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Posted: Jan 5th, 2007
Fullerenes shown to penetrate healthy skin
(Nanowerk Spotlight) Nanoparticles exhibit unique properties that make them ideal for a wide-variety of applications. Also unique, and largely unknown, are the interactions that occur between the biological environment and nanoparticles. On the upside, the ability of quantum dots and fullerenes to penetrate intact skin provides potential benefits for the development of nanomaterial applications involving drug delivery. On the downside, this ability poses potential risks associated with manufacturing and handling such nanoparticles. A new study now confirms that fullerene-based peptides can penetrate intact skin and that mechanical stressors, such as those associated with a repetitive flexing motion, increase the rate at which these particles traverse into the dermis. These results are important for identifying external factors that increase the risks associated with nanoparticle exposure during manufacturing or consumer processes. Future assessments of nanoparticle safety should recognize and take into account the effect that repetitive motion and mechanical stressors have on nanoparticle interactions with the biological environment. Additionally, these results could have profound implications for the development of nanoparticle use in drug delivery, specifically in understanding mechanisms by which nanoparticles penetrate intact skin.
"Our work investigates how a specific type of nanoparticle, an amino acid-derivatized fullerene, interacts with the biological environment both at the tissue and cellular levels" Jillian Rouse, a student at the Center for Chemical Toxicology Research and Pharmacokinetics (CCTRP) at North Carolina State University, explains to Nanowerk. "It is important to identify those nanoparticles that can penetrate intact skin and to determine the potential risk for toxicity once the particles are in the body. Nanoparticles have the potential to provide great scientific developments; however, if there are inherent biological risks associated with their use, then safety evaluations need to be conducted."
Rouse, Dr. Nancy Monteiro-Riviere, professor of investigative dermatology and toxicology at NC State's College of Veterinary Medicine, and Dr. Andrew R. Barron, professor of chemistry and materials science at Rice University are the first to show the ability of fullerenes to penetrate intact skin and to relate nanoparticle penetration to biomechanical stimuli.
"Our work was motivated by the discovery that mechanical stimuli applied during standard physiological processes, such as walking, increase the
penetration of particles found in soil and result in a higher prevalence of podoconiosis in the African rift valleys" says Rouse. "The ability of normal biomechanical movements to increase nanoparticle penetration solidifies the need for risk assessment, especially in occupational settings where constant, repetitive motions are involved."
The researchers have previously reported the synthesis of the phenylalanine-based fullerene amino acid, Bucky amino acid (Baa), and the uptake and interaction of Baa with human epidermal keratinocytes ("Fullerene-based amino acid nanoparticle interactions with human epidermal keratinocytes"). The presence of the fullerene
substituent has a significant effect on the intracellular transport of peptides containing Baa. The addition of a fullerene-derived amino acid to a cationic peptide results in the peptide showing cellular uptake, whereas the same peptide sequence in the absence of Baa shows no transport across the cell membrane.
The peptide sequence used is based on on the nuclear localization sequence (NLS). After the NLS sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) was completed, a Lys(Mtt) residue was coupled to the end to allow attachment of the fluorescein isothiocyanate (FITC) fluorescent marker. The individual Baa-Lys(FITC)-NLS particles measured ca. 3.5 nm in size.
Because of its physiological and structural similarities to human skin, porcine skin was used a model for human skin in this study. To determine the effects of flexing on skin penetration, the dermatomed skin was dosed with 20 µL of Baa-Lys-(FITC)-NLS in 1% PBS, and the dosed areas
were subsequently flexed for 60 or 90 min or left unflexed (control) to investigate nanoparticle penetration.
Confocal scanning microscopy images of skin dosed with Baa-Lys(FITC)-NLS for 24 h. Top row: confocal-DIC channel image shows an intact stratum corneum (SC) and underlying epidermal (E) and dermal layers (D). Middle row: Baa-Lys(FITC)-NLS fluorescence channel (green) and confocal-DIC channel show fullerene penetration through the skin. Bottom row: fluorescence intensity scan of Baa-Lys(FITC)-NLS. All scale bars represent 50 µm. (Reprinted with permission from the American Chemical Society)
"After 24 h of Baa-Lys(FITC)-NLS treatment, skin penetration was greater in all experimental groups" explains Rouse. "The DIC (Differential Interference Contrast) images reveal a thick, intact stratum corneum and the intensity maps show the highest concentration of particles in the upper epidermal layers and a lower concentration as the fullerenes penetrate into the dermis. Skin flexed for 90 min showed the greatest amount of dermal penetration, evident by the higher fluorescence intensity of the nanoparticles in this group (bottom row in above image)."
The mechanical loading regimes used in this study attempt to mimic physiological forces that can occur during nanoparticle manufacturing processes or conditions involved in consumer use. The external forces applied to the skin while flexing proves to have a significant effect on both the rate
and extent of fullerene penetration. Skin flexed for 90 min shows evidence of dermal penetration after 8 h of nanoparticle exposure, whereas control specimens show evidence of fullerenes primarily localized in the epidermis and only a slight amount in the dermis after the 24 h treatment.
These results suggest that the action of a flexing procedure increases the rate at which fullerenes can penetrate through the skin. Furthermore, flexing increased the amount of fullerenes that were capable of penetrating into the dermal layers of skin, indicated by the higher fluorescence intensity of fullerenes for both 60 and 90 min flexed skin.
"It is important to note that for all treatments nanoparticle penetration was non-homogeneous probably due to a nonuniform distribution of the dose over the dose region and/or differences in the thickness of the epidermis" Rouse points out.
The route of nanoparticle penetration through the skin is of great interest, especially in the nanomedicine field. In this study, TEM depicted derivatized fullerenes localized within intercellular spaces of the epidermis, suggesting that migration through the skin occurs intercellularly as opposed to movement through cells. The findings also indicate that fullerene penetration occurs via a mechanism of passive diffusion. Therefore, movement of the derivatized nanoparticles through the skin is dependent on the hydrophobic lipid entities that are present between the epidermal
For drug-delivery applications, the ability of nanoparticles to have access to systemic circulation has important implications. However, because some nanoparticles have been shown to initiate adverse biological responses, there are potential risks for systemic toxicity to occur and, therefore, the need arises for risk assessment and the establishment of safety regulations.
An important conclusion from this research is that investigations of the interactions that occur between nanoparticles and the biological environment should take into account external stimuli, such as physiologically relevant biomechanical forces. Whether for nanoparticle risk assessment or for the development of nano-drug delivery systems, it is important to identify all factors that could influence how these particles interact with the body.