Nazemiyeh Amirreza, Dadashi Hamed, Mashinchian Milad, Karimian-Shaddel Alireza, Mohabbat Aria, Eskandani Morteza, Vandghanooni Somayeh
Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran.
Hematology and Oncology Research Center, Tabriz university of Medical Sciences, Tabriz, Iran.
Sci Rep. 2025 Jul 2;15(1):22657. doi: 10.1038/s41598-025-07996-2.
In recent years the efforts for devising effective methods to confront cancer have grasped significant attention. Since conventional methods have failed to fulfill the favorable therapeutic outcome, efforts have been made to develop novel methods. Currently, incorporating drugs in nanocarriers are considered as an effective method. Herein, we prepared chitosan nanoparticles (Cs NPs) loaded with doxorubicin (DOX) and metformin (Met) and evaluated their physicochemical and anticancer in vitro properties. The drug loaded Cs NPs were prepared using the ionic gelation method. The size and surface charge of the Cs NPs were evaluated with DLS. Further characterization was made with SEM imaging. Drug loading was confirmed with FTIR analysis and assessed with calculating the UV absorbance of the unloaded drug. Cs NPs stability was also examined with DLS and XRD results. Drug release was investigated ultimately using semi-permeable membrane. Initially, the biosafety of the blank Cs NPs was assessed on A549 and NIH-3 cells with MTT assay. Flowcytometry was used to test the NPs uptake by A549 cells. Similarly, the cytotoxic effect of Met and DOX Cs NPs was explored. The results were analyzed with CompuSyn software and the highest synergistic dose combination (Fa50) was obtained. Using Fa50 concentrations, apoptosis, scratch, 3D cultures, anti-angiogenesis, and western blot assays were performed. The size of DOX-Cs NPs and Met-Cs NPs were 51.20 nm and 48.20 nm, and zeta potentials were 23.26 mV and 25.05 mV, respectively. The EE were 59.03% for DOX and 86.39% for Met. Drug release investigation demonstrated a pH-independent release for DOX, achieving 80% release, while Met exhibited a pH-dependent release, with 70% release. Synergistic CI value of 0.91 was achieved in the toxicity assay and nanoparticulation lowered the IC of both drugs. An increase in early (35%) and late (25%) apoptosis with the combination therapy compared to controls (15% and 10%) was achieved. 3D spheroid models demonstrated that the treatment of A549 spheroids with DOX-Met-CS NPs inhibited tumor growth by 60% compared to untreated controls. Western blot analyses revealed downregulation of oncogenes ERBB2 and PI3K in response to the combination treatment. In conclusion, it was found that the combination of DOX/Met-Cs NPs significantly inhibits A549 cells growth, highlighting the proposed approach as an efficient treatment strategy against lung cancer disease.
近年来，致力于设计有效对抗癌症方法的努力备受关注。由于传统方法未能实现良好的治疗效果，人们已着手开发新方法。目前，将药物载入纳米载体被视为一种有效方法。在此，我们制备了负载阿霉素（DOX）和二甲双胍（Met）的壳聚糖纳米颗粒（Cs NPs），并评估了它们的理化性质和体外抗癌特性。采用离子凝胶法制备载药Cs NPs。用动态光散射（DLS）评估Cs NPs的尺寸和表面电荷。通过扫描电子显微镜（SEM）成像进行进一步表征。用傅里叶变换红外光谱（FTIR）分析确认载药情况，并通过计算未载药的紫外吸光度进行评估。还用DLS和X射线衍射（XRD）结果检测Cs NPs的稳定性。最终使用半透膜研究药物释放。最初，用MTT法在A549和NIH - 3细胞上评估空白Cs NPs的生物安全性。用流式细胞术检测A549细胞对纳米颗粒的摄取。同样，探究了Met和DOX Cs NPs的细胞毒性作用。用CompuSyn软件分析结果，获得最高协同剂量组合（Fa50）。使用Fa50浓度进行凋亡、划痕、三维培养、抗血管生成和蛋白质免疫印迹分析。DOX - Cs NPs和Met - Cs NPs的尺寸分别为51.20 nm和48.20 nm，zeta电位分别为23.26 mV和25.05 mV。DOX的包封率为59.03%，Met的包封率为86.39%。药物释放研究表明DOX的释放与pH无关，释放率达80%，而Met表现出pH依赖性释放，释放率为70%。在毒性试验中协同作用指数（CI）值为0.91，纳米颗粒化降低了两种药物的半数抑制浓度（IC）。与对照组（15%和10%）相比，联合治疗使早期（35%）和晚期（25%）凋亡增加。三维球体模型表明，用DOX - Met - CS NPs处理A549球体与未处理的对照组相比，肿瘤生长抑制了60%。蛋白质免疫印迹分析显示，联合治疗后癌基因ERBB2和PI3K下调。总之，发现DOX/Met - Cs NPs联合显著抑制A549细胞生长，突出了所提出的方法作为一种对抗肺癌疾病的有效治疗策略。
