基于多功能聚电解质修饰的氧化石墨烯作为抗癌药物口服缓控释载体的制备及其胃肠道转运机制研究

The recent discovery of graphene has been accompanied by increasing research attention to explore this new material for drug delivery applications. Graphene, a single layer of sp2-hybridized carbon atoms arranged in a honeycomb two-dimensional (2-D) crystal lattice, has evoked enormous interest throughout the scientific community since its first appearance in 2004. Due to its unique structure and geometry, graphene possesses remarkable physical–chemical properties, large specific surface area and biocompatibility. GO consists of aromaticplanes and polar functional groups which consequently provide itwith an outstanding potential to adsorb aromatic compounds via p–p stacking and hydrogen bonding. These properties enable graphene to be considered as an ideal material for a broad range of applications, ranging from quantum physics, nanoelectronics, energy research, catalysis and engineering of nanocomposites and biomaterials. The pharmacokinetics and toxicology of a drug are closely associated with its administration routes. Although researchers have gained substantial understanding regarding the behaviors and biological effects of GO and functionalized GO after inhalation and intravenous injection into mice. Up to now, the in vivo biodistribution and toxicity of GO derivatives in animals via other different administration routes such as oral feeding remain unclear. Therefore, in this work, we want to systematically study the in vivo biodistribution and long-term toxicity of as-made GO and Polyelectrolyte (such as PAA, PAH) coated GO functionalized by different ways, after oral administration into healthy mice. In this study, a polyelectrolyte-coated multifunctional DDS of BAM (PAA-cys/PAH-BAM-GO) was prepared through layer by layer deposition of poly(acrylic acid) (PAA) and polyallyl amine hydrochloride (PAH) on BAM-loaded GO. In addition to optimization of process variables, extensive physicochemical characterization, pharmacokinetic, and pharmacodynamic profiles of PAA-cys/PAH-BAM-GO were evaluated in vitro and in vivo, respectively. After the evaluation of the drug loading capacity and pH sensitive release, the uptake of PAA-cys/PAH-BAM-GO and cellular localization were tested in Caco-2/HT29-MTX cells. We believe that our study would remarkably improve our understanding regarding the behaviors of functionalized graphene materials in animals.

石墨烯类材料是新出现的一种药物载体，具有价格低廉、易规模化生产和性质稳定等特点。研究发现石墨烯能通过p-p共轭吸附作用对水溶性和脂溶性药物都具有极高的装载率。其细胞毒性主要体现在由机械力损伤诱发的细胞凋亡。由于口服后石墨烯不能穿透肠道黏液层和上皮细胞，目前主要用于静脉注射药物载体使用。本研究提出将氧化石墨烯作为水溶性抗癌药物的口服载体，利用其不能有效透过肠黏膜的特点，采用巯基化聚电解质包裹延长载体在肠道内的滞留时间、同时改变上皮细胞通透性，提高药物在肠道内的释放及生物利用度，达到缓控释的效果。采用Caco-2/HT29-MTX共培养体系，模拟体外肠道细胞的黏液环境，在器官和细胞水平上探讨氧化石墨烯作为水溶性抗癌药物口服载体的肠内转运、分布、药物释放及肠道清除等特征；解释聚电解质在打开细胞紧密连接及提高水溶性药物肠道细胞摄取的作用机制。为将其作为蛋白多肽类水溶性大分子药物口服载体使用奠定基础

本课题采用改良后的Hummers法制得氧化石墨，相较于以前的制备GO的方法，改良之后不仅简化了实验操作步骤，也获得了安全性方面的提升。研究了其对阿霉素（DOX）和平阳霉素（PYM）的包载和体外释放行为。通过载药实验数据分析，GO对DOX的包封率达87.58%；对PYM的包封率达到82.05%。制剂组（GO-DOX和GO-PYM）在不同的pH条件下表现出不同的释放效果，两组的实验结果又表现了相同的释放趋势，且两组都于60 h时在pH 2.3的条件下达到最高的累计释放量。说明其释放行为表现出一定的pH依赖性，但是未表现出明显的缓释性。通过单因素考察法对聚丙烯酸（PAA）结合半胱氨酸（CYs）的制备工艺的最优方法进行了研究。通过Ellman法检测游离巯基含量，结果表明反应时加入的EDC含量、pH和温度等都会影响PAA与CYs的结合，影响高分子聚合物PAA-CYs上二硫键的含量。将PAA-CYs/ PAH-GO-DOX和PAA-CYs/ PAH-GO-PYM进行了透射电镜的测定，结果呈现出相对稳定的结构。粒径考察结果可知，两种药物的制剂组的粒径分别为700 nm与750 nm左右。通过Zeta电位的考察可以看出Zeta电位为-39.8mV与-34.1mV，一方面表明出制剂的稳定性好，另一方面也证明制剂组带负电荷，与之前的理论分析相符合；通过检测自由巯基可看出两种制剂组都不含有游离的巯基，可以从侧面的证实PAA与CYs结合完全。.最后将DOX组，GO-DOX组和PAA-CYs/PAH-GO-DOX组与PYM组、GO-PYM组和PAA-CYs/PAH-GO-PYM组分别进行了体外释放考察。结果可知，制剂组全都表现出了极高的耐强酸稳定性，证明通过两层聚电解质的包裹，可使药物在强酸性环境得到保护。在肠吸收实验中，PAA-CYs/PAH-GO-PYM制剂组相比于PYM组和GO-PYM组表现出了更好的肠道吸收效果。推测主要是由于其表面有PAA-CYs的包裹，使PCPGP与肠道内壁相粘附，所以达到了更好的肠吸收效果。PCPGP制剂组在药动学研究方面，T1/2延长了0.519 h，表现出一定的缓释效果。MTT实验结果表明，PCPGP制剂组对于癌细胞A549的致死率相比于PYM组降低了2.40%。说明在24h内，PCPGP制剂组在相同浓度24 h的时间内表现出了一定的缓释效果。