Penetration of normal, damaged and diseased skin--an in vitro study on dendritic core-multishell nanotransporters.

COREL-07112; No of Pages 8 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

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Article history: Received 27 January 2014 Accepted 2 April 2014 Available online xxxx

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Keywords: Dendritic core–multishell nanotransporters Skin absorption Nanotoxicology Reconstructed human skin Non-melanoma skin cancer Peeling skin syndrome

Institute for Pharmacy (Pharmacology and Toxicology), Freie Universität Berlin, Berlin, Germany Institute of Chemistry and Biochemistry (Organic Chemistry), Freie Universität Berlin, Berlin, Germany c Institute of Experimental Physics, Freie Universität Berlin, Berlin, Germany d Institute of Chemistry and Biochemistry (Physical and Theoretical Chemistry), Freie Universität Berlin, Berlin, Germany e University of Cologne, Cologne Center for Genomics, Cologne, Germany f Dermatogenetics, Div. of Human Genetics, Dept. of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria g Department of Dermatology and Allergology, University Hospital RWTH Aachen, Aachen, Germany

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Nesrin Alnasif a, Christian Zoschke a, Emanuel Fleige b, Robert Brodwolf c, Alexander Boreham c, Eckart Rühl d, Katja-Martina Eckl e,f, Hans-Friedrich Merk g, Hans Christian Hennies e,f, Ulrike Alexiev c, Rainer Haag b, Sarah Küchler a, Monika Schäfer-Korting a,⁎

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A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core–multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6 h but penetrated into deeper skin layers after 24 h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter–skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases. © 2014 Published by Elsevier B.V.

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Penetration of normal, damaged and diseased skin — An in vitro study on dendritic core–multishell nanotransporters

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1. Introduction

A local approach is the favored treatment option in skin diseases as it bears low risk of systemic adverse effects, yet drug access to viable skin is only a few percent. Aiming to overcome the stratum corneum barrier more efficiently a wide variety of nanoparticles such as liposomes, lipid nanoparticles, dendritic carriers, and microemulsions have been developed and tested (for review see: [1,2]). However, today only very few drugs loaded onto such carrier systems have been introduced into the pharmaceutical market world-wide which is often due to limited stability and for safety reasons. Safety is also a matter of concern with respect to the increasing use of nanoparticles in consumer products [3,4] including cosmetics. Moreover, occupational exposure provokes concern [5].

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⁎ Corresponding author at: Freie Universität Berlin, Institute for Pharmacy (Pharmacology and Toxicology), Königin-Luise-Str. 2+4, 14195 Berlin, Germany. Tel.: + 49 30 838 53283; fax: + 49 30 838 470 871. E-mail address: [email protected] (M. Schäfer-Korting).

The magnitude of skin absorption of nanoparticles is the subject of ongoing research. Yet, the results are controversial. Some nanoparticles appear to pass the intact stratum corneum and reach the viable epidermis, whereas others obviously fail to access viable skin [6–8]. Very small particles with sizes ≤30 nm might penetrate into deeper skin layers via the intercellular route or aqueous pores of the skin [4], although, again, controversial results are being published [7]. The Scientific Committee on Consumer Products (SCCP) reviewed the likelihood of cutaneous absorption of nanoparticles [9] and summarized it as follows: There is some evidence for penetration into deeper skin layers of nanoparticles with sizes ≤ 10 nm. Nanoparticles ≥ 20 nm in size do not penetrate into viable skin layers in normal skin. Despite increasing research efforts almost nothing is known about the cutaneous absorption of nanoparticles or loaded drugs in diseased skin. Skin diseases, however, most likely influence the cutaneous absorption, especially diseases which are associated with either damages of the outermost barrier (scratching, wounding, inflammation) or those linked to disturbed epidermal differentiation resulting in a thickened stratum corneum; e.g. various forms of ichthyosis and frequent in

http://dx.doi.org/10.1016/j.jconrel.2014.04.006 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: N. Alnasif, et al., Penetration of normal, damaged and diseased skin — An in vitro study on dendritic core–multishell nanotransporters, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.006

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2. Materials and methods

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2.1. Particle preparation and characterization

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CMS nanotransporters (PG 10000 (NH 2) 0.7 (C 18 mPEG 6) 1.0 ; GPC: Mw 74,000 g/mol; Mn 92,000 g/mol) were synthesized and loaded with Nile red (0.004%; ABCR, Karlsruhe, Germany) as described previously [18]. Nile red loading was checked by UV/vis measurement (entrapment efficiency 83%). Alternatively, CMS nanotransporters were tagged by the fluorescent dye indocarbocyanine (ICC; Fig. 1A). Here, an amide coupling was chosen in order to achieve high linkage stability. For the dye-tagged CMS nanotransporters (CMS–ICC nanotransporters), hyperbranched polyglycerol amine was dissolved in methanol, ICC-N-hydroxysuccinimide ester (absorbance: 550 nm, fluorescence: 580 nm; mivenion, Berlin, Germany) was added and the mixture was stirred at room temperature for 6 h. Subsequently, 1-(2,5-dioxopyrrolidin-1-yl)-18-methoxy-poly(ethylene glycol)yl octadecanedioate was added dropwise and the mixture was stirred

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non-melanoma skin cancer. In order to reflect a disturbed barrier function, current approaches make use of stripped skin. Yet, lesional skin may be more accurately reflected by in vitro models mimicking rare or common skin diseases such as constructs reflecting autosomal recessive generalized peeling skin disease (PSD) [10], autosomal recessive congenital ichthyosis [11] and non-melanoma skin cancer (NMSC) [12]. The flexible dendrimer-type carrier, core–multishell (CMS) nanotransporters, can enhance skin penetration of loaded agents, are devoid of cytotoxicity in keratinocytes [13–15] and thus may offer the horizon for the treatment of severe and recalcitrant skin diseases, such as ichthyoses and recurrent NMSC [16,17]. Here, we compared the penetration of a loaded model dye and dye-tagged CMS nanotransporters, respectively, in normal human skin ex vivo as well as following the almost complete removal of the stratum corneum by stripping. The penetration of nanotransporters and loaded cargo in diseased skin was investigated using normal reconstructed human skin (RHS) and models of the hyperkeratotic PSD and NMSC, respectively.

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Fig. 1. Structure of CMS nanotransporters an the dye tag. A, indocarbocyanine (ICC) B, CMS–ICC nanotransporter. C, AFM height image (500 × 500 nm) and D, AFM height profile.


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2.3. Skin penetration studies, fluorescence reading

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Penetration of CMS–ICC nanotransporters or Nile red loaded CMS nanotransporters, respectively, was investigated according to previously published in vitro protocol using the static-type Franz cell setup close

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to the finite dose approach [20]. Tape stripping (n = 30 times; Tesafilm®, Beiersdorf, Hamburg, Germany) was performed in order to induce barrier impairment [22]. 20 μL/cm2 of test formulations was applied onto the skin surface and remained there for 6 h or 24 h, respectively [14]. The study of skin penetration in normal RHS and the disease models basically followed the same procedure. Here, the constructs were kept at 37 °C, 5% CO2 while still being placed in the inserts. 30 μL/cm2 of test formulation (5 g/L CMS nanotransporters in water, respectively) was applied onto the tissue surface reflecting the finite dose approach commonly used in skin penetration studies [23,24]. Exposure time was 3 h (all constructs) and 6 h (in-house constructs, two week culture), respectively. Fluorescence imaging (BZ-8000; Keyence, Neu-Isenburg, Germany) and analysis (BZ Analyzer©, Keyence, Neu-Isenburg, Germany) were performed according to previously published procedures [25]. The detection limit of CMS–ICC nanotransporters in (reconstructed) viable epidermis or dermis was 10 μg/mL.

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Human skin was obtained from 6 females who underwent abdominal reduction surgery (all patients signed written informed consent). Following removal of subcutaneous fat tissue the skin was stored at −20 °C for up to 6 months until usage [20,21]. The reconstructed normal human skin (RHS) used in these studies was EpiDermFT™ (MatTek, Ashland, MA, USA). Models of NMSC [12] and generalized PSD [10] as well as the respective normal in-house constructs were grown using fibroblasts and keratinocytes from the same donors for each disease. Briefly, for the induction of PSD in RHS, keratinocytes were transfected with an established set of three siRNA addressing the corneodesmosin gene (Stealth Select RNAi, Invitrogen, Paisley, UK; [11]). Fibroblasts (passages 2–4, 2.5 × 106 cells) mixed with 2.5 mL collagen I solution were incubated in 6 well inserts at 37 °C for 2–4 h, then normal or transfected keratinocytes (passage 2, 5 × 106/cm2 growth area) were seeded onto the dermis equivalent. The system was cultivated in keratinocyte growth medium for 24 h, raised to the air–liquid interface, and the medium was changed to a keratinocyte differentiation medium. Penetration experiments were performed 7 days after the airlift ([10]; 1-week culture). NMSC models were built according to [12]: fibroblasts (passages 1–3, 1 × 106 cells) were mixed with 4 mL collagen I solution, then normal keratinocytes (passages 2–3, 1 × 106 cells/cm2 growth area) were seeded onto the dermis equivalent and raised five days later to the air–liquid interface. Tumor growth was induced at day 14 by seeding SCC-12 cells (passage b100, 1 × 104 cells/cm2 growth area) onto the RHS. Penetration experiments were performed three days after the SCC co-culture (2-week culture). For human skin and the RHS, the stratum corneum thickness was measured using histological slices. The data are summarized in Table 1.

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Table 1 Stratum corneum thickness of human skin ex vivo +/− tape-stripping (3 donors), the disease models (PSD, NMSC) and the respective normal constructs plus EpiDermFT (2–3 batches).

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for 18 h. Methanol was evaporated and the crude residue was dissolved in water. After purification using size-exclusion chromatography the product was freeze-dried for storage upon use (yield: 95%; GPC: Mw 66,000 g/mol; Mn 91,000 g/mol). Size and polydispersity index were measured using photon correlation spectroscopy (PCS, Malvern Zetasizer ZS, Malvern Instruments, Malvern, UK) revealing nanoparticle unimers of 16 nm in size ( PDI 0.2). The unloaded CMS nanotransporter and CMS–ICC nanotransporter unimers form aggregates up to 140–160 nm (PDI 0.34) depending on the polymer concentration [18]. Aggregates of Nile red loaded CMS nanocarriers are up to 200–240 nm in size (PDI 0.18, can vary with the amount of Nile red) [19]. These aggregates are stable under normal conditions. However, when experiencing high shear stress disaggregation into unimers was observed [18,19]. Furthermore, the particles' surface was analyzed by atomic force microscopy (AFM) to gain information about their flexibility/rigidity. The AFM measurements have been performed using a Nanoscope MultiMode 8 (Veeco, now Bruker AXS, Karlsruhe). The microscope was operated in the tapping and soft-tapping mode using silicone probes PPP-NCL-R (NanoAndMore GmbH, Wetzlar) with a length of 225 μm and width of 38 μm and a tip radius of b 7 nm at resonance frequencies of 146–236 kHz under ambient conditions. The force constant was 21–98 N/m. The cantilever was forced to oscillate near its resonance frequency. The sample was prepared by spin coating (Spin Coater SCV-2) at 33 rps for 300 s on freshly cleaved mica. All images were flattened previous to height analysis using algorithms contained in the software NanoScope 8.10. Tip convolution makes lateral dimension analysis difficult.

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2.4. Fluorescence lifetime imaging microscopy (FLIM)

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Vertical slices of normal and stripped skin were also placed on a 0.17 mm thick cleaned cover glass and subjected to FLIM. The timeresolved fluorescence decay curves in each image pixel were obtained from time-correlated single photon counting within a confocal laser scanning FLIM setup. The setup consists of an Olympus IX71 inverted microscope equipped with a 40 × objective lens, a confocal scanning unit (DCS-120; Becker & Hickl, Berlin, Germany), and a Ti:sapphire laser system (Spectra Physics, Santa Clara, CA, USA) in the modelocked picosecond-pulsed regime [26,27]. The Ti:sapphire tsunami laser is pumped by a solid state Millennia V laser and the laser output is frequency doubled to obtain the excitation wavelength of 498 nm for the ICC dye. A pulse picker reduced the repetition rate to 4 MHz. An optical glass long pass filter (OG515) was placed in the emission path. Single photon counting is performed using a time correlated single photon counting module (SPC-150; Becker & Hickl, Berlin, Germany). The time range was set to 20 ns for 256 channels resulting in a channel width of 78 ps. The instrument response function (IRF) had a width of 70–100 ps (FWHM). FLIM images were analyzed using self-written routines in C++. Fluorescence decay curves were partitioned into classes (i.e. clusters) using a multivariate pattern recognition method. The fluorescence decays of the individual clusters were fitted with a sum of exponentials using χ2 minimization. False-color images were generated by assigning a distinct color to all pixels containing a fluorescence decay curve that belongs to one cluster.

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2.5. Data analysis and statistics

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The results are depicted as the mean ± SEM from 2 (EpiDermFT, PSD models plus control RHS) or 3 (human skin, NMSC models plus control RHS) independent experiments with two replicates, respectively. Statistical analysis was performed using the one-tailed Mann–Whitney U test, p ≤ 0.05 indicates statistically significant differences.

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3.1. Penetration in intact and stripped human skin

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Fluorescence analysis demonstrated that the cargo Nile red, loaded onto CMS nanotransporters, penetrated well into normal human skin after 6 h and 24 h. Profound removal of stratum corneum by tape stripping (Table 1) facilitated Nile red penetration into viable epidermis and dermis after 6 h (Fig. 2; p ≤ 0.05). After 24 h Nile red related fluorescence was enhanced by about 30% and even more in the dermis (Fig. 2). In order to determine whether the CMS nanotransporters themselves penetrate into the skin, the dye ICC was covalently bound to the CMS nanotransporter (Fig. 1); the skin penetration was tracked by fluorescence microscopy (Fig. 2) and FLIM (Fig. 3). In normal skin, the CMS–ICC nanotransporters remained exclusively in the stratum corneum and did not penetrate into deeper skin layers within 6 h. Yet, penetration into viable skin was seen after 24 h. In contrast, in stripped skin CMS–ICC nanotransporters were already found in the viable skin layers after 6 h and penetration was even more pronounced after 24 h (Fig. 2; p ≤ 0.05). This is well in line with the major barrier disruption of this skin sample.

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FLIM measurements were performed in order to obtain a more detailed insight into the CMS–ICC nanotransporter penetration and the interactions of CMS–ICC nanotransporters with the skin surface. Obvious differences in the fluorescence lifetime curves of ICC alone and of CMS–ICC nanotransporters, both in aqueous solution (Fig. 3G) and in normal skin (Fig. 3F), were detected. This observation implicated a stable dye-tag and ensured that the signals observed in the stratum corneum and viable skin layers of normal and stripped skin after 24 h incubation (Fig. 3A–C) are due to the fluorescent nanotransporters. Most interestingly, penetration through intact stratum corneum (Fig. 3A, cyan colored areas and Fig. 3D) resulted in distinct changes of the CMS–ICC fluorescence lifetime curve compared to those observed in aqueous solution (Fig. 3G) or deeper skin layers (Fig. 3A, red colored areas and Fig. 3E). We concluded that these changes in the fluorescence lifetime curve of ICC covalently bound to CMS nanotransporters indicate striking interactions of the nanotransporters with the lipids and/or proteins of the stratum corneum. These interactions differ from those experienced by the nanotransporters in deeper skin layers (Fig. 3A). The changes to the CMS nanotransporter during passage through the stratum corneum are gradually reversible with further penetration into the dermis but still persist in the viable epidermis (Fig. 3A, D). These stratum corneum specific changes to the fluorescence lifetime

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Fig. 2. Penetration in normal and stripped skin. CMS–ICC nanotransporters and Nile red (0.004%) loaded CMS nanotransporters were applied onto normal ( ) and stripped ( ) human skin ex vivo for 6 h (open columns) and for 24 h (striped columns). A, Pictures show representative overlay images (fluorescence and bright field) of the same area. B, Fluorescence intensity (arbitrary pixel brightness values, ABU) was quantified in the respective skin layers and depicted as mean ± SEM. Skin was from 3 donors, scale bar: 100 μm, *p ≤ 0.05.


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curve (Fig. 3A, cyan), however, are absent in stripped skin where the stratum corneum is removed (Fig. 3B).

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Next we investigated, if RHS can replace excised human skin for in vitro studies of nanoparticulate systems. Experiments were based on the validated, commercially available RHS (EpiDermFT) and normal in-house constructs (grown for 1 and 2 weeks) depicting normal morphology (Fig. 4) but varying in thickness of the stratum corneum. Whereas EpiDermFT has a rather thick stratum corneum, our in-house constructs are closer to normal skin (Table 1). The exposure time was adjusted to 3 h and 6 h, respectively, as in vitro skin models exhibit a higher permeability compared to normal human skin [20,21,28]. After 3 h exposure, no CMS–ICC nanotransporters were detected in viable layers of any construct (Fig. 4). Analog to normal human skin which was exposed for 24 h (Fig. 2), CMS–ICC nanotransporters were detectable in low amounts in the viable epidermis (23.7 ± 2.6 ABU) and dermis (7.7 ± 1.8 ABU) of normal RHS after 6 h. In contrast, the cargo Nile red was detected in viable layers of all constructs already after 3 h (Fig. 4). Following 6 h exposure, Nile red related fluorescence further increased to 71.8 ± 38.1 ABU and 32.4 ± 8.5 ABU in the epidermis and dermis, respectively.

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As shown in Table 1, the thickness of the stratum corneum in the reconstructed PSD model (18.5 μm) clearly exceeds the thickness of the normal 1-week construct (13.4 μm) and thus reflects the hyperkeratotic skin disease. Applying CMS–ICC nanotransporters for 3 h, the carrier did not surmount the stratum corneum, penetration into the viable epidermis and dermis was not observed (Fig. 4). Once more, the cargo Nile red (loaded onto CMS nanotransporters) did penetrate into deeper skin layers. Semiquantification of the fluorescence intensities in the respective skin layers reveals a slightly reduced Nile red penetration

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(epidermis 63.7 ABU) compared to the respective normal construct 291 (epidermis 76.4 ABU). 292

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Fig. 3. Fluorescence lifetime microscopy (FLIM) of CMS–ICC nanotransporters. CMS–ICC nanotransporters were applied onto normal and stripped human skin for 24 h. Representative overlay images (FLIM and bright field) of the same area for (A) normal and (B) stripped skin are shown. As a control, an aqueous ICC solution was applied to normal skin (C). The false color coding is based on the unique fluorescence lifetimes of CMS–ICC nanotransporters (cyan, red) or ICC (blue) in the different skin layers. D–F, The corresponding fluorescence lifetime traces are shown in the same colors as used in the overlay images. G, for comparison, the fluorescence lifetime traces of CMS–ICC nanotransporters and ICC in aqueous solution are shown. Scale bar: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Penetration in the NMSC model

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Stratum corneum thickness of the NMSC model (Table 1) is well in accordance with the control construct, yet clearly thinner than the PSD model. Other than the reference construct, we observed a remarkable penetration of CMS–ICC nanotransporters into the viable epidermis as well as into the dermal equivalent of NMSC constructs already after 3 h (Fig. 4) and even more pronounced after 6 h. After 6 h, the CMS–ICC nanotransporter related fluorescence in the viable epidermis was 93.1 ± 4.9 ABU (dermis: 7.7 ± 1.8 ABU). Also the cargo Nile red (loaded onto CMS nanoparticles) was delivered more efficiently compared to the normal construct. Nile red penetration in the NMSC model exceeded the penetration of the respective control about 3-fold (p ≤ 0.05) after 3 h and about 2-fold after 6 h (NMSC model: epidermis 114.3 ± 21.8 ABU, dermis 17.1 ± 3.3 ABU).

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4. Discussion

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The growing relevance of nanoparticles in dermatology, inevitable leading to contact with diseased skin underlines the importance of safety studies. Currently, no general conclusions can be drawn as controversial results on the magnitude of penetration of small sized particles are published [7,8,29]. The disparity of results is most likely due to different nanoparticles (e.g. size, surface properties, rigidity) and test methods [9]. Small rigid nanoparticles (20–50 (− 200) nm) do not surmount the stratum corneum [6,29], or only if massage is applied [7]. Various pathways into or through the skin are possible, such as the transepidermal or transappendageal route. Since the use of excised human skin in the Franz-cell setup excludes the transfollicular pathway and RHS are free from hair follicles we investigated selectively the transepidermal route. In this study, we focused on cutaneous absorption of CMS nanotransporters and Nile red loaded CMS nanotransporters.

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Fig. 4. Penetration in normal and diseased reconstructed skin. CMS–ICC nanotransporters and Nile red (0.004%) loaded to CMS nanotransporters were applied onto EpiDermFT ( ), NMSC model ( ; normal construct ) and PSD model ( ; normal construct ) for 3 h. A, Pictures show representative overlay images (fluorescence and bright field) of the same area. B, Fluorescence intensity (arbitrary pixel brightness values, ABU) was quantified in the respective skin layers and depicted as mean ± SEM. Constructs were from 2–3 batches, scale bar: 100 μm, *p ≤ 0.05.

4.1. Intact skin

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As expected and previously shown [14], a significant amount of Nile red penetrated into viable skin layers. CMS nanotransporters proved to deliver their cargo efficiently in viable layers of human skin. Since CMS nanotransporters are found in high amounts in the stratum corneum (Figs. 2 and 4) it appeared intriguing to study whether the soft and flexible nanoparticles themselves permeate the stratum corneum despite their molecular mass of approximately 70.000 g mol−1 and their size. The general absolute cut-off level for percutaneous absorption (800 g mol−1) holds true for skin and the constructs [14], respectively. However, after 24 h exposure for human skin ex vivo and 6 h exposure for RHS, CMS–ICC nanotransporters themselves were detected in viable skin layers. When applying the nanotransporters onto the skin, it is still ambiguous if the aggregates disintegrate into unimers or if they retain

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their agglomerate structure. This is the subject of ongoing research. In any case, the penetration of CMS nanotransporters into normal skin was unexpected, as even the size of the unimers (16 nm) is close to the cut-off level defined by the SCCP [9]. Tracking the CMS nanotransporters by conventional fluorescence microscopy and FLIM was made possible through the covalent coupling of the fluorescent dye ICC to the carrier molecule (Fig. 1). FLIM verified the passage of the stratum corneum and – importantly – allowed a first insight into the transport mechanism. Differences between the fluorescence lifetime curves in stratum corneum and deeper skin layers indicate striking interactions of the carrier with the lipids or proteins of the stratum corneum (Fig. 3). AFM measurements showed heights of below 1 nm for the CMS nanotransporters on the surface (see Fig. 1) and therefore indicates highly flexible and squeezable CMS nanotransporters which stretch out on the surface.


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Skin diseases specifically influenced the penetration of the CMS nanotransporters themselves (CMS–ICC nanotransporters) and its cargo Nile red (Fig. 4) as observed in disease models generated by gene silencing (PSD model [10]) and by co-culture with tumor cells (NMSC model [12]). The skin cultivation protocols vary in culture durations. The NMSC model is grown for two weeks, whereas the PSD model is only grown for one week. Aiming to discriminate disease associated effects from an influence of culture duration, we performed the same penetration experiments with RHS for each in vitro skin disease model. The PSD is a keratinization disorder linked to a corneodesmosin deficiency. Histopathologically, it is characterized by orthokeratotic hyperkeratosis and increased detachment of corneocytes from the granular layer. Penetration of CMS–ICC nanotransporters in viable skin layers was not seen after 3 h and slightly lower Nile red amounts in the viable layers of the hyperkeratotic skin model were detected compared to the normal constructs (Fig. 4). The permeation of standard compounds (caffeine, testosterone) applied in aqueous solution, however, exceeded the permeation of the normal constructs in a model grown from keratinocytes isolated from skin biopsies of corneodesmosin-deficient patients [10]. The differences might be due to vehicle effects or due to differences in the keratinocytes used for the model (from lesional skin and corneodesmosin knock-down, respectively). Further studies will be needed to elucidate this difference. NMSC clinically presents both hyperkeratosis and epidermal atrophy [33]. The effect of hyperkeratosis, a thicker stratum corneum but probably less functional barrier, on skin absorption is not yet investigated. Our NMSC constructs [12] did not present hyperkeratosis (Table 1) but a less functional barrier which is in accordance with the clinical signs of early actinic keratosis [34]. The penetration of the CMS–ICC nanotransporters and the cargo Nile red into the viable skin layers was significantly enhanced compared to the normal construct

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In contrast to rigid nanoparticles, the flexible CMS nanotransporters gain access to viable layers of human skin after prolonged exposure time. Cutaneous uptake of nanoparticles and agents loaded onto the nanocarriers increases significantly with skin barrier impairment due to either physical damage or due to skin diseases. Striking interactions of fluorescent nanoparticles with the skin barrier can be addressed by FLIM. For the first time, comprehensive studies on the behavior of nanoparticles and loaded agents in the severe skin disorders peeling skin disease and non-melanoma skin cancer became feasible using human cell-based skin models and revealing major differences between normal and diseased skin.

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The removal of most layers of the stratum corneum (Table 1) caused a major barrier disruption in human skin ex vivo (Fig. 2). The facilitated absorption due to physical barrier impairment following tape-stripping is well in accordance with previous studies (Figs. 2 and 3) [32]. Importantly, spatial changes in fluorescence lifetimes that are gradually reversible in normal skin and absent in stripped skin (Fig. 3B) support our hypothesis of protein or lipid interactions with CMS nanotransporters.

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This work was financially supported by the German Ministry of Education and Research Nanoderm Project (13N9062), the Deutsche Forschungsgemeinschaft (HE3119/9-1), the ERA-Net for Research Programs on Rare Diseases E-Rare-2 (01GM1201), the Leibniz Graduate School of Molecular Biophysics and the Helmholtz Virtual Institute on “Multifunctional Polymers for Medicine”. Nesrin Alnasif is a scholarship holder of the University of Damascus (Damascus, Syria). Christian Zoschke gratefully acknowledges a doctoral scholarship of the German National Academic Foundation. Provision of human skin by Dr. Uwe von Fritschen, HELIOS Klinikum Emil von Behring, Berlin is gratefully acknowledged. The performance of AFM measurements by Andrea Schulz and excellent technical assistance of Hannelore Gonska are gratefully acknowledged.

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(Fig. 4). Although the NMSC model allows to reflect the efficacy of 414 a photodynamic therapy [12], further investigations are needed to 415 prove the suitability of the NMSC model for nonclinical testing in full. 416

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Thus, currently, there are two hypotheses for skin penetration — structural flexibility and penetration enhancing effects of the nanocarriers. Further investigations will be performed to elucidate the proteins and lipids involved. Furthermore, the penetration of CMS–ICC nanotransporters in viable skin layers may be linked to a penetration enhancing effect of the carrier itself. This effect was described for less well tolerated dendrimers without a PEG shell [30]. The slow penetration of the nanoparticles, however, is not relevant for the delivery of the cargo. In the stratum corneum CMS nanotransporter aggregates release Nile red encapsulated in the particles' matrix and located between the unimers in CMS aggregates [14]. Considering the second aim of the study, our results demonstrate that RHS can replace human skin for skin absorption studies of small molecules [14] and nanoparticles. Most pronounced epidermal penetration of Nile red in constructs grown for one week compared to constructs grown for two weeks (Fig. 4) indicates a less developed barrier function in constructs grown for one week only. This can be explained by the improved order of stratum corneum lipids after two weeks which form the major penetration barrier [31]. A correlation of stratum corneum thickness with altered RHS absorption of Nile red was not observed (Fig. 4).

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Cardiac telocytes in normal and diseased hearts.

Vasa vasorum in normal and diseased arteries.

Arylamidases in normal and diseased human muscle.

In vitro modulation of normal and diseased human neutrophil function by pentoxifylline.

In vitro ablation of normal and diseased vascular tissue by a fiber-transmitted holmium laser.

The noncontact tonometer. Clinical evaluation on normal and diseased eyes.

Expression of VCAM-1 in the normal and diseased kidney.

Pattern of electrical activity in normal and diseased muscle.

Anatomic correlates of normal and diseased adenoids in children.

RPE Cell and Sheet Properties in Normal and Diseased Eyes.

A gene expression study of normal and damaged cartilage in anteromedial gonarthrosis, a phenotype of osteoarthritis.

Sigma receptors [σRs]: biology in normal and diseased states.

Carcinoembryonic antigen in normal and diseased liver tissue.

Soluble proteins in normal and diseased human brain.

Endothelial cell metabolism in normal and diseased vasculature.

Intracellular [Ca2+] in normal and diseased human myocardium.

Plasma antithrombin activity: a comparative study in normal and diseased animals.

Mineral density volume gradients in normal and diseased human tissues.

Guanylate cyclase activity in normal and diseased human muscle.

Expression of complement receptor CR2 (CD21) on human subcorneal keratinocytes in normal and diseased skin.

Radiologic evaluation of the normal and diseased posterior cervical space.

Data on expression of lipoxygenases-5 and -12 in the normal and acetaminophen-damaged liver.

Intravenous cholangiography in normal and subsequently liver-damaged dogs.

Classification of normal and diseased liver shapes based on Spherical Harmonics coefficients.