CN103145976B - For compound and the application thereof of cell-targeting genophore - Google Patents
For compound and the application thereof of cell-targeting genophore Download PDFInfo
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- CN103145976B CN103145976B CN201210064929.1A CN201210064929A CN103145976B CN 103145976 B CN103145976 B CN 103145976B CN 201210064929 A CN201210064929 A CN 201210064929A CN 103145976 B CN103145976 B CN 103145976B
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Abstract
The invention provides a kind of compound for cell-targeting genophore, gene targeting vector prepared by described compound and the mixture for gene therapy, described compound has the structure as shown in structural formula (I).Genophore prepared by the present invention has well liver cell targeted, very high transfection activity and less cytotoxicity.
Description
Technical field
The present invention relates to a kind of compound and application thereof, particularly relate to a kind of compound for cell-targeting genophore, described compound synthetic method and application.
Background technology
Gene therapy, be by allogenic gene material (DNA or RNA) transfered cell, facilitate or suppress the expression of specific protein, or replace, repair problematic gene, with the disease of correcting or compensator gene defect causes with exception, thus reach the object of disease treatment.Namely foreign gene is integrated in the suitable recipient cell of patient by gene transfer technique, makes the genetic products of exogenous gene expression can treat certain disease; The gene therapy of generalized concept, to refer to some transfer of genetic material in patient body, makes it express in vivo, finally reach the object of disease therapy.As far back as 1991, first Chinese Scientists has carried out the first haemophiliachemophiliac clinical gene therapy experiment in the world, subsequently, China scientist utilizes thymidine kinase gene therapy malignant glioma gene therapy approach to get permission to enter I clinical trial phase, preliminary observation shows: lifetime accounts for 55% more than more than 1 year person, wherein 1 example is more than three and half, has not yet to see tumor recurrence so far.In addition, vascellum esoderma growth factor gene treatment periphery infarctional lower limb vascular ospc gene treatment plan is adopted also to get permission to enter clinical trial.
Gene therapy encounters a series of technical bottleneck in its evolution, and wherein one of most important bottleneck is genetic stew safety, effective, targeting conveying.
Gene therapy technology needs genetic material to be assembled on genophore, general adopts virus mediated gene transfer, is namely carrier with virus, by genetic material by Technical forms such as gene recombination in virus, then make recombinant virus infection recipient host cell.Gene delivery carrier conventional at present can be divided into recombinant viral vector and synthetic carrier (i.e. non-virus carrier).
Wherein, recombinant viral vector has the ability of penetration cell, and the recipient cell of nearly 100% can be made infected, and transformant efficiency is high; Secondly, it can infect wide spectrum animal species and cell type and without strict tissue specificity; Moreover the virus of random integration can longer-term persistence, does not generally harm cell; As (" XI AN JIAOTONG UNIVERSITY Subject Index (medicines) " such as Cui Yuanyuan, 6th phase in 2007,624th ~ 625 pages) disclose the pLSXN retrovirus vector coming from moloney mouse leukaemia virus, prepare the retrovirus vector of mediation beta-secretase peptide substrate genetic expression; Patent WO01/90390A1 discloses a kind of baculovirus vector, can be used for the gene therapy of vascular disease; Patent US20020064520A1 also discloses a kind of composition for gene therapy, is core, makes gene delivery system with recombinant virus particle.Although recombinant viral vector shows high transfection efficiency, because the variation of virus can cause potential pathogenic risk, and virus surface becomes branch to cause Human immune responses, the simultaneously preparation of virus and purification difficult and carrying gene capacity is little.
Therefore non-viral gene vector is considered to more preferably genetic stew delivery vehicles.If Poly-cation (as PEI25kDa) is compared with virus and liposome vectors, there is gene and support that density and loading are all large, preparation is simple, be convenient to the advantages such as chemically modified, if Zhao Danjun is at " synthesis of Floxuridine bonding cation carrier and antitumor activity thereof " (journal of Zhejiang university medicine, 1st phase in 2009,53 ~ 58 pages) disclosed in polymine-beta-cyclodextrin polymer carrier, and Floxuridine be bonded in Soviet Union send on polymer carrier.But non-viral gene vector also also exists, and chemical toxicity is large, the shortcoming in body-internal-circulation cycle short (being unfavorable for target).
Summary of the invention
For the above-mentioned defect that genophore in gene therapy exists, the invention provides a kind of macromolecular compound for cell-targeting genophore and synthetic method thereof, and the preparation method of the genophore prepared of described macromolecular compound and described genophore, and comprise the gene therapy mixture of described genophore.
First aspect of the present invention is to provide a kind of compound for cell-targeting genophore (being called for short GPE), and described compound has the molecular structure as shown in structural formula (I):
Wherein:
A
1and A
2independently be-O-or-S-;
R
1for:
But should be understood that, R
1unit structure formula only represents the number ratio of two kinds of repeating units, does not represent the position of two kinds of unit, R
1in two kinds of repeating units can be random distribution, be alternately distributed or block distributed;
R
2for group structural formula (II) Suo Shi;
R
3, R
4, R
5, R
6independently be-CH
2-,-HCR-or-C (R)
2-in any one group, R is the alkyl containing C1 ~ C10 carbon chain lengths, and is preferably alkyl, as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, the tertiary butyl, phenyl ring, benzyl, cyclohexyl, cyclopentyl etc.;
N, x and y all refer to the polymerization degree.
Wherein R
1group Molecule amount is preferably Mw=500 ~ 1200, and more preferably 800 ~ 1000; Mn=400 ~ 1000, more preferably 500 ~ 800, as 600,650,700,750.
Described compound molecular weight is preferably: Mn is 5000 ~ 10000Da, Mw is 8000 ~ 15000Da.
Second aspect of the present invention is to provide a kind of synthetic method of above-claimed cpd, and step comprises:
Step 1, lactobionic acid (LA) carries out amidate action with starting compound (polyoxyethylene glycol, PEG) shown in structural formula (III), amidation intermediate A (Gal-PEG) shown in generating structure formula (IV);
Step 2, shown in amidation intermediate A and structure formula V, compound reacts, and prepares compound shown in structural formula (I);
Wherein,
R
1for:
R
2for group structural formula (II) Suo Shi;
A
1and A
2independently be-O-or-S-;
R
3, R
4, R
5, R
6independently be-CH
2-,-HCR-or-C (R)
2-in any one group, R is the alkyl containing C1 ~ C10 carbon chain lengths.
Wherein, lactobionic acid and polyoxyethylene glycol mol ratio are preferably 5 ~ 20:1
According in a kind of preferred implementation of above-claimed cpd of the present invention or its synthetic method, A
1and A
2independently be-O-, R
3, R
4, R
5, R
6independently be-CH
2-.
In described preferred implementation, described compou nd synthesis step comprises:
Step 1, lactobionic acid is dissolved in 2-(N-morpholine)-ethyl sulfonic acid (MES) damping fluid, N-hydroxy-succinamide (NHS) and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloride or its salt (EDC) activation, be then obtained by reacting the shown intermediate of structural formula (IV) with polyoxyethylene glycol structural formula (III) Suo Shi;
Step 2, described intermediate is dissolved in MES damping fluid, NHS and EDC activates, and then adds structural formula (V-1) described compound (PEI-Et) and reacts, prepare the compound shown in structural formula (I-1).
Wherein, described polyoxyethylene glycol (not comprising capping group) molecular weight is preferably 1500 ~ 4000, and more preferably 2000 ~ 4000.R
1group Molecule amount as previously mentioned.
Wherein, described MES pH of cushioning fluid is preferably 6 ~ 7, as 6.2,6.4,6.5,6.6,6.7,6.8.Described MES buffer concentration is preferably 0.05 ~ 0.2M, as 0.08M, 0.1M, 0.12M, 0.15M.
In above-mentioned steps 1 and step 2, described soak time is preferably 20 ~ 50min, more preferably 30 ~ 50min.
Reaction in above-mentioned steps 1 of the present invention and step 2 is all preferably first to be carried out at-10 DEG C ~ 5 DEG C, more preferably-5 DEG C ~ 5 DEG C, as under ice bath or ice-water bath, cryosel bath condition, reaction times is preferably 5 ~ 30 hours, more preferably 10 ~ 30 hours, as 12h, 15h, 20h, 24h etc.; Then react under 15 ~ 40 DEG C of conditions, as 20 DEG C, 25 DEG C, 30 DEG C etc., the reaction times is preferably 5 ~ 30 hours, more preferably 10 ~ 30 hours, as 12h, 15h, 20h, 24h etc.
3rd aspect of the present invention is to provide cell-targeting genophore prepared by a kind of above-claimed cpd, and the 4th aspect is to provide the application of a kind of described compound in cell-targeting genophore.Step comprises:
Step 1, provides compound according to claim 1;
Step 2, by described compound dissolution, dialysis, then millipore filtration, freeze-drying.
Described dialysis can be any dialysis process known in the art, and as used dialysis tubing, the molecular weight cut-off of dialysis tubing is preferably 1000Da ~ 3500Da.Dialysis time is preferably 10 ~ 50 hours, as 12h, 15h, 18h, 24h, 35h, 48h etc.
Described millipore filtration can be any microporous filtering method known in the art, and as millipore filtration, the aperture of millipore filtration is preferably 0.20 ~ 0.5 μm, more preferably 0.22 ~ 0.45 μm.
The present invention the 5th aspect is to provide a kind of mixture (i.e. gene therapy mixture) of described cell-targeting genophore, comprises described cell-targeting genophore and genetic stew.
Wherein, described genetic stew can be DNA and/or RNA, and/or DNA, RNA fragment, and is preferably DNA plasmid.
Described cell-targeting genophore and genetic stew weight ratio are preferably 1 ~ 100, and more preferably 5 ~ 100, more preferably 5 ~ 70.
6th aspect of the present invention is to provide a kind of preparation method of described mixture, is joined in the solution containing genetic stew by described gene targeting vector, hatches, dry.
Wherein, described in hatch and preferably carry out under 15 ~ 40 DEG C of conditions, as 20 DEG C, 25 DEG C, 30 DEG C etc., the reaction times is preferably 20 ~ 150min, more preferably 30 ~ 120min.
Compared with prior art, the present invention has following beneficial effect:
(1) the cell-targeting genophore structure prepared of the present invention simple, be easy to synthesis; (2) the cell-targeting genophore that prepared by the present invention has well liver cell targeted, is particularly useful for the gene therapy of liver; (3) the cell-targeting genophore that prepared by the present invention has very high transfection activity and less cytotoxicity.
Accompanying drawing explanation
Fig. 1 is the synthetic route schematic diagram of the preparation method of GPE of the present invention;
Fig. 2 be in embodiment 2 GPE of the present invention and plasmid different mass than time the grain-size graph of mixture that formed;
Fig. 3 be in embodiment 2 GPE of the present invention and plasmid different mass than time the potential energy diagram of mixture that formed;
Fig. 4 be in embodiment 2 GPE and DNA plasmid of the present invention different mass than time the agarose gel electrophoresis figure of mixture that formed;
Fig. 5 is the atomic force microscope figure of the mixture formed when GPE and DNA plasmid mass ratio of the present invention is 70 in embodiment 2;
Fig. 6 be in embodiment 2 GPE of the present invention and plasmid different mass than time the transfection activity schematic diagram of mixture in BRL-3A cell that formed, wherein, * is compared to PEI25k, P<0.05, * is compared to PEI25k, P<0.01;
Fig. 7 be mixture (w/w70) that in embodiment 2, GPE of the present invention and plasmid are formed to liver cell targeted experiment schematic diagram, wherein * is P<0.01;
Fig. 8 is the semi-lactosi Inhibition test schematic diagram of mixture (w/w70) in BRL-3A cell that in example 2, GPE of the present invention and plasmid are formed, and wherein * * is P<0.01;
Fig. 9 is that the BRL-3A cytotoxicity of GPE and the PEI25kDa of the present invention of different concns in embodiment 2 compares schematic diagram, and wherein * * is compared to PEI25k, P<0.01.
Embodiment
The invention provides a kind of compound for cell-targeting genophore and preparation method thereof, and the cell-targeting genophore prepared of described compound and the application of described compound in cell-targeting genophore, on this basis, present invention also offers a kind of mixture for gene therapy.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usual conveniently condition, such as Pehanorm Brooker equimolecular clone: the condition described in the laboratory manual third edition (Science Press, 2002), or according to the condition that each manufacturers advises.
the preparation of embodiment 1, cell-targeting genophore compound (GPE)
Fig. 1 is the synthetic route schematic diagram of the preparation method of compound for cell-targeting genophore, as shown in Figure 1, comprises the steps:
Step 1, is dissolved in MES damping fluid (0.1M, pH6.5) by LA, activate 30min, be then that the ratio of 10:1 is reacted (ice bath 12h, room temperature 12h) in molar ratio with PEG-2000, obtain intermediate product Gal-PEG with NHS and EDC.
Step 2, is dissolved in MES damping fluid (0.1M, pH6.5) by Gal-PEG, activate 30min, be then that the ratio of 1:1 is reacted (ice bath 12h, room temperature 12h) in molar ratio with PEI-Et, obtain target product GPE with NHS and EDC.Wherein, in PEI-Et, R1 group Molecule amount is Mw=800 (laser light scattering method mensuration), Mn=600 (gel chromatography).
record GPE molecular weight:measuring method is gel permeation chromatography (GPC) method, GPE prepared by polyoxyethylene glycol (PEG) standard substance and embodiment 1 is sample, the solution obtaining 10mg/ml is dissolved respectively with pure water, shake up standing, with the filtering with microporous membrane of 0.45 μm, get subsequent filtrate, sample introduction 20 μ L records color atlas.
The logarithmic value lgMw of the weight-average molecular weight of PEG standard substance and corresponding retention time (tR) are carried out linear regression, obtains regression equation.GPE sample passes through formulae discovery molecular weight (Mn and Mw) and the distribution (D) of this regression equation:
Mn＝ΣRIi/Σ(RIi/Mi)；
Mw＝Σ(RIiMi)/ΣRIi；
D＝Mw/Mn；
In above formula, Mn, Mw are respectively number-average molecular weight and weight-average molecular weight; D refers to distribution coefficient; RIi is the peak height of trial-product when retention time i; Mi is the molecular weight of trial-product when retention time i.
Calculate: the molecular weight Mn=6550Da of GPE, Mw=9489Da.
the preparation of embodiment 2, cell-targeting genophore
Step 1, prepares compound GPE according to method described in embodiment 1.
Step 2, after being dissolved by described Gal-PEG and GPE ultrapure water, is placed in dialysis tubing dialysis (can be the arbitrary value of 12 ~ 48 hours) that molecular weight cut-off is 1000Da and 3500Da respectively; After dialysis terminates, filter with millipore filtration (can be the arbitrary value in 0.22 ~ 0.45 μm), then transfer to respectively in preprepared cillin bottle, product after-20 DEG C of refrigerator pre-freezes are spent the night by freeze drier low-temperature freeze drying vacuum except anhydrating, stop freeze-drying after 24h, obtain product polymer GPE.
embodiment 3, prepares the mixture of cell-targeting genophore and genetic stew
Take quantitative GPE polymkeric substance, add the solution that ultrapure water is configured to 2mg/mL, then filter with the aseptic filter of 0.45 μm, the concentration dilution of plasmid becomes 1mg/mL;
The complex solution of configuration different mass ratio, keep the concentration of plasmid solution constant, then the concentration of macromolecular solution is diluted according to the mass ratio of different GPE and plasmid, keep the volume of the macromolecular solution after dilution and plasmid solution equal, finally macromolecular solution is joined in plasmid solution fast and mix, incubated at room temperature 30 ~ 120min, so just obtains the mixture of a series of mass ratio, can be used as further physico-chemical property and measures.
described mixture particle size determination:the sample size of the mensuration of mixture particle diameter is 1.6mL, the volume of EGFP (green fluorescent protein) plasmid and macromolecular solution is respectively 800 μ L, the concentration of plasmid is 20 μ g/mL, dilute macromolecular solution (original concentration is 2mg/mL) according to mass ratio, in required mensuration mixture, the mass ratio of GPE and EGFP plasmid is respectively 1,5,10,20,50,70.During mixing, added by macromolecular solution in plasmid solution, piping and druming is even, incubated at room 30min.Detect the particle instrument adopting BrookhavenInstruments company, each sample determination 3 times, mapping of averaging; As shown in Figure 2, after testing, Polyplex can form the particle that particle diameter is 79 ~ 100nm.
described mixture Zeta potential measures:the sample size of the mensuration of mixture zeta-potential is 1.6mL, the volume of EGFP plasmid and macromolecular solution is respectively 800 μ L, the concentration of plasmid is 20 μ g/mL, dilute macromolecular solution (original concentration is 2mg/mL) according to mass ratio, in required mensuration mixture, polymer (GPE) is respectively 1,5 with the mass ratio of EGFP plasmid, 10,20,50,70.During mixing, added by macromolecular solution in plasmid solution, evenly, incubated at room 30min, then detects in piping and druming.Detect the particle instrument adopting BrookhavenInstruments company, each sample determination 3 times, mapping of averaging; As shown in Figure 3, when mass ratio is more than 5, the Zeta electric potential of Polyplex is just, polymer can the DNA of wrap negative charge.
described mixture agarose gel electrophoresis:configuration quality is than 1.0% agarose solution, heating for dissolving in microwave oven, get 40mL solution, pour in the beaker of special EB pollution, add the EB solution of about 4 μ L, pour into after stirring evenly in mould, plug comb, the after coagulation of about 30min, adds appropriate TAE damping fluid in electrophoresis chamber, sepharose is put into electrophoresis chamber and wait for loading.Then the complex solution of different mass ratio is configured, incubated at room 30min.The Marker of loading selects the plasmid Marker of 1000 ~ 10000kb, first gets the sample-loading buffer of 1 μ L during loading, adds the sample of 5 μ L, after mixing, joins in gel pore.Add the voltage of 80 volts, after blue tetrabromophenol sulfonphthalein can move to the bottom of glue fast, about 40min, takes pictures with ultraviolet gel imaging system.The Gel electrophoresis results of mixture, as shown in Figure 4: band 1 is Marker, 2 is naked DNA, and 3-10 respectively corresponding mixture mass ratio is 1,3,5,10,20,30,50,70.When the mixture of GPE and DNA is when mass ratio is 1, because carrier does not wrap the migration that therefore DNA cannot block DNA, therefore demonstrate bright band.When mass ratio >=3 because DNA wraps by carrier completely, therefore DNA is non-migratory when electrophoresis, does not have band in swimming lane, and this shows that, mass ratio more than 3 time, polymkeric substance has the ability of very strong complex gene material.
described mixture cell transfection assays:
In 48 porocyte culture plates, add the cell suspension (BRL-3A or HeLa cell) of 0.5mL, density is 5.0 × 10
4~ 10 × 10
4/ mL, overnight incubation.During 48 orifice plate transfection, the amount that every hole adds plasmid is 500ng, volume 25 μ L, is configured to the solution of 2mg/mL by polymkeric substance, and with the filter membrane sterile filtration of 0.45 μm, according to the testing sample of setting and the mass ratio of plasmid, be diluted to required ratio, the cumulative volume of polymers soln is 25 μ L, in the middle of the solution then polymers soln being joined plasmid, quick mixing, hatches 30min.The volume adding the mixture in every hole is like this 50 μ L, is 1/10th of cumulative volume (500 μ L), conforms with the regulations.Each mass ratio does three multiple holes.Positive controls PEI25kDa (25K), result when being 2 with its optimum quality ratio respectively does three control wells, hatch during this period of time in, cell is taken out from incubator, removing has the substratum of serum, wash one time by the PBS solution of 200 μ L again, substratum changes the substratum of the serum-free of 250 μ L into, is then sequentially added into in cell by the mixture of hatching.
After 4 hours, the substratum of removing serum-free, every hole adds the perfect medium containing 10% foetal calf serum, cultivate 44 hours again, detect its transfection results in BRL-3A and HeLa cell, as shown in Fig. 6,7,8, result shows, GPE has the very strong ability supporting genetic stew in BRL-3A cell, be that within the scope of 30-70, transfection efficiency all exceedes positive control PEI25kDa (P<0.05) at mass ratio, and the high transfection activity of GPE in BRL-3A cell is with it, and to have this liver cell targeting group of galactose residue relevant.
the cytotoxicity experiment of GPE
Inoculation BRL-3A cell, becomes density to be 5 × 10 by cell dilution
4~ 10 × 10
4the cell suspension of/mL, in 96 orifice plates, every hole adds 100 μ L, overnight incubation.
The polymer DMEM solution dilution of 2mg/mL is become different concentration gradients, and final volume is that 100 μ L, positive controls PEI25kDa also dilute for the concentration gradient consistent with testing sample group.
After taking out cell, remove the substratum of serum, wash one time with PBS (phosphoric acid salt) damping fluid of 100 μ L, directly the polymer DMEM solution prepared is added in each cell hole, add 100 μ L serum-frees in negative control group without phenol red DMEM.After 4 hours, removing nutrient solution and macromolecular solution, every hole adds the serum-free of 100 μ L without phenol red medium, MTT solution (the 3-(4 of 25 μ L is added again under lucifuge condition, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salts solution solution, this solution PBS buffer becomes 5mg/mL), be placed in cell culture incubator and cultivate 6 hours.
The crystallization situation of basis of microscopic observation viable cell, if also there is no complete crystallization, can proper extension storage period.If crystallization completely, the liquid in cautious sucking-off 96 orifice plate, then adds the DMSO (methyl-sulphoxide) of 150 μ L in every hole, and slight wobble 96 orifice plate makes first a ceremonial jade-ladle, used in libation crystal fully dissolve.Because solution colour after adding DMSO can change in time, therefore preferably the detection of microplate reader is carried out in 20min, and determined wavelength is 570nm, does ratio, thus obtain the surviving rate of cell by the absorption value at this wavelength of sample sets and blank group.The toxicity comparison diagram of the BRL-3A cell of GPE and the PEI25kDa of different concns as shown in Figure 9, cell viability test shows: in 5 ~ 100 μ g/mL concentration ranges, cell survival rate is more than 91%, GPE almost no cytotoxicity is described, not and its cytotoxicity much smaller than positive control PEI25kDa (P<0.01).
Although only mention A in above-described embodiment
1, A
2be-O-, R
3, R
4, R
5, R
6be respectively-CH
2-etc. situation, but in compound of the present invention, target group is semi-lactosi, therefore, works as A
1, A
2, R
3, R
4, R
5, R
6for mention in content of the present invention other group time, also can obtain similar technique effect, and by being similar to the experiment of above-described embodiment, also can confirm this effect.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. for a compound for cell-targeting genophore, it is characterized in that, described compound has the molecular structure as shown in structural formula (I):
wherein:
A
1and A
2independently be-O-or-S-;
R
1for:
R
2for group structural formula (II) Suo Shi;
R
3, R
4, R
5, R
6independently be-CH
2-,-HCR-or-C (R)
2-in any one group, R is the alkyl containing C1 ~ C10 carbon chain lengths.
2. a method for synthesis compound as shown in claim 1, it is characterized in that, step comprises:
Step 1, shown in lactobionic acid and structural formula (III), starting compound carries out amidate action, amidation intermediate A shown in generating structure formula (IV);
Step 2, shown in amidation intermediate A and structure formula V, compound reacts, and prepares compound shown in structural formula (I);
Wherein,
R
1for:
R
2for group structural formula (II) Suo Shi;
A
1and A
2independently be-O-or-S-;
R
3, R
4, R
5, R
6independently be-CH
2-,-HCR-or-C (R)
2-in any one group, R is the alkyl containing C1 ~ C10 carbon chain lengths.
3. the application of compound in cell-targeting genophore as claimed in claim 1.
4. application according to claim 3, is characterized in that, the preparation method of described cell-targeting genophore, and step comprises:
Step 1, provides compound according to claim 1;
Step 2, by described compound dissolution, dialysis, then millipore filtration, freeze-drying.
5. application according to claim 4, is characterized in that, described dialysis procedure molecular weight cut-off is 1000Da ~ 3500Da.
6. application according to claim 4, is characterized in that, described millipore filtration millipore filtration aperture used is 0.2 μm ~ 0.5 μm.
7. the application of compound in the mixture preparing cell-targeting genophore as claimed in claim 1, is characterized in that, the mixture of described cell-targeting genophore comprises cell-targeting genophore according to claim 4 and genetic stew.
8. application according to claim 7, is characterized in that, described genetic stew is DNA plasmid.
9. application according to claim 7, is characterized in that, the preparation method of described mixture, for being joined in the solution containing genetic stew by gene targeting vector described in claim 3, is hatched, dry.
10. application according to claim 9, is characterized in that, described in hatch be carry out under 15 ~ 40 DEG C of conditions.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101259284A (en) * | 2008-04-15 | 2008-09-10 | 华东师范大学 | Liver target anticancer nano prodrug system based on tree shaped polymer, preparation and use |
| CN101270168A (en) * | 2008-05-13 | 2008-09-24 | 中国药科大学 | Hyaluronic acid stem grafting polyethylene imine copolymer, preparing method and application as genophore |
| CN101574527A (en) * | 2007-12-12 | 2009-11-11 | 华东师范大学 | Liver-targeting intelligent nano-micelle prodrug system and preparation thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101574527A (en) * | 2007-12-12 | 2009-11-11 | 华东师范大学 | Liver-targeting intelligent nano-micelle prodrug system and preparation thereof |
| CN101259284A (en) * | 2008-04-15 | 2008-09-10 | 华东师范大学 | Liver target anticancer nano prodrug system based on tree shaped polymer, preparation and use |
| CN101270168A (en) * | 2008-05-13 | 2008-09-24 | 中国药科大学 | Hyaluronic acid stem grafting polyethylene imine copolymer, preparing method and application as genophore |
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