CN103071161A - Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector - Google Patents
Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector Download PDFInfo
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Abstract
The invention discloses a preparation method of a gene drug delivery vector with polycationic liposome coating calcium phosphate nanoparticles. The calcium phosphate nanoparticles are prepared with an improved coprecipitation method or a reverse microemulsion method, and the stability of the calcium phosphate nanoparticles is improved; and an amphiphilic compound polyethyleneimine-cholesterol, phosphatide and/or cholesterin are/is taken as liposome membrane material(s), and incubation is performed by adopting both the lipidosome and the calcium phosphate nanoparticles, so that the gene drug delivery vector is obtained. According to the preparation method, the stability of the calcium phosphate nanoparticles is improved through improvements of a calcium phosphate nanoparticle prescription and a preparation process; the trend that the calcium phosphate nanoparticles can gather and precipitate easily is further overcome by a lipidosome coating technology; and polycations on the surface of the lipidosome increase the uptake of a cell for the vector and improve the endosomal escape capability. The prepared compound gene drug delivery vector improves the cellular uptake and transfection efficiencies on the basis of safely and economically reservation of the calcium phosphate vector, and has a broad application prospect.
Description
(1) technical field
The present invention relates to a kind of preparation field of gene drug carriers, relate in particular to the method for the drug administration carrier of gene of polycation lipesome parcel calcium phosphate nano grain preparation, i.e. the preparation method of polycation lipesome/calcium phosphate nano grain drug administration carrier.
(2) background technology
Gene therapy provides good prospect for the treatment of hereditary, infectious disease and cancer etc., and gene delivery system will become one of greatest factor of gene therapy success and develop safely and effectively.As the non-virus carrier of study hotspot, preparation is simple, carry in the unrestricted and body of gene size and use without specific immune response, has a good application prospect.Wherein cationic-liposome and cationic polymer are to study the most widely two kinds of non-virus carriers, because they can form complex by electrostatic interaction and gene, make it the easier cell that enters, and protect it to avoid the degraded of nuclease.Liposome-mediated gene enters the existing about 20 years history of research of cell, because cationic-liposome has higher transfection efficiency, it uses more general in the research of gene therapy.But bibliographical information is also arranged, and when improving the cationic-liposome transfection activity, its surperficial positive charge causes corresponding cytotoxicity, has limited its application in gene therapy.
Calcium phosphate is as the carrier of gene transfection, and because its technique is simple, bio-compatibility and safety are high, still are widely used at present outer-gene transfection research.But the same with most of non-virus carrier-gene composite, calcium phosphate mainly combines with gene by electrostatic interaction, the aggregation of particles precipitation appears easily, less stable, thereby cause cellular uptake to reduce, use separately calcium phosphate as genophore, its transfection efficiency will be lower than liposome vectors usually.
(3) summary of the invention
The preparation method that the purpose of this invention is to provide a kind of drug administration carrier of gene of polycation lipesome parcel calcium phosphate nano grain, adopt improved coprecipitation or reverse microemulsion to prepare the calcium phosphate nano grain, improve the stability of calcium phosphate nano grain,, adopt liposome and calcium phosphate nano grain jointly to hatch and obtain described drug administration carrier of gene as the liposome membrane material with amphiphilic compound polymine-cholesterol, phospholipid and/or cholesterol.
The technical solution used in the present invention is:
The invention provides the preparation method of two kinds of polycation lipesomes/calcium phosphate nano grain drug administration carrier:
The preparation method of the first polycation lipesome/calcium phosphate nano grain drug administration carrier: described method is: the preparation of (1) calcium phosphate nano grain solution: surfactant A aqueous solution, gene aqueous solution and calcium chloride water are mixed and made into mixed liquor A 1, then surfactant B aqueous solution, aqueous phosphatic and sodium citrate aqueous solution are mixed and made into mixed liquor A 2, under the condition that stirs, mixed liquor A 2 is dropwise splashed in the mixed liquor A 1 at last, after dropwising, stirring at room 0.5 ~ 2h, obtain mixed liquor, i.e. calcium phosphate nano grain solution; In the described calcium chloride water in calcium ion and the aqueous phosphatic ratio of the amount of substance of phosphate anion be 1:0.001～1, in the calcium chloride water in calcium ion and the gene aqueous solution mass ratio of gene be 1～100:1, the concentration of described calcium chloride water is 5 ~ 500 mmol/L, the gross mass consumption of surfactant A and surfactant B is 0.01～2% of final calcium phosphate nano grain liquor capacity, and the gross mass consumption of sodium citrate is 0.001%～1% of final calcium phosphate nano grain liquor capacity in the sodium citrate aqueous solution; Described surfactant A is the mixing of one or more arbitrary proportions in polyoxyethylene poly-oxygen propylene aether block copolymer (being poloxamer), sodium lauryl sulphate, polysorbate, Polyethylene Glycol or the polyethylene glycol mono stearate, and described surfactant B is identical surfactant with surfactant A; Phosphate is a kind of in sodium hydrogen phosphate, sodium dihydrogen phosphate, DAP, diphosphate or the sodium tripolyphosphate in the described aqueous phosphatic; Described gene comprises for genetic stews such as the common plasmid DNA of gene therapy, small molecules interference RNA, antisense oligonucleotides but is not limited to above-mentioned example; (2) preparation of polycation lipesome solution: with polymine-C/PL and/or cholesterol with the organic solvent A dissolving after, rotary evaporation in vacuo is removed organic solvent A to doing, obtain lipid membrane, in lipid membrane, add deionized water, water-bath supersound process 5～30min, and then after utilizing ultrasound probe to process 1～10min under ice bath, the 100～400W condition, adopt at last the filter membrane of aperture 0.1～0.8 μ m to process, get filtrate, obtain polycation lipesome solution; Polymine molecular weight in described polymine-cholesterol is 600～50000Da; Described organic solvent A is the mixture of one or more arbitrary proportions in chloroform, dichloromethane, ether, petroleum ether or the ethanol; Described phospholipid is natural phospholipid or synthetic phospholipid; The mass ratio of described polymine-C/PL is 1:0.01～100, and the mass ratio of described polymine-cholesterol and cholesterol is 1:0～10; (3) preparation of polycation lipesome/calcium phosphate nano grain drug administration carrier: the calcium phosphate nano grain solution of step (1) preparation is mixed with equal-volume after the deionized water dilution respectively with the polycation lipesome solution of step (2) preparation, behind incubated at room 5～60min, obtain described polycation lipesome/calcium phosphate nano grain drug administration carrier; The quality consumption of described polycation lipesome solution is in amount of substance amino in polymine-cholesterol molecule, the quality consumption of described calcium phosphate nano grain solution is in the amount of substance of phosphate radical in the gene molecule of original adding, the feed intake ratio of amount of substance of described amino and described phosphate radical is calculated as 0.01～1000:1, it is the ratio of polymine-cholesterol and gene in the final drug administration carrier, press nitrogen/phosphorus than (N/P, in polymine-cholesterol molecule in amino molal quantity and the gene molecule ratio of the molal quantity of phosphate radical).
Further, the ultrasound probe processing method is in the described step (2): be under the condition of 400W at power, every interval 3s utilizes ultrasound probe to carry out supersound process 2s, repeats 60 times.
Further, described step (2) phospholipid is the mixing of one or more arbitrary proportions in soybean phospholipid, lecithin, DOPE (DOPE) or 1, the 2-stearyl PHOSPHATIDYL ETHANOLAMINE.
Further, the feed intake ratio 0.1～100:1 of amount of substance of described amino and phosphate radical, it is the ratio of polymine-cholesterol and gene in the final complex carrier of described step (2), pressing nitrogen/phosphorus is 0.1～100:1 than (N/P, in polymine-cholesterol molecule in amino molal quantity and the gene molecule ratio of the molal quantity of phosphate radical).
The preparation method of the second polycation lipesome/calcium phosphate nano grain drug administration carrier is: (I) preparation of calcium phosphate nano grain solution: organic solvent B, surfactant C and cosurfactant A are mixed in proportion, make the oil phase ternary system, wherein the volume ratio of surfactant C and cosurfactant A is 1～10:1, and the cumulative volume of surfactant C and cosurfactant A and the volume ratio of organic solvent B are 0.1～10:1; Again calcium chloride water, gene aqueous solution, aqueous phosphatic and sodium citrate aqueous solution are mixed and made into mixed liquor A 3, under the condition that stirs, mixed liquor A 3 is dropwise splashed in the oil phase ternary system at last, after dropwising, stirring at room 0.5 ~ 2h, obtain mixed liquor, mixed liquor dialysed with dialyzer remove organic facies, get trapped fluid and be scattered in and be calcium phosphate nano grain solution in the deionized water; In the described calcium chloride water in calcium ion and the aqueous phosphatic ratio of the amount of substance of phosphate anion be 1:0.001～1, in the calcium chloride water in calcium ion and the gene aqueous solution mass ratio of gene be 1～100:1, the gross mass consumption of sodium citrate is 0.001%～1% of final calcium phosphate nano grain liquor capacity in the sodium citrate aqueous solution; The volume ratio of described oil phase ternary system and mixed liquor A 3 is 1:0～0.5, and the concentration of described calcium chloride water is 5 ~ 500 mmol/L; Described surfactant C is the mixing of one or more arbitrary proportions in polyoxyethylene poly-oxygen propylene aether block copolymer (being poloxamer), Triton X-100 (i.e. (Triton X-1)), sodium lauryl sulphate or the polysorbate; Described cosurfactant A is a kind of in ethanol, normal propyl alcohol, isopropyl alcohol, ethylene glycol, 1,2-PD, glycerol or the hexanol; Described organic solvent B is the microemulsion oil phases commonly used such as cyclohexane extraction, normal hexane, normal octane; Phosphate is a kind of in sodium hydrogen phosphate, sodium dihydrogen phosphate, DAP, diphosphate or the sodium tripolyphosphate in the described aqueous phosphatic; Described gene comprises for genetic stews such as the common plasmid DNA of gene therapy, small molecules interference RNA, antisense oligonucleotides but is not limited to above-mentioned example; (II) preparation of polycation lipesome solution: after polymine-C/PL and/or cholesterol usefulness organic solvent C dissolving, rotary evaporation in vacuo is removed organic solvent C to doing, obtain lipid membrane, in lipid membrane, add deionized water, water-bath supersound process 5～30min, and then after utilizing ultrasound probe to process 1～10min under ice bath, the 100～400W condition, adopt at last the filter membrane of aperture 0.1～0.8 μ m to process, get filtrate, be polycation lipesome solution; Polymine molecular weight in described polymine-cholesterol is 600～50000Da; Described organic solvent C is a kind of or mixture in the volatile organic solvents such as chloroform, dichloromethane, ethanol; Described phospholipid is natural phospholipid or synthetic phospholipid; The mass ratio of described polymine-C/PL is 1:0.01～100, and the mass ratio of described polymine-cholesterol and cholesterol is 1:0～10; (III) preparation of polycation lipesome/calcium phosphate nano grain drug administration carrier: the calcium phosphate nano grain solution of step (I) preparation is mixed with equal-volume after the deionized water dilution respectively with the polycation lipesome solution of step (II) preparation, behind incubated at room 5～60min, obtain described polycation lipesome/calcium phosphate nano grain drug administration carrier; The consumption of described polycation lipesome solution is in amount of substance amino in polymine-cholesterol molecule, the quality consumption of described calcium phosphate nano grain solution is in the amount of substance of phosphate radical in the gene molecule of original adding, described amino is 0.01～1000:1 with the ratio of phosphate radical amount of substance, it is the ratio of polymine-cholesterol and gene in the final complex carrier of described step (II), press nitrogen/phosphorus than (N/P, in polymine-cholesterol molecule in amino molal quantity and the gene molecule ratio of the molal quantity of phosphate radical).
Further, the ultrasound probe processing method is in the described step (II): be under the condition of 400W at power, every interval 3s utilizes ultrasound probe to carry out supersound process 2s, repeats 60 times.
Further, described step (II) phospholipid is the mixing of one or more arbitrary proportions in soybean phospholipid, lecithin, DOPE or 1, the 2-stearyl PHOSPHATIDYL ETHANOLAMINE.
Further, the described amino of described step (II) is 0.1～100:1 with the ratio of phosphate radical amount of substance, it is the ratio of polymine-cholesterol and gene in the final complex carrier of described step (II), pressing nitrogen/phosphorus is 0.1～100:1 than (N/P, in polymine-cholesterol molecule in amino molal quantity and the gene molecule ratio of the molal quantity of phosphate radical).
Surfactant A of the present invention, surfactant B, surfactant C is surfactant, name for ease of distinguishing the used surfactant difference of distinct methods, cosurfactant A, cosurfactant B is cosurfactant, name for ease of distinguishing the used cosurfactant difference of distinct methods, organic solvent A, organic solvent B, organic solvent C is organic solvent, name for ease of distinguishing distinct methods solvent for use difference, mixed liquor A 1, mixed liquor A 2, mixed liquor A 3 is the mixed liquor of different step preparation, name for ease of distinguishing statement, letter itself is without implication.
Poloxamer of the present invention is PLURONICS F87, chemical formula: H (C
2H
4O) a (C
3H
6O) b (C
2H
4O) aOH, this product is poly-(oxygen second is rare) a block copolymer of poly-(oxypropylene) b-of alpha-hydro-omega-hydroxypoly (oxygen second is rare) a-.By expoxy propane and propylene glycol reaction, form polyoxypropylene diols, then add oxirane and form block copolymer.Oxygen ethylene unit (a) is 75～85 in copolymer, and oxypropylene unit (b) is 25～30, oxygen ethylene (EO) content 79.9%～83.7%, and mean molecule quantity is 7680～9510.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
The present invention combines the advantage of calcium phosphate nano grain and polycation lipesome, and a kind of novel gene drug administration carrier is provided; By the improvement to calcium phosphate nano grain prescription and preparation technology, improved the stability of calcium phosphate nano grain, utilize the liposome technology further to overcome the tendency of the easy aggregate and precipitate of calcium phosphate nano grain, the polycation of surface of liposome has increased the ability that cell is escaped to picked-up and the endosome of carrier; The prepared gene drug delivery complex carrier of the present invention has improved cellular uptake and transfection efficiency keeping on calcium phosphate carrier safety, the economic basis, has broad application prospects.
(4) description of drawings
Fig. 1 Triton X-100 (Triton X-1)+hexanol/cyclohexane extraction/aqueous systems pseudoternary phase diagram (30 ℃);
The cytotoxicity of Fig. 2 polycation lipesome/calcium phosphate nano grain drug administration carrier, solid squares (
) be polycation lipesome/calcium phosphate nano grain drug administration carrier, solid circles (
) be polymine/gene mixture (molecular weight, 25kDa) contrast;
The fluorescence inverted microscope photo of Fig. 3 polycation lipesome/calcium phosphate nano grain drug administration carrier mediation siRNA transfection,
A1) and A2) Lipofectamine 2000; B1) and B2) calcium phosphate nano grain; C1) and C2) polycation lipesome/calcium phosphate nano grain drug administration carrier.Wherein 1 is the bright visual field, and 2 is the fluorescence visual field.
The fluorescence inverted microscope photo of the gene expression of Fig. 4 polycation lipesome/calcium phosphate nano grain drug administration carrier mediation;
A) polymine/gene mixture (molecular weight, 25kDa); B) polycation lipesome/calcium phosphate nano grain drug administration carrier.
(5) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
The synthetic method reference of described polymine-cholesterol (PEI-Chol): polycation lipesome telomere enzyme antiseuse oligonucleotide complex and preparation (publication number CN1936011).
Embodiment 1
(1) gets 75 mmol/L calcium chloride water 1ml, add 0.1mg/ml oligonucleotide (ODN, 5 '-CTCAGTTAGGGTTAG-3 ', the phosphate radical mole is 0.3nmol) solution 1ml, 0.4ml(4mg) 1% PLURONICS F87, hatched after the jolting gently 30 minutes, as mixed liquor A.Other gets 6 mmol/L sodium hydrogen phosphate aqueous solution 2ml, adds 25 mmol/L sodium citrate aqueous solution 0.5ml, and 0.6ml (6mg) 1% PLURONICS F87 mixes, as mixed liquid B.Mixed liquid B is dropwise joined in the mixed liquor A, and room temperature (25 ℃) stirs 30min, and obtaining calcium phosphate nano grain solution 5.5ml(ODN quality is 0.1mg).(2) get soybean phospholipid 10mg, cholesterol 5mg, polymine-cholesterol (PEI-Chol) 1mg(PEI molecular weight 800 Da, amino mole is 20nmol) dissolve with the 10ml chloroform, after rotary evaporation in vacuo volatilizes chloroform, add the 10ml deionized water, aquation 10min under the water-bath ultrasound condition, (the new sesame in Ningbo JY-92) carries out Probe Ultrasonic Searching and processes then further to utilize the ultrasonic grinding instrument under condition of ice bath, power 400W, ultrasonic 2 seconds, be 3 seconds off time, and cycle-index is 60 times, in the filter membrane of aperture 0.22 μ m, push film, obtained polycation lipesome solution (the PEI-Chol quality is 1mg).(3) according to the nitrogen/phosphorus of polymine-cholesterol and gene than (N/P, the ratio of the molal quantity of phosphate radical in amino molal quantity and the gene molecule in polymine-cholesterol molecule), get not commensurability calcium phosphate nano grain solution and polycation lipesome solution, after diluting respectively with deionized water, the room temperature condition volumetric mixes, and hatches the polycation lipesome that obtains different N/P ratio behind the 30min/calcium phosphate nano grain drug administration carrier.
The calcium phosphate nano grain solution of step (1) preparation is diluted to 10 ml with deionized water, getting successively 100 μ l(ODN quality from 10 ml is 1 μ g) as the calcium phosphate nano grain solution after the dilution, get respectively the polycation lipesome solution 1.5 μ l of step (2) preparation, 3.75 μ l, 7.5 μ l, 15 μ l, 30 μ l, 45 μ l, 60 μ l and 75 μ l also are diluted to 100 μ l, will obtain respectively N/P=1 after the polycation lipesome solution incubated at room after the calcium phosphate nano grain solution after the dilution and the dilution, 2.5,5,10,20,30,40 and 50 polycation lipesome/calcium phosphate nano grain drug administration carrier.
Embodiment 2
(1) gets 50 mmol/L calcium chloride water 1ml, add 0.1mg/ml green fluorescent protein plasmid (pEGFP-N1, GenBank Accession #U55762,4.7kb, the phosphate radical mole is 0.3nmol) solution 1ml, 0.4ml(4mg) 1% PLURONICS F87, hatched after the jolting gently 30 minutes, as mixed liquor A.Other gets 25 mmol/L sodium hydrogen phosphate aqueous solution 2ml, adds 25 mmol/L sodium citrate aqueous solution 2ml, and 0.8ml (8mg) 1% PLURONICS F87 mixes, as mixed liquid B.Mixed liquid B is dropwise joined in the mixed liquor A, and stirring at room 30min obtains calcium phosphate nano grain solution (pEGFP-N1 plasmid quality is 0.1mg).(2) get polymine-cholesterol 5mg(PEI molecular weight 800 Da, amino mole is 100nmol), DOPE 2.5mg dissolves with the 10ml chloroform, after rotary evaporation in vacuo volatilizes chloroform, add the 5ml deionized water, aquation 10min under the water-bath ultrasound condition, then further under condition of ice bath, utilize ultrasonic grinding instrument (the new sesame in Ningbo, JY-92) carry out Probe Ultrasonic Searching and process power 400W, ultrasonic 2 seconds, be 3 seconds off time, cycle-index is 60 times, pushes film in the filter membrane of aperture 0.22 μ m, obtains polycation lipesome solution (the PEI-Chol quality is 5mg).(3) according to the nitrogen/phosphorus of polymine-cholesterol and gene than (N/P, the ratio of the molal quantity of phosphate radical in amino molal quantity and the gene molecule in polymine-cholesterol molecule), get not commensurability calcium phosphate nano grain solution and polycation lipesome solution, after diluting respectively with deionized water, the room temperature condition volumetric mixes, and hatches the polycation lipesome that obtains different N/P ratio behind the 30min/calcium phosphate nano grain drug administration carrier.
Calcium phosphate nano grain solution is diluted to 10 ml with deionized water, getting 100 μ l(pEGFP-N1 quality from 10 ml is 1 μ g again) as the calcium phosphate nano grain solution after the dilution, polycation lipesome solution (2) is got 45 μ l and is diluted to 100 μ l with after 10 times of the deionized water dilution, will obtain the polycation lipesome of N/P=30/calcium phosphate nano grain drug administration carrier after the polycation lipesome solution incubated at room after the calcium phosphate nano grain solution after the dilution and the dilution.
Embodiment 3
(1) Triton X-100 (Triton X-1) 2.52ml, hexanol 1.68ml, cyclohexane extraction 2.8ml are mixed, consist of oil phase ternary system 7ml.With 250 mmol/L calcium chloride waters, 100 μ L, 25 mmol/L sodium hydrogen phosphate aqueous solutions, 100 μ L, 0.1mg/ml pEGFP-N1 plasmid 500 μ L(phosphate radical moles are 0.15nmol) and 15 mmol/L sodium citrate aqueous solutions, 100 μ L mixing, mixed liquor made.Under agitation dropwise splash into mixed liquor in the ternary system, stirring at room 0.5 hour, mixed liquor was removed organic facies in 72 hours with bag filter (molecular cut off 14000 Da) dialysis, dialysis solution is dehydrated alcohol, gets trapped fluid and be scattered in the 5ml deionized water to be calcium phosphate nano grain solution (pEGFP-N1 plasmid quality is 0.05mg).(2) get PEI-Chol 5mg(PEI molecular weight 800 Da, amino mole is 100nmol), DOPE 2.5mg dissolves with the 10ml chloroform, after rotary evaporation in vacuo volatilizes chloroform, add the 5ml deionized water, aquation 10min under the water-bath ultrasound condition, then further under condition of ice bath, utilize ultrasonic grinding instrument (the new sesame in Ningbo, JY-92) carry out Probe Ultrasonic Searching and process power 400W, ultrasonic 2 seconds, be 3 seconds off time, cycle-index is 60 times, pushes film in the filter membrane of aperture 0.22 μ m, obtains polycation lipesome solution (the PEI-Chol quality is 5mg).(3) according to the nitrogen/phosphorus of polymine-cholesterol and gene than (N/P, the ratio of the molal quantity of phosphate radical in amino molal quantity and the gene molecule in polymine-cholesterol molecule), get not commensurability calcium phosphate nano grain solution and polycation lipesome solution, after diluting respectively with deionized water, the room temperature condition volumetric mixes, and hatches the polycation lipesome that obtains different N/P ratio behind the 30min/calcium phosphate nano grain drug administration carrier.
Calcium phosphate nano grain liquor capacity is that 100 μ l(pEGFP-N1 quality are 1 μ g), polycation lipesome solution (2) reaches respectively 15 μ l and 45 μ l and is diluted to 100 μ l with after 10 times of the deionized water dilution, will obtain the polycation lipesome of N/P=10 and 30/calcium phosphate nano grain drug administration carrier after the polycation lipesome incubated at room after calcium phosphate nano grain and the dilution.
Embodiment 4
Replace pEGFP-N1 plasmid among the embodiment 3 with fluorescent labeling siRNA random sequence, obtain the polycation lipesome of different N/P ratio/calcium phosphate nano grain drug administration carrier.
Fluorescent labeling siRNA random sequence (5 ' end FAM modifies molecular weight 13300):
（5
’- 3
’）：UCC UCC GAA CGU GUC ACG UTT
ACG UGA CAC GUU CGG AGA ATT
The drafting of embodiment 5 pseudo-ternary phase diagrams
Make a series of oil-based system (cumulative volume of Triton X-100 and hexanol and the volume ratio of cyclohexane extraction are respectively 0.11:1,0.25:1,0.43:1,0.67:1,1:1,1.5:1,2.33:1,4:1,9:1) with Triton X-100 (Triton X-1) 2.52ml, hexanol 1.68ml mixing and with a certain amount of cyclohexane extraction, in this system, drip deionized water, the stable region of ocular estimate observe system microemulsion, the water yield that the record microemulsion region forms and disappears and add.The data obtained carries out pseudo-ternary phase diagram by ORIGIN software to be drawn, and the results are shown in accompanying drawing 1, and dash area is microemulsion region.
The particle size determination of embodiment 6 polycation lipesomes/calcium phosphate nano grain complex carrier
Get calcium phosphate nano grain or the polycation lipesome/calcium phosphate nano grain drug administration carrier of certain volume embodiment 2 and 3; be that 7.4 phosphate buffer is suitably after the dilution with pH; measure the particle diameter of carrier with laser diffraction granularity analyser (Malvern, Zetasizer 3000HS).The results are shown in Table shown in 1
The particle diameter of table 1 polycation lipesome/calcium phosphate nano grain drug administration carrier
The cytotoxicity experiment of embodiment 7 polycation lipesomes/calcium phosphate nano grain drug administration carrier
(1) material
Oligonucleotide (ODN, 5 '-CTCAGTTAGGGTTAG-3 ')
MCF-7 cell: human breast cancer cell
Serum-free medium: DMEM hangs down sugar culture-medium
Complete medium: the low sugar culture-medium of DMEM that contains 10% calf serum
(2) the MCF-7 cell (1 * 10
4Individual/hole) be inoculated in 96 well culture plates, 37 ℃ of incubated overnight of cell culture incubator (Thermo Scientific Forma, 3110) are fully adherent to cell, and degrees of fusion is 60 ~ 80%.Be to add serum-free medium 100 μ l/ holes after the washing of 7.4 phosphate buffer with pH; and the embodiment 1 that adds different N/P ratio prepares polycation lipesome/calcium phosphate nano grain drug administration carrier 40 μ l/ holes (ODN=0.2 μ g/ hole) or polymine/plasmid mixture (positive control; polymine molecular weight 25kDa; ODN=0.2 μ g/ hole), the blank group then adds isopyknic serum-free medium.Place cell culture incubator to cultivate 4h in cell and remove culture medium afterwards, change complete medium into after the phosphate buffer washing, continuation is cultivated 24h under similarity condition, then add 20 μ l MTT(5mg/ml in every hole), suck culture fluid behind the 4h, every hole adds 150 μ l DMSO, jolting 10min, on microplate reader (Bio-Tek, ELx800), measure light absorption value in 570nm.(1) calculates the cells survival rate as follows, the results are shown in shown in Figure 2.
Cells survival rate (%)=(A
Sample/ A
Blank) * 100% formula (1)
Polymine (molecular weight 25kDa) is a kind of non-viral gene vector commonly used, has good gene transfection ability, is usually used in the positive control as gene transfection research, but cytotoxicity is the principal element that suppresses its application.
The result shows that the cytotoxicity of polycation lipesome/calcium phosphate nano grain drug administration carrier is less than polymine (molecular weight 25kDa)/gene mixture.
Embodiment 8 polycation lipesomes/calcium phosphate nano grain drug administration carrier mediation siRNA transfection
(1) material
Fluorescent labeling siRNA random sequence (5 ' end FAM modifies molecular weight 13300):
（5
’- 3
’）：UCC UCC GAA CGU GUC ACG UTT
ACG UGA CAC GUU CGG AGA ATT
MCF-7 cell: human breast cancer cell
Serum-free medium: DMEM hangs down sugar culture-medium
Complete medium: the low sugar culture-medium of DMEM that contains 10% calf serum
(2) the MCF-7 cell (3 * 10
4Individual/hole) be inoculated in 96 well culture plates, 37 ℃ of incubated overnight of cell culture incubator are fully adherent to cell, and degrees of fusion is 60 ~ 80%.Be to add serum-free medium 100 μ l/ holes after the washing of 7.4 phosphate buffer with pH, and adding embodiment 4 preparation polycation lipesome/(siRNA=0.2 μ g/ holes, calcium phosphate nano grain drug administration carrier 40 μ l/ holes, N/P=10), Lipofectamine 2000/ siRNA mixture (Lipofectamine 2000=50 μ l/ hole, siRNA=0.2 μ g/ hole) and the calcium phosphate nano grain (siRNA=0.2 μ g/ hole, calcium ion and siRNA mass ratio 20:1) of embodiment 4 preparation.Cell and transfection liquid discard culture medium after 37 ℃ hatch 6h jointly, with changing complete medium into behind the phosphate buffer flushing cell, continue to cultivate 24h under similarity condition.Cell is placed observation siRNA transfection situation under the fluorescence inverted microscope (Carl Zeiss, HBO100).The results are shown in shown in Figure 3ly, the result shows that the transfection efficiency of drug administration carrier will be higher than independent calcium phosphate nano grain or cationic-liposome.
The gene expression of embodiment 9 polycation lipesomes/calcium phosphate nano grain drug administration carrier mediation
(1) material
Green fluorescent protein plasmid: pEGFP-N1, GenBank Accession #U55762,4.7kb
MCF-7 cell: human breast cancer cell
Serum-free medium: DMEM hangs down sugar culture-medium
Complete medium: the low sugar culture-medium of DMEM that contains 10% calf serum
(2) take the pEGFP-N1 plasmid as reporter gene, MCF-7 cell (1 * 10
5Individual/hole) be inoculated in 24 well culture plates, 37 ℃ of incubated overnight of cell culture incubator are fully adherent to cell, and degrees of fusion is 60 ~ 80%.Be to add serum-free medium 500 μ l/ holes after the washing of 7.4 phosphate buffer with pH; the polycation lipesome of adding embodiment 2 preparations/calcium phosphate nano grain complex carrier (200 μ l/ holes; pEGFP-N1 plasmid=1 μ g/ hole wherein; N/P=30); polymine/plasmid mixture (polymine molecular weight 25kDa; pEGFP-N1 plasmid=1 μ g/ hole is N/P=5) as positive control.Cell and transfection liquid discard culture medium after 37 ℃ hatch 6h jointly, with changing complete medium into behind the phosphate buffer flushing cell, continue to cultivate 24h under similarity condition.Cell is placed the expression of observing the green fluorescent protein plasmid under the fluorescence inverted microscope (Carl Zeiss, HBO100).The results are shown in shown in Figure 4ly, the result shows: the transfection efficiency of polycation lipesome/calcium phosphate nano grain drug administration carrier and positive control polymine (molecular weight 25kDa)/the gene mixture is suitable.
Claims (8)
1. the preparation method of polycation lipesome/calcium phosphate nano grain drug administration carrier, it is characterized in that described method is: the preparation of (1) calcium phosphate nano grain solution: surfactant A aqueous solution, gene aqueous solution and calcium chloride water are mixed and made into mixed liquor A 1, then surfactant B aqueous solution, aqueous phosphatic and sodium citrate aqueous solution are mixed and made into mixed liquor A 2, under the condition that stirs, mixed liquor A 2 is dropwise splashed in the mixed liquor A 1 at last, after dropwising, stirring at room 0.5 ~ 2h, obtain mixed liquor, i.e. calcium phosphate nano grain solution; In the described calcium chloride water in calcium ion and the aqueous phosphatic ratio of the amount of substance of phosphate anion be 1:0.001～1, in the calcium chloride water in calcium ion and the gene aqueous solution mass ratio of gene be 1～100:1, the concentration of described calcium chloride water is 5 ~ 500mmol/L, the gross mass consumption of surfactant A and surfactant B is 0.01～2% of final calcium phosphate nano grain liquor capacity, and the gross mass consumption of sodium citrate is 0.001%～1% of final calcium phosphate nano grain liquor capacity in the sodium citrate aqueous solution; Described surfactant A is the mixing of one or more arbitrary proportions in polyoxyethylene poly-oxygen propylene aether block copolymer, sodium lauryl sulphate, polysorbate, Polyethylene Glycol or the polyethylene glycol mono stearate, and described surfactant B is identical surfactant with surfactant A; Phosphate is a kind of in sodium hydrogen phosphate, sodium dihydrogen phosphate, DAP, diphosphate or the sodium tripolyphosphate in the described aqueous phosphatic; Described gene comprises plasmid DNA, small molecules interference RNA and antisense oligonucleotide; (2) preparation of polycation lipesome solution: with polymine-C/PL and/or cholesterol with the organic solvent A dissolving after, rotary evaporation in vacuo is removed organic solvent A to doing, obtain lipid membrane, in lipid membrane, add deionized water, water-bath supersound process 5～30min, and then after utilizing ultrasound probe to process 1～10min under ice bath, the 100～400W condition, adopt at last the filter membrane of aperture 0.1～0.8 μ m to process, get filtrate, obtain polycation lipesome solution; Polymine molecular weight in described polymine-cholesterol is 600～50000Da; Described organic solvent A is the mixture of one or more arbitrary proportions in chloroform, dichloromethane, ether, petroleum ether or the ethanol; Described phospholipid is natural phospholipid or synthetic phospholipid; The mass ratio of described polymine-C/PL is 1:0.01～100, and the mass ratio of described polymine-cholesterol and cholesterol is 1:0～10; (3) preparation of polycation lipesome/calcium phosphate nano grain drug administration carrier: the calcium phosphate nano grain solution of step (1) preparation is mixed with equal-volume after the deionized water dilution respectively with the polycation lipesome solution of step (2) preparation, behind incubated at room 5～60min, obtain described polycation lipesome/calcium phosphate nano grain drug administration carrier; The quality consumption of described polycation lipesome solution is in amount of substance amino in polymine-cholesterol molecule, the quality consumption of described calcium phosphate nano grain solution is in the amount of substance of phosphate radical in the gene molecule of original adding, and the feed intake ratio of amount of substance of described amino and described phosphate radical is 0.01～1000:1.
2. the preparation method of polycation lipesome as claimed in claim 1/calcium phosphate nano grain drug administration carrier, it is characterized in that the ultrasound probe processing method is in the described step (2): be under the condition of 400W at power, every interval 3s, utilize ultrasound probe to carry out supersound process 2s, repeat 60 times.
3. the preparation method of polycation lipesome as claimed in claim 1/calcium phosphate nano grain drug administration carrier; it is characterized in that described step (2) phospholipid is the mixing of one or more arbitrary proportions in soybean phospholipid, lecithin, DOPE or 1, the 2-stearyl PHOSPHATIDYL ETHANOLAMINE.
4. the preparation method of polycation lipesome as claimed in claim 1/calcium phosphate nano grain drug administration carrier is characterized in that the feed intake ratio 0.1～100:1 of amount of substance of described amino and described phosphate radical.
5. the preparation method of polycation lipesome/calcium phosphate nano grain drug administration carrier, it is characterized in that described method is: (I) preparation of calcium phosphate nano grain solution: organic solvent B, surfactant C and cosurfactant A are mixed, make the oil phase ternary system, the volume ratio of described surfactant C and cosurfactant A is 1～10:1, and the cumulative volume of surfactant C and cosurfactant A and the volume ratio of organic solvent B are 0.1～10:1; Again calcium chloride water, gene aqueous solution, aqueous phosphatic and sodium citrate aqueous solution are mixed and made into mixed liquor A 3, under the condition that stirs, mixed liquor A 3 is dropwise splashed in the oil phase ternary system at last, after dropwising, stirring at room 0.5 ~ 2h, obtain mixed liquor, mixed liquor dialysed with dialyzer remove organic facies, get trapped fluid and be scattered in and be calcium phosphate nano grain solution in the deionized water; In the described calcium chloride water in calcium ion and the aqueous phosphatic ratio of the amount of substance of phosphate anion be 1:0.001～1, the mass ratio of calcium ion and gene is 1～100:1 in the calcium chloride water, and the gross mass consumption of sodium citrate is 0.001%～1% of final calcium phosphate nano grain liquor capacity; The volume ratio of described oil phase ternary system and mixed liquor A 3 is 1:0～0.5, and the concentration of described calcium chloride water is 5 ~ 500mmol/L; Described surfactant C is the mixing of one or more arbitrary proportions in polyoxyethylene poly-oxygen propylene aether block copolymer, Triton X-100, sodium lauryl sulphate or the polysorbate; Described cosurfactant A is a kind of in ethanol, normal propyl alcohol, isopropyl alcohol, ethylene glycol, 1,2-PD, glycerol or the hexanol; Described organic solvent B is cyclohexane extraction, normal hexane or normal octane; Phosphate is a kind of in sodium hydrogen phosphate, sodium dihydrogen phosphate, DAP, diphosphate or the sodium tripolyphosphate in the described aqueous phosphatic; Described gene comprises plasmid DNA, small molecules interference RNA and antisense oligonucleotide; (II) preparation of polycation lipesome solution: after polymine-C/PL and/or cholesterol usefulness organic solvent C dissolving, rotary evaporation in vacuo is removed organic solvent C to doing, obtain lipid membrane, in lipid membrane, add deionized water, water-bath supersound process 5～30min, and then after utilizing ultrasound probe to process 1～10min under ice bath, the 100～400W condition, adopt at last the filter membrane of aperture 0.1～0.8 μ m to process, get filtrate, obtain polycation lipesome solution; Polymine molecular weight in described polymine-cholesterol is 600～50000Da; Described organic solvent C is the mixture of one or more arbitrary proportions in chloroform, dichloromethane, ether, petroleum ether or the ethanol; Described phospholipid is natural phospholipid or synthetic phospholipid; The mass ratio of described polymine-C/PL is 1:0.01～100, and the mass ratio of described polymine-cholesterol and cholesterol is 1:0～10; (III) preparation of polycation lipesome/calcium phosphate nano grain drug administration carrier: the calcium phosphate nano grain solution of step (I) preparation is mixed with equal-volume after the deionized water dilution respectively with the polycation lipesome solution of step (II) preparation, behind incubated at room 5～60min, obtain described polycation lipesome/calcium phosphate nano grain drug administration carrier; The consumption of described polycation lipesome solution is in amount of substance amino in polymine-cholesterol molecule, the quality consumption of described calcium phosphate nano grain solution is in the amount of substance of phosphate radical in the gene molecule of original adding, and described amino is 0.01～1000:1 with the ratio of described phosphate radical amount of substance.
6. the preparation method of polycation lipesome as claimed in claim 5/calcium phosphate nano grain drug administration carrier, it is characterized in that the ultrasound probe processing method is in the described step (II): be under the condition of 400W at power, every interval 3s, utilize ultrasound probe to carry out supersound process 2s, repeat 60 times.
7. the preparation method of polycation lipesome as claimed in claim 5/calcium phosphate nano grain drug administration carrier; it is characterized in that described step (II) phospholipid is the mixing of one or more arbitrary proportions in soybean phospholipid, lecithin, DOPE or 1, the 2-stearyl PHOSPHATIDYL ETHANOLAMINE.
8. the preparation method of polycation lipesome as claimed in claim 5/calcium phosphate nano grain drug administration carrier is characterized in that described amino and the ratio of described phosphate radical amount of substance are 0.1～100:1.
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