CN103060379A - Preparation method and application of protamine-nanometer diamond composite materials - Google Patents
Preparation method and application of protamine-nanometer diamond composite materials Download PDFInfo
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Abstract
The invention relates to a preparation method and an application of protamine-nanometer diamond composite materials, and belongs to the field of biological nanomaterials. Mixing post-surface-oxidation-treatment nanometer diamonds with protamine sulfate solutions to obtain mixed solutions; obtaining deposits after the mixed solutions are centrifuged, and dissolving the obtained deposit in deionized water to form the protamine-nanometer diamond composite materials. After nanometer diamonds absorb protamine sulfate by polarity interaction, the dynamic size of the protamine-nanometer diamond composite materials is 115nm-117nm, and the surface zeta potential is 31mv-34.1mv. Mixing siRNA with the protamine-nanometer diamond composite materials, by mass, with the proportion of 1 to 3, and siRNA-nanometer diamond-protamine transfection reagents are obtained. The transfection reagents are applied to transfection reagents. The protamine-nanometer diamond composite materials have the advantages of being good in biocompatibility, smaller in cell toxicity, and good in stability and in siRNA load capacity, and having proper physical and chemical properties.
Description
Technical field:
The invention belongs to the nano material field of biology.The present invention relates to a kind of siRNA genophore and preparation method thereof, be specifically related to genophore that a kind of nano material combines with protein and preparation method thereof.
Background technology:
It is a kind of natural mechanism that is present in the gene silencing in plant and mammalian cell that RNA disturbs (RNAi).MicroRNA is exactly the endogenous little RNA with RNA interferon activity of a class that people have just found in recent years.Little RNA like this is commonly referred to as siRNA.External source double-stranded RNA (double strand RNA by size in the 21-25bpd left and right, dsRNA) transfered cell can be formed to siRNA (the small interfering RNA of strand, siRNA), can be degraded and suppress its accurate translation with the mRNA of strand siRNA complementation and cause gene silencing.RNAi has specificity preferably, and the application of RNAi technology will be opened up a revolutionary field for the gene therapy of human diseases, will be not second to the PCR technology on the impact of life science.Having at present 3 kinds of methods can trigger RNA disturbs: the DNA plasmid of introducing transcribed generation RNA interfering in target cell; Introduce the RNA interfering precursor molecule; Introduce synthetic small molecules interference RNA (siRNA) in target cell.Wherein, the third method is because of little the attracting wide attention of fairly simple, the non-specific untoward reaction of chemical reaction step of synthetic siRNA.
SiRNA is unstable, easily degraded by enzymes, is difficult to by cytophagy, and transfection carrier is most important for the performance of its application efficiently.Although utilize at present recombinant virus high as transmitting the carrier transfer efficiency in the siRNA body, potential biological safety problem has limited its application.Non-viral type siRNA carrier generally includes cationic-liposome and the large class of cationic polymers two, and the cationic-liposome transfection efficiency is high, but its biocompatibility is poor, cytotoxicity is large.Although cationic polymers is higher to the plasmid transfection efficiency, for the siRNA of small molecules amount, its transfection ability a little less than.Therefore select suitable siRNA to transmit carrier, preparation safely, stable, carrier/the siRNA composite particles is significant efficiently.
Nano diamond is a kind of carbon nanomaterial, good biocompatibility, and cytotoxicity is little, and it and the contour interaction of molecules of PEI are used as genophore.But Nano diamond and protein interaction enter the carrier of cell as siRNA, up to now, there is not yet report.
Summary of the invention:
The purpose of this invention is to provide a kind of nano particle of usining Nano diamond and Protamine sulfates interaction formation and transmit carrier as siRNA, this carrier preparation process is quick, biological safety is good, cytotoxicity is lower, Gene silencing efficacy is good, this carrier and siRNA molecule by electrostatic interaction, have obtained carrying the nanometer delivery vehicles of siRNA in water.
The invention provides a kind of siRNA delivery vehicles, its activeconstituents is the nano particle that Nano diamond and Protamine sulfates form by polar interaction.
The preparation method of a kind of protamine-Nano diamond matrix material, is characterized in that,
Nano diamond, through surface oxidation treatment, is obtained to Nano diamond solution; Nano diamond solution and Protamine sulfates solution are mixed to get mixing solutions; Be precipitated thing after the mixing solutions obtained is centrifugal, the throw out obtained is dissolved in deionized water and forms protamine-Nano diamond matrix material, after Nano diamond adsorbs Protamine sulfates by polar interaction, the kinetics of protamine-Nano diamond matrix material is of a size of 115-117nm, and the surface zeta potential current potential is 31-34.1mv.
Further, described Protamine sulfates derives from salmon.
SiRNA and protamine-Nano diamond matrix material are mixed to get to siRNA-Nano diamond-protamine transfection reagent according to solute mass ratio 1:3.
SiRNA-Nano diamond-protamine transfection reagent is in the application of field of biology.
Described Protamine sulfates derives from salmon, and water miscible Protamine sulfates, by hydrogen bond, surface charge effect, is adsorbed in the Nano diamond surface.
Wherein, the single particle diameter of Nano diamond is 4-6nm, and aqueous solution medium power is learned and is of a size of 40-60nm, and the surface zeta potential current potential is 15-17mv.Nano diamond is through surface oxidation treatment, and, after adsorbing Protamine sulfates by polar interaction, kinetics is of a size of 115-117nm, and the surface zeta potential current potential is 31-34.1mv.
From molecular structure, analyze, siRNA carrier provided by the invention is formed by polar interaction by Protamine sulfates and Nano diamond, and protamine is adsorbed in the Nano diamond surface.Protamine is a kind of water miscible strong cation protein, can with the negative charge neutralization of the phosphate group band of nucleic acid, produce powerful affinity nucleic acid.But independent protamine is because molecular weight is large, the siRNA ability of transfection molecular weight a little less than, and Nano diamond is a kind of carbon nanomaterial, with other carbon nanomaterial, compare, as carbon nanotube, its toxicity is little, biocompatibility is better, the a large amount of hydroxyl of its surface enrichment, carboxyl, carbonyl etc., easily and the amino acid in protamine by hydrogen bond and charge effect, attract each other, form the nano particle of protamine parcel Nano diamond, its surface, with strong positive charge, is easily adsorbed siRNA and is formed the gene transfection carrier.
SiRNA delivery vehicles of the present invention has good biocompatibility; cytotoxicity is less; can form nano particle by the self-assembly mode, there is suitable physico-chemical property, preferably stability; the preparation method is simple; repeatable high, have good siRNA carrying capacity simultaneously, as nano-carrier, both can protect siRNA to avoid degraded; having again the distinctive dimensional effect of nano material, is a kind of good siRNA genophore.
In the present invention, along with the variation of siRNA and nano-carrier mass ratio, siRNA-Nano diamond-protamine mixture median size and surface zeta potential potential variation change thereupon.In the situation that mass ratio siRNA: Nano diamond/protamine=1:3, Nano diamond/protamine mixture can be combined with siRNA and be formed stable nano-complex.
The accompanying drawing explanation
The size that Fig. 1 is ND and ND/PRO;
The surface zeta potential current potential that Fig. 2 is ND and ND/PRO;
Fig. 3 is that different mass is than the size of lower ND/PRO/siRA mixture;
Fig. 4 is that different mass is than the surface zeta potential current potential of lower ND/PRO/siRNA mixture;
The agarose denaturing formaldehyde gel electrophoresis figure of the ND/PRO/siRNA that Fig. 5 is different ratios.Wherein the A swimming lane is simple siRNA, and the H swimming lane is simple ND/PRO mixture, and the B-G swimming lane is respectively mass ratio siRNA:ND/PRO=1:1,1:2,1:3,1:5,1:10, the ND/PRO/siRNA mixture of 1:20;
The siRNA for EGFP of the Cy3 mark that Fig. 6 is the ND/PRO load observed under laser confocal microscope enters EC109 cell situation.A figure is the white light visual field; B figure is the 364nm excitation wavelength, the blue light visual field under the 454nm emission wavelength; C figure is the 550nm excitation wavelength, the ruddiness visual field under the 615nm emission wavelength; D figure is the image that A, B, tri-groups of fusions of C are obtained;
Fig. 7 is the ND/PRO/siRNA that observes under the fluorescent microscope inhibition situation to the EGFP protein expression, and excitation wavelength is 488nm, 400 times of magnifications.A figure is ND/PRO/siRNA(+) group, B figure is for adding ND/PROsiRNA(-) group;
Fig. 8 is the suppression efficiency of ND/PRO/siRNA to the EGFP protein expression.Wherein the Notreat group is ND/PRO/siRNA(-), Naked siRNA group is siRNA(+), the siRNA/NP group is ND/PRO/siRNA(+);
Fig. 9 is ND/PRO and the lipofectamine that the CCK-8 method is measured
?2000 Cytotoxic comparisons.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment
1, the preparation of Nano diamond
By concentration, be 150mg/ml Nano diamond (Nanodiamond, ND, hereinafter to be referred as ND) hydrogel (catalog number: 386-8567, Tokida, Ueda, Nagano) be diluted to deionized water the aqueous solution that ND concentration is 1mg/ml, get KQ-100DB supersonic cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd.) ultrasonic 2h under 100W power for 2ml, obtain the aqueous solution that ND concentration is 1mg/ml.
2, Nano diamond surface oxidation
By massfraction, be that 98% nitric acid and massfraction are 98% sulfuric acid nitric acid in mass ratio: sulfuric acid=3:1 mixes, under 140 ° of C, the mixed solution that the aqueous solution that is 1mg/ml by the ND concentration that obtains in 1 is placed in above-mentioned nitric acid and sulfuric acid reacts 12h, reaction product is centrifugal, the precipitate with deionized water obtained is cleaned and is centrifugal, repeat 3 times, the precipitation obtained finally is dissolved in deionized water and is placed in 60 ° of C dried overnight of vacuum drying oven, the dried product of weighing, after weighing, product is dissolved in deionized water, makes the aqueous solution that ND concentration is 1mg/ml.
3, the preparation of the nano combined carrier of Nano diamond-protamine
Get 40mg Protamine sulfates (catalog number: PS4020, Sigma Adrich company), be dissolved in the 4ml deionized water, ultrasonic 10 minutes, accelerate dissolution, made the protamine solution of 10mg/ml.In order to prepare protamine-Nano diamond mixture (hereinafter to be referred as ND/PRO), press the solute quality than Nano diamond: protamine=1:20, the ND solution that is 2ml 1mg/ml mixes with the protamine solution of 4ml 10mg/ml, eddy mixer (Scientific Industries Inc) vortex 2 minutes, protamine and ND are fully interacted, by the mixing solutions that obtains with the centrifugal 15min of the centrifugal speed of 13000 rpm, remove supernatant liquor, obtain centrifugal sediment, and again use deionized water resuspended, centrifugal being precipitated, repeat 3 times.Centrifugal product freeze-drying, weighing by finally obtaining, be dissolved in product in deionized water after weighing, makes the solution that ND/PRO concentration is 0.6mg/ml.
4, the detection of ND, ND/PRO mixture size and surface zeta potential current potential
The ND/PRO solution dilution obtained in 3 is become to make to the concentration of 0.06mg/ml, by particle diameter and the surface zeta potential current potential of Zetasizer Nano ZS (Malvern, Worcestershire, U.K.) test ND/PRO.The parameter of test particle diameter is 25 ° of C, 173 ° of scattering angle.The mensuration of surface potential is based on the electrophoretic mobility of nanoparticle in solution, under automatic mode, measures.Make to use the same method and measure size and the surface zeta potential current potential of the ND that does not pass through surface oxidation treatment, compare both size of ND, ND/PRO and surface zeta potential current potential.
As shown in Figure 1 and Figure 2, ND learns and is of a size of 40-60nm at the aqueous solution medium power result, and the surface zeta potential current potential is 15-17mv.By surface oxidation, after by polarity, Protamine sulfates is adsorbed in left and right mutually, the kinetics in the aqueous solution of the ND/PRO of formation is of a size of 115-117nm, and the surface zeta potential current potential is 31-34.1mv.
5, the preparation of siRNA-Nano diamond-protamine transfection reagent
By 40 μ g for enhanced green fluorescence protein (Enhanced Green Fluorescent Protein, EGFP) siRNA(catalog number siP05815122144-1, Guangzhou Rui Bo biotech company) be dissolved in the water of 500 μ l without the RNA enzyme, making concentration is the siRNA working fluid of 80 μ g/ml.The ND/PRO that freeze-drying is obtained is dissolved in the water without the RNA enzyme, makes the solution that concentration is 0.6mg/ml, gets 20 μ l ND/PRO solution (i.e. 1.2 μ g) and is diluted in the water of 500 μ l without RNase, makes the working fluid that concentration is 2.4 μ g/ml.Get the i.e. 0.4 μ g of 10 μ l siRNA() working fluid, by the solute mass ratio, siRNA:ND/RPO is respectively 1:1,1:2,1:3,1:5,1:10,1:20 gets the ND/PRO aqueous solution of corresponding amount, and the siRNA working fluid is added in the ND/PRO working fluid, in the process added, use the concussion of vortex shaker to mix, after mixing, 25 ° of C are hatched 20min, make solute mass ratio siRNA:ND/RPO and are respectively 1:1,1:2,1:3,1:5,1:10, the siRNA-Nano diamond of 1:20-protamine mixture (hereinafter to be referred as ND/PRO/siRNA).
6, the detection of ND/PRO/siRNA mixture size and surface zeta potential current potential
The quality of the ND/RPO that adds of take is benchmark, the solute mass ratio siRNA:ND/RPO obtained is respectively to 1:1,1:2,1:3,1:5,1:10, the ND/PRO/siRNA of 1:20 is diluted to respectively the concentration of concentration 60 μ g/ml, particle diameter and surface zeta potential current potential by Zetasizer Nano ZS (Malvern, Worcestershire, U.K.) test compound thing.The parameter of test particle diameter is 25 ° of C, 173 ° of scattering angle.The mensuration of surface potential is based on the electrophoretic mobility of nanoparticle in solution, under automatic mode, measures.
Result as shown in Figure 3, Figure 4, shows the increase along with ND/PRO proportion in ND/PRO/siRNA, and the grain diameter size of formation increases gradually, and it is maximum that size reaches when mass ratio siRNA:ND/RPO=1:5, reduces gradually again subsequently.And surface zeta potential current potential particle surface electric charge when mass ratio siRNA:ND/RPO=1:1 of the particle formed is positive charge, then along with the increase of ND/PRO proportion in ND/PRO/siRNA, become gradually negative charge, and, when mass ratio siRNA:ND/RPO=1:10, again become positive charge.
7, the detection of the ability of Nano diamond-protamine load siRNA
(1), agarose electrophoresis
Prepare agarose denaturing formaldehyde glue (agarose concentration 12mg/ml, 100ml), 1.2g agarose and 87ml are mixed without RNA enzyme water, heating and melting, add 10ml 10 * TAE damping fluid, treat that glue is cooled to 60 ° of C, add 3ml deionization formaldehyde and 10 μ l 10000 * GelRed
tM(catalog number 41003, Biotium company) dyestuff, fall glue after mixing, use after solidifying half an hour.
Get 15 μ l different masies than (siRNA:ND/PRO is respectively 1:1,1:2,1:3,1:5,1:10,1:20) the ND/PRO/siRNA aqueous solution with 5 μ l glycerine, mix, take pure siRNA as contrast, carry out electrophoresis in the agarose denaturing formaldehyde glue prepared, deposition condition is constant voltage 110V, 15min, observe, take pictures.
Result as shown in Figure 5, show the increase along with the amount of ND/PRO, its carrying capacity to siRNA is more and more stronger, and reached threshold value in the time of mass ratio siRNA:ND/PRO=1:5, all siRNA all are attracted on the ND/PRO material, meanwhile, when mass ratio siRNA:ND/PRO=1:3, the ability of ND/PRO load siRNA is also stronger, the trend changed in conjunction with changing than ratio along with quality of ND/PRO/siRNA grain diameter size and surface zeta potential current potential, the present invention selects the ND/RPO/siRNA prepared when mass ratio siRNA:ND/PRO=1:3 as the experimental subjects of estimating ND/PRO/siRNA biological effect on cell levels.
(2) cellular uptake ND/PRO/siRNA mixture ability detects
1, cell cultures and inoculation
The present invention uses esophageal squamous cell carcinoma clone EC109 as the cell experiment object, the EC109 cell is provided by Chinese Disease Control and Prevention Center virus, use 10% (v/v, ml/ml) foetal calf serum, 1%(v/v, ml/ml) the RPIM 1640(Roswell Park Memorial Institute 1640 of glutamine, catalog number 22400089, Invitrogen company) substratum, in 37 ° of C, 5% CO2 (v/v), cultivate in the incubator (Thermo Fischer Scient Inc.) of 70% (v/v) humidity.By 2 * 10
5individual EC109 cell is inoculated in diameter 20mm, Confocal special culture dish (the catalog number 801001 of slide thickness 0.13-0.17mm, the Wuxi bio tech ltd of anti-the think of) in, use 10%(v/v, ml/ml) foetal calf serum, 1% (v/v, ml/ml) glutamine, antibiotic-free RPIM 1640 cultivate, and spend the night adherent.
2, ND/PRO/siRNA mixture preparation
SiRNA in mass ratio: the ratio of ND/PRO=1:3 prepares the ND/PRO/siRNA mixture.The present invention uses the siRNA(catalog number for enhanced green fluorescence protein (Enhanced Green Fluorescent Protein, EGFP) of Cy3 mark: siP05815122144-1, Guangzhou Rui Bo biotech company).40 μ g are dissolved in the water of 1ml without the RNA enzyme for the siRNA of EGFP, make the working fluid that concentration is 40 μ g/ml.1.2 μ g ND/PRO are dissolved in the water of 2ml l without the RNA enzyme, make the working fluid that concentration is 0.6 μ g/ml.Get the i.e. 1.6 μ g of 40 μ l siRNA() working fluid, add in 8 μ l (i.e. 4.8 μ g) ND/PRO solution, in the process added, use the vortex shaker fully to mix, mix rear 25 ° of C and hatch 20min.
3, ND/RPO/siRNA adds substratum and observation
The ND/PRO/siRNA that obtains in above-mentioned 2 and 1ml serum-free RPIM 1640 are cultivated and mix in the doffer's pipe without the RNA enzyme based on 1.5ml, add in the Confocal culture dish that is covered with EC109, the final concentration that makes siRNA is 100nM, only to add the negative contrast of ND/PRO, be placed in 37 ° of C, 5% CO
2(v/v), in the incubator of 70% (v/v) humidity, cultivate, after 6h, substratum is changed into to 10% (v/v) foetal calf serum, 1% (v/v) glutamine, antibiotic-free RPIM 1640 perfect mediums, in 37 ° of C, 5% CO
2(v/v), the condition of 70% (v/v) humidity is removed old substratum after cultivating 24h, PBS washed cell 3 times, each culture dish adds 1ml 4% (v/v) paraformaldehyde stationary liquid, fixedly 20min, remove stationary liquid, PBS washing 3 times, each culture dish adds 1ml DAPI staining fluid (catalog number C1005, green skies biotechnology research institute), dyeing 10min, remove the DAPI staining fluid, PBS washing 3 times, each 2min.Laser confocal microscope (German Lycra company) is observed siRNA in the intracellular distribution situation of EC109.
Result as shown in Figure 6, show to use the ND/PRO material can be by mark the siRNA of Cy3 be delivered in cell.
ND/RPO/siRNA knocks out the enhanced green fluorescence protein ability and detects
The siRNA of the inhibition EGFP that the present invention uses is purchased from Guangzhou Rui Bo biotech company, catalog number siP05815122144-1.
1, pEGFP-C3 plasmid and EGFP-siRNA cotransfection
By 6 * 10
4individual EC109 cell is inoculated in 24 orifice plates (Greiner Bio-one company), uses RPIM 1640 substratum of 10% (v/v) foetal calf serum, 1% (v/v) glutamine, antibiotic-free, and the adherent rear cell degree of converging that spends the night reaches 70%-80%.
By lipofectamine
?2000(catalog number: 11668-019, Life Technologies company) with the plasmid pEGFP-C3(Clontech company of expressing enhanced green fluorescence protein) plasmid mixes, formation pEGFP-C3/lipofectamine
?add in serum-free RPIM 1640 substratum with ND/PRO/siRNA after 2000 mixtures simultaneously, carry out cell transfecting.Concrete steps are as follows: (1), get the pEGFP-C3 plasmid of 400ng adds 50 μ l Opti-MEM
?subtract blood serum medium (catalog number: 31985070, Life Technologies company); (2), get 1.5 μ l lipofectamine
?2000 transfection reagents, add 50 μ l Opti-MEM
?subtract in blood serum medium incubated at room 5min; (3), will be diluted in Opti-MEM
?subtract pEGFP-C3 plasmid and lipofectamine in blood serum medium
?2000 mix, and 25 ° of C are hatched 20min; (4), siRNA in mass ratio: the ratio of ND/PRO=1:3 prepares the ND/PRO/siRNA mixture, and making the siRNA final concentration is 100nM; (5), by ND/PRO/siRNA mixture and pEGFP-C3/lipofectamine
?2000 mixtures add in RPIM 1640 substratum of 1ml serum-free, 1% (v/v) glutamine, antibiotic-free simultaneously, fully mix; (6), remove the old substratum in 24 orifice plates, add the substratum that contains pEGFP-C3 and ND/PRO/siRNA in (5), control group is set simultaneously, last grouping situation is: A: negative control is for being used lipofectamine
?2000 transfection pEGFP-C3, B: positive control is for being used lipofectamine
?2000 cotransfection pEGFP-C3 and siRNA, C group: directly pure siRNA is added in substratum.37 ° of C, 5% CO
2(v/v), change RPIM 1640 substratum of 10% (v/v) foetal calf serum, 1% (v/v) glutamine, antibiotic-free after 70% (v/v) humidity cultivation 6h into, continue to cultivate 24h; (7), to use fluorescent microscope (Zeiss, Germany company limited) after 24h be the expression that EGFP is observed at the 488nm place in excitation wavelength, take pictures.
Result as shown in Figure 7, can obviously find out that from figure the siRNA for EGFP of ND/PRO load can suppress the expression of a part of EGFP albumen, further understand that ND/PRO can effectively be delivered to siRNA in cell, and be delivered to the effect that intracellular siRNA can effectively bring into play the expression of inhibition corresponding gene.
2, EGFP is in the detection of protein expression amount
For the suppression efficiency of detection by quantitative ND/PRO/siRNA to EGFP, extract the total protein of each experimental group in above-mentioned 1, in the 488nm excitation wavelength, detect the expression amount of EGFP at protein level under the 528nm emission wavelength, concrete steps are as follows: (1), preparation cell pyrolysis liquid: use lysis liquid level RIPA lysate (by force) (catalog number: P0013B, green skies biotechnology research institute) lysing cell, within several minutes, add PMSF(catalog number ST506 before using, green skies biotechnology research institute), the final concentration that makes PMSF is 1mM; (2), remove the substratum in each hole, PBS washing 3 times, the RIPA lysate (by force) that adds 200 μ l to configure in each hole.Under several with rifle piping and druming, lysate is fully contacted with cell, hatch 5min on ice; (3), total protein is quantitative: use BCA protein quantification test kit (catalog number PA115, it root biochemical technology company limited) carry out protein quantification, 2., preparation BCA working fluid 1., the preparation standard product concrete steps are as follows:: use RIPA lysate (by force) according to the form below configured to be diluted the BSA standard substance:: reagent A is fully mixed, and then according to the volume ratio reagent A: reagent B=50:1 prepares the 10ml working fluid; 3., draw the total protein of 25 μ l by each experimental group of the freshly prepared standard substance of table 1 and extraction, add 96 orifice plates (Greiner Bio-one company), 5 every group multiple holes; 4., add 200 μ l BCA working fluids in every hole, and fully mix; 5., add a cover, be cooled to room temperature after 37 ° of C are hatched 30min; 6., detect light absorption value in the 562nm place at multi-functional microwell plate detector (U.S. Perkinelmer Inc.); 7., by standard substance, make typical curve, and calculate the protein content in each experimental group according to typical curve; (4), the EGFP expressing quantity detects: get the total protein of 100 each experimental group of μ l, use multi-functional microwell plate detector, detect the expression amount of EGFP albumen, testing conditions is: excitation wavelength 488nm, emission wavelength 528nm
Table 1 BSA normal concentration preparation table
As shown in Figure 8, after adding ND/PRO/EGFP-siRNA, the expression amount of EGFP teaches negative group to reduce by 22% to result, illustrate that ND/PRO is effectively by having proceeded in cell of EGFP-siRNA success, and EGFP-siRNA has brought into play the effect that suppresses EGFP genetic expression.
The ND/PRO cytotoxicity detects
The present invention adopts CCK-8 cell viability test kit (catalog number: CK-04, eastern Renhua subject skill company limited) to detect the cytotoxicity that ND/PRO produces the EC109 cell.Concrete implementation step is as follows:
With every hole 1 * 10
4the inoculum size of individual/100 μ l is inoculated in 96 orifice plates (Greiner Bio-one company) by EC109, RPIM 1640 culture medium culturing of 10% (v/v) foetal calf serum, 1% (v/v) glutamine, antibiotic-free, 37 ° of C, 5%CO
2(v/v), 70% (v/v) humidity cultivates, spend the night adherent.Remove the old substratum in 96 orifice plates, add RPIM 1640 substratum of 100 μ l serum-frees, 1% (v/v) glutamine, antibiotic-free.By the final concentration decibel of ND/PRO be 7.2 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 100 μ g/ml add corresponding amount ND/PRO to each hole, each concentration arranges 5 multiple holes.With lipofectamine
?2000 for contrast, considers lipofectamine
?2000 toxicity are larger, lipofectamine
?the lipofectamine of 2000 control groups
?2000 concentration is set to: 2.5 μ g/ml, 5 μ g/ml, 7.5 μ g/ml, 10 μ g/ml, 12.5 μ g/ml, 15 μ g/ml, 17.5 μ g/ml, 20 μ g/ml, each concentration arranges 5 multiple holes.Negative control is set simultaneously.37 ° of C, 5% CO
2(v/v), 70% (v/v) humidity cultivates after 6h RPIM 1640 substratum that change 10% (v/v) foetal calf serum, 1% (v/v) glutamine, antibiotic-free into, continues to cultivate 24h.CCK-8 is added in each hole with 10% amount of every hole culture volume, in 100 μ l substratum, add 10 μ l CCK-8,37 ° of C, 5% CO
2(v/v), 70% (v/v) humidity is hatched 2h.Use multi-functional microwell plate detector (U.S. Perkinelmer Inc.) to measure the absorbancy of every hole at the 450nm place.
As shown in Figure 9, when ND/PRO reaches 20 μ g/ml in concentration, cell viability still can remain on more than 85% result, and lipofectamine
?2000 when concentration reaches 20 μ g/ml cell viability be only 40%, relative lipofectamine is described
?the weak cytotoxicity that 2000, ND/PRO shows.
Claims (4)
1. the preparation method of protamine-Nano diamond matrix material, is characterized in that,
Nano diamond, through surface oxidation treatment, is obtained to Nano diamond solution; Nano diamond solution and Protamine sulfates solution are mixed to get mixing solutions; Be precipitated thing after the mixing solutions obtained is centrifugal, the throw out obtained is dissolved in deionized water and forms protamine-Nano diamond matrix material, after Nano diamond adsorbs Protamine sulfates by polar interaction, the kinetics of protamine-Nano diamond matrix material is of a size of 115-117nm, and the surface zeta potential current potential is 31-34.1mv.
2. the preparation method of a kind of protamine-Nano diamond matrix material according to claim 1, it is characterized in that: described Protamine sulfates derives from salmon.
3. the application of the matrix material that according to claim 1 prepared by method is characterized in that:
SiRNA and protamine-Nano diamond matrix material are mixed to get to siRNA-Nano diamond-protamine transfection reagent according to solute mass ratio 1:3.
4. the application of the matrix material that according to claim 1 prepared by method is characterized in that:
SiRNA-Nano diamond-protamine transfection reagent is in the application of field of biology.
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| CN106583747A (en) * | 2016-12-06 | 2017-04-26 | 南华大学 | Preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection |
| WO2021253470A1 (en) * | 2020-06-17 | 2021-12-23 | 广东量子墨滴生物科技有限公司 | Carbon nanocomposite biological agent and preparation method and application therefor |
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