Cationic super-deformation liposome capable of realizing gene transdermal transfection as well as preparation and application of cationic super-deformation liposome

Abstract

The invention discloses a cationic super-deformation liposome capable of realizing gene transdermal transfection as well as preparation and application of the cationic super-deformation liposome, and belongs to the field of gene transdermal delivery. Materials such as low-molecular-weight polyethyleneimine, lipidosome and Tween 80 are adopted, and the components are safe and simple; polyethyleneimine is used as a conjugation carrier of the gene to improve the binding capacity of the gene, liposome is used as a delivery carrier to improve the skin delivery capacity of the gene, and Tween 80 is used for improving the stability and transdermal transfection capacity of the carrier again, so that non-invasive targeted transdermal delivery of the gene is realized; in vitro stability is high, gene degradation can be effectively inhibited, and meanwhile in vitro cell experiments, high transfection efficiency and low toxicity are achieved; the cationic super-deformed liposome can successfully realize high-efficiency transdermal penetration of the gene and effectively promote transfection expression of the gene in the skin at the same time, a new idea is provided for gene delivery, and the advantage of treating skin diseases through non-invasive delivery of the gene is expected to be fully exerted.

Description

Technical Field

[0001] The invention belongs to the field of transdermal gene delivery, and specifically relates to a cationic super-deformable liposome capable of realizing transdermal gene transfection, and a preparation method and application thereof. Background Art

[0002] As an important treatment method, gene therapy treats genetic diseases by delivering genetic material to target cells to compensate or correct gene defects, which has completely changed the traditional medical methods of vaccination, cancer and autoimmune diseases. In recent years, with the progress of gene editing technology and immunotherapy, gene therapy has broad application prospects. However, the current gene delivery methods are limited to intravenous administration or direct local injection, which cannot achieve good biosafety, tissue targeting and transfection efficiency, resulting in certain limitations in the use of gene therapy, and the development of gene carriers is urgently needed. Among the gene delivery pathways, non-invasive transdermal delivery is an extremely attractive method and has made an increasing contribution to medical clinical applications. It has continuous and steady-state pharmacokinetics and its use is non-invasive and painless, which can bring compliance and convenience to patients and has broad application prospects. For example, Zhang et al. combined tannic acid (TA), RNase H response sequence, siRNA and tFNA into a new transdermal system STT with controllable assembly and disassembly. After transdermal administration, STT can specifically silence NF-κB p65, thereby maintaining the stability of the skin microenvironment and reshaping normal skin immune defense. This work demonstrates the advantages of STT in RNAi therapy and its potential for treating skin-related diseases in the future (Advanced Science, 2023, 10(33): 2303706). Gene carriers should achieve the following points: (1) stability during delivery; (2) efficient transdermal delivery capability; (3) efficient cell uptake; (4) successful release of plasmids into cells and exert their effects. Therefore, how to construct a new type of material that can effectively penetrate the skin and promote safe transfection of target genes through structural design of the material and the advantages of transdermal delivery combined with gene therapy to achieve non-invasive and efficient treatment of skin diseases has become an important scientific issue that needs to be solved urgently.

[0003] In recent years, researchers have proposed a variety of methods to improve the transdermal penetration of genes, mainly by changing the barrier function of the skin, external force driving the carrier, modifying or encapsulating the gene, etc. However, most of these methods will cause skin damage and affect the normal physiological function of the skin. Therefore, the development of non-invasive carriers for transdermal delivery has incomparable advantages. Liposome nanocarriers, as a kind of artificially prepared transdermal delivery system, have received extensive attention and research. They can encapsulate hydrophilic or lipophilic drugs and are widely used in intravenous, skin, lung and oral administration. Liposomes have been widely studied as carriers for transdermal gene delivery. For example, DOTAP lipid nanoparticles developed by Li et al. were successfully loaded with siRNA for targeted delivery and applied to pig skin. The transdermal permeability of siRNA was examined to evaluate its possible use in gene therapy (Biotechnology Journal, 2020, 15(12): 2000079). Arruda et al. used a simple nanoprecipitation method to develop a new type of hybrid lipid polymer nanoparticles. Through this method, hybrid lipid polymer nanoparticles were successfully prepared, which proved that they can be stably and efficiently used for specific silencing of target genes and achieved excellent results in oral delivery treatment of ulcerative colitis mice (Journal of Controlled Release, 2022, 350:228-243). Many studies at home and abroad have demonstrated the effectiveness of liposome nanocarriers in delivering gene therapy for diseases, but due to the poor transdermal effect of ordinary liposomes, most treatment methods still use injections or microneedles, and the difficulties of achieving non-invasive transdermal treatment of liposomes still need to be overcome.

[0004] Polyethyleneimine (PEI), as a typical representative of cationic polymers, is the most commonly used non-viral gene carrier. It can carry genes through electrostatic action and can successfully escape genes through the "proton sponge" effect. It is known as the gold standard for polymer gene delivery. In recent years, a series of studies have been conducted on the role of this polycation in gene therapy, and its ability to transfect a variety of cells has been fully confirmed. For example, Hasanzadeh et al. used nanoparticles prepared by cerium oxide coating containing PEI as a gene delivery carrier. The transfection results showed that the gene delivery carrier has low cytotoxicity and can be used as a combination therapy with conventional chemotherapy drugs to treat cancer (Life sciences, 2019, 232: 116661). Domestic and foreign studies have shown that it is difficult to achieve both high-efficiency gene transfection and low cytotoxicity of PEI due to the relationship between molecular weight. Therefore, when constructing an ideal gene carrier, PEI should have good biocompatibility, specific targeting and high buffering capacity. As a surfactant, Tween 80 is widely used in the development of transdermal systems due to its low toxicity, high biocompatibility, protein stability, and penetration-promoting properties. For example, Ni et al. added Tween 80 to multi-level targeted liposomes to give them super-deformability and constructed a delivery system for melanoma treatment. Studies have shown that the modified carrier has good penetrability, stability and permeability, and can achieve effective treatment of melanoma (Journal of Controlled Release, 2022, 351: 245-254).

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a cationic super-deformable liposome capable of achieving transdermal gene transfection and its preparation method and application. The cationic super-deformable liposome is a liposome with high-efficiency transdermal and targeted transfection functions.

[0007] The present invention aims at realizing non-invasive transdermal delivery and efficient transfection of genes, using cationic polymer polyethyleneimine (PEI) as a gene conjugation carrier to enhance its binding ability, using liposomes as gene delivery carriers to enhance its skin delivery ability, using Tween 80 to reduce the toxicity of the carrier and further enhance the transdermal delivery ability of the carrier while promoting its transfection in the skin, and embedding the cationic polymer into the liposome structure through chemical modification to enhance the delivery stability, and finally constructing a lipid nanocarrier that can realize non-invasive and efficient transdermal gene delivery and transfection.

[0008] The purpose of the present invention is achieved through the following technical solutions: One of the technical schemes of the present invention is a method for preparing a cationic super-deformable liposome, comprising the following steps: dissolving a phospholipid and a cationic polymer donor material in an organic solvent at room temperature to obtain a solution D, dissolving Tween 80 in enzyme-free water to obtain a solution E, then slowly adding the solution E to the solution D, using a high-shear dispersing emulsifier to mix the two uniformly, using a rotary evaporator to evaporate and remove the mixed solvent to obtain a solid F, finally adding enzyme-free water to the solid F for ultrasonic hydration, and finally obtaining the cationic super-deformable liposome suspension capable of loading genes through a micro-jet high-pressure process.

[0009] Preferably, the phospholipid comprises soybean lecithin, and the mass ratio of the phospholipid to the cationic polymer donor material is (1-1.5):1; the organic solvent comprises a mixed solution of ethanol and chloroform in a volume ratio of 1:(0.5-1); the amount of the organic solvent is calculated by adding 0.3-0.5 mg (preferably 0.45 mg) of soybean lecithin per 1 mL; the mass ratio of Tween 80 to the cationic polymer donor material is 1:(4-5); the molecular weight of Tween 80 is 604; the mass volume ratio of Tween 80 to enzyme-free water is (2-5) mg:2 mL; the slow addition speed is 4-5 mL / min; the power of the high shear dispersing emulsifier is 200-300 W, the speed is 8000 rpm, and the time for each time is 5 min; the speed of the rotary evaporation is 80-120 rpm (preferably 100 rpm), the temperature is 50-60°C (preferably 60°C), and the vacuum degree of the evaporating flask is 70-100 kPa (preferably 100 kPa); the mass volume ratio of the solid F to the enzyme-free water is (20-30) mg: 20 mL; the ultrasonic time is 20-30 min (preferably 20 min); the microjet high pressure condition is extrusion at a pressure of 500-800 bar for 3-5 times (preferably extrusion at a pressure of 500 bar for 5 times).

[0010] Preferably, the cationic polymer donor material is cholesterol modified with a cationic polymer; further, cholesterol modified with polyethyleneimine.

[0011] The method for preparing the cationic polymer donor material comprises the following steps: The cationic polymer is dissolved in an organic solvent A, cholesterol chloroformate and a catalyst triethylamine are added, and the reaction is carried out at 0-4°C, and then the temperature is heated to 5-35°C, and the reaction is continued. After the reaction is completed, the concentrate is concentrated, and hydrochloric acid is added to the obtained concentrate for redissolution, washed, precipitated, and dried to obtain cationic polymer-modified cholesterol, wherein the mass ratio of the cationic polymer to cholesterol chloroformate is 1: (5-10).

[0012] Preferably, the cationic polymer includes at least one hyperbranched polyethyleneimine with a molecular weight of 600 to 3000 (preferably 1000 to 2500; more preferably 1800±300); the organic solvent A includes dichloromethane; the amount of the organic solvent A is calculated by adding 1 to 4 g (preferably 1 g) of cholesterol chloroformate per 10 mL; the amount of the triethylamine is calculated by adding 0.1 to 0.5 mol (preferably 0.1 mol) of cholesterol chloroformate per 1 mol; the concentration of the hydrochloric acid is 0.1 to 0.4 M (preferably 0.1 M); the amount of the hydrochloric acid is added by adding 1 to 2 g of concentrate per 5 to 10 mL; the reaction time at 0 to 4°C is 10 to 30 min (preferably 20 min), and the continued reaction time is 10 to 14 h (preferably 12 h).

[0013] The second technical solution of the present invention: a cationic super-deformable liposome prepared according to the above preparation method.

[0014] The cationic super-deformable liposome can be used as a carrier for preparing gene transdermal transfection delivery.

[0015] The third technical solution of the present invention: a cationic super-deformable liposome complex loaded with a gene, comprising a gene and the above cationic super-deformable liposome. Furthermore, the mass ratio of the cationic super-deformable liposome to the gene is (5-80):1.

[0016] The preparation method of the gene-loaded cationic super-deformable liposome complex comprises the following steps: the complex solution comprises the above-mentioned cationic super-deformable liposome suspension and the gene solution; specifically, a certain amount of the cationic super-deformable liposome suspension is added to the gene solution, vortexed and allowed to stand for mixing and incubation for a certain period of time to obtain the gene-loaded cationic super-deformable liposome complex.

[0017] Preferably, the gene is at least one of DNA, siRNA, shRNA, gRNA, sgRNA and mRNA and genes for CRISPR-Cas9 gene editing; the DNA is preferably plasmid DNA; the vortex speed is 1000-1500 rpm (preferably 1000 rpm); the vortex time is 3-5 min (preferably 3 min); the static mixing incubation time is 25-30 min (preferably 30 min).

[0018] The fourth technical solution of the present invention: application of the above-mentioned gene-loaded cationic super-deformable liposome complex in the preparation of products that improve gene stability.

[0019] The fifth technical solution of the present invention: the use of the above-mentioned gene-loaded cationic super-deformable liposome complex in the preparation of a product for improving the in vitro transfection efficiency of genes in skin keratinocytes.

[0020] Technical solution six of the present invention: Application of the above-mentioned gene-loaded cationic super-deformable liposome complex in the preparation of products for improving gene transdermal efficiency.

[0021] Technical solution seven of the present invention: Use of the above-mentioned gene-loaded cationic super-deformable liposome complex in the preparation of products for enhancing gene transdermal transfection.

[0022] Technical solution eight of the present invention: Use of the above-mentioned gene-loaded cationic super-deformable liposome complex in the preparation of products for treating skin diseases through non-invasive gene delivery.

[0023] Compared with the prior art, the present invention has the following advantages and effects: (1) Compared with traditional gene carriers, the present invention uses low molecular weight polyethyleneimine, liposomes, Tween 80 and other materials, which are safe and simple in composition; cationic polymer polyethyleneimine is used as a gene conjugation carrier to enhance its binding ability, liposomes are used as delivery carriers to enhance its skin delivery ability, and Tween 80 is used to further enhance the stability and transdermal transfection ability of the carrier, thereby achieving non-invasive targeted transdermal delivery of genes.

[0024] (2) Compared with traditional gene vectors, the vector of the present invention has higher stability in vitro and can effectively inhibit gene degradation. At the same time, the vector has higher transfection efficiency and lower toxicity in in vitro cell experiments, which solves the contradiction between the high transfection efficiency and high cytotoxicity of polyethyleneimine.

[0025] (3) The vector of the present invention not only successfully achieves efficient gene transdermal delivery, but also effectively promotes its transfection expression in the skin. For example, carrying siRNA can exert a better effect in treating skin diseases. It provides a new idea for gene delivery and is expected to give full play to the advantages of non-invasive gene delivery in treating skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 Schematic diagram of the construction and gene loading of cationic superdeformable liposomes.

[0028] Figure 2 The characterization diagram of the cationic super deformable liposomes prepared in Example 2; A is the particle size and potential diagram; B is the deformation index result diagram, **** indicates that the difference is extremely significant ( P <0.0001).

[0029] Figure 3 The diagrams are for the determination of the plasmid-loaded cationic super-deformable liposome complex obtained in Example 3; wherein A is an agarose gel electrophoresis diagram, Nake represents a plasmid control, and B is a potential determination diagram.

[0030] Figure 4 This is an analysis diagram of the inhibition of gene degradation by the cationic superdeformable liposome complex loaded with plasmid; wherein, "+" represents the TCPL / Plasmid group, and "-" represents the CPL / Plasmid group.

[0031] Figure 5 The in vitro transfection verification of the cationic super-deformable liposome complex loaded with plasmid in human keratinocytes; A is the transfection efficiency diagram, and *** indicates a very significant difference ( P <0.001); B is the cytotoxicity graph; C is the expression graph of fluorescent protein in cells after treatment with TCPL / Plasmid complex.

[0032] Figure 6 This is a diagram of the in vivo transdermal verification of the Cy5 fluorescently labeled plasmid in mice; wherein A is the plasmid labeling result diagram; and B is the plasmid penetration diagram in the skin tissue.

[0033] Figure 7 This is a diagram showing the in vitro transdermal validation of Cy5 fluorescently labeled plasmid using a Franz diffusion cell.

[0034] Figure 8 The red fluorescent protein expression diagram of mice; A is the in vivo imaging diagram of mice; B is the fluorescent protein expression diagram of skin tissue. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0036] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0038] In the examples, the preparation method of anhydrous dichloromethane, chloroform or anhydrous ethanol refers to the document "CN115400084B, A liposome capable of releasing NO, its preparation method and application".

[0039] In the embodiment, the purity of the acetone used is ACS, ≥99.5%; the number average molecular weight of the polyethyleneimine used is 1800; the purity of the cholesterol used is AR, 98%; In the examples, room temperature refers to 10 to 35°C.

[0040] The description will not be repeated below.

[0041] The schematic diagram of the preparation of cationic superdeformable liposomes and gene loading is shown in Figure 1 shown.

[0042] Example 1 Cationic polymer donor material: Preparation of polyethyleneimine-modified cholesterol (Cho-PEI): Under ice-water bath conditions, polyethyleneimine (PEI) with a molecular weight of 1800 was dissolved in anhydrous dichloromethane. Triethylamine was used as a catalyst, and cholesterol chloroformate was added in a mass ratio of 1:10 to PEI. 1 g of cholesterol chloroformate was added to every 10 mL of anhydrous dichloromethane. The amount of triethylamine was 0.1 mol of cholesterol chloroformate per 1 mol. The reaction was carried out at 0-4 °C for 20 min and then warmed to room temperature (25 °C). After continuing the reaction for 12 h, it was concentrated under reduced pressure and the concentrate was redissolved with 10 mL of 0.1 M hydrochloric acid solution. Then, it was washed with dichloromethane, precipitated with acetone, and dried in vacuum at room temperature at 25 °C to obtain a white solid, namely, cholesterol Cho-PEI modified with polyethyleneimine.

[0043] Example 2 A preparation method of cationic super deformable liposomes comprises the following steps: Under room temperature, soybean lecithin and the cationic polymer donor material Cho-PEI prepared in Example 1 were dissolved in a mixed solution of anhydrous ethanol and chloroform, heated and stirred until completely dissolved, wherein the mass ratio of soybean lecithin to the cationic polymer donor material Cho-PEI was 1:1, the volume ratio of anhydrous ethanol to chloroform was 1:1, and the amount of the mixed solution was 0.45 mg soybean lecithin per 1 mL; Tween 80 with a molecular weight of 604 was dissolved in enzyme-free water, the mass volume ratio of Tween 80 to enzyme-free water was 1 mg / mL, and the mass ratio of Tween 80 to the cationic polymer donor material Cho-PEI was 1:5, and it was slowly (5 mL / min) added to the above organic mixed solution, and a high shear dispersing emulsifier (power of 300 W, speed of 8000 rpm, each time for 5 min) was used to mix the two evenly and then perform rotary evaporation, the set temperature was 60°C, the speed was 100 rpm, the vacuum degree of the evaporating flask was 100 kPa, and the rotary evaporation was 0.5~1 h, wait for the organic solvent to evaporate completely to obtain a white film solid, then dissolve it with enzyme-free water at a mass volume ratio of 1 mg:1 mL, and ultrasonically hydrate it for 20 min to obtain a milky white liposome suspension, which is extruded 5 times under a microfluidizer pressure of 500 bar to obtain a cationic super deformable liposome suspension, denoted as TCPL, and stored at 4°C.

[0044] Referring to the preparation steps of cationic super deformable liposomes in this example, the cationic polymer donor material Cho-PEI in this example is replaced by cholesterol chloroformate Cho in Example 1, which is denoted as TCL.

[0045] Referring to the preparation steps of cationic super deformable liposomes in this example, Tween 80 was not added and it was recorded as CPL.

[0046] Example 3 A preparation method of a gene-loaded cationic super-deformable liposome complex comprises the following steps: At room temperature, a plasmid expressing green fluorescent protein (pcDNA3.1-GFP, purchased from GenePharma) was selected as a model gene (Plasmid), and the cationic super deformable liposome suspension prepared in Example 2 was added. The mass ratio (w / w) of the cationic super deformable liposome to the plasmid was set to 1, 5, 10, 20, 40, and 80, respectively. After vortexing at 1000 rpm for 3 min, the mixture was allowed to stand and incubate for 30 min to obtain cationic super deformable liposome complexes loaded with plasmids at different mass ratios.

[0047] Example 4 100 μL of TCL, TCPL, and CPL obtained in Example 2 were dispersed into 1 mL of pure water respectively. After being evenly dispersed by ultrasonic, the potential and particle size were measured using a Malvern laser particle size analyzer. Three replicates were set for each group. The results are as follows: Figure 2 As shown in A, it can be seen that the hydrated particle size of cationic super deformable liposomes TCPL and CPL are both below 500 nm and are uniform in size; however, the addition of Tween 80 increases the potential of TCPL, which fully demonstrates that the surfactant can maintain the electrical layer state of the liposomes, keeping it at a higher potential and thus maintaining its stability. 1 mL of TCPL and CPL were added to the liposome extruder (Avanti®Polar Lipids, USA) respectively. The liposomes were passed through a 50 nm polycarbonate membrane at a pressure of 1 MPa. The extrusion volume was recorded after 5 min, and the particle size of each formulation before and after extrusion was measured using a laser particle size analyzer. The deformation index was calculated according to the formula. Figure 2 As shown in B, the deformation index of TCPL liposomes with added Tween 80 reached 0.37, which was significantly higher than that of CPL liposomes without added Tween 80 (0.15), which laid the foundation for the subsequent gene loading and transdermal transfection applications of TCPL liposomes.

[0048] Example 5 The cationic super-deformable liposome complexes with different mass ratios of loaded plasmids obtained in Example 3 were subjected to potential measurement and agarose gel electrophoresis experiments to verify the composite ability of the material and the gene. Weigh 0.7 g of agarose in a beaker, add 70 mL of TAE × 1 solution, and after fully dissolving, put it in a microwave oven to heat and boil, add 3 μL of Goldview thereto, and shake it well to dissolve it completely. Set up the gel tank, pour all of it into the gel tank while hot, and after removing all the bubbles, fix it in the groove on the left, and cool it for 30 min until the gel is formed. After the jack plate is pulled out, add 3 μL of Loading Buffer to 10 μL of the prepared materials of different concentrations. After fully mixing, add the gel wells and transfer them to the gel electrophoresis instrument, and then add some TAE solution to make it completely submerged. Run at 120V for 25 min. After the operation is completed, use a UV gel imager to image at a UV wavelength of 365 nm to characterize the gene load. Three replicates are set for each group. The results are as follows Figure 3As shown in A, as the mass of liposomes increases, the migration of DNA varies. In the groups with a w / w ratio of less than 5, obvious bright bands can be seen migrating from the pores, proving that the material cannot combine well with the plasmid when the w / w ratio is less than 5. When the mass ratio is greater than or equal to 5, no bright bands migrate out of the pores, indicating that the material has a good combination with the gene and forms a stable complex. This result was also verified in the potential measurement. When the mass ratio is greater than or equal to 5, the complex begins to show a positive potential, which can achieve effective combination with the gene ( Figure 3 B in the figure).

[0049] Example 6 The TCPL and CPL obtained in Example 2 were loaded with the plasmids described in Example 3 for enzyme degradation analysis to verify the inhibitory gene degradation function of TCPL and CPL, wherein the mass ratio of TCPL, CPL and plasmid was 10:1. At 37°C, CPL / Plasmid and TCPL / Plasmid were mixed with DNA enzymes, respectively, and then incubated for 2 h and 6 h, respectively, and then agarose gel electrophoresis was performed to detect the enzyme stability of the material. The total volume of the mixed system was 30 μL, wherein the final concentration of the plasmid Plasmid was 2 μg / 30 μL, and the final enzyme activity of the DNA enzyme was 5 U / μL. Figure 4 As shown in the figure, after 6 hours, the plasmid in the CPL / Plasmid group had been completely degraded by DNA enzyme, and no bands were visible in agarose gel electrophoresis, while the TCPL / Plasmid group still had bands. The enzyme degradation experiment fully demonstrated the protective function of cationic super deformable liposome TCPL when loading genes, laying the foundation for subsequent transdermal transfection.

[0050] Example 7 The TCPL and CPL obtained in Example 2 were loaded with the plasmid expressing green fluorescent protein described in Example 3 for transfection of human skin keratinocytes (HaCat). This cell is difficult to transfect and is often used as a model cell for skin application. In order to maximize the in vitro transfection efficiency and control the toxicity of the material, the mass ratio (w / w) of liposomes to green fluorescent protein plasmids was 80. First, HaCat cells were plated at a cell number of 5×10 4The cells were inoculated at a density of 100 μL in a 24-well plate. When the cell fusion rate reached 70%, TCPL / Plasmid and CPL / Plasmid complexes (pcDNA3.1-GFP plasmid 1 μg / well) were added respectively. PEI-25000 was used as a positive control (w / w=1.3:1, that is, the mass ratio of PEI-25000 to plasmid was 1.3:1). The blank group cells were used as negative controls (Control). Three replicates were set up for each group. After 4 h of culture, the culture medium in the wells was discarded, and 500 μL of fresh complete culture medium was added to continue culturing for 20 h. The expression of green fluorescent protein was qualitatively observed under an inverted fluorescence microscope and photographed. Subsequently, the culture medium was discarded, washed with PBS, digested with trypsin, and collected by centrifugation (1000 rpm, 5 min). Finally, the cells were resuspended with an appropriate amount of PBS, and the transfection efficiency was quantitatively analyzed by flow cytometry, as shown in the following figure. Figure 5 As shown in A, when TCPL and CPL were loaded with the same mass of plasmid, the transfection efficiency of TCPL reached about 55%, which was significantly higher than that of CPL liposomes (45.7%) and positive control (36.6%). This fully shows that the addition of Tween 80 is conducive to "stabilizing" the liposome system and significantly improving the transfection effect of cells. The expression of green fluorescence in cells treated with TCPL / Plasmid complex was clearly seen using a fluorescence microscope ( Figure 5 C), cytotoxicity results are shown in Figure 5 As shown in B, the TCPL / Plasmid and CPL / Plasmid complexes at this ratio have lower toxicity and are significantly superior to the positive control PEI-25000. This transdermal transfection system has good biosafety.

[0051] Example 8 The plasmid described in Example 3 was fluorescently labeled with Cy5 using Label IT Tracker reagent and then compounded with TCPL or CPL obtained in Example 2 according to the operation method of Example 3. The mass ratio of TCPL, CPL and Cy5 fluorescently labeled plasmid (CY5-Plasmid) was 80. Figure 6 As shown in A, laser confocal microscopy shows that the plasmid was successfully labeled with Cy5 fluorescence. After the mice (6-8 weeks old Balb / c mice, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.) were anesthetized, the backs were depilated, and 100 μL of the liposome complex was evenly applied to the back skin of the mice, covering an area of ​​1.5 cm 2At the same time, a CY5-Plasmid group was set up, with 3 mice in each group. After 24 hours, the skin tissue on the back of the mice was peeled off, and the residual material on the skin surface was washed with PBS (pH = 7.4) and dried. The frozen skin tissue was further cut vertically into skin slices with a thickness of about 10 μm using a freezing microtome. The distribution of Cy5 fluorescent labeling in the skin was observed using a fluorescence microscope. The results are shown in Figure 2. Figure 6 As shown in B, the plasmid loaded by cationic superdeformable liposomes successfully passed through the stratum corneum and reached the epidermis and dermis.

[0052] Example 9 The plasmid described in Example 3 was labeled with Cy5 fluorescence using Label IT Tracker reagent and then compounded with TCPL and CPL liposomes obtained in Example 2 according to the operation method of Example 3. The mass ratio of TCPL and CPL liposomes to the Cy5 fluorescently labeled plasmid (CY5-Plasmid) was 80. In this example, a Franz diffusion cell (TP-6) was used to conduct an in vitro skin penetration experiment simulating the surface temperature of the human body. The receiving chamber was filled with PBS buffer solution (pH = 7.4) and stirred continuously at a speed of 200 rpm. The diffusion cell was always kept at 37°C by a circulating water bath. A mouse skin of appropriate size (the mouse skin was prepared with reference to Example 7 of “CN115590774A-Hyaluronic acid liposome assembly and its preparation method and application”) was sandwiched between the supply chamber and the receiving chamber, wherein the surface of the skin faced the supply chamber; 1 mL of Cy5 fluorescently labeled plasmid and TCPL and CPL loaded plasmid were respectively transferred into the upper supply chamber of the Franz diffusion cell and sealed; after 24 hours, the mouse back skin tissue was removed, the residual material on the skin surface was washed with PBS (pH= 7.4) and dried, and the frozen skin tissue was further cut vertically into skin slices with a thickness of about 10 μm using a freezing microtome, and the distribution of Cy5 fluorescent labeling in the skin was observed using a fluorescence microscope. The results are as follows: Figure 7 As shown, cationic superdeformable liposomes loaded with plasmids successfully penetrated the mouse skin.

[0053] Example 10 The TCPL and CPL liposomes obtained in Example 2 were respectively compounded with a plasmid expressing red fluorescent protein (pcDNA3.1-RFP, purchased from GenePharma) at a mass ratio of 80. 100 μL of the liposome complex was evenly applied (once every 24 h) to the back of mice (6-8 week old Balb / c nude mice, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.) after back hair removal until completely absorbed. The area was 1.5 cm 2Plasmid group and blank control (Blank) were set up at the same time, with 3 mice in each group. The red fluorescence expression of the back of the mice was detected by small animal living imaging at 24 h and 48 h, respectively, with an excitation wavelength of 520 nm and an emission wavelength of 570 nm. Figure 8 As shown in A, the red fluorescent protein plasmid loaded with TCPL was successfully expressed in the mouse skin at 24 h and the fluorescence expression gradually became stronger after 48 h. After 48 h, the mice were euthanized, and the back skin was taken to observe the expression of fluorescent protein in the skin using a live imaging device. The skin tissue was fixed with 4% paraformaldehyde and frozen, and the penetration of fluorescent protein in the tissue was observed using a fluorescence microscope. Figure 8 As shown in B, it can be clearly seen that neither the simple plasmid nor the CPL-loaded plasmid expressed fluorescent protein, while the cationic super deformable liposome TCPL-loaded plasmid successfully expressed red fluorescent protein in the mouse skin, which fully proves that the vector has the ability to load genes for transdermal transfection.

[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a cationic super deformable liposome, characterized in that: The method comprises the following steps: dissolving phospholipid and cationic polymer donor material in an organic solvent at room temperature to obtain solution D, dissolving Tween 80 in enzyme-free water to obtain solution E, then slowly adding solution E to solution D, mixing the two evenly, removing the solvent to obtain solid F, finally adding enzyme-free water to solid F for ultrasonic hydration, and finally obtaining a cationic super-deformable liposome suspension capable of loading genes through a micro-jet high-pressure process; The cationic polymer donor material is cholesterol modified by a cationic polymer; further, it is cholesterol modified by polyethyleneimine.

2. The preparation method according to claim 1, characterized in that: The phospholipid comprises soybean lecithin, and the mass ratio of the phospholipid to the cationic polymer donor material is (1-1.5):1; the organic solvent comprises a mixed solution of ethanol and chloroform in a volume ratio of 1:(0.5-1); the amount of the organic solvent is calculated by adding 0.3-0.5 mg soybean lecithin per 1 mL; the mass ratio of Tween 80 to the cationic polymer donor material is 1:(4-5); the mass volume ratio of Tween 80 to enzyme-free water is (2-5) mg:2 mL; the slow addition speed is 4-5 mL / min; the mass volume ratio of the solid F to enzyme-free water is (20-30) mg:20 mL; the ultrasonic time is 20-30 min; and the microjet high pressure condition is extrusion at a pressure of 500-800 bar for 3-5 times.

3. The preparation method according to claim 1, characterized in that: The cationic polymer includes at least one of hyperbranched polyethyleneimines having a molecular weight of 600 to 3000; Furthermore, the method for preparing the cationic polymer donor material comprises the following steps: The cationic polymer is dissolved in an organic solvent A, cholesterol chloroformate and a catalyst triethylamine are added, and the reaction is carried out at 0-4°C, and then the temperature is heated to 5-35°C, and the reaction is continued. After the reaction is completed, the concentrate is concentrated, and hydrochloric acid is added to the obtained concentrate for redissolution, washed, precipitated, and dried to obtain cationic polymer-modified cholesterol, wherein the mass ratio of the cationic polymer to cholesterol chloroformate is 1: (5-10).

4. The preparation method according to claim 3, characterized in that: The organic solvent A includes dichloromethane; the amount of the organic solvent A is calculated by adding 1 to 4 g of cholesterol chloroformate per 10 mL; the amount of triethylamine is calculated by adding 0.1 to 0.5 mol of cholesterol chloroformate per 1 mol; the reaction time at 0 to 4° C. is 10 to 30 min, and the continued reaction time is 10 to 14 h.

5. A cationic super deformable liposome, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the cationic super-deformable liposomes according to claim 5 as a carrier for preparing gene transdermal transfection delivery.

7. A gene-loaded cationic super-deformable liposome complex, characterized in that: Comprising a gene and the cationic super deformable liposome according to claim 5; Furthermore, the mass ratio of the cationic super deformable liposome to the gene is (5-80):

1.

8. The method for preparing the gene-loaded cationic super-deformable liposome complex according to claim 7, characterized in that: The following steps are involved: The complex solution comprises a cationic super-deformable liposome suspension prepared by the preparation method according to any one of claims 1 to 4 and a gene solution; specifically, a certain amount of cationic super-deformable liposome suspension is added to the gene solution, vortexed and allowed to stand for mixing and incubation for a certain period of time to obtain a cationic super-deformable liposome complex loaded with a gene.

9. The preparation method according to claim 8, characterized in that: The gene is at least one of DNA, siRNA, shRNA, gRNA, sgRNA and mRNA; and the static mixing incubation time is 25 to 30 minutes.

10. The use of the gene-loaded cationic super-deformable liposome complex according to claim 7, characterized in that: Apply one or more combinations of the following: (1) Application in the preparation of products that improve gene stability; (2) Application in the preparation of products for improving the efficiency of gene transfection in skin keratinocytes in vitro; (3) Application in the preparation of products that improve the efficiency of gene transdermal penetration; (4) Application in the preparation of products that enhance transdermal gene delivery; (5) Application in the preparation of products for treating skin diseases through non-invasive gene delivery.

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