Study of autophagy in HepG2 cancer cells after MicroRNA-34a and methyl adenin treatment loaded with cellulose lysine particles and depicting bioinformatic’s map of related genes

Abstract

Introduction: Liver cancer is one of the most dangerous types of cancer today and includes two types: primary and secondary. Primary cancer starts in the liver tissue itself, while secondary cancer has metastasized to the liver from another organ. Hepatocellular carcinoma (HCC) is known as the most common type of cancer in adults and is currently one of the leading causes of death among people with cirrhosis caused by hepatitis B or C. Current treatments include surgery and chemotherapy, but these methods have many side effects and may reduce survival rates. Therefore, researchers are trying to find alternative treatments with fewer side effects and greater efficacy. One promising option in this regard is the use of nanocarriers. Cellulose, a natural polymer material, has attracted much attention in recent years in the field of drug delivery due to its properties such as biocompatibility, biodegradability, and easy availability. The aim of this study is to synthesize nanoparticles based on microcrystalline cellulose derivatives that have the ability to load polyplexes containing miR-34a and 3MA and simultaneously affect cancer cells in vitro. Method: Microcrystalline cellulose is oxidized and cellulose dialdehyde is synthesized. The synthesized cellulose dialdehyde in combination with oleylamine provides a dual-functional cellulose-based nanoparticle with aldehyde and amine functional groups. The poly-L-lysine polymer forms a polyplex through electrostatic bonding with miR-34a. Dual-functional cellulose nanoparticles simultaneously load miR-34a and 3MA through self-assembly. The physicochemical properties of this nanoparticle loaded with therapeutic agents were evaluated. In addition, MTT, cell uptake, apoptosis, cell cycle, Western blot and Real-time PCR tests were performed to investigate the effect of this nanoparticle on the HepG2 cell line. Findings: In the present study, a modified dialdehyde cellulose (MDAC) nanocarrier that responds to lysosomal pH was designed to simultaneously load polyplexes of hsa-miR-34a and 3MA and evaluate its antitumor effect against HCC cells. The polyplexes containing hsa-miR-34a and poly L-lysine (PLL) with an optimal N/P ratio of 1:20 exhibited a zeta potential of +9.28. These polycations significantly modulated the surface charge of 3MA MDAC for optimal cell membrane transport and significantly increased their stability. The PLLmiR34a/3MA MDAC nanoparticles had a loading efficiency of about 99.7% for miR-34a and 35% for 3MA. In accordance with pH dependence, PLLmiR34a/3MA MDAC polyplex nanoparticles inhibited the expression ofautophagy genes (p<0.05), prevented the formation of autophagosomal vacuoles and were highly effective in reducing cell survival, anti-migratory effects (>100% scratch) and scratch blockage (early + late apoptosis = 67.15%) in HepG2 cells. Our cellulose-based nanocarrier may show the potential to enhance the combination therapies for future clinical translation in therapeutic efficacy of HCC. Conclusion: This is the first study in which 3MA and miR-34a were coloaded onto dual-drug cellulose nanoparticles. The cellulose-based nanocarrier enhanced the anticancer efficacy of the loaded therapeutic agents compared to the free state. Keywords: Hepatocellular carcinoma, cellulose-based nanocarrier, 3MA, miR-34a, autophagy, apoptosis