CN110638788A - siRNA capable of silencing Pcsk9 protein, nano delivery system and application thereof - Google Patents
siRNA capable of silencing Pcsk9 protein, nano delivery system and application thereof Download PDFInfo
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Abstract
The invention provides siRNA capable of silencing PCSK9 protein, wherein the sense strand amino acid sequence of the siRNA is 5 '-UUCCGAAAUACUCCAGGCdTdT-3', the antisense strand amino acid sequence of the siRNA is 5 '-GCCUGGAGUUAUUCCGGAAdTdT-3', and a nano delivery system, a preparation method and application thereof. The method has higher transfection effect, can maintain long-term effect and has better treatment effect on reducing blood fat. The invention is suitable for the gene therapy of reducing blood fat. The method of the present invention is believed to have broad application prospects in cardiovascular diseases, genetic diseases, and the like.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to siRNA capable of silencing Pcsk9 protein, a nano delivery system and application thereof.
Background
With the continuous improvement of living standard and the change of dietary structure and life style of people, the cases of hyperglycemia, hypercholesterolemia and hypertriglyceridemia are on the trend of increasing year by year, and the diseases such as diabetes, arteriosclerosis, cardiovascular and cerebrovascular diseases and the like are generally caused. The World Health Organization (WHO) published in 2002, and the cholesterol-too-high disease in ten major risk factors of human health is positioned eighth.
The common familial hypercholesterolemia is mainly caused by Apolipoprotein (APOB), low-density lipoprotein receptor (LDL-R), and proprotein convertase bacillus subtilis convertase (PCSK9) gene mutation, PCSK9 is mainly distributed in the liver, and PCSK9 secreted to the outside of cells can be combined with LDL-R to be targeted to lysosome for degradation. As the number of LDL receptors on the cell surface decreases, the overall low-density lipoprotein cholesterol (LDL-C) levels in the blood increase, and studies have shown that loss-of-function mutations in PCSK9 result in decreased LDL cholesterol levels, and are therefore expected to be potentially useful therapeutic targets for the control of hypercholesterolemia and its complications.
At present, the clinical treatment means mainly comprises statins, and researches show that patients who are intolerant to statins can cause muscle diseases and other adverse reactions, such as myalgia and rhabdomyolysis. Although ilouxuzumab (available under the trade name of reberan) is currently marketed in china, PCSK9 mab is expensive to manufacture and needs to be injected every 2-4 weeks when metabolized by the reticuloendothelial system. The research shows that the small interfering RNA (siRNA) can specifically silence the Pcsk9 gene, thereby inhibiting the protein expression of the Pcsk9 gene and reducing the low-density lipoprotein. But sirnas used to silence Pcsk9 inevitably suffer from the difficulties faced with gene delivery: 1) the unmodified siRNA is easy to degrade under the action of nuclease; 2) the siRNA is modified, the structure is complex, the difficulty is high, and the specificity of the siRNA is possibly reduced; 3) the siRNA is randomly distributed in the body, so that the accumulation of the siRNA at a target position is reduced; 4) siRNA exhibits strong electronegativity due to the presence of phosphate residues in the backbone and is difficult to be taken up by cells carrying the same surface charge. siRNA delivery needs to be assisted by a highly efficient transduction system, and viral vectors are currently most widely used. Viral vectors can efficiently deliver hairpin RNA, effectively silencing Pcsk9, but they are potentially immunogenic, tumorigenic, and teratogenic, raising concerns about their biological safety. The non-viral vectors are mainly liposomes and high molecular materials. However, the current non-viral vectors suffer from problems including high toxicity, poor stability and low transfection efficiency. One often has difficulty deciding on delivery efficiency and safety. Due to their potential immunogenicity, tumorigenicity and teratogenicity, current viral vectors are banned for clinical therapy, which greatly limits their use. The non-viral vector is used as a system with great potential, makes up the deficiency of the viral vector and has great development prospect. However, the current non-viral vectors suffer from problems including high toxicity, poor stability and low transfection efficiency. Specifically for the treatment of hypercholesterolemia, it is desirable to deliver siRNA to the liver site efficiently, to be taken up by hepatocytes, and to successfully escape lysosomes, thereby producing a gene silencing effect. How to effectively solve the problem of non-viral vectors in siRNA delivery becomes a hot spot of research. Therefore, the development of a highly effective delivery system to silence Pcsk9 would provide an effective means for long-term treatment of hypercholesterolemia.
Disclosure of Invention
Therefore, the invention aims to overcome the defects in the prior art and find a siPcsk9 nanocapsule high-efficiency transfection based on platelet membrane camouflage to reduce the level of PCSK9 protein, thereby achieving the treatment effect of efficiently and permanently reducing the cholesterol level.
Before the technical solution of the present invention is explained, the terms used herein are defined as follows:
the term "siRNA" refers to: small interfering RNA.
The term "PLGA" refers to: polylactic acid-glycolic acid copolymer.
The term "DOTAP" refers to: (2, 3-dioleoyl-propyl) trimethylammonium chloride.
The term "PVA" refers to: polyvinyl alcohol.
The term "siRNA" refers to: small interfering RNA.
The term "PBS" refers to: phosphate buffered saline solution.
The term "PM/PDC/siPcsk 9" refers to: platelet membranes encapsulate nanocapsules with a PLGA/DOTAP core containing siPcsk 9.
The term "LDL-C" means: low density lipoprotein cholesterol.
The term "HDL-C" refers to: high density lipoprotein cholesterol.
The term "TG" means: a triglyceride.
The term "TBIL" refers to: total bilirubin.
The term "ALT" refers to: glutamate pyruvate transaminase.
The term "AST" means: glutamic-oxalacetic transaminase.
The term "PBAE" refers to: polyaminoester poly-beta-amino esters.
The term "HA" refers to: hyaluronic acid.
The term "PLA" refers to: polylactic acid.
In order to achieve the aim, the first aspect of the invention provides siRNA capable of silencing PCSK9 protein, wherein the sense strand amino acid sequence of the siRNA is 5 '-UUCCGAAACUCCAGGCdTdT-3', and the antisense strand amino acid sequence of the siRNA is 5 '-GCCUGGAGUUAUUCCGGAAdTdT-3'.
A second aspect of the invention provides a nanosupport system capable of silencing PCSK9 protein, the nanosupport being a biofilm-camouflaged nanocapsule encapsulating siRNA according to the first aspect.
The nano-delivery system according to the second aspect of the present invention, wherein the biofilm is selected from one or more of: platelet membrane, erythrocyte membrane, mesenchymal stem cell of marrow, mesenchymal stem cell of umbilical cord, macrophage; platelet membranes are preferred.
The nano-delivery system according to the second aspect of the present invention, wherein the material of the nanocapsule is selected from one or more of: PLGA, DOTAP, polyamino ester, hyaluronic acid, chitosan and polylactic acid; preferably PLGA and DOTAP.
The nano delivery system according to the second aspect of the invention, wherein the mass ratio of PLGA to DOTAP in the nano capsule is 20: 1-20: 11, preferably 20: 1-20: 5, and most preferably 20: 3.
The nano delivery system according to the second aspect of the present invention, wherein the biological membrane, the nanocapsule and the siRNA are in parts by mass: 1-50 parts of a biological membrane, 1-50 parts of a nanocapsule and 0.1-10 parts of siRNA0; preferably 10-30 parts of biological membrane, 10-30 parts of nanocapsule and 0.5-2 parts of siRNA; most preferably 23 parts of biomembrane, 23 parts of nanocapsule and 1 part of siRNA;
preferably, when the material of the nanocapsule is selected from PLGA and DOTAP, the mass ratio of the biofilm to the nanocapsule is 1: 1.
A third aspect of the present invention provides a method of preparing the nano delivery system of the second aspect, which may comprise the steps of:
(1) preparing a siRNA solution;
(2) dripping the siRNA solution prepared in the step (1) into the solution of the nanocapsule material;
(3) dropwise adding the mixed solution obtained in the step (2) into a solvent, ultrasonically stirring, centrifuging to remove supernatant, and re-suspending the obtained precipitate;
(4) and (4) mixing the resuspension obtained in the step (3) with a biological membrane, and performing ultrasonic treatment to obtain the nano delivery system.
The production method according to the third aspect of the present invention, wherein, in the step (3), the solvent is selected from one or more of: aqueous PVA solution, aqueous Tween 20 solution and aqueous Tween 80 solution; preferably an aqueous solution of PVA, more preferably from 1% to 10% aqueous solution of PVA, most preferably a 2% aqueous solution of PVA.
In a fourth aspect, the present invention provides a medicament for down-regulating low density lipoprotein cholesterol, the medicament comprising:
the siRNA of the first aspect; and/or
The nano-delivery system of the second aspect;
preferably, the drug is a drug that down-regulates low density lipoprotein cholesterol.
A fifth aspect of the invention provides the use of an siRNA of the first aspect and/or a nano-delivery system of the second aspect in the manufacture of a medicament for the treatment of hypercholesterolemia.
The invention aims to find a siPcsk9 nanocapsule high-efficiency transfection based on platelet membrane camouflage and knock down the level of PCSK9 protein, so that the treatment effect of efficiently and permanently reducing cholesterol level is achieved.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to the invention, polylactic acid-polyglycolic acid copolymer (PLGA) with biocompatibility and degradability is adopted to wrap siPcsk9, and cationic liposome DOTAP is used to overcome the defects that PLGA and nucleic acid have small adsorption force and lysosome escape cannot be completed to construct a nanocapsule, and then a platelet membrane from a donor is adopted to wrap the nanocapsule, so that a siPcsk9 nanocapsule system is efficiently delivered into cells under the condition of no need of any other transfection reagent, the cell uptake rate and the transfection efficiency are improved, the level of PCSK9 protein is low, and the treatment effect of continuously reducing the blood cholesterol level is achieved, and the application value is important. The specific flow is shown in figure 1.
The invention provides a gene capable of silencing a PCSK9 protein, which comprises the following components in part by weight:
siRNAs against PCSK9(siPCSK9),
a sense strand of 5 '-UUCCGAAAUAAACCUCCAGGCdtdT-3',
antisense strand 5 '-GCCUGGAGUUAUUCCGGAAdTdT-3'
The ratio of platelet membrane, PLGA, siPcsk9 was fixed in the present invention.
The nano capsule core of the invention comprises PLGA and DOTAP, and the proportion is fixed.
The method adopts platelet membrane with good biocompatibility to modify PLGA/DOTAP core. The structure endows the nanoparticles with the capacity of loading siRNA with high efficiency, and improves the stability of the particles in vivo. And the DOTAP modified nucleus can promote siRNA to escape from lysosome successfully, so that the silencing efficiency is improved. This structure ultimately allows for safe and efficient down-regulation of LDL-C in vivo.
The delivery system of the present invention may have the following beneficial effects, but is not limited to:
the method has higher transfection effect, can maintain long-term effect and has better treatment effect on reducing blood fat. The invention is suitable for the gene therapy of reducing blood fat. The method of the present invention is believed to have broad application prospects in cardiovascular diseases, genetic diseases, and the like.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
figure 1 shows the platelet membrane camouflaged siPcsk9 nanocapsule preparation process. Wherein, FIG. 1A shows polylactic-co-glycolic acid (PLGA) and cationic liposome DOTAP to wrap siPcsk9 to construct nanocapsules, and FIG. 1B shows the process of preparing platelet membrane disguised siPcsk9 nanocapsules by wrapping the nanocapsules with platelet membrane.
Fig. 2 shows the cytotoxicity of the nanocapsules of example 1. In PDC, the ratio of PLGA to DOTAP is 20/0, 20/1, 20/3, 20/5, 20/7, 20/9 and 20/11, respectively. The concentrations of siRNA were 0,9.38,18.75,37.5,75,150, and 300nM, respectively.
Figure 3 shows the loading efficiency of the nanocapsules on siRNA of example 1. In the PDC/siRNA, the mass ratio of PDC to siRNA is 23/0.1, 23/0.2, 23/0.5, 23/1 and 23/2 respectively.
Fig. 4 shows the case of platelet membrane-encapsulated siRNA loaded nanocapsules of example 1. Wherein the mass ratio of the platelet membrane to the nanocapsule is 0/1, 1/5, 1/1 and 5/1.
Fig. 5 shows the nanoparticles obtained in example 1. Wherein FIG. 5A shows plasma obtained after centrifugation of a blood taken from a mouse, and FIG. 5B shows flow-type quantitative determination of platelet proportion; FIG. 5C shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein components (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP nuclei); FIG. 5D shows the platelet membrane encapsulating the PLGA/DOTAP core before FIG. 5E shows the platelet membrane encapsulating the PLGA/DOTAP core.
Figure 6 shows the levels of PCSK9 in mouse liver tissue detected by Western Blot in experimental example 1.
FIG. 7 shows the expression levels of indices PCSK9, LDL-C, HDL, TG and the like in the blood of mice in Experimental example 1, wherein FIG. 7A shows the PCSK9 level, FIG. 7B shows the LDL-C level, FIG. 7C shows the HDL-C level, FIG. 7D shows the TG level, FIG. 7E shows the TBIL level, FIG. 7F shows the ALT level, and FIG. 7G shows the AST level.
Figure 8 shows the level of cellular PCSK9 detected by Western Blot in experimental example 2.
FIG. 9 shows the in vitro Hepa1-6 PCSK9 band changes in test example 3.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and instrumentation used in the following examples are as follows:
reagent:
ribozyme-free water, siPcsk9 dry powder, purchased from Takara japan;
PLGA, available from sika, guangzhou;
DOTAP, available from Avanti, usa;
dichloromethane, purchased from Guangzhou chemical industries, China;
PVA, available from Sigma-Aldrich, USA;
PBS, available from Life technology, usa;
phalloidin, purchased from Life, usa;
TritonX-100, BSA, available from Sigma-Aldrich, USA;
c57BL/6 mice, purchased from Sayborno Biotech, Guangzhou;
ELISA kit, Hepa1-6 cells, was purchased from Changsheng biotechnology, Inc., Beijing ancient cooking.
The instrument comprises the following steps:
confocal laser microscopy, available from Zeiss germany.
Example 1
This example serves to illustrate the synthesis of the nanoparticles of the invention.
Constructing a method for preparing biomembrane-coated nanoparticles by using the nano-materials and the extracted platelet membrane.
1) 56nmol of dry powder of siPcsk9 (0.75mg) was dissolved in 100. mu.L of ribozyme-free water, added dropwise to a solution (250. mu.L) of PLGA/DOTAP (w/w 20/0, 20/1, 20/3, 20/5, 20/7, 20/9 and 20/11) in methylene chloride containing 69mg/mL, and sonicated in ice bath for 90 s.
2) The colostrum obtained above was added dropwise to 1mL of 2% aqueous PVA solution and sonicated in ice bath for 1 min.
3) The prepared mixed solution is completely added into 10mL of 2% PVA aqueous solution and stirred for 3h at room temperature, the organic solvent is volatilized, the mixed solution is centrifuged for 15 min at the temperature of 4 ℃ and 18000g, the supernatant is removed, the mixed solution is repeatedly washed for three times, and the obtained precipitate is re-suspended by sterile PBS.
4) And mixing the mixed solution with a platelet membrane (the platelet membrane is quantified by the mass of membrane protein) according to the mass ratio of protein on the platelet membrane to nanocapsule of 0/1, 1/1, 1/5, 1/1 and 5/1), and ultrasonically wrapping the nanocapsule containing the siPcsk9 to obtain the nano-particle PM/PDC/siPcsk 9.
Wherein, the synthesis of the platelet membrane is as follows:
1) the preparation method comprises the steps of collecting animal whole blood, centrifuging at 1500rpm at room temperature for 20 minutes to obtain platelet-rich plasma, centrifuging at 1500rpm at room temperature for 1 minute to remove residual red blood cells and white blood cells, centrifuging at 3500rpm again for 12 minutes to obtain platelet precipitates, preparing suspension, and storing in a refrigerator at-80 ℃.
2) Extracting platelet membrane: and (3) melting the suspension at room temperature, repeatedly freezing and thawing for 3 times, washing with PBS, centrifuging at 5000rpm for 5 minutes, and performing water bath ultrasound for 5 minutes until platelet membrane vesicles are formed.
Figure 1 shows the platelet membrane camouflaged siPcsk9 nanocapsule preparation process. Wherein, FIG. 1A shows polylactic-co-glycolic acid (PLGA) and cationic liposome DOTAP to wrap siPcsk9 to construct nanocapsules, and FIG. 1B shows the process of preparing platelet membrane disguised siPcsk9 nanocapsules by wrapping the nanocapsules with platelet membrane.
Fig. 2 shows the cytotoxicity of the nanocapsules of example 1. In PDC, the ratio of PLGA to DOTAP is 20/0, 20/1, 20/3, 20/5, 20/7, 20/9 and 20/11, respectively. The concentrations of siRNA were 0,9.38,18.75,37.5,75,150, and 300nM, respectively.
Figure 3 shows the loading efficiency of the nanocapsules on siRNA of example 1. In the PDC/siRNA, the mass ratio of PDC to siRNA is 23/0.1, 23/0.2, 23/0.5, 23/1 and 23/2 respectively.
Fig. 4 shows the case of platelet membrane-encapsulated siRNA loaded nanocapsules of example 1. Wherein the mass ratio of the platelet membrane protein to the nanocapsule is 0/1, 1/5, 1/1 and 5/1 respectively.
Fig. 5 shows the nanoparticles obtained in example 1. Wherein FIG. 5A shows plasma obtained after centrifugation of a blood taken from a mouse, and FIG. 5B shows flow-type quantitative determination of platelet proportion; FIG. 5C shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein components (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP nuclei); FIG. 5D shows the platelet membrane encapsulating the PLGA/DOTAP core before FIG. 5E shows the platelet membrane encapsulating the PLGA/DOTAP core.
Test example 1
This test example is intended to illustrate the treatment of hypercholesterolemia with the nanoparticles of the invention.
Nanoparticles PM/PDC/siPcsk9 were synthesized as in example 1 to treat hypercholesterolemia. PM/PDC/siPcsk9 is the experimental group, Lipo2000/siPcsk9 is the positive control group, while PM/PDC/siLuc is the control group used to detect siCPSK9 specific silencing, siPcsk9 is the unmodified drug control group, PM/PDC is the drug-free no-load control group, and PBS is the blank control group.
Lipo2000/SiPcsk9 preparation:
1) dissolving 5nmol of dry powder of siPcsk9 in 250. mu.L of ribozyme-free water, collecting 15. mu.L of the dry powder, adding to 150. mu.L of LOpti-MEM, and mixing
2) At the same time, 22.5. mu.L of Lipofectamine 2000(Life, USA) was added to 150. mu.L of LOpti-MEM and gently mixed
3) Adding all the solution prepared in the step 1) into the solution prepared in the step 2), slightly inverting and uniformly mixing, and incubating for 5 minutes at room temperature.
PM/PDC/siLuc preparation:
1) 56nmol of dry powder of siLuciferase was dissolved in 100. mu.L of ribozyme-free water, added dropwise to a solution of PLGA/DOTAP (w/w 20:3) in dichloromethane (250. mu.L) containing 69mg/mL, and sonicated in ice bath for 90 s.
2) The colostrum prepared above is added dropwise into 1mL of 2% PVA water solution, and ultrasonic treatment is carried out in ice bath for 1 min.
3) The prepared mixed solution is completely added into 10mL of 2% PVA aqueous solution and stirred for 1h at room temperature, the organic solvent is volatilized in vacuum for 3h, the mixture is centrifuged for 15 min at 4 ℃ and 18000g, the supernatant is removed, the mixture is repeatedly washed for three times, and the obtained precipitate is re-suspended by sterile PBS.
4) And mixing the mixed solution with a platelet membrane (the mass ratio of PLGA to protein on the platelet membrane is 1:1), and ultrasonically coating the nanocapsule containing the siLuciferase.
siPcsk9 preparation:
5nmol of dry powder of siPcsk9 was dissolved in 25. mu.L of ribozyme-free water and diluted to 200. mu.M with sterile PBS solution.
Preparing PM/PDC:
1) 100 μ L of ribozyme-free water was added dropwise to a solution (250 μ L) containing 69mg/mL of PLGA/DOTAP (w/w 20:3) in methylene chloride and sonicated in ice for 90 s.
2) The colostrum prepared above is added dropwise into 1mL of 2% PVA water solution, and ultrasonic treatment is carried out in ice bath for 1 min.
3) And (3) completely adding the prepared mixed solution into 10mL of 2% PVA aqueous solution, stirring for 3h at room temperature, volatilizing the organic solvent, centrifuging for 15 min at the temperature of 4 ℃ at 18000g, removing the supernatant, repeatedly washing for three times, and re-suspending the obtained precipitate with sterile PBS.
4) And mixing the mixed solution with a platelet membrane (the mass ratio of PLGA to protein on the platelet membrane is 1:1), and ultrasonically coating the nanocapsule containing the siPcsk 9.
Fig. 6 and 7 are in contrast to other treatment methods. FIG. 6 shows the expression level change of PCSK9 in mouse liver tissue, and FIG. 7 shows the change of biochemical factors in mouse body.
1) 30 female C57BL/6 mice (6-8 weeks old, body weight 20 g) were divided randomly into five groups, and after two weeks of administration, the mice livers were separated, proteins were extracted, and the levels of PCSK9 in the mice were detected by Western immunoblotting (Western Blot), as shown in FIG. 6. The nanocapsules synthesized by the invention have higher effect of knocking down the protein level of Pcsk9 in mice compared with Lipo2000/siPcsk9 group, while the groups of PM/PDC/siLuc, siPcsk9 and PM/PDC hardly affect the expression level of Pcsk9 protein in vivo.
2) Mouse serum is extracted according to the method, and expression levels of indexes such as PCSK9, LDL-C, HDL, TG and the like in blood are detected by using an ELISA kit, as shown in figure 7.
As shown in fig. 7, the silencing effect was stronger in the experimental group and the low-density lipoprotein-cholesterol (LDL-C) level was significantly reduced compared to the control group.
Test example 2
This experimental example serves to illustrate the in vitro transfection experiment of hepatocytes with the nanoparticles of the present invention.
Nanoparticles were synthesized as in example 1, and mouse-derived liver cancer cells (Hepa1-6) were transfected in vitro.
Cell uptake was observed by laser confocal microscopy (CLSM).
1) Taking Hepa1-6 cells in logarithmic growth phase, digesting with pancreatin, washing, centrifuging and preparing into cell suspension.
2) Inoculating 1mL of the suspension into a confocal small dish, culturing 1.0 x 10^5 cells in each dish at 37 ℃ overnight, and adding the medicament for continuous culture after the adherent growth density of the cells under a microscope reaches more than 80%.
3) After the incubation time was reached, the stock culture was aspirated, washed with pre-warmed PBS, fixed for 10 minutes at room temperature by adding 4% paraformaldehyde, and then washed with PBS 2 times.
4) The solution was discarded, 0.1% TritonX-100 was added, membranes were broken for 5 minutes, washed and then blocked with 1% BSA for 30 minutes, followed by incubation with 0.2% Phalloidin (FITC-Phalloidin) for 30 minutes.
5) The solution was discarded and then stained with Hoechst for 15 minutes at room temperature, washed and photographed under CLSM as shown in fig. 8. As shown in FIG. 8, the cell uptake of the cy 5-labeled nanocapsule was low at 1 hour, gradually increased with time, and reached the maximum uptake at 9 hours.
Test example 3
This experimental example serves to demonstrate that protein immunoblot (Western Blot) of the nanoparticles of the invention detects levels of cellular PCSK 9.
1) The nanoparticles PM/PDC/siPcsk9 were synthesized as described in example 1, and the groups Lipo2000/siPcsk9, PM/PDC/siLuc, siPcsk9 and PM/PDC were synthesized as described in Experimental example 1, respectively.
2) A cell suspension was prepared according to the method of test example 2, 2mL of the suspension was cultured in six-well plates containing 3.0 x 10^5 cells per well, and transfection was performed by administration when the cells grew adherent and the density reached 80%.
3) After 24 hours of culture, the original culture medium is discarded, cell total protein is extracted by RIPA lysate, and the change of PCSK9 bands is detected by WB, as shown in figure 9, the synthesized nanocapsules have the effect of reducing the protein level of Pcsk9 in vitro similar to the Lipo2000/siPcsk9 group, while the PM/PDC/siLuc, siPcsk9 and PM/PDC groups hardly influence the expression level of the Pcsk9 protein in vivo.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (10)
1. An siRNA capable of silencing PCSK9 protein, characterized in that the sense strand amino acid sequence of the siRNA is 5 '-UUCCGAAAUACUCCAGGCdTdT-3', and the antisense strand amino acid sequence is 5 '-GCCUGGAGUUAUUCCGGAAdTdT-3'.
2. A nanosupport system capable of silencing PCSK9 protein, wherein the nanosupport is a nanocapsule camouflaged by a biological membrane coated with the siRNA of claim 1.
3. The nano-delivery system according to claim 2, wherein the biofilm is selected from one or more of the following: platelet membrane, erythrocyte membrane, mesenchymal stem cell of marrow, mesenchymal stem cell of umbilical cord, macrophage; platelet membranes are preferred.
4. The nano-delivery system according to claim 2 or 3, wherein the material of the nanocapsule is selected from one or more of the following: PLGA, DOTAP, polyamino ester, hyaluronic acid, chitosan and polylactic acid; preferably PLGA and DOTAP.
5. The nano-delivery system according to claim 4, wherein the nanocapsule has a mass ratio of PLGA to DOTAP of 20:1 to 20:11, preferably of 20:1 to 20:5, most preferably of 20: 3.
6. The nano delivery system according to any one of claims 2 to 5, wherein the parts by mass of the biofilm protein, the nanocapsule and the siRNA are 1-50 parts of the biofilm protein, 1-50 parts of the nanocapsule, and 0.1-10 parts of SiRNA; preferably 10-30 parts of biological membrane, 10-30 parts of nanocapsule and 0.5-2 parts of siRNA; most preferably 23 parts of biomembrane protein, 23 parts of nanocapsule and 1 part of siRNA;
preferably, when the material of the nanocapsule is selected from PLGA and DOTAP, the mass ratio of the biofilm to the nanocapsule is 1: 1.
7. Method for the preparation of a nano-delivery system according to any of claims 2 to 6, characterized in that it comprises the following steps:
(1) preparing a siRNA solution;
(2) dripping the siRNA solution prepared in the step (1) into the solution of the nanocapsule material;
(3) dropwise adding the mixed solution obtained in the step (2) into a solvent, ultrasonically stirring, centrifuging to remove supernatant, and re-suspending the obtained precipitate;
(4) and (4) mixing the resuspension obtained in the step (3) with a biological membrane, and performing ultrasonic treatment to obtain the nano delivery system.
8. The method according to claim 7, wherein in the step (3), the solvent is selected from one or more of: PVA water solution, Tween 20 water solution and Tween 80 water solution; preferably an aqueous PVA solution, more preferably a 1% to 10% aqueous PVA solution, and most preferably a 2% aqueous PVA solution.
9. A medicament for down-regulating low density lipoprotein cholesterol, the medicament comprising:
the siRNA of claim 1; and/or
The nano-delivery system of any one of claims 2 to 6;
preferably, the drug is a drug that down-regulates low density lipoprotein cholesterol.
10. Use of the siRNA of claim 1 and/or the nano delivery system of any one of claims 2 to 6 for the preparation of a medicament for the treatment of hypercholesterolemia.
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CN117018229A (en) * | 2023-08-14 | 2023-11-10 | 暨南大学附属第一医院(广州华侨医院) | Application of over-expressed Clec1b protein cell membrane in preparation of knee osteoarthritis drugs |
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