Nucleic acid delivery carrier based on natural lipid as well as preparation method and application of nucleic acid delivery carrier

Abstract

The invention discloses a nucleic acid delivery carrier based on natural lipids and a preparation method and application thereof, the natural lipids of the nucleic acid delivery carrier comprise coix seed oil, brucea javanica oil and soybean oil, and other components comprise DOTAP, DOPE, glycerin and deionized water. The nucleic acid delivery carrier for delivering the siRNA drug has difference in cellular uptake and transfection efficiencies, and the lysosome escape condition and lipid droplet co-localization result taking the coix seed oil and the brucea javanica oil as lipids are superior to those of soybean oil; and the in-vitro transfection efficiency of the coix seed oil nucleic acid delivery vector on AsPC-1 and human embryo kidney cell strain HEK293T cells has no significant difference with the cell transfection efficiency of a commercially available nucleic acid delivery vector. The constructed natural nucleic acid delivery carrier is prepared into a freeze-dried preparation, so that the stability of the nucleic acid delivery carrier can be improved; the natural nucleic acid delivery carrier which is redissolved after being freeze-dried still keeps relatively good transfection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a natural lipid-based nucleic acid delivery vector and a preparation method and application thereof. Background Art

[0002] siRNA is a double-stranded RNA composed of approximately 19-23 nucleotides, which participates in the post-translational gene silencing mechanism. When siRNA is effectively delivered to the target site, its antisense strand will interact with the RNA-induced silencing complex (RISC) and decompose the complementary mRNA sequence under the action of Argonaut. Ultimately, this method will block the expression of specific genes. However, free siRNA has certain limitations. It is not easy to penetrate the cell membrane and is easily degraded by nucleases. Among the current vectors for delivering siRNA, viral vectors and non-viral vectors are the main ones. Compared with viral vectors, non-viral vectors have lower immunogenicity and high drug loading, but non-viral vectors also have two major problems. One is that the targeting is not strong, and siRNA is easily taken up by the reticuloendothelial system and enriched in the liver and spleen; the other is that the lysosomal escape is not strong. If siRNA cannot escape from the lysosome, it will be degraded by nucleases. Therefore, the development of an effective and safe siRNA delivery vector remains a major challenge.

[0003] Lipid nanoparticles (LNPs) are currently the most important non-viral carriers for delivering siRNA drugs. Their key component, ionizable lipids, have great advantages in nucleic acid delivery, such as high nucleic acid encapsulation efficiency, effective cell transfection, and strong tissue penetration. However, they also have the problem of low lysosomal escape efficiency. In addition, ionizable lipids also face complex structures, high prices, and potential clinical safety issues. Natural lipids such as coix seed lipids and brucea javanica lipids have the characteristics of abundant resources, high biocompatibility, and high human safety.

[0004] Coix seed oil, Brucea javanica oil and soybean oil are all injection-grade pharmaceutical excipients. Among them, coix seed oil is the main component of the traditional Chinese medicine coix seed, and has been proven to have anti-tumor, antioxidant, and immunity-enhancing effects. In addition to the already marketed Kanglaite injection, nanocarriers with coix seed oil as one of the components have been shown to be able to synergize with other drugs to enhance lysosomal escape and anti-tumor efficacy. Brucea javanica oil is a complex mixture of fatty acids and fatty acid derivatives obtained by extracting the fruit of Brucea javanica, a plant of the Simaroubaceae family, with petroleum ether. It has anti-inflammatory, antimalarial and anti-tumor activities. Soybean oil is a fatty oil extracted from the seeds of the leguminous plant soybean. Due to its abundant resources and high compatibility, it has been widely used as the oil phase for the preparation of lipid emulsions, such as marketed drugs such as aprepitant emulsion and propofol medium-chain fat emulsion.

[0005] Lysosomal escape efficiency and biocompatibility are key issues that need to be addressed for nucleic acid delivery vectors. In addition, the currently marketed nucleic acid delivery vector preparations are mostly in the form of suspension injections, which have the problems of poor formulation stability and strict storage conditions. How to improve the storage stability of nucleic acid delivery vectors is also an issue that needs to be addressed urgently. Freeze drying, as a drying technology, is an attractive method to improve the thermal stability of nucleic acid delivery vectors. Summary of the invention

[0006] The purpose of the present invention is to provide a natural lipid-based nucleic acid delivery vector and a preparation method and application thereof in view of the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A nucleic acid delivery vector based on natural lipids comprises the following components: natural lipids, DOTAP, DOPE, glycerol and deionized water; the natural lipids are coix seed oil, javanica oil or soybean oil.

[0009] A method for preparing a nucleic acid delivery vector based on natural lipids comprises the following steps:

[0010] 1. DOTAP, DOPE and natural lipids are mixed and dissolved in a water bath at 25°C-70°C to obtain an oil phase, wherein the natural lipids are coix seed oil, Brucea javanica oil or soybean oil;

[0011] 2. Mix glycerol and water and place in a 25℃-70℃ water bath to obtain the aqueous phase;

[0012] 3. When the temperature of the water phase is consistent with that of the oil phase, the water phase is slowly added dropwise to the oil phase under the action of magnetic stirring; colostrum is then prepared by a high-pressure homogenization method, and then the colostrum is prepared into a nucleic acid delivery vector by a probe ultrasound method; the nucleic acid delivery vector has a potential of 40-55mV and a particle size of 160nm-700nm, and a more preferred particle size of 170nm-190nm.

[0013] Furthermore, in the step 1, the mass percentage of DOTAP is 0.18%-1.06%, the mass percentage of DOPE is 0.06%-0.12%, and the mass percentage of natural lipids is 1.79%-15.26%; in the step 2, the mass percentage of glycerol is 2.11%, and the mass percentage of deionized water is 82.16%-95.63%.

[0014] Furthermore, the mass percentage of DOTAP is 0.35%, the mass percentage of DOPE is 0.12%, the mass percentage of natural lipids is 3.52%, the mass percentage of glycerol is 2.11%, and the mass percentage of deionized water is 93.9%.

[0015] Furthermore, in steps 1 and 2, the water bath temperature is 25°C-70°C.

[0016] Furthermore, the water bath temperature was 70°C.

[0017] Furthermore, in step 3, the high-pressure homogenization conditions are 2000r / min-20000r / min, 5min; the ultrasonic conditions are 50W-200W, 5min, and the ultrasonic operation is repeated for 2s and the interval is 2s.

[0018] Furthermore, the high-pressure homogenization conditions are 20000 r / min, 5 min; and the ultrasonic conditions are 200 W, 5 min.

[0019] Furthermore, the nucleic acid delivery vector obtained in step 3 is dissolved in a lyophilization protectant, and a lyophilized powder preparation is obtained by freeze-drying technology.

[0020] Furthermore, the lyoprotectant is mannose, glucose or sucrose, and the mass ratio of the nucleic acid delivery vector to the lyoprotectant is 1:2, 1:4, 1:6, 1:8 or 1:10.

[0021] Furthermore, the lyophilization protectant is glucose, and the mass ratio of the nucleic acid delivery vector to the lyophilization protectant is 1:8.

[0022] The present invention also provides a lyophilized powder preparation prepared according to the above preparation method, which improves the stability of the nucleic acid delivery vector and facilitates the storage and transportation of the nucleic acid delivery vector.

[0023] A natural lipid-based nucleic acid delivery vector is used in the delivery of siRNA. The nucleic acid delivery vector can be combined with siRNA through electrostatic interaction, and lipid droplet targeting is used to enhance the lysosomal escape efficiency of siRNA and reduce the degradation of siRNA.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses natural lipids as pharmaceutical excipients instead of synthetic lipids as one of the key components of nucleic acid delivery vectors, which can improve the safety and biocompatibility of nucleic acid delivery vectors, and natural lipids have the characteristics of simple preparation and abundant resources compared with synthetic lipids, which is conducive to accelerating the development of nucleic acid delivery vectors in the field of nucleic acid delivery. At the same time, the natural lipid nucleic acid delivery vector can enhance the lysosomal escape of siRNA through lipid droplet targeting, and the transfection efficiency of coix seed oil nucleic acid delivery vector is not significantly different from that of commercially available transfection reagents.

[0026] The present invention prepares a natural lipid-based nucleic acid delivery vector into a lyophilized preparation through freeze-drying technology, thereby improving the stability of the nucleic acid delivery vector, facilitating the storage and transportation of the nucleic acid delivery vector, and at the same time the lyophilized preparation still maintains a relatively good transfection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The electron micrographs of three natural lipid nucleic acid delivery vectors are shown in Figure 1, wherein (a) is an electron micrograph of a coix seed oil nucleic acid delivery vector, (b) is an electron micrograph of a javanica oil nucleic acid delivery vector, and (c) is an electron micrograph of a soybean seed oil nucleic acid delivery vector.

[0028] Figure 2 Schematic diagram of the adsorption capacity of three natural lipid nucleic acid delivery carriers for siRNA at different N / P ratios;

[0029] Figure 3 The figures are the cytotoxicity experimental diagrams of three natural lipid nucleic acid delivery vectors on three types of cells, wherein (a) is the cytotoxicity diagram of three natural lipid nucleic acid delivery vectors on AsPC-1, (b) is the cytotoxicity diagram of three natural lipid nucleic acid delivery vectors on human normal epithelial cell line HPDE6-C7, and (c) is the cytotoxicity diagram of three natural lipid nucleic acid delivery vectors on human embryonic kidney cell line HEK293T;

[0030] Figure 4 The cellular uptake diagram of three natural lipid nucleic acid delivery vectors loaded with FAM-siRNA in the human tumor cell line AsPC-1;

[0031] Figure 5 Figures 2 and 3 are diagrams showing the lysosomal escape of three natural lipid nucleic acid delivery vectors loaded with FAM-siRNA in the human tumor cell line AsPC-1, wherein (a) is a diagram showing the lysosomal escape after 6 h, (b) is a representative co-localization analysis curve of Lipo in (a), (c) is a representative co-localization analysis curve of coix seed oil nucleic acid delivery vector in (a), (d) is a representative co-localization analysis curve of javanica oil nucleic acid delivery vector in (a), (e) is a representative co-localization analysis curve of soybean oil nucleic acid delivery vector in (a), (f) is a diagram showing the lysosomal escape after 12 h, (g) is a representative co-localization analysis curve of Lipo in (f), (h) is a representative co-localization analysis curve of coix seed oil nucleic acid delivery vector in (f), (i) is a representative co-localization analysis curve of javanica oil nucleic acid delivery vector in (f), and (j) is a representative co-localization analysis curve of soybean oil nucleic acid delivery vector in (f);

[0032] Figure 6Figures are of the co-localization of three natural lipid nucleic acid delivery vectors loaded with FAM-siRNA and lipid droplets in the human tumor cell line AsPC-1, wherein (a) is a representative laser confocal image, (b) is a representative co-localization analysis curve of Lipo in (a), (c) is a representative co-localization analysis curve of coix seed oil nucleic acid delivery vector in (a), (d) is a representative co-localization analysis curve of javanica oil nucleic acid delivery vector in (a), and (e) is a representative co-localization analysis curve of soybean oil nucleic acid delivery vector in (a);

[0033] Figure 7 The results of immunoblotting of three natural lipid nucleic acid delivery vectors mediated siRNA knockdown of target protein expression levels in different types of cell lines; (a) shows the results of three natural lipid nucleic acid delivery vectors mediated siRNA knockdown of Kras in human tumor cell line AsPC-1 G12D Schematic diagram of protein expression levels, (b) is a schematic diagram of three natural lipid nucleic acid delivery vectors mediated siRNA knockdown of Kras protein expression levels in human normal epithelial cell line HPDE6-C7, (c) is a schematic diagram of three natural lipid nucleic acid delivery vectors mediated siRNA knockdown of Kras protein expression levels in human embryonic kidney cell line HEK293T;

[0034] Figure 8 The electron micrographs of three natural lipid nucleic acid delivery vectors after freeze-drying and reconstitution are shown, wherein (a) is an electron micrograph of coix seed oil nucleic acid delivery vector, (b) is an electron micrograph of javanica oil nucleic acid delivery vector, and (c) is an electron micrograph of soybean seed oil nucleic acid delivery vector;

[0035] Fig. 9 The results of the siRNA adsorption capacity of three natural lipid nucleic acid delivery carriers after freeze-drying and reconstitution at different N / P ratios;

[0036] Fig.10 The figure shows the immunoblotting results of three natural lipid nucleic acid delivery vectors after lyophilization. DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention, and the protection scope of the present invention is not limited to the following embodiments.

[0038] Example 1: Preparation of natural lipid nucleic acid delivery vector

[0039] The specific preparation steps are as follows: 0.35% DOTAP, 0.12% DOPE and 3.52% coix seed oil / brucea javanica oil / soybean oil are mixed and dissolved in a 70°C water bath to obtain an oil phase. Glycerin and water are mixed and placed in a 70°C water bath to obtain an aqueous phase. When the temperature of the aqueous phase is consistent with that of the oil phase, the aqueous phase is slowly added dropwise to the oil phase under the action of magnetic stirring. First, high-pressure homogenization is performed at 20,000 r / min for 5 min, and then the nucleic acid delivery vector is ultrasonically treated with an ultrasonic cell disruptor probe for 5 min (200 W, working for 2 s, interval for 2 s) to obtain the final nucleic acid delivery vector.

[0040] Comparative Example 1:

[0041] 0.35% DOTAP, 0.12% DOPE and 1.79% coix seed oil / javanica oil / soybean oil in a mass percentage were mixed and dissolved in a 70° C. water bath to obtain an oil phase. Other operations were the same as in Example 1.

[0042] Comparative Example 2:

[0043] 0.35% DOTAP, 0.12% DOPE and 6.80% coix seed oil / javanica oil / soybean oil were mixed and dissolved in a 55° C. water bath to obtain an oil phase. Other operations were the same as in Example 1.

[0044] Comparative Example 3:

[0045] 0.35% DOTAP, 0.12% DOPE and 15.26% coix seed oil / javanica oil / soybean oil were mixed and dissolved in a 25° C. water bath to obtain an oil phase. Other operations were the same as in Example 1.

[0046] Comparative Example 4:

[0047] 0.18% DOTAP, 0.06% DOPE and 3.52% coix seed oil / javanica oil / soybean oil in a mass percentage were mixed and dissolved in a 45° C. water bath to obtain an oil phase. Other operations were the same as in Example 1.

[0048] Comparative Example 5:

[0049] 1.05% DOTAP, 0.12% DOPE and 3.52% coix seed oil / javanica oil / soybean oil in a mass percentage were mixed and dissolved in a 60° C. water bath to obtain an oil phase. Other operations were the same as in Example 1.

[0050] Example 2: Determination of particle size, polydispersity coefficient and potential of nucleic acid delivery vector

[0051] After the nucleic acid delivery vectors prepared in Example 1 and Comparative Examples 1-5 were diluted 100 times, the particle size, polydispersity coefficient and potential of the nucleic acid delivery vectors were detected using a Malvern laser particle size analyzer NanoZS90. The results are shown in Tables 1-3.

[0052] Table 1: Effects of the mass percentage of coix seed oil, DOTAP, and DOPE on the particle size, polydispersity coefficient, and Zeta potential of nucleic acid delivery vectors

[0053]

[0054] Table 2: Effects of the mass percentage of Brucea javanica oil, DOTAP, and DOPE on the particle size, polydispersity coefficient, and Zeta potential of nucleic acid delivery vectors

[0055]

[0056] Table 3: Effects of the mass percentage of soybean oil, DOTAP, and DOPE on the particle size, polydispersity coefficient, and Zeta potential of nucleic acid delivery vectors

[0057]

[0058] It can be found from Tables 1-3 that the polydispersity coefficients of the three natural lipid nucleic acid delivery vectors in Example 1 are less than 0.2, and the particle size is about 160-180 nm, which is better than Comparative Examples 2-5. Although Comparative Example 1 has a better polydispersity coefficient and particle size, considering that natural lipids play a role of drug-excipient in nucleic acid delivery vectors, it is more preferred to increase the mass percentage of natural lipids under the premise that both the particle size and the polydispersity coefficient are better, so the prescription of Example 1 is used as the final prescription for subsequent research.

[0059] Example 3: Transmission electron microscopy (TEM) detection of nucleic acid delivery vector

[0060] The three prepared natural lipid nucleic acid delivery vectors were subjected to transmission electron microscopy detection using transmission electron microscopy negative staining technology, in order to observe the morphology of the three natural lipid nucleic acid delivery vectors prepared in Example 1.

[0061] The results are as follows Figure 1 As shown, it was observed that the prepared nucleic acid delivery vector had a spherical appearance, a good polydispersity coefficient, and a particle size of about 160-180 nm.

[0062] Example 4: Agarose gel electrophoresis blocking experiment

[0063] The ability of three natural lipid nucleic acid delivery vectors to load siRNA was characterized by agarose gel electrophoresis experiments. In short, take 0.4g of low melting point agarose, add 40mLl×TAE buffer, and heat for 2min to completely dissolve it. After the solution is slightly cooled, add 4μL of gel red nucleic acid dye and mix well. Pour the solution into a gel plate of appropriate size. Set different N / P ratios to configure the corresponding concentration of the nucleic acid delivery vector obtained in Example 1 and incubate it with siRNA. After the gel is formed, run the samples of different N / P ratio groups at 120V for 15min. Then observe the siRNA bands in the gel imaging system.

[0064] like Figure 2 As shown, there is no free siRNA in the three natural lipid nucleic acid delivery carriers when the N / P ratio is 2:1, proving that siRNA can be completely adsorbed at this N / P ratio.

[0065] NOTE: The ratio between the cationic nitrogen groups (N) from the ionizable / cationic lipids of the delivery system and the negative charges of the phosphate groups (P) from the RNA is defined as the N / P ratio.

[0066] Example 5: Toxicity study of three natural lipid nucleic acid delivery vectors

[0067] Cells in the logarithmic growth phase (human tumor cell line AsPC-1, human embryonic kidney cell line HEK293T, human normal epithelial cell line HPDE6-C7) were inoculated into 96-well cell culture plates at appropriate densities, incubated overnight in an incubator at 37°C and 5% CO2 by volume, and then a series of nucleic acid delivery vectors of different concentrations were added. After incubation in the incubator for 48 hours, the original culture medium was discarded, and the cells were washed 2-3 times with phosphate buffer. After adding culture medium containing MTS and PMS (20:1 ratio) to each well, the cells were incubated for another 2-3 hours, and the absorbance value was measured at 490nm using an ELISA reader.

[0068] The results are as follows Figure 3 As shown, within the set concentration range (0-200μg / mL), when the highest concentration of the three nucleic acid delivery vectors reached 200μg / ml, the survival rates of the three cells were all 80% and above. The administration concentration of the three nucleic acid delivery vectors was ≤50μg / ml, and the survival rates of the three cells were all greater than 90% and above. This shows that the toxicity test results of the three new nucleic acid delivery vectors on the three cells are consistent. Within the set concentration range, there is no significant change in cell activity, and they all have high safety, and are transfection preparations with high safety.

[0069] Example 6: Cellular Uptake of Three Natural Lipid Nucleic Acid Delivery Vectors

[0070] Human tumor cell line AsPC-1 in logarithmic growth phase was cultured at 1×10 4 The cells were inoculated into cell slides at a density of 10 cells / mL and incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded 4 h before administration and serum-free culture medium was added for pretreatment. Lipofectamine RNAiMAX (Lipo) was used to prepare Lipo / FAM-siRNA complexes according to the instructions. Coix seed oil nucleic acid delivery vector, Brucea javanica oil nucleic acid delivery vector, and soybean oil nucleic acid delivery vector were also diluted with serum-free culture medium and added to FAM-siRNA solutions of the same concentration for standby use. The cells were rinsed twice with phosphate buffer, and the drug-containing culture medium of free FAM-siRNA, Lipo / FAM-siRNA, coix seed oil nucleic acid delivery vector / FAM-siRNA, Brucea javanica oil nucleic acid delivery vector / FAM-siRNA, and soybean oil nucleic acid delivery vector / FAM-siRNA (containing 200 nM siRNA in each group) was incubated in a cell culture incubator for 6 h. The culture medium was discarded, and the cells were washed three times with phosphate buffer. After fixing the cells with 4% paraformaldehyde for 10 minutes, the cells were washed three times with phosphate buffer, and then 0.2 μg / mL 4',6-diamidino-2-phenylindole (DAPI) solution was added and incubated at room temperature for 10 minutes. The cells were washed three times with phosphate buffer. The slides were taken out and sealed with anti-fluorescence quenching sealing liquid, and the cell uptake of FAM-siRNA was observed using a laser confocal microscope. Among them, the excitation wavelength of DAPI is λ = 358nm, and the emission wavelength is λ = 461nm; the excitation wavelength of FAM is λ = 494nm, and the emission wavelength is λ = 519nm.

[0071] The results are as follows Figure 4 As shown in the figure, the fluorescence intensity of coix seed oil nucleic acid delivery vector / FAM-siRNA, brucea javanica oil nucleic acid delivery vector / FAM-siRNA, and soybean oil nucleic acid delivery vector / FAM-siRNA groups were significantly higher than that of the free FAM-siRNA group, and a large amount of FAM fluorescence signals were visible in the cells. At the same time, the fluorescence signal values ​​of coix seed oil nucleic acid delivery vector / FAM-siRNA, brucea javanica oil nucleic acid delivery vector / FAM-siRNA and Lipo / FAM-siRNA groups were close, indicating that the transfection efficiency of nucleic acid delivery vectors is similar to that of Lipo, and siRNA can be effectively delivered into cells.

[0072] Example 7: Lysosomal escape of three natural lipid nucleic acid delivery vectors

[0073] siRNA needs to reach the cytoplasm to exert its therapeutic effect, and lysosomes are the key obstacle to its reaching the cytoplasm. The efficiency of lysosomal escape is the key evaluation point for the delivery of siRNA vectors. Therefore, the present invention conducts a lysosomal escape experiment on the prepared novel nucleic acid delivery vector. The human tumor cell line AsPC-1 in the logarithmic growth phase was inoculated in a confocal microplate (1×10 4 The cells were incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded 4 h before administration and serum-free culture medium was added for pretreatment. The cells were rinsed twice with phosphate buffer, and the drug-containing culture medium of Lipo / FAM-siRNA, coix seed oil nucleic acid delivery vector / FAM-siRNA, Brucea javanica oil nucleic acid delivery vector / FAM-siRNA, and soybean oil nucleic acid delivery vector / FAM-siRNA (200 nM siRNA in each group) was incubated in a cell culture incubator for 6 and 12 h. The culture medium was removed, the cells were washed 3 times with phosphate buffer, and then stained with lysosomal fluorescent probe LysoTracker Red preheated at 37°C for 30 min at 37°C; the dye working solution was aspirated, the cells were washed 3 times with precooled phosphate buffer, and the cells were incubated with Hoechst33342 nuclear dye (2 μg / ml) at room temperature for 10 min. After the cells were washed 3 times with phosphate buffer, they were placed under a laser confocal microscope (Zeiss LSM880) for observation. Among them, the excitation wavelength λ of Hoechst33342 is 346 nm, and the emission wavelength λ is 460 nm; the excitation wavelength λ of Ly soybean oil Tracker Red is 577 nm, and the emission wavelength λ is 590 nm.

[0074] The results are as follows Figure 5 As shown in the figure, when Lipo, coix seed oil nucleic acid delivery vector, Brucea oil nucleic acid delivery vector, and soybean oil nucleic acid delivery vector carrying FAM-siRNA were co-incubated with human tumor cell line AsPC-1 for 6 hours, the co-localization results showed that the results of the four groups of preparations showed that most of the green light emitted by FAM-siRNA overlapped with the red light emitted by the lysosome to show a yellow signal, indicating that the vector entered the lysosome through the endocytosis pathway, and the carried FAM-siRNA had not completely escaped from the lysosome at 6 hours. When the incubation time was extended to 12 hours, most of the green fluorescence in the coix seed oil nucleic acid delivery vector and Brucea oil nucleic acid delivery vector groups was separated from the red fluorescence, indicating that the siRNA basically escaped from the lysosome and the siRNA was successfully delivered. However, part of the green fluorescence in the Lipo group and the soybean oil nucleic acid delivery vector group was separated from the red fluorescence.

[0075] Example 8: Experiment on co-localization of siRNA and lipid droplets with three natural lipid nucleic acid delivery vectors

[0076] The human tumor cell line AsPC-1 was inoculated in a confocal microplate (1×10 4 100 μL of Bodipy was added. TM 558 / 568c12 dye working solution, gently shake it to completely cover the cells, and incubate for 10 minutes. Aspirate the dye working solution and wash the cells three times with phosphate buffer. Rinse the cells twice with phosphate buffer, add Lipo / FAM-siRNA, coix seed oil nucleic acid delivery vector / FAM-siRNA, Brucea javanica oil nucleic acid delivery vector / FAM-siRNA, and soybean oil nucleic acid delivery vector / FAM-siRNA drug-containing culture medium (each group contains 200nM siRNA), and incubate in a cell culture incubator for 12 hours. Discard the culture medium, wash the cells three times with phosphate buffer, fix the cells with 4% paraformaldehyde for 10 minutes, wash the cells three times with phosphate buffer, and then add 0.2μg / mL DAPI solution and incubate at room temperature for 10 minutes. After washing the cells three times with phosphate buffer, observe under a laser confocal microscope (Zeiss LSM880). Among them, Bodipy TM The excitation wavelength λ of 558 / 568c12 is 558nm, and the emission wavelength λ is 568nm.

[0077] The results are as follows Figure 6 As shown, the FAM green fluorescence and Bodipy TM The yellow signal of the overlapping red fluorescence of 558 / 568c12 was strong, indicating that the adsorbed FAM-siRNA in the coix seed oil nucleic acid delivery vector group and the Brucea javanica oil nucleic acid delivery vector group had more obvious co-localization with lipid droplets. However, there was no obvious co-localization phenomenon in the Lipo group and the soybean oil nucleic acid delivery vector group. The above results show that Chinese medicine lipids (CSO and BJO) have natural lipid droplet targeting compared with natural lipids (SO), and can mediate and enhance the lysosomal escape of siRNA.

[0078] Example 9: Transfection efficiency experiment of three natural lipid nucleic acid delivery vectors

[0079] In order to verify the transfection effect of the novel nucleic acid delivery vector prepared by the present invention, the present invention selected KRAS G12D The three nucleic acid delivery vectors prepared in Example 1 of the present invention were used to transfect siKRAS into the human tumor cell line AsPC-1. G12D, transfected siKRAS into human embryonic kidney cell line HEK293T and human normal epithelial cell line HPDE6-C7 cells. The target gene was selected because KRAS has been considered an "undruggable" target for decades, and there are relatively few targeted drugs directly targeting the KRAS gene on the market. The present invention envisions using siRNA to knock down the KRAS gene to achieve the corresponding therapeutic effect.

[0080] After 48h and 72h of siRNA transfection, the expression of Kras in human tumor cell line AsPC-1 was detected by real-time fluorescence quantitative PCR and immunoblotting WB, respectively. G12D The relative expression of mRNA, the relative expression of KRAS in human embryonic kidney cell line HEK293T, human normal epithelial cell line HPDE6-C7 cells and the corresponding protein expression level. The specific methods are as follows:

[0081] 1. Extraction of cellular RNA and reverse transcription of cDNA

[0082] Discard the cell culture medium, wash with phosphate buffer 2-3 times, lyse the cells with AG RNAex Pro RNA extraction reagent, let stand on ice for 10 minutes, and transfer to a nuclease-free 1.5mL EP tube. Add an appropriate amount of chloroform to 0.2mL chloroform per 1mL Trizol, shake vigorously for 15-30s, let stand for 2-3min, and then centrifuge at 12000rpm and 4℃ for 15min. Take the upper colorless liquid and put it in a new nuclease-free 1.5mL EP tube, add an equal volume of isopropanol and mix, let stand at room temperature for 5min to precipitate RNA, and then centrifuge at 12000rpm and 4℃ for 15min. Discard the supernatant, take the precipitate and wash it with 500μL of 75% ethanol by volume, and centrifuge it at 12000rpm and 4℃ for 15min. Repeat the wash 2-3 times, dry the RNA at room temperature, and add an appropriate amount of DEPC water to dissolve it. The concentration and purity of RNA were tested using a micro-nucleic acid protein detector (Nanodrop 2000). An appropriate amount of extracted RNA was reverse transcribed into cDNA using the Evo M-MLV reverse transcription premix kit. The relative expression of mRNA of related genes was detected using an RT-PCR instrument (Applied BiosystemsTM 7500).

[0083] 2. qPCR detection

[0084] (1) The reaction system was 20 μL, 10 μL BrightCycle Universal SYBR Green qPCR Mix with UDG (Abclonal, RK21219), 1 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, and 8.2 μL DEPC water.

[0085] (2) The reaction was carried out on an Applied BiosystemsTM 7500 instrument.

[0086] (3) The reaction conditions were as follows: Step 1: pre-denaturation at 95°C for 3 min; Step 2: collecting fluorescence signals at 95°C for 5 s and 60°C for 30 s, for a total of 40 cycles.

[0087] (4) Data analysis: KRAS was analyzed using the ΔΔCT method G12D mRNA and KRAS mRNA expression in cells.

[0088] 3. Extraction and content determination of total protein

[0089] Discard the cell culture medium and wash 2-3 times with pre-cooled phosphate buffer. Add 100 μL of lysis solution containing 1% protease inhibitor and 1% phosphatase inhibitor to each well and place on ice for 30 minutes. Use a clean cell scraper to scrape the cells on ice and gather them on one side of the culture dish, and then use a pipette to transfer the resulting cell lysate to a 1.5 ml EP tube. Move the EP tube containing the sample into an ultra-speed low-temperature centrifuge, centrifuge at 4°C and 12000rpm for 15 minutes, and use a pipette to aspirate the supernatant to obtain a total protein extract.

[0090] According to the instructions of the BCA protein quantification kit, a standard protein curve was prepared. The absorbance was detected at 562nm using an enzyme reader, and the standard curve was analyzed, calculated, and drawn. According to the standard curve, the total protein content in the extracted protein sample was calculated. According to the calculation results in the previous step, appropriate lysis buffer was added to the protein sample to unify the protein content between samples. The protein mixture and 5× loading buffer were mixed at a volume ratio of 4:1, vortexed on a dry vortex instrument, centrifuged gently, and vortexed gently again to mix the two thoroughly. Place the EP tube in a dry thermostatic heater, boil at 100℃ for 5min, open the lid to release air, cool on ice, and store in a -20℃ refrigerator.

[0091] 4. Immunoblotting

[0092] Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk for 2 h, and incubated with primary antibodies (Anti-KRAS G12D: AB221163, 1:1000, Anti-KRAS: HA601059, 1:1000, Anti GAPDH: AM8539b, 1:2000) at 4°C overnight, and then incubated with secondary antibodies (Goat Anti-Rabbit IgG: B2001RA45, 1:2000, Goat Anti Mouse Ig G: RS23710, 1:2000) at room temperature for 2 h. Exposure was performed using the LI-corOdyssey Clx near-infrared imaging system.

[0093] This experiment set up a normal control group, a positive control Lipo group, an experimental group, and a negative control NC siRNA group. The results are shown in Table 4. The transfection results of the human tumor cell line AsPC-1 showed that all natural nucleic acid delivery vectors could successfully mediate siRNA knockdown of KRAS in the human tumor cell line AsPC-1. G12D At the gene and protein levels. Coix seed oil nucleic acid delivery vector, Brucea javanica oil nucleic acid delivery vector, and soybean oil nucleic acid delivery vector respectively induced KRAS in the human tumor cell line AsPC-1. G12D mRNA levels decreased by 67.87%, 47.18%, and 42.52%. The three nucleic acid delivery vectors and the positive control vector Lipo reduced KRAS G12D Compared with Lipo mRNA 73.43%, there was no significant difference in the knockdown efficiency of coix seed oil nucleic acid delivery vector. Although the knockdown efficiency of javanica oil nucleic acid delivery vector and soybean oil nucleic acid delivery vector was weaker than that of Lipo, they also reached 64.25% and 57.91% of Lipo, respectively.

[0094] The transfection results of human normal epithelial cell line HPDE6-C7 and human embryonic kidney cell line HEK293T cells are shown in Tables 5-6. All natural nucleic acid delivery vectors can also successfully mediate siRNA to knock down the KRAS gene and protein levels of human normal epithelial cell line HPDE6-C7 and human embryonic kidney cell line HEK293T cells. Coix seed oil nucleic acid delivery vector, Brucea javanica oil nucleic acid delivery vector, and soybean oil nucleic acid delivery vector reduced the KRAS mRNA level of human normal epithelial cell line HPDE6-C7 cells by 64.31%, 43.10%, and 31.51%, respectively. The KRAS mRNA level of human embryonic kidney cell line HEK293T cells was reduced by 63.46%, 54.58%, and 46.52%, respectively. For human normal epithelial cell line HPDE6-C7 cells, the knockdown efficiency of coix seed oil nucleic acid delivery vector, javanica oil nucleic acid delivery vector, and soybean oil nucleic acid delivery vector reached 88.52%, 59.33%, and 43.37%, respectively, compared with the positive control vector Lipo, which reduced KRAS mRNA by 72.65%. For human embryonic kidney cell line HEK293T cells, the transfection results were similar to those of human tumor cell line AsPC-1. Compared with the positive control vector Lipo, which reduced KRAS mRNA by 69.07%, there was no significant difference in the knockdown efficiency of coix seed oil nucleic acid delivery vector. Although the knockdown efficiency of javanica oil nucleic acid delivery vector and soybean oil nucleic acid delivery vector was weaker than Lipo, they also reached 79.02% and 67.35%, respectively. The results of immunoblotting are shown in Figure 7 The results further prove the above conclusions.

[0095] Table 4 AsPC-1 cell transfection effect test data

[0096] Group (ASPC-1 cells) <![CDATA[KRAS G12D Relative mRNA expression (%)]]> Control 100.54±9.52 Lipo 26.57±2.87 NC siRNA 96.63±8.65 Coix seed oil nucleic acid delivery vector 32.13±3.89 Brucea javanica oil nucleic acid delivery vector 52.82±7.65 Soybean Oil Nucleic Acid Delivery Vector 57.48±8.90

[0097] Table 5 HPDE6-C7 cell transfection effect test data

[0098] Group (HPDE6-C7 cells) KRAS mRNA relative expression (%) Control 101.81±10.38 Lipo 27.35±2.45 NC siRNA 108.06±8.11 Coix seed oil nucleic acid delivery vector 35.69±3.59 Brucea javanica oil nucleic acid delivery vector 56.90±9.82 Soybean Oil Nucleic Acid Delivery Vector 68.49±8.61

[0099] Table 6 HEK293T cell transfection effect test data

[0100] Group (HEK293T cells) KRAS mRNA relative expression (%) Control 100.02±9.80 Lipo 30.93±4.64 NC siRNA 101.77±8.85 Coix seed oil nucleic acid delivery vector 36.54±4.69 Brucea javanica oil nucleic acid delivery vector 45.42±3.71 Soybean Oil Nucleic Acid Delivery Vector 53.48±7.10

[0101] Example 10: Lyoprotectants affect the physicochemical properties of lyophilized nucleic acid delivery vectors

[0102] At present, the strict storage conditions of nucleic acid delivery vectors limit the further development of nucleic acid delivery vectors. In order to improve the storage conditions of nucleic acid delivery vectors, the present invention starts from improving the problem of instability of nucleic acid delivery vectors, adopts freeze-drying method, and adds different lyophilization protective agents to improve the stability of nucleic acid delivery vectors.

[0103] Take 0.4 mL of the coix seed oil nucleic acid delivery vector prepared in Example 1 in a 5 mL vial. Add lyophilization protectant (lyophilization protectant includes mannitol, glucose and sucrose) according to a certain mass ratio of nucleic acid delivery vector and lyophilization protectant, and vortex to fully dissolve it. Freeze the mixture at -20 ° C for 12 h in advance, and then vacuum freeze-dry for 48 h. Observe the appearance after lyophilization and measure the particle size and polydispersity coefficient after reconstitution.

[0104] Combined with Table 7, it can be seen that the blank control group without the addition of lyophilization protectant has a yellow oily appearance, and the particle size and polydispersity coefficient are significantly increased to 953.4±48.6nm and 0.898 respectively. When glucose is selected as a lyophilization protectant and the ratio of the oil phase to the lyophilization protectant is 1:8, the appearance of the freeze-dried coix seed oil nucleic acid delivery vector is relatively ideal, snow-white, smooth, and full, and the particle size and polydispersity coefficient are not significantly increased to 204.4±1.9nm and 0.180 respectively. When mannitol is used as a lyophilization protectant, the appearance of the coix seed oil nucleic acid delivery vector is collapsed and the particle size and polydispersity coefficient are significantly increased. Sucrose as a lyophilization protectant has no significant increase in particle size and polydispersity coefficient compared to before lyophilization, but the appearance of the lyophilized product fails to achieve the ideal lyophilization effect. Therefore, the mass ratio of nucleic acid delivery vector and lyophilization protectant is selected to be 1:8, and glucose is used as a lyophilization protectant.

[0105] After determining the ratio of nucleic acid delivery vector and lyoprotectant and the type of lyoprotectant, the present invention applies the lyophilization conditions to the lyophilization of brucea oil nucleic acid delivery vector and soybean oil nucleic acid delivery vector, and finds that the lyophilization conditions are also suitable for brucea oil nucleic acid delivery vector and soybean oil nucleic acid delivery vector. After lyophilization, it has an ideal appearance of snow-white, smooth, and full state, and the particle size and potential have not changed significantly, as shown in Table 8. The particle size and potential of brucea oil nucleic acid delivery vector are 192.5±1.1nm and 42.2±0.4mV, respectively, and the particle size and potential of soybean oil nucleic acid delivery vector are 198.4±2.8nm and 48.8±0.3mV, respectively.

[0106] Table 7: Effects of lyophilization protectant, nucleic acid delivery vector and lyophilization protectant mass ratio on particle size and polydispersity coefficient of coix seed oil nucleic acid delivery vector

[0107]

[0108]

[0109] Table 8: Average particle size and polydispersity coefficient of nucleic acid delivery vector after lyophilization

[0110] Average particle size / nm Polydispersity coefficient Coix seed oil nucleic acid delivery vector 204.4±1.9 0.180 Brucea javanica oil nucleic acid delivery vector 192.5±1.1 0.152 Soybean Oil Nucleic Acid Delivery Vector 198.4±2.8 0.172

[0111] Example 11: Electron micrograph of freeze-dried nucleic acid delivery vector

[0112] Freeze drying may destroy the morphology of the nanocarriers. Therefore, the present invention conducted electron microscopic observation on the three nucleic acid delivery vectors after freeze drying. Figure 8 As shown in the figure, the three nucleic acid delivery vectors after freeze-drying all maintain the same morphology as before freeze-drying, which is spherical. Electron microscopy shows that the freeze-drying conditions can stably preserve the nucleic acid delivery vectors and do not destroy the morphological characteristics of the nucleic acid delivery vectors.

[0113] Example 12: Evaluation of siRNA adsorption capacity and transfection efficiency of freeze-dried nucleic acid delivery vectors

[0114] The present invention conducted agarose gel retardation, fluorescence quantitative PCR and immunoblotting experiments on the freeze-dried nucleic acid delivery vector to investigate the ability of the freeze-dried nucleic acid delivery vector to adsorb siRNA and the in vitro transfection efficiency. The three nucleic acid delivery vectors after reconstitution still had good ability to adsorb siRNA. Fig. 9 As shown, siRNA can be completely adsorbed when the N / P ratio is 2:1, and the siRNA adsorption capacity is maintained the same as that before lyophilization.

[0115] Finally, the in vitro transfection efficiency of the three nucleic acid delivery vectors after freeze-drying was evaluated. In this experiment, the present invention set up a normal control group, a positive control Lipo group, an experimental group (a freeze-dried group of coix seed oil nucleic acid delivery vector, a freeze-dried group of soybean oil nucleic acid delivery vector, a freeze-dried group of Brucea javanica oil nucleic acid delivery vector) and a Lipo freeze-dried group. The results are shown in Table 9. The coix seed oil nucleic acid delivery vector and the Brucea javanica oil nucleic acid delivery vector after freeze-drying and re-dissolving still have a certain transfection efficiency, knocking down Kras G12D The mRNA levels were 58.47% and 51.83%, respectively. The transfection efficiency of soybean oil nucleic acid delivery vector was relatively low, only 38.00%. At the same time, the commercially available transfection reagent Lipo had almost no transfection ability after freeze-drying. The results of immunoblotting were consistent with the results of fluorescence quantification. Fig.10 As shown, it is further proved that the new nucleic acid delivery vector still has the ability to transfect siRNA after freeze-drying and reconstitution.

[0116] Table 9: AsPC-1 cell transfection effect test data

[0117]

[0118]

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nucleic acid delivery vector based on natural lipids, It is characterized in that The invention comprises the following components: natural lipid, DOTAP, DOPE, glycerol and deionized water; the natural lipid is coix seed oil, javanica oil or soybean oil.

2. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 1, It is characterized in that The steps include: (1) mixing and dissolving DOTAP, DOPE and natural lipid in a water bath at 50° C. to 70° C. to obtain an oil phase, wherein the natural lipid is coix seed oil, javanica oil or soybean oil; (2) mixing glycerol and water and placing in a 50°C-70°C water bath to obtain an aqueous phase; (3) When the temperature of the water phase is consistent with that of the oil phase, the water phase is slowly added dropwise to the oil phase under the action of magnetic stirring; colostrum is then prepared by a high-pressure homogenization method, and then a probe ultrasound method is used to prepare the colostrum into a nucleic acid delivery vector.

3. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 2, Features: In the step (1), the mass percentage of DOTAP is 0.18%-1.06%, the mass percentage of DOPE is 0.06%-0.12%, and the mass percentage of natural lipids is 1.79%-15.26%; in the step (2), the mass percentage of glycerol is 2.11%, and the mass percentage of deionized water is 82.16%-95.63%.

4. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 3, Features: The mass percentage of DOTAP is 0.35%, the mass percentage of DOPE is 0.12%, the mass percentage of natural lipids is 3.52%, the mass percentage of glycerol is 2.11%, and the mass percentage of deionized water is 93.9%.

5. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 2, Features: In the step (3), the high-pressure homogenization condition is 2000 r / min-20000 r / min, 5 min; the ultrasonic condition is 50 W-200 W, 5 min.

6. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 5, Features: The high-pressure homogenization conditions were 20000 r / min, 5 min; the ultrasonic conditions were 200 W, 5 min.

7. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 2, It is characterized in that include: The nucleic acid delivery vector obtained in step (3) is dissolved in a lyophilization protective agent, and a lyophilized powder preparation is obtained by freeze-drying technology.

8. A method for preparing a natural lipid-based nucleic acid delivery vector according to claim 7, It is characterized in that The lyoprotectant is mannose, glucose or sucrose, and the mass ratio of the nucleic acid delivery vector to the lyoprotectant is 1:2, 1:4, 1:6, 1:8 or 1:

10.

9. The method for preparing a natural lipid-based nucleic acid delivery vector according to claim 8, It is characterized in that The lyophilization protective agent is glucose, and the mass ratio of the nucleic acid delivery vector to the lyophilization protective agent is 1:

8.

10. A lyophilized powder preparation prepared according to the method for preparing a natural lipid-based nucleic acid delivery vector according to any one of claims 7 to 9.

11. Use of a natural lipid-based nucleic acid delivery vector according to claim 1 in delivering siRNA.

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