CN102698290A - Gene transfection method based on polyamide arborescent macromolecule carrier - Google Patents
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- CN102698290A CN102698290A CN201210183556XA CN201210183556A CN102698290A CN 102698290 A CN102698290 A CN 102698290A CN 201210183556X A CN201210183556X A CN 201210183556XA CN 201210183556 A CN201210183556 A CN 201210183556A CN 102698290 A CN102698290 A CN 102698290A
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Abstract
The invention relates to a gene transfection method based on a polyamide arborescent macromolecule carrier. The gene transfection method comprises the followings: lactobionic acid decoration to the surface of polyamide arborescent macromolecule to obtain G5. NH2-La; (2), preparation of lactobionic acid decorated polyamide arborescent macromolecule carrier and plasmid compound so as to obtain G5.NH2-La and plasmid compound G5.NH2-La/p DNA; and (3), lactobionic acid decorated polyamide arborescent macromolecule plasmid compound targeting gene transfer and expression to hepatoma carcinoma cells. The functionalized polyamide arborescent macromolecule plays a targeting effect on the hepatoma carcinoma cells; and the gene transfection method provided by the invention has the advantages of temperate transfection conditions, easiness in operation, high transfection efficiency, excellent specificity and the like, and has a wide application prospect.
Description
Technical field
The invention belongs to high molecular nanometer carrier target gene transfection field, particularly relate to a kind of gene transfection method based on the polyamide-amine dendrimer carrier.
Background technology
At present, the expression regulation of cell comes into one's own day by day, and wherein gene transfection is a kind of important means that realizes the cellular expression regulation and control.The carrier that is used for gene transfection is divided into viral vector and non-virus carrier.Though viral vector has higher transfection efficiency, because of itself exist immunogenicity, high toxicity and can not large-scale production etc. shortcoming be restricted.Non-virus carrier, especially polyamide-amine dendrimer (PAMAM) because its low toxicity, efficient, can large-scale production etc. advantage and coming into one's own day by day.In the expression regulation of specific part (like hepatic tissue), many genophores can reduce the efficient of cellular expression regulation and control, even can cause unnecessary trouble owing to can't be targeted to particular organization.It is reported that the PAMAM dendrimer not only can improve transfection efficiency can also improve its transfection specificity after modifying.[Santos?JL.,Pandita?D,Rodrigues?J,et?al.,Receptor-mediated?gene?delivery?using?PAMAM?dendrimers?conjugated?with?peptides?recognized?by?mesenchymal?stem?cells.Mol?Pharmaceutics,2010,7,763-774.]。In recent years, develop a kind of nano-carrier, realize the targeted expression regulation and control of HCC, become the focus and the difficult point of research with HCC target function.
(asialoglycoprotein receptor ASGPR) can discern galactose residue and the combination with it that molecular end has by specificity to the asialoglycoprotein receptor that HCC film surface specific is expressed.[Guo R, Yao Y, Cheng G such as Guo R.; Et al.; Synthesis of glycoconjugated poly (amindoamine) dendrimers for targeting human liver cancer cells, RSC Advances, 2012; 2,99-102.] reported that the polyamide-amine dendrimer that lactobionic acid is modified has good targeting to HCC.
Daiamid (PAMAM) dendrimer is a kind of highly branched single macromole that disperses, and it has good water-solubility, under using dosage, does not have cytotoxicity and immunogenicity.There are a large amount of controllable function groups on PAMAM dendrimer surface, can connect or some targeting groups of physical absorption, antibody or drug molecule etc. by chemistry.Lactobionic acid (La) is a kind of micromolecule that contains galactose residue, and the surface that can be modified at nano-carrier or macromolecular material is to improve its targeting ability to HCC.Therefore, lactobionic acid is modified at PAMAM dendrimer surface, is expected to prepare a kind of genophore, realize the targeted expression regulation and control of HCC with HCC target function.
Retrieving domestic and international pertinent literature and patent results shows: the polyamide-amine dendrimer of modifying with lactobionic acid is a targeting vector, is used for the method for hepatoma-targeting gene transfection, does not appear in the newspapers as yet.
Summary of the invention
Technical problem to be solved by this invention provides a kind of gene transfection method based on the polyamide-amine dendrimer carrier, and this gene transfection method has the transfection conditions gentleness, easy operating, transfection efficiency advantages of higher.
A kind of gene transfection method based on the polyamide-amine dendrimer carrier of the present invention comprises:
(1) lactobionic acid on polyamide-amine dendrimer (PAMAM) surface is modified:
Lactobionic acid La is dissolved in the phosphate buffer, adds 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy thiosuccinimide NHS, strong magnetic agitation reaction 1-5 hour obtains the good lactobionic acid solution of activation; With the 5th generation polyamide-amide dendrimer G5.NH
2(Sigma company) is dissolved in the distilled water, obtains G5.NH
2Solution, then that above-mentioned activation is good lactobionic acid solution adds G5.NH
2In the solution, the magnetic agitation reaction is 1-5 days in the room temperature; After reaction finishes,, carry out lyophilization at last, obtain product G5.NH the product dialysis
2-La;
(2) polyamide-amine dendrimer (G5.NH of lactobionic acid modification
2-La) with the preparation of plasmid composite:
The G5.NH that step (1) is obtained
2-La dilutes with sterilized water, obtains G5.NH
2The sterilized water diluent of-La; With sterilized water pDNA is diluted, obtain the sterilized water diluent of pDNA, then with described G5.NH
2The sterilized water diluent mix homogeneously of the sterilized water diluent of-La and pDNA was hatched 20-30 minute in 37 ℃ again, obtained G5.NH
2The complex G5.NH of-La and plasmid
2-La/pDNA;
(3) cell transfecting of the polyamide-amine dendrimer plasmid composite of lactobionic acid modification:
HepG2 cell (HCC) is laid in the 6-96 well culture plate; Adopt the MEM culture medium do not contain FBS (contain Earle ' s salt-mixture, 2mM glutamine, the nonessential aminoacid of 0.1mM, 100U/mL penicillin, 100 μ g/mL streptomycins), in 37 ℃, 5%CO
2(CO in the incubator
2Volumetric concentration) cultivated 20-30 hour, evenly be added dropwise to G5.NH then
2-La/pDNA complex solution shakes up, and cultivates after 2-5 hour for 37 ℃ to be replaced by the MEM culture medium that contains 10% (mass concentration) FBS, continues to cultivate 20-30 hour, gets final product.
The pH value of the phosphate buffer described in the step (1) is 6.0.
The amount ratio of lactobionic acid described in the step (1) and phosphate buffer is 15.7mg:2mL.
The mol ratio of the EDC described in the step (1), NHS and lactobionic acid carboxyl is about 5:5:1.
G5.NH described in the step (1)
2G5.NH in the solution
2Concentration be 10.4mg/mL.
Lactobionic acid and G5.NH described in the step (1)
2Mol ratio be 7:1～15:1.
Dialysis described in the step (1) is that to be transferred to molecular cut off be in 14000 the bag filter, and dialysis is 2 days in distilled water (2L * 6, every day 3 times).
The concentration of pDNA is 0.02-0.10g/L in the sterilized water diluent of pDNA described in the step (2).
Resulting G5.NH in the step (2)
2G5.NH among the-La/pDNA
2Primary amino radical and pDNA skeleton on phosphate group mol ratio (N/P) be 0.25-5:1.
Expression time is 24～48 hours behind the cell transfecting described in the step (3).
The end group that the present invention modifies with lactobionic acid is that the 5th generation daiamid (PAMAM) dendrimer of amino is the gene transfection carrier, as target cell, carries out gene transfection with HCC HepG2.The present invention characterizes through the type and the quantity of nuclear magnetic resonance, NMR (NMR) to the dendrimer surface group.Gel retardation assasy, AFM (AFM), hydrodynamics particle diameter and surface potential characterize the ability of gene transfection carrier and pDNA formation complex.In addition, with the plasmid (Luc) of luciferase labelling and the plasmid (EGFP) of green fluorescent protein labelling the gene transfection ability of gene transfection carrier is measured.G5.NH with contrast without the lactobionic acid modification
2The transfection comparison of carrier; The genophore of the present invention's preparation can improve the transfection ability to HCC significantly, so the polyamide-amine dendrimer with hepatoma-targeting function of the present invention's preparation can be used for the target gene of HCC is carried as genophore.
The result of nuclear magnetic resonance, NMR (NMR), gel retardation assasy, AFM (AFM), hydrodynamics particle diameter and surface potential, luciferase gene and green fluorescence protein gene transfection experiment distinguishes as follows:
(1)
1H NMR test result
1H NMR collection of illustrative plates is used to characterize the modification of lactobionic acid to the dendrimer surface.Corresponding to the lactobionic acid molecule, corresponding chemical shift peak is carried out integration referring to the proton peak in the Figure of description 1:3.40-4.10ppm scope, 10.4 the lactobionic acid molecules that shown each dendrimer finishing.Provable thus lactobionic acid successfully is modified at the dendrimer surface, has obtained the synthetic G5.NH of design
2-La targeting vector.
(2) gel retardation assasy test result
Gel retardation assasy is the result show, G5.NH
2-La and G5.NH
2All has good compound ability with DNA.Referring to Figure of description 2.The result shows: G5.NH
2-La and G5.NH
2All can be fine compound with DNA down at low N/P (N/P=1), with the DNA retardance, and then possibility is provided for DNA transfection entering cell.
(3) AFM test result
AFM tests the surface topography of dendrimer/plasmid composite sample, referring to Figure of description 3.The result shows: complex is all spherical in shape, shows G5.NH
2-La and G5.NH
2All can complex plasmid DNA, make gene transfection is got into cell to become possibility.In addition, G5.NH
2The size ratio G5.NH of/DNA complex
2The particle diameter of-La/DNA complex is big, shows G5.NH
2-La has higher compressed capability to DNA.
(4) hydrodynamics particle diameter and surface potential test result
Test result is shown in Figure of description 4.The result shows, G5.NH
2/ DNA complex and G5.NH
2The size of-La/DNA complex is in the size range of suitable transfection.Since the introducing of lactobionic acid, under the identical N/P, G5.NH
2The electromotive force of-La/DNA complex is than G5.NH
2The electromotive force of/DNA complex is low, but still in the potential range that is fit to transfection.
(5) luciferase gene transfection experiment
With the plasmid of luciferase labelling and to have the human liver cancer cell HepG2 cell of expressing than strong ASGPR is that model cell is checked two kinds of material G5.NH
2With G5.NH
2-La is to the transfection effect of HCC.Referring to Figure of description 5.Test result shows: the transfection efficiency of two kinds of materials is all relevant with N/P, and under identical N/P, G5.NH
2The transfection efficiency of-La is apparently higher than G5.NH
2Transfection efficiency.When N/P=2.5, G5.NH
2The transfection efficiency of-La is the highest.Show G5.NH
2-La has better targeting transfection effect to the HepG2 cell.
(6) enhanced green fluorescent protein (EGFP) gene transfection experiment
With the plasmid of green fluorescent protein labelling and to have the human liver cancer cell HepG2 that expresses than strong ASGPR is that model cell is further checked two kinds of material G5.NH
2With G5.NH
2-La is to the targeting transfection effect of HCC.Referring to Figure of description 6.For having the HepG2 cell that strong ASGPR expresses, modified the material G5.NH of lactobionic acid
2-La can get into cell with the EGFP plasmid transfection better, and cell has stronger green fluorescence signal representation; And the material G5.NH of unmodified lactobionic acid
2Efficient to the EGFP plasmid transfection is lower, and cell does not almost have tangible fluorescence signal.This result proves: the G5.NH that has only lactobionic acid to modify
2-La has specific targeting effect to HCC, and the material of unmodified does not have the targeting effect to HCC.
Beneficial effect
(1) the functionalization polyamide-amine dendrimer of this method preparation has targeting to HCC, can be used for the targeted of gene molecule.
(2) gene transfection method of the present invention has the transfection conditions gentleness, easy operating, and advantage such as transfection efficiency is high, specificity is good has good application prospects at the aspects such as targeted expression regulation and control of HCC.
Description of drawings
Fig. 1 is the G5.NH of the present invention's preparation
2The hydrogen nuclear magnetic resonance spectrogram of-La;
Fig. 2 is the G5.NH of the present invention's preparation
2/ DNA complex (a) and G5.NH
2The gel retardation assasy electrophoresis pattern of-La/DNA complex (b);
Fig. 3 is the G5.NH of the present invention's preparation
2/ pDNA complex AFM collection of illustrative plates (a) and G5.NH
2-La/pDNA complex AFM collection of illustrative plates (b);
Fig. 4 is the G5.NH of the present invention's preparation
2-La and pDNA form the particle diameter (a) and the electromotive force collection of illustrative plates (b) of complex; G5.NH
2/ pDNA complex is a matched group.
Fig. 5 is G5.NH
2With G5.NH
2Two kinds of materials of-La under different N/P to the luciferase gene transfection efficiency comparison diagram of HepG2 cell;
Fig. 6 is G5.NH
2(a, b) and G5.NH
2-La (c, d) two kinds of materials to the light field photo of the green fluorescence protein gene transfection of HepG2 cell (a, c) and fluorescence photo (b, d).
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Take by weighing lactobionic acid (La) 15.7mg, be dissolved in (pH=6.0) in the 2mL phosphate buffer.Add five times of normal EDC of lactobionic acid carboxyl molal quantity (0.30M, 0.726mL), NHS (0.25M, 0.892mL), strong magnetic agitation reaction 3 hours.
Take by weighing the 5th generation polyamide-amine dendrimer (G5.NH
2) 41.5mg, be dissolved in the 4mL distilled water, be mixed with the solution that concentration is 10.4mg/mL.According to La/G5.NH
2Mol ratio is 11/1, and the lactobionic acid solution that activation is good adds G5.NH
2In the solution, magnetic agitation in the room temperature was reacted 3 days.
After reaction finished, it was that dialysis is 2 days in distilled water (2L * 6, every day 3 times) in 14000 the bag filter that product is transferred to molecular cut off.Carry out lyophilization then and handle, obtain G5.NH
2-La product.To the product G 5.NH that obtains
2-La carries out nuclear-magnetism and characterizes, and the result sees description of drawings book 1.Proton peak in the 3.40-4.10ppm scope is carried out integral and calculating to corresponding proton peak and can be known corresponding to the lactobionic acid molecule, the dendrimer finishing 10.4 lactobionic acid molecules.Therefore, lactobionic acid successfully is modified at the dendrimer surface, has obtained the synthetic G5.NH of design
2-La targeting vector.
G5.NH according to the preparation of the method for embodiment 1
2-La and the 5th generation end group be amino polyamide-amine dendrimer G5.NH
2Carry out gel retardation assasy with pDNA.Prepare the agarose gel (1.0%w/v) that contains ethidium bromide (0.1 μ g/mL) in 8 holes, room temperature is placed and is treated that agarose gel solidifies.With 1 μ g DNA is example, is contrast respectively according to different N/P (0.25,0.5,1,2,3,4,5) formulation vehicle/DNA complex, and with the naked DNA.Get the G5.NH that embodiment 1 obtains
2-La is diluted to 50 μ L with sterilized water, with sterilized water 1 μ g DNA is diluted to 50 μ L then, mix homogeneously then, and final volume is 100 μ L, hatches 30 minutes for 37 ℃.Then corresponding vectors/DNA complex is added in the hole of agarose gel voltage 80V, time 35min respectively.(UVP, USA) migration in gel is analyzed to DNA to use the gel imaging appearance.The result is shown in description of drawings book 2.The result shows, G5.NH
2-La and G5.NH
2All can be fine compound with DNA down at low N/P (N/P=1), with the DNA retardance, and then possibility is provided for DNA changes cell over to.
G5.NH according to the preparation of the method for embodiment 1
2-La and G5.NH
2With 1 μ g DNA (N/P=2.5) compound after, get the drips of solution of 5 μ L after compound to the mica sheet of suitable size, air drying.(Veeco Instruments) observes the surface topography of complex with AFM, and the result is shown in description of drawings book 3.The result shows that complex is all spherical in shape, shows G5.NH
2-La and G5.NH
2All can complex plasmid DNA, make gene transfection is got into cell to become possibility.In addition, G5.NH
2The size ratio G5.NH of/DNA complex
2The particle diameter of-La/DNA complex is big, shows G5.NH
2-La is better to the compression effectiveness of DNA.
G5.NH according to the preparation of the method for embodiment 1
2-La and G5.NH
2With 5 μ g DNA (N/P=1,2.5,5) compound after, use the deionized water adjusted volume to be 1mL respectively.Adopt Ma Erwen laser particle analyzer (Malvern, UK, 633nm exciting light) that its particle diameter and surface potential are characterized, the result is shown in description of drawings book 4.The result shows that along with the increase of N/P, the size of all complex all reduces.Under the identical N/P, G5.NH
2-La/DNA complex size is than G5.NH
2The size of/DNA complex is little.In addition along with the increase of N/P, G5.NH
2-La/DNA complex and G5.NH
2The surface potential of/DNA complex all increases to some extent.Since the introducing of lactobionic acid, under the identical N/P, G5.NH
2The electromotive force of-La/DNA complex is than G5.NH
2The electromotive force of/DNA complex is low, but still in the potential range that is fit to transfection.
With the plasmid of luciferase labelling and to have the human liver cancer cell HepG2 cell of expressing than strong ASGPR is that model cell is checked two kinds of material G5.NH
2With G5.NH
2-La is to the targeting transfection effect of HCC.Model cell is incubated in the culture fluid that does not contain FBS, adds certain density G5.NH
2/ DNA and G5.NH
2-La/DNA complex and co-culture of cells 4 hours are changed the culture medium that contains 10%FBS subsequently, continue to cultivate 24 hours.Uciferase activity is measured in lysis, and the result sees description of drawings book 5.Test result shows: the transfection efficiency of two kinds of materials is all relevant with N/P, and under identical N/P, G5.NH
2The transfection efficiency of-La is apparently higher than G5.NH
2Transfection efficiency.When N/P=2.5, G5.NH
2The transfection efficiency of-La is the highest.
With the plasmid of enhanced green fluorescent protein (EGFP) labelling and to have the human liver cancer cell HepG2 that expresses than strong ASGPR is that model cell is further checked two kinds of material G5.NH
2With G5.NH
2-La is to the targeting transfection effect of HCC.HepG2 cell (HCC) is laid on 24 orifice plates with proper density, in 37 ℃, 5% CO
2Cultivated 24 hours, with 100 μ L G5.NH
2-La/pDNA complex solution evenly is added drop-wise in 24 orifice plates, shakes up, and cultivates after 4 hours for 37 ℃ and is replaced by the MEM culture medium that contains 10%FBS (hyclone), continues to cultivate 24 hours.Observe with inverted fluorescence microscope, the result sees description of drawings book 6.For having the HepG2 cell that strong ASGPR expresses, modified the material G5.NH of lactobionic acid
2-La can get into cell with the EGFP plasmid transfection better, and cell has stronger green fluorescence signal representation; And the material G5.NH of unmodified lactobionic acid
2Efficient to the EGFP plasmid transfection is lower, and cell does not almost have tangible fluorescence signal.This result proves: the G5.NH that has only lactobionic acid to modify
2-La has specific targeting effect to HCC, and the material of unmodified does not have the targeting effect to HCC.
Claims (9)
1. gene transfection method based on the polyamide-amine dendrimer carrier comprises:
(1) lactobionic acid La is dissolved in the phosphate buffer, adds 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, stirring reaction 1-5 hour, obtain the good lactobionic acid solution of activation; With the 5th generation polyamide-amide dendrimer G5.NH
2Be dissolved in the distilled water, obtain G5.NH
2Solution, then that above-mentioned activation is good lactobionic acid solution adds G5.NH
2In the solution, in the room temperature stirring reaction 1-5 days; After reaction finishes,, carry out lyophilization at last, obtain product G5.NH the product dialysis
2-La;
(2) G5.NH that step (1) is obtained
2-La dilutes with sterilized water, obtains G5.NH
2The sterilized water diluent of-La; With sterilized water pDNA is diluted, obtain the sterilized water diluent of pDNA, then with described G5.NH
2The sterilized water diluent mix homogeneously of the sterilized water diluent of-La and pDNA was hatched 20-30 minute in 37 ℃ again, obtained G5.NH
2The complex G5.NH of-La and plasmid
2-La/pDNA;
(3) the HepG2 cell is laid in the 6-96 well culture plate, adopts the MEM culture medium that does not contain FBS, in 37 ℃, 5%CO
2Cultivated 20-30 hour, and be added dropwise to G5.NH then
2-La/pDNA complex solution shakes up, and cultivates after 2-5 hour for 37 ℃ to be replaced by the MEM culture medium that contains 10%FBS, continues to cultivate 20-30 hour, gets final product.
2. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the pH value of the phosphate buffer described in the step (1) is 6.0.
3. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the amount ratio of lactobionic acid described in the step (1) and phosphate buffer is 15.7mg:2mL.
4. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the mol ratio of the EDC described in the step (1), NHS and lactobionic acid carboxyl is 5:5:1.
5. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the G5.NH described in the step (1)
2G5.NH in the solution
2Concentration be 10.4mg/mL.
6. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: lactobionic acid and the G5.NH described in the step (1)
2Mol ratio be 7:1～15:1.
7. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the dialysis described in the step (1) is that to be transferred to molecular cut off be in 14000 the bag filter, and dialysis is 2 days in distilled water.
8. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: the concentration of pDNA is 0.02-0.10g/L in the sterilized water diluent of the pDNA described in the step (2).
9. a kind of gene transfection method based on the polyamide-amine dendrimer carrier according to claim 1 is characterized in that: resulting G5.NH in the step (2)
2G5.NH among the-La/pDNA
2Primary amino radical and pDNA skeleton on the phosphate group mol ratio be 0.25-5:1.
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| CN103435815B (en) * | 2013-07-11 | 2015-11-18 | 东华大学 | A kind of functionalization Polyamidoamine Dendrimers and nano-complex thereof are used for the method for gene transfection |
| CN106632748A (en) * | 2016-09-21 | 2017-05-10 | 武汉理工大学 | Lactose-based intelligent polymer and application thereof |
| CN106620701A (en) * | 2016-12-30 | 2017-05-10 | 东华大学 | Preparation method of G5-MoS2/Bcl-2 siRNA compound |
| CN107142281A (en) * | 2017-06-07 | 2017-09-08 | 东华大学 | The compound of polyamide-amine dendrimer and nanogold particle carries out the application process of gene transfection as non-virus carrier |
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