Toxicity of Water-Soluble D-g-PNIPAM Polymers in a Complex with Chemotherapy Drugs and Mechanism of Their Action In Vitro.

The application of a biocompatible polymer nanocarrier can provide target delivery to tumor tissues, improved pharmacokinetics, controlled drug release, etc. Therefore, the proposed strategy was to use the water-soluble star-like copolymers with a Dextran core and Poly(N-isopropylacrylamide) grafts (D-g-PNIPAM) for conjugation with the widely used chemotherapy drugs in oncology-Cisplatin (Cis-Pt) and Doxorubicin (Dox). The molecular characteristics of the copolymer were received using size-exclusion chromatography. The physicochemical characterization of the D-g-PNIPAM-Cis-Pt (or Dox) nanosystem was conducted using dynamic light scattering and FTIR spectroscopy. Using traditional biochemical methods, a comparative analysis of the enhancement of the cytotoxic effect of free Cis-Pt and Dox in combination with D-g-PNIPAM copolymers was performed in cancer cells of the Lewis lung carcinoma line, which are both sensitive and resistant to Dox; in addition, the mechanism of their action in vitro was evaluated.

Comparison of Sonodynamic Treatment Set-Ups for Cancer Cells with Organic Sonosensitizers and Nanosonosensitizers.

Cancer sonodynamic therapy (SDT) is the therapeutic strategy of a high-frequency ultrasound (US) combined with a special sonosensitizer that becomes cytotoxic upon US exposure. The growing number of newly discovered sonosensitizers and custom US in vitro treatment solutions push the SDT field into a need for systemic studies and reproducible in vitro experimental set-ups. In the current research, we aimed to compare two of the most used and suitable SDT in vitro set-ups-"sealed well" and "transducer in well"-in one systematic study. We assessed US pressure, intensity, and temperature distribution in wells under US irradiation. Treatment efficacy was evaluated for both set-ups towards cancer cell lines of different origins, treated with two promising sonosensitizer candidates-carbon nanoparticle C60 fullerene (C60) and herbal alkaloid berberine. C60 was found to exhibit higher sonotoxicity toward cancer cells than berberine. The higher efficacy of sonodynamic treatment with a "transducer in well" set-up than a "sealed well" set-up underlined its promising application for SDT in vitro studies. The "transducer in well" set-up is recommended for in vitro US treatment investigations based on its US-field homogeneity and pronounced cellular effects. Moreover, SDT with C60 and berberine could be exploited as a promising combinative approach for cancer treatment.

Silent Death by Sound: C60 Fullerene Sonodynamic Treatment of Cancer Cells.

The acoustic pressure waves of ultrasound (US) not only penetrate biological tissues deeper than light, but they also generate light emission, termed sonoluminescence. This promoted the idea of its use as an alternative energy source for photosensitizer excitation. Pristine C60 fullerene (C60), an excellent photosensitizer, was explored in the frame of cancer sonodynamic therapy (SDT). For that purpose, we analyzed C60 effects on human cervix carcinoma HeLa cells in combination with a low-intensity US treatment. The time-dependent accumulation of C60 in HeLa cells reached its maximum at 24 h (800 ± 66 ng/106 cells). Half of extranuclear C60 is localized within mitochondria. The efficiency of the C60 nanostructure's sonoexcitation with 1 MHz US was tested with cell-based assays. A significant proapoptotic sonotoxic effect of C60 was found for HeLa cells. C60's ability to induce apoptosis of carcinoma cells after sonoexcitation with US provides a promising novel approach for cancer treatment.

FLASHlab@PITZ: New R&D platform with unique capabilities for electron FLASH and VHEE radiation therapy and radiation biology under preparation at PITZ.

At the Photo Injector Test facility at DESY in Zeuthen (PITZ), an R&D platform for electron FLASH and very high energy electron radiation therapy and radiation biology is being prepared (FLASHlab@PITZ). The beam parameters available at PITZ are worldwide unique. They are based on experiences from 20 + years of developing high brightness beam sources and an ultra-intensive THz light source demonstrator for ps scale electron bunches with up to 5 nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms length, currently 22 MeV (upgrade to 250 MeV planned). Individual bunches can provide peak dose rates up to 1014 Gy/s, and 10 Gy can be delivered within picoseconds. Upon demand, each bunch of the bunch train can be guided to a different transverse location, so that either a "painting" with micro beams (comparable to pencil beam scanning in proton therapy) or a cumulative increase of absorbed dose, using a wide beam distribution, can be realized at the tumor. Full tumor treatment can hence be completed within 1 ms, mitigating organ movement issues. With extremely flexible beam manipulation capabilities, FLASHlab@PITZ will cover the current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexploited short treatment times and high dose rates. A summary of the plans for FLASHlab@PITZ and the status of its realization will be presented.

Drug delivery with a pH-sensitive star-like dextran-graft polyacrylamide copolymer.

The development of precision cancer medicine relies on novel formulation strategies for targeted drug delivery to increase the therapeutic outcome. Biocompatible polymer nanoparticles, namely dextran-graft-polyacrylamide (D-g-PAA) copolymers, represent one of the innovative non-invasive approaches for drug delivery applications in cancer therapy. In this study, the star-like D-g-PAA copolymer in anionic form (D-g-PAAan) was developed for pH-triggered targeted drug delivery of the common chemotherapeutic drugs - doxorubicin (Dox) and cisplatin (Cis). The initial D-g-PAA copolymer was synthesized by the radical graft polymerization method, and then alkaline-hydrolyzed to get this polymer in anionic form for further use for drug encapsulation. The acidification of the buffer promoted the release of loaded drugs. D-g-PAAan nanoparticles increased the toxic potential of the drugs against human and mouse lung carcinoma cells (A549 and LLC), but not against normal human lung cells (HEL299). The drug-loaded D-g-PAAan-nanoparticles promoted further oxidative stress and apoptosis induction in LLC cells. D-g-PAAan-nanoparticles improved Dox accumulation and drugs' toxicity in a 3D LLC multi-cellular spheroid model. The data obtained indicate that the strategy of chemotherapeutic drug encapsulation within the branched D-g-PAAan nanoparticle allows not only to realize pH-triggered drug release but also to potentiate its cytotoxic, prooxidant and proapoptotic effects against lung carcinoma 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.

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.

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.