论文标题:α，β-聚[（N-羟丙基/氨乙基）-DL-天冬酰胺-CO-L-赖氨酸]：作为潜在的非病毒性基因载体的研究
α,β-poly [(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine]: Potential Non-viral Vector for Gene Delivery
论文作者 杨直
论文导师 汤谷平,论文学位 硕士,论文专业 药物化学
论文单位 浙江大学,点击次数 158,论文页数 83页File Size3482k
2003-05-01论文网 http://www.lw23.com/lunwen_1931072/ 热共聚;非病毒性基因载体;聚[DL-天冬氨酸-CO-L-赖氨酸];α,β-聚[(N-羟丙基/氨乙基)DL-天冬酰胺-CO-L-赖氨酸]
thermal copolycondensation;non-viral gene vector;Poly[Aspartic acid-co-L-lysine];αβ-poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine]
聚氨基酸材料由于可生物降解性，低毒性等特点而被广泛的作为非病毒性基因载体材料的研究。其中研究较多的有聚赖氨酸，聚天冬氨酸，聚鸟氨酸，聚谷氨酸等。聚赖氨酸和聚鸟氨酸在生理条件下带有正电荷，可以通过静电吸引结合DNA。聚天冬氨酸具有酰亚胺环状结构，可被碱性试剂开环而引入结合DNA的功能性侧链。根据共聚氨基酸酶识别位点多，降解速度快的优点，通过磷酸催化的热共聚反应合成了一类新的以天冬氨酸和赖氨酸的共聚物为骨架的基因载体材料。在这方面的同类研究目前还比较少。DL-天冬氨酸和L-赖氨酸通过不同的比例在不同的条件下合成了一系列的聚[DL-天冬氨酸-CO-L-赖氨酸](Poly[DL-Aspartic acid-CO-L-Lysine],PAL)，经过~1H-NMR、FT-IR、X-Ray、TGA方法确认了结构。通过毛细管黏度法测量各个PAL聚合物的特性黏度。PAL在碱性的氨基丙醇和乙二胺开环下合成α，β-聚[(N-羟丙基／氨乙基)-DL-天冬酰胺-CO-L-赖氨酸](α β-poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine],PHAAL)。PHAAL在磷酸缓冲液(ph=7.4，0.01M)和酶(木瓜蛋白酶，胰蛋白酶)溶液中，温度在37±0.1℃条件下降解，经过GPC分析具有良好的降解能力。在磷酸溶液中水解4天，PHAAL的平均分子量降为原来的70％。在木瓜蛋白酶溶液(1mg／ml)中降解12小时，PHAAL的平均分子量下降了32％，24小时内完全降解。而在胰浙江大学硕卜研究生论文蛋白酶溶液(lmg/ml)中12小时内就完全降解了。利用MTT方法测PHAAL在Hela，E.C.V一304，BcaP37细胞中的细胞毒性，实验结果表明PHAAL的细胞毒性比聚乙烯亚胺(polyethylenimine，pEI(25kD))、聚左旋赖氨酸(poly一L一Lysine，pLL(15.7kD)嘟要低，而且PHAAL在浓度达到40ug/ml时也没有明显的细胞毒性。利用含嗅乙睫(0.25mg/ml)的琼脂糖凝胶(l .0%，w八)电泳检测PHAAL的DNA结合能力，发现赖氨酸聚合比例高的PHAAL结合DNA的能力强，其中PHAAL一T25(DL一天冬氨酸:L一赖氨酸=5:5，mol/mol):DNA一150:1(w/w)时能完全结合DNA，阻滞其迁移。综合各种实验结果分析，PHAAL是一类可作为非病毒性基因载体的聚氨基酸类高分子材料。
Polyaminoacid materials are widely researched as non-viral gene vectors because of the advantages, such as biodegradation, low cytotoxicity and so on. Polylysine, poly(aspartic acid), polyornithine and polyglutamide of them are researched in a lot of articles. Polylysine and polyornithine have positively charge in the physiological condition and can condense DNA by the static force. Poly(aspartic acid) has the structure of the imide ring and can be grafted with the functional side chains of condensing DNA by ring-opening with alkaline reagents. According to the advantages of many sites recognized by enzymes and high speed of degradation of copolyaminoacid materials, a new type of gene vector material, copolymers of aspartic acid and lysine, is synthesized by thermal copolycondensation with phosphoric acid catalysis. Now the similar researches are little. A series of Poly[Aspartic acid-co-L-lysine](PAL) are copolycondensed by DL-aspartice acid and L-lysine with different ratios in different conditions and their constructions are affirmed by the spectra of "H-NMR, FT-IR, X-Ray, TGA. Characteristic viscosity of the series of PAL are measured with the method of capillary viscosity, αβ -poly [(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine] (PHAAL) is synthesized by ring-opening poly[aspartic acid-co-lysine](PAL) with the alkaline n-propanolamine and ethylene diamine. PHAAL performed good degradability in the phosphoric acid buffer solution(PBS)(0.01M, ph=7.4) and in the solution of enzyme(Papain, Trypsine) at 37?0.1 by GPC experiment. After degradation in PBS for four days, the mean molecule of PHAAL fell to the 70% of origin. After degradation in the solution of Papain for 12 hours, the mean molecule of PHAAL dropped 32% and degradation was complete in 24 hours. Moreover PHAAL degraded completely in the solution of Trypsin in 12 hours. PHAAL appeared less cytotoxicity than poly(ethylenimine)(25KDa) and poly-L-lysine(15.7KDa) in Hela, E.C.V-304, Bcap37 cell lines, which was quantified by MTT assay. Furthermore PHAAL didn"t perform distinct cytotoxicity up to 40ug/ml. The combination ability of PHAAL and plasmid DNA was evaluated by agarose gel electrophoresis with agarose gel(1.0% w/v) containing ethidium bromide(0.25ug/ml). The PHAAL with higher ratios of lysine in the copolymers have higher ability of condensing DNA. Among them, the PHAAL-T25(DL-aspartic acid: L-lysine=5:5, mol/mol) can condense and regard DNA completely. In a word, the polyaminoacid materials, PHAAL, is the kind of macromolecule materials as the non-viral gene vector.
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