Amphiphilic nucleic acid nano-carrier as well as preparation method and application thereof

Abstract

The invention discloses an amphiphilic nucleic acid nano-carrier as well as a preparation method and application thereof. The invention discloses a preparation method of an amphiphilic nucleic acid nano-carrier, which comprises the following steps: mixing DHA-NHS and polylysine hydrochloride, then adding an organic solvent and triethylamine, reacting and assembling in a weak base environment, after the reaction is finished, removing the organic solvent, and drying to obtain solid powder, namely the amphiphilic nucleic acid nano-carrier. The invention also discloses a pharmaceutical composition, which comprises the amphiphilic nucleic acid nano-carrier and a negatively charged nucleic acid drug. The amphiphilic nucleic acid nano-carrier provided by the invention can load nucleic acid drugs with negative charges through an electrostatic adsorption effect, has very strong corneal tissue permeability and extremely strong antibacterial performance, can relatively prolong the retention time of the drugs on ocular surfaces while exerting the antibacterial function, and can be used for preparing the nano-carrier for treating the corneal diseases, such as the corneal diseases, the corneal diseases, the corneal diseases, the corneal diseases and the like. The medicine effect of the medicine is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an amphiphilic nucleic acid nanocarrier and a preparation method and application thereof. Background Art

[0002] MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs with a length of about 20-22 nucleotides and gene regulatory functions. miRNAs play an important role in the normal development and function of all tissues and organ systems and the pathogenesis of diseases. Although the connection between miRNAs and infectious eye lesions remains to be studied, there are literature reports that miRNAs play an important role in inhibiting ocular neovascularization and in the repair of skin wounds. However, miRNAs are single-stranded small RNAs with unstable properties and are easily degraded by factors such as enzymes, temperature, and ultraviolet rays. The gene regulation efficiency of miRNAs depends on the carrier, and cationic nanocarriers are currently one of the nucleic acid nanocarriers with higher loading efficiency. Therefore, in order to realize the clinical application of miRNA, it is necessary to develop miRNA transport carriers with high tissue penetration, low toxicity and high efficiency. Summary of the invention

[0003] In order to solve the deficiencies in the prior art, the present invention aims to provide an amphiphilic nucleic acid nanocarrier and a preparation method and application thereof.

[0004] The specific technical solutions of the present invention are as follows:

[0005] The present invention provides a method for preparing an amphiphilic nucleic acid nanocarrier, comprising the steps of: mixing DHA-NHS with polylysine hydrochloride, then adding an organic solvent and triethylamine, allowing DHA-NHS and polylysine hydrochloride to react and assemble in a weakly alkaline environment, and after the reaction is completed, removing the organic solvent and drying to obtain a solid powder, namely the amphiphilic nucleic acid nanocarrier; wherein the DHA-NHS is shown in the following formula I:

[0006]

[0007] Furthermore, the molar ratio of DHA-NHS to polylysine hydrochloride is 1:2-1:20, preferably 1:10.

[0008] Furthermore, the Mw of the polylysine hydrochloride is not particularly limited, and is preferably 5000Da.

[0009] Further, the organic solvent is anhydrous DMF;

[0010] The drying is freeze drying.

[0011] Furthermore, the method for removing the organic solvent is: dialyzing the reaction liquid for 1-2 days using a dialysis bag with a molecular weight cutoff of 3500Da to remove the residual organic solvent in the solution.

[0012] The present invention also provides an amphiphilic nucleic acid nanocarrier prepared by the preparation method.

[0013] The present invention also provides a pharmaceutical composition, which comprises the amphiphilic nucleic acid nanocarrier and a negatively charged nucleic acid drug; the pharmaceutical composition is prepared by mixing the amphiphilic nucleic acid nanocarrier and the negatively charged nucleic acid drug.

[0014] Furthermore, the negatively charged nucleic acid drug is miRNA.

[0015] Furthermore, the mass ratio of the amphiphilic nucleic acid nanocarrier to the negatively charged nucleic acid drug is 3:1-8:1.

[0016] The present invention also provides the use of the amphiphilic nucleic acid nanocarrier or the pharmaceutical composition in preparing a drug for treating keratitis.

[0017] Furthermore, the keratitis is bacterial keratitis.

[0018] The beneficial effects of the present invention are:

[0019] The amphiphilic nucleic acid nanocarrier provided by the present invention is a nanostructure (DHA-lys) formed by self-assembly of ω-3 unsaturated fatty acids (DHA, docosahexaenoic acid) and polylysine (ε-PL), and can load negatively charged nucleic acid drugs, such as miRNAs, through electrostatic adsorption. In addition, the amphiphilic nucleic acid nanocarrier particles of the present invention have strong corneal tissue penetration and extremely strong antibacterial properties. While exerting the antibacterial function, the residence time of the drug on the ocular surface can be relatively prolonged, thereby enhancing the efficacy of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis of DHA-lys and the spectral determination of DHA-lys.

[0021] Figure 2 Schematic diagram of DHA-lys loading miRNAs through electrostatic adsorption.

[0022] Figure 3 A is the diameter of the nanocarrier measured by dynamic light scattering; Figure 3 B is a scanning electron microscope and Figure 3 C is the mesoporous structure on the surface of the nanocarrier shown by transmission electron microscopy; Figure 3 D. Gel electrophoresis was used to determine the binding capacity of nanocarriers to miRNA (N / P ratio).

[0023] Figure 4 The changes in particle size and Zeta potential of DHA-lys@miRNAs with different mass ratios.

[0024] Figure 5 A is the uptake of DHA-lys@miR-Cy3 by cells at different time points measured by flow cytometry; Figure 5 B. Statistical analysis of the results of flow cytometry detection; Figure 5 C Confocal microscopy was used to observe the cellular uptake of amphiphilic nucleic acid nanocarriers (DHA-lys@miR-Cy3) at different time points.

[0025] Figure 6 A shows the changes in the cytoskeleton morphology of RAW 264.7 cells; Figure 6 B shows the changes in the cytoskeleton morphology of HCECs.

[0026] Figure 7 A is the quantification of DHA-lys@Cy 5.5 and Cy 5.5 by UV; Figure 7 B is the rabbit cornea treated with DHA-lys@Cy 5.5 and Cy 5.5 for different time periods, photographed by confocal laser scanning (CLSM) to evaluate the retention time of nanoparticles in the cornea, scale bar = 100 μm; Figure 7 C is the fluorescence intensity measurement of Cy 5.5 and DHA-lys@Cy 5.5 in the rabbit aqueous humor to evaluate the permeability of nanoparticles in the cornea.

[0027] Figure 8 A is the antibacterial mechanism of DHA-lys against Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus); Figure 8 B is the morphological changes of S. aureus and E. coli after being treated with high concentrations of ε-PL (2.0 mg / mL, 1.0 mg / mL) and DHA-lys (2.0 mg / mL, 1.0 mg / mL) for 12 hours, photographed by SEM; Figure 8 C is the agar plate colony image of S. aureus and E. coli after being treated with different concentrations of DHA-lys for 12 hours. DETAILED DESCRIPTION

[0028] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0029] Example 1

[0030] This embodiment provides a method for preparing an amphiphilic nucleic acid nanocarrier (DHA-lys), such as Figure 1 As shown, the following steps are included:

[0031] (1) First, add 250 mg of NHS powder and 450 mg of DCC powder into a 150 mL three-necked round-bottom flask. After sealing, introduce nitrogen to exhaust the air in the flask. Then use a 1 mL syringe to draw 200 μL of DHA stock solution and quickly put it into the flask to prevent DHA from being oxidized due to long exposure to the air. After the raw materials are added, use a 10 mL syringe to draw 5-6 mL of anhydrous chloroform as the reaction solvent. After the powder is completely dissolved, stir overnight in a water bath at room temperature away from light, and let it stand in a refrigerator at 4 ° C for 4 hours until stratification occurs. After purification by chromatography on a silica gel column, the intermediate product DHA-NHS is obtained for the next reaction.

[0032] (2) The above reaction product DHA-NHS (yield of about 140-150 mg / time, Mw of about 425.58) and ε-PL (polylysine hydrochloride, total Mw of about 5000 Da, monomer molecular weight of about 164.67, feed amount of about 410-480 mg / time) were mixed in a molar ratio of 1:10, and after exhausting the air in the bottle, anhydrous DMF and triethylamine were added to react and assemble in a weak alkaline environment. Finally, the DHA-lys liquid synthesized by the reaction was dialyzed for 1-2 days using a dialysis bag with a molecular weight cutoff of 3500 Da to remove the residual organic solvent in the solution, and then the dialyzed solution was placed at -80°C for pre-freezing for more than 12 hours. Finally, the DHA-lys solid powder was obtained by freeze drying, and the powder was dried and packaged and stored in the dark at -20°C. When used, the powder was weighed and dissolved in DDW (deionized water). After it was completely dissolved, it can be stored in a refrigerator at 4°C for a long time in the dark, or used for subsequent experiments.

[0033] The amphiphilic nucleic acid nanocarrier provided by the present invention can load miRNAs through electrostatic adsorption. Since the nucleic acid nanocarrier is amphiphilic, polylysine (ε-PL) carries positive ions that can carry negatively charged miRNAs. Therefore, the nanocarrier DHA-lys and miRNAs are mixed at a certain mass ratio for 30 minutes to prepare DHA-lys@miRNAs nanoparticles ( Figure 2 ). It also helps the drug to stay in the conjunctival layer, enhancing the absorption and penetration of the corneal epithelium. At the same time, the fat-soluble ω-3 unsaturated fat (DHA) can help the drug to penetrate the corneal stroma. The diameter and mesoporous structure of the amphiphilic nucleic acid nanocarrier prepared in this example can be detected by dynamic light scattering and transmission electron microscopy ( Figure 3 A, 3B, 3C), the binding capacity (N / P ratio) of the nanocarrier and miRNA (miR-Negative Contro is used here) can be displayed by gel electrophoresis ( Figure 3 D), Figure 4 The particle size and Zeta potential changes of DHA-lys@miRNAs with different mass ratios were shown. When the mass ratio was 8:1, the nanocarrier was the most stable, so 8:1 was determined to be the optimal mass ratio of the two.

[0034] Example 2

[0035] Amphiphilic nucleic acid nanocarrier particles can effectively promote the cellular uptake of drugs at the cellular level. The cyanine dye (Cy3) was labeled with the miRNA molecule (the miRNA used here was miR-Negative Contro), and human corneal epithelial cells (HCECs) were inoculated in 6-well plates at a density of 1×10 5 cells / well, and cultured in a 37°C incubator for 24 hours before use. Use DHA-lys prepared in Example 1, and use the method of Example 1 to prepare DHA-lys@miR-Cy 3. Then, DHA-lys@miR-Cy 3 (250 μg / mL) was added to the complete culture medium and incubated with the cells. The cells were collected at 0h, 2h, 4h, 8h, 12h, and 24h after administration and washed with PBS. Flow cytometry was used to detect the uptake of nanoparticles by HCECs after treatment with DHA-lys@miR-Cy 3 nanoparticles for different periods of time, and the uptake of nanocarriers by cells was quantified by the detected intracellular fluorescence intensity. Figure 5 A is the fluorescence intensity detected in cells at different times after being processed by FlowJo software. Figure 5 B is to quantify the detected intracellular fluorescence intensity (relative to the fluorescence intensity detected at 0h), and then statistically analyze it. The statistical results show that compared with the 0h treatment group, HCECs cells can take up about 68.6% of the nanoparticles after being treated with DHA-lys@miR-Cy3 nanoparticles for 4h, and about 96.3% after 12h, which is not much different from the uptake after 24h treatment. At the same time, confocal microscopy was used to shoot the uptake of nanoparticles by HCECs cells after being treated with DHA-lys@miR-Cy3 nanoparticles for different time periods, which also corresponds to the results of flow cytometry detection ( Figure 5 C) These results indicate that the amphiphilic nucleic acid nanocarriers can effectively transfect miRNA into corneal cells.

[0036] Example 3

[0037] The amphiphilic nucleic acid nanocarrier particles do not cause changes in the cytoskeleton and can protect the normal physiological functions of the cells. The DHA-lys used in this embodiment is the DHA-lys prepared in Example 1. After the RAW 264.7 and HCECs cells were placed in a special laser confocal culture dish and cultured for 24 hours, the RAW 264.7 cells were cultured with LPS+IFN-γ (100 ng / mL), IL-4 (40ng / mL), DHA-lys (250μg / mL), and ε-PL (250μg / mL) for 24 hours, and the HCECs cells were cultured with DHA-lys (250μg / mL) and ε-PL (250μg / mL) for 24 hours. The culture medium was then aspirated, and then rinsed with PBS 3 times and fixed with 4% paraformaldehyde at room temperature for 10-15 minutes. After fixation, wash with PBS 2-3 times, each time for 5-10 min. Then, the cells were permeabilized with 0.5% TritonX-100 solution for 5 minutes, and then washed with PBS 2-3 times, each time for 5-10 minutes. 200 μL of the phalloidin working solution prepared with 1% BSA blocking solution was taken and incubated in a 37°C incubator in the dark for 30 minutes. DAPI was used to stain the cell nucleus, and then observed with a confocal laser scanning microscope (CLSM) to obtain fluorescence images.

[0038] Figure 6 A The results showed that compared with the cell morphology and skeleton changes of M1 phenotype macrophages RAW 264.7, the experimental groups (DHA-lys group and ε-PL group) did not cause cell stress, and their morphology was more inclined to M0 resting macrophages. In addition, the observation of the effect of phalloidin on cytoskeleton microfilaments was used to simply verify whether the positively charged nanomaterial would affect the physiological structure of the epithelial cell line. Figure 6 B The results show that under the same concentration incubation conditions, ε-PL has a significant effect on the cytoskeleton and the microfilament protein is significantly reduced, while the nanocarrier DHA-lys has a smaller effect on the cytoskeleton and will have a weaker effect on the normal physiological functions of epithelial cells.

[0039] Example 4

[0040] The amphiphilic nucleic acid nanocarrier particles have strong corneal tissue penetration performance. The DHA-lys@Cy 5.5 used in this example was prepared according to the method in Example 1, except that NHS was replaced with Cy 5.5-NHS (commercially available), the input amount was proportionally reduced, and the purified sample was obtained by dialysis. First, the concentrations of DHA-lys@Cy 5.5 and Cy 5.5 were consistent by ultraviolet quantitative method, as shown in FIG. Figure 7A. Then, DHA-lys@Cy 5.5 and Cy 5.5 were diluted in the same proportion with PBS solution (both 1.0 mg / mL). After the solutions were prepared, they were dripped onto the rabbit cornea (50 μL / eye, dripped 3 times). The rabbit cornea was photographed using confocal laser scanning microscopy (CLSM). Figure 7 B The results show that DHA-lys@Cy 5.5 can effectively prolong the ocular surface retention time compared with Cy 5.5. Purple fluorescence can still be observed in the corneal epithelium after 24 hours, and lavender fluorescence can also be observed in the corneal endothelium. Finally, the rabbits were killed 12 hours and 24 hours after the nanoparticles were added, and the cornea and anterior chamber fluid were collected. The collected rabbit anterior chamber fluid was measured for fluorescence. Figure 7 As shown in Figure C, the amount of DHA-lys@Cy 5.5 detected in the anterior chamber fluid is greater than that of Cy 5.5, indicating that the permeability of DHA-lys@Cy 5.5 in the cornea is better than that of a simple aqueous solution, i.e., Cy 5.5 solution. These results indicate that nanocarrier particles can prolong the duration of drug action in the cornea and can efficiently deliver drugs to penetrate into tissues.

[0041] Example 5

[0042] The amphiphilic nucleic acid nanocarrier particles have extremely strong antibacterial properties. The DHA-lys used in this example is the DHA-lys prepared in Example 1. Figure 8 A is a schematic diagram of the antibacterial mechanism of DHA-lys. DHA-lys mainly relies on the free amino group (-NH2) to dissociate positive charges and act on the bacterial cell wall. It will adsorb on the bacterial cell wall, affect the formation of the bacterial cell wall structure, hinder bacterial growth, and achieve antibacterial effect. The bacteria treated with different concentrations of DHA-lys for 12 hours were observed for colony growth on agar plates. Figure 8 B From the morphology of bacteria, it can be seen that the antibacterial efficiency of DHA-lys is not much different from that of ε-PL. Both can damage the surface cell wall and cell membrane of S. aureus and E. coli bacteria, causing the bacteria to autolyze and inhibit their growth activity, thus achieving an antibacterial effect. Figure 8 C results showed that DHA-lys still had an antibacterial effect at a concentration of 0.25 mg / mL within 12 hours. The above results all confirmed that amphiphilic nucleic acid nanocarriers have a strong antibacterial effect.

[0043] 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 present invention.

Claims

1. A method for preparing an amphiphilic nucleic acid nanocarrier, characterized in that: The method comprises the steps of: mixing DHA-NHS with polylysine hydrochloride, then adding an organic solvent and triethylamine, allowing DHA-NHS and polylysine hydrochloride to react and assemble in a weakly alkaline environment, removing the organic solvent after the reaction, and drying to obtain a solid powder, which is an amphiphilic nucleic acid nanocarrier; wherein the DHA-NHS is shown in the following formula I; 2. The preparation method according to claim 1, characterized in that: The molar ratio of DHA-NHS to polylysine hydrochloride is 1:2-1:

20.

3. The preparation method according to claim 1, characterized in that: The organic solvent is anhydrous DMF; The drying is freeze drying.

4. The preparation method according to claim 1, characterized in that: The method for removing the organic solvent is: using a dialysis bag with a molecular weight cutoff of 3500Da to dialyze the reaction liquid for 1-2 days to remove the residual organic solvent in the solution.

5. The amphiphilic nucleic acid nanocarrier prepared by the preparation method according to any one of claims 1 to 4.

6. A pharmaceutical composition, characterized in that It comprises the amphiphilic nucleic acid nanocarrier according to claim 5 and a negatively charged nucleic acid drug; the pharmaceutical composition is prepared by mixing the amphiphilic nucleic acid nanocarrier with the negatively charged nucleic acid drug.

7. The pharmaceutical composition according to claim 6, characterized in that The negatively charged nucleic acid drug is miRNA.

8. The pharmaceutical composition according to claim 6, characterized in that The mass ratio of the amphiphilic nucleic acid nanocarrier to the negatively charged nucleic acid drug is 3:1-8:

1.

9. Use of the amphiphilic nucleic acid nanocarrier according to claim 5 or the pharmaceutical composition according to claim 6 in preparing a drug for treating keratitis.

10. The use according to claim 6, characterized in that: The keratitis is bacterial keratitis.

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