Application of silicon dioxide nanosphere drug delivery system in drug preparation

Abstract

The invention discloses application of a silicon dioxide nanosphere drug delivery system in drug preparation. The silicon dioxide nanosphere drug delivery system is constructed by silicon dioxide nanoparticles with surface spines, and the silicon dioxide nanoparticles are nanospheres which take silicon dioxide as a matrix, have solid cores of 80-120 nanometers and are coated with hard spines of 40-60 nanometers and are used for delivering RNA to a target part through skin or mucous membranes. Imiquimod is used for inducing a psoriasis mouse model, it is proved that a silicon dioxide nanosphere drug delivery system can deliver small nucleic acid RNA to epidermis and dermal cells, the pharmacological action is achieved, then the silicon dioxide nanosphere drug delivery system is used for treating diseases such as psoriasis, skin cancer, HPV virus infection or androgenetic alopecia, and the wide market application prospect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an application of a silicon dioxide nanosphere drug delivery system in preparing drugs. Background Art

[0002] In recent years, small nucleic acids such as siRNA, miRNA and ASO nucleic acids have been favored because they can precisely regulate gene expression, act on genes that traditional small molecule drugs cannot target, and have a relatively simple drug design process. In 2018, the United States Food and Drug Administration (USFDA) approved the first RNAi-based drug Patisiran for the treatment of hereditary thyroid hormone amyloidosis (HATTR), marking a new era in nucleic acid drug therapy. Since small nucleic acids cannot penetrate cell membranes and are unstable in the circulatory system, the delivery system has always been a key link in the clinical application of nucleic acid drugs. At present, researchers are exploring various materials to meet the challenges of in vivo delivery. These materials include lipid nanoparticles, conjugates, polymers, etc. The delivery carriers currently used in clinical applications are mainly viral vectors and liquid-based nanoparticle carriers (LNPs), and there is little research on solid nanoparticle delivery carriers. Due to the instability of LNPs-based delivery formulations, the current method of administration of nucleic acid drugs is mainly intramuscular or intravenous injection, and there is almost no clinical application of transdermal administration.

[0003] Silica is a low-toxic, highly biocompatible material that is widely used in the food and pharmaceutical fields. For example, the U.S. Food and Drug Administration (USFDA) and the China Food and Drug Administration (CFDA) allow the addition of silica to infant formula; silica particles with a diameter of 100 nanometers are legal food additives; and at least 11 types of silica-containing nanoparticles have been used in clinical practice. Summary of the invention

[0004] The present invention provides an application of a silicon dioxide nanosphere drug delivery system in the preparation of drugs. The delivery system of the present invention is constructed of nanoparticles with silicon dioxide as a matrix and surface spikes. The delivery system can deliver small nucleic acids to target sites after being physically mixed with small nucleic acids and applied to the skin surface to regulate the expression of target genes. It can be used to treat skin diseases such as psoriasis, skin tumors, skin infections, and androgenic alopecia (male baldness). Local administration through the skin is a convenient, safe and effective treatment method.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The invention provides an application of a silicon dioxide nanosphere drug delivery system in preparing drugs.

[0007] Furthermore, the drug is a drug based on small nucleic acid RNA.

[0008] Furthermore, the drug is a drug for treating psoriasis, skin cancer, HPV virus infection or androgenic alopecia.

[0009] Furthermore, the drug is administered by transdermal application.

[0010] Furthermore, the silica nanosphere drug delivery system is constructed from silica nanoparticles with surface spikes.

[0011] Furthermore, the core of the silicon dioxide nanoparticles is a solid sphere with a diameter of 80-120 nanometers, the surface is a silicon dioxide spike with a length of 40-60 nanometers, and the total particle size is 160-240 nanometers.

[0012] Furthermore, the preparation steps of the silica nanoparticles are: adding ethylamine, phenol, formaldehyde, tetraethyl orthosilicate, and sodium hydroxide into an ethanol aqueous solution and stirring evenly; then adding ammonia water, tetraethyl orthosilicate and ethylamine to react, and then adding phenol and formaldehyde thereto, continuing to stir the reaction, centrifuging, drying, and calcining after the reaction is completed to obtain silica nanoparticles.

[0013] Furthermore, the mass ratio of ethylamine:phenol:formaldehyde:tetraethylorthosilicate:sodium hydroxide:ethanol aqueous solution is 0.05-0.1:0.1-0.3:0.1-0.9:0.5-1:05-0.8:10-20.

[0014] Furthermore, the silica nanoparticles can deliver small nucleic acid RNA to epidermal and dermal cells and exert pharmacological effects.

[0015] Furthermore, the silica nanoparticles are used at a concentration of 10 mg / mL to 20 mg / mL.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: the silica nanosphere drug delivery system described in the present invention is more stable at room temperature than the prior art and does not rely on low-temperature storage and transportation; it can block the degradation of small nucleic acids by nucleases; unlike the prior art which is mainly used for intramuscular or intravenous injection, the silica nanosphere drug delivery system can be directly applied to the surface of the skin (mucosa) to deliver the small nucleic acid of therapeutic purpose to the target location, which has higher convenience and safety. It has a good therapeutic effect on skin diseases such as psoriasis, skin tumors, skin infections, and androgenic alopecia (male baldness), and has a wide market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Transmission electron microscopy image of silica nanoparticles SSN-2 with surface spikes.

[0018] Figure 2 Microscope photos of silica nanoparticles delivering FAM-siRNA into RAW 264.7 and BMDM cells.

[0019] Figure 3 This is a diagram showing the transdermal effect of silica nanoparticles SSN-2 delivering FAM-siRNA.

[0020] Figure 4 Figure 2 shows the cytotoxicity of SSN-2 silica nanoparticles with surface spikes.

[0021] Figure 5 This is a diagram of the RAW 264.7 cell model of psoriasis inflammation.

[0022] Figure 6 This is a diagram of the BMDM cell model of psoriasis inflammation.

[0023] Figure 7 This is a diagram showing the effect of SSN-2 delivery of TNF-α-siRNA in RAW 264.7 cells.

[0024] Figure 8 This is a diagram showing the effect of SSN-2 delivery of TNF-α-siRNA in BMDM cells.

[0025] Fig. 9 This is a picture of the skin lesions on the back of BALB / c psoriasis model mice.

[0026] Fig.10 This is a graph showing weight changes in BALB / c psoriasis model mice.

[0027] Fig.11 This is a graph showing the PASI score results on the 6th day of modeling of the BALB / c psoriasis model.

[0028] Fig.12 This is a graph showing the expression of inflammatory factors in the skin lesions of the BALB / c psoriasis model.

[0029] Fig.13 This is the H&E staining image of the skin lesion tissue of the BALB / c psoriasis model.

[0030] Fig.14 This is a diagram showing the therapeutic effect of SSN-2 delivered TNF-α-siRNA on the back lesions of BALB / c psoriasis model mice.

[0031] Fig.15 This is a graph showing the effect of SSN-2 delivered TNF-α-siRNA on body weight changes in BALB / c psoriasis model mice.

[0032] Fig.16 This is the PASI score result of SSN-2 delivered TNF-α-siRNA in the BALB / c psoriasis model on day 6.

[0033] Fig.17 This is a graph showing the expression of inflammatory factors in the lesional tissue of the BALB / c psoriasis model on the 6th day after SSN-2 delivered TNF-α-siRNA to treat the disease.

[0034] Fig.18 H&E staining of skin lesion tissue on day 6 in the BALB / c psoriasis model treated with SSN-2 delivered TNF-α-siRNA.

[0035] Fig.19 Microscope photos of SSN-2 delivered FAM-siRNA into HFK cells.

[0036] Fig. 20 This is a diagram showing the effect of delivering HPV-siRNA to SSN-2 to silence HPV target genes. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail with reference to the following specific examples. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0038] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0039] The present invention will be further described in detail below in conjunction with specific preparation examples and application examples and with reference to data. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0040] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0041] The silica nanosphere drug delivery system provided by the present invention is realized by the following method:

[0042] S1: Ethyl silicate is added into the reactor to generate smooth spherical SiO2 through hydrolysis and condensation reaction;

[0043] Specifically, ethyl silicate, ammonia water and ethylenediamine are added into the ethanol aqueous solution in the reactor; the mixture is stirred evenly at 60° C. to react and generate smooth spherical SiO2.

[0044] S2: Establish the relationship between the total mass M of Si elements in ethyl silicate and the mass m(t) of Si ions participating in hydrolysis and condensation at reaction time t;

[0045] have to ;

[0046] S3: Assume the density of the smooth spherical SiO2 is ρ and the radius is R; then ;

[0047] S4: Phenolic resin is added at reaction time t to form l(t) long burrs (gene delivery vector) on the smooth spherical surface of SiO2;

[0048] Specifically, at time t after ethyl silicate is added to the reactor, phenol and formaldehyde (used to produce phenolic resin in the reactor, or phenolic resin can be directly added) are added; the reaction is stirred (stirring speed is 500 rpm) to form l (t) long burrs on the smooth spherical surface of SiO2. After the addition of phenolic resin, the Si element (mass Mm (t)) of ethyl silicate that does not form smooth spherical SiO2 in the reactor will grow uniformly on the surface of the smooth spherical SiO2 in the form of burrs.

[0049] S5: Assume that the density of burrs on the smooth spherical surface of SiO2 is , then the number of burrs is , the mass of each burr is ;

[0050] S6: Establish the relationship between the length of the burr on the smooth spherical surface of SiO2 and the mass of the burr: ;

[0051] S7: Establish the equation relationship between the length of the burr on the smooth spherical surface of SiO2 and the reaction time t: ;

[0052] Where k is a constant,

[0053] S8: The reaction solution was centrifuged at 8000 rpm for 10 min, and the obtained precipitate was calcined at 500° C. for 300 min to obtain silica nanoparticles with surface spikes.

[0054] Example 1: Transmission electron microscopy experiment of silica nanoparticles SSN-2 with surface spikes

[0055] Preparation steps of silica nanoparticles SSN-2 with surface spikes: prepare 100 g of ethanol aqueous solution with a mass fraction of 25%, add 3.12 mL of ammonia water, 5 g of tetraethyl orthosilicate, 500 mg of ethylamine and 3 g of sodium hydroxide to the ethanol aqueous solution, stir evenly at 60°C, the stirring speed is 500 rpm, after reacting for 5 minutes, add 1 g of phenol and 2 g of formaldehyde, continue stirring and reacting for 300 minutes, the stirring speed is 500 rpm. After the reaction is completed, centrifuge at 8000 rpm for 10 minutes, dry the precipitate and calcine at 500°C for 300 minutes to obtain silica nanoparticles SSN-2 with surface spikes, such as Figure 1 shown.

[0056] The invention adopts a one-pot reaction method, and mixes ethanol aqueous solution, ammonia water, ethylamine, phenol, formaldehyde, tetraethyl orthosilicate and sodium hydroxide, and then reacts to prepare silicon dioxide nanoparticles SSN-2 with surface spikes.

[0057] The surface of SSN-2 is modified to be positively charged. Its core is a solid sphere with a diameter of 80-120 nanometers, and the surface is a silica spike with a length of 40-60 nanometers. The total particle size is 160-240 nanometers. The optimized core diameter is 110±13 nanometers and the spike length is 45±8 nanometers.

[0058] Example 2: Experiment of delivering FAM-siRNA to SSN-2

[0059] RAW 264.7 and BMDM cells were cultured at 1 × 10 5 The cells were inoculated into 24-well plates at a density of 1 mL / well. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured overnight at 37 °C. Two 1.5 mL RNase-free and DNase-free sterile centrifuge tubes were prepared. 25 μL Opti-MEM and 1 μL FAM-siRNA (20 μM) were added to one tube and mixed with a pipette. 15 μL Opti-MEM and 10 μL SSN-2 (10 mg / mL) were added to another 1.5 mL tube and mixed with a pipette. The mixture was added to the siRNA-medium mixture and mixed. After standing at room temperature for 20 min, transfection was performed immediately. Photos were taken under a fluorescence microscope 6 h later.

[0060] The results are as follows Figure 2 As shown, the silica nanoparticles SSN-2 with surface spikes in the silica nanosphere drug delivery system for delivering small interfering RNA used in this experiment have good siRNA delivery efficiency in macrophages RAW264.7 and primary BMDM cells, indicating that SSN-2 is a good siRNA delivery system.

[0061] Example 3: Transdermal delivery of FAM-siRNA by SSN-2

[0062] Six-week-old male BALB / c mice were randomly divided into blank control group (NTC) and SSN-2 transfected FAM-siRNA group, with 3 mice in each group. Mice were anesthetized by intraperitoneal injection of 1.25% avertin at a dose of 0.2 mL / 10 g one day in advance, and the back hair was shaved with a pet shaver to expose the back skin of about 2 cm×3 cm. Prepare two 1.5 mL sterile centrifuge tubes, add 12.5 μL Opti-MEM and 37.5 μL FAM-siRNA (20 μM) to one tube for a total of 5 μg, and mix with a pipette; add 50 μL SSN-2 (10 mg / mL) to another 1.5 mL tube for a total of 500 μg, mix with a pipette, add to the small interfering RNA-medium mixture, mix, let stand at room temperature for 20 minutes, and apply to the mouse skin with a pipette tip or silicone brush until absorbed. The mice were euthanized after being protected from light for 6-8 h, and the skin tissues were frozen and sectioned with a thickness of 6 μm, and photographed under a fluorescence microscope.

[0063] The results are as follows Figure 3 As shown, the fluorescence signal is distributed in the dermis. Compared with the NTC group, the skin tissue itself has a weak fluorescence signal, while the fluorescence intensity of the FAM-siRNA group is significantly higher than that of the control group. This shows that SSN-2 successfully delivers FAM-siRNA into the skin and shows good permeability. These results provide a theoretical basis for SSN-2 as a delivery carrier for animal skin tissue.

[0064] Example 4: Cytotoxicity assay of SSN-2 cells with silica nanoparticles with surface spikes

[0065] RAW 264.7, M-BMDM, and GM-BMDM cells were cultured at 1×10 4The cells were inoculated into 96-well plates at a density of 100 μL per well. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured overnight at 37 °C. The cells were incubated with different concentrations of SSN-2 (50, 100, 200, 400, 800, 1000 μg / mL) for 48 h, with 3 replicates per group. MTT (5 mg / mL) was added to each well at a ratio of MTT: culture medium = 1:10. After continued culture in a cell culture incubator for 4 h, the supernatant was discarded and dimethyl sulfoxide (150 μL) was added to each well. The cells were placed on a shaker at 100 r / min for 10 min to repeatedly dissolve the purple formazan crystals generated in the cells. The absorbance value at 490 nm was read using a SpectraMax M3 microplate reader, and the cell viability was calculated as (blank control group OD value-experimental group OD value) / blank control group OD value × 100%.

[0066] The results are as follows Figure 4 As shown, different concentrations of SSN-2 had no obvious cytotoxicity in RAW 264.7 cells, and the viability of M-BMDM cells decreased when the SSN-2 concentration was higher than 400 μg / mL, and the viability of GM-BMDM cells decreased when the SSN-2 concentration was higher than 800 μg / mL. The concentration of SSN-2 used in Example 3 to deliver siRNA was 200 μg / mL, which had a good delivery effect. Therefore, the concentration of SSN-2 used in the present invention had no obvious effect on cell viability.

[0067] Example 5: Experiment on the construction of psoriasis RAW 264.7 cell inflammation model

[0068] RAW 264.7 cells were cultured at 50 × 10 4 The cells were inoculated into 24-well plates at a density of 100 cells / well, and the inoculation volume was 2 mL. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured at 37°C overnight. After washing the cells with phosphate buffered saline (PBS), monophosphoryl lipid A (MPL) was added at a concentration of 1 μg / mL. The cells were treated with MPL at a concentration of 1 μg / mL for 24 h, 48 h, and 72 h in RAW 264.7 cells. RNA was collected for qRT-PCR detection of TNF-α, and the culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) detection of TNF-α. The experimental steps of qRT-PCR and ELISA are as follows.

[0069] qRT-PCR:

[0070] 1) Extraction of total RNA:

[0071] The whole process should be performed on ice. After collecting the cells, wash them twice with PBS, add 1 mL of Trizol, and place them on ice for about 15 minutes. After that, collect the lysate into a RNase-free, sterile 1.5 mL centrifuge tube, add 200 μL of chloroform to the tube, shake it up and down vigorously, let it stand for 10 minutes, and centrifuge it at 13000 rpm at 4°C for 20 minutes. At this time, the liquid in the tube is layered, and the upper layer is a colorless and transparent aqueous phase. Transfer 500 μL of the upper layer to a new EP tube, add 500 μL of isopropanol, mix it and let it stand for 10 minutes, and centrifuge it at 13000 rpm at 4°C for 20 minutes. Carefully discard the supernatant, and white precipitate can be seen on the tube wall. Use DEPC water to prepare 75% ethanol solution to wash the precipitate, centrifuge it at 12000 rpm for 10 minutes, 4°C, discard the supernatant, and dry it at room temperature. Add DEPC water to dissolve the RNA precipitate, and measure the RNA concentration in Nanodrop.

[0072] 2) cDNA synthesis:

[0073] To remove genomic DNA (gDNA), prepare the following mixture in an RNase-free tube and incubate at 42°C for 2 min.

[0074] Table 1 gDNA removal reaction system

[0075]

[0076] Add 4 μL 5×HiScriptIII qRT SuperMix to the product of the previous step, and the total reaction system is 20 μL. Mix by pipetting, and incubate at 37°C for 15 min and 85°C for 5 s. The reaction product can be used for qPCR reaction or stored at -20°C.

[0077] 3) Real-time fluorescence quantitative PCR (qPCR):

[0078] The cDNA obtained by reverse transcription was diluted 40 times with ddH2O, and the reaction system was prepared according to Table 2.

[0079] Table 2 qPCR reaction system

[0080]

[0081] The cells were placed in a fluorescence quantitative PCR instrument for real-time quantitative PCR. The amplification program was as follows: Preincubation 1 cycle, 95 ℃ 600 s; 3 step Amplification 45 cycles, 95 ℃ 10 s; 60 ℃ 10 s; 72 ℃ 10 s; Melting 1 cycle, 95 ℃ 10 s, 65 ℃ 60 s; 97 ℃ 1 s. 18S rRNA was used as the internal reference gene, and the accumulation index (2 -Ct ) represents data.

[0082] Enzyme-linked immunosorbent assay (ELISA)

[0083] 1) Sample collection: Collect the cell culture supernatant, centrifuge at 2000 rpm, 4 ℃ for 20 min, and carefully collect the supernatant.

[0084] 2) Sample addition: dilute the standard sample in series to concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 0 (mg / mL). Add 100 μL of Cytokine standard to the standard well, 100 μL of sample to the sample well, and 100 μL of Dilution buffer R (1×) to the blank well.

[0085] 3) Add detection antibody: Add 50 μL of Biotinylated antibody working solution to each well. Incubate at 37°C for 90 minutes.

[0086] 4) Washing: Discard the liquid in the wells, add 300 μL 1× Washing buffer to the wells, and discard after 2 min. Repeat 4 times, patting dry on filter paper each time.

[0087] 5) Add enzyme: Add 100 μL Streptavidin-HRP working solution to each well. Incubate at 37°C for 30 min.

[0088] 6) Wash the plate: Repeat step 4.

[0089] 7) Color development: Add 100 μL TMB to each well and incubate at 37°C in the dark for 10-20 min. The reaction is terminated based on the depth of the color in the well.

[0090] 8) Stop the reaction: Add 100 μL Stop solution to each well to stop the reaction.

[0091] The results are as follows Figure 5As shown, MPL can effectively stimulate the expression of inflammatory factor TNF-α at the mRNA level and protein level in RAW 264.7 cells after 24 hours of action, and the expression levels at 48 hours and 72 hours are almost the same. In the present invention, it is desirable to use MPL to induce a psoriasis cell model for 24 hours.

[0092] Example 6: Experiment on the construction of psoriasis DMDM ​​cell inflammation model

[0093] DMDM cell extraction: Euthanize C57BL / 6J mice and soak them in 75% alcohol for 5 minutes. Peel off the muscle tissue, separate the femur and tibia, and soak them in PBS. Cut off both ends of the leg bone and use a 10 mL syringe to flush the bone marrow in the leg bone into a 200 μm filter. Grind the bone marrow remaining on the filter thoroughly, transfer the slurry to a 50 mL centrifuge tube, and centrifuge at 1000 rpm for 5 min. Discard the upper PBS, resuspend the precipitate in culture medium (high-glucose DMEM culture medium supplemented with 10% FBS and 1% penicillin-streptomycin), and count 7.5×10 5 The colony stimulating factor GM-CSF (final concentration of 20 ng / mL) or M-CSF (final concentration of 50 ng / mL) was added and the cells were cultured for 7 days to induce GM-BMDM and M-BMDM cells.

[0094] After washing the cells with PBS, monophosphoryl lipid A (MPL) was added at a concentration of 1 μg / mL and allowed to act in BMDM cells for 24 h. RNA was collected for qRT-PCR detection of TNF-α, and the culture supernatant was collected for ELISA detection of TNF-α.

[0095] The results are as follows Figure 6 As shown, MPL can also effectively stimulate the expression of inflammatory factor TNF-α at both mRNA and protein levels in primary cells.

[0096] Example 7: Experimental study on the effect of SSN-2 delivery of TNF-α-siRNA in RAW 264.7 cells

[0097] RAW 264.7 cells were cultured at 50 × 10 4The cells were inoculated into a 24-well plate at a density of 100 cells / well, and the inoculation volume was 2 mL. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured overnight at 37 °C. After washing the cells with PBS, monophosphoryl lipid A (MPL) was added at a concentration of 1 μg / mL. Prepare two 1.5 mL sterile centrifuge tubes, add 25 μL Opti-MEM to one tube, add 1 μL siRNA (20 μM), and mix with a pipette; add 15 μL Opti-MEM and 10 μL SSN-2 (10 mg / mL) to another 1.5 mL tube, mix with a pipette, add to the siRNA-culture medium mixture, mix, let stand at room temperature for 20 min, and transfect immediately. TNF-α-siRNAs were numbered 138#, 200#, and 228#, and control-siRNA was set, and the sequence was shown in Table 3. MPL induction and transfection were performed simultaneously, and RNA was collected 24 hours later for qRT-PCR detection of TNF-α, and culture supernatant was collected for ELISA detection of TNF-α.

[0098] Table 3 siRNA sequences

[0099]

[0100] The results are as follows Figure 7 As shown, compared with the negative control, the results at the mRNA level showed that SSN-2 delivered TNFα-siRNA 138#, 200#, and 228# all had an inhibitory effect on TNFα. The ELISA results showed that SSN-2 delivered TNFα-siRNA 138#, 200#, and 228# also achieved a good gene silencing effect on TNFα at the protein level. In RAW 264.7 cells, the silica nanosphere drug delivery system that delivers small interfering RNA has a good therapeutic effect.

[0101] Example 8: Experimental study on the effect of SSN-2 delivery of TNF-α-siRNA in BMDM cells

[0102] After washing BMDM cells with PBS, add monophosphoryl lipid A (MPL) at a concentration of 1 μg / mL. Prepare two 1.5 mL sterile centrifuge tubes, add 25 μL Opti-MEM to one tube, add 1 μL siRNA (20 μM), and mix with a pipette; add 15 μL Opti-MEM and 10 μL SSN-2 (10 mg / mL) to another 1.5 mL tube, mix with a pipette, add to the siRNA-medium mixture, mix, let stand at room temperature for 20 min, and transfect immediately. TNF-α-siRNA number 138#, 200#, 228#, set control-siRNA, MPL induction and transfection are carried out simultaneously, collect RNA after 24 hours for qRT-PCR detection of TNF-α, and collect culture supernatant for ELISA detection of TNF-α.

[0103] The results are as follows Figure 8 As shown, compared with the negative control, the results at the mRNA level showed that SSN-2 delivered TNFα-siRNA 138#, 200#, and 228# all had an inhibitory effect on TNFα. The ELISA results showed that SSN-2 delivered TNFα-siRNA 138#, 200#, and 228# also achieved a good gene silencing effect on TNFα at the protein level. In BMDM cells, the silica nanosphere drug delivery system that delivers small interfering RNA has a good therapeutic effect.

[0104] Example 9: Construction of BALB / c psoriasis model and detection of mouse indicators

[0105] 1. Construction of BALB / c psoriasis model and observation experiment of skin lesions on the back of mice

[0106] Six-week-old male BALB / c mice were randomly divided into a saline control group (Saline) and a model group (IMQ), with 3 mice in each group. Local hair removal was performed on the back skin of mice one day before modeling. 1.25% Avertin was applied intraperitoneally at a dose of 0.2 mL / 10 g for anesthesia. The hair on the back skin of mice was completely removed using a pet shaver combined with a mild depilatory cream to expose an area of ​​about 2 cm×3 cm of back skin. Vaseline was applied to the back of the Saline group, and 5% IMQ62.5 mg was applied to the back of the model group. After application of the medicine, local massage was performed 30 to 60 times, once a day, for 5 days and 7 days after modeling. The back skin lesions were photographed on the sixth and eighth days of modeling, respectively.

[0107] The results are as follows Fig. 9 As shown in the figure, on the sixth day of modeling, mice had obvious erythema, obvious red skin phenomenon, thickened skin, scales, and obvious skin lesions. On the eighth day, scales were obvious, red skin phenomenon was not obvious, and skin thickened.

[0108] 2. Construction of BALB / c psoriasis model and weight monitoring experiment in mice

[0109] The body weight of the above mice was monitored.

[0110] The results are as follows Fig.10 As shown, the body weight of the model group decreased continuously in the first three days of modeling and then slowly increased.

[0111] 3. PASI score on the 6th day of BALB / c psoriasis model establishment

[0112] The skin lesions on the backs of the mice were monitored daily, and PASI scores were performed according to the standards in Table 4.

[0113] Table 4 PASI scoring criteria

[0114]

[0115] The results are as follows Fig.11 As shown in the figure, mice developed erythema on the fourth, fifth, and sixth days of modeling, showing obvious red skin phenomenon. Scaly skin began to appear on the second day and lasted until the sixth day, and the skin thickened, with individual differences. The overall skin lesions were more obvious, and the PASI score was higher. Scaly skin began to appear on the second day of modeling in the D8 group and lasted until the eighth day. The red skin phenomenon was not obvious, the skin thickened, and there were individual differences. The overall skin lesions were more obvious, and the PASI score was higher.

[0116] 4. Experiment on the expression of inflammatory factors in lesional tissue of BALB / c psoriasis model

[0117] The mice were euthanized on the 6th and 8th days of modeling, and the exposed skin on the back was taken and divided into three parts. One part was washed with PBS and then ground in trizol. RNA was extracted and qRT-PCR was performed to detect the expression of inflammatory factors, including TNF-α, IL-1β, IL17F, and IL23.

[0118] The results are as follows Fig.12 As shown, the expression of inflammatory factors TNFα, IL1β, and IL23 in the modeling group was upregulated, but there was no significant difference.

[0119] 5. H&E staining of lesional tissue of BALB / c psoriasis model

[0120] The mice were euthanized on the 6th and 8th days after modeling, and the exposed skin on the back was taken and divided into three parts. One part was washed with PBS and then immersed in 4% PFA. The skin was stained with H&E and pathologically analyzed. The other part was washed with PBS and stored in PBS at -80°C for future use.

[0121] The results are as follows Fig.13 As shown, IMQ induced typical psoriasis pathological changes in the skin. HE staining results showed that after modeling, the epidermis was thickened, the granular layer was reduced, the spinous layer was thickened, keratinization was excessive, and inflammatory cells were infiltrated. The inflammation was more significant on the sixth day, and there were individual differences within the group.

[0122] Example 10: Effect of SSN-2 delivery of TNF-α-siRNA on BALB / c psoriasis model mice

[0123] 1. Experiment on monitoring the therapeutic effect of SSN-2-delivered TNF-α-siRNA on the back lesions of BALB / c psoriasis model mice

[0124] The BALB / c back skin was locally depilated one day before modeling, and 1.25% avertin was applied at a dose of 0.2 mL / 10 g for intraperitoneal injection anesthesia. The hair on the back skin of the mice was completely removed using a pet shaver combined with a mild depilatory cream, exposing an area of ​​about 2 cm×3 cm of the back skin. There were 5 mice in each group, including a saline control group (Saline), a model group (IMQ), a positive control group (dexamethasone, DEX), a siControl group (Neg.), a siRNA2 (138#+200#) smearing group, and a siRNA3 (138#+200#+228#) smearing group. The NC group was smeared with 62.5 mg of vaseline on the back, the model group was smeared with 5% IMQ62.5 mg on the back, and the positive control group was smeared with 0.75% dexamethasone ointment 100 mg. The dosage of siRNA2 and siRNA3 was 0.25 mg / kg (20 μM), and the administration method was divided into smearing and subcutaneous injection. On the first day of modeling, only IMQ was applied. From the second day, the drug was administered 1 h after IMQ application. In the smear administration group, siRNA was mixed with 200 μg SSN-2 and allowed to stand at room temperature for 5 min before administration. Modeling was continued for five consecutive days, drug administration was continued for four consecutive days, and the back lesions were photographed on the sixth day.

[0125] 2. Experiment on weight monitoring of BALB / c psoriasis model mice by delivery of TNF-α-siRNA by SSN-2

[0126] The body weight of the above mice was monitored.

[0127] 3. PASI score on day 6 of BALB / c psoriasis model treated with TNF-α-siRNA delivered by SSN-2

[0128] The skin lesions on the backs of the mice were monitored daily and PASI scores were performed.

[0129] 4. Experiment on the expression of inflammatory factors in skin lesions of BALB / c psoriasis model treated with TNF-α-siRNA delivered by SSN-2 on day 6

[0130] The mice were euthanized on the 6th day of modeling, and the exposed skin on the back was taken and divided into three parts. One part was washed with PBS and then ground in trizol. RNA was extracted and qRT-PCR was performed to detect the expression of inflammatory factors, including TNF-α, IL-1β, IL17F, and IL23.

[0131] 5. H&E staining of skin lesions in BALB / c psoriasis model treated with SSN-2 delivered TNF-α-siRNA on day 6

[0132] The mice were euthanized on the 6th day of modeling, and the exposed skin on the back was taken and divided into three parts. One part was washed with PBS and then immersed in 4% PFA. The skin was stained with H&E and pathologically analyzed. The other part was washed with PBS and stored in PBS at -80°C for future use.

[0133] The results are as follows Figure 14-18 As shown in the figure, the weight of mice in the Saline group increased in the first three days and then leveled off. The weight of mice in the model group and the drug administration group showed a decreasing trend in the first three days, and the weight began to rise on the fourth day. The weight of mice in the DEX group showed an overall decreasing trend. The modeling and drug administration may have stimulated the mice to a certain extent and caused weight changes, but the daily weight change did not exceed 20% of the original weight, which was within the normal fluctuation range. From the PASI score, the back skin of mice in each group began to show lesions on the second day of modeling. The IMQ group and the Neg. group reached the peak of lesions on the fourth day and continued to have lesions. The scales in the DEX positive control group and the siRNA drug administration group were significantly reduced on the fourth day. The use of two sequences was better than the use of three sequences. On the sixth day, the skin lesions in the DEX positive control group and the smear drug administration group basically returned to normal. The expression of inflammatory factors in mice in the IMQ group and the Neg. group was different from each other. There was no significant difference in the expression of each inflammatory factor between the IMQ group and the Saline group. The expression levels of inflammatory factors in the siRNA drug administration group and the DEX positive control group were low, among which the expression of IL23 was significantly reduced compared with the IMQ group. Compared with the IMQ group, the expression of IL22 in the two-sequence co-administration group was significantly reduced, and there was no significant difference in the expression of other inflammatory factors. Histopathological results showed that the Saline group had no obvious inflammatory manifestations, while the IMQ group and the Neg. group had thickened epidermis, hyperkeratosis, reduced or disappeared granular layer, thickened acanthosis, irregular upward protrusions and hyperplasia of the dermal papillary layer, and infiltration of inflammatory cells. The epidermis of the DEX positive control group and the drug administration group was significantly thinner than that of the IMQ group, indicating that the positive control group and the siRNA administration group had certain anti-inflammatory effects.

[0134] From the appearance of the skin lesions, the effect of two sequences of siRNA drugs was better than that of three sequences. The symptoms of mouse skin erythema and scaling were significantly alleviated, and the epidermal thickening, epidermal ridge elongation and dermal lymphocyte infiltration caused by IMQ were also improved to varying degrees. During the experiment, the PASI score showed that transdermal administration had a significant improvement on IMQ-induced psoriasis-like lesions, and the score decreased after administration. The positive control drug DEX also showed a good therapeutic effect on IMQ-induced psoriasis-like skin lesions, but due to its potential side effects, it is not suitable for long-term use. The results of qRT-PCR showed that the expression of inflammatory factors was not significantly different from that of the normal group, which was speculated to be related to the improper method of sample collection. The results of H&E staining showed that transdermal administration significantly alleviated the symptoms of parakeratosis, epidermal thickening and obvious hypertrophy of the spinous layer caused by IMQ stimulation, and the effect was very close to that of the DEX group.

[0135] In summary, TNFα-siRNA drugs 138#, 200#, and 228# still have good efficacy in the psoriasis model, and the effect is better when the two sequences are used together. Transdermal administration has been proven to be an effective way to treat psoriasis, and these results provide a certain basis for further research and development of new treatment methods. The silica nanosphere drug delivery system for delivering small interfering RNA used in the present invention has certain potential for treating psoriasis.

[0136] Example 11: Experiment of delivering FAM-siRNA to SSN-2 in HFK (Human Foreskin Keratinocytes) cells

[0137] HFK cells were cultured at 1×10 5 The cells were inoculated into a 24-well plate at a density of 1 / well, and the inoculation volume was 1 mL. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured overnight at 37°C. Prepare two 1.5 mL sterile centrifuge tubes, add 25 μL Opti-MEM and 1 μL FAM-siRNA (20 μM) to one tube, and mix with a pipette; add 15 μL Opti-MEM and 10 μL SSN-2 (10 mg / mL) to another 1.5 mL tube, mix with a pipette, add to the siRNA-culture medium mixture, mix, let stand at room temperature for 20 min, and then transfect immediately. Take pictures under a fluorescence microscope 6 h later.

[0138] The results are as follows Fig.19As shown, the delivery efficiency of the silica nanospheres SSN-2 with surface spikes in the silica nanosphere drug delivery system for delivering small interfering RNA used in this experiment in primary HFK cells is much higher than that of RNAi-MAX, a commonly used siRNA drug delivery vector on the market, in primary HFK cells.

[0139] Example 12: Experiment on silencing HPV target genes by delivering HPV-siRNA using SSN-2

[0140] HFK cells were cultured at 1×10 5 The cells were inoculated into 24-well plates at a density of 1 / well, and the inoculation volume was 1 mL. The inoculated cells were placed in a cell culture incubator with 5% CO2 and cultured overnight at 37°C. HFK cells were infected with the assembled HPV pseudovirus for 48 h, and two 1.5 mL sterile centrifuge tubes were prepared. 25 μL Opti-MEM and 1 μL HBV-siRNA (20 μM) were added to one tube and mixed with a pipette; 15 μL Opti-MEM and 10 μL SSN-2 (10 mg / mL) were added to the other 1.5 mL tube, mixed with a pipette, added to the siRNA-medium mixture, and allowed to stand at room temperature for 20 min before transfection. HBV-siRNA numbers are 864#, 869#, 1016#, and 1783#. After 24 h of action, viral genomic DNA was extracted from the cell culture supernatant and the cells were lysed to extract viral genomic DNA for qPCR detection.

[0141] The results are as follows Fig. 20 As shown in the figure, HBV-siRNA No. 864#, 869#, 1783# can effectively silence the expression of HPV31 E1 gene, which is significantly different from the siControl group, but has not yet affected the expression of E2 gene related to viral replication and L1 gene related to assembly. The amount of DNA extracted from the cell culture supernatant is small and has no gene silencing effect, indicating that the virus particles have not been released into the supernatant for amplification. These results suggest the potential of silica nanosphere drug delivery system for delivering small interfering RNA in silencing HPV target genes.

[0142] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Application of a silica nanosphere drug delivery system in the preparation of drugs.

2. The use according to claim 1, characterized in that: The drug is a small nucleic acid RNA-based drug.

3. The use according to claim 1, characterized in that: The medicine is a medicine for treating psoriasis, skin cancer, skin infection or androgenic alopecia.

4. The use according to claim 1, characterized in that: The drug is administered via transdermal application.

5. The use according to claim 1, characterized in that: The silica nanosphere drug delivery system is constructed by silica nanoparticles with surface spikes.

6. The use according to claim 5, characterized in that: The core of the silicon dioxide nanoparticles is a solid sphere with a diameter of 80-120 nanometers, the surface is a silicon dioxide spike with a length of 40-60 nanometers, and the total particle size is 160-240 nanometers.

7. The use according to claim 5, characterized in that: The preparation steps of the silicon dioxide nanoparticles are: adding ammonia water, ethylamine, tetraethyl orthosilicate and sodium hydroxide into an ethanol aqueous solution, stirring and reacting uniformly; then adding phenol and formaldehyde thereto, continuing stirring and reacting, centrifuging, drying and roasting after the reaction is completed, so as to obtain the silicon dioxide nanoparticles.

8. The use according to claim 7, characterized in that: The mass ratio of ammonia water: ethylamine: phenol: formaldehyde: tetraethyl orthosilicate: sodium hydroxide: ethanol aqueous solution is 0.5-5: 0.05-0.1: 0.1-0.3: 0.1-0.9: 0.5-1: 05-0.8: 10-20.

9. The use according to claim 5, characterized in that: The silica nanoparticles can deliver small nucleic acid RNA to epidermal and dermal cells and exert pharmacological effects.

10. The use according to claim 5, characterized in that: The silica nanoparticles are used at a concentration of 10 mg / mL to 20 mg / mL.

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