Preparation, Characterization And In Vitro Cytotoxicity Study Of Adriamycin-loaded PLGA Nanoparticles On Hepatocellular Carcinoma Cells

HCC is a common malignant tumor with a high mortality rate, but the main treatment in advanced HCC patients, systemic chemotherapy, has a poor efficacy due to the low selectivity and toxicity of chemotherapy drugs. Drug-loaded nanoparticle delivery system is very popular in transporting drug-targeting agents in recent years. The system is prepared by incorporating drugs with a variety of non-toxic carriers, producing nano-level microspheres with targeted delivery. With controlled drug release, enhanced efficacy, reduced side effects and low toxicity characteristics, the system is expected to be a efficient targeted anti-cancer chemotherapeutic agents. This study includes the preparation, characterization and in vitro cytotoxicity evaluation of adriamycin-loaded nanoparticles. More importantly, the purpose of this study is to provide some experimental basis for the development of drug-loaded nanoparticle delivery system. ObjectiveThis study is aimed to prepare and characterize adriamycin-loaded PLGA copolymer nanoparticles(ADM-PLGA-NP) and investigate the cytotoxicity of released adriamycin on human hepatocellular carcinoma cell line HepG2.Methods1. PLGA nanoparticles containing adriamycin were formulated by w/o/w improved emulsification-solvent evaporation method. The morphology was observed by scanning electron microscopy (SEM) and the size distribution was investigated by laser diffraction analyzer. The encapsulating efficiency of ADM was determined by ultraviolet spectrophotometry. ADM-PLGA-NP was put in a dialysis bag to observe the releasing characteristics of ADM from ADM-PLGA nanoparticles in vitro.2. Hepatocellular carcinoma cell line HepG2 was cultured with ADM-PLGA nanoparticles of different concentrations (the released drug doses were calculated according to the release curve in vitro) and the original ADM drug for a certain amount of time. Then, the morphology of HepG2 cells were photographed by using phase-contrast microscope. MTT assay was used to observe the cell growth inhibition rate. The cellular uptake of nanoparticles was evaluated by fluorescence microscopy. Flow cytometry was employed to quantify red fluorescence intensity in HepG2 cells.Results1. The prepared ADM-PLGA nanoparticles were of spherical or elliptical shape with average size of 237±12.7nm. The loading efficiency and entrapment efficiency were about 1.42±0.67% and 23.82±8.34%, respectively. The releasing test in vitro manifested that over 85 percent of ADM encapsulated in ADM-PLGA nanoparticles was sustained released in about 5 days, with a burst release in the first 12 hours. 2. Co-culture of HepG2 cells with ADM-PLGA-NPs showed that the cells were extremely spreaded with weakening cell adhesion and cytoplasmic condensation. MTT test showed that the average cell growth inhibition rate of blank nanoparticles was 5.98±2.96%; while the cell growth inhibition rate of ADM-PLGA-NP was higher than that of original drug (p <0.05). The cellular uptake of nanoparticles was observed by fluorescence microscopy. Flow cytometry illustrated that red fluorescence intensity in HepG2 cells incubated with ADM-PLGA-NP was stronger than that of incubated with original drug.ConclusionsThe ADM-loaded PLGA nanoparticles formulated by w/o/w emulsification solvent evaporation method had satisfactory characterization with sustained release of ADM drugs. ADM-PLGA-NP can be swallowed by HepG2 cells and showed more significant effects in inhibiting the growth of HepG2 cells than the original ADM drug did. ADM-PLGA-NPs are expected to become a new targeting chemotherapy agents.