CN108841822A - Nanometer selenium supported V P1 gene siRNA and the preparation method and application thereof - Google Patents

Abstract

The invention discloses a nano-selenium-loaded VP1 gene siRNA, the nano-selenium-loaded VP1 gene siRNA has a small particle size, can avoid the immune system to reach the smallest capillary, and prolong the residence time in the blood flow; and it has Higher target selectivity, longer half-life. The gene site of the siRNA interference is the 157th nucleotide; the siRNA sequence is: Sense 5'-ggcaucaucaaaugcuagutt-3', Antisense 5'-acuagcauuugaugaugcctt-3'. The present invention also provides the preparation method and application of the VP1 gene siRNA loaded with nanometer selenium.

Description

Nano selenium loaded VP1 gene siRNA and its preparation method and application

technical field

The invention relates to VP1 gene siRNA loaded with nano-selenium, in particular to VP1 gene siRNA loaded with nano-selenium and its preparation method and application.

Background technique

Hand, foot and mouth disease is a common infectious disease caused by enteroviruses, mainly in infants and young children, with fever and rashes or herpes on the hands, feet, mouth and other parts as the main features. Most patients have mild symptoms, but a small number of patients may develop fatal complications such as myocarditis, pulmonary edema, acute flaccid paralysis, aseptic meningitis, encephalitis, and some severely ill children progress rapidly and easily cause death. Since the first large-scale outbreak of hand, foot and mouth disease in China in 2008, it has become popular every year to varying degrees, often causing kindergartens in various places to be forced to suspend classes and kindergartens, bringing huge negative impacts on society and the economy. Enterovirus 71 (EV71) is the main pathogen causing HFMD. The main reason why the morbidity and mortality of hand, foot and mouth disease have remained high in the past ten years is that the EV71 virus has multiple genotypes, which are constantly changing during the epidemic. The prevention and treatment of hand, foot and mouth disease is facing enormous pressure. At present, there is no effective drug treatment, and clinical treatment is mainly symptomatic and supportive. Therefore, vaccines and effective drugs for the prevention and treatment of hand, foot and mouth disease are problems to be solved urgently.

The EV71 virus inactivated vaccine obtained clinical approval in 2015, but the incidence of EV71 virus has not decreased in recent years. The main reasons are: the vaccine strain uses the C4 subtype EV71 virus strain, and the clinical epidemic strain may have mutation characteristics that cause the vaccine In addition, the titer of neutralizing antibodies began to decline in the 6th month after vaccine immunization, and the protection period was limited. At present, there is no effective drug for HFMD, and clinically still focus on symptomatic and supportive treatment. Therefore, the research on effective drugs against HFMD pathogens, especially the research on broad-spectrum anti-HFMD pathogenic drugs is of great significance.

RNA interference technology can target the conserved region of the virus genome, thereby limiting the ability of the virus to produce escape mutants to a certain extent. The specificity, high efficiency, stability of the target gene silencing effect of RNA interference technology, and the characteristics of not changing the host genome provide the possibility for the application of RNA interference technology in antiviral therapy and determine its use in the treatment of viruses. The characteristics of strong drug efficacy and small adverse reactions can appear during the disease. However, siRNA is a hydrophilic negatively charged macromolecule, which limits its uptake by tissue cells, and is extremely unstable in vivo and easily degraded by RNases. Moreover, commonly used siRNA transfer carriers such as adenovirus are easy to cause cell mutation and immune response, and cationic liposomes have low transfection efficiency to primary cells and immune cells. Therefore, finding a suitable carrier to safely, efficiently and stably deliver siRNA into cells is a key issue for the clinical application of siRNA drugs. Nanoparticles have excellent characteristics such as small size effect, surface effect, and macroscopic quantum tunneling effect. Due to their special physical and chemical properties, when used as a drug transport carrier, they can wrap the drug inside, reducing the degradation, hydrolysis and degradation of the drug after entering the body. Oxidation-reduction reactions enhance drug stability.

Selenium is an essential trace element for the human body. It mainly plays biological functions and participates in various physiological links in the form of selenoprotein in the living body. The mechanism of selenium and selenoprotein involved in the development of viral diseases is also a research hotspot of current interest. Studies have shown that selenium is involved in the occurrence and virulence of many viral infections and the occurrence and development of viral diseases; the selenium content in host cells will affect the mutation, replication and virulence of many invading viruses. Deletion of selenium causes the accumulation of genome mutations in some RNA viruses, such as coxsackievirus B3, AIDS, influenza A virus, atypical pneumonia coronavirus and Ebola virus, resulting in changes in the gene structure related to viral virulence .

So far, the antiviral research of nano-selenium carrying siRNA has not been reported.

Contents of the invention

One of the objects of the present invention is to provide the above-mentioned nano-selenium-loaded VP1 gene siRNA, the particle size of the nano-selenium-loaded VP1 gene siRNA is small, which can avoid the immune system to reach the smallest capillaries, and prolong the residence time in the blood stream; And it has high target selectivity and long half-life.

The second object of the present invention is to provide a method for preparing the VP1 gene siRNA loaded with nanometer selenium.

The third object of the present invention is to provide the application of the VP1 gene siRNA loaded with nanometer selenium.

One of the objects of the present invention is achieved by the following method: a nano-selenium-loaded VP1 gene siRNA, the gene site of the siRNA interference is the 157th nucleotide;

Described siRNA sequence is:

Sense 5'-ggcaucaucaaaugcuagutt-3',

Antisense 5'-acuagcauuugaugaugcctt-3'.

The second object of the present invention is achieved by the following method: a method for preparing the nano-selenium-loaded VP1 gene siRNA as described above, comprising the following steps:

Step 1: Mix vitamin C solution and sodium selenite solution in a dialysis bag, stir, place in water for dialysis, and obtain purified nano-selenium solution;

Step 2: Add polyethyleneimine (PEI) and siRNA to the nano-selenium solution, stir, centrifuge to discard the supernatant, and freeze-dry the precipitate to obtain the product.

Wherein, the concentration of the vitamin C solution in the step 1 is 400 μg/ml; the concentration of the sodium selenite solution is 400 μg/ml; the volume ratio of the vitamin C solution to the sodium selenite solution is 1:40.

Wherein, the stirring speed in the step 1 is 200-300rpm, and the stirring time is 0.5-1h. The stirring speed of the step 2 is 200-300rpm, and the stirring time is 0.5-1h.

Wherein, the centrifugation speed in the step 2 is 8000-12000rpm, and the centrifugation time is 15-30min.

Wherein, the way of mixing the vitamin C solution and the sodium selenite solution in the step 1 is: adding the vitamin C solution to the sodium selenite solution drop by drop.

Wherein, the reaction condition of the step 1 is 23°C-27°C.

The third object of the present invention is achieved by the following method: a nano-selenium-loaded VP1 gene siRNA as described above is applied to prepare a drug for inhibiting EV71 virus infection.

Wherein, the present invention has the following advantages with respect to the prior art:

The inhibitory site of the siRNA prepared by the present invention capable of inhibiting the VP1 gene of EV71 virus is located at the 157th site, and it is verified by PCR and Western blot that the siRNA designed for this site has a relatively high efficiency for silencing the VP1 gene. Among them, the PCR experiment confirmed that the nucleic acid expression of the VP1 gene was significantly inhibited by 62%, that is, the transfection efficiency of the siRNA was 38%. The Western blot experiment confirmed that the expression of the VP1 protein decreased after the siRNA was transfected by nano-selenium.

siRNA is a hydrophilic negatively charged macromolecule, which limits its uptake by tissue cells, and is extremely unstable in vivo and easily degraded by RNase. Moreover, commonly used siRNA transfer carriers such as adenovirus are easy to cause cell mutation and immune response, and cationic liposomes have low transfection efficiency to primary cells and immune cells. Nanoparticles have excellent characteristics such as small size effect, surface effect, and macroscopic quantum tunneling effect. Due to their special physical and chemical properties, when used as a drug transport carrier, they can wrap the drug inside, reducing the degradation, hydrolysis and degradation of the drug after entering the body. Oxidation-reduction reactions enhance drug stability.

Because the nano-selenium-loaded VP1 gene siRNA has a small particle size, it can avoid the immune system to reach the smallest capillaries and prolong the residence time in the blood stream; secondly, the nano-selenium-siRNA carrier is modified to enhance the target selectivity of siRNA Or improve the siRNA to overcome the barriers of organisms and reach the target efficiently; thirdly, the outer layer of the nanocarrier is chemically modified to improve its solubility and biocompatibility, which can avoid the clearance of the reticuloendothelial system and have a longer Half-life, and even controllable release of siRNA; Fourth, improve the utilization efficiency of loaded siRNA.

Description of drawings

Fig. 1 is the preparation and characterization diagram of nano-selenium loaded VP1 gene siRNA;

Figure 2 is a graph showing the detection of Vero cell viability by nano-selenium-loaded VP1 gene siRNA;

Fig. 3 is the interference efficiency figure of using nanometer selenium, PEI transfection siRNA;

Figure 4 is the potential distribution diagram of SeNPs, Se@PEI and Se@PEI@siRNA;

Fig. 5 is the result figure that measures nano-selenium carrying siRNA to Vero cell toxicity;

Fig. 6 is a graph showing the interference effect of measuring nanometer selenium-carrying siRNA.

Detailed ways

Below in conjunction with specific embodiment, the claims of the present invention are described in further detail, but do not constitute any restriction to the present invention, any limited number of modifications done within the protection scope of the claims of the present invention are still protected by the claims of the present invention within range.

Example 1

(1) Design and synthesis of siRNA targeting EV71 virus VP1 gene

The VP1 gene region of the C4 subtype EV71 virus popular in Asia was selected, and siRNA was designed and chemically synthesized.

The interfering gene site is the 157th nucleotide, and the siRNA sequence is:

Sense 5'-ggcaucaucaaaugcuagutt-3',

Antisense 5'-acuagcauuugaugaugcctt-3'.

The scrambled negative control siRNA (scramble siRNA) sequence is:

Sense 5'-gcaagaauggugcacccautt-3';

Antisense 5'-augggugcaccauucuugctt-3'.

(2) Preparation and characterization of nano-selenium system

0.1ml of 400μg/ml vitamin C solution was added dropwise to 4ml of 400μg/ml sodium selenite solution, magnetically stirred for 2 hours, and purified nano-selenium solution was obtained after dialysis. Take the above nano-selenium solution, add PEI and siRNA, stir magnetically for 1 h, centrifuge the reacted solution at 10,000 rpm for 10 minutes, wash with deionized water three times, and freeze-dry the sample to obtain a nano-selenium-carrying siRNA nano-drug loading system.

After resuspending the nano-selenium-siRNA in PBS buffer, observe the morphology characteristics through a transmission electron microscope, and detect it through a particle size analyzer. The results are shown in Figure 1-4. In the figure:

In Figure 1, a and b are the results of nano-selenium (SeNPs), polyethyleneimine-modified nano-selenium solution (Se@PEI) and polyethyleneimine-modified siRNA carried by nano-selenium (Se@PEI@siRNA), respectively. Transmission electron microscope image. It can be seen from the figure that unmodified SeNPs are prone to agglomeration, and the functionalized nano-selenium (ie Se@PEI, Se@PEI@siRNA) is more uniformly dispersed than SeNPs.

Figure 2 is a diagram of the Tyndall effect of Se@PEI@siRNA.

Figure 3 shows the particle size distribution of SeNPs, Se@PEI and Se@PEI@siRNA. It can be seen from the figure that the average particle size of SeNPs is 200nm, the average particle size of Se@PEI is 100nm, and the average particle size of Se@PEI@siRNA is 80nm. Obviously, the particle size of Se@PEI@siRNA is smaller and it is easier to enter cells.

Figure 4 shows the potential distribution of SeNPs, Se@PEI and Se@PEI@siRNA. It can be seen from the figure that the potential of SeNPs is -25mv, the potential of Se@PEI is 6mv, and the potential of Se@PEI@siRNA is 12mv. From the data analysis of the potential, the absolute value of the potential of Se@PEI@siRNA is larger than that of SeNPs, indicating that the stability is better than that of SeNPs.

(3) Determination of toxicity of nano-selenium-carrying siRNA to Vero cells

Cell viability was determined by MTT method:

The cells were digested with 0.25% trypsin and seeded in 96-well plates at ⁴ ×10 cells/ml, 100 μl per well.

After culturing in a 37°C, 5% CO ₂ incubator for 24 hours, 100 μl each of SeNPs, Se@PEI, and Se@PEI@siRNA solutions were added, and the culture was continued for 12 hours.

Add 5mg/ml 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT), 20μl/well, incubate at 37°C for 5h, then aspirate into the wells of the culture plate Add dimethyl sulfoxide (DMSO), 150 μl/well, and shake for 10 minutes. After the purple crystals are fully dissolved, measure the OD ₅₇₀ value of each well. Taking the OD ₅₇₀ of the control group as 100%, the cell survival rate of the drug treatment group was calculated. Cell survival rate (%)=(OD ₅₇₀ experimental group/OD ₅₇₀ control group)×100%. The results are shown in Figure 5, in which:

The effect of different drug components (control group, virus-infected cell group, virus-infected cell+SeNPs group, virus-infected cell Se@PEI, virus-infected cell+Se@PEI@siRNA group) on cells after treatment was measured by MTT method.

It was shown that after the virus infected the cells, the cell survival rate was 37%, which was significantly lower than that of the control group. After the virus infected the cells, the cell survival rates were 42%, 40%, and 73% after treatment with SeNPs, Se@PEI, and Se@PEI@siRNA, respectively. The above data show that after the virus infected the cells, the cell survival rate decreased significantly compared with the control group. After the virus infected the cells, they were treated with nano-selenium and PEI-modified nano-selenium respectively, and the levels increased compared with the control group. Nano-selenium carries siRNA, and the cell survival rate is significantly increased compared with other components.

(4) Interference effect of nano-selenium carrying siRNA

Before the EV71 virus infection, Vero cells were not treated with other components (EV71 group), or were added with nano-selenium SeNPs, SeNPs@PEI, negative control siRNA modified by polyethyleneimine and carried by nano-selenium (Se@PEI@scramblesiRNA ), Se@PEI@siRNA, after 12 hours, discard the supernatant, wash with PBS for 3 times, add EV71 virus solution with a virus titer of 100TCID50 and incubate for 2 hours, replace with fresh DMEM medium containing 10% fetal bovine serum and continue to cultivate for 48 hours , extract cellular RNA, amplify VP1 gene, and calculate the inhibitory efficiency of Se@PEI@siRNA on VP1 gene mRNA expression according to the CT value.

The results show that: see Figure 6 for details, the expression of VP1 gene nucleic acid after transfection of Se@PEI@scramble siRNA was 94% of that of the non-transfection control group (EV71 group), and the results were not statistically different; after transfection of SeNPs@PEI siRNA, the expression of VP1 gene The reduction of nucleic acid expression was 38%, which was significantly different from that of the non-transfected control group (EV71 group), that is, the interference efficiency was 62%.

In summary, the nano-selenium modified by PEI can effectively carry siRNA into cells and play a highly efficient interference effect, thereby resisting cell apoptosis caused by virus infection.

sequence listing

<110> Guangzhou Women and Children's Medical Center

<120> Nano selenium-loaded VP1 gene siRNA and its preparation method and application

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Claims (10)

1. a kind of nanometer selenium supported V P1 gene siRNA, which is characterized in that the gene loci of the siRNA interference is the 157th Nucleotide；

The siRNA sequence is：

Sense 5 '-ggcaucaucaaaugcuagutt-3 ',

Antisense 5’-acuagcauuugaugaugcctt-3’。

2. a kind of preparation method of nanometer selenium supported V P1 gene siRNA as described in claim 1, which is characterized in that including with Lower step：

Step 1：Vitamin c solution is mixed in bag filter with sodium selenite solution, is stirred, is placed in water and dialyses, obtain Nanometer selenium solution after purification；

Step 2：By in the nanometer selenium solution, polyethyleneimine and siRNA is added, both make final concentration be respectively 40 μM and Supernatant is abandoned in 2.5nM, stirring, centrifugation, by pellet frozen it is dry to get.

3. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step The concentration of vitamin c solution is 400 μ g/ml in 1.

4. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step The molten concentration of sodium selenite is 400 μ g/ml in 1.

5. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 3, which is characterized in that the step Vitamin c solution and the molten volume ratio of sodium selenite are 1 in 1:40.

6. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step 1 mixing speed is 200-300rpm, mixing time 0.5-1h.

7. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step 2 mixing speed is 200-300rpm, mixing time 0.5-1h.

8. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step 2 centrifugal speed is 8000-12000rpm, centrifugation time 15-30min.

9. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step The mode that 1 vitamin c solution is mixed with sodium selenite solution is：Vitamin c solution is added dropwise to sodium selenite solution In.

10. a kind of nanometer selenium supported V P1 gene siRNA as described in claim 1, which is applied to preparation, inhibits EV71 virus infection Drug.