CN103641106A - Preparation method of nano sulfonated graphene and application of nano sulfonated graphene as gene transfer material - Google Patents
Preparation method of nano sulfonated graphene and application of nano sulfonated graphene as gene transfer material Download PDFInfo
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Abstract
The invention relates to the technical field of gene transfer materials, particularly a preparation method of nano sulfonated graphene and application of nano sulfonated graphene as a gene transfer material. The preparation method of the nano sulfonated graphene comprises the following steps: 1. preparing graphene oxide; 2. preparing reduced graphene oxide; 3. preparing diazonium salt of sulfanilic acid; and 4. preparing the nano sulfonated graphene. The nano sulfonated graphene can be used as a gene transfer material since positive charges of ions on particle surfaces can be combined with green fluorescent proteins in a non-covalent mode to perform transfection; and when being used as a gene transfer material, the nano sulfonated graphene has the advantages of high transfection effect, favorable cell compatibility and high cell survival rate. The transfection efficiency of the nano sulfonated graphene in human breast cancer cells is up to 40% or so.
Description
Technical field
The present invention relates to gene transfer material technical field, be specifically related to a kind of preparation method of nanometer sulfonated graphene and the application that nanometer sulfonated graphene is used as gene transfer material.
Background technology
Method of gene introduction can be divided into two classes: the first kind is virus type method of gene introduction, is that to take retrovirus, adenovirus, adeno-associated virus be carrier; Equations of The Second Kind is non-virus type method of gene introduction, as microinjection, particle gun, coprecipitation of calcium phosphate, cationic-liposome method and utilize emerging nanometer gene transfer material to carry out gene transfection.
There are many serious deficiencies in virus type method of gene introduction, for example, likely activates proto-oncogene during virus transfection.Therefore, non-viral type method of gene introduction is current study hotspot, still, and the equal Shortcomings of non-viral type method of gene introduction described above: microinjection once can only be processed a cell, and its transfection efficiency is very low; The penetration power of particle gun is very limited; The transfection efficiency of calcium phosphate precipitation is affected by the many factors such as temperature, concentration, operating environment, and transfection results is very unstable; Although cationic-liposome method shows good transfection efficiency, cationic-liposome, because toxicity is high, is restricted the application of cationic-liposome method; Nanometer gene transfer material of the prior art exists that production cost is high, preparation is not easy to obtain, is difficult to the shortcoming of penetration and promotion application with raw material.
Summary of the invention
One of object of the present invention is for the deficiencies in the prior art, a kind of preparation method of nanometer sulfonated graphene is provided, the prepared nanometer sulfonated graphene of preparation method of this nanometer sulfonated graphene can be used as gene transfer material, and this preparation method has the advantages that production cost is low, preparation is easy to get with raw material.
Two of object of the present invention is for the deficiencies in the prior art, and the application of nanometer sulfonated graphene as gene transfer material is provided, and when this nanometer sulfonated graphene is applied to gene transfer material, has the advantages that transfection effect is high, cell compatibility is good.
One of to achieve these goals, the present invention adopts following technical scheme:
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9 ~ 10;
(3) dispersion system of the graphene oxide that is 9 ~ 10 toward above-mentioned pH value adds sodium borohydride solution, then stir at a certain temperature certain hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and stir certain hour under condition of ice bath, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, the certain hour that then refluxes at a certain temperature, obtains sulfonated graphene;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out to vacuum-drying at a certain temperature, obtain nanometer sulfonated graphene.
In technique scheme, (1) step of described step 2, in the dispersion system of described graphene oxide, the mass ratio of described deionized water and described graphene oxide is 980 ~ 1020:1.
In technique scheme, (3) step of described step 2, the dispersion system of described graphene oxide and the volume ratio of described sodium borohydride solution are 70 ~ 80:10 ~ 20; The quality concentration of volume percent (W/V) of described sodium borohydride solution is 3% ~ 5%.
In technique scheme, (3) step of described step 2, whipping temp is set to 75 ℃ ~ 85 ℃, and churning time is set to 0.8 hour ~ and 1.2 hours.
In technique scheme, in described step 3, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 40 ~ 50:8 ~ 12:10 ~ 15; The volumetric molar concentration of described hydrochloric acid soln is 1mol/L ~ 2mol/L.
In technique scheme, (2) step of described step 4 stirs 1.5 hours ~ 2.5 hours under condition of ice bath.
In technique scheme, (3) step of described step 4, the volume ratio of hydrazine hydrate and deionized water is 1.5 ~ 3:95 ~ 110; Reflux temperature is 95 ℃ ~ 105 ℃, and return time is 23 hours ~ 25 hours.
In technique scheme, (4) step of described step 4, vacuum drying temperature setting is set to 55 ℃ ~ 65 ℃.
To achieve these goals two, the present invention adopts following technical scheme:
The prepared nanometer sulfonated graphene of preparation method of a kind of nanometer sulfonated graphene described above is as the application of gene transfer material.
The prepared nanometer sulfonated graphene of preparation method of a kind of nanometer sulfonated graphene described above is for making the application of antitumor drug.
Compared with prior art, beneficial effect is in the present invention:
1) preparation method of a kind of nanometer sulfonated graphene provided by the invention, prepared nanometer sulfonated graphene is spheroidal particle, the surface ion positively charged of this nanometer sulfonated graphene particle, and the median size of nanometer sulfonated graphene is 95nm ~ 105nm, and sulfonated graphene of the prior art generally can only reach micron level.
2) preparation method of a kind of nanometer sulfonated graphene provided by the invention, owing to using Sulphanilic Acid, prepare Diazosalt of sulfanilic acid, recycling Diazosalt of sulfanilic acid and redox graphene generation coupling reaction, the couplings generating is finally reduced to sulfonated graphene, and the nanometer sulfonated graphene particle that the contained sulfonic acid group of the Sulphanilic Acid in preparation process, Diazosalt of sulfanilic acid and couplings can be controlled final generation is spherical.
3) preparation method of a kind of nanometer sulfonated graphene provided by the invention, the cost of the raw material that preparation nanometer sulfonated graphene is used is lower, and preparation is easy to get with raw material, and therefore, this preparation method has advantages of that production cost is low.
4) preparation method of a kind of nanometer sulfonated graphene provided by the invention, prepared nanometer sulfonated graphene is hypotoxicity, and the ion band positive electricity of nanometer sulfonated graphene particle surface, thereby make prepared nanometer sulfonated graphene possess the potentiality that become gene transfer material.
5) preparation method of a kind of nanometer sulfonated graphene provided by the invention, this preparation method simply, easily operation, favorable repeatability.
6) preparation method of a kind of nanometer sulfonated graphene provided by the invention, prepared nanometer sulfonated graphene can be used as gene transfer material.
7) nanometer sulfonated graphene provided by the invention can be used as gene transfer material, the positive charge that this nanometer sulfonated graphene relies on its particle surface ion is combined in non-covalent mode with green fluorescent protein plasmid, carry out transfection, application as gene transfer material, when this nanometer sulfonated graphene is applied to gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.
Accompanying drawing explanation
Fig. 1 is the preparation method's of a kind of nanometer sulfonated graphene of the present invention scanning electron microscope (SEM) photograph of the prepared nanometer sulfonated graphene of embodiment 1.
Fig. 2 fluorescence picture under the inverted fluorescence microscope after transfection that is nanometer sulfonated graphene of the present invention as the association of nanometer sulfonated graphene and pEGFP-C1 in the transfection experiment of the application of gene transfer material.
Fig. 3 cell survival rate picture after transfection that is nanometer sulfonated graphene of the present invention as the association of nanometer sulfonated graphene and pEGFP-C1 in the transfection experiment of the application of gene transfer material.
Fig. 4 is that nanometer sulfonated graphene of the present invention is as the transfection efficiency figure of the association of nanometer sulfonated graphene and pEGFP-C1 in the transfection experiment of the application of gene transfer material.
Wherein, in Fig. 3 to Fig. 4, cell viability(%) represent cell survival rate, transfection efficiency (%) represents transfection efficiency.
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention; but not limiting the scope of the invention; although the present invention has been done to explain with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 1000:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9;
(3) dispersion system of the graphene oxide that is 9 toward above-mentioned pH value adds sodium borohydride solution, then at 80 ℃, stir 1 hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 75:15; The quality concentration of volume percent (W/V) of sodium borohydride solution is 4%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 46:10:11; The volumetric molar concentration of hydrochloric acid soln is 1.5mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 2 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 100 ℃, reflux 24 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 2:100;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 60 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 1 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 100nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 1 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 2.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 980:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 10;
(3) dispersion system of the graphene oxide that is 10 toward above-mentioned pH value adds sodium borohydride solution, then at 75 ℃, stir 1.2 hours, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 70:10; The quality concentration of volume percent (W/V) of sodium borohydride solution is 3%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 40:8:10; The volumetric molar concentration of hydrochloric acid soln is 1mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 1.5 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 95 ℃, reflux 25 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 1.5:95;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 58 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 2 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 95nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 2 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 3.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 1020:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.5;
(3) dispersion system of the graphene oxide that is 9.5 toward above-mentioned pH value adds sodium borohydride solution, then at 85 ℃, stir 0.8 hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 80:20; The quality concentration of volume percent (W/V) of sodium borohydride solution is 5%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 50:12:15; The volumetric molar concentration of hydrochloric acid soln is 2mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 2.5 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 105 ℃, reflux 23 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 3:110;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 55 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 3 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 105nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 3 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 4.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 990:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.8;
(3) dispersion system of the graphene oxide that is 9.8 toward above-mentioned pH value adds sodium borohydride solution, then at 83 ℃, stir 0.9 hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 72:13; The quality concentration of volume percent (W/V) of sodium borohydride solution is 3.5%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 42:9:12; The volumetric molar concentration of hydrochloric acid soln is 1.8mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 2.2 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 98 ℃, reflux 24.7 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 1.8:97;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 65 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 4 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 98nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 4 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 5.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 1010:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.2;
(3) dispersion system of the graphene oxide that is 9.2 toward above-mentioned pH value adds sodium borohydride solution, then at 78 ℃, stir 1.1 hours, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 78:17; The quality concentration of volume percent (W/V) of sodium borohydride solution is 4.5%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 48:11:14; The volumetric molar concentration of hydrochloric acid soln is 1.3mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 1.8 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 102 ℃, reflux 23.5 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 2.5:105;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 62 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 5 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 97nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 5 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 6.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 1015:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.6;
(3) dispersion system of the graphene oxide that is 9.6 toward above-mentioned pH value adds sodium borohydride solution, then at 76 ℃, stir 1.1 hours, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 73:12; The quality concentration of volume percent (W/V) of sodium borohydride solution is 4.2%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 45:10:13; The volumetric molar concentration of hydrochloric acid soln is 1.6mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 2.3 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 97 ℃, reflux 24.8 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 2.2:102;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 59 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 6 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 102nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 6 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 7.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 995:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.3;
(3) dispersion system of the graphene oxide that is 9.3 toward above-mentioned pH value adds sodium borohydride solution, then at 84 ℃, stir 0.8 hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 76:16; The quality concentration of volume percent (W/V) of sodium borohydride solution is 3.8%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 47:10:11; The volumetric molar concentration of hydrochloric acid soln is 1.1mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 1.9 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 103 ℃, reflux 23.3 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 1.7:98;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 61 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 7 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 101nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 7 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
embodiment 8.
A preparation method for nanometer sulfonated graphene, it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide; Wherein, the mass ratio of deionized water and graphene oxide is 985:1;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9.4;
(3) dispersion system of the graphene oxide that is 9.4 toward above-mentioned pH value adds sodium borohydride solution, then at 82 ℃, stir 0.9 hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene; Wherein, the dispersion system of graphene oxide and the volume ratio of sodium borohydride solution are 74:14; The quality concentration of volume percent (W/V) of sodium borohydride solution is 4.7%;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid; Wherein, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 44:9:11; The volumetric molar concentration of hydrochloric acid soln is 1.9mol/L;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and under condition of ice bath, stir 2.1 hours, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, then at 96 ℃, reflux 24.9 hours, obtain sulfonated graphene; Wherein, the volume ratio of hydrazine hydrate and deionized water is 1.7:98;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out at 57 ℃ to vacuum-drying, obtain nanometer sulfonated graphene.
The nanometer sulfonated graphene that the present embodiment 8 makes is spheroidal particle, and the median size of nanometer sulfonated graphene is 99nm, and, the surface ion positively charged of nanometer sulfonated graphene particle.
The nanometer sulfonated graphene that the present embodiment 8 makes can be used as gene transfer material, this nanometer sulfonated graphene relies on positive charge and the green fluorescent protein plasmid of its particle surface ion and has an effect, carry out transfection, when nanometer sulfonated graphene is used as gene transfer material, have advantages of that transfection effect is high, cell compatibility good, cells survival rate is high; In human breast cancer cell, the transfection efficiency of this nanometer sulfonated graphene reaches 40% left and right.And prepared nanometer sulfonated graphene can be applied to make antitumor drug.
the mensuration of bonding properties
1, main raw
The nanometer sulfonated graphene (see figure 1) that embodiment 1 makes; Green fluorescent protein plasmid (pEGFP-C1); HEPES balanced salt solution (autogamy); Electrophoretic buffer (0.5 * TBE, autogamy); DNA sample-loading buffer; Ethidum Eremide (EB).
2, main method
The nanometer sulfonated graphene solution of 1 μ L (5mg nanometer sulfonated graphene is dissolved in 500 μ L distilled waters) mixes in 1.5mL centrifuge tube with the 1 μ LpEGFP-C1 aqueous solution (0.1 mg/mL) and 8 ml distilled waters (pH=7.4), is placed under room temperature and allows it fully in conjunction with 30 minutes.Then under the speed of 5000rpm centrifugal 5 minutes, then throw out is loaded in 1% agarose (EB0.1 mg/mL), and run 40 minutes under the buffering of TAE damping fluid and under 100V voltage, band then at 320nm place, observed.
Wherein, five following samples are formulated in this experiment: the mass ratio of nanometer sulfonated graphene and pEGFP-C1 is used respectively 100:1,50:1, and 30:1,10:1,1:1, above-mentioned sample is also labeled as respectively sample 1, sample 2, sample 3, sample 4 and sample 5.
Wherein, the compound method of above-mentioned TAE is as follows:
(1) weigh tromethane 242g, Na2EDTA2H2O 37.2g and be placed in 1L beaker;
(2) in beaker, add about 800ml deionized water, stir;
(3) again to the glacial acetic acid that adds 57.1ml in beaker, fully dissolve;
(4) then in beaker, add NaOH that the pH value of the mixed solution in beaker is adjusted to 8.3, then add deionized water and be settled to after 1L, room temperature preservation, during use, redilution is 50 times, obtains TAE damping fluid.
3, result
Result is observed, and has multiple band to occur, this is because pEGFP-C1 has due to multiple configuration.Band at sample 3, sample 4 and sample 5 places is clear obviously, the mass ratio that nanometer sulfonated graphene and pEGFP-C1 are described is 30:1, when 10:1 and 1:1, the combination of nanometer sulfonated graphene and green fluorescent protein plasmid is not fine, yet the band at sample 1 and sample 2 places disappears, when the mass ratio that nanometer sulfonated graphene and pEGFP-C1 be described is 100:1 and 50:1, the combination of nanometer sulfonated graphene and green fluorescent protein plasmid is complete.
The above results shows: pEGFP-C1 and nanometer sulfonated graphene exist a best combination ratio, when the mass ratio of nanometer sulfonated graphene and pEGFP-C1 is greater than 50:1, continue to add nanometer sulfonated graphene, green fluorescent protein plasmid can be combined with unnecessary nanometer sulfonated graphene more yet, therefore, this experimental result draws, the combination ratio that the mass ratio of nanometer sulfonated graphene and pEGFP-C1 of take is 50:1 carries out transfection.
transfection experiment
1, main raw
Human breast cancer cell; Mass ratio is the nanometer sulfonated graphene of 50:1 and the association of pEGFP-C1; DMEM substratum; Foetal calf serum FBS; 6 well culture plates.
2, main method
(1) cultivation of human breast cancer cell: human breast cancer cell is digested out with trypsinase-EDTA, after counting, join in (5 * 106/ hole) in 6 orifice plates, then with containing the DMEM solution of FBS10% and add transfection and optimize reagent to be cultured to cell density be 60%～70% degrees of fusion.
(2) cell transfecting experiment: the supernatant of cultured human breast cancer cell is removed, and change new nutrient solution, then be divided into three parts, be respectively the first sample, the second sample and the 3rd sample, in the first sample, do not add any other material, toward adding mass ratio in the second sample, be the nanometer sulfonated graphene of 50:1 and the association of pEGFP-C1, toward the association that adds liposome 2000 and pEGFP-C1 in the 3rd sample, then to the first sample, the second sample and the 3rd sample are cultivated respectively 48 hours, then the second sample is carried out to fluorometric assay (see figure 2), and to the first sample, the second sample and the 3rd sample carry out respectively cell survival rate and measure (see figure 3), transfection efficiency is measured (see figure 4).Wherein, when cell survival rate is measured, be to identify with iodate pyridine, and use flow cytometer to detect.It is to use flow cytometer to detect that transfection efficiency is measured.
3, interpretation of result
Fluorescence picture from Fig. 2 can find out there is obvious green fluorescence, and the coverage rate of green fluorescence is larger, illustrates that the association of nanometer sulfonated graphene and pEGFP-C1 dyes merit in human breast cancer cell transfer.
In the cell survival rate picture of Fig. 3, by the left-to-right survival rate that represents respectively the first sample (control), the second sample (ACC nanoparticles) and the 3rd sample (lipofectamine 2000), wherein, the survival rate of the first sample is 100%, the survival rate of the second sample is that the survival rate of 98%, the three sample is 88%.Therefore, as can see from Figure 3, mass ratio is that the nanometer sulfonated graphene of 50:1 and the association of pEGFP-C1 are very little on the impact of the survival rate of human breast cancer cell, and by contrast, the association of liposome 2000 and pEGFP-C1 is larger on the impact of the survival rate of human breast cancer cell.
In the transfection efficiency picture of Fig. 4, by the left-to-right transfection efficiency that represents respectively the first sample (control), the second sample (ACC nanoparticles) and the 3rd sample (lipofectamine 2000).In Fig. 4, can see, the association of nanometer sulfonated graphene and pEGFP-C1 can reach approximately 40% transfection efficiency, and this is at the transfection efficiency that has guaranteed that prerequisite that human breast cancer cell survival rate is 98% is issued to.Although the transfection efficiency of the association of nanometer sulfonated graphene and pEGFP-C1 is high not as good as the transfection efficiency of liposome 2000,, the high cell compatibility of the association of nanometer sulfonated graphene and pEGFP-C1 is that liposome 2000 is incomparable.
Eventually the above, from above-mentioned experimental result and analysis, nanometer sulfonated graphene has huge potentiality as transfection reagent.
Claims (10)
1. a preparation method for nanometer sulfonated graphene, is characterized in that: it comprises the following steps:
Step 1, Hummers legal system are for graphene oxide: with powdered graphite, potassium permanganate, SODIUMNITRATE and the vitriol oil by Hummers legal system for graphene oxide;
The preparation of step 2, redox graphene:
(1) graphene oxide is scattered in deionized water, then carries out, after supersound process certain hour, obtaining the dispersion system of graphene oxide;
(2) dispersion system toward graphene oxide adds sodium carbonate solution, so that the pH value of the dispersion system of graphene oxide is 9 ~ 10;
(3) dispersion system of the graphene oxide that is 9 ~ 10 toward above-mentioned pH value adds sodium borohydride solution, then stir at a certain temperature certain hour, obtain redox graphene, then by redox graphene with distilled water wash to pH value be 7, obtain neutral redox graphene;
The preparation of step 3, Diazosalt of sulfanilic acid: Sulphanilic Acid and SODIUMNITRATE are dissolved in hydrochloric acid soln, under the cooling condition of ice bath, make Diazosalt of sulfanilic acid;
The preparation of step 4, nanometer sulfonated graphene:
(1) redox graphene is scattered in deionized water, obtains redox graphene suspension;
(2) Diazosalt of sulfanilic acid is joined to redox graphene suspension, and stir certain hour under condition of ice bath, make redox graphene and Diazosalt of sulfanilic acid generation coupling reaction generate couplings, then carry out centrifugal treating, again with the couplings that distilled water generates reaction wash to pH value be 7, obtain the couplings that is neutral;
(3) couplings of above-mentioned gained is scattered in the mixing solutions of hydrazine hydrate and deionized water and carries out reduction reaction, obtain reaction mixture, the certain hour that then refluxes at a certain temperature, obtains sulfonated graphene;
(4) the above-mentioned sulfonated graphene making is thoroughly washed with distilled water, then sulfonated graphene is carried out to vacuum-drying at a certain temperature, obtain nanometer sulfonated graphene.
2. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, it is characterized in that: (1) step of described step 2, in the dispersion system of described graphene oxide, the mass ratio of described deionized water and described graphene oxide is 980 ~ 1020:1.
3. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: (3) step of described step 2, and the dispersion system of described graphene oxide and the volume ratio of described sodium borohydride solution are 70 ~ 80:10 ~ 20; The quality concentration of volume percent (W/V) of described sodium borohydride solution is 3% ~ 5%.
4. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: (3) step of described step 2, and whipping temp is set to 75 ℃ ~ 85 ℃, and churning time is set to 0.8 hour ~ and 1.2 hours.
5. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: in described step 3, the mass ratio of Sulphanilic Acid, SODIUMNITRATE and hydrochloric acid soln is 40 ~ 50:8 ~ 12:10 ~ 15; The volumetric molar concentration of described hydrochloric acid soln is 1mol/L ~ 2mol/L.
6. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: (2) step of described step 4 stirs 1.5 hours ~ 2.5 hours under condition of ice bath.
7. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: (3) step of described step 4, and the volume ratio of hydrazine hydrate and deionized water is 1.5 ~ 3:95 ~ 110; Reflux temperature is 95 ℃ ~ 105 ℃, and return time is 23 hours ~ 25 hours.
8. the preparation method of a kind of nanometer sulfonated graphene according to claim 1, is characterized in that: (4) step of described step 4, vacuum drying temperature setting is set to 55 ℃ ~ 65 ℃.
9. the prepared nanometer sulfonated graphene of the preparation method of a kind of nanometer sulfonated graphene described in claim 1 to 8 any one is as the application of gene transfer material.
10. the prepared nanometer sulfonated graphene of the preparation method of a kind of nanometer sulfonated graphene described in claim 1 to 8 any one is for making the application of antitumor drug.
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