Preparation method of miR-143-3p-loaded flexible hollow mesoporous organic silicon oxide nano material

Abstract

The invention discloses a preparation method of a miR-143-3p loaded flexible hollow mesoporous organic silicon oxide nano material, and relates to the technical field of nano materials. The method comprises the following steps: step 1, dissolving 0.16 g of CTAB in a mixture of 70mL of water, 30mL of absolute ethyl alcohol and 1mL of ammonia water, and uniformly stirring to obtain a mixed solution; and step 2, mixing 0.125 mL of TEOS and 0.1 mL of LTETS, adding the mixture into the mixed solution in the step 1, uniformly stirring, centrifugally collecting, and washing with absolute ethyl alcohol for 3 times to obtain a precipitate. The flexible hollow mesoporous organic silicon oxide nano material is of a hollow collapsed structure, has a large cavity, a thin shell thickness and rich organic components, and is more easily absorbed by cells; modifying PEG-MAL on the surface to enable the surface to be soluble in water; according to the present invention, the synthesized flexible hollow mesoporous silica nano-material is used for loading miR-143-3p, such that miR-143-3p can be protected from being degraded by RNA enzyme, the cell internalization rate of miR-143-3p can be improved, and the stable delivery system can be provided for miR-143-3p.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a method for preparing a flexible hollow mesoporous organic silicon oxide nanomaterial carrying miR-143-3p. Background Art

[0002] Previous high-throughput analysis found that compared with control mice, the expression of miR-143-3p in mice with acute lung injury was significantly reduced, and experimental results showed that miR-143-3p is a potential target for the treatment of acute lung injury, but miR-143-3p is easily degraded by RNases and has a low cellular internalization rate; commonly used silica nanomaterials are prone to agglomeration and precipitation in water, and have a low cellular internalization rate. Therefore, we proposed a method for preparing flexible hollow mesoporous organic silica nanomaterials loaded with miR-143-3p to solve the above problems.

[0003] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a flexible hollow mesoporous organic silicon oxide nanomaterial carrying miR-143-3p, so as to solve the problems raised by the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a flexible hollow mesoporous organic silicon oxide nanomaterial carrying miR-143-3p, comprising the following steps:

[0006] Step 1, dissolve 0.16 g CTAB in a mixture of 70 mL water + 30 mL anhydrous ethanol + 1 mL ammonia water, stir evenly to obtain a mixed solution;

[0007] Step 2, 0.125 ml of LTEOS and 0.1 ml of LTETS were mixed and added to the mixed solution of step 1, stirred evenly, collected by centrifugation, and washed with anhydrous ethanol for 3 times to obtain a precipitate;

[0008] Step 3, placing the precipitate of step 2 in a NaOH aqueous solution, etching at room temperature for 20 minutes, collecting by centrifugation, and washing with grade I water for 3 times to obtain product 1;

[0009] Step 4, adding the product of step 3 to a mixed solution of 50 μL concentrated hydrochloric acid and 100 mL ethanol, subjecting to high temperature for a period of time, repeating this operation 3 times, washing with anhydrous ethanol 3 times, and collecting by centrifugation to obtain mesoporous organic silicon oxide nanospheres;

[0010] Step 5, 0.065 g of mesoporous organic silicon oxide nanospheres were dispersed in a mixed solution containing 1.1 mL of dioxane, 0.3 mL of water and 0.10 g of triphenylphosphine, and then washed three times with deionized water to obtain flexible mesoporous organic silicon oxide nanospheres (HMONs), washed three times with deionized water, and collected by centrifugation at 12000 rpm;

[0011] Step 6, 12 mg of reduced HMONs was mixed with 1.2 mL of N, N-dimethylformamide and 3.24 mg of PEG-MAL at room temperature, and then 1.2 mg of Cy5.5-MAL was added and mixed at room temperature to obtain flexible mesoporous organic silica nanospheres modified with PEG and Cy5.5 (HMONs-PEG@Cy5.5, Nps);

[0012] Step 7, after dispersing 1 mg of flexible mesoporous organic silica nanospheres in 1 mL of water, adding a polyethyleneimine (PEI) aqueous solution, and performing ultrasonic dispersion for 30 minutes to obtain NPs@PEI;

[0013] Step 8: Centrifuge the NPs@PEI and wash it three times in ethanol and water, disperse the NPs@PEI in 125 μL miR-143-3p solution, incubate at room temperature, and finally obtain NPs@PEI@miR-143-3p.

[0014] Preferably, in step 1, stirring is performed at 35° C. for at least 60 min.

[0015] Preferably, in step 2, stirring is performed at 35° C. for 24 hours and centrifugation is performed at 12000 rpm for collection.

[0016] Preferably, in step 3, the concentration of the NaOH aqueous solution is 0.48 mol / L, and the solution is collected by centrifugation at 12000 rpm.

[0017] Preferably, in step 4, the reaction is carried out at 60° C. for 3 h and the mixture is collected by centrifugation at 12000 rpm.

[0018] Preferably, in step 5, the dispersion is carried out for 120 minutes.

[0019] Preferably, in step 6, the former is mixed at room temperature for 12 h, and the latter is mixed at room temperature for 12 h.

[0020] Preferably, in step 7, the mass ratio of PEI to NPs is 2:0.5.

[0021] Preferably, in step 8, NPs@PEI and miR-143-3p are incubated at room temperature for 20 min, and the surface of NPs is successfully loaded with miR-143-3p by modifying PEI, and the concentration of the miR-143-3p solution is 20 μmol / L.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The flexible hollow mesoporous organic silicon oxide nanomaterial of the present invention is a hollow collapsed structure with a large cavity, a thin shell thickness and rich organic components, and is more easily absorbed by cells; PEG-MAL is modified on the surface to make it soluble in water; the synthetic flexible hollow mesoporous silicon dioxide nanomaterial of the present invention is used to load miR-143-3p, which can protect miR-143-3p from being degraded by RNAse, improve the cellular internalization rate of miR-143-3p, and provide a more stable delivery system for miR-143-3p.

[0024] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A flow chart showing the principle of preparation of the present invention;

[0026] Figure 2 This is an electron microscopy result of the flexible mesoporous organic silicon oxide nanoparticles (HMONs) of the present invention;

[0027] Figure 3 The infrared spectra of the flexible mesoporous organic silicon oxide nanoparticles (HMONs) and the flexible mesoporous organic silicon oxide nanoparticles modified with PEG and Cy5.5 (HMONs-PEG@Cy5.5, NPs) of the present invention are shown;

[0028] Figure 4 This is a zeta potential detection diagram of PEI-modified NPs (NPs-PEI) of the present invention;

[0029] Figure 5 This is a schematic diagram of the cytotoxicity test results of HMONs and NPs of the present invention;

[0030] Figure 6 Schematic diagram of laser confocal microscopy detection of cellular uptake of NPs at different time periods;

[0031] Figure 7 Schematic diagram of flow cytometry detection of NPs cellular uptake at different time periods;

[0032] Figure 8 Schematic diagram of the expression detection results of NPs-PEI loaded with miR-143-3p (NPs-PEI@miR-143-3p) in cells and mouse lung tissues. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] See also Figure 1 , a method for preparing a flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p comprises the following steps:

[0035] Step 1, dissolve 0.16 g CTAB in a mixture of 70 mL water + 30 mL anhydrous ethanol + 1 mL ammonia water, and stir evenly at 35° C. for at least 60 min to obtain a mixed solution;

[0036] Step 2, 0.125 mL of TEOS (tetraethyl orthosilicate) and 0.1 mL of TETS were mixed and added to the mixed solution of step 1, stirred at 35° C. for 24 h, centrifuged at 12000 rpm, and washed with anhydrous ethanol three times to obtain a precipitate;

[0037] Step 3, placing the precipitate of step 2 in a NaOH aqueous solution (0.48 M), etching at room temperature for 20 min, collecting by centrifugation at 12000 rpm, and washing with grade I water for 3 times to obtain product 1;

[0038] Step 4, adding the product of step 3 to a mixed solution of 50 μL concentrated hydrochloric acid and 100 mL ethanol, reacting at 60° C. for 3 h, repeating this operation 3 times, washing with anhydrous ethanol 3 times, and collecting by centrifugation at 12000 rpm to obtain mesoporous organic silicon oxide nanospheres;

[0039] Step 5, dispersing 0.065 g of mesoporous organic silicon oxide nanospheres in a mixed solution containing 1.1 mL of dioxane, 0.3 mL of water and 0.10 g of triphenylphosphine for 120 min, then washing three times with deionized water to obtain flexible mesoporous organic silicon oxide nanospheres (HMONs), washing three times with deionized water, and collecting by centrifugation at 12000 rpm;

[0040] Step 6, 12 mg of reduced HMONs was mixed with 1.2 mL of N, N-dimethylformamide and 3.24 mg of PEG-MAL at room temperature for 12 h, and then 1.2 mg of Cy5.5-MAL was added and mixed at room temperature for 12 h to obtain flexible mesoporous organic silica nanospheres modified with PEG and Cy5.5 (HMONs-PEG@Cy5.5, Nps);

[0041] Step 7, after dispersing 1 mg of flexible mesoporous organic silica nanospheres in 1 mL of water, a polyethyleneimine (PEI) aqueous solution (the mass ratio of PEI to NPs is 2:0.5) is added, and ultrasonic dispersion is performed for 30 minutes to obtain NPs@PEI;

[0042] Step 8. Centrifuge the NPs@PEI and wash it three times in ethanol and water. Disperse the NPs@PEI in 125 μL miR-143-3p solution (20 μM). Incubate the NPs@PEI with miR-143-3p at room temperature for 20 min. The surface of the NPs is successfully loaded with miR-143-3p by modifying the PEI. Finally, NPs@PEI@miR-143-3p is obtained.

[0043] 1. The flexible mesoporous organic silicon oxide nanoparticles (HMONs) of the present invention were scanned by electron microscope. The results are as follows: Figure 2 As shown, it is a flexible structure that is wrinkled and collapsed.

[0044] 2. Infrared spectroscopy experiments were performed on the flexible mesoporous organic silicon oxide nanoparticles (HMONs) and the flexible mesoporous organic silicon oxide nanoparticles modified with PEG and Cy5.5 (HMONs-PEG@Cy5.5, NPs) of the present invention. The results are as follows: Figure 3 shown.

[0045] 3. Zeta potential detection: Detect the zeta potential of PEI-modified NPs (NPs-PEI). The results are as follows: Figure 4 shown.

[0046] 4. HMONs and NPs cytotoxicity test: The results are as follows Figure 5 As shown, the experimental results showed no obvious cytotoxicity.

[0047] 5. Cellular uptake: Laser confocal microscopy was used to detect the cellular uptake of NPs at different time periods. Figure 6 As shown, it was found that cellular uptake increased significantly over time.

[0048] 6. Cellular uptake: Flow cytometry was used to detect the cellular uptake of NPs at different time periods. The results are as follows: Figure 7 As shown, it was also found that the cellular uptake increased significantly with the extension of time.

[0049] 7. qPCR experiment: Detect the expression of NPs-PEI loaded miR-143-3p (NPs-PEI@miR-143-3p) in cells and mouse lung tissues, such as Figure 8 As shown, the expression of miR-143-3p in cells and mouse tissues treated with NPs-PEI@miR-143-3p was significantly increased.

[0050] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0051] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p, characterized in that: The steps include: Step 1, dissolve 0.16 g CTAB in a mixture of 70 mL water + 30 mL anhydrous ethanol + 1 mL ammonia water, stir evenly to obtain a mixed solution; Step 2, add 0.125 mL TEOS and 0.1 mL LTETS to the mixed solution of step 1, stir evenly, collect by centrifugation, and wash with anhydrous ethanol three times to obtain a precipitate; Step 3, placing the precipitate of step 2 in a NaOH aqueous solution, etching at room temperature for 20 minutes, collecting by centrifugation, and washing with grade I water for 3 times to obtain product 1; Step 4, adding the product of step 3 to a mixed solution of 50 μL concentrated hydrochloric acid and 100 mL ethanol, reacting at 60° C. for 3 h, repeating this operation 3 times, washing with anhydrous ethanol 3 times, and obtaining mesoporous organic silicon oxide nanospheres; Step 5: Disperse 0.065 g of mesoporous organic silica nanospheres in a mixed solution containing 1.1 mL of dioxane, 0.3 mL of water and 0.10 g of triphenylphosphine, and then wash three times with deionized water to obtain flexible mesoporous organic silica nanospheres (HMONs), wash three times with deionized water, and collect by centrifugation at 12,000 rpm. Step 6, 12 mg of reduced HMONs was mixed with 1.2 mL of N, N-dimethylformamide and 3.24 mg of PEG-MAL at room temperature, and then 1.2 mg of Cy5.5-MAL was added and mixed at room temperature to obtain flexible mesoporous organic silica nanospheres modified with PEG and Cy5.5 (HMONs-PEG@Cy5.5, Nps); Step 7, after dispersing 1 mg of flexible mesoporous organic silica nanospheres in 1 mL of water, adding a polyethyleneimine (PEI) aqueous solution, and performing ultrasonic dispersion for 30 minutes to obtain NPs@PEI; Step 8: Centrifuge the NPs@PEI and wash it three times in ethanol and water, disperse the NPs@PEI in 125 μL miR-143-3p solution, incubate at room temperature, and finally obtain NPs@PEI@miR-143-3p.

2. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 1, stirring is performed at 35° C. for at least 60 min.

3. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 2, stirring is performed at 35° C. for 24 hours and centrifugation is performed at 12000 rpm for collection.

4. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 3, the concentration of the NaOH aqueous solution is 0.48 mol / L, and the solution is collected by centrifugation at 12000 rpm.

5. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 4, the reaction was carried out at 60° C. for 3 h and the mixture was collected by centrifugation at 12000 rpm.

6. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 5, the dispersion is carried out for 120 minutes.

7. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 6, the former is mixed at room temperature for 12 h, and the latter is mixed at room temperature for 12 h.

8. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In step 7, the mass ratio of PEI to NPs is 2:0.

5.

9. The method for preparing the flexible hollow mesoporous organic silicon oxide nanomaterial loaded with miR-143-3p according to claim 1, characterized in that: In the step 8, NPs@PEI and miR-143-3p were incubated at room temperature for 20 min, and the surface of NPs was successfully loaded with miR-143-3p by modifying PEI, and the concentration of the miR-143-3p solution was 20 μmol / L.

Images