Generation of toxic degradation products by sonication of Pluronic® dispersants: implications for nanotoxicity testing.

Poloxamers (known by the trade name Pluronic®) are triblock copolymer surfactants that contain two polyethylene glycol blocks and one polypropylene glycol block of various sizes. Poloxamers are widely used as nanoparticle dispersants for nanotoxicity studies wherein nanoparticles are sonicated with a dispersant to prepare suspensions. It is known that poloxamers can be degraded during sonication and that reactive oxygen species contribute to the degradation process. However, the possibility that poloxamer degradation products are toxic to mammalian cells has not been well studied. We report here that aqueous solutions of poloxamer 188 (Pluronic® F-68) and poloxamer 407 (Pluronic® F-127) sonicated in the presence or absence of multi-walled carbon nanotubes (MWNTs) can became highly toxic to cultured cells. Moreover, toxicity correlated with the sonolytic degradation of the polymers. These findings suggest that caution should be used in interpreting the results of nanotoxicity studies where the potential sonolytic degradation of dispersants was not controlled.

STUDY OF THE NEAR INFRARED-MEDIATED HEATING OF DISPERSIONS OF PROTEIN-COATED PRISTINE AND CARBOXYLATED SINGLE-WALLED CARBON NANOTUBES.

Previously, we demonstrated the selective NIR-mediated ablation of tumor cells in vitro using pristine single-walled carbon nanotubes (SWNTs) with adsorbed tumor-targeting ligands and carboxylated SWNTs with covalently-attached ligands. The covalent approach is advantageous in ensuring that protein ligands remain associated with the NIR-absorbing SWNTs in biological matrices and the noncovalent approach has the advantage of enabling SWNT functionalization without perturbation of the SWNT lattice and photothermal properties. Herein, we compare the ability of moderately-carboxylated (~4 at.% carboxylic acid groups) and pristine SWNT materials to absorb 808 nm radiation and convert it to heat. Under conditions of a constant 808 nm laser power density, the approach involved measuring the temperature of aqueous dispersions of protein-coated SWNTs as a function of the irradiation time. Nearly identical temperature profiles were observed for dispersions of moderately-carboxylated and pristine SWNTs possessing matched 808 nm optical densities and equivalent concentrations of carbonaceous species (i.e., SWNTs and amorphous carbon impurities). The results indicate that the amount of carbonaceous species in purified dispersions of protein-coated SWNTs is more important for converting absorbed 808 nm radiation into heat than whether or not the SWNTs were moderately carboxylated, and that moderately-carboxylated SWNTs could be the SWNT-material of choice for the targeted photothermal ablation of tumor cells.

Cytotoxicity screening of single-walled carbon nanotubes: detection and removal of cytotoxic contaminants from carboxylated carbon nanotubes.

This study compares the cytotoxicity to cultured mammalian cells of nine different single-walled carbon nanotube (SWNT) products synthesized by a variety of methods and obtained from a cross section of vendors. A standard procedure involving sonication and centrifugation in buffered bovine serum albumin was developed to disperse all the SWNTs in a biocompatible solution to facilitate comparisons. The effect of the SWNTs on the proliferative ability of a standard cell line was then assessed. Of the nine different SWNT materials tested, only two were significantly toxic, and both were functionalized by carboxylation from different vendors. This was unexpected because carboxylation makes SWNTs more water-soluble, which would presumably correlate with better biocompatibility. However, additional purification work demonstrated that the toxic material in the carboxylated SWNT preparations could be separated from the SWNTs by filtration. The filtrate that contained the toxic activity also contained abundant small carbon fragments that had Raman signatures characteristic of amorphous carbon species, suggesting a correlation between toxicity and oxidized carbon fragments. The removal of a toxic contaminant associated with carboxylated SWNTs is important in the development of carboxylated SWNTs for pharmacological applications.

Gel electrophoresis method to measure the concentration of single-walled carbon nanotubes extracted from biological tissue.

A rapid and sensitive method to detect single-walled carbon nanotubes (SWNTs) in biological samples is presented. The method uses polyacrylamide gel electrophoresis (PAGE) followed by quantification of SWNT bands. SWNTs dispersed in bovine serum albumin (BSA) were used to develop the method. When BSA-SWNT dispersions were subjected to sodium dodecyl sulfate (SDS)-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected. The SWNTs, however, accumulated in a sharp band at the interface between the loading well and the stacking gel. The intensities from digitized images of these bands were proportional to the amount of SWNTs loaded onto the gel with a detection limit of 5 ng of SWNTs. To test the method, normal rat kidney (NRK) cells in culture were allowed to take up SWNTs upon exposure to medium containing various concentrations of BSA-SWNTs for different times and temperatures. The SDS-PAGE analyses of cell lysate samples suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis at a rate of 30 fg/day/cell upon exposure to medium containing 98 microg/mL SWNTs.