WO2021077856A1 - Sirna and nano delivery system capable of silencing pcsk9 protein, and application of nano delivery system - Google Patents
Sirna and nano delivery system capable of silencing pcsk9 protein, and application of nano delivery system Download PDFInfo
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Definitions
- the invention belongs to the field of medicine, and specifically relates to a siRNA capable of silencing Pcsk9 protein, a nano-delivery system and application thereof.
- PCSK9 is mainly caused by It is distributed in the liver, and PCSK9 secreted outside the cell can bind to LDL-R and target to the lysosome for degradation.
- LDL-C low-density lipoprotein cholesterol
- statins The current clinical treatment is mainly based on statins. Studies have shown that patients who are intolerant to statins can cause adverse reactions such as muscle pain and rhabdomyolysis. Although evolocumab (trade name Ruibaian) is currently on the market in China, due to its high cost and the metabolism of PCSK9 monoclonal antibody via the reticuloendothelial system, injections are required every 2-4 weeks. Studies have shown that small interfering RNA (siRNA) can specifically silence the Pcsk9 gene, thereby inhibiting its protein expression and reducing low-density lipoprotein.
- siRNA small interfering RNA
- siRNA used to silence Pcsk9 inevitably encounters the problems faced by gene delivery: 1) Unmodified siRNA is easily degraded under the action of nuclease; 2) Modification of siRNA is complicated in structure, difficult, and may be reduced. The specificity of siRNA; 3) siRNA is randomly distributed in the body, reducing its accumulation at the target site; 4) siRNA is strongly negative due to the presence of phosphate residues in the backbone, and it is difficult to be taken up by cells with the same surface charge.
- the delivery of siRNA requires the help of an efficient transduction system, and viral vectors are currently the most widely used.
- Viral vectors can efficiently deliver hairpin RNA and effectively silence Pcsk9, but it has potential immunogenicity, tumorigenicity and teratogenicity, which raises concerns about its biological safety.
- Non-viral vectors are mainly liposomes and polymer materials.
- current non-viral vectors also encounter problems including high toxicity, poor stability, and low transfection efficiency. It is often difficult for people to make a choice between delivery efficiency and safety. Due to potential immunogenicity, tumorigenicity and teratogenicity, current viral vectors are forbidden for clinical treatment, which greatly limits their applications. As a system with great potential, non-viral vectors make up for the deficiencies of viral vectors and have great development prospects.
- the purpose of the present invention is to overcome the shortcomings in the prior art and find a siPcsk9 nanocapsule based on biomembrane camouflage (such as platelet membrane) to efficiently transfect siPcsk9 nanocapsules to reduce the level of PCSK9 protein, so as to achieve an efficient and long-term treatment of lowering cholesterol levels. effect.
- biomembrane camouflage such as platelet membrane
- the first aspect of the present invention provides a siRNA capable of silencing PCSK9 protein.
- the sense chain amino acid sequence of the siRNA is 5′-UUCCGAAUAAACUCCAGGC-3′, and the antisense chain amino acid sequence is 5′-GCCUGGAGUUUAUUCGGAA- 3'.
- the 3'end of the siRNA sense strand and/or antisense strand is further modified with dTdT.
- the second aspect of the present invention provides a nano-delivery system capable of silencing PCSK9 protein.
- the nano-system is a nanocapsule camouflaged by a biological membrane and wrapped with siRNA capable of silencing PCSK9 protein.
- the siRNA capable of silencing the PCSK9 protein may be, for example, the siRNA described in the first aspect.
- biofilm is selected from one or more of the following: platelet membrane, red blood cell membrane, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, macrophages; preferably Platelet membrane.
- nano delivery system wherein the material of the nanocapsule is selected from one or more of the following: PLGA, DOTAP, polyurethane, hyaluronic acid, chitosan, polylactic acid; preferably PLGA And DOTAP.
- the nano delivery system according to the second aspect of the present invention wherein the mass ratio of PLGA to DOTAP in the nanocapsule is 20:1-20:11, preferably 20:1-20:5, most preferably 20:3 .
- the nano-delivery system according to the second aspect of the present invention wherein the mass parts of the biofilm, nanocapsules and siRNA are: 1-50 parts of biofilm, 1-50 parts of nanocapsules, and 0.1-10 parts of siRNA; preferably 10-30 parts of biofilm, 10-30 parts of nanocapsule, 0.5-2 parts of siRNA; most preferably 23 parts of biofilm, 23 parts of nanocapsule, and 1 part of siRNA;
- the mass ratio of the biofilm to the nanocapsule is 1:1.
- the third aspect of the present invention provides a preparation method of the nano delivery system described in the second aspect, and the preparation method may include the following steps:
- step (3) Add the mixed solution obtained in step (2) dropwise to the solvent, ultrasonically stir and centrifuge to remove the supernatant, resuspend the obtained precipitate, and resuspend to obtain a resuspension;
- step (3) Mix the resuspension solution obtained in step (3) with the biofilm, and sonicate to obtain the nano-delivery system.
- the solvent is selected from one or more of the following: PVA aqueous solution, Tween 20 aqueous solution, Tween 80 aqueous solution; preferably PVA aqueous solution, It is more preferably a 1%-10% PVA aqueous solution, and most preferably a 2% PVA aqueous solution.
- the PLGA/DOTAP solution is preferably a dichloromethane solution in which PLGA and DOTAP are mixed according to a ratio, and more preferably PLGA and DOTAP are mixed according to a ratio of 20:1-15.
- the fourth aspect of the present invention provides a medicine for lowering low-density lipoprotein cholesterol, the medicine comprising:
- the nucleic acid or small molecule compound may be siPCSK9
- the fifth aspect of the present invention provides the application of the siRNA described in the first aspect and/or the nano-delivery system described in the second aspect in the preparation of drugs for the treatment of hypercholesterolemia.
- the sixth aspect of the present invention provides a drug nano-delivery system, which can be used to deliver drugs into relevant cells, improve the uptake rate of the drugs by the cells, and the stability of the drugs in the body.
- a drug nano-delivery system is composed of a nanocapsule loaded with a drug and a biomembrane enveloping the nanocapsule.
- the biomembrane is a platelet membrane or a red blood cell membrane.
- the drug may be small interfering RNA, for example, a type of siRNA that can silence the PCSK9 protein, for example, the siRNA described in the first aspect, or CRISPR/Cas9, CRISPR/dCas9, and other shRNAs.
- siRNA for example, a type of siRNA that can silence the PCSK9 protein, for example, the siRNA described in the first aspect, or CRISPR/Cas9, CRISPR/dCas9, and other shRNAs.
- the method of the present invention uses a platelet membrane with good biocompatibility to modify the PLGA/DOTAP core (PDC).
- PDC PLGA/DOTAP core
- This structure gives the nanoparticles the ability to efficiently load siRNA and improves the stability of the particles in the body.
- the DOTAP modified nucleus can promote the successful escape of siRNA from the lysosome, thereby improving the efficiency of silencing. This structure can ultimately achieve safe and efficient down-regulation of LDL-C in the body.
- the method of the present invention has a high transfection effect and can maintain a long-term effect, and has a good therapeutic effect on lowering blood lipids.
- the invention is suitable for gene therapy for lowering blood fat. It is believed that the method of the present invention has broad application prospects in cardiovascular diseases, genetic diseases and the like.
- Figure 1 shows the preparation process of siPcsk9 nanocapsules camouflaged by platelet membrane.
- Figure 1A shows polylactic acid-glycolic acid copolymer (PLGA) and cationic liposome DOTAP encapsulating siPcsk9 to construct nanocapsules
- Figure 1B shows the use of platelet membrane to wrap nanocapsules to prepare siPcsk9 camouflaged by platelet membrane. Nanocapsule process.
- PLGA polylactic acid-glycolic acid copolymer
- DOTAP cationic liposome DOTAP
- Figure 2 shows the cytotoxicity of the nanocapsules in Example 1.
- the ratios of PLGA and DOTAP are 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 300 nM.
- Figure 3 shows the loading efficiency of Example 1 nanocapsules on siRNA.
- the mass ratios of PDC to siRNA are 23/0.1, 23/0.2, 23/0.5, 23/1, 23/2, respectively.
- Figure 4 shows the case of Example 1 where the platelet membrane is wrapped and loaded with siRNA nanocapsules. Among them, the mass ratio of platelet membrane to nanocapsule is 0/1, 1/5, 1/1 and 5/1, respectively.
- FIG. 5 shows the nanoparticles obtained in Example 1.
- Figure 5A shows the plasma obtained after blood collection and centrifugation in mice
- Figure 5B shows the flow cytometric quantitative determination of the proportion of platelets
- Figure 5C shows the analysis of protein components by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP core)
- TPP total platelet protein
- PM platelet membrane
- PDC PLGA/DOTAP core
- Figure 5D shows the platelet membrane before encapsulating the PLGA/DOTAP core
- Figure 5E shows the platelet membrane encapsulating the PDC.
- Figure 6 shows the expression levels of PCSK9, LDL-C, HDL, TG and other indicators in the blood of mice in Test Example 1.
- Figure 6A shows the PCSK9 level
- Figure 6B shows the LDL-C level
- Figure 6C shows Figure 6D shows the TG level
- Figure 6E shows the TBIL level
- Figure 6F shows the ALT level
- Figure 6G shows the AST level.
- Figure 7 shows the in vitro uptake of PPDP in Test Example 2, where Figure 7A shows the cellular uptake levels of PPDP (PM/PDC/siPcsk9) at different concentrations, and Figure 7B shows the uptake levels of PPDP cells at different times Fig. 7C shows the PPDP cell uptake levels of different cell lines, and Fig. 7D shows the PPDP cell fluorescence uptake levels of different cell lines.
- Figure 7A shows the cellular uptake levels of PPDP (PM/PDC/siPcsk9) at different concentrations
- Figure 7B shows the uptake levels of PPDP cells at different times
- Fig. 7C shows the PPDP cell uptake levels of different cell lines
- Fig. 7D shows the PPDP cell fluorescence uptake levels of different cell lines.
- Fig. 8 shows the regulation level of Hepa 1-6PCSK9 in vitro in Test Example 3.
- Fig. 8A shows the lysosomal escape ability of PPDP
- Fig. 8B shows the in vitro mRNA level of PCSK9
- Fig. 8C shows the in vitro protein level of PCSK9.
- Figure 9 shows the hemolysis and cytotoxicity of PPDP in vitro in Test Example 4.
- Figure 9A shows the hemolysis caused by different concentrations of PPDP
- Figure 9B shows the hemolysis rate caused by different concentrations of PPDP
- Figure 9C shows the living and dead cells caused by different concentrations of PPDP under CLSM observation
- Figure 9D shows the different Grouping the hemolysis caused by PPDP
- Fig. 9E shows the hemolysis rate caused by different groups of PPDP
- Fig. 9F shows the status of live and dead cells caused by different groups of PPDP under CLSM observation.
- Figure 10 shows the biodistribution and circulation of PPDP in the body in Test Example 4
- Figure 9A shows the distribution of PPDP in the body over time
- Figure 10B shows the distribution of PPDP in various organs after 72 hours
- Figure 10C shows the circulation of PPDP in the body
- Figure 11 shows the protein expression levels of PCSK9 and LDLR in Test Example 4 in vivo.
- Figure 11A shows the protein level of PCSK9 in vivo
- Figure 11B shows the protein level of LDLR in vivo.
- Figure 12 shows the protein expression levels of LDLR in hepatocytes and Kupffer cells in Test Example 4.
- Fig. 13 shows the histopathological changes of the heart, liver, spleen, lung and kidney of mice after administration in Test Example 4.
- RNA refers to: small interfering RNA.
- PLGA refers to: polylactic acid-glycolic acid copolymer.
- DOTAP refers to: (2,3-dioleoyl-propyl)trimethylammonium chloride.
- PVA polyvinyl alcohol
- RNA refers to: small interfering RNA.
- PBS phosphate buffered saline solution
- PM/PDC/siPcsk9 refers to: the platelet membrane encapsulates the PLGA/DOTAP core containing siPcsk9 nanocapsules.
- LDL-C low-density lipoprotein cholesterol
- HDL-C refers to: high-density lipoprotein cholesterol.
- TG means: triglycerides
- TBIL refers to: total bilirubin.
- ALT refers to: alanine aminotransferase.
- AST means: aspartate aminotransferase.
- PBAE poly- ⁇ -aminoesters
- HA means: hyaluronic acid
- PVA polylactic acid
- PLGA purchased from Sigma-Aldrich, USA;
- DOTAP purchased from Avanti, USA;
- PBS purchased from Lifetechnology, USA;
- Phalloidin purchased from Life, USA;
- TritonX-100 BSA, purchased from Sigma-Aldrich, USA;
- the present invention aims to find a high-efficiency transfection of siPcsk9 nanocapsules based on platelet membrane camouflage to knock down the PCSK9 protein level, so as to achieve a high-efficiency and long-term therapeutic effect of reducing cholesterol levels.
- the present invention adopts the following technical solutions:
- the present invention adopts biocompatibility and degradability polylactic acid-polyglycolic acid copolymer (PLGA) to encapsulate small interfering RNA, and at the same time, cationic liposome DOTAP is used to solve the problem that the adsorption force of PLGA and nucleic acid is small, and the lysosome cannot be completed.
- the platelet membrane from the donor is used to wrap the nanocapsules. Without any other transfection reagents, the small interfering RNA nanocapsule system can be efficiently delivered into the cells, which improves the cellular uptake. Intake rate and transfection efficiency are low in PCSK9 protein level, so as to achieve the therapeutic effect of continuously lowering blood cholesterol level, which has important application value.
- the specific process is shown in Figure 1.
- the present invention exemplifies a gene that can silence the PCSK9 protein:
- Sense chain 5′-UUCCGAAUAAACUCCAGGCT-3′ (SEQ ID NO.1),
- Antisense strand 5'-GCCUGGAGUUUAUUCGGAA-3' (SEQ ID NO. 2).
- This example is used to illustrate the synthesis of nanoparticles of the present invention.
- a method for preparing biofilm-encapsulated nanoparticles using nanomaterials and platelet membranes extracted is constructed.
- Figure 1 shows the preparation process of siPcsk9 nanocapsules camouflaged by platelet membrane.
- Figure 1A shows polylactic acid-glycolic acid copolymer (PLGA) and cationic liposome DOTAP encapsulating siPcsk9 to construct nanocapsules
- Figure 1B shows the use of platelet membrane to wrap nanocapsules to prepare siPcsk9 camouflaged by platelet membrane. Nanocapsule process.
- PLGA polylactic acid-glycolic acid copolymer
- DOTAP cationic liposome DOTAP
- Figure 2 shows the cytotoxicity of the nanocapsules in Example 1.
- the ratios of PLGA and DOTAP are 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 300 nM.
- Figure 3 shows the loading efficiency of Example 1 nanocapsules on siRNA.
- the mass ratios of PDC to siRNA are 23/0.1, 23/0.2, 23/0.5, 23/1, 23/2, respectively.
- the efficiency of loading siRNA on PLGA/DOTAP nanoparticles can be close to 100%.
- FIG. 4 shows the case of Example 1 where the platelet membrane is wrapped and loaded with siRNA nanocapsules.
- the mass ratios of platelet membrane protein to nanocapsules are 0/1, 1/5, 1/1, and 5/1, respectively.
- TEM Transmission electron microscopy
- PPDP PM/PDC/siPcsk9, referred to as PPDP, which is PLGA/DOTAP/siPCSK9 nanoparticles wrapped in the final synthesized platelet membrane
- the effect is the best.
- a smaller or larger PPDP coating quality ratio will cause the coating to be incomplete or greatly increase the size of the coating.
- FIG. 5 shows the nanoparticles obtained in Example 1.
- Figure 5A shows the plasma obtained after blood collection and centrifugation in mice
- Figure 5B shows the flow cytometric quantitative determination of the proportion of platelets
- Figure 5C shows the analysis of protein components by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP nucleus)
- TPP total platelet protein
- PM platelet membrane
- PDC PLGA/DOTAP nucleus
- Figure 5D shows the platelet membrane before encapsulating the PLGA/DOTAP nucleus
- Figure 5E shows the platelet membrane encapsulating the PLGA/DOTAP nucleus.
- Whole blood was collected from mice, and platelets were extracted from serum (Figure 5A).
- This test example is used to illustrate the treatment of hypercholesterolemia by the nanoparticles of the present invention.
- the nanoparticles PM/PDC/siPcsk9 were synthesized to treat hypercholesterolemia.
- PM/PDC/siPcsk9 is the experimental group
- Lipo2000/siPcsk9 is the positive control group
- siLuciferase is used as the internal control, according to the preparation method of PM/PDC/siPcsk9, after loading siLuciferase, PM/PDC/siLuc is formed
- siPcsk9 is the unmodified drug control
- PM/PDC is the no-load control group without drugs
- PBS is the blank control group.
- Figure 6 compares with other treatment methods.
- Figure 6 shows the changes of biochemical factors in mice.
- the experimental group has a stronger silencing effect and significantly reduces the level of low-density lipoprotein-cholesterol (LDL-C).
- LDL-C low-density lipoprotein-cholesterol
- siRNA delivery using this optimized material will result in an effective down-regulation of LDL-C (approximately 56%), previous studies have also shown that the use of lipid nanoparticles can lead to impaired liver function.
- Our work contributes to the safe delivery of therapeutic siRNA based on PM camouflage nanoparticles without significant impact on liver function.
- This test example is used to illustrate the in vitro transfection experiment of the nanoparticle hepatocytes of the present invention.
- nanoparticles PM/PDC/siPcsk9 synthesized by the method in Example 1 were transfected in vitro to mouse-derived liver cancer cells (Hepa1-6).
- CLSM Confocal laser microscope
- the platelet membrane is coated on the PDP to form Cy5-PPDP, as described previously. Hepa1-6 cells were seeded in a confocal culture dish with a density of 10 ⁇ 5 cells/well.
- the medium was removed, the cells were gently washed with PBS 3 times, and fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice with PBS; ruptured with 0.1% Triton for 1 min, PBS Wash twice; then block with 3% BSA for 30 min, wash twice with PBS; then stain the cytoskeleton with Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, USA), and then use DAPI (4,6-diamino-2) -Phenylindole) dye counterstain the nucleus, and wash repeatedly with PBS.
- Cell imaging was performed on CLSM (LSM 880, Zeiss).
- Nanoparticles were added at the last 150 nM Cy5-siPcsk9 and incubated for 1, 3, 6, 9 and 12h ( Figure 7B). Then, the cells were stained with FITC-Phalloidin and Hoechst in the same way. Cell lines of alveolar macrophages, mononuclear macrophages, and umbilical cord mesenchymal stem cells were cultivated to prepare Cy5-PPDP, and different nano-preparations with a Cy5-siPcsk9 concentration of 150 nM were incubated with the cells for 9 hours at 37°C. The medium was removed, and the cells were washed three times with PBS.
- Cy5-siPcsk9 is used to evaluate the endoplasmic/lysosome escape of CLSM.
- Nanodrop measures RNA concentration.
- RNA for reverse transcription Take total RNA for reverse transcription, use the stem-loop primers synthesized by Ruibo Company, use TaKaRa Reverse Transcription Kit (RR037A, Japan) for reverse transcription, and use SYBR reagent (Cat.172-5124, BioRad) for reverse transcription. Perform quantitative PCR analysis. The operation steps are the same as the instructions, and the Light cycler 480 system is used for operation.
- ⁇ Ct (Ct target gene-Ct internal reference gene)
- ⁇ Ct ⁇ Ct processed sample- ⁇ Ct control sample
- 2- ⁇ Ct 2- ⁇ Ct
- siRNA sequence is as follows:
- siPcsk9 siRNA targeting Pcsk9mRNA
- siLuc siLuc (siRNA targeting luciferase mRNA):
- the primer sequences of the target gene PCSK9 and the internal reference gene GAPDH are as follows:
- GAPDH Forward TGCACCACCACCAACTGCTTAGC, (SEQ ID NO.7)
- GAPDH Reverse GGCATGGACTGTGGTCATGAG (SEQ ID NO. 8).
- Glue preparation Glue is prepared according to the size of the protein, 10% rapid glue (BIO-RAD) is prepared and inserted into the comb, and it is allowed to stand at room temperature until solidified.
- BIO-RAD 10% rapid glue
- Transfer membrane After the electrophoresis is completed, take out the glue, discard the excess glue, cut the PVDF membrane to the same size as the glue, and soak the membrane in methanol for 5 minutes to activate the membrane. Put the sponge, filter paper, gel, PVDF membrane, filter paper, sponge in the transfer box in sequence to make a "sandwich", then drive away the bubbles, place it in the electrophoresis tank after clamping, and add an appropriate amount of freshly prepared Transfer membrane solution, place the electrophoresis tank on ice, and transfer membrane at a constant pressure of 100V for 2 hours.
- Exposure Mix the solutions A and B in the developing reagent at a ratio of 1:1, and add them evenly to the surface of the membrane. After incubating for about 1 min, place in the exposure machine Amersham Imager 600 system (GE, USA) for automatic exposure, and save the data. The results are shown in Figure 8C.
- 0.50 mL of red blood cell suspension was mixed with 0.50 mL of analogs, and 0.50 mL of PPDP, PPDL, PDP and Lipo2000/siPcsk9 were added to 0.50 mL of red blood cell suspension.
- the added PBS and 1.0% Triton X-100 solution were used as negative control and positive control, respectively.
- the sample was incubated at 37°C for 1 h, centrifuged at 10 ⁇ 4 rpm for 10 min, and then 0.10 ml of supernatant was added to a 96-well plate. The absorbance of hemoglobin at 540nm was recorded.
- hemolysis rate% [(At-Anc)/(Apc-Anc)] ⁇ 100%.
- Anc the absorbance of the negative control
- Apc the absorbance of the positive control
- the medium was replaced with freshly prepared complete DMEM containing PPDP, and the final siRNA concentration ranged from 18.75 to 300 nM.
- Cells treated with PBS or 1% Triton X-100 were used as negative control or positive control.
- the cells were stained with a live/dead viability/cytotoxicity kit and imaged with CLSM.
- the cytotoxicity of other nanoparticles was also evaluated, such as Lipo2000/siPCSK9, PPDL, and PDP, where the siRNA concentration is equivalent to 150nM. See Figure 9 for the result.
- PDP and PPDP The purchased 10 female C57BL/6 (weight 18-20g) were randomly divided into 2 groups (PDP and PPDP). PDP and PPDP are synthesized in the above manner, and both are labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate (DiD), which stimulates The wavelength and emission wavelength are 644/665nm. Observe whether the C57BL/6 mice have hunched back, sneezing, convulsions and other abnormal conditions for a week.
- DID 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate
- Processing the blood sample Mix the sample with 0.1 mM EDTA and perform three repeated freezing and thawing processes to ensure that the cells are completely destroyed and the nanoparticles are released. Then, add 500 ⁇ L of dichloromethane and immediately vortex for 30s, centrifuge at 20000g at 4°C for 30min, and transfer the supernatant to a new test tube. The blank blood sample treatment is consistent with this method. Subsequently, the fluorescence intensity was measured with an RF-6000 fluorescence spectrophotometer (Shimadzu, Japan).
- DiD standard curve DiD blood solution was diluted to 20, 15, 10, 7.5, 5, 0.75 ⁇ g/mL, and then the fluorescence intensity was measured with an RF-6000 fluorescence spectrophotometer (Shimadzu, Japan).
- Organ treatment Put the organs of each mouse in each group into 2mL EP tubes, add 1mL water and homogenize, centrifuge at 20000g at 4°C for 30min, transfer the supernatant to a new EP tube, and use RF-6000 fluorescence spectrophotometry A meter (Shimadzu, Japan) detects the fluorescence intensity. See Figure 10 for the results.
- 4.6 is the expression levels of PCSK9 and LDLR in the liver of mice, and the specific steps are the same as the Western blot analysis in Figure 11.
- Tail vein administration is administered every 5 days.
- Figure 11 We evaluated the effects of PPDPs in vivo. WB analysis showed that after PPDP treatment, the level of PCSK9 in liver tissue was effectively suppressed (Figure 11A). Consistent with this, the level of low-density lipoprotein receptors in liver tissue was high. In the control group ( Figure 11B).
- 4.7 is the LDLR western blot analysis of isolated hepatocytes and Kupffer cells in mice. The specific steps are as follows:
- mice were anesthetized, and the liver was perfused through the portal vein with 20 mL 0.5 mM EDTA-free Hank balanced salt solution.
- Figure 9 To evaluate the biocompatibility, we analyzed the hemolysis and cytotoxicity of PPDP. Neither PPDP nor uncoated nanoparticles caused any significant hemolytic toxicity at working concentrations ( Figures 9A and 9B), which can be confirmed by the live/dead assay of CLSM ( Figure 9C). However, the use of commercially available Lipofectamine 2000 will have significant hemolysis ( Figure 9D and 9E), which indicates that PPDP causes lower hemolysis and cytotoxicity, which is essential for in vivo applications. In vitro CLSM also shows that the commercially available Lipofectamine 2000 phase It caused higher cytotoxicity than PPDP ( Figure 9F).
- Figure 10 After injection of PPDPs containing Cy5-siPcsk9 into mice, the imaging system was used to track the distribution of nanoparticles in the body. We found that after 24h, the cumulative amount of PPDP in the liver was greater than that of naked nanoparticles (Figure 10A). In vitro imaging of major organs showed that the PPDP retained in the liver was about 20 times higher than that of other organs ( Figure 10B). In addition, the liver of PPDP-administered mice increased twice as much as that of PDP mice ( Figure 10B). Interestingly, PPDP showed that the circulation time of mice was much longer than that of PDP ( Figure 10C).
- Figure 11 We evaluated the effects of PPDPs in vivo. WB analysis showed that after PPDP treatment, the level of PCSK9 in liver tissue was effectively suppressed (Figure 11A). Consistent with this, the level of low-density lipoprotein receptors in liver tissue was high. In the control group ( Figure 11B).
- Figure 12 In order to clarify whether PPDP is taken up by immune cells such as liver macrophages, Kupffer cells, we analyzed LDLR in two cell lines with WB and found that LDLR in liver cells increased significantly after PPDP administration ( Figure 12). But the LDLR of Kupffer cells did not change much.
- HE Hematoxylin-eosin
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Abstract
siRNA capable of silencing a Pcsk9 protein. A sense strand amino acid sequence of the siRNA is 5'-UUCCGAAUAAACUCCAGGC-3', and an anti-sense strand amino acid sequence is 5'-GCCUGGAGUUUAUUCGGAA-3'. Further provided are a nano delivery system capable of silencing the Pcsk9 protein, a preparation method therefor, and an application thereof. The method has a higher transfection effect, can maintain long-term action, has a better treatment effect of reducing blood lipid, and is applicable to gene therapy for reducing blood lipid.
Description
本发明属于医药领域,具体涉及一种能够沉默Pcsk9蛋白的siRNA、其纳米递送系统及应用。The invention belongs to the field of medicine, and specifically relates to a siRNA capable of silencing Pcsk9 protein, a nano-delivery system and application thereof.
随着人们生活水平的不断提高和饮食结构及生活方式的变化,高血糖、高胆固醇以及高甘油三酯类的病例呈逐年上升趋势,而这又通常会导致糖尿病、动脉硬化、心脑血管等疾病的发生。2002年世界卫生组织(WHO)就已公布,人类健康十大危险因素中胆固醇过高疾患位居第八位。With the continuous improvement of people's living standards and changes in dietary structure and lifestyle, cases of high blood sugar, high cholesterol and high triglycerides are on the rise year by year, which in turn usually leads to diabetes, arteriosclerosis, cardiovascular and cerebrovascular diseases, etc. The occurrence of the disease. In 2002, the World Health Organization (WHO) announced that among the top ten risk factors for human health, hypercholesterolemia ranked eighth.
常见的家族性高胆固醇血症主要是由载脂蛋白(APOB),低密度脂蛋白受体(LDL-R),与前蛋白转化酶枯草杆菌转化酶(PCSK9)基因突变引起的,PCSK9主要在肝脏分布,分泌到细胞外的PCSK9可以与LDL-R结合,靶向到溶酶体中降解。当细胞表面的LDL受体数量减少,血液中总体低密度脂蛋白胆固醇(LDL-C)水平则随之提高,研究表明PCSK9的功能丧失突变导致LDL胆固醇水平降低,因此有望成为控制高胆固醇血症及其并发症的潜在有意义的治疗靶点。Common familial hypercholesterolemia is mainly caused by apolipoprotein (APOB), low-density lipoprotein receptor (LDL-R), and proprotein convertase Bacillus subtilis convertase (PCSK9) gene mutations. PCSK9 is mainly caused by It is distributed in the liver, and PCSK9 secreted outside the cell can bind to LDL-R and target to the lysosome for degradation. When the number of LDL receptors on the cell surface decreases, the overall low-density lipoprotein cholesterol (LDL-C) level in the blood increases accordingly. Studies have shown that the loss of function mutation of PCSK9 leads to a decrease in LDL cholesterol levels, so it is expected to be a control for hypercholesterolemia Potentially meaningful therapeutic targets for its complications.
目前的临床治疗手段主要还是以他汀类药物为主,研究表明对他汀类药物不耐受患者,可引起肌肉病等不良反应,如肌肉痛、横纹肌溶解。虽然目前中国已有依洛尤单抗(evolocumab,商品名瑞百安)上市,但是由于其造价昂贵,且PCSK9单抗经网状内皮系统代谢每隔2-4周就需要注射。研究表明,小干扰RNA(siRNA)可特异性沉默Pcsk9基因,从而抑制其蛋白表达,降低低密度脂蛋白。但用于沉默Pcsk9的siRNA不可避免地遭遇到基因递送面临的难题:1)未做修饰的siRNA在核酸酶的作用下易降解;2)对siRNA修饰,结构复杂,难度高,且有可能降低siRNA的特异性;3)siRNA在体内随机分布,降低了其在目标部位的蓄积;4)siRNA由于骨架中磷酸残基的存在,呈现强负电性,难以被携带相同表面电荷的细胞摄入。siRNA的递送需借助于高效的转导系统,当前应用最广泛的是病毒载体。病毒载体可高效递送发夹RNA,有效沉默Pcsk9,但其具有潜在的免疫原性、致瘤性和致畸性,引起人们对其生物安全性的担忧。非病毒载体主要为脂质体和高分子材料。但当前的非病毒载体也遇到包括毒性大、稳定性差及转染效率低等问题。人们往往在递送效率和安全性方面难以作出抉择。由于具有潜在的免疫原性、致瘤性和致畸性,目前的病毒载体被禁止用于临床治疗,这极大地限制了它们的应用。非病毒载体作为潜力 巨大的系统,弥补了病毒载体的不足,具有很大的开发前景。但当前的非病毒载体也遇到包括毒性大、稳定性差及转染效率低等问题。具体到高胆固醇血症的治疗,人们需要将siRNA高效地递送到肝脏部位,被肝细胞摄入,并成功逃逸溶酶体,从而产生基因沉默效应。如何有效地解决非病毒载体在siRNA递送中问题,成为研究的热点。因此,开发一种高效的沉默Pcsk9的递送体系,将为长效治疗高胆固醇血症提供有效的手段。The current clinical treatment is mainly based on statins. Studies have shown that patients who are intolerant to statins can cause adverse reactions such as muscle pain and rhabdomyolysis. Although evolocumab (trade name Ruibaian) is currently on the market in China, due to its high cost and the metabolism of PCSK9 monoclonal antibody via the reticuloendothelial system, injections are required every 2-4 weeks. Studies have shown that small interfering RNA (siRNA) can specifically silence the Pcsk9 gene, thereby inhibiting its protein expression and reducing low-density lipoprotein. However, the siRNA used to silence Pcsk9 inevitably encounters the problems faced by gene delivery: 1) Unmodified siRNA is easily degraded under the action of nuclease; 2) Modification of siRNA is complicated in structure, difficult, and may be reduced. The specificity of siRNA; 3) siRNA is randomly distributed in the body, reducing its accumulation at the target site; 4) siRNA is strongly negative due to the presence of phosphate residues in the backbone, and it is difficult to be taken up by cells with the same surface charge. The delivery of siRNA requires the help of an efficient transduction system, and viral vectors are currently the most widely used. Viral vectors can efficiently deliver hairpin RNA and effectively silence Pcsk9, but it has potential immunogenicity, tumorigenicity and teratogenicity, which raises concerns about its biological safety. Non-viral vectors are mainly liposomes and polymer materials. However, current non-viral vectors also encounter problems including high toxicity, poor stability, and low transfection efficiency. It is often difficult for people to make a choice between delivery efficiency and safety. Due to potential immunogenicity, tumorigenicity and teratogenicity, current viral vectors are forbidden for clinical treatment, which greatly limits their applications. As a system with great potential, non-viral vectors make up for the deficiencies of viral vectors and have great development prospects. However, current non-viral vectors also encounter problems including high toxicity, poor stability, and low transfection efficiency. Specifically for the treatment of hypercholesterolemia, people need to efficiently deliver siRNA to the liver, be taken up by hepatocytes, and successfully escape the lysosome, thereby producing a gene silencing effect. How to effectively solve the problem of non-viral vectors in siRNA delivery has become a research hotspot. Therefore, the development of an efficient delivery system for silencing Pcsk9 will provide an effective means for long-term treatment of hypercholesterolemia.
发明内容Summary of the invention
因此,本发明的目的在于克服现有技术中的缺陷,寻找一种基于生物膜伪装(例如血小板膜)伪装的siPcsk9纳米囊高效转染,下调PCSK9蛋白水平,从而达到高效长久降低胆固醇水平的治疗效果。Therefore, the purpose of the present invention is to overcome the shortcomings in the prior art and find a siPcsk9 nanocapsule based on biomembrane camouflage (such as platelet membrane) to efficiently transfect siPcsk9 nanocapsules to reduce the level of PCSK9 protein, so as to achieve an efficient and long-term treatment of lowering cholesterol levels. effect.
为实现上述目的,本发明的第一方面提供了一种能够沉默PCSK9蛋白的siRNA,所述siRNA的正义链氨基酸序列为5′-UUCCGAAUAAACUCCAGGC-3′,反义链氨基酸序列为5′-GCCUGGAGUUUAUUCGGAA-3′。In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a siRNA capable of silencing PCSK9 protein. The sense chain amino acid sequence of the siRNA is 5′-UUCCGAAUAAACUCCAGGC-3′, and the antisense chain amino acid sequence is 5′-GCCUGGAGUUUAUUCGGAA- 3'.
优选地,所述siRNA正义链和/或反义链的3′端还修饰有dTdT。Preferably, the 3'end of the siRNA sense strand and/or antisense strand is further modified with dTdT.
本发明的第二方面提供了一种能够沉默PCSK9蛋白的纳米递送系统,所述纳米系统为生物膜伪装的包裹有能够沉默PCSK9蛋白的siRNA的纳米囊。The second aspect of the present invention provides a nano-delivery system capable of silencing PCSK9 protein. The nano-system is a nanocapsule camouflaged by a biological membrane and wrapped with siRNA capable of silencing PCSK9 protein.
其中,能够沉默PCSK9蛋白的siRNA,例如可以是第一方面所述的siRNA。Among them, the siRNA capable of silencing the PCSK9 protein may be, for example, the siRNA described in the first aspect.
根据本发明第二方面的纳米递送系统,其中,所述生物膜选自以下一种或多种:血小板膜,红细胞膜,骨髓间充质干细胞,脐带间充质干细胞,巨噬细胞;优选为血小板膜。The nano-delivery system according to the second aspect of the present invention, wherein the biofilm is selected from one or more of the following: platelet membrane, red blood cell membrane, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, macrophages; preferably Platelet membrane.
根据本发明第二方面的纳米递送系统,其中,所述纳米囊的材料选自以下一种或多种:PLGA、DOTAP、聚氨基酯、透明质酸、壳聚糖、聚乳酸;优选为PLGA和DOTAP。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 the following: PLGA, DOTAP, polyurethane, hyaluronic acid, chitosan, polylactic acid; preferably PLGA And DOTAP.
根据本发明第二方面的纳米递送系统,其中,所述纳米囊中,PLGA与DOTAP的质量比为20:1~20:11,优选为20:1~20:5,最优选为20:3。根据本发明第二方面的纳米递送系统,其中,所述生物膜、纳米囊与siRNA的质量份数为:生物膜1~50份,纳米囊1~50份,siRNA0.1~10份;优选为生物膜10~30份,纳米囊10~30份,siRNA 0.5~2份;最优选为生物膜23份,纳米囊23份,siRNA 1份;The nano delivery system according to the second aspect of the present invention, wherein the mass ratio of PLGA to DOTAP in the nanocapsule is 20:1-20:11, preferably 20:1-20:5, most preferably 20:3 . The nano-delivery system according to the second aspect of the present invention, wherein the mass parts of the biofilm, nanocapsules and siRNA are: 1-50 parts of biofilm, 1-50 parts of nanocapsules, and 0.1-10 parts of siRNA; preferably 10-30 parts of biofilm, 10-30 parts of nanocapsule, 0.5-2 parts of siRNA; most preferably 23 parts of biofilm, 23 parts of nanocapsule, and 1 part of siRNA;
优选地,当纳米囊材料选自PLGA与DOTAP时,所述生物膜与所述纳米囊的质量比为1:1。Preferably, when the nanocapsule material is selected from PLGA and DOTAP, the mass ratio of the biofilm to the nanocapsule is 1:1.
本发明的第三方面提供了第二方面所述的纳米递送系统的制备方法,该制备方法可以包括以下步骤:The third aspect of the present invention provides a preparation method of the nano delivery system described in the second aspect, and the preparation method may include the following steps:
(1)制备siRNA溶液;(1) Prepare siRNA solution;
(2)将步骤(1)制备的siRNA溶液滴加入PLGA/DOTAP溶液中;(2) Drop the siRNA solution prepared in step (1) into the PLGA/DOTAP solution;
(3)将步骤(2)所得混合溶液滴加入溶剂中,超声搅拌离心去上清,重悬所得沉淀,重悬,得重悬液;(3) Add the mixed solution obtained in step (2) dropwise to the solvent, ultrasonically stir and centrifuge to remove the supernatant, resuspend the obtained precipitate, and resuspend to obtain a resuspension;
(4)将步骤(3)所得重悬液与生物膜混合,超声,得到所述纳米递送系统。(4) Mix the resuspension solution obtained in step (3) with the biofilm, and sonicate to obtain the nano-delivery system.
根据本发明第三方面的制备方法,其中,所述步骤(3)中,所述溶剂选自以下一种或多种:PVA水溶液、吐温20水溶液、吐温80水溶液;优选为PVA水溶液,更优选为1%-10%的PVA水溶液,最优选为2%PVA水溶液。其中,PLGA/DOTAP溶液优选为PLGA和DOTAP按照比例混合的二氯甲烷溶液,进一步优选为PLGA和DOTAP按照20:1-15的比例混合。According to the preparation method of the third aspect of the present invention, in the step (3), the solvent is selected from one or more of the following: PVA aqueous solution, Tween 20 aqueous solution, Tween 80 aqueous solution; preferably PVA aqueous solution, It is more preferably a 1%-10% PVA aqueous solution, and most preferably a 2% PVA aqueous solution. Among them, the PLGA/DOTAP solution is preferably a dichloromethane solution in which PLGA and DOTAP are mixed according to a ratio, and more preferably PLGA and DOTAP are mixed according to a ratio of 20:1-15.
本发明的第四方面提供了一种下调低密度脂蛋白胆固醇的药物,所述药物包括:The fourth aspect of the present invention provides a medicine for lowering low-density lipoprotein cholesterol, the medicine comprising:
核酸或小分子化合物;和/或Nucleic acid or small molecule compound; and/or
第二方面所述的纳米递送系统。The nano delivery system described in the second aspect.
所述核酸或者小分子化合物可以是siPCSK9The nucleic acid or small molecule compound may be siPCSK9
本发明的第五方面提供了第一方面所述的siRNA和/或第二方面所述的纳米递送系统在制备用于治疗高胆固醇血症的药物中的应用。The fifth aspect of the present invention provides the application of the siRNA described in the first aspect and/or the nano-delivery system described in the second aspect in the preparation of drugs for the treatment of hypercholesterolemia.
本发明的第六方面提供了一种药物纳米递送系统,该纳米递送系统可以用于递送药物到相关细胞内,提高细胞对药物的摄入率,以及药物在体内的稳定性。The sixth aspect of the present invention provides a drug nano-delivery system, which can be used to deliver drugs into relevant cells, improve the uptake rate of the drugs by the cells, and the stability of the drugs in the body.
一种药物纳米递送系统,其由包括负载有药物的纳米囊,以及将纳米囊包裹的生物膜构成,所述生物膜为血小板膜或红细胞膜。A drug nano-delivery system is composed of a nanocapsule loaded with a drug and a biomembrane enveloping the nanocapsule. The biomembrane is a platelet membrane or a red blood cell membrane.
所述药物可以是小干扰RNA,例如是能够沉默PCSK9蛋白的一类siRNA,例如可以是第一方面所述的siRNA,也可以是CRISPR/Cas9,CRISPR/dCas9,及其他shRNA。The drug may be small interfering RNA, for example, a type of siRNA that can silence the PCSK9 protein, for example, the siRNA described in the first aspect, or CRISPR/Cas9, CRISPR/dCas9, and other shRNAs.
本发明所述方法采用生物相容性良好的血小板膜修饰PLGA/DOTAP核(PDC)。这一结构赋予纳米颗粒具有高效负载siRNA的能力,提高了颗粒在体内的稳定性。而DOTAP修饰的核,能促进siRNA从溶酶体中成功逃逸,从而提高沉默效率。这一结构最终可实现在体内安全高效地下调LDL-C。The method of the present invention uses a platelet membrane with good biocompatibility to modify the PLGA/DOTAP core (PDC). This structure gives the nanoparticles the ability to efficiently load siRNA and improves the stability of the particles in the body. The DOTAP modified nucleus can promote the successful escape of siRNA from the lysosome, thereby improving the efficiency of silencing. This structure can ultimately achieve safe and efficient down-regulation of LDL-C in the body.
本发明的递送系统可以具有但不限于以下有益效果:The delivery system of the present invention can have but not limited to the following beneficial effects:
本发明所述方法具有较高的转染效果并能维持长时间作用,对于降血脂具有较好的治疗效果。本发明适用于降血脂的基因治疗。相信本发明的方法在心血管疾病、遗传性疾病等方面具有广泛的应用前景。The method of the present invention has a high transfection effect and can maintain a long-term effect, and has a good therapeutic effect on lowering blood lipids. The invention is suitable for gene therapy for lowering blood fat. It is believed that the method of the present invention has broad application prospects in cardiovascular diseases, genetic diseases and the like.
以下,结合附图来详细说明本发明的实施方案,其中:Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:
图1示出了血小板膜伪装的siPcsk9纳米囊制备过程。其中,图1A示出了聚乳酸-羟基乙酸共聚物(PLGA)和阳离子脂质体DOTAP包裹siPcsk9,构建纳米囊,图1B示出了采用血小板膜包裹纳米囊,制备血小板膜血小板膜伪装的siPcsk9纳米囊过程。Figure 1 shows the preparation process of siPcsk9 nanocapsules camouflaged by platelet membrane. Among them, Figure 1A shows polylactic acid-glycolic acid copolymer (PLGA) and cationic liposome DOTAP encapsulating siPcsk9 to construct nanocapsules, and Figure 1B shows the use of platelet membrane to wrap nanocapsules to prepare siPcsk9 camouflaged by platelet membrane. Nanocapsule process.
图2示出了实施例1纳米囊的细胞毒性情况。PDC中,PLGA与DOTAP的比例分别为20/0,20/1,20/3,20/5,20/7,20/9和20/11。siRNA的浓度分别是0,9.38,18.75,37.5,75,150,和300nM。Figure 2 shows the cytotoxicity of the nanocapsules in Example 1. In PDC, the ratios of PLGA and DOTAP are 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 300 nM.
图3示出了实施例1纳米囊对siRNA的负载效率。PDC/siRNA中,PDC与siRNA的质量比分别为23/0.1,23/0.2,23/0.5,23/1,23/2。Figure 3 shows the loading efficiency of Example 1 nanocapsules on siRNA. In PDC/siRNA, the mass ratios of PDC to siRNA are 23/0.1, 23/0.2, 23/0.5, 23/1, 23/2, respectively.
图4示出了实施例1血小板膜包裹负载siRNA纳米囊的情况。其中,血小板膜与纳米囊的质量比分别为0/1,1/5,1/1和5/1。Figure 4 shows the case of Example 1 where the platelet membrane is wrapped and loaded with siRNA nanocapsules. Among them, the mass ratio of platelet membrane to nanocapsule is 0/1, 1/5, 1/1 and 5/1, respectively.
图5示出了实施例1所得到的纳米颗粒。其中图5A示出了小鼠采血离心后得到的血浆,图5B示出了流式定量测定血小板比例;图5C示出了十二烷基磺酸钠-聚丙烯酰胺凝胶电泳分析蛋白组分(TPP,血小板总蛋白;PM,血小板膜;PDC,PLGA/DOTAP核);图5D示出了血小板膜包裹PLGA/DOTAP核之前,图5E示出了血小板膜包裹PDC。FIG. 5 shows the nanoparticles obtained in Example 1. Figure 5A shows the plasma obtained after blood collection and centrifugation in mice, Figure 5B shows the flow cytometric quantitative determination of the proportion of platelets; Figure 5C shows the analysis of protein components by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP core); Figure 5D shows the platelet membrane before encapsulating the PLGA/DOTAP core, and Figure 5E shows the platelet membrane encapsulating the PDC.
图6示出了试验例1中小鼠血液中PCSK9、LDL-C、HDL、TG等指标的表达水平,其中图6A示出了PCSK9水平,图6B示出了LDL-C水平,图6C示出了HDL-C水平,图6D示出了TG水平,图6E示出了TBIL水平,图6F是示出了ALT水平,图6G示出了AST水平。Figure 6 shows the expression levels of PCSK9, LDL-C, HDL, TG and other indicators in the blood of mice in Test Example 1. Figure 6A shows the PCSK9 level, Figure 6B shows the LDL-C level, and Figure 6C shows Figure 6D shows the TG level, Figure 6E shows the TBIL level, Figure 6F shows the ALT level, and Figure 6G shows the AST level.
图7示出了试验例2中PPDP在体外的摄入情况,其中图7A示出了不同浓度的PPDP(PM/PDC/siPcsk9)细胞摄取水平,图7B示出了不同时间的PPDP细胞摄取水平,图7C示出了不同细胞系的PPDP细胞摄取水平,图7D示出了不同细胞系的PPDP细胞荧光摄取水平。Figure 7 shows the in vitro uptake of PPDP in Test Example 2, where Figure 7A shows the cellular uptake levels of PPDP (PM/PDC/siPcsk9) at different concentrations, and Figure 7B shows the uptake levels of PPDP cells at different times Fig. 7C shows the PPDP cell uptake levels of different cell lines, and Fig. 7D shows the PPDP cell fluorescence uptake levels of different cell lines.
图8示出了试验例3中体外Hepa 1-6PCSK9调节水平。图8A示出了PPDP的溶酶体逃逸能力,图8B示出了PCSK9体外mRNA水平,图8C示出了PCSK9体外蛋白水平。Fig. 8 shows the regulation level of Hepa 1-6PCSK9 in vitro in Test Example 3. Fig. 8A shows the lysosomal escape ability of PPDP, Fig. 8B shows the in vitro mRNA level of PCSK9, and Fig. 8C shows the in vitro protein level of PCSK9.
图9示出了试验例4中PPDP在体外的溶血与细胞毒性情况。图9A示出了不同浓度PPDP引起的溶血情况,图9B示出了不同浓度PPDP引起的溶血率,图9C示出了CLSM观察下不同浓度PPDP引起的活死细胞情况,图9D示出了不同分组PPDP引起的溶血情况,图9E示出了不同分组PPDP引起的溶血率,图9F示出了CLSM观察下不同分组PPDP引起的活死细胞情况。Figure 9 shows the hemolysis and cytotoxicity of PPDP in vitro in Test Example 4. Figure 9A shows the hemolysis caused by different concentrations of PPDP, Figure 9B shows the hemolysis rate caused by different concentrations of PPDP, Figure 9C shows the living and dead cells caused by different concentrations of PPDP under CLSM observation, and Figure 9D shows the different Grouping the hemolysis caused by PPDP, Fig. 9E shows the hemolysis rate caused by different groups of PPDP, and Fig. 9F shows the status of live and dead cells caused by different groups of PPDP under CLSM observation.
图10示出了试验例4中PPDP在体内的生物分布和循环,图9A示出了PPDP随着不同时间变化在体内的分布变化,图10B示出了PPDP给药72h后在各个器官的分布量,图 10C示出了PPDP在体内的循环性Figure 10 shows the biodistribution and circulation of PPDP in the body in Test Example 4, Figure 9A shows the distribution of PPDP in the body over time, and Figure 10B shows the distribution of PPDP in various organs after 72 hours. Figure 10C shows the circulation of PPDP in the body
图11示出了试验例4中PCSK9与LDLR在体内的蛋白表达水平。图11A示出了PCSK9体内蛋白水平,图11B示出了LDLR体内蛋白水平。Figure 11 shows the protein expression levels of PCSK9 and LDLR in Test Example 4 in vivo. Figure 11A shows the protein level of PCSK9 in vivo, and Figure 11B shows the protein level of LDLR in vivo.
图12示出了试验例4中LDLR在肝实质细胞与Kupffer细胞的蛋白表达水平。Figure 12 shows the protein expression levels of LDLR in hepatocytes and Kupffer cells in Test Example 4.
图13示出了试验例4中给药后小鼠心肝脾肺肾的组织病理变化情况。Fig. 13 shows the histopathological changes of the heart, liver, spleen, lung and kidney of mice after administration in Test Example 4.
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细具体地说明之用,而不应理解为用于以任何形式限制本发明。The present invention will be further explained by specific examples below. However, it should be understood that these examples are only used for more detailed and specific description, and should not be understood as limiting the present invention in any form.
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚,在上下文中,如果未特别说明,本发明所用材料和操作方法是本领域公知的。This section gives a general description of the materials and test methods used in the test of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described here in as much detail as possible. It is clear to those skilled in the art that, in the context, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art.
在具体阐述本发明的技术方案之前,定义本文中所使用的术语如下:Before describing the technical solution of the present invention in detail, the terms used in this article are defined as follows:
术语“siRNA”是指:小干扰RNA。The term "siRNA" refers to: small interfering RNA.
术语“PLGA”是指:聚乳酸-羟基乙酸共聚物。The term "PLGA" refers to: polylactic acid-glycolic acid copolymer.
术语“DOTAP”是指:(2,3-二油酰基-丙基)三甲基氯化铵。The term "DOTAP" refers to: (2,3-dioleoyl-propyl)trimethylammonium chloride.
术语“PVA”是指:聚乙烯醇。The term "PVA" means: polyvinyl alcohol.
术语“siRNA”是指:小干扰RNA。The term "siRNA" refers to: small interfering RNA.
术语“PBS”是指:磷酸缓冲盐溶液。The term "PBS" means: phosphate buffered saline solution.
术语“PM/PDC/siPcsk9”是指:血小板膜包裹PLGA/DOTAP核含siPcsk9的纳米囊。The term "PM/PDC/siPcsk9" refers to: the platelet membrane encapsulates the PLGA/DOTAP core containing siPcsk9 nanocapsules.
术语“LDL-C”是指:低密度脂蛋白胆固醇。The term "LDL-C" refers to: low-density lipoprotein cholesterol.
术语“HDL-C”是指:高密度脂蛋白胆固醇。The term "HDL-C" refers to: high-density lipoprotein cholesterol.
术语“TG”是指:甘油三酯。The term "TG" means: triglycerides.
术语“TBIL”是指:总胆红素。The term "TBIL" refers to: total bilirubin.
术语“ALT”是指:谷丙转氨酶。The term "ALT" refers to: alanine aminotransferase.
术语“AST”是指:谷草转氨酶。The term "AST" means: aspartate aminotransferase.
术语“PBAE”是指:聚氨基酯poly-β-amino esters。The term "PBAE" refers to: poly-β-aminoesters.
术语“HA”是指:透明质酸。The term "HA" means: hyaluronic acid.
术语“PLA”是指:聚乳酸。The term "PLA" means: polylactic acid.
以下实施例中使用的试剂和仪器如下:The reagents and instruments used in the following examples are as follows:
试剂:Reagents:
无核酶水,siPcsk9干粉,购自Takara日本;Nuclease-free water, siPcsk9 dry powder, purchased from Takara Japan;
PLGA,购自Sigma-Aldrich,美国;PLGA, purchased from Sigma-Aldrich, USA;
DOTAP,购自Avanti,美国;DOTAP, purchased from Avanti, USA;
二氯甲烷,购自广州化学试剂厂,中国;Dichloromethane, purchased from Guangzhou Chemical Reagent Factory, China;
PVA,购自Sigma-Aldrich,美国;PVA, purchased from Sigma-Aldrich, USA;
PBS,购自Life technology,美国;PBS, purchased from Lifetechnology, USA;
鬼笔环肽,购自Life,美国;Phalloidin, purchased from Life, USA;
TritonX-100,BSA,购自Sigma-Aldrich,美国;TritonX-100, BSA, purchased from Sigma-Aldrich, USA;
C57BL/6小鼠,购自广州市赛柏诺生物科技有限公司;C57BL/6 mice, purchased from Guangzhou Cybino Biotechnology Co., Ltd.;
ELISA试剂盒,Hepa1-6细胞,购自北京鼎国昌盛生物技术有限责任公司。ELISA kit, Hepa1-6 cells, purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd.
仪器:instrument:
激光共聚焦显微镜,购自Zeiss德国。Confocal laser microscope, purchased from Zeiss Germany.
本发明一方面旨在寻找一种基于血小板膜伪装的siPcsk9纳米囊高效转染,敲低PCSK9蛋白水平,从而达到高效长久降低胆固醇水平的治疗效果。On the one hand, the present invention aims to find a high-efficiency transfection of siPcsk9 nanocapsules based on platelet membrane camouflage to knock down the PCSK9 protein level, so as to achieve a high-efficiency and long-term therapeutic effect of reducing cholesterol levels.
为达到目的,本发明采用以下技术方案:To achieve the goal, the present invention adopts the following technical solutions:
本发明采用具有生物相容性、可降解性的聚乳酸-聚乙醇酸共聚物(PLGA)包裹小干扰RNA,同时用阳离子脂质体DOTAP解决PLGA与核酸的吸附力小、不能完成溶酶体逃逸等缺陷构建纳米囊后,采用供体自身来源的血小板膜包裹纳米囊,在不需要任何其他转染试剂的情况下,高效的将小干扰RNA纳米囊系统递送到细胞内,提高了细胞摄入率和转染效率低PCSK9蛋白水平,从而达到持续降低血液胆固醇水平的治疗效果,具有重要的应用价值。具体流程如图1所示。The present invention adopts biocompatibility and degradability polylactic acid-polyglycolic acid copolymer (PLGA) to encapsulate small interfering RNA, and at the same time, cationic liposome DOTAP is used to solve the problem that the adsorption force of PLGA and nucleic acid is small, and the lysosome cannot be completed. After the nanocapsules are constructed from defects such as escape, the platelet membrane from the donor is used to wrap the nanocapsules. Without any other transfection reagents, the small interfering RNA nanocapsule system can be efficiently delivered into the cells, which improves the cellular uptake. Intake rate and transfection efficiency are low in PCSK9 protein level, so as to achieve the therapeutic effect of continuously lowering blood cholesterol level, which has important application value. The specific process is shown in Figure 1.
本发明例举了一种可以使PCSK9蛋白沉默的基因:The present invention exemplifies a gene that can silence the PCSK9 protein:
siRNAs against PCSK9,siRNAs against PCSK9,
正义链:5′-UUCCGAAUAAACUCCAGGCT-3′(SEQ ID NO.1),Sense chain: 5′-UUCCGAAUAAACUCCAGGCT-3′ (SEQ ID NO.1),
反义链:5′-GCCUGGAGUUUAUUCGGAA-3′(SEQ ID NO.2)。Antisense strand: 5'-GCCUGGAGUUUAUUCGGAA-3' (SEQ ID NO. 2).
并在上述正义链和反义链的3′修饰有悬挂碱基dTdT(siPCSK9)。And the 3'of the above-mentioned sense strand and antisense strand are modified with a hanging base dTdT (siPCSK9).
实施例1Example 1
本实施例用于说明本发明纳米颗粒的合成。This example is used to illustrate the synthesis of nanoparticles of the present invention.
构建利用纳米材料和提取出的血小板膜制备生物膜包裹纳米粒子的方法。A method for preparing biofilm-encapsulated nanoparticles using nanomaterials and platelet membranes extracted is constructed.
1)用100μL无核酶水溶解56nmol的siPcsk9干粉(0.75mg),逐滴加入到含69mg/mL含PLGA/DOTAP(PLGA与DOTAP按照w/w 20/0,20/1,20/3,20/5,20/7,20/9和20/11 的比例混合)的二氯甲烷溶液(250μL)中,冰浴超声90s。1) Dissolve 56nmol of siPcsk9 dry powder (0.75mg) with 100μL of ribozyme-free water, and add it dropwise to 69mg/mL containing PLGA/DOTAP (PLGA and DOTAP follow w/w 20/0, 20/1, 20/3, 20/5, 20/7, 20/9 and 20/11) in dichloromethane solution (250μL), ice bath ultrasonic for 90s.
2)将上述制得初乳全部逐滴加入到1mL2%的PVA水溶液中,冰浴超声1min。2) All the colostrum prepared above was added dropwise to 1 mL of 2% PVA aqueous solution, and ultrasonicated in an ice bath for 1 min.
3)将上述制得混合液全部加入到10mL2%PVA水溶液中室温搅拌3h,挥发有机溶剂,4℃,18000g离心15min,去掉上清,再反复洗涤三次,得到的沉淀用无菌PBS重悬,得重悬液。3) Add all the above-prepared mixture to 10mL of 2% PVA aqueous solution and stir for 3h at room temperature, evaporate the organic solvent, centrifuge at 4℃, 18000g for 15min, remove the supernatant, and wash three times repeatedly. The obtained precipitate is resuspended in sterile PBS. Get resuspension.
4)将上述得重悬液混合液与血小板膜混合(按血小板膜上蛋白与纳米囊质量比0/1,1/1,1/5,1/1和5/1)(血小板膜以膜蛋白质量定量),超声包裹含siPcsk9的纳米囊,得到纳米颗粒PM/PDC/siPcsk9。4) Mix the above-mentioned resuspension mixture with the platelet membrane (according to the mass ratio of the protein on the platelet membrane to the nanocapsules 0/1, 1/1, 1/5, 1/1 and 5/1) (platelet membrane with membrane Quantitative protein quality), ultrasonically wrap the nanocapsules containing siPcsk9 to obtain nanoparticles PM/PDC/siPcsk9.
其中,血小板膜的合成为:Among them, the synthesis of platelet membrane is:
1)通过收集动物全血,然后室温下1500rpm,离心20min,得到富血小板血浆,室温下1500rpm,离心1min,去除残余的红细胞与白细胞,再次3500rpm,离心12min,得到血小板沉淀,制成混悬液后-80℃冰箱保存。1) Collect animal whole blood, then centrifuge at 1500 rpm for 20 min at room temperature to obtain platelet rich plasma, centrifuge at 1500 rpm at room temperature for 1 min to remove residual red blood cells and white blood cells, and centrifuge again at 3500 rpm for 12 min to obtain platelet pellets and prepare suspensions Store in the refrigerator at -80℃.
2)血小板膜提取:将上述混悬液放至室温融解,反复冻融3次,PBS洗涤,5000rpm离心5min,水浴超声5min至形成血小板膜囊泡。2) Platelet membrane extraction: the above suspension was thawed at room temperature, repeatedly frozen and thawed 3 times, washed with PBS, centrifuged at 5000 rpm for 5 minutes, and ultrasonicated in a water bath for 5 minutes until platelet membrane vesicles were formed.
图1示出了血小板膜伪装的siPcsk9纳米囊制备过程。其中,图1A示出了聚乳酸-羟基乙酸共聚物(PLGA)和阳离子脂质体DOTAP包裹siPcsk9,构建纳米囊,图1B示出了采用血小板膜包裹纳米囊,制备血小板膜血小板膜伪装的siPcsk9纳米囊过程。Figure 1 shows the preparation process of siPcsk9 nanocapsules camouflaged by platelet membrane. Among them, Figure 1A shows polylactic acid-glycolic acid copolymer (PLGA) and cationic liposome DOTAP encapsulating siPcsk9 to construct nanocapsules, and Figure 1B shows the use of platelet membrane to wrap nanocapsules to prepare siPcsk9 camouflaged by platelet membrane. Nanocapsule process.
图2示出了实施例1纳米囊的细胞毒性情况。PDC中,PLGA与DOTAP的比例分别为20/0,20/1,20/3,20/5,20/7,20/9和20/11。siRNA的浓度分别是0,9.38,18.75,37.5,75,150,和300nM。我们优化了PLGA与DOTAP的比值,发现当比值在20/3时显示高细胞活力(>80%)。Figure 2 shows the cytotoxicity of the nanocapsules in Example 1. In PDC, the ratios of PLGA and DOTAP are 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 300 nM. We optimized the ratio of PLGA to DOTAP and found that when the ratio was 20/3, it showed high cell viability (>80%).
图3示出了实施例1纳米囊对siRNA的负载效率。PDC/siRNA中,PDC与siRNA的质量比分别为23/0.1,23/0.2,23/0.5,23/1,23/2。其中,PLGA/DOTAP纳米粒负载siRNA的效率可接近100%。Figure 3 shows the loading efficiency of Example 1 nanocapsules on siRNA. In PDC/siRNA, the mass ratios of PDC to siRNA are 23/0.1, 23/0.2, 23/0.5, 23/1, 23/2, respectively. Among them, the efficiency of loading siRNA on PLGA/DOTAP nanoparticles can be close to 100%.
图4示出了实施例1血小板膜包裹负载siRNA纳米囊的情况。其中,血小板膜蛋白与纳米囊的质量比分别为0/1,1/5,1/1和5/1。通过透射电镜(TEM)分析表明,PM/PDP质量比为1/1的PPDP(PM/PDC/siPcsk9,简称PPDP,即最终合成的血小板膜包裹后的PLGA/DOTAP/siPCSK9的纳米颗粒)涂层效果最好,较小或较大的PPDP涂层质量比会导致涂层不完整或大大增大涂层尺寸。Figure 4 shows the case of Example 1 where the platelet membrane is wrapped and loaded with siRNA nanocapsules. Among them, the mass ratios of platelet membrane protein to nanocapsules are 0/1, 1/5, 1/1, and 5/1, respectively. Transmission electron microscopy (TEM) analysis shows that PPDP (PM/PDC/siPcsk9, referred to as PPDP, which is PLGA/DOTAP/siPCSK9 nanoparticles wrapped in the final synthesized platelet membrane) coating with a PM/PDP mass ratio of 1/1 The effect is the best. A smaller or larger PPDP coating quality ratio will cause the coating to be incomplete or greatly increase the size of the coating.
图5示出了实施例1所得到的纳米颗粒。其中图5A示出了小鼠采血离心后得到的血浆,图5B示出了流式定量测定血小板比例;图5C示出了十二烷基磺酸钠-聚丙烯酰胺凝 胶电泳分析蛋白组分(TPP,血小板总蛋白;PM,血小板膜;PDC,PLGA/DOTAP核);图5D示出了血小板膜包裹PLGA/DOTAP核之前,图5E示出了血小板膜包裹PLGA/DOTAP核。从小鼠采集全血,从血清中提取血小板(图5A)。在用抗CD42d抗体标记细胞并用流式细胞术(FACS)分析后,高达99%的细胞为CD42d阳性(图5B)。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明,PPDP保留了血小板膜上的大部分蛋白质(图5C),使纳米粒子具有类血小板样功能。此外,TEM还显示,PDP的核心约为130nm(图5D)。包裹上血小板膜后,尺寸增加到约180纳米(图5E)。FIG. 5 shows the nanoparticles obtained in Example 1. Figure 5A shows the plasma obtained after blood collection and centrifugation in mice, Figure 5B shows the flow cytometric quantitative determination of the proportion of platelets; Figure 5C shows the analysis of protein components by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (TPP, total platelet protein; PM, platelet membrane; PDC, PLGA/DOTAP nucleus); Figure 5D shows the platelet membrane before encapsulating the PLGA/DOTAP nucleus, and Figure 5E shows the platelet membrane encapsulating the PLGA/DOTAP nucleus. Whole blood was collected from mice, and platelets were extracted from serum (Figure 5A). After labeling the cells with anti-CD42d antibody and analyzing by flow cytometry (FACS), up to 99% of the cells were CD42d positive (Figure 5B). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that PPDP retained most of the protein on the platelet membrane (Figure 5C), making the nanoparticles have platelet-like functions. In addition, TEM also showed that the core of the PDP is about 130nm (Figure 5D). After wrapping the platelet membrane, the size increased to about 180 nanometers (Figure 5E).
试验例1Test example 1
本试验例用于说明本发明的纳米颗粒治疗高胆固醇血症。This test example is used to illustrate the treatment of hypercholesterolemia by the nanoparticles of the present invention.
如实施例1方法合成纳米颗粒PM/PDC/siPcsk9,治疗高胆固醇血症。PM/PDC/siPcsk9是实验组,Lipo2000/siPcsk9是阳性对照组,siLuciferase作为内参,按照PM/PDC/siPcsk9的制备方法,负载siLuciferase后,形成PM/PDC/siLuc,siPcsk9是未加修饰的药物对照组,PM/PDC是不含药物的空载对照组,PBS是空白对照组。As in Example 1, the nanoparticles PM/PDC/siPcsk9 were synthesized to treat hypercholesterolemia. PM/PDC/siPcsk9 is the experimental group, Lipo2000/siPcsk9 is the positive control group, siLuciferase is used as the internal control, according to the preparation method of PM/PDC/siPcsk9, after loading siLuciferase, PM/PDC/siLuc is formed, siPcsk9 is the unmodified drug control In the group, PM/PDC is the no-load control group without drugs, and PBS is the blank control group.
Lipo2000/siPcsk9制备:Preparation of Lipo2000/siPcsk9:
1)用250μL无核酶水溶解5nmol的siPcsk9干粉,取其中15μL加入到150μLOpti-MEM轻轻混匀1) Dissolve 5nmol of siPcsk9 dry powder with 250μL ribozyme-free water, take 15μL of it and add it to 150μL Opti-MEM and mix gently
2)同时取22.5μL Lipofectamine 2000(Life,美国)加入到150μL Opti-MEM轻轻混匀2) At the same time, take 22.5μL Lipofectamine 2000 (Life, USA) and add it to 150μL Opti-MEM and mix gently
3)将上述步骤1)制得溶液全部加入到上述步骤2)制得溶液,轻轻颠倒混合均匀,室温下孵育5min。3) Add all the solution prepared in the above step 1) to the solution prepared in the above step 2), gently invert to mix evenly, and incubate at room temperature for 5 minutes.
PM/PDC/siLuc制备:PM/PDC/siLuc preparation:
1)用100μL无核酶水溶解56nmol的siLuciferase(Luciferase的siRNA)干粉,逐滴加入到含69mg/mL含PLGA/DOTAP(w/w 20:3)的二氯甲烷溶液(250μL)中,冰浴超声90s。1) Dissolve 56 nmol of siLuciferase (Luciferase siRNA) dry powder with 100 μL ribozyme-free water, and add it dropwise to 69 mg/mL dichloromethane solution (250 μL) containing PLGA/DOTAP (w/w 20:3), ice Bath ultrasound for 90s.
2)将上述制得初乳全部逐滴加入到1mL 2%的PVA水溶液中,冰浴超声1min。2) Add all the colostrum prepared above dropwise to 1 mL 2% PVA aqueous solution, and ultrasonicate for 1 min in an ice bath.
3)将上述制得混合液全部加入到10mL2%PVA水溶液中室温搅拌1h,真空中挥发有机溶剂3h,4℃,18000g离心15min,去掉上清,再反复洗涤三次,得到的沉淀用无菌PBS重悬。3) Add all the above-prepared mixed solution to 10mL 2% PVA aqueous solution and stir at room temperature for 1h, volatilize the organic solvent in vacuum for 3h, 4℃, 18000g centrifugation for 15min, remove the supernatant, and wash three times repeatedly. The precipitate obtained is sterile PBS Resuspended.
4)将上述混合液与血小板膜混合(按PLGA与血小板膜上蛋白质量比1:1),超声包裹含siLuciferase的纳米囊。4) Mix the above mixture with the platelet membrane (according to the ratio of PLGA to the protein on the platelet membrane at 1:1), and ultrasonically wrap the nanocapsules containing siLuciferase.
siPcsk9制备:siPcsk9 preparation:
用25μL无核酶水溶解5nmol的siPcsk9干粉,用无菌PBS溶液稀释至200μM。Dissolve 5nmol of siPcsk9 dry powder with 25μL of ribozyme-free water and dilute to 200μM with sterile PBS solution.
PM/PDC制备:PM/PDC preparation:
1)取100μL无核酶水逐滴加入到含69mg/mL含PLGA/DOTAP(w/w 20:3)的二氯甲烷溶液(250μL)中,冰浴超声90s。1) Take 100 μL of ribozyme-free water and add it dropwise to a dichloromethane solution (250 μL) containing 69 mg/mL PLGA/DOTAP (w/w 20:3), and ultrasonication in an ice bath for 90 seconds.
2)将上述制得初乳全部逐滴加入到1mL 2%的PVA水溶液中,冰浴超声1min。2) Add all the colostrum prepared above dropwise to 1 mL 2% PVA aqueous solution, and ultrasonicate for 1 min in an ice bath.
3)将上述制得混合液全部加入到10mL 2%PVA水溶液中室温搅拌3h,挥发有机溶剂,4℃,18000g离心15min,去掉上清,再反复洗涤三次,得到的沉淀用无菌PBS重悬。3) Add all the above-prepared mixture to 10mL 2% PVA aqueous solution and stir for 3h at room temperature, evaporate the organic solvent, 4℃, centrifuge at 18000g for 15min, remove the supernatant, and wash three times repeatedly. The obtained precipitate is resuspended in sterile PBS .
4)将上述混合液与血小板膜混合(按PLGA与血小板膜上蛋白质量比1:1),超声包裹含siPcsk9的纳米囊。4) Mix the above mixture with the platelet membrane (according to the ratio of PLGA and the protein on the platelet membrane at 1:1), and ultrasonically wrap the nanocapsules containing siPcsk9.
图6与其它治疗方法对比。图6为小鼠体内生化因子的变化。Figure 6 compares with other treatment methods. Figure 6 shows the changes of biochemical factors in mice.
1)取30只雌性C57BL/6小鼠(6-8周龄,体重在20g左右),随机均分成以上所述五组,给药两周后,分离小鼠肝脏,提取小鼠血清同时用ELISA试剂盒检测血液中PCSK9、LDL-C、HDL、TG等指标的表达水平,如图6所示。1) Take 30 female C57BL/6 mice (6-8 weeks old, weighing about 20g) and randomly divide them into the above five groups. After two weeks of administration, separate mouse liver and extract mouse serum for simultaneous use The ELISA kit detects the expression levels of PCSK9, LDL-C, HDL, TG and other indicators in the blood, as shown in Figure 6.
如图6所示,与对照组相比,实验组的沉默效果较强,明显降低低密度脂蛋白-胆固醇(LDL-C)水平。As shown in Figure 6, compared with the control group, the experimental group has a stronger silencing effect and significantly reduces the level of low-density lipoprotein-cholesterol (LDL-C).
请参见图6,ELISA结果表明,PCSK9水平为499.38±129.61pg/ml,比生理盐水处理的小鼠(840.00±48.07pg/mL)低约40%(图6A)。然而,对照组Lipo2000/siPCSK9(615.77±209.20pg/ml)比PPDP组显示约20%的PCSK9减少。其余各组PPDL、siPcsk9和PPD对PCSK9抑制作用均无显著影响(图6A)。在抑制PCSK9的同时,PPDP对小鼠LDL-C水平也有约28%的下调作用。对照组无明显变化。PPDP和Lipo2000/siPcsk9诱导的PCSK9和LDL-C水平在本研究中无统计学意义,但Lipo2000/siPcsk9诱导的PCSK9和LDL-C水平存在离散的数据点,说明Lipo2000在体内的应用表现不稳定。而非特异性生化指标HDL、TG与对照组相比无明显变化。肝功能评价显示,Lipo2000/siPcsk9组治疗后TBIL、ALT、AST明显高于生理盐水组,暗示其具有潜在的毒性(图6)。尽管使用这种优化材料的siRNA传递会导致LDL-C的有效下调(约56%),然而先前的研究也表明使用脂质纳米粒会导致肝功能受损。我们的工作有助于基于PM伪装纳米颗粒的治疗siRNA的安全递送,而对肝功能没有显著影响。Please refer to Figure 6. ELISA results show that the PCSK9 level is 499.38±129.61 pg/ml, which is about 40% lower than that of normal saline-treated mice (840.00±48.07 pg/mL) (Figure 6A). However, the control group Lipo2000/siPCSK9 (615.77±209.20pg/ml) showed about 20% reduction in PCSK9 than the PPDP group. The other groups of PPDL, siPcsk9 and PPD had no significant effect on the inhibition of PCSK9 (Figure 6A). While inhibiting PCSK9, PPDP also had a 28% down-regulation effect on LDL-C levels in mice. There was no significant change in the control group. The levels of PCSK9 and LDL-C induced by PPDP and Lipo2000/siPcsk9 were not statistically significant in this study, but there are discrete data points in the levels of PCSK9 and LDL-C induced by Lipo2000/siPcsk9, indicating that the application of Lipo2000 in vivo is unstable. Compared with the control group, the non-specific biochemical indicators HDL and TG did not change significantly. Liver function evaluation showed that TBIL, ALT, and AST in the Lipo2000/siPcsk9 group were significantly higher than those in the normal saline group after treatment, suggesting that it has potential toxicity (Figure 6). Although siRNA delivery using this optimized material will result in an effective down-regulation of LDL-C (approximately 56%), previous studies have also shown that the use of lipid nanoparticles can lead to impaired liver function. Our work contributes to the safe delivery of therapeutic siRNA based on PM camouflage nanoparticles without significant impact on liver function.
试验例2Test example 2
本试验例用于说明本发明的纳米颗粒的肝细胞的体外转染实验。This test example is used to illustrate the in vitro transfection experiment of the nanoparticle hepatocytes of the present invention.
如实施例1方法合成的纳米颗粒PM/PDC/siPcsk9,对小鼠来源的肝癌细胞(Hepa1-6)进行体外转染。The nanoparticles PM/PDC/siPcsk9 synthesized by the method in Example 1 were transfected in vitro to mouse-derived liver cancer cells (Hepa1-6).
激光共聚焦显微镜(CLSM)观察细胞摄入。Confocal laser microscope (CLSM) observes cell uptake.
为了研究细胞摄取,采用荧光染料2-((1E,3E,5E)-5-(1-(5-羧基戊基)-3,3-二甲基吲哚-2-亚立德)戊-1,3-二烯基)-1-乙基-3,3-二甲基-3H-吲哚氯(Cy5)-标记的siPcsk9(Cy5-siPcsk9)加载到PLGA/DOTAP核(Cy5-PDP)中,血小板膜涂覆在PDP上以形成Cy5-PPDP,如前所述。将Hepa1-6细胞接种于共聚焦培养皿中,密度为10^5个细胞/孔。一旦细胞生长到约70%的汇合,将其洗涤并在含有Cy5PPDP的新制备的Opti-mem培养基中培养,Cy5-siPcsk9的浓度相当于18.75、37.5、75、150和300nM(图7A)。在规定的培养时间后,除去培养基,用PBS轻轻洗涤细胞3次,并在室温下用4%多聚甲醛固定15min,用PBS洗两次;用0.1%曲拉通破膜1min,PBS洗两次;接着3%BSA封闭30min,PBS洗涤两次;再用Alexa Fluor 488鬼笔环肽(Invitrogen,Carlsbad,USA)染色于细胞骨架,并然后用DAPI(4,6-二氨基-2-苯基吲哚)染料复染细胞核,PBS反复洗涤。在CLSM(LSM 880,蔡司)上进行细胞成像。此外,通过CLSM评估PPDP随时间的细胞摄取。在最后一个150nM的Cy5-siPcsk9处加入纳米粒并孵育1、3、6、9和12h(图7B)。然后,同法用FITC-鬼笔环肽和Hoechst对细胞进行染色。分别培育肺泡巨噬细胞、单核巨噬细胞、脐带间充质干细胞的细胞系,制备Cy5-PPDP,将Cy5-siPcsk9浓度为150nM的不同纳米制剂与细胞在37℃下孵育9h。去除培养基,并用PBS洗涤细胞三次。对于CLSM,同样用FITC-标记的鬼笔环肽和DAPI固定和染色图(7C)。为了观测图(7C)的细胞系对于Cy5-PPDP的低摄取量,同时与图10C一样,分别给不同的细胞系与合成的Cy5-PPDP孵育9h后,分别取每组的培养基1mL,测荧光图(7D)In order to study cellular uptake, the fluorescent dye 2-((1E, 3E, 5E)-5-(1-(5-carboxypentyl)-3,3-dimethylindole-2-ylide)pentan- 1,3-dienyl)-1-ethyl-3,3-dimethyl-3H-indole chloride (Cy5)-labeled siPcsk9 (Cy5-siPcsk9) loaded onto PLGA/DOTAP core (Cy5-PDP) In, the platelet membrane is coated on the PDP to form Cy5-PPDP, as described previously. Hepa1-6 cells were seeded in a confocal culture dish with a density of 10^5 cells/well. Once the cells had grown to about 70% confluence, they were washed and cultured in freshly prepared Opti-mem medium containing Cy5PPDP, the concentration of Cy5-siPcsk9 was equivalent to 18.75, 37.5, 75, 150 and 300 nM (Figure 7A). After the specified incubation time, the medium was removed, the cells were gently washed with PBS 3 times, and fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice with PBS; ruptured with 0.1% Triton for 1 min, PBS Wash twice; then block with 3% BSA for 30 min, wash twice with PBS; then stain the cytoskeleton with Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, USA), and then use DAPI (4,6-diamino-2) -Phenylindole) dye counterstain the nucleus, and wash repeatedly with PBS. Cell imaging was performed on CLSM (LSM 880, Zeiss). In addition, the cellular uptake of PPDP over time was evaluated by CLSM. Nanoparticles were added at the last 150 nM Cy5-siPcsk9 and incubated for 1, 3, 6, 9 and 12h (Figure 7B). Then, the cells were stained with FITC-Phalloidin and Hoechst in the same way. Cell lines of alveolar macrophages, mononuclear macrophages, and umbilical cord mesenchymal stem cells were cultivated to prepare Cy5-PPDP, and different nano-preparations with a Cy5-siPcsk9 concentration of 150 nM were incubated with the cells for 9 hours at 37°C. The medium was removed, and the cells were washed three times with PBS. For CLSM, FITC-labeled phalloidin and DAPI were also fixed and stained (7C). In order to observe the low uptake of Cy5-PPDP by the cell line in Figure (7C), and at the same time, as in Figure 10C, different cell lines were incubated with synthetic Cy5-PPDP for 9 hours, and 1 mL of the culture medium of each group was taken. Fluorescence image (7D)
请参见图7。我们通过调整剂量和时间来优化有效转染的条件。CLSM和FACS分析表明,siPcsk9浓度为18.75nM的PPDP的转染效率为较低,150nM在孵育6h后的转染效率为几乎达到百分百,较高浓度的PPDP并未进一步显著提高转染效率(图7A)。同时,CLSM和FACS分析表明转染时间为9h,转染效率最高为99%,转染时间越长,转染效率越高(图7B)。因此,我们在优化的siRNA浓度为150nM,转染9h的条件下进行了进一步的实验。我们还分析了其他细胞系的细胞摄取,即肺泡巨噬细胞(AMs)、小鼠白血病单核细胞巨噬细胞(Raw 264.7)和小鼠骨髓间充质干细胞(MBMSCs)。这些细胞的细胞摄取率分别为9.04%、23.0%和16%(图7C)。同时为了探索免疫细胞摄取带荧光的PPDP较弱是否归因于其降解功能,我们测试了免疫细胞(AMs或Raw264.7细胞)上清液中的PDPP,发现与Hepa1-6细胞相比,Cy5阳性纳米颗粒高出约4倍(图7D)。值得注意的是,AMs和Raw 264.7细胞都是免疫细胞系,细胞摄取量低,表明生物诱导人工血小板可以避免免疫细胞的清除,有利于体内应用。See Figure 7. We optimize the conditions for effective transfection by adjusting the dose and time. CLSM and FACS analysis showed that the transfection efficiency of PPDP with a siPcsk9 concentration of 18.75nM was low, and the transfection efficiency of 150nM after 6h incubation was almost 100%. Higher concentrations of PPDP did not further significantly improve the transfection efficiency. (Figure 7A). At the same time, CLSM and FACS analysis showed that the transfection time was 9h, and the transfection efficiency was 99% at the highest. The longer the transfection time, the higher the transfection efficiency (Figure 7B). Therefore, we conducted further experiments under the conditions of an optimized siRNA concentration of 150 nM and 9 hours of transfection. We also analyzed the cellular uptake of other cell lines, namely alveolar macrophages (AMs), mouse leukemia monocyte macrophages (Raw 264.7) and mouse bone marrow mesenchymal stem cells (MBMSCs). The cell uptake rates of these cells were 9.04%, 23.0%, and 16%, respectively (Figure 7C). At the same time, in order to explore whether the weaker uptake of fluorescent PPDP by immune cells is due to its degradation function, we tested the PDPP in the supernatant of immune cells (AMs or Raw264.7 cells) and found that compared with Hepa1-6 cells, Cy5 The positive nanoparticles were about 4 times higher (Figure 7D). It is worth noting that both AMs and Raw 264.7 cells are immune cell lines with low cell uptake, indicating that biologically induced artificial platelets can avoid immune cell clearance, which is beneficial for in vivo applications.
实验例3Experimental example 3
4.1 Cy5-siPcsk9用于评价CLSM的内质/溶酶体逃逸。4.1 Cy5-siPcsk9 is used to evaluate the endoplasmic/lysosome escape of CLSM.
将Hepa1-6(1×10^5细胞/孔)接种在共聚焦小皿中并孵育24h后,给含150nM Cy5-PPDP或PM/PLGA/Cy5-siPcsk9进行细胞转染,培养不同的时间点(1、3、6、9和12h)后,然后用PBS洗涤两次同时然后用LysoTracker染色
TM蓝色DND-22(ThermoFisher Scientific,美国)根据说明书室温染色30分钟,通过共聚焦激光扫描显示纳米颗粒的细胞在不同时间点纳米材料的细胞分布情况(结果请参见图8A)。
After inoculating Hepa1-6 (1×10^5 cells/well) in a confocal small dish and incubating for 24h, cells containing 150nM Cy5-PPDP or PM/PLGA/Cy5-siPcsk9 were transfected and cultured at different time points ( 1, 3, 6, 9 and 12h), then washed twice with PBS and dyed with LysoTracker TM blue DND-22 (ThermoFisher Scientific, USA) at room temperature for 30 minutes according to the instructions, and the nanoparticles were displayed by confocal laser scanning The cell distribution of nanomaterials at different time points of the cells (see Figure 8A for the results).
4.2实时荧光定量PCR(Quantitative Real-time PCR,qPCR)实验,具体步骤如下:在qPCR实验中,首先将Hepa1-6细胞接种到6孔板中,密度为每孔10^6个细胞,在2.0mL补充有10%FBS和1%青霉素/链霉素的DMEM中,并在37℃,5%CO
2湿润的气氛中培养24h。用含有PBS,siPcsk9,PPDL、PDP和Lipo2000/siPcsk9的Opti-men减血清培养基替换培养基,其中siRNA浓度相当于150nM,将细胞再培养48h。
4.2 Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) experiment, the specific steps are as follows: In the qPCR experiment, first inoculate Hepa1-6 cells into a 6-well plate with a density of 10^6 cells per well, at 2.0 mL was supplemented with 10% FBS and 1% penicillin/streptomycin in DMEM, and cultured at 37°C in a 5% CO 2 humidified atmosphere for 24 hours. The medium was replaced with Opti-men serum-reduced medium containing PBS, siPcsk9, PPDL, PDP and Lipo2000/siPcsk9, where the siRNA concentration was equivalent to 150nM, and the cells were cultured for another 48h.
(1)使用Trizol(Invitrogen,USA)从细胞中分离总RNA(1) Use Trizol (Invitrogen, USA) to isolate total RNA from cells
1)首先预冷PBS洗涤细胞2次,然后尽可能吸去残留液体,加入400μL的Trizol-reagent,尽可能将细胞冲洗下来,然后转移至1.5mL EP管中,再向每管加入预冷氯仿80μL,盖紧EP管并涡旋30s(注:氯仿:Trizol=1:5,v/v);1) First, wash the cells twice with pre-cooled PBS, and then aspirate the remaining liquid as much as possible, add 400μL of Trizol-reagent, wash down the cells as much as possible, then transfer to a 1.5mL EP tube, and then add pre-cooled chloroform to each tube 80μL, cap the EP tube tightly and vortex for 30s (Note: chloroform: Trizol=1:5, v/v);
2)室温静置2-3min,出现明显分层后,12000rpm,4℃离心15min;2) Leave it at room temperature for 2-3 minutes, after obvious stratification occurs, centrifuge at 12000rpm, 4℃ for 15min;
3)用200μL量程移液枪转移上清液至新的无核酶EP管中(切勿吸到中间或下层的蛋白等杂质,否则重新离心分离),然后加入等体积的异丙醇(约250μL),反复颠倒十几次,室温静置10min;3) Use a 200μL volume pipette to transfer the supernatant to a new ribozyme-free EP tube (do not suck the protein and other impurities in the middle or lower layer, or re-centrifuge), and then add an equal volume of isopropanol (about 250μL), inverted more than ten times, let stand at room temperature for 10min;
4)12000rpm,4℃离心10min,EP管底部可见有白色沉淀,除去上清,倒扣在滤纸上小心沉淀被倒出);4) Centrifuge at 12000 rpm and 4°C for 10 min. A white precipitate can be seen at the bottom of the EP tube. Remove the supernatant and place it on the filter paper carefully.
5)然后每管加入预冷400μL 75%酒精(DEPC水:无水乙醇=1:3,v/v配制),用移液枪小心重悬沉淀;5) Then add 400μL of pre-cooled 75% alcohol to each tube (DEPC water: absolute ethanol = 1:3, v/v preparation), and carefully resuspend the pellet with a pipette;
6)12000rpm,4℃离心5min,吸去上清,注意不要将白色沉淀吸出;6) Centrifuge at 12000rpm for 5min at 4℃, aspirate the supernatant, and be careful not to aspirate the white precipitate;
7)然后将EP管开盖置于通风橱中几分钟,晾干后每管加入25μL无核酶水;7) Then open the lid of the EP tube and place it in a fume hood for a few minutes. After drying, add 25μL of ribozyme-free water to each tube;
9)Nanodrop测量RNA浓度。9) Nanodrop measures RNA concentration.
(2)取总RNA进行逆转录,使用锐博公司合成的茎环法引物,用TaKaRa逆转录试剂盒(RR037A,日本)进行逆转录,用SYBR试剂(Cat.172-5124,BioRad)对其进行quantitative PCR分析。操作步骤同说明书,应用Light cycler 480system进行操作。(2) Take total RNA for reverse transcription, use the stem-loop primers synthesized by Ruibo Company, use TaKaRa Reverse Transcription Kit (RR037A, Japan) for reverse transcription, and use SYBR reagent (Cat.172-5124, BioRad) for reverse transcription. Perform quantitative PCR analysis. The operation steps are the same as the instructions, and the Light cycler 480 system is used for operation.
(3)数据处理(3) Data processing
采用ΔΔCt的方法,ΔCt=(Ct目的基因-Ct内参基因),ΔΔCt=ΔCt处理样本-ΔCt对照样本,最后的结果取2-ΔΔCt.确定药物对细胞内特定PCSK9miRNAs表达水平的影响。结果参见图8B。Using the ΔΔCt method, ΔCt=(Ct target gene-Ct internal reference gene), ΔΔCt=ΔCt processed sample-ΔCt control sample, and the final result is 2-ΔΔCt. Determine the effect of the drug on the expression of specific PCSK9miRNAs in the cell. See Figure 8B for the results.
(4)siRNA序列如下:(4) The siRNA sequence is as follows:
siPcsk9(siRNA靶向Pcsk9mRNA):siPcsk9 (siRNA targeting Pcsk9mRNA):
sense:5′-UUCCGAAUAAACUCCAGGCdTdT-3′,(SEQ ID NO.1)sense:5′-UUCCGAAUAAACUCCAGGCdTdT-3′, (SEQ ID NO.1)
anti-sense:5′-GCCUGGAGUUUAUUCGGAAdTdT-3′,(SEQ ID NO.2)anti-sense: 5′-GCCUGGAGUUUAUUCGGAAdTdT-3′, (SEQ ID NO.2)
siLuc(siRNA靶向luciferase mRNA):siLuc (siRNA targeting luciferase mRNA):
sense:5′-CUUACGCUGAGUACUUCGAdTdT-3′,(SEQ ID NO.3)sense:5′-CUUACGCUGAGUACUUCGAdTdT-3′,(SEQ ID NO.3)
anti-sense:5′-UCGAAGUACUCAGCGUAAGdTdT-3′,(SEQ ID NO.4)anti-sense: 5′-UCGAAGUACUCAGCGUAAGdTdT-3′, (SEQ ID NO.4)
目的基因PCSK9与内参基因GAPDH引物序列如下:The primer sequences of the target gene PCSK9 and the internal reference gene GAPDH are as follows:
Pcsk9 Forward:GAGACCCAGAGGCTACAGATT,(SEQ ID NO.5)Pcsk9 Forward: GAGACCCAGAGGCTACAGATT, (SEQ ID NO.5)
Pcsk9 Reverse:AATGTACTCCACATGGGGCAA;(SEQ ID NO.6)Pcsk9 Reverse: AATGTACTCCACATGGGGCAA; (SEQ ID NO.6)
GAPDH Forward:TGCACCACCAACTGCTTAGC,(SEQ ID NO.7)GAPDH Forward: TGCACCACCAACTGCTTAGC, (SEQ ID NO.7)
GAPDH Reverse:GGCATGGACTGTGGTCATGAG(SEQ ID NO.8)。GAPDH Reverse: GGCATGGACTGTGGTCATGAG (SEQ ID NO. 8).
4.3为蛋白印迹分析实验,具体步骤如下:4.3 is a Western blot analysis experiment, the specific steps are as follows:
蛋白印迹分析(Western Blot)Western Blot Analysis (Western Blot)
首先将Hepa1-6细胞接种到6孔板中,密度为每孔10^6个细胞,在2.0mL补充有10%FBS和1%青霉素/链霉素的DMEM中,并在37℃,5%CO
2湿润的气氛中培养24h。用含有PBS,siPcsk9,PPDL、PDP和Lipo2000/siPcsk9的Opti-men减血清培养基替换培养基,其中siRNA浓度相当于150nM,将细胞再培养48h。
First, inoculate Hepa1-6 cells into a 6-well plate with a density of 10^6 cells per well, in 2.0 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and at 37°C, 5% Cultivate for 24h in a CO 2 humidified atmosphere. The medium was replaced with Opti-men serum-reduced medium containing PBS, siPcsk9, PPDL, PDP and Lipo2000/siPcsk9, where the siRNA concentration was equivalent to 150nM, and the cells were cultured for another 48h.
(1)蛋白提取和浓度测试(1) Protein extraction and concentration test
首先用预冷PBS轻柔洗涤细胞,加入90μL RIPA裂解缓冲液(临用前加入完全蛋白酶抑制剂)。使用细胞刮轻柔将细胞刮落,转移到1.5mL的EP管中。置冰上孵育30min(期间可以每次涡旋震荡5s后迅速重置冰上,重复3-5次),然后12000rpm,4℃离心15min,取上清80μL转移到新的EP管中。按上述BCA试剂法测出每组蛋白浓度,并加入5×loading buffer混匀(终浓度为1×),煮沸5min,放于冰上冷却后-20℃保存。First, wash the cells gently with pre-chilled PBS, and add 90μL of RIPA Lysis Buffer (add complete protease inhibitor just before use). Use a cell scraper to gently scrape off the cells and transfer to a 1.5mL EP tube. Incubate on ice for 30 min (during this period, you can quickly reset on ice after vortexing for 5 seconds each time, repeat 3-5 times), then centrifuge at 12000 rpm, 4°C for 15 min, and transfer 80 μL of the supernatant to a new EP tube. Measure the concentration of each group of proteins according to the above-mentioned BCA reagent method, and add 5×loading buffer to mix well (final concentration is 1×), boil for 5min, cool on ice and store at -20°C.
(2)SDS-PAGE跑胶、转膜和曝光(2) SDS-PAGE running glue, transfer film and exposure
1)制胶:根据蛋白大小制胶,配制10%快速胶(BIO-RAD)插入梳子,室温静置至凝固。1) Glue preparation: Glue is prepared according to the size of the protein, 10% rapid glue (BIO-RAD) is prepared and inserted into the comb, and it is allowed to stand at room temperature until solidified.
2)上样:将已凝固的胶装进电泳槽内,加入新鲜配制的电泳液。轻拔梳子,向加样孔中加入Marker和样品,保持恒压200V进行电泳,当溴酚蓝到达分离胶底端时停止电泳。2) Sample loading: Put the solidified gel into the electrophoresis tank, and add the freshly prepared electrophoresis solution. Pull the comb gently, add Marker and sample to the sample hole, keep constant voltage 200V for electrophoresis, stop electrophoresis when bromophenol blue reaches the bottom of the separation gel.
3)转膜:电泳完毕后取出胶,弃去多余胶部分,将PVDF膜裁至与胶相同的大小,甲醇中浸润5min活化膜。在转移盒中依次放入(负极开始)海绵、滤纸、凝胶、PVDF膜、滤纸、海绵顺序制作“三明治”,然后赶走气泡,夹紧后置于电泳槽中,加入适量的新鲜配制的转膜液,电泳槽置于冰上,恒压100V转膜2h。3) Transfer membrane: After the electrophoresis is completed, take out the glue, discard the excess glue, cut the PVDF membrane to the same size as the glue, and soak the membrane in methanol for 5 minutes to activate the membrane. Put the sponge, filter paper, gel, PVDF membrane, filter paper, sponge in the transfer box in sequence to make a "sandwich", then drive away the bubbles, place it in the electrophoresis tank after clamping, and add an appropriate amount of freshly prepared Transfer membrane solution, place the electrophoresis tank on ice, and transfer membrane at a constant pressure of 100V for 2 hours.
4)封闭:将转入蛋白的PVDF膜用TBST润洗,转移至适量的5%脱脂奶粉(用TBST,至少覆盖膜),在摇床下室温封闭1h。4) Sealing: Rinse the protein-transferred PVDF membrane with TBST, transfer to an appropriate amount of 5% skimmed milk powder (with TBST, at least covering the membrane), and seal at room temperature under a shaker for 1 hour.
5)敷抗体:弃去封闭液,用TBST在摇床上洗膜3次,每次10min,将PVDF膜转移至预先稀释好的一抗溶液中,PCSK9(Abcam,1:1000),GAPDH(Abcam,1:1000)于摇床上4℃孵育,过夜。孵育结束后回收一抗溶液,用TBST洗膜3次,每次10min;再将PVDF膜转移至稀释好的二抗溶液(1:5000)中,置于摇床常温孵育2h,而后用TBST洗涤。5) Apply antibody: Discard the blocking solution, wash the membrane 3 times with TBST on a shaker, 10 minutes each time, transfer the PVDF membrane to the pre-diluted primary antibody solution, PCSK9 (Abcam, 1:1000), GAPDH (Abcam) , 1:1000) incubate overnight at 4°C on a shaker. After the incubation, recover the primary antibody solution, wash the membrane with TBST 3 times, 10 min each time; then transfer the PVDF membrane to the diluted secondary antibody solution (1:5000), incubate on a shaker at room temperature for 2 hours, and then wash with TBST .
6)曝光:用显影试剂中的溶液A和B以1:1混合,均匀加到膜表面,孵育约1min后,置于曝光机Amersham Imager 600system(GE,USA)等待自动曝光,将数据保存。结果请见图8C。6) Exposure: Mix the solutions A and B in the developing reagent at a ratio of 1:1, and add them evenly to the surface of the membrane. After incubating for about 1 min, place in the exposure machine Amersham Imager 600 system (GE, USA) for automatic exposure, and save the data. The results are shown in Figure 8C.
由于siRNA的溶酶体逃逸是有效基因沉默的关键过程,我们评估了Pcsk9的溶酶体逃逸和由此产生的基因沉默。细胞摄取的PPDP在1h后被溶酶体捕获(Cy5-siPcsk9)(红色荧光与溶酶体蓝标记的红色荧光重叠)(图8A),其中剖面分析也证实Cy5-siPcsk9与溶酶体重叠。9h后,溶酶体中分离出大量Cy5-siPcsk9(红色荧光与蓝色荧光分离),相应的剖面分析也显示了这一点。而无DOTAP(PLGA/siPcsk9)的核结构在9h内溶酶体逃逸效率较低,DOTAP修饰使siRNA具有更好的溶酶体逃逸能力。实时PCR检测表明PCSK9 mRNA通过PPDP(图8B)下调约66%。然而,对照组,PPDL,siPcsk9,PPD和PBS,在Pcsk9mRNA下调方面变化不明显(图8B)。与Lipo2000/siPcsk9相比,PPDP还显示出令人印象深刻的基因沉默效果(66%对74%)(图8B)。WB分析证实PPDP显著抑制PCSK9的表达(图8C)。我们设计了一种纳米结构来改善溶酶体逃逸,并特异性沉默靶向基因Pcsk9。Since the lysosomal escape of siRNA is a key process for effective gene silencing, we evaluated the lysosomal escape of Pcsk9 and the resulting gene silencing. The PPDP taken up by the cells was captured by lysosomes (Cy5-siPcsk9) (red fluorescence overlapped with lysosomal blue-labeled red fluorescence) after 1 h (Figure 8A). The profile analysis also confirmed that Cy5-siPcsk9 overlapped with lysosomes. After 9h, a large amount of Cy5-siPcsk9 (separation of red fluorescence and blue fluorescence) was separated from the lysosome, and the corresponding profile analysis also showed this. However, the nuclear structure without DOTAP (PLGA/siPcsk9) has a lower lysosomal escape efficiency within 9 hours, and DOTAP modification makes siRNA have better lysosomal escape ability. Real-time PCR detection showed that PCSK9 mRNA was down-regulated by about 66% through PPDP (Figure 8B). However, the control group, PPDL, siPcsk9, PPD and PBS, did not change significantly in the down-regulation of Pcsk9 mRNA (Figure 8B). Compared with Lipo2000/siPcsk9, PPDP also showed an impressive gene silencing effect (66% vs. 74%) (Figure 8B). WB analysis confirmed that PPDP significantly inhibited the expression of PCSK9 (Figure 8C). We designed a nanostructure to improve lysosomal escape and specifically silence the targeted gene Pcsk9.
4.4溶血实验与活死细胞染色,具体步骤如下:4.4 Hemolysis experiment and live and dead cell staining, the specific steps are as follows:
溶血实验具体步骤如下:The specific steps of the hemolysis experiment are as follows:
取C57小鼠新鲜全血,加入EDTA-2Na抗凝剂。血液以5000转/分离心5min。弃去上层血浆及白细胞,收集红细胞,用PBS缓冲液多次洗涤红细胞,直到上清中没有发现颜色。收集底部RBC沉淀,用PBS稀释成2%的溶液,如20μL加入到980μL的PBS中。将红细胞溶液与上述制备好的的PPDP溶液混合成1%RBC溶液。最终的siPcsk9浓度分别为18.75、37.5、75、150和300nM。将0.50mL红细胞悬液与0.50mL类似物混合,0.50mL PPDP、PPDL、PDP和Lipo2000/siPcsk9加入0.50mL红细胞悬液中。加入的PBS和1.0%Triton X-100溶液 分别作为阴性对照和阳性对照。将样品在37℃下孵育1h,以10^4rpm离心10min,然后将0.10ml上清液加入96孔板中。记录了540nm处血红蛋白的吸光度。计算溶血率如下:溶血率%=[(At–Anc)/(Apc–Anc)]×100%。At,供试品吸光度;Anc,阴性对照吸光度;Apc,阳性对照吸光度Take fresh whole blood from C57 mice and add EDTA-2Na anticoagulant. The blood was separated at 5000 rpm for 5 minutes. Discard the upper layer of plasma and white blood cells, collect the red blood cells, wash the red blood cells with PBS buffer for several times until no color is found in the supernatant. Collect the bottom RBC pellet and dilute it with PBS to a 2% solution, such as adding 20 μL to 980 μL of PBS. The red blood cell solution and the PPDP solution prepared above are mixed to form a 1% RBC solution. The final siPcsk9 concentrations were 18.75, 37.5, 75, 150 and 300nM, respectively. 0.50 mL of red blood cell suspension was mixed with 0.50 mL of analogs, and 0.50 mL of PPDP, PPDL, PDP and Lipo2000/siPcsk9 were added to 0.50 mL of red blood cell suspension. The added PBS and 1.0% Triton X-100 solution were used as negative control and positive control, respectively. The sample was incubated at 37°C for 1 h, centrifuged at 10^4 rpm for 10 min, and then 0.10 ml of supernatant was added to a 96-well plate. The absorbance of hemoglobin at 540nm was recorded. The hemolysis rate is calculated as follows: hemolysis rate%=[(At-Anc)/(Apc-Anc)]×100%. At, the absorbance of the test substance; Anc, the absorbance of the negative control; Apc, the absorbance of the positive control
活死细胞染色具体步骤如下:The specific steps for staining live and dead cells are as follows:
为了评价PPDP的细胞毒性,用新鲜制备的含有PPDP的完全DMEM替换培养基,最终的siRNA浓度范围为18.75至300nM。分别用PBS或1%Triton X-100处理的细胞作为阴性对照或阳性对照。孵育24小时后,用活/死活力/细胞毒性试剂盒对细胞进行染色,并用CLSM成像。同时评价了其他纳米颗粒的细胞毒性,如Lipo2000/siPCSK9,PPDL,和PDP,其中siRNA浓度相当于150nM。结果请参见图9。In order to evaluate the cytotoxicity of PPDP, the medium was replaced with freshly prepared complete DMEM containing PPDP, and the final siRNA concentration ranged from 18.75 to 300 nM. Cells treated with PBS or 1% Triton X-100 were used as negative control or positive control. After 24 hours of incubation, the cells were stained with a live/dead viability/cytotoxicity kit and imaged with CLSM. The cytotoxicity of other nanoparticles was also evaluated, such as Lipo2000/siPCSK9, PPDL, and PDP, where the siRNA concentration is equivalent to 150nM. See Figure 9 for the result.
为了评价生物相容性,我们分析了PPDP的溶血和细胞毒性。无论是PPDP还是非包覆纳米颗粒在工作浓度均未引起任何显著的溶血毒性(图9A和9B),同时用CLSM(图9C)的活/死测定可以证实。然而,使用市售的Lipofectamine 2000会有明显溶血(图9D和9E),这表明PPDP引起较低的溶血和细胞毒性,对于体内应用是至关重要的,体外CLSM同时表明市售的Lipofectamine 2000相较于PPDP引起较高的细胞毒性(图9F)。To evaluate the biocompatibility, we analyzed the hemolysis and cytotoxicity of PPDP. Neither PPDP nor uncoated nanoparticles caused any significant hemolytic toxicity at working concentrations (Figures 9A and 9B), which can be confirmed by the live/dead assay of CLSM (Figure 9C). However, the use of commercially available Lipofectamine 2000 will have significant hemolysis (Figure 9D and 9E), which indicates that PPDP causes lower hemolysis and cytotoxicity, which is essential for in vivo applications. In vitro CLSM also shows that the commercially available Lipofectamine 2000 phase It caused higher cytotoxicity than PPDP (Figure 9F).
4.5为体内追踪研究与体内药物代谢动力学研究,具体步骤如下:4.5 is the in vivo tracking study and the in vivo pharmacokinetic study, the specific steps are as follows:
体内追踪研究具体步骤如下:The specific steps of the in vivo tracking study are as follows:
为了考察材料的体内富集性,我们制备了Cy5-PDP、Cy5-PPDP制剂以进行体内示踪研究。通过C57BL/6小鼠尾静脉静脉内注射材料制剂。在所有操作过程中,使用2%异氟烷麻醉小鼠。并用成像系统(CRi Maestro,美国)进行跟踪。时间点分别设为1、6、12、24、48、72h。体内追踪后,处死小鼠,提取主要器官并成像。荧光分析采用Maestro V3.0.A软件。In order to investigate the in vivo enrichment of materials, we prepared Cy5-PDP and Cy5-PPDP preparations for in vivo tracing studies. The material preparation was injected intravenously through the tail vein of C57BL/6 mice. During all operations, the mice were anesthetized with 2% isoflurane. The imaging system (CRi Maestro, USA) is also used for tracking. The time points were set to 1, 6, 12, 24, 48, 72h. After in vivo tracking, the mice were sacrificed, and the main organs were extracted and imaged. Fluorescence analysis uses Maestro V3.0.A software.
体内药物代谢动力学研究,具体步骤如下:In vivo pharmacokinetics study, the specific steps are as follows:
所购买的10只雌性C57BL/6(体重在18-20g)随机分成2组(PDP跟PPDP)。PDP跟PPDP按上述方式合成,均以1,1'-双十八烷基-3,3,3',3'-四甲基吲哚二碳菁高氯酸盐(DiD)标记,其激发波长与发射波长为644/665nm。观察C57BL/6鼠有无弓背、打喷嚏、抽搐等异常状况一周,待观测无异常后,通过尾静脉注射(每只含DiD 20μg)设置时间点0.5,1,2,4,8,24,and 48h,在每个时间节点取小鼠体内全血50μL至含EDTA-2Na的抗凝管中,在第48h完成取血然后立即进行动物的安乐死,随后剥取下每只鼠的脏器(包括心、肝、脾、肺与肾)。The purchased 10 female C57BL/6 (weight 18-20g) were randomly divided into 2 groups (PDP and PPDP). PDP and PPDP are synthesized in the above manner, and both are labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate (DiD), which stimulates The wavelength and emission wavelength are 644/665nm. Observe whether the C57BL/6 mice have hunched back, sneezing, convulsions and other abnormal conditions for a week. After the observation is not abnormal, set the time point 0.5,1,2,4,8,24 through tail vein injection (each containing DiD 20μg) ,and 48h, at each time node, take 50μL of mouse whole blood into an anticoagulation tube containing EDTA-2Na, complete the blood collection at 48h, and then immediately euthanize the animal, and then remove the organs of each mouse (Including heart, liver, spleen, lung and kidney).
处理血样:将样品与0.1mM的EDTA混合,并进行三次反复冻融过程,以确保细胞完全被破坏,纳米颗粒被释放。然后,加入500μL二氯甲烷立即震荡涡旋30s,4℃ 20000g离心30min,将上清液转移到新试管中,空白血样处理与本法一致。随后用RF-6000荧光分光 光度计(日本岛津)检测荧光强度。Processing the blood sample: Mix the sample with 0.1 mM EDTA and perform three repeated freezing and thawing processes to ensure that the cells are completely destroyed and the nanoparticles are released. Then, add 500μL of dichloromethane and immediately vortex for 30s, centrifuge at 20000g at 4°C for 30min, and transfer the supernatant to a new test tube. The blank blood sample treatment is consistent with this method. Subsequently, the fluorescence intensity was measured with an RF-6000 fluorescence spectrophotometer (Shimadzu, Japan).
DiD标准曲线的建立:将DiD血溶液稀释成20,15,10,7.5,5,0.75μg/mL,随后用RF-6000荧光分光光度计(日本岛津)检测荧光强度。The establishment of the DiD standard curve: DiD blood solution was diluted to 20, 15, 10, 7.5, 5, 0.75 μg/mL, and then the fluorescence intensity was measured with an RF-6000 fluorescence spectrophotometer (Shimadzu, Japan).
脏器处理:将每组每只鼠的脏器分别装入2mLEP管中,加入1mL水后匀浆,4℃ 20000g离心30min,转移上清至新的EP管中,用RF-6000荧光分光光度计(日本岛津)检测荧光强度。结果参见图10。Organ treatment: Put the organs of each mouse in each group into 2mL EP tubes, add 1mL water and homogenize, centrifuge at 20000g at 4℃ for 30min, transfer the supernatant to a new EP tube, and use RF-6000 fluorescence spectrophotometry A meter (Shimadzu, Japan) detects the fluorescence intensity. See Figure 10 for the results.
给小鼠注射含Cy5-siPcsk9的PPDPs后,用成像系统跟踪纳米粒在体内的分布。我们发现,在24h后,PPDP在肝脏中的累积量大于裸纳米粒子(图10A)。主要器官的体外成像表明,与其他器官(图10B)相比,保留在肝脏中的PPDP高约20倍。此外,PPDP给药的小鼠肝脏比PDP小鼠增加了2倍(图10B)。有趣的是,PPDP显示小鼠的循环时间比PDP长得多(图10C)。After injecting the PPDPs containing Cy5-siPcsk9 into the mice, the imaging system was used to track the distribution of nanoparticles in the body. We found that after 24h, the cumulative amount of PPDP in the liver was greater than that of naked nanoparticles (Figure 10A). In vitro imaging of major organs showed that the PPDP retained in the liver was about 20 times higher than that of other organs (Figure 10B). In addition, the liver of PPDP-administered mice increased twice as much as that of PDP mice (Figure 10B). Interestingly, PPDP showed that the circulation time of mice was much longer than that of PDP (Figure 10C).
4.6为小鼠体内肝脏PCSK9与LDLR的表达水平,具体步骤与图11的蛋白印迹分析相同。购买的30只雌性C57BL/6小鼠(体重在18-20g),随机分成6组:Saline组、siPcsk9组、PP组、PPDL组、Lipo2000/siPcsk9组、PPDP组,PPDP是实验组,Lipo2000/siPcsk9是阳性对照组,而PPDL是用于检测siCPSK9特异性沉默作用的对照组,siPcsk9是未加修饰的药物对照组,PP组是不含药物的空载对照组,PBS是空白对照组。尾静脉给药每隔5天给药一次,尾静脉给药32天后,将每只鼠采全血200μL至1.5mLEP管中,待其凝固后3500rpm离心20min,取上层血清至新的离心管中,-80℃保存备用,随后给予安乐死,剥离下每只鼠的心肝脾肺肾。结果请参见图11。4.6 is the expression levels of PCSK9 and LDLR in the liver of mice, and the specific steps are the same as the Western blot analysis in Figure 11. Purchased 30 female C57BL/6 mice (weight 18-20g), randomly divided into 6 groups: Saline group, siPcsk9 group, PP group, PPDL group, Lipo2000/siPcsk9 group, PPDP group, PPDP is the experimental group, Lipo2000/ siPcsk9 is the positive control group, while PPDL is the control group used to detect the specific silencing effect of siCPSK9, siPcsk9 is the unmodified drug control group, the PP group is the drug-free control group, and PBS is the blank control group. Tail vein administration is administered every 5 days. After 32 days of tail vein administration, 200 μL of whole blood from each mouse is collected into a 1.5 mL EP tube, and after it has clotted, it is centrifuged at 3500 rpm for 20 minutes, and the upper layer of serum is transferred to a new centrifuge tube. , Preserved at -80°C for later use, and then euthanized. The heart, liver, spleen, lung and kidney of each mouse were stripped off. See Figure 11 for the result.
图11:我们评估了PPDPs在体内的影响,WB分析表明,PPDP治疗后,肝组织中的PCSK9水平有效抑制(图11A),与此一致的是肝组织中的低密度脂蛋白受体水平高于对照组(图11B)。Figure 11: We evaluated the effects of PPDPs in vivo. WB analysis showed that after PPDP treatment, the level of PCSK9 in liver tissue was effectively suppressed (Figure 11A). Consistent with this, the level of low-density lipoprotein receptors in liver tissue was high. In the control group (Figure 11B).
4.7为小鼠体内分离肝细胞与Kupffer细胞的LDLR蛋白印迹分析,具体步骤如下:4.7 is the LDLR western blot analysis of isolated hepatocytes and Kupffer cells in mice. The specific steps are as follows:
1)取给药PPDP 32d后C57BL/6小鼠以及生理盐水处理的C57BL/6小鼠为对照。1) C57BL/6 mice and C57BL/6 mice treated with normal saline were taken as controls 32 days after PPDP administration.
2)对小鼠进行麻醉,用含20mL 0.5mM EDTA的无钙Hank平衡盐溶液通过门静脉灌注肝脏。接着,将温预过后的含有0.05%胶原酶和5mM钙的20mL Hanks平衡盐溶液灌入肝脏。2) The mice were anesthetized, and the liver was perfused through the portal vein with 20 mL 0.5 mM EDTA-free Hank balanced salt solution. Next, 20 mL of Hanks' balanced salt solution containing 0.05% collagenase and 5 mM calcium, which has been pre-warmed, was infused into the liver.
3)采集肝组织并剪切,然后在37℃下胶原酶溶液中浸泡20min,使用细胞过滤器(美国BD Biosciences)过滤悬浮液。滤液以50g的速度离心5min,获得颗粒中的肝细胞。3) Collect liver tissue and cut it, then soak it in collagenase solution at 37°C for 20 minutes, and filter the suspension with a cell filter (BD Biosciences, USA). The filtrate was centrifuged at a speed of 50 g for 5 min to obtain hepatocytes in the pellet.
4)为了获得Kupffer细胞,上清液在330g下离心10min。使用PBS重新悬浮细胞颗粒,然后转移到Percoll混合溶液中(PBS中30%和70%Percoll)。混合物在1600g下离心30min,在30%和70%组分的界面处,回收Kupffer细胞,用冰冷PBS洗涤三次。用白细胞计数法分 析肝细胞和枯否细胞。结果请见图12和图13。4) In order to obtain Kupffer cells, the supernatant was centrifuged at 330g for 10 min. The cell pellet was resuspended in PBS, and then transferred to Percoll mixed solution (30% and 70% Percoll in PBS). The mixture was centrifuged at 1600 g for 30 min, and Kupffer cells were recovered at the interface of 30% and 70% components, and washed three times with ice-cold PBS. Analyze hepatocytes and Kupffer cells by white blood cell counting method. The results are shown in Figure 12 and Figure 13.
相应的剖面分析也显示了这一点。而无DOTAP(PLGA/siPcsk9)的核结构在9h内溶酶体逃逸效率较低,DOTAP修饰使siRNA具有更好的溶酶体逃逸能力。实时PCR检测表明PCSK9 mRNA通过PPDP(图8B)下调约66%。然而,对照组,PPDL,siPcsk9,PPD和PBS,在Pcsk9mRNA下调方面变化不明显(图8B)。与Lipo2000/siPcsk9相比,PPDP还显示出令人印象深刻的基因沉默效果(66%对74%)(图8B)。WB分析证实PPDP显著抑制PCSK9的表达(图8C)。我们设计了一种纳米结构来改善溶酶体逃逸,并特异性沉默靶向基因Pcsk9。The corresponding profile analysis also shows this. However, the nuclear structure without DOTAP (PLGA/siPcsk9) has a lower lysosomal escape efficiency within 9 hours, and DOTAP modification makes siRNA have better lysosomal escape ability. Real-time PCR detection showed that PCSK9 mRNA was down-regulated by about 66% through PPDP (Figure 8B). However, the control group, PPDL, siPcsk9, PPD and PBS, did not change significantly in the down-regulation of Pcsk9 mRNA (Figure 8B). Compared with Lipo2000/siPcsk9, PPDP also showed an impressive gene silencing effect (66% vs. 74%) (Figure 8B). WB analysis confirmed that PPDP significantly inhibited the expression of PCSK9 (Figure 8C). We designed a nanostructure to improve lysosomal escape and specifically silence the targeted gene Pcsk9.
图9:为了评价生物相容性,我们分析了PPDP的溶血和细胞毒性。无论是PPDP还是非包覆纳米颗粒在工作浓度均未引起任何显著的溶血毒性(图9A和9B),同时用CLSM(图9C)的活/死测定可以证实。然而,使用市售的Lipofectamine 2000会有明显溶血(图9D和9E),这表明PPDP引起较低的溶血和细胞毒性,对于体内应用是至关重要的,体外CLSM同时表明市售的Lipofectamine 2000相较于PPDP引起较高的细胞毒性(图9F)。Figure 9: To evaluate the biocompatibility, we analyzed the hemolysis and cytotoxicity of PPDP. Neither PPDP nor uncoated nanoparticles caused any significant hemolytic toxicity at working concentrations (Figures 9A and 9B), which can be confirmed by the live/dead assay of CLSM (Figure 9C). However, the use of commercially available Lipofectamine 2000 will have significant hemolysis (Figure 9D and 9E), which indicates that PPDP causes lower hemolysis and cytotoxicity, which is essential for in vivo applications. In vitro CLSM also shows that the commercially available Lipofectamine 2000 phase It caused higher cytotoxicity than PPDP (Figure 9F).
图10:给小鼠注射含Cy5-siPcsk9的PPDPs后,用成像系统跟踪纳米粒在体内的分布。我们发现,在24h后,PPDP在肝脏中的累积量大于裸纳米粒子(图10A)。主要器官的体外成像表明,与其他器官(图10B)相比,保留在肝脏中的PPDP高约20倍。此外,PPDP给药的小鼠肝脏比PDP小鼠增加了2倍(图10B)。有趣的是,PPDP显示小鼠的循环时间比PDP长得多(图10C)。Figure 10: After injection of PPDPs containing Cy5-siPcsk9 into mice, the imaging system was used to track the distribution of nanoparticles in the body. We found that after 24h, the cumulative amount of PPDP in the liver was greater than that of naked nanoparticles (Figure 10A). In vitro imaging of major organs showed that the PPDP retained in the liver was about 20 times higher than that of other organs (Figure 10B). In addition, the liver of PPDP-administered mice increased twice as much as that of PDP mice (Figure 10B). Interestingly, PPDP showed that the circulation time of mice was much longer than that of PDP (Figure 10C).
图11:我们评估了PPDPs在体内的影响,WB分析表明,PPDP治疗后,肝组织中的PCSK9水平有效抑制(图11A),与此一致的是肝组织中的低密度脂蛋白受体水平高于对照组(图11B)。Figure 11: We evaluated the effects of PPDPs in vivo. WB analysis showed that after PPDP treatment, the level of PCSK9 in liver tissue was effectively suppressed (Figure 11A). Consistent with this, the level of low-density lipoprotein receptors in liver tissue was high. In the control group (Figure 11B).
图12:为了阐明PPDP是否被肝脏巨噬细胞Kupffer细胞等免疫细胞摄取,我们用WB分析了两种细胞系中的LDLR,发现PPDP给药后肝细胞中的LDLR显著增加(图12)。但Kupffer细胞LDLR变化不大。Figure 12: In order to clarify whether PPDP is taken up by immune cells such as liver macrophages, Kupffer cells, we analyzed LDLR in two cell lines with WB and found that LDLR in liver cells increased significantly after PPDP administration (Figure 12). But the LDLR of Kupffer cells did not change much.
提取的组织的苏木素-伊红染色(hematoxylin eosin,HE)染色表明PPDP对小鼠没有明显的毒性(图13)。体内实验表明,PPDP可安全有效地降低PCSK9的表达,其作用机制与LDLR的上调和LDL-C的降低有关。Hematoxylin-eosin (HE) staining of the extracted tissues showed that PPDP has no obvious toxicity to mice (Figure 13). In vivo experiments have shown that PPDP can safely and effectively reduce the expression of PCSK9, and its mechanism of action is related to the up-regulation of LDLR and the decrease of LDL-C.
尽管本发明已进行了一定程度的描述,明显地,在不脱离本发明的精神和范围的条件下,可进行各个条件的适当变化。可以理解,本发明不限于所述实施方案,而归于权利要求的范围,其包括所述每个因素的等同替换。Although the present invention has been described to a certain extent, it is obvious that various conditions can be appropriately changed without departing from the spirit and scope of the present invention. It can be understood that the present invention is not limited to the embodiments, but belongs to the scope of the claims, which includes equivalent substitutions of each of the factors.
Claims (18)
- 一种能够沉默PCSK9蛋白的siRNA,其特征在于,所述siRNA的正义链氨基酸序列为5′-UUCCGAAUAAACUCCAGGC-3′,反义链氨基酸序列为5′-GCCUGGAGUUUAUUCGGAA-3′。An siRNA capable of silencing the PCSK9 protein is characterized in that the amino acid sequence of the sense strand of the siRNA is 5'-UUCCGAAUAAACUCCAGGC-3', and the amino acid sequence of the antisense strand is 5'-GCCUGGAGUUUAUUCGGAA-3'.
- 根据权利要求1所述的能够沉默PCSK9蛋白的siRNA,其特征在于,所述siRNA正义链和/或反义链的3′端还修饰有dTdT。The siRNA capable of silencing PCSK9 protein according to claim 1, wherein the 3'end of the sense strand and/or antisense strand of the siRNA is further modified with dTdT.
- 一种能够沉默PCSK9蛋白的纳米递送系统,其特征在于,所述纳米系统为生物膜伪装的包裹能够沉默PCSK9蛋白的siRNA的纳米囊。A nano-delivery system capable of silencing PCSK9 protein is characterized in that the nano-system is a nanocapsule camouflaged by a biological membrane that wraps siRNA capable of silencing PCSK9 protein.
- 根据权利要求3所述的能够沉默PCSK9蛋白的纳米递送系统,其特征在于,所述siRNA如权利要求1或2所述。The nano-delivery system capable of silencing PCSK9 protein according to claim 3, wherein the siRNA is according to claim 1 or 2.
- 根据权利要求3所述的纳米递送系统,其特征在于,所述生物膜选自以下一种或多种:血小板膜,红细胞膜,骨髓间充质干细胞,脐带间充质干细胞,巨噬细胞;优选为血小板膜。The nano-delivery system according to claim 3, wherein the biofilm is selected from one or more of the following: platelet membrane, red blood cell membrane, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and macrophages; Preferably, it is a platelet membrane.
- 根据权利要求3所述的纳米递送系统,其特征在于,所述纳米囊的材料选自以下一种或多种:PLGA、DOTAP、聚氨基酯、透明质酸、壳聚糖、聚乳酸;优选为PLGA和DOTAP。The nano delivery system according to claim 3, wherein the material of the nanocapsule is selected from one or more of the following: PLGA, DOTAP, polyurethane, hyaluronic acid, chitosan, polylactic acid; preferably For PLGA and DOTAP.
- 根据权利要求6所述的纳米递送系统,其特征在于,所述纳米囊中,PLGA与DOTAP的质量比为20:1~20:11,优选为20:1~20:5,最优选为20:3。The nano-delivery system according to claim 6, wherein the mass ratio of PLGA to DOTAP in the nanocapsule is 20:1-20:11, preferably 20:1-20:5, most preferably 20 :3.
- 根据权利要求3至7中任一项所述的纳米递送系统,其特征在于,所述生物膜蛋白、纳米囊与siRNA的质量份数为生物膜蛋白1~50份,纳米囊1~50份,siRNA0.1~10份;优选为生物膜10~30份,纳米囊10~30份,siRNA 0.5~2份;最优选为生物膜23份,纳米囊23份,siRNA 1份;The nano-delivery system according to any one of claims 3 to 7, wherein the mass parts of the biomembrane protein, nanocapsules and siRNA are 1-50 parts of biomembrane protein, and 1-50 parts of nanocapsules. , 0.1-10 parts of siRNA; preferably 10-30 parts of biofilm, 10-30 parts of nanocapsule, 0.5-2 parts of siRNA; most preferably 23 parts of biofilm, 23 parts of nanocapsule, and 1 part of siRNA;优选地,当纳米囊材料选自PLGA与DOTAP时,所述生物膜与所述纳米囊的质量比为1:1。Preferably, when the nanocapsule material is selected from PLGA and DOTAP, the mass ratio of the biofilm to the nanocapsule is 1:1.
- 根据权利要求3至7中任一项所述的纳米递送系统的制备方法,其特征在于,所述方法包括以下步骤:The method for preparing a nano delivery system according to any one of claims 3 to 7, wherein the method comprises the following steps:(1)制备siRNA溶液;(1) Prepare siRNA solution;(2)将步骤(1)制备的siRNA溶液滴加入纳米囊材料的溶液中;(2) Drop the siRNA solution prepared in step (1) into the solution of nanocapsule material;(3)将步骤(2)所得混合溶液滴加入溶剂中,超声搅拌离心去上清,重悬所得沉淀,重悬,得重悬液;(3) Add the mixed solution obtained in step (2) dropwise to the solvent, ultrasonically stir and centrifuge to remove the supernatant, resuspend the obtained precipitate, and resuspend to obtain a resuspension;(4)将步骤(3)所得重悬液与生物膜混合,超声,得到所述纳米递送系统。(4) Mix the resuspension solution obtained in step (3) with the biofilm, and sonicate to obtain the nano-delivery system.
- 根据权利要求9所述的制备方法,其特征在于,所述步骤(3)中,所述溶剂选自 以下一种或多种:PVA水溶液、吐温20水溶液、吐温80水溶液;优选为PVA水溶液,更优选为1%~10%的PVA水溶液,最优选为2%PVA水溶液。The preparation method according to claim 9, wherein in the step (3), the solvent is selected from one or more of the following: PVA aqueous solution, Tween 20 aqueous solution, Tween 80 aqueous solution; preferably PVA The aqueous solution is more preferably a 1%-10% PVA aqueous solution, and most preferably a 2% PVA aqueous solution.
- 一种下调低密度脂蛋白胆固醇的药物,其特征在于,所述药物包括:A medicine for lowering low-density lipoprotein cholesterol, characterized in that the medicine comprises:能够沉默PCSK9蛋白的siRNA;和/或SiRNA capable of silencing PCSK9 protein; and/or权利要求3至7中任一项所述的纳米递送系统。The nano delivery system of any one of claims 3 to 7.
- 根据权利要求11所述的下调低密度脂蛋白胆固醇的药物,其特征在于,所述siRNA如权利要求1或2所述。The medicine for down-regulating low-density lipoprotein cholesterol according to claim 11, wherein the siRNA is according to claim 1 or 2.
- 权利要求1或2所述的siRNA和/或权利要求3至7中任一项所述的纳米递送系统在制备用于治疗高胆固醇血症的药物中的应用。Application of the siRNA according to claim 1 or 2 and/or the nano delivery system according to any one of claims 3 to 7 in the preparation of a medicine for treating hypercholesterolemia.
- 一种药物纳米递送系统,其特征在于,其由包括负载有药物的纳米囊,以及将纳米囊包裹的生物膜构成,所述生物膜为血小板膜或红细胞膜。A drug nano-delivery system is characterized in that it is composed of a drug-loaded nanocapsule and a biofilm that wraps the nanocapsule, and the biofilm is a platelet membrane or a red blood cell membrane.
- 根据权利要求14所述的药物纳米递送系统,其特征在于,所述药物为siRNA,进一步优选为能够沉默PCSK9蛋白的siRNA,更优选为如SEQ ID NO.1和SEQ ID NO.2所示的siRNA。The drug nano-delivery system according to claim 14, wherein the drug is siRNA, further preferably siRNA capable of silencing PCSK9 protein, more preferably as shown in SEQ ID NO. 1 and SEQ ID NO. 2. siRNA.
- 根据权利要求14或15所述的药物纳米递送系统,其特征在于,制备所述纳米囊的材料选自以下一种或多种:PLGA、DOTAP、聚氨基酯、透明质酸、壳聚糖、聚乳酸;优选为PLGA和DOTAP。The drug nano-delivery system according to claim 14 or 15, wherein the material for preparing the nanocapsules is selected from one or more of the following: PLGA, DOTAP, polyurethane, hyaluronic acid, chitosan, Polylactic acid; preferably PLGA and DOTAP.
- 根据权利要求16所述的纳米递送系统,其特征在于,所述纳米囊中,PLGA与DOTAP的质量比为20:1~20:11,优选为20:1~20:5,最优选为20:3。The nano delivery system according to claim 16, wherein the mass ratio of PLGA to DOTAP in the nanocapsule is 20:1 to 20:11, preferably 20:1 to 20:5, most preferably 20 :3.
- 根据权利要求14所述的纳米递送系统,其特征在于,所述生物膜蛋白、纳米囊与药物的质量份数为生物膜1~50份,纳米囊1~50份,siRNA0.1~10份;优选为生物膜10~30份,纳米囊10~30份,siRNA 0.5~2份;最优选为生物膜23份,纳米囊23份,siRNA 1份;The nano-delivery system according to claim 14, wherein the mass parts of the biofilm protein, nanocapsules and drugs are 1-50 parts of biofilm, 1-50 parts of nanocapsules, and 0.1-10 parts of siRNA ; Preferably it is 10-30 parts of biomembrane, 10-30 parts of nanocapsule, 0.5-2 parts of siRNA; most preferably 23 parts of biomembrane, 23 parts of nanocapsule, and 1 part of siRNA;优选地,当纳米囊材料选自PLGA与DOTAP时,所述生物膜与所述纳米囊的质量比为1:1。Preferably, when the nanocapsule material is selected from PLGA and DOTAP, the mass ratio of the biofilm to the nanocapsule is 1:1.
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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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CN111803653B (en) * | 2020-06-27 | 2022-04-15 | 苏州大学 | Gene delivery system capable of removing mixed cell membrane coating and preparation method and application thereof |
CN112451504B (en) * | 2020-11-09 | 2022-10-18 | 四川大学华西医院 | Preparation method and application of core-shell nanoparticles carrying EBV-LMP2 mRNA |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011038031A1 (en) * | 2009-09-22 | 2011-03-31 | Alnylam Pharmaceuticals, Inc. | Dual targeting sirna agents |
WO2011119852A1 (en) * | 2010-03-24 | 2011-09-29 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering rnai compounds |
CN104651408A (en) * | 2009-06-15 | 2015-05-27 | 阿尔尼拉姆医药品有限公司 | Lipid formulated siRNA targeted to PCSK9 gene |
CN104854242A (en) * | 2012-12-05 | 2015-08-19 | 阿尔尼拉姆医药品有限公司 | PCSK9 iRNA compositions and methods of use thereof |
CN105164143A (en) * | 2013-03-14 | 2015-12-16 | 杰罗米.J.申塔格 | Cholestosome vesicles for incorporation of molecules into chylomicrons |
CN105497912A (en) * | 2016-01-21 | 2016-04-20 | 华东师范大学 | Preparation method and application of erythrocyte membrane-coated acid-sensitive polymer prodrug nano drug delivery system |
CN108350085A (en) * | 2015-10-16 | 2018-07-31 | 钜川生物医药 | Anti- PCSK9 antibody and its application |
CN108553650A (en) * | 2018-03-28 | 2018-09-21 | 暨南大学 | A kind of bionic nano red blood cell genophore and the preparation method and application thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010147992A1 (en) * | 2009-06-15 | 2010-12-23 | Alnylam Pharmaceuticals, Inc. | Methods for increasing efficacy of lipid formulated sirna |
US11224577B2 (en) * | 2015-06-19 | 2022-01-18 | The Regents Of The University Of California | Treating infection by a platelet-targeting microbe using nanoparticles |
CN108066763A (en) * | 2017-12-21 | 2018-05-25 | 陈敏 | Application of the PCSK9 inhibitor in the inflammatory-immune diseases medicine for preparing treatment T cell mediation |
CN109432048B (en) * | 2018-11-22 | 2021-03-19 | 东华大学 | Platelet membrane-coated drug-loaded porous nanoparticle and preparation method thereof |
-
2019
- 2019-10-25 CN CN201911022385.0A patent/CN110638788A/en active Pending
-
2020
- 2020-08-07 WO PCT/CN2020/107917 patent/WO2021077856A1/en active Application Filing
- 2020-08-07 CN CN202080072674.6A patent/CN114641279A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104651408A (en) * | 2009-06-15 | 2015-05-27 | 阿尔尼拉姆医药品有限公司 | Lipid formulated siRNA targeted to PCSK9 gene |
WO2011038031A1 (en) * | 2009-09-22 | 2011-03-31 | Alnylam Pharmaceuticals, Inc. | Dual targeting sirna agents |
WO2011119852A1 (en) * | 2010-03-24 | 2011-09-29 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering rnai compounds |
CN104854242A (en) * | 2012-12-05 | 2015-08-19 | 阿尔尼拉姆医药品有限公司 | PCSK9 iRNA compositions and methods of use thereof |
CN105164143A (en) * | 2013-03-14 | 2015-12-16 | 杰罗米.J.申塔格 | Cholestosome vesicles for incorporation of molecules into chylomicrons |
CN108350085A (en) * | 2015-10-16 | 2018-07-31 | 钜川生物医药 | Anti- PCSK9 antibody and its application |
CN105497912A (en) * | 2016-01-21 | 2016-04-20 | 华东师范大学 | Preparation method and application of erythrocyte membrane-coated acid-sensitive polymer prodrug nano drug delivery system |
CN108553650A (en) * | 2018-03-28 | 2018-09-21 | 暨南大学 | A kind of bionic nano red blood cell genophore and the preparation method and application thereof |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023051822A1 (en) * | 2021-09-30 | 2023-04-06 | 北京安龙生物医药有限公司 | Targeting oligonucleotide for treating diseases associated with pcsk9 |
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 |
CN117018229B (en) * | 2023-08-14 | 2024-05-24 | 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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