CN114557964B - RNA-loadable cationic shuttle-type flexible liposome and preparation method and application thereof - Google Patents
RNA-loadable cationic shuttle-type flexible liposome and preparation method and application thereof Download PDFInfo
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- CN114557964B CN114557964B CN202210266123.4A CN202210266123A CN114557964B CN 114557964 B CN114557964 B CN 114557964B CN 202210266123 A CN202210266123 A CN 202210266123A CN 114557964 B CN114557964 B CN 114557964B
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Abstract
The invention discloses a cationic shuttle-type flexible liposome capable of carrying RNA, which has the particle size of 90-120 nm and comprises the following components in parts by weight: 40 to 50 parts of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 12 to 15 parts of dioleoyl phosphatidylethanolamine, 0.8 to 1 part of cholesterol, 8 to 10 parts of sodium deoxycholate and 8 to 10 parts of dextran. In addition, the invention also discloses a preparation method and application of the cationic shuttle-type flexible liposome. The cationic shuttle-type flexible liposome has high entrapment rate, small particle size, high deformability, high transdermal efficiency, good stability, particle size range of 109-122 nm in 3 months, surface potential of 85.65-94.02 mV, high RNA entrapment rate of 17.2%, good transfection effect and strong RNA protection effect.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a cationic shuttle-type flexible liposome capable of carrying RNA, and a preparation method and application thereof.
Background
Flexible liposomes are an improvement over the prescription of conventional liposomes. The flexible liposome is characterized in that different additives such as surfactant Tween, cholate, deoxycholate and other softening agents are added into a bilayer of the liposome, so that the liposome membrane has excellent flexibility and deformability, can penetrate through skin and mucous membrane easily, reaches dermis layers or even deeper tissues, has higher permeation efficiency, and keeps the composition unchanged.
The research proves that the flexible liposome can effectively enhance the percutaneous permeation of the medicine, enhance the therapeutic effect of the medicine, and avoid the potential safety hazard caused by the in-vivo administration of the medicine. Flexible liposomes are expected to be effective in treating skin disorders after drug entrapment.
Nucleic acid drugs are a class of DNA or RNA with diverse functions that have specific targets and mechanisms of action, typically at the level of the gene or its expression. Typically include Aptamer (Aptamer), anti-gene (anti-gene), ribozyme (Ribozyme), antisense oligonucleotide (ASO), RNA-interfering agent, and the like. These drugs have high specificity and can target selected genes, mRNA or non-coding RNA, thereby acting. At present, 13 nucleic acid medicines are obtained at home and abroad, and the effectiveness of the nucleic acid medicines is also verified by adenovirus recombinant mRNA vaccine which is put into use in a new coronal epidemic situation. With the rapid development of research and development technology, nucleic acid drugs are expected to become the third largest type of drugs following traditional chemical drugs and antibody protein drugs.
RNA drugs are easily hydrolyzed during the circulation of the human body due to their own instability. Most RNA drugs are negatively charged nucleic acid macromolecules that are difficult to access to the intracellular environment through the plasma membrane. The chemical modification of RNA can improve the stability of nucleic acid drugs, and the cationic liposome, polycation and lipid ion nano-particles can be used as a delivery carrier of nucleic acid drugs to overcome the problem of low RNA permeability.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the cationic shuttle-type flexible liposome capable of carrying RNA aiming at the defects of the prior art. The cationic shuttle-type flexible liposome has flexible property, good deformability and good elasticity and deformation, so that the cationic shuttle-type flexible liposome can better penetrate into the deep layer of skin, and the percutaneous absorption effect is greatly improved; meanwhile, the method has good stability, the grain diameter range is between 109nm and 122nm within 3 months, the surface potential is between 85.65mV and 94.02mV, the RNA entrapment rate is up to 17.2 percent, the transfection effect is good, and the protection effect on RNA is strong.
In order to solve the technical problems, the invention adopts the following technical scheme: the cationic shuttle-type flexible liposome capable of carrying RNA is characterized in that the particle size of the flexible liposome is 90-120 nm, and the cationic shuttle-type flexible liposome comprises the following components in parts by weight: 40 to 50 parts of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 12 to 15 parts of dioleoyl phosphatidylethanolamine, 0.8 to 1 part of cholesterol, 8 to 10 parts of sodium deoxycholate and 8 to 10 parts of dextran.
In addition, the invention also provides a method for preparing the RNA-loadable cationic shuttle-type flexible liposome, which is characterized by comprising the following steps:
step one, dissolving (2, 3-dioleoyl-propyl) -trimethylammonium chloride, dioleoyl phosphatidylethanolamine and cholesterol with chloroform; then vacuum rotary evaporator is used under the water bath condition, the chloroform in the solution is removed by decompression evaporation, a uniform film is formed on the bottle wall, and then the bottle wall is cooled at the temperature of minus 20 ℃;
adding a sodium deoxycholate solution into the cooled product in the first step, and performing rotary hydration with DEPC water until eluting to obtain a cationic flexible liposome suspension primary solution;
and thirdly, adding glucan into the suspension primary liquid of the cationic flexible liposome in the second step, stirring and mixing at room temperature, and filtering by a microporous filter membrane to obtain the cationic shuttle-type flexible liposome capable of carrying RNA.
The method is characterized in that the chloroform is added in the first step in such an amount that the final concentration of (2, 3-dioleoyl-propyl) -trimethylammonium chloride is 1 mg/mL-3 mg/mL.
The method is characterized in that the temperature of the water bath in the first step is 35-40 ℃, the rotation condition is 80-100 rpm, and the cooling time at-20 ℃ is not less than 6h.
The method is characterized in that the water bath temperature of the rotary hydration in the second step is 40-50 ℃, and the rotation condition is 80-100 rpm.
The method is characterized in that the pore diameter of the microporous filter membrane in the step three is 0.22 mu m.
The method is characterized in that the mass ratio of RNA to (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride in the cationic shuttle-type flexible liposome capable of carrying RNA is 1 (4-5), the incubation environment is RNase-free, and the incubation time is 15-20 min.
Further, the invention provides application of the RNA-loadable cationic shuttle-type flexible liposome in preparing a skin external cosmetic for delivering siRNA medicine.
Furthermore, the invention provides application of the RNA-loadable cationic shuttle-type flexible liposome in preparing external skin medicines for delivering RNA medicines.
The parts by weight may be in units of micrograms, milligrams, grams, and kilograms.
Compared with common liposome, the invention has the following advantages:
1. the cationic shuttle-type flexible liposome has high entrapment rate, small particle size, flexible property, high deformability and high transdermal efficiency, and good elasticity and deformation can enable the cationic shuttle-type flexible liposome to better penetrate into the deep layer of skin, so that the transdermal absorption effect is greatly improved; meanwhile, the method has good stability, the grain diameter range is between 109nm and 122nm within 3 months, the surface potential is between 85.65mV and 94.02mV, the RNA entrapment rate is up to 17.2 percent, the transfection effect is good, and the protection effect on RNA is strong.
2. The preparation method provided by the invention is simple and easy to operate, the reaction process is mild, the whole process is high in mechanization degree, and the preparation method has excellent reproducibility and stability under the same process, and is easy for industrial production.
3. The invention adopts DEPC water for hydration, which can reduce degradation of RNA.
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and the examples.
Drawings
FIG. 1 is a particle size diagram of a cationic shuttle-type flexible liposome according to example 1 of the present invention.
FIG. 2 is a Zeta potential diagram of a cationic shuttle type flexible liposome according to example 1 of the present invention.
FIG. 3 is a Transmission Electron Microscope (TEM) photograph of a cationic shuttle-type flexible liposome according to example 1 of the present invention.
FIG. 4 is a graph showing the deformability of the cationic shuttle-type flexible liposome according to example 4 of the present invention.
FIG. 5 is a graph showing the results of the transdermal properties of the cationic shuttle-type flexible liposome of example 6 of the present invention.
FIG. 6 is a nucleic acid electrophoresis chart of the optimal incubation ratio of cationic shuttle-type flexible liposomes and RNA drugs according to example 7 of the present invention.
FIG. 7 is a graph showing the transfection efficiency of RNA-carrying cationic shuttle-type flexible liposomes according to example 8 of the present invention.
FIG. 8 is a qPCR analysis of eGFP gene expression levels of HEK293T cells transfected with RNA-carrying cationic shuttle-type flexible liposomes according to example 8 of the present invention.
FIG. 9 is a graph showing the results of the optimal RNA concentration search at the time of transfection of RNA-loaded cationic shuttle-type flexible liposomes according to example 8 of the present invention.
FIG. 10 is an agarose gel electrophoresis chart of example 9 of the present invention.
FIG. 11 shows the results of RNA transdermal depth detection according to example 10 of the present invention.
FIG. 12 is a graph showing comparison of hair growth in mice of example 11 of the present invention.
Detailed Description
Example 1: cationic shuttle type flexible liposome prepared by film dispersion method
Quantitatively taking 4mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.2mg of dioleoyl phosphatidylethanolamine and 0.08mg of cholesterol, placing the materials into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 2mL, evaporating the materials for 1h under reduced pressure by a rotary evaporator under a constant-temperature water bath at 40 ℃, completely removing chloroform, and enabling film-forming materials such as cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation rotating speed is 90rpm; sealing the bottle mouth after film forming, and cooling and storing for more than 6 hours at the temperature of minus 20 ℃ in a refrigerator;
adding 4.9mL of DEPC water and 0.8mg of sodium deoxycholate after cooling, hydrating in a water bath at 50 ℃ for 1h, and obtaining a milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted, wherein the rotation speed is 90rpm;
and step three, adding 0.8mg of glucan into the suspension primary liquid of the cationic flexible liposome in the step two, stirring for more than 1h at room temperature, and then passing through a microporous filter membrane with the aperture of 0.22 mu m to obtain the cationic shuttle-type flexible liposome capable of carrying RNA.
As shown in fig. 1, the particle size of the cationic shuttle-type flexible liposome prepared in the example is 130.5nm, as shown in fig. 2, the Zeta potential of the cationic shuttle-type flexible liposome prepared in the example is 12.23mV, and as shown in fig. 3, the cationic shuttle-type flexible liposome prepared in the example is a vesicle structure with complete morphology, nonuniform size and irregular morphology as measured by a Transmission Electron Microscope (TEM).
Example 2: cationic shuttle type flexible liposome prepared by film dispersion method
Quantitatively taking 4.5mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.35mg of dioleoyl phosphatidylethanolamine and 0.09mg of cholesterol, placing the materials into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 1.5mL, evaporating the materials for 1h under reduced pressure by a rotary evaporator under a constant temperature water bath of 35 ℃, completely removing chloroform, and enabling film-forming materials such as cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation rotating speed is 80rpm; sealing the bottle mouth after film forming, and cooling and storing for more than 6 hours at the temperature of minus 20 ℃ in a refrigerator;
adding 4.8mL of DEPC water and 0.9mg of deoxycholate after cooling, hydrating in a water bath at 40 ℃ for 1h, and obtaining a milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted, wherein the rotation speed is 80rpm;
and step three, adding 0.9mg of glucan into the suspension primary liquid of the cationic flexible liposome in the step two, stirring for more than 1h at room temperature, and then passing through a microporous filter membrane with the aperture of 0.22 mu m to obtain the cationic shuttle-type flexible liposome capable of carrying RNA.
The cationic shuttle-type flexible liposome prepared in this example was similar in particle size, zeta potential and morphology to example 1.
Example 3: cationic shuttle type flexible liposome prepared by film dispersion method
Quantitatively taking 5mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.5mg of dioleoyl phosphatidylethanolamine and 0.1mg of cholesterol, placing the materials into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 5mL, evaporating the materials for 0.5h under the constant-temperature water bath at 38 ℃ under reduced pressure by a rotary evaporator, completely removing chloroform, and enabling film-forming materials such as cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation rotating speed is 100rpm; sealing the bottle mouth after film forming, and cooling and storing for more than 6 hours at the temperature of minus 20 ℃ in a refrigerator;
adding 4.8mL of DEPC water and 1mg of sodium deoxycholate after cooling, hydrating in a water bath at 45 ℃ for 1h, and obtaining a milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted, wherein the rotation speed is 100rpm;
and step three, adding 1mg of glucan into the suspension primary liquid of the cationic flexible liposome in the step two, stirring for more than 1h at room temperature, and then passing through a microporous filter membrane with the aperture of 0.22 mu m to obtain the cationic shuttle-type flexible liposome capable of carrying RNA.
The cationic shuttle-type flexible liposome prepared in this example was similar in particle size, zeta potential and morphology to example 1.
Example 4: deformability examination was performed by taking the cationic shuttle type flexible liposome prepared in example 1 as an example
Under the action of external pressure, the cationic shuttle-type flexible liposome prepared in the example 1 which is coated with coumarin and the water suspension are passed through a microporous filter membrane with the pore diameter of 0.22 mu m, and the passing performance of the cationic shuttle-type flexible liposome is observed. The grouping is as follows:
group 1:1mL coumarin aqueous solution.
Group 2:1mL of cationic shuttle-type flexible liposome and water suspension which are loaded with coumarin.
Group 3:1mL of normal cationic liposome and water suspension containing coumarin.
And respectively extruding the solution through a microporous filter membrane with the thickness of 0.22 mu m by using an injector, observing whether the cationic shuttle type flexible liposome which is coated with coumarin after extruding and passing through the microporous filter membrane with the thickness of 0.22 mu m changes to cause leakage of coumarin, and simultaneously recording the time of the liquid with the same volume passing through the microporous filter membrane with the thickness of 0.22 mu m.
As shown in FIG. 4, the results indicate that the cationic liposome suspension containing sodium deoxycholate can permeate through the 0.22 μm microporous filter membrane, and the permeation rate increases with the pressure. The cationic shuttle-type flexible liposome prepared by the invention is easier to penetrate through a microporous filter membrane with the thickness of 0.22 mu m than normal cationic liposome, and the blocking feeling during extrusion is similar to that of coumarin aqueous solution.
Example 5: stability examination of the cationic shuttle type Flexible Liposome prepared in example 1
As shown in Table 1, the cationic shuttle type flexible liposome prepared in example 1 was left to stand at a low temperature (4 ℃) for 3 months, and the average particle size, the polydispersity index (PDI) and the Zeta potential of the cationic shuttle type flexible liposome were measured by sampling at intervals, and the Zeta potential was found to be not changed basically, which indicates that the cationic shuttle type flexible liposome has good stability at a low temperature (4 ℃)
TABLE 1 stability investigation of cationic shuttle-type flexible liposomes
Example 6: the transdermal properties of the cationic shuttle-type flexible liposomes prepared in example 1 were examined
The percutaneous results of the liposomes were observed using a biopsy instrument. As shown in FIG. 5, the cationic shuttle-type flexible liposome and the cationic liposome prepared in example 1 both penetrate the back skin of the rat, and the transmittance increases with the time. The cationic shuttle-type flexible liposome has high deformability, and the transmittance is obviously higher than that of the cationic liposome.
The specific operation steps are as follows:
(1) Treatment of laboratory animals
Healthy SD rats are weighed, anesthetized by intraperitoneal injection of 0.3% barbital (0.2 ml/10 g), the hair on the back and abdomen is removed, the hair is scraped to about 0.5-1mm by using a Jilin scraper, the depilating area is 4cm x 2cm, the skin is washed by normal saline, and the experiment is carried out after the skin is dried in the air.
(2) Preparation of samples
Taking 400ul of cationic liposome and 400ul of cationic shuttle-type flexible liposome respectively, adding 8ul of Dir dye respectively, and incubating for 30min at 37 ℃. After incubation, the mixture was centrifuged at 2000rpm/min for 10min in a centrifuge. The precipitate was discarded and centrifuged at 3000rpm/min for another 10min. Taking the supernatant, and passing through a 0.22um microporous filter membrane to obtain the fluorescent cationic liposome and the cationic shuttle-type flexible liposome, wherein the particle size range of the cationic liposome and the cationic shuttle-type flexible liposome is 120nm-160nm according to experimental requirements.
(3) Applying the medicine and observing
Preparing gel from the fluorescent-labeled cationic liposome and cationic shuttle-type flexible liposome in (2), cleaning the surface of rat skin with physiological saline, and adjusting the concentration to 20mg/cm 2 The administration dosage of the drug is that the cationic shuttle type flexible liposome gel and the cationic liposome gel are respectively and evenly smeared on the skin of the dehairing part on the back of the rat, and are respectively administered for 2 hours and 24 hours, and the distribution of the liposome which is coated with coumarin at different time points in the skin tissue of the rat after administration is observed under a living body imager. SD rats without any treatment were negative control group.
Example 7: the optimal incubation ratio of the cationic shuttle-type flexible liposome prepared in example 1 and RNA drug was selected and prepared
As shown in fig. 6, according to the mass ratio (N: P) of RNA to (2, 3-dioleoyl-propyl) -trimethylammonium chloride in the cationic shuttle-type flexible liposome prepared in example 1 being an incubation ratio of 5:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:5, incubating for 15min to 20min in an RNase-free environment, preparing the RNA-carrying cationic shuttle-type flexible liposome, and performing agarose gel electrophoresis experiment on the incubated RNA-carrying cationic shuttle-type flexible liposome to screen the optimal incubation ratio, thereby obtaining an optimal incubation ratio, i.e., N: p=1 (4 to 5). The preparation can be incubated on demand in the RNase-free environment.
Example 8: the transfection efficiency of RNA-carrying cationic shuttle-type flexible liposomes prepared in example 1 was examined
The siRNA-eGFP capable of inhibiting eGFP protein expression is used for co-incubation with the cationic shuttle type flexible liposome prepared in the embodiment 1 according to the optimal proportion, so that the siRNA-eGFP shuttle type flexible liposome is obtained, HEK-293T cell strain capable of stably expressing green fluorescent protein is transfected, the intensity of green fluorescence is observed under a fluorescent microscope, and the transfection efficiency is examined by comparison. Meanwhile, quantitative comparison is carried out on eGFP gene expression in HEK293T through qPCR experiments, and the quantitative comparison is divided into a Control group, a Lip2000 group, a Normal low group, a Normal high group, a Soft low group and a Soft high group, wherein the RNA dosage of the Normal high group and the Soft high group is the same as the Lip2000 dosage and is 5 times of the RNA dosage of the Normal low group and the Soft low group.
As shown in fig. 7, 8 and 9, the transfection efficiency of the RNA-loaded cationic shuttle-type flexible liposome was slightly weaker than that of commercially available lip2000, but superior to that of cationic liposome. lip2000 could not be used in animal experiments.
Example 9: the protection effect of the RNA-carrying cationic shuttle-type flexible liposome on RNA is examined
Four groups of environments of-20 ℃, 4 ℃, 45 ℃ and 25 ℃ are arranged, and RNA and cationic shuttle-type flexible liposome are incubated together according to the optimal ratio of RNA to liposome=1:4. After 15min incubation at room temperature, the samples were placed in the four groups of environments and stored in a sealed manner. A set of samples were set every 5 days. And after the incubation time is over, membrane rupture treatment is carried out on the shuttle-type flexible liposome, and an agarose gel electrophoresis experiment is carried out to observe the degradation condition of RNA. As shown in FIG. 10, the RNA-carrying cationic shuttle-type flexible liposome can effectively protect RNA in two months at-20 ℃ and 4 ℃. Can effectively protect RNA in 20-25 days under the conditions of 25 ℃ and 45 ℃.
Example 10: the transdermal depth of the RNA-carrying cationic shuttle-type flexible liposome is examined
Delivery RNA transdermal depth detection was performed on cationic shuttle-type flexible liposomes using Franz diffusion cells. As shown in FIG. 11, after 24 hours of continuous administration, naked RNA, RNA-entrapped cationic liposomes, and RNA-entrapped cationic shuttle-type flexible liposomes were effective to penetrate the skin to the dermis. The transmittance is obviously higher than that of the cationic liposome.
The specific operation steps are as follows:
(1) Treatment of experimental skin
The back skin of SD rat killed after abdominal aorta blood collection is cut off, hair on skin is removed by depilatory cream, skin is cleaned by normal saline, and skin moisture is removed for standby.
(2) Preparation of samples
Respectively taking 1ml of cationic liposome and 1ml of cationic shuttle-type flexible liposome, and mixing RNA according to the optimal ratio: 4 FITC-labeled miRNA-34a was added and incubated for 15min at room temperature. To obtain the cationic liposome which is coated with the RNA carrying the fluorescent label and the cationic shuttle-type flexible liposome which is coated with the RNA carrying the fluorescent label. The naked RNA group for administration is obtained by diluting miRNA-34a with DEPC water in the same amount as the liposome solution.
(3) Establishment of Franz diffusion cell
The SD rat dorsal skin treated in (1) was sandwiched between Franz diffusion cells with the epidermis facing the supply cell and kept dry. RNase-free PBS buffer was added to the receiving well. The naked RNA solution in (2), the cationic liposome coated with the RNA carrying the fluorescent tag and the cationic shuttle-type flexible liposome coated with the RNA carrying the fluorescent tag were spread on the epidermis of the supply tank uniformly in 200 ul. Incubate in the dark at 37℃for 24h.
(4) Collection and detection of skin samples
The skin of the rat after 24 hours of administration in (3) was removed. After the PBS buffer solution of RNase-free is rinsed lightly, the mixture is quickly placed in a refrigerator at-80 ℃ and stored in a dark place. Frozen sections were cut longitudinally along the central long axis of the epidermis. The depth and state of fluorescence into the skin were observed under a fluorescence microscope.
Example 11: shuttle-type flexible liposome delivery recombinant mir218 promotes mouse hair growth
The experiment is divided into: blank (PBS solution) and treated (recombinant mir-218). 32 healthy C57BL6 male mice of 2 months of age were purchased as experimental animals, and 8 animals were randomly selected from each group. The skin parts on two sides of the back spine are selected for dehairing without damaging the epidermis. After the molding is finished, PBS is sprayed on the blank control group, shuttle-type flexible liposome and recombinant miR-218 solution are sprayed on the treatment group, and the concentration of the recombinant miR-218 is 2.5 mug/mL, 5 mug/mL and 10 mug/mL respectively. Each mouse is administrated with a dose of 1mL, and after being sprayed on the back of the experimental animal to remove hair skin, the skin is massaged for 1 minute to promote skin absorption, 1 time a day for 20 days, and hair growth is counted.
As shown in FIG. 12, the effect of promoting hair regeneration to a different extent and for 20 days was evident in the treated group of recombinant mir-218 as compared to the PBS blank group. The coverage of the new hair at 20d was above 60% in the treatment group of recombinant mir-218, which was significantly different from the coverage of the new hair in the control group mice, P <0.05, P <0.01, and P <0.001.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification and variation of the above embodiment according to the technical substance of the present invention still falls within the scope of the technical solution of the present invention.
Claims (1)
1. A preparation method of RNA-loadable cationic shuttle-type flexible liposome comprises the following steps:
quantitatively taking 4mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.2mg of dioleoyl phosphatidylethanolamine and 0.08mg of cholesterol, placing the materials into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 2mL, evaporating the materials for 1h under reduced pressure by a rotary evaporator under a constant-temperature water bath at 40 ℃, completely removing chloroform, and enabling film-forming materials such as cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation rotating speed is 90rpm; sealing the bottle mouth after film forming, and cooling and storing for more than 6 hours at the temperature of minus 20 ℃ in a refrigerator;
adding 4.9mL of DEPC water and 0.8mg of sodium deoxycholate after cooling, hydrating in a water bath at 50 ℃ for 1h, and obtaining a milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted, wherein the rotation speed is 90rpm;
and step three, adding 0.8mg of glucan into the suspension primary liquid of the cationic flexible liposome in the step two, stirring for more than 1h at room temperature, and then passing through a microporous filter membrane with the aperture of 0.22 mu m to obtain the cationic shuttle-type flexible liposome capable of carrying RNA.
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