Nanoparticle for delivering siRNA and application thereof

Abstract

The invention discloses a nanoparticle for delivering siRNA, the nanoparticle is composed of an inner core and a shell, the inner core comprises API and a cationic polymer, and the shell is composed of a three-component lipid or a four-component lipid. It is preferable that the API is a siRNA of 15 to 60 nucleotides. The prepared nanoparticles can reduce expression of target genes in Hela cells, the inhibition rate reaches 95% or above, and the nanoparticles have great significance in application of tumor treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a nanoparticle for delivering siRNA and its application. Background Art

[0002] RNA interference refers to a phenomenon of gene silencing induced by double-stranded RNA in molecular biology. Its mechanism is to inhibit gene expression by hindering the transcription or translation of specific genes. When double-stranded RNA homologous to the coding region of endogenous messenger RNA (mRNA) is introduced into cells, the mRNA is degraded, resulting in gene expression silencing. Small interfering RNAs with a length of 20 - 25 nt can trigger RNAi, specifically down-regulate or turn off the expression of specific genes, and have the characteristics of high efficiency, easy synthesis, and easy operation. Therefore, this technology has been widely used in the fields of exploring gene functions and gene therapy for infectious diseases and malignant tumors.

[0003] In 2018, the first siRNA drug, Patisiran, was approved by the FDA for clinical use. However, the delivery of siRNA is difficult and it is easily degraded in vivo, significantly reducing its therapeutic effect. Therefore, a drug delivery system that can effectively deliver siRNA into cells to play a role plays an important role. The biggest problem currently existing in the application of drugs such as siRNA is the low delivery efficiency of siRNA at the cellular level, and even lower at the animal level. If the therapeutic effect is to be improved, the amount of siRNA needs to be increased, which will increase the toxicity of siRNA. Therefore, currently, the key to the application of drugs such as siRNA is how to improve the delivery efficiency of siRNA and other drugs and reduce their toxicity and side effects. Summary of the Invention

[0004] The object of the present invention is to provide a nanoparticle for delivering siRNA.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A nanoparticle for delivering siRNA, the nanoparticle is composed of a core and a shell, the core includes API and a cationic polymer, and the shell is composed of a three-component lipid or a four-component lipid.

[0007] Preferably, the API is siRNA.

[0008] Preferably, the siRNA is a modified or unmodified siRNA with 15 - 60 nucleotides.

[0009] Preferably, the cationic polymer includes polyethyleneimine, chitosan, poly-L-lysine, poly-L-arginine, poly-L-histidine, polyamidoamine, protamine sulfate, polycaprolactone, polylactic acid, and dicyandiamide.

[0010] Preferably, the three-component lipid includes a phospholipid or its derivative, cholesterol or its derivative, and a conjugated lipid.

[0011] Preferably, the four-component lipid includes a cationic lipid or an ionizable lipid, a phospholipid or its derivative, cholesterol or its derivative, and a conjugated lipid.

[0012] Preferably, the phospholipid or its derivative is one or more of DSPC (dipalmitoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), and DMPC (dimyristoylphosphatidylcholine).

[0013] Preferably, the cholesterol or its derivative is one or more of cholesterol, vitamin D derivatives, and steroid substances.

[0014] Preferably, the conjugated lipid is one or more of PEG-DAG (polyethylene glycol-diacylglycerol) conjugate, PEG-DMA (polyethylene glycol-dimyristyloxypropyl) conjugate, PEG-DSA (polyethylene glycol-distearyloxypropyl) conjugate, and DMG-PEG2000 (dimyristoyl glycerol-polyethylene glycol 2000) conjugate.

[0015] Preferably, the cationic lipid is one or more of DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride) and DOTMA (1,2-dioctadecyloxy-3-methylammonium propane chloride); or the ionizable lipid is one or more of DLin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (dilinoleoyl) methyl ester), DLin-KC2-DMA (2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), ALC-0315 (((4-hydroxybutyl)aza dialkyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate)), and SM-102 (heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)).

[0016] Preferably, the nitrogen-to-phosphorus ratio of the cationic polymer to siRNA is 1 to 40.

[0017] Preferably, in the three-component lipid, the phospholipid or its derivative accounts for 10 mol% to 90 mol% of the total lipid; cholesterol or its derivative accounts for 10 mol% to 90 mol% of the total lipid; and the conjugated lipid accounts for 0.2 mol% to 5 mol% of the total lipid.

[0018] Preferably, in the four-component lipid, the cationic lipid or ionizable lipid accounts for 55 mol% to 70 mol% of the total lipid; the phospholipid or its derivative accounts for 10 mol% to 21.3 mol% of the total lipid; the cholesterol or its derivative accounts for 10 mol% to 50 mol% of the total lipid; and the conjugated lipid accounts for 0.2 mol% to 1.7 mol% of the total lipid.

[0019] Preferably, the application of the nanoparticles in anti-tumor drugs.

[0020] The beneficial effects that can be achieved by the present invention are as follows:

[0021] 1. The nanoparticles obtained by the present invention are applicable to the delivery of siRNA;

[0022] 2. The nanoparticles obtained by the present invention can specifically deliver siRNA to the tumor site;

[0023] 3. The three-component lipid nanoparticles obtained by the present invention can avoid the use of cationic lipids, reducing immune stimulation and inflammatory reactions. Description of the Drawings

[0024] The following further describes in detail the specific embodiments of the present invention with reference to the drawings:

[0025] Figure 1 It is the KD effect diagram in Hela cells for Example 1;

[0026] Figure 2 It is the KD effect diagram in Hela cells for three-component lipid nanoparticles with different nitrogen-phosphorus ratios;

[0027] Figure 3 It is the KD effect diagram in Hela cells for three-component lipid nanoparticles with different lipid ratio compositions;

[0028] Figure 4 It is the KD effect diagram in Hela cells for four-component lipid nanoparticles with different lipid ratio compositions;

[0029] Figure 5 It is the encapsulation effect diagram for three-component lipid nanoparticles with different concentrations. Specific Embodiments

[0030] Cationic polymers include substances that can electrostatically adsorb and self-assemble with siRNA and have delivery functions, which are naturally extracted or artificially synthesized. In this application, polyethyleneimine is selected for the three-component lipid, and protamine sulfate is selected for the four-component lipid. One or more of polyethyleneimine, protamine sulfate, chitosan, poly-L-lysine, poly-L-arginine, poly-L-histidine, polyamidoamine, polycaprolactone, polylactic acid, and dicyandiamide can also be selected.

[0031] The phospholipid or its derivative is one or more of DSPC (dipalmitoyl phosphatidylcholine), DPPC (dipalmitoyl phosphatidylcholine), and DMPC (dimyristoyl phosphatidylcholine). DSPC is selected in this application.

[0032] The cholesterol or its derivative is one or more of cholesterol, vitamin D derivative, and steroid substance. Cholesterol is selected in this application.

[0033] The conjugated lipid is one or more of PEG-DAG (polyethylene glycol-diacylglycerol) conjugate, PEG-DMA (polyethylene glycol-dimyristyloxypropyl) conjugate, PEG-DSA (polyethylene glycol-distearyloxypropyl) conjugate, and DMG-PEG2000 (dimyristoyl glycerol-polyethylene glycol 2000) conjugate. DMG-PEG2000 is selected in this application.

[0034] The cationic lipid is one or more of DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride) and DOTMA (1,2-dioctadecyloxy-3-methylammonium propane chloride); or the ionizable lipid is one or more of DLin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (dilinoleoyl) methyl ester), DLin-KC2-DMA (2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), ALC-0315 (((4-hydroxybutyl)azaalkanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and SM-102 (heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)). Dlin-MC3-DMA is selected for the four-component lipid in this application.

[0035] Example 1: Preparation of three-component lipid and four-component lipid nanoparticles and investigation of cell KD effect

[0036] 1. Preparation of three-component lipid nanoparticles and investigation of cell KD effect

[0037] 1) Take 1.316 mg of API (API refers to the active pharmaceutical ingredient, preferably siRNA), add 4 ml of enzyme-free and sterile water, measure the absorbance to be 325.089 ng / µl, and then add 9 ml of enzyme-free and sterile water to dilute to 0.1 mg / ml (the concentration is measured by Nano-300) to prepare an API solution for storage.

[0038] 2) Weigh 1.329 mg of polyethyleneimine, add 1.3 ml of enzyme-free and sterile water to dissolve until clear and transparent, with a concentration of 1 mg / ml, to prepare a polyethyleneimine solution for storage at room temperature.

[0039] 3) Weigh 8.319 mg of DSPC, 4.178 mg of cholesterol, and 1.889 mg of DMG-PEG2000 in sequence, dissolve them in 3.9 ml of absolute ethanol until clear and transparent to prepare a lipid phase solution, and store it for later use.

[0040] 4) Manually mix 0.5 ml of the API solution and 0.5 ml of the polyethyleneimine solution, vortex to obtain a preliminary mixed intermediate solution, and let it stand at room temperature for incubation for 15 minutes.

[0041] 5) Then manually mix 0.9 ml of the preliminary mixed intermediate solution and 0.3 ml of the lipid phase solution, vortex to obtain a total mixed intermediate solution.

[0042] 6) Dilute the total mixed intermediate solution by 3 times according to the theoretical concentration, and then filter it through a bacteria-removing filter to obtain the sample.

[0043] As Figure 1 shown, the prepared sample has a KD effect of more than 95% on Hela cells.

[0044] 2. Preparation of nanoparticles of four-component lipids and investigation of cell KD effect

[0045] 1) Take 2.255 mg of API (siRNA), add 3 ml of enzyme-free and sterile water to measure the absorbance of 776.322 ng / µl, and then add 8 ml of enzyme-free and sterile water to dilute it to 0.2 mg / ml (measure the concentration with Nano-300) to prepare an API solution, and store it for later use.

[0046] 2) Weigh 3.233 mg of protamine sulfate, add 1.35 ml of enzyme-free and sterile water to dissolve it until clear and transparent, with a concentration of 2.4 mg / ml, to prepare a protamine sulfate solution, and store it at room temperature for later use.

[0047] 3) Weigh 8.475 mg of Dlin-MC3-DMA, 4.313 mg of DSPC, 2.099 mg of cholesterol, and 1.040 mg of DMG-PEG2000 in sequence, dissolve them in 1.9 ml of absolute ethanol until clear and transparent to prepare a lipid phase solution, and store it for later use.

[0048] 4) Manually mix 0.5 ml of the API solution and 0.5 ml of the protamine sulfate solution, vortex to obtain a preliminary mixed intermediate solution, and let it stand at room temperature for incubation for 15 minutes.

[0049] 5) Then manually mix 0.9 ml of the preliminary mixed intermediate solution and 0.3 ml of the lipid phase solution, vortex to obtain a total mixed intermediate solution.

[0050] 6) Dilute the total mixed intermediate solution by 3 times according to the theoretical concentration, and then filter it through a bacteria-removing filter to obtain the sample.

[0051] As Figure 1 shown, the prepared sample was tested and had a KD effect of more than 90% on Hela cells.

[0052] The results showed that, as Figure 1 shown, both the three-component lipid and the four-component lipid had good inhibitory effects, and the three-component lipid was slightly better than the four-component lipid. In addition, since the three-component lipid does not contain cationic lipids, it can reduce immune stimulation and inflammatory responses.

[0053] Example 2: Preparation of three-component lipid nanoparticle samples with different nitrogen-phosphorus ratios and investigation of cell KD effects (N / P = 1 - 40)

[0054] 1) Take 2.255 mg of API (siRNA), add 3 ml of enzyme-free and sterile water to measure the absorbance of 776.322 ng / µl, and then add 8 ml of enzyme-free and sterile water to dilute to 0.2 mg / ml (the concentration is measured with Nano-300) to prepare an API solution for storage.

[0055] 2) Preparation for the N / P = 40 group: Weigh 5.556 mg of polyethyleneimine, add 2.778 ml of enzyme-free and sterile water to dissolve until it is clear and transparent, with a concentration of 2 mg / ml, and store at room temperature for use;

[0056] Preparation for the N / P = 30 group: Take another 0.75 ml of 2 mg / ml polyethyleneimine solution, add 0.25 ml of enzyme-free and sterile water to dilute to 1.5 mg / ml;

[0057] Preparation for the N / P = 20 group: Take 1 ml of 2 mg / ml polyethyleneimine solution, add 1 ml of enzyme-free and sterile water to dilute to 1 mg / ml;

[0058] Preparation for the N / P = 10 group: Take 1 ml of 1 mg / ml polyethyleneimine solution, add 1 ml of enzyme-free and sterile water to dilute to 0.5 mg / ml;

[0059] Preparation for the N / P = 5 group: Take 1 ml of 0.5 mg / ml polyethyleneimine solution, add 1 ml of enzyme-free and sterile water to dilute to 0.25 mg / ml;

[0060] Preparation for the N / P = 1 group: Take 0.2 ml of 0.25 mg / ml polyethyleneimine solution, add 0.8 ml of enzyme-free and sterile water to dilute to 0.05 mg / ml, and store the above polyethyleneimine solutions with different concentrations for use.

[0061] 3) Weigh 4.709 mg of DSPC, 2.46 mg of cholesterol, and 1.166 mg of DMG-PEG2000 in sequence, dissolve them in 2.2 ml of absolute ethanol until it is clear and transparent to prepare a lipid phase solution for storage.

[0062] 4) Prepare samples according to different N / P ratios. Manually mix 0.5 ml of the API solution and 0.5 ml of the delivery substance solution, and vortex to obtain a preliminary mixed intermediate solution. Let it stand at room temperature for incubation for 15 minutes.

[0063] 5) Manually mix 0.9 ml of the preliminary mixed intermediate solution and 0.3 ml of the lipid phase solution according to the groups, and vortex to obtain the total mixed intermediate solution.

[0064] 6) Dilute the total mixed intermediate solution 3 times according to the theoretical concentration, and then sterilize and filter to obtain the preparation samples with different N / P ratios.

[0065] 7) Detect the KD effect of the prepared samples on Hela cells.

[0066] As Figure 2 shown, the experimental results show that good KD effects are achieved when N / P = 10 - 40, especially when N / P is above 20, the KD effects all reach more than 90%.

[0067] Example 3: Preparation of nanoparticle samples with different lipid ratios and investigation of cell KD effects

[0068] 1) Weigh 2.521 mg of API (siRNA), add 6 ml of enzyme-free and sterile water, measure the absorbance to be 379.103 ng / µl, and then add 5 ml of enzyme-free and sterile water to dilute to 0.2 mg / ml (measure the concentration with Nano-300) to prepare the API solution for storage.

[0069] 2) Weigh 4.185 mg of polyethyleneimine, add 4.185 ml of enzyme-free and sterile water to dissolve until clear and transparent, with a concentration of 1 mg / ml, to prepare the polyethyleneimine solution for storage at room temperature.

[0070] 3) Weigh 6.176 mg of protamine sulfate, add 5.147 ml of enzyme-free and sterile water to dissolve until clear and transparent, with a concentration of 1.2 mg / ml, to prepare the protamine sulfate solution for storage at room temperature.

[0071] 4) Prepare the lipid phase solutions of ternary lipids and quaternary lipids

[0072] Durable range of ternary lipids (molar ratio) (0820-F001 (22)-Lip)

[0073] ① DSPC (10%)

[0074] Weigh 0.988 mg of DSPC, 4.063 mg of cholesterol, and 2.101 mg of DMG-PEG2000 in sequence, dissolve them in 1.907 ml of absolute ethanol until clear and transparent, and store for use.

[0075] ② DSPC (47%)

[0076] Weigh out 8.319 mg of DSPC, 4.178 mg of cholesterol, and 1.889 mg of DMG-PEG2000 successively, and dissolve them in 3.9 ml of absolute ethanol until clear and transparent, then store for later use.

[0077] ③ DSPC (90%)

[0078] Weigh out 39.655 mg of DSPC, 2.069 mg of cholesterol, and 1.039 mg of DMG-PEG2000 successively, and dissolve them in 11.404 ml of absolute ethanol until clear and transparent, then store for later use.

[0079] ④ Cholesterol (10%)

[0080] Weigh out 16.062 mg of DSPC, 0.948 mg of cholesterol, and 4.010 mg of DMG-PEG2000 successively, and dissolve them in 5.605 ml of absolute ethanol until clear and transparent, then store for later use.

[0081] ⑤ Cholesterol (49%)

[0082] Weigh out 8.319 mg of DSPC, 4.178 mg of cholesterol, and 1.889 mg of DMG-PEG2000 successively, and dissolve them in 3.9 ml of absolute ethanol until clear and transparent, then store for later use.

[0083] ⑥ Cholesterol (90%)

[0084] Weigh out 4.055 mg of DSPC, 19.053 mg of cholesterol, and 1.019 mg of DMG-PEG2000 successively, and dissolve them in 6.434 ml of absolute ethanol until clear and transparent, then store for later use.

[0085] ⑦ DMG-PEG2000 (0.2%)

[0086] Weigh out 79.983 mg of DSPC, 40.049 mg of cholesterol, and 1.035 mg of DMG-PEG2000 successively, and dissolve them in 32.285 ml of absolute ethanol until clear and transparent, then store for later use.

[0087] ⑧ DMG-PEG2000 (4%)

[0088] Weigh out 8.319 mg of DSPC, 4.178 mg of cholesterol, and 1.889 mg of DMG-PEG2000 successively, and dissolve them in 3.9 ml of absolute ethanol until clear and transparent, then store for later use.

[0089] ⑨ DMG-PEG2000 (5%)

[0090] Weigh 4.126 mg of DSPC, 2.014 mg of cholesterol, and 1.471 mg of DMG-PEG2000 in sequence, dissolve them in 2.030 ml of absolute ethanol until clear and transparent, and store for later use.

[0091] Four-component lipid durability range (molar ratio) (0820-PRTM-Lip)

[0092] ① Dlin-MC3-DMA (55%)

[0093] Weigh 8.475 mg of Dlin-MC3-DMA, 4.313 mg of DSPC, 2.099 mg of cholesterol, and 1.040 mg of DMG-PEG2000 in sequence, dissolve them in 1.9 ml of absolute ethanol until clear and transparent, and prepare a lipid phase solution for storage.

[0094] ② Dlin-MC3-DMA (70%)

[0095] Weigh 15.624 mg of Dlin-MC3-DMA, 4.140 mg of DSPC, 1.945 mg of cholesterol, and 1.158 mg of DMG-PEG2000 in sequence, dissolve them in 6.098 ml of absolute ethanol until clear and transparent, and store for later use.

[0096] ③ DSPC (10%)

[0097] Weigh 8.434 mg of Dlin-MC3-DMA, 1.615 mg of DSPC, 2.048 mg of cholesterol, and 1.059 mg of DMG-PEG2000 in sequence, dissolve them in 3.508 ml of absolute ethanol until clear and transparent, and store for later use.

[0098] ④ DSPC (21.3%)

[0099] Weigh 8.475 mg of Dlin-MC3-DMA, 4.313 mg of DSPC, 2.099 mg of cholesterol, and 1.040 mg of DMG-PEG2000 in sequence, dissolve them in 1.9 ml of absolute ethanol until clear and transparent, and prepare a lipid phase solution for storage.

[0100] ⑤ Cholesterol (10%)

[0101] Weigh 12.747 mg of Dlin-MC3-DMA, 6.098 mg of DSPC, 1.247 mg of cholesterol, and 1.626 mg of DMG-PEG2000 in sequence, dissolve them in 5.791 ml of absolute ethanol until clear and transparent, and store for later use.

[0102] ⑥ Cholesterol (22%)

[0103] Weigh 8.475 mg of Dlin-MC3-DMA, 4.313 mg of DSPC, 2.099 mg of cholesterol, and 1.040 mg of DMG-PEG2000 in sequence. Dissolve them in 1.9 ml of absolute ethanol until clear and transparent to obtain a lipid phase solution, which is stored for later use.

[0104] ⑦ Cholesterol (50%)

[0105] Weigh 8.498 mg of Dlin-MC3-DMA, 4.170 mg of DSPC, 7.174 mg of cholesterol, and 1.279 mg of DMG-PEG2000 in sequence. Dissolve them in 5.632 ml of absolute ethanol until clear and transparent, and store for later use.

[0106] ⑧ DMG-PEG2000 (0.2%)

[0107] Weigh 83.922 mg of Dlin-MC3-DMA, 40.004 mg of DSPC, 20.146 mg of cholesterol, and 1.212 mg of DMG-PEG2000 in sequence. Dissolve them in 38.742 ml of absolute ethanol until clear and transparent, and store for later use.

[0108] ⑨ DMG-PEG2000 (1.7%)

[0109] Weigh 8.475 mg of Dlin-MC3-DMA, 4.313 mg of DSPC, 2.099 mg of cholesterol, and 1.040 mg of DMG-PEG2000 in sequence. Dissolve them in 1.9 ml of absolute ethanol until clear and transparent to obtain a lipid phase solution, which is stored for later use.

[0110] 5) Preparation of the preliminary mixing intermediate solution

[0111] Prepare the samples in sequence according to the groups. Manually mix 0.5 ml of the API solution with 0.5 ml of the polyethyleneimine solution and 0.5 ml of the protamine sulfate solution respectively, vortex, and incubate at room temperature for 15 minutes.

[0112] 6) Preparation of the total mixing intermediate solution

[0113] Manually mix 0.9 ml of the preliminary mixing intermediate solution and 0.3 ml of the lipid phase solution according to the groups respectively, and vortex.

[0114] 7) Dilution and filtration

[0115] Dilute the total mixing intermediate solution 3 times according to the theoretical concentration, and then filter it through bacteria removal to obtain the sample.

[0116] 8) Detect the KD effect of the prepared sample on Hela cells

[0117] As Figures 3 - 4As shown, the results indicate that both lipid formulations have good KD effects within a certain range, and the durability of the three-component lipid formulation is significantly better than that of the four-component lipid formulation.

[0118] Example 4: Preparation of nanoparticles by microfluidics and investigation of stability

[0119] 1) Preparation of API (siRNA) solution

[0120] Weigh 1.499 mg of API, add 7 ml of enzyme-free and sterile water, measure the absorbance to be 200.715 ng / µl (concentration determined by Nano-300), and store for later use.

[0121] 2) Preparation of polyethyleneimine solution

[0122] Weigh 5.140 mg of polyethyleneimine, add 5.14 ml of enzyme-free and sterile water to dissolve until clear and transparent, with a concentration of 1 mg / ml, and store for later use.

[0123] 3) Preparation of lipid solution

[0124] Weigh 6.462 mg of DSPC, 3.258 mg of cholesterol, and 1.671 mg of DMG-PEG2000 in sequence, dissolve them in 3 ml of absolute ethanol until clear and transparent, and store for later use.

[0125] 4) Preparation of primary mixed intermediate solution

[0126] Take 5 ml each of the API solution and the polyethyleneimine solution, mix them at a microfluidic flow rate between 2 ml / min and 12 ml / min, and then let them stand at room temperature for incubation for 15 minutes.

[0127] 5) Preparation of total mixed intermediate solution

[0128] Mix 9 ml of the primary mixed intermediate solution and 3 ml of the lipid phase solution at a microfluidic flow rate between 2 ml / min and 12 ml / min.

[0129] 6) Ultrafiltration

[0130] Take the total mixed intermediate solution for ultrafiltration, control the shear force between 1000 s -1 ~4000 s -1 After concentration, wash and filter with a 10-fold volume.

[0131] 7) Investigation of sample stability

[0132] The total mixed intermediate solution after ultrafiltration is sterilized by filtration to obtain the final product. Generally, the particle size of the nanoparticles is 40 - 200 nm. Measure the particle size and distribution and the potential, and investigate the sample stability under the two conditions of 5°C and 25°C. The specific data are shown in the following table:

[0134] It can be seen from the data that the particle size, distribution and potential of the nanoparticles show almost no obvious changes under the two conditions of 5°C and 25°C, indicating good stability.

[0135] Example 5: Gel electrophoresis was used to investigate the encapsulation effect of the ternary lipid nanoparticles.

[0136] 1) Prepare a 4 µg / ml control API solution.

[0137] 2) Prepare nanoparticle samples with different concentrations: Take 100 µl of the stability sample (the theoretical concentration of API is 75 µg / ml) and dilute it to 200 µl with enzyme-free and sterile water to obtain Sample 1 (the theoretical concentration of API is 37.5 µg / ml).

[0138] Take 107 µl from the Sample 1 solution and dilute it to 200 µl with enzyme-free and sterile water to obtain Sample 2 solution (the theoretical concentration of API is 20 µg / ml).

[0139] Take 100 µl from the Sample 2 solution and dilute it to 200 µl with enzyme-free and sterile water to obtain Sample 3 solution (the theoretical concentration of API is 10 µg / ml).

[0140] Take 100 µl from the Sample 3 solution and dilute it to 200 µl with enzyme-free and sterile water to obtain Sample 4 solution (the theoretical concentration of API is 5 µg / ml).

[0141] 3) Prepare the gel: Weigh 0.5021 g of agarose into a conical flask, add 50 ml of 1×TAE buffer, heat to dissolve the agarose, then add 2.5 µl of nucleic acid dye and mix well. Pour it out while it is hot and let it cool to form a gel.

[0142] 4) Load the sample and perform electrophoresis: Prepare the loading solution by mixing 10 µl of the above four sample solutions with 10 µl of RNA loading buffer. Load 18 µl into each well and perform electrophoresis using a gel electrophoresis apparatus at 120 V for 25 min. After electrophoresis, use a gel imaging system to take pictures.

[0143] As Figure 5 shown, the results show that the encapsulation effects of the 4 groups of samples are all good, and there is almost no free drug.

[0144] The foregoing detailed description of a series of embodiments is merely a specific description of the feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Those skilled in the art can design many other modifications and embodiments, and these modifications and embodiments will fall within the scope of the principles and spirit disclosed in this application. More specifically, within the scope of the disclosure, drawings and claims of this application, various variations and improvements can be made to the components and / or layout of the subject combination layout. In addition to the variations and improvements made to the components and / or layout, other uses will also be obvious to those skilled in the art.

Claims

1. A nanoparticle for delivering siRNA, characterized in that: The nanoparticle consists of an inner core and an outer shell, wherein the inner core comprises siRNA and a cationic polymer, and the outer shell consists of three-component lipids or four-component lipids.

2. The nanoparticle according to claim 1, characterized in that The cationic polymer includes polyethyleneimine, chitosan, poly-L-lysine, poly-L-arginine, poly-L-histidine, polyamidoamine, protamine sulfate, polycaprolactone, polylactic acid, and polycyanamide.

3. The nanoparticles according to claim 1, characterized in that The three-component lipid comprises phospholipid or its derivative, cholesterol or its derivative and conjugated lipid.

4. The nanoparticles according to claim 1, characterized in that The four-component lipid comprises a cationic lipid or an ionizable lipid, a phospholipid or a derivative thereof, cholesterol or a derivative thereof, and a conjugated lipid.

5. The nanoparticles according to claim 3 or 4, characterized in that The phospholipid or its derivative is one or more of DSPC (distearylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), and DMPC (dimyristoylphosphatidylcholine).

6. The nanoparticles according to claim 3 or 4, characterized in that The cholesterol or its derivative is one or more of cholesterol, vitamin D derivatives, and steroid substances.

7. The nanoparticles according to claim 3 or 4, characterized in that The conjugated lipid is one or more of a PEG-DAG (polyethylene glycol-diacylglycerol) conjugate, a PEG-DMA (polyethylene glycol-dimyristyloxypropyl) conjugate, a PEG-DSA (polyethylene glycol-distearyloxypropyl) conjugate, and a DMG-PEG2000 (dimyristoylglycerol-polyethylene glycol 2000) conjugate.

8. The nanoparticles according to claim 4, characterized in that The cationic lipid is one or more of DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride), DOTMA (1,2-dioctadeceneoxy-3-methylammonium propane chloride); or the ionizable lipid is one or more of DLin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), ALC-0315 (((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), SM-102 (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate)).

9. The nanoparticle according to claim 1, characterized in that The nitrogen-phosphorus ratio of the cationic polymer to the siRNA is 1-40.

10. The nanoparticles according to claim 3, characterized in that Among the three components of lipids, phospholipids or their derivatives account for 10 mol% to 90 mol% of the total lipids; cholesterol or its derivatives account for 10 mol% to 90 mol% of the total lipids; and conjugated lipids account for 0.2 mol% to 5 mol% of the total lipids.

11. The nanoparticle according to claim 4, characterized in that Among the four components of lipids, cationic lipids or ionizable lipids account for 55 mol% to 70 mol% of the total lipids; phospholipids or their derivatives account for 10 mol% to 21.3 mol% of the total lipids; cholesterol or its derivatives account for 10 mol% to 50 mol% of the total lipids; and conjugated lipids account for 0.2 mol% to 1.7 mol% of the total lipids.

12. The nanoparticle according to any one of claims 1 to 11, characterized in that The nanoparticles are used in tumor treatment drugs.

Images