CARBON NANOTUBES FILLED WITH CARBOPLATIN – A NANOCARRIER FOR DRUG TRANSPORT

1. FIG.8 Induction of apoptosis in EJ28 cells 72h after treatment with carboplatin (CP) or carboplatin-filled CNTs (CNT-CP). CNT concentration: 0.3µg/µl. B A FIG.1 Schematic sketch of a proposed multi-functional nanocontainer. The filling could contain a therapeutic, a magnet (iron) and a temperature sensor. Iron can be used to generate heat by application of an external magnetic field to use the container for hyperthermia which could enhance the effects of the chemotherapeutics delivered in parallel. FIG.4 Release of carboplatin from CNT-CP. RepresentativeHRTEM pictures were shown after incubation of CNT-CP for A) 30min or B) 24h in cell culture medium at 37°C. FIG.2 HRTEM image of a CNT filled with carbo-platin. Insert: carboplatin clusters at higher magni-fication. FIG.3 EDX-spectrum of CNT-CP. Elemental analysis confirmed the presence of platinum within CNT-CP (copper is derived from the TEM-grid). FIG.5 Inhibition of EJ28 viability by carboplatin-filled CNTs. Relative viability 72h after treatment with different concentrations of empty CNT, carboplatin-filled CNT (CNT-CP) or free carboplatin (CP). Values are normalized to untreated cells (=100 %). Error bars correspond to the standard deviation. FIG.6 Decrease in EJ28 cell count 72h after treatment caused by carboplatin-filled CNTs. CNT concentration: 0.3µg/µl; CP - carboplatin, CNT-CP - carboplatin filled CNT. FIG.7 G2/M arrest of EJ28 cells 72h after treatment. Concentration of CNT 0.3µg/µl. CP - carboplatin, CNT-CP - carboplatin filled CNT, n.d. - not determinable (cell cycle distribution could not be determined due to high apoptosis). • CARBON NANOTUBES FILLED WITH CARBOPLATIN – • A NANOCARRIER FOR DRUG TRANSPORT Kai Kraemera, Doreen Kunzea, Silke Hampelb, Diana Haaseb, Susanne Fuessela, Albrecht Leonhardtb, Axel Meyea, Bernd Buechnerb, Manfred P. Wirtha aDepartment of Urology, Technical University Dresden, Fetscherstrasse 74, 01307 Dresden, Germany bLeibniz Institute of Solid State and Materials Research (IFW), Helmholtzstrasse 20, 01069 Dresden, Germany • Material & Methods • production of multi-walled, empty CNTs: chemical vapor deposition (CVD) with Fe as the catalyst in a solid source CVD-system • purification of CNTs from catalyst particles and impurities: thermal treatment (450°C in air, 1h) and subsequent wash in hydrochloric acid • characterization of CNTs: size and purity were evaluated by Scanning and Transmission Electron Microscopy (SEM, TEM) • filling of CNTs with carboplatin: opening of CNTs by nitric acid, suspension of 1mg/ml opened CNTs in carboplatin solution (10mg/ml) by sonication (10min) and agitation (24h) • purification of CNT-CP-surfaces from carboplatin particles: washing with A.dest. and ethanol, drying at 100°C • characterization of CNT-CP: High Resolution Transmission Electron Microscopy (HRTEM) for morphology and Energy dispersive X-ray-Analysis (EDX) for elemental analysis • cell culture experiments: EJ28 BCa cells were incubated for 24h with: • empty CNT • carboplatin alone (10-150µg/ml) • empty CNT (0.3µg/µl) + carboplatin (40µg/ml) • CNT-CP (0.1, 0.3 or 0.5µg/µl) • investigation of cellular viability, apoptosis and cell cycle: • WST-1 viability assay (Roche) in quadruplicates 48 and 72h after treatment • Annexin V/propidium iodide staining (BD Bio- sciences) to quantify apoptosis • CycleTest Plus DNA Reagent Kit • (BD Biosciences) to investigate cell cycle • Results • Characterization of CNT-CP (IFW Dresden) • electron microscopy revealed many spherical carboplatin clusters with a mean diameter of 1–2nm incorporated • into opened CNTs [Fig.2] • the filling yield was about 30% (w/w) • element analysis of CNT-CP detected a high amount of carbon (derived from CNTs) and the presence of • platinum [Fig.3] • In vitro experiments (Dept. of Urology) • CNT-CP release carboplatin into culture medium within 24h [Fig.4] • empty CNTs had no (0.1µg/µl) or minor (0.3µg/µl, 0.5µg/µl) influence on viability of EJ28, PC-3 and fibroblasts despite the use of relative high concentrations [Fig.5] • free carboplatin inhibited the cellular viability of EJ28 cells in a concentration dependent manner [Fig.4] • CNT-CP in concentrations of >0.1µg/µl clearly reduced the viability; e.g. 0.5µg/µl CNT-CP caused a decreased viability to <5% of the control whereas the same concentration of empty CNTs caused a minor decrease of viability to 70% [Fig.5] • empty CNTs did not cause alterations in cell count [Fig.6], cell cycle [Fig.7] and apoptosis [Fig.8] • CNT-CP caused a 3-fold reduction in cell count [Fig.6], a G2/M arrest in cell cycle [Fig.7] and an increase in apoptosis to almost 70% [Fig.8] in comparison to untreated cells • a combination of empty CNTs and free carboplatin induced additive effects on cell count [Fig.6] and apoptosis [Fig.8] in comparison to the separate agents • Introduction • multiwalled carbonanotubes (CNTs) are hollow tubes composed of multiple carbon layers • diameters of 20 to 60nm, length of up to 10µm • high mechanical and chemical stability • their surface, which is naturally inert, can be functionalized by introduction of reactive groups • interesting material for applications in biology and medicine • incorporation of carboplatin into CNTs (CNT-CP) to use them as container/transporter for cytotoxic drugs • potential advantages of a multifunctional nano- scaled container [FIG.1]: • slow drug release (depot function), • controlled drug release (e.g. temperature sensitive cap for controlled opening of CNT-CP) • tumor targeting (magnet targeting using Fe-filling; functionalization of CNT-CP with antibodies) • parallel application of chemotherapy and hyperthermia if CNTs were filled with both chemotherapeutics and iron (Fe) • Futher Questions • Which method is suitable for the dispersion of naturally hydrophobic CNT-CP? • What is the amount of chemotherapeutic drug that can be transported by CNTs? • Is the drug completely released from CNT-CP over the time? • Can CNT-CP be closed and opened in a controlled way? • Can CNTs be filled with Fe and chemotherpeutics in parallel? • Conclusions • This preliminary study describes the synthesis and function of carboplatin-filled CNTs (CNT-CP). • carboplatin can be incorporated into CNTs by wet-chemical techniques • CNT-CP are suitable to transport carboplatin in vitro • CNT-CP released carboplatin as shown by their inhibiting effects on tumor cell growth http://urologie.uniklinikum-dresden.de