CN111254162A

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

Info

Publication number: CN111254162A
Application number: CN202010056548.3A
Authority: CN
Inventors: 陈智毅; 王奕; 张辉; 刘付春; 李悦
Original assignee: Third Affiliated Hospital of Guangzhou Medical University
Current assignee: Third Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-01-18
Filing date: 2020-01-18
Publication date: 2020-06-09
Anticipated expiration: 2040-01-18
Also published as: CN111254162B

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

Preparation of cationic lipid microvesicle and mediated gene delivery method thereof

Technical Field

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

Background

Ultrasonic targeted delivery technology (UTMD) provides a safe and effective gene delivery tool for us. With the continuous development of ultrasound contrast agents, there are increasing examples of the application of UTMD to gene delivery. UTMD-mediated gene delivery has the advantages of being relatively simple to operate and non-invasive relative to other non-viral gene delivery techniques. However, the key to successful gene delivery is to protect the gene from shear forces in the blood stream and degradation by dnase during delivery, and to effect site-directed, targeted release of the carried gene to the target cell. The basic principle of UTMD is: the gene is carried on the surface or inside of the ultrasonic microbubble, and the ultrasonic irradiation is locally applied in vitro to generate cavitation, improve the permeability of cell membranes and effectively promote the gene to be delivered to the cells. The ultrasonic microvesicle is used as a novel non-viral vector for gene therapy, has been adopted by numerous scholars at home and abroad, and compared with a viral vector, the artificially synthesized gene 'transport tool' has the advantages of low immunogenicity, large gene loading capacity, easiness in preparation, low cost and the like. The miR-126-3p is delivered by utilizing a UTMD technology, so that the expression of a target gene is effectively reduced, and the blood vessel density in a skeletal muscle model with chronic anemia is improved. In the aspect of tumor, the UTMD-mediated siRNA targeting lncRNA-ATB is used for transfecting liver cancer cells, so that the expression of lncRNA-ATB is successfully reduced, the transfer and invasion of the liver cancer cells are inhibited, and in addition, research shows that the efficiency of the UTMD-mediated siRNA transfection is higher than that of liposome transfection.

Therefore, the development of a gene vector for improving the gene delivery efficiency and a related preparation method thereof have application prospects.

Disclosure of Invention

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

Another objective of the invention is to provide a safe, effective, time-saving and labor-saving gene in vitro delivery method, which meets the requirements of in vitro gene function research.

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

a method for preparing cationic lipid microvesicles, comprising the steps of:

(1) dissolving DSPC, DSPE-PEG2000 and DOTAP in organic solvent to obtain solution, treating the solution with ultrasound, and bottling;

(2) removing the organic solvent of the solution prepared in the step (1), and adding a buffer solution into the bottle;

(3) and (3) replacing the air in the bottle added with the buffer solution in the step (2) with perfluoropropane, shaking the solution in the bottle to obtain the cationic lipid microbubble, and storing at 4 ℃.

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

the organic solvent in the step (1) is chloroform.

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

Further, the solution prepared in the step (1) is divided into 4 vials, in the step (2), the 4 vials are placed in a rotary evaporator and are subjected to rotary evaporation at 65 ℃ for 1h to completely volatilize the organic solvent, and 800 μ l of PBS buffer containing glycerol is added into each vial;

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

Further, the solution in the step (3) is Vialmix^TMThe silver mercury mixer was shaken vigorously for 45s to obtain a suspension of cationic lipid microbubbles.

A method of cationic lipid microbubble mediated gene delivery comprising the steps of:

(a) one day before gene transfection, 1X 10 cells were transfected⁵Inoculating MDA-MB-231 cells/hole into a DMEM medium containing 10% fetal bovine serum, placing the DMEM medium in a 24-hole plate, incubating for 24 hours, and changing the medium to 300 mu l of serum-free medium OPTI-MEM when the cell confluency reaches 80%;

(b) incubating 10 mul of cationic lipid microvesicle with 10ug of plasmid pEGFP or 300mM miR-34a mimics for 10min to obtain a cationic lipid microvesicle-target gene mixture;

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

(d) filling disinfected distilled water in a gap between the bottom of the 24-hole plate and the ultrasonic probe, setting ultrasonic intensity, duty ratio and irradiation time parameters, starting a Sonovitro instrument, and performing ultrasonic irradiation transfection;

(e) after transfection, cells were placed in CO₂Culturing at 37 deg.C for 10min in 5% cell culture box, and culturing for 24h in fresh DMEM medium containing 10% fetal calf serum.

Preferably, the ultrasonic intensity of step (d) is 0.6W/cm²。

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

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

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

the ultrasound microbubble-mediated gene transfection process involves a variety of mechanisms in which cavitation plays a key role. Under the action of cavitation, strong standing waves and microjets are generated, and pores with nanometer scale are formed on the surface of cells, so that the permeability of cell membranes is improved, and the gene is favorably introduced into the cells. After the ultrasonic microbubble treatment, the cell membrane immediately appears small holes, and is closed after about 30min, and the cell morphology returns to normal. Of course, the ultrasonic parameters of the ultrasonic intensity (AI), Duty Cycle (DC) and irradiation time (ET) are optimized. Especially when the AI exceeds 1.0W/cm²When the cells are damaged irreversibly, the survival rate of the cells is reduced remarkably.

Based on systematic study of different factors, pEGFP plasmid DNA was transfected into MDA-MB-231 cells to evaluate the efficiency of UTMD system. According to the invention, through the ultrasonic microbubble delivery method, pEGFP can be effectively transferred into cells to express green fluorescent protein. The detection result of the flow cytometry shows that the transfection rate reaches more than 40 percent.

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

In conclusion, the invention provides a method for delivering genes by using ultrasonic microvesicles, and the invention transfects pEGFP and miR-34a mimics by using a cationic lipid microvesicle + ultrasonic method. Wherein, the miR-34amimics can effectively inhibit MDA-MB-231 cells. In addition, downstream proteins including Notch1 and Hes1 were down-regulated by miR-34 a. The system has the potential for further in vivo gene therapy studies.

Drawings

FIG. 1 is a schematic diagram showing the installation of an ultrasonic microbubble transfection system, wherein a Sonovitro apparatus is composed of a cover (1), a water tank (2), an ultrasonic probe (3) and a control panel (5), and cells cultured in a 24-well plate (4) are placed in the center of the probe; filling the lower surface of the culture plate with sterilized distilled water;

FIG. 2 is a flow chart of a UTMD transfection experiment;

FIG. 3 is a graph of the effect of different ultrasound parameters on cell viability, wherein A is the effect of irradiation time on cell viability; graph B shows the effect of ultrasound intensity on cell viability and graph C shows the effect of duty cycle on cell viability;

FIG. 4 is an experimental graph of the effect of ultrasound and cationic lipid microbubbles on MDA-MB-231 apoptosis, wherein, the graph A shows the experimental graph of detecting and comparing the apoptosis induced by Untreated (Untreated group), US (ultrasonic treated group), CMBs (cationic lipid microbubble treated group) and US + CMBs (ultrasonic treated + cationic lipid microbubble group) by flow cytometry; panel B is an experimental plot of the rate of apoptosis expressed as a percentage of Annexin V positive cells to total cells.

FIG. 5 is an experimental diagram of the structural change of cell membrane at different times after the ultrasonic microvesicle acts on the cell. Wherein, figure A: experimental plots for the control group; and B: experimental graphs 5min after the action of pure ultrasound; and (C) figure: experimental plot 0min after UTMD; FIG. D: experimental plots 5min after UTMD action; FIG. E: experimental plots 15min after UTMD action; FIG. F: experimental plots 30min after UTMD action;

FIG. 6 is an experimental picture of the effect of different sound intensities UTMD treatments on cell membranes.

FIG. 7 is a diagram showing the results of the pEGFP plasmid DNA transfection. FIG. A is an experimental chart of the observation result under a fluorescence microscope; panel B is a flow quantification experimental plot.

FIG. 8 is a diagram of miR-34a mimics positioning analysis and quantification experiment. Panel A is a graph of the experiment of cell uptake of cy5 labeled miR-34amimics (red) 24h after treatment of different experimental groups; and the picture B is an RT-qPCR quantitative experiment picture of miR-34 a.

FIG. 9 is a diagram of MDA-MB-231 cell proliferation and apoptosis analysis experiment. Wherein, the graph A is an experimental graph of the inhibition effect of miR-34amimics on MDA-MB-231 cell proliferation; FIG. B is an experimental graph for detecting apoptosis induced by miR-34a mimics through flow cytometry; panel C is an experimental plot of the percentage of annexin V positive cells to total cells.

FIG. 10 is a graph showing an analysis experiment of the expression of Notch1 and hes1 in MDA-MB-231 cells. After 48h of UTMD transfection of microRNA-34a mimics, western blot is used for detecting the expression conditions of Notch1 and hes1 proteins.

Detailed Description

The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.

Example one preparation of cationic lipid microvesicles and detection of characterization thereof

The invention provides a method for preparing cationic lipid microvesicles and detecting the characterization of the cationic lipid microvesicles, which comprises the following steps:

(1) accurately weighing 9mg of DSPC, 2mg of DSPE-PEG2000 and 1mg of DOTAP, dissolving in 1ml of chloroform to prepare a solution, placing the solution in an ultrasonic water bath, carrying out ultrasonic treatment for 30s, and then subpackaging the solution in 4 small bottles, wherein each bottle is 250 mu l;

(2) putting the 4 vials prepared in the step (1) into a rotary evaporator, and carrying out rotary evaporation for 1h at 65 ℃ to completely volatilize chloroform; 800. mu.l of PBS buffer containing glycerol was added to each vial, the ratio of the mass of glycerol to the volume of PBS buffer being 0.2%;

(3) replacing the air in the vial of step (2) with perfluoropropane; the solution is in Vialmix^TMViolently shaking the silver-mercury mixer for 45s to obtain cationic lipid microbubble suspension;

(4) placing the cationic lipid microbubble suspension obtained in the step (3) in a refrigerator at 4 ℃ for about 1h, taking a proper amount of cationic lipid microbubble suspension, adding 0.5ml of PBS for dilution,

example two optimization of ultrasound parameters during cell transfection

In order to determine the influence of the ultrasonic irradiation time, the ultrasonic intensity and the duty ratio on the cell activity in the transfection process, the MDA-MB-231 cell transfection experiment is carried out by adopting different ultrasonic irradiation times, ultrasonic intensities and duty ratios, the cell activity is detected by adopting a CCK-8 experiment, and the parameters of the ultrasonic irradiation time, the ultrasonic intensity and the duty ratio are shown in the following table 1-table 3:

TABLE 1

Time of ultrasonic irradiation	Blank space	20s	40s	60s
					Cellular activity	100％	95％	93％	92％

TABLE 2

Sound intensity

Blank space

CMB

0.6

0.8

1.0

1.2

Cellular activity

100％

95％

94％

92％

89％

80％

TABLE 3

Duty cycle

Blank space

15％

20％

25％

30％

35％

Cellular activity

100％

98％

93％

92％

90％

The parameters that have the least effect on cell viability are seen as follows: the ultrasonic irradiation is carried out for 20s, and the cell activity is strongest; sound intensity 0.6W/cm²The cell activity is strongest; the duty cycle was 20% and the cell activity was strongest, and the results are shown in FIG. 3. Therefore, the ultrasonic irradiation parameters used in the subsequent experiments were the parameters that had the least effect on the cell viability.

EXAMPLE III apoptosis assay

To determine whether the ultrasound in combination with cationic lipid microvesicles transfected cells had an effect on apoptosis, the experiments were divided into 4 experimental groups, each consisting of:

untreated group, MDA-MB-231 cells were not treated at all;

ultrasonic treatment group (US), 0.6W/cm²AI. Ultrasonically treating MDA-MB-231 cells under the conditions of 20% DC and 20s ET;

cationic lipid microbubble treatment groups (CMBs): the cationic lipid microbubble prepared by the invention is mixed with MDA-MB-231 cells for transfection;

ultrasound + cationic lipid microbubble treated groups (US + CMBs): the cationic lipid microbubble prepared by the invention is 0.6W/cm²AI. Ultrasonically treating MDA-MB-231 cells under the conditions of 20% DC and 20s ET;

the above experimental groups were evaluated for apoptosis by Annexin-V/PI staining and flow cytometry. As shown in fig. 4, the differences were not statistically significant, and thus the sonication + cationic lipid microbubble groups (US + CMBs) had no significant effect on apoptosis compared to the Untreated group (Untreated), the sonication group (US) and the cationic lipid microbubble treated group (CMBs).

Example four ultrasonic microbubble sonoporation

To analyze the sonoporation of cationic lipid microbubbles + ultrasound, the experiments were divided into 3 experimental groups, each consisting of:

blank control group: MDA-MB-231 cells were not treated;

an ultrasonic group: 0.6W/cm²AI. Ultrasonically treating MDA-MB-231 cells under the conditions of 20% DC and 20s ET;

ultrasound + microbubble group: the cationic lipid microbubble prepared by the invention is 0.6W/cm²AI. Treating MDA-MB-231 cells by ultrasonic treatment under 20% DC and 20s ET conditions, namely UTMD treatment;

and observing the change of the cell membrane structures of the control group, the ultrasonic group after ultrasonic irradiation and the ultrasonic + microbubble group after ultrasonic irradiation at different time points by adopting an electron microscope. As shown in fig. 5, a in the figure is the state of the control group; b is the state 5min after the ultrasonic action of the ultrasonic group, C is the state 0min after the UTMD action of the ultrasonic + microbubble group; d is the state 5min after the UTMD action of the ultrasonic and microbubble group, E is the state 15min after the UTMD action of the ultrasonic and microbubble group, and F is the state 30min after the UTMD action of the ultrasonic and microbubble group, and the following can be known in the figure: compared with the control group and the ultrasonic group, the ultrasonic + microbubble group can obviously observe a large number of small holes on the cell membrane, particularly the number of the small holes on the cell membrane is the largest within 5min after the UTMD effect, and the small holes on the cell are gradually reduced after the time exceeds 15 min. The visible cationic lipid microbubble combined with the ultrasound has a certain sonoporation effect, can perforate a cell membrane, improves the permeability of the cell membrane, and is beneficial to the gene entering the cell. With the increase of the sound intensity, the number of the small holes on the cell membrane is increased, and as shown in fig. 6, the number is an experimental diagram of different experimental groups after 5min of ultrasonic action, wherein A: a control group; b: an ultrasonic group; c: 0.4W/cm2+ 30. mu.l microbubbles; d: 0.6W/cm2+ 30. mu.l microbubbles; e: 0.8W/cm2+ 30. mu.l microbubbles; f: 1.0W/cm2+ 30. mu.l microbubbles. It should be noted that the size shown in FIGS. 5 and 6 is 5 μm

Example five transfection of plasmid DNA

MDA-MB-231 cells were now transfected using the transfection method of the invention (UTMD), and the general scheme is shown in FIG. 2. The detailed operation process is as follows:

(1) before transfection, 1X 10 cells were transfected⁵The MDA-MB-231 cells/well are inoculated in DMEM medium containing 10% fetal bovine serum, placed in a 24-well plate and incubated for 24h, when the cell confluence reaches about 80%, the medium is changed to 300. mu.l of serum-free medium (OPTI-MEM);

(2) mixing 10 μ l of cationic lipid microbubble and 10 μ g of pEGFP plasmid DNA, and incubating for 10min to obtain a cationic lipid microbubble-plasmid mixture;

(3) adding 10 μ g of the cationic lipid microbubble-plasmid mixture of step (2) to the MDA-MB-231 cells incubated in step (1), supplementing the medium to a final volume of 500 μ l, gently shaking the 24-well plate, and placing on the ultrasound probe of a Sonovitro apparatus;

(4) filling disinfected distilled water in a gap between the bottom of the 24-hole plate and the ultrasonic probe, setting ultrasonic intensity, duty ratio and irradiation time parameters according to the ultrasonic parameters obtained by optimization in the second embodiment, and starting a Sonovitro instrument to perform ultrasonic irradiation transfection;

as shown in fig. 1, the Sonovitro apparatus is composed of a lid 1, a water tank 2, an ultrasonic probe 3, and a control panel 5, and cells cultured in a 24-well plate 4 are placed in the center of the probe. The underside of the 24-well plate 4 is filled with sterilized distilled water.

(5) After transfection, cells were placed in CO₂Culturing in an incubator at 37 deg.C and 5% concentration; after 10min, changing the culture solution to 500 mu l of DMEM medium containing 10% fetal calf serum, and placing the DMEM medium in an incubator to continue culturing for 24 h; transfection efficiency was observed under a fluorescent microscope. As shown in FIG. 7, under the condition, the MDA-MB-231 cells are transfected, the number of cells expressing green fluorescent protein is obviously increased, and the quantitative result of flow cytometry shows that the transfection rate can reach 46.8%.

Example six ultrasound + cationic lipid microvesicles delivery of miR-34a mimics

In order to study whether the miR-34a mimics can be used for efficiently transfecting MDA-MB-231 cells or not, the experiment synthesizes the miR-34 mimics marked by cy5, and the miR-34 mimics are divided into 4 experiment groups:

untreated group: MDA-MB-231 cells were not treated;

an ultrasonic group: treating MDA-MB-231 cells by using miR-34a mimics labeled by ultrasound + cy5, referring to the operation process of five pEGFP plasmid DNA transfection in the above example, except that only 300mM miR-34 mimics labeled by cy5 are added into the MDA-MB-231 cells in the operation step (3);

group of microvesicles: treating MDA-MB-231 cells by cationic lipid microvesicle + cy5 labeled miR-34a mimics; referring to the procedure of the five pEGFP plasmid DNA transfection of the above example, except that only a mixture of cationic lipid microbubbles and 300mM cy 5-labeled miR-34amimics was added to MDA-MB-231 cells, no sonication was performed.

Ultrasound + microbubble group: MDA-MB-231 cells were treated with ultrasound, cationic lipid microbubbles, and miR-34a mimics labeled with cy5, and the transfection method was performed according to the procedure of the five pEGFP plasmid DNA transfection described in the above example. In contrast, a mixture of cationic lipid microbubbles and 300mM cy 5-labeled miR-34amimics was added to MDA-MB-231 cells and sonicated. Experiments prove that as shown in A of FIG. 8, the cy 5-labeled miR-34 amics can enter cells with the help of ultrasound and cationic lipid microbubbles, and miR-34a mimics cannot enter cells by relying on ultrasound or cationic lipid microbubbles alone. The miR-34a is further quantitatively detected by using RT-qPCR, and as shown in B of figure 8, the miR-34a expression of the UTMD group is increased by about 2 times compared with the miR-34a expression of other groups.

EXAMPLE VII Effect of miR-34a mimics on MDA-MB-231 cells

To verify the function of the miR-34 amics transfected by the method (UTMD) of the invention, CCK-8 was used to detect cell proliferation. 3 days after UTMD transfection, MDA-MB-231 cell proliferation is obviously inhibited compared with the miR-NC group. The flow cytometry is adopted to detect the miR-34a induced apoptosis, and the result is shown in figure 9, compared with the miR-NC group, the miR-34a mimics transfected by UTMD obviously promotes the apoptosis, and the apoptosis rate is about 25%. And finally, detecting the expression conditions of the target protein Notch1 of the miR-34a and the target protein hes1 thereof by using western blot, wherein the expression of the Notch1 and the target protein hes1 is reduced by the miR-34amimics as shown in figure 10.

In the above, pEGFP plasmid DNA or miR-34amimics was introduced into MDA-MB-231 cells, and the relevant factors were studied. As can be known, the UTMD can transfect pEGFP and miR-34a mimics with high efficiency. Moreover, the function of miR-34a delivered by UTMD was also studied in MDA-MB-231 cells. The results show that the method has the potential for further in vivo gene therapy research.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Claims

1. A method for preparing cationic lipid microbubbles, comprising the following steps:

2. The method of claim 1, wherein the lipid microbubble is a cationic lipid microbubble,

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

the organic solvent in the step (1) is chloroform.

3. The method of claim 1, wherein the solution of step (1) is sonicated in a water bath for 30 seconds.

4. The method of claim 1, wherein the lipid microbubble is a cationic lipid microbubble,

the solution prepared in the step (1) is divided into 4 vials, in the step (2), the 4 vials are placed in a rotary evaporator and are subjected to rotary evaporation at 65 ℃ for 1 hour to completely volatilize the organic solvent, and 800 mu l of PBS buffer solution containing glycerol is added into each vial;

5. The method of claim 1, wherein the lipid microbubble is a cationic lipid microbubble,

the solution in the step (3) is Vialmix^TMThe silver mercury mixer was shaken vigorously for 45s to obtain a suspension of cationic lipid microbubbles.

6. The method of cationic lipid microbubble mediated gene delivery according to claim 1, comprising the steps of:

(b) incubating 10 mul of cationic lipid microvesicle with 10ug of plasmid pEGFP or 300mM miR-34 amics or 300mM miR-34a expressing green fluorescent protein for 10min to obtain a cationic lipid microvesicle-target gene mixture;

7. The method of cationic lipid microbubble mediated gene delivery according to claim 6,

the ultrasonic intensity of the step (d) is 0.6W/cm²。

8. The method of cationic lipid microbubble mediated gene delivery according to claim 6,

the step (d) duty cycle is 20%.

9. The method of cationic lipid microbubble mediated gene delivery according to claim 6,

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