In this study, we describe the penetration of nanoparticles (QD) through skin in vivo following application in a cream similar to those used in skin lotions or sunscreens. The PEG-coated QD were topically applied at a dose of 9.4μM at a cream dose rate of 2 mg/cm2. When the QD were applied to intact skin or skin where the stratum corneum was removed by tape stripping, we found no evidence of the migration of the QD into and through the skin to the regional lymph nodes or liver, based on the measurement of cadmium in tissues and fluorescent QD by confocal microscopy. We did detect elevated cadmium in the liver from one group of mice at 48 hr after application of QD to intact skin. However, taking into consideration the lack of significant increase of cadmium in the lymph nodes and liver of other groups (e.g., tape stripped, acetone treated, intact skin covered with occlusion patch), we conclude that this one result is probably spurious and that there is no evidence of QD penetration through skin containing an intact epidermis and biodistribution to the lymph nodes or liver. The same cannot be said for application of the QD to the skin of animals that had the skin damaged through dermabrasion. We found a significant increase in the level of cadmium in the lymph nodes and livers of mice pretreated with dermabrasion. Considering the amount of cadmium contained in the applied QD, we were able to detect the migration of 1.98 and 1.96% of the topically applied dose in the livers in 24 h in dermabraded skin with and without the occlusion patch, respectively. This also indicates that of the approximately 2.8 × 1013 QD that were applied to the mice, approximately 5.6 × 1011 penetrated the skin and migrated to the liver. To the best of our knowledge, this is the first quantitative determination of the migration of QD from the skin into internal sentinel organs.
Tape stripping is the most common method to remove the outermost barrier of the skin to environment. Studies have shown that the greater the number of tape strippings can increase the degree of corneocyte removal and epidermis damage can occur; however, these results are dependent on the tape, pressure, and time on the skin (Breternitz et al., 2007
). As an example, Oudshoorn et al. (2009)
have shown that the application of four tape strippings with fingertip pressure to SKH-1 mice resulted in transepidermal water loss (TEWL), but not complete removal of corneocytes. After six, seven, and eight tape strippings, the TEWL was increased, and the corneocyte removal was complete at eight tape strippings. With eight or less tape strippings the skin recovered within 24 or 48 h, however, with 12 tape strippings, the skin was damaged to the point that it did not recover in 48 h. In our study we quantified the number of tape strips required to achieve stratum corneum removal without damaging the epidermis using histology. Our results that 10 tape strippings are adequate are consistent with those of Oudshoorn et al. (2009)
In a prior study we established that the lymph nodes and liver were appropriate sentinel organs for distribution of PEG-coated QD (37 nm diameter) following intradermal injection (Gopee et al., 2007
). The QD disbursed throughout the viable subcutis at the injection site (visualized using fluorescence microscopy) and were additionally visible within minutes migrating through the lymphatics to the regional draining lymph nodes (Gopee et al., 2007
). The migration of the QD was quantitatively determined using ICP-MS and was based on the cadmium content of the CdSe-based QD. At 24 h following injection, approximately 6% of the injected dose of QD was contained in the liver, with ~1 and ~0.5% present in the regional lymph nodes and kidney, respectively. This study established that if PEG-coated QD were present in the dermis, the migration of the QD from the dermis could be monitored using the regional draining lymph nodes, liver, and kidney as sentinel organs. These results were very similar to those reported by Manolova et al. (2008)
following injection of virus-like particles (30 nm) and polystyrene spheres (20, 500, 1000, and 2000 nm). They found that the 20-nm virus-like particles and 20-nm polystyrene spheres migrated from the footpad (injection site) to the popliteal lymph nodes by free drainage and were taken up by macrophages, whereas the larger polystyrene spheres (500–2000 nm) were transported to the lymph nodes by dendritic cells. Fluorescence microscopic analysis of tissue sections revealed a similar pattern of initial deposition of free nanoparticles through the lymph system to the sinuses (, Gopee et al., 2007
; , Manolova et al., 2008
) where they interact with either subcapsular macrophages or lymph node resident CD8α+
The skin can be a difficult organ in which to assess penetration of drugs and materials; however, because the skin is an important first line of defense against many environmental and topically applied materials, it is very important to understand its potential as a barrier, especially to nanoscale materials. As a result, several methods have been developed to assess epidermal penetration and possible distribution to the rest of the body of drugs and chemicals: (1) application to skin in vitro and examination of skin for test material or flow-through fluid for test article; (2) application to skin in vivo and detection of the test material either (a) in the skin or (b) in sentinel organs following biodistribution.
The first method for determining skin penetration, an in vitro
–based method, involves removal of the skin from an animal, the skin is stretched over a holding apparatus (e.g., flow-through diffusion cell; Bronaugh, 2000
; Bronaugh and Stewart, 1985
), the test article applied to the epidermis side of the skin, and penetration is assessed by detecting nanoparticle presence in the epidermis or dermis, or by detection of the nanoparticle in the fluid that passed underneath the skin. Baroli et al. (2007)
studied the penetration of nanoscale tetramethylammonium hydroxide stabilized maghemite (6 nm) and sodium bis(2-ethylhexyl)sulfosuccinate stabilized iron (predominantly 5 nm) as aqueous suspensions in a diffusion cell in vitro
using full thickness human skin. Skin samples were exposed to the nanoparticles and removed, frozen, and analyzed using scanning electron microscopy with backscatter electron imaging and energy dispersive X-ray analysis (EDS). Using this method, the authors were able to demonstrate that the iron nanoparticles were contained in the stratum corneum, sometimes in the uppermost sections of the viable epidermis, and in rare cases below the stratum corneum and epidermal junction. This study clearly demonstrated the integrity of the skin barrier to a particular nanoparticle, but also demonstrated the necessity of robust methods to analyze the skin for nanoparticles (e.g., EDS).
Tinkle and colleagues (Tinkle et al., 2003
) used human skin samples, and determined the penetration of 0.5-, 1-, 2-, or 4-μm-diameter fluorescent dextran spheres into the skin following continual skin flexing (45°, 0.33 Hz). Sections of the skin were analyzed using confocal fluorescence microscopy for fluorescence particle penetration. The 0.5- and 1-μm beads penetrated to the stratum corneum and epidermal interface, with some particles penetrating into the epidermis after 30 and 60 min. Most notable, discontinuous stratum corneum allowed significantly more penetration of the particles into the epidermis.
In another study, gold nanoparticles (15, 102, and 198 nm) were suspended in 0.15M phosphate, pH 7.4, were applied to rat skin using the in vitro
skin system (e.g., Franz diffusion cell) and quantified using electron microscopy with X-ray energy dispersive spectrometry (Sonavane et al., 2008
). In 24 h, the 15-nm gold penetrated the skin to an extent that was ~125 and ~3800 times greater that of the 102- and 198-nm gold, respectively. Using constantly perfused rat intestine, at 6 h the 15-nm gold had permeated the intestines to an extent that was ~75 and ~980 times greater than the 102 and 198-nm gold, respectively.
In a study by Ryman-Rasmussen et al. (2006)
, commercially available QD were applied to porcine skin using an in vitro
flow-through diffusion cell. The QD were PEG coated (35 and 45 nm), PEG-coated amine terminated (15 and 20 nm), or PEG-coated carboxylic acid terminated (14 and 18 nm) and suspended in a borate buffer. After application, a physiological-equivalent perfusate was applied to facilitate diffusion into and through the skin. Using confocal microscopy the authors demonstrated that PEG-coated QD penetrated the intact stratum corneum and into the epidermis (Ryman-Rasmussen et al., 2006
). The amine terminated QD penetrated the stratum corneum into the epidermis, and some were found in the dermis by 8 h. The carboxylic acid terminated QD remained in the stratum corneum at 8 h, with some evidence of penetration at 24 h. These studies were extended, applying the same PEG-coated QD as used in the current study as an aqueous suspension to porcine skin in vitro
(Zhang et al., 2008b
). Using confocal fluorescence microscopy, the QD did not penetrate the stratum corneum or were found at the stratum corneum stratum granulosum interface, but were not detected in the epidermis.
Together the studies of Tinkle et al. (2003)
, Ryman-Rasmussen et al. (2006)
, Baroli et al. (2007)
, Zhang et al. (2008b)
, Rouse et al. (2007)
using fullerene derivatized peptides, and Sonavane et al. (2008)
demonstrate that nanoparticle penetration of skin is dependent on the particle size and chemistry, and that assumptions should not be made regarding skin penetration by nanomaterials. In addition, the power of the observations and conclusions regarding skin penetration is dependent on the methodology used to analyze the samples for the nanoparticles, where methods such as EDS show a tremendous ability to qualitatively measure the penetration of nanoparticles into skin. The studies by Tinkle et al. (2003)
suggested that skin integrity might provide a route of entry of particles into the epidermis.
Our results, showing that a significant percentage of the dose of topically applied QD penetrated the skin following damage (e.g., dermabrasion) and migrated to the lymph nodes and liver, is consistent with recent observations by others. Zhang and Monteiro-Riviere (2008)
applied an aqueous solution of commercially available QD to rat skin (hair clipped) that was either tape stripped or abraded by sandpaper. The penetration of the QD into the skin was assessed using confocal microscopy and there was no evidence of epidermal penetration following tape stripping; however, abrasion using sandpaper led to penetration of the QD into the dermis (Zhang and Monteiro-Riviere, 2008
). Our results using confocal fluorescence microscopy are consistent with those of Zhang and Monteiro-Riviere (2008)
, but more importantly, reinforce their microscopic observations of QD in the skin, by detecting and quantifying the distribution of QD to regional lymph nodes and liver. Zhang and Monteiro-Riviere (2008)
replicated the in vitro
skin flexing studies reported by Tinkle et al. (2003)
using PEG-coated QD and rat skin. They were not able to visualize QD penetrating into the viable epidermis following 60 min of flexing.
In a recent study, Mortensen et al. (2008)
applied carboxylic acid terminated PEG-coated QD in a 75% glycerol solution to the backs of hairless mice. The mouse skin was either normal or pretreated 1 h prior with an erythemic dose of UV (270 mJ/cm2
UVB). Using confocal fluorescence microscopy and narrow band-pass filters, Mortensen et al. (2008)
demonstrated low levels of QD penetration through the stratum corneum and into the epidermis of skin from control mice at 24 h, and the effect of ultraviolet light was to increase the extent of penetration. The presence of the QD in the epidermis was confirmed using transmission electron microscopy with EDS.
Our observations along with those of Zhang and Monteiro-Riviere (2008)
and Mortensen et al. (2008)
demonstrate that damaged skin (either dermabrasion or UV-induced damage) could be a portal for entry of nanoparticles into the viable epidermis and dermis, and our studies extend these observations to show that the penetrating particles can biodistribute to other organs, thereby increasing the exposure and potential for adverse effects.
In conclusion, we were able to quantify the dermal penetration of PEG-coated QD nanoparticles into and through the skin of mice following skin damage (removal of epidermis, dermabrasion). Skin penetration did not occur in mouse skin (which is considerably thinner than human skin) when the skin was untreated, when the stratum corneum was removed by tape stripping, or when the skin was pretreated with acetone (dry skin). Skin penetration did not occur in The QD biodistributed and were detected in the regional lymph nodes and liver. We propose that proper assessment of the dermal penetration of nanoparticles should require quantitative determination of the distribution of the nanoparticles to internal organs (e.g., sentinel lymph nodes and liver) in addition to the examination of the presence of nanoparticles in the skin (see Baroli et al., 2007
; Zhang et al., 2008b
). In order to conduct proper risk assessments for dermal exposure to nanomaterials, in addition to knowing the toxicity of the nanoparticles, the condition of the skin and the internalization of the nanoparticles need to be quantified.