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DOI: 10.1002/ ((please add manuscript number)) Article type: Full Paper, 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* Ms Rute Fernandes, Ms Amelie Heuer-Jungemann, Prof Otto L Muskens, Dr Antonios G Kanaras Institute of Life Sciences, Physics and Astronomy, Faculty of Applied and Physical Sciences, University of Southampton, Southampton, SO171BJ, UK E-mail: a.kanaras@soton.ac.uk Mr Simone Nitti Istituto Italiano di Technologia, Via Morego 30, 16163 Genova, Italy Dr Neil R Smyth, Faculty of Natural and Environmental Sciences, University of Southampton, SO17 1BJ, UK Dr Michael R Ardern-Jones Faculty of Medicine, Southampton General Hospital, University of Southampton, Southampton, SO17 1BJ, UK Keywords: gold nanoparticles, peptides, functionality, skin penetration Abstract We investigate the interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics By systematically varying the charge, shape and functionality of gold nanoparticles, we assess the nanoparticle penetration through the different skin layers 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 penetrated into 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 Our studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R 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 can be of strong interest for new developments in transdermal administration of drugs and therapy 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 [2021] 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 the subcellular level is of critical importance for the development of new biomedical applications exploiting inorganic nanoparticles However, equally important and not yet well-understood, 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 15nm, 102nm and 198nm citrate-coated gold nanoparticles through rat skin using Franz diffusion cells Their TEM, EDS and ICP analyses showed that the penetration of gold nanoparticles through rat skin is sizeindependent however the 15nm 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 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 results further showed that a significantly higher gold amount was found in damaged skin compared to intact skin Huang and coworkers showed that 5nm PVP-coated gold nanoparticles are skin permeable [32] They attributed the permeability to the nano-bio 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 co-workers published a number of studies on the penetration of gold nanoparticles with human skin [33-38] Their data showed that 15nm citrate-coated gold nanoparticles in aqueous solution tended to aggregate on the superficial stratum corneum after 24 h exposure, while 6nm 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 (15nm citrate-coated in water, 6nm dodecanethiol-coated in toluene, 6nm lecithin-coated in water and 15nm cetrimide-coated in toluene through human skin was investigated using multiphoton microscopy They found that the correct skin exposure time (>6h) 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 or 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 exvivo 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 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 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 24h using histological experiments In our experiments, the human skin retained its integrity for a maximum of 24h and the mouse skin for a maximum of 6h as shown in skin cross sections in Figure For comparison, a figure where the skin has lost its integrity with signs of cellular degeneration is shown in supporting information (Figure S2) 2.2 Nanoparticles 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 multi-step coating as previously reported for similar systems.[42] To assign a particular functionality to the particles we coated gold nanospheres with three different types of peptides These are the CALNN, CALNNTat, and CALNNR The first batch of particles was coated only with CALNN peptide, the second with a mixed monolayer of CALNN/CALNNTat and the third with a mixed monolayer of CALNN/CALNNR 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) 2.3 Experimental configuration 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 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 oC 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 the skin for the maximum possible time that the skin retained its structure (Here, 24h for the human skin and 6h 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 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 tape-stripped 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 dermis and viable epidermis ICP-OES results were obtained from three independent experiments for each type of nanoparticles Figure shows the percentage of nanoparticles found in the skin for nanospheres (NSs) and nanorods (NRs) of opposite charge (Fig 3a), and for the three types of peptide-coated nanoparticles in comparison to the non-functional pegylated nanospheres [(fig 3b)-see also supporting info 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 not give information related to the location of nanoparticles in the skin structure 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 90nm 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 shows different TEM images of skin thinsections 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) 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 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 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 ICPMS 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 10 Table of Contents Interactions of Skin with Gold Nanoparticles of Different Surface Charge, Shape and Functionality An investigation about the interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics It is found that gold nanoparticles functionalized with cell penetrating peptides enter the skin in larger numbers than pegylated nanoparticles Rute Fernandes, Neil R Smyth, Otto L Muskens, Simone Nitti, Amelie Heuer-Jungemann, Michael R Ardern-Jones, and Antonios G Kanaras* ToC figure 28 Supporting Information 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* Table of contents: S1 Skin structure S2 TEM Images of degenerating skin after prolonged incubation S3 Schematic illustration of the different types of gold nanoparticles used in the skin penetration experiments S4 Physicochemical characterization of the different types of nanoparticles S5 The two experimental set-ups when incubating nanoparticles with skin S6 ICP-OES measurements of number of nanoparticles found in skin S7 TEM images of thin-sectioned skin with various types of nanoparticles S8 EDX analysis of TEM images of thin-sectioned skin and gold nanoparticles S9 Optical and TPPL images of mouse skin S10 References 29 S1 Skin Structure Figure S1 A schematic illustration of human skin structure (courtesy of www.medicalartist.com) The major layers of skin (epidermis, dermis,and underlying hypodermis) are shown Reprinted from www.webmd.com Copyright 2009 WebMD, LLC The skin is made up of three major layers: epidermis, dermis and hypodermis The hypodermis mainly consists of collagen and adipose tissue and its role is to insulate the body and to withstand sudden mechanical shock The dermis varies in thickness but averages of about mm and carries blood and lymphatic vessels as well as nerves innervating the skin It has an abundant loose extracellular matrix containing collagen, elastic microfilaments fibronectin and proteoglycans In addition, the dermis contains various cell types such as endothelial cells, fibroblasts, lymphocytes and macrophages These are accompanied by various other microstructures such nerves, sweat and sebaceous glands, lymph and blood vessels, hair follicles as well as fibers A major role of the dermis is to support the epidermis by providing nutrients The dermis plays also important in wound healing as it provides some tools for skin regeneration The basal lamina separates the dermis from the epidermis The 30 epidermis is the outermost layer, which contains predominantly keratinocytes but also immunological cells such as Langerhans cells, melanocytes and others such as Merkel cells The maintenance of epidermis involves the replication of active cells at the bottom layer of the epidermis, which slowly migrate to the top skin layers, differentiating and changing morphology The general transfer of fluid in vivo occurs through the skin from dermis to epidermis, however the epidermis acts as a very efficient barrier reducing fluid loss In the absence of vascular pressure and temperature gradients, combined with the high humidity environment, organ culture systems will have negligible vascular flow of fluid through the tissue.[1,2] The outermost part of the epidermis, known as the stratum corneum is constructed of enucleated keratinocytes surrounded by a lipid layer The main role of the epidermis is its barrier function providing protection from the external environment and reduction in water loss, however it also acts as a sensor for the detection of external stimuli 31 S2 TEM Images of degenerating skin after prolonged incubation Figure S2 TEM images of cross sectioned human (A) and mouse (B) skin The skin was incubated in our experimental set-up more Nuthan 24h (human skin) and 6h (mouse skin) Degeneration of skin structures (i.e disintegration of the nucleous) is clearly observed in both skin types Nu Nu 32 S3 Schematic illustration of the different types of gold nanoparticles used in the skin penetration experiments Figure S3a Schematic illustration of gold nanoparticles of different shape and charge These types of particles were used in our skin penetration experiments to assess if the charge and shape of nanoparticles influence their penetration through skin 33 Figure S3b Schematic illustration of gold nanospheres of different functionalities These types of particles were used in our skin penetration experiments to assess if particular functions on the ligand shell of nanoparticles influence their penetration through skin S4 Physicochemical characterization of the different types of nanoparticles 34 Figure S4a Physicochemical characterization of gold nanoparticles of different shape and charge (A) type of nanoparticle, (B) Relevant TEM images, (C) UV-Vis spectra, (D) net charge of each type of nanoparticles, (E) Histograms of the size distribution for spheres and nanorods Scale bars are 100 nm 35 Figure S4b Physicochemical characterization of gold nanospheres with different functionalities (A) Type of nanoparticle, (B) Representative TEM images, (C) UV-Vis spectra, (D) Histogram of the size distribution of the nanospheres Scale bars are 100 nm S5 The two experimental set-ups when incubating nanoparticles with skin 36 Figure S5 Images from different angles showing the experimental configuration when incubating nanoparticles with human (A and B) and mouse (C and D) skin The red solution in the middle of the O-ring are the nanoparticles Scale bars are cm S6 ICP-OES measurements of number of nanoparticles found in skin 37 Figure S6 ICP-OES measurements showing the calculated number of particles found in the skin For nanorods the initial concentration of incubation with the skin was 14 nM For all the types of nanospheres the initial concentration of incubation with the skin was 100 nM In (A), nanoparticles of different shape and charge are compared, while in (B) nanoparticles of similar size and shape but of different functionalities are shown S7 TEM images of thin-sectioned skin with various types of nanoparticles 38 Figure S7a TEM images of thin sections of mouse skin incubated with nanoparticles The color coded stars indicate the regions that the nanoparticles are allocated in the skin (BL: Basal Lamina, ED: Epidermis, D: Dermis) ED BL D 10 μm 39 Figure S7b TEM images of thin sections human skin incubated with nanoparticles The color coded stars indicate the regions that the nanoparticles are allocated in the skin M: CF Mitochodrion, CF: Collagen Fibers) M 10 μm 40 S8 EDX analysis of TEM images of thin-sectioned skin and gold nanoparticles Figure S8 EDX analysis of TEM thin –sectioned human skin containing gold nanoparticles The elemental analysis of the dark spots in red circles indicates the presence of gold nanoparticles In (A), AuNSs-PEG-COOH particles and in (B) AuNSs-PEG-NH2 Scale bars are 200 nm 41 S9 Optical and TPPL images of mouse skin Figure S9 Optical and TPPL images of mouse skin without nanoparticles (control) and with different types of functionalized nanoparticles Structures of particles are found across all the layers of the skin Some small bubbles in the brightfield (taken after TPPL) resulted from local excessive heating of the section by the gold nanoparticles during TPPL S10 References [1] L Steinstraesser, A Rittig, K Gevers, M Sorkin, T Hirsch, M Kesting, M Sand, S AlBenna, S Langer, H.U Steinau, and F Jacobsen, Eplasty, 2009, 9; e5 [2] E.K Turksen, Epidermal Cells – Methods in Molecular Biology, 2010, Vol 585 42