CN111254162B

CN111254162B - Preparation of cationic lipid microvesicles and mediated gene delivery method thereof

Info

Publication number: CN111254162B
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: 2023-09-19
Anticipated expiration: 2040-01-18
Also published as: CN111254162A

Abstract

The invention discloses a preparation method of cationic lipid microbubbles and a mediated gene delivery method thereof, the preparation method comprises the steps of taking DSPC, DSPE-PEG2000 and DOTAP, dissolving 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, oscillating to obtain cationic lipid microbubbles, and preserving at 4deg.C; the delivery method is as follows: cells were cultured before transfection, cationic lipid microbubbles were incubated with the target gene, then cells were added, transfected by ultrasonic irradiation, and cultured overnight after transfection. The invention has the advantages that: the microbubble with high gene delivery efficiency is provided, the advantages of relatively simple and noninvasive solution operation are provided for UTMD mediated gene in-vitro delivery, and the gene transfection efficiency is improved.

Description

Preparation of cationic lipid microvesicles 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

Ultrasound targeted delivery technology (Ultrasound targeted microbubble destruction, UTMD) provides us with a safe and effective gene delivery tool. With the continued development of ultrasound contrast agents, the application of UTMD to gene delivery has been increasingly exemplified. UTMD mediated gene delivery has two major advantages of relatively simple operation and non-invasiveness over other non-viral gene delivery techniques. However, the key to successful gene delivery is to protect the gene from the effects of blood flow shear forces and degradation of dnase during delivery, and to site-directed, targeted release of the carried gene into the target cell. The basic principle of UTMD is: the genes are carried on the surface or inside of the ultrasonic microbubbles, and the ultrasonic irradiation is locally applied in vitro to generate cavitation, so that the permeability of cell membranes is improved, and the delivery of the genes into cells is effectively promoted. Ultrasonic microvesicles are used as a novel non-viral vector for gene therapy, have been adopted by a plurality of scholars at home and abroad, and compared with viral vectors, the artificially synthesized gene "transport means" has the advantages of low immunogenicity, large gene load, easy preparation, low cost and the like. The technology of UTMD is used for delivering miR-126-3p, so that the expression of target genes is effectively reduced, and the blood vessel density in the chronic anemia skeletal muscle model is improved. In terms of tumors, the expression of lncRNA-ATB is successfully down-regulated by the UTMD-mediated siRNA targeting lncRNA-ATB to inhibit the metastasis and invasion of the liver cancer cells, and in addition, the research shows that the efficiency of UTMD-mediated siRNA transfection is higher than that of liposome transfection.

Therefore, it is promising to develop a gene vector for improving gene delivery efficiency and a related preparation method thereof.

Disclosure of Invention

It is an object of the present invention to establish a cationic lipid vesicle preparation method 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 in vitro delivery method, which meets the needs of in vitro gene function research.

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

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

(1) Taking DSPC, DSPE-PEG2000 and DOTAP, dissolving in an organic solvent to prepare a solution, carrying out ultrasonic treatment on the solution, and filling the solution into a bottle;

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

(3) And (3) replacing air in the bottle after the buffer solution is added in the step (2) with perfluoropropane, and vibrating the solution in the bottle to obtain the cationic lipid microbubbles, and preserving at 4 ℃.

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

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

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

Further, the solution prepared in the step (1) is packaged in 4 vials, the 4 vials are placed in a rotary evaporator in the step (2), the organic solvent is completely volatilized by rotary evaporation for 1h at 65 ℃, and 800 μl of PBS buffer solution 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 the PBS buffer was 0.2%.

Further, the solution in the step (3) is in Vialmix ^TM And (5) vigorously shaking for 45s in a silver mercury blender to obtain cationic lipid microbubble suspension.

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

(a) The day before gene transfection, 1X 10 ⁵ Inoculating MDA-MB-231 cells/hole into DMEM culture medium containing 10% fetal bovine serum, placing into a 24-hole plate for incubation for 24 hours, and changing the culture medium to 300 μl serum-free culture medium OPTI-MEM when the cell confluence reaches 80%;

(b) Incubating 10 mu l of cationic lipid microvesicles with 10ug of plasmid pEGFP or 300mM miR-34a micrometers or 300mM miR-34a expressing green fluorescent protein for 10min to obtain a cationic lipid microvesicle-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 on an ultrasound probe;

(d) Filling sterilized distilled water in a gap between the bottom of the 24 pore plate and the ultrasonic probe, setting ultrasonic intensity, duty ratio and irradiation time parameters, starting a Sonovitro instrument, and carrying out ultrasonic irradiation transfection;

(e) After transfection, the cells were placed in CO ₂ The cells were cultured in a cell incubator at a concentration of 5% and a temperature of 37℃for 10min, and then replaced with fresh DMEM medium containing 10% fetal bovine serum for 24h.

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

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

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

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 cavitation, strong standing waves and microjet are generated, and nanoscale pores are formed on the surface of the cell, so that the permeability of the cell membrane is improved, and the gene transfer into the cell is facilitated. The cell membrane immediately appears small holes after ultrasonic micro-bubble treatment, and is closed after about 30min, so that the cell morphology is recovered to be normal. Of course, the ultrasound parameters of ultrasound intensity (acoustic intensity, AI), duty Cycle (DC) and irradiation time (ET) are optimized. Especially when AI exceeds 1.0W/cm ² When the cells are subjected to obvious irreversible damage, the cell survival rate is obviously reduced.

The efficiency of the UTMD system was evaluated by transfecting the pEGFP plasmid DNA into MDA-MB-231 cells based on a systematic study of the different factors. According to the ultrasonic pico-bubble 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 efficiency reaches more than 40%.

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

In summary, the invention provides a method for delivering genes by ultrasonic microbubbles, which adopts a cationic lipid microbubble and ultrasonic method to transfect pEGFP and miR-34a micrometers. Wherein, miR-34 amides 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 of an ultrasonic microbubble transfection system installation, wherein the Sonovitro device consists 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; the lower surface of the culture plate is filled with sterilized distilled water;

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

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

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

FIG. 5 is an experimental graph of the structural changes of cell membranes at different times after the action of ultrasonic microbubbles on the cells. Wherein, diagram a: experimental plots of control group; graph B: experimental plot 5min after pure ultrasound action; graph C: experimental plot 0min after UTMD action; graph D: experimental plot 5min after UTMD action; diagram E: experimental plot 15min after UTMD action; drawing F: experimental plot 30min after UTMD action;

FIG. 6 is a graph of experiments showing the effect of different acoustic UTMD treatments on cell membranes.

FIG. 7 is a diagram showing the results of experiments after transfection of pEGFP plasmid DNA. FIG. A is a diagram of experimental observation results under a fluorescence microscope; FIG. B is a flow chart of quantitative results.

FIG. 8 is a diagram of miR-34a micrometers localization analysis and quantification experiments. FIG. A is a diagram of experiments with cy 5-labeled miR-34 amides taken up by cells 24h after treatment in different experimental groups; panel B is an RT-qPCR quantitative experimental plot of miR-34 a.

FIG. 9 is a diagram showing MDA-MB-231 cell proliferation and apoptosis analysis. Wherein, the diagram A is an experimental diagram of the inhibition of miR-34 amides on MDA-MB-231 cell proliferation; FIG. B is an experimental diagram of flow cytometry detection of miR-34a micrometers-induced apoptosis; panel C is an experimental plot of the percentage of AnnexinV positive cells to total cells.

FIG. 10 is a graph showing analytical experiments of Notch1 and hes1 expression in MDA-MB-231 cells. After UTMD transfects microRNA-34a micrometers for 48 hours, western blot detects the expression of Notch1 and hes1 proteins.

Detailed Description

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

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

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

(1) Accurately weighing 9mg DSPC,2mg DSPE-PEG2000 and 1mg DOTAP, dissolving in 1ml chloroform to prepare a solution, placing the solution in an ultrasonic water bath kettle, carrying out ultrasonic treatment for 30s, and then separating the solution into 4 small bottles of 250 μl each;

(2) Placing the 4 small bottle solutions prepared in the step (1) in a rotary evaporator, and performing 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; solution in Vialmix ^TM Violently oscillating for 45s in a silver mercury mixer to obtain cationic lipid microbubble suspension;

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

example two ultrasound parameter optimization during cell transfection

In order to clearly determine the influence of ultrasonic irradiation time, ultrasonic intensity and duty cycle on cell activity in the transfection process, MDA-MB-231 cell transfection experiments are carried out by adopting different ultrasonic irradiation time, ultrasonic intensity and duty cycle, and CCK-8 experiments are adopted to detect cell activity, and the parameters of ultrasonic irradiation time, ultrasonic intensity and duty cycle on each item are shown in the following tables 1-3:

TABLE 1

Ultrasonic irradiation time	Blank space	20s	40s	60s
					Cell Activity	100％	95％	93％	92％

TABLE 2

Sound intensity

Blank space

CMB

0.6

0.8

1.0

1.2

Cell Activity

100％

95％

94％

92％

89％

80％

TABLE 3 Table 3

Duty cycle

Blank space

15％

20％

25％

30％

35％

Cell Activity

100％

98％

93％

92％

90％

The parameters that have minimal effect on cell viability are seen as follows: ultrasonic irradiation for 20s, wherein the cell activity is strongest; sound intensity 0.6W/cm ² The 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 use the parameters that have the least influence on the cell viability.

Example three, apoptosis detection

To determine whether ultrasound-conjugated cationic lipid microbubble transfected cells had an effect on apoptosis, the experiment was divided into 4 experimental groups, each:

untreated (Untreated), MDA-MB-231 cells were not subjected to any treatment;

ultrasonic treatment group (US), 0.6W/cm ² AI. Sonicating MDA-MB-231 cells under 20% DC and 20s ET conditions;

cationic lipid microvesicle treatment group (Cationic microbubbles, CMBs): the cationic lipid microbubbles prepared by the invention are mixed with MDA-MB-231 cells for transfection;

ultrasound+cationic lipid microbubble treatment group (us+cmbs): cationic lipid microbubbles prepared by the invention, 0.6W/cm ² AI. Sonicating MDA-MB-231 cells under 20% DC and 20s ET conditions;

apoptosis in each of the above groups was assessed by Annexin-V/PI staining and flow cytometry. As shown in fig. 4, the differences were not statistically significant, so that the sonication+cationic lipid microbubble group (us+cmbs) had no significant effect on apoptosis compared to the Untreated group (un-treated), the sonication group (US) and the cationic lipid microbubble group (CMBs).

Fourth example, ultrasonic microbubble Acoustic pore Effect analysis

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

blank control group: MDA-MB-231 cells were not subjected to any treatment;

ultrasonic group: 0.6W/cm ² AI. Sonicating MDA-MB-231 cells under 20% DC and 20s ET conditions;

ultrasound + microbubble group: cationic lipid microbubbles prepared by the invention, 0.6W/cm ² AI. Sonicating MDA-MB-231 cells under 20% DC and 20s ET conditions, i.e., UTMD treatment;

and observing the changes of 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 is a state of the control group; b is a state 5min after the ultrasonic action of the ultrasonic group, C is a state 0min after the ultrasonic and microbubble group UTMD action; d is a state 5min after the action of the ultrasonic and microbubble group UTMD, E is a state 15min after the action of the ultrasonic and microbubble group UTMD, and F is a state 30min after the action of the ultrasonic and microbubble group UTMD, as can be seen from the figure: compared with the control group and the ultrasonic group, the ultrasonic+microbubble group can obviously observe that a large number of small holes appear on the cell membrane, especially the number of small holes on the cell membrane is the largest within 5min after UTMD action, and the small holes on the cells gradually decrease after time exceeds 15 min. The cation lipid microbubble has a certain sound hole effect in combination with ultrasound, can perforate a cell membrane, improves the permeability of the cell membrane, and is beneficial to the gene entering the cell. With increasing sound intensity, the number of small holes on the cell membrane increases, as shown in fig. 6, which is an experimental diagram of different experimental groups after 5min of ultrasonic action, wherein a: a control group; b: an ultrasound group; c:0.4W/cm < 2+ > 30 μl microbubbles; d:0.6W/cm < 2+ > 30 μl microbubbles; e:0.8W/cm < 2+ > 30 μl microbubbles; f:1.0W/cm2+ 30. Mu.l microbubbles. The dimensions shown in the figures 5 and 6 were 5. Mu.m

Example five, plasmid DNA transfection

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

(1) 1X 10 before transfection ⁵ Inoculating MDA-MB-231 cells/well into DMEM medium containing 10% fetal bovine serum, placing into a 24-well plate, incubating for 24h, and changing the medium to 300 μl serum-free medium (OPTI-MEM) when the cell confluence reaches about 80%;

(2) 10. Mu.l of cationic lipid microbubbles were incubated with 10. Mu.g of pEGFP plasmid DNA for 10min to give cationic lipid microbubble-plasmid mixtures;

(3) Adding 10 μg of the cationic lipid microbubble-plasmid mixture of the step (2) into the MDA-MB-231 cells incubated in the step (1), supplementing the culture medium to a final volume of 500 μl, gently shaking the 24-well plate, and placing on an ultrasonic probe of a Sonovitro instrument;

(4) Filling sterilized distilled water in a gap between the bottom of the 24 pore plate and the ultrasonic probe, setting ultrasonic intensity, duty cycle and irradiation time parameters according to ultrasonic parameters obtained by optimizing in the second embodiment, starting a Sonovitro instrument, and carrying out ultrasonic irradiation transfection;

as shown in fig. 1, the 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 at the center of the probe. The bottom of the 24-well plate 4 is filled with sterilized distilled water.

(5) After transfection, the cells were placed in CO ₂ Culturing in an incubator with concentration of 5% and temperature of 37 ℃; after 10min, the solution is changed to 500 mu l of DMEM culture medium containing 10% of fetal calf serum, and the culture is continued in an incubator for 24h; transfection efficiency was observed under a fluorescence microscope. Microscopic observation shows that under the condition, MDA-MB-231 cells are transfected, the number of cells expressing green fluorescent protein is significantly increased, and the quantitative result of flow cytometry shows that the transfection efficiency can reach 46.8%.

Example six ultrasound+cationic lipid microvesicles deliver miR-34a micrometers

In order to study whether the method can efficiently transfect miR-34a micrometers into MDA-MB-231 cells, the experiment synthesizes cy5 marked miR-34 micrometers, and the experiment is divided into 4 experiment groups:

untreated group: MDA-MB-231 cells were not subjected to any treatment;

ultrasonic group: the MDA-MB-231 cells are treated by the miR-34a micrometers marked by ultrasonic +cy5, and the operation procedure of the five pEGFP plasmid DNA transfection in the embodiment is referred to, except that the miR-34 micrometers marked by the cy5 only of the MDA-MB-231 cells is added in the operation step in the step (3);

group of microbubbles: treating MDA-MB-231 cells by using miR-34a micrometers marked by cationic lipid microbubbles and cy 5; reference was made 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-34 amics was added to MDA-MB-231 cells, without sonication.

Ultrasound + microbubble group: MDA-MB-231 cells were treated with ultrasound + cationic lipid microbubbles + cy 5-labeled miR-34a micrometers, and the transfection method was referred to the procedure for five pEGFP plasmid DNA transfection in the above example. In contrast, a mixture of cationic lipid microbubbles and 300mM cy 5-labeled miR-34 amides was added to MDA-MB-231 cells for sonication. Experiments show that, as shown in A of FIG. 8, cy 5-labeled miR-34 amics can enter cells with the help of ultrasound and cationic lipid microbubbles, and miR-34a amics cannot enter cells by simply relying on ultrasound or cationic lipid microbubbles. Further quantitative detection of miR-34a using RT-qPCR, as shown in FIG. 8B, UTMD group miR-34a expression is increased by about 2-fold over other groups.

Example seven Effect of miR-34a micrometers on MDA-MB-231 cells

To verify the function of miR-34 amides transfected by the method (UTMD), CCK-8 was used to detect cell proliferation. MDA-MB-231 cell proliferation was significantly inhibited 3 days after UTMD transfection compared to the miR-NC group. The apoptosis induced by miR-34a is detected by flow cytometry, and the result is shown in figure 9, and compared with miR-NC group, the apoptosis is obviously promoted by miR-34a micrometers transfected by UTMD, and the apoptosis rate is about 25%. Finally, the expression of the target protein Notch1 of miR-34a and the target protein hes1 thereof is detected by using a western blot, and as shown in FIG. 10, the expression of Notch1 and hes1 is reduced by miR-34 amides.

Among the above, pEGFP plasmid DNA or miR-34 amics was introduced into MDA-MB-231 cells, and the related factors were studied. It can be known that the UTMD of the invention can efficiently transfect pEGFP and miR-34a micrometers. Furthermore, in MDA-MB-231 cells, the function of miR-34a transmitted by UTMD was also studied. The results show that the method has potential for further in vivo gene therapy research.

The foregoing has described in detail the technical solutions provided by the embodiments of the present invention, and specific examples have been applied to illustrate the principles and implementations of the embodiments of the present invention, where the above description of the embodiments is only suitable for helping to understand the principles of the embodiments of the present invention; meanwhile, as for those skilled in the art, according to the embodiments of the present invention, there are variations in the specific embodiments and the application scope, and the present description should not be construed as limiting the present invention.

Claims

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

(d) The space between the bottom of the 24-well plate and the ultrasonic probe is filled with sterilized distilled water, and the ultrasonic intensity is set to be 0.6W/cm ² The duty ratio is 20%, the irradiation time is 20s, and a Sonovitro instrument is started to carry out ultrasonic irradiation transfection;

(e) After transfection, the cells were placed in CO ₂ The concentration is 5 percent,culturing in a cell incubator at 37deg.C for 10min, and culturing for 24 hr with fresh DMEM medium containing 10% foetus calf serum;

the preparation of the cationic lipid microbubbles comprises the following steps:

(1) Dissolving DSPC, DSPE-PEG2000 and DOTAP in chloroform to obtain solution, ultrasonic treating for 30s, and bottling; the ratio of the dosage of DSPC, DSPE-PEG2000 and DOTAP, and the dosage of the organic solvent is 9mg:2mg:1mg:1mL;

(2) Removing the organic solvent of the solution prepared in the step (1), and then adding a buffer solution into the bottle; the buffer solution is PBS buffer solution containing glycerol, and the ratio of the mass of the glycerol to the volume of the PBS buffer solution is 0.2%;

2. A method of cationic lipid microbubble mediated gene delivery according to claim 1,

the cationic lipid microsphere preparation step (1) further comprises the step of separating the prepared solution into 4 small bottles, the step (2) further comprises the step of placing the 4 small bottles in a rotary evaporator, and performing rotary evaporation for 1h at 65 ℃ to completely volatilize the organic solvent, wherein 800 mu l of PBS buffer solution containing glycerol is added into each small bottle.

3. A method of cationic lipid microbubble mediated gene delivery according to claim 1,

the detailed process of oscillating the solution in the bottle in the step (3) comprises the following steps: solution in Vialmix ^TM And (5) vigorously shaking for 45s in a silver mercury blender to obtain cationic lipid microbubble suspension.