CN104059231A - Preparation and purpose of novel dendritic polymer gene carrier - Google Patents
Preparation and purpose of novel dendritic polymer gene carrier Download PDFInfo
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- CN104059231A CN104059231A CN201310092038.1A CN201310092038A CN104059231A CN 104059231 A CN104059231 A CN 104059231A CN 201310092038 A CN201310092038 A CN 201310092038A CN 104059231 A CN104059231 A CN 104059231A
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Abstract
The invention relates to a preparation and a purpose of a novel dendritic polymer gene carrier. According to the invention, the a tertiary amino group and a primary amino group-containing dendritic polyamide-amine high-molecular polymer which is characterized in that polyethylene glycol is taken as a core and the primary amino group is taken as the surface, a preparation method and a purpose of the high-molecular polymer as the gene carrier. The dendritic high-molecular polymer is taken as the gene carrier, enables transfection of gene with high efficiency or is used for gene treatment, so that the dendritic high-molecular polymer is used for treatment, diagnosis and prevention of various diseases. The effect of the gene carrier is better than the effect of polyethylene glycol or polyamidoamine-containing gene carrier during a gene transhipment period for treating disease.
Description
Technical field
The invention discloses taking polyoxyethylene glycol as core, taking primary amino as surface, the dendroid polyamide-amide high molecular polymer that contains the amino and secondary amino group of uncle using and preparation method thereof with its purposes as genophore.
Background technology
In following clinical treatment, personalized gene therapy substituted chemistry pharmacological agent disease has become the common recognition of scientific circles, how maximum obstacle imports to the gene of external source in target cell efficiently at present, or in corresponding environment, making it to correct or compensator gene defect or the disease extremely causing, is exactly briefly how foreign gene or genetic information is inserted in recipient cell by gene transfer technique.Key problem is fallen on genophore so.
Genophore technology is growing at present, is mainly divided into virus vector and non-virus carrier.Due to problems such as immunity of organism problem and host's integration risks, virus vector can not be served as the first-selection of following clinical treatment substantially.The exploitation of non-virus carrier is scientist's major fields of work at present.Emphasis is high transfection efficiency and hypotoxicity.
The genophore that this patent is developed is taking polyoxyethylene glycol as core, and taking primary amino as surface, taking secondary amino group and the amino dendroid polyamide-amide high molecular polymer as " branch " of uncle.This patent discloses Preparation Method And The Use simultaneously.This dendrimer polymkeric substance is as genophore, rotaring redyeing gene efficiently, do not contain amino dendrimer with containing polyoxyethylene glycol or contain amino and the dendrimer that do not contain polyoxyethylene glycol is compared, its feature is greatly to have strengthened its through performance and security in vivo, thereby has significantly improved transfection efficiency.
Summary of the invention
Of the present invention theing contents are as follows:
The invention discloses the structure shown in the formula shown in following formula, its structure is as follows:
Wherein the molecular weight polyethylene glycol in this branch-shape polymer is 100-200000, the integer between n=1-500, the preferably integer between n=1-300.
Preparation method characteristic is:
1) will be decomposed into taking S-S as core taking hydroxyl as surperficial polyamide-amide branch-shape polymer A with half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer B;
2) obtain with half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer with polyoxyethylene glycol C(OPSS-PEG-OPSS with active group) react and obtain the hydroxyl taking polyoxyethylene glycol as core as surperficial branch-shape polymer D;
3) Boc-GABA-OH E reacts with D and obtains taking NHBoc as surperficial compound.
4) reaction of upper step gained compound for catalysis obtains the polyamide-amide branch-shape polymer genophore F that contains uncle's amino and secondary amino group taking polyoxyethylene glycol as core taking primary amino as surface;
Compd A;
Compd B;
Compound C;
Compound D;
Compd E;
Compound F 17-hydroxy-corticosterone
Wherein said chemical step 1-4 selects solvent to be selected from: one or more in benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
End product can be prepared into the carrier that is suitable for gene drug delivery.The carrier of described gene drug delivery is portability range gene or carries range gene simultaneously.Purposes is for being used for the treatment of, the genophore of diagnosis and prevention.
Preparation method of the present invention is specific as follows:
By taking S-S as core taking hydroxyl as surperficial polyamide-amide branch-shape polymer 3-20g in the phosphate buffer solution (ph7-8) of three (2-chloroethyl) phosphoric acid ester of 50-1000 volumetric molar concentration reaction 1-30 hour.Be decomposed into half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer.
What obtain reacts with the polyoxyethylene glycol (OPSS-PEG-OPSS) with active group taking hydroxyl as surperficial polyamide-amide branch-shape polymer for half with the strong activity of SH, normal temperature spends the night, and the hydroxyl obtaining taking polyoxyethylene glycol as core is surperficial branch-shape polymer.
Boc-GABA-OH0.5g-20g reacts the compound of gained under EDC and DMAP catalysis with upper step, in the mixing solutions of DMF and DMSO, react and obtain taking NHBoc as surperficial compound.
Upper step gained compound at normal temperatures, reacts 10-2000 minute in TFA:DCM mixing solutions, obtains the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group taking polyoxyethylene glycol as core taking primary amino as surface.
The gene 0.1-1mg/ml of 100-5000ul is mixed with the upper step compound solution of 10-2000ul concussion both carrying genes novel taking primary amino as surface, taking polyoxyethylene glycol as core, the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group.
The present invention obtain taking primary amino as surface, taking polyoxyethylene glycol as core, containing uncle's polyamide-amide branch-shape polymer genophore amino and secondary amino group does not contain amino dendrimer or contains amino and the dendrimer that do not contain polyoxyethylene glycol is compared with containing polyoxyethylene glycol, its feature is greatly to have strengthened its through performance and security in vivo, thereby has significantly improved transfection efficiency.Surmount other forms of genophore completely.
brief description of the drawings:
fig. 1the nuclear magnetic resonance map of this genophore.
Embodiment
Specific embodiment is described in further detail the present invention below, but the present invention not only limits to following examples.
Preparation Example is as follows:
embodiment 1
1) by the reaction two hours in the phosphate buffer solution (ph7.3) of three (2-chloroethyl) phosphoric acid ester of 100 volumetric molar concentrations taking S-S as core taking hydroxyl as surperficial polyamide-amide branch-shape polymer 3g.Be decomposed into half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer.
2) what obtain reacts with the polyoxyethylene glycol (OPSS-PEG-OPSS) with active group taking hydroxyl as surperficial polyamide-amide branch-shape polymer for half with the strong activity of SH, normal temperature spends the night, and the hydroxyl obtaining taking polyoxyethylene glycol as core is surperficial branch-shape polymer.
3) compound that Boc-GABA-OH1g reacts gained with upper step, under EDC and DMAP catalysis, reacts and obtains taking NHBoc as surperficial compound in the mixing solutions of DMF and DMSO.
4) upper step gained compound at normal temperatures, is in 6:4 solution, to react 10 minutes at TFA:DCM, obtains the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group taking polyoxyethylene glycol as core taking primary amino as surface.
5) gene of 1000ul (IL-21) 0.2mg/ml is mixed with the upper step compound solution of 100ul concussion both carrying genes (IL-21) novel taking primary amino as surface, taking polyoxyethylene glycol as core, the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group.
embodiment 2
1) by the reaction 4 hours in the phosphate buffer solution (ph7.4) of three (2-chloroethyl) phosphoric acid ester of 200 volumetric molar concentrations taking S-S as core taking hydroxyl as surperficial polyamide-amide branch-shape polymer 5g.Be decomposed into half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer.
2) what obtain reacts with the polyoxyethylene glycol (OPSS-PEG-OPSS) with active group taking hydroxyl as surperficial polyamide-amide branch-shape polymer for half with the strong activity of SH, normal temperature spends the night, and the hydroxyl obtaining taking polyoxyethylene glycol as core is surperficial branch-shape polymer.
3) compound that Boc-GABA-OH1g reacts gained with upper step, under EDC and DMAP catalysis, reacts and obtains taking NHBoc as surperficial compound in the mixing solutions of DMF and DMSO.
4) upper step gained compound at normal temperatures, is in 5:5 solution, to react 20 minutes at TFA:DCM, obtains the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group taking polyoxyethylene glycol as core taking primary amino as surface.
5) gene of 500ul (IL-21) 0.3mg/ml is mixed with the upper step compound solution of 80ul concussion both carrying genes (IL-21) novel taking primary amino as surface, taking polyoxyethylene glycol as core, the polyamide-amide branch-shape polymer genophore that contains uncle's amino and secondary amino group.
effect experiment is as follows:
Sample prepared by embodiment 1-2 and polyoxyethylene glycol are the dendrimer with hydroxyl surface of core, and PAMAM 2 generation dendrimer carries IL21 treatment H22 tumor-bearing mice situation and carries out pharmacodynamics comparison.
Preparation H22 liver cancer mouse model, according to traditional method, H22 cell is implanted to mouse oxter, prepare after model, be divided into 5 groups, blank group, the dendrimer with hydroxyl surface that sample prepared by embodiment 1-2 and polyoxyethylene glycol are core, PAMAM 2 generation dendrimer carries IL21 group, every group of 20 mouse.
Sample prepared by embodiment 1-2 and polyoxyethylene glycol are the dendrimer with hydroxyl surface of core, and PAMAM 2 generation dendrimer carries IL21.(carry equivalent IL21, every group containing 10ulIL21), intratumor injection is respectively organized in Mice Body respectively, tests after 2 weeks, puts to death mouse, gets knurl kind of calliper volume, and result is as follows.
Table 1 knurl size relatively
Table 1 gross tumor volume (unit: cm2)
| Group | Knurl size |
| Control group | 16.04 2.18 |
| Polyoxyethylene glycol is the dendrimer with hydroxyl surface of core | 10.82 1.08* # |
| PAMAM 2 generation dendrimer | 9.98 1.59* # |
| Embodiment 1 | 5.84 0.87** ## |
| Embodiment 2 | 5.46 0.69** ## |
| Direct injection IL21 ordinary preparation | 13.07 1.29* # |
With relatively * p<0.05 of control group group, * * p<0.01, with the comparison of ordinary preparation group
#p<0.05,
##p<0.01.
Claims (7)
1. the structure being shown below, its structure is as follows:
。
2. the branch-shape polymer of claim 1, wherein the molecular weight polyethylene glycol in this branch-shape polymer is 100-200000, the integer between n=1-500, the preferably integer between n=1-300.
3. the preparation method of polymkeric substance as claimed in claim 1, is characterized in that:
1) will be decomposed into taking S-S as core taking hydroxyl as surperficial polyamide-amide branch-shape polymer A with half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer B;
2) obtain with half of the strong activity of SH taking hydroxyl as surperficial polyamide-amide branch-shape polymer with polyoxyethylene glycol C(OPSS-PEG-OPSS with active group) react and obtain the hydroxyl taking polyoxyethylene glycol as core as surperficial branch-shape polymer D;
3) Boc-GABA-OH E reacts with D and obtains taking NHBoc as surperficial compound;
4) reaction of upper step gained compound for catalysis obtains the polyamide-amide branch-shape polymer genophore F that contains uncle's amino and secondary amino group taking polyoxyethylene glycol as core taking primary amino as surface;
Compd A;
Compd B;
Compound C;
Compound D;
Compd E;
Compound F 17-hydroxy-corticosterone
。
4. the method for claim 3, wherein said chemical step 1-4 selects solvent to be selected from: one or more in benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
5. the compound of claim 1 can be prepared into the carrier that is suitable for gene drug delivery.
6. described in claim 5, the carrier of gene drug delivery is portability range gene or carries range gene simultaneously.
7. the purposes of the compound of claim 1, purposes is for being used for the treatment of, the genophore of diagnosis and prevention.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101327328A (en) * | 2008-07-29 | 2008-12-24 | 华东师范大学 | Dendrimers targeting nano particle and preparation and application thereof |
| CN102503869A (en) * | 2011-10-19 | 2012-06-20 | 沈阳药科大学 | 2-Hydroxylimine hydrocinnamamide ethanedithiol derivatives and application thereof |
-
2013
- 2013-03-21 CN CN201310092038.1A patent/CN104059231A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101327328A (en) * | 2008-07-29 | 2008-12-24 | 华东师范大学 | Dendrimers targeting nano particle and preparation and application thereof |
| CN102503869A (en) * | 2011-10-19 | 2012-06-20 | 沈阳药科大学 | 2-Hydroxylimine hydrocinnamamide ethanedithiol derivatives and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王持，等: "聚酰胺-胺树枝状高分子的聚乙二醇改性及作为基因载体的性能", 《药学学报》 * |
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