Abstract：
The high incidence rate coupled with significant mortality makes colorectal cancer one of the most prevalent and devastating cancers worldwide. Research is currently underway to explore new forms of treatment that could potentially maximize treatment outcomes while minimizing the side effects associated with conventional chemotherapy. Metformin, a natural biguanide drug, has anti-cancer properties that can inhibit the growth and proliferation of cancer cells. However, due to its short half-life and low bioavailability, the efficacy of Metf as an anti-cancer agent is limited. The purpose of this research is to assess the potency of PEGylated niosomes as a nano-delivery system for Metf, with the aim of increasing its anti-cancer effects on CaCo2 colorectal cancer cells through the effect on the expression of genes, including GPR75, hTERT, Bax, Bcl2, and Cyclin D1. Metf-loaded niosomal NPs (N-Metf) were synthesized using the thin-film hydration method and then characterized using SEM, FTIR, AFM, and DLS techniques. The release pattern of the drug from the nanoparticles (NPS) was determined using the dialysis membrane method. Furthermore, the cytotoxic effect of the metformin-loaded PEGylated niosome on the CaCo2 cell line was evaluated by the MTT test. Additionally, an apoptosis assay was conducted to assess the effect of free Metf and Metf-loaded NPS on the programmed death of the CaCo2 cells, and the impact on the cell cycle was studied through a cell cycle test. Finally, the expression levels of hTERT, Cyclin D1, BCL2, GPR75, and BAX genes were assessed in the presence of free Metf and Metf-loaded NPs by RT-PCR. Characterization experiments showed successful loading of metformin into PEGylated niosomes. The results of cytotoxicity evaluation showed that Metf-NPs had more cytotoxicity than free Metf in a dose-dependent manner. Furthermore, nuclear fragmentation and the percentage of apoptotic cells induced by Metf-NPs were significantly higher than those induced by free Metf. Additionally, Metf-NPs were found to induce more cell cycle arrest at the sub-G1 checkpoint than free Metf did. Compared with Metf-treated cells, the mRNA expression levels of GPR75, Cyclin D1, and hTERT were significantly changed in cells treated with Metf-NPs. Ultimately, it is hypothesized the nano-encapsulation of Metf into PEGylated niosomal NPs could be a worthwhile drug delivery system to enhance its effectiveness in treating colorectal cancer cells.
摘要：
高发病率加上显著的死亡率使结直肠癌成为全球最普遍和最具破坏性的癌症之一。目前正在进行研究，以探索新的治疗形式，这些治疗形式可能会最大程度地提高治疗效果，同时最大程度地减少与常规化疗相关的副作用。二甲双胍,一种天然的双胍药物，具有抗癌特性，可以抑制癌细胞的生长和增殖。然而,由于其半衰期短和生物利用度低，Metf作为抗癌剂的功效是有限的。这项研究的目的是评估PEG化的脂质体作为Metf纳米递送系统的效力，目的是通过对基因表达的影响来增加其对CaCo2结直肠癌细胞的抗癌作用，包括GPR75，hTERT，Bax,Bcl2和细胞周期蛋白D1。采用薄膜水化法合成了负载Metf的NPs(N-Metf),FTIR,AFM,DLS技术。使用透析膜方法确定药物从纳米颗粒（NPS）的释放模式。此外，通过MTT试验评估了装载二甲双胍的聚乙二醇化的脂质体对CaCo2细胞系的细胞毒性作用。此外,进行了细胞凋亡试验，以评估游离的Metf和Metf负载的NPS对CaCo2细胞程序性死亡的影响，并通过细胞周期试验研究了对细胞周期的影响。最后,hTERT的表达水平，通过RT-PCR在游离Metf和Metf负载的NP存在下评估细胞周期蛋白D1、BCL2、GPR75和BAX基因。表征实验显示二甲双胍成功加载到PEG化的囊泡中。细胞毒性评价结果显示,Metf-NP以剂量依赖性方式比游离Metf具有更大的细胞毒性。此外，Metf-NP诱导的细胞核碎裂和凋亡细胞百分比显著高于游离Metf诱导的细胞核碎裂和凋亡细胞百分比。此外,发现Metf-NP在sub-G1检查点比游离Metf诱导更多的细胞周期停滞。与Metf处理的细胞相比，在Metf-NP处理的细胞中，GPR75，CyclinD1和hTERT的mRNA表达水平显着变化。最终,假设将Metf纳米封装到PEG化的脂质体NP中可能是一种有价值的药物递送系统，以增强其治疗结直肠癌细胞的有效性。
