Cationic superdeformable liposome capable of realizing gene transdermal transfection and preparation and application thereof

Abstract

The application discloses cationic super-deformable liposomes capable of realizing gene transdermal transfection and a preparation and application thereof, and belongs to the field of gene transdermal delivery. The application adopts low-molecular-weight polyethyleneimine, liposomes, Tween 80 and other materials, and the components are safe and simple; polyethyleneimine is used as a conjugated carrier of the gene to improve the combination capacity of the gene; liposomes are used as a delivery carrier to improve the skin delivery capacity of the liposomes; Tween 80 is used to improve the stability and transdermal transfection capacity of the carrier again, so that non-invasive targeted transdermal delivery of the gene is realized; the cationic super-deformable liposomes have high stability in vitro and can effectively inhibit gene degradation, and have high transfection efficiency and low toxicity in in-vitro cell experiments; the cationic super-deformable liposomes successfully realize efficient transdermal delivery of the gene and effectively promote transdermal transfection and expression of the gene in the skin, so that a new thought is provided for gene delivery, and the cationic super-deformable liposomes are expected to fully play the advantages of treating skin diseases through non-invasive gene delivery.

Description

Technical Field

[0001] This invention belongs to the field of transdermal gene delivery, specifically relating to a cationic hyperdeformable liposome capable of transdermal gene transfection, its preparation method, and its application. Background Technology

[0002] Gene therapy, as an important treatment modality, treats hereditary diseases by delivering genetic material to target cells to compensate for or correct gene defects, revolutionizing traditional medical approaches to vaccination, cancer, and autoimmune diseases. In recent years, with advancements in gene editing technology and immunotherapy, gene therapy has shown great promise. However, current gene delivery methods are limited to intravenous administration or direct local injection, failing to achieve good biosafety, tissue targeting, and transfection efficiency, thus limiting the use of gene therapy and necessitating the development of gene vectors. Among gene delivery routes, non-invasive transdermal delivery is a highly attractive approach, making increasingly significant contributions to clinical medical applications. It possesses continuous and steady-state pharmacokinetics, and its non-invasive and painless nature enhances patient compliance and convenience, demonstrating broad application prospects. For example, Zhang et al. combined tannic acid (TA), RNase H-responsive sequences, siRNA, and tFNA to create a novel 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 remodeling normal skin immune defense. This work demonstrates the advantages of STT in RNAi therapy and its potential for future treatment of skin-related diseases (Advanced Science, 2023, 10(33): 2303706). Gene vectors should achieve the following: (1) stability during delivery; (2) efficient transdermal delivery capability; (3) efficient cellular uptake; (4) successful release of plasmids into cells and their function. Therefore, how to construct a new type of material that can effectively transdermally deliver and promote the safe transfection of target genes to achieve non-invasive and efficient treatment of skin diseases by designing the structure of the material and utilizing the advantages of transdermal delivery combined with gene therapy has become an important scientific problem that urgently needs to be solved.

[0003] In recent years, researchers have proposed various methods to improve the transdermal penetration of genes, mainly by altering the skin barrier function, using externally driven carriers, modifying or encapsulating genes. However, most of these methods cause skin damage and affect the normal physiological function of the skin. Therefore, developing non-invasive carriers for transdermal delivery has unparalleled advantages. Liposome nanocarriers, as artificially prepared transdermal delivery systems, have received widespread attention and research. They can encapsulate hydrophilic or lipophilic drugs and are widely used in intravenous, dermal, pulmonary, and oral drug delivery. Liposomes have been extensively studied as carriers for transdermal gene delivery. For example, Li et al. successfully loaded siRNA onto DOTAP lipid nanoparticles for targeted delivery and applied them to the skin of pigs to examine the transdermal permeability of siRNA to evaluate its potential use in gene therapy (Biotechnology Journal, 2020, 15(12): 2000079). Arruda et al. developed novel hybrid lipid polymer nanoparticles using a simple nanoprecipitation method. This method successfully prepared hybrid lipid polymer nanoparticles, demonstrating their stable and efficient use for specific silencing of target genes and achieving excellent results in oral delivery therapy in mice with ulcerative colitis (Journal of Controlled Release, 2022, 350:228-243). Numerous studies both domestically and internationally have demonstrated the effectiveness of liposome nanocarriers in delivering gene therapy for diseases. However, due to the poor transdermal permeability of ordinary liposomes, most treatment methods still rely on injection or microneedles. The challenge of achieving non-invasive transdermal liposome therapy remains to be overcome.

[0004] Polyethyleneimine (PEI), a typical representative of cationic polymers, is one of the most commonly used non-viral gene carriers. It can carry genes through electrostatic interactions and can successfully escape genes through the "proton sponge" effect, making it considered 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 various cell types has been fully demonstrated. For example, Hasanzadeh et al. used nanoparticles prepared with a cerium oxide coating containing PEI as a gene delivery carrier. Transfection results showed that this gene delivery carrier had low cytotoxicity and could be used as a combination therapy in synergy with conventional chemotherapy drugs for cancer treatment (Life Sciences, 2019, 232: 116661). Domestic and international research indicates that it is difficult to simultaneously achieve both high efficiency in gene transfection and low cytotoxicity with PEI due to its molecular weight. Therefore, when constructing an ideal gene carrier, PEI should possess good biocompatibility, specific targeting, and high buffering capacity. Tween 80, as a surfactant, is widely used in the development of transdermal systems due to its low toxicity, high biocompatibility, protein stability, and permeation-enhancing properties. For example, Ni et al. added Tween 80 to multi-level targeted liposomes to give them superdeformability and constructed a delivery system for the treatment of melanoma. 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] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a cationic hyperdeformable liposome capable of transdermal gene transfection, its preparation method, and its applications. This cationic hyperdeformable liposome is a liposome with highly efficient transdermal and targeted transfection functions.

[0007] This invention addresses how to achieve non-invasive transdermal gene delivery and efficient transfection. It utilizes the cationic polymer polyethyleneimine (PEI) as a gene conjugation carrier to enhance its binding ability, uses liposomes as a gene delivery carrier to enhance its skin delivery ability, utilizes Tween 80 to reduce the toxicity of the carrier and further enhance its transdermal delivery ability while promoting its transfection in the skin, and improves the delivery stability by chemically modifying the cationic polymer into the liposome structure. Finally, it constructs a lipid nanocarrier that can achieve non-invasive and efficient transdermal gene delivery and transfection.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] One of the technical solutions of the present invention is a method for preparing cationic hyperdeformable liposomes, comprising the following steps: dissolving phospholipids and cationic polymer donor materials 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 using a high-shear dispersing emulsifier; evaporating the mixed solvent using a rotary evaporator to obtain solid F; finally adding enzyme-free water to solid F for ultrasonic hydration; and finally obtaining the gene-loadable cationic hyperdeformable liposome suspension through a microfluidic high-pressure process.

[0010] 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 as 0.3-0.5 mg (preferably 0.45 mg) of soybean lecithin added 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-to-volume ratio of Tween 80 to enzyme-free water is (2-5) mg:2 mL; the slow addition rate is 4-5 mL / min; the high-shear dispersing emulsifier has a power of 200-300 W, a rotation speed of 8000 rpm, and a cycle time of 5 min; the rotary evaporator has a rotation speed of 80-120 rpm (preferably 100 rpm), a temperature of 50-60℃ (preferably 60℃), and a vacuum degree of 70-100 kPa (preferably 100 kPa). kPa); the mass-to-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 to squeeze 3-5 times at a pressure of 500-800 bar (preferably squeeze 5 times at a pressure of 500 bar).

[0011] Preferably, the cationic polymer donor material is cholesterol modified with a cationic polymer; more preferably, it is cholesterol modified with polyethyleneimine.

[0012] The preparation method of the cationic polymer donor material includes the following steps:

[0013] The cationic polymer was dissolved in organic solvent A, cholesterol chloroformate and triethylamine catalyst were added, and the reaction was carried out at 0-4°C. Then the mixture was heated to 5-35°C and the reaction was continued. After the reaction was completed, the mixture was concentrated, and hydrochloric acid was added to the concentrate to re-dissolve it. The concentrate was washed, precipitated and dried to obtain cholesterol modified by the cationic polymer. The mass ratio of the cationic polymer to cholesterol chloroformate was 1:(5-10).

[0014] Preferably, the cationic polymer comprises at least one of hyperbranched polyethyleneimine with a molecular weight of 600-3000 (preferably 1000-2500; more preferably 1800±300); the organic solvent A comprises dichloromethane; the amount of organic solvent A is calculated as 1-4 g (preferably 1 g) of cholesterol chloroformate added per 10 mL; the amount of triethylamine is calculated as 0.1-0.5 mol (preferably 0.1 mol) of cholesterol chloroformate added per 1 mol; the concentration of hydrochloric acid is 0.1-0.4 M (preferably 0.1 M); the amount of hydrochloric acid is 1-2 g of concentrate added per 5-10 mL; the reaction time at 0-4°C is 10-30 min (preferably 20 min), and the reaction time continues for 10-14 h (preferably 12 h).

[0015] The second technical solution of the present invention: a cationic hyperdeformable liposome prepared according to the above preparation method.

[0016] The cationic hyperdeformable liposomes can be used as vectors for preparing transdermal gene transfection.

[0017] The third technical solution of the present invention: a gene-loaded cationic hyperdeformable liposome complex, comprising the gene and the aforementioned cationic hyperdeformable liposomes. Further, the mass ratio of the cationic hyperdeformable liposomes to the gene is (5-80):1.

[0018] The method for preparing the gene-loaded cationic hyperdeformable liposome complex includes the following steps: the complex solution comprises the above-mentioned cationic hyperdeformable liposome suspension and the gene solution; specifically, a certain amount of cationic hyperdeformable liposome suspension is added to the gene solution, vortexed and allowed to stand for mixing and incubation for a certain time to obtain the gene-loaded cationic hyperdeformable liposome complex.

[0019] Preferably, the gene is at least one of DNA, siRNA, shRNA, gRNA, sgRNA, and mRNA, as well as genes used 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); and the static mixing incubation time is 25–30 min (preferably 30 min).

[0020] The fourth technical solution of the present invention: the application of the above-described gene-loaded cationic hyperdeformable liposome complex in the preparation of products that enhance gene stability.

[0021] The fifth technical solution of the present invention: the application of the above-described gene-loaded cationic hyperdeformable liposome complex in the preparation of products that enhance the efficiency of gene transfection in keratinocytes.

[0022] The sixth technical solution of the present invention: the application of the above-described gene-loaded cationic hyperdeformable liposome complex in the preparation of products that enhance gene transdermal efficiency.

[0023] The seventh technical solution of the present invention: the application of the above-described gene-loaded cationic hyperdeformable liposome complex in the preparation of products that enhance transdermal gene transfection.

[0024] The eighth technical solution of the present invention: the application of the above-described gene-loaded cationic hyperdeformable liposome complex in the preparation of products for treating skin diseases through non-invasive gene delivery.

[0025] The present invention has the following advantages and effects compared with the prior art:

[0026] (1) Compared with traditional gene vectors, this invention uses low molecular weight polyethyleneimine, liposomes, Tween 80 and other materials, which are safe and simple in composition; it uses cationic polymer polyethyleneimine as a gene conjugation vector to enhance its binding ability, uses liposomes as a delivery vector to enhance its skin delivery ability, and uses Tween 80 to further enhance the stability of the vector and transdermal transfection ability, so as to achieve non-invasive targeted transdermal delivery of genes.

[0027] (2) Compared with traditional gene vectors, the vector of the present invention has high stability in vitro and can effectively inhibit gene degradation. At the same time, the vector has high transfection efficiency and low toxicity in in vitro cell experiments, which solves the contradiction between high transfection efficiency and high cytotoxicity of polyethyleneimine.

[0028] (3) The vector of the present invention can effectively promote the transfection and expression of genes in the skin while achieving efficient transdermal transmission. For example, carrying siRNA can exert a better therapeutic effect on 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. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram illustrating the construction and gene loading of cationic hyperdeformable liposomes.

[0031] Figure 2 Characterization diagrams of the cationic hyperdeformable liposomes prepared in Example 2 are shown; where A is the particle size and potential diagram; B is the deformation index result diagram, **** indicates extremely significant differences (…). P <0.0001).

[0032] Figure 3 The image shows the assay results of the cationic hyperdeformable liposome complex loaded with plasmid obtained in Example 3; where A is an agarose gel electrophoresis image, and Nake represents the plasmid control; B is a potential assay image.

[0033] Figure 4 This is a diagram showing the analysis of gene degradation inhibition by cationic hyperdeformable liposome complexes loaded with plasmids; where "+" indicates the TCPL / Plasmid group and "-" indicates the CPL / Plasmid group.

[0034] Figure 5 To validate the in vitro transfection of plasmid-loaded cationic hyperdeformable liposome complexes into human keratinocytes; where A represents the transfection efficiency, and *** indicates extremely significant differences (…). P <0.001); B is the cytotoxicity graph; C is the expression graph of fluorescent proteins in cells after treatment with the TCPL / Plasmid complex.

[0035] Figure 6 The images show the transdermal validation of the Cy5 fluorescently labeled plasmid in mice; where A is the plasmid labeling result and B is the plasmid penetration in skin tissue.

[0036] Figure 7 This image shows the in vitro transdermal validation of Cy5 fluorescently labeled plasmids using the Franz diffusion cell.

[0037] Figure 8 The images show the expression of red fluorescent protein in mice; where A is an in vivo image of a mouse, and B is an image of fluorescent protein expression in skin tissue. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0041] In the examples, the preparation methods of anhydrous dichloromethane, chloroform or anhydrous ethanol are referred to in the literature "CN115400084B, A liposome that can release NO and its preparation method and application".

[0042] In the examples, the acetone used had a purity of ACS, ≥99.5%; the polyethyleneimine used had a number-average molecular weight of 1800; and the cholesterol used had a purity of AR, 98%.

[0043] In the embodiments, room temperature refers to 10–35°C.

[0044] The following description will not be repeated.

[0045] The schematic diagram of the preparation and gene loading of cationic hyperdeformable liposomes is shown below. Figure 1 As shown.

[0046] Example 1

[0047] Preparation of cationic polymer donor material: polyethyleneimine-modified cholesterol (Cho-PEI):

[0048] Under ice-water bath conditions, polyethyleneimine (PEI) with a molecular weight of 1800 was dissolved in anhydrous dichloromethane. Using triethylamine as a catalyst, cholesterol chloroformate was added at a mass ratio of 1:10 to PEI, with 1 g of cholesterol chloroformate added per 10 mL of anhydrous dichloromethane. The amount of triethylamine added was 0.1 mol of cholesterol chloroformate per 1 mol. The reaction was carried out at 0–4 °C for 20 min, then raised to room temperature (25 °C), and the reaction was continued for 12 h. The mixture was then concentrated under reduced pressure, and the concentrate was redissolved with 10 mL of 0.1 M hydrochloric acid solution. After washing with dichloromethane and precipitation with acetone, the mixture was dried under vacuum at room temperature (25 °C) to obtain a white solid, namely polyethyleneimine-modified cholesterol Cho-PEI.

[0049] Example 2

[0050] The preparation of a cationic hyperdeformable liposome includes the following steps:

[0051] At 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. The solution was heated and stirred until completely dissolved. The mass ratio of soybean lecithin to cationic polymer donor material Cho-PEI was 1:1, and the volume ratio of anhydrous ethanol to chloroform was 1:1. The amount of mixed solution added was 0.45 mg of soybean lecithin per 1 mL. Tween 80 with a molecular weight of 604 was dissolved in enzyme-free water. The mass-to-volume ratio of Tween 80 to enzyme-free water was 1 mg / mL, and the mass ratio of Tween 80 to cationic polymer donor material Cho-PEI was 1:5. This solution was slowly added (5 mL / min) to the above organic mixed solution. The two were mixed evenly using a high-shear dispersing emulsifier (power 300 W, speed 8000 rpm, time 5 min each time) and then rotary evaporated. The temperature was set at 60℃, the speed at 100 rpm, the vacuum degree of the evaporation flask was 100 kPa, and the rotary evaporation time was 0.5–100 min. h, after the organic solvent has completely evaporated to obtain a white thin film solid, then it is dissolved in enzyme-free water at a mass-volume ratio of 1 mg:1 mL, and hydrated by sonication for 20 min to obtain a milky white liposome suspension. After being squeezed 5 times under a microfluidic pressure of 500 bar, it becomes a cationic hyperdeformable liposome suspension, denoted as TCPL, and stored at 4℃.

[0052] Referring to the preparation steps of cationic hyperdeformable liposomes in this embodiment, the cationic polymer donor material Cho-PEI in this embodiment is replaced with cholesterol chloroformate Cho in Example 1, denoted as TCL.

[0053] Following the preparation steps of cationic hyperdeformable liposomes in this embodiment, without adding Tween 80, it is denoted as CPL.

[0054] Example 3

[0055] The preparation of a gene-loaded cationic hyperdeformable liposome complex includes the following steps:

[0056] At room temperature, a plasmid expressing green fluorescent protein (pcDNA3.1-GFP, purchased from GenePharma) was selected as the model gene. The cationic hyperdeformable liposome suspension prepared in Example 2 was added, and the mass ratio (w / w) of cationic hyperdeformable liposomes to plasmid was set to 1, 5, 10, 20, 40 and 80, respectively. After vortexing at 1000 rpm for 3 min, the mixture was incubated for 30 min to obtain cationic hyperdeformable liposome complexes loaded with plasmids at different mass ratios.

[0057] Example 4

[0058] 100 μL of TCL, TCPL, and CPL obtained in Example 2 were dispersed into 1 mL of pure water, respectively. After uniform ultrasonic dispersion, 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 Figure A, the hydrated particle sizes of both cationic hyperdeformable liposomes TCPL and CPL are below 500 nm and uniform in size. However, the addition of Tween 80 increased the potential of TCPL, demonstrating that this surfactant can maintain the electrical layer of the liposomes, keeping them at a higher potential and thus maintaining their stability. 1 mL of TCPL and CPL were added to a liposome extruder (Avanti® Polar Lipids, USA). The liposomes were passed through a 50 nm polycarbonate membrane at a pressure of 1 MPa. After 5 min, the extrusion volume was recorded, 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 deformability 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). This lays the foundation for the subsequent gene loading and transdermal transfection applications of TCPL liposomes.

[0059]

[0060] Example 5

[0061] The cationic hyperdeformable liposome complexes with different mass ratios of plasmids obtained in Example 3 were subjected to potentiometry and agarose gel electrophoresis to verify the material's ability to bind with genes. 0.7 g of agarose was weighed into a beaker, and 70 mL of TAE × 1 solution was added. After complete dissolution, the solution was heated to boiling in a microwave oven. 3 μL of Goldview solution was added and shaken until completely dissolved. The gel casting tank was prepared, and the mixture was poured into the tank while still hot. After removing air bubbles, the tank was fixed in the left groove and cooled for 30 min until gel formation. After removing the well plate, 3 μL of Loading Buffer was added to 10 μL of the prepared material at different concentrations. After thorough mixing, the mixture was transferred to the gel electrophoresis apparatus, and some TAE solution was added to completely submerge the gel. The gel was run at 120V for 25 min. After the run, the gene loading was characterized by imaging at 365 nm using a UV gel imager. Each group was set up with 3 replicates. The results are shown below. Figure 3As shown in Figure A, the DNA migration varies with increasing liposome mass. In groups with a w / w ratio less than 5, distinct bright bands migrate from the wells, indicating that the material cannot bind well to the plasmid at a w / w ratio less than 5. However, when the mass ratio is greater than or equal to 5, no bright bands migrate from the wells, indicating that the material binds well to the gene and forms a stable complex. This result was also verified by potentiometry; when the mass ratio is greater than or equal to 5, the complex begins to show a positive potential, enabling effective binding to the gene. Figure 3 (B in the middle).

[0062] Example 6

[0063] The TCPL and CPL obtained in Example 2 were loaded with the plasmid described in Example 3 for enzymatic degradation analysis to verify the gene degradation inhibition function of TCPL and CPL. The mass ratio of TCPL, CPL, and plasmid was 10:1. At 37°C, CPL / Plasmid and TCPL / Plasmid were mixed with DNase, and incubated for 2 h and 6 h respectively. Agarose gel electrophoresis was then performed to detect the enzyme stability of the materials. The total volume of the mixture was 30 μL, with a final concentration of 2 μg / 30 μL for plasmid and a final DNase activity of 5 U / μL. Figure 4 As shown, after 6 h, the plasmids in the CPL / Plasmid group were completely degraded by DNase, and no bands were observed on agarose gel electrophoresis, while bands were still observed in the TCPL / Plasmid group. The enzymatic degradation experiment fully demonstrated the protective function of cationic hyperdeformable liposomes TCPL when loaded with genes, laying the foundation for subsequent transdermal transfection.

[0064] Example 7

[0065] The TCPL and CPL obtained in Example 2 were loaded with the plasmid expressing green fluorescent protein described in Example 3 and transfected into human skin keratinocytes (HaCat). HaCat cells are difficult to transfect and are commonly used as model cells for skin applications. To maximize in vitro transfection efficiency and control material toxicity, the mass ratio (w / w) of liposomes to green fluorescent protein plasmids was 80. HaCat cells were first divided into groups of 5 x 10 cells per well. 4Cells were seeded at a density of [number] cells / well in 24-well plates. When the cell confluence 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, i.e., the mass ratio of PEI-25000 to plasmid was 1.3:1). A blank control group was used as the negative control. Each group was set up in triplicate. After culturing for 4 h, the culture medium in the wells was aspirated, and 500 μL of fresh complete culture medium was added for further 20 h of culture. Green fluorescent protein expression was qualitatively observed and photographed using an inverted fluorescence microscope. Subsequently, the culture medium was aspirated, the cells were washed with PBS, digested with trypsin, and collected by centrifugation (1000 rpm, 5 min). Finally, the cells were resuspended in an appropriate amount of PBS, and the transfection efficiency was quantitatively analyzed using flow cytometry. Figure 5 As shown in Figure A, when TCPL and CPL were loaded with the same mass of plasmid, the transfection efficiency of TCPL reached approximately 55%, significantly higher than that of CPL liposomes (45.7%) and the positive control (36.6%). This fully demonstrates that the addition of Tween 80 is beneficial for "stabilizing" the liposome system and significantly improves the transfection effect of cells. The expression of green fluorescence in cells treated with the TCPL / Plasmid complex was clearly observed using a fluorescence microscope. Figure 5 (C in the text), cytotoxicity results as follows Figure 5 As shown in B, the TCPL / Plasmid and CPL / Plasmid complexes at this ratio have low toxicity and show significant advantages over the positive control PEI-25000, indicating that this transdermal transfection system has good biosafety.

[0066] Example 8

[0067] After labeling the plasmid described in Example 3 with Cy5 fluorescence using Label IT Tracker reagent, it was combined with TCPL or CPL obtained in Example 2 following the procedure described in Example 3. The mass ratio of TCPL, CPL, and Cy5-labeled plasmid (CY5-Plasmid) was 80. Figure 6 As shown in Figure A, laser confocal microscopy revealed that the plasmid was successfully labeled with Cy5 fluorescence. After anesthetizing the mice (6-8 week old Balb / c mice, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.), the backs were shaved. 100 μL of the liposome complex was evenly applied to the mouse's back skin, covering an area of ​​1.5 cm². 2Simultaneously, a CY5-Plasmid group was set up, with 3 mice in each group; 24 h later, the skin tissue on the back of the mice was dissected, and residual material on the skin surface was washed with PBS (pH = 7.4) and dried. The frozen skin tissue was then vertically sectioned into skin sections approximately 10 μm thick using a cryostat. The distribution of Cy5 fluorescent labeling in the skin was observed using a fluorescence microscope. The results are as follows: Figure 6 As shown in B, the plasmid loaded with cationic hyperdeformable liposomes successfully penetrated the stratum corneum and reached the epidermis and dermis.

[0068] Example 9

[0069] The plasmid described in Example 3 was labeled with Cy5 fluorescence using Label IT Tracker reagent and then combined with the TCPL and CPL liposomes obtained in Example 2, following the procedure described in Example 3. The mass ratio of TCPL / CPL liposomes to the Cy5-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 human surface temperature. The receiving chamber was filled with PBS buffer solution (pH = 7.4) and continuously stirred at 200 rpm. The diffusion cell was kept at 37°C by circulating water bath. A mouse skin of appropriate size (prepared according 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, with the surface of the skin facing the supply chamber. 1 mL of Cy5 fluorescently labeled plasmid and TCPL and CPL-loaded plasmids were respectively transferred and added to the upper supply chamber of the Franz diffusion cell and sealed. After 24 h, the mouse back skin tissue was removed, and residual material on the skin surface was washed with PBS (pH=7.4) and dried. The frozen skin tissue was then vertically sectioned into sections approximately 10 μm thick using a cryostat. The distribution of Cy5 fluorescent labeling in the skin was observed using a fluorescence microscope. The results are as follows: Figure 7 As shown, the plasmid loaded with cationic hyperdeformable liposomes successfully penetrated mouse skin.

[0070] Example 10

[0071] The TCPL and CPL liposomes obtained in Example 2 were 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 (every 24 hours) to the back of mice (6-8 week old Balb / c nude mice, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.) after dorsal hair removal treatment until complete absorption, covering an area of ​​1.5 cm². 2Simultaneously, a Plasmid group and a blank control group (3 mice) were set up, with each group containing 3 mice; at 24 h and 48 h, the red fluorescence expression on the back of the mice was detected using a small animal in vivo imaging system, with an excitation wavelength of 520 nm and an emission wavelength of 570 nm. Figure 8 As shown in Figure A, the plasmid expressing red fluorescent protein loaded with TCPL was successfully expressed in mouse skin at 24 h, and the fluorescence expression gradually intensified after 48 h. Mice were euthanized after 48 h, and skin from the back was harvested for in vivo imaging to observe the expression of fluorescent protein in the skin. Skin tissue was fixed with 4% paraformaldehyde and frozen sectioned; the penetration of fluorescent protein into the tissue was observed using a fluorescence microscope. Figure 8 As shown in Figure B, it can be clearly seen that neither the simple plasmid nor the CPL-loaded plasmid expressed fluorescent protein, while the cationic hyperdeformable liposome TCPL-loaded plasmid successfully expressed red fluorescent protein in mouse skin. This fully demonstrates that the vector has the ability to carry genes for transdermal transfection.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing cationic hyperdeformable liposomes, characterized in that: The process includes the following steps: dissolving phospholipids and cationic polymer donor materials 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 thoroughly; removing the solvent to obtain solid F; finally adding enzyme-free water to solid F for ultrasonic hydration; and finally obtaining a gene-loadable cationic hyperdeformable liposome suspension through a microfluidic high-pressure process. The cationic polymer donor material is cholesterol modified with a cationic polymer; the cationic polymer is at least one of hyperbranched polyethyleneimine with a molecular weight of 1000-2500. The phospholipids include soybean lecithin, and the mass ratio of the phospholipids to the cationic polymer donor material is (1-1.5):1; the mass ratio of Tween 80 to the cationic polymer donor material is 1:(4-5). The mass-to-volume ratio of solid F to enzyme-free water is (20-30) mg: 20 mL; the ultrasonic time is 20-30 min.

2. The preparation method according to claim 1, characterized in that: 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 as 0.3-0.5 mg of soybean lecithin added per 1 mL; the mass-volume ratio of Tween 80 to enzyme-free water is (2-5) mg:2 mL; the slow addition rate is 4-5 mL / min; the microjet high pressure condition is extrusion 3-5 times at a pressure of 500-800 bar.

3. The preparation method according to claim 1, characterized in that: The cationic polymer is at least one of hyperbranched polyethyleneimine with a molecular weight of 1800±300.

4. The preparation method according to claim 1, characterized in that: The preparation method of the cationic polymer donor material includes the following steps: The cationic polymer was dissolved in organic solvent A, cholesterol chloroformate and triethylamine catalyst were added, and the reaction was carried out at 0-4°C. Then the mixture was heated to 5-35°C and the reaction was continued. After the reaction was completed, the mixture was concentrated, and hydrochloric acid was added to the concentrate to re-dissolve it. The concentrate was washed, precipitated and dried to obtain cholesterol modified by the cationic polymer. The mass ratio of the cationic polymer to cholesterol chloroformate was 1:(5-10).

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

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

7. The use of the cationic hyperdeformable liposomes of claim 6 as a vector for preparing transdermal gene transfection delivery.

8. A gene-loaded cationic hyperdeformable liposome complex, characterized in that: It includes genes and the cationic hyperdeformable liposomes of claim 6; the genes are at least one of DNA, siRNA, shRNA, gRNA, sgRNA and mRNA.

9. The gene-loaded cationic hyperdeformable liposome complex according to claim 8, characterized in that: The mass ratio of the cationic hyperdeformable liposomes to the gene is (5-80):

1.

10. A method for preparing the gene-loaded cationic hyperdeformable liposome complex according to claim 8 or 9, characterized in that: Includes the following steps: The complex solution comprises a cationic hyperdeformable liposome suspension prepared by the preparation method according to any one of claims 1 to 5 and a gene solution; specifically, a certain amount of cationic hyperdeformable liposome suspension is added to the gene solution, vortexed and allowed to stand for mixing and incubation for a certain time to obtain a gene-loaded cationic hyperdeformable liposome complex.

11. The preparation method according to claim 10, characterized in that: The static mixing incubation time is 25–30 min.

12. The use of the gene-loaded cationic hyperdeformable liposome complex of claim 8 or 9 in the preparation of products for treating skin diseases via non-invasive gene delivery.

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