CN108451908B

CN108451908B - Hyaluronic acid modified cationic liposome, preparation method and application thereof

Info

Publication number: CN108451908B
Application number: CN201810618189.9A
Authority: CN
Inventors: 甘莉; 秦艳梅; 张华�; 田永丰; 刘阳
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2018-06-15
Filing date: 2018-06-15
Publication date: 2021-08-31
Anticipated expiration: 2038-06-15
Also published as: CN108451908A

Abstract

The invention discloses a hyaluronic acid modified cationic liposome, a preparation method and application thereof. The invention takes nonionic surfactant, auxiliary phospholipid and cationic phospholipid as main film forming materials, and the outer layer is coated and modified with hyaluronic acid; wherein the nonionic surfactant is Tween 80 or span 80, and the auxiliary phospholipid is squalene or cholesterol. The invention has the beneficial effects that: the hyaluronic acid modified cationic lipoid gene obtained by the invention has high transfection efficiency, can be specifically targeted to an ocular retina layer, and is used as an ocular gene delivery vector.

Description

Hyaluronic acid modified cationic liposome, preparation method and application thereof

Technical Field

The invention relates to the technical field of lipoid and gene delivery, in particular to a cationic lipoid modified by hyaluronic acid, a preparation method and application thereof.

Background

Among ocular diseases, ocular fundus diseases are clinically common and relatively difficult to treat. Fundus-related diseases such as diabetic retinopathy, non-glaucoma optic nerve damage, and the like, are largely associated with mortality. At present, eyeground diseases seriously affect the life quality of people. At present, the treatment of fundus diseases mainly comprises two modes of surgical operation treatment and drug treatment. Generally, surgical treatment is associated with a certain risk and may cause a certain irreversible damage to the tissue surrounding the lesion. The traditional drug therapy has the defects of temporary solution, permanent cure and more side effects, and the disease can only be controlled by inhibition because the disease cannot be fundamentally solved, so that the patients need frequent and long-term administration, and the economic burden is increased.

Based on many of the drawbacks of existing drug therapies, many scholars have turned their eyes to the field of gene therapy in recent years. The gene therapy of fundus diseases is adopted, and the expression of specific proteins is hindered or promoted by correcting gene misordering, repairing genetic errors or generating therapeutic factors, so that the fundus diseases are ultimately treated fundamentally from the gene level.

In recent years, niosomes have been considered as potential alternative delivery systems to conventional liposomes. Compared with phospholipid vesicles, the niosomes have a liposome-like bilayer structure, but have the advantages of greater accessibility, stability, lower cost and the like. Furthermore, the higher cell survival rates of niosomes compared to liposomes and polymers indicate lower cytotoxicity. As a gene vector, the lipoid has better transfection efficiency. Using squalene as a helper lipid, it has been reported that niosomes enhance the escape capacity of lysosomes by structure transfer. Meanwhile, aiming at the condition of high expression of CD44 on RPE cells, hyaluronic acid modified on the outer layer of the niosome to be capable of targeting CD44 can realize the effect of cell targeting. Therefore, the hyaluronic acid modified cationic liposome is a novel gene delivery vector with great potential.

Disclosure of Invention

In order to overcome the defects of the existing surgical treatment and drug treatment, the invention aims to provide a hyaluronic acid modified cationic liposome for researching ocular gene delivery. The gene delivery vector has the characteristics of low cytotoxicity, high stability and strong transfection effect, and can be used for effectively delivering genes in ocular retinal cells.

The invention also aims to provide a preparation method of the hyaluronic acid modified cationic lipoid liposome serving as the ocular gene delivery carrier.

The technical scheme of the invention is specifically introduced as follows.

The invention provides a hyaluronic acid modified cationic liposome, which takes a nonionic surfactant, auxiliary phospholipid and cationic phospholipid as main film forming materials, and the outer layer is coated and modified with hyaluronic acid; wherein the nonionic surfactant is Tween 80 or span 80, and the auxiliary phospholipid is squalene or cholesterol.

In the invention, the particle size is 100-300nm, and the Zeta potential is-5 to-80 mV.

The invention also provides a preparation method of the cationic liposome, which comprises the following specific steps:

(1) mixing the nonionic surfactant, the auxiliary phospholipid and the cationic phospholipid, and completely dissolving in absolute ethyl alcohol;

(2) injecting ethanol solution into buffer salt solution at 40-80 deg.C at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain cationic liposome;

(3) adding hyaluronic acid into the cationic liposome obtained in the step (2), and incubating for 20-40min at 37 ℃ to obtain the hyaluronic acid modified cationic liposome.

According to the invention, in the step (1), the molar ratio of the nonionic surfactant to the auxiliary phospholipid is 5: 2-5: 8, and the mass ratio of the cationic phospholipid to the nonionic surfactant is 7: 100-13: 100.

In the present invention, in step (3), the molecular weight range of hyaluronic acid is 500-3900 kDa.

In the invention, the mass ratio of HA-DOPE to cationic liposome is 1: 10-1: 5.

compared with the prior art, the invention has the beneficial effects that:

(1) the cationic liposome prepared by the method has the advantages that the nonionic surfactant has good stability, is not easy to oxidize and has low cytotoxicity; the squalene is used as auxiliary lipid, can play a certain supporting role, and has a certain auxiliary capacity for forming a bilayer; the cationic modifier is added to change the potential from negative to positive, can specifically combine with the gene with negative polarity, and realizes the encapsulation through electrostatic adsorption. The finally formed cationic liposome can successfully realize gene entrapment.

(2) In the invention, after the vitreous body is injected into the fundus, the cationic liposome modified by hyaluronic acid can be targeted to RPE cells with high CD44 distribution, and the endocytosis is realized through the affinity of HA-CD 44; after entering lysosome, hyaluronic acid is degraded by internal hyaluronidase to expose internal gene-carrying cationic liposome, so that effective escape of lysosome is realized under the action of cationic DOTAP, and the gene is released into cytoplasm to complete delivery.

(3) The invention utilizes the characteristic that retinal cells can highly express CD44 and the specific affinity capacity between HA and CD44 to modify the outer layer of the prepared cationic liposome with a certain amount of hyaluronic acid, thereby realizing the effect of targeted delivery to RPE cells.

Drawings

FIG. 1: transmission electron micrographs of C-Niosomes and HA-C-Niosomes.

FIG. 2: results of MTT cytotoxicity experiments (a: niosomes, b: C-niosomes, C: 10% HA-C-niosomes, d: 20% HA-C-niosomes).

FIG. 3: results of cellular uptake of the Gene vector (a: C-niosomes/Cy3-pDNA, b: 10% HA-C-niosomes/Cy3-pDNA, C: 20% HA-C-niosomes/Cy 3-pDNA).

FIG. 4: lysosomal co-localization events.

FIG. 5: gene vector cell transfection results (a: C-niosomes/pEGFP, b: 10% HA-C-niosomes/pEGFP, C: 20% HA-C-niosomes/pEGFP).

Detailed Description

The technical solution of the present invention will be described in detail with reference to the accompanying drawings and embodiments.

The invention adopts an ethanol injection method to prepare the lipoid, and then cationic phospholipid is added to carry out potential modification to obtain the cationic lipoid. And adding hyaluronic acid into the vector, wrapping, and constructing a vector which can target to RPE cells with high expression of CD44 receptors and realize effective gene transfection.

Examples 1 to 5

The prescription composition is as follows: see Table 1

The preparation method comprises the following steps: (1) mixing tween 80 and squalene in different prescriptions, and completely dissolving in anhydrous ethanol; (2) injecting the ethanol solution into 40-80 deg.C buffer salt solution at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain liposome. The particle size, PDI and potential of the niosomes were measured at different molar ratios using a Malvern particle sizer. See table 1.

TABLE 1

Example 3 reduced the amount of the auxiliary phospholipid used in the formulation composition compared to examples 1 and 2. In contrast, examples 1 and 2 added the sample of helper phospholipid and were hazy in appearance, whereas example 3 appeared clear and blue opalescence was evident. Compared with examples 3 and 4, in example 3, the amount of the auxiliary phospholipid used was increased in the formulation composition, and PDI was small, indicating that the particle size was uniform. More co-phospholipids can help the nonionic surfactant to make its critical encapsulation parameter greater than 1/2, thereby forming a bilayer structure.

Example 6

The preparation method comprises the following steps: (1) mixing the Tween 80 and cholesterol in the prescription amount, and completely dissolving in absolute ethyl alcohol; (2) injecting the ethanol solution into 40-80 deg.C buffer salt solution at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain liposome. The particle size, PDI and potential of the niosomes were measured using a Malvern particle sizer, and the results are shown in Table 2.

TABLE 2

Example 7

The preparation method comprises the following steps: (1) mixing the prescription amount of span 80 and squalene, and completely dissolving in absolute ethyl alcohol; (2) injecting the ethanol solution into 40-80 deg.C buffer salt solution at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain liposome. The particle size, PDI and potential of the niosomes were measured using a Malvern particle sizer, and the results are shown in Table 3.

TABLE 3

Example 8

The preparation method comprises the following steps: (1) mixing the prescription amount of span 80 and cholesterol, and completely dissolving in absolute ethyl alcohol; (2) injecting the ethanol solution into 40-80 deg.C buffer salt solution at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain liposome. The particle size, PDI and potential of the niosomes were measured using a Malvern particle sizer, and the results are shown in Table 4.

TABLE 4

Examples 9 to 12

The preparation method comprises the following steps: (1) mixing tween 80 and squalene according to the prescription amount, adding a certain amount of cationic phospholipid according to the following table, and completely dissolving in absolute ethyl alcohol; (2) injecting the ethanol solution into buffer salt solution at 40-80 deg.C at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain cationic liposome. The cationic liposome was measured for particle size, PDI and potential using a Malvern particle sizer, and the results are shown in Table 5.

TABLE 5

Examples 13 to 16

The preparation method comprises the following steps: (1) mixing the Tween 80, squalene and cationic phospholipid in the prescribed amount, and completely dissolving in anhydrous ethanol; (2) injecting ethanol solution into buffer salt solution at 40-80 deg.C at constant speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain cationic liposome; (3) mixing HA-DOPE and cationic liposome according to the mass ratio of 1: 10-1: 5 (see table below), adding into cationic liposome, and incubating at 37 deg.C for 20-40min to obtain hyaluronic acid modified cationic liposome (HA-C-Niosomes). The particle diameter, PDI and Zeta potential were measured. The results are shown in Table 6. FIG. 1: the spherical structure of the basic niosome can be clearly seen in panel a. While the basic structure of the bilayer vesicles can be seen in the magnified TEM image of the individual niosomes in figure B. And the figure C shows that a layer of hyaluronic acid is obviously coated on the outer layer of the lipoid.

TABLE 6

Application examples

Cytological experimental evaluation is carried out on RPE by applying example 3(Niosomes), 10 (C-niomes), 14 (10% HA-C-niomes) and 16 (20% HA-C-niomes) of the invention to confirm that hyaluronic acid modification HAs obvious targeting effect on RPE cells with high expression of CD44, and can specifically deliver genes to enter target cells, lysosome escape is carried out, and final gene transfection is realized.

First, evaluation of toxicity of vector (MTT method)

The test procedure was as follows:

(1) the cells are at a ratio of 10X 10³The culture medium is inoculated into a 96-well plate at a density of one well and cultured for 24 hours. C-niosomes with concentrations of 2.5, 2.0, 1.5, 1.0 and 0.5mM/L were incubated at 37 ℃ for 4h, washed 3 times with isotonic PBS, 100. mu.L of serum-free medium and 10. mu.L of LMTT working solution (5mg/ml) were added to each well, the culture was terminated after 4h incubation in an incubator, and the medium was removed from the wells. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. And (3) measuring the light absorption value at the wavelength of 570nm of the microplate reader, and calculating the cell activity of the carrier under different concentrations by taking the cell group without preparation treatment as a negative control group.

(2) The cells are at a ratio of 10X 10³The culture medium is inoculated into a 96-well plate at a density of one well and cultured for 24 hours. Each group of vectors (niosomes, C-niosomes, 10% HA-C-niosomes and 20% HA-C-niosomes) was incubated at a concentration of 0.5mM/L at 37 ℃ for 4h, the cells were washed 3 times with isotonic PBS, 100. mu.L of serum-free medium and 10. mu.L of LMTT working solution (5mg/ml) were added to each well, the culture was terminated after 4h incubation in an incubator, and the medium was removed from the wells. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. And (3) measuring the light absorption value at the wavelength of 570nm of the microplate reader, and calculating the cell activity of the carrier under different concentrations by taking the cell group without preparation treatment as a negative control group.

The results of the experiment are shown in FIG. 2 (A). At an initial concentration of 2.5mM/L, the cell viability was only 15%, indicating that the vector was more toxic at this time; after the cell is diluted by a certain multiple, the cell activity is suddenly increased and can reach more than 80 percent. When diluted 5-fold, the cells reached 91.91%, indicating that the vector was almost non-toxic at this time. Thus the final selected concentration of vector was 0.5mM/L, i.e., diluted 1/5-fold. FIG. 2(B) is the cell viability of the vectors of each group after 5-fold dilution. After 5 times dilution, the cytotoxicity of each group of carriers is more than 90%, and the carriers are considered to be safe and nontoxic. Experimental data in groups 2 and 3 show that the addition of cationic phospholipid DOTAP can increase the toxicity of the carrier slightly. That is, the amount of DOTAP added needs to be controlled within a certain range, and too much will increase cytotoxicity. This also meets our initial concerns regarding the amount of DOTAP added. Observing data in groups 3, 4 and 5, the modification of HA did serve to encapsulate the carrier periphery. Furthermore, as the amount of HA modification increased, the cellular viability value of the vector changed, from 95.0% initially (0% HA modification) to 97.5% (10% HA modification) and finally to 99.6% (20% HA modification). On the one hand, it is demonstrated that the modification of HA can reduce the cytotoxicity of the vector, and on the other hand, it is demonstrated that the higher the amount of HA modification, the lower the cytotoxicity.

Second, study of vector cell uptake

The test procedure was as follows:

(1) preparation of a gene-carrying vector: the gene-loaded vector was prepared using the principle of electrostatic adsorption between negatively charged cy3-pDNA and positively charged C-niosomes. Preparing C-niosomes according to the optimized prescription proportion and process. C-niosomes (C (DOTAP) ═ 0.3mg/ml) were vortexed at 30:1 with cy3-pDNA (0.5mg/ml) for 200s, and allowed to stand at room temperature for 30min, allowing cy3-pDNA to bind completely to the C-niosomes. Storing at 4 deg.C for use. Finally, HA-DOPE was added dropwise to the above solution at 10% (w/w), 20%, and incubated for 40min at 37 ℃ on a shaker at 180rpm to form HA-C-niosomes/DNA complexes.

(2) Sterile-processed round coverslips (15#) were placed in a 24-well plate and cells were plated at 10 × 10⁴The cells were seeded in a well plate at a density of one well, and 300. mu. l D-MEM/F-12 (containing 10% bovine serum) was added and incubated at 37 ℃ in a 5% CO2 environment. Culturing for 24h until the cells grow to be about 80-90% full of the pore plate. The medium was discarded and 5-fold dilutions of serum-free medium were added to each group of vectors containing Cy 3-labeled plasmid (C-niosomes/Cy3-pDNA, 10% HA-C-niosomes/Cy3-pDNA and 20% HA-C-niosomes/Cy 3-pD)NA), blank medium containing only the cy 3-labeled plasmid as a negative control, was incubated at 37 ℃ for 4 hours. Discarding the carrier suspension, washing the cells for 3 times by isotonic PBS, fixing the cells for 10min by 4% paraformaldehyde, dyeing the cell nucleus for 5min by DAPI, washing the cells for 3 times by isotonic PBS after dyeing, taking out the cell, sealing the cell by 90% glycerol sealing solution, drying in the sun, storing at-20 ℃, and observing the uptake condition of the cells to each group of carriers by CLSM. The remaining cells were detached from the microplate by adding 200ul of trypsin/EDTA. Once the cells were separated, the cell suspension was collected, resuspended by adding 400ul of complete medium, and then subjected to flow cytometry to detect Cy 3-labeled positive cells. 10000 cells per assay.

The results are shown in FIG. 3(A), cellular uptake of 10% HA-C-niosomes and 20% HA-C-niosomes is significantly higher than that of C-niosomes, and with increasing HA percentage, the cells exhibit gradually increasing red fluorescence, indicating increased uptake of HA-C-niosomes by ARPE-19 cells. In addition, the red fluorescence in cells of the C-niosomes group is stronger than that of the naked plasmid group, probably because the cationic carriers can be electrostatically adsorbed to the negatively charged cell surface, making it easy to be taken up by the cells. Fig. 3(B) is a flow cytometer quantitative analysis of cellular uptake results, consistent with the results of CLSM detection. With 100% C-niosomes, the uptake for the 10% HA-C-niosomes group was 1.3 times that of the naked plasmid group, whereas the uptake for the 20% HA-C-niosomes group was 1.5 times that of the naked plasmid group, due to the modification of HA. In the ligand targeting nano-delivery system, the modification amount of the ligand on the surface of the nanoparticle is an important factor for adjusting the targeting property of the system. Therefore, the modification amount of the ligand is properly increased, so that the contact probability of the vector and cells can be increased, and the targeting property of the vector is improved. The experiment can find that the targeting property of the HA modified carrier is positively correlated with the HA modification amount.

Co-localization experiment of three lysosomes

The test procedure was as follows:

the cells were cultured at 20X 10⁴The cells were seeded in 12-well plates at a density of one well, and 600. mu. l D-MEM/F-12 (containing 10% bovine serum) was added and incubated at 37 ℃ in a 5% CO2 environment. Culturing for 24h until the cell grows to reach 80-90% of the pore plate, discarding the culture medium, adding 100ul of a proper amount of pre-incubation at 37 deg.CLysotracker Red (50nM, PBS) stained lysosomes for 90min at 37 ℃. Then removing the staining solution, washing the cells for 3 times by isotonic PBS, then adding various groups of coumarin (C6) fluorescence labeling carriers (niosomes, 20% HA-C-niosomes) diluted by serum-free culture medium, incubating for 1h and 2h at 37 ℃, removing the carrier solution, washing the cells for 3 times by PBS, fixing the cells for 10min by 4% paraformaldehyde, performing cell nucleus staining by DAPI, washing the cells for 3 times by isotonic PBS after the staining is finished, sealing by 90% glycerol sealing solution, and storing in the dark at 20 ℃. And observing the co-localization condition of lysosome and the carrier in the cells under a 60-fold oil microscope.

As shown in fig. 4, red fluorescence in the cytoplasm indicates specifically stained lysosomes, while green fluorescence is a coumarin-labeled vector. Yellow fluorescence indicates a vector residing in the lysosome. After 2h of co-incubation, the 20% HA-C-Niosomes and Niosomes both exhibited a partial yellow color within the cells. The yellow color indicates that HA-C-Niosomes and Niosomes are now predominantly located in the lysosomes. In contrast, the HA-C-niosomes group showed very low cytoplasmic yellow fluorescence, indicating only partial co-presence in lysosomes. The green color is obviously enhanced, which indicates that a large amount of carriers escape from lysosomes. The yellow fluorescence in cytoplasm of the Niosomes group is strong, and the green color of a small part of regions is obvious, which indicates that a large amount of carriers coexist with lysosomes, namely the lysosome escape capacity of pure Niosomes is far inferior to that of HA-C-niomes.

Fourth, gene vector cell transfection experiment

The test procedure was as follows:

(1) the gene-carrying vector is prepared by utilizing the principle of positive and negative charge interaction of cationic lipoid (C-niosomes) and pEGFP. C-niosomes (C (DOTAP)) were prepared at 0.3mg/ml following the optimized recipe. The concentration of pEGFP was 200. mu.g/ml. C-niosomes were mixed with pEGFP according to the following table, as shown in the table below, in 12: 1, vortexed for 200sec, and allowed to stand at room temperature for 15min to allow complete binding of C-niosomes to pEGFP. Adding a certain amount of HA-DOPE, and shaking in a shaker at 37 ℃ for 30min to obtain HA-C-niosomes/pEGFP.

(2) ARPE-19 cells in a number of 5X 10⁵And inoculating the seeds in 12-well plates for 24 h. The plasmid-containing vector C-niosomes, 10% HA-C-niosomes and 20% HA-C-n were addedTransfecting cells for 4h by using an iosomes solution with naked pEGFP as a control, removing a carrier solution, replacing a fresh culture medium, continuously culturing for 24h, and observing the transfection condition by using a laser confocal microscope after the cells are fixed. And preparing cell suspension, and detecting the cell transfection rate by using a flow cytometer.

The results of the experiment (FIG. 5) show that the green fluorescence intensity of the cationic liposome is enhanced to a certain extent compared with that of the naked plasmid. After the cells are treated by the HA-C-niosomes vector and transfected for 48 hours, the green fluorescence intensity of intracellular pEGFP expression is obviously stronger than that of an unmodified vector group. Furthermore, as the amount of HA modification increases, the fluorescence intensity also increases. Flow cytometry analysis showed that C-niosomes had 1.5-fold (150.4. + -. 8.5%) green fluorescence intensity 48h after transfection, as compared to the transfection efficiency of the naked plasmid group (100%). Whereas, the transfection efficiency of 10% HA-C-niosomes/pEGFP group (157.5. + -. 2.1%) and 20% HA-C-niosomes (194.9. + -. 1.4%) was significantly higher than that of the naked plasmid group by modification of HA. And the transfection efficiency is improved along with the increase of the HA modification amount. The results show that the nano-carrier modified by HA can obviously improve the gene transfection efficiency.

In summary, the gene delivery vector can effectively target RPE cells, and achieves the effect of gene transfection. Therefore, for the related treatment of the fundus disease, the vector can be used for effectively delivering the gene, and has obvious advantages for the treatment.

Claims

1. A hyaluronic acid modified gene-entrapped cationic liposome is characterized in that a nonionic surfactant, auxiliary phospholipid and cationic phospholipid are used as main film forming materials, and the outer layer is coated with modified hyaluronic acid; wherein the nonionic surfactant is tween 80 or span 80, and the auxiliary phospholipid is squalene or cholesterol; the particle size of the hyaluronic acid modified gene-entrapped cationic liposome is 100-300nm, and the Zeta potential is-5 to-80 mV;

the mol ratio of the nonionic surfactant to the auxiliary phospholipid is 5: 2-5: 8, and the mass ratio of the cationic phospholipid to the nonionic surfactant is 7: 100-13: 100;

the auxiliary phospholipid can play a certain supporting role, has certain auxiliary capacity for forming a bilayer, is added with cationic phospholipid to ensure that the potential is changed from negative to positive, is specifically combined with a negative gene, is wrapped by electrostatic adsorption, and finally forms a cationic liposome to realize gene entrapment;

the entrapped gene is an electronegative gene for treating fundus diseases;

the cationic phospholipid is 2, 3-dioleoyl-propyl-trimethylamine.

2. The method for preparing the hyaluronic acid modified gene-entrapped cationic niosome according to claim 1, which comprises the following steps:

(2) injecting the ethanol solution into buffer salt solution at 40-80 deg.C at uniform speed, stirring for 30-50nin, and ultrafiltering under ultrafiltration membrane for 1-3 times to obtain cationic liposome;

(3) mixing the negatively charged gene and the positively charged cationic liposome, vortexing for 200S, and standing at room temperature;

(4) adding hyaluronic acid-dioleoyl phosphatidylethanolamine into the product obtained in the step (3), and incubating for 20-40min at 37 ℃ to obtain the hyaluronic acid modified cationic lipoid.

3. The method for preparing the hyaluronic acid modified gene-entrapped cationic niosome according to claim 2, wherein the molecular weight of hyaluronic acid in step (3) is in the range of 500-3900 kDa.

4. The method for preparing the cationic liposome carrying the gene modified by the hyaluronic acid according to claim 2, wherein the mass ratio of the hyaluronic acid-dioleoyl phosphatidylethanolamine to the cationic liposome is 1: 10-1: 5.

5. use of the hyaluronic acid-modified gene-entrapped cationic niosome of claim 1 for the preparation of an ocular gene delivery vehicle.