Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Tracing upconversion nanoparticle penetration in human skin Zahra Khabir a,b , Anna E. Guller a,b,c,e , Vlada S. Rozova a,b,d , Liuen Liang a,b , Yi-Jen Lai b , Ewa M. Goldys a,b,e , Honghua Hu b , Karen Vickery b , Andrei V. Zvyagin b,c,d, ⁎ a ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, 2109, Australia b Macquarie University, Sydney, 2109, Australia c Sechenov University, Moscow, 119991, Russia d Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, 603022, Russia e University of New South Wales, Sydney, 2052, Australia ARTICLE INFO Keywords: Upconversion nanoparticles Cytotoxicity Transdermal delivery Penetration enhancer Background-free optical imaging Tissue engineering ABSTRACT Due to their unique optical properties upconversion nanoparticles (UCNPs) provide exceptionally high contrast for imaging of true nanoparticle distribution in excised human skin. It makes possible to show penetration of solid nanoparticles in skin treated with chemical enhancers. We demonstrated tracing upconversion nano- particles in excised human skin by means of optical microscopy at the discrete particle level sensitivity to obtain their penetration profles, which was validated by laser-ablation inductively-coupled-plasma mass-spectrometry. To demonstrate utilities of our method, UCNPs were coated with polymers, formulated in water and chemical enhancers, and applied on excised human skin mounted on Franz cells, followed by imaging using a custom-built laser-scanning microscope. To evaluate the toxicity impact on skin by polymer-coated UCNPs, we introduced a tissue engineering model of viable epidermis made of decellularized chick embryo skin seeded with keratino- cytes. UCNPs formulated in water stopped in stratum corneum, whereas UCNPs formulated in ethanol-water solution crossed stratum corneum and reached viable epidermis – hence, the enhancement efect for solid nano- particles was detected by optical microscopy. All polymer-coated UCNPs were found nontoxic within the ac- cepted safety levels. The keratinocyte resilience to polyethyleneimine-coated UCNPs was surprising considering cytotoxicity of polyethyleneimine to two-dimensional cell cultures. 1. Introduction The growing cosmetics market continues to develop sophisticated nanomaterial formulations and delivery vehicles with new functional- ities [1]. Transdermal drug delivery, an alternative to the existing oral and intravenous drug administration, represents another feld for na- nomaterials’ usage, with its simplicity, pain-free well-controlled drug release, and the absence of frst-pass drug-degradation efects [2,3]. Drug and gene delivery via nanoparticle vehicles coupled with targeting molecules, including receptor ligands, antibodies, peptides or metabo- lites represent another emerging area in pharmacology [4]. However, the ability of nanoparticles (NPs) to bring therapeutic agents to the target is limited and determined by various biological barriers pre- sented by the body. Skin, the largest organ of the body, is the most evident of these barriers and its barrier function is mainly due to its topmost layer, the stratum corneum (SC) [5,6]. Human skin SC has a thickness ranging from 10 μm to 50 μm and it consists of dead kerati- nocytes enveloped in intercellular stacked lipid bilayers, or lipid la- mellae, which are the main pathway for the difusion of exogenous li- pophilic compounds. The lipid lamellae are composed of ceramides, cholesterol, and free fatty acids [7–9]. Skin penetration of NPs occurs primarily through intercellular and/ or transappendageal routes and the physicochemical properties of NPs including size, composition, morphology, surface chemistry, and skin https://doi.org/10.1016/j.colsurfb.2019.110480 Received 24 May 2019; Received in revised form 9 August 2019; Accepted 30 August 2019 Abbreviations: NPs, Nanoparticles; SC, stratum corneum; VE, viable epidermis; EtOH, ethanol; CLSM, confocal laser scanning microscopy; TEM, transmission electron microscopy; UCNPs, upconversion nanoparticles; FT-IR, Fourier transform infrared; LA-ICP-MS, laser ablation inductively coupled plasma mass spectro- scopy; PEI, polyethyleneimine; PAA, poly(acrylic acid); PEG, poly(ethylene glycol)-amine; HaCaT, immortalized human keratinocytes cell line; ADM, acellular dermal matrix; DLS, dynamic size scattering; TECs, tissue engineering constructs ⁎ Corresponding author at: Department of Physics and Astronomy, Macquarie University, Balaclava Road, North Ryde, NSW 2109, Sydney, Australia. E-mail address: andrei.zvyagin@mq.edu.au (A.V. Zvyagin). Colloids and Surfaces B: Biointerfaces 184 (2019) 110480 Available online 05 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved. T