新型基因载体-聚酰胺-胺树状大分子聚合物

利用重组DNA技术进行疾病预防和治疗是近几年来的研究热点,如何提高重组DNA的转染效率一直是研究的难题之一,新型阳离子多聚物--聚酰胺-胺型树状大分子(Starburst PAMAM dendrimers,PAMAM-D)的出现为解决现存的难题带来希望.PAMAM-D最早由美国人Tomalia于1985年合成,目前已广泛应用于各个领域:药物载体、废水处理、催化剂、光电传感、基因载体等.由于PAMAM-D具有对称的刚性球体结构,表面带有大量正电荷,容易与带负电荷的核酸形成复合物,故作为基因载体具有许多优于其他现有转染试剂的优良特性.本文主要介绍了PAMAM-D的结构、性质、应用以及作为基因载体的优越性,并对今后的研究方向进行了概述.     

聚酰胺-胺树状大分子作为药物载体的体外增溶、缓释试验

目的:考察以聚酰胺-胺树状大分子(PAMAM)作为喹诺酮类药物载体在增溶、缓释方面的作用。方法:根据文献合成PAMAM,并作结构分析;以巴洛沙星为模型药物,检测不同代数不同浓度的PAMAM对巴洛沙星的增溶作用;同时用不同代数的PAMAM与巴洛沙星复合,检测PAMAM对巴洛沙星体外释放的影响,以及在水及模拟肠液中的释放情况。结果:巴洛沙星在水中溶解度约为1.5g·L-1,完全释药时间约为4h。随着PAMAM代数和浓度的增加巴洛沙星溶解度增加(2～5.25g·L-1),PAMAM与巴洛沙星复合物在水中24h释放80%,在模拟肠液中72h释放73%。结论:PAMAM对巴洛沙星具有增溶、缓释作用,有可能促进其制成具有缓释作用的溶液剂或注射剂。     

阿霉素聚酰胺-胺树状大分子脂质体包覆物的制备与评价

目的制备阿霉素聚酰胺-胺树状大分子脂质体包覆物(LLDs-PAMAM-DOX),并对制剂性质进行评价。方法采用逆向蒸发结合pH梯度法制备LLDs-PAMAM-DOX,观察形态,测定粒径分布及zeta电位;采用凝胶柱-UV法测定包封率;采用透析法测定体外释药;采用MTT法,考察其对人乳腺癌细胞MCF-7的细胞毒作用。结果LLDs-PAMAM-DOX的粒径约为80¹20nm;zeta电位约为22.5120nm;zeta电位约为22.5³2.4mV;包封率大于85%,缓释作用明显。LLDs-PAMAM-DOX呈剂量依赖性抑制肿瘤细胞的生长,对MCF-7的抑制作用明显优于普通脂质体L-DOX。结论聚酰胺-胺树状大分子脂质体包覆物(LLDs-PAMAM)作为阿霉素(doxorubicin,DOX)的载体,具有较好的缓释作用及良好的抑制肿瘤细胞作用,有望成为一种有潜力的新型药物载体。     

G2.0聚酰胺-胺树状大分子及其水杨醛修饰物体外释药性能的研究

目的考察G2.0聚酰胺-胺树状大分子(PAMAM)及水杨醛修饰物(PAMAM-SA)的释药机制。方法用紫外可见分光光度法检测2种大分子环境敏感性及对氧氟沙星的增溶作用。氧氟沙星含量测定采用HPLC法:ODS-C18色谱柱(150mm×4.6mm,5μm);流动相为乙腈、三乙胺溶液(磷酸调pH 4.0)(14∶86);检测波长294nm;流速1.0mL·min-1;柱温40℃。结果氧氟沙星单独释放80%需要2h,G2.0PAMAM-氧氟沙星复合物释放氧氟沙星80%需要5h,G2.0PAMAM-SA-氧氟沙星复合物释放氧氟沙星80%需要2.5h。结论 2种大分子具有环境敏感性,对氧氟沙星具有增溶、缓释作用,2种复合物形成是大分子的氨基阳离子与氧氟沙星的羧基阴离子之间静电作用的结果。     

聚酰胺-胺树状大分子及其PEG修饰物的合成、表征与载药性能

目的合成并表征各代聚酰胺-胺(polyamide-amine,PAMAM)树状大分子及聚乙二醇(poly-ethylene glycol,PEG)修饰PAMAM,研究其对难溶性药物的包载及释放行为。方法用发散法合成1-5代PAMAM大分子及PEG化PAMAM,采用IR、NMR、端基滴定、GPC等方法对其进行表征,并以阿霉素为模型药物,比较PAMAM和PEG-PAMAM载药能力及释放效果。结果FTIR、NMR、端基滴定、GPC测定结果表明所合成的产物确为1-5代PAMAM树状大分子,IR测定结果表征PEG化PAMAM合成,PAMAM大分子及PEG-PAMAM对难溶性药物具有较强包载能力,其中PEG-PAMAM能够更好地延缓药物释放。结论PEG-PAMAM大分子具有良好的作为难溶性药物载体的潜力。     

聚乙二醇化聚酰胺-胺树状大分子的合成及作为基因载体的研究

目的研究聚乙二醇(polyethylene glycol,PEG)化聚酰胺-胺树状大分子(polyamidoamine dendrimer,PAMAM)的理化性质及其作为质粒DNA载体。方法用1H-NMR考察PAMAM-PEG的化学结构;用琼脂糖凝胶电泳法、圆二色谱法和透射电镜法考察复合物的形成;用动态光散射法测定复合物的粒径和zeta电位;用MTT法测定复合物的毒性;用萤光素酶报告基因的表达表征复合物的转染效率。结果结果表明,载体化合物合成成功,PEG修饰度为9%;琼脂糖凝胶电泳结果表明,在N/P比大于2时,复合物能阻滞DNA的电泳;圆二色谱、透射电镜结果表明复合物已经形成,其形态规整,呈球形。随着N/P比的增加,复合物的粒径减小,zeta电位升高;MTT结果表明复合物的细胞毒性小;萤光素酶实验结果表明复合物的转染效率高。结论作为DNA载体,PAMAM-PEG的细胞毒性小、转染效率高,具有很大的研究前景。     

叶酸-聚酰胺-胺聚合物靶向给药系统的研究进展

简要介绍了聚酰胺-胺树状大分子(PAMAM-D)的特点,叶酸修饰的PAMAM-D复合物的主动靶向性机制,及其在抗肿瘤、基因治疗等领域的研究进展.     

树状大分子作为新型药物载体的研究进展

树状大分子是一类高度枝化的单分散性大分子，其内部结构呈疏水性，外表面呈亲水性，可称为“单分子胶束”。本文在简介树状大分子的发展及结构特点的基础上，阐述了树状大分子作为药物载体的作用特点及其与药物的结合方式。目前，树状大分子在介导药物靶向传递及基因转染等方面的应用也备受关注，是一种颇有发展潜力的新型载体。     

PAMAM树状大分子对阿霉素在HepG 2细胞中的摄取动力学的影响

目的利用聚酰胺-胺(polyamidoamine,PAMAM)树枝状大分子包裹抗癌药物阿霉素,建立细胞内测定阿霉素浓度的HPLC检测方法,研究PAMAM对阿霉素在HepG 2细胞内的摄取行为的影响。方法将盐酸阿霉素脱去盐酸后利用疏水作用进入PAMAM内腔制备载药胶束,以柔红霉素为内标,粉碎细胞后提取细胞内的阿霉素进行浓度测定。测定细胞摄取、外排期间的药物浓度,绘制时间-浓度曲线,并计算动力学参数。结果 PAMAM包裹脱盐酸阿霉素后得到阿霉素的质量浓度为1.034g·L-1的溶液,成功建立胞内阿霉素的含量测定方法。细胞摄取动力学结果显示PAMAM使得阿霉素胞内达峰时间为1h,胞内浓度明显增高;消除动力学结果显示,k减小为原来的0.347倍,t1/2延长为原来的2.878倍,MRT增加为原来的2倍。结论经PAMAM包裹后阿霉素能够在HepG 2细胞内快速达峰,胞内浓度增加,消除速率减慢,滞留时间延长,有利于药物药效的发挥。     

DNA桥接的Hela细胞靶向组装型树状大分子的制备

目的:设计一种叶酸受体介导的经DNA桥接的靶向组装型聚酰胺-胺树状大分子,探讨靶向树状大分子库及功能树状大分子库经DNA组合可行性。方法:将第五代聚酰胺-胺树状大分子(G5 PAMAM)部分酰化后,分2组分别连接叶酸(FA)以及异硫氰酸荧光素(FITC),再分别与经EDC活化的互补单链DNA相连,最后完成DNA杂交合成终产物,荧光显微镜观察其对HELA细胞的靶向性。结果:(1)G5 PAMAM的酰化按投料比获得末端酰化率约为70%的PAMAM。(2)氢谱显示FA和FITC与部分酰化G5 PAMAM连接后,产物出现相应的苯环氢,通过积分计算出每个部分酰化的G5 PAMAM连接约5个FA或2.5个FITC,且经TLC检测,产物经葡聚糖凝胶层析和透析后可达色谱纯。(3)经DNA桥接的PAMAM-FA及PAMAM-FITC加入HELA细胞后相对于没有连接叶酸的桥接产物荧光显微镜在细胞内可观察到明显的荧光,并且FA可竞争性阻断。结论:由DNA桥接的肿瘤靶向组装型树状大分子具一定的可行性。     

pH敏感PAMAM树状大分子复合物胶束的制备及性质研究

目的利用聚乙二醇-聚磺胺二甲氧嘧啶(PEG-b-PSD)和PAMAM树状大分子制备具有pH敏感性的复合物胶束并对其制剂学性质进行研究。方法利用正负电荷的相互作用使PEG-b-PSD与PAMAM树状大分子在pH值为7.4条件下形成复合物胶束。考察正负电荷比、离子强度等对zeta电位、载药效果和粒径的影响。利用透射电镜观察制剂形态,考察制剂在不同pH缓冲液和血浆中的释放行为。结果采用正负电荷比为1∶1制备PAMAM/PEG-b-PSD复合物,制剂粒径为50 nm左右,形态为类球形,体外释放速率在pH值为6.5的PBS水溶液中要明显慢于在pH值为7.4的PBS水溶液和血浆中。结论 PEG-b-PSD与PAMAM利用正负电荷相互吸引的方式形成复合物,该复合物具有明显的pH值敏感性,适用于在低pH值部位定位释放药物。     

Determination of puerarin in rabbit aqueous humor by liquid chromatography tandem mass spectrometry using microdialysis sampling after topical administration of puerarin PAMAM dendrimer complex

To study pharmacokinetic properties of puerarin poly(amido amine) (PAMAM) dendrimer complex, a sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine puerarin in rabbit aqueous humor using microdialysis sampling. Astilbin was used as the internal standard. The linear range for puerarin was from 2 to 1000 ng/mL (r = 0.9986) based on 20 μL of aqueous humor. The coefficients of variations for intra-day and inter-day precisions were less than 10.0%, and the relative error of accuracy was within ±6.3%. The mean extraction recovery of puerarin varied from 80.4% to 85.5%. Microdialysis provides a complete concentration versus time profile. A significant difference was observed in main pharmacokinetic parameters of C_max, AUC and t_1/2 between puerarin solution and puerarin PAMAM dendrimer complex. Complex formation resulted in an obvious increase in bioavailability of puerarin after topical administration to rabbit according to the above LC-MS/MS assay method.     

植入用缓释MTX体外释放度测定方法

目的建立植入用缓释MTX体外释放方法.方法选择体外释放的重要影响因素,采用四因素三水平的正交试验,以3个综合考核指标进行优选.结果释放介质为偏碱性等渗液;介质浓度为25%;介质体积为200ml;搅拌转速为50 r·min-1.结论所定方法可用于植入用缓释MTX的体外释放测试.     

Developmental toxicity of low generation PAMAM dendrimers in zebrafish

Biological molecules and intracellular structures operate at the nanoscale; therefore, development of nanomedicines shows great promise for the treatment of disease by using targeted drug delivery and gene therapies. PAMAM dendrimers, which are highly branched polymers with low polydispersity and high functionality, provide an ideal architecture for construction of effective drug carriers, gene transfer devices and imaging of biological systems. For example, dendrimers bioconjugated with selective ligands such as Arg-Gly-Asp (RGD) would theoretically target cells that contain integrin receptors and show potential for use as drug delivery devices. While RGD-conjugated dendrimers are generally considered not to be cytotoxic, there currently exists little information on the risks that such materials pose to human health. In an effort to compliment and extend the knowledge gleaned from cell culture assays, we have used the zebrafish embryo as a rapid, medium throughput, cost-effective whole-animal model to provide a more comprehensive and predictive developmental toxicity screen for nanomaterials such as PAMAM dendrimers. Using the zebrafish embryo, we have assessed the developmental toxicity of low generation (G3.5 and G4) PAMAM dendrimers, as well as RGD-conjugated forms for comparison. Our results demonstrate that G4 dendrimers, which have amino functional groups, are toxic and attenuate growth and development of zebrafish embryos at sublethal concentrations; however, G3.5 dendrimers, with carboxylic acid terminal functional groups, are not toxic to zebrafish embryos. Furthermore, RGD-conjugated G4 dendrimers are less potent in causing embryo toxicity than G4 dendrimers. RGD-conjugated G3.5 dendrimers do not elicit toxicity at the highest concentrations tested and warrant further study for use as a drug delivery device.     

甲氨蝶呤缓释植入剂体外释放方法的研究

目的:建立甲氨蝶呤缓释植入剂体外释放方法。方法:选择影响甲氨蝶呤缓释植入剂体外释放的重要影响因素进行正交试验,优选释放条件。结果:最优参数如下:介质为偏碱性等渗液,介质浓度为25%,介质体积为200mL,搅拌转速为50r·min~(-1)。结论:所定方法可用于甲氨蝶呤植入剂的体外释放测试。     

溴新斯的明多囊脂质体的制备及其体外释放行为的初步研究

目的:制备溴新斯的明多囊脂质体(NB-MVL),并对其体外药物释放行为进行考察。方法:采用复乳法制备载药多囊脂质体,并用单因素试验优化处方,以动态透析技术与ƒ₂相似因子评价法对NB-MVL与溴新斯的明在体外不同释放介质中的释放行为进行研究,计算其药物释放度与相似因子。结果:NB-MVL的包封率受投料比、药物浓度等因素影响;NB-MVL在不同的释放介质中所获得的释放模型均符合Weibull模型,同时与溴新斯的明溶液在不同释放介质中所得累积释放曲线间的相似因子小于50。结论:本试验制备所得NB-MVL包封率较高,且体外药物释放行为与溴新斯的明存在显著性差异,表现出良好的缓释作用。     

First evidences of PAMAM dendrimer internalization in microorganisms of environmental relevance: A linkage with toxicity and oxidative stress

Abstract

This article reports novel results on the toxic mechanisms of action of amine- and hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers toward microorganisms of environmental relevance, namely a cyanobacterium of the genus Anabaena and the green alga Chlamydomonas reinhardtii. We used PAMAM ethylenediamine core dendrimers from generations G2 to G4, which displayed a positive charge, measured as ζ-potential, in culture media. All amine-terminated and most remarkably the G4 hydroxyl-terminated dendrimer inhibited the growth of both microorganisms. The effect on the growth of the green alga was significantly higher than that on the cyanobacterium. With concentrations expressed in terms of molarity, there was a clear relationship between dendrimer generation and toxicity, with higher toxicity for higher generation. Hormesis was observed for hydroxyl-terminated dendrimers at low concentrations. The cationic dendrimers and G4-OH significantly increased the formation of reactive oxygen species (ROS) in both organisms. ROS formation was not related with the chloroplast or photosynthetic membranes and photosystem II photochemistry was unaffected. Cell damage resulted in cytoplasm disorganization and cell deformities and was associated to an increase in ROS formation and lipid peroxidation in mitochondria in the green alga; cell wall and membrane disruption with apparent loss of cytoplasmic contents was found in the cyanobacterium. It was determined for the first time that cationic PAMAM dendrimers were quickly and largely internalized by both organisms. These results warn against the generalization of the use of dendrimers, which may pose significant risk for the environment and particularly for primary producers which are determinant for the health of natural ecosystems.