CN106924746A - Complex gene carrier and its application - Google Patents
Complex gene carrier and its application Download PDFInfo
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- CN106924746A CN106924746A CN201710124855.9A CN201710124855A CN106924746A CN 106924746 A CN106924746 A CN 106924746A CN 201710124855 A CN201710124855 A CN 201710124855A CN 106924746 A CN106924746 A CN 106924746A
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- cdg2
- dna
- liposome
- dope
- complex gene
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
Abstract
The present invention relates to a kind of complex gene carrier and its application.The complex gene carrier is composited by CDG2 and liposome, and the mass ratio of the CDG2 and liposome is 15:1, the CDG2 are prepared by the outside grafting PAMAM G2 in α cyclodextrin.Complex gene carrier of the invention, can significantly improve cell transfection rate, be conducive to promoting the development of gene therapy medicament.
Description
Technical field
The present invention relates to pharmaceutical technology field, more particularly to a kind of complex gene carrier and its application.
Background technology
With 21 century protein science, biotechnology, chemical genomics fast development, gene therapy has turned into life section
One study hotspot in field.At present, the conventional therapy in the field such as tumour, angiocardiopathy still lacks active drug, therefore,
Disease treatment is carried out from gene level has turned into a study hotspot of medical domain.And during gene therapy, it is necessary to
Foreign gene is incorporated into cell, but DNA molecular generally exists with electronegative rarefaction, and volume is larger, full negative electricity
There is repulsive interaction between lotus and cell surface, hardly enter in cell, DNA can be degraded by internal nuclease in addition, not
Into target cell, in addition not up to target organ when be just degraded into micromolecule nucleotide, so as to lose therapeutic action, so for base
Because of the high efficiency treated, it is desirable to which therapeutic gene need to be protected preferably in transportation in vivo, therefore genophore meets the tendency of
And give birth to, genes of interest is combined with carrier so that foreign gene or functional nucleic acid fragment are imported into cell, realize gene
Expression regulation, gene function even gene therapy purpose.Efficient genophore is developed to be treated for whole gene or even base
Because being all a particularly important ring in engineering, will also transition of the gene therapy to conventional therapy be promoted.The efficiency of genophore can
Represented with the expression quantity of cell transfection rate and encoding foreign proteins, the carrier of only high transfection efficiency is only and can really be applied to
During gene therapy, therefore the carrier of exploitation high transfection efficiency is an emphasis in gene therapy research.
Two kinds of current genophore partitivirus carrier and non-virus carrier.Viral vectors transfection efficiency is very high, but exists
Such as serious safety issue of immunogenicity, and it is small to carry genes of interest capacity, prepares complicated, somewhat expensive.It is non-viral
Vector immunogenic is low, low toxicity, prepare easy and be adapted to large-scale production, but universal transfection efficiency is all relatively low.
Liposome vectors (such as DC-Chol-DOPE, DOSPER-DOPE, DOTMA-DOPE and LipofectamineTM2000
Etc.) it is to study a more deep class carrier in non-virus carrier, cationic-liposome is easy to operate because of its, the safety of transfection
Property, high efficiency and versatility and be used widely and existing procucts are applied to clinical test.LipofectamineTM2000 (with
Down be referred to as Lipo 2000) be liposome vectors a Typical Representative, be the star's product in present commercial vectors, can
For 517 kinds of cells provide high transfection efficiency (percentage of express transgenic cell) and activity (transgene in cell extract
Enzyme product activity).The transfection procedures of Lipo 2000 are simple and convenient, and the transfection efficiency to many cells is also higher.But
The high transfection efficiency of Lipo2000 can not be directed to all cells, and for some cells, its transfection efficiency can not reach treatment
The result that research process is satisfied with.
In addition, cationic polymer is also a class of non-virus carrier.Polyamide-amide (PAMAM) dendrimer is one
Class synthesis, mono-dispersed nano level macromolecular compound, with hyperbranched, polyvalency, easily be modified, the spy such as widely used
Point.Many documents have the derivative obtained to PAMAM and its modification to study, and research finds, polyamide-amide is tree-shaped high poly-
Thing can be used as cationic genophore, and it can effectively wrap up nucleic acid, the good (bibliography of security：[1]Huang H,Tang
G,Wang Q,et al.Two novel non-viral gene delivery vectors:low molecular weight
polyethylenimine cross-linked by(2-hydroxypropyl)-beta-cyclodextrinor(2-
hydroxypropyl)-gamma-cyclodextrin.Chem Commun(Camb),2006,22:2382-2384.[2]Yang
C,Wang X,Li H,et al.Synthesis and characterization of polyrotaxanes
consisting of cationic alpha-cyclodextrins threaded on poly[(ethylene oxide)-
ran-(propylene oxide)]as gene carriers.Biomacromolecules,2007,8:3365-3374.[3]
Yang C,Li H,Goh SH,et al.Cationic star polymers consisting of alpha-
cyclodextrin core and oligoethylenimine arms as nonviral gene delivery
vectors.Biomaterials,2007,28:3245-3254.).It is low in its outside scion grafting using alpha-cyclodextrin as kernel
The Polyamidoamine Dendrimers second generation (PAMAM-G2) obtains high molecular polymer CDG2.CDG2 carriers are used as polycation
Some cells are had certain transfection efficiency by one kind of type genophore, the ability for having certain parcel and compression nucleic acid, but
It is to equally exist the low problem of transfection efficiency caused by cell category otherness, it is extremely low for some cells even transfection efficiency.
The content of the invention
Based on this, the invention provides a kind of complex gene carrier, the complex gene carrier to various kinds of cell all have compared with
Transfection efficiency high.
Concrete technical scheme is as follows：
A kind of complex gene carrier, is composited by CDG2 and liposome, and the mass ratio of the CDG2 and liposome is 1-
5:1, the CDG2 are prepared by the outside grafting PAMAM-G2 in alpha-cyclodextrin.
Wherein in some embodiments, the mass ratio of the CDG2 and liposome is 1.5-4:1.
Wherein in some embodiments, the liposome is selected from DC-Chol-DOPE, DOSPER-DOPE, DOTMA-DOPE
And LipofectamineTMAt least one in 2000.
Wherein in some embodiments, the preparation method of the CDG2 is comprised the following steps：By alpha-cyclodextrin and coupling agent
Reaction, obtains being connected with the alpha-cyclodextrin of coupling agent；PAMAM-G2 is reacted with the alpha-cyclodextrin for being connected with coupling agent, institute is obtained final product
State CDG2.
Wherein in some embodiments, the coupling agent is N, N- carbonyl dimidazoles.
Wherein in some embodiments, the alpha-cyclodextrin, N, N- carbonyl dimidazoles are 1 with the mass ratio of PAMAM-G2：
11-15：50-55.
Present invention also offers the application of above-mentioned complex gene carrier.
Concrete technical scheme is as follows：
Application of the above-mentioned complex gene carrier in gene therapy medicament is prepared.
Present invention also offers a kind of DNA/ carrier complexes.
Concrete technical scheme is as follows：
A kind of DNA/ carrier complexes, are composited by above-mentioned complex gene carrier with DNA.
Wherein in some embodiments, the complex gene carrier is 2.5-5 with the mass ratio of DNA:1.
Present invention also offers a kind of preparation method of DNA/ carrier complexes.
Concrete technical scheme is as follows：
A kind of method for preparing DNA/ carrier complexes, comprises the following steps：CDG2 is configured to concentration for 0.7-
The CDG2 aqueous solution of 0.9mg/mL, liposome formulation into the liposome aqueous solution that concentration is 0.9-1.1mg/mL prepares DNA
It is the aqueous dna of 75-85ng/ μ L into concentration；Take the 18.5-19 μ LCDG2 aqueous solution, 5-7 μ L liposome aqueous solutions, 70-80 μ
The LDNA aqueous solution and 45-55 μ L water, stationary incubation 25-35min at room temperature after mixing, obtain final product the DNA/ carrier complexes；
The CDG2 is prepared by the outside grafting PAMAM-G2 in alpha-cyclodextrin.
Wherein in some embodiments, the liposome is selected from DC-Chol-DOPE, DOSPER-DOPE, DOTMA-DOPE
And LipofectamineTMAt least one in 2000.
Wherein in some embodiments, the preparation method of the CDG2 is comprised the following steps：By alpha-cyclodextrin and coupling agent
Reaction, obtains being connected with the alpha-cyclodextrin of coupling agent；PAMAM-G2 is reacted with the alpha-cyclodextrin for being connected with coupling agent, institute is obtained final product
State CDG2.
Wherein in some embodiments, the coupling agent is N, N- carbonyl dimidazoles.
Wherein in some embodiments, the alpha-cyclodextrin, N, N- carbonyl dimidazoles are 1 with the mass ratio of PAMAM-G2：
11-15：50-55.
Complex gene carrier of the invention and its application have advantages below and beneficial effect：
The present inventor is had found by lot of experiments, CDG2 and liposome gene carrier is obtained after compound
Complex gene carrier is compared to single CDG2 and single liposome gene carrier can significantly improve cell transfecting efficiency.Pass through
The conventional experimental cell in several laboratories, including Hek293T cells, A549 cells, LOVO cells and Hek293A cells are transfected,
Confirm the result of its high transfection efficiency.Due to experiment needs or the individual difference of cell, gene delivery vector is turned
Dye efficiency can propose requirement higher, and complex gene carrier of the invention can significantly improve cell transfection rate, use it for base
Because of the preparation of medicine, be conducive to promoting the development of gene therapy medicament.
Brief description of the drawings
Fig. 1 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections Hek293T cells in embodiment 1；
Fig. 2 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections A549 cells in embodiment 1；
Fig. 3 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections LOVO cells in embodiment 1；
Fig. 4 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections Hek293A cells in embodiment 1；
Fig. 5 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection Hek293T cells in embodiment 1；
Fig. 6 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection A549 cells in embodiment 1；
Fig. 7 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection LOVO cells in embodiment 1；
Fig. 8 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection Hek293A cells in embodiment 1；
Fig. 9 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections Hek293T cells in embodiment 2；
Figure 10 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection Hek293T cells in embodiment 2；
Figure 11 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections Hek293T cells in embodiment 3；
Figure 12 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection Hek293T cells in embodiment 3；
Figure 13 is three kinds of fluorescence pictures of carrier mediated pEGPF-C1 transfections Hek293T cells in embodiment 4；
Figure 14 is the transfection efficiency of the three kinds of pDNA/ carriers nano-complexes transfection Hek293T cells in embodiment 4.
Specific embodiment
Complex gene carrier of the invention and its application are further described in detail below in conjunction with specific embodiment.
LipofectamineTM2000 is Invitrogen companies market commodity, referred to as Lipo2000；
DC-Chol-DOPE：Self-control, wherein DC-Chol is purchased from Shanghai Advanced viecle Technology Co., Ltd., and DOPE is purchased from
Cordenpharma companies of Switzerland.
Its preparation method is as follows：1mg DC-Chol and 1mgDOPE are weighed, is dissolved in 1mL chloroform/methanols.Nitrogen is dried up,
Film is formed, 10h is vacuum dried.1mL sterilized waters, 45 degrees Celsius of ultrasound 1.5h of constant temperature are added to the inside.Transparent clarification is obtained to produce
Product, that is, be obtained DC-Chol-DOPE.
DOSPER-DOPE：Self-control, wherein DOSPER is purchased from Shanghai Bei Zhuo bio tech ltd, and DOPE is purchased from Switzerland
Cordenpharma companies.
Its preparation method is as follows：1mg DOSPER and 1mgDOPE are weighed, is dissolved in 1mL chloroform/methanols.Nitrogen is dried up, shape
Into film, 10h is vacuum dried.1mL sterilized waters, 45 degrees Celsius of ultrasound 1.5h of constant temperature are added to the inside.Transparent clarified product is obtained,
DOSPER-DOPE is obtained.
DOTMA-DOPE：Self-control, wherein DOTMA is purchased from Shanghai Advanced viecle Technology Co., Ltd., and DOPE is purchased from Switzerland
Cordenpharma companies.
Its preparation method is as follows：1mg DOTMA and 1mgDOPE are weighed, is dissolved in 1mL chloroform/methanols.Nitrogen is dried up, shape
Into film, 10h is vacuum dried.1mL sterilized waters, 45 degrees Celsius of ultrasound 1.5h of constant temperature are added to the inside.Transparent clarified product is obtained,
DOTMA-DOPE is obtained.
CDG2 is made products, and wherein preparing raw material PAMAM-G2 (oligoamide-amine dendrimer second generation) is purchased from：
Xi'an Rui Xi bio tech ltd；The preparation method of CDG2 is comprised the following steps：
(1) by coupling agent N, N- carbonyl dimidazoles (CDI) are connected on alpha-cyclodextrin (CD)：Weigh respectively 6.35gCDI and
0.47gCD, dries 24 hours in 35 DEG C of vacuum drying chambers, is dissolved in respectively in the anhydrous DMSO solutions of 25mL, by the anhydrous DMSO of CD
Solution ultrasound is transparent to solution, under stirring and nitrogen protection, CD solution syringes is slowly added in CDI solution, and room temperature is stirred
Mix reaction 24 hours.With mixed solvent (200mL THF:400mL Et2O) reaction solution is precipitated, is centrifuged, in careful removal
Clear liquid, washed for several times with 300mL THF, centrifugation obtains clear yellow viscous liquid, dried 24 hours in 90 DEG C of vacuum drying chambers,
Thick white thing is obtained, the alpha-cyclodextrin (CD-CDI) of coupling agent is as connected with.
(2) PAMAM G2 purifying：Accurately take 24.8g PAMAM-G2 to be dissolved in 100mL methanol solutions, use mixed solvent
(400mL THF:200mL Et2O) precipitate, centrifugation removes supernatant, uses 400mL Et2O wash for several times, centrifugation, precipitation, obtain
Yellow, viscous material, 6h, PAMAM G2 as after purification are dried in 60 DEG C of vacuum drying chambers.
(3) PAMAM G2 after purification are grafted to and CDG2 is prepared on CD-CDI：The CD-CDI that to prepare in step (2) and
The PAMAM G2 for having purified are dissolved in the anhydrous DMSO solutions of 25mL and 100mL respectively, under stirring and nitrogen protection, by CD-CDI
In slowly adding PAMAM G2 solution with syringe (it is 30 minutes to add the time), lower reaction 24 hours is stirred at room temperature, with mixed
Bonding solvent (300mL THF:600mL Et2O liquid precipitate, centrifugation) will be reacted, supernatant, 400mL THF washing numbers will carefully be removed
Secondary, centrifugation obtains clear yellow viscous liquid, clear yellow viscous liquid is dissolved in 20mL deionized waters and is dialysed 3 days, obtains final product CDG2.
PDNA used is pEGPF-C1 plasmids in following examples, and its preparation method is carried out by kit explanation, specifically such as
Under：
(1) preparation of 100mL LB culture mediums：1.0g peptones, 0.5g yeast, and 1.0g sodium chloride are weighed respectively, are dissolved in
In 100mL deionized waters, steam sterilizing 20min under 15psi high pressures.
(2) 100 μ L kanamycins are added in Biohazard Safety Equipment to sterilized LB culture mediums, 1mL is added and has been turned
Change has the Escherichia coli of green fluorescent protein plasmid gene, in 37 DEG C, 14~16h of 270rpm shaking cultures.
(3) to 2.5mL equilibrium liquid BL, 1000rpm is added in adsorption column CP5,2min is centrifuged, outwells useless in collecting pipe
Liquid, and adsorption column withdraws collecting pipe again.
(4) by the bacterium solution addition 50mL centrifuge tubes of shaking in 24 hours, 4 DEG C in supercentrifuge, 12000rpm is centrifuged
3min, abandoning supernatant, and centrifuge tube is inverted on filter paper to blot the culture medium remained on tube wall completely.
(5) to the P1 solution for adding 7mL to contain RNaseA in the centrifuge tube of the thalline being collected into, it is vortexed thin to be thoroughly suspended
Bacterium, is subsequently adding 7mL solution P2, leniently spins upside down 6 to 8 times immediately, is stored at room temperature 5min, adds 7mL solution P4, then
Secondary excitation is gentle to be spun upside down 6 to 8 times, is stored at room temperature 10min, and 4 DEG C, 1000rpm is centrifuged 20~30min so that thalline egg
It is white to be deposited in centrifugation bottom of the tube completely, then carefully all pour into filter CS solution, push handle filtering is slowly promoted, finally
Filtrate is collected in clean 50mL centrifuge tubes.
(6) to 0.3 times of isopropanol of filtrate volume is added in filtrate, slowly absorption is transferred to after spinning upside down reverse mixing
In post CP5,4 DEG C, 1000rpm, room temperature centrifugation 4min, outwell the waste liquid in collecting pipe, according still further to above-mentioned centrifugal condition be centrifuged to
Whole filtrates cross post, during adsorption column CP5 placed back in into collecting pipe.
(7) to 10ML rinsing liquids PW is added in adsorption column CP5,4 DEG C, 10000rpm is centrifuged 2min, in outwelling collecting pipe
Waste liquid, during adsorption column placed back in into collecting pipe, and repeats rinsing once.
(8) to 3mL absolute ethyl alcohols are added in adsorption column, 4 DEG C, 12000rpm is centrifuged 5min, discards useless in collecting pipe
Liquid, then adsorption column is placed back in into collecting pipe, 4 DEG C, 12000rpm is centrifuged 5min, adsorption column is uncapped and is placed as room temperature
5min, fully to dry the ethanol solution remained in adsorption column.
(9) by adsorption column as a clean 50ml centrifuge tube in, to be slowly added dropwise 1.5mL wash-outs slow to hanging in adsorption column
Fliud flushing TB, room temperature places 8min, and then 1000rpm centrifugations 3min, collects the eluent in collecting pipe, as plasmid pEGPF-
C1, the TB elution buffers for adding 2mL preheatings are repeated once.
(10) careful collection filtrate, with elution buffer TB or deionized water as blank, in Nanodrop2000 is ultraviolet can
See and DNA concentration is determined in spectrophotometer, and record the value of OD260 and OD280.Wherein OD280/OD260 ratios are in 1.8-2.0
Between then DNA purity it is qualified, DNA solution finally is diluted into working concentration with deionized water, and to be positioned over -20 DEG C of storages standby.
Embodiment 1
1st, the preparation of pDNA/ carriers nano-complex
(1) CDG2 is configured to the aqueous solution that concentration is 0.8mg/mL；It is the water-soluble of 80ng/ μ L that DNA is configured into concentration
Liquid；Lipo2000 is configured to the aqueous solution that concentration is 1mg/mL.
(2) prepare complex solution according to the following ratio, then by complex solution stationary incubation 30min at room temperature, that is, divide
It is nano combined that pDNA/CDG2 nano-complexes, pDNA/Lipo2000 nano-complexes, pDNA/CDG2/Lipo2000 are not obtained
Thing.
The composition of pDNA/CDG2 complex solutions:37.5μL CDG2,75μL DNA,37.5μL H2O。
The composition of pDNA/Lipo2000 complex solutions:12μL Lipo2000,75μL DNA,63μL H2O。
The composition of pDNA/CDG2/Lipo2000 complex solutions:6μL Lipo2000,18.8μL CDG2,75μL DNA,
50.2μL H2O。
2nd, cell culture
By Hek293T cells, Hek293A cells, A549 cells and LOVO cells respectively containing 10% hyclone (FBS)
DMEM in high glucose culture medium in, cultivated in 37 DEG C, 5% CO2gas incubator.
3rd, transfection experiment
(1) inoculating cell
With the Trypsin Induced containing 0.25%EDTA be in growth logarithmic phase Hek293T, Hek293A, A549 and
LOVO cells, are prepared into single cell suspension, then seed cells into 24 orifice plates, and 6.0 × 105Individual cells/well, in containing serum
24h is cultivated in complete medium, cell is rinsed with PBS, DMEM culture mediums of the 600 μ L without serum is added per hole, wait cell to converge
It is right when being 80% by transfected.
(2) cell transfecting
By being inoculated with 24 orifice plates of cell for above-mentioned pDNA/ carriers nano-complex addition step (1) for preparing, per hole
50 μ L (amount of DNA is 2 μ g) are added, every group of three multiple holes after transfecting 4h in 37 DEG C, the incubator of 5% carbon dioxide, discard training
Nutrient solution, adds DMEMs of the 1mL containing 10% hyclone, after continuing to cultivate 44h, discards culture medium.
With the transfection efficiency in vitro of inverted phase contrast fluorescence microscope qualitative observation pDNA/ carrier nano-complexes.In inversion
The expression of observation of cell state and green fluorescent protein under difference fluorescence microscope, and with graphics software Image Pro Plus
6.0 shoot record fluorescence picture, as a result see Fig. 1-Fig. 4.As can be seen from the figure：CDG2's and Lipo2000 is compound-mediated
The egfp expression amount highest of pEGPF-C1 transfectional cells (Hek293T, Hek293A, A549 and LOVO), is significantly higher than
Single CDG2 and single Lipo2000.
The transfection efficiency in vitro of pDNA/ carrier nano-complexes is quantitative determined with flow cytometry.Previous step is taken pictures completion
Afterwards, each hole cell in plate is washed 2 times with PBS immediately, adds Trypsin Induceds of the 300 μ L containing 0.25%EDTA,
Add DMEM culture mediums of the 600 μ L containing 10% serum to terminate digesting, 1000rpm centrifugation 5min, careful abandoning supernatant, finally
Add 300 μ L0.5% paraformaldehyde solution re-suspended cells.Express green glimmering in determining every 10000 cells with flow cytometer
The percentage of cells of photoprotein, obtains the cell transfecting efficiency of pDNA/ carrier nano-complexes, as a result sees Fig. 5-Fig. 8.From figure
In it can be seen that：The transfection efficiency of CDG2 and Lipo2000 compounds is significantly higher than single CDG2 and Lipo2000.
Embodiment 2
1st, the preparation of pDNA/ carriers nano-complex
(1) CDG2 is configured to the aqueous solution that concentration is 0.8mg/mL；It is the water-soluble of 80ng/ μ L that DNA is configured into concentration
Liquid；DC-Chol-DOPE is configured to the aqueous solution that concentration is 1mg/mL.
(2) prepare complex solution according to the following ratio, then by complex solution stationary incubation 30min at room temperature, that is, divide
PDNA/CDG2 nano-complexes, pDNA/DC-Chol-DOPE nano-complexes, pDNA/CDG2/DC-Chol-DOPE are not obtained
Nano-complex.
The composition of pDNA/CDG2 complex solutions:37.5μL CDG2,75μL DNA,37.5μL H2O。
The composition of pDNA/DC-Chol-DOPE complex solutions:12μLDC-Chol-DOPE,75μL DNA,63μL H2O。
The composition of pDNA/CDG2/DC-Chol-DOPE complex solutions:6μL DC-Chol-DOPE,18.8μL CDG2,
75μL DNA,50.2μL H2O。
Hek293T cell culture and transfection experiment with embodiment 1, with the result of inverted phase contrast fluorescence microscope qualitative observation
See Fig. 9, the result quantitative determined with flow cytometer is shown in Figure 10, as can be seen from the figure：CDG2 and DC-Chol-DOPE is combined
The transfection efficiency of thing is significantly higher than single CDG2 and DC-Chol-DOPE.
Embodiment 3
1st, the preparation of pDNA/ carriers nano-complex
(1) CDG2 is configured to the aqueous solution that concentration is 0.8mg/mL；It is the water-soluble of 80ng/ μ L that DNA is configured into concentration
Liquid；DOSPER-DOPE is configured to the aqueous solution that concentration is 1mg/mL.
(2) prepare complex solution according to the following ratio, then by complex solution stationary incubation 30min at room temperature, that is, divide
PDNA/CDG2 nano-complexes, pDNA/DOSPER-DOPE nano-complexes, pDNA/CDG2/DOSPER-DOPE is not obtained to receive
Rice compound.
The composition of pDNA/CDG2 complex solutions:37.5μL CDG2,75μL DNA,37.5μL H2O。
The composition of pDNA/DOSPER-DOPE complex solutions:12μDOSPER-DOPE,75μL DNA,63μL H2O。
The composition of pDNA/CDG2/DOSPER-DOPE complex solutions:6μL DOSPER-DOPE,18.8μL CDG2,75μ
L DNA,50.2μL H2O。
Hek293T cell culture and transfection experiment with embodiment 1, with the result of inverted phase contrast fluorescence microscope qualitative observation
See Figure 11, the result quantitative determined with flow cytometer is shown in Figure 12, as can be seen from the figure：CDG2 and DOSPER-DOPE is combined
The transfection efficiency of thing is significantly higher than single CDG2 and DOSPER-DOPE.
Embodiment 4
1st, the preparation of pDNA/ carriers nano-complex
(1) CDG2 is configured to the aqueous solution that concentration is 0.8mg/mL；It is the water-soluble of 80ng/ μ L that DNA is configured into concentration
Liquid；DOTMA-DOPE is configured to the aqueous solution that concentration is 1mg/mL.
(2) prepare complex solution according to the following ratio, then by complex solution stationary incubation 30min at room temperature, that is, divide
Do not obtain pDNA/CDG2 nano-complexes, pDNA/DOTMA-DOPE nano-complexes, pDNA/CDG2/DOTMA-DOPE nanometers
Compound.
The composition of pDNA/CDG2 complex solutions:37.5μL CDG2,75μL DNA,37.5μL H2O。
The composition of pDNA/DOTMA-DOPE complex solutions:12μDOTMA-DOPE,75μL DNA,63μL H2O。
The composition of pDNA/CDG2/DOTMA-DOPE complex solutions:6μL DOTMA-DOPE,18.8μL CDG2,75μL
DNA,50.2μL H2O。
Hek293T cell culture and transfection experiment with embodiment 1, with the result of inverted phase contrast fluorescence microscope qualitative observation
See Figure 13, the result quantitative determined with flow cytometer is shown in Figure 14, as can be seen from the figure：CDG2 and DOTMA-DOPE compounds
Transfection efficiency be significantly higher than single CDG2 and DOTMA-DOPE.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of complex gene carrier, it is characterised in that be composited by CDG2 and liposome, the matter of the CDG2 and liposome
Amount is than being 1-5:1, the CDG2 are prepared by the outside grafting PAMAM-G2 in alpha-cyclodextrin.
2. complex gene carrier according to claim 1, it is characterised in that the mass ratio of CDG2 and liposome is 1.5-4:
1。
3. complex gene carrier according to claim 1, it is characterised in that the liposome be selected from DC-Chol-DOPE,
DOSPER-DOPE, DOTMA-DOPE and LipofectamineTMAt least one in 2000.
4. the complex gene carrier according to claim any one of 1-3, it is characterised in that the preparation method bag of the CDG2
Include following steps：Alpha-cyclodextrin and coupling agent are reacted, obtains being connected with the alpha-cyclodextrin of coupling agent；By PAMAM-G2 be connected
The alpha-cyclodextrin for having coupling agent reacts, and obtains final product the CDG2.
5. complex gene carrier according to claim 4, it is characterised in that the coupling agent is N, N- carbonyl dimidazoles.
6. complex gene carrier according to claim 5, it is characterised in that the alpha-cyclodextrin, N, N- carbonyl dimidazoles
It is 1 with the mass ratio of PAMAM-G2：11-15：50-55.
7. application of the complex gene carrier described in any one of claim 1-6 in gene therapy medicament is prepared.
8. a kind of DNA/ carrier complexes, it is characterised in that complex gene carrier as described in claim any one of 1-6 with
DNA is composited.
9. DNA/ carrier complexes according to claim 8, it is characterised in that the matter of the complex gene carrier and DNA
Amount is than being 2.5-5:1.
10. a kind of method for preparing DNA/ carrier complexes, it is characterised in that comprise the following steps：CDG2 is configured to concentration
It is the CDG2 aqueous solution of 0.7-0.9mg/mL, by liposome formulation into the liposome aqueous solution that concentration is 0.9-1.1mg/mL, will
DNA is configured to the aqueous dna that concentration is 75-85ng/ μ L；Take the 18.5-19 μ LCDG2 aqueous solution, 5-7 μ L liposome aqueous solutions,
The 70-80 μ LDNA aqueous solution and 45-55 μ L water, stationary incubation 25-35min at room temperature after mixing obtain final product the DNA/ carriers and answer
Compound；The CDG2 is prepared by the outside grafting PAMAM-G2 in alpha-cyclodextrin.
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