CN102813942A - Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof - Google Patents
Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof Download PDFInfo
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- CN102813942A CN102813942A CN2012102902519A CN201210290251A CN102813942A CN 102813942 A CN102813942 A CN 102813942A CN 2012102902519 A CN2012102902519 A CN 2012102902519A CN 201210290251 A CN201210290251 A CN 201210290251A CN 102813942 A CN102813942 A CN 102813942A
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Abstract
The invention relates to an adeno-associated virus based and lipid ultrasound microbubble mediated novel preparation capable of obviously improving gene transfection efficiency on cells with compact structure. A structure of the novel preparation is shown as a figure 1, and consists of three parts: an adeno-associated virus, perfluoropropane, and a lipid bilayer. According to the invention, the advantage of efficient gene transfection of the adeno-associated virus vector is utilized; under the effect of ultrasound, the ''cavitation effect'' generated by the ultrasound microbubbles acts on cell membranes of the cells with compact structure to generate ''overflow pores'' and increase permeability of the cell membranes, so that adeno-associated viruses released after destruction of the microbubbles can enter blood vessel walls and even tissue spaces, thereby promoting transmembrane gene transfection. The novel preparation provided by the invention is simple for preparation and convenient for usage, and does not require special production conditions.
Description
One, technical field
Patent of the present invention relate to a kind of based on adeno-associated virus by the new formulation of the microbubble mediated remarkable enhancing of lipid ultrasonic to the gene transfer efficient of tool compact texture cell, belong to the biological medicine technology field.
Two, background technology
At present, in the research of gene therapy, gene delivery system is the focus that people pay close attention to safely and effectively.Usually, according to the difference of carrier, can gene delivery system be divided into virus carrier system and non-virus carrier system.Non-virus carrier such as liposome, nanoparticle, microcapsule, microsphere etc. have easy to use, can be mass-produced and advantage such as non-immunogenicity, but its shortcoming are that body planted agent time spent transfection efficiency is lower.In the viral vector, use to such an extent that be gland relevant viral vector comparatively widely.Researcher finds that (Adeno-associated virus AAV) stablizes various physics and chemistry processing adeno-associated virus, is easy to separation and purification; Host range is wide, can infect Unseparated Cell; No pathogenicity, and in host, can not cause immunoreation; Can fixed point integration of foreign gene is long-armed to human No. 19 chromosomes, gene expression is stable.Gland relevant viral vector has been applied to the clinical treatment cystic fibrosis.But as carrier, though gene transfection efficient is higher relatively, its safety and immunogenicity are the problems that needs people to consider with virus.This shows that viral vector and non-virus carrier respectively have its merits and demerits,, will be expected to impel separately maximize favourable factors and minimize unfavourable ones, respectively execute its ability, each most chief if can both be combined through certain mode.
In recent years, along with the appearance of targeting acoustic contrast, edify people and set up a kind of novel therapeutic genes targeting transmission system by means of ultransonic directional transmissions non-invasive and collaborative microvesicle with orthoselection effect.Research shows; Carry the activity that gene can keep these therapeutic substances on the one hand with microbubble contrast agent as carrier; Avoid they from blood circulation by clear, especially when gene delivery, because the endogenous endonuclease liver relevant with electric charge known system; Make naked DNA and other carriers at endovascular poor stability, very soon by metabolism; On the other hand; For the cell that forms compact texture (closely connecting) and tissue like airway epithelial cell; Adeno-associated virus is unable to provide ideal transduction efficiency; We by means of ultransonic non-invasive with utilize ultrasonic taking to set up a kind of novel adeno-associated virus gene delivery systems, i.e. ultrasonic microbubble gene delivery system with the directional transmissions of microvesicle.Wherein, Under the ultrasound wave effect, " cavitation effect " that ultrasonic microbubble produces acts on cell membrane, produces " outflow hole "; Permeability of cell membrane is increased, thus make the gene that discharges after the microbubble destruction can the intravasation wall in addition interstice promote gene to stride the film transfection.
Three, goal of the invention
The objective of the invention is to make full use of the advantage of gland relevant viral vector high efficiency transducible gene; Avoid its immunogenicity and toxic generation; Prepare a kind of lipid ultrasonic microbubble mediated " cavitation effect " that pass through the ultrasonic microbubble generation that utilizes and act on the adeno-associated virus transfecting formulations of the cell membrane of fine and close cell, thereby significantly strengthen gene transfection efficient tool compact texture cell.
Four, summary of the invention
The present invention relates to a kind of based on adeno-associated virus, by the new formulation of the microbubble mediated remarkable enhancing of lipid ultrasonic to the gene transfer efficient of tool compact texture cell.This new formulation structure is formed as shown in Figure 1, comprises three parts: adeno-associated virus, perfluoropropane, double-layer of lipoid.The present invention utilizes the advantage of the efficient transducible gene of gland relevant viral vector; Under the ultrasound wave effect; Act on the cell membrane of tool compact texture cell through " cavitation effect " of ultrasonic microbubble generation; Produce " outflow hole ", permeability of cell membrane increases, thus make the adeno-associated virus that discharges after the microbubble destruction can the intravasation wall in addition interstice promote gene to stride the film transfection.
The specific embodiment is following:
Embodiment 1: the preparation of carrying adeno-associated virus lipid ultrasonic microvesicle
Precision is measured a certain proportion of AAV, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE; Cholesterol derivative CH (CHEMS) and glycerol are dissolved in PBS, in 20 ℃ of water, mix 10 min, pour perfluoropropane gas; Ultrasonic with the 100W ultrasonic cleaner in room-temperature water bath; Every ultrasonic 20s is 10s at interval, and totally 10 circulations promptly make the lipid ultrasonic microvesicle that carries AAV.
Embodiment 2: the morphologic observation of medicine carrying lipid microbubble, particle size distribution detect
Place 4 ℃ of refrigerators to preserve freshly prepd lipid ultrasonic microvesicle sealing, respectively at 1h, 24h, 48h and 2 months points take out, and are placed on the suitable dilution of PBS do and are inverted optical microscope its mode of appearance of observation down.For investigating the concentration of lipid ultrasonic microvesicle, diluent is got 1 place on the blood counting chamber, the microvesicle number of 8 big lattice of counting average then (surveying at least 3 times).And utilize Malvern Sizer Nano ZS90 particle size analyzer to analyze microvesicle particle diameter and distribution according to operating instruction.The inversion observation by light microscope shows that the lipid ultrasonic microvesicle of this Experiment Preparation is spheroidal, and size evenly.Place 1h, observed result consistent (Fig. 2) under the lipid microbubble mirror behind 24h and the 48h; Place that lipid microbubble after February is a large amount of assembles agglomerating (Fig. 3), explain that thus the lipide supersonic microvesicle of this Experiment Preparation is stable in 48h at least, but can not long-term storage.Detecting microbubble concentration with blood cell numeration appearance is 1.89 ± 0.5 * 10
9Individual/ml.Marlven particle size analyzer testing result shows (Fig. 4), and the lipid ultrasonic microvesicle mean diameter of this Experiment Preparation is 2.46 ± 1.2 μ m, and particle size distribution is normal distribution, and its result is consistent with the result of observation by light microscope.
Embodiment 3: cell culture
Human bronchial epithelial cell 16HBE14o-grows in and contains 10% hyclone, in DMEM/F12 (1:1) culture medium of 100u/ml penicillin, 100 μ g/ml streptomycins, at 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.Behind the frozen 16HBE14o-cell recovery in the aseptic Tissue Culture Dish of 150mm normal cultured.
The Transwell cell (transparent polyester film, 0.4 μ m) of individual packaging is placed the aseptic Tissue Culture Plate in 12-hole.Get and be in the good 16HBE14o-cell of exponential phase growth conditions,, be made into the individual cells suspension with DMEM/F12 (1:1) culture medium that contains 10% hyclone, with every hole 0.5ml, 3.5 * 10 with 0.25% trypsinization single-layer culturing cell, 2 min
6Individual cell inoculation is in the Transwell inner room, and the mistress adds 1.5 ml complete mediums.Culture plate is moved into CO
2In the incubator, at 37 ℃, 5% CO
2And cultivate under the saturated humidity condition, make the cell attachment growth, and change liquid every other day.
Embodiment 4: the mensuration of cell transmembrane resistance (TEER)
Can stride the detection of membrane resistance behind the cell attachment.At first regulate and stride membrane resistance mensuration system,, immerse serum-free medium balance 15 min again with 70% soak with ethanol electrode, 15 min.Discard original culture medium of Transwell mistress, preheating DMEM/F12 (1:1) culture medium to 37 ℃, the inner room of Transwell adds 0.5 ml mistress and adds 1.5 ml and balance 30 min, inserts electrode, measures and strides membrane resistance; Three readings are averaged, and deduct the blank culture hole reading that does not have cell, are the membrane resistance of striding of tested cell.The result shows: striding increases slowlyer in 1 week of membrane resistance pro-, at this moment cell division is described and is not reached fusion as yet; Cell begins to increase rapidly at the 7th day resistance after beginning to merge; Reached 225 Ω cm by the 12nd day
2, then slowly increase; Basically maintained a definite value 130 Ω cm in two days subsequently
2About, explain that this moment, cell reached good fusion state, can be used for subsequent experimental.Each is organized cell and handles according to design grouping administration: blank control group (A), and AAV organizes (B) separately, and AAV takes with supersound process group (C), AAV microvesicle group (D), the AAV microvesicle is taken with supersound process group (E); Then, detect cell transmembrane resistance once more.The result shows, the cell average transmembrane resistance that A, B and D are three groups does not change; C group cell average transmembrane resistance is handled the back in administration and is reduced by 50 Ω * cm
2The average transmembrane resistance of E group reduces by 120 Ω * cm
2Explain that thus supersound process can be at the compactness extent that to a certain degree reduces cell (like C group), to produce the influence that " cavitation effect " caused with supersound process big but the influence degree of single ultrasonic pair cell is taken not as good as the AAV microvesicle far away.
Embodiment 5: the cells in vitro Transduction Study
Two weeks of cell inoculation are divided into five groups (n=5) with the cell in the Transwell cell, remove stock solution, and with PBS washed cell 3 times, according to experimental design, pair cell is bestowed following disposal respectively: (1) blank; (2) AAV; (3) AAV+ is ultrasonic; (4) AAV microvesicle; (5) AAV microvesicle+ultrasonic.Bestow ultransonic culture hole for needs, with single another the aseptic 12-hole aseptic culture plate that moves into successively of corresponding Transwell cell, the Ultrasound Instrument probe stretches into liquid level 0.4-0.6cm, the ultrasonic 10s of 50w, interval 10s, two circulations.Continue to cultivate 8h then, remove liquid, change DMEM/F12 (1:1) complete medium into; Behind the 48h, stop to cultivate.
With each group cell, place and observe luciferase expression under the inverted fluorescence microscope and gather picture.The cell of respectively organizing of administration being handled back 56h places qualitative observation under the inverted fluorescence microscope; The result shows (Fig. 5): blank control group (A) does not have fluorescence to show; AAV organizes (B) separately and shows more weak fluorescence with AAV microvesicle group (D); AAV takes with supersound process group (C) and shows the fluorescence to a certain degree strengthened, and the AAV microvesicle takes with supersound process group (E) and show obvious enhanced fluorescence.
Be further detection by quantitative transfection efficiency, respectively organize cell with the digestion of 2.0% trypsin solution, PBS washing and re-suspended cell are processed 16HBE14o-individual cells suspension, detect with flow cytometer immediately and respectively organize fluorescence intensity.The detection by quantitative result of flow cytometer (Fig. 6) shows, compares with blank control group A, and all the other each groups comprise: the B group (
★p<0.05), the C group (
★ ★p<0.01), the D group (
★p<0.05) and the E group (
★ ★ ★P<0.001) all having significant difference, this is consistent with above-mentioned fluorescence qualitative observation result.
According to each variation of group cell average transmembrane resistance before and after administration is handled,, explain that the difference of cell compactness extent directly influences its transduction efficiency in conjunction with respectively organizing cell transduction efficient.The AAV lipid microbubble that carries that this experiment is adopted is taken the transduction efficiency to the compact texture cell that can significantly strengthen the AAV mediation with the transduction mode of supersound process.
Five, beneficial effect
The present invention utilizes the advantage of the efficient transducible gene of gland relevant viral vector; Under the ultrasound wave effect; " cavitation effect " that produces through ultrasonic microbubble acts on the cell membrane of the cell of tool compact texture, produces " outflow hole ", and permeability of cell membrane increases; Thereby make the adeno-associated virus that discharges after the microbubble destruction can the intravasation wall in addition interstice promote gene to stride the film transfection, this invention will provide effective way for the gene therapy of compact texture tissue.
Explanation to accompanying drawing:
Fig. 1 target new formulation structure composition diagram
The mode of appearance of Fig. 2 lipid ultrasonic microvesicle (depositing 48 hours bar=5 μ m for 4 ℃)
The mode of appearance of Fig. 3 lipid ultrasonic microvesicle (depositing 2 months bar=5 μ m for 4 ℃)
Fig. 4 Malvern laser particle analyzer detects the particle diameter and the distribution of lipid ultrasonic microvesicle
Fig. 5 fluorescence inverted microscope qualitative observation is respectively organized cell transduction efficient (200 *).Be divided into five groups: blank control group (A:Blank); AAV organizes (B:AAV alone) separately; AAV+ ultrasonic (C:AAV+Ultrasonic exposure); AAV microvesicle (D:AAV/Microbubble); AAV microvesicle+ultrasonic (E:AAV/Microbubble+Ultrasonic exposure)
Fig. 6 flow cytometer detection by quantitative is respectively organized cell transduction efficient.Totally five groups: blank control group (A:Blank); AAV organizes (B:AAV alone) separately; AAV+ ultrasonic (C:AAV+Ultrasonic exposure); AAV microvesicle (D:AAV/Microbubble); AAV microvesicle+ultrasonic (E:AAV/Microbubble+Ultrasonic exposure).
Claims (4)
1. one kind based on adeno-associated virus, and by the new formulation of the microbubble mediated remarkable enhancing of lipid ultrasonic to the gene transfer efficient of tool compact texture cell, its structure is formed and comprised three parts: adeno-associated virus, perfluoropropane, double-layer of lipoid.
2. the method for preparing of new formulation according to claim 1, it is characterized in that: precision is measured a certain proportion of adeno-associated virus, phosphatidylcholine; PHOSPHATIDYL ETHANOLAMINE, cholesterol derivative CH (CHEMS) and glycerol are dissolved in PBS, in 20 ℃ of water, mix 10 min; Pour perfluoropropane gas, ultrasonic with the 100W ultrasonic cleaner in room-temperature water bath, every ultrasonic 20s is 10s at interval; Totally 10 circulations promptly make the lipid ultrasonic microvesicle that carries adeno-associated virus.
3. in the element of new formulation according to claim 1, lipid components is a phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, one or more in the cholesterol derivative CH (CHEMS).
4. the method for preparing of new formulation according to claim 2, it is characterized in that: the ratio of described adeno-associated virus is 1:1～1:1000.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652456A (en) * | 2019-01-22 | 2019-04-19 | 北京大学深圳医院 | Receptor transfection microvesicle, ligand transfection microvesicle and two sections of conduction cell transfecting methods processed for cell transfecting |
| CN111529718A (en) * | 2020-04-01 | 2020-08-14 | 四川大学华西医院 | Cationic microbubble-rAAV-miRNA virus compound and preparation method and application thereof |
| CN112955185A (en) * | 2018-06-22 | 2021-06-11 | 医福斯治疗有限公司 | Combination gene therapy |
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| CN1177342A (en) * | 1995-02-21 | 1998-03-25 | ImaRx药物公司 | Novel cationic lipids and the use thereof |
| CN1216925A (en) * | 1996-04-30 | 1999-05-19 | ImaRx药物公司 | Novel Dispersible lipid blends and uses therefor |
| EP1046394A2 (en) * | 1999-04-19 | 2000-10-25 | ImaRx Pharmaceutical Corp. | Novel compositions useful for delivering compounds into a cell |
| US20020102217A1 (en) * | 1996-10-28 | 2002-08-01 | Nycomed Imaging As | Diagnostic/therapeutic agents |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177342A (en) * | 1995-02-21 | 1998-03-25 | ImaRx药物公司 | Novel cationic lipids and the use thereof |
| CN1216925A (en) * | 1996-04-30 | 1999-05-19 | ImaRx药物公司 | Novel Dispersible lipid blends and uses therefor |
| US20020102217A1 (en) * | 1996-10-28 | 2002-08-01 | Nycomed Imaging As | Diagnostic/therapeutic agents |
| EP1046394A2 (en) * | 1999-04-19 | 2000-10-25 | ImaRx Pharmaceutical Corp. | Novel compositions useful for delivering compounds into a cell |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112955185A (en) * | 2018-06-22 | 2021-06-11 | 医福斯治疗有限公司 | Combination gene therapy |
| CN109652456A (en) * | 2019-01-22 | 2019-04-19 | 北京大学深圳医院 | Receptor transfection microvesicle, ligand transfection microvesicle and two sections of conduction cell transfecting methods processed for cell transfecting |
| CN111529718A (en) * | 2020-04-01 | 2020-08-14 | 四川大学华西医院 | Cationic microbubble-rAAV-miRNA virus compound and preparation method and application thereof |
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