CN104109684A - Functionalized nano hydroxyapatite gene delivery material, preparation method, and applications thereof - Google Patents
Functionalized nano hydroxyapatite gene delivery material, preparation method, and applications thereof Download PDFInfo
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- CN104109684A CN104109684A CN201410274374.2A CN201410274374A CN104109684A CN 104109684 A CN104109684 A CN 104109684A CN 201410274374 A CN201410274374 A CN 201410274374A CN 104109684 A CN104109684 A CN 104109684A
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Abstract
The invention relates to the technical field of gene delivery materials, and specifically relates to a functionalized nano hydroxyapatite gene delivery material, a preparation method and applications thereof. The preparation method comprises the following steps: step one, preparing needle-shaped hydroxyapatite; and step two, making the hydroxyapatite absorb cis-platinum so as to obtain the nano hydroxyapatite gene delivery material. The prepared nano hydroxyapatite gene delivery material is a nano spherical material with an average diameter of around 100 nm, is capable of combining with green fluorescent protein plasmid gene pGFP to carry out transfection, and has the advantages of high transfection efficiency, good cell compatibility, and high cell survival rate. For instance, in the human breast cancer cells, the transfection efficiency of combined nano hydroxyapatite gene delivery material and pEGFP-C1 can reach 40% more or less.
Description
Technical field
The present invention relates to gene transfer material technical field, be specifically related to gene transfer material of a kind of functionalized nano hydroxyapatite and its preparation method and application.
Background technology
Method of gene introduction can be divided into two classes: the first kind is virus type method of gene introduction, is taking retrovirus, adenovirus, adeno-associated virus as 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 when 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; There is the shortcoming that production cost is high, preparation raw material is not easy to obtain, is difficult to penetration and promotion application in nanometer gene transfer material of the prior art.
Summary of the invention
One of object of the present invention is for the deficiencies in the prior art, and a kind of preparation method of gene transfer material of functionalized nano hydroxyapatite is provided.
Two of object of the present invention is for the deficiencies in the prior art, and the gene transfer material of the functionalized nano hydroxyapatite that a kind of preparation method of the gene transfer material that utilizes functionalized nano hydroxyapatite makes is provided.
Three of object of the present invention is for the deficiencies in the prior art, provides a kind of gene transfer material of functionalized nano hydroxyapatite for making the application of antitumor drug.
One of to achieve these goals, the present invention adopts following technical scheme:
A preparation method for the gene transfer material of functionalized nano hydroxyapatite, it comprises the following steps:
Step 1, the preparation of needle-like hydroxyapatite: the aqueous solution of calcium hydroxide is slowly joined in the aqueous solution of dilute phosphoric acid, and stir at a certain temperature certain hour, then after leaving standstill certain hour, filter, obtain particulate matter, then particulate matter is washed after also vacuum-drying, obtain needle-like hydroxyapatite;
Step 2, absorption cis-platinum: cis diamino dichloro network platinum is dissolved in ultrapure water, then after sonic oscillation certain hour, the needle-like hydroxyapatite that adds step 1 to make, and stir at a certain temperature after certain hour, carry out centrifugal and washing, obtain the gene transfer material of nanometer hydroxyapatite.
In technique scheme, in step 1, the mol ratio of the phosphoric acid in the calcium hydroxide in the aqueous solution of described calcium hydroxide and the aqueous solution of described dilute phosphoric acid is 4 ~ 6:2 ~ 4.
In technique scheme, in step 1, in the aqueous solution of described calcium hydroxide, the volumetric molar concentration of described calcium hydroxide is 0.1mol/L ~ 0.3 mol/L.
In technique scheme, in step 1, in the aqueous solution of described dilute phosphoric acid, the volumetric molar concentration of described dilute phosphoric acid is 0.1mol/L ~ 0.3 mol/L.
In technique scheme, in step 1, described whipping temp is 33 DEG C ~ 40 DEG C, and described churning time is 22 hours ~ 26 hours; Described time of repose is 1.5 hours ~ 2.5 hours.
In technique scheme, in step 2, the mass ratio of described needle-like hydroxyapatite and described cis diamino dichloro network platinum is 40 ~ 60:1 ~ 5.
In technique scheme, in step 2, quality-volumetric concentration that described cis diamino dichloro network platinum is dissolved in ultrapure water is 0.2mg/mL ~ 0.6 mg/mL.
In technique scheme, in step 2, the time of described sonic oscillation is 20 minutes ~ 40 minutes; Described whipping temp is 33 DEG C ~ 40 DEG C, and described churning time is 45 hours ~ 50 hours.
To achieve these goals two, the present invention adopts following technical scheme:
The gene transfer material that utilizes the functionalized nano hydroxyapatite that the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite described above makes, the gene transfer material of described nanometer hydroxyapatite is that a kind of mean diameter is the nanometer needle-like material of 80nm ~ 100nm.
To achieve these goals three, the present invention adopts following technical scheme:
The gene transfer material of the prepared functionalized nano hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite described above is for making the application of antitumor drug.
Compared with prior art, beneficial effect is in the present invention:
The gene transfer material of the prepared nanometer hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite provided by the invention is that a kind of mean diameter is the nanometer needle-like material of 80nm ~ 100nm left and right, and the particle diameter of this nanometer needle-like material can pass through the control in reaction times and then control the particle diameter of nano particle, thereby expand the range of choice of particle diameter.The gene transfer material energy of this nanometer hydroxyapatite and green fluorescent protein plasmid gene pGFP, with non-covalent mode combination, form inorganic nano Gene delivery system, for gene transfection.The gene transfer material of this nanometer hydroxyapatite after being combined with green fluorescent protein plasmid gene pGFP again with DNA in calcium hydroxide and the positive polarity root of phosphate ion have an effect; can reach stable transfection efficiency; and can reduce the activity that high concentration calcium ion may cause, thereby the gene transfer material of the prepared nanometer hydroxyapatite of the present invention can further be applied in human body.Compared with prior art, the gene transfer material of the nanometer hydroxyapatite making of the present invention has the following advantages:
(1) there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right;
(2) hypotoxicity, because the gene transfer material of the nanometer hydroxyapatite of needle-like has good biocompatibility, cells survival rate is very high;
(3) favorable dispersity of the gene transfer material of this nanometer hydroxyapatite, meets the requirement to transfection;
(4) preparation cost is extremely low, the main active ingredient of extracting because of the gene transfer material of this nanometer hydroxyapatite---polyphenolic compound cheap; And reaction is all the common pharmaceutical chemicals being easy to get with reagent, if calcium hydroxide and phosphoric acid are all cheap reagent;
(5) material preparation feedback is simple, easily operation, favorable repeatability;
(6) application prospect is good, can be applied to and prepare antitumor drug.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of the gene transfer material of the preparation method's of the gene transfer material of a kind of functionalized nano hydroxyapatite of the present invention embodiment 1 prepared nanometer hydroxyapatite.
Fig. 2 is the fluorogram under the gene transfer material of the nanometer hydroxyapatite of the present invention in transfection experiment and the association of the pEGFP-C1 inverted fluorescence microscope after transfection.
Fig. 3 is the gene transfer material of the nanometer hydroxyapatite of the present invention in transfection experiment and the cell survival rate figure of the association of pEGFP-C1 after transfection.
Fig. 4 is the transfection efficiency figure of the gene transfer material of the nanometer hydroxyapatite of the present invention in transfection experiment and the association of pEGFP-C1.
Wherein, in Fig. 3 to Fig. 4, cell viability(%) represent cell survival rate, transfection efficiency (%) represents transfection efficiency.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1.
A preparation method for the gene transfer material of functionalized nano hydroxyapatite, it comprises the following steps:
Step 1, the preparation of needle-like hydroxyapatite: the aqueous solution of calcium hydroxide is slowly joined in the aqueous solution of dilute phosphoric acid, and at the temperature of 37 DEG C, stir 24 hours, then leave standstill and filter after 2 hours, obtain particulate matter, then particulate matter is washed after also vacuum-drying, obtain needle-like hydroxyapatite.
Wherein, in step 1, the mol ratio of the phosphoric acid in the calcium hydroxide in the aqueous solution of calcium hydroxide and the aqueous solution of dilute phosphoric acid is 5:3.Wherein, in the aqueous solution of calcium hydroxide, the volumetric molar concentration of calcium hydroxide is 0.17mol/L.In the aqueous solution of dilute phosphoric acid, the volumetric molar concentration of dilute phosphoric acid is 0.15mol/L.
Step 2, absorption cis-platinum: cis diamino dichloro network platinum is dissolved in ultrapure water, then sonic oscillation is after 30 minutes, the needle-like hydroxyapatite that adds step 1 to make, and at the temperature of 37 DEG C, stir after 48 hours, carry out centrifugal and washing, obtain the gene transfer material of nanometer hydroxyapatite.
Wherein, the mass ratio of the needle-like hydroxyapatite in step 2 and cis diamino dichloro network platinum is 50:3.Wherein, to be dissolved in the quality-volumetric concentration of ultrapure water be 0.4mg/mL to cis diamino dichloro network platinum.
The gene transfer material that utilizes the functionalized nano hydroxyapatite that the preparation method of the gene transfer material of 1 one kinds of functionalized nano hydroxyapatites of the present embodiment makes, the gene transfer material of this nanometer hydroxyapatite is that a kind of mean diameter is the nanometer needle-like material of 100nm.
The gene transfer material of the prepared functionalized nano hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite of the present embodiment 1 is for making the application of antitumor drug, there is hypotoxicity, good dispersiveness and there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right.
embodiment 2.
A preparation method for the gene transfer material of functionalized nano hydroxyapatite, it comprises the following steps:
Step 1, the preparation of needle-like hydroxyapatite: the aqueous solution of calcium hydroxide is slowly joined in the aqueous solution of dilute phosphoric acid, and at the temperature of 33 DEG C, stir 26 hours, then leave standstill and filter after 1.5 hours, obtain particulate matter, then particulate matter is washed after also vacuum-drying, obtain needle-like hydroxyapatite.
Wherein, in step 1, the mol ratio of the phosphoric acid in the calcium hydroxide in the aqueous solution of calcium hydroxide and the aqueous solution of dilute phosphoric acid is 4:2.Wherein, in the aqueous solution of calcium hydroxide, the volumetric molar concentration of calcium hydroxide is 0.1mol/L.In the aqueous solution of dilute phosphoric acid, the volumetric molar concentration of dilute phosphoric acid is 0.1mol/L.
Step 2, absorption cis-platinum: cis diamino dichloro network platinum is dissolved in ultrapure water, then sonic oscillation is after 20 minutes, the needle-like hydroxyapatite that adds step 1 to make, and at the temperature of 33 DEG C, stir after 50 hours, carry out centrifugal and washing, obtain the gene transfer material of nanometer hydroxyapatite.
Wherein, the mass ratio of the needle-like hydroxyapatite in step 2 and cis diamino dichloro network platinum is 40:1.Wherein, to be dissolved in the quality-volumetric concentration of ultrapure water be 0.2mg/mL to cis diamino dichloro network platinum.
The gene transfer material that utilizes the functionalized nano hydroxyapatite that the preparation method of the gene transfer material of 2 one kinds of functionalized nano hydroxyapatites of the present embodiment makes, the gene transfer material of this nanometer hydroxyapatite is that a kind of mean diameter is the nanometer needle-like material of 80nm.
The gene transfer material of the prepared functionalized nano hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite of the present embodiment 2 is for making the application of antitumor drug, there is hypotoxicity, good dispersiveness and there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right.
embodiment 3.
A preparation method for the gene transfer material of functionalized nano hydroxyapatite, it comprises the following steps:
Step 1, the preparation of needle-like hydroxyapatite: the aqueous solution of calcium hydroxide is slowly joined in the aqueous solution of dilute phosphoric acid, and at the temperature of 40 DEG C, stir 22 hours, then leave standstill and filter after 2.5 hours, obtain particulate matter, then particulate matter is washed after also vacuum-drying, obtain needle-like hydroxyapatite.
Wherein, in step 1, the mol ratio of the phosphoric acid in the calcium hydroxide in the aqueous solution of calcium hydroxide and the aqueous solution of dilute phosphoric acid is 6:4.Wherein, in the aqueous solution of calcium hydroxide, the volumetric molar concentration of calcium hydroxide is 0.3mol/L.In the aqueous solution of dilute phosphoric acid, the volumetric molar concentration of dilute phosphoric acid is 0.3mol/L.
Step 2, absorption cis-platinum: cis diamino dichloro network platinum is dissolved in ultrapure water, then sonic oscillation is after 40 minutes, the needle-like hydroxyapatite that adds step 1 to make, and at the temperature of 40 DEG C, stir after 45 hours, carry out centrifugal and washing, obtain the gene transfer material of nanometer hydroxyapatite.
Wherein, the mass ratio of the needle-like hydroxyapatite in step 2 and cis diamino dichloro network platinum is 60:5.Wherein, to be dissolved in the quality-volumetric concentration of ultrapure water be 0.6mg/mL to cis diamino dichloro network platinum.
The gene transfer material that utilizes the functionalized nano hydroxyapatite that the preparation method of the gene transfer material of 3 one kinds of functionalized nano hydroxyapatites of the present embodiment makes, the gene transfer material of this nanometer hydroxyapatite is that a kind of mean diameter is the nanometer needle-like material of 90nm.
The gene transfer material of the prepared functionalized nano hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite of the present embodiment 3 is for making the application of antitumor drug, there is hypotoxicity, good dispersiveness and there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right.
The gene transfer material of the nanometer hydroxyapatite that above-described embodiment 1 is made is for following experiment.
the mensuration of bonding properties
1, main raw
The gene transfer material (see figure 1) of the nanometer hydroxyapatite 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 solution (gene transfer material of 5mg needle nano-hydroxy apatite is dissolved in 500 μ L distilled waters) of the gene transfer material of 1 μ L needle nano-hydroxy apatite 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 in room temperature and within lower 30 minutes, allows its abundant combination.The gene transfer material of needle nano-hydroxy apatite and the mass ratio of pEGFP-C1 are respectively 100:1,50:1,30:1,10:1,5:1,1:1.Under the speed of 5000rpm, centrifugal 5min, is precipitated thing.Throw out is loaded in 1% agarose (EB0.1 mg/mL), and under the buffering of TAE, under 100V voltage, runs 40min, then observe band at 320nm place.
3, result
Result is observed, and has multiple band to occur, this is because pEGFP-C1 has due to multiple configuration.At mass ratio 30:1,10:1,5:1,1:1 place band is clear obviously, illustrate under these mass ratioes, the gene transfer material particle of DNA and nanometer hydroxyapatite, in conjunction with being not fine, has arrived 100:1,50:1 band disappears, and illustrates that the gene transfer material particle of DNA and nanometer hydroxyapatite is in conjunction with complete.
The above results shows: positively charged pEGFP-C1 and the gene transfer material of nanometer hydroxyapatite exist a best combination ratio, when the gene transfer material of nanometer hydroxyapatite and the mass ratio of pEGFP-C1 are greater than 50:1, continue to add the gene transfer material of nanometer hydroxyapatite, green fluorescent protein plasmid can be not again combined with the gene transfer material of unnecessary nanometer hydroxyapatite yet, therefore, this experimental result draws, carries out transfection taking the gene transfer material of nanometer hydroxyapatite and the mass ratio of pEGFP-C1 as the combination ratio of 50:1.
transfection experiment
1, main raw
Human breast cancer cell; The gene transfer material of needle nano-hydroxy apatite and pEGFP-C1 association (above-mentioned preparation); DMEM substratum; Foetal calf serum FBS; 6 well culture plates.
2, main method
(1) cultivation of cell: human breast cancer cell is digested with trypsinase-EDTA, join after counting in (5 × 106/ hole) in 6 orifice plates, 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: cultured cell conditioned medium 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, be the gene transfer material of needle nano-hydroxy apatite and the association of pEGFP-C1 of 50:1 toward adding mass ratio in the second sample, 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
Fluorogram from Fig. 2 can be found out obvious green fluorescence, and the coverage rate of green fluorescence is larger, illustrates that the gene transfer material of needle nano-hydroxy apatite and the association of pEGFP-C1 dye 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 (nano) and the 3rd sample (lipofectamine 2000), wherein, the survival rate of the first sample is 98%, the survival rate that the survival rate of the second sample is about 87%, the three sample is 75%.Therefore, as can see from Figure 3, mass ratio is that the impact of the gene transfer material of needle nano-hydroxy apatite and the association of the pEGFP-C1 survival rate on human breast cancer cell of 50:1 is very little, by contrast, the impact of the survival rate of the association of liposome 2000 and pEGFP-C1 on human breast cancer cell is larger.
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 (nano) and the 3rd sample (lipofectamine 2000).By seeing in Fig. 4, the gene transfer material of needle nano-hydroxy apatite and the association of pEGFP-C1 can reach approximately 40% transfection efficiency, and this is to ensure that human breast cancer cell survival rate is about the transfection efficiency that 87% prerequisite is issued to.Although the transfection efficiency of the gene transfer material of needle nano-hydroxy apatite and the association of pEGFP-C1 is high not as good as the transfection efficiency of liposome 2000, but the high cell compatibility of the gene transfer material of needle nano-hydroxy apatite and the association of pEGFP-C1 is that liposome 2000 is incomparable.
Eventually the above, from above-mentioned experimental result and analysis, the gene transfer material of needle nano-hydroxy apatite has huge potentiality as transfection reagent.
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.
Claims (10)
1. a preparation method for the gene transfer material of functionalized nano hydroxyapatite, is characterized in that: it comprises the following steps:
Step 1, the preparation of needle-like hydroxyapatite: the aqueous solution of calcium hydroxide is slowly joined in the aqueous solution of dilute phosphoric acid, and stir at a certain temperature certain hour, then after leaving standstill certain hour, filter, obtain particulate matter, then particulate matter is washed after also vacuum-drying, obtain needle-like hydroxyapatite;
Step 2, absorption cis-platinum: cis diamino dichloro network platinum is dissolved in ultrapure water, then after sonic oscillation certain hour, the needle-like hydroxyapatite that adds step 1 to make, and stir at a certain temperature after certain hour, carry out centrifugal and washing, obtain the gene transfer material of nanometer hydroxyapatite.
2. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, it is characterized in that: in step 1, the mol ratio of the phosphoric acid in the calcium hydroxide in the aqueous solution of described calcium hydroxide and the aqueous solution of described dilute phosphoric acid is 4 ~ 6:2 ~ 4.
3. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, is characterized in that: in step 1, in the aqueous solution of described calcium hydroxide, the volumetric molar concentration of described calcium hydroxide is 0.1mol/L ~ 0.3 mol/L.
4. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, is characterized in that: in step 1, in the aqueous solution of described dilute phosphoric acid, the volumetric molar concentration of described dilute phosphoric acid is 0.1mol/L ~ 0.3 mol/L.
5. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, is characterized in that: in step 1, described whipping temp is 33 DEG C ~ 40 DEG C, and described churning time is 22 hours ~ 26 hours; Described time of repose is 1.5 hours ~ 2.5 hours.
6. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, is characterized in that: in step 2, the mass ratio of described needle-like hydroxyapatite and described cis diamino dichloro network platinum is 40 ~ 60:1 ~ 5.
7. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, it is characterized in that: in step 2, quality-volumetric concentration that described cis diamino dichloro network platinum is dissolved in ultrapure water is 0.2mg/mL ~ 0.6 mg/mL.
8. the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite according to claim 1, is characterized in that: in step 2, the time of described sonic oscillation is 20 minutes ~ 40 minutes; Described whipping temp is 33 DEG C ~ 40 DEG C, and described churning time is 45 hours ~ 50 hours.
9. the gene transfer material that utilizes the functionalized nano hydroxyapatite that the preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite described in claim 1-8 any one makes, is characterized in that: the gene transfer material of described nanometer hydroxyapatite is that a kind of mean diameter is the nanometer needle-like material of 80nm ~ 100nm.
10. the gene transfer material of the prepared functionalized nano hydroxyapatite of preparation method of the gene transfer material of a kind of functionalized nano hydroxyapatite described in claim 1 to 8 any one is for making the application of antitumor drug.
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