group of Example 4. Before the test, the hair on the left and right sides of the guinea pigs' backs was removed 24 h in advance, and the hair removal area was 3 cm × 4 cm. Then, the guinea pigs were sensitized 3 times on days 0, 7, and 14. On the depilated area on the left side of the guinea pigs, 0.2 mL of vehicle, sample solution, and 1% 2,4-dinitrofluorobenzene were applied respectively, covered with a layer of cellophane and two layers of gauze, fixed with adhesive tape, and lasted for 6 h. Then, on day 28, the guinea pigs were challenged. The hair on the right side of the guinea pigs was removed, and the area was the same as that on the left side. 0.2 mL of vehicle, sample solution, and 0.1% 2,4-dinitrofluorobenzene were applied respectively, covered with a layer of cellophane and two layers of gauze, fixed with adhesive tape, and lasted for 6 h. After the test substance was removed, the skin hypersensitivity reaction was observed immediately, and then the skin hypersensitivity reaction was observed again at 24, 48, and 72 h to evaluate the sensitization grade. The incidence of sensitization reaction and the average reaction score were calculated according to the following formulas.

[0073] Incidence of sensitization reaction (%) = Number of animals with allergic reaction ÷ Total number of animals × 100% Average reaction score = Total score of all animals ÷ Total number of animals.

[0074] The results are shown in Table 8. As can be seen from Table 8, the sensitization rate of the 50 ppm sample solution is 0%.

[0075] Table 8 Effect of Sample Solution on Guinea Pig Skin Hypersensitivity Reaction

[0076]

[0077] Example 6 Detection of siRNA Nanoparticle Integrity

[0078] The working solutions configured at three concentrations of 200 U, 100 U, and 50 U in Table 3 (1 ppm = 1 U) were placed in sterilized test tubes. A sterilized PVA (polyvinyl alcohol) sponge of 0.5×0.5×0.5 cm was placed inside the test tubes. 500 μL of each of the 3 working solutions was pipetted onto the PVA sponge, and the operation was carried out once every 24 h. By 96 h, it was the 5th time. Throughout the process, the test tubes were placed in an incubator at 40 °C. At the same time, a cryopreservation control of siRNA nanoparticles and a treatment of the uncoated working solution were set up. After 96 h, it was moistened with 200 μL of TE dissolution solution to dissolve the contents inside the sponge. The PVA sponge was transferred to a 1.5 mL centrifuge tube in a laminar flow hood. After centrifugation at 10,000 rpm for 3 min, the centrifugate was obtained. After the above centrifugate was loaded, agarose gel electrophoresis was carried out to obtain the basic RNA bands. The agarose gel electrophoresis was as Figure 4 shown. The results showed that for the working solutions at a concentration of 50 U and above, after 96 h of incubation and repeated drying and rewetting, the siRNA nanoparticles still remained intact.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A superoxidation protection preparation for stable storage of siRNA nanoparticles, characterized in that: Including superoxide protection agent and superoxide protection solution; The superoxide protective agent includes metal chelators, mannitol, protein freeze-thaw stabilizers, disintegrants, povidone, tubulin and redox agents; the metal chelators include ethylenediaminetetraacetic acid and / or disodium ethylenediaminetetraacetate; the protein freeze-thaw stabilizers include trehalose and / or sucrose; the disintegrants include one or more of sodium carboxymethyl starch, effervescent disintegrants, sodium carboxymethyl cellulose and polyvinyl pyrrolidone; the effervescent disintegrants include citric acid and NaHCO3; the redox agents include chlorine dioxide; The superoxidized protective liquid comprises a metal chelator, a pH buffer, a film-forming agent, an oxidation-maintaining agent, chitosan, sodium chloride and a polypeptide; the metal chelator comprises ethylenediaminetetraacetic acid and / or disodium ethylenediaminetetraacetic acid; the pH buffer comprises one or more of acetic acid, α-hydroxy acid, A acid, disodium hydrogen phosphate, potassium dihydrogen phosphate and salicylic acid; the film-forming agent comprises one or more of urea, ceramide and glycerol; the oxidation-maintaining agent comprises hydrogen peroxide and / or sodium hypochlorite; the polypeptide comprises cecropin and / or bacitracin.

2. The superoxidation protective preparation according to claim 1, characterized in that The concentration of the metal chelator in the superoxide protective agent is 10-40 mg / g, the concentration of the mannitol is 100-400 mg / g, the concentration of the protein freeze-thaw stabilizer is 200 mg / g, the mass percentage of the disintegrant is 2-8%, the mass percentage of the povidone is 0.5-2%, the mass percentage of the microtubule protein is 1-4%; and the chlorine dioxide is 0.03 g-0.12 g / g.

3. The superoxidation protective preparation according to claim 1, characterized in that When the pH buffer in the superoxidized protective solution is acetic acid, disodium hydrogen phosphate and potassium dihydrogen phosphate, the film-forming agent is urea, and the oxidation maintenance agent is hydrogen peroxide, the concentration of the metal chelating agent in the superoxidized protective solution is 5-20 mM, the concentration of the acetic acid is 50-200 mM, the concentration of the disodium hydrogen phosphate is 1.44 g / L, the concentration of the potassium dihydrogen phosphate is 0.24 g / L, the concentration of the film-forming agent is 1.8-7.2 mM, the mass percentage of the oxidation maintenance agent is 1-4%, the concentration of the chitosan is 50-200 mg / L, the mass percentage of the sodium chloride is 0.9%, and the mass percentage of the polypeptide is 0.05-0.2%; When the pH buffer in the superoxidized protective solution is three of α-hydroxy acid, A acid, disodium hydrogen phosphate, potassium dihydrogen phosphate and salicylic acid, the film-forming agent is ceramide or glycerol, and the oxidation-maintaining agent is sodium hypochlorite, the concentration of the metal chelating agent in the superoxidized protective solution is 5-20 mM, the mass percentage of the α-hydroxy acid, A acid or salicylic acid is 1-4%, the concentration of disodium hydrogen phosphate is 1.44 g / L, the concentration of potassium dihydrogen phosphate is 0.24 g / L, the mass percentage of the film-forming agent is 2.5-10%, the concentration of the oxidation-maintaining agent is 50-200 ppm, the concentration of chitosan is 50-200 mg / L, the mass percentage of sodium chloride is 0.9%; and the mass percentage of the polypeptide is 0.05-0.2%.

4. The superoxidation protective preparation according to claim 1, characterized in that The mass volume ratio of the superoxidation protective agent to the superoxidation protective solution is 1g:(0.25L-1L).

5. Use of the superoxidation protection agent according to any one of claims 1 to 4 in maintaining the properties of siRNA.

6. The use according to claim 5, characterized in that: The properties include one or more of stability, interference, antioxidant and antifungal properties.

7. Use of the superoxidation protection preparation according to any one of claims 1 to 4 in the preparation of siRNA nanoparticle preparations.

8. A siRNA nanoparticle preparation, characterized in that: The siRNA nanoparticle preparation comprises the superoxidation protection preparation according to claims 1 to 4 and siRNA nanoparticles.

9. The siRNA nanoparticle preparation according to claim 8, characterized in that: The mass volume ratio of the superoxidation protective agent, the superoxidation protective solution and the siRNA particles is 1g:(0.25L-1L):1g.

10. A siRNA nanoparticle preparation storage bottle, characterized in that: The siRNA nanoparticle preparation storage bottle comprises a bottle cap, a barrel wall, a drug loading groove and a bottle body; the drug loading groove comprises a groove A for loading the superoxidation protective agent in the superoxidation protective preparation described in any one of claims 1 to 4 and a groove B for loading the siRNA nanoparticles; the bottle body is loaded with the superoxidation protective solution in the superoxidation protective preparation described in any one of claims 1 to 4.

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