CN114557964A - Cationic shuttle-type flexible liposome capable of carrying RNA (ribonucleic acid), and preparation method and application thereof - Google Patents
Cationic shuttle-type flexible liposome capable of carrying RNA (ribonucleic acid), and preparation method and application thereof Download PDFInfo
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- CN114557964A CN114557964A CN202210266123.4A CN202210266123A CN114557964A CN 114557964 A CN114557964 A CN 114557964A CN 202210266123 A CN202210266123 A CN 202210266123A CN 114557964 A CN114557964 A CN 114557964A
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Abstract
The invention discloses a cationic shuttle-type flexible liposome capable of carrying RNA, the particle size of the flexible liposome is 90 nm-120 nm, and the raw materials comprise the following components in parts by weight: 40-50 parts of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 12-15 parts of dioleoyl phosphatidylethanolamine, 0.8-1 part of cholesterol, 8-10 parts of sodium deoxycholate and 8-10 parts of glucan. In addition, the invention also discloses a preparation method and application of the cation shuttle-type flexible liposome. The cation fusiform flexible liposome has the advantages of high encapsulation efficiency, small particle size, high deformability, high transdermal efficiency and good stability, the particle size range is 109-122 nm within 3 months, the surface potential is 85.65-94.02 mV, the RNA encapsulation rate is up to 17.2%, the transfection effect is good, and the RNA protection effect is strong.
Description
Technical Field
The invention belongs to the technical field of biological medicines, 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 developed over the general liposome formulation. The flexible liposome is prepared by adding different additives such as surfactants Tween, cholate, deoxycholate and other softeners into a bilayer of the liposome, so that a liposome membrane has excellent flexibility and deformability, can easily penetrate through skin and a mucous membrane to reach tissues in a dermis layer or even deeper, has high permeation efficiency and keeps the composition of the liposome unchanged.
Researches prove that the flexible liposome can effectively enhance the percutaneous permeation of the medicine and enhance the treatment effect of the medicine, and the external administration avoids the potential safety hazard caused by the medicine entering the body. The flexible liposome is expected to effectively treat skin diseases after encapsulating the drug.
Nucleic acid drugs are a class of DNA or RNA with diverse functions, which have specific targets and mechanisms of action, usually acting on a gene or its expression level. Typically, aptamers (aptamers), antigenes (antibiotics), ribozymes (ribozymes), antisense oligonucleotides (ASOs), RNA interference agents, and the like are included. These drugs have high specificity and can target selected genes, mRNA or non-coding RNA to further function. At present, 13 types of nucleic acid medicaments are approved at home and abroad, and the adenovirus recombinant mRNA vaccine put into use during the new crown epidemic situation also verifies the effectiveness of the nucleic acid medicaments. With the rapid development of research and development technologies, nucleic acid drugs are expected to become the third major class of drugs following traditional chemical drugs and antibody protein drugs.
RNA drugs are easily hydrolyzed during the human circulation due to their own instability. Most RNA drugs are negatively charged nucleic acid macromolecules that are difficult to access 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 nanoparticles can overcome the problem of low RNA permeability when used as delivery carriers of nucleic acid drugs.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a cationic shuttle-type flexible liposome capable of carrying RNA, which addresses the above-mentioned deficiencies of the prior art. The cation shuttle-shaped flexible liposome has the advantages of flexibility, good deformability, good elasticity and deformation, and capability of better penetrating into the deep layer of skin, thereby greatly increasing the transdermal absorption effect; meanwhile, the composite has good stability, the particle size range is between 109nm and 122nm within 3 months, the surface potential is 85.65mV to 94.02mV, the RNA entrapment rate is up to 17.2 percent, the transfection effect is good, and the RNA protection effect is strong.
In order to solve the technical problems, the invention adopts the technical scheme that: the cationic shuttle-type flexible liposome capable of carrying RNA is characterized in that the particle size of the flexible liposome is 90 nm-120 nm, and the flexible liposome comprises the following components in parts by weight: 40-50 parts of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 12-15 parts of dioleoyl phosphatidylethanolamine, 0.8-1 part of cholesterol, 8-10 parts of sodium deoxycholate and 8-10 parts of glucan.
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:
dissolving (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, dioleoyl phosphatidylethanolamine and cholesterol in chloroform; then using a vacuum rotary evaporator under the condition of water bath, removing chloroform in the solution by reduced pressure evaporation, forming a uniform film on the bottle wall, and then placing at-20 ℃ for cooling;
step two, adding a sodium deoxycholate solution into the product cooled in the step one, and carrying out rotary hydration by DEPC water until elution is carried out to obtain a suspension primary liquid of the cationic flexible liposome;
and step three, adding glucan into the primary suspension liquid of the cationic flexible liposome in the step two, stirring and mixing at room temperature, and filtering by using a microporous filter membrane to obtain the cationic fusiform flexible liposome capable of carrying RNA.
The method described above, wherein chloroform is added in the first step in an amount such that the final concentration of (2, 3-dioleoyl-propyl) -trimethylammonium chloride is 1mg/mL to 3 mg/mL.
The method is characterized in that the temperature of the water bath in the step one is 35-40 ℃, the rotation condition is 80-100 rpm, and the cooling time at-20 ℃ is not less than 6 h.
The method is characterized in that the temperature of the water bath for the rotary hydration in the step two 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 μm.
The method is characterized in that when the cationic fusiform flexible liposome capable of carrying RNA is used, the mass ratio of the RNA to the (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride in the cationic fusiform flexible liposome 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 cationic shuttle-type flexible liposome capable of carrying the RNA in preparing cosmetics for external application of skin for delivering siRNA medicaments.
Further, the invention provides application of the RNA-loadable cationic fusiform flexible liposome in preparing an external skin medicament for delivering an RNA medicament.
The parts by weight may be microgram, milligram, gram, kilogram, etc. units by weight.
Compared with the common liposome, the invention has the following advantages:
1. the cation fusiform flexible liposome has the advantages of high entrapment rate, small particle size, flexible property, high deformability, high transdermal efficiency, good elasticity and deformation, and capability of better penetrating into the deep layer of skin, thereby greatly increasing the transdermal absorption effect; meanwhile, the composite has good stability, the particle size range is between 109nm and 122nm within 3 months, the surface potential is 85.65mV to 94.02mV, the RNA entrapment rate is up to 17.2 percent, the transfection effect is good, and the RNA protection effect is strong.
2. The preparation method is simple and easy to operate, the reaction process is mild, the whole process has high mechanization degree, and the same process has excellent reproducibility and stability and is easy for industrial production.
3. The invention adopts DEPC water for hydration, and can reduce the degradation of RNA.
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.
Drawings
FIG. 1 is a particle size diagram of the flexible liposome of the cationic shuttle type in example 1 of the present invention.
FIG. 2 is Zeta potential diagram of cationic fusiform flexible liposomes of example 1 of the present invention.
FIG. 3 is a Transmission Electron Microscope (TEM) photograph of the flexible cationic shuttle-type liposome of example 1 of the present invention.
FIG. 4 is a graph showing the results of deformability of the cationic shuttle-type flexible liposomes of example 4 of the present invention.
Fig. 5 is a graph showing the results of the transdermal performance of the cationic shuttle-type flexible liposome of example 6 of the present invention.
FIG. 6 is the nucleic acid electrophoresis chart of the optimal incubation ratio of the cationic shuttle-type flexible liposome and the RNA drug in example 7 of the present invention.
FIG. 7 is a graph showing the results of transfection efficiency of RNA-loaded cationic shuttle-type flexible liposomes of example 8 of the present invention.
FIG. 8 is a qPCR analysis graph of eGFP gene expression levels in HEK293T cells after transfection of RNA-loaded cationic shuttle-type flexible liposomes of example 8 of the present invention.
FIG. 9 is a graph showing the results of searching for the optimal RNA concentration in transfection of RNA-loaded cationic shuttle-type flexible liposomes of example 8.
FIG. 10 is an agarose gel electrophoresis image of example 9 of the present invention.
FIG. 11 shows the results of the depth measurement of RNA penetration in example 10 of the present invention.
FIG. 12 is a graph showing a comparison of hair growth in groups of mice according to example 11 of the present invention.
Detailed Description
Example 1: preparation of cation shuttle-type flexible liposome by thin film dispersion method
Step one, quantitatively taking 4mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.2mg of dioleoyl phosphatidylethanolamine and 0.08mg of cholesterol, placing the (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.2mg of dioleoyl phosphatidylethanolamine and 0.08mg of cholesterol into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 2mL, performing reduced pressure evaporation for 1h by using a rotary evaporator under a constant-temperature water bath at 40 ℃, completely removing chloroform, and enabling film-forming materials such as the cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation speed is 90 rpm; sealing the bottle mouth after film forming is finished, and placing the bottle mouth in a refrigerator for cooling and storing for more than 6 hours at the temperature of 20 ℃ below zero;
step two, adding 4.9mL of DEPC water and 0.8mg of sodium deoxycholate after cooling, hydrating in a water bath at 50 ℃ for 1h, wherein the rotating speed is 90rpm, and obtaining milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted;
and step three, adding 0.8mg of glucan into the suspension primary solution 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 pore diameter 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 flexible liposome of the cation shuttle type prepared by the example is 130.5nm, as shown in FIG. 2, the Zeta potential of the flexible liposome of the cation shuttle type prepared by the example is 12.23mV, as shown in FIG. 3, the flexible liposome of the cation shuttle type prepared by the example is a vesicle structure with complete morphology, non-uniform size and irregular morphology as measured by Transmission Electron Microscope (TEM).
Example 2: preparation of cation shuttle-type flexible liposome by thin 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 (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.35mg of dioleoyl phosphatidylethanolamine and 0.09mg of cholesterol into a 50mL eggplant-shaped bottle, adding a chloroform solution into the bottle until the total volume of the bottle is 1.5mL, performing reduced-pressure evaporation for 1h by using a rotary evaporator under a constant-temperature water bath at 35 ℃, completely removing the chloroform, and enabling film-forming materials such as the cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation speed is 80 rpm; sealing the bottle mouth after film forming, and placing in a refrigerator for cooling and storing for more than 6h at the temperature of 20 ℃ below zero;
step two, adding 4.8mL of DEPC water and 0.9mg of sodium deoxycholate after cooling, hydrating in water bath at 40 ℃ for 1h, wherein the rotating speed is 80rpm, and obtaining milky semitransparent cation flexible liposome suspension primary liquid after the film is completely eluted;
and step three, adding 0.9mg of glucan into the primary suspension of the cationic flexible liposome in the step two, stirring at room temperature for more than 1h, 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 particle size, Zeta potential and morphology of the cationic fusiform flexible liposomes prepared in this example were similar to those of example 1.
Example 3: preparation of cation shuttle-type flexible liposome by thin film dispersion method
Step one, quantitatively taking 5mg of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.5mg of dioleoyl phosphatidylethanolamine and 0.1mg of cholesterol, placing the (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 1.5mg of dioleoyl phosphatidylethanolamine and 0.1mg of cholesterol into a 50mL eggplant-shaped bottle, adding a chloroform solution until the total volume in the bottle is 5mL, performing reduced pressure evaporation for 0.5h by using a rotary evaporator in a constant-temperature water bath at 38 ℃, completely removing chloroform, and enabling film-forming materials such as the cholesterol and the like to form a uniform semitransparent lipid film on the inner wall of the eggplant-shaped bottle, wherein the rotary evaporation speed is 100 rpm; sealing the bottle mouth after film forming is finished, and placing the bottle mouth in a refrigerator for cooling and storing for more than 6 hours at the temperature of 20 ℃ below zero;
step two, adding 4.8mL of DEPC water and 1mg of sodium deoxycholate after cooling, hydrating in a water bath at 45 ℃ for 1h, and obtaining milky semitransparent cation flexible liposome suspension initial solution after the film is completely eluted, wherein the rotating speed is 100 rpm;
and step three, adding 1mg of glucan into the primary suspension of the cationic flexible liposome in the step two, stirring at room temperature for more than 1h, and then passing through a microporous filter membrane with the aperture of 0.22 mu m to obtain the cationic fusiform flexible liposome capable of carrying RNA.
The particle size, Zeta potential and morphology of the cationic fusiform flexible liposomes prepared in this example were similar to those of example 1.
Example 4: deformability examination was conducted by taking the cationic shuttle-type flexible liposome prepared in example 1 as an example
The cation shuttle-type flexible liposome prepared in example 1 and coated with coumarin and the aqueous suspension were passed through a microfiltration membrane having a pore size of 0.22 μm under external pressure, and the passage performance thereof was observed. The grouping is as follows:
group 1: 1mL of an aqueous coumarin solution.
Group 2: 1mL of coumarin-loaded cationic fusiform flexible liposome and water suspension.
Group 3: 1mL of normal cationic liposome loaded with coumarin and aqueous suspension.
Respectively extruding the solution through a 0.22 mu m microporous filter membrane by using a syringe, observing whether the coumarin leaks due to the change of the cation shuttle-type flexible liposome coated with the coumarin after the solution is extruded through the 0.22 mu m microporous filter membrane, and simultaneously recording the time of the liquid with the same volume passing through the 0.22 mu m microporous filter membrane.
As shown in FIG. 4, the results show that the cationic liposome suspension containing sodium deoxycholate can pass through a 0.22 μm microporous membrane, and the permeation rate increases with the increase of pressure. Compared with the normal cationic liposome, the cationic fusiform flexible liposome prepared by the invention is easier to permeate a 0.22 mu m microporous filter membrane, and the blocking feeling is similar to that of coumarin aqueous solution during extrusion.
Example 5: stability examination of the cationic shuttle-type flexible liposomes prepared in example 1
As shown in Table 1, the cationic shuttle-type flexible liposome prepared in example 1 was allowed 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 substantially unchanged by measuring the particle size and the Zeta potential at intervals, which indicates that the cationic shuttle-type flexible liposome has good stability at a low temperature (4 ℃)
TABLE 1 cationic spindle-type Flexible Liposome stability Studies
Example 6: examination of the skin penetration of the cationic shuttle-type flexible liposomes prepared in example 1
The transdermal results of the liposomes were observed using a live imager. As shown in FIG. 5, both the cationic shuttle-type flexible liposomes and the cationic liposomes prepared in example 1 were able to penetrate the dorsal skin of rats and the permeability increased with the passage of time. Because the cation shuttle-type flexible liposome has high deformability, the permeability is obviously higher than that of the cation liposome.
The specific operation steps are as follows:
(1) treatment of laboratory animals
Healthy SD rats were weighed, anesthetized with 0.3% barbiturate (0.2ml/10g) by intraperitoneal injection, the back and abdomen were debrided, the hair was scraped to about 0.5-1mm with a gill blade, the area of debridement was 4 cm. times.2 cm, the skin was washed with physiological saline, and the experiment was performed after the skin was dried in the air.
(2) Preparation of samples
Respectively taking 400ul of the cationic liposome and the cationic spindle-type flexible liposome, respectively adding 8ul of Dir dye, and incubating at 37 ℃ for 30 min. After the incubation, the mixture is centrifuged at 2000rpm/min for 10min in a centrifuge. The precipitate was discarded and centrifuged at 3000rpm/min for 10 min. Taking the supernatant, passing through a 0.22um microporous filter membrane to obtain fluorescent cationic liposome and cationic shuttle-type flexible liposome, and measuring the particle size of the cationic liposome and the cationic shuttle-type flexible liposome to be 120-160 nm, thereby meeting the experimental requirements.
(3) Applying the medicine and observing
Preparing the cationic liposome with the fluorescent label in the step (2) and the cationic shuttle-type flexible liposome into gel, cleaning the skin surface of the rat by using normal saline according to the ratio of 20mg/cm2The cationic spindle-type flexible liposome gel and the cationic liposome gel are respectively and uniformly coated on the skin of a depilatory part on the back of a rat and are respectively administeredThe distribution of the liposome coated with coumarin in rat skin tissues at different time points after administration is observed under a living body imaging instrument for 2h and 24 h. SD rats without any treatment were negative control group.
Example 7: the optimal incubation ratio of the cationic fusiform flexible liposome prepared in example 1 and the RNA drug is screened and prepared
As shown in fig. 6, the RNA and the (2, 3-dioleoyl-propyl) -trimethylammonium chloride in the cationic shuttle-type flexible liposome prepared in example 1 were incubated at an incubation ratio (N: P) of 5:1, 4:1, 2:1, 1:2, 1:4, and 1:5 for 15min to 20min in an RNase-free environment to prepare the RNA-loaded cationic shuttle-type flexible liposome, and the incubated RNA-loaded cationic shuttle-type flexible liposome was subjected to agarose gel electrophoresis to screen the optimum incubation ratio, i.e., N: P ═ 1 (4 to 5). Can be prepared by incubation in the RNase-free environment as required.
Example 8: examination of transfection efficiency of the RNA-loaded cationic shuttle-type flexible liposomes prepared in example 1
And (2) co-incubating the siRNA-eGFP capable of inhibiting the expression of the eGFP protein and the cation shuttle-type flexible liposome prepared in the example 1 according to the optimal proportion to obtain the siRNA-eGFP-loaded shuttle-type flexible liposome, transfecting the HEK-293T cell strain capable of stably expressing the green fluorescent protein, observing the intensity of green fluorescence under a fluorescence microscope, and comparing to examine the transfection efficiency. Meanwhile, quantitative comparison is carried out on the expression of the eGFP gene in the HEK293T through a qPCR experiment, and the eGFP gene 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 dosage of RNA in the Normal high group and the Soft high group is the same as that of the Lip2000, and is 5 times that of the RNA in the Normal low group and the Soft low group.
As shown in FIGS. 7, 8 and 9, the transfection efficiency of the RNA-loaded cationic shuttle-type flexible liposomes was slightly weaker than that of the commercial lip2000, but better than that of the cationic liposomes. lip2000 cannot be used in animal experiments.
Example 9: the protection effect of the RNA-loaded cationic shuttle-type flexible liposome on RNA is investigated
Setting four groups of environments of-20 ℃, 4 ℃, 45 ℃ and 25 ℃, and co-incubating the RNA and the cation shuttle-type flexible liposome according to the optimal ratio of RNA to liposome as 1: 4. After incubation for 15min at room temperature, the cells were stored in four groups of the above-described environments under sealed conditions. One set of samples was set every 5 days. And (3) after the incubation time is over, performing membrane rupture treatment on the shuttle-shaped flexible liposome, and performing an agarose gel electrophoresis experiment to observe the RNA degradation condition. As shown in FIG. 10, the RNA-loaded cationic shuttle-type flexible liposome can effectively protect RNA within two months at the temperature of-20 ℃ and 4 ℃. The RNA can be effectively protected within 20-25 days in the environment of 25 ℃ and 45 ℃.
Example 10: the transdermal depth of the RNA-loaded cation shuttle-type flexible liposome is investigated
And (3) carrying out transdermal depth detection on the delivered RNA by adopting a Franz diffusion cell on the cation shuttle-type flexible liposome. As shown in FIG. 11, after 24h of continuous administration, naked RNA, RNA-entrapped cationic liposome and RNA-entrapped cationic shuttle-type flexible liposome were all effective in penetrating the skin to the dermal layer. The permeability is obviously higher than that of the cationic liposome.
The specific operation steps are as follows:
(1) treatment of experimental skin
The dorsal skin of the sacrificed SD rat after the abdominal aorta blood sampling was cut, the hair on the skin was removed with depilatory cream, the skin was washed with physiological saline, and the moisture on the epidermis was wiped off for use.
(2) Preparation of samples
Respectively taking 1ml of cationic liposome and 1ml of cationic shuttle-type flexible liposome, and mixing the cationic shuttle-type flexible liposome and the cationic shuttle-type flexible liposome according to the optimal ratio of RNA: 4 adding the miRNA-34a after the FITC labeling, and incubating for 15min at room temperature. Obtaining the cationic liposome coated with the RNA carrying the fluorescent label and the cationic shuttle-type flexible liposome coated with the RNA carrying the fluorescent label. The administration-use naked RNA group was obtained by diluting miRNA-34a at the same dose with DEPC water in an amount equivalent to that of the liposome solution.
(3) Franz diffusion cell establishment
The SD rat dorsal skin treated in (1) was sandwiched between Franz diffusion cells with the skin facing the donor cell and kept dry. The receiving cell was filled with RNase-free PBS buffer. Uniformly spreading the naked RNA solution in the step (2), the cationic liposome coated with the RNA carrying the fluorescent label and 200ul of the cationic shuttle-type flexible liposome coated with the RNA carrying the fluorescent label on the epidermis of the supply pool. Incubation was carried out at 37 ℃ for 24h in the absence of light.
(4) Skin sample collection and testing
The skin of the rat in (3) after 24h of administration was removed. After being slightly rinsed by RNase-free PBS buffer solution, the mixture is quickly placed in a refrigerator at minus 80 ℃ and kept away from light. Cryosections were taken by longitudinal sectioning along the central long axis of the epidermis. The depth and state of the fluorescence entering the skin were observed under a fluorescence microscope.
Example 11: shuttle-type flexible liposome delivery recombinant mir218 for promoting mouse hair growth
The experiment is divided into: blank control group (PBS solution) and treatment group (recombinant mir-218). 32 healthy C57BL6 male mice at 2 months of age were purchased as experimental animals, and 8 mice were randomly selected per group. Selecting skin parts on two sides of the back spine to remove hairs under the condition of not damaging epidermis. After the modeling is finished, the blank control group is sprayed with PBS, the treatment group is respectively sprayed with fusiform flexible liposome and recombinant miR-218 solution, and the concentrations of the recombinant miR-218 are respectively 2.5 mug/mL, 5 mug/mL and 10 mug/mL. The dosage of each mouse is 1mL, the medicine is sprayed on the back of the experimental animal to remove hair, then the medicine is massaged for 1 minute to promote skin absorption, the medicine is taken 1 time a day and lasts for 20 days, and the hair growth condition is counted.
As shown in FIG. 12, the treatment group of recombinant mir-218 promoted hair regrowth to a different extent over time than the PBS blank control group, with a very significant effect at 20 days. The treatment group of recombinant mir-218 had more than 60% coverage of new hairs at 20d, with a significant difference from the control group mice in coverage of new hairs,. P <0.05,. P <0.01, and. P < 0.001.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications and changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (9)
1. The cationic shuttle-type flexible liposome capable of carrying RNA is characterized in that the particle size of the flexible liposome is 90 nm-120 nm, and the raw materials comprise the following components in parts by weight: 40-50 parts of (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, 12-15 parts of dioleoyl phosphatidylethanolamine, 0.8-1 part of cholesterol, 8-10 parts of sodium deoxycholate and 8-10 parts of glucan.
2. A method of preparing the RNA loadable cationic fusiform flexible liposome of claim 1, comprising the steps of:
dissolving (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride, dioleoyl phosphatidylethanolamine and cholesterol in chloroform; then using a vacuum rotary evaporator under the condition of water bath, removing chloroform in the solution by reduced pressure evaporation, forming a uniform film on the bottle wall, and then placing at-20 ℃ for cooling;
step two, adding a sodium deoxycholate solution into the product cooled in the step one, and carrying out rotary hydration by DEPC water until elution is carried out to obtain a suspension primary liquid of the cationic flexible liposome;
and step three, adding glucan into the primary suspension liquid of the cationic flexible liposome in the step two, stirring and mixing at room temperature, and filtering by using a microporous filter membrane to obtain the cationic fusiform flexible liposome capable of carrying RNA.
3. The method of claim 2, wherein chloroform is added in step one in an amount to provide a final concentration of (2, 3-dioleoyl-propyl) -trimethylammonium chloride of 1mg/mL to 3 mg/mL.
4. The method as claimed in claim 2, wherein 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 6 h.
5. The method according to claim 2, wherein the temperature of the water bath for the rotary hydration in the second step is 40-50 ℃, and the rotation condition is 80-100 rpm.
6. The method as claimed in claim 2, wherein the pore size of the microfiltration membrane in step three is 0.22 μm.
7. The method of claim 2, wherein the mass ratio of the RNA to the (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride in the cationic shuttle-type flexible liposome is 1 (4-5), the incubation environment is RNase-free, and the incubation time is 15-20 min.
8. Use of the RNA loadable cationic fusiform flexible liposome of claim 1 for the preparation of a cosmetic for external application to the skin for delivering siRNA drugs.
9. Use of the RNA loadable cationic fusiform flexible liposome of claim 1 for the preparation of a medicament for external application to the skin for delivering an RNA drug.
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