table1_In vitro Toxico-genomics of Etoposide Loaded Gelatin Nanoparticles and Its in-vivo Therapeutic Potential: Pharmacokinetics, Biodistribution and Tumor Regression in Ehrlich Ascites Carcinoma (EAC) Mice Model.pdf

Globally, breast cancer is the foremost cause of mortality among women detected with cancer, with 21% diagnosed in India alone. Etoposide loaded gelatin nanoparticles (EGNP) were prepared and its physical characterization (size:150nm±0.241; zeta potential −29.32 mV) was done along with in-vitro studies to assess biotoxicity, intracellular ROS, cell cycle arrest and death caused by EGNPs. We report the molecular pathways induced by EGNP in-vitro, pharmacokinetics, biodistribution and tumor regression in-vivo in Balb/c mice.Gene expression profiling of Bax, Bcl2, p53, Caspase-3, RIPK1, RIPK3 and ß-actin as internal control were done by RT-PCR wherein Etoposide and EGNP treated MCF-7 cells showed higher expressions of apoptotic genes-Bax, p53, caspase-3, lower expression of anti-apoptotic gene-Bcl2 when compared to control. Enhanced expression of necroptosis-RIPK1 were observed, while RIPK3 was insignificant. Since, RIPK1 regulates necroptosis and apoptosis, expression of apoptotic markers confirmed apoptotic molecular mechanisms. Negligible hemolysis of Gelatin nanoparticles (GNP), and EGNP at selected dosages confirmed biocompatibility. In vivo pharmacokinetics and biodistribution were done by 99Tc-labelled nanoparticles indicating increased circulation of EGNPs, allowing accumulation at the tumor site by Enhanced permeability and retention (EPR) phenomena. Tumor regression indicates the efficacy of EGNP by reducing the tumor burden when compared to void GNP and Etop per se, resulting in increased life span. High biocompatibility and bio-efficacy of EGNPs prove their therapeutic potential in cancer treatment.

在全球范围内，乳腺癌是女性癌症患者死亡的首要诱因，仅印度一国的确诊病例便占全球总量的21%。本研究制备了依托泊苷（Etoposide）负载明胶纳米颗粒（EGNP），对其进行了物理表征：粒径为150nm±0.241，zeta电位为-29.32 mV；同时开展体外实验，以评估EGNP的生物毒性、细胞内活性氧（Reactive Oxygen Species, ROS）水平、细胞周期阻滞效应及诱导的细胞死亡类型。本研究还报道了EGNP在体外诱导的分子通路，以及Balb/c小鼠体内的药代动力学、生物分布与肿瘤消退情况。采用逆转录聚合酶链式反应（Reverse Transcription Polymerase Chain Reaction, RT-PCR）对作为内参的β-肌动蛋白（ß-actin）以及Bax、Bcl2、p53、半胱天冬酶-3（Caspase-3）、受体相互作用蛋白激酶1（RIPK1）、受体相互作用蛋白激酶3（RIPK3）进行基因表达谱分析，结果显示：与对照组相比，经依托泊苷和EGNP处理的MCF-7细胞中，促凋亡基因Bax、p53、Caspase-3的表达水平显著上调，而抗凋亡基因Bcl2的表达水平显著下调。研究观察到坏死性凋亡（Necroptosis）相关的RIPK1表达上调，而RIPK3的表达无显著差异。鉴于RIPK1可同时调控坏死性凋亡与凋亡过程，上述凋亡标志物的表达结果证实了EGNP诱导的凋亡分子机制。在选定剂量下，明胶纳米颗粒（GNP）与EGNP均未引发显著溶血反应，证实了其良好的生物相容性。采用锝-99（99Tc）标记的纳米颗粒开展体内药代动力学与生物分布实验，结果显示EGNP的循环时间延长，可通过增强渗透滞留（Enhanced Permeability and Retention, EPR）效应在肿瘤部位富集。与空白GNP组及单纯依托泊苷组相比，EGNP可通过降低肿瘤负荷实现肿瘤消退，进而延长小鼠生存期，证实了其抗肿瘤疗效。EGNP优异的生物相容性与生物活性证实了其在癌症治疗中的应用潜力。