2.1.

NP Penetration in Skin Depends on NP Physiochemical Properties

Fluorescent dyes such as fluorescein 5-isothiocyanate (FITC) can be conjugated to NP, allowing researchers to investigate NP interactions with skin. However, traditional fluorescent dyes are not stable and may quench under the exposure of the lasers utilized with the confocal microscope. Another source of quenching may be due to the NP itself. Quantum dot (QD) NP have potential use in diagnostics, drug delivery and imaging in biomedicine or therapeutic applications due to their optical characteristics that result in strong fluorescence without photobleaching.¹⁹ QD are a very good model to determine the penetration of NP in skin. For such models, QD potential for toxicity and interactions within biological systems must be determined before nanomaterial risk assessments can be made. For imaging and optical sectioning, CLSM can precisely localize the QD (or other appropriate fluorophore-conjugated NP) within the optical sections to localize the NP in the different skin layers.

The penetration of QD through skin has been addressed by our research group employing an ex vivo porcine skin model. QD565 and QD655 have a cadmium/selenide (CdSe) core surrounded by a zinc sulfide (ZnS) shell. By TEM, QD565 are spherical with a diameter of 4.6 nm, while QD655 are ellipsoid with a size of $6\times 12{\mathrm{nm}}^2$. The hydrodynamic diameters for QD565 are 35 nm for the QD coated with polyethylene glycol (PEG), uncharged, 14 nm for the QD coated with carboxylic acid (COOH), negatively charged, and 15 nm for the QD coated with PEG-amine (${\mathrm{NH}}_2$), positively charged. The hydrodynamic diameters of the coated QD655 were 45 nm (PEG), 18 nm (COOH), and 20 nm (${\mathrm{NH}}_2$). The penetration of QD 565 and 655 with the three different coatings were investigated in porcine flow-through diffusion cells, followed by skin sectioning and CLSM imaging.¹² QD were excited by ultraviolet light or by 488 nm argon laser in CLSM; QD565 can be filtered and imaged at the emission wavelength of 565 nm, while QD655 image at the emission wavelength of 655 nm. It was noted that PEG and COOH coated QD 565 were localized within the epidermis at 8 h, while $\mathrm{PEG}\text{-}{\mathrm{NH}}_2$ coated QD 565 were localized within the dermis. QD 655 with PEG and $\mathrm{PEG}\text{-}{\mathrm{NH}}_2$ coatings were found within the epidermal layers after 8 h. In comparison, the penetration of QD655COOH coating was relatively slow and present within the epidermis at 24 h.¹² Therefore, the surface coatings can greatly influence the depth of skin penetration of the NP.

Also, we have performed in vitro flow-through cell diffusion studies with QD621, which has a different shape (nail shaped) and different shell coating (CdS shell).²⁰ At the low concentration of 1 μM, the QD621 were located primarily in the normal intact stratum corneum layers of the skin, with no fluorescence detected in the stratum granulosum, stratum spinosum, or stratum basale layers of the epidermis. At the high concentration of 10 μM (Fig. 2), QD621 were primarily present within the stratum corneum layers or between the stratum granulosum-stratum corneum interface although a focal area of fluorescence was detected in the upper epidermal layers. Occasionally, QD621 were noted in the outer root sheath of the hair follicle.¹⁵

Fig. 2

Topically applied QD621 to skin as imaged by CLSM. QD remained on the surface of the stratum corneum layer or within the outer root sheath of the hair follicle.¹⁵

A recent study with poly (amidoamine) (PAMAM) dendrimers conjugated with rhodamine B isothiocyanate reported penetration in porcine skin.²¹ The authors used a Zeiss LSM 510 Meta confocal laser scanning microscope with a 1 mW helium—neon laser at 543 nm to image the localization of PAMAM in the skin. They hypothesized that these dendrimers at smaller sizes can penetrate the skin layers more efficiently than the larger dendrimers. In addition, dendrimers with surface-modification of either acetylation or carboxylation can increase skin permeation. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. Compared to the QD studies performed in our lab, these results show differences between surface coatings and the ability to skin penetration. However, the authors claim that PAMAM dendrimers are skin-penetration enhancers, which could result in differences in skin penetration compared to QD. This illustrates that using CLSM, NP penetration in the skin is dependent on the composition, size, shape and surface coating of the NP.

2.2.

NP Penetration in Skin Depends on Skin Conditions

In the work place, a compromised skin barrier due to physical and/or chemical damage may increase dermal absorption of chemicals. The impaired skin barrier might lead not only to enhanced absorption of a specific chemical, but may increase the skin penetration and absorption of large molecules such as proteins and NP which are normally unable to penetrate intact skin.²² Additional studies investigated the penetration of QD655 and QD565 coated with COOH in rat skin for 8 and 24 h in flow-through diffusion skin cells.¹⁴ The skin was flexed, tape-stripped and abraded to determine if these mechanical actions could perturb the barrier and affect penetration. The QD655 or QD565 were localized on the surface of the stratum corneum in a homogeneous and continuous pattern, with minimal difference in QD penetration between flexed skin and intact skin. QD were also noted to be bound to the hairs. Rat skin was tape-stripped 10 times to remove the stratum corneum layers. In tape-stripped skin, QD were deposited evenly and homogeneously on the surface of the viable epidermal layers. Rat skin was abraded 60 times with sandpaper until the skin was slightly red but not bleeding. This mechanical action removed the stratum corneum layers and some of the viable epidermis so that penetration of QD was facilitated through skin. QD655 and QD565 showed slight penetration through the damaged skin into the dermis at both 8 and 24 h. Additional studies with QD in tape-stripped and intact human skin in flow-through cells found similar results, with QD penetration minimal.²³ A previous study showed a fullerene amino acid-derivatized peptide NP of 3.5 nm was capable of penetrating the dermal layers of porcine skin flexed for 60 min and placed in flow-through diffusion cells for 8 h, while nonflexed control skin showed limited penetration to the upper epidermal layers.¹³ In another in vitro skin model, the isolated perfused porcine skin flap, QD were assessed to localize distribution in the skin by CLSM. Arterial extraction of QD was spectroscopically assayed for fluorescence after infusion into the perfused skin. QD-COOH had greater tissue deposition than did QD-PEG as assessed by CLSM.²⁴ All of the above studies demonstrate that there are species differences, model differences and the condition of the skin barrier may play a role in the penetration of NP in skin.

2.3.

NP Penetration into the Skin from Top to Bottom by CLSM

Alvarez-Roman et al.⁸ investigated the skin distribution of fluorescent NP from the skin surface to deeper areas of the skin using CLMS at excitation wavelengths of 568 nm to scan the skin and at 488 nm to image the fluorescent NP, which reflected the NP interaction with the surface of the skin. This method allowed for the direct visualization of NP distribution along the skin surface and within follicular openings. Recently, investigators used a Zeiss CLSM to illuminate QD dosed on human skin in Franz static diffusion cells with a 488 nm argon excitation laser The QD were topically applied on the human skin followed by optical sectioning with the CLSM from the top (surface of the skin) to the bottom of the skin, a depth of more than 50 μm.¹⁶ This technique detected the presence of QD on the outer surface of the skin and determined whether the QD penetrated the skin. The authors concluded that some penetration occurred into the intact viable epidermis of skin by the QD-PEG with a pH 8.3, but not with QD-PEG with a pH 7.0. But with tape stripping, the stratum corneum layer was removed and QD penetrated through the viable epidermis and into the upper dermis within 24 h. This study showed that CLSM can determine the depth of NP interaction with the skin by sequentially analyzing optical sections in the stacked skin volume.

2.4.

NP Interaction with Skin Cells

In vitro studies for NP interaction with cells have been well documented in recent years. Our lab has conducted numerous experiments with NP such as QD and fullerene (${\mathrm{C}}_{60}$) derivatives to dose human epidermal keratinocytes (HEK) and analyze the NP uptake by CLSM.^25,26 Using CLSM, QD interactions with HEK is dependent upon the surface coating.^27,28 Other than the traditional detection of fluorophore conjugated NP by CLSM, MWCNT can be visualized within HEK stained with cytoskeleton antibodies in the reflection mode to allow detection of individual MWCNT in the focal plane (Fig. 3).²⁹ Additional in vitro models have utilized dendritic cells (DC) to study the cellular uptake of NP. Langerhans cells located within the epidermis of skin have similar functions to DC. They are a type of antigen-presenting immune cell for innate immunoresponse in the skin. Therefore, we established a technique using porcine monocyte-derived DC as an in vitro model. Pigs, unlike rodents, are genetically and physiologically similar to humans. Peripheral blood mononuclear cells were isolated from porcine blood by gradient centrifugation and purified to retain the CD14 positive monocytes. The monocytes were differentiated into DC with granulocyte macrophage colony-stimulating factor and interleukin-4 and then incubated with QD655-COOH. These DC were studied by CLSM and found to efficiently take up the QD655-COOH (Fig. 4).³⁰

Fig. 3

MWCNT interaction with human epidermal keratinocytes (HEK). MWCNT (yellow) at $0.1\mathrm{mg}/\mathrm{ml}$ were dosed in HEK culture and visualized in the reflection mode of CLSM. F-actin (green) was stained to visualize the cell cytoskeleton.²⁶

Fig. 4

QD interaction with monocyte-derived dendritic cells (DC). QD655-COOH at 0.05 nM were incubated with DC for 30 min and viewed in CLSM. Note that QD can be internalized into the cell very quickly.³⁰