Grantee Research Project Results
Nancy A. Monteiro-Riviere and Jim E. Riviere
Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University, Raleigh, NC
The field of nanoscience has experienced unprecedented growth during the last few years and has received a great deal of attention because of the many potential applications. Nanomaterials possess unique physicochemical properties compared to larger microparticles that enable novel engineering applications. Their large surface area and increase in reactivity can enhance the transport of nanomaterials in the environment and in biological systems. However, there are many challenges that must be overcome before we can apply nanotechnology to the field of nanomedicine or conduct science based occupational or environmental exposure risk assessments. There is a serious lack of information about human health and environmental implications of manufactured nanomaterials. This new field of nanotoxicology will continue to grow and emerge as new products are produced. Insufficient data has been collected so far to allow for full interpretation or thorough understanding of the toxicological implications of occupational exposure or potential environmental impact of nanomaterials.
Skin is a primary route of potential exposure to toxicants and nanomaterials, and the ability to traverse the stratum corneum and viable epidermal layers is a primary determinant of the dermatotoxic potential. Currently, there is no information on whether carbon nanomaterials, or nanomaterials in general, can penetrate intact skin and cause adverse effects.
The goal of this project was to study the effects of several different types of nanomaterial interactions with skin, including dermal absorption and cutaneous toxicity and the ability to distribute to skin after systemic exposure. To date, we have evaluated several types of nanomaterials in two different in vitro model systems under different conditions (concentration, surfactants, and vehicles); multi-walled carbon nanotubes (MWCNT) in human epidermal keratinocytes (HEK), the fullerenes nanoC60 and derivatized C60 (C60(OH)24) in HEK. Also, we have evaluated fullerene based amino acids (BAA) and fullerene C60 peptide with a fluorescently tagged nuclear localization signal (NLS-FITC) for 24h and 48h in HEK. In addition, we have evaluated quantum dots (QD) of two different sizes (6 nm and 10 nm), and three different surface coatings (polyethylene glycol, carboxylic acid, or amine). All of these studies were conducted in HEK to assess cellular uptake, cytotoxicity, and inflammatory potential. The fullerene NLS-FITC and QD were also evaluated in flow though diffusion cells to assess skin penetration.
HEK were exposed to 0.1, 0.2, and 0.4 mg/ml of multi-walled carbon nanotubes (MWCNT) for 1, 2, 4, 8, 12, 24, and 48 h. HEK were examined by transmission electron microscopy (TEM) for the presence of MWCNT. This study showed that chemically unmodified MWCNT were present within cytoplasmic vacuoles of the HEK at all time points. The MWCNT also induced the release of the proinflammatory cytokine interleukin 8 (IL-8), a biomarker for skin irritation, from HEK in a time dependent manner. These data clearly show that MWCNT, neither derivatized nor optimized for biological applications, are capable of both localizing within and initiating an irritation response.
Studies with the fullerenes nanoC60, and derivatized C60 (C60(OH)24) were conducted in HEK. HEK were exposed to nanoC60 ranging in concentrations (0.0000025 - 0.0005 mg/ml), derivatized C60 (C60(OH)24) ranging in concentrations (0.000005 - 0.001 mg/ml)(n=8/treatment) for 24 h and 48 h. MTT viability showed nano-C60 ranging from 0.00025 to 0.0005 mg/ml decreased HEK viability significantly (p<0.05) by 24 h. The inflammatory mediators IL-8 and IL-6 were significantly (p<0.05) greater than controls, while IL-10, IL-1 , and TNF- were below detectable limits. (C60(OH)24) showed no toxicity to HEK at all concentrations. At high concentrations of nano-C60 (0.003 mg/ml), MTT viability was reduced by 48 h and IL-8 increased from 4 hr to 48 hr. Additional studies with 0.5 percent Pluronic F127 surfactant found that nano-C60 (0.0005 mg/ml) caused a significant (p<0.05) decrease in viability and a significant increase in IL-8 production by 48 h. In contrast,C60(OH)24 treated with the surfactant showed no statistical differences in HEK viability or cytokine production. Finally, the addition of 1% DMSO to nano-C60 (0.0005 mg/ml and 0.003 mg/ml) significantly decreased MTT viability, and the 0.003 mg/ml significantly increased the release of IL-8 by 48 h. These results show that while derivatizedC60(OH)24 are nontoxic in the tested range, nano-C60 are toxic at concentrations as low as 0.00025 mg/ml in HEK.
The amino acid derivatized fullerene C60 BAA has the potential to provide greater interaction between the fullerene and the biological environment yielding potential new medical and pharmacological applications. BAA can serve as a nanoparticle platform for conjugation of targeting peptides and therapeutic biomolecules. HEK were exposed to fullerene BAA concentrations of 0.4-0.00004 mg/ml for 1, 4, 8, 12, 24, and 48h. MTT cell viability after 48 h significantly decreased (p<0.05) at concentrations of 0.4 and 0.04mg/ml. Proinflammatory cytokines IL-6, IL-8, TNF-α, IL-1, and IL-10 were assessed at concentrations ranging from 0.4 - 0.004 mg/ml. IL-8 concentrations for the 0.04 mg/ml treatment were significantly greater (p<0.05) than all other concentrations at 8, 12, 24, and 48h. IL-6 and IL-1 activities were greater at the 24h and 48h for 0.4 and 0.04 mg/ml. No TNF-? or IL-10 activity existed at any time points for any of the concentrations. These results indicate that concentrations lower than 0.04 mg/ml initiate less cytokine activity and maintain cell viability at control levels. BAA concentrations of 0.4 and 0.04 mg/ml in HEK decreases cell viability and can initiate a proinflammatory response in the dominant cell type of skin.
HEK were also exposed to concentrations from 0.4-.001 mg/ml (n=8/treatment) of C60 BAA conjugated to a peptide with a fluorescently tagged nuclear localization signal (NLS-FITC) for 24 h and 48 h. MTT viability and the proinflammatory mediator's interleukin IL-8, IL-6, IL-1β, IL-10, and TNF-α were assessed. In addition, 4.4 g/cm2 and 1.1 g/cm2 of C60-NLS-FITC with and without 1 percent Pluronic surfactant or NLS-FITC alone was topically applied to porcine flow through diffusion cells for 24 h. MTT viability decreased and was statistically significant (p<0.05) in HEK at 0.4 mg/ml at both 24 h and 48 h. IL-8 and IL-6 statistically increased at 0.2 mg/ml and 0.4 mg/ml. IL-1? (0.4 mg/ml) was low but was significantly different from controls, while IL-10 and TNF-α were below detectable limits. Confocal microscopy of the flow through skin at both concentrations for C60-NLS-FITC and NLS-FITC depicted penetration through all epidermal layers. Surfactant greatly enhanced the permeability for all treatments. These results showed that the substituted fullerenes can penetrate through intact skin and can elicit an inflammatory response.
QD of two different sizes (6 nm and 10 nm) and three different surface coatings (polyethylene glycol, carboxylic acid, or amine) were topically applied to porcine skin in flow-through diffusion cells at a concentration of 62.5 pmoles/cm2. Confocal microscopy showed QD were localized within the epidermis and dermis by 24 h for all QD preparations. Additional studies utilized HEK to investigate the cellular uptake, cytotoxicity, and inflammatory potential of these nanomaterials. Live cell confocal imaging showed that all types of QD were localized within the cell by 24 h. MTT viability and multiplex ELISA for the inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α revealed that carboxylic acid-coated QD at a concentration of 20 nM exhibited significant cytotoxicity and increased release of IL-8 by 24 h. This study showed that commercially available QD with diverse surface chemistries can penetrate the skin and be incorporated within HEK by 24 h, and that QD surface coatings can influence cytotoxic and inflammatory effects.
These ongoing studies and the results so far indicate that skin cells and intact skin exposed to nanoscale materials of different physicochemical properties such as size and surface charge, and different vehicles may result in localized toxicity. The dermatotoxic potential of nanomaterials is based on whether they can traverse through the stratum corneum layers, penetrate through the viable epidermal layers to the dermis, and be absorbed by the capillaries in the dermal papillary layer to have a systemic effect. This data is necessary to define the doses for systemic exposure after topical administration as well as the cutaneous hazard after either topical or systemic exposure, the two essential components of any risk assessment. If nanomaterials are inadvertently modified or if exposure occurs before cleansing, they could have untoward consequences if they gain entry through the skin. A single study will not definitively answer all of the pertinent questions relative to dermal risk assessment of nanomaterials, but they provide a foundation for future work. Ultimately, they should be able to provide an insight into the nature of the potential hazard to nanomaterials of certain concentrations and under various conditions and should provide an initial estimate of the dermal exposure parameters that can be used to design more definitive studies.