Nanocomplex of Berberine with C60 Fullerene Is a Potent Suppressor of Lewis Lung Carcinoma Cells Invasion In Vitro and Metastatic Activity In Vivo.

Effective targeting of metastasis is considered the main problem in cancer therapy. The development of herbal alkaloid Berberine (Ber)-based anticancer drugs is limited due to Ber' low effective concentration, poor membrane permeability, and short plasma half-life. To overcome these limitations, we used Ber noncovalently bound to C60 fullerene (C60). The complexation between C60 and Ber molecules was evidenced with computer simulation. The aim of the present study was to estimate the effect of the free Ber and C60-Ber nanocomplex in a low Ber equivalent concentration on Lewis lung carcinoma cells (LLC) invasion potential, expression of epithelial-to-mesenchymal transition (EMT) markers in vitro, and the ability of cancer cells to form distant lung metastases in vivo in a mice model of LLC. It was shown that in contrast to free Ber its nanocomplex with C60 demonstrated significantly higher efficiency to suppress invasion potential, to downregulate the level of EMT-inducing transcription factors SNAI1, ZEB1, and TWIST1, to unblock expression of epithelial marker E-cadherin, and to repress cancer stem cells-like markers. More importantly, a relatively low dose of C60-Ber nanocomplex was able to suppress lung metastasis in vivo. These findings indicated that сomplexation of natural alkaloid Ber with C60 can be used as an additional therapeutic strategy against aggressive lung cancer.

Mode of photoexcited C60 fullerene involvement in potentiating cisplatin toxicity against drug-resistant L1210 cells.

Introduction: C60 fullerene has received great attention as a candidate for biomedical applications. Due to unique structure and properties, C60 fullerene nanoparticles are supposed to be useful in drug delivery, photodynamic therapy (PDT) of cancer, and reversion of tumor cells' multidrug resistance. The aim of this study was to elucidate the possible molecular mechanisms involved in photoexcited C60 fullerene-dependent enhancement of cisplatin toxicity against leukemic cells resistant to cisplatin. Methods: Stable homogeneous pristine C60 fullerene aqueous colloid solution (10-4 М, purity 99.5%) was used in the study. The photoactivation of C60 fullerene accumulated by L1210R cells was done by irradiation in microplates with light-emitting diode lamp (420-700 nm light, 100 mW·cm-2). Cells were further incubated with the addition of Cis-Pt to a final concentration of 1 µg/mL. Activation of p38 MAPK was visualized by Western blot analysis. Flow cytometry was used for the estimation of cells distribution on cell cycle. Mitochondrial membrane potential (Δψm) was estimated with the use of fluorescent potential-sensitive probe TMRE (Tetramethylrhodamine Ethyl Ester). Results: Cis-Pt applied alone at 1 µg/mL concentration failed to affect mitochondrial membrane potential in L1210R cells or cell cycle distribution as compared with untreated cells. Activation of ROS-sensitive proapoptotic p38 kinase and enhanced content of cells in subG1 phase were detected after irradiation of L1210R cells treated with 10-5M C60 fullerene. Combined treatment with photoexcited C60 fullerene and Cis-Pt was followed by the dissipation of Δψm at early-term period, blockage of cell transition into S phase, and considerable accumulation of cells in proapoptotic subG1 phase at prolonged incubation. Conclusion: The effect of the synergic cytotoxic activity of both agents allowed to suppose that photoexcited C60 fullerene promoted Cis-Pt accumulation in leukemic cells resistant to Cis-Pt. The data obtained could be useful for the development of new approaches to overcome drug-resistance of leukemic cells.

C60 Fullerene as an Effective Nanoplatform of Alkaloid Berberine Delivery into Leukemic Cells.

A herbal alkaloid Berberine (Ber), used for centuries in Ayurvedic, Chinese, Middle-Eastern, and native American folk medicines, is nowadays proved to function as a safe anticancer agent. Yet, its poor water solubility, stability, and bioavailability hinder clinical application. In this study, we have explored a nanosized carbon nanoparticle-C60 fullerene (C60)-for optimized Ber delivery into leukemic cells. Water dispersions of noncovalent C60-Ber nanocomplexes in the 1:2, 1:1, and 2:1 molar ratios were prepared. UV-Vis spectroscopy, dynamic light scattering (DLS), and atomic force microscopy (AFM) evidenced a complexation of the Ber cation with the negatively charged C60 molecule. The computer simulation showed that π-stacking dominates in Ber and C60 binding in an aqueous solution. Complexation with C60 was found to promote Ber intracellular uptake. By increasing C60 concentration, the C60-Ber nanocomplexes exhibited higher antiproliferative potential towards CCRF-CEM cells, in accordance with the following order: free Ber < 1:2 < 1:1 < 2:1 (the most toxic). The activation of caspase 3/7 and accumulation in the sub-G1 phase of CCRF-CEM cells treated with C60-Ber nanocomplexes evidenced apoptosis induction. Thus, this study indicates that the fast and easy noncovalent complexation of alkaloid Ber with C60 improved its in vitro efficiency against cancer cells.

Synergy of Chemo- and Photodynamic Therapies with C60 Fullerene-Doxorubicin Nanocomplex.

A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C60 fullerene (C60) were applied in 1:1 and 2:1 molar ratio, exploiting C60 both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox's nuclear and C60's extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox's antiproliferative activity and C60's photoinduced pro-oxidant activity. When cells were treated with 2:1 C60-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C60-Dox enabled a nanomolar concentration of Dox and C60 to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC50 16, 9 and 7 × 103-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C60's photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C60-mediated Dox delivery and C60 photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C60-Dox nanoformulation provides a promising synergetic approach for cancer treatment.

Toxicity of C60 fullerene-cisplatin nanocomplex against Lewis lung carcinoma cells.

Cisplatin (Cis-Pt) is the cytotoxic agent widely used against tumors of various origin, but its therapeutic efficiency is substantially limited by a non-selective effect and high toxicity. Conjugation of Cis-Pt with nanocarriers is thought to be one option to enable drug targeting. The aim of this study was to estimate toxic effects of the nanocomplex formed by noncovalent interaction of C60 fullerene with Cis-Pt against Lewis lung carcinoma (LLC) cells in comparison with free drug. Scanning tunneling microscopy showed that the minimum size of C60-Cis-Pt nanoparticles in aqueous colloid solution was 1.1 nm whereas that of C60 fullerene was 0.72 nm, thus confirming formation of the nanocomplex. The cytotoxic effect of C60-Cis-Pt nanocomplex against LLC cells was shown to be higher with IC50 values 3.3 and 4.5 times lower at 48 h and 72 h, respectively, as compared to the free drug. 12.5 µM Cis-Pt had no effect on LLC cell viability and morphology while C60-Cis-Pt nanocomplex in Cis-Pt-equivalent concentration substantially decreased the cell viability, impaired their shape and adhesion, inhibited migration and induced accumulation in proapoptotic subG1 phase. Apoptosis induced by the C60-Cis-Pt nanocomplex was confirmed by caspase 3/7 activation and externalization of phosphatidylserine on the outer surface of LLC cells with the double Annexin V-FITC/PI staining. We assume that C60 fullerene as a component of the C60-Cis-Pt nanocomplex promoted Cis-Pt entry and intracellular accumulation thus contributing to intensification of the drug's toxic effect against lung cancer cells.

Correction to: Complexation with C60 Fullerene Increases Doxorubicin Efficiency against Leukemic Cells In Vitro.

Following publication of the original article [1], the authors flagged that there was unfortunately an error with Fig. 3 of the article.

Complexation with C60 Fullerene Increases Doxorubicin Efficiency against Leukemic Cells In Vitro.

Conventional anticancer chemotherapy is limited because of severe side effects as well as a quickly evolving multidrug resistance of the tumor cells. To address this problem, we have explored a C60 fullerene-based nanosized system as a carrier for anticancer drugs for an optimized drug delivery to leukemic cells.Here, we studied the physicochemical properties and anticancer activity of C60 fullerene noncovalent complexes with the commonly used anticancer drug doxorubicin. C60-Doxorubicin complexes in a ratio 1:1 and 2:1 were characterized with UV/Vis spectrometry, dynamic light scattering, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The obtained analytical data indicated that the 140-nm complexes were stable and could be used for biological applications. In leukemic cell lines (CCRF-CEM, Jurkat, THP1 and Molt-16), the nanocomplexes revealed ≤ 3.5 higher cytotoxic potential in comparison with the free drug in a range of nanomolar concentrations. Also, the intracellular drug's level evidenced C60 fullerene considerable nanocarrier function.The results of this study indicated that C60 fullerene-based delivery nanocomplexes had a potential value for optimization of doxorubicin efficiency against leukemic cells.

HPLC-ESI-MS method for C60 fullerene mitochondrial content quantification.

The presented dataset describes the quantification of carbon nanoparticle C60 fullerene accumulated in mitochondria of human leukemic cells treated with nanostructure. Firstly, the high performance liquid chromatography-electro spray ionization-mass spectrometry (HPLC-ESI-MS) method was developed for quantitative analysis of pristine C60 fullerene. Then, human leukemic cells were incubated with C60 fullerene, homogenized and subjected to the differential centrifugation to retrieve mitochondrial fraction. The C60 fullerene content was quantified by HPLC-ESI-MS in extracts of cellular fractions. This data article refers to the research article "C60 Fullerene Accumulation in Human Leukemic Cells and Perspectives of LED-mediated Photodynamic Therapy" by Grebinyk et al. [1].

C60 fullerene accumulation in human leukemic cells and perspectives of LED-mediated photodynamic therapy.

Recent progress in nanobiotechnology has attracted interest to a biomedical application of the carbon nanostructure C60 fullerene since it possesses a unique structure and versatile biological activity. C60 fullerene potential application in the frame of cancer photodynamic therapy (PDT) relies on rapid development of new light sources as well as on better understanding of the fullerene interaction with cells. The aim of this study was to analyze C60 fullerene effects on human leukemic cells (CCRF-CEM) in combination with high power single chip light-emitting diodes (LEDs) light irradiation of different wavelengths: ultraviolet (UV, 365 nm), violet (405 nm), green (515 nm) and red (632 nm). The time-dependent accumulation of fullerene C60 in CCRF-CEM cells up to 250 ng/106 cells at 24 h with predominant localization within mitochondria was demonstrated with immunocytochemical staining and liquid chromatography mass spectrometry. In a cell viability assay we studied photoexcitation of the accumulated C60 nanostructures with ultraviolet or violet LEDs and could prove that significant phototoxic effects did arise. A less pronounced C60 fullerene phototoxic effect was observed after irradiation with green, and no effect was detected with red light. A C60 fullerene photoactivation with violet light induced substantial ROS generation and apoptotic cell death, confirmed by caspase3/7 activation and plasma membrane phosphatidylserine externalization. Our work proved C60 fullerene ability to induce apoptosis of leukemic cells after photoexcitation with high power single chip 405 nm LED as a light source. This underlined the potential for application of C60 nanostructure as a photosensitizer for anticancer therapy.

C60 fullerene as synergistic agent in tumor-inhibitory Doxorubicin treatment.

BACKGROUND: Doxorubicin (Dox) is one of the most potent anticancer drugs, but its successful use is hampered by high toxicity caused mainly by generation of reactive oxygen species. One approach to protect against Dox-dependent chemical insult is combined use of the cytostatic drug with antioxidants. C60 fullerene has a nanostructure with both antioxidant and antitumor potential and may be useful in modulating cell responses to Dox. OBJECTIVE: The aim of this study was to estimate the antitumor effect and antioxidant enzyme activity of combined C60 fullerene and Dox (C60 + Dox) in the liver and heart of mice with Lewis lung carcinoma compared with Dox treatment alone. METHODS: Highly stable pristine C60 fullerene aqueous colloid solution (concentration 1.0 mg/ml, average hydrodynamic diameter of nanoparticles 50 nm) was used in the study and characterized by means of atomic force microscopy (AFM). The in vivo investigation of C60-Dox action was performed via the standard methods of histological and enzyme activity analyses. RESULTS: Dox (total dose 2.5 mg/kg) combined with C60 fullerene (total dose 25 mg/kg) in tumor-bearing animals resulted in tumor growth inhibition, prolongation of life, metastasis inhibition, and increased number of apoptotic tumor cells and was more effective than the corresponding course of Dox treatment alone. C60 fullerene demonstrated a protective effect against superoxide dismutase and glutathione peroxidase inhibition induced by Dox-dependent oxidative insult in the liver and heart. CONCLUSION: Combined treatment with C60 + Dox is considered to be a promising approach for cancer chemotherapy.

Therapeutic reactive oxygen generation.

An increase of the intracellular reactive oxygen species (ROS) concentration leads to the development of oxidative stress and, thus, to the damage of cell components. The cause-and-effect relations between these processes have not been fully established yet. The ability of photo excited supramolecular composites containing fullerenes C60 immobilized at nanosilica particles to generate reactive oxygen species (ROS) in cells of two types (rat thymocytes, and transformed cells of ascite Erlich carcinoma, EAC, and leucosis L1210) is demonstrated. The damaging effect of photo excited C60-composites are shown, which appeared to be selective and manifested in transformed cells, but not in thymocytes. It has been shown that after the irradiation of aqueous solutions or cell suspensions in the presence of fullerene C60, the generation of reactive oxygen species is observed. It has been shown that the influence of photo excited fullerene C60 on metabolic processes depends on the composition of C60-containing complex and on the type of the cells. The damaging effects of photo excited fullerene C60-containing composites were demonstrated to be selective. The data presented suggest that the application of fullerene C60-containing composites for the selective activation of ROS-dependent death program in certain types of tumor cells is very promising.

Catalytic system of the reactive oxygen species on the C60 fullerene basis.

AIM: To study the ability of fullerenes C(60) to catalyse the reactions of generation of reactive oxygen species (ROS) in water solution after photoexcitation and to affect the vitality of tumor cells in vitro. METHODS: The number and vitality of cultured Ehrlich carcinoma cells or rat thymocytes were determined using tripane blue, ROS levels were registered using the methods of electron paramagnetic resonance (EPR) spectroscopy and spin traps, photoirradiation of water solution of fullerenes C(60) with visible light was carried out using mercury lamp. RESULTS: Irradiation of water solution of fullerenes C(60) (10(-5) M) was accompanied with generation of ROS with the rate of 10 nMol/ml/min. After addition of irradiated C(60) solution to suspension of thymocytes or ascite cells, the decrease of the number of vital cells by 67 and 58%, respectively, has been registered. CONCLUSION: Photoirradiated water solutions of fullerenes C(60) may be potentially useful for photodynamic therapy of tumors as ROS catalytic system.