CN107142281A - The compound of polyamide-amine dendrimer and nanogold particle carries out the application process of gene transfection as non-virus carrier - Google Patents
The compound of polyamide-amine dendrimer and nanogold particle carries out the application process of gene transfection as non-virus carrier Download PDFInfo
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- CN107142281A CN107142281A CN201710422901.3A CN201710422901A CN107142281A CN 107142281 A CN107142281 A CN 107142281A CN 201710422901 A CN201710422901 A CN 201710422901A CN 107142281 A CN107142281 A CN 107142281A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/028—Polyamidoamines
Abstract
The invention discloses the application process that the compound of a kind of polyamide-amine dendrimer and nanogold particle carries out gene transfection as non-virus carrier, it is characterized in that, the surface of the dendrimer is amino terminal, and it, which is chemically modified, can reduce its toxicity and improve transfection efficiency；Its end connection targeting group includes folic acid, and RGD, hyaluronic acid can efficiently carry out gene delivery.Polyamide-amine dendrimer and its nanogold particle compound prepared by the present invention has good gene transfection；Transfection process is easily operated, and good biocompatibility, immunogenicity is low, and being non-viral gene vector, applying on gene delivery provides reference.
Description
Technical field
Enter the present invention relates to the compound of a kind of Polyamidoamine Dendrimers and nanogold particle as non-virus carrier
The application process of row gene transfection, the Polyamidoamine Dendrimers parcel of more particularly to a kind of polyethylene glycol and modified with folic acid
Nanogold particle carries out the application process of gene transfection and immunological safety research as non-virus carrier, belongs to functionalization high score
Sub- nano material Gene transfer vector technical field.
Background technology
Gene therapy refers to reach the method for treating or preventing disease by the operation of gene level.At present, gene is controlled
Treat and treated primarily directed to the serious disease for threatening human health, cancer is its main application fields.Gene therapy technology
Nowadays quickly grow, portion gene therapeutic scheme comes into clinical experimental stage.In clinical test, it was found that many safety
The problems such as hidden danger, such as its security, targeting, relatively low transfer efficiency, not yet solves.
At present, two parts, virus carrier system and non-viral carrier systems are broadly divided into gene vector system.Virus
Carrier is because its efficient transfection efficiency is so as to applied to gene therapy clinically.But it is due to its small volume, so delivery
The limited amount of gene, and easily produce gene mutation and immunogenicity.However, the biological safety of non-virus carrier is good,
Immunogenicity is low.The successive appearance of its product such as with liposome, polymer and DNA compounds, promotes non-virus carrier in gene
Extensive use in treatment.
In non-viral carrier systems, dendrimer can be as a kind of preferable Application of micron in gene delivery system
System.It is different from traditional linear polymerization species nano material, with unique highly branched 3-D solid structure, there is good
Monodispersity.Dendrimer passes through the various modifications that come to the surface, and such as Pegylation, acetylation and alkylation are generated not
Same physicochemical property.Research shows that the 5th generation dendrimer has certain cytotoxicity, but forms multiple with nanogold particle
Compound [Shan Y., Luo T., Peng C., et al., Gene delivery using dendrimer-entrapped
Gold nanoparticles as nonviral vectors [J] Biomaterials, 2012,33 (10)：p.3025-
3035.] toxicity of carrier can be reduced.[Xiao T., Hou W., Cao X., the et al., Dendrimer- such as Xiao
entrapped gold nanoparticles modified with folic acid for targeted gene
Delivery applications.Biomaterials Science, 2013,1 (11)：P.1172.] by modified with folic acid to
Five generation dendrimer (PAMAM) surfaces, higher efficiency gene transfection is generated with gene under special ratios.
The immunological safety transfected from molecular biology angle analysis gene, wherein real time fluorescent quantitative reverse transcription PCR skill
Art is that the important detection means of gene expression amount is detected on gene transcription level, is widely used in biomedical sector.It is glimmering in real time
Light Quantitative Reverse Transcription round pcr typically uses 2-△△CtMethod, experiment with computing group and the relative expression quantity of gene in control group.Formula
It is △ △ Ct=(Ct target gene-Ct crt genes) experimental group-(Ct target gene-Ct crt genes) control group, Ct values are
Fluorescence signal reaches system number of cycles during threshold value.
Retrieve domestic and international pertinent literature and patent results show：Polyethylene glycol and the common modification polyamide-amide of folic acid are tree-shaped
Macromolecular wraps up nanogold particle as genophore and carries out gene transfection, with real time fluorescent quantitative reverse transcription PCR technology pair
There is not been reported for immunological safety detection after gene transfection.
The content of the invention
The technical problems to be solved by the invention are to provide answering for a kind of Polyamidoamine Dendrimers and nanogold particle
Compound carries out the application process of gene transfection as non-virus carrier, and obtained compound can have good biocompatibility, thin
Born of the same parents' immunological safety and high efficiency gene transfection efficiency, there is preferable application prospect in terms of the gene therapy of malignant tumour.
In order to solve the above problems, the invention provides answering for a kind of Polyamidoamine Dendrimers and nanogold particle
Compound carries out the application process of gene transfection as non-virus carrier, it is characterised in that the surface of the dendrimer is ammonia
Base end, it, which is chemically modified, can reduce its toxicity and improve transfection efficiency；Its end connection targeting group includes folic acid,
RGD, hyaluronic acid can efficiently carry out gene delivery.
Preferably, the method for the chemical modification is modification polyethylene glycol, modification cyclodextrin, acetylation and the side of alkylation
Any one or a few in method.
Preferably, Au and G5.NH in the compound2Mol ratio be 25: 1 and more than.
It is highly preferred that Au and G5.NH in the compound2Mol ratio is 25: 1,50: 1,75: 1 or 100: 1.
Preferably, concentration of the compound in gene rotaring redyeing system is 1.0~2.0mg/mL.
Preferably, the transmission exogenous therapeutic gene of the gene delivery include pDNA, CpG DNA, GEM 132,
Any one or a few in siRNA and ssDNA.
It is highly preferred that the target cell of the exogenous therapeutic gene of the gene delivery includes cancer cell and body cell.
It is highly preferred that the cancer cell includes HeLa, U87 and A549；The body cell includes macrophage, and T lymphs are thin
Born of the same parents, bone-marrow-derived lymphocyte, BMDC.
It is highly preferred that the compound and DNA N/P ratios be 0.5: 1 and more than.
It is highly preferred that the compound and DNA N/P ratios are 0.5: 1,1: 1,2.5: 1,5: 1 or 7: 1.
It is highly preferred that the pDNA is extracted from Escherichia coli (E.coli DH5 α bacterial strains), plasmid blocks that to carry
Mycin resistant gene and the plasmid with Green fluorescent protein fusion vector；The CpG DNA are that by artificial synthesized, sequence is
5’-TCCATGACGTTCCTGATGCT-3’。
Preferably, the transfection time of the gene transfection is 4~12h.
Preferably, the preparation method of the compound of the Polyamidoamine Dendrimers and nanogold particle includes following
Specific steps：
Step 1)：{(Au0)50-G5.NH2-mPEG10Synthesis：MPEG (relative molecular mass is 2000) is dissolved in
In DMSO, EDC is mixed with mPEG according to the ratio uniform of mol ratio 10: 1, is reacted 30-40 min, is added and EDC equal proportions
NHS reaction 3-4h；By the mPEG activated and G5.NH2DMSO solution according to mol ratio 10: 1 ratio mix 72-
84h；In the product of above-mentioned reaction gold chloride HAuCl is added according to dendrimer with golden mol ratio for 1: 50 amount4Reaction
30min；Then NaBH is added4It is reduced, 3-4h is reacted under stirring condition；Reaction solution is dialysed 3-4 days, it is last chilled dry
It is dry to obtain sample { (Au0)50-G5.NH2-mPEG10, it is denoted as S1；
Step 2)：{(Au0)50-G5.NH2-PEG10-FA5Synthesis：FA and EDC is equal according to the ratio of mol ratio 1: 0.9
Even mixing, reacts 30-40min, and the NHS added with EDC equal proportions reacts 3-4h；By the PEG and FA that has activated according to mole
G5.NH is added to than 1: 2 ratio2In, lucifuge stirring 72-84h；It is 1: 50 addition according still further to dendrimer and golden mol ratio
HAuCl430min is reacted, then is rapidly added the NaBH of 5 times of amounts4It is reduced, 3-4h is stirred under the conditions of lucifuge；Reaction solution is saturating
Analysis 3-4 days, eventually passes freeze-drying and obtains the { (Au of sample 20)50-G5.NH2-PEG10-FA5, it is denoted as S2；Wherein HAuCl4It is molten
The concentration of liquid is 30.15mg/mL；NaBH4The concentration of solution is 10mg/mL；EDC concentration is 9.7mg/mL；NHS concentration is
5.05mg/mL；MPEG concentration is 4mg/mL；G5.NH2The concentration of solution is 0.44 μm of ol/mL, and FA concentration is 2mg/mL.
It is highly preferred that the step 1) with step 2) synthesis two kinds of samples as carrier be applied to HeLa gene turn
The method of dye (is set according to different N/P with pDNA from the compound of nanogold particle according to Polyamidoamine Dendrimers
The primary amino radical of shape macromolecular and the phosphate group mol ratio in pDNA molecules) be respectively 1: 1,2.5: 1,5: 1 prepare carrier with
PDNA compound；In 37 DEG C, 5%CO2Under the conditions of cultivate HeLa cells, add foregoing three kinds of N/P than compound/
PDNA complexs gene in cell transfects 4h.
Preferably, using real-time fluorescence quantitative PCR instrument or enzyme-linked immunosorbent assay gene transfection after compound or
Compound stimulates the relative expression quantity of immunocyte secrete cytokines with DNA.
It is highly preferred that the cell factor that PCR is expanded in the real-time fluorescence quantitative PCR instrument is IFN-α, IFN-β, IFN-
Any one or a few cell factor in γ, IL-1 β, IL-6, IL-10, TNF-α and IL-12.
It is highly preferred that the compound carries out cell factor IL- in RAW 264.7 (macrophage) after gene transfection 4h
6 and TNF-α cytokines measurement, step 1) with step 2) two kinds of samples preparing enter with pDNA and CpG DNA compounds respectively
Row gene is transfected, and RAW 264.7 cytokine TNF-α and IL-6 gene are detected with real time fluorescent quantitative reverse transcription PCR
Expression, to detect whether gene transfection causes cell immune response.
It is highly preferred that whether the detection gene transfection causes comprising the following steps that for cell immune response：
Step a)：The extraction of macrophage total nucleic acid：With every 1 × 106Individual macrophage kind adds into 6 orifice plates to every hole
Enter after compound or compound/DNA complex transfection 4h, be separately added into Total RNA Extractor 1mL；The sample of cracking
5-10min is placed at room temperature, 0.2mL chloroforms are added, and is acutely placed 3min after vibration, is then centrifuged under the conditions of 12000rpm
10min；Take upper strata aqueous phase to centrifuge tube, add isometric isopropanol, mix and place 20min；Centrifuged under the conditions of 12000rpm
10min, removes supernatant；1mL 75% ethanol washing precipitation is added, 3min is centrifuged in 12000rpm, removes supernatant；
Finally plus 30~50 μ L RNase-free ddH2O fully dissolves RNA, obtains RNA solution；
Step b)：Prepare reverse transcription sample：Following reagent is added into ice bath with reference to commercially available reverse transcription reagent box specification
PCR pipe：1 μ L RNA templates, 1 μ L primers, 1 μ L dNTP (0.5mM), RNase-free ddH2The μ L of O 10, gently mix after from
Heart 5s；Reactant mixture is after 65 DEG C of warm bath 5min, and ice bath 2min is then centrifuged for 3~5s；By PCR pipe ice bath, reagent is added
4 μ L 5 times of reverse transcription buffers, 0.5 μ L ribonuclease inhibitors and 1 μ L reverse transcriptases；Again gently mix after centrifugation 3~
5s；Reverse transcription reaction is carried out in PCR instrument：25 DEG C of incubations 10min, 50 DEG C of reaction 30min carry out cDNA synthesis, 85 DEG C of reactions
5min carries out terminating reaction, obtains reverse transcription sample；
Step c)：PCR is expanded：Following reactant mixture, 10 μ L2 × SuperReal PreMix are added under condition of ice bath
Plus, 0.6 μ L sense primers (10 μM), 0.6 μ L anti-sense primers (10 μM), 2.5 μ L cDNA templates (being diluted to 10pM), 0.4 μ
L 50 × Rox, RNase-free ddH2O adds to 20 μ L；In 95 DEG C of pre-degenerations 15min, 95 DEG C of denaturation 10s, 62 DEG C are annealed and prolonged
32s is stretched, 40 circulations are reacted；Last Instrumental Analysis goes out the Standard kinetic curve and solubility curve of amplified production.
The present invention carries DNA using two kinds of Polyamidoamine Dendrimers and its nanogold particle compound as carrier
Gene transfection is carried out to HeLa cells.By nuclear-magnetism, determination of uv absorption, transmission electron microscope to the physical chemistry of compound
Feature is characterized；The terminal amino group number of carrier is determined using terminal amino group quantification kit；CCK-8 detects carrier and pDNA
The cytotoxicity of compound；Agarose gel electrophoresis technology determines that carrier is completely combined the N/P ratio of gene；Pass through surface potential
The potential and particle diameter of carrier and pDNA compounds are analyzed with hydrodynamics particle diameter；The expression of green fluorescent protein is used
Carry out the efficiency that Study of Support transmits DNA；Flow Cytometry and Laser confocal scanning microscope technique be used for Study of Support with
The cell endocytic efficiency and cellular localization of gene composite；Examined using real time fluorescent quantitative inverse transcription polymerase chain reaction method
Survey the immunogenicity of carrier and gene composite.The present invention, can be with by mPEG and G5 PAMAM surface amino groups group covalent bonds
By neutralizing its part surface amino to reduce the cytotoxicity of compound, and improve the biocompatibility of compound.By even
Folic acid is connected, can make compound that there is targeting, improve efficiency gene transfection.
Polyamidoamine Dendrimers and its nanogold particle compound prepared by the present invention have good gene transfection
Effect；Transfection process is easily operated, and good biocompatibility, immunogenicity is low, is non-viral gene vector on gene delivery
Using offer reference.
Brief description of the drawings
Fig. 1 be embodiment 2 in compound nuclear magnetic spectrum；
Fig. 2 be embodiment 3 in compound ultraviolet absorpting spectrum；
Fig. 3 be embodiment 4 in compound transmission electron microscope figure and average particle diameter size figure.3a is compound S1
(upper left) and S2 (lower-left) transmission electron micrograph；3b is compound S1 (upper right) and S2 (bottom right) average grain diameter chi
Very little figure；
Fig. 4 schemes for the HeLa cytotoxicities detection of compound/pDNA complexs in embodiment 6；
Fig. 5 be embodiment 7 in compound/pDNA complexs agarose gel electrophoretogram；
Fig. 6 be embodiment 8 in compound/pDNA complexs surface potential figure；
Fig. 7 be embodiment 8 in compound/pDNA complexs hydrodynamics grain-size graph；
Fig. 8 is that compound/pDNA complexs in embodiment 9 detect figure to the egfp expression of HeLa cells；
Fig. 9 is that compound/pDNA complexs in embodiment 10 swallow efficiency chart to the cell of HeLa cells；
Figure 10 is inner cellular localization result figure of the compound/pDNA complexs in embodiment 11 to HeLa cells；
Figure 11 is that compound/pDNA complexs in embodiment 12 detect figure to RAW264.7 cytotoxicities；
Figure 12 is that compound/pDNA complexs in embodiment 13 are bent to RAW264.7 immunogenicity examination criterias dynamics
Line；
Figure 13 is that compound/pDNA complexs in embodiment 13 detect solubility curve to RAW264.7 immunogenicities；
Figure 14 is that the compound in embodiment 13 detects figure to RAW264.7 cells to RAW264.7 immunogenicity；
Figure 15 examines for the compound and compound/pDNA complexs in embodiment 13 to the immunogenicity of RAW264.7 cells
Mapping；
Figure 16 is immunogene of the compound and compound/CpG DNA complexs in embodiment 13 to RAW264.7 cells
Property detection figure；
Figure 17 is the compound synthesis step schematic diagram in embodiment 1, and a is compound S1 synthesis step, and b is compound
Thing S2 synthesis step.
Embodiment
To become apparent the present invention, hereby with preferred embodiment, and accompanying drawing is coordinated to be described in detail below.
Embodiment 1
The mPEG (molecular weight is 2000) for weighing 20.25mg is dissolved in DMSO, 9.59mg EDC and mPEG solution according to
The ratio uniform mixing of mol ratio 10: 1, adds 5.25mgNHS reactions 3h.By 47.34mg G5.NH2It is dissolved in DMSO molten
Liquid is sufficiently mixed according to the ratio of mol ratio 10: 1, reacts 72h.Rubbed in the product of above-mentioned reaction according to dendrimer with gold
You for 1: 50 amount than adding 41.39 mg HAuCl430min is reacted, 18.9mg NaBH are added43h is reacted under stirring condition.
Dialysis obtains { (Au after 3 days through freeze-drying0)50-G5.NH2-mPEG10, it is recorded as S1 (such as Figure 17 a).Weigh 11.26 mg's
FA, 4.4mg EDC and 2.64mg NHS are mixed according to 1: 0.9: 0.9 ratio uniform.By 20.25mg PEG and activate
FA is added to 47.34mg G5.NH according to the ratio of mol ratio 1: 22In, lucifuge stirring 72h.Add HAuCl4React 30min,
It is rapidly added the NaBH of 5 times of amounts4It is reduced, 3h is stirred under the conditions of lucifuge.Reaction solution is dialysed 3 days, it is last freeze-dried
Obtain { (Au0)50-G5.NH2-PEG10-FA5, it is recorded as S2 (such as Figure 17 b).
Embodiment 2
The S1 and S2 for weighing 5mg respectively are dissolved in 700mL D respectively2In O, ultrasound is carried out to two kinds of solution, then using core
Resonance spectrometer determines the nucleus magnetic hydrogen spectrum of two kinds of compounds.Nuclear-magnetism result as shown in figure 1,1H NMR spectras are connected to for sign
G5.NH2On carbochain number, G5.NH2The proton peak of characteristic group is between chemical shift 2.0-3.5ppm, in 3.4-
The proton uptake peak occurred between 3.6ppm, illustrates that mPEG is successfully connected to G5.NH2On, go out between 6.6-8.5ppm
Existing proton uptake peak illustrates that FA is successfully connected to G5.NH2On.The integration face of proton peak is calculated using origin softwares
Product, obtains averagely each G5.NH2Surface connects about 10 mPEG, about 3.2 FA.As a result show according to default target
{ (Au is synthesized0)50-G5.NH2-mPEG10And { (Au0)50-G5.NH2-PEG10-FA5}。
Embodiment 3
Material S1 and material S2 are dissolved separately in ultra-pure water, concentration is configured to for 200 μ g/mL solution, using it is ultraviolet-
Visible spectrophotometer detects the ultraviolet absorption value in 300nm-800nm wavelength bands.Spectrophotometry detection knot
Fruit is as shown in Fig. 2 G5.NH in compound2There is absworption peak left and right at 520nm, illustrates that nanogold particle is successfully wrapped up.
There is absworption peak to illustrate that FA has successfully been connected to G5.NH at 280nm2On.
Embodiment 4
S1 the and S2 aqueous solution (1mg/mL) prepared is dripped to the copper mesh surface of carbon film, is placed in and dries at room temperature, is made
Obtain sample.Then sample (Fig. 3 a) is detected under 200kV using JEM-2010F types transmission electron microscope.It is soft using Image J
Part measures and calculates sample size, and S1 average-size (Fig. 3 b) about 2.9nm and S2 average-size (Fig. 3 b) is about
3.1nm.It can be seen that the compound of preparation is spherical in shape in figure, dispersiveness is preferably.
Embodiment 5
With cervical cancer cell (HeLa) for pattern cell, two kinds of compound terminal amino groups that embodiment 1 is prepared determine nitrogen
Detection.S1 and S2 are dissolved in water to the solution for being configured to 2mg/mL, further according to primary amino detection kit
(PANOPA) the surface amino groups number of determination of experimental method compound.Experimental procedure is：A grain slice medicine is taken from reagent 1
Product, are dissolved, wiring solution-forming 1 with 3mL ultra-pure waters.Take 50 μ L ultra-pure waters to add solution 1 in control group fully to mix, taken in experimental group
The μ L of compound S1 or S2 50 to be measured add solution 1 and fully mixed, in absorbance to be measured at 340nm after reaction 3min, respectively
Record absorbance A0And A1；100 μ L solution 2 are separately added into again fully to mix and react 15min, are to remember at 340 nm in absorbance
Record data A2And A3.The concentration (mg/L) of terminal amino group nitrogen in sample is calculated according to formula.As a result show, after being modified through mPEG,
The number of terminal amino group is 29.9.And after modification mPEG reconnects FA, the number of terminal amino group is 21.6, this is probably
Because FA is connected with amino covalence, so that the number of PAMAM surface amino groups is reduced, to reduce the toxicity of compound.
Embodiment 6
With cervical cancer cell (HeLa) for pattern cell, two kinds of compounds/pDNA complexs that embodiment 1 is prepared
Cytotoxicity detection.Collected after the HeLa cell pancreatin in exponential phase of growth digests in blake bottle, with 6 × 103Cell
Density per hole is by HeLa cells kind in 96 orifice plates.In the 200 μ L DMEM culture mediums containing 10%FBS at 37 DEG C and
5%CO2Under the conditions of cultivate 12h, change into containing 50nM, 100 nM, 500nM, 1000nM, 2000nM, 3000nM various concentrations answer
The culture medium of compound, adds 1 μ g pDNA compounds and continues to cultivate 4h.Change culture medium into serum free medium culture 24h,
After culture terminates, 10 μ L CCK-8 (5.0mg/mL) solution are added per hole, and be incubated in incubator 4h.After incubation terminates, use
The absorbance in each hole at enzyme-linked immunosorbent assay instrument measurement 450nm wavelength.Fig. 4 results are shown, are increased with the concentration of compound, carefully
Born of the same parents' vigor is gradually reduced.But carrier and pDNA compounds in cell survival rate intracellular HeLa more than 75%, show
The cytotoxicity of two kinds of compound/pDNA complexs is very low, is conducive to subsequent experimental.
Embodiment 7
With cervical cancer cell (HeLa) for pattern cell, the combination of compound and DNA that embodiment 1 is prepared
Ability is typically measured using agarose gel electrophoresis experiment.It is that 0.25,0.5,1,2.5,5,7 preparation is carried according to N/P ratio
Body and pDNA compounds, single pDNA are used as control.0.5g agaroses are weighed to add to 50mL Tris- boric acid running buffers
Mixed in liquid (0.5 × TBE).Heat three times repeatedly in micro-wave oven, every time about 1min, until agarose dissolving, and nothing in bottle
Bubble.Treat that temperature is reduced to 50-60 DEG C in bottle, ethidium bromide (EB) dyestuff that 4 μ L concentration are 1mg/mL is added into glue, is shaken up
Pour into and set in the glue groove of comb, wait and comb is pulled out after about 15min, prepare loading.By carrier and pDNA compounds and loading
Buffer solution (6 × loading buffer) is added separately in the hole of Ago-Gel after mixing, voltage 80V, time 30min.Run
Migration situations of the cementing Shu Houyong gel imagers analysis pDNA in gel.Under the conditions of different N/P, bar in each swimming lane
The separate condition of band represents that the compound of various concentrations wraps up the ability of gene, and Fig. 5 is the agarose of carrier and pDNA compounds
Show that compound/pDNA complexs do not observe obvious DNA bands, explanation when N/P is 2.5 in gel electrophoresis spectrum, figure
Compound/DNA complex is not moved to positive pole, with stronger parcel ability, can compress DNA.
Embodiment 8
With cervical cancer cell (HeLa) for pattern cell, compound prepared by the method for DNA and embodiment 1 according to
Three kinds of N/P ratios are 1: 1,2.5: 1,5: 1, be incubated altogether with 1 μ g pDNA obtain carrier and pDNA compounds respectively.Compound S1 and
S2 is with pDNA complexs as experimental group, and compound S1 and S2 are as a control group.1 is added after being incubated 30min at ambient temperature
Milliliter PBS, then determines the hydrodynamics particle diameter and surface electricity of compound using laser particle analyzer (Malvern, UK)
Gesture.Test is that, using He-Ne as lasing light emitter, optical maser wavelength is 633nm, and incidence angle is 90 °, and it is experiment bar that measurement temperature, which is 25 DEG C,
Part, is characterized to compound.As a result show, the surface potential (Fig. 6) of carrier and pDNA compounds is that just, pDNA's is negative
Charging neutrality compound positive charge, as the potential of two kinds of carriers in figure and DNA compounds is respectively smaller than the electricity of two kinds of carriers
Gesture.With the increase of N/P ratio, the surface potential of compound also has increased trend, and potential range has in normal range (NR)
Beneficial to compound and cell-cell interaction, it is easy to transmission of the carrier to target gene.Carrier is with pDNA compounds with nitrogen phosphorus
Than increase, Interaction enhanced, particle diameter is sequentially reduced (Fig. 7).When N/P ratio be 2.5 when, particle size range be in 200nm with
Between 300nm, now compound is easily accessible cell, is transmitted beneficial to genes within cells.
Embodiment 9
With cervical cancer cell (HeLa) for pattern cell, the compound transmission that embodiment 1 is prepared (increases containing EGFP
Strong type green fluorescent protein) gene DNA Efficiency testing, the size of fluorescence intensity reflects the transfection effect of EGFP gene
Rate.
It is 3.5 × 10 that density is planted on 24 orifice plates4HeLa cell per wells, in 37 DEG C and 5%CO2Under the conditions of cultivate 12h, abandon
Old culture medium is removed, fresh serum-free media culture is added.Plasmid and compound are to mix under the conditions of 1,2.5,5 according to N/P,
Avoid light place 20min, adds in orifice plate and carries out gene transfection 4h at room temperature.With fluorescence microscope green fluorescent protein
Expression.Fig. 8 is compound and egfp expression detection figure of the pDNA complexs in HeLa cells.As a result show, S2
Under the conditions of above-mentioned three kinds of N/P, the expression quantity of green fluorescent protein is all more than S1.Illustrate that the targeting mechanism for connecting folic acid can be effective
Raising compound gene transfection abilities.Cell transfecting efficiency high when than N/P being 5 when N/P is 2.5, it may be possible to due to N/
When P increases, compound and pDNA interaction increase, so as to cause pDNA, hardly possible is discharged from compound in the cell,
Be beyond expression out corresponding protein product.When connecting FA, egfp expression amount increases, and can be FA targets with initial guess
To the result of effect.
Embodiment 10
With cervical cancer cell (HeLa) for pattern cell, the compound that the method for embodiment 1 is prepared transmits pDNA's
Cell endocytic efficiency.With 5 × 104The density kind of HeLa cell per wells is in 24 orifice plates, in 37 DEG C and 5%CO2Under the conditions of cultivate
12h.Under the conditions of the above three N/P of embodiment 8, the pDNA and compound for adding 1g Cy3 marks according to every hole are mixed, room
The lower avoid light place 20min of temperature, adds in orifice plate and carries out gene transfection 4h, make carrier with Cy3-pDNA compounds well into thin
Born of the same parents.Then, the PBS sterilized with 1mL suspension cells, and detected sample is placed on ice again.Three parallel realities of every group of setting
Test, each sample is detected with flow cytometer.It is 1 × 10 to determine cell quantity every time4Often manage, detection cell release
Red fluorescence, sets FSH-H/SSC-H as cell door, sets FL2-H as Cy3 fluorescence channels, every group of sample is repeated three times.
Fig. 9 is that compound marks cell endocytic of the pDNA complexs in HeLa cells to test with Cy3, is as a result shown in N/P for 2.5
Under part, the red fluorescent intensity that Cy3 is excited is most strong.Show that S1, S2 show good cell endocytic when N/P is 2.5
Efficiency.
Embodiment 11
With cervical cancer cell (HeLa) for pattern cell, the method for embodiment 1 prepares compound S1, S2.Pass through born of the same parents
Interior common location tests pipeline and inner cellular localization feelings that can be to Au DENPs when as Gene transfer vector in intracellular
Condition is studied, and with Cy3 dyestuff specific marker pDNA, transfection HeLa cell is carried out with compound formation complex, so as to
Determine whether they have identical intracellular pipeline and enter intracellular later whereabouts.With 1 × 105The each hole of cell
Density kind on 12 orifice plates, in 37 DEG C and 5%CO2Under the conditions of cultivate 24h.Change serum free medium into, add compound with
PDNA complexs, hatch 4h.After hatching, compound and pDNA complexs are suctioned out, washed with sterile PBS 2 times, 2.5% is added
Glutaraldehyde, fixes 30min, DAPI dye marker nucleus 7min at 4 DEG C.Then cleaned with PBS after cell three times, with swashing
Experimental result is observed and recorded to light Laser Scanning Confocal Microscope.Figure 10 is each compound and Cy3-pDNA complexs in HeLa cells
Cellular localization figure.As a result show that compound after 4h gene transfection, is passed gene by intracellular pipeline with cell
It is delivered to around nucleus, especially under the conditions of N/P conditions is 2.5, the pDNA complexs that S2 and Cy3 is marked largely are concentrated on
Around cytoplasmic nucleus, show that having modified PEG and FA does not influence the biological function of compound, also improve gene and turn
Contaminate efficiency.On the whole, cell endocytic efficiency of the S2 than S1 is slightly higher, consistent with the result of embodiment 10.
Embodiment 12
With macrophage (RAW264.7) for pattern cell, the compound that detection embodiment 1 is prepared is formed with pDNA
Complex cytotoxicity.With 6 × 103The density of cell per well by RAW264.7 cells kind in 96 orifice plates, containing
In 37 DEG C and 5%CO in 10%FBS 200 μ L DMEM culture mediums2Under the conditions of cultivate 12h, change into containing 50nM, 100nM,
The fresh culture of 500nM, 1000nM, 2000nM, 3000nM various concentrations graded composite thing, adds 1 μ g pDNA and is combined
Thing continues to cultivate 4h, and each gradient sets 3 multiple holes.Change culture medium into serum free medium culture 24h, after culture terminates,
10 μ L CCK-8 (5.0mg/mL) solution are added per hole, and are incubated in incubator 4h.After incubation terminates, enzyme linked immunosorbent detection is used
The absorbance in each hole at instrument measurement 450nm wavelength.Figure 11 results show that, with the concentration increase of compound, cell viability is gradually
Reduction.On the whole, compound shows compound with cell survival rate of the pDNA complexs in macrophage more than 75%
Cytotoxicity with pDNA complexs is very low, is conducive to subsequent experimental.
Embodiment 13
Macrophage culture is collected to exponential phase of growth after being digested with pancreatin, with 6 × 10 after rolling counters forward5Cell/
The density kind in hole adds culture medium DMEM about 2mL into 6 orifice plates；According to default N/P values be 2.5 prepare two kinds of compounds and
DNA complex.The cell that liposome adds serum free medium is used as positive controls.Experimental procedure is broadly divided into total serum IgE
Extraction, reverse transcription, two-step method pcr amplification reaction.Specifically include：
(1) extraction of macrophage total nucleic acid：With every 1 × 106Individual macrophage kind is added multiple into 6 orifice plates to every hole
Compound or compound/DNA complex are transfected after 4h, are separately added into the mL of Total RNA Extractor 1.The sample of cracking exists
5-10min is placed at room temperature, 0.2mL chloroforms are added, and is acutely placed 3 min after vibration, is then centrifuged under the conditions of 12000rpm
10min.Take upper strata aqueous phase to centrifuge tube, add isometric isopropanol, mix and place 20min.Centrifuged under the conditions of 12000rpm
10min, removes supernatant.1mL 75% ethanol washing precipitation is added, 3min is centrifuged in 12000rpm, removes supernatant.
Finally plus 30~50 μ L RNase-free ddH2O fully dissolves RNA, obtains RNA solution.
(2) reverse transcription sample is prepared：Following reagent will be added in the PCR pipe of ice bath：The RNA of extraction, 1 μ L primers, 1 μ L
DNTP (0.5mM), RNase-free ddH2The μ L of O about 10,5 s are centrifuged after gently mixing.Reactant mixture is in 65 DEG C of warm bath 5min
Afterwards, ice bath 2min, is then centrifuged for 3~5s.By PCR pipe ice bath, the μ L of reagent 45 times of reverse transcription buffers, 0.5 μ L cores are added
Ribonuclease T. inhibitor and 1 μ L reverse transcriptases.3~5s is centrifuged after gently mixing again.Reverse transcription reaction is carried out in PCR instrument：25
DEG C be incubated 10min, 50 DEG C reaction 30min carry out cDNA synthesis, 85 DEG C reaction 5min carry out terminating reactions, obtain reverse transcription sample
Product.
(3) PCR is expanded：Following reactant mixture, 10 μ L2 × SuperReal PreMix are added under condition of ice bath
Plus, 0.6 μ L sense primers (10 μM), 0.6 μ L anti-sense primers (10 μM), 2.5 μ L cDNA templates (being diluted to 10pM), 0.4 μ
L 50 × Rox, RNase-free ddH2O adds to 20 μ L.In 95 DEG C of pre-degenerations 15min, 95 DEG C of denaturation 10s, 62 DEG C are annealed and prolonged
32s is stretched, 40 circulations are reacted.Finally product is analyzed with the instruments of ABI 7500, system is according to the changing rule of fluorescent value
Generate Standard kinetic curve (Figure 12) and solubility curve (Figure 13).The S types of rule are presented in its standard curve.Three groups of dissolvings are bent
Line detects internal reference GAPDH, cell factor IL-6 and TNF-α respectively, and each solubility curve shows a peak and without miscellaneous peak, shown
Amplified production is homogeneous.As a result the relative intensity of fluorescence of experimental group and control group is calculated using △ △ Ct methods.Figure 14 is to use qRT-
PCR detection compounds act on cytokines mRNA expression quantity in immunocyte.The normal cell not dealt with is as control
Group.Two kinds of compounds are co-cultured with macrophage, then are expanded with reverse transcription real-time quantitative PCR kit using two-step method response procedures
Increase and PCR primer.Result shows that independent compound will not cause immunocyte to secret out of both the above cell factor, explanation in figure
The compound of preparation will not cause corresponding immune response, and immunological safety is high.Figure 15 is qRT-PCR detection compounds and pDNA
Complex acts on mRNA expression of cytokines amount in macrophage.PDNA is added in macrophage and is used as negative control group.
N/P is that under the conditions of 2.5, compound and pDNA are incubated 4h and transfected, transfection terminate after again with the amplification of reverse transcription real-time quantitative PCR
Go out PCR primer, calculate relative intensity of fluorescence.As a result show after DNA gene is transfected, macrophage is not also produced
Cell factor, illustrates that the gene transfection process immunological safety of compound and pDNA complexs is high, immunogenicity is good.Figure 16 is
QRT-PCR detects that compound acts on mRNA expression of cytokines amount in macrophage with CpG DNA complexs.In macrophage
Add CpG DNA and be used as negative control group.Compound and CpG DNA are incubated 4h and transfected, and transfection uses reverse transcription again after terminating
Real-time quantitative PCR amplifies PCR primer.Because CpG DNA are strong immunoactivators, compared with above-mentioned two groups of experiments, phase
It is high to luciferase expression amount, further illustrate that compound and the immunogenicity of pDNA complex genes transfection process are low, bio-safety
Property it is high.
Claims (18)
1. the compound of a kind of Polyamidoamine Dendrimers and nanogold particle carries out gene transfection as non-virus carrier
Application process, it is characterised in that the surface of the dendrimer is amino terminal, it, which is chemically modified, can reduce its poison
Property simultaneously improves transfection efficiency；Its end connection targeting group includes folic acid, and RGD, hyaluronic acid can efficiently carry out gene biography
Pass.
2. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the method for the chemical modification is modification polyethylene glycol, trimming loop paste
Any one or a few in essence, the method for acetylation and alkylation.
3. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that Au and G5.NH in the compound2Mol ratio be 25: 1 and more than.
4. the compound of Polyamidoamine Dendrimers as claimed in claim 3 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that Au and G5.NH in the compound2Mol ratio is 25: 1,50: 1,75:
1 or 100: 1.
5. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out gene transfection application process, it is characterised in that concentration of the compound in gene rotaring redyeing system be 1.0~
2.0mg/mL。
6. the compound conduct of the Polyamidoamine Dendrimers and nanogold particle as described in claim 1-5 any one
Non-virus carrier carries out the application process of gene transfection, it is characterised in that the transmission exogenous therapeutic gene bag of the gene delivery
Include pDNA, CpG DNA, GEM 132, any one or a few in siRNA and ssDNA.
7. the compound of Polyamidoamine Dendrimers as claimed in claim 6 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the target cell of the exogenous therapeutic gene includes cancer cell and body is thin
Born of the same parents.
8. the compound of Polyamidoamine Dendrimers as claimed in claim 7 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the cancer cell includes HeLa, U87 and A549；The body cell includes
Macrophage, T lymphocytes, bone-marrow-derived lymphocyte, BMDC.
9. the compound of Polyamidoamine Dendrimers as claimed in claim 6 and nanogold particle is used as non-virus carrier
Carry out gene transfection application process, it is characterised in that the N/P ratios of the compound and DNA be 0.5: 1 and more than.
10. the compound of Polyamidoamine Dendrimers as claimed in claim 9 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the N/P ratios of the compound and DNA are 0.5: 1,1: 1,2.5: 1,5: 1
Or 7: 1.
11. the compound of Polyamidoamine Dendrimers as claimed in claim 6 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the pDNA is extracted from Escherichia coli, plasmid blocks that to carry
Mycin resistant gene and the plasmid with Green fluorescent protein fusion vector；The CpG DNA are that by artificial synthesized, sequence is
5’-TCCATGACGTTCCTGATGCT-3’。
12. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the transfection time of the gene transfection is 4~12h.
13. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that the compound of the Polyamidoamine Dendrimers and nanogold particle
Preparation method include step in detail below：
Step 1)：{(Au0)50-G5.NH2-mPEG10Synthesis：MPEG is dissolved in DMSO, EDC and mPEG is according to 10: 1
Ratio uniform is mixed, and reacts 30-40min, and the NHS added with EDC equal proportions reacts 3-4h；By the mPEG activated with
G5.NH2DMSO solution according to mol ratio 10: 1 ratio mix 72-84h；According to tree-shaped big point in the product of above-mentioned reaction
Son adds gold chloride HAuCl with golden mol ratio for 1: 50 amount4React 30min；Then NaBH is added4Reduce it, stirring condition
Lower reaction 3-4h；Reaction solution is dialysed 3-4 days, it is finally freeze-dried to obtain sample { (Au0)50-G5.NH2-mPEG10, it is denoted as
S1；
Step 2)：{(Au0)50-G5.NH2-PEG10-FA5Synthesis：FA and EDC is mixed according to 1: 0.9 ratio uniform, reacted
30-40min, the NHS added with EDC equal proportions reacts 3-4h；PEG and the FA activated are added according to the ratio of mol ratio 1: 2
Enter to G5.NH2In, lucifuge stirring 72-84h；It is 1: 50 addition HAuCl according still further to dendrimer and golden mol ratio4Reaction
30min, then it is rapidly added the NaBH of 5 times of amounts4It is reduced, 3-4h is stirred under the conditions of lucifuge；Reaction solution is dialysed 3-4 days, most
Afterwards the { (Au of sample 2 is obtained by freeze-drying0)50-G5.NH2-PEG10-FA5, it is denoted as S2；Wherein HAuCl4The concentration of solution is
30.15mg/mL；NaBH4The concentration of solution is 10mg/mL；EDC concentration is 9.7mg/mL；NHS concentration is 5.05mg/mL；mPEG
Concentration be 4mg/mL；The concentration of G5.NH2 solution is 0.44 μm of ol/mL, and FA concentration is 2mg/mL.
14. the compound of Polyamidoamine Dendrimers as claimed in claim 13 and nanogold particle is carried as non-viral
Body carry out gene transfection application process, it is characterised in that the step 1) with step 2) synthesis two kinds of samples be used as carrier
The method that gene applied to HeLa is transfected is compound and the pDNA according to Polyamidoamine Dendrimers and nanogold particle
It is respectively 1: 1,2.5: 1,5: 1 to prepare carrier and pDNA compound according to different N/P；In 37 DEG C, 5%CO2Under the conditions of train
Support HeLa cells, add foregoing three kinds of N/P than compound the gene in cell transfects 4h with pDNA.
15. the compound of Polyamidoamine Dendrimers as claimed in claim 1 and nanogold particle is used as non-virus carrier
Carry out the application process of gene transfection, it is characterised in that use real-time fluorescence quantitative PCR instrument or enzyme-linked immunosorbent assay base
Because the compound or compound after transfection and DNA stimulate the relative expression quantity of immunocyte secrete cytokines.
16. the compound of Polyamidoamine Dendrimers as claimed in claim 15 and nanogold particle is carried as non-viral
Body carries out the application process of gene transfection, it is characterised in that the cell factor that PCR is expanded in the real-time fluorescence quantitative PCR instrument
For any one or a few cell factor in IFN-α, IFN-β, IFN-γ, IL-1 β, IL-6, IL-10, TNF-α and IL-12.
17. the compound of Polyamidoamine Dendrimers as claimed in claim 13 and nanogold particle is carried as non-viral
Body carries out the application process of gene transfection, it is characterised in that the compound carries out thin in RAW 264.7 after gene transfection 4h
Intracellular cytokine IL-6 and TNF-α cytokines measurement, step 1) with step 2) prepare two kinds of samples respectively with pDNA and CpG DNA
Compound carries out gene transfection, and RAW 264.7 cytokine TNF-α and IL- are detected with real time fluorescent quantitative reverse transcription PCR
6 gene expression dose, to detect whether gene transfection causes cell immune response.
18. the compound of Polyamidoamine Dendrimers as claimed in claim 17 and nanogold particle is carried as non-viral
Body carries out the application process of gene transfection, it is characterised in that whether the detection gene transfection causes the tool of cell immune response
Body step is as follows：
Step a)：The extraction of macrophage total nucleic acid：With every 1 × 106Individual macrophage kind adds two kinds into 6 orifice plates to every hole
After compound S1 and S2 or two kinds of compound S1 and S2 and DNA complex transfection 4h, Total RNA are separately added into
Extractor 1mL；The sample of cracking places 5-10min at room temperature, adds 0.2mL chloroforms, acutely places 3min after vibration,
Then 10min is centrifuged under the conditions of 12000rpm；Take upper strata aqueous phase to centrifuge tube, add isometric isopropanol, mix and place
20min；10min is centrifuged under the conditions of 12000rpm, supernatant is removed；1mL 75% ethanol washing precipitation is added,
12000rpm centrifuges 3min, removes supernatant；Finally plus 30~50 μ L RNase-free ddH2O fully dissolves RNA, obtains
RNA solution；
Step b)：Prepare reverse transcription sample：Following reagent will be added in the PCR pipe of ice bath：1 μ L RNA templates, 1 μ L primers, 1 μ
L dNTP, RNase-free ddH2O10 μ L, 5s is centrifuged after gently mixing；Reactant mixture is after 65 DEG C of warm bath 5min, ice bath
2min, is then centrifuged for 3~5s；By PCR pipe ice bath, the μ L of reagent 45 times of reverse transcription buffers, 0.5 μ L ribalgilases are added
Inhibitor and 1 μ L reverse transcriptases；3~5s is centrifuged after gently mixing again；Reverse transcription reaction is carried out in PCR instrument：25 DEG C of incubations
10min, 50 DEG C of reaction 30min carry out cDNA synthesis, and 85 DEG C of reaction 5min carry out terminating reaction, obtain reverse transcription sample；
Step c)：PCR is expanded：Following reactant mixture, 10 μ 2 × SuperReal of L PreMix are added under condition of ice bath
Plus, 0.6 μ L sense primers, 0.6 μ L anti-sense primers, 2.5 μ L cDNA templates, 0.4 μ L 50 × Rox, RNase-free
ddH2O adds to 20 μ L；In 95 DEG C of pre-degenerations 15min, 95 DEG C of denaturation 10s, 62 DEG C are annealed and extension 32s, react 40 circulations；Most
Instrumental Analysis goes out the Standard kinetic curve and solubility curve of amplified production afterwards.
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