用于靶向质粒DNA输送的美聚氨酸功能化生物相容性块共聚物
Summary
本作品通过可逆添加-碎片链转移(RAFT)方法介绍了蛋氨酸功能化生物相容性块共聚物(mBG)的制备。研究了获得mBG的质粒DNA复合能力及其转染效率。RAFT方法对聚合含有特殊功能组的单体非常有益。
Abstract
可逆添加-碎片链转移(RAFT)聚合融合了基聚合和活聚合的优点。本作品介绍了通过RAFT聚合制备蛋氨酸功能化生物相容性块共聚物。首先,N,N-二乙二醇(2-羟基苯甲酸酯)甲酰胺-b-N-(3-氨基丙烯酰胺)甲酰胺(BNHEMA-b-APMA,BA)通过RAFT聚合合成,使用4,4′-亚索比(4-青黄酸)(ACVA)作为启动剂和4-氰化二苯甲酸二甲苯甲酸(CTP)作为链转移剂。随后,N,N-bis(2-羟基)甲酰胺-b-N-(3-关尼诺丙基)甲酰胺(美聚氨酸嫁接BNHEMA-b-GPMA,mBG)通过用甲胺和瓜尼丁修饰APMA中的胺组制备组。合成了三种块状聚合物,mBG1、mBG2和mBG3进行比较。使用宁西林反应来量化APMA含量;mBG1、mBG2 和 mBG3 分别占 APMA 的 21%、37% 和 52%。凝胶渗透色谱(GPC)结果表明,BA共聚物的分子量为16,200(BA1)、20,900(BA2)和27,200(BA3)克/摩尔。研究了获得块共聚物基因载体的质粒DNA(pDNA)复合能力。当pDNA分别与mBG1、mBG2、mBG3完全复合时,电荷比(N/P)为8、16和4。当mBG/pDNA多聚体N/P比高于1时,mBG的Zeta电位为正。在 16 和 32 之间的 N/P 比率下,mBG/pDNA 聚丛的平均颗粒大小在 100-200 nm 之间。总体而言,这项工作说明了块共聚载体合成的简单和方便的方案。
Introduction
近年来,基因疗法已出现以核酸的治疗性输送为治疗各种疾病的药物。包括质粒DNA(pDNA)和小干扰RNA(siRNA)在内的基因药物的开发依赖于药物输送系统(DDS)2的稳定性和效率。在所有DDS中,阳离子聚合物载体具有稳定性好、免疫原性低、易制制剂和改性等优点,使阳离子聚合物载体具有广阔的应用前景。为了在生物医学中的实际应用,研究人员必须找到一种高效、低毒性、具有良好的靶向能力的阳离子聚合物载体。在所有聚合物载体中,块共聚物是使用最广泛的药物输送系统之一。块共聚物被深入研究,因为它们的自组装特性和在药物输送中形成云母、微球和纳米粒子的能力5。块共聚物可以通过活聚合或点击化学方法合成。
1956年,Szwarc等人提出了活聚合的课题,将其定义为无链断裂反应的6,7。从那时起,已经开发出多种技术,用这种方法合成聚合物;因此,活聚合被认为是聚合物科学的一个里程碑。活聚合可分为活阴离子聚合、活阳离子聚合和可逆失活基聚合(RDRP)9。活阴离子/阳离子聚合具有有限的应用范围,由于其严格的反应条件10。受控/活基聚合(CRP)反应条件温和,处置方便,产量好,是近年来的主要研究重点。在CRP中,活性传播链可逆钝化为休眠链,以降低自由基的浓度,避免传播链基的双分子反应。只有当非活性休眠传播链可逆地动画化为链基时,添加聚合才能继续。可逆添加-裂变链转移(RAFT)聚合作为最有前途的活基聚合形式之一,是一种适用于分子量和结构可控、分子量窄的块状聚合物的屈服方法。分布,并携带功能组12。成功进行RAFT聚合的关键是链转移剂(通常是二硫酯)的作用,后者具有非常高的链传递常数。
本文设计了一种RAFT聚合法,制备BNHEMA-b-APMA块状聚合物,以4,4′-亚索比(4-青黄酸)(ACVA)作为起始剂,4-氰化二苯甲酸二硫苯甲酸(CTP)作为链转移剂。RAFT聚合被两次用于将BNHEMA引入阳离子聚合物载体。随后,用甲胺磷和甲胺酰胺试剂1-酰胺盐酸基苯甲酸酯对APMA链中的胺组进行了修饰。利用关尼酰胺试剂和甲酰胺聚合物骨架结构的正电荷,提高了所得块状聚合物载体的细胞吸收效率。
Protocol
Representative Results
Discussion
本研究介绍了一系列BNHEMA-b-APMA块状聚合物阳离子基因载体。这些块状聚合物是通过可逆的添加-碎片链转移(RAFT)方法合成的。引入亲水段BNHEMA以提高溶解度。对美氨酸和瓜尼丁组进行了改造,以提高靶能力和转染效率5.MBG共聚物中的APMA链含量增加,核化减少了mBG/pDNA聚体颗粒尺寸。颗粒大小和表面电位使复合物易于穿过细胞膜,适用于转染。实验的关键在于严格控制BNHEMA单体和CTP的摩…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家重点研究发展计划(2016YFC0905900号)、国家自然科学基金(第81801827号、81872365号)、江苏省基础研究计划(自然科学基金,No.BK20181086)和江苏省肿瘤医院科研基金(No.ZK201605)。
Materials
| 1-hydroxybenzotriazole | Macklin Biochemical Co., Ltd,China | H810970 | ≥97.0% |
| 1,4-dioxane | Sinopharm chemical reagent Co., Ltd, China | 10008918 | AR |
| 1-amidinopyrazole Hydrochloride | Aladdin Co., Ltd., China | A107935 | 98% |
| 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride | Aladdin Co., Ltd., China | E106172 | AR |
| 4,4’-azobis(4-cyanovaleric acid) | Aladdin Co., Ltd., China | A106307 | Analytical reagent (AR) |
| 4-cyano-4-(phenylcarbonothioylthio)pentanoic Acid | Aladdin Co., Ltd., China | C132316 | >97%(HPLC) |
| Acetate | Sinopharm chemical reagent Co., Ltd, China | 81014818 | AR |
| Acetone | Sinopharm chemical reagent Co., Ltd, China | 10000418 | AR |
| Agarose | Aladdin Co., Ltd., China | A118881 | High resolution |
| Ascorbic acid | Aladdin Co., Ltd., China | A103533 | AR |
| DMSO | Aladdin Co., Ltd., China | D103272 | AR |
| Ethylene glycol | Aladdin Co., Ltd., China | E103319 | AR |
| N-(3-aminopropyl)methacrylamide hydrochloride | Aladdin Co., Ltd., China | N129096 | ≥98.0%(HPLC) |
| N,N-bis(2-hydroxyethyl)methacrylamide | ZaiQi Bio-Tech Co.,Ltd, China | CF259748 | ≥98.0%(HPLC) |
| Ninhydrin | Aladdin Co., Ltd., China | N105629 | AR |
| PBS buffer | Aladdin Co., Ltd., China | P196986 | pH 7.4 |
| Plasmid DNA | BIOGOT Co., Ltd, China | pDNA-EGFP | pDNA-EGFP |
| Plasmid DNA | BIOGOT Co., Ltd, China | Pdna | pDNA |
| Sodium carbonate decahydrate | Aladdin Co., Ltd., China | S112589 | AR |
| Trimethylamine | Aladdin Co., Ltd., China | T103285 | AR |
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