CN103721269B - Nm of gold genophore of liposome protection and preparation method thereof - Google Patents
Nm of gold genophore of liposome protection and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of the nm of gold genophore of liposome protection：Octacosyl ditallowdimethyl ammonium bromide and DOPE are dissolved in organic solvent, water is dissolved in after then removing the organic solvent, ultrasound obtains phosphatide vesicle solution；The octacosyl ditallowdimethyl ammonium bromide is 1 with the mol ratio of DOPE:10~10:1；After the phosphatide vesicle solution is mixed with the compound containing gold ion, reducing agent is added, reaction obtains the nm of gold genophore of liposome protection.After the nm of gold genophore of invented liposomes protection is compound with gene, transfection efficiency is high, and Gene releaser speed is fast in specific environment；The good stability in serum, without clustering phenomena, in vitro without by the serum removal in nutrient solution, simplifying transfection process in transfection process；Loading to DNA is higher, reduces DNA usage quantities, reduces exogenous DNA to cytotoxicity and the immunogenicity to body.
Description
Technical field
The present invention relates to genophore field, more particularly to the nm of gold genophore and its method of liposome protection.
Background technology
Gene therapy is a kind for the treatment of method of emerging gene aspect, can treat single gene inheritance disease, polygenes disease
Disease, cancer, infectious diseases（Such as AIDS）, angiocardiopathy etc..Gene therapy is different from conventional treatments：General significance
The treatment of upper disease is directed to because of various symptoms caused by gene unconventionality, and gene therapy be directed to the root of disease-
Abnormal gene in itself, thus is a kind of new method of thorough radical curing of disease, with great medical value.November nineteen ninety is beautiful
The NIH of state has carried out the clinical trial of the first Human Gene Therapy, by retroviruse by adenosine deaminase（ADA）Gene turns
In moving on to the leucocyte of patient.After eight months, ADA levels reach the 25% of normal value in infant body, have no apparent side effect.
1991, China scientist carried out the Gene Therapy Clinical Trials of the first hemophilia B in the world, and 4 hemophilia are had at present
Patient receives gene therapy, and bleeding mitigates after treatment, achieves safely and effectively therapeutic effect.
By must be by certain technical method or carrier, but physics method in the channel genes biological cell of external source
As electroporation and direct microinjection cannot be applied on a large scale because or efficiency larger to cellular damage is low.
Gene therapy is easier to be achieved by genophore.Current genophore can be broadly divided into viral vectors and non-viral load
The major class of body two.The characteristics of viral vectors is because with transduction efficiency and expression efficiency high, but serious side effect is easily caused, such as poison
Property, immunological effect, carcinogenicity and inflammatory reaction etc..Non-virus carrier is emerging gene transduction system, with low toxicity, low is exempted from
Epidemic disease reaction, exogenous origin gene integrator probability be low, without the limitation of gene insertion size, and using it is simple, prepare it is convenient, be easy to protect
The advantage such as deposit and check.Non-viral gene vector mainly has a cationic-liposome, cationic polymer, dendrimer and
Inorganic nano material of surface modification etc..Liposome is easily prepared as conventional non-viral carrier, safe, so far for
Only, more than 30 are had in terms of gene transfer and plants new cationic-liposome appearance, some of them have been enter into clinical research.Liposome
Although showing preferable transgene efficiency, expression time is short in vivo, be difficult targeting, and cationic-liposome reaches necessarily
Concentration can also produce serious cytotoxicity, therefore have certain limitation in clinical practice.
Gold nano-material has the advantages that strong stable chemical nature, affinity, good biocompatibility, is easy to surface modification,
It is thus the earliest of research, and the nano material being most widely used in biomedical sector.Nanogold particle prepares simple, grain
Footpath is adjustable, exists with the form of colloid in aqueous.Gold nano grain self property stabilization, good biocompatibility, synthesis side
Method is simple and size tunable, easily carries out surface modification, and these advantages cause that it, as a kind of ideal carrier material, draws
The very big interest of people is played.
Wang Er health seminar has synthesized two-Dodecydimethylammonium bronides of cation lipid first（DDAB）With two-
Octadecyldimethyl ammonium bromide（DODAB）Two kinds of gold nano grains of phosphatide protection.But the gold nano grain of DDAB protections
Because it interacts very strong with genes of interest, it is impossible to realizes genes of interest release in the cell, and cannot make genes of interest
Express (Journal of Controlled Release2008,129,128-134) in the cell.DODAB protects gold nano
Grain can realize the expression of genes of interest, and also DODAB improves 4 times to its transfection efficiency, but it is relatively low to be still present transfection efficiency,
Genes of interest discharges relatively slow in the cell, genes of interest start expression in the cell required for time it is long the shortcomings of
（Biomaterials2008,29,3617-3624）.
The content of the invention
Present invention solves the technical problem that being nm of gold genophore and its preparation side for providing a kind of liposome protection
Method, the nm of gold genophore transfection efficiency of the liposome protection is high, and Gene releaser speed is fast.
The invention discloses a kind of nm of gold genophore of liposome protection, including：Two-octadecyldimethyl bromination
Ammonium, DOPE and nanogold particle；
Two-octadecyldimethyl ammonium bromide and DOPE are wrapped in outside nanogold particle；
Two-octadecyldimethyl ammonium bromide is 1 with the mol ratio of DOPE:10~10:1.
Preferably, two-octadecyldimethyl ammonium bromide and the mol ratio of DOPE are 1:3~
3:1。
The invention discloses a kind of preparation method of the nm of gold genophore of liposome protection, comprise the following steps：
（A）Two-octadecyldimethyl ammonium bromide and DOPE are dissolved in organic solvent, are then removed
Water is dissolved in after removing the organic solvent, it is ultrasonically treated to obtain phosphatide vesicle solution；
Two-octadecyldimethyl ammonium bromide is 1 with the mol ratio of DOPE:10~10:1；
（B）After the phosphatide vesicle solution is mixed with the compound containing gold ion, reducing agent is added, reaction obtains fat
The nm of gold genophore of plastid protection.
Preferably, the concentration of the vesicle solution is 0.1~20mM.
Preferably, two-octadecyldimethyl ammonium bromide and the mol ratio of DOPE are 1:3~
3:1。
Preferably, the compound containing gold ion is gold chloride, gold bromide or auric iodide.
Preferably, the reducing agent is sodium borohydride solution, ascorbic acid or hydroxylamine hydrochloride that concentration is 0.1~0.6M.
Preferably, the mol ratio of the phosphatide in the reducing agent and phosphatide vesicle solution is 1:10~10:1.
Preferably, the organic solvent is chloroform, ethanol and one or more therein of methyl alcohol.
Preferably, the step（B）In, also include：It is centrifuged after the reaction.
Compared with prior art, the preparation method of the nm of gold genophore of invented liposomes protection is：By two-ten eight
Alkyl dimethyl ammonium bromide is dissolved in organic solvent with DOPE, is dissolved in after then removing the organic solvent
Water, ultrasound obtains phosphatide vesicle solution；Two-octadecyldimethyl ammonium bromide and DOPE mole
Than being 1:10~10:1；After the phosphatide vesicle solution is mixed with the compound containing gold ion, reducing agent is added, reacted
To the nm of gold genophore of liposome protection.DOPE（DOPE）With very strong cell membrane destabilization
Effect, the incorporation of DOPE can make the low pH of the nm of gold genophore and genes of interest of liposome protection in endosome（5.0）
In environment, the structure of phospholipid in compound is by Lα CLamella phase in version is HⅡ CHexagon phase, promotes genes of interest release, transfection effect
Rate is improved.Secondly as two-octadecyldimethyl ammonium bromide（DODAB）With using cooperatively for DOPE,
Make genophore and the transfection composite of genes of interest the formation good stability in serum, without clustering phenomena, transfected in vitro
Without by the serum removal in nutrient solution, simplifying transfection process in journey.In addition, the positive electricity that genophore of the present invention is carried
Lotus is more, and the loading of DNA is higher, reduces target DNA usage quantity, reduces exogenous DNA to cytotoxicity and to body
Immunogenicity.
Brief description of the drawings
The ultraviolet absorpting spectrum of the nm of gold genophore of the liposome protection that Fig. 1 embodiments 1 are obtained；
Fig. 2 is the electron microscope of the nm of gold genophore of liposome protection prepared by embodiment 1；
Fig. 3 is that genophore prepared by embodiment 1 carries the laser co-focusing fluorescence microscopy figure after the transfection of DNA；
Fig. 4 is the ultraviolet absorpting spectrum of the nm of gold genophore of the liposome protection that embodiment 2 is obtained；
Fig. 5 is the electron microscope of the nm of gold genophore of liposome protection prepared by embodiment 2；
Fig. 6 is that genophore prepared by embodiment 2 carries the laser co-focusing fluorescence microscopy figure after the transfection of DNA；
Fig. 7 is the ultraviolet absorpting spectrum of the nm of gold genophore of the liposome protection that embodiment 3 is obtained；
Fig. 8 is the electron microscope of the nm of gold genophore of liposome protection prepared by embodiment 3；
Fig. 9 is that genophore prepared by embodiment 3 carries the laser co-focusing fluorescence microscopy figure after the transfection of DNA；
Figure 10 is the cytotoxicity analysis figure of the genophore of embodiment and comparative example；
Figure 11 is the column diagram of the transfection efficiency that genophore prepared by each embodiment and comparative example carries DNA；
Figure 12 is the phase that genophore prepared by each embodiment and comparative example carries luciferase in the Transfected cells of DNA
To fluorescence activity figure；
Figure 13 is the electrophoresis of compound released dna after incubation of genophore and the DNA formation of embodiment and comparative example
Figure.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described with reference to embodiment, but
It should be appreciated that these descriptions are simply to further illustrate the features and advantages of the present invention, rather than to the claims in the present invention
Limitation.
The embodiment of the invention discloses a kind of nm of gold genophore of liposome protection, including：Two-hexadecyldimethylamine
Base ammonium bromide, DOPE and nanogold particle；
Two-octadecyldimethyl ammonium bromide and DOPE are wrapped in outside nanogold particle；
Two-octadecyldimethyl ammonium bromide is 1 with the mol ratio of DOPE:10~10:1,
Preferably 1:3~3:1.
In the present invention, there is mating reaction in two-octadecyldimethyl ammonium bromide and DOPE, and
And be wrapped in outside nanogold particle jointly, the nm of gold genophore good stability of the liposome protection for thus constituting, particle
Small, transfection efficiency is high.
The invention discloses a kind of preparation method of the nm of gold genophore of liposome protection, comprise the following steps：
（A）Two-octadecyldimethyl ammonium bromide and DOPE are dissolved in organic solvent, are then removed
Water is dissolved in after removing the organic solvent, it is ultrasonically treated to obtain phosphatide vesicle solution；
Two-octadecyldimethyl ammonium bromide is 1 with the mol ratio of DOPE:10~10:1；
（B）After the phosphatide vesicle solution is mixed with the compound containing gold ion, reducing agent is added, reaction obtains fat
The nm of gold genophore of plastid protection.
In the present invention, two-octadecyldimethyl ammonium bromide and DOPE have been dissolved in first
Machine solvent, then removes the organic solvent and is dissolved in water, and ultrasound obtains phosphatide vesicle solution.Two-the octadecyldimethyl
Ammonium bromide is 1 with the mol ratio of DOPE:10~10:1, preferably 1:3~3:1.The organic solvent is excellent
Elect readily volatilized organic solvent as, more preferably chloroform, ethanol and one or more therein of methyl alcohol.The present invention is for ultrasound
The time for the treatment of is not particularly limited, ultrasonically treated to solution clarification.The concentration of the vesicle solution is preferably 0.1~
20mM, more preferably 0.5~5mM.
After obtaining the phosphatide vesicle solution, after the phosphatide vesicle solution is mixed with the compound containing gold ion,
Reducing agent is added, reaction obtains the nm of gold genophore of liposome protection.The compound containing gold ion is preferably chlorine
Auric acid, gold bromide or auric iodide, more preferably gold chloride.The reducing agent is preferably the sodium borohydride that concentration is 0.1~0.6M
Solution, ascorbic acid or hydroxylamine hydrochloride.The mol ratio of the phosphatide in the reducing agent and phosphatide vesicle solution is preferably 1:10~
10:1, more preferably（5~2）：（2~5）.The reducing agent reacts with the compound containing gold ion, will containing gold from
The compound of son is reduced to nano Au particle.The time of the reaction is preferably 1~5 hour.The temperature of the reaction is without spy
Different limitation, room temperature.It is preferred by centrifugation after the reaction terminates, remove unnecessary phosphatide vesicle solution.The centrifugation
Speed is preferably 15000~25000rpm, more preferably 18000~21000rpm.
In the present invention, DOPE（DOPE）Acted on very strong cell membrane destabilizationization, DOPE
Incorporation can make the low pH in endosome of nm of gold genophore and genes of interest that liposome protects（5.0）It is multiple in environment
Structure of phospholipid in compound is by Lα CLamella phase in version is HⅡ CHexagon phase, promotes genes of interest release, and transfection efficiency is improved.Its
It is secondary, due to two-octadecyldimethyl ammonium bromide（DODAB）With using cooperatively for DOPE, carry gene
The transfection composite that body is formed with genes of interest good stability in serum, without clustering phenomena, in vitro need not in transfection process
By the serum removal in nutrient solution, transfection process is simplified.In addition, the positive charge that genophore of the present invention is carried is more,
The loading of DNA is higher, reduces target DNA usage quantity, reduces exogenous DNA to cytotoxicity and the immunogene to body
Property.
For a further understanding of the present invention, with reference to the nanometer auri that embodiment is protected to the liposome that the present invention is provided
Because carrier and preparation method thereof is illustrated, protection scope of the present invention is not limited by the following examples.
Embodiment 1
By 0.0032g DODAB and 0.0018g DOPE to be dissolved into 10mL chloroforms, most of chlorine is removed with nitrogen stream
After imitative, it is vacuum dried 2 hours, removes trace chloroform.After adding the 7mL aqueous solution, ultrasound to clarification prepares DODAB/DOPE
Vesicle solution.Then mixing is stirred to the chlorauric acid solutions of 7 microlitres of 0.1M of addition in the vesicle solution for preparing.Added after mixing
8 microlitres of sodium borohydride solutions of 0.4M.Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour.
Take out 500 microlitres and record ultraviolet-visible absorption spectroscopy at room temperature, referring to Fig. 1, Fig. 1 is the fat that embodiment 1 is obtained
The ultraviolet absorpting spectrum of the nm of gold genophore of plastid protection.Fig. 2 is the nanometer auri of liposome protection prepared by embodiment 1
Because of the electron microscope of carrier.From Fig. 1 and Fig. 2, the present invention has obtained the nm of gold genophore of liposome protection.
Gold nano grain will be prepared 20, be centrifuged 30 minutes under the conditions of 000 centrifugal force.After cleaning 2 times with secondary water, will receive
Rice grain is used standby after concentrating 10 times.By the DNA 100ng containing egfp expression gene and 1 microlitre of gold nano
Particle solution mixes and places 15 minutes at room temperature, is then added directly into the cell culture medium containing HEK293 cells.
Placed 4 hours at 37 DEG C, cell is fully absorbed transfection reagent, then carefully suction out culture medium, added thin containing HEK293
The fresh culture of born of the same parents and after continuing at 37 DEG C to cultivate 24 hours, green fluorescence egg is observed with laser confocal fluorescence microscope
White expression, referring to Fig. 3, Fig. 3 is that the laser co-focusing that genophore prepared by embodiment 1 is carried after the transfection of DNA is glimmering
Light micrograph, from the figure 3, it may be seen that the nano-gene carrier transfection efficiency that liposome prepared by embodiment 1 is protected is high.
Embodiment 2
By 0.0032g DODAB and 0.0037g DOPE to be dissolved into 10mL chloroforms, most of chlorine is removed with nitrogen stream
After imitative, it is vacuum dried 2 hours, removes trace chloroform.After adding the 10mL aqueous solution, ultrasound to clarification prepares DODAB/DOPE
Vesicle solution.Then to 10 microlitres of 0.1M chlorauric acid solutions stirring mixing of addition in the vesicle solution for preparing.Added after mixing
12 microlitres of 0.4M sodium borohydride solutions.Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour.
Take out 500 microlitres and record ultraviolet-visible absorption spectroscopy at room temperature.Referring to Fig. 4, Fig. 4 is the fat that embodiment 2 is obtained
The ultraviolet absorpting spectrum of the nm of gold genophore of plastid protection.Fig. 5 is the nanometer auri of liposome protection prepared by embodiment 2
Because of the electron microscope of carrier.From Fig. 4 and Fig. 5, the present invention has obtained the nm of gold genophore of liposome protection.
The gold nano grain that will be prepared is centrifuged 30 minutes 20 under the conditions of 000 centrifugal force.After 2 times being cleaned with secondary water, will
Nano particle is used standby after concentrating 10 times.By the DNA 100ng containing egfp expression gene and 0.5 microlitre of gold
Nanoparticles solution mixes and is added directly into the cell culture medium containing HEK293 cells.Continue culture 24 at 37 DEG C small
Shi Hou, with the expression of confocal laser scanning microscope green fluorescent protein, referring to Fig. 6, Fig. 6 is prepared by embodiment 2
Genophore carries the laser co-focusing fluorescence microscopy figure after the transfection of DNA, it will be appreciated from fig. 6 that liposome prepared by embodiment 2 is protected
The nano-gene carrier transfection efficiency of shield is high.
Embodiment 3
By 0.0032g DODAB and 0.0111g DOPE to be dissolved into 10mL chloroforms, most of chlorine is removed with nitrogen stream
After imitative, it is vacuum dried 2 hours, removes trace chloroform.After adding the 20mL aqueous solution, ultrasound to clarification prepares DODAB/DOPE
Vesicle solution.Then to 20 microlitres of 0.1M chlorauric acid solutions stirring mixing of addition in the vesicle solution for preparing.Added after mixing
24 microlitres of 0.4M sodium borohydride solutions.Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour.
Take out 500 microlitres and record gold nano genophore ultraviolet-visible absorption spectroscopy at room temperature.Referring to Fig. 7, Fig. 7 is real
Apply the ultraviolet absorpting spectrum of the nm of gold genophore of the liposome protection that example 3 is obtained.Fig. 8 is liposome prepared by embodiment 3
The electron microscope of the nm of gold genophore of protection.From Fig. 7 and Fig. 8, the present invention has obtained the nanometer auri of liposome protection
Because of carrier.
Gold nano grain will be prepared 20, be centrifuged 30 minutes under the conditions of 000 centrifugal force, it will be unnecessary to be removed more with as far as possible
DODAB/DOPE vesicles.
After cleaning 2 times with secondary water, nano particle is used standby after concentrating 10 times.Egfp expression base will be contained
The DNA 100ng of cause and the mixing of 0.2 microlitre of gold nano grain solution, and will prepare after placing 15 minutes at room temperature
Mixed solution is added directly into the cell culture medium containing HEK293 cells.After continuing at 37 DEG C to cultivate 24 hours, with swashing
The expression of light confocal microscopy green fluorescent protein, referring to Fig. 9, Fig. 9 is that genophore prepared by embodiment 3 is taken
Laser co-focusing fluorescence microscopy figure after transfection with DNA, as shown in Figure 9, the nanometer base of liposome protection prepared by embodiment 3
Because carrier transfection efficiency is high.
Embodiment 4
0.0032g DODAB and 0.00124g DOPE are dissolved into 10mL chloroforms, most of chloroform is removed with nitrogen stream
Afterwards, it is vacuum dried 2 hours, removes trace chloroform.After adding the 10mL aqueous solution, ultrasound to clarification prepares DODAB/DOPE bubbles
Capsule solution.Then to 22 microlitres of 0.1M chlorauric acid solutions stirring mixing of addition in the vesicle solution for preparing.28 are added after mixing
Microlitre 0.4M sodium borohydride solutions.Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour.
Embodiment 5
0.0032g DODAB and 0.00744g DOPE are dissolved into 10mL chloroforms, most of chloroform is removed with nitrogen stream
Afterwards, it is vacuum dried 2 hours, removes trace chloroform.After adding the 10mL aqueous solution, ultrasound to clarification prepares DODAB/DOPE bubbles
Capsule solution.Then to 24 microlitres of 0.1M chlorauric acid solutions stirring mixing of addition in the vesicle solution for preparing.30 are added after mixing
Microlitre 0.4M sodium borohydride solutions.Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour.
Comparative example 1
0.0032g DODAB are dissolved into 10mL chloroforms, after removing most of chloroform with nitrogen stream, vacuum drying 2 is small
When, remove trace chloroform.After adding the 20mL aqueous solution, ultrasound to clarification prepares DODAB vesicle solution.Then as preparing
Vesicle solution in add the stirring mixing of 6 microlitres of 0.1M chlorauric acid solutions.7 microlitres of 0.4M sodium borohydride solutions are added after mixing.
Stirring reaction obtains the gold nano grain of DODAB protections after 1 hour.
Embodiment 6
Toxicity test is carried out to the genophore that embodiment 1~5 and comparative example 1 are obtained.By embodiment 1~5 and comparative example 1
The genophore for obtaining is respectively configured as the solution that nanometer gold concentration is 2,4,6,8,10nM.Liposome without nm of gold is sky
White control, no matter with DODAB or with DOPE as carrier, the survival rate of cell is 100%.Put culture in incubator（37 DEG C,
5%CO2）After 24 hours, 10 μ L MTT are added per hole（5mg/mL）Reagent；Continue to cultivate 4 hours, it is then careful to suction out culture medium,
100 μ L DMSO are added in per hole.Room temperature lucifuge jog is fully dissolved out purple crystal in 15 minutes；Finally determined using ELIASA
Light absorption value of each hole at 490nm.Each sample does 10 parallel laboratory tests, results averaged.Result is referring to Figure 10, Tu10Wei
The cytotoxicity analysis figure of the genophore of embodiment and comparative example.In Figure 10,It is the genophore of comparative example 1 to thin
The toxicity of born of the same parents；It is the genophore of embodiment 4 to the toxicity of cell；It is the genophore of embodiment 1 to cell
Toxicity；It is the genophore of embodiment 2 to the toxicity of cell；It is the genophore of embodiment 5 to the poison of cell
Property；It is the genophore of embodiment 3 to the toxicity of cell.
Embodiment 7
Transfection efficiency comparing is carried out to the genophore that embodiment 1~5 and comparative example 1 are obtained.By 0.2 microlitre of embodiment 1
~5 and DNA 100ng mixing of the genophore that obtains of comparative example 1 respectively and containing egfp expression gene, and
The mixed solution that will be prepared after placing 15 minutes at room temperature is added directly into the cell culture medium containing HEK293 cells.
After continuing to cultivate 24 hours at 37 DEG C, the cell of egfp expression is quantified with flow cytometer, as a result joined
See the column diagram that Figure 11, Figure 11 are the transfection efficiency that genophore prepared by each embodiment and comparative example carries DNA；In Figure 11, A
It is the transfection efficiency of the genophore of comparative example 1；B is the transfection efficiency of the genophore of embodiment 4；C is the gene of embodiment 1
Transfection efficiency；D is the transfection efficiency of the genophore of embodiment 2；E is the transfection efficiency of the genophore of embodiment 5；F is
The transfection efficiency of the genophore of embodiment 3.
Embodiment 8
Transfection efficiency comparing is carried out to the genophore that embodiment 1~5 and comparative example 1 are obtained.By 0.2 microlitre of embodiment 1
~5 and the genophore that obtains of comparative example 1 mix with the DNA 100ng of luciferase reporter gene respectively, and at room temperature
The mixed solution that will be prepared after placing 15 minutes is added directly into the cell culture medium containing HEK293 cells.At 37 DEG C
After continuing to cultivate 24 hours, luciferase assays kit is used, coordinate fluorescence detector to determine enzymatic activity, as a result referring to figure
12, Figure 12 is that genophore prepared by each embodiment and comparative example carries the relatively glimmering of luciferase in the Transfected cells of DNA
Photolytic activity figure；In Figure 12, A lives for the relative fluorescence of luciferase in the Transfected cells of the genophore carrying DNA of comparative example 1
Property；B is the luciferase activity of luciferase in the Transfected cells of the genophore carrying DNA of embodiment 4；C is embodiment 1
Genophore carry DNA Transfected cells in luciferase luciferase activity；D takes for the genophore of embodiment 2
The luciferase activity of luciferase in Transfected cells with DNA；After E carries the transfection of DNA for the genophore of embodiment 5
The luciferase activity of luciferase in cell；F is fluorescein in the Transfected cells of the genophore carrying DNA of embodiment 3
The luciferase activity of enzyme.
Embodiment 9
Agarose gel electrophoresis experimental simulation DNA discharges from lysosome.0.2 microlitre of embodiment 2 and comparative example 1 are obtained
Genophore mixes with DNA 100ng respectively, and the mixed solution that placement will be prepared after 15 minutes at room temperature is added
After 0.175mM dodecyl sodium sulfates, it is incubated 10,30,60 and 120 minutes respectively.Product after incubation is carried out into agarose to coagulate
Gel electrophoresis.Result is released for the compound that the genophore of embodiment and comparative example is formed with DNA referring to Figure 13, Figure 13 after incubation
Put the electrophoretogram of DNA.In Figure 13, M swimming lanes are DNA marker；DNA swimming lanes are compareed for DNA；1 swimming lane is the gene of comparative example 1
Carrier and DNA compounds；2-5 swimming lanes add 0.175mM dodecyl sodium sulfonates for the genophore of comparative example 1 with DNA compounds
After sodium, the result after being incubated 10,30,60 and 120 minutes respectively.6 swimming lanes are the genophore and DNA compounds of embodiment 2;7-
After 10 swimming lanes add 0.175mM dodecyl sodium sulfates for the genophore of embodiment 2 with DNA compounds, 10 are incubated respectively,
30,60 and 120 minutes.Result shows, after genophore of the invention supports gene, in suitable environment, Gene releaser
Speed is fast.
The explanation of above example is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that right
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or uses the present invention.
Various modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The scope most wide for causing.
Claims (1)
1. the nm of gold genophore that a kind of liposome is protected, including：Two-octadecyldimethyl ammonium bromide, dioleoyl phospholipid
Acyl monoethanolamine and nanogold particle；
Two-octadecyldimethyl ammonium bromide and DOPE are wrapped in outside nanogold particle；
The nm of gold genophore is prepared by following steps：
0.0032g DODAB and 0.0111g DOPE are dissolved into 10mL chloroforms, after removing most of chloroform with nitrogen stream, very
Sky is dried 2 hours, removes trace chloroform；It is ultrasonically treated to clarifying after adding the 20mL aqueous solution, prepare DODAB/DOPE bubbles
Capsule solution；Then to 20 microlitres of 0.1M chlorauric acid solutions stirring mixing of addition in the DODAB/DOPE vesicles solution；After mixing
Add 24 microlitres of 0.4M sodium borohydride solutions；Stirring reaction obtains the gold nano grain of DODAB/DOPE protections after 1 hour；
The nm of gold genophore is formulated as the solution that nanometer gold concentration is 2nmol/L, the solution is added and contains HEK
In the fresh culture of 293 cells, it is placed in incubator, in CO2Volume fraction is culture, the culture under conditions of 5%
Temperature be 37 DEG C, time of culture is 24h, and then, it is the MTT reagents of 5mg/mL to add 10 μ L mass concentrations, continues to cultivate
4h, then suctions out culture medium, adds 100 μ LDMSO, room temperature lucifuge jog 15min, is fully dissolved out purple crystal, finally, profit
The light absorption value at 490nm is determined with ELIASA, so as to draw versus cell survival rate.
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| CN106434751B (en) * | 2016-10-08 | 2019-12-24 | 西安交通大学 | miRNA delivery vector based on ultra-small gold nanoparticles and preparation method and application thereof |
| CN106581695A (en) * | 2016-10-27 | 2017-04-26 | 山东省分析测试中心 | Cationic liposome, preparation method and applications thereof |
| CN111759808B (en) * | 2020-07-09 | 2021-08-17 | 中国科学院长春应用化学研究所 | Liposome-graphene-gold composite nano material and preparation method and application thereof |
| CN113398281B (en) * | 2021-07-13 | 2022-05-06 | 中国科学院长春应用化学研究所 | Gold nanoflower polypeptide compound, preparation method thereof and application thereof in tumor diagnosis and treatment |
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2014
- 2014-01-22 CN CN201410029448.6A patent/CN103721269B/en active Active
Non-Patent Citations (3)
| Title |
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| Anionic Lipid, pH-Sensitive Liposome-Gold Nanoparticle Hybrids for Gene Delivery – Quantitative Research of the Mechanism;Baoji Du ,et al;《small》;20150131;第1-8页 * |
| Lipoplexes Formed by DNA and Ferrocenyl Lipids: Effect of Lipid Oxidation State on Size, Internal Dynamics, and z-Potential;Melissa E. Hays，et al;《Biophysical Journal》;20071231;第93卷;4414-4424 * |
| The Effect of a Neutral Lipid on the Transfection Efficiency of Cationic Lipid Coated Gold Nanoparticles;Dan LI，et al;《14th ISEAC Poster Presentations》;20130820;图1，第128页左栏倒数第二段 * |
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