NSTI BioNano 2010

Ultra-Structural Analysis of the Interaction of Carboxylated Multi-Walled Carbon Nanotubes with Human Blood Platelets

S.H. Lacerda, J. Semberova., K. Holada., O. Simakova, M.P. Gelderman-Fuhrmann, J. Simak

Keywords: MWCNT, carbon nanotubes, platelet, blood


Carbon nanotubes (CNTs) are a promising new class of materials with many potential biomedical applications. CNTs have profound impact on the development of diagnostic biosensors, drug delivery nanosystems, imaging nanoprobes for intravascular use or other devices that come in contact with blood. Therefore, the biocompatibility/toxicity of CNTs in blood have to be assessed carefully.1 It has been shown that some carbon nanoparticles activate platelets and enhance arterial thrombosis.2 Therefore, an understanding of the effects of CNTs on platelets is a critical safety issue. We have previously shown that different agglomerates of non-functionalized CNTs activate human platelets by inducing extracellular Ca2+ influx.3 Functionalization of CNTs with carboxylic groups is a procedure that not only reduces impurities of the pristine samples, but also provides water solubility to the CNTs. Moreover, carboxylated CNTs allow for easy bioconjugation. Here we investigate the effects of carboxylated multi-walled carbon nanotubes (MWCNTs) on human platelets in vitro in comparison to amorphous carbon nanoparticles (ACNs). Both Transmission Electron Microscopy (TEM) and Field-Emission Scanning Electron Microscopy (FESEM) were used to evaluate their specific interaction with platelets. In a pilot experiment using light transmission platelet aggregometry we showed that carboxy-MWCNTs from NanoLab (>95% purity, outer diameter 15  5 nm) at 1mg/mL induced marked platelet aggregation similarly to non-functionalized MWCNTs. A significantly lower platelet aggregation response was observed with ACNs, while fullerene (nC60) and polystyrene beads (20 nm and 200 nm) did not induce platelet aggregating activity. Due to the limited dispersability of commercial carboxy-MWCNTs, we have prepared carboxy-MWCNTs in house by refluxing pristine MWCNTs in a concentrated 3:1 (v/v) H2SO4 and HNO3 mixture at 70oC, and following washing and dialysis in water. Our preparation of carboxy-MWCNTs was well dispersed in PBS, but was unstable in citrate blood plasma, and formed bundles and larger agglomerates. FESEM showed that platelets in contact with carboxy-MWCNTs undergo morphological changes from the resting discoid state to an activated state with pseudopodia. TEM revealed that platelets are able to internalize carboxy-MWCNTs. This effect was not as pronounced with ACNs. In conclusion, carboxy-MWCNTs activate blood platelets similarly like their pristine counterparts. Role of nanopenetration and internalization of carboxy-MWCNTs in platelet activation remains to be elucidated. The findings and conclusions in this study have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
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