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).

Objective: The aim of this study was to optimize the formulation of a C60-modified self-microemulsifying drug delivery system loaded with triptolide (C60-SMEDDS/TP) and evaluate the cytoprotective effect of the C60-SMEDDS/TP on normal human cells. Results: The C60-SMEDDS/TP exhibited rapid emulsification, an optimal particle size distribution of 50 ± 0.19 nm (PDI 0.211 ± 0.049), and a near-neutral zeta potential of −1.60 mV. The release kinetics of TP from the C60-SMEDDS/TP exhibited a sustained release profile and followed pseudo-first-order release kinetics. Cellular proliferation and apoptosis analysis indicated that the C60-SMEDDS/TP (with a mass ratio of TP: DSPE-PEG-C60 = 1:10) exhibited lower toxicity towards L02 and GES-1 cells. This was demonstrated by a higher IC50 (40.88 nM on L02 cells and 17.22 nM on GES-1 cells) compared to free TP (21.3 nM and 11.1 nM), and a lower apoptosis rate (20.8% on L02 cells and 26.3% on GES-1 cells, respectively) compared to free TP (50.5% and 47.0%) at a concentration of 50 nM. In comparison to the free TP group, L02 cells and GES-1 cells exposed to the C60-SMEDDS/TP exhibited a significant decrease in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM). On the other hand, the C60-SMEDDS/TP demonstrated a similar inhibitory effect on BEL-7402 cells (IC50 = 28.9 nM) and HepG2 cells (IC50 = 107.6 nM), comparable to that of the free TP (27.2 nM and 90.4 nM). The C60-SMEDDS/TP group also exhibited a similar intracellular level of ROS and mitochondrial membrane potential compared to the SMEDDS/TP and free TP groups. Method: Fullerenol-Grafted Distearoyl Phosphatidylethanolamine-Polyethylene Glycol (DSPE-PEG-C60) was synthesized and applied in the self-microemulsifying drug delivery system. The C60-SMEDDS/TP was formulated using Cremophor EL, medium-chain triglycerides (MCT), PEG-400, and DSPE-PEG-C60, and loaded with triptolide (TP). The toxicity and bioactivity of the C60-SMEDDS/TP were assessed using normal human liver cell lines (L02 cells), normal human gastric mucosal epithelial cell lines (GES-1 cells), and liver cancer cell lines (BEL-7402 cells and HepG2 cells). The production of reactive oxygen species (ROS) after the C60-SMEDDS/TP treatment was assessed using 2′,7′-dichlorofluorescein diacetate (DCFDA) staining. The alterations in mitochondrial membrane potential (ΔψM) were assessed by measuring JC-1 fluorescence. Conclusions: The cytoprotection provided by the C60-SMEDDS/TP favored normal cells (L02 and GES-1) over tumor cells (BEL-7402 and HepG2 cells) in vitro. This suggests a promising approach for the safe and effective treatment of TP.