CN104311830A - Dendritic gene and drug carrier, and preparation and application thereof - Google Patents
Dendritic gene and drug carrier, and preparation and application thereof Download PDFInfo
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Abstract
The invention provides biodegradable dendritic cationic polymer with a high efficiency and low toxicity. Low-algebraic dendritic cationic polymer with low toxicity is linked to a water-soluble core with good biocompatibility through disulfide bonds to form polymer with a high molecular weight. The biodegradable dendritic cationic polymer is high in gene and drug delivery efficiency, is degradable in cells and effectively relieves contradiction between the delivery efficiency and toxicity of the dendritic cationic polymer. Multi-branch water-soluble polymer and the low-algebraic dendritic cationic polymer which are easily available and simple are adopted as raw materials, so that the cost is low. A synthetic method is simple, mostly comprises room-temperature reactions, and is free of rigorous reaction conditions such as no water and no oxygen. Post-treatment generally adopts extraction or dialysis as a separation method, and operation is simple. A structure formula of the biodegradable dendritic cationic polymer is shown in the specification.
Description
Technical field
The invention belongs to biological technical field, relate to the biodegradable dendroid gene drug carriers of a class high-efficiency low-toxicity, be specifically related to class dendroid polycationic material and its preparation method and application.
Background technology
Genomic medicine therapy is the method for the Therapeutic cancer that recent two decades rises, and its elementary tactics foreign gene or cancer therapy drug is imported object cell by the mode that carrier transports and plays its effect, thus reach the object of Therapeutic cancer.In genomic medicine therapy, the transport of gene and medicine is very part and parcel, for the carrier of transport, by a certain amount of goal gene with medicine is efficient must be transported in target cell, makes its safety, effectively, stable playing a role be the most basic requirement.The carrier being applied to genomic medicine therapy at present is mainly divided into two large classes: viral vectors and non-viral carrier.
Viral vectors, can natural infection cell because of its natural preferendum, so have very high conveying efficiency, has a lot of conventional virus vector, and enter clinical experimental stage in current gene therapy.The virus now used has retrovirus, adenovirus, poxvirus, hsv, adeno-associated virus etc.Research in recent years in virus vector achieves major progress, but because viral vectors still also exists biological safety deficiency, targeting specific not, easily induce the defects such as host immune response to some cell transduction incomplete sum, limit viral vectors and further develop (Thomas C.E., Ehrhardt A., Kay M.A. Nat Rev Genet. 2003,4:346-358).Non-viral gene vector is high less than viral vectors on the delivery efficiency of gene and medicine, but they have unique advantage, as hypotoxicity, reduced immunogenicity, without (AL-Dosari M.S., the GAO X. such as infecting, synthesis preparation is easy, flexible structure is controlled
aaps J,
2009, 11 (4): 671-681), therefore obtain the widespread use of investigator, and polycationic material is wherein main and research one of non-viral carrier the most widely.
Polycationic material due in structure with positive charge, form mixture easily via electrostatic interaction with the nucleic acid (DNA, RNA, PNA) of negative charge, nucleic acid compressed, thus avoids degraded by enzymes.Because mixture is under normal conditions with positive net charge, contribute to the process being attached to cell surface and endocytosis subsequently and lysosome escape.This seminar once delivered discusses (TANG G.P., LU X. about the special topic of polycationic material
journal of Zhejiang University:Medical Sciences.
2009, 38 (1): 1-6), there are again many breakthroughs to the research of polycationic material in recent years.Polycationic material conventional at present comprises (the Morille M. such as chitosan (chitosan), polymine (PEI), polylysine (PLL), branch-shape polymer (dendrimer), Passirani C., Vonarbourg A., etal.
biomaterials,
2008, 29 (24-25): 3477-3496).
Branch-shape polymer is as a kind of special polymkeric substance, it has the branched structure of exact placement, surface also exists a large amount of cationic group, may be used for the transport of gene, simultaneously due to the cladon that its internal symmetry arranges, make whole molecule present a kind of strict 3-D solid structure, which forms a lot of supramolecule cavity, for carrying medicine (Menjoge A.R., Kannan R.M., Tomalia D.A.
drug Discov. Today.
2010, 15:171 – 185).But branch-shape polymer also exists certain contradiction in conveying efficiency and self toxicity, and the dendrimer of high algebraically has good conveying efficiency, but show obvious cytotoxicity due to degradation property difference.Synthesis aspect also very difficult (Cheng Y.Y., Zhao L.B., Li Y.W., the Xu T.W. of high algebraically dendrimer simultaneously
chem. Soc. Rev. 2011,40:2673 2703; Kukowska-latallo J.F., Bielinska A.U., Johnson J., Spindler R., Tomalia D.A., Baker J.R.,
jr. Proc. Natl. Acad. Sci. USA.1996,93:4897 4902).
This research is coupled together by the dendroid cationic polymers of degradable chemical key by water-soluble polymers controlled for a class size and some low algebraically, thus obtains the dendroid polycationic material of the biodegradable high-efficiency low-toxicity of series of new.By research, we find that this kind of material has gene and medicine and well carry and delivery capability, and efficiency is high, and toxicity is little, and biological degradability is good, are the very potential gene drug carriers of a class.
Summary of the invention
First object of the present invention is to provide a kind of dendroid gene drug carriers, i.e. the dendroid polycationic compounds (Dendritic cationic polymer) of the biodegradable high-efficiency low-toxicity of a class, has following structural formula:
Wherein:
R is two generation AB
3type dendroid cationic polymers (AB
3-Dendeimer G2), structural formula is:
N is the number of the repeating unit in each side chain of eight branch's polyoxyethylene glycol;
M is the number of the repeating unit in straight chain polyoxyethylene glycol.
Second object of the present invention is to provide the preparation method of this class novel dendritic polycationic compounds, realizes (for 8armPEG-SS-PEG-Dendrimer G2) by following steps:
1, the preparation in two generations dendrimer (compound 4): take a certain amount of compound 1 and be dissolved in mass volume ratio 20-40 methylene dichloride doubly, add 3-5 activator 1-hydroxyl-7-azo benzotriazole (HOAt) doubly that consumption is compound 1 amount of substance wherein and add the catalyst of triethylamine of HOAt amount of substance 2-4 times amount, after stirring at normal temperature 5-20 minute, add the 5-10 N-tertbutyloxycarbonyl quadrol doubly that consumption is compound 1 amount of substance, then under the condition of 0-5 degree Celsius slowly, add 2-4 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (EDCI) doubly that consumption is HOAt amount of substance, add rear maintenance 0-5 degree Celsius reaction 0.5-2 hour, normal-temperature reaction 3-5 hour again.After completion of the reaction, the ethyl acetate of certain volume is added in reaction solution, organic phase salt pickling 2-4 time of 0.1-0.5 mol/L, wash 2-4 time with the saturated sodium bicarbonate solution of certain volume, wash 1-2 time with the saturated nacl aqueous solution of certain volume, use a certain amount of anhydrous sodium sulfate drying again, revolve steaming.
Then gained intermediate product is dissolved in mass volume ratio 10-20 methyl alcohol doubly, add the 1-2 Nickel dichloride hexahydrate doubly of compound 2 amount of substance wherein, under the condition of 0-5 degree Celsius, then slowly add the 3-10 sodium borohydride doubly of compound 2 amount of substance.After adding, at 0-5 degree Celsius of reaction 0.3-0.5 hour, then room temperature reaction 3-5 hour.After completion of the reaction, with the hydrochloric acid soln of 0.5-2 mol/L, pH value is adjusted to acidity, and then with saturated sodium bicarbonate solution, pH value is adjusted to alkalescence.Revolved by methyl alcohol in solution and evaporate, then be extracted with ethyl acetate 2-4 time, organic phase saturated nacl aqueous solution is washed 1-2 time, with anhydrous sodium sulfate drying, then concentrates, and the method used column chromatography obtains compound 2.
Taking a certain amount of compound 2, the compound 3 of 0.05-0.08 times of compound 2 amount of substance and the 1.5-3 HOAt doubly of compound 2 amount of substance is dissolved in mass volume ratio 20-40 methylene dichloride doubly, add the catalyst of triethylamine of HOAt amount of substance 2-4 times amount, then under the condition of 0-5 degree Celsius, add the EDCI of HOAt amount of substance 2-4 times amount, add rear control 0-5 degree Celsius of reaction 1-3 hour, afterwards normal-temperature reaction 30-50 hour.After completion of the reaction, certain volume ethyl acetate is added in reaction solution, the salt pickling of the 0.1-0.5 mol/L of organic phase certain volume 2-4 time, wash 2-4 time with the saturated sodium bicarbonate solution of certain volume, wash 1-2 time with the saturated nacl aqueous solution of certain volume, use a certain amount of anhydrous sodium sulfate drying again, filter and revolve steaming, the method that residual residue uses column chromatography obtains intermediate product.
Then gained intermediate product is dissolved under 0-5 degrees celsius in the mixed solvent of mass volume ratio 30-50 times trifluoroacetic acid and methylene dichloride, stirring at normal temperature 10-20 hour, to dialyse in water 20-40 hour with the dialysis tubing of certain molecular weight cut-off afterwards, after lyophilize, obtained for two generations dendrimer (compound 4).
The preparation of the water-soluble inner core molecule (compound 6) 2, containing cystine linkage: water-soluble for a certain amount of multiple-limb kernelised compound 5 is dissolved in mass volume ratio 3-5 dimethyl sulfoxide (DMSO) doubly; then the linking agent of activated hydroxyl groups is taken; consumption is 10-30 times of the amount of multiple-limb combinations of materials; linking agent is dissolved in mass volume ratio 1-5 dimethyl sulfoxide (DMSO) doubly; and add linking agent amount of substance than being 1-2 triethylamine doubly; the solution of multiple-limb compound drops in the solution of linking agent by lucifuge under nitrogen protection, continues stirring reaction 3-5 hour afterwards.Afterwards reaction solution is added to volume ratio 20-50 ether doubly: in the mixing solutions of tetrahydrofuran (THF)=3:1-5:1 volume ratio, under 0-5 degrees celsius, leaves standstill 1-3 hour, filter and be precipitated i.e. intermediate product.
Then be dissolved in mass volume ratio 5-10 dimethyl sulfoxide (DMSO) doubly by filtering the intermediate product obtained, take 2-aminoethyl disulfide dihydrochloride again, consumption is 15-30 times of this intermediate product amount of substance, be dissolved in mass volume ratio 3-10 dimethyl sulfoxide (DMSO) doubly, and add the 3-5 triethylamine doubly taking 2-aminoethyl disulfide dihydrochloride amount of substance, stir a moment to dissolving completely.Slowly dropped in the solution of cystamine by the solution of this intermediate product afterwards, drip 3-5 hour, continue reaction 3-5 hour afterwards, after completion of the reaction, solution is dialysed 24-48 hour in dialysis tubing, and then frost drying obtains intermediate product.
Again gained intermediate product is dissolved in mass volume ratio 5-10 dimethyl sulfoxide (DMSO) doubly, take the straight chain polyoxyethylene glycol of one-sided activation again, consumption is 5-10 times of this intermediate product amount of substance, be dissolved in mass volume ratio 5-10 dimethyl sulfoxide (DMSO) doubly, the solution of this intermediate product is added dropwise in the solution of straight chain polyoxyethylene glycol, stirring reaction 2-5 hour, afterwards reaction solution is added to volume ratio 20-50 ether doubly: in the mixing solutions of tetrahydrofuran (THF)=3:1-5:1 volume ratio, 1-3 hour is left standstill under 0-5 degrees celsius, filter afterwards and obtain white powder and be water-soluble inner core molecule containing cystine linkage, i.e. compound 6.
3, the preparation of final product dendroid polycationic compounds (compound 7): the water-soluble inner core molecule compound 6 containing cystine linkage is dissolved in mass volume ratio 5-10 dimethyl sulfoxide (DMSO) doubly; then the linking agent of activated hydroxyl groups is taken; consumption is 15-30 times of compound 6 amount of substance; linking agent is dissolved in mass volume ratio 5-10 dimethyl sulfoxide (DMSO) doubly; and add linking agent amount of substance than being 1-2 triethylamine doubly; two kinds of solution mix by lucifuge under nitrogen protection, afterwards stirring reaction 4-8 hour.Afterwards reaction solution is added to volume ratio 10-50 ether doubly: in the mixing solutions of tetrahydrofuran (THF)=3:1-5:1 volume ratio, under 0-5 degrees celsius, leaves standstill 1-2 hour, filter and be precipitated i.e. intermediate product.
Then be dissolved in mass volume ratio 10-20 dimethyl sulfoxide (DMSO) doubly by filtering the intermediate product obtained, take a certain amount of compound 4 again, consumption is 5-10 times of this intermediate product amount of substance, is dissolved in mass volume ratio 10-20 dimethyl sulfoxide (DMSO) doubly.The solution afterwards solution of this intermediate product slowly being dropped to compound 4 has suffered, drip 2-4 hour, continue reaction 12-18 hour afterwards, after completion of the reaction, solution is dialysed 24-48 hour in dialysis tubing, then frost drying, obtains powdery substance, i.e. final product dendroid polycationic material (compound 7).
Reaction formula is as follows:
In reaction formula, compound 1 is known compound (Newkome G.R.
journal of Organic Chemistry,
1988, V53 (23): P5552-5554; Leonard N.J.
journal of the American Chemical Society,
1949, V71:P1762-1764); Compound 2 is the reduzates after compound 1 and the condensation of N-tertbutyloxycarbonyl quadrol; Compound 3 is known compound (Newkome G.R., Young J.K., Baker G.R., Potter R, Audoly L.P., Cooper D., and Weis C.D.
macromolecules,
1993, 26:2394-2396; Huang Q.R.
langmuir,
2005, V21 (7): P2737-2742); Compound 4 is the hydrolysates after compound 2 and compound 3 condensation, i.e. two generation AB
3type dendroid cationic polymers (AB
3-Dendeimer G2); Compound 5 is eight branch's polyoxyethylene glycol; Compound 6 is the water-soluble inner core molecules containing cystine linkage, is linked to eight branch's polyoxyethylene glycol (8arm-PEG-SS-PEG) of straight chain polyoxyethylene glycol with disulfide linkage; Compound 7 is condensation products of compound 4 and compound 6, i.e. final product dendroid polycationic material (8arm-PEG-SS-PEG-Dendrimer G2 is called for short PSPG2s).
In addition, the n in reaction formula is the number of the repeating unit in each side chain of eight branch's polyoxyethylene glycol, and m is the number of the repeating unit in straight chain polyoxyethylene glycol, n and m is determined by the molecular weight of polyoxyethylene glycol raw material.HOAT in reaction conditions represents 1-hydroxyl-7-azo benzotriazole, EDCI represents 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, TFA represents trifluoroacetic acid, CDI represents N, N'-carbonyl dimidazoles, the R group in compound 7 represents compound 4 group (Dendrimer G2).
Dendroid polycationic compounds of the present invention, namely compound 7 is: AB
3type polyamidoamine dendritic macromole-G2 (Dendrimer G2), AB
2type polyamidoamine dendritic macromole-G2, G3, G4 (PAMAM G2, G3, G4).
Water-soluble dendroid kernel (compound 5) of the present invention is: multiple-limb polyoxyethylene glycol (3arm, 4arm, 8arm), molecular weight is 10000Da-40000Da.
The straight chain molecular weight polyethylene glycol of one-sided N-hydroxysuccinimide activation of the present invention is MW 2000Da-5000Da.
Linking agent of the present invention is: in N, N'-carbonyl dimidazoles (CDI), N, N'-bis-succinimidyl carbonate (DSC), N-hydroxy-succinamide chloro-formic ester, 9-fluorenyl methyl 1-benzotriazole base carbonic ether (FMOC-OBT) any one.
Linking agent consumption of the present invention is 10-30 times amount with the amount of substance ratio of corresponding water-soluble dendroid kernel.
The mass volume ratio of water-soluble dendroid kernel consumption of the present invention and dimethyl sulfoxide (DMSO) is 3-5 times amount.
The mass volume ratio of linking agent consumption of the present invention and dimethyl sulfoxide (DMSO) is 1-5 times amount.
Catalyzer of the present invention is triethylamine, and consumption and activator HOAT amount of substance, than being 2-4 times amount, are 1-2 times amount with linking agent amount of substance ratio; With 2-aminoethyl disulfide dihydrochloride amount of substance than being 3-5 times amount.
2-aminoethyl disulfide dihydrochloride consumption of the present invention is 15-30 times amount with the amount of substance ratio of corresponding water-soluble dendroid kernel.
Straight chain of the present invention water-soluble dendroid kernel (straight chain polyoxyethylene glycol) consumption (NHS-PEG-OH) is 5-10 times amount with the amount of substance ratio of corresponding compound 5.
Dendroid micromolecular compound of the present invention (compound 4) consumption and corresponding PEG(compound 6) amount of substance than for 5-10 times amount.
3rd object of the present invention is to provide such novel dendritic polycationic material described as the application in gene drug carriers, and wherein said medicine is the medicine with hydrophobic property.
Material of the present invention is as non-viral gene pharmaceutical carrier, this carrier is kernel by the water-soluble polymers (as polyoxyethylene glycol) of a class multiple-limb, by degradable disulfide linkage, at multiple low algebraically (G2-G4) dendroid cationic polymers (AB synthesized in as the present invention of end link
3type PAMAM and commercial AB
2type PAMAM), define the dendroid polycationic material (Dendritic cationic polymer) of series of new.This kind of material not only can transporter gene and medicine efficiently, and have hypotoxicity and degradability, and synthetic method is simple, cost is lower, is the material that a class has applications well prospect simultaneously.
The compounds of this invention is in preparation, and have chosen the dendroid cationic polymers of multiple-limb water-soluble polymers simple and easy to get and low algebraically as raw material, cost compare is cheap; And synthetic method is simple and easy, be normal-temperature reaction mostly, do not need the reaction conditions of the harshnesses such as anhydrous and oxygen-free, aftertreatment generally adopts the separation method of extraction or dialysis, simple to operate.
The present invention has used water-soluble kernel and dendroid cationic polymers feature separately dexterously, by simple connection, reaches good effect.First, because dendroid cationic polymers surface is with a large amount of positive charges, mutually repel to each other, so whole solid support material presents radial form in water, spherical dendroid cationic polymers is dispersed in around water-soluble kernel, utilize the distinctive hydrophobic cavity of dendroid cationic polymers therein simultaneously, easily can carry fat-soluble medicine; Secondly, the outer field positive charge of dendroid cationic polymers can combine with the gene (DNA or RNA) with negative charge effectively, when positive charge is neutralized, due to water miscible deficiency, can be wrapped up by water-soluble better inner core, formed close to electric neutrality and the size suitable cell nanoparticle of engulfing.3rd, owing to linking with disulfide linkage between water-soluble inner core molecule with dendroid cationic polymers, and disulfide linkage is easy to be reduced degraded in cell, thus discharges and be wrapped in interior gene and medicine, makes it play a role.
The present invention utilizes the hydrophilic and hydrophobic of the feature of this body structure and compound each several part different, replaced by the upset of compound ectonexine structure, reach the function of effectively carrying gene (DNA or RNA) and medicine, form the nanoparticle of stable in properties, about size 200nm simultaneously.This nanoparticle has very high gene delivery efficiency in cell, and medicine also can stablize slowly-releasing constantly in cell, improves the bioavailability of gene and medicine.In addition, traditional low algebraically dendroid cationic polymers delivery of gene and the efficiency of medicine very low, and although high algebraically dendroid cationic polymers has certain delivery efficiency, but because self-molecules present amount is very high, and cannot degradation in vivo, cytotoxicity is very large, the present invention utilizes disulfide linkage to be linked on the good water-soluble kernel of biocompatibility by low algebraically dendroid cationic polymers very low for toxicity, define the polymkeric substance of a high molecular, not only there are good gene and drug delivery efficiency, and can at intracellular degradation, effectively alleviate the contradiction between dendroid cationic polymers delivery efficiency and toxicity.
Accompanying drawing explanation
Fig. 1 is the perspective view of novel dendroid polycationic compounds.
Fig. 2 is AB
3the nucleus magnetic hydrogen spectrum phenogram of-Dendrimer G2.
Fig. 3 is AB
3the high resolution mass spectrum phenogram of-Dendrimer G2.
Fig. 4 is that the nucleus magnetic hydrogen spectrum of 8armPEG-SS-PEG-Dendrimer G2 and building-up process product characterizes.
Fig. 5 is that the nucleus magnetic hydrogen spectrum of the PSPG2s of four kinds of different ratioss characterizes.
Fig. 6 is the agarose gel electrophoresis blockade test that material carries DNA and RNA.
Fig. 7 is the attenuated total reflectance attenuated total refraction FTIR spectrum of PSPG2s-6 and PSPG2s-6/Dox/DNA.
Fig. 8 is the surface X-ray photoelectron spectrum figure of PSPG2s-6 and PSPG2s-6/Dox/DNA.
Fig. 9 is the Electronic Speculum figure of PSPG2s-6/DOX/DNA, and wherein (a) is transmission electron microscope picture, and (b) is scanning electron microscope (SEM) photograph.
Figure 10 (a-h) is PSPG2s-6, particle diameter after Dendrimer G2 and medicine carrying thereof and gene and potential ph diagram ph, wherein scheme particle diameter and current potential that a is Dendrimer G2 and 4 kind of different ratios PSPG2s, figure b is particle diameter and the current potential that Dendrimer G2 and PSPG2s-6 carries different ratios Zorubicin, figure c and figure d be Dendrimer G2 and 4 kind of different ratios PSPG2s respectively in conjunction with after DNA different N/P than under particle diameter and current potential, figure e and figure f is that Dendrimer G2 and PSPG2s-6 to carry after Zorubicin at different N/P than the lower particle diameter in conjunction with DNA and current potential respectively, figure g and figure h is Dendrimer G2 respectively, 2 kinds of different ratios PSPG2s and PSPG2s-6 to carry in 2 after different ratios Zorubicin at different N/P than the lower particle diameter in conjunction with siRNA and current potential.
Figure 11 is that Dendrimer G2 and PSPG2s-6 is in conjunction with DNA or in conjunction with the heparin interference experiment after Zorubicin and DNA.
Figure 12 be PSPG2s-6 PH=7.4,6.5 and 5.0 PBS solution in degradation experiment.
Figure 13 is the cytotoxicities of 6 kinds of materials under 4 hours different concns conditions.
Figure 14 is the cytotoxicities of 6 kinds of materials under 24 hours different concns conditions.
Figure 15 is that 4 kinds of different ratios PSPG2s and 2 kind of different ratios PPG2s carries luciferase protein plasmid gene (Luciferase) transfection results on Hek293 cell.
Figure 16 is 4 kinds of different ratios PSPG2s and 2 kinds of different ratios PPG2s Carrying Green Fluorescent Proteins RNA interfering (GFP-siRNA), in the green fluorescent protein silencing experiments result of people's renal epithelial cell (293T-GFP) in upper 24 hour of expressing green fluorescent protein.
Figure 17 be PSPG2s-6/Dox9% PH=5.0,7.4 PBS in Zorubicin release result.
Figure 18 is the TEM picture of PSPG2s-6/Dox9% under the acidic conditions of PH=5.0 during 0h and 12h, and PSPG2s-6/Dox9% PH=5.0,7.4 condition under the white light photo of 0h and 12h.
Dox, siRNA and PSPG2s/Dox9%/siRNA living body fluorescent figure in the different time points of nude mouse tumor position metabolism in Figure 19.
Figure 20 is the relative intensity change curve of red green fluorescence in Figure 19.
Figure 21 is the tumor biopsy figure of Dox and PSPG2s-6/Dox9%/siRNA after nude mouse tumor position metabolism 24h.
Figure 22 be 2 kinds access ratios PSPP3s/DNA(PSPP3s-4/6 represent that the amount of substance ratio of 8arm-PEG and PAMAM G3 in dendroid polycationic compounds PSPG2s is 4/6 respectively) different N/P than under the agarose gel electrophoresis blockade test carrying DNA.
Figure 23 be PAMAM G3, PEI25KD, PSPP3s-6 under optimum N/P ratio, be respectively the transfection experiment result of the luciferase reporter gene Luciferase under the condition of 0%, 10%, 25%, 50% at serum-concentration.
Figure 24 is the test experience result of differing materials lysosome escape capability.
Figure 25 is that different carrying drug ratio affects experimental result to transfection.
Figure 26 be 2 kinds access ratios 4PSPP4s/DNA(4PSPP4s-2/4 represent that the amount of substance ratio of 4arm-PEG and PAMAM G4 in dendroid polycationic compounds 4PSPP4s is 2/4 respectively) different N/P than under the agarose gel electrophoresis blockade test carrying DNA.
Figure 27 is 4PSPP4s(a) and atomic force microscope (AFM) figure 4PSPP4s/Gef/DNA(b).
Figure 28 is that Buthionine sulfoximine (BSO) is tested the impact of Materials Cell toxicity.
Figure 29 is that Buthionine sulfoximine (BSO) is tested the impact of Materials Cell transfection.
Figure 30 is PAMAM G4,4PSPP4s-4 and 4PPP4s-4 after in conjunction with Gefitinib and DNA, to dissociate the potential variation in experiment at heparin (heparin).
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments, but is not limited to the content disclosed in embodiment.
embodiment 1: solid support material 8armPEG-SS-PEG-Dendrimer G2(PSPG2s) preparation and carry medicine and gene.(branch-shape polymer is AB3-Dendrimer G2,8armPEG be 15000Da, NHS-PEG-OH be 2000Da, linking agent is CDI, and medicine is Zorubicin, and gene is blank report gene DNA)
(1) AB
3the synthesis of-Dendeimer G2
Taking 445.6 milligrams of compounds 1 is dissolved in 15 milliliters of methylene dichloride, add 871 milligrams of 1-hydroxyl-7-azo benzotriazole (HOAt) and 3 milliliters of triethylamines wherein, stirring at normal temperature is after 10 minutes, add 2.1 grams of N-tertbutyloxycarbonyl quadrols, then under the condition of 0 degree Celsius, slowly add 4.05 grams of 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochlorides (EDCI), add rear maintenance 0 degree Celsius reaction 1 hour, then normal-temperature reaction 4 hours.After completion of the reaction, 30 milliliters of ethyl acetate are added in reaction solution, the salt pickling of organic phase 20 milliliters of use 0.4 mol/L three times, three times are washed with 20 milliliters of saturated sodium bicarbonate solutions, wash once with 20 milliliters of saturated nacl aqueous solutions, use a small amount of anhydrous sodium sulfate drying again, filter and revolve steaming, obtain white solid.
Taking 703.8 milligrams of these white solids is dissolved in 10 ml methanol, adds the Nickel dichloride hexahydrate of 356.7 milligrams wherein, under the condition of 0 degree Celsius, then slowly add the sodium borohydride of 189.4 milligrams.After adding, 0 degree Celsius of reaction 0.3 hour, then room temperature reaction 4 hours.After completion of the reaction, with the hydrochloric acid soln of 1 mol/L, pH value is adjusted to 5, and then with saturated sodium bicarbonate solution, pH value is adjusted to 9.Revolved by methyl alcohol in solution and evaporate, then be extracted with ethyl acetate three times, organic phase saturated nacl aqueous solution is washed once, with anhydrous sodium sulfate drying, filter and then concentrate, the method (n-hexane/ethyl acetate=1/1) used column chromatography obtains colourless oily mater, i.e. compound 2.
Take 337 milligrams of compounds, 2,47 milligrams of compounds 3 and 136 milligrams of HOAt are dissolved in 10 milliliters of methylene dichloride, add 0.5 milliliter of triethylamine, then under the condition of 0 degree Celsius, slowly add 575 milligrams of EDCI, add rear control 0 degree Celsius reaction 2 hours, normal-temperature reaction 36 hours afterwards.After completion of the reaction, 75 milliliters of ethyl acetate are added in reaction solution, the organic phase salt pickling twice of 50 milliliter of 0.4 mol/L, twice is washed with 50 milliliters of saturated sodium bicarbonate solutions, wash once with 50 milliliters of saturated nacl aqueous solutions, use anhydrous sodium sulfate drying again, filter and revolve steaming, the method (methanol/ethyl acetate=1/4) that residual residue uses column chromatography obtains white solid.
Take this white solid 460 milligrams, in 0 degree Celsius of mixed solvent being dissolved in 10 milliliters of trifluoroacetic acids and 10 milliliters of methylene dichloride, stirring at normal temperature 12 hours, dialyse in water 24 hours with the dialysis tubing of molecular weight 2000 afterwards, white powder is obtained, i.e. two generation dendrimer 4(AB after lyophilize
3-Dendeimer G2).
(2) synthesis of 8armPEG-SS-PEG
The 8armPEG taking 6 grams of 15000Da is dissolved in 18 milliliters of dimethyl sulfoxide (DMSO); take 19 grams of N; N'-carbonyl dimidazoles (CDI); be dissolved in 20 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 1.6 milliliters; the solution of 8armPEG drops in the solution of CDI by lucifuge under nitrogen protection, continues stirring reaction 3 hours afterwards.Reaction solution is added to the ether that volume is 2 liters afterwards: in the mixing solutions of tetrahydrofuran (THF)=4:1 volume ratio, under 0 degrees celsius, leaves standstill 1 hour, filter afterwards and obtain white powder.
Then taking 3 grams of these white powders to be dissolved in 15 milliliters of dimethyl sulfoxide (DMSO), take 1.35 grams of 2-aminoethyl disulfide dihydrochlorides and be dissolved in 5 milliliters of dimethyl sulfoxide (DMSO), and add 2.5 milliliters of triethylamines, stirring a moment to dissolving completely.Slowly dropped in the solution of cystamine by the solution of PEG afterwards, drip 3 hours, continue reaction 3 hours afterwards, after completion of the reaction, solution is dialysed 24 hours in MW14000 dialysis tubing, and then frost drying, obtains white powder.
Then taking 1.75 grams of these white powders is dissolved in 10 milliliters of dimethyl sulfoxide (DMSO), the straight chain polyoxyethylene glycol (NHS-PEG-OH) taking the one-sided N-hydroxysuccinimide activation of 2 grams of 2000Da is again dissolved in 10 milliliters of dimethyl sulfoxide (DMSO), the solution of white powder is added dropwise in the solution of NHS-PEG-OH, stirring reaction 2 hours, afterwards reaction solution is joined the ether of 400 milliliters: in the mixing solutions of tetrahydrofuran (THF)=4:1 volume ratio, leave standstill 1 hour under 0 degrees celsius, filtration afterwards obtains white powder and is 8armPEG-SS-PEG.
(3) 8armPEG-SS-PEG-Dendrimer G2(PSPG2s) synthesis
3.4 grams of 8armPEG-SS-PEG are dissolved in 35 milliliters of dimethyl sulfoxide (DMSO); then 500 milligrams of N are taken; N'-carbonyl dimidazoles (CDI) is dissolved in 5 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 400 microlitre; two kinds of solution mix by lucifuge under nitrogen protection, afterwards stirring reaction 6 hours.Afterwards reaction solution is added to the ether of 400 milliliters: in the mixing solutions of tetrahydrofuran (THF)=4:1 volume ratio, under 0 degrees celsius, leaves standstill 2 hours, filter afterwards and obtain white powder.
Then take 1 gram of this white powder to be dissolved in 10 milliliters of dimethyl sulfoxide (DMSO), then take 1.5 grams of AB
3-Dendrimer G2 branch-shape polymer is dissolved in 15 milliliters of dimethyl sulfoxide (DMSO), the solution afterwards solution of white powder slowly being dropped to branch-shape polymer has suffered, drip 3 hours, continue reaction 12 hours afterwards, after completion of the reaction, solution is dialysed 36 hours in the dialysis tubing of MW14000, then frost drying, obtain white powder material, i.e. final product dendroid polycationic material PSPG2s.
Reaction formula is the same, and compound 7 is structural formulas of final product dendroid polycationic compounds, and wherein R group is compound 4, and the three-dimensional arrangement of compound 7 is see Fig. 1.
(4) preparation of nanoparticle PSPG2s-6/Dox
4 milligrams of Zorubicins (Doxorubicin, Dox) are dissolved in 1 milliliter of CH
2cl
2in, add 3 microlitre triethylamines, 36 milligrams of PSPG2s-6 are dissolved in 10 milliliters of DMSO, then both are mixed, and the uncovered stirring of room temperature is spent the night.Then the solution dialysis tubing dialysis 4h of molecular weight 14000, lyophilize, obtains PSPG2s-6/Dox.
(5) preparation of nanoparticle PSPG2s-6/DNA, PSPG2s-6/Dox/DNA
Blank gene DNA water dissolution is made into the solution of 0.1 microgram/microlitre, get this solution of 10 microlitres, again the PSPG2s-6 of 10 micrograms/microlitre or the PSPG2s-6/Dox aqueous solution equal-volume containing 10 micrograms/microlitre PSPG2s-6 are joined in DNA solution, static 30 minutes of room temperature, namely nanoparticle PSPG2s-6/DNA or PSPG2s-6/Dox/DNA is formed, can for follow-up.
embodiment 2: the sign of material
(1) AB
3the sign of-Dendrimer G2
Fig. 2 is AB
3the spectrogram of the nucleus magnetic hydrogen spectrum of-Dendrimer G2 and carbon spectrum, Fig. 3 is AB
3the spectrogram of the high resolution mass spectrum (MALDI-TOF MS) of-Dendrimer G2, substantially identical with theoretical molecular 5604, AB is described
3the preparation method of-Dendrimer G2 is feasible.
(2) synthesis characterization of 8armPEG-SS-PEG-Dendrimer G2
Fig. 4 is eight branch's polyoxyethylene glycol (8arm-PEG), eight branch's polyoxyethylene glycol (8arm-PEG-SS-PEG), the two generation AB linking straight chain polyoxyethylene glycol with disulfide linkage
3type branch-shape polymer (AB
3-Dendrimer G2), final product dendroid polycationic material (8armPEG-SS-PEG-Dendrimer G2, PSPG2s) hydrogen nuclear magnetic resonance spectrogram, in figure, each nonactive hydrogen atom displacement as shown in the figure, the proton nmr spectra of final product PSPG2s shows that polyoxyethylene glycol and branch-shape polymer are an entirety, the feasibility of the preparation method that sufficient proof provides.Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of the PSPG2s of four kinds of different ratioss, and wherein the ratio of kernel 8armPEG-SS-PEG and Dendrimer G2 is approximate 1:2,1:4,1:6,1:8 respectively, illustrates that this synthetic method can control to synthesize the product of different ratios.
embodiment 3: compound carries the ability of gene
Fig. 6 (a) be Dendrimer G2/DNA and 4 kind access ratio PSPG2s/DNA(PSPG2s-2/4/6/8 represent that the amount of substance ratio of 8arm-PEG and Dendrimer G2 in dendroid polycationic compounds PSPG2s is 2/4/6/8 respectively) different N/P than under the agarose gel electrophoresis blockade test carrying DNA.Illustrate shown in figure that this compounds of PSPG2s effectively can carry DNA, and the efficiency of carrying of the Dendrimer G2 of efficiency ratio Individual existence wants high.And to scheme (b) be PSPG2s-2/4/6/8 different N/P than under the agarose gel electrophoresis blockade test carrying RNA.Illustrate shown in figure that this compounds of PSPG2s also effectively can carry RNA.
embodiment 4: compound carries the structural characterization of gene and medicine
(1) the infrared experiment of attenuated total reflectance attenuated total refraction Fourier of PSPG2s-6 and PSPG2s-6/Dox/DNA
Fig. 7 is the attenuated total reflectance attenuated total refraction FTIR spectrum (PSPG2s-6 represents 8arm-PEG-SS-PEG-Dendrimer G2-6, and namely average each 8armPEG accesses 6 Dendrimer G2) of PSPG2s-6 and PSPG2s-6/Dox/DNA.In figure shown in two bands of a spectrum; PSPG2s-6 material is after carrying DOX and DNA; in bands of a spectrum, the peak area of Dendrimer G2 significantly reduces; the upset that there occurs structure when carrying DOX and DNA is described; originally the positively charged ion Dendrimer being exposed to outer ring combines the DNA of negative charge; be wrapped in Nano microsphere by PEG inner, PEG then defines layer protective layer outside.
(2) the surface X-ray Photoelectron Spectroscopy Experiment of PSPG2s-6 and PSPG2s-6/Dox/DNA
Fig. 8 is C1s(a, the b of PSPG2s-6 and PSPG2s-6/Dox/DNA) and O1s(c, d) high resolution x-ray photoelectron energy spectrogram, the lower ownership explanation for each peak of figure.In figure corresponding C1s swarming result in, a figure and the contrast of b figure show that PSPG2s-6 is after carrying DOX and DNA, and the surperficial peak belonging to the C=O of Dendrimer G2 disappears substantially originally; And in the swarming result of corresponding O1s, the overwhelming majority is the peak of the C=O belonging to Dendrimer G2 in c figure, and in d figure, be almost the peak of the C-O in PEG completely.Above-mentioned foundation all illustrates, PSPG2s-6 is after carrying DOX and DNA, and its surface presents the character of PEG substantially, and Dendrimer G2 is wrapped in Nano microsphere inside together with DOX and DNA carried.
embodiment 5: material carries the electron microscope experiment of gene and medicine
Fig. 9 is transmission electron microscope picture (a) and the scanning electron microscope (SEM) photograph (b) of PSPG2s-6/DOX/DNA.The particle diameter that the figure of two kinds of Electronic Speculum all describes Nano microsphere is about 200nm, belongs to applicable cytophagic scope, is conducive to effect that it plays gene drug carriers.
embodiment 6: compound and the point of the particle diameter after carrying gene and medicine bit table are levied
In Figure 10, figure a is particle diameter and the current potential of Dendrimer G2 and 4 kind of different ratios PSPG2s.Figure b is particle diameter and the current potential that Dendrimer G2 and PSPG2s-6 carries different ratios Zorubicin.Figure c and figure d be Dendrimer G2 and 4 kind of different ratios PSPG2s respectively in conjunction with after DNA different N/P than under particle diameter and current potential.Figure e and figure f is that Dendrimer G2 and PSPG2s-6 to carry after Zorubicin at different N/P than the lower particle diameter in conjunction with DNA and current potential respectively.Figure g and figure h is Dendrimer G2 respectively, 2 kinds of different ratios PSPG2s and PSPG2s-6 to carry in 2 after different ratios Zorubicin at different N/P than the lower particle diameter in conjunction with siRNA and current potential.Comprehensive the above results can draw to draw a conclusion: PSPG2s and PSPG2s/DOX particle diameter after in conjunction with DNA or siRNA is stabilized in about 200nm, surface charge there occurs wide variation simultaneously, become close to neutral from obvious positive polarity, this point and independent Dendrimer G2 and Dendrimer G2/DOX are comparing in conjunction with the potential variation after DNA or siRNA, this again testimonial material itself structurally there occurs the upset of ectonexine, the Dendrimer of positive charge is wrapped in Nano microsphere inside, and surface is the containment structure that PEG is formed.
embodiment 7: the heparin interference test of compound
Figure 11 be Dendrimer G2 and PSPG2s-6 in conjunction with DNA or in conjunction with after Zorubicin and DNA, to dissociate the change of size in experiment at heparin, wherein the effect of DTT is degraded disulfide linkage, namely disconnects the link between PEG and Dendrimer.Result in figure illustrates is carrying gene formation Nano microsphere due to the protection of PEG, and effectively can avoid the interference of extraneous electric charge, its structure of protection losing PEG is then no longer stable.
embodiment 8: the degradation experiment of material
Figure 12 be PSPG2s-6 PH=7.4,6.5 and 5.0 PBS solution in degradation experiment.Result shows that compound PSPG2s-6 can degrade in acid condition, can reduce its cytotoxicity in use like this.
embodiment 9: the cytotoxicity experiment of compound
Figure 13 and Figure 14 is 4 kinds of different ratios PSPG2s and 2 kinds of different ratios PPG2s(8arm-PEG-PEG-Dendrimer G2, expression Putriscine replaces 2-aminoethyl disulfide dihydrochloride synthesize the dendroid polycationic compounds not having disulfide linkage obtained) in the cytotoxicity of Hek293 cell upper 4 hour and 24 hours.Result shows, PSPG2s did not almost have cytotoxicity in 24 hours, does not have the compound of disulfide linkage then can show certain toxicity.Because disulfide linkage can be reduced in cell, cause compound to be broken down into and almost there is no Cytotoxic small molecules, so overall toxicity is low, then toxicity is comparatively large not have the PPG2s of disulfide linkage, illustrates that the use of disulfide linkage is conducive to reducing the cytotoxicity of compound.
embodiment 10:
Figure 15 is that 4 kinds of different ratios PSPG2s and 2 kind of different ratios PPG2s carries luciferase protein plasmid gene (Luciferase) transfection results on Hek293 cell.Result shows that disulfide linkage discharges in the process that gene makes it play a role and has vital effect after compound enters cell, and this compounds of PSPG2s has the ability of good delivery of gene.
embodiment 11:
Figure 16: 4 kinds of different ratios PSPG2s and 2 kind of different ratios PPG2s Carrying Green Fluorescent Proteins RNA interfering (GFP-siRNA), in the green fluorescent protein silencing experiments result of people's renal epithelial cell (293T-GFP) in upper 24 hour of expressing green fluorescent protein.Result shows that disulfide linkage discharges in the process that gene makes it play a role and has vital effect after compound enters cell, and this compounds of PSPG2s effectively can also carry siRNA and enter cells play effect.
Embodiment 12:
Figure 17 be PSPG2s-6/Dox9% PH=5.0,7.4 PBS in Zorubicin release result.Result shows, compound can effectively carry and discharge medicine, and release efficiency is in acid condition than high a lot of under neutrallty condition, and this is conducive to compound and discharges medicine under the sour environment of tumor section, reaches curative effect.Figure 18 is the TEM picture of PSPG2s-6/Dox9% under the acidic conditions of PH=5.0 during 0h and 12h, and PSPG2s-6/Dox9% PH=5.0,7.4 condition under the white light photo of 0h and 12h, the above results shows, the structure of PSPG2s-6/Dox9% Nano microsphere in acid condition can be scattered, medicine can discharge, and drug release can be suppressed in neutral conditions.This is because the compound of Nano microsphere inside and the solvability of medicine can improve in acid condition, result can fluff loose, thus accelerates the release of medicine.
embodiment 13:
Figure 19 is Dox, siRNA and PSPG2s/Dox9%/siRNA living body fluorescent figure in the different time points of nude mouse tumor position metabolism; Figure 20 is the relative intensity change curve of red green fluorescence in Figure 19; Figure 21 is the tumor biopsy figure of Dox and PSPG2s-6/Dox9%/siRNA after nude mouse tumor position metabolism 24h.Result shows, material can effectively carry in vivo and discharge gene and medicine simultaneously, has good slow release effect at tumor locus simultaneously.
embodiment 14:
Solid support material 8armPEG-SS-PEG-PAMAM G3(PSPP3s) preparation and carry medicine and gene.(branch-shape polymer is PAMAM G3,8armPEG be 20000Da, NHS-PEG-OH be 3500Da, linking agent is DSC, and medicine is taxol (Paclitaxel, Taxol), and gene is luciferase reporter gene Luciferase)
(1) synthesis of 8armPEG-SS-PEG
The 8armPEG taking 8 grams of 20000Da is dissolved in 40 milliliters of dimethyl sulfoxide (DMSO); take 2 grams of N; N'-bis-succinimidyl carbonate (DSC); be dissolved in 6 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 2.2 milliliters; the solution of 8armPEG drops in the solution of DSC by lucifuge under nitrogen protection, continues stirring reaction 5 hours afterwards.Reaction solution is added to the ether that volume is 1 liter afterwards: in the mixing solutions of tetrahydrofuran (THF)=5:1 volume ratio, under 5 degrees celsius, leaves standstill 3 hours, filter afterwards and obtain white powder.
Then taking 4 grams of these white powders to be dissolved in 40 milliliters of dimethyl sulfoxide (DMSO), take 1 gram of 2-aminoethyl disulfide dihydrochloride and be dissolved in 10 milliliters of dimethyl sulfoxide (DMSO), and add 2.7 milliliters of triethylamines, stirring a moment to dissolving completely.Slowly dropped in the solution of cystamine by the solution of PEG afterwards, drip 5 hours, continue reaction 5 hours afterwards, after completion of the reaction, solution is dialysed 48 hours in MW14000 dialysis tubing, and then frost drying, obtains white powder.
Then taking 2.25 grams of these white powders is dissolved in 20 milliliters of dimethyl sulfoxide (DMSO), the straight chain polyoxyethylene glycol (NHS-PEG-OH) taking the one-sided N-hydroxysuccinimide activation of 2.8 grams of 3500Da is again dissolved in 20 milliliters of dimethyl sulfoxide (DMSO), the solution of white powder is added dropwise in the solution of NHS-PEG-OH, stirring reaction 5 hours, afterwards reaction solution is joined the ether of 2 liters: in the mixing solutions of tetrahydrofuran (THF)=3:1 volume ratio, leave standstill 3 hours under 5 degrees celsius, filtration afterwards obtains white powder and is 8armPEG-SS-PEG.
(2) 8armPEG-SS-PEG-PAMAM G3(PSPP3s) synthesis
4.5 grams of 8armPEG-SS-PEG are dissolved in 25 milliliters of dimethyl sulfoxide (DMSO); then 650 milligrams of N are taken; N'-bis-succinimidyl carbonate (DSC) is dissolved in 4 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 650 microlitre; two kinds of solution mix by lucifuge under nitrogen protection, afterwards stirring reaction 8 hours.Afterwards reaction solution is added to the ether of 1.2 liters: in the mixing solutions of tetrahydrofuran (THF)=3:1 volume ratio, under 5 degrees celsius, leaves standstill 1.5 hours, filter afterwards and obtain white powder.
Then taking 1.3 grams of these white powders is dissolved in 25 milliliters of dimethyl sulfoxide (DMSO), taking 1.7 grams of PAMAM G3 branch-shape polymers is again dissolved in 35 milliliters of dimethyl sulfoxide (DMSO), the solution afterwards solution of white powder slowly being dropped to branch-shape polymer has suffered, drip 4 hours, continue reaction 18 hours afterwards, after completion of the reaction, solution is dialysed 48 hours in the dialysis tubing of MW14000, then frost drying, obtain white powder material, i.e. final product dendroid polycationic compounds PSPP3s.
(3) preparation of nanoparticle PSPP3s-6/Taxol
4 milligrams of taxols (Paclitaxel, Taxol) are dissolved in 1 milliliter of CH
2cl
2in, add 3 microlitre triethylamines, 36 milligrams of PSPP3s-6 are dissolved in 10 milliliters of DMSO, then both are mixed, and the uncovered stirring of room temperature is spent the night.Then the solution dialysis tubing dialysis 4h of molecular weight 14000, lyophilize, obtains PSPP3s-6/Taxol.
(4) preparation of nanoparticle PSPP3s-6/DNA, PSPP3s-6/Taxol/DNA
Luciferase reporter gene Luciferase water dissolution is made into the solution of 0.1 microgram/microlitre, get this solution of 10 microlitres, again the PSPP3s-6 of 10 micrograms/microlitre or the PSPP3s-6/Taxol aqueous solution equal-volume containing 10 micrograms/microlitre PSPP3s-6 are joined in DNA solution, static 30 minutes of room temperature, namely nanoparticle PSPP3s-6/DNA or PSPP3s-6/Taxol/DNA is formed, can for follow-up.
embodiment 15: compound carries the ability of gene
Figure 22 be 2 kinds access ratios PSPP3s/DNA(PSPP3s-4/6 represent that the amount of substance ratio of 8arm-PEG and PAMAM G3 in dendroid polycationic compounds PSPG2s is 4/6 respectively) different N/P than under the agarose gel electrophoresis blockade test carrying DNA.Illustrate shown in figure that this compounds of PSPP3s effectively can carry DNA.
embodiment 16: serum-concentration is on the impact of transfection
Figure 23 be PAMAM G3, PEI25KD, PSPP3s-6 under optimum N/P ratio, be respectively the transfection experiment result of the luciferase reporter gene Luciferase under the condition of 0%, 10%, 25%, 50% at serum-concentration.Shown in figure, the transfection efficiency of PAMAM G3 and PEI25KD all presents significantly downtrending along with the rising of serum-concentration, and PSPP3s-6 is then influenced hardly.This is because the material such as albumen in serum can affect the stability of Nano microsphere, and the Nano microsphere surface that PSPP3s-6/DNA is formed is the neutral material of PEG, be subject to the impact of albumen hardly, improve the stability of microballoon, thus transfection efficiency is not significantly affected.It is worth mentioning that PAMAM G3 is that the transfection efficiency of the compound of branch is substantially identical with PEI25KD simultaneously, and the transfection efficiency that the Dendrimer G2 that we independently synthesize is the PSPG2s-6 of branch is 1.13 times (embodiments 10) of PEI25KD, there is better delivery capability.
embodiment 17: the test experience of lysosome escape capability
Figure 24 be PAMAM G3, PEI25KD, PSPP3s-6 under the condition of optimum N/P ratio, the transfection experiment result of the luciferase reporter gene Luciferase respectively under existing with or without chloroquine and Bafilomycin A1.The effect of chloroquine promotes that the lysosome of endocytosis material is escaped, and the effect of Bafilomycin A1 is by suppressing the normal work of ionic pump thus suppressing lysosome to be escaped.Experimental result shows, PSPP3s-6 has the ability that good lysosome is escaped, and chloroquine is not obvious to its promoter action under the same conditions, and then escape capability is poor for PAMAM G3 monomer, under the condition of chloroquine, have obvious lifting; On the other hand, illustrate that the lysosome escape of PSPP3s-6 is the proton sponge effect due to self, inhibit the function of ionic pump, significantly will suppress its escape capability.
embodiment 18:
Figure 25 is the transfection experiment result that PSPP3s-6 carries the luciferase reporter gene Luciferase of the taxol (Taxol) of different ratios, and carrying drug ratio is respectively 0%, 9%, 20%, 30%.Result shows, along with the increase of carrying drug ratio, transfection efficiency can decrease to some degree, this is because medicine itself has cell certain lethal, makes cell normal function be subject to certain suppression, indirectly causes transfection efficiency to decline.
embodiment 19: solid support material 4armPEG-SS-PEG-PAMAM G4(4PSPP4s) preparation and carry medicine and gene.(branch-shape polymer is PAMAM G4,8armPEG be 40000Da, NHS-PEG-OH be 5000Da, linking agent is FMOC-OBT, and medicine is Gefitinib (Gefitinib, Gef), and gene is luciferase reporter gene Luciferase)
(1) synthesis of 4armPEG-SS-PEG
The 4armPEG taking 10 grams of 40000Da is dissolved in 40 milliliters of dimethyl sulfoxide (DMSO); take 0.9 gram of 9-fluorenyl methyl 1-benzotriazole base carbonic ether (FMOC-OBT); be dissolved in 5 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 0.5 milliliter; the solution of 8armPEG drops in the solution of FMOC-OBT by lucifuge under nitrogen protection, continues stirring reaction 4 hours afterwards.Reaction solution is added to the ether that volume is 2 liters afterwards: in the mixing solutions of tetrahydrofuran (THF)=3:1 volume ratio, under 0 degrees celsius, leaves standstill 2 hours, filter afterwards and obtain pulverulent solids.
Then taking 8 grams of these powder dissolutions in 60 milliliters of dimethyl sulfoxide (DMSO), take 0.7 gram of 2-aminoethyl disulfide dihydrochloride and be dissolved in 5 milliliters of dimethyl sulfoxide (DMSO), and add 1.6 milliliters of triethylamines, stirring a moment to dissolving completely.Slowly dropped in the solution of cystamine by the solution of PEG afterwards, drip 4 hours, continue reaction 4 hours afterwards, after completion of the reaction, solution is dialysed 36 hours in MW14000 dialysis tubing, and then frost drying, obtains white powder.
Then taking 4 grams of these white powders is dissolved in 30 milliliters of dimethyl sulfoxide (DMSO), the straight chain polyoxyethylene glycol (NHS-PEG-OH) taking the one-sided N-hydroxysuccinimide activation of 2.5 grams of 5000Da is again dissolved in 25 milliliters of dimethyl sulfoxide (DMSO), the solution of white powder is added dropwise in the solution of NHS-PEG-OH, stirring reaction 3.5 hours, afterwards reaction solution is joined the ether of 2 liters: in the mixing solutions of tetrahydrofuran (THF)=5:1 volume ratio, leave standstill 2 hours under 0 degrees celsius, filtration afterwards obtains white powder and is 4armPEG-SS-PEG.
(2) 4armPEG-SS-PEG-PAMAM G4(4PSPP4s) synthesis
3 grams of 8armPEG-SS-PEG are dissolved in 25 milliliters of dimethyl sulfoxide (DMSO); then taking 270 milligrams of 9-fluorenyl methyl 1-benzotriazole base carbonic ether (FMOC-OBT) is dissolved in 2 milliliters of dimethyl sulfoxide (DMSO); and add catalyst of triethylamine 100 microlitre; two kinds of solution mix by lucifuge under nitrogen protection, afterwards stirring reaction 4 hours.Afterwards reaction solution is added to the ether of 1 liter: in the mixing solutions of tetrahydrofuran (THF)=5:1 volume ratio, under 0 degrees celsius, leaves standstill 1 hour, filter afterwards and obtain white powder.
Then taking 1.8 grams of these white powders is dissolved in 25 milliliters of dimethyl sulfoxide (DMSO), taking 2.7 grams of PAMAM G4 branch-shape polymers is again dissolved in 40 milliliters of dimethyl sulfoxide (DMSO), the solution afterwards solution of white powder slowly being dropped to branch-shape polymer has suffered, drip 2 hours, continue reaction 15 hours afterwards, after completion of the reaction, solution is dialysed 24 hours in the dialysis tubing of MW50000, then frost drying, obtain white powder material, i.e. final product dendroid polycationic compounds 4PSPP4s.
(3) preparation of nanoparticle 4PSPP4s-4/Gef
4 milligrams of Gefitinib (Gefitinib, Gef) are dissolved in 1 milliliter of CH
2cl
2in, add 3 microlitre triethylamines, 36 milligrams of 4PSPP4s-4 are dissolved in 10 milliliters of DMSO, then both are mixed, and the uncovered stirring of room temperature is spent the night.Then the solution dialysis tubing dialysis 4h of molecular weight 14000, lyophilize, obtains 4PSPP4s-4/Gef.
(4) preparation of nanoparticle 4PSPP4s-4/DNA, 4PSPP4s-4/Gef/DNA
Be the solution that luciferase reporter gene Luciferase water dissolution is made into 0.1 microgram/microlitre by gene, get this solution of 10 microlitres, again the 4PSPP4s-4 of 10 micrograms/microlitre or the 4PSPP4s-4/Gef aqueous solution equal-volume containing 10 micrograms/microlitre 4PSPP4s-4 are joined in DNA solution, static 30 minutes of room temperature, namely nanoparticle 4PSPP4s-4/DNA or 4PSPP4s-4/Gef/DNA is formed, can for follow-up.
embodiment 20: material carries the ability of gene
Figure 26 be 2 kinds access ratios 4PSPP4s/DNA(4PSPP4s-2/4 represent that the amount of substance ratio of 4arm-PEG and PAMAM G4 in dendroid polycationic compounds 4PSPP4s is 2/4 respectively) different N/P than under the agarose gel electrophoresis blockade test carrying DNA.Illustrate shown in figure that this kind of material of 4PSPP4s effectively can carry DNA.
embodiment 21:
Figure 27 is 4PSPP4s(a) and atomic force microscope (AFM) figure 4PSPP4s/Gef/DNA(b).Show in figure, 4PSPP4s can form the Nano microsphere that particle diameter is about 200nm after carrying gene and medicine, belongs to applicable cytophagic scope, is conducive to effect that it plays gene drug carriers.
embodiment 22: disulfide linkage is on the impact of Materials Cell toxicity
Figure 28 is the research that disulfide linkage affects Compound Cytotoxicity, be specially 4PSPP4s-4 and 4PPP4s-4(4arm-PEG-PEG-PAMAM G4, expression Putriscine replaces 2-aminoethyl disulfide dihydrochloride to synthesize the dendroid polycationic compounds not having disulfide linkage obtained) with or without the cytotoxicity experiment under Buthionine sulfoximine (BSO) existent condition.Buthionine sulfoximine is the synthetic inhibitor of the regulation and control application specific gsh (GSH) of cellular oxidation-reduction-state, and it can by the degraded suppressing the synthesis of GSH to carry out disulfide linkage in Inhibitor.Experimental result shows, the degraded of disulfide linkage serves vital effect in the hypotoxicity of compound, the 4PSPP4s-4 of BSO is not had almost not have toxic manifestations in cell, and under having BSO existent condition, the cytotoxicity of 4PSPP4s-4 is similar to not having the 4PPP4s-4 of disulfide linkage.
embodiment 23: disulfide linkage is on the impact of Compound cellular transfection
Figure 29 is the research that disulfide linkage affects Compound cellular transfection, is specially 4PSPP4s-4 and 4PPP4s-4 with or without the luciferase reporter gene Luciferase transfection experiment under Buthionine sulfoximine (BSO) existent condition.Buthionine sulfoximine is the synthetic inhibitor of the regulation and control application specific gsh (GSH) of cellular oxidation-reduction-state, and it can by the degraded suppressing the synthesis of GSH to carry out disulfide linkage in Inhibitor.Experimental result shows, the degraded of disulfide linkage serves vital effect in the transfection of compound, this is because the degraded of disulfide linkage facilitates the release of DNA, thus the effective transfection efficiency improved.
embodiment 24: the heparin interference test of compound
Figure 30 is PAMAM G4,4PSPP4s-4 and 4PPP4s-4 after in conjunction with Gefitinib and DNA, heparin (heparin) dissociate experiment in potential variation, wherein the effect of dithiothreitol (DTT) (DTT) is degraded disulfide linkage, namely disconnects the link between 4arm-PEG and PAMAM G4.Result display in figure, the current potential of PAMAM G4 can be subject to the impact of heparin, 4PSPP4s-4 and 4PPP4s-4 is under the protection of PEG; substantially not by the impact of heparin; and 4PSPP4s-4 disulfide linkage degraded under DTT effect, after PEG leaves away, also can be subject to the impact of heparin.This illustrates that carrying gene and medicine at 4PSPP4s-4 forms Nano microsphere and only have, can due to the protection of self PEG, and effectively can avoid the interference of extraneous electric charge, its structure of protection losing PEG is then no longer stable.
Claims (10)
1. a class dendroid polycationic compounds, it is characterized in that, structural formula is as follows:
Wherein:
R is two generation AB
3type dendroid cationic polymers, structural formula is:
N is the number of the repeating unit in each side chain of eight branch's polyoxyethylene glycol;
M is the number of the repeating unit in straight chain polyoxyethylene glycol.
2. the preparation method of a class dendroid polycationic compounds according to claim 1, be is characterized in that, realized by following steps, for 8armPEG-SS-PEG-Dendrimer G2:
(1) preparation of compound 4: compound 1 is dissolved in methylene dichloride, add activator 1-hydroxyl-7-azo benzotriazole wherein and add catalyst of triethylamine, stirring at normal temperature, add N-tertbutyloxycarbonyl quadrol afterwards, then 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride is slowly added, add rear continuation reaction, after completion of the reaction, ethyl acetate is added in reaction solution, organic phase uses hydrochloric acid successively, saturated sodium bicarbonate solution, saturated nacl aqueous solution washs, use anhydrous sodium sulfate drying again, revolve steaming, obtain intermediate product, intermediate product is dissolved in methyl alcohol, add Nickel dichloride hexahydrate wherein, then slowly sodium borohydride is added, add rear continuation reaction, after completion of the reaction, with hydrochloric acid soln, pH value is adjusted to acidity, with saturated sodium bicarbonate solution, pH value is adjusted to alkalescence again, methyl alcohol in solution is revolved and evaporates, be extracted with ethyl acetate again, organic phase saturated nacl aqueous solution washs, with anhydrous sodium sulfate drying, then concentrate, the method used column chromatography obtains compound 2,
By compound 2, compound 3 and 1-hydroxyl-7-azo benzotriazole are dissolved in methylene dichloride, add catalyst of triethylamine, then 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride is added, add rear continuation reaction, after completion of the reaction, ethyl acetate is added in reaction solution, organic phase uses hydrochloric acid successively, saturated sodium bicarbonate solution, saturated nacl aqueous solution washs, use anhydrous sodium sulfate drying again, steaming is revolved in filtration, the method that residual residue uses column chromatography obtains intermediate product, then gained intermediate product is dissolved in the mixed solvent of trifluoroacetic acid and methylene dichloride, stirring at normal temperature, dialyse with water afterwards, compound 4 is obtained after lyophilize,
(2) preparation method of compound 6: compound 5 is dissolved in dimethyl sulfoxide (DMSO), then linking agent is dissolved in dimethyl sulfoxide (DMSO), and add triethylamine, the solution of compound 5 drops in the solution of linking agent by lucifuge under nitrogen protection, continue reaction, afterwards reaction solution is added in ether and tetrahydrofuran (THF) mixing solutions, leave standstill, filtration is precipitated i.e. intermediate product, intermediate product is dissolved in dimethyl sulfoxide (DMSO), again 2-aminoethyl disulfide dihydrochloride is dissolved in dimethyl sulfoxide (DMSO), and add triethylamine, be stirred to and dissolve completely, the solution of this intermediate product is slowly dropped in the solution of cystamine, continue reaction, after completion of the reaction, solution with water is dialysed, frost drying obtains intermediate product, again this intermediate product is dissolved in dimethyl sulfoxide (DMSO), again the straight chain polyoxyethylene glycol of one-sided activation is dissolved in dimethyl sulfoxide (DMSO), the solution of this intermediate product is added dropwise in the solution of polyoxyethylene glycol, stirring reaction, afterwards reaction solution is added in the mixing solutions of ether and tetrahydrofuran (THF), leave standstill, filtration obtains white powder and is compound 6,
(3) preparation of final product compound 7: compound 6 is dissolved in dimethyl sulfoxide (DMSO), then linking agent is dissolved in dimethyl sulfoxide (DMSO), and add triethylamine, two kinds of solution mix by lucifuge under nitrogen protection, stirring reaction, reaction solution is added in the mixing solutions of ether and tetrahydrofuran (THF), leave standstill, filtration is precipitated i.e. intermediate product, then this intermediate product and compound 4 are dissolved in dimethyl sulfoxide (DMSO) respectively, the solution of intermediate product is slowly dropped in the solution of compound 4, continue reaction afterwards, after completion of the reaction, solution with water is dialysed, then frost drying, obtain powdery substance, i.e. final product compound 7, reaction formula is as follows:
In reaction formula, n is the number of the repeating unit in each side chain of eight branch's polyoxyethylene glycol, and m is the number of the repeating unit in straight chain polyoxyethylene glycol, n and m is determined by the molecular weight of polyoxyethylene glycol raw material; HOAT in reaction conditions represents 1-hydroxyl-7-azo benzotriazole, EDCI represents 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, and TFA represents trifluoroacetic acid, and CDI represents N, N'-carbonyl dimidazoles, the R group in compound 7 represents compound 4.
3. the preparation method of a class dendroid polycationic compounds according to claim 2, it is characterized in that, described dendroid polycationic compounds, namely compound 7 is: AB
3type polyamidoamine dendritic macromole-G2, AB
2type polyamidoamine dendritic macromole-G2, G3, G4.
4. the preparation method of a class dendroid polycationic compounds according to claim 2, is characterized in that, described compound 5 is: 3arm, 4arm, 8arm multiple-limb polyoxyethylene glycol, and molecular weight is 10000Da-40000Da; Described straight chain molecular weight polyethylene glycol is MW 2000Da-5000Da.
5. the preparation method of a class dendroid polycationic compounds according to claim 2, it is characterized in that, described linking agent is: N, in N'-carbonyl dimidazoles, N, N'-bis-succinimidyl carbonate, N-hydroxy-succinamide chloro-formic ester, 9-fluorenyl methyl 1-benzotriazole base carbonic ether any one.
6. the preparation method of a class dendroid polycationic compounds according to claim 2, it is characterized in that, described linking agent consumption is 10-30 times amount with the amount of substance ratio of corresponding compound 5, and described compound 5 consumption and the mass volume ratio of dimethyl sulfoxide (DMSO) are 3-5 times amount.
7. the preparation method of a class dendroid polycationic compounds according to claim 2, is characterized in that, described linking agent consumption and the mass volume ratio of dimethyl sulfoxide (DMSO) are 1-5 times amount.
8. the preparation method of a class dendroid polycationic compounds according to claim 2, it is characterized in that, described catalyzer is triethylamine, consumption and activator HOAT amount of substance are than being 2-4 times amount, with linking agent amount of substance than being 1-2 times amount, with 2-aminoethyl disulfide dihydrochloride amount of substance than being 3-5 times amount.
9. the preparation method of a class dendroid polycationic compounds according to claim 2, is characterized in that, described 2-aminoethyl disulfide dihydrochloride consumption is 15-30 times amount with the amount of substance ratio of corresponding compound 5; Straight chain polyoxyethylene glycol consumption is 5-10 times amount with the amount of substance ratio of corresponding compound 5; Compound 4 consumption is 5-10 times amount with the amount of substance ratio of corresponding compound 6.
10. a class dendroid polycationic compounds according to claim 1 is as the application in gene drug carriers, and wherein said medicine is the medicine with hydrophobic property.
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| CN111607078A (en) * | 2020-06-17 | 2020-09-01 | 苏州大学 | Hypoxic responsive cationic polymer and preparation method and application thereof |
| CN111607078B (en) * | 2020-06-17 | 2022-06-03 | 苏州大学 | Hypoxic responsive cationic polymer and preparation method and application thereof |
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