Institute of Life Sciences, Nalco Square, Bhubaneswar, 751023, India.
Nanomedicine (Lond). 2011 Apr;6(3):489-507. doi: 10.2217/nnm.10.102.
The objective of the present study is to prepare and characterize nutlin-3a loaded polymeric poly(lactide-co-glycolide) nanoparticles (NPs) surface functionalized with transferrin ligand, to deliver the encapsulated drug in a targeted manner to its site of action and to evaluate the efficacy of the nanoformulation in terms of its cellular uptake, cell cytotoxicity, cell cycle arrest, apoptosis and activation of p53 pathway at molecular level in MCF-7 breast cancer cell line.
Nutlin-3a loaded poly(lactide-co-glycolide) NPs were prepared following the single oil-in-water emulsion method. Physicochemical characterization of the formulation included size and surface charge measurement, transmission electron microscopy characterization, study of surface morphology using scanning electron microscopy, Fourier-transform infrared spectral analysis and in vitro release kinetics studies. Furthermore, targeting ability of the conjugated system was assessed by cellular uptake and cell cytotoxicity studies in an in vitro cell model. Molecular basis of nutlin-3a-mediated p53 activation pathway was investigated by western blot analysis. Inhibition of cell cycle progression and apoptosis was evaluated by flow cytometry.
Physiochemical characterization of the formulations revealed that nutlin-3a was efficiently encapsulated in the nanoparticulate system, reaching an encapsulation efficiency of approximately 80% with size of approximately 220 nm and negative zeta potential of approximately -10.4 mV. Higher cellular uptake efficiency of the conjugated system proved the effectiveness of targeted therapy. IC(50) values, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, showed superior antiproliferative activity of transferrin-conjugated NPs over unconjugated NPs and native nutlin-3a, owing to enhanced cellular uptake by cancer cells. At the molecular level the conjugated system showed enhanced activation of p53 pathway in comparison to native drug as evident from western blot analysis. Augmented cell cycle arrest and apoptosis was exhibited by the conjugated system. Thus, our results suggest that transferrin-conjugated nutlin-3a loaded NPs could be a potential drug carrier system for targeted delivery of potent anticancer drug nutlin-3a for breast cancer therapy.
本研究的目的是制备并表征载有 nutlin-3a 的聚合物聚（乳酸-共-乙醇酸）纳米粒（NPs），该纳米粒表面用转铁蛋白配体功能化，以靶向方式将包封的药物递送至作用部位，并评估纳米制剂在 MCF-7 乳腺癌细胞系中的细胞摄取、细胞毒性、细胞周期停滞、细胞凋亡和激活 p53 通路的分子水平方面的疗效。
采用单油包水乳液法制备载有 nutlin-3a 的聚（乳酸-共-乙醇酸） NPs。制剂的理化特性包括粒径和表面电荷测量、透射电子显微镜表征、扫描电子显微镜研究表面形态、傅里叶变换红外光谱分析和体外释放动力学研究。此外，通过体外细胞模型中的细胞摄取和细胞毒性研究评估了共轭系统的靶向能力。通过 Western blot 分析研究了 nutlin-3a 介导的 p53 激活通路的分子基础。通过流式细胞术评估了细胞周期进展和凋亡的抑制。
制剂的理化特性研究表明，nutlin-3a 被有效地包封在纳米颗粒系统中，包封效率约为 80%，粒径约为 220nm，zeta 电位约为-10.4mV。共轭系统的更高细胞摄取效率证明了靶向治疗的有效性。3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐法测定的 IC50 值表明，与未共轭 NPs 和天然 nutlin-3a 相比，转铁蛋白共轭 NPs 具有更好的抗增殖活性，这归因于癌细胞对药物的摄取增强。在分子水平上，与天然药物相比，Western blot 分析显示共轭系统对 p53 通路的激活增强。共轭系统表现出增强的细胞周期停滞和凋亡。因此，我们的结果表明，转铁蛋白共轭 nutlin-3a 载药 NPs 可能是一种有前途的药物载体系统，可用于靶向递送有效的抗癌药物 nutlin-3a 进行乳腺癌治疗。
