C60 Fullerene Nanocomplexes Boost Doxorubicin Potency Against Leukemia Cells In Vitro

Abstract

Traditional chemotherapy often fails due to severe side effects and the rapid emergence of multidrug resistance. We investigated C₆₀ fullerene as a nanoscale carrier for doxorubicin (Dox) to enhance drug delivery specifically to leukemic cells. Non‑covalent C₆₀‑Dox complexes (1:1 and 2:1 ratios) were characterized by UV/Vis, dynamic light scattering, and HPLC‑MS/MS, confirming a stable 140‑nm nanoparticle size suitable for biological use. In leukemic cell lines (CCRF‑CEM, Jurkat, THP‑1, Molt‑16), these complexes displayed up to 3.5‑fold greater cytotoxicity than free Dox at nanomolar concentrations, with intracellular accumulation evidence supporting enhanced delivery. These findings suggest C₆₀ fullerene as a promising nanocarrier to improve Dox efficacy against leukemia.

Introduction

Nanobiotechnology offers a pathway to more selective and potent anticancer therapies. C₆₀ fullerene, a spherical carbon nanostructure, exhibits unique physicochemical properties, intrinsic antioxidant activity, and proven drug‑loading capabilities. Doxorubicin, while effective, is limited by cardiotoxicity and oxidative stress. Combining Dox with C₆₀ leverages the fullerene’s antioxidant properties and drug‑delivery potential to mitigate side effects and improve tumor targeting. Prior studies have shown that non‑covalent C₆₀ complexes can enhance anticancer activity in vitro and in vivo. This study evaluates the physicochemical characteristics, cellular uptake, and cytotoxic potency of C₆₀‑Dox complexes against human leukemic cell lines.

Methods

Materials

Chemicals and cell lines were sourced from standard suppliers (Biochrom, Sigma‑Aldrich, Leibniz Institute DSMZ). Cell culture conditions adhered to ISO‑standard protocols.

Complex Preparation

Pristine C₆₀ aqueous colloids were prepared via ultrasound sonication, achieving 150 µg mL⁻¹ purity and 100 nm hydrodynamic diameter. Dox was dissolved at 150 µg mL⁻¹ and mixed with C₆₀ in 1:1 or 2:1 weight ratios, sonicated for 30 min, and stirred 24 h at room temperature. Excess free drug was removed by dialysis. Final complex concentrations were 75 µg mL⁻¹ (1:1) and 100/50 µg mL⁻¹ (2:1). Stability was confirmed over 6 months in saline.

Analytical Characterization

HPLC‑MS/MS (Shimadzu LCMS‑8040) quantified Dox release, using a C18 column and 80:20 acetonitrile:0.1% formic acid water mobile phase. UV/Vis absorption and fluorescence spectra were recorded with a Tecan Infinite M200 Pro. Dynamic light scattering (Malvern Zetasizer Nano S) measured particle size in PBS and RPMI‑1640 +10% FBS.

Cell Viability

MTT assays assessed viability of 10⁴ cells/well after 24, 48, and 72 h exposure to free Dox or C₆₀‑Dox complexes at 0.1–100 µM. IC₅₀ values were derived via nonlinear regression (GraphPad Prism 7).

Uptake Studies

Fluorescence microscopy (Keyence BZ‑9000) and flow cytometry (BD FACSJazz) quantified intracellular Dox after 1, 3, and 6 h exposure to 1 µM free or complexed drug in CCRF‑CEM cells.

Results

Complex Stability and Size

HPLC‑MS/MS chromatograms revealed distinct retention times for free Dox (11.66 min) versus complex‑bound Dox (9.44 min), confirming complex formation. UV/Vis spectra showed a 30% hypochromic shift in absorbance, while fluorescence quenching (~50% at 3 µM) indicated close π‑π stacking between Dox and C₆₀. DLS determined an average hydrodynamic diameter of ~140 nm, stable in PBS and after 72 h incubation in RPMI‑1640 +10% FBS.

Enhanced Cytotoxicity

All four leukemic lines exhibited nanomolar sensitivity to Dox, with IC₅₀ values ranging from 2 nM (Molt‑16) to 80 nM (CCRF‑CEM). C₆₀‑Dox complexes consistently lowered IC₅₀ values by up to 3.5‑fold compared to free drug, with the 1:1 complex outperforming the 2:1 ratio. The higher C₆₀ content in the 2:1 complex may confer antioxidant protection, mitigating Dox‑induced oxidative stress.

Increased Intracellular Uptake

Fluorescence microscopy and flow cytometry demonstrated that C₆₀‑Dox complexes entered cells more rapidly than free Dox, achieving a 1.5–2.2‑fold increase in intracellular fluorescence by 6 h. Dox localized to the nucleus in both formulations, as confirmed by colocalization with Hoechst 33342 staining.

Conclusion

C₆₀ fullerene forms stable, 140‑nm non‑covalent complexes with doxorubicin that markedly enhance its cytotoxicity against human leukemic cells. The nanocarrier facilitates faster cellular uptake without altering drug localization, achieving up to 3.5‑fold potency improvement at equivalent doses. These results support further development of C₆₀ nanocomplexes to lower effective Dox concentrations, potentially reducing cardiotoxicity and other adverse effects in clinical settings.