Tadini Maraine Catarina, de Freitas Pinheiro Ana Maria, Carrão Daniel Blascke, Aguillera Forte Ana Luiza Scarano, Nikolaou Sofia, de Oliveira Anderson R M, Berretta Andresa Aparecida, Marquele-Oliveira Franciane
Laboratory of Research, Development and Innovation, ELEVE Research and Development, Ribeirão Preto, SP, Brazil.
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil.
J Pharm Biomed Anal. 2017 Oct 25;145:576-585. doi: 10.1016/j.jpba.2017.06.034. Epub 2017 Jul 8.
Drug Delivery Systems (DDS) of known drugs are prominent candidates towards new and more-effective treatments of various infectious diseases as they may increase drug bioavailability, control drug delivery and target the site of action. In this sense, the encapsulation of Amphotericin B (AmB) in Nanostructured Lipid Carriers (NLCs) designed with pH-sensible phospholipids to target infectious tissues was proposed and suitable analytical methods were validated, as well as, proper nanoparticle characterization were conducted. Characterization assays by Dinamic Light Scattering (DLS) and Atomic Force Microscopy demonstrated spherical particles with nanometric size 268.0±11.8nm and Zeta Potential -42.5±1.5mV suggestive of important stability. DSC/TGA and FT-IR assessments suggested mechanical encapsulation of AmB. The AmB aggregation study indicated that the encapsulation provided AmB at the lowest cytotoxic form, polyaggregate. Analytical methods were developed and validated according to regulatory agencies in order to fast and assertively determine AmB in nanoparticle suspension and, in Drug Encapsulation Efficiency (EE%), release and stability studies. The quantification method for AmB in NLC suspension presented linearity in 5.05-60.60μgmL range (y=0.07659x+0.05344) and for AmB in receptor solution presented linearity in 0.15-10.00μgmL range (y=54609x+263.1), both with r≥0.999. EE% was approximately 100% and according to the release results, at pH 7.4, a sustained controlled profile was observed for up 46h. In the meantime, a micellar AmB solution demonstrated an instability pattern after 7h of contact with the medium. Degradation and release studies under acid conditions (infectious condition) firstly depicted a prominent degradation of AmB (raw-material), with 20.3±3.5% at the first hour, reaching 43.3±7.0% after 7h of study. Next, particles faster disruption in acid environment was evidenced by measuring the NLC size variation by DLS and by the loss of the bluish sheen, characteristic of the nanostructured system macroscopically observed. Finally, safety studies depicted that NLC-AmB presented reduced toxicity in fibroblast cells, corroborating with AmB aggregated form study. Therefore, an innovative AmB formulation was fully characterized and it is a new proposal for in vivo investigations.
已知药物的给药系统（DDS）是各种传染病新的、更有效治疗方法的突出候选者，因为它们可以提高药物生物利用度、控制药物递送并靶向作用部位。从这个意义上讲，提出了将两性霉素B（AmB）包裹在由pH敏感磷脂设计的纳米结构脂质载体（NLC）中以靶向感染组织，并验证了合适的分析方法，同时进行了适当的纳米颗粒表征。动态光散射（DLS）和原子力显微镜表征分析表明，纳米颗粒呈球形，尺寸为268.0±11.8nm，zeta电位为-42.5±1.5mV，表明具有重要的稳定性。差示扫描量热法/热重分析法（DSC/TGA）和傅里叶变换红外光谱（FT-IR）评估表明AmB为机械包裹。AmB聚集研究表明，包裹使AmB呈细胞毒性最低的多聚集体形式。根据监管机构的要求开发并验证了分析方法，以便快速、准确地测定纳米颗粒悬浮液中的AmB以及进行药物包封率（EE%）、释放和稳定性研究。NLC悬浮液中AmB的定量方法在5.05-60.60μg/mL范围内呈线性（y=0.07659x+0.05344），受体溶液中AmB的定量方法在0.15-10.00μg/mL范围内呈线性（y=54609x+263.1），两者的r均≥0.999。EE%约为100%，根据释放结果，在pH 7.4时，观察到长达46小时的持续控释曲线。同时，胶束状AmB溶液与介质接触7小时后表现出不稳定模式。酸性条件（感染条件）下的降解和释放研究首先表明AmB（原料药）有显著降解，第1小时降解率为20.3±3.5%，研究7小时后达到43.3±7.0%。接下来，通过DLS测量NLC尺寸变化以及宏观观察到的纳米结构系统特有的蓝色光泽消失，证明了颗粒在酸性环境中更快地破裂。最后，安全性研究表明NLC-AmB在成纤维细胞中的毒性降低，这与AmB聚集形式研究结果一致。因此，一种创新的AmB制剂得到了全面表征，是体内研究的新方案。
