CN111254162A - Preparation of cationic lipid microvesicle and mediated gene delivery method thereof - Google Patents

Abstract

The invention discloses a preparation method of cationic lipid microbubbles and a mediated gene delivery method thereof, wherein the preparation method comprises the steps of dissolving DSPC, DSPE-PEG2000 and DOTAP in an organic solvent to prepare a solution, carrying out ultrasonic treatment on the solution, filling the solution into a bottle, removing the organic solvent and adding a buffer solution; replacing air in the bottle with perfluoropropane, shaking to obtain cationic lipid microbubble, and storing at 4 deg.C; the delivery method comprises the following steps: culturing cells before transfection, incubating the cationic lipid microvesicle and the target gene, adding the cells, performing ultrasonic irradiation transfection, and culturing overnight after transfection. The invention has the advantages that: provides a microbubble with high gene delivery efficiency, provides two advantages of simple and noninvasive solution scheme operation for UTMD-mediated gene in-vitro delivery, and improves the gene transfection efficiency.

Description

A kind of preparation of cationic lipid microbubble and its mediated gene delivery method

technical field

The invention belongs to the technical field of gene delivery, and in particular relates to a method for gene delivery.

Background technique

Ultrasound targeted microbubble destruction (UTMD) provides us with a safe and effective gene delivery tool. With the continuous development of ultrasound contrast agents, the application of UTMD to gene delivery has gradually increased. Compared with other non-viral gene delivery technologies, UTMD-mediated gene delivery has the advantages of relatively simple operation and non-invasiveness. However, the key to successful gene delivery is to protect the gene from the shear force of blood flow and the degradation of DNase during the delivery process, and to release the carried gene to target cells in a targeted and directional manner. The basic principle of UTMD is: carry genes on the surface or inside of ultrasonic microbubbles, and locally apply ultrasonic irradiation in vitro to generate cavitation, improve the permeability of cell membranes, and effectively promote gene delivery into cells. Ultrasonic microbubbles, as a new type of non-viral vector used in gene therapy, have been adopted by many scholars at home and abroad. It has the advantages of large quantity, easy preparation and low cost. Some studies have used UTMD technology to deliver miR-126-3p, which can effectively reduce the expression of target genes and increase the blood vessel density in a chronic anemia skeletal muscle model. In terms of tumors, UTMD-mediated siRNA targeting lncRNA-ATB was transfected into liver cancer cells, which successfully down-regulated the expression of lncRNA-ATB and inhibited the metastasis and invasion of liver cancer cells. The efficiency is higher than that of lipofection.

Therefore, it is promising to develop a gene vector that improves gene delivery efficiency and its related preparation method.

SUMMARY OF THE INVENTION

One object of the present invention is to establish a method for preparing cationic lipid microvesicles to obtain microvesicles with high gene delivery efficiency.

Another object of the present invention is to provide a safe, effective, time-saving and labor-saving gene delivery method in vitro to meet the needs of in vitro gene function research.

In order to achieve the above object, the technical scheme provided by the invention is as follows:

A preparation method of cationic lipid microbubbles, comprising the following steps:

(1) get DSPC, DSPE-PEG2000, DOTAP, dissolve in organic solvent to prepare solution, ultrasonically process the solution, put into bottle;

(2) remove the organic solvent of the solution prepared in step (1), then add buffer to the bottle;

(3) Replace the air in the bottle after adding the buffer in step (2) with perfluoropropane, shake the solution in the bottle, obtain cationic lipid microbubbles, and store at 4°C.

Further, in described step (1), DSPC, DSPE-PEG2000, DOTAP, the ratio of the consumption of organic solvent is 9mg: 2mg: 1mg: 1mL;

In the step (1), the organic solvent is chloroform.

Further, in the step (1), the solution is placed in an ultrasonic water bath, and ultrasonically treated for 30s.

Further, the solution prepared in step (1) is divided into 4 vials, and in the step (2), the 4 vials are placed in a rotary evaporator, and rotary evaporated at 65° C. for 1 h to completely volatilize the organic solvent. , 800 μl of glycerol-containing PBS buffer was added to each vial;

In step (2), the buffer is a PBS buffer containing glycerol, and the ratio of the mass of glycerol to the volume of the PBS buffer is 0.2%.

Further, in the step (3), the solution was vigorously shaken in a Vialmix ^TM amalgam blender for 45s to obtain a cationic lipid microbubble suspension.

A method for gene delivery mediated by cationic lipid microvesicles, comprising the following steps:

(a) One day before gene transfection, 1×10 ⁵ MDA-MB-231 cells/well were seeded in DMEM medium containing 10% fetal bovine serum, and incubated in a 24-well plate for 24 hours. When the cells were confluent When it reaches 80%, change the medium to 300 μl serum-free medium OPTI-MEM;

(b) 10 μl of cationic lipid microbubbles and 10 ug of plasmid pEGFP expressing green fluorescent protein or 300 mM miR-34a mimics or 300 mM miR-34a were incubated for 10 min to obtain a cationic lipid microbubble-target gene mixture;

(c) adding the mixture obtained in step (b) to the cells incubated in step (a), supplementing the medium to a final volume of 500 μl, gently shaking the 24-well plate, and placing it on the ultrasonic probe;

(d) filling the space between the bottom of the 24-well plate and the ultrasonic probe with sterilized distilled water, setting parameters of ultrasonic intensity, duty cycle and irradiation time, starting the Sonovitro instrument, and carrying out ultrasonic irradiation transfection;

(e) After transfection, the cells were cultured in a cell incubator with a _CO concentration of 5% and a temperature of 37°C for 10 min, and then the medium was changed to fresh DMEM medium containing 10% fetal bovine serum for 24 h.

Preferably, the ultrasonic intensity of the step (d) is 0.6 W/cm ² .

Preferably, the duty cycle of the step (d) is 20%.

Preferably, the irradiation time of the step (d) is 20s.

Compared with the prior art, the beneficial effects of the present invention are:

The ultrasonic microbubble-mediated gene transfection process involves multiple mechanisms, and cavitation plays a key role in it. Under the action of cavitation, strong standing waves and micro-jets are generated, and nano-scale pores are formed on the cell surface, thereby improving the permeability of the cell membrane and facilitating the introduction of genes into cells. After ultrasonic microbubble treatment, small pores appeared in the cell membrane immediately, which were closed after about 30 minutes, and the cell morphology returned to normal. Of course, ultrasonic parameters such as ultrasonic intensity (AI), duty cycle (DC) and exposure time (ET) are optimized. Especially when the AI exceeds 1.0W/cm ² , the cells are obviously irreversibly damaged, and the cell viability decreases significantly.

On the basis of systematically studying different factors, pEGFP plasmid DNA was transfected into MDA-MB-231 cells to evaluate the efficiency of UTMD system. Through this ultrasonic microbubble delivery method in the present invention, pEGFP can be effectively transferred into cells to express green fluorescent protein. The results of flow cytometry showed that the transfection rate was over 40%.

Furthermore, miR-34amimics were used to demonstrate the flexibility of the method. miR-34amimics can effectively inhibit breast cancer cell proliferation and induce apoptosis. In addition, downstream proteins including Notch1 and hes1 are down-regulated by miR-34a.

To sum up, the present invention provides a method for gene delivery by ultrasonic microbubbles. The present invention adopts the method of cationic lipid microbubbles + ultrasonic to transfect pEGFP and miR-34a mimics. Among them, miR-34amimics can effectively inhibit MDA-MB-231 cells. In addition, downstream proteins including Notch1 and Hes1 were down-regulated by miR-34a. This system has the potential for further in vivo gene therapy studies.

Description of drawings

Figure 1 is a schematic diagram of the installation of the ultrasonic microbubble transfection system, wherein the Sonovitro equipment consists of a cover (1), a water tank (2), an ultrasonic probe (3) and a control panel (5). The center of the probe is placed in a 24-well plate (4). Cultured cells; the bottom of the culture plate is filled with sterilized distilled water;

Fig. 2 is the flow chart of UTMD transfection experiment;

Figure 3 is the experimental graph of the effect of different ultrasonic parameters on cell viability, in which, Figure A represents the effect of irradiation time on cell viability; Figure B represents the effect of ultrasonic intensity on cell viability, and Figure C represents the effect of duty cycle on cell viability ;

Figure 4 is the experimental graph of the effects of ultrasound and cationic lipid microbubbles on the apoptosis of MDA-MB-231 cells, in which, Figure A shows the detection of Untreated (untreated group), US (ultrasound-treated group), and CMBs by flow cytometry (Cationic lipid microbubble treatment group) and US+CMBs (ultrasonic treatment + cationic lipid microbubble group)-induced apoptosis and comparison of the experimental graph; Figure B is the apoptosis rate, Annexin V positive cells accounted for the total cells The percentage of the experimental graph.

Figure 5 is an experimental diagram of changes in cell membrane structure at different times after ultrasonic microbubbles act on cells. Among them, Figure A: the experimental map of the control group; Figure B: the experimental map of 5 minutes after the action of pure ultrasound; Figure C: the experimental map of 0min after the effect of UTMD; Figure D: the experimental map of 5 minutes after the effect of UTMD; Figure E: the effect of UTMD The experimental map of the last 15min; Figure F: the experimental map of 30min after UTMD;

Figure 6 is an experimental graph of the effect of UTMD treatment with different sound intensities on cell membranes.

Figure 7 is an experimental diagram of the results after pEGFP plasmid DNA transfection. Figure A is the experimental diagram of the observation results under a fluorescence microscope; Figure B is the experimental diagram of the flow quantification results.

Figure 8 shows the localization analysis and quantitative experiments of miR-34a mimics. Figure A is the experimental figure of the cellular uptake of cy5-labeled miR-34amimics (red) after 24 hours of treatment in different experimental groups; Figure B is the RT-qPCR quantitative experimental figure of miR-34a.

FIG. 9 is an experimental diagram of the analysis of proliferation and apoptosis of MDA-MB-231 cells. Among them, Figure A is the experimental graph of the inhibitory effect of miR-34amimics on the proliferation of MDA-MB-231 cells; Figure B is the experimental graph of the apoptosis induced by miR-34a mimics detected by flow cytometry; Figure C is the proportion of AnnexinV positive cells in the total Percentage of cells experimental plot.

Fig. 10 is a graph of the analysis experiment of Notch1 and hes1 expression in MDA-MB-231 cells. 48h after UTMD was transfected with microRNA-34a mimics, the protein expressions of Notch1 and hes1 were detected by western blot.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

Embodiment 1. Preparation and detection of cationic lipid microbubbles and their characterization

The present invention provides a method for the preparation of cationic lipid microbubbles and a method for detecting their characterization, comprising the following steps:

(1) Accurately weigh 9mg DSPC, 2mg DSPE-PEG2000 and 1mg DOTAP, dissolve them in 1ml chloroform to prepare a solution, place the solution in an ultrasonic water bath, ultrasonically treat it for 30s, and then divide the solution into 4 vials, 250μl per bottle;

(2) Place the 4 vials of solution prepared in step (1) in a rotary evaporator, and rotate at 65°C for 1 h to completely volatilize chloroform; add 800 μl of glycerol-containing PBS buffer to each vial, and glycerol The mass to volume ratio of PBS buffer is 0.2%;

(3) replacing the air in the vial of step (2) with perfluoropropane; the solution was vigorously shaken for 45s in a Vialmix ^TM amalgam blender to obtain a cationic lipid microbubble suspension;

(4) After placing the cationic lipid microbubble suspension in step (3) in a refrigerator at 4°C for about 1 hour, take an appropriate amount of the cationic lipid microbubble suspension, add 0.5 ml of PBS to dilute,

Example 2. Optimization of ultrasonic parameters during cell transfection

In order to clarify the effects of ultrasonic irradiation time, ultrasonic intensity and duty cycle on cell viability during the transfection process, different ultrasonic irradiation time, ultrasonic intensity and duty cycle were used to conduct MDA-MB-231 cell transfection experiments. -8 Experiments to detect cell activity, the parameters of ultrasonic irradiation time, ultrasonic intensity and duty cycle are shown in Tables 1 to 3 below:

Table 1

Ultrasound irradiation time blank 20s 40s 60s cell activity 100% 95% 93% 92%

Table 2

sound intensity blank CMB 0.6 0.8 1.0 1.2 cell activity 100% 95% 94% 92% 89% 80%

table 3

duty cycle blank 15% 20% 25% 30% 35% cell activity 100% 98% 98% 93% 92% 90%

It can be seen that the parameters that have the least effect on cell viability are as follows: ultrasonic irradiation for 20s, cell viability is the strongest; sound intensity 0.6W/cm ² , cell viability is the strongest; duty cycle 20%, cell viability is the strongest, the results are shown in Figure 3 . Therefore, the ultrasonic irradiation parameters used in the subsequent experiments adopted the parameters with the least effect on cell viability.

Example 3. Apoptosis detection

In order to determine whether ultrasound combined with cationic lipid microbubble transfection has an effect on cell apoptosis, the experiment was divided into 4 experimental groups, namely:

Untreated group (Untreated), MDA-MB-231 cells did not undergo any treatment;

Ultrasonic treatment group (Ultrasound, US), MDA-MB-231 cells were sonicated under the conditions of 0.6W/cm ² AI, 20% DC and 20s ET;

Cationic lipid microbubbles (Cationic microbubbles, CMBs): the cationic lipid microbubbles prepared by the present invention were mixed with MDA-MB-231 cells for transfection;

Ultrasound+cationic lipid microbubble treatment group (US+CMBs): MDA-MB-231 cells were ultrasonically treated under the conditions of 0.6W/cm ² AI, 20% DC and 20s ET using the cationic lipid microbubbles prepared by the present invention;

The apoptosis of the above experimental groups was evaluated by Annexin-V/PI staining and flow cytometry. As shown in Figure 4, the difference was not statistically significant, so the sonication + cationic lipid microbubble group (US+CMBs) was compared with the untreated group (Untreated), the sonication group (US) and the cationic lipid microbubble treatment Compared with the control group (CMBs), the ultrasonic treatment + cationic ultrasonic microbubble group (US + CMBs) had no significant effect on apoptosis.

Example 4. Analysis of sonoporation effect of ultrasonic microbubbles

In order to analyze the sonoporation effect of cationic lipid microbubbles + ultrasound, the experiment was divided into 3 experimental groups, namely:

Blank control group: MDA-MB-231 cells did not undergo any treatment;

Ultrasound group: MDA-MB-231 cells were sonicated under the conditions of 0.6W/cm ² AI, 20% DC and 20s ET;

Ultrasound+microbubble group: using the cationic lipid microbubbles prepared by the present invention, MDA-MB-231 cells were ultrasonically treated under the conditions of 0.6W/cm ² AI, 20% DC and 20s ET, that is, UTMD treatment;

Electron microscope was used to observe the changes of cell membrane structure at different time points in the control group, the ultrasonic group after ultrasonic irradiation, and the ultrasonic + microbubble group after ultrasonic irradiation. As shown in Figure 5, A is the state of the control group; B is the state of the ultrasound group after 5 minutes of ultrasound, C is the state of the ultrasound + microbubble group at 0 minutes after UTMD; D is the ultrasound + microbubble group after UTMD 5min state, E is the state of ultrasound + microbubble group 15min after UTMD action, F is the state of ultrasound + microbubble group 30min after UTMD action, it can be seen from the figure: compared with the control group and ultrasound group, ultrasound + microbubble group In the group, a large number of small pores appeared on the cell membrane, especially within 5 min after UTMD, the number of pores on the cell membrane was the largest, when the time exceeded 15 min, the pores on the cells gradually decreased. It can be seen that cationic lipid microbubbles combined with ultrasound have a certain sonoporous effect, which can perforate the cell membrane, improve the permeability of the cell membrane, and help genes enter cells. With the increase of the sound intensity, the number of pores on the cell membrane increased, as shown in Figure 6, which are the experimental pictures of different experimental groups after 5 minutes of ultrasonication, in which A: control group; B: ultrasonic group; C: 0.4W/cm2+30μl microbubbles; D: 0.6W/cm2+30μl microbubbles; E: 0.8W/cm2+30μl microbubbles; F: 1.0W/cm2+30μl microbubbles. It should be noted that the size shown in Figure 5 and Figure 6 is 5μm

Embodiment 5. Plasmid DNA transfection

MDA-MB-231 cells are now transfected by the transfection method of the present invention (UTMD), and the general process is shown in Figure 2. The detailed operation process is as follows:

(1) Before transfection, inoculate 1×10 ⁵ MDA-MB-231 cells/well in DMEM medium containing 10% fetal bovine serum, and incubate in a 24-well plate for 24 hours. 80%, change the medium to 300 μl serum-free medium (OPTI-MEM);

(2) 10 μl of cationic lipid microvesicles and 10 μg of pEGFP plasmid DNA were mixed and incubated for 10 min to obtain a cationic lipid microvesicle-plasmid mixture;

(3) Add 10 μg of the cationic lipid microbubble-plasmid mixture from step (2) to the MDA-MB-231 cells incubated in step (1), supplement the medium to a final volume of 500 μl, and gently shake the 24-well plate , placed on the ultrasound probe of the Sonovitro instrument;

(4) be filled with sterilized distilled water in the gap between the bottom of the 24-well plate and the ultrasonic probe, set ultrasonic intensity, duty cycle and irradiation time parameters according to the ultrasonic parameters of the optimization gained in Example 2, start the Sonovitro instrument, carry out ultrasonic Irradiation transfection;

As shown in Figure 1, the Sonovitro device consists of a lid 1, a water tank 2, an ultrasonic probe 3, and a control panel 5, with the cells cultured in a 24-well plate 4 placed in the center of the probe. The bottom of 24-well plate 4 is filled with sterilized distilled water.

(5) After transfection, the cells were cultured in an incubator with a _CO concentration of 5% and a temperature of 37°C; after 10 min, the medium was changed to 500 μl of DMEM medium containing 10% fetal bovine serum, and placed in the incubator Incubate for 24h; observe the transfection efficiency under a fluorescence microscope. Microscopic observation showed that, as shown in Figure 7, under this condition, MDA-MB-231 cells were transfected, and the number of cells expressing green fluorescent protein increased significantly. The quantitative results of flow cytometry showed that the transfection rate could reach 46.8%.

Example 6. Ultrasound+cationic lipid microbubbles deliver miR-34a mimics

In order to study whether the method of the present invention can efficiently transfect miR-34a mimics into MDA-MB-231 cells, cy5-labeled miR-34amimics were synthesized in this experiment and divided into 4 experimental groups:

Untreated group: MDA-MB-231 cells did not undergo any treatment;

Ultrasound group: Ultrasound + cy5-labeled miR-34a mimics were used to treat MDA-MB-231 cells. Refer to the operation process of pEGFP plasmid DNA transfection in Example 5 above. The difference is that MDA was added to the operation step in step (3). -MB-231 cells with only 300mM cy5-labeled miR-34amimics;

Microbubble group: MDA-MB-231 cells were treated with cationic lipid microbubbles + cy5-labeled miR-34a mimics; refer to the operation process of pEGFP plasmid DNA transfection in Example 5 above, the difference is that only MDA-MB -231 cells were added with a mixture of cationic lipid microbubbles and 300 mM cy5-labeled miR-34 amimics without sonication.

Ultrasound+microbubble group: Ultrasound+cationic lipid microbubbles+cy5-labeled miR-34a mimics were used to treat MDA-MB-231 cells, and the transfection method was referred to the operation process of pEGFP plasmid DNA transfection in Example 5 above. In contrast, a mixture of cationic lipid microbubbles and 300 mM cy5-labeled miR-34 amimics was added to MDA-MB-231 cells for sonication. Experiments proved that, as shown in Figure 8A, cy5-labeled miR-34amimics could enter cells with the help of ultrasound + cationic lipid microbubbles, but ultrasound or cationic lipid microbubbles alone could not make mir-34a mimics enter cells . RT-qPCR was used to further quantitatively detect miR-34a. As shown in Figure 8B, the expression of miR-34a in the UTMD group was approximately 2-fold higher than that in the other groups.

Example 7. The effect of miR-34a mimics on MDA-MB-231 cells

To verify the function of miR-34 amimics transfected by the method of the present invention (UTMD), CCK-8 was used to detect cell proliferation. At 3 days after UTMD transfection, the proliferation of MDA-MB-231 cells was significantly inhibited compared with the miR-NC group. Flow cytometry was used to detect the apoptosis induced by miR-34a. The results are shown in Figure 9. Compared with the miR-NC group, the miR-34a mimics transfected by UTMD significantly promoted cell apoptosis, and the apoptosis rate was about 25%. %. Finally, the expression of miR-34a target protein Notch1 and its target protein hes1 was detected by western blot. As shown in Figure 10, miR-34amimics reduced the expression of Notch1 and hes1.

In the above, pEGFP plasmid DNA or miR-34amimics were introduced into MDA-MB-231 cells, and related factors were studied. It can be known that the UTMD of the present invention can efficiently transfect pEGFP and miR-34a mimics. Also, in MDA-MB-231 cells, the function of UTMD-delivered miR-34a was also investigated. The results suggest that this approach has the potential for further in vivo gene therapy studies.

The technical solutions provided by the embodiments of the present invention have been introduced in detail above. The principles and implementations of the embodiments of the present invention are described in this paper by using specific examples. The descriptions of the above embodiments are only applicable to help understand the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation and application scope. To sum up, the content of this specification should not be construed as a limitation of the present invention.

Claims (9)

1. a preparation method of cationic lipid microbubbles, is characterized in that, comprises the steps: (1) get DSPC, DSPE-PEG2000, DOTAP, dissolve in organic solvent to prepare solution, ultrasonically process the solution, put into bottle; (2) remove the organic solvent of the solution prepared in step (1), then add buffer to the bottle; (3) Replace the air in the bottle after adding the buffer in step (2) with perfluoropropane, shake the solution in the bottle, obtain cationic lipid microbubbles, and store at 4°C. 2. the preparation method of a kind of cationic lipid microbubble as claimed in claim 1, is characterized in that, In described step (1), DSPC, DSPE-PEG2000, DOTAP, the ratio of the consumption of organic solvent is 9mg: 2mg: 1mg: 1mL; In the step (1), the organic solvent is chloroform. 3. the preparation method of a kind of cationic lipid microbubble as claimed in claim 1, is characterized in that, in the described step (1), the solution is placed in an ultrasonic water bath, and ultrasonically treated for 30s. 4. the preparation method of a kind of cationic lipid microbubble as claimed in claim 1, is characterized in that, The solution prepared in step (1) is divided into 4 vials, and in step (2), the 4 vials are placed in a rotary evaporator, and rotary evaporated at 65° C. for 1 h to completely volatilize the organic solvent. 800 μl of glycerol-containing PBS buffer was added to each vial; In step (2), the buffer is a PBS buffer containing glycerol, and the ratio of the mass of glycerol to the volume of the PBS buffer is 0.2%. 5. the preparation method of a kind of cationic lipid microbubble as claimed in claim 1, is characterized in that, In the step (3), the solution was vigorously shaken in a Vialmix ^TM amalgam blender for 45s to obtain a cationic lipid microbubble suspension. 6. The gene delivery method mediated by cationic lipid microvesicles as claimed in claim 1, wherein the method comprises the steps: (a) One day before gene transfection, 1×10 ⁵ MDA-MB-231 cells/well were seeded in DMEM medium containing 10% fetal bovine serum, and incubated in a 24-well plate for 24 hours. When the cells were confluent When it reaches 80%, change the medium to 300 μl serum-free medium OPTI-MEM; (b) 10 μl of cationic lipid microbubbles and 10 ug of plasmid pEGFP expressing green fluorescent protein or 300 mM miR-34amimics or 300 mM miR-34a were incubated for 10 min to obtain a cationic lipid microbubble-target gene mixture; (c) adding the mixture obtained in step (b) to the cells incubated in step (a), supplementing the medium to a final volume of 500 μl, gently shaking the 24-well plate, and placing it on the ultrasonic probe; (d) filling the space between the bottom of the 24-well plate and the ultrasonic probe with sterilized distilled water, setting parameters of ultrasonic intensity, duty cycle and irradiation time, starting the Sonovitro instrument, and carrying out ultrasonic irradiation transfection; (e) After transfection, the cells were cultured in a cell incubator with a _CO concentration of 5% and a temperature of 37°C for 10 min, and then the medium was changed to fresh DMEM medium containing 10% fetal bovine serum for 24 h. 7. The gene delivery method mediated by cationic lipid microvesicles as claimed in claim 6, characterized in that, The ultrasonic intensity of the step (d) was 0.6 W/cm ² . 8. The gene delivery method mediated by cationic lipid microvesicles as claimed in claim 6, wherein, The step (d) has a duty cycle of 20%. 9. The gene delivery method mediated by cationic lipid microvesicles as claimed in claim 6, characterized in that, The irradiation time of the step (d) is 20s.

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