CN103012803A - Polyamidoamine derivative and preparation method and application thereof - Google Patents
Polyamidoamine derivative and preparation method and application thereof Download PDFInfo
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- CN103012803A CN103012803A CN2012100559651A CN201210055965A CN103012803A CN 103012803 A CN103012803 A CN 103012803A CN 2012100559651 A CN2012100559651 A CN 2012100559651A CN 201210055965 A CN201210055965 A CN 201210055965A CN 103012803 A CN103012803 A CN 103012803A
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Abstract
The invention discloses a polyamidoamine derivative which is pluronic modified polyamidoamine. The invention also discloses a preparation method and application of the polyamidoamine derivative. The polyamidoamine derivative disclosed by the invention has low cell toxicity and high transfection efficiency, and is very suitable to be used as a gene carrier material.
Description
Technical field
The present invention relates to medical technical field, be specifically related to daiamid derivative of a kind of pluronic (Pluronic has another name called poloxamer Poloxamer) modification and its preparation method and application.
Background technology
Gene therapy is that normal gene or medicative gene are imported target cell to correct genetic flaw or performance therapeutic action by certain way.Because of exist in the body multiple can degradation of dna or the enzyme of RNA and dissimilar biological barriers, so therapeutic gene wants effectively to arrive target site, must be by means of certain carrier transportation.But owing to lack safely and effectively genophore, limited its clinical application.The genophore that uses at present generally is divided into virus vector and non-virus carrier, and the virus vector transfection efficiency is higher but to exist carrying capacity low, the problems such as potential security threat is arranged, so non-virus carrier has caused people's attention gradually.In numerous non-virus carriers, daiamid (polyamidoamine, PAMAM) has higher transfection efficiency in vivo because of it, low toxicity and without advantages such as immunogenicity, good stabilities and enjoy people's attention.The transfection effect of PAMAM and its algebraically, namely molecular weight is relevant, and algebraically is larger, and the transfection effect is better, however along with the increase of molecular weight, its cytotoxicity also increases thereupon, and this has just limited its application clinically.In order to overcome the shortcoming of PAMAM, the hydrophilic polymer of normal operation biocompatibility, for example polyoxyethylene glycol (PEG) is to improve the stability of PAMAM/DNA mixture, reduce its toxicity, because the avidity that PAMAM is modified rear and cell by hydrophilic polymer reduces, so that gene can't enter cell, transfection efficiency descends.
Therefore how further PAMAM being transformed into the gene vector material that cytotoxicity is little and transfection efficiency is high, is to solve the breach that PAMAM uses a difficult problem.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of daiamid derivative, and this daiamid derivative is the daiamid that pluronic is modified, and it is as non-viral gene vector, and cytotoxicity is little, and transfection efficiency is high.
In addition, also need to provide a kind of preparation method of daiamid derivative and as the application of genophore.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of daiamid derivative, this daiamid derivative is the daiamid that pluronic is modified, and its chemical structural formula is as follows:
In the formula, PAMAM is daiamid, x=20-100, y=20-100.
Pluronic is amphipathic nature polyalcohol, is comprised of hydrophilic polyoxyethylene chain (EO) and lipophilic polyoxypropylene chains (PO), and its chemical structural formula is as follows:
EO chain formation wetting ability shell can increase the colloidal stability of solid support material, reduces the cytotoxicity of PAMAM; And PO chain formation lipotropy kernel can increase the lipotropy of material, improves the avidity to cell, and then improves transfection efficiency.
Because the wetting ability of polyoxyethylene chain and the lipotropy of polyoxypropylene chains make this analog copolymer of pluronic have surfactivity far from it, belong to nonionic surface active agent.
Preferably, pluronic of the present invention is selected from: model is respectively the pluronic of F127, P123, P105, P85 (Mw=12220,5780,6500,4600).Marque for pluronic, its identify code is the letter (L: liquid of physical aspect under its room temperature of expression, P: pasty state, F: sheet (solid)) two of suffix or three bit digital, the first figure place (and three bit digital situations under front two number) multiply by the approximate molecular mass of 300 expression hydrophobic materials polyoxypropylenes, and rear one digit number x10 is the shared per-cent of polyoxyethylene (for example P85 represents that molecular mass is the pluronic that the polyoxypropylene of 2300g/mol consists of with 50% polyoxyethylene).
In another aspect of this invention, provide a kind of preparation method of above-mentioned daiamid derivative, may further comprise the steps:
With triphosgene and N-hydroxy-succinamide activation pluronic two terminal hydroxy group;
Pluronic and daiamid reaction with activation get the daiamid that pluronic is modified.
In another aspect of this invention, also provide the application of above-mentioned daiamid derivative as gene vector material.
Described genophore carrying DNA or RNA.
Daiamid derivative of the present invention confirms that through cytotoxicity experiment and in-vitro transfection cell experiment not only cytotoxicity is little, and transfection efficiency is high, is suitable for using as genophore.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the synthetic reaction scheme figure of daiamid derivative that pluronic of the present invention is modified;
Fig. 2 is PAMAM after the embodiment of the invention 5 is modified and the PAMAM cytotoxicity experiment measurement result figure of unmodified;
Fig. 3 is the PAMAM in-vitro transfection measuring figure as a result of PAMAM after the embodiment of the invention 6 is modified and unmodified.
Embodiment
Daiamid derivative of the present invention is the polymkeric substance that utilizes pluronic to form as bridge chain molecule series connection low molecular polyamides amine, and it is as genophore, and not only cytotoxicity is little, and transfection efficiency is high.
The preparation method of daiamid derivative of the present invention following (seeing Fig. 1):
(A) activation of pluronic
Four kinds of pluronics are dissolved in respectively in the mixing solutions of toluene and methylene dichloride, add triphosgene, magnetic agitation is spent the night, with the organic solvent evaporate to dryness, residue with the mixed solvent dissolving of toluene and methylene dichloride, adds N-hydroxy-succinamide again, under magnetic agitation, slowly drip anhydrous triethylamine, react after 3-8 hour, with solution filter, obtain the pluronic formic acid succinimide ester of four kinds of activation behind the filtrate evaporate to dryness;
The activation method of pluronic also comprises the method for mentioning in the following document: Nguyen.H.K., Lemieux.P., Vinogradov.S.V., etc.Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents.Gene Therapy, 2000,7 (2): 126-138; Vinogradov S.V., Batrakova E.V., Li, S., etc.Mixed polymer micel les of amphiphilic and cationic copolymers for delivery of antisense oligonucleotides.J Drug Target, 12,517-526 (2004).
(B) the pluronic modified daiamid after the activation
Daiamid is dissolved in the ethanolic soln of 0%-20%, under agitation add the pluronic after activating, reacted 16-48 hour, reaction solution is crossed the glucose gel post, unreacted daiamid is removed, pass through ultrafiltration purification, the reaction solution lyophilize behind the purifying obtains the daiamid through the pluronic modification again.
The preparation of the daiamid (PAMAM) that embodiment 1 pluronic F127 modifies
Get 1.459g pluronic F127 (U.S. Aldrich company) at 50 ℃ of lower vacuum-drying 24h, be dissolved in 40ml toluene and methylene chloride volume ratio and be in 3: 1 the mixed solvent, add triphosgene 0.356g, magnetic agitation is spent the night, use Rotary Evaporators with organic solvent at 50 ℃ of lower evaporates to dryness, it is in 2: 1 the mixing solutions that residuum is dissolved in 30ml toluene and methylene chloride volume ratio again, add the 0.137g N-hydroxy-succinamide, be added dropwise to anhydrous triethylamine 0.200ml, behind the reaction 4h, reacting liquid filtering is removed insolubles, and rotary evaporation is removed organic solvent, the pluronic F127 that obtains activating.
Get 4 parts of PAMAM (G5,0.28g, 0.01mmol, sigma company) is dissolved in respectively in 10% ethanolic soln of 10ml, continuing to add respectively 0.01 under the stirring, 0.02,0.05, pluronic F127 with the activation of 0.1mmol amount, stirring at room 24h, the centrifugal insolubles of removing of reaction solution is crossed Sephadex G-75 glucose gel post and is removed unreacted raw material, after lyophilize, obtain the reactant of purifying, with reactant difference called after F127-1g-PAMAM, F127-2g-PAMAM, F127-5g-PAMAM, the F127-10g-PAMAM of different modifying degree.
The preparation of the daiamid (PAMAM) that embodiment 2 pluronic P123 modify
Get 0.694g pluronic P123 (U.S. Aldrich company) at 50 ℃ of lower vacuum-drying 24h, be dissolved in 40ml toluene and methylene chloride volume ratio and be in 3: 1 the mixed solvent, add triphosgene 0.356g, magnetic agitation is spent the night, use Rotary Evaporators with organic solvent at 50 ℃ of lower evaporates to dryness, it is in 2: 1 the mixing solutions that residuum is dissolved in 30ml toluene and methylene chloride volume ratio again, add the 0.137g N-hydroxy-succinamide, be added dropwise to anhydrous triethylamine 0.200ml, behind the reaction 4h, reacting liquid filtering is removed insolubles, and rotary evaporation is removed organic solvent, the pluronic P123 that obtains activating.
Get 4 parts of PAMAM (G5,0.28g, 0.01mmol, sigma company) is dissolved in respectively in 10% ethanolic soln of 10ml, continuing to add respectively 0.01 under the stirring, 0.02,0.05, pluronic P123 with the activation of 0.1mmol amount, stirring at room 24h, the centrifugal insolubles of removing of reaction solution is crossed Sephadex G-75 glucose gel post and is removed unreacted raw material, after lyophilize, obtain the reactant of purifying, with reactant difference called after P123-1g-PAMAM, P123-2g-PAMAM, P123-5g-PAMAM, the P123-10g-PAMAM of different modifying degree.
The preparation of the daiamid (PAMAM) that embodiment 3 pluronic P105 modify
Get 0.785g pluronic P105 (U.S. Aldrich company) at 50 ℃ of lower vacuum-drying 24h, be dissolved in 40ml toluene and methylene chloride volume ratio and be in 3: 1 the mixed solvent, add triphosgene 0.356g, magnetic agitation is spent the night, use Rotary Evaporators with organic solvent at 50 ℃ of lower evaporates to dryness, it is in 2: 1 the mixing solutions that residuum is dissolved in 30ml toluene and methylene chloride volume ratio again, add the 0.137g N-hydroxy-succinamide, be added dropwise to anhydrous triethylamine 0.200ml, behind the reaction 4h, reacting liquid filtering is removed insolubles, and rotary evaporation is removed organic solvent, the pluronic P105 that obtains activating.
Get 4 parts of PAMAM (G5,0.28g, 0.01mmol, sigma company) is dissolved in respectively in the distilled water of 10ml, continuing to add respectively 0.01 under the stirring, 0.02,0.05, pluronic P105 with the activation of 0.1mmol amount, stirring at room 24h, the centrifugal insolubles of removing of reaction solution is crossed Sephadex G-75 glucose gel post and is removed unreacted raw material, after lyophilize, obtain the reactant of purifying, with reactant difference called after P105-1g-PAMAM, P105-2g-PAMAM, P105-5g-PAMAM, the P105-10g-PAMAM of different modifying degree.
The preparation of the daiamid (PAMAM) that embodiment 4 pluronic P85 modify
Get 0.510g pluronic P85 (U.S. Aldrich company) at 50 ℃ of lower vacuum-drying 24h, be dissolved in 40ml toluene and methylene chloride volume ratio and be in 3: 1 the mixed solvent, add triphosgene 0.356g, magnetic agitation is spent the night, use Rotary Evaporators with organic solvent at 50 ℃ of lower evaporates to dryness, it is in 2: 1 the mixing solutions that residuum is dissolved in 30ml toluene and methylene chloride volume ratio again, add the 0.137g N-hydroxy-succinamide, be added dropwise to anhydrous triethylamine 0.200ml, behind the reaction 4h, reacting liquid filtering is removed insolubles, and rotary evaporation is removed organic solvent, the pluronic P85 that obtains activating.
Get 4 parts of PAMAM (G5,0.28g, 0.01mmol, sigma company) is dissolved in respectively in the distilled water of 10ml, continuing to add respectively 0.01 under the stirring, 0.02,0.05, pluronic P85 with the activation of 0.1mmol amount, stirring at room 24h, the centrifugal insolubles of removing of reaction solution is crossed Sephadex G-75 glucose gel post and is removed unreacted raw material, after lyophilize, obtain the reactant of purifying, with reactant difference called after P85-1g-PAMAM, P85-2g-PAMAM, P85-5g-PAMAM, the P85-10g-PAMAM of different modifying degree.
The Cytotoxic evaluation experiment of the daiamid that embodiment 5 pluronics are modified
The HepG2 cell is seeded on 96 orifice plates, every hole about 10,000 cell, cultivate the PAMAM that adds respectively a series of concentration (20,40,60,80,120,160 μ g/ml) after 24 hours and modify after PAMAM (F127-g-PAMAM), continue to cultivate 24h, mtt assay is measured cytotoxicity.From Fig. 2 A, can find out through modifying PAMAM (F127-g-PAMAM) and compare with the PAMAM of unmodified, all demonstrate stronger cytoactive in each concentration.
The HepG2 cell is seeded on 96 orifice plates, every hole about 10,000 cell, cultivate the PAMAM that adds respectively a series of concentration (20,40,60,80,120,160 μ g/ml) after 24 hours and modify after PAMAM (P123-g-PAMAM), continue to cultivate 24h, mtt assay is measured cytotoxicity.From Fig. 2 B, can find out through modifying PAMAM (P123-g-PAMAM) and compare with the PAMAM of unmodified, all demonstrate stronger cytoactive in each concentration.
The HepG2 cell is seeded on 96 orifice plates, every hole about 10,000 cell, cultivate the PAMAM that adds respectively a series of concentration (20,40,60,80,120,160 μ g/ml) after 24 hours and modify after PAMAM (P105-g-PAMAM), continue to cultivate 24h, mtt assay is measured cytotoxicity.From Fig. 2 C, can find out through modifying PAMAM (P105-g-PAMAM) and compare with the PAMAM of unmodified, all demonstrate stronger cytoactive in each concentration.
The HepG2 cell is seeded on 96 orifice plates, every hole about 10,000 cell, cultivate the PAMAM that adds respectively a series of concentration (20,40,60,80,120,160 μ g/ml) after 24 hours and modify after PAMAM (P85-g-PAMAM), continue to cultivate 24h, mtt assay is measured cytotoxicity.From Fig. 2 D, can find out through modifying PAMAM (P85-g-PAMAM) and compare with the PAMAM of unmodified, all demonstrate stronger cytoactive in each concentration.
The cell in vitro transfection experiment of the daiamid that embodiment 6 pluronics are modified
The HepG2 cell is seeded on 24 orifice plates, and about 10,000 cells in every hole are cultivated 24h.PAMAM and modified PAMAM (F127-g-PAMAM) (can be expressed Luci with plasmid pGL3, available from Promega company) form mixture at different N/P than (N/P=6,9,12,18) lower mixing, incubated at room 30min, mixture is dispersed in the nutrient solution of serum-free and joins 24 orifice plates, cultivate 5h, the nutrient solution that replacement contains serum continues to cultivate 48h, measures the expression of Luci in the cell.Can see that from Fig. 3 A under different N/P values, F127-1g-PAMAM, F127-2g-PAMAM have shown the transfection efficiency suitable with the PAMAM of unmodified, the PAMAM transfection efficiency of other modification degree all is lower than the PAMAM transfection efficiency of unmodified.
The HepG2 cell is seeded on 24 orifice plates, and about 10,000 cells in every hole are cultivated 24h.PAMAM and modified PAMAM (P123-g-PAMAM) (can be expressed Luci with plasmid pGL3, available from Promega company) form mixture at different N/P than (N/P=6,9,12,18) lower mixing, incubated at room 30min, mixture is dispersed in the nutrient solution of serum-free and joins 24 orifice plates, cultivate 5h, the nutrient solution that replacement contains serum continues to cultivate 48h, measures the expression of Luci in the cell.Can see from Fig. 3 B, be that the PAMAM of 9,12,18 o'clock various modification degree demonstrates stronger transfection efficiency than the PAMAM of unmodified in the N/P value, is that 6 o'clock P123-1g-PAMAM, P123-2g-PAMAM have demonstrated stronger transfection efficiency at N/P.
The HepG2 cell is seeded on 24 orifice plates, and about 10,000 cells in every hole are cultivated 24h.PAMAM and modified PAMAM (P105-g-PAMAM) (can be expressed Luci with plasmid pGL3, available from Promega company) form mixture at different N/P than (N/P=6,9,12,18) lower mixing, incubated at room 30min, mixture is dispersed in the nutrient solution of serum-free and joins 24 orifice plates, cultivate 5h, the nutrient solution that replacement contains serum continues to cultivate 48h, measures the expression of Luci in the cell.Can see from Fig. 3 C, be that the PAMAM of 12,18 o'clock various modification degree demonstrates stronger transfection efficiency than the PAMAM of unmodified in the N/P value, is that 6,9 o'clock P105-1g-PAMAM, P105-2g-PAMAM have demonstrated stronger transfection efficiency at N/P.
The HepG2 cell is seeded on 24 orifice plates, and about 10,000 cells in every hole are cultivated 24h.PAMAM and modified PAMAM (P85-g-PAMAM) (can be expressed Luci with plasmid pGL3, available from Promega company) form mixture at different N/P than (N/P=6,9,12,18) lower mixing, incubated at room 30min, mixture is dispersed in the nutrient solution of serum-free and joins 24 orifice plates, cultivate 5h, the nutrient solution that replacement contains serum continues to cultivate 48h, measures the expression of Luci in the cell.Can see from Fig. 3 D, be that the PAMAM of 12,18 o'clock various modification degree demonstrates stronger transfection efficiency than the PAMAM of unmodified in the N/P value, is that 6,9 o'clock P85-1g-PAMAM, P85-2g-PAMAM have demonstrated stronger transfection efficiency at N/P.
Above experimental result shows the PAMAM that modifies through the PAMAM, particularly pluronic P123, P105 of pluronic modification, P85 as genophore, and toxicity is little, and transfection efficiency is high, and application prospect is extensive.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. a daiamid derivative is characterized in that, this daiamid derivative is the daiamid that pluronic is modified, and its chemical structural formula is as follows:
In the formula, PAMAM is daiamid, x=20-100, y=20-100.
2. daiamid derivative according to claim 1 is characterized in that, described pluronic is selected from: model is the pluronic of F127, P123, P105 or P85.
3. the preparation method of the described daiamid derivative of claim 1 is characterized in that, may further comprise the steps:
With triphosgene and N-hydroxy-succinamide activation pluronic two terminal hydroxy group;
Pluronic and daiamid reaction with activation get the daiamid that pluronic is modified.
4. the described daiamid derivative of claim 1 is as the application of gene vector material.
5. application according to claim 4 is characterized in that, described genophore carrying DNA or RNA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289097A (en) * | 2013-05-15 | 2013-09-11 | 浙江大学 | Pluronic-modified PAMAM (polyamide-amine) graft polymer and application |
| CN106496585A (en) * | 2016-10-21 | 2017-03-15 | 浙江大学 | Sensitive graphene oxide based nano-materials of a kind of ROS and its preparation method and application |
-
2012
- 2012-03-06 CN CN2012100559651A patent/CN103012803A/en active Pending
Non-Patent Citations (1)
| Title |
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| 王翔: "Pluronic修饰的聚阳离子非病毒基因载体的构建、表征及体外转染效率评价", 《万方数据 知识服务平台》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289097A (en) * | 2013-05-15 | 2013-09-11 | 浙江大学 | Pluronic-modified PAMAM (polyamide-amine) graft polymer and application |
| CN106496585A (en) * | 2016-10-21 | 2017-03-15 | 浙江大学 | Sensitive graphene oxide based nano-materials of a kind of ROS and its preparation method and application |
| CN106496585B (en) * | 2016-10-21 | 2019-02-19 | 浙江大学 | A kind of graphene oxide based nano-material and its preparation method and application of ROS sensitivity |
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Inventor after: Gao Shen Inventor after: Wang Xiaoyu Inventor after: Zhang Muzihe Inventor after: Ding Baoyue Inventor after: Wang Xiang Inventor after: Wu Xin Inventor after: Fan Wei Inventor after: Zhu Quangang Inventor after: Ding Xueying Inventor after: Gao Jing Inventor after: Liu Jiyong Inventor after: Zhang Min Inventor before: Gao Shen Inventor before: Wang Xiaoyu Inventor before: Zhang Muzihe Inventor before: Wang Xiang Inventor before: Wu Xin Inventor before: Fan Wei Inventor before: Zhu Quangang Inventor before: Ding Xueying Inventor before: Gao Jing Inventor before: Liu Jiyong Inventor before: Zhang Min |
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Free format text: CORRECT: INVENTOR; FROM: GAO SHEN WANG XIANG WU XIN FAN WEI ZHU QUANGANG DING XUEYING GAO JING LIU JIYONG ZHANG MIN WANG XIAOYU ZHANG MUZIHE TO: GAO SHEN WANG XIANG WU XIN FAN WEI ZHU QUANGANG DING XUEYING GAO JING LIU JIYONG ZHANG MIN WANG XIAOYU ZHANG MUZIHE DING BAOYUE |
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Application publication date: 20130403 |