Gold Nanoparticles

Interactions of Skin with Gold Nanoparticles of Different Surface Charge, Shape, and Functionality Rute Fernandes, Neil R. Smyth, Otto L. Muskens, Simone Nitti, Amelie Heuer-Jungemann, Michael R. Ardern-Jones, and Antonios G. Kanaras*

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP-OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R7 are found in the skin in larger quantities than polyethylene glycol-functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.

R. Fernandes, Prof. O. L. Muskens, A. Heuer-Jungemann, Dr. A. G. Kanaras Institute of Life Sciences Physics and Astronomy Faculty of Applied and Physical Sciences University of Southampton Southampton, SO171BJ, UK E-mail: [email protected] S. Nitti Istituto Italiano di Technologia Via Morego 30 16163, Genova, Italy Dr. N. R. Smyth Faculty of Natural and Environmental Sciences University of Southampton SO17 1BJ, UK Dr. M. R. Ardern-Jones Faculty of Medicine Southampton General Hospital University of Southampton Southampton, SO17 1BJ, UK DOI: 10.1002/smll.201401913

small 2014, DOI: 10.1002/smll.201401913

1. Introduction The utilization of nanoparticles in biomedicine holds potential for important developments in drug delivery, imaging, diagnosis and therapy.[1–4] This is further fueled by significant advancements in nanoparticle chemical synthesis and surface functionalization which in many cases allow pre-designing the properties of the functional nanomaterial.[5–12] Having available a rich library of nanomaterials, one of the biggest challenges is to understand the nanoparticle behavior when introduced to biological structures.[13,14] Currently many studies focus on how the morphology, charge and ligand capping of nanoparticles influence their cellular fate.[15–19] For example, in earlier work it was shown how the physicochemical characteristics of gold nanoparticles impact on the number of particles taken up or exocytosed by endothelial cells.[20,21] It was also recently discussed how functional nanoparticles can prevent or accelerate the organization of endothelial cells to create blood vessels.[22] Understanding the basic rules that govern nanoparticle-cell interactions at

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the subcellular level is of critical importance for the development of new biomedical applications exploiting inorganic nanoparticles. However, equally important and not yet wellunderstood, is how the physicochemical characteristics of nanoparticles influence their interactions with complex tissues, which extend at larger scales than the cells. A typical example of such a complex tissue structure is the skin. Understanding how the morphology, charge and function of nanoparticles influences their penetration through the different layers of the skin would lead to better design of nanoparticles and the development of new transdermal drug delivery methods. On the other hand, nanoparticle design rules to minimize skin penetration would be of great interest to the cosmetic industry and health and safety regulations in an industrial environment. Many studies have recently targeted the subject of nanoparticle-skin interactions.[23–28] For example, Sonavane et al.[29] studied the penetration of 15 nm, 102 nm and 198 nm citrate-coated gold nanoparticles through rat skin using Franz diffusion cells. Their transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), and inductively coupled plasma-optical emission spectrometry (ICP-OES) analyses showed that the penetration of gold nanoparticles through rat skin is size-independent. however the 15 nm gold nanoparticles showed higher permeation compared to the larger particles. It is noteworthy to say here that Franz diffusion cell set-ups, although very commonly used for skin experiments, result in the skin being exposed to excessive pressure and shear stress, which can affect the penetration. Krishnan et al.[30] and Filon et al.[31] induced the penetration of citrate-coated gold nanoparticles through human skin by dermaportation using a pulsed electromagnetic field and by dermabrasion, respectively. Krishnan et al.[30] concluded that 10 nm gold nanoparticles do not penetrate intact human skin; however the stratum corneum penetration was enhanced by the pulsed electromagnetic field. On the other hand, Filon et al.[31] reported on the penetration of 12.9 nm gold nanoparticles into both the epidermis and dermis of human skin. Their ICP-OES results further showed that a significantly higher gold amount was found in damaged skin compared to intact skin. Huang and co-workers showed that 5 nm polyvinylpyrrolidone (PVP)-coated gold nanoparticles are skin permeable.[32] They attributed the permeability to the nanobio interaction with skin lipids and the consequent induction of transient and reversible openings on the stratum corneum. Furthermore, when they applied a mixture of gold nanoparticles and protein drugs, both were able to penetrate the skin barrier and migrate into the deep layers. Labouta and coworkers published a number of studies on the penetration of gold nanoparticles with human skin.[33–38] Their data showed that 15 nm citrate-coated gold nanoparticles in aqueous solution tended to aggregate on the superficial stratum corneum after 24 h exposure, while 6 nm dodecanethiol-coated gold nanoparticles in toluene penetrated through the stratum corneum and into viable epidermal layers of human skin. In another study by the same group[34] the penetration of four model gold nanoparticles (15 nm citrate-coated in water, 6 nm dodecanethiol-coated in toluene, 6 nm lecithin-coated in water and 15 nm cetrimide-coated in toluene through

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human skin was investigated using multiphoton microscopy. They found that the correct skin exposure time (>6 h) was crucial in order to have a significant penetration extent for studying the effect of the different physicochemical, formulation and environmental factors. Due to the complexity of the skin penetration experiments and in some cases the lack of a systematic experimental approach, often the conclusions of different studies have been contradictory as to whether nanoparticles do or do not penetrate the skin.[39,40] Experimental parameters such as skin type and condition (e.g. intact skin, and skin chemically or mechanically treated to enhance penetration), skin surface application area, exposure time, skin maintenance during the experiment, application vehicle (e.g. solvent, emulsion) concentration and type of nanoparticles (chemical composition, size, shape and functionality) play a key role in the evaluation of nanoparticle penetration through the skin and should be carefully considered in order to obtain reliable conclusions. In this paper we report a systematic study of the interactions of gold nanoparticles with ex-vivo mouse and human skin. Utilizing complementary characterization techniques we evaluate for the first time how the penetration of gold nanoparticles through skin is influenced by the charge, morphology and function of the nanoparticles. For these experiments, we employed gold nanospheres and nanorods of a well-defined size distribution and a well-understood pegylated coating as well as spherical gold nanoparticles containing cell penetration peptides.

2. Results and Discussion 2.1. Skin Structure In general, the skin structure consists of three major layers, the epidermis (which is the top layer of the skin), dermis, and the hypodermis (see a schematic illustration and detailed explanation in Supporting Information and Figure S1). In our experiments we utilized two types of skin: Human skin explants from the breast area of an adult and skin from the back of neonatal mice. The choice of skin substrates is very important and can influence the level of penetration. Although the main skin structure is universal in mammalians, there are variations to the skin characteristics related to the species and the part of the body where skin is coming from. For example, mouse skin has a markedly thinner epidermis than human skin, but in adults has a greater number of hair follicles. The age of the subject is also critical with the skin having a higher possibility to be scarred in aged organisms. All these parameters must be taken into account when conclusions are drawn and they also determine the design of the experiment as it will be discussed later on. The human skin used in our experiments has a low density of hair follicles while the skin from the newly born mouse is in the early phases of hair follicle development. Another parameter, which plays a critical role and it is not always taken into account is the integrity of the skin during culture. When ex-vivo experiments are conducted, it is critical that the skin remains in a good condition during the course

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Interactions of Skin with Gold Nanoparticles

To assign a particular functionality to the particles we coated gold nanospheres with three different types of peptides. These are the CALNN, CALNNTat, and CALNNR7. The first batch of particles was coated only with CALNN peptide, the second with a mixed monolayer of CALNN/ CALNNT at and the third with a mixed monolayer of CALNN/CALNNR7. While CALNN is a well-studied peptide,[43] which facilitates the stabilization of gold nanoparticles, Tat and R7 have been reported to have important penetration properties.[44–46] Tat has been successfully applied for intracellular delivery of a broad variety of cargoes including various nanosized carriers as liposomes,[47] micelles,[48] and nanoparticles,[49] whilst drugs conjugated to R7 have been shown to cross along the skin barrier.[50] Schematic illustrations of the different types of particles and their physicochemical characteristics are shown in the Supporting Information (Figure S3 and Figure S4).

Figure 1. Cross-sections of viable human (A) and mouse (B) skin under a light microscope (1) and a transmission electron microscope (2). The human skin and mouse skin used in our experiments remain intact for 24 h and 6 h, respectively (Nu: Nucleus).

2.3. Experimental Configuration

2.2. Nanoparticles

The configuration of the experimental set up is very important. On one hand it must be appropriate to preserve the skin viability and structure, while on the other hand the area where the particle sample is applied must be limited to avoid leakage of nanoparticles outside of the application domain. Two different configurations, employed in our experiments, are shown in Figure 2 for the human and mouse skin (see also Supporting Information Figure S5). For the thicker human skin, the skin was placed on a microplate well at the top of the medium at 37 °C while in the case of the thinner mouse skin, it was placed in a transwell insert. In these experiments aqueous droplets of the different types of nanoparticles were applied to the skin surface within the area of an O-ring. In order to assess to which extent the different types of nanoparticles diffuse through the skin we used high initial concentrations of colloidal particles (see experimental section). The nanoparticles were incubated with

Seven different types of gold nanoparticles were employed to study their penetration through skin. Gold nanoparticles can be easily synthesized with different morphologies in narrow distributions and their surface chemistry is well-established. We systematically tuned three major nanoparticle characteristics namely: charge, shape and function. For this purpose, we synthesized gold nanospheres (NSs) with a narrow size distribution (15 nm ±1) following the Turkevich method[57] and we functionalized them with thiol containing polyethyelene glycols with either a terminal amine (positively charged particles) or a carboxylic group (negatively charged particles). High molecular weight polyethyelene glycols (5000 Da) were chosen based on their widely accepted biocompatibility. Gold nanorods (NRs) with an aspect ratio 2.8 ± 0.5 were prepared following well-established protocols.[41] For consistency, these nanorods were coated with the same types of polyethylene glycols as the gold nanospheres. To make sure that both nanorods and nanospheres had a compact layer of polyethylene glycols on their surface we performed a multistep coating as previously reported for similar systems.[42]

Figure 2. Schematic illustration and images of the experimental configuration for human (A, C) and mouse (B, D) skin, respectively. For the thinner mouse skin a transwell insert was used. The nanoparticle droplet is placed in the o-ring on the top of the skin. In both cases the skin is remained viable over the time course of the experiment.

of the experiment. Cultured skin can start to degenerate relatively fast, which can falsify the experimental observations. Thus, prior to any experiments it is important to identify the maximum incubation period for which the skin sample retains its structure. For this purpose the skin morphology was monitored for 24 h using histological experiments. In our experiments, the human skin retained its integrity for a maximum of 24 h and the mouse skin for a maximum of 6 h as shown in skin cross sections in Figure 1. For comparison, a figure where the skin has lost its integrity with signs of cellular degeneration is shown in Supporting Information (Figure S2).

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the skin for the maximum possible time that the skin retained its structure (Here, 24 h for the human skin and 6 h for the mouse skin). The selection of the longest incubation time, in which skin maintained its integrity, and the highest nanoparticle concentration applied, allowed us to draw conclusions for the maximum nanoparticle penetration through skin, under the chosen experimental conditions. It is also important to note here that our experimental configurations facilitate the suppression of vascular pressure and evaporation due to the high humidity conditions in the incubation chamber. Moreover, as the skin is fully hydrated the capillary forces are static and the movement of fluid is at equilibrium.

2.4. Characterization A powerful quantitative method for evaluating the penetration of the different types of nanoparticles through skin is the inductively coupled plasma optical emission spectometry (ICP-OES), where the skin is dissolved, decomposed and ionized and then the amount of gold content is calculated from the photoemission spectra. Prior to this type of analysis the gold nanoparticles were incubated with the skin and then the remaining nanoparticle droplet was removed. The skin was washed several times and it was subsequently tapestripped six times to ensure that any nanoparticles attached to the skin surface and the uppermost layers of the stratum corneum were removed. Thus, the ICP-OES results refer mainly to the amount of nanoparticles present in the deeper layers of the stratum corneum as well as the dermis and viable epidermis. ICP-OES results were obtained from three independent experiments for each type of nanoparticles. Figure 3 shows the percentage of nanoparticles found in the skin for nanospheres (NSs) and nanorods (NRs) of opposite charge (Figure 3a), and for the three types of peptide-coated nanoparticles in comparison to the non-functional pegylated nanospheres [(Figure 3b)-see also Supporting Information Figure S6]. As can be seen there is a clear overall trend that positively charged pegylated nanoparticles are found in the skin in higher numbers (2–6 times) than their negatively charged counterparts. This observation is in agreement with recent studies of liposomes. There, it was shown that cationic liposomes penetrate the skin more efficiently than anionic ones.[51] The enhanced skin permeation of cationic liposomes was attributed to the “Donnan exclusion effect” and is related to the more efficient interaction of cationic particles with the negatively charged skin cells. The second observation is that the percentage of NRs found in the skin is higher than for NSs, especially in mouse skin. Several studies have demonstrated an effect of nanoparticle geometry on uptake in cells, related to the radius curvature.[21,52–54] Our results hint at a similar possible contribution of particle geometry on transport through tissue. Thirdly, there is an overall trend that peptide coated NSs are found in skin in larger numbers (up to 10 times more in some cases) compared to pegylated NSs. Although ICP-OES measurements are quantitative, they do not give information related to the location of nanoparticles in the skin structure.

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Figure 3. ICP-OES measurements showing the amount of gold nanoparticles found in the skin in respect to the initial concentration of nanoparticles applied to the skin surface. In (A) pegylated gold nanospheres and gold nanorods are compared in respect to their shape and charge. In (B) the gold nanospheres are compared in respect to their function.

Therefore to qualitatively assess the spatial distribution of nanoparticles found inside the skin, we employed transmission electron microscopy and two photon photoluminescence microscopy. These imaging based methods allow identification of areas of the skin where nanoparticles can be found and together with ICP-OES offer more reliable conclusions as to the interactions of particles with the skin. To perform these techniques skin was thin-sectioned after the interaction with the nanoparticles. For TEM observations, the skin was cut to sections of 90 nm thickness. While these sections are very thin and it is not expected to see a large number of particles we can extract information about the nanoparticle location within the sectioned domain. It is worth noting here that the sectioning of the skin is a quite delicate process and needs to be performed carefully to avoid contamination of the deeper layers of the skin with nanoparticles found in the surface of the skin. For this reason, the epidermis was orientated perpendicular to the blade when cutting ultra-thin sections. Figure 4 shows different TEM images of skin thin-sections containing nanoparticles (more images are provided in the Supporting Information Figure S7 and S8). Nanoparticles were found in all layers of the skin, including the deeper layers i.e. epidermis and dermis. However, information regarding the localization of nanoparticles in certain compartments of the skin cannot be extracted from TEM because of the very small dimensions of the tissue sections. Compared to TEM sectioning, two photon photoluminescence microscopy allows characterization of thicker (14 µm)

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Interactions of Skin with Gold Nanoparticles

Figure 5. Optical and TPPL images of gold nanoparticles in sections of mouse skin. In (A) gold nanoparticles containing the CALNNR7 peptide and in (B) gold nanoparticles containing the CALNNTat peptide. The particles are shown in intense red areas and are found in all layers of the skin.

Figure 4. Representative TEM images of thin-sectioned skin after incubation with different types of spherical gold nanoparticles for human (A, B) and mouse (C, D) skin. In figure B and C the nanoparticles are coated with HS-PEG-COOH. In figure A with HS-PEG-NH2 and in figure D with CALNN/CALNNR7. All images represent nanoparticles found in the deeper stratum corneum, epidermis and dermis. Scale bar is 200 nm. (CF: Collagen Fibers, M: Mitochondrion, BL: Basal Lamina, D: Dermis, ED: Epidermis, V: Cellular Vesicle. White arrow indicates characteristic christa in mitochondria).

and larger (250 µm) sections of skin. While it does not provide detailed information on the number of particles in skin, TPPL gives a global overview of the distribution of densities of particles with micrometer resolution. For our experiment, a dedicated TPPL set up was built utilizing a femtosecond pulsed laser excitation at a wavelength of 515 nm. Compared to most TPPL studies using near-infrared laser excitation, our setup is well matched to the localized surface plasmon resonance (LSPR) of individual gold NSs. Figure 5 shows optical and TPPL images of sectioned mouse skin incubated with the nanoparticles that contain the penetrating peptides Tat and R7 (see also Supporting Information Figure S9). These types of particles were chosen due to their particular functionality and because they have been shown in our ICP-OES studies to penetrate in higher numbers through the skin. Thus, there is a particular interest about their spatial distribution within the skin structure. The dark blue background outlining the tissue corresponds to a small scattering background which was not completely suppressed by the fluorescence filter. The bright spots indicate the presence of the nanoparticles, which penetrate deep in the dermis in both cases. Organization of >10 nanoparticles into domains is shown, indicating that macroscale localization takes place in certain areas of the dermis. Some of these domains are also visible as characteristic darker regions in small 2014, DOI: 10.1002/smll.201401913

the bright field images and indicate that large densities of particles were collected into specific domains. Similar clustering of nanoparticles was found in varying amounts in sections incubated with the different types of nanoparticles, as is shown in the Supporting Information Figure S9. We emphasize that the TPPL maps represent relatively thin sections of tissue and therefore only provide qualitative information on nanoparticle localization and not on their quantitative penetration levels, as is obtained from ICP-OES. The increased penetration of Tat and R7 functionalized nanoparticles is attributed to the key role of Tat and R7 cell penetrating peptides. This is further indicated in Figure 4D which depicts peptide-coated particles inside cellular compartments. As reported elsewhere, cell penetrating peptides can penetrate through the non-viable and viable part of the skin.[55] Both samples were not tape-stripped and for this reason high densities of nanoparticles are also observed at the surface of the skin. While at this stage no conclusive answer can be given regarding the macroscale organization of nanoparticles in the deeper layers of the skin, it is well possible that these are gathered in cells, specially aimed at harvesting infiltrating specimens. Indeed, studies of intradermally-injected quantum dots have indicated evidence of translocation of nanoparticles to adjacent lymph nodes via skin macrophages and dendritic (Langerhans) cells.[56] Such translocation mechanisms could explain the observation of concentrated nanoparticle clustering deep inside the dermis layer. Our studies show that nanoparticle penetration through skin is dependent on the individual physicochemical characteristics of the nanoparticles and these parameters must be considered in future studies especially when transdermal drug delivery is chosen as an administration tool. While the skin explant experiments show a systematic trend which qualitatively confirms the functionality of advanced nanoparticle coatings, we emphasize that nanoparticle-skin interactions under in-vivo conditions may differ in the amounts of nanoparticle penetration and dynamical processes mediated by the living body.

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3. Conclusion In this work we investigated the penetration of different types of gold nanoparticles through human and mouse skin. We evaluated how the charge, shape and functionality of nanoparticles can critically influence the interaction of the nanoparticles with the different types of skin. Our systematic studies include quantitative and qualitative characterization of our samples and concluded that there is an overall trend that positively charged nanoparticles penetrated the skin in larger numbers (2–6 times) in comparison to their negatively charged counterparts. Also, it was observed that rod-shape nanoparticles are found in the skin in higher numbers than spheres, thus the morphology of the particle has to be taken into account in such studies. Moreover, there is an overall trend that the peptide-coated nanospheres employed in our experiments penetrate the skin in larger numbers (up to 10 times more in some cases) in comparison to pegylated nanospheres, highlighting the important role of cell penetrating peptides. Thin sections (90 nm) of samples under TEM show that the particles are found both in epidermis and dermis. In thick sections of skin (14 µm) TPPL studies showed that Tat and R7 containing nanoparticles migrate in large numbers in the deeper layers of the skin and present a microscale organization.

4. Experimental Section Materials and Methods: Reagents were purchased from the following suppliers: trisodium citrate, sodium tetrachloroaurate (III) dehydrate, sodium chloride, L-ascorbic acid, bis(p-sulfonatophenyl)–phenyl phosphine dehydrate dipotassium salt (BSPP), hexadecyltrimethylammonium bromide (CTAB) sodium borohydride, and tween20 were purchased from Sigma-Aldrich. Silver nitrate was purchased from Fisher Scientific. Alpha-Aminoomega-mercapto poly (ethylene glycol) hydrochloride, MW 5000 Dalton (SH-PEG-NH2) and alpha-Thio-omega-carboxy poly(ethylene glycol), MW 5000 Dalton (SH-PEG-COOH) were purchased from Iris Biotech GmbH. The peptides CALNN, CALNNTat and CALNNR7 were purchased from PeptideSynthetics. Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium, fetal bovine serum (FCS) were purchased from Life Technologies Ltd. Penicillin-Streptomycin solution and trypsin were purchased from sigma Aldrich. Osmium (VIII) Oxide was purchased from OXKEM Limited. Acetonitrile was purchased from Fisher Scientific. 0.45 µm pore size, 25 mm diameter cellulose acetate membrane syringe filter was purchased from VWR international Lda. 3.05 mm diameter Carbon coated 400 mesh Copper grids and 3.05 mm diameter Palladium coated 200 mesh Copper grids were purchased from Agar Scientific. 12 well costar transwell-clear support tissue culture treated sterile polyester membrane (0.4 um pore size, 12 mm membrane diameter) and cell culture 6-well and 96-well plates were purchased from Fisher Scientific. 3M Micropore Medical Tape and O-rings (I.D. = 4.47 mm, W = 1.76 mm) were purchase from amazon.co.uk. 26 × 76 mm 1 mm–1.2 mm thick microscope glass slides and 22 × 22mm n°1 cover glasses were purchased from VWR International. UV-visible spectra of colloidal gold nanoparticles were collected using a Cary 300 Bio UV-vis spectrophotometer

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over the range from 350 to 800 nm. Electrophoretic light scattering and dynamic light scattering measurements were obtained with a Malvern Zetasizer Nano E0248. TEM images were obtained with a FEI Technai12 Transmission electron microscope operating at a voltage of 80 kV. Ultrathin sections were cut on a Reichurt Om-U3 ultramicrotome and the glass knifes were made with a LKB knife maker. EDAX X-ray spectrometer was used on the EDS analysis (see Supporting Information). Two-photon photoluminescence (TPPL) were obtained with a TPPL microscope. ICP-OES measurements were made with an ICAP 6300 duo Spectrophotometer. Milli-Q water was used in all experiments. Synthesis of Spherical Gold Nanoparticles: Sodium citrate stabilized spherical gold nanoparticles were prepared using the Turkevich method.[57] In detail, a solution of trisodium citrate (19.5 mM, 2.5 mL) was brought to boil and quickly added into a boiling solution of sodium tetrachloroaurate (III) dihydrate (0.5 mM, 25 mL) while stirring vigorously. The colour of the solution changed from pale yellow to colourless, then to purple and finally to deep red indicating the formation of nanospheres. The reaction mixture was boiled and stirred for an additional 5 min, cooled down to room temperature while stirring and purified by filtration through a 0.45 µm syringe filter. Citrate coated gold nanoparticles were capped with BSPP via ligand exchange reaction. Basically, BSPP (10 mg; MW = 498.6) was added to a solution of citrate stabilized gold nanoparticles (27.5 mL, 4.32 nM) and the mixture was stirred overnight at room temperature. BSPP-coated nanospheres were precipitated with sodium chloride (50 mg), purified by one step centrifugation (5000 rpm, 5 min, 22 °C) and redispersed by sonication in 100 µL of Milli-Q water. Synthesis of Gold Nanorods: Gold nanorods were synthesized using an optimized seed mediated growth method.[31] In detail, a seed solution was prepared by mixing CTAB (0.2 M, 1 mL) with sodium tetrachloroaurate (III) dihydrate (5 mM, 1 mL). Then, an ice-cold solution of sodium borohydride (0.01 M, 0.5 mL) was added dropwise to mixture while stirring vigorously. The colour of the solution changed from dark yellow to colourless and then to light brown, indicating the formation of the nanospheres. The solution was stirred for 2 min and used immediately after. A growth solution was prepared by mixing an aqueous solution of CTAB (0.2 M, 14.24 mL) with sodium tetrachloroaurate (III) dihydrate (5 mM, 2 mL) and silver nitrate (5 mM, 0.18 mL) at 35 °C. While stirring at 250 rpm, a L-ascorbic acid solution (78.8 mM, 160 µL) was added to the mixture. The colour of the growth solution changes from dark yellow to colourless. 30 s after the L-ascorbic acid addition, 16 µL of the seed solution were injected into the growth solution. The solution was kept unstirred at 35 °C for 4 h. The colour of the solution changes from colourless to blue greenish over that period. As-made gold nanorods solution was purified by two steps of centrifugation (8500 rpm, 20 min, 22 °C) and redispersed in 5 mL of Milli-Q water. Surface Functionalization of Gold Nanospheres with PEGContaining Molecules: A freshly prepared SH-PEG-COOH or SHPEG-NH2 aqueous solution (5 mg/mL, 200 µL, MW = 5000 Da) was added to a solution of BSPP coated gold nanospheres (5 nM, 10 mL), while stirring. The mixture was incubated for 2 h at room temperature while shaking and then overnight at 4 °C. Functionalised gold nanoparticles were purified by three steps of centrifugation (16400 rpm, 15 min, 10 °C) and redispersed by sonication in 100 µL of Milli-Q water.

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Interactions of Skin with Gold Nanoparticles

Surface Functionalization of Gold Nanorods with PEG-Containing Molecules: A freshly prepared SH-PEG-COOH or SH-PEGNH2 aqueous solution (0.5 mg/mL, 2 ml, MW = 5000 Da) was added to a solution of gold nanorods (as-prepared, 2 mL), while stirring at 500 rpm. The mixture was sonicated for 30 s and kept overnight at 500 rpm at room temperature. After, the solution was centrifuged (8500 rpm, 16 min, 22 °C) and redispersed in a freshly prepared SH-PEG-COOH or SH-PEG-NH2 aqueous solution (0.25 mg/mL, 4 mL, MW = 5000 Da). The mixture was incubated for 4 h at room temperature while shaking at 500 rpm. Capped gold nanorods were purified by two steps of centrifugation/decantation (8500 rpm, 16 min, 22 °C) and redispersed by sonication in 100 µL of Milli-Q water. The concentration of gold nanorods was calculated by ICP-OES. Surface Functionalization of Gold Nanospheres with CALNN Peptide and CALNN Peptide Derivatives: The surface of spherical gold nanoparticles was functionalized with CALNN, 99% CALNN – 1% CALNNTat and 99% CALNN – 1% CALNNR7 peptides via the thiol group of the cysteine. In all the cases a 5000:1 peptide to nanoparticle molar ratio was used. Briefly, three solutions of BSPP coated gold nanospheres (5 nM, 5 ml) were injected with the following aqueous solutions, respectively: CALNN (0.5 mg/mL, 133.4 µL), CALNN – CALNNTat mixture [CALNN (0.5 mg/mL, 132.0 µL) + CALNNTat (0.1 mg/mL, 26.7 µL)] and CALNN – CALNNR7 mixture (CALNN (0.5 mg/mL, 132.0 µL) + CALNNR7 (0.1 mg/mL, 20.35 µL)) whilst shaking at 500 rpm. In order to avoid unspecific biding, an aqueous solution of tween 20 (1 wt%, 150 µl) was added to the solution. After 4 h shaking at room temperature, the reaction mixture was centrifuged (16400 rpm, 10 min, 22 °C) and redispersed in 5 mL of water with CALNN (0.5 mg/ml, 133.4 µL), CALNNTat (CALNN (0.5 mg/mL, 132.0 µL) + CALNNTat (0.1 mg/mL, 26.7 µL) and CALNNTat (CALNN (0.5 mg/mL, 132.0 µL) + CALNNR7 (0.1 mg/mL, 20.35 µL). An aqueous solution of tween 20 (1 wt%, 150 µL) was then added to the solution. The solutions were incubated overnight at room temperature while shaking at 500 rpm and purified from the excess peptide by 3 centrifugation steps (16400 rpm, 15 min) and redispersed in 100 µL of phosphate buffer (0.01 M, pH 7.2). Human Skin Preparation: Human full thickness skin was obtained from surgical resection after receiving written consent with approval by the Southampton and South West Hampshire Research Ethics Committee in adherence to Helsinki Guidelines (NRES 07/Q1704/59). The experiments were carried on immediately after skin was excised. The subcutaneous fat was carefully removed with a blade and the skin was washed with PBS. For the entire study skin samples from four different donors were utilized. The skin was equally divided for experiments into pieces of ∼133 mg each. Skin samples from 2 donors were used in the histological studies; 2–4 sections were investigated for each sample. Mouse Skin Preparation: The dorsal skin from newborn hairless mice was excised. C57/BL6 mice were bred at the University of Southampton (Certificate of designation number 70/2906). Tissue was removed from day old mice after euthanasia by a schedule one method following the UK Animals (Scientific Procedures) Act 1986. Briefly, a cut was made at the base of the mouse torso with sterile scissors. The scissors were then used to gently peel away the skin toward the frontal part of the mouse. Mouse skin was then washed with PBS buffer and used in further experiments. For the entire study, the skin of 22 mice was used. The skin small 2014, DOI: 10.1002/smll.201401913

was equally divided for experiments into pieces of ∼19 mg each. Skin samples from 6 mice were used in the integrity studies; 2–4 sections were investigated for each sample. Histological Analysis of Untreated Human Skin: The cleaned human skin was then evenly cut into smaller pieces and placed in a 6-well microplate and RPMI media (RPMI + 5% Penicillin/Streptimycin + 10% Foetal Calf Serum; 600 uL per well) was added in order to immerse the dermis and leave the epidermis at the airmedium interface. Samples were then incubated for 12, 24 and 48 h at 37 °C in 5% CO2/air. After incubation time, the skin was minced into ∼3–4 mm3 portions and fixed with 10% formaldehyde. The samples were then embedded in paraffin, hematoxylin and eosin stained, cut into 0.5 µm sections and observed by light microscopy. Histological Analysis of Untreated Mouse Skin: The cleaned mouse skin was evenly cut into smaller pieces and placed in 12 mm transwell insert with DMEM media (DMEM + 5% Penicillin/Streptomycin + 10% Foetal Calf Serum; 400 µL per well) in the basolateral chamber. Samples were then incubated for 6 and 24 h at 37 °C in 5% CO2/air. After incubation time, the skin was minced into ∼3–4 mm3 portions and fixed with 10% formaldehyde. The samples were then embedded in paraffin, hematoxylin and eosin stained, cut into 0.5 µm sections and observed by light microscopy. Assessment of Human Skin Penetration: The cleaned human skin was then evenly cut into smaller pieces and rubber O-rings (I.D. = 4.47 mm; W = 1.76 mm) were clamped and sealed with vaseline on the top of each piece of skin. The skin pieces were then placed in a 6-well microplate and RPMI media (RPMI + 5% Penicillin/Streptomycin + 10% Foetal Calf Serum; 600 uL per well) was added in order to immerse the dermis and leave the epidermis at the air-medium interface. Each O-ring was filled with 20 µL of gold NPs solution (100 nM for gold nanospheres and 14 nM for gold nanorods). (One of the skin pieces was used as a control where the skin was not incubated with nanoparticles and exposed to the same conditions as the NP-treated skin pieces). The skin was then incubated for 24 h at 37 °C in 5% CO2/air. After incubation, the gold NPs solution that remained in the ring was collected and the skin was washed 2 times with 20 µL of Milli-Q water. The ring was carefully removed from the skin and the skin underneath the ring was collected for characterization. Assessment of Mouse Skin Penetration: The cleaned mouse skin was evenly cut into smaller pieces and rubber O-rings (I.D. = 4.47 mm; W = 1.76 mm) were clamped and sealed with vaseline on the top of each piece of skin. The skin pieces were then placed in 12 mm transwell insert with DMEM media (DMEM + 5% Penicillin/Streptomycin + 10% Foetal Calf Serum; 400 µL per well) in the basolateral chamber. Each O-ring was filled with 20 µL of gold NPs solution (100 nM for gold nanospheres and 14 nM for gold nanorods). (As in the case of human skin, one of the skin pieces was used as a control where the skin was not incubated with nanoparticles and exposed to the same conditions as the NP-treated skin pieces). The skin was then incubated for 24 hours (human skin) 6 hours (mouse skin) at 37 °C in 5% CO2/air. After incubation, the gold NPs solution that remained in the ring was collected and the skin was washed 2 times with 20 µL of Milli-Q water. The ring was carefully removed from the skin and the skin underneath the ring was collected for characterization. TEM and EDX of Gold Nanoparticle-Treated Skin: After the 24 hours gold (for human skin) or 6 hours (for mouse skin) of

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gold nanoparticles treatment, the skin underneath the O-ring was minced into ∼3–4 mm3 pieces. For electron microscopy, tissue specimens were fixed overnight in a solution containing glutaraldehyde (3%) and formaldehyde (4%) in PIPES buffered (0.1 M, pH 7.2). Tissues were then washed twice in PIPES (0.1 M, pH 7.2) and postfixed for 1 hour in a solution containing osmium tetraoxide buffered (2%) with PIPES (0.1 M, pH 7.2). Next, tissues were washed twice in PIPES buffer (0.1 M, pH 7.2) and treated with uranyl acetate for 20 min. Tissues were dehydrated in an ascending ethanol series [30% (10 minutes), 50% (10 minutes), 75% (10 minutes), 95% (10 minutes) and 100% ethanol (twice 20 minutes)] and embedded in acetonitrile for 10 min, 50:50 acetonitrile:Spurr resin overnight and Spurr resin for 6 hours. Samples were then polymerized in fresh Spurr resin for 16 hours at 60 °C. Specimens were subsequently thin-sectioned into 90 nm slices, mounted on 400 mesh copper/palladium 3.05 mm grids and observed on a FEI Technai12 TEM, operating at 80 kV. EDX analysis of the TEM grids was performed to evaluate the gold content of the samples. ICP-OES for the Human and Mouse Skin Treated with Nanoparticles: After the skin samples were tape-stripped sic times for each case, they were dissolved in aqua regia (10%). For the human skin, a microwave digester was used to facilitate the process. Once the samples were digested, Milli-Q water was used to bring the final volume to 10 mL. Finally, ICAP 6300 duo Spectrophotometer was used to measure the amount of gold present in the skin specimens in ppm. Two-Photon Microscopy of Gold Nanoparticle-Treated Mouse Skin: After the mouse skin was treated for 6 hours with gold NP the skin samples were fixed for 24 hours in a 10% buffered formalin solution at room temperature, using 10 times the volume of fixative to the volume of specimen. Tissues were dehydrated in an ascending ethanol series (50% (1 hour), 70% (1 hour), 90% (1 hour), 100% (twice 1 hour) and 100% ethanol (twice 2 hours)) and then placed in Xylene (three times 1 hour and 30 minutes each) and then in paraffin (1 hour and 30 min followed by three times 1 hour each). Finally samples were embedded into a block al let to set for 1 hour in a freezer plate. Specimens were subsequently thin-sectioned into 14 µm slices, mounted in cover slips and let to dry overnight. Sections were then dewaxed in xylene (twice 10 minutes), 100% ethanol (twice 5 minutes), 70% ethanol (twice 5 minutes) and water (5 minutes). Slides were then dried at room temperature and coverslips were mounted on the top of the glass slides with Mowiol. Samples were imaged on a two-photon photoluminescence microscope. Illumination was done at 515 nm using the second-harmonic of a 1030 nm femtosecond laser (Pharos, Light Conversion) producing short pulses of 100 fs duration at 76 MHz repetition frequency. Emitted light was spectrally filtered using a bandpass filter between 500–510 nm to detect the antiStokes fluorescence emission mediated by the surface plasmon resonance of the gold nanospheres. Emission from a single gold nanosphere corresponds to approx. 100 counts in the image.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

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Acknowledgements The University of Southampton, Royal Society and EPSRC are gratefully acknowledged for partial support of this project. A.G.K. would also like to thank the EU COST actions MP1202, MP1005, TD1003 and TD1004 for networking opportunities associated with this work. R.F. thanks the School of Physics and Astronomy for a mayflower studentship. NRS was funded by MRC (grant number G0501515).

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Interactions of Skin with Gold Nanoparticles

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Received: June 30, 2014 Revised: August 27, 2014 Published online:

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Interactions of skin with gold nanoparticles of different surface charge, shape, and functionality.

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varyin...
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