Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

OBJECTIVE

The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD.

METHOD

The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining.

RESULTS

The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 μM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 μM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group.

CONCLUSIONS

The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).