Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab, India.
James Graham Brown Cancer Centre, University of Louisville, Louisville, Kentucky, USA.
Pharm Res. 2017 Nov;34(11):2295-2311. doi: 10.1007/s11095-017-2238-8. Epub 2017 Aug 9.
The present study reports a novel conjugate of gemcitabine (GEM) with bovine serum albumin (BSA) and thereof nanoparticles (GEM-BSA NPs) to potentiate the therapeutic efficacy by altering physicochemical properties, improving cellular uptake and stability of GEM.
The synthesized GEM-BSA conjugate was extensively characterized by NMR, FTIR, MALDI-TOF and elemental analysis. Conjugation mediated changes in structural conformation and physicochemical properties were analysed by fluorescence, Raman and CD spectroscopy, DSC and contact angle analysis. Further, BSA nanoparticles were developed from BSA-GEM conjugate and extensively evaluated against in-vitro pancreatic cancer cell lines to explore cellular uptake pathways and therapeutic efficacy.
Various characterization techniques confirmed covalent conjugation of GEM with BSA. GEM-BSA conjugate was then transformed into NPs via high pressure homogenization technique with particle size 147.2 ± 7.3, PDI 0.16 ± 0.06 and ZP -19.2 ± 1.4. The morphological analysis by SEM and AFM revealed the formation of smooth surface spherical nanoparticles. Cellular uptake studies in MIA PaCa-2 (GEM sensitive) and PANC-1 (GEM resistant) pancreatic cell lines confirmed energy dependent clathrin internalization/endocytosis as a primary mechanism of NPs uptake. In-vitro cytotoxicity studies confirmed the hNTs independent transport of GEM in MIA PaCa-2 and PANC-1 cells. Moreover, DNA damage and annexin-V assay revealed significantly higher apoptosis level in case of cells treated with GEM-BSA NPs as compared to free GEM.
GEM-BSA NPs were found to potentiate the therapeutic efficacy by altering physicochemical properties, improving cellular uptake and stability of GEM and thus demonstrated promising therapeutic potential over free drug. Graphical Abstract ᅟ.
本研究报告了一种新型吉西他滨（GEM）与牛血清白蛋白（BSA）的缀合物及其纳米颗粒（GEM-BSA NPs），通过改变理化性质、提高 GEM 的细胞摄取和稳定性来增强治疗效果。
通过 NMR、FTIR、MALDI-TOF 和元素分析对合成的 GEM-BSA 缀合物进行了广泛的表征。通过荧光、拉曼和 CD 光谱、差示扫描量热法和接触角分析分析了缀合介导的结构构象和理化性质的变化。进一步，从 BSA-GEM 缀合物中开发出 BSA 纳米颗粒，并对其进行了广泛的评估，以研究其对胰腺癌细胞系的细胞摄取途径和治疗效果。
各种表征技术证实了 GEM 与 BSA 的共价缀合。然后，通过高压匀质技术将 GEM-BSA 缀合物转化为 NPs，粒径为 147.2±7.3nm，PDI 为 0.16±0.06，Zeta 电位为-19.2±1.4mV。SEM 和 AFM 的形态分析表明形成了光滑表面的球形纳米颗粒。在 MIA PaCa-2（GEM 敏感）和 PANC-1（GEM 耐药）胰腺细胞系中的细胞摄取研究证实，能量依赖的网格蛋白内吞/内吞作用是 NPs 摄取的主要机制。体外细胞毒性研究证实，GEM 在 MIA PaCa-2 和 PANC-1 细胞中的转运不依赖于 hNTs。此外，与游离 GEM 相比，用 GEM-BSA NPs 处理的细胞中 DNA 损伤和 Annexin-V 检测显示出更高的凋亡水平。
GEM-BSA NPs 通过改变理化性质、提高 GEM 的细胞摄取和稳定性来增强治疗效果，因此与游离药物相比显示出有希望的治疗潜力。
