CN109771669A - A kind of DNA release carrier of Dopamine nano silver particles and preparation method thereof - Google Patents
A kind of DNA release carrier of Dopamine nano silver particles and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of DNA release carrier and preparation method thereof of Dopamine nano silver particles, the DNA release carrier of the Dopamine nano silver particles includes nano silver particles, poly-dopamine and cationic polymer, wherein, the poly-dopamine is coated on the surface of the nano silver particles and forms the nano silver particles of Dopamine, the cationic polymer grafts on the surface of the nano silver particles of the Dopamine, and forms the DNA release carrier of the Dopamine nano silver particles.The present invention significantly reduces its cytotoxicity by the way that cationic polymer to be grafted on to the nano silver particles surface of Dopamine.
Description
Technical field
The present invention relates to biomedicine technical field, in particular to a kind of DNA of Dopamine nano silver particles is released
Put carrier and preparation method thereof.
Background technique
In recent years, in rising trend with the disease incidence of gene-associated diseases, therefore, find a kind of at low cost and effective
Treatment method become particularly important.Gene therapy is one of most effective one method, and either acquired disease is (such as
AIDS or cancer) or genetic disease.Gene therapy includes turning Plasmid DNA (pDNA) or small RNA interfering (siRNA)
It moves on in target cell, it is necessary to pass it to the inside of target cell, while also to be deposited in the biological defensive system of human body
It survives, therefore its success or not depends on the development of genophore.
Genophore is broadly divided into two classes: viral vectors and non-virus carrier at present.Although viral vectors transfection efficiency phase
To higher, but developmental limitation due to its biggish toxicity.Therefore, non-viral gene vector is studied by higher and higher attention,
It has many advantages, such as largely to prepare compared to viral vectors, without infectiousness, do not limit bearer capabilities and chemical structure is controllable,
Therefore researcher is to seek new material to have carried out extensive work, including polycation organic polymer chemical combination as carrier
Object, cationic-liposome, inorganic nano-particle etc., but at present used in non-virus carrier generally existing cytotoxicity is big asks
Topic.
Summary of the invention
The main object of the present invention is to propose that a kind of DNA of Dopamine nano silver particles discharges carrier and its preparation
Method, it is intended to reduce the cytotoxicity of DNA vector.
To achieve the above object, the present invention proposes a kind of DNA release carrier of Dopamine nano silver particles, including
Nano silver particles, poly-dopamine and cationic polymer, wherein the poly-dopamine is coated on the table of the nano silver particles
Face and the nano silver particles for forming Dopamine, the cationic polymer graft on the nanometer of the Dopamine
The surface of silver particles, and form the DNA release carrier of the Dopamine nano silver particles.
Preferably, the cationic polymer is in polyethyleneimine, PLL and polyamidoamine dendrimer
Any one.
Preferably, the cationic polymer is polyethyleneimine.
To achieve the above object, the present invention also proposes a kind of DNA of Dopamine nano silver particles as described above
Discharge the preparation method of carrier, comprising the following steps:
Step S10, it disperses nano silver particles in Tris-HCl buffer, dopamine hydrochloride is then added, stir
After reacting 46~50h, the sediment after reacting is isolated, Ag/ poly-dopamine composite nanoparticle is obtained；
Step S20, it disperses the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, is then added
Cationic polymer is stirred to react to making the cationic polymer graft on the Ag/ poly-dopamine composite nano-granule sublist
Behind face, the solid product after reacting is isolated, Ag/ poly-dopamine/cationic polymer composite nanoparticle, as dopamine is obtained
The DNA of functionalized nano silver particles discharges carrier.
Preferably, in step slo: the molar ratio of the nano silver particles and the dopamine hydrochloride be 1:(1~
2)。
Preferably, step S10 includes:
It under ultrasonication, disperses the nano silver particles in Tris-HCl buffer, is ultrasonically treated 25~35min
Afterwards, dopamine hydrochloride is added and continues 25~35min of ultrasound, obtains to reaction solution；
By described after 46~50h of reaction is stirred at room temperature in reaction solution, sediment is collected using Magneto separate, by washing
It washs, be dried, obtain Ag/ poly-dopamine composite nanoparticle.
Preferably, in step S20: the Ag/ poly-dopamine composite nanoparticle rubs with the cationic polymer
You are than being 1:(1~2).
Preferably, the cationic polymer in step S20 is polyethyleneimine, and step S20 includes:
It under ultrasonication, disperses the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, ultrasound
After handling 25~35min, polyethyleneimine is added and continues 25~35min of ultrasound, obtains to reaction solution；
By described after 46~50h of reaction is stirred at room temperature in reaction solution, sediment is collected using Magneto separate, by washing
It washs, be dried, obtain Ag/ poly-dopamine/polyethyleneimine composite nanoparticle, as Dopamine nano silver particles
DNA discharges carrier.
Preferably, the cationic polymer in step S20 is polyamidoamine dendrimer, and step S20
Include:
It under ultrasonication, disperses the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, ultrasound
After handling 25~35min, suspension is obtained；
After dispersing polyamidoamine dendrimer in Tris-HCl buffer, it is added in the suspension,
It obtains to reaction solution；
Will be described after reaction solution at room temperature 22~26h of ultrasonic reaction, sediment is collected using Magneto separate, by washing
It washs, be dried, obtain Ag/ poly-dopamine/polyethyleneimine composite nanoparticle, as Dopamine nano silver particles
DNA discharges carrier.
In technical solution provided by the invention, dopamine is made with nano silver particles, poly-dopamine and cationic polymer
The DNA of functionalized nano silver particles discharges carrier, which discharges in carrier, and poly-dopamine is coated on the surface of nano silver particles
And the nano silver particles of Dopamine are formed, then cationic polymer grafts on the nanometer silver granuel of the Dopamine
The surface of son, and the DNA release carrier of the Dopamine nano silver particles is formed, by the way that cationic polymer is grafted
In the nano silver particles surface of Dopamine, its cytotoxicity for being used as DNA vector is significantly reduced, is improved using peace
Quan Xing.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the reality that the DNA of Dopamine nano silver particles provided by the invention discharges the preparation method of carrier
Apply the flow diagram of example；
Fig. 2 to Fig. 7 is that the DNA of the Dopamine nano silver particles of each embodiment preparation discharges the electrophoresis test of carrier
Result figure；
Fig. 8 to Figure 10 is that the DNA of the Dopamine nano silver particles of each embodiment preparation discharges the cell toxicant of carrier
Property testing result figure；
Figure 11 to Figure 13 is that the DNA of the Dopamine nano silver particles of each embodiment preparation discharges the transfection effect of carrier
Fruit testing result figure.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Existing non-virus carrier includes cationic-liposome, cationic polymer, dendrimer and nanoparticle etc.,
But that there are stability is poor for above-mentioned non-virus carrier, synthesis step is cumbersome, toxicity is big, synthesis cost is high and structural unstable asks
Topic, requirement needed for present Research is far from satisfying field of gene.It is big to improve existing non-virus carrier cytotoxicity
The problem of, the present invention proposes a kind of DNA release carrier of Dopamine nano silver particles, including nano silver particles, poly- more
Bar amine and cationic polymer, wherein the poly-dopamine is coated on the surface of the nano silver particles and forms dopamine
The nano silver particles of functionalization, the cationic polymer graft on the surface of the nano silver particles of the Dopamine,
And form the DNA release carrier of the Dopamine nano silver particles.
In technical solution provided by the invention, dopamine is made with nano silver particles, poly-dopamine and cationic polymer
The DNA of functionalized nano silver particles discharges carrier, which discharges in carrier, and poly-dopamine is coated on the surface of nano silver particles
And the nano silver particles of Dopamine are formed, then cationic polymer grafts on the nanometer silver granuel of the Dopamine
The surface of son, and the DNA release carrier of the Dopamine nano silver particles is formed, by the way that cationic polymer is grafted
In the nano silver particles surface of Dopamine, its cytotoxicity for being used as DNA vector is significantly reduced, is improved using peace
Quan Xing.
Further, the cationic polymer be polyethyleneimine (abbreviation PEI), polylysine (abbreviation PLL) and gather
Any one in amide-amine type dendrimer (abbreviation PAMAM), the more preferably described cationic polymer are PEI, institute
The cytotoxicity of the DNA release carrier of Dopamine nano silver particles obtained is lower, for example, in specific preparation, it can
Selection molecular weight is that the PEI (hereinafter referred to as PEI-30k) of 30000Da (namely 30kDa) is used as the cationic polymer (at this
It is illustrated so that the cationic polymer is PEI-30k as an example in following embodiments of text).
Carrier is discharged for the DNA of Dopamine nano silver particles set forth above, it is more that the present invention also provides this
The preparation method of the DNA release carrier of bar amino-functionalization nano silver particles, Fig. 1 show Dopamine provided by the invention
One embodiment of the preparation method of the DNA release carrier of nano silver particles.Referring to Fig. 1, in the present embodiment, the dopamine
Functionalized nano silver particles DNA release carrier preparation method the following steps are included:
Step S10, it disperses nano silver particles in Tris-HCl buffer, dopamine hydrochloride is then added, stir
After reacting 46~50h, the sediment after reacting is isolated, Ag/ poly-dopamine composite nanoparticle is obtained；
In step S10 during being stirred to react, dopamine hydrochloride (3-Hydroxytyramine
Hydrochloride, abbreviation DA, molecular formula C8H11NO2HCl, molecular weight 189.64, No. CAS is 62-31-7) occur
Auto polymerization, and poly-dopamine (abbreviation PDA) film is formed on the nano silver particles surface, that is, as made from step S10
In Ag/ poly-dopamine composite nanoparticle (abbreviation Ag/PDA composite nanoparticle below), the PDA is coated on the nanometer
The surface of silver particles.To make to increase the PDA in the cladding area on the nano silver particles surface, the dopamine hydrochloride
Molal quantity should be no less than the molal quantity of the nano silver particles, in the present embodiment: the nano silver particles and the dopamine
The molar ratio of hydrochloride is 1:(1~2), within this range, the PDA can wrap completely the surface of the nano silver particles
It covers, and not will lead to excessive waste.
Agitating mode when it is stirred to react can be using usual manners such as mechanical stirring or ultrasounds, in the present embodiment
Be illustrated by taking mechanical stirring as an example, step S10 in the specific implementation the following steps are included:
Step S11, it under ultrasonication, disperses the nano silver particles in Tris-HCl buffer, is ultrasonically treated
After 25~35min, dopamine hydrochloride is added and continues 25~35min of ultrasound, obtains to reaction solution；
It is acted on by means of ultrasonic disperse, is conducive to improve the nano silver particles and the dopamine hydrochloride described
Dispersion efficiency and dispersed homogeneous degree in Tris-HCl buffer, when specific operation, the frequency of settable ultrasound is 35~
45Hz, more preferably 40Hz.Wherein, the pH value of selected Tris-HCl buffer is 8~9, the more preferably described Tris-
The pH value of HCl buffer is 8.5.
Step S12, by described after 46~50h of reaction is stirred at room temperature in reaction solution, sediment is collected using Magneto separate,
By washing, being dried, Ag/PDA composite nanoparticle is obtained.
After the completion of ultrasonic disperse, the reaction vessels such as three-necked flask, conical flask or reaction kettle are transferred to reaction solution by described
Middle carry out mechanical stirring, and 46~50h of reaction is stirred at room temperature, to after the reaction was completed, because containing in the sediment after reaction
The nano silver particles and band is magnetic, at this time using by reaction after solution be placed under magnetic fields by the way of, i.e., it is available
Magneto separate collects sediment therein, compared to conventional solid-liquid separation methods such as filtering, centrifugations, separative efficiency it is higher and
It operates more easy.After the completion of separation, be freeze-dried again after repeatedly washing using ethyl alcohol and deionized water, it is multiple to obtain Ag/PDA
Close nanoparticle.In other embodiments of the invention, the drying side of the routine such as vacuum drying, microwave drying can also be selected
Formula, and have the characteristics that drying efficiency is high using freeze-drying, be not easy that substance self property is caused excessively to influence, below
It is illustrated so that the drying mode is freeze-drying as an example, in the specific implementation, drying of being subject to thoroughly is volatilized to moisture, example
Such as, it can be set to be freeze-dried 10~14h at -50~-60 DEG C.
Step S20, it disperses the Ag/PDA composite nanoparticle in Tris-HCl buffer, cation is then added
Polymer is stirred to react to after making the cationic polymer graft on the Ag/ poly-dopamine composite nanoparticle surface, point
Solid product after separating out reaction, obtains Ag/PDA/ cationic polymer composite nanoparticle, as Dopamine nano silver
The DNA of particle discharges carrier.
In step S20 during being stirred to react, the cationic polymer grafts on the Ag/PDA composite nano-granule
Sublist face, that is, in the Ag/PDA/ cationic polymer composite nanoparticle as made from step S20, the cationic polymerization
Object is coated on the surface of the Ag/PDA composite nanoparticle.It is compound in the Ag/PDA to make to increase the cationic polymer
The cladding area of nanoparticle surface, the molal quantity of the cationic polymer should be no less than the Ag/PDA composite nanoparticle
Molal quantity, in the present embodiment: the molar ratio of the Ag/PDA composite nanoparticle and the cationic polymer is 1:(1
~2), within this range, the surface of the nano silver particles can be coated and be will lead to completely by the cationic polymer
Excessive waste.
Selected cationic polymer is the PEI (Ag/ prepared using PEI as cationic polymer in the present embodiment
PDA/ cationic polymer composite nanoparticle is referred to as Ag/PDA/PEI composite nanoparticle below), accordingly, step
S20 specifically includes the following steps:
Step S21a, it under ultrasonication, disperses the Ag/PDA composite nanoparticle in Tris-HCl buffer,
After being ultrasonically treated 25~35min, PEI is added and continues 25~35min of ultrasound, obtains to reaction solution；
Similarly, it is acted on by means of ultrasonic disperse, is conducive to improve the Ag/PDA composite nanoparticle in the Tris-
Dispersion efficiency and dispersed homogeneous degree in HCl buffer, it equally can be set to 35 in supersonic frequency when specific operation~
45Hz, more preferably 40Hz.Wherein, the pH value of selected Tris-HCl buffer is 8~9, the more preferably described Tris-
The pH value of HCl buffer is 8.5.
Step S22a, it by described after 46~50h of reaction is stirred at room temperature in reaction solution, is collected and is precipitated using Magneto separate
Object obtains Ag/PDA/PEI composite nanoparticle, the as DNA of Dopamine nano silver particles by washing, being dried
Discharge carrier.
After the completion of ultrasonic disperse, the reaction vessels such as three-necked flask, conical flask or reaction kettle are transferred to reaction solution by described
Middle carry out mechanical stirring, and 46~50h of reaction is stirred at room temperature, to after the reaction was completed, equally be received by the way of Magneto separate
Collect sediment therein, after the completion of separation, be freeze-dried again after repeatedly washing using ethyl alcohol and deionized water, obtain Ag/
The DNA of PDA/PEI composite nanoparticle, the as described Dopamine nano silver particles discharges carrier.
In other embodiments of the invention, it when the cationic polymer of selection is PLL or PAMAM, needs to step
Some processes in S20 are adjusted, for example, (the Ag/PDA/ prepared using PAMAM as cationic polymer when selecting PAMAM
Cationic polymer composite nanoparticle is referred to as Ag/PDA/PAMAM composite nanoparticle below), step S20 can be according to
Following manner carries out:
Step S21b, under ultrasonication, the Tris- that pH value is 8.5 is dispersed by the Ag/PDA composite nanoparticle
In HCl buffer, continues 25~35min of ultrasound and obtain suspension；
Step S22b, it disperses PAMAM in the Tris-HCl buffer that pH value is 8.5 and forms mixed liquor, then by institute
It states mixed liquor to be added in above-mentioned suspension, obtain to reaction solution；
Step S23b, under room temperature environment, described 22~26h will be reacted under ultrasonic wave effect to reaction solution, then adopted
Ag/PDA/PAMAM composite nanoparticle is obtained, as by washing, being dried with the sediment that Magneto separate is collected after reaction
The DNA of Dopamine nano silver particles discharges carrier.
Wherein, the ultrasonic treatment in step S21b is to improve the Ag/PDA composite nanoparticle in the Tris-
Dispersion efficiency and the uniformity in HCl buffer, and the ultrasonic treatment in step S23b is acted on being by ultrasonic agitation
Promote the PAMAM reactive grafting in the surface of the Ag/PDA composite nanoparticle, in the present embodiment, supersonic frequency is equal
It can be set to 35~45Hz, more preferably 40Hz.
It, can be according to the step S21a to S22a or step of above-mentioned offer when selecting PLL as the cationic polymer
Rapid S21b to step S23b same or like operating method carries out, and the PLL is made to graft on the Ag/PDA composite Nano
The surface of particle, this will not be repeated here.
The preparation method of the DNA release carrier of Dopamine nano silver particles provided by the invention, passes through dopamine
Auto polymerization, the nano silver particles surface formed PDA film, stability is not high, easy to reunite, can save for a long time, improve
It is currently used for poor, the easy to reunite problem of the nano silver particles stability of DNA vector；The nano silver particles are embedded in dopamine
Afterwards, further surface-functionalized modification is carried out to the nano silver particles using the group on the surface PDA, to nano silver particles table
The embodiment that face carries out functional modification is simple and easy to do, avoids the nano silver particles function of surface for being currently used for DNA vector
Change the problem of step complexity；Pass through the magnetic nano particle sublist for coating the high dendrimer grafting materials in dopamine
Face significantly reduces the cytotoxicity of the DNA vector, and it is high to improve the cationic polymer for being currently used for DNA vector, dendroid
The problem that the molecular weight of molecular material is larger and causes its cytotoxicity big, improves the Dopamine nano silver particles
The safety in utilization of DNA release carrier.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
(1) it at ultrasonication (40Hz), disperses nano silver particles in the Tris-HCl buffer that pH value is 8.5,
After being ultrasonically treated 30min, it is ultrasonic afterwards that dopamine hydrochloride (molar ratio of nano silver particles and dopamine hydrochloride is 1:1) is added
30min is handled, then the solution after ultrasound is transferred in three-necked flask and carries out mechanical stirring, reaction 48h is stirred at room temperature
Afterwards, sediment is collected by the way of Magneto separate, after being rinsed three times respectively with ethyl alcohol and deionized water, is freezed at -55 DEG C dry
Dry 14h obtains Ag/PDA composite nanoparticle；
(2) at ultrasonication (40Hz), dispersing pH value for Ag/PDA composite nanoparticle made from step (1) is
In 8.5 Tris-HCl buffer, after being ultrasonically treated 30min, PEI-30k is added, and (Ag/PDA composite nanoparticle and PEI's rubs
You are than being 1:1) it is ultrasonically treated 30min afterwards, then the solution after ultrasound is transferred in three-necked flask and carries out mechanical stirring, in room
After being stirred to react 48h under temperature, sediment is collected by the way of Magneto separate, after being rinsed three times respectively with ethyl alcohol and deionized water,
It is freeze-dried 14h at -55 DEG C, Ag/PDA/PEI composite nanoparticle is obtained, that is, obtains Dopamine nano silver particles
DNA discharges carrier.
Embodiment 2
(1) it at ultrasonication (40Hz), disperses nano silver particles in the Tris-HCl buffer that pH value is 8.5,
After continuing ultrasound 25min, it is subsequent that dopamine hydrochloride (molar ratio of nano silver particles and dopamine hydrochloride is 1:1.2) is added
Solution after ultrasound, is then transferred in three-necked flask and carries out mechanical stirring, reaction is stirred at room temperature by continuous ultrasound 25min
After 46h, sediment is collected by the way of Magneto separate, after being rinsed three times respectively with ethyl alcohol and deionized water, is freezed at -60 DEG C
Dry 12h, obtains Ag/PDA composite nanoparticle；
(2) at ultrasonication (40Hz), dispersing pH value for Ag/PDA composite nanoparticle made from step (1) is
In 8.5 Tris-HCl buffer, after continuing ultrasound 25min, (Ag/PDA composite nanoparticle and PEI's rubs addition PEI-30k
You are than being 1:1.2) continue ultrasound 35min afterwards, then the solution after ultrasound is transferred in three-necked flask and carries out mechanical stirring,
After being stirred to react 50h at room temperature, sediment is collected by the way of Magneto separate, is rinsed respectively three times with ethyl alcohol and deionized water
Afterwards, it is freeze-dried 12h at -60 DEG C, obtains Ag/PDA/PEI composite nanoparticle, is i.e. acquisition Dopamine nano silver particles
DNA discharge carrier.
Embodiment 3
(1) it at ultrasonication (40Hz), disperses nano silver particles in the Tris-HCl buffer that pH value is 8.5,
After continuing ultrasound 25min, dopamine hydrochloride (molar ratio of nano silver particles and dopamine hydrochloride is 1:2) is added and continues afterwards
Then solution after ultrasound is transferred in three-necked flask and carries out mechanical stirring by ultrasonic 35min, reaction 50h is stirred at room temperature
Afterwards, sediment is collected by the way of Magneto separate, after being rinsed three times respectively with ethyl alcohol and deionized water, is freezed at -60 DEG C dry
Dry 10h obtains Ag/PDA composite nanoparticle；
(2) at ultrasonication (40Hz), dispersing pH value for Ag/PDA composite nanoparticle made from step (1) is
In 9.0 Tris-HCl buffer, after continuing ultrasound 35min, (Ag/PDA composite nanoparticle and PEI's rubs addition PEI-30k
You are than being 1:2) continue ultrasound 25min afterwards, then the solution after ultrasound is transferred in three-necked flask and carries out mechanical stirring, in room
After being stirred to react 46h under temperature, sediment is collected by the way of Magneto separate, after being rinsed three times respectively with ethyl alcohol and deionized water,
It is freeze-dried 10h at -60 DEG C, Ag/PDA/PEI composite nanoparticle is obtained, that is, obtains Dopamine nano silver particles
DNA discharges carrier.
The DNA of Dopamine nano silver particles made from above-described embodiment 1 to 3 is taken to discharge carrier as DNA respectively
Carrier sample is discharged, measure of merit is carried out, test method and result are as follows:
(1) condensation ability and protective capability of the carrier to DNA
DNA release support samples are added in suitable deionized water and are dissolved, certain density sample solution is made.
1. being condensed using the DNA release carrier of agarose gel electrophoresis evaluation Dopamine nano silver particles to DNA
Ability: by appropriate amount of sample solution and 0.3 μ g Plasmid DNA (such as green fluorescent protein plasmid, GFP plasmid DNA, abbreviation
PDNA it) is mixed by different mass ratioes, obtains (the quality score of DNA release carrier and pDNA of DNA release carrier/pDNA compound
It Wei 0.5:1,1:1,1.5:1,2:1 and 2.5:1)；After reacting 40min, sample-loading buffer is added, is loaded onto Ago-Gel
(1%w/v), and gel is placed in Tris- boric acid (TBE) buffer that pH is 7.4, carry out agarose gel electrophoresis, setting
Voltage is 90V, time 40min；Naked DNA and PEI-30k control are set simultaneously.After electrophoresis, contaminated with ethidium bromide (EB)
Color gel shows DNA band.It is observed and is taken pictures with gel imager after dyeing, as a result as shown in Figures 2 to 4, wherein figure
2, Fig. 3, Fig. 4 are respectively the electrophoresis test result figure of DNA release support samples prepared by embodiment 1,2,3, and Fig. 2 is into Fig. 4,
0.5:1,1:1,1.5:1,2:1 and 2.5:1 above electrophoretogram indicate the mass ratio of DNA release carrier and pDNA.
Fig. 2 to Fig. 4's the result shows that, DNA can with embodiment prepare Dopamine nano silver particles DNA discharge
Carrier occurs cohesion and is trapped in gel lane.
2. being protected using the DNA release carrier of agarose gel electrophoresis evaluation Dopamine nano silver particles to DNA
Ability: setting with 1. in identical mass ratio DNA discharge carrier/pDNA compound, react 40min after, addition nuclease
(DNase I, 40 μ/mL) processing carries out agarose gel electrophoresis using with 1. identical method, as a result as shown in Figures 5 to 7,
Wherein, Fig. 5, Fig. 6, Fig. 7 are respectively that the DNA of the preparation of embodiment 1,2,3 discharges the electrophoresis test result figure of support samples, and Fig. 5
Into Fig. 7,0.5:1,1:1,1.5:1,2:1 and 2.5:1 above electrophoretogram indicate the mass ratio of DNA release carrier and pDNA.
Fig. 5 to Fig. 7's the result shows that, embodiment preparation Dopamine nano silver particles DNA release carrier can
To protect DNA not by the hydrolysis of nuclease.
(2) cytotoxicity detects
Using thiazolyl blue (MTT) colorimetric method to the safety of the DNA of Dopamine nano silver particles release carrier into
Row evaluation, while it is control group that comparable sodium PEI-30k, which is arranged,.By a series of DNA of concentration gradients release support samples and
After PEI-30k is incubated for for 24 hours with A2780 cell (Proliferation of Human Ovarian Cell) jointly respectively, MTT solution (0.5mg/mL) is added and continues to train
4h is supported, culture solution is drawn, dimethyl sulfoxide is added, using the absorbance at microplate reader measurement 570nm, and calculates the survival of cell
Rate, as a result as shown in Fig. 8 to Figure 10 and table 1, wherein the DNA release that Fig. 8 to Figure 10 is followed successively by the preparation of embodiment 1,2,3 carries
The cytotoxicity testing result figure of body sample, and Ag@PDA@PEI-30K indicates that the DNA of embodiment preparation discharges carrier in figure,
PEI-30K indicates control group.
The cytotoxicity testing result of the DNA release carrier of each embodiment of table 1 preparation
Fig. 8 to Figure 10 and table 1 the result shows that, the DNA release carrier of Dopamine nano silver particles is without obvious
Cytotoxicity, and compared with PEI-30k, cytotoxicity is substantially reduced, and illustrates the Dopamine of preparation of the embodiment of the present invention
The DNA release carrier of nano silver particles can be used as a kind of safe genophore.
(3) transfection detects
DNA by investigating Dopamine nano silver particles discharges carrier in HEK-293T into the cell to enhancing green
The expression of fluorescin (EGFP), evaluates the transfection abilities of the carrier:
It is that 5:1 is mixed that DNA is discharged support samples with EGFP in mass ratio, total with HEK-293T cell after being incubated for 40min
With culture 4h, then uses the fresh culture solution containing 10% fetal calf serum instead and continue to cultivate 48h.Fluorescence microscopy is used after culture
Mirror amplifies the expression of 100 times of observation EGFP under blue light excitation, as a result such as (the dotted portion of white in figure Figure 11 to Figure 13
Point be observe green fluorescence expression) and table 2 shown in, wherein Figure 11 to Figure 13 be followed successively by embodiment 1,2,3 prepare
The transfection testing result figure of DNA release support samples.
The transfection testing result of the DNA release carrier of each embodiment of table 2 preparation
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Transfection efficiency (%) | 11.28 | 10.33 | 9.89 |
Figure 11 to Figure 13 and table 2 the result shows that, green fluorescent protein can realize expression in the cell, illustrate this hair
The DNA release carrier of the Dopamine nano silver particles of bright embodiment preparation can be used as efficient gene carrier.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
Claims (9)
1. the DNA of Dopamine nano silver particles a kind of discharges carrier, which is characterized in that including nano silver particles, poly- more
Bar amine and cationic polymer, wherein the poly-dopamine is coated on the surface of the nano silver particles and forms dopamine
The nano silver particles of functionalization, the cationic polymer graft on the surface of the nano silver particles of the Dopamine,
And form the DNA release carrier of the Dopamine nano silver particles.
2. the DNA of Dopamine nano silver particles as described in claim 1 discharges carrier, which is characterized in that the sun
Ionomer is any one in polyethyleneimine, polylysine and polyamidoamine dendrimer.
3. the DNA of Dopamine nano silver particles as claimed in claim 2 discharges carrier, which is characterized in that the sun
Ionomer is polyethyleneimine.
4. a kind of system of the DNA release carrier of Dopamine nano silver particles as described in claims 1 to 3 any one
Preparation Method, which comprises the following steps:
Step S10, it disperses nano silver particles in Tris-HCl buffer, dopamine hydrochloride is then added, be stirred to react
After 46~50h, the sediment after reacting is isolated, Ag/ poly-dopamine composite nanoparticle is obtained；
Step S20, disperse the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, then be added sun from
Sub- polymer is stirred to react to after making the cationic polymer graft on the Ag/ poly-dopamine composite nanoparticle surface,
Solid product after isolating reaction, obtains Ag/ poly-dopamine/cationic polymer composite nanoparticle, as Dopamine
The DNA for changing nano silver particles discharges carrier.
5. the preparation method of the DNA release carrier of Dopamine nano silver particles as claimed in claim 4, feature exist
In in step slo: the molar ratio of the nano silver particles and the dopamine hydrochloride is 1:(1~2).
6. the preparation method of the DNA release carrier of Dopamine nano silver particles as claimed in claim 4, feature exist
In step S10 includes:
Under ultrasonication, disperse the nano silver particles in Tris-HCl buffer, after being ultrasonically treated 25~35min,
Dopamine hydrochloride is added and continues 25~35min of ultrasound, obtains to reaction solution；
By described after 46~50h of reaction is stirred at room temperature in reaction solution, sediment is collected using Magneto separate, by washing, doing
Dry processing obtains Ag/ poly-dopamine composite nanoparticle.
7. the preparation method of the DNA release carrier of Dopamine nano silver particles as claimed in claim 4, feature exist
In in step S20: the molar ratio of the Ag/ poly-dopamine composite nanoparticle and the cationic polymer be 1:(1~
2)。
8. the preparation method of the DNA release carrier of Dopamine nano silver particles as claimed in claim 4, feature exist
In the cationic polymer in step S20 is polyethyleneimine, and step S20 includes:
It under ultrasonication, disperses the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, is ultrasonically treated
After 25~35min, polyethyleneimine is added and continues 25~35min of ultrasound, obtains to reaction solution；
By described after 46~50h of reaction is stirred at room temperature in reaction solution, sediment is collected using Magneto separate, by washing, doing
Dry processing, obtains Ag/ poly-dopamine/polyethyleneimine composite nanoparticle, and the DNA of as Dopamine nano silver particles is released
Put carrier.
9. the preparation method of the DNA release carrier of Dopamine nano silver particles as described in claim 1, feature exist
In the cationic polymer in step S20 is polyamidoamine dendrimer, and step S20 includes:
It under ultrasonication, disperses the Ag/ poly-dopamine composite nanoparticle in Tris-HCl buffer, is ultrasonically treated
After 25~35min, suspension is obtained；
After dispersing polyamidoamine dendrimer in Tris-HCl buffer, be added in the suspension, obtain to
Reaction solution；
Will be described after reaction solution at room temperature 22~26h of ultrasonic reaction, sediment is collected using Magneto separate, by washing, doing
Dry processing, obtains Ag/ poly-dopamine/polyethyleneimine composite nanoparticle, and the DNA of as Dopamine nano silver particles is released
Put carrier.
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