Torres-Martinez Zally, Pérez Daraishka, Torres Grace, Estrada Sthephanie, Correa Clarissa, Mederos Natasha, Velazquez Kimberly, Castillo Betzaida, Griebenow Kai, Delgado Yamixa
Chemistry Department, University of Puerto Rico, Rio Piedras Campus, San Juan 00925, Puerto Rico.
Neuroscience Department, Universidad Central del Caribe, Bayamon 00960, Puerto Rico.
BioTech (Basel). 2023 Jan 26;12(1):13. doi: 10.3390/biotech12010013.
Nanosized drug delivery systems (DDS) have been studied as a novel strategy against cancer due to their potential to simultaneously decrease drug inactivation and systemic toxicity and increase passive and/or active drug accumulation within the tumor(s). Triterpenes are plant-derived compounds with interesting therapeutic properties. Betulinic acid (BeA) is a pentacyclic triterpene that has great cytotoxic activity against different cancer types. Herein, we developed a nanosized protein-based DDS of bovine serum albumin (BSA) as the drug carrier combining two compounds, doxorubicin (Dox) and the triterpene BeA, using an oil-water-like micro-emulsion method. We used spectrophotometric assays to determine protein and drug concentrations in the DDS. The biophysical properties of these DDS were characterized using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy, confirming nanoparticle (NP) formation and drug loading into the protein structure, respectively. The encapsulation efficiency was 77% for Dox and 18% for BeA. More than 50% of both drugs were released within 24 h at pH 6.8, while less drug was released at pH 7.4 in this period. Co-incubation viability assays of Dox and BeA alone for 24 h demonstrated synergistic cytotoxic activity in the low μM range against non-small-cell lung carcinoma (NSCLC) A549 cells. Viability assays of the BSA-(Dox+BeA) DDS demonstrated a higher synergistic cytotoxic activity than the two drugs with no carrier. Moreover, confocal microscopy analysis confirmed the cellular internalization of the DDS and the accumulation of the Dox in the nucleus. We determined the mechanism of action of the BSA-(Dox+BeA) DDS, confirming S-phase cell cycle arrest, DNA damage, caspase cascade activation, and downregulation of epidermal growth factor receptor (EGFR) expression. This DDS has the potential to synergistically maximize the therapeutic effect of Dox and diminish chemoresistance induced by EGFR expression using a natural triterpene against NSCLC.
纳米级药物递送系统（DDS）因其具有同时降低药物失活和全身毒性以及增加药物在肿瘤内被动和/或主动蓄积的潜力，已作为一种抗癌新策略进行研究。三萜类化合物是具有有趣治疗特性的植物源化合物。桦木酸（BeA）是一种五环三萜，对不同癌症类型具有强大的细胞毒性活性。在此，我们使用类似油包水微乳液法，开发了一种基于纳米级蛋白质的牛血清白蛋白（BSA）药物递送系统，将两种化合物，即阿霉素（Dox）和三萜类化合物BeA结合在一起。我们使用分光光度法测定药物递送系统中的蛋白质和药物浓度。使用动态光散射（DLS）和圆二色性（CD）光谱对这些药物递送系统的生物物理性质进行了表征，分别证实了纳米颗粒（NP）的形成以及药物负载到蛋白质结构中。阿霉素的包封率为77%，桦木酸的包封率为18%。在pH 6.8时，两种药物在24小时内释放超过50%，而在此期间，在pH 7.4时释放的药物较少。单独的阿霉素和桦木酸共孵育24小时的活力测定表明，在低μM范围内对非小细胞肺癌（NSCLC）A549细胞具有协同细胞毒性活性。BSA-（阿霉素+桦木酸）药物递送系统的活力测定表明，其协同细胞毒性活性高于无载体的两种药物。此外，共聚焦显微镜分析证实了药物递送系统的细胞内化以及阿霉素在细胞核中的蓄积。我们确定了BSA-（阿霉素+桦木酸）药物递送系统的作用机制，证实了S期细胞周期停滞、DNA损伤、半胱天冬酶级联激活以及表皮生长因子受体（EGFR）表达下调。这种药物递送系统有可能通过使用天然三萜类化合物协同最大化阿霉素的治疗效果，并减少由EGFR表达诱导的化疗耐药性，用于治疗非小细胞肺癌。
