KYRIAKI EVANGELATOU Diploma in Chemical Engineering

Home , Epithelial polarity

UNIVERSITY OF PATRAS SCHOOL OF HEALTH SCIENCES DEPARTMENT OF PHARMACY

MASTER OF SCIENCE (MSc) in “DRUG DISCOVERY and DEVELOPMENT” Specialization in “Industrial Pharmaceutics”

Title: “Preparation and characterization of 3D- reconstructed human skin”

Patras 2020

ΔΙΠΛΩΜΑ ΜΕΤΑΠΤΥΧΙΑΚΩΝ ΣΠΟΥΔΩΝ στην “ΑΝΑΚΑΛΥΨΗ ΚΑΙ ΑΝΑΠΤΥΞΗ ΦΑΡΜΑΚΩΝ” Ειδίκευση στην «Βιομηχανική Φαρμακευτική»

Τίτλος: “Παρασκευή σε καλλιέργεια και χαρακτηρισμός τρισδιάστατου υποκατάστατου δέρματος ανθρώπου”

ΚΥΡΙΑΚΗ ΕΥΑΓΓΕΛΑΤΟΥ Χημικός Μηχανικός

Πάτρα 2020

Πνευματικά Δικαιώματα

Κυριακή Ευαγγελάτου Πανεπιστήμιο Πατρών, Τμήμα Φαρμακευτικής, 2020 – Με την επιφύλαξη παντός δικαιώματος Σύμφωνα με τις διατάξεις του ν.2121/1993 απαγορεύεται η αναδιατύπωση ή αναπαραγωγή του παρόντος διπλώματος μεταπτυχιακών σπουδών στο σύνολό του ή τμημάτων του με οποιονδήποτε τρόπο.

Έλεγχος πρωτοτυπίας για αποφυγή λογοκλοπής:

Η συγγραφέας του παρόντος Διπλώματος Μεταπτυχιακών Σπουδών, Κυριακή Ευαγγελάτου, φέρει αποκλειστικά την ευθύνη ότι το περιεχόμενο αυτής, στο σύνολό της, δεν αποτελεί προϊόν λογοκλοπής. Η Επιβλέπουσα Καθηγήτρια Γεωργία Σωτηροπούλου, καθώς και τα λοιπά μέλη της Τριμελούς Εξεταστικής Επιτροπής δεν φέρουν καμία ευθύνη για το περιεχόμενο του παρόντος διπλώματος Μεταπτυχιακών Σπουδών. Το κείμενο στο σύνολό του ελέγχθηκε επιτυχώς με τη διαδικτυακή εφαρμογή Turnitin που διατίθεται/συνιστάται από το Πανεπιστήμιο Πατρών για την εξακρίβωση της πρωτοτυπίας. Το αποτέλεσμα του ελέγχου Turnitin παρατίθεται στο τέλος του παρόντος Διπλώματος Μεταπτυχιακών Σπουδών ADVISORY COMMITTEE

Georgia Sotiropoulou, Professor, Supervisor Department of Pharmacy, School of Health Sciences, University of Patras

Sophia Antimisiaris, Professor Department of Pharmacy, School of Health Sciences, University of Patras

Georgios Pampalakis, Assistant Professor Department of Pharmacognosy-Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki

ΤΡΙΜΕΛΗΣ ΣΥΜΒΟΥΛΕΥΤΙΚΗ ΕΠΙΤΡΟΠΗ

Γεωργία Σωτηροπούλου, Καθηγήτρια, Επιβλέπουσα Τμήμα Φαρμακευτικής, Σχολή Επιστημών Υγείας, Πανεπιστήμιο Πατρών

Σοφία Αντιμησιάρη, Καθηγήτρια Τμήμα Φαρμακευτικής, Σχολή Επιστημών Υγείας, Πανεπιστήμιο Πατρών

Γεώργιος Παμπαλάκης, Επίκουρος Καθηγητής Τμήμα Φαρμακογνωσίας και Φαρμακολογίας, Σχολή Φαρμακευτικής, Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης

Στον Βενέδικτο και την Ασημίνα,

iii

ΕΥΧΑΡΙΣΤΙΕΣ

Η παρούσα διατριβή ΜΔΕ εκπονήθηκε στο Εργαστήριο Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων - Ομάδα Φαρμακευτικής Βιοτεχνολογίας και Μοριακής Διαγνωστικής του Τμήματος Φαρμακευτικής του Πανεπιστημίου Πατρών.

Αρχικά, ευχαριστώ θερμά την Επιβλέπουσα Καθηγήτριά μου, Κυρία Γεωργία Σωτηροπούλου για την ευκαιρία που μου έδωσε να γίνω μέλος της Ερευνητικής Ομάδας, παρόλο που προερχόμουν από ένα διαφορετικό επιστημονικό πεδίο. Την ευχαριστώ για την καθοδήγηση και τις χρήσιμες συμβουλές και υποδείξεις που μου προσέφερε για την επίλυση πολλών επιστημονικών προβλημάτων όλο τον καιρό που ήμουν στο Εργαστήριό της. Επιπλέον, την ευχαριστώ για τη πολύτιμη βοήθειά της στην συγγραφή του παρόντος ΜΔΕ και για το ευχάριστο κλίμα που δημιούργησε καθ’ όλη τη διάρκεια της συνεργασίας μας.

Θα ήθελα να ευχαριστήσω και τα υπόλοιπα μέλη της Τριμελούς Συμβουλευτικής Επιτροπής, την Καθηγήτρια Σοφία Αντιμησιάρη και τον Επίκουρο Καθηγητή Γεώργιο Παμπαλάκη για την κριτική ανάγνωση του παρόντος ΜΔΕ και τα χρήσιμα σχόλιά τους.

Επιπλέον, θα ήθελα να ευχαριστήσω ιδιαίτερα την Δρ. Ελένη Ζήνγκου για την άρτια εκπαίδευση, την υποστήριξη και τις χρήσιμες συμβουλές της. Επίσης, την ευχαριστώ θερμά για την πολύτιμη βοήθεια της τόσο στη διεξαγωγή των πειραμάτων όσο και στην τελειοποίηση του παρόντος κειμένου και φυσικά για την υπομονή και το ευχάριστο περιβάλλον συνεργασίας που δημιούργησε το διάστημα που δουλέψαμε μαζί.

Θα ήθελα να ευχαριστήσω, επίσης, την Δρ. Γκόλφω Κορδοπάτη, για τις χρήσιμες συμβουλές και τη βοήθεια της. Τέλος, ευχαριστώ τα υπόλοιπα μέλη του Εργαστηρίου, τον Υποψήφιο Διδάκτορα Ευάγγελo Μπισύρη και τις Μεταπτυχιακές Φοιτήτριες Γιαννακοπούλου Χριστίνα και Τσιαούση Ελένη για το ευχάριστο κλίμα συνεργασίας που δημιούργησαν.

Τέλος οφείλω ένα μεγάλο ευχαριστώ στους γονείς μου, Νικόλαο και Μαρία, και στα αδέρφια μου Ασημίνα, Αναστασία, Δημήτρη, Ευαγγελία, Ελισσάβετ, καθώς και στην υπόλοιπη οικογένειά μου και στους φίλους μου για την αγάπη, την υπομονή και την στήριξή τους.

TABLE OF CONTENTS

TABLE OF FIGURES ...... 5

LIST OF TABLES ...... 7

LIST OF ABBREVIATIONS ...... 9

ABSTRACT ...... 11

ΠΕΡΙΛΗΨΗ ...... 13

INTRODUCTION ...... 17

SKIN ANATOMY ...... 19

Skin structure ...... 19

Epidermal appendages ...... 22

SKIN BARRIER FUNCTION ...... 23

Epidermal differentiation ...... 23

The brick and mortar model ...... 24

SKIN PROTEASES ...... 28

Tissue kallikrein related peptidases ...... 28

HUMAN SKIN EQUIVALENTS ...... 29

Reconstructed human epidermis ...... 29

Full thickness HSEs ...... 31

Applications of RHEs and full thickness HSEs ...... 32

Applications of the 3D RHS in the study of drug permeation ...... 37

Applications of 3D RHS in irritation and corrosion ...... 39

Applications of 3D RHS in sensitization ...... 39

MICRO- AND NANO-EMULSIONS ...... 40

Properties and characteristics of micro- and nano-emulsions ...... 41

Applications of micro- and nano-emulsions in drug delivery ...... 41

STATE-OF-THE ART ...... 42

SPECIFIC AIMS OF THE STUDY ...... 43

MATERIALS, METHODS AND INSTUMENTATION ...... 44

Instruments ...... 44

Plastic or glass laboratory consumables ...... 44

Media and Reagents ...... 45

Antibodies ...... 46

Cell lines ...... 47

Micro- and nano-emulsions, oils and surfactants ...... 47

METHODS ...... 48

Cell culture ...... 48

Cell cryopreservation ...... 48

Cell viability ...... 48

Embedding tissue into paraffin blocks ...... 51

Hematoxylin and eosin staining ...... 52

Immunohistochemistry ...... 52

Neutral red uptake assay ...... 53

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ..... 54

RESULTS ...... 55

CHAPTER 1: Generation of 3D RHS ...... 55

Morphology of immortalized normal human dermal fibroblasts ...... 55

Morphology of immortalized normal human epidermal keratinocytes ...... 56

Preparation of cellularized collagen gel ...... 57

Harvesting of 3D reconstructed tissues for analysis ...... 58

CHAPTER 2: Characterization of 3D RHS ...... 59

The 3D RHS displays a microstructure very similar to normal skin tissue ...... 59

IHC assessment showed that 3D RHS mimics biochemically the native human skin ...... 59

The 3D RHS shows increased proliferation index of keratinocytes in the epidermis ...... 63

The 3D RHS maintains structural integrity similar to normal skin tissue ...... 63

Cell differentiation is normal in the 3D RHS ...... 63

Normal expression pattern of structural (corneo)desmosomal proteins in 3D

RHS ...... 64

The skin proteases KLK5, KLK6, and KLK7 are comparably expressed in 3D

RHS and native skin ...... 64

CHAPTER 3: Toxicity tests of micro- and nano-emulsions, EOs and surfactants, on iNHKs ...... 68

NRU and MTT assays revealed that EOs at concentrations 0.1 %, 0.01% have low and no cytotoxicity respectively ...... 70

Toxicity tests of surfactants on iNHKs assessed by the NRU and MTT assay, showed that the surfactants do not reduce cellular viability at specific concentrations ...... 78

Treatment with micro-emulsion at concentration 10% and 1% could significantly reduce cellular viability according to NRU assay, while treatment with micro-emulsions at concentration 0.1% does not reduce cellular viability . 82

DISCUSSION ...... 91

APPENDIX ...... 107

Immortalization of keratinocytes ...... 107

Immortalization of fibroblasts ...... 107

CURRICULUM VITAE ...... 108

TABLE OF FIGURES

Figure 1. Schematic illustration of skin structure...... 19

Figure 2. Schematic depiction of the epidermis...... 21

Figure 3. Epidermal differentiation...... 23

Figure 4. Bricks and mortar model for the structure of the SC...... 26

Figure 5. Generation of 3D-skin models...... 30

Figure 6. Histological images of RHE...... 31

Figure 7. Depiction of 3D tissue construction...... 32

Figure 8. Applications of full thickness skin models...... 33

Figure 9. Cell number calculation and cell viability estimation...... 50

Figure 10. Chemical structure of NR dye...... 54

Figure 11. Structure of both MTT and formazan...... 54

Figure 12. Immortalized normal human dermal fibroblasts...... 55

Figure 13. Immortalized normal human epidermal keratinocytes...... 56

Figure 14. Preparation of 3D RHS...... 57

Figure 15. Macroscopic appearance of 3D RHS at air-liquid interface...... 58

Figure 16. The 3D RHS forms a stratified squamous epithelium...... 60

Figure 17. Epidermis of 3D RHS is hyperproliferative...... 61

Figure 18. Immunohistochemical analysis of E-cadherin...... 62

Figure 19. Immunohistochemical analyses of skin differentiation markers...... 65

Figure 20. Immunohistochemical analyses of the structural proteins of desmosomes and corneodesmosomes...... 66

Figure 21. Immunohistochemical analyses of human KLK proteases in skin. .... 67

Figure 22. Indicative photographs of iNHKs before treatment...... 70

Figure 23. Viability testing of iNHKs following treatment with 0.1% EOs by

NRU assay...... 72

Figure 24. Viability testing of iNHKs following treatment with 0.01% EOs by

NRU assay...... 74

Figure 25. Morphology of iNHKs before treatment with EOs...... 75

Figure 26. Viability testing of iNHKs following treatment with 0.01% EOs by

MTT assay...... 76

Figure 27. Toxicity testing of surfactants on iNHKs by NRU assay...... 79

Figure 28. Toxicity testing of surfactants on iNHKs by MTT assay...... 81

Figure 29. Viability testing of iNHKs following treatment with 10% o/w micro- emulsions by NRU assay...... 83

Figure 30. Viability testing of iNHKs following treatment with 1% o/w micro- emulsions by NRU assay...... 84

Figure 31. Toxicity testing of 0.1% o/w micro and nano-emulsions on iNHKs by

NRU assay...... 86

Figure 32. Toxicity testing of 0.1% o/w micro- and nano-emulsions on iNHKs by

MTT assay...... 89

LIST OF TABLES

Table 1. Full thickness HSEs...... 34

Table 2. Reconstructed 3D skin disease models...... 35

Table 3. RHE used for drug permeation testing...... 38

Table 4. Reconstructed Full thickness skin used for drug permeation testing. ... 38

Table 5. Comparison of size, shape, stability, method of preparation and polydispersity in different kinds of emulsions...... 40

Table 6. Different micro-emulsions and their potential substances tested for in vitro cytotoxicity on iNHKs...... 69

Table 7. Viability percentage values of iNHKs treated with 0.1% EOs determined by NRU assay...... 73

Table 8. Viability percentage values of iNHKs treated with 0.01% EOs determined by NRU assay...... 73

Table 9. Viability percentage values of iNHKs treated with 0.01% EOs determined by MTT assay...... 77

Table 10. The % viability values of the surfactants tested for cytotoxicity on iNHKs determined by NRU assay...... 80

Table 11. Viability percentage values of iNHKs treated with surfactants determined by MTT assay...... 80

Table 12. Viability percentage values of iNHKs following treatment with 10%,

1% and 0.1% micro-emulsions determined by NRU assay...... 87

Table 13. Viability percentage values of iNHKs treated with 0.1% micro- and nano-emulsions determined by MTT assay...... 88

LIST OF ABBREVIATIONS

2D, two dimensional 3D, three dimensional BSA, bovine serum albumin CaCl2, calcium chloride

CO2, Carbon dioxide CE, cornified envelope CSDN, corneodesmosin DAB, 3,3-diaminobenzidine DED, de-epidermized dermis DF, dilution factor DSC1, desmocollin1 DSG1, desmoglein1 DMEM, dulbecco’s modified eagle medium DMSO, dimethyl sulfoxide DNFB, 1-fluoro-2,4-dinitrobenzene D-PBS, dulbecco’s phosphate saline EDTA, ethylenediaminetetraacetic acid EU, european union EOs, essential oils FDA, food and drug administration FBS, fetal bovine serum

H2O2, hydrogen peroxide HBSS, hank’s balanced salt solution HEEs, human epidermal equivalents HCl, hydrochloric acid HSEs, human skin equivalents HSV1, herpes simplex virus 1 HUVEC, Human Umbilical Vein Endothelial Cells PBS, phosphate-buffered saline

PGE2, prostaglandin E2 RDEB, recessive dystrophic epidermolysis bullosa RH, relative humidity

9 rhEGF, recombinant human epidermal growth factor RHS, reconstructed human skin RHE, reconstructed human epidermis RT, room temperature IHC, immunohistochemistry iNHKs, immortalized normal human keratinocytes iNHFs, immortalized normal human fibroblasts IL-1a, interleukin-1a IL-6, interleukin 6 IL-8, interleukin 8 IL-17, interleukin 17 IPM, isopropyl myristate K1, keratin 1 K5, keratin 5 K10, keratin 10 K14, keratin 14 KLK, kallikrein-related peptidase KSFM, keratinocyte serum-free medium MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide OECD, organisation for economic co-operation and development OTC, organotypic cultures o/w, oil in water SB, stratum basale SC, stratum corneum SDS, sodium dodecyl sulfate SG, stratum granulosum SP, stratum spinosum TEWL, transepidermal water loss NR, neutral red NRU, neutral red uptake NMF, natural moisturizing factor UV, ultraviolet light w/o, water in oil

ABSTRACT

Three-dimensional (3D, 3 Dimensional) reconstructed human skin equivalents (HSEs, Human Skin Equivalents), (i.e. epidermal models, 3D reconstructed full-thickness skin) are extremely valuable tools for in vitrο research, as they are scientifically valid alternatives to animal experimentation. Consequently, in 2013, testing cosmetic products and ingredients in animals was banned by the European Commission (Niehues et al., 2018). HSEs mimic the native skin, structurally and biochemically, with comparable cellular heterogeneity and structural complexity (Niehues et al., 2018). Thus, they play a key role in preclinical research for drug development (Mathes et al., 2014), while they are also absolutely essential to the cosmetics industries (Gabbanini et al., 2009). Importantly, 3D reconstructed human skin equivalents (RHS, Reconstructed Human Skin) are very useful in studying rare skin diseases, such as the recessive dystrophic epidermolysis bullosa (RDEB, Recessive Dystrophic Epidermolysis Bullosa) (Mittapalli et al., 2016) but also common skin diseases like psoriasis (Tjabringa and Bergers, 2008; Chiricozzi et al., 2014).

The present study aims to develop and characterize a 3D RHS model by use of immortalized normal human dermal fibroblasts (iNHFs, immortalized Normal Human Fibroblasts) and immortalized normal human epidermal keratinocytes (iNHKs, immortalized Normal Human Keratinocytes) and, subsequently, characterize the developed RHS, structurally and biochemically, by parallel comparison with native skin tissue. Furthermore, the generated RHS was exploited for testing cosmetic micro- and nano-emulsion formulations, as well as their constituent essential oils (EOs) and surfactants. These were initially tested here in two dimensional (2D, 2 Dimensional) cultures for potential cytotoxicity and will be subsequently evaluated in our “in-house” 3D RHS.

For generation of the 3D RHS, iNHFs were mixed with collagen and placed into a 6- well plate that allowed the intake of growth factors, hormones and nutrients suitable for the development of the 3D RHS, then, iNHKs were seeded on the reconstructed dermis. After four weeks in culture, the 3D RHS was ready to be characterized using established microscopic, histological and molecular assays.

Staining of the prepared 3D RHS with hematoxylin and eosin (H&E) showed that its microstructure mimics that of the native skin, and it had a physiological epidermal

11 architecture containing the characteristic distinct epidermal layers, i.e. the stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS) and stratum basale (SB). Consistently, normal expression of key skin proteins was confirmed by immunohistochemistry (IHC) using specific antibodies. In particular, structural proteins of desmosomes, such as desmoglein1 (DSG1), desmocolin1 (DSC1), and corneodesmosin (CDSN), as well as the characteristic cell adhesion molecule E- cadherin displayed similar expression profiles in the 3D RHS and native skin. Furthermore, proteins related to the regulation of skin’s exfoliation/desquamation, such as the epidermal proteases KLK5, KLK6, and KLK7, as well as proteins related to the skin differentiation process, such as the involucrin, loricrin and keratin 5, were similarly expressed in both 3D RHS and native skin. Elevated expression of Ki67 in 3D RHS indicated increased proliferation compared to native skin as also observed by others. Cumulatively, the developed 3D RHS mimics native skin, thus, it can be used for the evaluation of cytotoxicity and moisturizing action of cosmetic oil in water (o/w, oil in water) micro- and nano-emulsion formulations (ΕΥΔΕ ΕΤΑΚ-ΕΥΔ ΕΠΑνΕΚ ΕΣΠΑ 2014-2020/ QFytoTera-Τ1ΕΔΚ-00996).

The neutral red uptake (ΝRU, Neutral Red Uptake) assay and colorimetric MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were applied for testing the cytotoxicity of EOs, surfactants and micro- and nano-emulsions on 2D cultures of iNHKs. The majority of EOs and cosmetic micro-emulsion formulations tested were found non-cytotoxic, with values of iNHKs viability higher than 95% at the lower EOs concentration range. Therefore, their cosmetic properties, such as their moisturizing action, will be evaluated by application on the 3D RHS but also to test whether they may cause undesired skin irritation and corrosion, as part of their integrated assessment in vitro.

Keywords:

3D reconstructed human skin, histology, immunohistochemistry, microemulsions, nanoemulsions, essential oils, cytotoxicity, neutral red uptake assay, MTT assay

ΠΕΡΙΛΗΨΗ

Η καθιέρωση της χρήσης νέων, αλλά επιστημονικά τεκμηριωμένων και αξιόπιστων in vitro πειραματικών προτύπων που προσομοιάζουν καλύτερα και με ακρίβεια τους ανθρώπινους ιστούς είναι μεγάλης σημασίας. Τα τρισδιάστατα (3D, 3 Dimensional) υποκατάστατα ανθρωπίνου δέρματος (HSEs, Human Skin Equivalents), τα οποία είναι υποκατάστατα ολοκλήρου ιστού δέρματος ή μόνον της επιδερμίδας, αποτελούν πρότυπα, τα οποία προσομοιάζουν το ανθρώπινο δέρμα, δομικά και βιοχημικά, δεδομένου ότι διαθέτουν κυτταρική ετερογένεια και μιμούνται τις κυτταρικές αλληλεπιδράσεις που υφίστανται στο φυσιολογικό δέρμα (Niehues et al., 2018). Παράλληλα, ένα πολύ σημαντικό πλεονέκτημα που αποφέρουν είναι ο περιορισμός της χρήσης πειραματόζωων, των οποίων η χρήση, ειδικά στον τομέα των καλλυντικών, έχει απαγορευτεί από την Ευρωπαϊκή Επιτροπή από το έτος 2013. Συνεπώς, η ανάπτυξη τέτοιου είδους προτύπων είναι καθοριστική για τις εταιρείες καλλυντικών. Ωστόσο, τα υποκατάστατα ανθρωπίνου 3D δέρματος (RHS, Reconstructed Human Skin) συμβάλλουν, επίσης, στην προκλινική αξιολόγηση διαδερμικά χορηγούμενων φαρμακευτικών ουσιών (Mathes et al., 2014) και συνεισφέρουν παράλληλα στην μελέτη κοινών αλλά και σπανίων δερματικών ασθενειών, όπως η ψωρίαση (Tjabringa and Bergers 2008; Chiricozzi et al., 2014) και η πομφoλυγώδης επιδερμόλυση (RDEB, Recessive Dystrophic Epidermolysis Bullosa) (Mittapalli et al., 2016).

Η παρούσα μελέτη στοχεύει στην δημιουργία προτύπου 3D RHS, το οποίο προσομοιάζει το φυσιολογικό δέρμα ανθρώπου, βιοχημικά και δομικά, συγκεκριμένα εκφράζει χαρακτηριστικές πρωτεΐνες του δέρματος σε συγκρίσιμα επίπεδα, και ιστολογικά διαθέτει χορίο και διαφοροποιημένη επιδερμίδα, αντίστοιχα. Επιπλέον, σκοπός της μελέτης είναι η αξιολόγηση της κυτταροτοξικότητας μικρο- και νάνο- γαλακτωμάτων ως υποψήφιων καλλυντικών, μεμονωμένων συστατικών τους, αλλά και των διαφόρων αιθέριων ελαίων, σε δυσδιάστατες (2D, 2 Dimensional) καλλιέργειες φυσιολογικών κερατινοκυττάρων ανθρώπου που έχουν αθανατοποιηθεί (iNHKs, immortalized Normal Human Keratinocytes), ως πρόδρομο βήμα, προκειμένου να αξιολογηθούν στη συνέχεια στο σύστημα 3D RHS, οι καλλυντικές ιδιότητες, όπως η ενυδάτωση, αλλά και να ελεγχθεί κατά πόσον προκαλούν ανεπιθύμητη ερεθιστικότητα ή διάβρωση στο δέρμα.

Για την παρασκευή του συστήματος 3D RHS, χρησιμοποιήθηκαν κερατινοκύτταρα και ινοβλάστες που απομονώθηκαν από βιοψία φυσιολογικού δέρματος ανθρώπου, καθιερώθηκαν σε καλλιέργεια και αθανατοποιήθηκαν (βλ. Παράρτημα), προκειμένου να διαιρούνται χωρίς περιορισμό στον αριθμό των κυτταρικών διαιρέσεων. Αρχικά, οι αθανατοποιημένοι ινοβλάστες (iNHFs, immortalized Normal Human Fibroblasts) ενσωματώθηκαν σε κολλαγόνο σε κατάλληλο ικρίωμα που επιτρέπει την πρόσληψη αυξητικών παραγόντων, ορμονών και θρεπτικών συστατικών και, στη συνέχεια, επιστρώθηκαν με iNHKs. Μετά από περίπου 4 εβδομάδες σε καλλιέργεια λήφθηκε το RHS, το οποίο αξιολογήθηκε με καθιερωμένες μικροσκοπικές, ιστολογικές και μοριακές μεθόδους συγκρινόμενο παράλληλα με φυσιολογικό ιστό δέρματος ανθρώπου.

Παρατηρήθηκε σημαντική δομική ομοιότητα του 3D RHS με το φυσιολογικό ανθρώπινο δέρμα, η οποία επιβεβαιώθηκε μικροσκοπικά με χρώση βιοψιών με ηωσίνη και αιματοξυλίνη (H&E) και διαπιστώθηκε φυσιολογική δομή δέρματος με διακριτές στιβάδες της επιδερμίδας από το υποκείμενο χόριο. Ανοσοϊστοχημική ανάλυση της έκφρασης βασικών δομικών πρωτεϊνών των δεσμοσωμάτων, όπως η δεσμογλεΐνη 1 (DSG1), η δεσμοκολλίνη 1 (DSC1), και η κορνεοδεσμοσίνη (CDSN), έδειξε ότι το 3D στο RHS παρουσιάζει φυσιολογικά επίπεδα έκφρασης των πρωτεϊνών αυτών, χωροταξικά κατανεμημένη όπως στον φυσιολογικό ιστό δέρματος. Εν συνεχεία, προσδιορίσθηκε η χωροταξιήά έκφραση σερινοπρωτεασών της οικογένειας των καλλικρεϊνών, όπως η KLK5, η KLK6, και η KLK7, σε βιοψίες 3D RHS και σε ανθρώπινο φυσιολογικό δέρμα. Οι πρωτεάσες αυτές αποτελούν κεντρικούς ρυθμιστές της φυσιολογικής αποφολίδωσης (απολέπισης) του δέρματος. Επιπλέον, στο σύστημα 3D RHS παρατηρήθηκε φυσιολογικό προφίλ έκφρασης δεικτών διαφοροποίησης, όπως η ινβολουκρίνη, η λορικρίνη, και η κερατίνη 5 υποδεικνύοντας ότι τα κερατινοκύτταρα στο 3D RHS έχουν φυσιολογική διαφοροποίηση. Επιπλέον, διαπιστώθηκε ότι τα επίπεδα της πρωτεΐνης Ki67 είναι υψηλότερα στο 3D RHS σε σχέση με την έκφραση της Ki67 σε βιοψία φυσιολογικού δέρματος, υποδεικνύοντας αυξημένο πολλαπλασιασμό των κερατινοκυττάρων, κάτι που έχει παρατηρηθεί και από άλλους ερευνητές.

Συνοπτικά, το 3D RHS που παρασκευάσθηκε προσομοιάζει το ανθρώπινο δέρμα με βάση την δομή, την διαφοροποίηση και την έκφραση λειτουργικών μορίων, και ως εκ τούτου μπορεί να αξιοποιηθεί σε τρέχουσες μελέτες κυτταροτοξικότητας και μελέτες

των ενυδατικών ιδιοτήτων μικρο- και νανο-γαλακτωμάτων ελαίου σε νερό (o/w, oil in water) φυτικών αιθέριων ελαίων (ΕΥΔΕ ΕΤΑΚ-ΕΥΔ ΕΠΑνΕΚ ΕΣΠΑ 2014-2020 / QFytoTera-Τ1ΕΔΚ-00996).

Στη συνέχεια, υπό ανάπτυξη καλλυντικά προϊόντα εφαρμόσθηκαν σε δυσδιάστατες καλλιέργειες iNHKs και η κυτταροτοξικότητα αυτών προσδιορίσθηκε με τη μέθοδο «πρόσληψης της χρωστικής ουδέτερου ερυθρού» (ΝRU, Neutral Red Uptake), καθώς και με τη χρωματομετρική μέθοδο MTT (βρωμιούχο 3-(4,5-διμεθυλοθειαζολ-2-υλο) - 2,5-διφαινυλοτετραζολίο).

Στην πλειοψηφία τους, τα υπό ανάπτυξη καλλυντικά προϊόντα και τα συστατικά τους, βρέθηκαν μη τοξικά. Συγκεκριμένα, η βιωσιμότητα των iNHKs, έπειτα από έκθεση σε αιθέρια έλαια χαμηλών συγκεντρώσεων, ήταν 95% κατ’ ελάχιστον. Συνεπώς, η αξιολόγηση της ευερεθιστικότητας και των καλλυντικών ιδιοτήτων των προϊόντων στο σύστημα 3D RHS είναι το επόμενο βήμα στη διαδικασία ανάπτυξης και αξιολόγησης των προϊόντων αυτών.

Λέξεις κλειδιά:

Τρισδιάστατο (3D) υποκατάστατο δέρματος, ιστολογία, ανοσοϊστοχημεία, μικρογαλακτώματα, νανογαλακτώματα, αιθέρια έλαια, κυτταροτοξικότητα, πρόσληψη χρωστικής ουδέτερου ερυθρού, MTT

INTRODUCTION

SKIN ANATOMY

Skin structure

The skin is the largest organ in the human body, which separates living organisms from the environment, while providing protection against a variety of external (physical, chemical and biological) insults (Baroni et al., 2012). Skin is composed of three distinct structural layers: the epidermis, the dermis and subcutaneous tissue, as illustrated in Figure 1 (Mathes et al., 2014). The epidermis constitutes the outermost layer of the skin. Its complex structure comprises four distinct layers of keratinocytes at increasing stages of differentiation going towards the outer side of the epidermis (Eckert et al., 2005). Dermis represents the collagenous connective tissue between the epidermis and the underlying subcutaneous tissue that provides structural protection for the underlying skeletal muscles and organs, while it reinforces skin toughness.

Figure 1. Schematic illustration of skin structure. Skin is comprised of three main layers: epidermis, dermis, and subcutaneous tissue. Skin appendages, like hair, sebaceous glands, sweat glands as well as blood vessels are embedded in skin (adapted from Mathes et al., 2014).

Epidermis represents the thinner layer of the skin consisting of a complex wall of stratified squamous epithelial cells organized in four distinct sublayers that exhibit varying characteristics depending on the corresponding degree of keratinocyte differentiation. The degree of differentiation increases across the four layers of epidermis, i.e. stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC) (Figure 2).

Stratum basale comprises basal keratinocytes, the least differentiated cells of the epidermis. Part of basal keratinocytes are stem cells hooked into the dermis by hemidesmosomes that are attached to collagen at the basement membrane (Treuting et al., 2017). Basal keratinocytes divide to form partially differentiated keratinocytes, which leave the basal layer moving outwards to the skin surface. Melanocytes and Merkel cells are also found in the basal layer, where they play vital roles in several functions in situ. Melanocytes are responsible for the production of melanosomes that are transferred to the keratinocytes (Treuting et al., 2017). Melanosomes, provide protection against ultraviolet (UV) light and determine skin color (Cichorek et al., 2013). Merkel cells are involved in terminal filaments of cutaneous nerves and they are associated with light touch sensation (Wever et al., 2015).

The SS consists of keratinocytes and Langerhans cells. Keratinocytes are interconnected by desmosmomes, intercellular structures involved in cell-to-cell adhesion, i.e. protein complexes that aid adjacent cells to attach to each other as depicted in Figure 2 (Green and Jones, 1996; Ross and Christiano, 2006). Langerhans cells, on the other hand, are dendritic immune cells that are found in the middle of SS. These cells participate in immune functions of the skin as antigen-presenting cells (Holikova et al., 2001).

The SG is the layer of the epidermis where final differentiation occurs, a process known as cornification. Keratohyalin granules, which contain the main structural proteins of the SC, such as the keratins, are also found in SG. During keratinization, proteins are crosslinked by transglutaminases inside the cytoplasmic membrane to form the cornified envelope (CE) (Candi et al., 2005).

In the transition zone between SG and SC, during the cornification process, keratinocytes lose their organelles, including the nucleus, their cytoplasm appears granular and they turn into dead, flattened corneocytes. Corneocytes are linked by

20 corneodesmosomes, which are modified desmosomes containing corneodesmosin protein (CDSN) (Ovaere et al., 2009).

The SC consists of apparently 15-20 layers of corneocytes that are embedded in a lipid envelope (Eckhart et al., 2013). The outermost layer of the SC, continuously sheds corneocytes, which are constantly replaced by newly formed corneocytes. To preserve homeostasis, the process of desquamation must be tightly regulated.

In the transitional zone between the SG and SC, around the CE, lipids are extruded to form a water repelling envelope, thus, ensuring an effective permeability barrier function of the epidermis, which offers adequate protection from external insults (Denecker et al., 2008).

Figure 2. Schematic depiction of the epidermis. The SB, SG, SS, and SC are illustrated as separate layers. Starting from the bottom, basal keratinocytes continue to multiply until they reach the SC, where they eventually undergo terminal differentiation to corneocytes, i.e. dead cells without a nucleus or other organelles. Desmosomes, composed of cadherins (desmogleins 1, 3 -extracellular connectors), aid intercellular adhesion of epidermal cells, while hemidesmosomes, which consist of a variety of proteins, maintain the basement membrane-keratinocyte junction (adapted from Ross et al., 2006).

Dermis is the collagenous conjoining tissue located precisely underneath the epidermis that confers toughness and strength to the skin and indispensable for protection of internal organs. Both collagen and elastin are extracellular matrix components produced by fibroblasts (mesenchymal cells). Elastin is important for skin flexibility and elasticity, while matrix components, like proteoglycans, maintain hydration in the extracellular matrix, thus, providing viscoelasticity (Smith and Melrose, 2015). Furthermore, the dermis hosts hair roots, sebaceous glands, sweat glands, nervous and mast cells, macrophages and dermal dendrocytes. Subcutaneous tissue, which is located underneath the dermis, is composed of white fat and loose connective tissue containing adipocytes (Treuting et al., 2017).

Epidermal appendages

Epidermal appendages include sweat glands, sebaceous and mammary glands, hair, hair follicles and nails. Sweat glands, which are located inside the dermis, are classified in two types: eccrine and apocrine. Eccrine glands exist almost everywhere in human skin and are responsible for secretion of sweat. Apocrine sweat glands are located mostly in axillae and perianal areas in humans (Kurosumi et al., 1984) and are related to emotional sweating (stress, fear, pain, sexual stimulation) (Wilke et al., 2007).

Sebaceous glands are exocrine glands that produce sebum. They cover the surface of the skin except for foot soles and hand palms (James et al., 2006). They are classified into those connected with hair follicles and those found in hairless areas of the body (i.e. hairless areas of nose, eyelids, nipples, mucosal membrane of the cheeks) (Young et al., 2006). Sebum plays a vital role in epidermal structure development and in normal skin barrier function (Pilgram et al., 2001), while it provides protection against microbes by production of antioxidants on skin surface (Packer et al., 1999).

Hair follicles are self-renewing structures composed of dermal papilla, root sheaths, and the bulge region. Hair follicles are renewed by a cyclic growth procedure of three distinct phases: the growing phase, the regression phase and the resting phase. Formation of hair occurs during the anagen-growth phase by proliferation of matrix keratinocytes in the bulb, and the duration of this phase usually depends on the hair type. Proliferation of matrix cells eventually ends during the catagen-regression phase, hair growth is terminated, while hair falling occurs during the telogen-resting phase, in preparation of a new anagen phase (Everts, 2012).

SKIN BARRIER FUNCTION

Skin functions as a protective and immunological barrier to external threats (Elias and Choi, 2005). It prevents skin invasion by allergens and pathogens, it protects the organism from dehydration, it aids temperature regulation, while it diminishes the destructive effects of UV radiation. Finally, skin provides protection against physical, chemical, thermal and mechanical injuries (Wickett and Visscher, 2006).

Epidermal differentiation

The detachment of keratinocytes from the SB, induces a change in their gene expression profile under the control of transcription factors. Keratin 5 (K5) and keratin 14 (K14) are expressed by proliferating keratinocytes throughout the SB, as shown in Figure 3, while keratin 1 (K1) and keratin 10 (K10) are expressed by differentiating keratinocytes (Eckhart et al., 2013). Later in the differentiation process, keratinocytes express differentiation markers, such as involucrin, loricrin, filaggrin, as well as (corneo) desmosomal proteins, which play a vital role in the formation of the skin barrier.

Figure 3. Epidermal layers contain keratinocytes of increasing degrees of differentiation and each expresses specific markers and other functional proteins. Epidermis contains keratinocytes, which multiply within the basal layer. As differentiation occurs, keratinocytes move from the lower layers upwards to the outermost surface, becoming increasingly compacted in size and unnucleated, and they are eventually shed from the skin surface, in a process named skin desquamation. Specific proteins are expressed in each stage of epidermal differentiation, as depicted.

Keratinocytes produce keratohyalin granules in the granular layer, which are composed of filaggrin mixed with keratin fibers to prevent the disintegration of filaggrin by proteolytic enzymes (Sandilands et al., 2009). Finally, keratin-filaggrin complexes are broken down by proteolytic enzymes, such as the caspase 14. Water-preserving keratins remain inside the corneocytes, while filaggrin forms the outer surface of corneocytes. During this process, the moisture content of the skin is decreased and specific proteolytic enzymes in the SC degrade filaggrin into free amino acids (Denecker et al., 2008).

The brick and mortar model

The bricks and mortar model has been used to describe the structural organization of the SC (Figure 4). Corneocytes with their cell envelopes represent the bricks and the lipids between the corneocytes represent the mortar (Elias et al., 1983). The SC consists of intermediate filaments, structural proteins in nails, hair and skin, which partially form the cytoskeleton of nucleated cells. Keratins comprise the two largest categories of intermediate filament proteins:

• The acidic type I keratin

• The neutral to basic type II keratin

Acidic type I keratin consists of proteins with negatively charged amino acids (e.g.; aspartic or glutamic acid). Neutral to basic type II keratin mostly contain amino acids with positively charged side chains (e.g. lysine, arginine or histidine). As a result, the alpha-helices of proteins interact with each other to form a structure known as coiled- coil (Fuchs, 1995; Steinert, 1993). Coiled-coils play an incredibly vital role for the structure of keratinocytes and corneocytes, moreover, they are considered to be important for preserving cohesion. Incorrect assembly of coiled coils can result in fragile keratinocytes, which smash easily and can lead to blistering diseases (Coulombe, 1991; Fuchs, 1995). During differentiation of keratinocytes to corneocytes, the coiled coils compound to form fibrils, which are microstructures located parallel to the surface of the skin, thus, reinforcing corneocytes (Norlén et al.,1997).

Profilaggrin, the main component of keratohyalin granules, participates in aggregation of keratin coiled coils (Dale et al., 1997). Profilaggrin has no keratin binding activity and is heavily phosphorylated. During terminal differentiation, proteolytic enzymes dephosphorylate and digest profilaggrin into multiple filaggrin monomers. Free filaggrin binds to keratin intermediate filaments, thus, provoking their aggregation into macrofibrils, which are subsequently, crosslinked by transglutaminases to create a highly insoluble keratin matrix. More specifically, the CE proteins and lipids from the SC attach to this keratin matrix, which acts as a protein scaffold (Sandilands et al., 2009). Natural moisturizing factors (NMFs) play a crucial role in the physiological maintenance of SC hydration, which offers flexibility and proper desquamation. NMFs are comprised mainly of lactic acid, urea and salts (Nakagawa et al., 2004; Rawlings et al., 1994).

Keratinocytes have a water impermeable phospholipid bilayer in the lower layers of the epidermis, while at the SG, the keratinocyte membrane is modified to the resistant envelope of the corneocytes (Candi et al., 2005). Transglutaminase (TGase) 1 and 3 are enzymes of the TGase family, that are considered to take part in the development of the CE (Thacher et al., 1985). Furthermore, various proteins, like loricrin and involucrin, are involved in the crosslinking reaction and are substrates for TGases. Loricrin is a spherical protein released by keratohyalin granules, composed mainly of hydrophobic amino acids and cysteine (Candi et al., 1995). Α preliminary step for the formation of the CE is the crosslinking between involucrin and loricrin (Steinert and Marekov, 1997). Keratin fibers are crosslinked to the CE as well, while lipids - which are highly important for ensuring a normal skin barrier function (Meguro et al., 2000) - are attached to involucrin on the external layer (Candi et al., 1998; Marekov and Steinert 1998). Ceramides, cholesterol, and free fatty acids form the intercellular lamellar lipid membrane (the “mortar”) and they are produced enzymatically in the SC from glycosylceramides, sphingomyelin and phospholipids, respectively. These precursor lipids, which are included in lamellar bodies in the granular layer, are released into the intercellular space at the cornified layer, where the glycosylceramides are converted to ceramides by β-glucosylcerebrosidase (Holleran et al., 1994). Stacked lipid structures that surround corneocytes are composed of ceramides and are capable of binding water molecules in their hydrophilic region, thus, preventing movement of water out of the surface layers of the skin by creating an impermeable barrier. In the SC, phospholipases

25 break down phospholipids of the keratinocytes in the living layers, to produce fatty acids, which play a vital role in normal skin barrier function and contribute to pH acidification of the SC. The acidic pH provides defense against bacterial infections (Fluhr et al., 2001).

Lipids have two vital roles: • To preserve skin homeostasis by maintaining the cornified layer’s content. • To ensure normal transepidermal water loss (TEWL).

Corneodesmosomes are the main intercellular adhesive structures that are found in the SC and other cornified squamous epithelia (Harding, 2004). These complexes contain three proteins, i.e. desmoglein1 (DSG1), desmocollin1 (DSC1), and CDSN, in the extracellular portion (Jonca et al., 2002). CDSN is encoded by the CDSN gene. During maturation of the cornified layers, CDSN undergoes a series of cleavages, which are thought to be required for desquamation.

Figure 4. Bricks and mortar model for the structure of the SC. “Bricks” represent the corneocytes and “mortar” depicts the intercellular lamellar lipid membrane.

Mechanisms that regulate skin barrier homeostasis

The integrity of the skin barrier is indispensable for normal skin function and is maintained by tightly regulated mechanisms (i.e. hydration of the SC, calcium ions gradient in the epidermis, skin surface acidity), which are responsible for preserving homeostasis of the skin barrier. Deregulation of these regulatory mechanisms has severe consequences, such as the deterioration of skin barrier functions. An important role of the SC barrier is to regulate and control water flow to the external environment, as water is fundamental for most of the functional processes in living organisms (Feingold et al., 2007).

Physiological properties, like skin elasticity, are closely related to water retention and hydration of the SC. Reduced hydration of the SC could result in deregulated degradation. As a result, corneocytes accumulate on the skin surface as in certain skin disorders like ichthyosis vulgaris and xerosis of the skin (Rawlings AV, 2003). Decreased hydration of the SC provokes degradation of filaggrin to hygroscopic amino acids that constitute the natural moisturizing factor (NMF), which preserves water in the SC (Scott et al., 1986). Modified humidity of the SC may lead to release of proinflammatory mediators, such as interleukin-1a (IL-1), which usually promotes the progression of inflammatory skin disorders (Wood et al., 1996).

A vertical Ca2+ concentration gradient is gradually increased at the SG and decreases in SB and SS (Cornelissen et al., 2007). Changes in Ca2+ concentrations may result in upregulation of KLK5 and KLK7 expression. For example, high levels of Ca2+ are related to the expression of KLK5 and KLK7 at mRNA and protein levels providing an indication that Ca2+ deposition gradient and production of KLKs in SG are related (Pampalakis and Sotiropoulou, 2017a). SC and tight junctions in the granular layer form and maintain Ca2+ gradient (Kurasawa et al., 2007). The Ca2+ gradient is important for preserving epidermal homeostasis and vanishes upon skin barrier disruption. The skin barrier is restored in parallel with the restoration of the Ca2+ gradient. Moreover, the Ca2+ gradient is strongly related to keratinocyte differentiation (Elias et al., 2002). Finally, the acidic pH of the skin surface layers is crucial for the defensive mechanisms of the epidermal barrier and for maintenance of its integrity (Hachem et al., 2003).

SKIN PROTEASES

The epidermis contains proteases and endogenous proteases inhibitors, that are necessary for the function of the skin barrier and for desquamation (de Veer et al., 2014). Proteases are classified as aspartate-, cysteine-, glutamate-, metallo-, serine- and threonine proteases, depending on their catalytic sequences. Different proteases and inhibitors exist throughout the epidermal layers where they play major roles in strictly controlled processes, having significant importance for preserving normal barrier function and skin homeostasis. Disruption of the epidermal barrier is followed by abnormal activities of serine protease (Hachem et al., 2006).

Tissue kallikrein related peptidases

Tissue kallikrein-related peptidases (KLKs) are a family of 15 serine proteases with trypsin- or chymotrypsin-like activity. They constitute the largest group of serine proteases in humans and their genes are clustered together in a non-interrupted way on chromosome 19q13.3-13.4 (Sotiropoulou et al., 2002). In skin, they are principally produced by epidermal keratinocytes of the SG and are released in the intercellular space between SG and SC. Specifically, KLK5, is a trypsin-like protease (Yousef et al., 2003; Michael et al., 2005), which was identified in SC and it plays a crucial role in the regulation of the desquamation process (Furio et al., 2015; Ekholm et al., 2000) due to its autocatalytic activation, which leads to the activation of other epidermal proteases that form a proteolytic cascade (Brattsand et al., 2005; Pampalakis et al., 2007; Michael et al., 2006). In addition, KLK5, KLK7, KLK14 are related to the regulation of skin’s exfoliation through the gradual proteolytic cleavage of corneodesmosomes (Brattsand et al., 2005; Borgoño et al., 2007; Sotiropoulou et al., 2009). Generally, activation of KLKs result in corneocytes’ shedding as they cleave the structural proteins of corneodesmosomes. In particular KLK5 cleaves DSG1, DSC1, and CDSN, and KLK7 cleaves DSC and CDSN at pH 5.6 (Caubet et al., 2004). In addition, abnormal activities of KLKs are connected to the underlying mechanisms of several skin diseases, for instance it was observed that levels of KLK6 are elevated in psoriatic skin (Komatsu et al., 2007).

HUMAN SKIN EQUIVALENTS

Human skin equivalents (HSEs) are in vitro skin substitutes consisting of primary human skin cells and collagen. HSEs resemble native skin in terms of structure and biochemistry (Zhang and Michniak-Kohn, 2012). In 1976, one of the first explant 3D model was designed when inverted dead pig skin was utilized to establish outgrowth of keratinocytes (Freeman et al., 1976). This method was further improved by utilization of collagen matrices to culture keratinocytes at the air-liquid interface (Lillie et al., 1980).

Ponec et al. have upgraded this DeEpiDermized method (DED) to culture keratinocytes that attach to the existing basement membrane (Ponec et al., 1988). During the eighties, culture protocols were improved and, as a result, many different types of HSEs and human epidermal equivalents (HEEs) were described, designated as organotypic cultures, skin substitutes or living skin equivalents, with different types of dermal substrates (inert filters, DED, collagen matrices etc.) (Figure 5). Extensive efforts for the development of HSEs led to numerous clinical products and novel skin models for both pharmaceutical and cosmetic companies. These skin models are essential for preclinical drug development as they are suitable for testing drug safety and efficacy with higher accuracy when compared to 2D cell cultures. Actually, the environment of cells in 3D cultures mimics better the in vivo state (Sceats, 2010), moreover this HSEs are used for basic research and toxicology screening (Flaten et al., 2015; Kandárová et al., 2004 and 2006). In many cases, they reduce the need for experiments in animals.

Reconstructed human epidermis

Reconstructed human epidermis (RHE) is composed of differentiated epidermal keratinocytes seeded on acellular inert filter substrates. Several commercial RHEs are available, e.g. SkinEthic™ (Episkin, France), made of normal human keratinocytes (NHKs) cultured on an inert polycarbonate filter. Episkin™ (Episkin, France) and EpiDerm™ (MatTek, Ashland, USA) produce RHEs composed of NHKs cultured on a collagen matrix (Figure 6). These models yield a stratified epidermis that expresses epidermal differentiation markers such as keratin 1 (K1), loricrin and filaggrin, as well as skin lipids like phospholipids, cholesterol, triglycerides and ceramides.

Figure 5. Generation of 3D skin models. Epidermis and dermis can be separated from a skin biopsy to isolate keratinocytes and fibroblasts, required to produce a 3D skin model. Keratinocytes can be seeded on three different type of matrixes: fibroblast-collagen matrix, acellular matrix (DED or inert plastic filter). Approximately 14 days later, a multilayered stratified epithelium is formed that mimics native human skin.

RHEs systems have been approved for in vitro skin irritation and corrosion studies, however, their usage for permeation testing in vitro is quite ambiguous because of the inferior barrier properties, compared to normal skin (Schäfer-Korting et al., 2008). Several studies were carried out to identify whether permeation testing is validated for skin equivalents. It was shown that normal human epidermis exhibits lower permeability compared to RHEs (Schmook et al., 2001; Zghoul et al., 2001). However, a validation study involving permeability testing of ten substances through RHE models (Episkin, EpiDerm™ and SkinEthic™) showed that RHE models mimic the permeation through human epidermis better than any other HSE (Schäfer-Korting et al., 2008).

Figure 6. Histological images of RHEs. Depiction of commercially available HSEs. SkinEthicTM (Episkin, France) is an in vitro RHE from NHKs cultured on an inert polycarbonate filter at the air-liquid interface. T-Skin™ (Episkin, France) consists of NHKs and NHFs, EpiDerm™ (MatTek, Ashland, USA) consists of NHKs cultured on tissue culture inserts (adapted from https://www.episkin.com, https://www.mattek.com/products/epiderm/).

Full-thickness HSEs

Human full-thickness skin models are more complex than RHEs, as a result they mimic native skin much better. They consist of keratinocytes cultured in dermal substrate mixed with fibroblast to form both epidermis and dermis. A basic protocol for the construction of a 3D RHS has been described from Carlson et al. (2008). According to this protocol a 3D model is composed of a stratified epithelium with differentiated keratinocytes that are seeded in a contracted matrix of collagen full of dermal fibroblasts as depicted in Figure 5. First, an acellular layer of collagen is constructed to help cellular collagen to attach. Then, cellular collagen populated with dermal fibroblasts is constructed and allowed to attach for approximately seven days, immersed in medium. When the matrix is stabilized, keratinocytes are seeded on the matrix surface and attach to the collagen substrate to produce a confluent cellular monolayer that will initiate the stratification of the tissue. Finally, as illustrated in Figure 7, tissues are exposed to an air-liquid interface, in order to complete stratification along with full morphological and biochemical differentiation (Carlson et al., 2008).

Figure 7. Depiction of 3D tissue construction. A, an acellular layer of collagen is constructed to help cellular collagen to attach B, a collagen gel full of dermal fibroblasts is constructed above the acellular layer. C, after seven days submerged in medium, fibroblasts remodel the matrix, causing the contraction away from the walls of insert forming a plateau. D, keratinocytes are seeded in surface of the matrix and attach to the collagen to create a confluent cellular monolayer that will form the SB of the tissue E, tissues are exposed to an air-liquid interface to complete stratification and full morphological and biochemical differentiation. F, further exposure to air-liquid interface in cornification medium produces SS, SG and SC.

In the recent years, quite sophisticated models of full-thickness HSEs have been developed, which incorporate Langerhans cells (MUTZ-3), neuron cells and melanocytes (Table 1). Moreover, models studying skin pigmentation and wound healing (Table 1) as well as skin-disease models (Table 2) have also been reported.

Applications of RHEs and full-thickness HSEs

There is a variety of in vitro applications for RHEs and full-thickness HSEs that range from investigation of cell interactions, cellular pathways, and mechanisms of disease, to studying drug permeation (Figure 8).

Reconstructed human 3D skin models of diseased skin

The 3D RHS models of diseased skin, include melanoma progression model, psoriasis model, wound healing model (Coolen et al., 2008) etc. Moreover, infections like Candida albicans, have been studied in RHE (Johannes et al., 2002) and herpes simplex virus 1 (HSV1) infections have been studied in full-thickness HSE model (Hogk et al., 2013). The majority of the reconstructed human 3D skin models of diseased skin are summarized in Table 2.

Figure 8. Applications of full-thickness skin models. Depiction of HSEs recapitulating human diseases like HSV1 infections model (adapted from Hogk et al., 2013), psoriatic model (adapted from Jean et al., 2009), epidermolysis bullosa model (adapted from Itoh et al., 2011), melanoma progression model (adapted from Li et al., 2011), atopic dermatitis (adapted from Rouaud-Tinguely et al., 2015) and wound healing (adapted from Rouaud-Tinguely et al., 2015) as well as applications of 3D RHS in drug permeation (adapted from Ackermann et al., 2010) and skin pigmentation (adapted from Duval et al., 2012).

Table 1. Full-thickness HSEs.

Epidermal cells and dermis substrate used to form HSEs for several applications.

Dermal cells Dermal cells Epidermal substrate Dermis Utilization Reference

Primary human human Primary MUTZ keratinocytes; LC 3 dermal human Primary fibroblasts Collagen cutaneous Study response immune 2011 al., et Laubach

HaCaT HaCaT human Primary fibroblasts Collagen model disease HSV 2013 al., et Hogk

Primary human human Primary keratinocytes human Primary human fibroblasts; melanocytes 1 type Bovine collagen skin Study pigmentation 2012 al., et Duval

Primary human human Primary keratinocytes fibroblasts Human 1 type Bovine collagen healing Wound model 2010 al., et Egles

)

HUVEC

(

Primary human human Primary keratinocytes fibroblasts; Human neurons; sensory Umbilical Human Endothelial Vein Cells 1,3 type Bovine sponge collagen of influence Study to wound neuron healing 2014 al., et Blais

Table 2. Reconstructed 3D skin disease models.

Generated disease models of human skin and their characteristics.

Disease model model Disease Characteristics Reference

Atopic dermatitis dermatitis Atopic show to used Itwas filaggrin the in downregulation of patients epidermis dermatitis withatopic or obtained either maybe inborn, for responsible directly disease the of some alterations related 2014 al., et Pendaries

related related

Tinguely et al., 2015 al., et Tinguely

topic dermatitis topic

econstructed epidermis epidermis econstructed

Atopic dermatitis Atopic R mimicking A invitro inflammation Rouaud

effector effector

3D model of dermatitis of model 3D foreskin Human fibroblasts cells HaCaT Memory cells T (CD45RO+) tat material: Scaffold I, type tailcollagen fibronectin 2005 al., et Engelhart

used to study to used

induced gene gene induced

HSEs HSEs

thickness skin skin thickness

soriatic

Psoriasis Full IL toidentify used inpsoriasis genes responsive P cytokine expression 2014 etal., Chiricozzi 2008 and Bergers Tjabringa

model model

RHS

D D

3D model of of model 3D melanoma 3 incorporating cells melanocytic 2011 al., et Li

as as

epidermal epidermal

reconstruction

skin

ollagen type I type ollagen

3D metastatic of model melano malignant Human (A375) cells melanoma human Normal keratinocytes (NHKs) fibroblasts human Normal (NHFs) C material scaffold 2007 al., et Mohapatra

LU LU

115

organotypic skin skin organotypic

at tail collagen type I type collagen attail

3D spheroid melanoma model cell melanoma Human (RGP), SBCL2 lines WM 451 and (VGP), (MM) primary Human keratinocytes primary Human fibroblasts R material asscaffold 2013 al., et Vörsmann

as as

at tail collagen type I type collagen attail

3D model of human of model 3D cell squamous cutaneous carcinoma NHKs Primary SCC12B2 NHFs, Primary lines cell SCC13 and R material scaffold EGF with Pretreatment 2012 al., et Commandeur

ing

fibrosis

screen

esting in vivo the invivo esting

3D model of of model 3D scleroderma T of progression and scleroderma for useful drugs antifibrotic 2016 al., et Luchetti

Applications of the 3D RHS in the study of drug permeation

As already mentioned, permeation testing on RHE is ambiguous due to the poorer barrier properties of 3D RHS compared to native skin. Nevertheless, many companies have made attempts to establish permeation tests on both RHE (Table 3) and in full- thickness HSEs (Table 4). Specifically, Schmook et al. (2001) studied the permeation ability of four different drugs (Tables 3 and 4) on commercially available RHE and full- thickness skin. The full-thickness Graftskin™ LSE™ has shown a sufficient barrier for salicylic acid (flux was only two times higher compared to human skin). However, application of more hydrophobic drugs like hydrocortisone and clotrimazole, on Graftskin™ LSE™ showed inferior barrier properties (flux was 200 and 1000 times higher compared to human skin for hydrocortisone and clotrimazole, respectively). In another investigation by Ackermann et al. (2010) percutaneous absorption of four different compounds (Table 4) has been tested in full-thickness Phenion FT® and compared with permeation of these compounds on pig skin and on RHEs (i.e. EpiDerm™, SkinEthic™, Episkin). In general, full-thickness skin showed poorer barrier properties and increased permeability in contrast to pig skin. In addition, RHE has shown similar barrier properties and permeability compared to human full- thickness skin. In summary, HSEs indicate overpredicted permeability parameters compared to human skin, that could be a result of different organization and lipid composition of the cornified layer of human epidermis.

Thakoersing et al. (2013) found increased monounsaturated fatty acids and hexagonal lipid packing in the cornified layer of their in-house HSEs, suggesting that this could be the reason of the poorer barrier properties of HSEs compared to native human skin. On the other hand, another study revealed that growth conditions of in-house collagen- based full-thickness HSEs can be upgraded and both their ceramide profile and their barrier properties can be optimized with the addition of clofibrate, fatty acids and ascorbic acid (Batheja et al., 2009). In addition, the lack of epidermal derivatives like hair follicles and sweat glands is another key factor responsible for the inadequate resemblance of HSEs with native human skin. In general, the drug delivery of nanoparticles, which activity is very important for topical and transdermal healing of a variety of dermatological diseases (Zhang et al., 2013), occurs through hair follicles penetration (Lademann et al., 2007; Otberg et al., 2008; Shim et al., 2004). Therefore, it is important to focus on the development of HSEs with epidermal appendages.

Table 3. RHE used for drug permeation testing. RHE developed by MaTek Corp and Episkin tested for the permeation of several pharmaceutical substances like flufenamic acid, clotrimazole, hydrocortisone, salicylic acid, terbinafine as well as caffeine and testosterone. Reconstructed Company Drug Reference human epidermis (RHE) EpiDerm™ MatTek Corp Flufenamic acid Zghoul et al., 2001 (Ashland, USA)

SkinEthic™ Episkin Clotrimazole Schmook., 2001 (Lyon, France) Hydrocortisone Salicylic acid Terbinafine

Episkin Episkin Caffeine Netzlaff et al., 2007 (Lyon, France) Testosterone

Table 4. Reconstructed full-thickness skin used for drug permeation testing. Reconstructed full-thickness skin developed by two different companies (Organogenesis and Henkel) for testing permeation of specific natural/ pharmaceutical compounds. Full-thickness Company Drug Reference skin GraftskinTM Organogenesis Clotrimazole Schmook et al., 2001 LSETM (MA, USA) Hydrocortisone Salicylic acid Terbinafine Phenion FT Henkel Benzoic acid Ackermann et al., 2010 (Dusseldorf, Caffeine Germany) Nicotine Testosterone

Applications of 3D RHS in irritation and corrosion

Generally, HSEs are scientifically valid alternatives to animal models for testing skin corrosion and irritation of chemical compounds (Alépée et al., 2018; Nomura et al., 2018). There are numerous epidermal and full-thickness skin equivalents commercially available for this purpose. Overall, Episkin, Epiderm™ and SkinEthic models referred to organisation for economic cooperation and development (OECD) test guideline 439 are accepted by European Union (EU) and Food and drug administration (FDA) for skin irritation on different chemical compounds measuring cell viability of the epidermis using MTT assay. Moreover, the secretion of IL-1α, IL-6, and IL-8 cytokines at epidermis and full-thickness skin models can be used to detect skin irritation.

Full-thickness skin models like TestSkin®, Apligraf®, AST-2000®, and Skin2® have been tested for their ability to predict skin irritation by evaluating the cell viability, cytotoxicity and secretion of soluble factors causing inflammation (Gibbs, 2009). More specifically these factors can be IL-1, IL-8, IL-6, and prostaglandin E2 (PGE2) after exposure to potential irritants. In conclusion, although HSEs are acceptable to the EU and FDA as an appropriate alternative method to in vivo animal testing (Draize albino rabbit test), it should be considered that there are still some problems to be solved including: batch-to-batch variation, higher cost, SC barrier that is more permeable than that of native skin and lack of epidermal appendages in 3D models such as sweat glands (irritation responses that may happen due to sweat cannot be predicted) (Matsumura et al., 1995).

Applications of 3D RHS in sensitization

Assessing skin sensitization using RHS can offer several benefits. Full-thickness HSE maintain very important cellular interactions between the fibroblasts and keratinocytes that occur during sensitization. Moreover, expression levels of mRNA for metabolic enzymes in full-thickness HSEs were found to be related with in vivo human skin more than cells in RHEs (Luu-The et al., 2009). EST-1000 epidermal model and AST-2000 full-thickness model from Cell Systems (St. Katharinen, Germany) have been evaluated for screening skin sensitizers like oxazolone, 1-fluoro-2,4-dinitrobenzene (DNFB) and irritants like SDS and Triton X-100. Furthermore, investigation of cell signaling pathways that transduce immune responses and regulate cytokines have been conducted (Koeper et al., 2007).

MICRO- AND NANO-EMULSIONS

Generally, emulsion technology has been utilized tremendously in the pharmaceutical industry. There are three different categories of emulsion depending on size, shape, stability, method of preparation and polydispersity: macro-emulsions, nano-emulsions and micro-emulsions (Zanatta et al., 2008) .

Macro-emulsions or opaque emulsions, have large droplet sizes and as a result they usually form cloudy solutions (Rosen and Kunjappu, 2012). They are classified in water in oil (w/o) and oil in water emulsions (o/w).

Nano-emulsions or mini-emulsions compared to macro-emulsions have smaller droplet size and are semi-opaque (Rosen and Kunjappu 2012). They are also classified as o/w and w/o.

Micro-emulsion solutions became famous in 1943 after Hoar and Schulman blended a milky solution and hexanol to produce a uniform single-phase and non-conducting solution (Gibaud and Attivi, 2012). Micro-emulsions are transparent, thermodynamically stable mixtures of oil and water stabilized by emulsifiers. They have classified in o/w and w/o micro-emulsions just like nano and macro-emulsions but they have significantly different properties (type, size, formation and stability) relative to nano and macro-emulsions (Table 5).

Table 5. Comparison of size, shape, stability, method of preparation and polydispersity in different kinds of emulsions.

Macro-emulsions Nano-emulsions Micro-emulsions

Size 1-100μm 20-500nm 10-100nm Shape Spherical Spherical Spherical, lamellar Stability Thermodynamically Thermodynamically Thermodynamically unstable, weakly unstable, kinetically stable kinetically stable stable Method of High & low energy High and low Low energy method preparation methods energy methods Polydispersity Often high (>40%) Typically, low Typically, low (<10-20%) (<10%)

Properties and characteristics of micro- and nano-emulsions

Micro-emulsions as well as nano-emulsions are composed of water, oil, and a surfactant. Surfactants are amphiphilic molecules, which are usually used in conjunction with a co-surfactant (Callender et al., 2017). Micro-emulsions are thermodynamically stable and form spontaneously. In contrast to nano-emulsions, micro-emulsions usually require a higher percentage of surfactant or emulsifier (i.e., 15–30% w/w of the oil phase) for formation. Additionally, the co-surfactant required is usually of a shorter carbon chain length than that required in nano-emulsions (Rosen and Kunjappu, 2012).

Nano-emulsions are kinetically resistant and require energy input for their formation. They require less emulsifier than microemulsions 1–3% of the volume of the oil phase (Rosen and Kunjappu, 2012). Ultrasonic agitation and use of high-pressure homogenization with microfluidic devices are used for the nano-emulsion preparation (Mason et al., 2006).

Applications of micro- and nano-emulsions in drug delivery

Composition of microemulsions improve the solubility, chemical stability, and oral bioavailability of many poorly water-soluble drugs, moreover they can encapsulate and deliver both hydrophilic and hydrophobic compounds. Furthermore, they have low interfacial tension, large interfacial area and capacity to solubilize both aqueous and oil soluble compounds (Callender et al., 2017). Microemulsions also have slow degradation, controlled drug release rate, and target specificity (Jadhav et al., 2006). Thus, microemulsions are very competitive candidates as drug delivery vehicles.

Nano-emulsions also have utility in drug delivery research, precisely a study of Zulli and his team revealed that encapsulation of the cosmetic substance coenzyme Q10 in nano-emulsions results in significantly enhanced bioavailability (Zulli, 2006). Additionally, nano-emulsions prepared according to a patented process, allow the administration of several incompatible substances at the same time, for example Vitamin E and coenzyme Q10, which are poorly water-soluble substances, form a “double” nano-emulsion that can be successfully used for cosmetic purposes (Guglielmini, 2008; Merisko et al., 2003).

STATE-OF-THE ART

The 3D reconstructed human dermal and epidermal models are considered a breakthrough in cosmetic industry since they are the best alternatives to animal testing (Sheasgreen et al., 2009). Consequently, they increasingly became an indispensable part for pharmaceutical investigation and development as well (Mathes et al., 2014). In addition, 3D models for rare and common skin diseases are valuable tools in studies aiming to decipher the molecular mechanisms underlying a variety of skin diseases such as melanoma progression (Li et al., 2011), recessive dystrophic epidermolysis bullosa (Mittapalli et al., 2016), atopic dermatitis (Pendaries et al., 2014) etc. In other words, 3D RHS models provide novel experimental systems that recapitulate adequately the in vivo state and could be used for a wealth of reasons.

The 3D reconstructed skin developed here, is a model of a full-thickness normal skin, which mimics native human skin both structurally and biochemically. More precisely, it contains a distinct dermis and a well-structured epidermis consisting of a cornified layer (SC), a granular layer (SG), a spinous layer (SS) and a basal layer (SB). Moreover, it expresses the typical skin differentiation markers (involucrin, loricrin, keratin 5), proliferation markers (Ki67), cell-adhesion molecules (E-cadherin), structural proteins (DSC1, DSG1, CDSN) and proteases (KLK5, KLK6, KLK7) with key functions in skin physiology.

Our in-house 3D skin substitute was generated to be exploited in the evaluation of the cosmetic properties and skin irritation of a variety of essential oils (EOs) and cosmetic micro-and nano- emulsion formulations with encapsulated natural EOs as bioactive compounds. This novel formulation could provide safety given that it contains only natural ingredients, controlled release of the bioactive compound as well as skin moisturizing action.

To evaluate safety, preliminary studies of cytotoxicity were performed in the frame of the current study prior to examination of cosmetic properties, such as the moisturizing action of these formulations, and the extent of potential skin irritation on the 3D RHS.

SPECIFIC AIMS OF THE STUDY

The main purpose of this study was the generation of a 3D skin model that simulates the structural and biochemical characteristics of the native human skin. For the achievement of this objective, a 3D reconstructed full-thickness human skin was developed. More accurately, immortalized human dermal fibroblasts (iNHFs) mixed with collagen were used for the development of the reconstructed dermis and immortalized human epidermal keratinocytes (iNHKs) were used for the development of the reconstructed epidermis. In addition, the key objective was to validate whether the keratinocytes seeded in the dermis substrate had the ability to multiply and differentiate to form a differentiated epithelium that mimics perfectly the in vivo state.

Histology was used to show that the in-house model resembles native skin remarkably, which was one of the main aims of the study. The investigation of the expression of proteins that exist principally in native human skin was the other basic goal to achieve for the proof-of-concept that our in-house 3D model mimics biochemically the native skin. Therefore, the expression of the proliferation marker Ki67, of the cell adhesion molecule E-cadherin, of the differentiation markers (involucrin, loririn, keratin 5) and structural proteins (DSC1, DSG1, CDSN) as well as of kallikrein-related peptidases (KLK5, KLK6, KLK7) was the key to validate the success of our 3D skin model.

Finally, this study’s purpose was to examine the cytotoxicity of EOs and novel cosmetic micro-and nano- emulsion formulations. Cytotoxicity of these cosmetic products was evaluated firstly on iNHKs. Studies of cytotoxicity on iNHKs were essential as a preliminary step before their evaluation on the in-house 3D RHS.

The developed products will be examined on the 3D RHS for cosmetic properties, such as their moisturizing action. Skin irritation and corrosion should also be tested on the 3D skin model after treatment with the specific cosmetic products.

MATERIALS, METHODS AND INSTUMENTATION

Instruments Equipment Supplier

Camera Olympus stylus Cold plate Leica Centrifuge, Rotofix 32 Hettich

CO2 water jacketed incubator series II Forma Scientific, Inc Diaphragm vacuum KNF Hemocytometer Neubauer Incubator MMM medcenter Inverted light microscope Hund wetzlar Microtiter plate reader - Infinite F50 Tecan Microflow biological safety cabinet Astec Microtome LEICA Orbital shaker Thermo Scinetific Upright light microscope with Axiocam ERc5s ZEISS Tissue embedder Leica Water bath Memmert

Plastic or glass laboratory consumables Material Company 6-well deep plate Falcon 96-well cell culture plate Corning Cell culture inserts Falcon Cryovials SDL Life Sciences Embedding cassettes Fisher Scientific Microtome blades Leica Microscope slides Thermo scientific Pasteur pipettes Kisher-Biotech G Serological pipettes SDL Life Sciences Sterile petri-dishes 100 x15 mm, 60 x16 mm Falcon Syringe filters 0.22 μm Sigma Aldrich 50 ml and 15 ml conical centrifuge tubes Falcon T25 and T75 tissue culture flask Falcon

Media and Reagents

Adenine, Sigma Aldrich, A2786-25G Agar, Invitrogen, 30391-023 Ascorbic acid, Sigma Aldrich, A1300000 Aquaguard-1 Solution, Promocell, PK-CC01-867-1B Bovine serum albumin BSA, Sigma Aldrich, A7906-50G

Calcium chloride CaCl2, Honeywell, C1016-100G Citrate sodium, Sigma Aldrich, C8532-500G Collagen, Corning, 354236 Dulbecco’s Modified Eagle Medium DMEM, Gibco, 41965-039 Dimethyl Sulfoxide DMSO, Sigma Aldrich, D2650 Ethanol, Sigma Aldrich, 24194 Eosin, Sigma Aldrich Eukitt Quick-Hardening mounting medium, Sigma Aldrich, 03989 Fetal Bovine Serum FBS, Gibco 12676029 Formaldehyde, Merck, K41839403 Hanks’ Balanced Salt Solution HBSS, Lonza, 10-527F Hematoxylin, Sigma Aldrich, MHS16 Hydrochloric acid HCl, Sigma Aldrich, 30721 Hydrocortisone, Millipore

Hydrogen peroxide H2O2, Sigma Aldrich, 31642 Insulin, Millipore, I-5500 Isopropanol, Sigma Aldrich, 33539 Glacial acetic acid, Sigma Aldrich, 33209 Keratinocyte SFM, Gibco, 17005-034 Keratinocytes supplements, Gibco, 37000-015 L-Glutamine 200mM, Gibco, 25030-081 Neutral red NR, Sigma Aldrich, N4638-1G MTT, Sigma Aldrich, M5655 Penicillin/streptomycin, Gibco, 15140-122 Recombinant human epidermal growth factor rhEGF, Millipore Sodium dodecyl sulfate SDS, Sigma Aldrich, L3771-100G Sodium hydroxide, SO04211000

Triton® X-100, Sigma Aldrich, X114 Trypan Blue, Sigma, T8154 Trypsin- Ethylenediaminetetracetic acid EDTA 0.05%, Gibco 25300-054 Tween® 20, Sigma Aldrich, P1379 Xylene, Sigma Aldrich, 16446

Antibodies

• Anti-KLK5, R&D Systems AF1108 (working dilution: IHC-P 1/100) • Anti-KLK6 IgY (Sotiropoulou et al., 2012) (working dilution: IHC-P 1/200) • Anti-KLK7, R&D Systems AF2624 (working dilution: IHC-P 1/100) • Anti-Cytokeratin 5, Abcam ab 24647 (working dilution: IHP 1/1000) • Anti-CDSN, Flarebio CSB-PA005124 (working dilution: IHC-P 1/500) • Anti-Loricrin, Abcam ab24722 (working dilution: IHC-P 1/1000) • DSC1 (L-15), Santa Cruz SC-20114 (working dilution: IHC-P 1/250) • DSG1 (H-290), Santa Cruz SC-15230 (working dilution: IHC-P 1/300) • Anti-E-cadherin (G10), Santa Cruz SC8426 (working dilution: IHC-P 1/1/200) • Anti-Ki67 Abcam 15580 (working dilution: IHC-P 1/1/1000) • Involucrin (M-15), Santa Cruz SC-15230 (working dilution: IHC-P 1/100) • Anti-chicken IgY HRP, Sigma Aldrich A9046 (working dilution: IHC-P 1/300) • Anti-mouse IgG HRP, Sigma Aldrich A9044 (working dilution: IHC-P 1/200) • Anti-rabbit HRP, Millipore AP132P (working dilution: IHC-P 1/200) • Donkey anti-goat IgG-HRP, Santa Cruz sc-2020 (working dilution: IHC-P 1/200)

Native human skin

Native human skin was kindly provided by Manthoula Valari, MD, PhD, i.e. ear skin tissue excised by plastic surgery on healthy donors for cosmetic purposes. Biopsy samples were embedded in paraffin for further analysis including microscopic observation of the skin structure and immunohistochemical analysis. Biopsies were taken with full informed consent and the study was approved by the Ethics Committee.

Cell lines

Human epidermal keratinocytes and dermal fibroblasts cell lines used in this study, were kindly offered by Dr. Med. Dimitra Kiritsi, Department of Dermatology, Faculty of Medicine, Medical Center-University of Freiburg, Freiburg, Germany.

Skin specimens were obtained from healthy donors after written informed consent and was approved by the Ethics Committee of Freiburg University (#521/13). Primary keratinocytes and fibroblasts were then isolated from human skin samples and cultured in serum-free keratinocyte growth medium and DMEM respectively, in the presence of o 5% CO2 at 37 C. Stable cell lines were generated upon immortalization of both epidermal keratinocytes and dermal fibroblasts with standard protocols.

Micro- and nano-emulsions, oils and surfactants

Micro- and nano-emulsions, EOs and surfactants used in terms of this study, were kindly offered by Dr. Vassiliki Papadimitriou, Department of Biomimetics and Nanobiotechnology, Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece.

METHODS

Cell culture

Normal human fibroblast cell lines were cultured at 37°C in 5% CO2 in DMEM medium containing 10% FBS, 2% L-glutamine and 1% penicillin/streptomycin.

Normal human keratinocyte cell lines were grown in KSFM, supplemented with growth factors and hormones necessary for cell growth including hEGF and pituitary bovine extract. Cells were passaged when more than ~70-80% confluence was observed.

For subculture, both fibroblasts and keratinocytes had to be detached from the bottom of the culture flask. Firstly, the culture medium was discarded and 1 or 2 ml trypsin/EDTA solution was added into 25 cm2 or 75 cm2 flask, respectively, following by 3 min incubation at 37oC. Action of trypsin was stopped when cells detached by addition of two volumes of phosphate-buffered saline (PBS), 10% FBS, then cell suspension was centrifuged at 1000 x rpm for 5 min, following by resuspension of cell pellet in fresh medium and finally cell suspension was transferred to culture flasks for cell growth.

Photographs of iNHFs and iNHKs were taken with an OLYMPUS stylus SP-100EE camera focused in 35 mm, optical microscope’s total magnification was 100x (10x objective lenses * 10x eyepieces).

Cell cryopreservation

Cells were observed under an inverted light microscope to assess % confluence, which should be close to 80-90% and to confirm the absence of contamination by bacteria and/or fungi. Then, the cells were detached with trypsin/EDTA, as described previously in “cell culture’’, centrifuged and finally, 1-2 x 106 cells were resuspended in 1ml freezing medium containing 80% DMEM, 10% FBS and 10% DMSO, which is the most commonly used cryoprotectant. Afterwards, ~0.5-1 x 106 cells were transferred into each cryovial, which was labeled and stored at -800C for 1-3 days and finally, transferred in liquid nitrogen for long-term storage.

Cell viability

Number and viability of the cells were estimated using a glass hemocytometer and trypan blue exclusion test according to Current Protocols in Immunology (Strober.,

2001). Cells were detached from the bottom of the flask by trypsinization, centrifuged at 1000 x rpm for 5 min and then, resuspend in the required prewarmed medium depending on the cell type. Once the cells were fully resuspended, a small amount of cell suspension was mixed with 0.4% trypan-blue at a ratio of 1:1 (i.e. dilution factor, 푣표푙푢푚푒 표푓 푐푒푙푙 푠푢푠푝푒푛푠푖표푛+푣표푙푢푚푒 표푓 푡푟푦푝푎푛 푏푙푢푒 푠푡푎푖푛 DF= ) and counting of both dead and 푣표푙푢푚푒 표푓 푐푒푙푙 푠푢푠푝푒푛푠푖표푛 alive cells was performed (Figure 9). Trypan blue exclusion test is based on the principle that live cells possess intact cell membranes that exclude trypan blue dye while dead cells do not. Viable cells have clear cytoplasm while a dead cell’s cytoplasm is blue. The percentage of cells’ viability was calculated using the following general 푁푢푚푏푒푟 표푓 푙푖푣푒 푐푒푙푙푠 equation: ∗ 100. 푁푢푚푏푒푟 표푓 푙푖푣푒 푐푒푙푙푠+푁푢푚푏푒푟 표푓 푑푒푎푑 푐푒푙푙푠

In addition, considering that the depth of a chamber is 0.1 mm and each large square’s surface is 1 mm2 (i.e. volume of one large square is 0.1 mm3 =10-4 ml) (Figure 9), the density of cells was estimated using the following equation:

푁푢푚푏푒푟 표푓 푙푖푣푒 푐푒푙푙푠 ∗ DF = 푁푢푚푏푒푟 표푓 푙푎푟푔푒 푠푞푢푎푟푒푠 푐표푢푛푡푒푑∗푉표푙푢푚푒 표푓 1 푠푞푢푎푟푒

푁푢푚푏푒푟 표푓 푙푖푣푒 푐푒푙푙푠 * 104 * DF cells/ml 푁푢푚푏푒푟 표푓 푙푎푟푔푒 푠푞푢푎푟푒푠 푐표푢푛푡푒푑

In conclusion, to calculate the total concentration of viable cells, the following equation was used:

cells cell density ( ) ∗ total volume of cell suspension ml

B C

Figure 9. Cell number calculation and cell viability estimation. A, chemical structure of the trypan blue dye. B, schematic representation of a hemocytometer used for cell number calculation and cell viability estimation; an enlarged area is shown on the right. C, microscopic appearance of cells stained with trypan blue. Viable cells have clear cytoplasm, while in dead cells the cytoplasm is stained blue, since their membranes are not intact allowing penetration of the trypan blue dye.

Development of the 3D RHS

Development of the 3D RHS was described previously (Mittapalli et al., 2016). Briefly, fibroblasts were detached from the bottom of the flask via trypsinization and then cells were measured using a glass hemocytometer and finally resuspended in FBS to a final concentration of 3,000,000 cells/ml. Τrypan blue exclusion assay confirmed that iNHFs had viability over 90%. The ice-cold rat tail collagen I solution (80% of total volume) was mixed with HBSS 10x (10% of total volume) to a final concentration of 3.5 mg/ml and then, pH was adjusted to 7.4 with 5 M NaOH under gently stirring on ice to avoid formation of air bubbles and premature gelation, following by addition of 750,000 fibroblasts/ml collagen gel. Using pre-cooled pipettes, 2.5 ml of the collagen I- fibroblasts mixture was transferred to each 1 μm pore size filter insert and then o incubated at 37 C, 5% CO2 for gelation.

Thereafter, a glass ring with 12 mm inner diameter and 10 mm height were placed on each gel and gently pushed down to confine the area for the epithelial cell growth. Gels, were placed for 1 hour at 370C in a humidified incubator and then, excess liquid was carefully aspirated and finally, the gels were submerged in 15 ml DMEM containing 2% FBS, 2% L-glutamine, 1% penicillin/streptomycin per well and incubated for 24 hours in an incubator (37°C, 5% CO2). After 1 day, medium was carefully removed and 1-2 * 106 keratinocytes were seeded on each gel. Four days later, the culture at the air–liquid interface is initiated therefore, the culture medium was replaced by 10 ml of organotypic culture -organotypic cultures (OTC) medium (2% L-glutamine, 5 ng/ml rhEGF, 1 μg/ml hydrocortisone, 5 μg/ml insulin, 0.18 mM adenine, 5% FBS and 1.8 mM Ca2+) containing 10 μl of 50 mg/ml ascorbic acid. Culture medium was renewed every 2-3 days and the 3D RHS equivalents were harvested four weeks after initiating the culture at the air-liquid interface.

Embedding tissue into paraffin blocks

Inserts with the reconstructed tissue were placed in a glass plate and cut out in pieces using sterilized surgical knifes, with caution not to disturb the epidermis. Tissue was placed in 3.7% formaldehyde in PBS and then embedded in 2% agar in PBS. Afterwards, reconstructed tissues were left 24 hours in PBS and then were embedded in paraffin.

More specifically, tissue was placed in embedding cassette and dehydrated through a series of ethanol dilutions (70, 85, 95, and 100%) and finally cleared with two washes with xylene, 45 min each at room temperature (RT). Subsequently, tissue was left to incorporate paraffin for approximately 2 hours. Then, liquid warmed paraffin was poured in stainless-steel base mold and the agar-tissue block was placed in the middle of the mold, which was then filled with paraffin and was left to cool and solidify on a cold plate.

Hematoxylin and eosin staining

Tissue paraffin sections of 7 μm were cut using a microtome and placed on a microscope slide. Sections were then air-dried for 1 hour and incubated in 600C for paraffin melting. Subsequently, sections were deparaffinized in xylene (20 dips), rehydrated in decreasing concentrations of ethanol (100, 75, and 50%) and finally in water (10 dips in each solution). Finally, sections were stained with hematoxylin, which stains the nucleus of the cells for approximately 3 minutes and then with eosin, which stains the cytoplasm. Afterwards, sections were dehydrated in increasing dilutions of ethanol (50, 75, and 100%), cleared in xylene (20 dips) and mounted with Eukitt.

Photographs of skin tissues were taken with Axiocam ERc5s under a light microscope using Zeiss Zen software. Total magnification was 200x (10x objective lenses * 20x eyepieces) and 400x (10x objective lenses * 40x eyepieces).

Immunohistochemistry

Paraffin sections of 7 μm were cut, air-dried and then paraffin was melted in a preheated incubator at 60oC. Then, sections were deparaffinized in xylene (20 dips) and rehydrated in a serial dilution of ethanol (100% to 0%). Afterwards, sections were boiled 20 min in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween pH 6.0). The sections were washed in PBS for 5 min and endogenous peroxidase activity was quenched by incubation in 3% H2O2 in PBS for 10 min. In addition, sections were immersed in blocking buffer (0.3% bovine serum albumin or BSA in PBS-Triton 0.1%) for 5 min. Consequently, the primary antibody was placed on sections at the appropriate concentration and time. Sections were washed twice with 0.3% BSA in PBS and the secondary antibody conjugated with a peroxide enzyme was added on sections for 45

52 min to bind to the 1st antibody. Sections were washed again, twice with 0.3% BSA in PBS and once with PBS. Then, 3,3-diaminobenzidine (DAB) substrate was placed upon sections. As already mentioned, 2nd antibody is conjugated with a peroxidase enzyme, which in the presence of hydrogen peroxide, oxidizes DAB to a brown product. Finally, sections were counterstained with hematoxylin, dehydrated and mounted with Eukitt. Axiocam ERc5s was used under a light microscope using Zeiss Zen software for capturing photographs under 400x (10x objective lenses*40x eyepieces) magnification.

Neutral red uptake assay

The NRU assay was carried out according to the experimental protocol reported by Repetto et al. (2008) . The basic principle of THE NRU assay is that viable cells are able to incorporate and bind the supravital dye NR in the lysosomes. The chemical structure of NR is shown in Figure 10.

Specifically, NHK were grown in keratinocyte serum-free medium (KSFM) and when reached 80% confluence, cells were detached via trypsinization and were counted using a hemocytometer. Subsequently, cell viability was checked using trypan blue exclusion test as detailed in Material and Methods. Finally, cells were diluted in order to form a suspension of 75 x 103 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate, which was incubated at 37±1°C, 5±1 % CO2, 95 % relative humidity (RH) for 24 hours. On the next day, cells were exposed to increasing concentrations of testing products in KSFM and incubated at 37±1°C, 5±1

% CO2, 95 % RH for the appropriate time. Alongside, a NR solution 40 μg/ml in water was prepared and incubated for an hour at 370C.

The testing medium was aspirated from the cells and the cells were washed twice with 150 μl of PBS. Subsequently 100 μl of NR medium was added in each well plate and incubated for two hours at 37±1°C, 5±1 % CO2, 95 % RH. Then, microscopic evaluation was conducted and cells were washed with 150 μl water. In addition, each well was filled with destaining solution (50% ethanol-49% deionized water and 1% glacial acetic acid) and plate was left under stirring for 15 min. Finally, the OD was measured at 490 nm in a microtiter plate spectrophotometer.

Figure 10. Chemical structure of the NR dye. Viable cells incorporate and bind the supravital dye NR in the lysosomes and are stained red.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

MTT assay was conducted following the recommended protocol from Abcam. The basic principle of MTT assay is that water soluble yellow MTT, is reduced to an insoluble purple formazan product by active mitochondrial dehydrogenases of viable cells (Figure 11). Briefly, iNHKs were grown in KSFM to 80% confluence, then, they were used in MTT assay. Cells were collected and measured as detailed in NRU assay. Approximately 75 x 10 cells/ml were dispersed per well in 96-well plates. Plates were then incubated at 37±1°C, 5±1 % CO2, 95 % RH for 24 hours.

Cells were exposed to different concentrations of testing products in KSFM and incubated at 37±1°C, 5±1 % CO2, 95 % RH for the appropriate time. Then, 50 μl of fresh KSFM and 50 μl of MTT (5 mg/ml in PBS) were added into each well and the o plate was incubated for 3 hours at 37 C, 5% CO2, followed by addition of 150 μl of MTT solvent (0.1 Ν ΗCl in isopropanol) into each well. Then, the plate was wrapped in foil and placed in an orbital shaker for 15 min and finally, the OD was measured at 590 nm in a microtiter plate spectrophotometer.

Figure 11. Structure of both MTT and formazan. A yellow tetrazole, is reduced to purple formazan in living cells.

RESULTS

CHAPTER 1: Generation of the 3D RHS

Development of 3D RHS could be summarized in 4 steps including, establishment of fibroblast and keratinocyte cell cultures, preparation of cellularized collagen with the appropriate concentration of dermal fibroblasts, seeding of keratinocytes and finally tissue harvesting and precipitation for further analysis (Carlson et al., 2008).

Morphology of immortalized normal human dermal fibroblasts

Cell lines of iNHFs were cultured in flasks with surface area 25 cm2 (T25) and 75 cm2

(T75) in feeding medium DMEM supplemented with 10% FBS, 2% L-glutamine and 1% penicillin/streptomycin and when they had reached ~80% confluence, they were trypsinized, counted and, finally, used for the formation of normal 3D RHS (Figure 12).

Figure 12. Immortalized normal human dermal fibroblasts. The 2D iNHF cultures were routinely observed under an optical microscope until 80% confluence, then cells were used for the 3D RHS, as described in Figure 14.

Morphology of immortalized normal human epidermal keratinocytes

Cell lines of iNHKs were firstly cultured in KSFM in T25 flasks, with keratinocytes supplements and consequently they were used for the formation of normal 3D RHS (Figure 13).

Figure 13. Immortalized normal human epidermal keratinocytes. The 2D iNHK cultures were routinely observed under an optical microscope until 80% confluence, then cells were used for the 3D RHS as described in Figure 14.

Preparation of cellularized collagen gel

Firstly, iNHFs viability was estimated higher than 95% using trypan blue exclusion test as detailed in Materials and Methods. The rat tail collagen 1 was mixed with iNHFs using chilled pipets on ice to prevent premature gelation. It is important to neutralize the acidic collagen with 5M NaOH to avoid cell damaging. The final concentration of iNHFs was 3 x 105 fibroblasts/ml gel.

Figure 14. Preparation of 3D RHS. Day 0, collagen gel mixed with iNHFs was placed above the culture insets. Day 5, iNHKs were seeded onto the surface of the matrix, attached to the collagen- fibroblast gel and allowed to be fully adhered. Day 10, tissues were exposed to the air-liquid interface in order to complete stratification and full morphological and biochemical differentiation, and the medium was changed from DMEM to OTC. Day 37, tissues were ready for paraffin embedding and sectioning followed by histological characterization.

Seeding of keratinocytes

During the second day of 3D’s reconstructed skin development, fibroblasts mixed with rat tail collagen I have begun to form a massive gel (the dermis). On day five, 2,000,000 iNHKs with viability of ~94% were seeded onto the collagen-fibroblast gel. Keratinocyte differentiation began a few days later and on day 10, tissues were exposed to an air liquid interface to complete stratification and full morphological and biochemical differentiation (Figure 14 and 15). Furthermore, medium was changed from DMEM containing, 2% FBS, 2% L-glutamine, 1% penicillin/streptomycin to OTC containing, 2% L-glutamine, 5 ng/ml rhEGF, 1 μg/ml hydrocortisone, 5 μg/ml insulin, 0.18 mM adenine, 5% FBS and 1.8 mM Ca+2.

Harvesting of 3D reconstructed tissues for analysis

Around day 37, reconstructed tissues were ready to be harvested and precipitated for further analysis including histological and immunohistochemical characterization. Using sterile scissors and forceps, tissues were carefully harvested and then embedded in paraffin as detailed in Materials and Methods. Briefly, they were placed in embedding cassettes, dehydrated, cleared with xylene and finally embedded in paraffin.

Figure 15. Macroscopic appearance of 3D RHS at air-liquid interface. Keratinocytes were seeded on the collagen-fibroblasts gel, after 28 days of growth at air-liquid interface, keratinocytes were completely differentiated and the squamous stratified epidermal epithelium was fully formed by day 37.

CHAPTER 2: Characterization of 3D RHS

The 3D RHS displays a microstructure very similar to normal skin tissue

The microstructure of 3D RHS was evaluated by eosin-hematoxylin (Η&Ε) staining. Eosin is an anionic and acidic stain that stains the cytoplasm while hematoxylin is a basic and cationic stain that stains the nucleus of the cells (Chan, 2014), as a result, cytoplasm appears pink while nucleus appears blue. Histological examination of skin paraffin sections revealed that skin microstructure was normal in 3D RHS collected upon 28 days culture at the air-liquid interface compared to native human skin (Figure 16A). Epidermis, the outermost layer of the skin, as well as dermis, the inner skin layer, were distinct both in native skin and 3D RHS paraffin sections. All epidermal layers, including from superficial to deep SC, SG, SS, and SB, were clearly observed (Figure 16A) suggesting that iNHKs were differentiated and formed the characteristic stratified squamous epithelium of the epidermis. Overall, the skin microstructure of 3D RHS appears very similar to native human skin. Nevertheless, in accordance with previous reports (Mc Govern et al., 2016; Vicanova et al., 1996), it was observed that epidermis of 3D RHS was almost twice as thick as native’s skin epidermis. In addition, hyperkeratosis is another characteristic observed in 3D RHS (Figure 16B). Therefore, epidermal thickness varies according to ethnic origin, age, gender, body region and smoking habits (Elder, 2009; Lee et al., 2002; Hoffmann et al., 1994; Seidenari et al., 1994; Sandby‑Møller et al., 2003; Dao et al., 2007; Shuster et al., 1975; Southwood et al., 1946; Chopra et al., 2015; Gültekin et al., 2011), which may have affected the results obtained in this study.

IHC assessment showed that 3D RHS mimics biochemically the native human skin

The method of IHC was utilized to evaluate the biochemical functions of the 3D RHS. More specifically, 3D RHS was characterized for the proliferation activity of keratinocytes, for dermo-epidermal junction, for competence in differentiation markers, for the expression of structural proteins of desmosomes and corneodesmosomes and finally for the expression of KLKs.

C e ll u l a r e p

i d B e

r 300 Stratum Corneum m

μ Cellular epidermis m 250 32,5 200 is 150 15 240 u 100 150 l 50 a Thickness, Epidermal 0 Native skin 3D RHS r e Figurep 16. The 3D RHS forms a stratified squamous epithelium. Αi, representative paraffin sections of 7 μm stained with H&E show that the structure of 3Dd RHS appears similar to native human skin. Epidermal and dermal layers of 3D RHS ande native skin are distinct. The SC, SS, SG and SB were also visible in the epidermis. The blue dashed line represents the epidermis-dermis junction. r B, quantifications of epidermal thickness. The graph shows the thickness of the SC and m of the viable layers of the epidermis both in 3D RHS and native skin. Native skin had smalleris total epidermal thickness than the 3D RHS. Data are shown as median ± SEM, n>14, n; measured in randomly selected fields.

30 23 20 10,33

10 %Ki67 positive nuclei positive %Ki67 0 Native skin 3D RHS

Figure 17. Epidermis of 3D RHS is hyperproliferative. A, Ki67 IHC staining in 3D RHS and native skin biopsies. The blue dashed line represents the epidermis-dermis junction. B, percentage of Ki67-positive cells was increased in epidermis of 3D RHS. Data are shown as median ± SEM, n>7, n=ratio of Ki67 positive cells/total cells, calculated for randomly chosen fields.

40x

Figure 18. Immunohistochemical analysis of E-cadherin. E-cadherin was expressed throughout the epidermis both in native skin and 3D RHS. Immunostained areas in the blue panel are enlarged and indicated by blue arrows. The blue dashed line represents the epidermis-dermis junction.

The 3D RHS shows increased proliferation index of keratinocytes in the epidermis

Ki67 is a proliferation marker, which is expressed in the nuclei of all keratinocytes that are not in the G0 resting phase of the cell cycle (Noszczyk et al., 2001). Immunohistochemical analysis of biopsy sections revealed that Ki67 immunoreactivity was restricted mostly to the basal keratinocytes both in native skin and 3D RHS (Figure 17A). Quantification of Ki67 positive cells showed that 3D RHS sustained a higher epidermal proliferative index than that observed in native human skin. Specifically, approximately 23% of keratinocytes were hyperproliferative in 3D RHS, while 10% were hyperproliferative in native human skin (Figure 17B). Generally, keratinocyte proliferation is important for the formation of the SC. Thus, hyperkeratosis observed in 3D RHS is compatible with its hyperproliferation.

The 3D RHS maintains structural integrity similar to normal skin tissue

To investigate skin integrity in 3D RHS, we next investigated the expression levels of E-cadherin, a calcium-dependent cell adhesion molecule whose function is a main contributor of epidermal epithelial polarity and skin structural integrity. It was observed that E-Cadherin was expressed in both native skin and 3D RHS and staining was localized to membrane of keratinocytes (Figure 18) indicating that cell-cell adhesions potentially exist in epidermis of 3D RHS.

Cell differentiation is normal in the 3D RHS

Epidermal differentiation process was investigated by immunohistochemical analysis of several differentiation markers since abnormal keratinocyte differentiation is linked with several epidermal abnormalities. Immunostaining of paraffin sections obtained from 3D RHS and native skin revealed that loricrin, a terminal differentiation marker, localized in the outer epidermal layers of native skin and 3D RHS. Involucrin, a differentiation marker, which appears in the early stages of keratinocyte terminal differentiation process, was expressed in the outer epidermal layers of the native skin and 3D RHS (Figure 19). Keratin 5, an intermediate filament protein, was expressed predominantly in the basal keratinocytes of the native human epidermis in the SB but was also slightly expressed in the other layers of the epidermis except for the SC, at the RHS (Figure 19).

Normal expression pattern of structural (corneo)desmosomal proteins in 3D RHS

The SC, the outer layer of the epidermis, contains DSG1, DSC1, which are the structural proteins of desmosomes and corneodesmosomes. Another structural protein is CDSN, which is only found in corneodesmosomes. Expression profile of all structural proteins (DSG1, DSC1, CDSN) was similar to both 3D RHS and native skin (Figure 20). Precisely, DSG1 was expressed mostly in the outer epidermal layers of native skin and 3D RHS while DSC1 and CDSN were predominately expressed in SC of both native human epidermis and 3D RHS (Figure 20).

The skin proteases KLK5, KLK6, and KLK7 are comparably expressed in 3D RHS and native skin

Generally, KLKs are related to the regulation of skin’s exfoliation and desquamation process. KLK5 and KLK7 are expressed by keratinocytes at the SG and their activity is confined at the SC (McGovern et al., 2006). Moreover, these KLKs cleave some of the structural proteins of desmosomes and corneodesmosomes, precisely, KLK5 cleaves DSC1, DSG1, CDSN, while KLK7 cleaves DSC1 and CDSN (Caubet et al., 2004; Descargues et al., 2006). The expression of KLK5 and KLK7 in native human skin was detected mostly in the SC. In addition, these proteins were weakly expressed in the other upper layers of the epidermis (Figure 21). KLK5 and KLK7 were expressed primarily in SC of 3D RHS and slightly in the other epidermal layers. KLK6, which is generally a protein that participates in the differentiation process of keratinocytes (Lin et al., 2002), localized predominantly in SC and SG epidermal layers of native human skin. In the epidermis of 3D RHS, KLK6 staining was strongly evident in the upper layers of keratinocytes (Figure 21).

Figure 19. Immunohistochemical analyses of skin differentiation markers. Immunoreactivity of differentiation markers loricrin, involucrin and keratin 5. Loricrin and involucrin were primarily expressed in the SC of both native human skin and 3D RHS however, keratin 5 was expressed predominantly in SB of native human epidermis and 3D RHS. Areas of loricrin and keratin 5 staining are indicated by arrows. The blue dashed line represents the epidermis-dermis junction.

Figure 20. Immunohistochemical analyses of the structural proteins of desmosomes and corneodesmosomes. Expression profiles of DSG1, DSC1 and CDSN was similar to both native epidermis and 3D RHS. Areas of intense staining are indicated by arrows. The blue dashed line represents the epidermis-dermis junction.

Figure 21. Immunohistochemical analyses of human KLK proteases in skin. KLK5 and KLK7 were expressed mostly in the SC of native human skin, while KLK6 appeared equally at the SC and the SG. KLK5, KLK6 and KLK7 appeared mostly at the cornified layer of the 3D RHS. The blue dashed line represents the epidermis-dermis junction.

CHAPTER 3: Toxicity tests of micro- and nano-emulsions, EOs and surfactants, on iNHKs

Micro- and nano-emulsions are promising drug delivery vehicles/systems, as detailed in the introduction, since they have a dual role: [1] to protect the bioactive ingredient from metabolism and physical barriers, and [2] to improve the delivery of bioactive compound to the skin. In addition, micro- and nano-emulsions have been developed for cosmetic purposes, offering unique advantages including improvement of product efficiency and enhancement of stability of the bioactive compounds.

Generally, the surfactants/co-surfactants used in the composition of micro- and nano- emulsions, are excipients that decrease the interfacial tension and expand the flexibility of the interfacial film, respectively (Boonme, 2007). Many different EOs were tested here as potential bioactive cosmetic compounds encapsulated in o/w micro- and nano- emulsions.

In this study, we present toxicity data from a number of different EOs, different types of surfactants and cosurfactants, which could be candidate compounds in o/w micro- emulsion cosmetic formulations (Table 6). Moreover, cytotoxicity of eight different micro-emulsions as well as one nano-emulsion, was examined on iNHKs (Table 6) using NRU and/or the MTT assay. Their exact formulation cannot be disclosed in this Master thesis.

Table 6. Different micro-emulsions and their potential substances tested for in vitro cytotoxicity on iNHKs. The EOs, surfactants and cosurfactants, which were tested as candidate substances for micro- and nano-emulsions. In addition, the micro- and nano-emulsions tested are shown.

Essential oils

Achillea millefolium-Lavandin Lavandin Achillea millefolium Lavender Citrepel Melissa officinalis Geraniol Mentha spicata Isopropyl myristate Oregano

Surfactants: Cosurfactants

Diethylene glycol monoethyl ether Span 85

Labrasol Tween 80

Micro- or Nano-emulsions

6% isopropyl palmitate encapsulated in micro-emulsion 10% citrepel encapsulated in micro-emulsion 20% citrepel encapsulated in micro-emulsion 30% citrepel encapsulated in micro-emulsion 10% geraniol encapsulated in micro-emulsion 20% geraniol encapsulated in micro-emulsion 30% geraniol encapsulated in micro-emulsion without encapsulated essential oil in micro-emulsion 1% geraniol encapsulated in nano-emulsion

Commercially available product with 15% citrodiol encapsulated

The NRU and MTT assays revealed that EOs at concentrations 0.1 %, 0.01% are not cytotoxic

Generally, EOs could be encapsulated in the micro-emulsions, creating a promising cosmetic micro-emulsion formulation as detailed previously. The in vitro EOs cytotoxicity effects were examined on iNHKs using the NRU and MTT assay for determination of cell viability.

The iNHKs were grown in KSFM until they reached a culture density of approximately 80% confluence. Cells were trypsinized and counted using a hemocytometer to estimate their exact number following by confirmation of a higher than 90% cellular viability using the trypan blue exclusion test, prior to treatment with EOs. Afterwards, cells were diluted to 7.5 x 104 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate resulting in a density of 1.5 x 103 cells/well. Finally, the plate was covered and incubated at 37±1°C, 5±1 % CO2, 95 % RH for 24 hours.

Next day, cell morphology and confluence in the wells of the 96-well tissue plate was evaluated under a phase contrast light microscope (Figure 22), cells were then exposed for 1 hour to the testing EOs in a working dilution of 0.1% EO in KSFM containing DMSO as solvent for EOs with the limitation that DMSO should not exceed 0.5% of the total volume since it is cytotoxic in higher concentrations.

Figure 22. Indicative photographs of iNHKs before treatment.

The iNHKs were > 80% confluent. Normal cellular morphology before treatment, no cellular stress observed.

It should be pinpointed that each plate encompasses control wells containing cells that were not treated with EOs but with KSFM (negative control), cells treated with cytotoxic 1% SDS (positive control) and wells containing growth medium without cells for use as assay blanks. At the end of the treatment period, examination of iNHKs under a phase contrast light microscope, revealed no changes in cell morphology indicative of no cytotoxic effects. Afterwards, cell viability was estimated by the NRU and/or MTT assay.

The NRU assay showed that viability of iNHKs upon treatment with 0.1% Achillea millefolium, citrepel, geraniol, lavandin and mentha spicata oil was between 70-80%, only viability of iNHKs treated with Melissa officinalis oil was around 60% while iNHKs treated with lavander and isopropyl myristate (IPM) oil reached viability around 85% (Figure 23B; Table 7). Viability results obtained after comparing untreated cells with iNHKs upon treatment with EOs. Finally, iNHKs treated with 0.5% DMSO showed viability higher than 95% assuring that 0.5% DMSO was not cytotoxic and can be definitely used as a solvent for EOs, while iNHKs exposed to 1% SDS showed viability equal to zero as expected.

In addition, a lower concentration of EOs was tested to determine a range of concentrations where EOs were not cytotoxic to iNHKs. Similarly, iNHKs were exposed for 1 hour to the testing EOs in a working dilution of 0.01% EO in KSFM containing 0.1% DMSO as solvent for EOs. Each plate contained control wells, as detailed previously. Microscopic examination after treatment with EOs and staining with NR showed absence of cellular stress and successful incorporation of NR in the lysosomes indicative of cell viability (Figure 24A). Αs evident in Figure 24B and Table 8, the tested EOs at concentration 0.01% were not cytotoxic and cell viability was higher than 90%.

Achillea millefolium Citrepel Geraniol

Isopropyl myristate Lavandin Lavander

Mellissa officinalis Mentha spicata Oregano

B 120 100 80 60 40

Viability, % Viability, 20 0

Figure 23. Viability testing of iNHKs following treatment with 0.1% EOs by NRU assay. A, microscopic observation of iNHKs following treatment with 0.1% EOs for 1 hour. Representative photographs showed no cellular stress response. B, cytotoxic effects of 0.1% EOs on iNHKs. The % viability compared to control cells treated only with KSFM. Data are shown as median ± SEM, n=2 independent experiments.

Table 7. Viability percentage values of iNHKs treated with 0.1% EOs determined by NRU assay. The highest viability was observed in iNHKs treated with 0.1% lavander or IPM, while the lowest viability was seen in iNHKs treated with 0.1% Melissa officinalis. Essential oils, 0.1% Viability, %

Achillea millefolium 73.9

Citrepel 70.0

Geraniol 71.2

Isopropyl myristate 87.1

Lavandin 77.0

Lavander 84.9

Melissa officinalis 59.6

Mentha spicata 76.6

Oregano 71.9

Table 8. Viability percentage values of iNHKs treated with 0.01% EOs determined by NRU assay. Observed viability in iNHKs treated with 0.01% EOs was higher than 90%, indicating that 0.01% EOs were not cytotoxic.

Essential oils, 0.01% Viability, %

Achillea millefolium-Lavandin 99.1

Achillea millefolium 96.1

Citrepel 97.8

Geraniol 94.5

Lavandin 96.1

Achillea millefolium-Lavandin Achillea millefolium Citrepel

GeraniolGeraniol Lavandin 0.1% DMSO

120 100 80 60 40

Viabiliy, % Viabiliy, 20 0

Figure 24. Viability testing of iNHKs following treatment with 0.01% EOs by NRU assay. A, visualization of iNHKs under the light microscope following treatment with 0.01% EOs for 1 hour and staining with NR. Cells treated with 0.01% EOs showed no cellular stress. B, the % viability of iNHKs treated with EOs compared to untreated control cells. Data are shown as median ± SEM, n=3 independent experiments.

Viability of iNHKs treated with 0.01% EOs was also determined by the MTT assay to assess the potential cytotoxicity of EOs used in the developed formulations. Morphology of iNHKs before treatment was analyzed under a phase contrast light microscope to assure that cells had normal morphology and higher than 80% confluence (Figure 25). Microscopic evaluation revealed that iNHKs treated with 0.01% EOs for 1 hour showed normal morphology resembling that of control-untreated iNHKs (Figure 26A). In accordance with the % viability values estimated by NRU assay, which were presented previously, it was found by MTT assay that 0.01% EOs were not cytotoxic (Figure 26B; Table 9). Τhis, may be correlated with the potential ability of EOs to enhance cellular proliferation, but this remains further investigation. Statistical analysis by Student’s t-tests revealed that differences between % viability values of control- untreated cells and those of treated iNHKs with 0.01% are not statistically significant, showing p values > 0.5.

Consequently, both NRU and MTT assays confirmed that EOs at low concentrations examined, do not have a negative impact on viability of iNHKs in 2D cell cultures. Thus, EOs tested could be encapsulated in micro- and nano-emulsions to enhance their properties such as moisturizing action.

Figure 25. Morphology of iNHKs before treatment with EOs. Cultured iNHKs were observed under the light microscope until they reached 80% confluence, then cells were used for the NRU and MTT cytotoxicity assays.

Achillea millefolium-Lavandin Achillea millefolium Citrepel

Geraniol Lavandin

140 120 100 80 60 40 Viability, % Viability, 20 0

Figure 26. Viability testing of iNHKs following treatment with 0.01% EOs by MTT assay. A, microscopic observation of iNHKs upon treatment with EOs was assessed by the MTT assay. MTT was successfully reduced in mitochondria of viable cells, thus insoluble blue formazan crystals are visible under a phase contrast light microscope. No cellular stress was observed. B, graph showing % viability of iNHKs upon treatment with 0.01% EOs. The % viability was estimated compared to % viability of control-untreated cells, which was 100%. Results are shown as median ± SEM n=3 independent experiments.

Table 9. Viability percentage values of iNHKs treated with 0.01% EOs determined by MTT assay. The EOs were not cytotoxic.

Essential oils, 0.01% Viability, %

Achillea millefolium-Lavandin 109.0

Achillea millefolium 100.8

Citrepel 109.7

Geraniol 109.0

Lavandin 102.9

Toxicity tests of surfactants on iNHKs assessed by the NRU and MTT assays, showed that the surfactants do not reduce cell viability at the concentrations tested

Commonly, micro-emulsions in cosmetic formulations compromised of oil, water, a surfactant, a cosurfactant and/or an active ingredient. It was considered necessary to study the viability of iNHKs after treatment with some different substances that could be used for the development of cosmetic micro-emulsion formulations. Thus, candidate surfactants-cosurfactants have been tested for cytotoxicity at different concentrations, range between 0.01-0.04% in KSFM.

Precisely, iNHKs were grown in KSFM, trypsinized, counted and cellular viability was always estimated higher than 95%, prior to 1 hour treatment with surfactants- cosurfactants. Subsequently, cells were diluted to 7.5 x 104 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate resulting in a density of 1.5 x 103 cells/well. Finally, the plate was covered and incubated at 37±1°C, 5±1 % CO2, 95 % RH for 24 hours.

Morphology of iNHKs before treatment with EOs was examined under a phase contrast light microscope (Figure 25). The iNHKs were grown in KSFM until they reached a culture density of approximately 80%. Subsequently, NRU assay was conducted, and microscopic evaluation of the iNHKs after treatment showed that cells did not face cellular stress and they successfully incorporated NR inside their lysosomes (Figure 27A). The % viability of iNHKs treated with surfactants was compared with the viability of cells treated only with KSFM while 1% SDS was used as an indicative cytotoxic substance. The viability of iNHKs was higher than 90%, showing that surfactants were not cytotoxic at the examined concentrations (Figure 27B; Table 10).

Diethylene glycol Labrasol Span 85 monoethyl ether

Tween 80 Untreated 1% SDS

B 140 120 100 80 60 40 20

viability, % viability, 0

Figure 27. Toxicity testing of surfactants on iNHKs by NRU assay. A, microscopic examination showed that iNHKs had successfully incorporated NR and were confluent. B, viability of iNHKs treated with surfactants compared to untreated cells, showed that surfactants were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments.

Table 10. The % viability values of the surfactants tested for cytotoxicity on iNHKs determined by NRU assay. Tested samples were not cytotoxic.

Surfactants Viability, %

Diethylene glycol monoethyl ether 0.04% 102.0

Labrasol 0.03% 90.7

Span 85 0.01% 102.7

Tween 80 0.01% 95.1

Evaluating the toxicity of the surfactants by the MTT assay was conducted to confirm the results obtained with the NRU assay. Visualization of iNHKs under the light microscope before treatment is shown in Figure 25, indicating that iNHKs were confluent. After treatment with surfactants, iNHKs where metabolically active as yellow tetrazole was successfully reduced to visible purple formazan crystals (Figure 28A). In addition, viability percentage values were higher than 95% for all of the surfactants (diethylene glycol monoethyl ether, labrasol, span 85, tween 80) examined (Figure 28B; Table 11).

In conclusion, this study indicates that constituents added to micro- and nano- emulsions, such as the diethylene glycol monoethyl ether, labrasol, tween 80 and span 85 at low concentrations do not affect the viability of iNHKs in 2D cell cultures, as shown in Table 11. These results were dually confirmed by the NRU and MTT assay.

Table 11. Viability percentage values of iNHKs treated with surfactants determined by MTT assay. Most of the samples had viability higher than 90%.

Surfactants Viability, %

Diethylene glycol monoethyl ether 0.04% 101.7

Labrasol 0.03% 95.2

Span 85 0.01% 98.1

Tween 80 0.01% 95.5

Diethylene glycol Labrasol Span 85 monoethyl ether

Tween 80 Untreated 1% SDS

B 120 100 80 60 40 viability, % viability, 20 0

Figure 28. Toxicity testing of surfactants on iNHKs by MTT assay. A, microscopic examination showed no cellular stress response. B, cellular viability was higher than 90% for iNHKs treated with surfactants indicating that samples were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments.

Treatment with micro-emulsion at concentration 10% and 1% could significantly reduce cell viability according to NRU assay but treatment with micro-emulsions at concentration 0.1% does not reduce cellular viability

Whilst it may be easier to characterize separately each ingredient of emulsions, the actual properties and the characteristics may be modified into the emulsion, during application or residence in the skin or affected by other environmental parameters. Consequently, it is crucial to examine whether micro- and nano-emulsions produced here, are cytototoxic. The toxicity of micro- and nano-emulsion, listed in Table 6 was assessed though different in vitro assays including NRU and MTT assay as detailed previously.

Microscopic evaluation of cells before the cytotoxicity assay was always necessary to evaluate their condition (Figure 25). Initially, 10, 1 and 0.1 % of micro- and nano- emulsions diluted in KSFM were examined for cytotoxicity with NRU. Microscopic evaluation after treatment with 10 and 1% of micro-emulsions for 1 hour, in comparison with microscopic evaluation before treatment (Figure 25) showed that micro-emulsions provoked cellular stress response. The viability percentage values were estimated compared to % viability of untreated cells, which was 100% while 1% SDS used as indicative cytotoxic substance (positive control). Especially, viability of iNHKs was estimated under 50% for 10% micro-emulsion having 6% isopropyl palmitate encapsulated, 10% micro-emulsion having 10, 20, 30% citrepel encapsulated, 10% micro-emulsiom having 10, 20, 30% geraniol encapsulated (Figure 29) and 10% of control micro-emulsion having no EO encapsulated, indicating that 10% micro- emulsions were cytotoxic.

Therefore, viability of iNHKs after 1 hour treatment with the same micro-emulsions was around 50-60% (Figure 30). Precisely, micro-emulsion having no EO encapsulated recorded the greatest viability (63%, micro-emulsion 8), while micro-emulsions with 10 and 20% geraniol encapsulated, do not reduce further the cell viability compared to micro-emulsion 8 (Table 12).

Isopropyl palmitate 10% 20% 30%

Citrepel

10% 20% 30% Without EO

Geraniol

Untreated 1% SDS

120

B 100 80 Citrepel Geraniol 60

Figure 29. Viability testing of iNHKs following treatment with 10% o/w micro- emulsions by the NRU assay. A, representative photographs of iNHKs after treatment with 10% micro-emulsions. Intense cellular stress response was observed. B, viability of iNHKs after treatment with micro-emulsions was estimated under 50% indicating that micro-emulsions were cytotoxic. Data are shown as median ± SEM n=2 independent experiments.

Isopropyl palmitate 10% 20% 30% Citrepel

10% 20% 30% Without EO Geraniol

Commercial product Untreated 1% SDS

B Citrepel Geraniol

Figure 30. Viability testing of iNHKs following treatment with 1% o/w micro- emulsions by the NRU assay. A, microscopic observation of iNHKs after treatment with 1% micro-emulsions. Changes in cells’ morphology were observed. B, viability of iNHKs after treatment with micro-emulsions was estimated around 50- 60% for most of the micro-emulsions. Data are shown as median.

Contrary, the NRU assay revealed that treatment for 1 hour with 0.1% of micro- emulsion having 10% citrepel encapsulated, 0.1% of micro-emulsion having 10% geraniol encapsulated and 0.1% nano-emulsion having 1% geraniol encapsulated, showed absence of cellular stress in iNHKs and viability of iNHKs was higher than 95%, showing that emulsions were not cytotoxic at low concentrations (Figure 31). In addition, the viability of iNHKs exposed to the commercially available product having 15% citrodiol encapsulated, was almost 100% and no cellular stress response was observed. Viability values are also represented at Table 12.

The cell viability to cytotoxicity ratio of iNHKs treated with 0.1% micro and nano- emulsions was also assessed by the MTT assay. Morphology of iNHKs before treatment was observed under a light microscope to confirm that cells had normal morphology and were confluent, a representative example is shown in Figure 25. Observation after treatment revealed that iNHKs were not stressed and they efficiently reduced MTT in their mitochondria (Figure 31A). In addition, viability of iNHKs treated with 0.1% micro-emulsion having 10% citrepel encapsulated as well as 0.1% micro-emulsion containing 1% geraniol encapsulated showed viability greater than 90%, while iNHKs treated with 0.1% micro-emulsion having 10% geraniol encapsulated showed viability 75%, however the difference is not statistically significant (t-test between micro- emulsion with 10% geraniol encapsulated and untreated cells) (Figure 31B; Table 13).

Across the novel emulsions tested here, the factor of concentration led to iNHKs toxicity with a concentration of 1% being less toxic than 10%, which was cytotoxic. Micro- and nano-emulsions with a concentration of 0.1% were not cytotoxic showing that concentration can have a profound effect on cells’ viability. As a result, a range of concentrations where o/w micro- and nano-emulsions are not cytotoxic to iNHKs was determined.

10% Citrepel 10% Geraniol 1% Geraniol

Commercial product Untreated 1% SDS

B 140 120 100 80 60 40 20

Viability, % Viability, 0

Figure 31. Toxicity testing of 0.1% o/w micro and nano-emulsions on iNHKs by the NRU assay. A, microscopic observation showed that iNHKs were successfully incorporated neutral red inside their lysosomes and were confluent. B, micro-emulsions’ and nano-emulsion’s viability was higher than 95%, indicating that samples were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments.

Table 12. Viability percentage values of iNHKs following treatment with 10%, 1% and 0.1% micro-emulsions determined by the NRU assay. The viability of cells treated with 10% micro-emulsions was determined to be below 50%, showing that micro-emulsions were cytotoxic. The viability of cells treated with 1% micro-emulsions was estimated between 50-60%. The viability of cells treated with 0.1% micro-emulsions and nano-emulsion were higher than 95%, thus those emulsions were not cytotoxic.

Sample Viability, %

1. 10% of 6% isopropyl palmitate encapsulated in micro-emulsion 48.1

2. 10% of 10% citrepel encapsulated in micro-emulsion 40.7

3. 10% of 20% citrepel encapsulated in micro-emulsion 42.6

4. 10% of 30% citrepel encapsulated in micro-emulsion 48.7

5. 10% of 10% geraniol encapsulated in micro-emulsion 51.7

6. 10% of 20% geraniol encapsulated in micro-emulsion 49.1

7. 10% of 30% geraniol encapsulated in micro-emulsion 48.6

8. 10% without encapsulated essential oil in micro-emulsion 53.1

1. 1% of 6% isopropyl palmitate encapsulated in micro-emulsion 58.8

2. 1% of 10% citrepel encapsulated in micro-emulsion 52.8

3. 1% of 20% citrepel encapsulated in micro-emulsion 51.8

4. 1% of 30% citrepel encapsulated in micro-emulsion 53.8

5. 1% of 10% geraniol encapsulated in micro-emulsion 60.0

6. 1% of 20% geraniol encapsulated in micro-emulsion 63.8

7. 1% of 30% geraniol encapsulated in micro-emulsion 53.8

8. 1% without encapsulated essential oil in micro-emulsion 63.8

0.1% of 10% citrepel encapsulated in micro-emulsion 104.8

0.1% of 10% geraniol encapsulated in micro-emulsion 100.9

0.1% of 1% geraniol encapsulated in nano-emulsion 98.9

Commercially available product with 15% citrodiol encapsulated* 97.1

*median; Figure 30D; Figure 31B.

Table 13. Viability percentage values of iNHKs treated with 0.1% micro- and nano-emulsions determined by MTT assay. Viability of iNHKs treated with 0.1% micro-emulsion with 10% citrepel encapsulated and nano-emulsion with 1% geraniol encapsulated were higher than 90% while iNHKs treated with 0.1% micro-emulsion with 10% geraniol encapsulated were 75.6%.

Emulsions Viability, %

0.1% of 10% citrepel encapsulated in micro-emulsion 97.6

0.1% of 10% geraniol encapsulated in micro-emulsion 75.6

0.1% of 1% geraniol encapsulated in nano-emulsion 91.2

Commercially available product with 15% citrodiol encapsulated 101.1

10 % Citrepel 10 % Geraniol 1% Geraniol

Commercial product Untreated 1% SDS

B 120 100 80 60 40

Viability, Viability, % 20 0

Figure 32. Toxicity testing of 0.1% o/w micro- and nano-emulsions on iNHKs by the MTT assay. A, microscopic evaluation showed that iNHKs were not affected by the treatment of micro-emulsions. B, viability was higher than 90%. Therefore, the samples were not cytotoxic. Data are shown as medians ± SEM n=3 independent experiments.

DISCUSSION

Overall, 3D RHS models are quite useful tools for the development and evaluation of pharmaceuticals and cosmetic products (Suhail et al., 2019) and for the study of skin diseases especially rare skin diseases (Carlson et al., 2008). Finally, RHS models are valid in vitro alternatives to animal testing as they mimic perfectly the in vivo state. Therefore, this kind of models play a pivotal role in cosmetic industry as animal welfare concerns are increasing globally while animal experimentation for cosmetic products in the EU has already be banned.

The main aim of this study was the generation of a 3D skin model that resembles native skin both structurally and biochemically. The iNHFs were mixed with collagen to form the dermis of the RHS and iNHKs were then seeded above the reconstructed “dermis” for the construction of the epidermis. Keratinocytes begun to differentiate until they formed a stratified squamous epithelium and the 3D skin model was ready to be characterized with histology and IHC.

Histology was the method used for studying the structure of the 3D RHS. Staining with eosin and hematoxylin (H&E) was enough to show that the generated 3D skin model, resembles excellently the native skin in structure. The layers of the reconstructed epidermis (SC, SG, SS, SB) as well as the reconstructed dermis were distinct and successfully developed. In addition, immunohistochemical analysis was used for determining the existence of skin proteins in the 3D RHS model. Proliferation marker (Ki67), protein related with epidermal junction (E-cadherin), differentiation markers (keratin 5, involucrin, loricrin), structural proteins of desmosomes and corneo desmosomes (DSG1, DSC1, CDSN) that are important for preserving normal skin structure and physiology, were well expressed throughout the epidermis. Even the activity of kallikrein-related peptidases has been evaluated in the epidermis of the in- house 3D RHS, showing that epidermal proteolytic enzymes like KLK5, KLK6, KLK7 that are key to normal skin function and to the desquamation process are expressed equally in 3D RHS and native skin. In conclusion, the developed 3D skin model resembles normal human skin in terms of structure and biochemistry/physiology and can be used as a reliable human skin substitute for the examination of toxicity and moisturizing action of cosmetic micro- and nano- emulsion formulations.

Generally, in vitro studies using 3D RHS in contrast to animal experimentation, offer higher speed, considering that animal testing can take months while in vitro testing in a skin substitute is a matter of days or weeks, and greater accuracy. Additionally, testing on 3D RHS helps gathering information in order to highlight the advantages and disadvantages of cosmetic products. 3D RHS has several advantages compared to 2D cultures in efficacy examination of a cosmetic product, because it gives the possibility of long term application of the testing product, it has cellular heterogeneity, structural complexity, and it resembles better the human skin considering the interactions between dermis and epidermis (Niehues et al., 2018; Schlotmann et al., 2001).

Nevertheless, 2D cultures could be a valuable tool for the preliminary evaluation of skin care cosmetic products and topically applied drugs before their examination on 3D RHS substitutes. For this reason, at this study EOs, surfactants and cosmetic micro- and nano-emulsion formulations were firstly tested for their cytotoxicity on 2D cultures as a preliminary step before the evaluation of their properties (cosmetic properties; skin moisturizing action, skin irritation, skin corrosion) on the 3D RHS. More specifically, a range of concentrations where EOs, surfactants and o/w micro- and nano-emulsions are not cytotoxic to iNHKs was determined by the NRU and/ or MTT assays.

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APPENDIX

Immortalization of keratinocytes

Primary keratinocytes were isolated from the skin of healthy donors and cultured in 0 serum-free keratinocyte growth medium (Gibco) in the presence of 5% CO2 at 37 C.

After immortalization with standard protocol stable cell lines with normal keratinocytes were generated by BIOSS Center for Biological Signaling Studies (University of Freiburg, Freiburg, Germany) with lentiviral infection, and infected keratinocytes were selected with puromycin.

Immortalization of fibroblasts

Primary fibroblasts were isolated from the skin of healthy donors and cultured in serum- 0 free keratinocyte growth medium (Gibco) in the presence of 5% CO2 at 37 C.

Following immortalization by an established protocol (Zingkou et al., 2019) the stable cell line of normal fibroblasts was generated by BIOSS Center for Biological Signaling Studies (University of Freiburg, Freiburg, Germany) with lentiviral infection, and infected fibroblasts were selected with puromycin.

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CURRICULUM VITAE

PERSONAL DETAILS

Last name : EVANGELATOU First name : KYRIAKI Date of Birth : 1995/07/04 Address : Agiou Nikolaou 27, Lagonissi, CA 19010, Greece Τelephone-Mobile : +30 2291079535, +30 6970550026 E-mail : [email protected]

EDUCATION

2018-present Μ.Sc. “Drug Discovery and Development” (GPA /10), Department of Pharmacy, School of Health Sciences, University of Patras.

2013-2018 SCD (Second Cycle Diploma) Chemical Engineering, Internationally accredited Engineering Master Degree by IChemE/EngC (GPA 8.2/10), Department of Chemical Engineering, School of Engineering, University of Patras.

LANGUAGES

Greek: Native English: Proficient (Level C2), ECPE, University of Michigan (2015) French: Basic (DELF B2) Diplôme d'études en langue française (2018)

SEMINARS AND OTHER CERTIFICATES

A series of lectures on "Plastics at Work", Speaker: Professor Dr. Rudy Koopmans, (2018/04/23-2018/04/27) Sanitary and safety seminar, University of Patras, (2019/05/06-2019/05/07) Music Theory Diploma (2015) Trampoline coach Diploma from General Secretariat of Sports-Greece (2018) National Trampoline Judge Member of Greek Guiding Association (SEO) Driving License B category (2013)

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RESEARCH EXPERIENCE

October M.Sc. Student, Group of Pharmaceutical Biotechnology and 2018-present Molecular Diagnostics, Department of Pharmacy, School of Health Sciences, University of Patras. Supervisor: Professor Georgia Sotiropoulou

Title of MSc Diploma Thesis: “Development and characterization of 3D human reconstructed skin”.

2017-2018 B.Sc Student, Laboratory of Polymers, Department of Chemical Engineering, School of Engineering, University of Patras. Supervisor: Professor Konstantinos Tsitsilianis Title of Diploma Thesis: “Layer by layer polymer coated mesoporous silica microparticles for drug delivery potential applications”

July 2017 Department of Research and Development (R&D), Pharmathen Pharmaceuticals, Athens. Main duties: • Buffer and Solution preparation • HPLC training (LabSolution software)

Academic year Design and Financial Evaluation of Biofuel Production Unit 2017-2018 from Biotechnological Exploitation of Agro-Industrial By- Products. Laboratory of the Undergraduate course: Plant Design. Department of Chemical Engineering, School of Engineering, University of Patras Supervisor: Associate professor Ioannis Koukos

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TEACHING EXPERIENCE

Teaching Assistance in the Department of Pharmacy, University of Patras Academic year: 2019-2020 For “Laboratory training of the undergraduate course “Pharmaceutical Biotechnology”

TECHNICAL EXPERTISE

Molecular Biology DNA extraction, PCR

Analytical Biochemistry SDS-PAGE, Western blotting, immunohistochemistry

Electron Micrtoscopy Scanning Electron Microscopy (SEM) Transmission Electron Microscopy (TEM)

Analytical Techniques UV-VIS spectroscopy, Thermogravimetric Analysis (TGA)

Histology-Histochemistry Histology of 3D reconstructed human skin, immunohistochemistry

Cell cultures

Informatics MS Office, Origin Pro 8, Honeywell Unisim, Matlab 2015, Fortran

PRICES/AWARDS

Pre-graduate scholarship from the State scholarships foundation (IKY), Academic years: 2016-2017, 2017-2018.

National Double mini Trampoline Champion, 2012, 2014, 2018

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PUBLICATIONS IN SCIENTIFIC INTERNATIONAL JOURNALS

Iatridi Z, Evangelatou K, Theodorakis N, Angelopoulou A, Avgoustakis K, Tsitsilianis C. (2019) “Multicompartmental mesoporous silica/polymer nanostructured hybrids: design capabilities by integrating linear and star-shaped block copolymers”. Polymers

51. [IF2018=3.771]

POSTER PRESENTATION

Evangelatou Kyriaki, Iatridi Zaharoula, Tsitsilianis Constantinos. “Layer by layer polymer coated mesoporous silica microparticles for drug delivery potential applications”. 12th Hellenic Polymer Society International Conference, University of Ioannina, Greece, 30 September- 3 October 2018.

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ORIGINALITY REPORT

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Turnitin Originality Report

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< 1% match (Internet from 10-Jan-2018) http://www.mdpi.com/1422-0067/19/1/145/htm

< 1% match (Internet from 18-Jun-2020) https://link.springer.com/article/10.1007/s11095-014-1580-3?code=9acb8585-46cd-4be0-a8d7-48767437fec4&error=cookies_not_supported

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< 1% match (Internet from 10-Apr-2020) https://www.oncotarget.com/article/21078/text/

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< 1% match (student papers from 08-Dec-2011) Submitted to University of Central Lancashire on 2011-12-08

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< 1% match (Internet from 30-Mar-2016) http://aut.researchgateway.ac.nz/bitstream/handle/10292/437/NagarS.pdf?isAllowed=y&sequence=4

< 1% match (student papers from 17-Dec-2018) Submitted to University of Hull on 2018-12-17

< 1% match (student papers from 13-May-2019) Submitted to University of Nottingham on 2019-05-13

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< 1% match (student papers from 13-Dec-2017) Submitted to Institute of Technology, Tralee on 2017-12-13

< 1% match (student papers from 01-Nov-2018) Submitted to University of Queensland on 2018-11-01

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< 1% match (publications) Giacomoni, P.U.. "Gender-linked differences in human skin", Journal of Dermatological Science, 200909

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< 1% match (publications) Choi, H.. "Sphingosylphosphorylcholine down-regulates filaggrin gene transcription through NOX5-based NADPH oxidase and cyclooxygenase-2 in human keratinocytes", Biochemical Pharmacology, 20100701

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< 1% match (student papers from 05-Sep-2019) Submitted to The University of Manchester on 2019-09-05

< 1% match (student papers from 23-Mar-2018) Submitted to Imperial College of Science, Technology and Medicine on 2018-03-23

< 1% match (student papers from 15-Jun-2018) Submitted to Medizinischen Universität Wien on 2018-06-15

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< 1% match (student papers from 12-Sep-2018) Submitted to University of Bath on 2018-09-12

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< 1% match (student papers from 06-Dec-2015) Submitted to University of Surrey on 2015-12-06

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< 1% match (publications) "Alternatives for Dermal Toxicity Testing", Springer Nature, 2017

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< 1% match (publications) "Electron Microscopy", Springer Science and Business Media LLC, 2014

< 1% match (publications) Jami, Mohammad-Saeid, Ramavati Pal, Esthelle Hoedt, Thomas A Neubert, Jan Larsen, and Simon Møller. "Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons", BMC Neuroscience, 2014.

< 1% match (publications) Wei Long Ng, Wai Yee Yeong. "The future of skin toxicology testing – 3D bioprinting meets microfluidics", International Journal of Bioprinting, 2019

< 1% match (student papers from 30-Jan-2017) Submitted to North West University on 2017-01-30

< 1% match (student papers from 27-Oct-2015) Submitted to University of Sheffield on 2015-10-27

< 1% match (student papers from 17-Apr-2013) Submitted to Univerza v Ljubljani on 2013-04-17

< 1% match (student papers from 09-May-2020) Submitted to University of California, Merced on 2020-05-09

< 1% match (student papers from 30-Apr-2013) Submitted to University of Durham on 2013-04-30

< 1% match (student papers from 08-Apr-2013) Submitted to National University of Singapore on 2013-04-08

i ii TABLE OF CONTENTS TABLE OF FIGURES...... 5 LIST OF TABLES ...... 7 LIST OF ABBREVIATIONS ...... 9 ABSTRACT ...... 11 ΠΕΡΙΛΗΨΗ...... 14 INTRODUCTION...... 18 SKIN ANATOMY...... 20 Skin structure ...... 20 Epidermal appendages ...... 23 SKIN BARRIER FUNCTION ...... 24 Epidermal differentiation...... 24 The brick and mortar model...... 26 SKIN PROTEASES ...... 30 Tissue kallikrein related peptidases ...... 30 HUMAN SKIN EQUIVALENTS ...... 31 Reconstructed human epidermis ...... 31 Full thickness HSEs ...... 33 Applications of RHEs and full thickness HSEs ...... 34 Applications of the 3D RHS in the study of drug permeation ...... 40 1 Applications of 3D RHS in irritation and corrosion ...... 42 Applications of 3D RHS in sensitization ...... 42 MICRO- AND NANO-EMULSIONS ...... 43 Properties and characteristics of micro- and nano- emulsions...... 44 Applications of micro- and nano-emulsions in drug delivery ...... 44 STATE-OF-THE ART ...... 45 SPECIFIC AIMS OF THE STUDY ...... 46 MATERIALS, METHODS AND INSTUMENTATION ...... 47 Instruments...... 47 Plastic or glass laboratory consumables ...... 47 Media and Reagents...... 48 Antibodies ...... 49 Cell lines ...... 50 Micro- and nano-emulsions, oils and surfactants ...... 50 METHODS

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...... 51 Cell culture ...... 51 Cell cryopreservation...... 51 Cell viability...... 52 Embedding tissue into paraffin blocks...... 54 Hematoxylin and eosin staining ...... 55 2 Immunohistochemistry ...... 55 Neutral red uptake assay ...... 56 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay ..... 57 RESULTS ...... 58 CHAPTER 1: Generation of 3D RHS ...... 58 Morphology of immortalized normal human dermal fibroblasts ...... 58 Morphology of immortalized normal human epidermal keratinocytes...... 59 Preparation of cellularized collagen gel ...... 60 Harvesting of 3D reconstructed tissues for analysis ...... 61 CHAPTER 2: Characterization of 3D RHS ...... 62 The 3D RHS displays a microstructure very similar to normal skin tissue ...... 62 IHC assessment showed that 3D RHS mimics biochemically the native human skin ...... 62 The 3D RHS shows increased proliferation index of keratinocytes in the epidermis...... 66 The 3D RHS maintains structural integrity similar to normal skin tissue...... 66 Cell differentiation is normal in the 3D RHS ...... 66 Normal expression pattern of structural (corneo)desmosomal proteins in 3D RHS ...... 67 The skin proteases KLK5, KLK6, and KLK7 are comparably expressed in 3D RHS and native skin ...... 67 3 CHAPTER 3: Toxicity tests of micro- and nano-emulsions, EOs and surfactants, on iNHKs...... 71 NRU and MTT assays revealed that EOs at concentrations 0.1 %, 0.01% have low and no cytotoxicity respectively ...... 73 Toxicity tests of surfactants on iNHKs assessed by the NRU and MTT assay, showed that the surfactants do not reduce cellular viability at specific concentrations ...... 81 Treatment with micro-emulsion at concentration 10% and 1% could significantly reduce cellular viability according to NRU assay, while treatment with micro-emulsions at concentration 0.1% does not reduce cellular viability . 85 DISCUSSION ...... 94 APPENDIX ...... 110 Immortalization of keratinocytes ...... 110 Immortalization of fibroblasts ...... 110 CURRICULUM VITAE...... Error! Bookmark not defined. TABLE OF FIGURES Figure 1. Schematic illustration of skin structure...... 20 Figure 2. Schematic depiction of the epidermis...... 22 Figure 3. Epidermal differentiation...... 24 Figure 4. Bricks and mortar model for the structure of the SC...... 28 Figure 5. Generation of 3D-skin models...... 32 Figure 6. Histological images of RHE...... 33 Figure 7. Depiction of 3D tissue construction...... 34 Figure 8. Applications of full thickness skin models...... 35 Figure 9. Cell number calculation and cell viability estimation...... 53 Figure 10. Chemical structure of NR dye...... 57 Figure 11. Structure of both MTT and formazan...... 57 Figure 12. Immortalized normal human dermal fibroblasts...... 58 Figure 13. Immortalized normal human epidermal keratinocytes...... 59 Figure 14. Preparation of 3D RHS...... 60 Figure 15. Macroscopic appearance of 3D RHS at air-liquid interface...... 61 Figure 16. The 3D RHS forms a stratified squamous epithelium...... 63 Figure 17. Epidermis of 3D RHS is hyperproliferative...... 64 Figure 18. Immunohistochemical analysis of E-cadherin...... 65 Figure 19. Immunohistochemical analyses of skin differentiation markers...... 68 Figure 20. Immunohistochemical analyses of the structural proteins of desmosomes and corneodesmosomes...... 69 Figure 21. Immunohistochemical analyses of human KLK proteases in skin. .... 70 Figure 22. Indicative photographs of iNHKs before treatment...... 73 Figure 23. Viability testing of iNHKs following treatment with 0.1% EOs by NRU assay...... 75 Figure 24. Viability testing of iNHKs following treatment with 0.01% EOs by NRU assay...... 77 Figure 25. Morphology of iNHKs before treatment with EOs...... 78 Figure 26. Viability testing of iNHKs following treatment with 0.01% EOs by MTT assay...... 79 Figure 27. Toxicity testing of surfactants on iNHKs by NRU assay...... 82 Figure 28. Toxicity testing of surfactants on iNHKs by MTT assay...... 84 Figure 29. Viability testing of iNHKs following treatment with 10% o/w micro- emulsions by NRU assay...... 86 Figure 30. Viability testing of iNHKs following treatment with 1% o/w micro- emulsions by NRU assay...... 87 Figure 31. Toxicity testing of 0.1% o/w micro and nano-emulsions on iNHKs by NRU assay...... 89 Figure 32. Toxicity testing of 0.1% o/w micro- and nano-emulsions on iNHKs by MTT assay...... 92 LIST OF TABLES Table 1. Full thickness HSEs...... 37 Table 2. Reconstructed 3D skin disease models...... 37 Table 3. RHE used for drug permeation testing...... 41 Table 4. Reconstructed Full thickness skin used for drug permeation testing. ... 41 Table 5. Comparison of size, shape, stability, method of preparation and polydispersity in different kinds of emulsions...... 43 Table 6. Different micro-emulsions and their potential substances tested for in vitro cytotoxicity on iNHKs...... 72 Table 7. Viability percentage values of iNHKs treated with 0.1% EOs determined by NRU assay...... 76 Table 8. Viability percentage values of iNHKs treated with 0.01% EOs determined by NRU assay...... 76 Table 9. Viability percentage values of iNHKs treated with 0.01% EOs determined by MTT assay...... 80 Table 10. The % viability values of the surfactants tested for cytotoxicity on iNHKs determined by NRU assay...... 83 Table 11. Viability percentage values of iNHKs treated with surfactants determined by MTT assay...... 83 Table 12. Viability percentage values of iNHKs following treatment with 10%, 1% and 0.1% micro-emulsions determined by NRU assay...... 90 Table 13. Viability percentage values of iNHKs treated with 0.1% micro- and nano-emulsions determined by MTT assay...... 91 LIST OF ABBREVIATIONS 2D, two dimensional 3D, three dimensional BSA, bovine serum albumin CaCl2, calcium chloride CO2, Carbon dioxide CE, cornified envelope CSDN, corneodesmosin DAB, 3,3-diaminobenzidine DED, deepidermized dermis DF, dilution factor DSC1, desmocollin1 DSG1, desmoglein1 DMEM, dulbecco’s modified eagle medium DMSO, dimethyl sulfoxide DNFB, 1-fluoro -2,4- dinitrobenzene D-PBS, dulbecco’s phosphate saline EDTA, ethylenediaminetetraacetic acid EU, european union EOs, essential oils FDA, food and drug administration FBS, fetal bovine serum H2O2, hydrogen peroxide HBSS, hank’s balanced salt solution HEEs, human epidermal equivalents HCl, hydrochloric acid HSEs, human skin equivalents HSV1, herpes simplex virus 1 HUVEC, Human Umbilical Vein Endothelial Cells PBS, phosphate- buffered saline PGE2, prostaglandin E2 RDEB, recessive dystrophic epidermolysis bullosa RH, relative humidity rhEGF, recombinant human epidermal growth factor RHS, reconstructed human skin RHE, reconstructed human epidermis RT, room temperature IHC, immunohistochemistry iNHKs, immortalized normal human keratinocytes iNHFs, immortalized normal human fibroblasts IL-1a, interleukin-1a IL-6, interleukin 6 IL-8, interleukin 8 IL-17, interleukin 17 IPM, isopropyl myristate K1, keratin 1 K5, keratin 5 K10, keratin 10 K14, keratin 14 KLK, kallikrein-related peptidase KSFM, keratinocyte serum-free medium MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide OECD, organisation for economic co-operation and development OTC, organotypic cultures o/w, oil in water SB, stratum basale SC, stratum corneum SDS, sodium dodecyl sulfate SG, stratum granulosum SP, stratum spinosum TEWL, transepidermal water loss NR, neutral red NRU, neutral red uptake NMF, natural moisturizing factor UV, ultraviolet light w/o, water in oil ABSTRACT Three-dimensional (3D, 3 Dimensional) reconstructed human skin equivalents (HSEs, Human Skin Equivalents), (i.e. epidermal models, 3D reconstructed full- thickness skin) are extremely valuable tools for in vitrο research, as they are scientifically valid alternatives to animal experimentation. Consequently, in 2013, testing cosmetic products and ingredients in animals was banned by the European Commission (Niehues et al., 2018). HSEs mimic the native skin, structurally and biochemically, with comparable cellular heterogeneity and structural complexity (Niehues et al., 2018). Thus, they play a key role in preclinical research for drug development (Mathes et al., 2014), while they are also absolutely essential to the cosmetics industries (Gabbanini et al., 2009). Importantly, 3D reconstructed human skin equivalents (RHS, Reconstructed Human Skin) are very useful in studying rare skin diseases, such as the recessive dystrophic epidermolysis bullosa (RDEB, Recessive Dystrophic Epidermolysis Bullosa) (Mittapalli et al., 2016) but also common skin diseases like psoriasis (Tjabringa and Bergers, 2008; Chiricozzi et al., 2014). The present study aims to develop and characterize a 3D RHS model by use of immortalized normal human dermal fibroblasts (iNHFs, immortalized Normal Human Fibroblasts) and immortalized normal human epidermal keratinocytes (iNHKs, immortalized Normal Human Keratinocytes) and, subsequently, characterize the developed RHS, structurally and biochemically, by parallel comparison with native skin tissue. Furthermore, the generated RHS was exploited for testing cosmetic micro- and nano-emulsion formulations, as well as their constituent essential oils (EOs) and surfactants. These were initially tested here in two dimensional (2D, 2 Dimensional) cultures for potential cytotoxicity and will be subsequently evaluated in our “in- house” 3D RHS. For generation of the 3D RHS, iNHFs were mixed with collagen and placed into a 6- well plate that allowed the intake of growth factors, hormones and nutrients suitable for the development of the 3D RHS, then, iNHKs were seeded on the reconstructed dermis. After four weeks in culture, the 3D RHS was ready to be characterized using established microscopic, histological and molecular assays. Staining of the prepared 3D RHS with hematoxylin and eosin (H&E) showed that its microstructure mimics that of the native skin, and it had a physiological epidermal architecture containing the characteristic distinct epidermal layers, i .e. the stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS) and stratum basale (SB). Consistently, normal expression of key skin proteins was confirmed by immunohistochemistry (IHC) using specific antibodies. In particular, structural proteins of desmosomes, such as desmoglein1 (DSG1), desmocolin1 (DSC1), and corneodesmosin (CDSN), as well as the characteristic cell adhesion molecule E- cadherin displayed similar expression profiles in the 3D RHS and native skin. Furthermore, proteins related to the regulation of skin’s exfoliation/desquamation, such as the epidermal proteases KLK5, KLK6, and KLK7, as well as proteins related to the skin differentiation process, such as the involucrin, loricrin and keratin 5, were similarly expressed in both 3D RHS and native skin. Elevated expression of Ki67 in 3D RHS indicated increased proliferation compared to native skin as also observed by others. Cumulatively, the developed 3D RHS mimics native skin, thus, it can be used for the evaluation of cytotoxicity and moisturizing action of cosmetic oil in water (o/w, oil in water) micro- and nano-emulsion formulations (ΕΥΔΕ ΕΤΑΚ-ΕΥΔ ΕΠΑνΕΚ ΕΣΠΑ 2014-2020/ QFytoTera-Τ1ΕΔΚ-00996). The neutral red uptake (ΝRU, Neutral Red Uptake) assay and colorimetric MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were applied for testing the cytotoxicity of EOs, surfactants and micro- and nano-emulsions on 2D cultures of iNHKs. The majority of EOs and cosmetic micro-emulsion formulations tested were found non-cytotoxic, with values of iNHKs viability higher than 95% at the lower EOs concentration range. Therefore, their cosmetic properties, such as their moisturizing action, will be evaluated by application on the 3D RHS but also to test whether they may cause undesired skin irritation and corrosion, as part of their integrated assessment in vitro. Keywords: 3D reconstructed human skin, histology, immunohistochemistry, microemulsions, nanoemulsions, essential oils, cytotoxicity, neutral red uptake assay, MTT assay ΠΕΡΙΛΗΨΗ Η καθιέρωση της χρήσης νέων, αλλά επιστημονικά τεκμηριωμένων και αξιόπιστων in vitro πειραματικών προτύπων που προσομοιάζουν καλύτερα και με ακρίβεια τους ανθρώπινους ιστούς είναι μεγάλης σημασίας. Τα τρισδιάστατα (3D, 3 Dimensional) υποκατάστατα ανθρωπίνου δέρματος (HSEs, Human Skin Equivalents), τα οποία είναι υποκατάστατα ολοκλήρου ιστού δέρματος ή μόνον της επιδερμίδας, αποτελούν πρότυπα, τα οποία προσομοιάζουν το ανθρώπινο δέρμα, δομικά και βιοχημικά, δεδομένου ότι διαθέτουν κυτταρική ετερογένεια και μιμούνται τις κυτταρικές αλληλεπιδράσεις που υφίστανται στο φυσιολογικό δέρμα (Niehues et al., 2018). Παράλληλα, ένα πολύ σημαντικό πλεονέκτημα που αποφέρουν είναι ο περιορισμός της χρήσης πειραματόζωων, των οποίων η χρήση, ειδικά στον τομέα των καλλυντικών, έχει απαγορευτεί από την Ευρωπαϊκή Επιτροπή από το έτος 2013. Συνεπώς, η ανάπτυξη τέτοιου είδους προτύπων είναι καθοριστική για τις εταιρείες καλλυντικών. Ωστόσο, τα υποκατάστατα ανθρωπίνου 3D δέρματος (RHS, Reconstructed Human Skin) συμβάλλουν, επίσης, στην προκλινική αξιολόγηση διαδερμικά χορηγούμενων φαρμακευτικών ουσιών (Mathes et al., 2014) και συνεισφέρουν παράλληλα στην μελέτη κοινών αλλά και σπανίων δερματικών ασθενειών, όπως η ψωρίαση (Tjabringa and Bergers 2008; Chiricozzi et al., 2014) και η πομφoλυγώδης επιδερμόλυση (RDEB, Recessive Dystrophic Epidermolysis Bullosa) (Mittapalli et al., 2016). Η παρούσα μελέτη στοχεύει στην δημιουργία προτύπου 3D RHS, το οποίο προσομοιάζει το φυσιολογικό

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δέρμα ανθρώπου, βιοχημικά και δομικά, συγκεκριμένα εκφράζει χαρακτηριστικές πρωτεΐνες του δέρματος σε συγκρίσιμα επίπεδα, και ιστολογικά διαθέτει χορίο και διαφοροποιημένη επιδερμίδα, αντίστοιχα. Επιπλέον, σκοπός της μελέτης είναι η αξιολόγηση της κυτταροτοξικότητας μικρο- και νάνο- γαλακτωμάτων ως υποψήφιων καλλυντικών, μεμονωμένων συστατικών τους, αλλά και των διαφόρων αιθέριων ελαίων, σε δυσδιάστατες (2D, 2 Dimensional) καλλιέργειες φυσιολογικών κερατινοκυττάρων ανθρώπου που έχουν αθανατοποιηθεί (iNHKs, immortalized Normal Human Keratinocytes), ως πρόδρομο βήμα, προκειμένου να αξιολογηθούν στη συνέχεια στο σύστημα 3D RHS, οι καλλυντικές ιδιότητες, όπως η ενυδάτωση, αλλά και να ελεγχθεί κατά πόσον προκαλούν ανεπιθύμητη ερεθιστικότητα ή διάβρωση στο δέρμα. Για την παρασκευή του συστήματος 3D RHS, χρησιμοποιήθηκαν κερατινοκύτταρα και ινοβλάστες που απομονώθηκαν από βιοψία φυσιολογικού δέρματος ανθρώπου, καθιερώθηκαν σε καλλιέργεια και αθανατοποιήθηκαν (βλ. Παράρτημα), προκειμένου να διαιρούνται χωρίς περιορισμό στον αριθμό των κυτταρικών διαιρέσεων. Αρχικά, οι αθανατοποιημένοι ινοβλάστες (iNHFs, immortalized Normal Human Fibroblasts) ενσωματώθηκαν σε κολλαγόνο σε κατάλληλο ικρίωμα που επιτρέπει την πρόσληψη αυξητικών παραγόντων, ορμονών και θρεπτικών συστατικών και, στη συνέχεια, επιστρώθηκαν με iNHKs. Μετά από περίπου 4 εβδομάδες σε καλλιέργεια λήφθηκε το RHS, το οποίο αξιολογήθηκε με καθιερωμένες μικροσκοπικές, ιστολογικές και μοριακές μεθόδους συγκρινόμενο παράλληλα με φυσιολογικό ιστό δέρματος ανθρώπου. Παρατηρήθηκε σημαντική δομική ομοιότητα του 3D RHS με το φυσιολογικό ανθρώπινο δέρμα, η οποία επιβεβαιώθηκε μικροσκοπικά με χρώση βιοψιών με ηωσίνη και αιματοξυλίνη (H&E) και διαπιστώθηκε φυσιολογική δομή δέρματος με διακριτές στιβάδες της επιδερμίδας από το υποκείμενο χόριο. Ανοσοϊστοχημική ανάλυση της έκφρασης βασικών δομικών πρωτεϊνών των δεσμοσωμάτων, όπως η δεσμογλεΐνη 1 (DSG1), η δεσμοκολλίνη 1 (DSC1), και η κορνεοδεσμοσίνη (CDSN), έδειξε ότι το 3D στο RHS παρουσιάζει φυσιολογικά επίπεδα έκφρασης των πρωτεϊνών αυτών, χωροταξικά κατανεμημένη όπως στον φυσιολογικό ιστό δέρματος. Εν συνεχεία, προσδιορίσθηκε η χωροταξιήά έκφραση σερινοπρωτεασών της οικογένειας των καλλικρεϊνών, όπως η KLK5, η KLK6, και η KLK7, σε βιοψίες 3D RHS και σε ανθρώπινο φυσιολογικό δέρμα. Οι πρωτεάσες αυτές αποτελούν κεντρικούς ρυθμιστές της φυσιολογικής αποφολίδωσης (απολέπισης) του δέρματος. Επιπλέον, στο σύστημα 3D RHS παρατηρήθηκε φυσιολογικό προφίλ έκφρασης δεικτών διαφοροποίησης, όπως η ινβολουκρίνη, η λορικρίνη, και η κερατίνη 5 υποδεικνύοντας ότι τα κερατινοκύτταρα στο 3D RHS έχουν φυσιολογική διαφοροποίηση. Επιπλέον, διαπιστώθηκε ότι τα επίπεδα της πρωτεΐνης Ki67 είναι υψηλότερα στο 3D RHS σε σχέση με την έκφραση της Ki67 σε βιοψία φυσιολογικού δέρματος, υποδεικνύοντας αυξημένο πολλαπλασιασμό των κερατινοκυττάρων, κάτι που έχει παρατηρηθεί και από άλλους ερευνητές. Συνοπτικά, το 3D RHS που παρασκευάσθηκε προσομοιάζει το ανθρώπινο δέρμα με βάση την δομή, την διαφοροποίηση και την έκφραση λειτουργικών μορίων, και ως εκ τούτου μπορεί να αξιοποιηθεί σε τρέχουσες μελέτες κυτταροτοξικότητας και μελέτες των ενυδατικών ιδιοτήτων μικρο- και νανο-γαλακτωμάτων ελαίου σε νερό (o/w, oil in water) φυτικών αιθέριων ελαίων (ΕΥΔΕ ΕΤΑΚ-ΕΥΔ ΕΠΑνΕΚ ΕΣΠΑ 2014-2020 / QFytoTera-Τ1ΕΔΚ-00996). Στη συνέχεια, υπό ανάπτυξη καλλυντικά προϊόντα εφαρμόσθηκαν σε δυσδιάστατες καλλιέργειες iNHKs και η κυτταροτοξικότητα αυτών προσδιορίσθηκε με τη μέθοδο «πρόσληψης της χρωστικής ουδέτερου ερυθρού» (ΝRU, Neutral Red Uptake), καθώς και με τη χρωματομετρική μέθοδο MTT (βρωμιούχο 3-(4,5-διμεθυλοθειαζολ-2-υλο) - 2,5- διφαινυλοτετραζολίο). Στην πλειοψηφία τους, τα υπό ανάπτυξη καλλυντικά προϊόντα και τα συστατικά τους, βρέθηκαν μη τοξικά. Συγκεκριμένα, η βιωσιμότητα των iNHKs, έπειτα από έκθεση σε αιθέρια έλαια χαμηλών συγκεντρώσεων, ήταν 95% κατ’ ελάχιστον. Συνεπώς, η αξιολόγηση της ευερεθιστικότητας και των καλλυντικών ιδιοτήτων των προϊόντων στο σύστημα 3D RHS είναι το επόμενο βήμα στη διαδικασία ανάπτυξης και αξιολόγησης των προϊόντων αυτών. Λέξεις κλειδιά: Τρισδιάστατο (3D) υποκατάστατο δέρματος, ιστολογία, ανοσοϊστοχημεία, μικρογαλακτώματα, νανογαλακτώματα, αιθέρια έλαια, κυτταροτοξικότητα, πρόσληψη χρωστικής ουδέτερου ερυθρού, MTT INTRODUCTION SKIN ANATOMY Skin structure The skin is the largest organ in the human body, which separates living organisms from the environment, while providing protection against a variety of external (physical, chemical and biological) insults (Baroni et al., 2012). Skin is composed of three distinct structural layers: the epidermis, the dermis and subcutaneous tissue, as illustrated in Figure 1 (Mathes et al., 2014). The epidermis constitutes the outermost layer of the skin. Its complex structure comprises four distinct layers of keratinocytes at increasing stages of differentiation going towards the outer side of the epidermis (Eckert et al., 2005). Dermis represents the collagenous connective tissue between the epidermis and the underlying subcutaneous tissue that provides structural protection for the underlying skeletal muscles and organs, while it reinforces skin toughness. Figure 1. Schematic illustration of skin structure. Skin is comprised of three main layers: epidermis, dermis, and subcutaneous tissue. Skin appendages, like hair, sebaceous glands, sweat glands as well as blood vessels are embedded in skin (adapted from Mathes et al., 2014). Epidermis represents the thinner layer of the skin consisting of a complex wall of stratified squamous epithelial cells organized in four distinct sublayers that exhibit varying characteristics depending on the corresponding degree of keratinocyte differentiation. The degree of differentiation increases across the four layers of epidermis, i.e. stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC) (Figure 2). Stratum basale comprises basal keratinocytes, the least differentiated cells of the epidermis. Part of basal keratinocytes are stem cells hooked into the dermis by hemidesmosomes that are attached to collagen at the basement membrane (Treuting et al., 2017). Basal keratinocytes divide to form partially differentiated keratinocytes, which leave the basal layer moving outwards to the skin surface. Melanocytes and Merkel cells are also found in the basal layer, where they play vital roles in several functions in situ. Melanocytes are responsible for the production of melanosomes that are transferred to the keratinocytes (Treuting et al., 2017). Melanosomes, provide protection against ultraviolet (UV) light and determine skin color (Cichorek et al., 2013). Merkel cells are involved in terminal filaments of cutaneous nerves and they are associated with light touch sensation (Wever et al., 2015). The SS consists of keratinocytes and Langerhans cells. Keratinocytes are interconnected by desmosmomes, intercellular structures involved in cell-to-cell adhesion, i.e. protein complexes that aid adjacent cells to attach to each other as depicted in Figure 2 (Green and Jones, 1996; Ross and Christiano, 2006). Langerhans cells, on the other hand, are dendritic immune cells that are found in the middle of SS. These cells participate in immune functions of the skin as antigen-presenting cells (Holikova et al., 2001). The SG is the layer of the epidermis where final differentiation occurs, a process known as cornification. Keratohyalin granules, which contain the main structural proteins of the SC, such as the keratins, are also found in SG. During keratinization, proteins are crosslinked by transglutaminases inside the cytoplasmic membrane to form the cornified envelope (CE) (Candi et al., 2005). In the transition zone between SG and SC, during the cornification process, keratinocytes lose their organelles, including the nucleus, their cytoplasm appears granular and they turn into dead, flattened corneocytes. Corneocytes are linked by corneodesmosomes, which are modified desmosomes containing corneodesmosin protein (CDSN) (Ovaere et al., 2009). The SC consists of apparently 15-20 layers of corneocytes that are embedded in a lipid envelope (Eckhart et al., 2013). The outermost layer of the SC, continuously sheds corneocytes, which are constantly replaced by newly formed corneocytes. To preserve homeostasis, the process of desquamation must be tightly regulated. In the transitional zone between the SG and SC, around the CE, lipids are extruded to form a water repelling envelope, thus, ensuring an effective permeability barrier function of the epidermis, which offers adequate protection from external insults (Denecker et al., 2008). Figure 2. Schematic depiction of the epidermis. The SB, SG, SS, and SC are illustrated as separate layers. Starting from the bottom, basal keratinocytes continue to multiply until they reach the SC, where they eventually undergo terminal differentiation to corneocytes, i.e. dead cells without a nucleus or other organelles. Desmosomes, composed of cadherins (desmogleins 1, 3 - extracellular connectors), aid intercellular adhesion of epidermal cells, while hemidesmosomes, which consist of a variety of proteins, maintain the basement membrane- keratinocyte junction (adapted from Ross et al., 2006). Dermis is the collagenous conjoining tissue located precisely underneath the epidermis that confers toughness and strength to the skin and indispensable for protection of internal organs. Both collagen and elastin are extracellular matrix components produced by fibroblasts (mesenchymal cells). Elastin is important for skin flexibility and elasticity, while matrix components, like proteoglycans, maintain hydration in the extracellular matrix, thus, providing viscoelasticity (Smith and Melrose, 2015). Furthermore, the dermis hosts hair roots, sebaceous glands, sweat glands, nervous and mast cells, macrophages and dermal dendrocytes. Subcutaneous tissue, which is located underneath the dermis, is composed of white fat and loose connective tissue containing adipocytes (Treuting et al., 2017). Epidermal appendages Epidermal appendages include sweat glands, sebaceous and mammary glands, hair, hair follicles and nails. Sweat glands, which are located inside the dermis, are classified in two types: eccrine and apocrine. Eccrine glands exist almost everywhere in human skin and are responsible for secretion of sweat. Apocrine sweat glands are located mostly in axillae and perianal areas in humans (Kurosumi et al., 1984) and are related to emotional sweating (stress, fear, pain, sexual stimulation) (Wilke et al., 2007). Sebaceous glands are exocrine glands that produce sebum. They cover the surface of the skin except for foot soles and hand palms (James et al., 2006). They are classified into those connected with hair follicles and those found in hairless areas of the body (i.e. hairless areas of nose, eyelids, nipples, mucosal membrane of the cheeks) (Young et al., 2006). Sebum plays a vital role in epidermal structure development and in normal skin barrier function (Pilgram et al., 2001), while it provides protection against microbes by production of antioxidants on skin surface (Packer et al., 1999). Hair follicles are self-renewing structures composed of dermal papilla, root sheaths, and the bulge region. Hair follicles are renewed by a cyclic growth procedure of three distinct phases: the growing phase, the regression phase and the resting phase. Formation of hair occurs during the anagen-growth phase by proliferation of matrix keratinocytes in the bulb, and the duration of this phase usually depends on the hair type. Proliferation of matrix cells eventually ends during the catagen-regression phase, hair growth is terminated, while hair falling occurs during the telogen-resting phase, in preparation of a new anagen phase (Everts, 2012). SKIN BARRIER FUNCTION Skin functions as a protective and immunological barrier to external threats (Elias and Choi, 2005). It prevents skin invasion by allergens and pathogens, it protects the organism from dehydration, it aids temperature regulation, while it diminishes the destructive effects of UV radiation. Finally, skin provides protection against physical, chemical, thermal and mechanical injuries (Wickett and Visscher, 2006). Epidermal differentiation The detachment of keratinocytes from the SB, induces a change in their gene expression profile under the control of transcription factors. Keratin 5 (K5) and keratin 14 (K14) are expressed by proliferating keratinocytes throughout the SB, as shown in Figure 3, while keratin 1 (K1) and keratin 10 (K10) are expressed by differentiating keratinocytes (Eckhart et al., 2013). Later in the differentiation process, keratinocytes express differentiation markers, such as involucrin, loricrin, filaggrin, as well as (corneo) desmosomal proteins, which play a vital role in the formation of the skin barrier. Figure 3. Epidermal layers contain keratinocytes of increasing degrees of differentiation and each expresses specific markers and other functional proteins. Epidermis contains keratinocytes, which multiply within the basal layer. As differentiation occurs, keratinocytes move from the lower layers upwards to the outermost surface, becoming increasingly compacted in size and unnucleated, and they are eventually shed from the skin surface, in a process named skin desquamation. Specific proteins are expressed in each stage of epidermal differentiation, as depicted. Keratinocytes produce keratohyalin granules in the granular layer, which are composed of filaggrin mixed with keratin fibers to prevent the disintegration of filaggrin by proteolytic enzymes (Sandilands et al., 2009). Finally, keratin-filaggrin complexes are broken down by proteolytic enzymes, such as the caspase 14. Water- preserving keratins remain inside the corneocytes, while filaggrin forms the outer surface of corneocytes. During this process, the moisture content of the skin is decreased and specific proteolytic enzymes in the SC degrade filaggrin into free amino acids (Denecker et al., 2008). The brick and mortar model The bricks and mortar model has been used to describe the structural organization of the SC (Figure 4). Corneocytes with their cell envelopes represent the bricks and the lipids between the corneocytes represent the mortar (Elias et al., 1983). The SC consists of intermediate filaments, structural proteins in nails, hair and skin, which partially form the cytoskeleton of nucleated cells. Keratins comprise the two largest categories of intermediate filament proteins: ? The acidic type I keratin ? The neutral to basic type II keratin Acidic type I keratin consists of proteins with negatively charged amino acids (e.g.; aspartic or glutamic acid). Neutral to basic type II keratin mostly contain amino acids with positively charged side chains (e.g. lysine, arginine or histidine). As a result, the alpha- helices of proteins interact with each other to form a structure known as coiled- coil (Fuchs, 1995; Steinert, 1993). Coiled-coils play an incredibly vital role for the structure of keratinocytes and corneocytes, moreover, they are considered to be important for preserving cohesion. Incorrect assembly of coiled coils can result in fragile keratinocytes, which smash easily and can lead to blistering diseases (Coulombe, 1991; Fuchs, 1995). During differentiation of keratinocytes to corneocytes, the coiled coils compound to form fibrils, which are microstructures located parallel to the surface of the skin, thus, reinforcing corneocytes (Norlén et al.,1997). Profilaggrin, the main component of keratohyalin granules, participates in aggregation of keratin coiled coils (Dale et al., 1997). Profilaggrin has no keratin binding activity and is heavily phosphorylated. During terminal differentiation, proteolytic enzymes dephosphorylate and digest profilaggrin into multiple filaggrin monomers. Free filaggrin binds to keratin intermediate filaments, thus, provoking their aggregation into macrofibrils, which are subsequently, crosslinked by transglutaminases to create a highly insoluble keratin matrix. More specifically, the CE proteins and lipids from the SC attach to this keratin matrix, which acts as a protein scaffold (Sandilands et al., 2009). Natural moisturizing factors (NMFs) play a crucial role in the physiological maintenance of SC hydration, which offers flexibility and proper desquamation. NMFs are comprised mainly of lactic acid, urea and salts (Nakagawa et al., 2004; Rawlings et al., 1994). Keratinocytes have a water impermeable phospholipid bilayer in the lower layers of the epidermis, while at the SG, the keratinocyte membrane is modified to the resistant envelope of the corneocytes (Candi et al., 2005). Transglutaminase (TGase) 1 and 3 are enzymes of the TGase family, that are considered to take part in the development of the CE (Thacher et al., 1985). Furthermore, various proteins, like loricrin and involucrin, are involved in the crosslinking reaction and are substrates for TGases. Loricrin is a spherical protein released by keratohyalin granules, composed mainly of hydrophobic amino acids and cysteine (Candi et al., 1995). Α preliminary step for the formation of the CE is the crosslinking between involucrin and loricrin (Steinert and Marekov, 1997). Keratin fibers are crosslinked to the CE as well, while lipids - which are highly important for ensuring a normal skin barrier function (Meguro et al., 2000) - are attached to involucrin on the external layer (Candi et al., 1998; Marekov and Steinert 1998). Ceramides, cholesterol, and free fatty acids form

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the intercellular lamellar lipid membrane (the “mortar”) and they are produced enzymatically in the SC from glycosylceramides, sphingomyelin and phospholipids, respectively. These precursor lipids, which are included in lamellar bodies in the granular layer, are released into the intercellular space at the cornified layer, where the glycosylceramides are converted to ceramides by β-glucosylcerebrosidase (Holleran et al., 1994). Stacked lipid structures that surround corneocytes are composed of ceramides and are capable of binding water molecules in their hydrophilic region, thus, preventing movement of water out of the surface layers of the skin by creating an impermeable barrier. In the SC, phospholipases break down phospholipids of the keratinocytes in the living layers, to produce fatty acids, which play a vital role in normal skin barrier function and contribute to pH acidification of the SC. The acidic pH provides defense against bacterial infections (Fluhr et al., 2001). Lipids have two vital roles: ? To preserve skin homeostasis by maintaining the cornified layer’s content. ? To ensure normal transepidermal water loss (TEWL). Corneodesmosomes are the main intercellular adhesive structures that are found in the SC and other cornified squamous epithelia (Harding, 2004). These complexes contain three proteins, i.e. desmoglein1 (DSG1), desmocollin1 (DSC1), and CDSN, in the extracellular portion (Jonca et al., 2002). CDSN is encoded by the CDSN gene. During maturation of the cornified layers, CDSN undergoes a series of cleavages, which are thought to be required for desquamation. Figure 4. Bricks and mortar model for the structure of the SC. “Bricks” represent the corneocytes and “mortar” depicts the intercellular lamellar lipid membrane. Mechanisms that regulate skin barrier homeostasis The integrity of the skin barrier is indispensable for normal skin function and is maintained by tightly regulated mechanisms (i.e. hydration of the SC, calcium ions gradient in the epidermis, skin surface acidity), which are responsible for preserving homeostasis of the skin barrier. Deregulation of these regulatory mechanisms has severe consequences, such as the deterioration of skin barrier functions. An important role of the SC barrier is to regulate and control water flow to the external environment, as water is fundamental for most of the functional processes in living organisms (Feingold et al., 2007). Physiological properties, like skin elasticity, are closely related to water retention and hydration of the SC. Reduced hydration of the SC could result in deregulated degradation. As a result, corneocytes accumulate on the skin surface as in certain skin disorders like ichthyosis vulgaris and xerosis of the skin (Rawlings AV, 2003). Decreased hydration of the SC provokes degradation of filaggrin to hygroscopic amino acids that constitute the natural moisturizing factor (NMF), which preserves water in the SC (Scott et al., 1986). Modified humidity of the SC may lead to release of proinflammatory mediators, such as interleukin-1a (IL-1), which usually promotes the progression of inflammatory skin disorders (Wood et al., 1996). A vertical Ca2+ concentration gradient is gradually increased at the SG and decreases in SB and SS (Cornelissen et al., 2007). Changes in Ca2+ concentrations may result in upregulation of KLK5 and KLK7 expression. For example, high levels of Ca2+ are related to the expression of KLK5 and KLK7 at mRNA and protein levels providing an indication that Ca2+ deposition gradient and production of KLKs in SG are related (Pampalakis and Sotiropoulou, 2017a). SC and tight junctions in the granular layer form and maintain Ca2+ gradient (Kurasawa et al., 2007). The Ca2+ gradient is important for preserving epidermal homeostasis and vanishes upon skin barrier disruption. The skin barrier is restored in parallel with the restoration of the Ca2+ gradient. Moreover, the Ca2+ gradient is strongly related to keratinocyte differentiation (Elias et al., 2002). Finally, the acidic pH of the skin surface layers is crucial for the defensive mechanisms of the epidermal barrier and for maintenance of its integrity (Hachem et al., 2003). SKIN PROTEASES The epidermis contains proteases and endogenous proteases inhibitors, that are necessary for the function of the skin barrier and for desquamation (de Veer et al., 2014). Proteases are classified as aspartate-, cysteine-, glutamate-, metallo-, serine- and threonine proteases, depending on their catalytic sequences. Different proteases and inhibitors exist throughout the epidermal layers where they play major roles in strictly controlled processes, having significant importance for preserving normal barrier function and skin homeostasis. Disruption of the epidermal barrier is followed by abnormal activities of serine protease (Hachem et al., 2006). Tissue kallikrein related peptidases Tissue kallikrein-related peptidases (KLKs) are a family of 15 serine proteases with trypsin- or chymotrypsin-like activity. They constitute the largest group of serine proteases in humans and their genes are clustered together in a non-interrupted way on chromosome 19q13.3-13.4 (Sotiropoulou et al., 2002). In skin, they are principally produced by epidermal keratinocytes of the SG and are released in the intercellular space between SG and SC. Specifically, KLK5, is a trypsin-like protease (Yousef et al., 2003; Michael et al., 2005), which was identified in SC and it plays a crucial role in the regulation of the desquamation process (Furio et al., 2015; Ekholm et al., 2000) due to its autocatalytic activation, which leads to the activation of other epidermal proteases that form a proteolytic cascade (Brattsand et al., 2005; Pampalakis et al., 2007; Michael et al., 2006). In addition, KLK5, KLK7, KLK14 are related to the regulation of skin’s exfoliation through the gradual proteolytic cleavage of corneodesmosomes (Brattsand et al., 2005; Borgoño et al., 2007; Sotiropoulou et al., 2009). Generally, activation of KLKs result in corneocytes’ shedding as they cleave the structural proteins of corneodesmosomes. In particular KLK5 cleaves DSG1, DSC1, and CDSN, and KLK7 cleaves DSC and CDSN at pH 5.6 (Caubet et al., 2004). In addition, abnormal activities of KLKs are connected to the underlying mechanisms of several skin diseases, for instance it was observed that levels of KLK6 are elevated in psoriatic skin (Komatsu et al., 2007). HUMAN SKIN EQUIVALENTS Human skin equivalents (HSEs) are in vitro skin substitutes consisting of primary human skin cells and collagen. HSEs resemble native skin in terms of structure and biochemistry (Zhang and Michniak-Kohn, 2012). In 1976, one of the first explant 3D model was designed when inverted dead pig skin was utilized to establish outgrowth of keratinocytes (Freeman et al., 1976). This method was further improved by utilization of collagen matrices to culture keratinocytes at the air-liquid interface (Lillie et al., 1980). Ponec et al. have upgraded this DeEpiDermized method (DED) to culture keratinocytes that attach to the existing basement membrane (Ponec et al., 1988). During the eighties, culture protocols were improved and, as a result, many different types of HSEs and human epidermal equivalents (HEEs) were described, designated as organotypic cultures, skin substitutes or living skin equivalents, with different types of dermal substrates (inert filters, DED, collagen matrices etc.) (Figure 5). Extensive efforts for the development of HSEs led to numerous clinical products and novel skin models for both pharmaceutical and cosmetic companies. These skin models are essential for preclinical drug development as they are suitable for testing drug safety and efficacy with higher accuracy when compared to 2D cell cultures. Actually, the environment of cells in 3D cultures mimics better the in vivo state (Sceats, 2010), moreover this HSEs are used for basic research and toxicology screening (Flaten et al., 2015; Kandárová et al., 2004 and 2006). In many cases, they reduce the need for experiments in animals. Reconstructed human epidermis Reconstructed human epidermis (RHE) is composed of differentiated epidermal keratinocytes seeded on acellular inert filter substrates. Several commercial RHEs are available, e.g. SkinEthic™ (Episkin, France), made of normal human keratinocytes (NHKs) cultured on an inert polycarbonate filter. Episkin™ (Episkin, France) and EpiDerm™ (MatTek, Ashland, USA) produce RHEs composed of NHKs cultured on a collagen matrix (Figure 6). These models yield a stratified epidermis that expresses epidermal differentiation markers such as keratin 1 (K1), loricrin and filaggrin, as well as skin lipids like phospholipids, cholesterol, triglycerides and ceramides. Figure 5. Generation of 3D skin models. Epidermis and dermis can be separated from a skin biopsy to isolate keratinocytes and fibroblasts, required to produce a 3D skin model. Keratinocytes can be seeded on three different type of matrixes: fibroblast-collagen matrix, acellular matrix (DED or inert plastic filter). Approximately 14 days later, a multilayered stratified epithelium is formed that mimics native human skin. RHEs systems have been approved for in vitro skin irritation and corrosion studies, however, their usage for permeation testing in vitro is quite ambiguous because of the inferior barrier properties, compared to normal skin (Schäfer-Korting et al., 2008). Several studies were carried out to identify whether permeation testing is validated for skin equivalents. It was shown that normal human epidermis exhibits lower permeability compared to RHEs (Schmook et al., 2001; Zghoul et al., 2001). However, a validation study involving permeability testing of ten substances through RHE models (Episkin, EpiDerm™ and SkinEthic™) showed that RHE models mimic the permeation through human epidermis better than any other HSE (Schäfer-Korting et al., 2008). Figure 6. Histological images of RHEs. Depiction of commercially available HSEs. SkinEthicTM (Episkin, France) is an in vitro RHE from NHKs cultured on an inert polycarbonate filter at the air-liquid interface. T-Skin™ (Episkin, France) consists of NHKs and NHFs, EpiDerm™ (MatTek, Ashland, USA) consists of NHKs cultured on tissue culture inserts (adapted from https://www.episkin.com, https://www.mattek.com/products/epiderm/). Full-thickness HSEs Human full-thickness skin models are more complex than RHEs, as a result they mimic native skin much better. They consist of keratinocytes cultured in dermal substrate mixed with fibroblast to form both epidermis and dermis. A basic protocol for the construction of a 3D RHS has been described from Carlson et al. (2008). According to this protocol a 3D model is composed of a stratified epithelium with differentiated keratinocytes that are seeded in a contracted matrix of collagen full of dermal fibroblasts as depicted in Figure 5. First, an acellular layer of collagen is constructed to help cellular collagen to attach. Then, cellular collagen populated with dermal fibroblasts is constructed and allowed to attach for approximately seven days, immersed in medium. When the matrix is stabilized, keratinocytes are seeded on the matrix surface and attach to the collagen substrate to produce a confluent cellular monolayer that will initiate the stratification of the tissue. Finally, as illustrated in Figure 7, tissues are exposed to an air-liquid interface, in order to complete stratification along with full morphological and biochemical differentiation (Carlson et al., 2008). Figure 7. Depiction of 3D tissue construction. A, an acellular layer of collagen is constructed to help cellular collagen to attach B, a collagen gel full of dermal fibroblasts is constructed above the acellular layer. C, after seven days submerged in medium, fibroblasts remodel the matrix, causing the contraction away from the walls of insert forming a plateau. D, keratinocytes are seeded in surface of the matrix and attach to the collagen to create a confluent cellular monolayer that will form the SB of the tissue E, tissues are exposed to an air-liquid interface to complete stratification and full morphological and biochemical differentiation. F, further exposure to air-liquid interface in cornification medium produces SS, SG and SC. In the recent years, quite sophisticated models of full-thickness HSEs have been developed, which incorporate Langerhans cells (MUTZ-3), neuron cells and melanocytes (Table 1). Moreover, models studying skin pigmentation and wound healing (Table 1) as well as skin-disease models (Table 2) have also been reported. Applications of RHEs and full-thickness HSEs There is a variety of in vitro applications for RHEs and full-thickness HSEs that range from investigation of cell interactions, cellular pathways, and mechanisms of disease, to studying drug permeation (Figure 8). 34 Reconstructed human 3D skin models of diseased skin The 3D RHS models of diseased skin, include melanoma progression model, psoriasis model, wound healing model (Coolen et al., 2008) etc. Moreover, infections like Candida albicans, have been studied in RHE (Johannes et al., 2002) and herpes simplex virus 1 (HSV1) infections have been studied in full-thickness HSE model (Hogk et al., 2013). The majority of the reconstructed human 3D skin models of diseased skin are summarized in Table 2. Figure 8. Applications of full-thickness skin models. Depiction of HSEs recapitulating human diseases like HSV1 infections model (adapted from Hogk et al., 2013), psoriatic model (adapted from Jean et al., 2009), epidermolysis bullosa model (adapted from Itoh et al., 2011), melanoma progression model (adapted from Li et al., 2011), atopic dermatitis (adapted from Rouaud- Tinguely et al., 2015) and wound healing (adapted from Rouaud-Tinguely et al., 2015) as well as applications of 3D RHS in drug permeation (adapted from Ackermann et al., 2010) and skin pigmentation (adapted from Duval et al., 2012). Primary human keratinocytes Primary human keratinocytes Primary human keratinocytes HaCaT Primary human keratinocytes; MUTZ- 3 LC Dermal cells Human fibroblasts; Human fibroblasts sensory neurons; Human Umbilical Vein Endothelial Cells (HUVEC) Primary human melanocytes Primary human fibroblasts; human fibroblasts Primary human dermal Epidermal cells fibroblasts Bovine type 1,3 collagen sponge Bovine type 1 collagen Bovine type 1 collagen Collagen Collagen Dermis substrate Study influence of neuron to wound healing Wound healing model Study skin pigmentation HSV disease model Study cutaneous immune response Utilization Blais et al., 2014 Egles et al., 2010 Duval et al., 2012 Hogk et al., 2013 Laubach et al., 2011 Reference 37 cs. risti acte char r thei and skin an hum of els mod ase dise ed erat Gen . dels mo ase dise skin 3D ed ruct onst Rec le 2. Tab Epidermal cells and dermis substrate used to form HSEs for several applications. Table 1. Full-thickness HSEs. Psoriasis 3D model of dermatitis Atopic dermatitis Atopic dermatitis Disease model Human foreskin Reconstructed epidermis It was used to show Characteristics Full-thickness skin fibroblasts mimicking filaggrin used to identify IL-17- HaCaT cells Atopic dermatitis-related downregulation in the responsive genes in psoriasis Memory-effector inflammation in vitro epidermis of patients (CD45RO+) T cells with atopic dermatitis Psoriatic HSEs used to study Scaffold material: tat either obtained or cytokine-induced gene tail collagen type I, inborn, may be expression fibronectin directly responsible for some of the disease- related alterations Chiricozzi et al., 2014 Engelhart et al., 2005 Rouaud-Tinguely et al., 2015 Pendaries et al., 2014 Reference Tjabringa and Bergers 2008 38 3D model of scleroderma fibrosis 3D model of human cutaneous squamous cell carcinoma 3D organotypic skin melanoma spheroid model 3D skin-reconstruction model of metastatic melanoma 3D model of melanoma Testing in vivo the progression of scleroderma and useful for screening antifibrotic drugs Primary NHKs Primary NHFs, SCC12B2 and SCC13 cell lines Rat tail collagen type I as scaffold material Pretreatment with EGF Human melanoma cell lines SBCL2 (RGP), WM-115 (VGP), and 451-LU (MM) Human primary keratinocytes Human primary fibroblasts Rat tail collagen type I as scaffold material Human malignant melanoma cells (A375) Normal human epidermal keratinocytes (NHKs) Normal human fibroblasts (NHFs) Collagen type I as scaffold material 3D RHS model incorporating melanocytic cells Luchetti et al., 2016 Commandeur et al., 2012 Vörsmann et al., 2013 Mohapatra et al., 2007 Li et al., 2011 39 Applications of the 3D RHS in the study of drug permeation As already mentioned, permeation testing on RHE is ambiguous due to the poorer barrier properties of 3D RHS compared to native skin. Nevertheless, many companies have made attempts to establish permeation tests on both RHE (Table 3) and in full- thickness HSEs (Table 4). Specifically, Schmook et al. (2001) studied the permeation ability of four different drugs (Tables 3 and 4) on commercially available RHE and full-thickness skin. The full-thickness Graftskin™ LSE™ has shown a sufficient barrier for salicylic acid (flux was only two times higher compared to human skin). However, application of more hydrophobic drugs like hydrocortisone and clotrimazole, on Graftskin™ LSE™ showed inferior barrier properties (flux was 200 and 1000 times higher compared to human skin for hydrocortisone and clotrimazole, respectively). In another investigation by Ackermann et al. (2010) percutaneous absorption of four different compounds (Table 4) has been tested in full-thickness Phenion FT® and compared with permeation of these

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compounds on pig skin and on RHEs (i.e. EpiDerm™, SkinEthic™, Episkin). In general, full-thickness skin showed poorer barrier properties and increased permeability in contrast to pig skin. In addition, RHE has shown similar barrier properties and permeability compared to human full-thickness skin. In summary, HSEs indicate overpredicted permeability parameters compared to human skin, that could be a result of different organization and lipid composition of the cornified layer of human epidermis. Thakoersing et al. (2013) found increased monounsaturated fatty acids and hexagonal lipid packing in the cornified layer of their in-house HSEs, suggesting that this could be the reason of the poorer barrier properties of HSEs compared to native human skin. On the other hand, another study revealed that growth conditions of in- house collagen-based full-thickness HSEs can be upgraded and both their ceramide profile and their barrier properties can be optimized with the addition of clofibrate, fatty acids and ascorbic acid (Batheja et al., 2009). In addition, the lack of epidermal derivatives like hair follicles and sweat glands is another key factor responsible for the inadequate resemblance of HSEs with native human skin. In general, the drug delivery of nanoparticles, which activity is very important for topical and transdermal healing of a variety of dermatological diseases (Zhang et al., 2013), occurs through hair follicles penetration (Lademann et al., 2007; Otberg et al., 2008; Shim et al., 2004). Therefore, it is important to focus on the development of HSEs with epidermal appendages. Table 3. RHE used for drug permeation testing. RHE developed by MaTek Corp and Episkin tested for the permeation of several pharmaceutical substances like flufenamic acid, clotrimazole, hydrocortisone, salicylic acid, terbinafine as well as caffeine and testosterone. Reconstructed human epidermis (RHE) Company Drug Reference EpiDerm™ MatTek Corp (Ashland, USA) Flufenamic acid Zghoul et al., 2001 SkinEthic™ Episkin (Lyon, France) Clotrimazole Hydrocortisone Salicylic acid Terbinafine Schmook., 2001 Episkin Episkin (Lyon, France) Caffeine Testosterone Netzlaff et al., 2007 Table 4. Reconstructed full-thickness skin used for drug permeation testing. Reconstructed full- thickness skin developed by two different companies (Organogenesis and Henkel) for testing permeation of specific natural/ pharmaceutical compounds. Full-thickness skin GraftskinTM LSETM Company Organogenesis (MA, USA) Drug Clotrimazole Hydrocortisone Salicylic acid Terbinafine Reference Schmook et al., 2001 Phenion FT Henkel (Dusseldorf, Germany) Benzoic acid Caffeine Nicotine Testosterone Ackermann et al., 2010 Applications of 3D RHS in irritation and corrosion Generally, HSEs are scientifically valid alternatives to animal models for testing skin corrosion and irritation of chemical compounds (Alépée et al., 2018; Nomura et al., 2018). There are numerous epidermal and full-thickness skin equivalents commercially available for this purpose. Overall, Episkin, Epiderm™ and SkinEthic models referred to organisation for economic cooperation and development (OECD) test guideline 439 are accepted by European Union (EU) and Food and drug administration (FDA) for skin irritation on different chemical compounds measuring cell viability of the epidermis using MTT assay. Moreover, the secretion of IL-1α, IL- 6, and IL-8 cytokines at epidermis and full-thickness skin models can be used to detect skin irritation. Full-thickness skin models like TestSkin®, Apligraf®, AST-2000®, and Skin2® have been tested for their ability to predict skin irritation by evaluating the cell viability, cytotoxicity and secretion of soluble factors causing inflammation (Gibbs, 2009). More specifically these factors can be IL-1, IL-8, IL-6, and prostaglandin E2 (PGE2) after exposure to potential irritants. In conclusion, although HSEs are acceptable to the EU and FDA as an appropriate alternative method to in vivo animal testing (Draize albino rabbit test), it should be considered that there are still some problems to be solved including: batch-to-batch variation, higher cost, SC barrier that is more permeable than that of native skin and lack of epidermal appendages in 3D models such as sweat glands (irritation responses that may happen due to sweat cannot be predicted) (Matsumura et al., 1995). Applications of 3D RHS in sensitization Assessing skin sensitization using RHS can offer several benefits. Full-thickness HSE maintain very important cellular interactions between the fibroblasts and keratinocytes that occur during sensitization. Moreover, expression levels of mRNA for metabolic enzymes in full-thickness HSEs were found to be related with in vivo human skin more than cells in RHEs (Luu-The et al., 2009). EST-1000 epidermal model and AST-2000 full-thickness model from Cell Systems (St. Katharinen, Germany) have been evaluated for screening skin sensitizers like oxazolone, 1-fluoro-2,4- dinitrobenzene (DNFB) and irritants like SDS and Triton X -100. Furthermore, investigation of cell signaling pathways that transduce immune responses and regulate cytokines have been conducted (Koeper et al., 2007). MICRO- AND NANO-EMULSIONS Generally, emulsion technology has been utilized tremendously in the pharmaceutical industry. There are three different categories of emulsion depending on size, shape, stability, method of preparation and polydispersity: macro-emulsions, nano-emulsions and micro -emulsions (Zanatta et al., 2008) . Macro-emulsions or opaque emulsions, have large droplet sizes and as a result they usually form cloudy solutions (Rosen and Kunjappu, 2012). They are classified in water in oil (w/o) and oil in water emulsions (o/w). Nano-emulsions or mini -emulsions compared to macro-emulsions have smaller droplet size and are semi-opaque (Rosen and Kunjappu 2012). They are also classified as o/w and w/o. Micro -emulsion solutions became famous in 1943 after Hoar and Schulman blended a milky solution and hexanol to produce a uniform single-phase and non-conducting solution (Gibaud and Attivi, 2012). Micro-emulsions are transparent, thermodynamically stable mixtures of oil and water stabilized by emulsifiers. They have classified in o/w and w/o micro-emulsions just like nano and macro-emulsions but they have significantly different properties (type, size, formation and stability) relative to nano and macro-emulsions (Table 5). Table 5. Comparison of size, shape, stability, method of preparation and polydispersity in different kinds of emulsions. Macro-emulsions Nano-emulsions Micro-emulsions Size 1-100μm 20-500nm 10-100nm Shape Spherical Spherical Spherical, lamellar Stability Thermodynamically unstable, weakly kinetically stable Thermodynamically unstable, kinetically stable Thermodynamically stable Method of preparation High & low energy methods High and low energy methods Low energy method Polydispersity Often high (>40%) Typically, low (<10-20%) Typically, low (<10%) Properties and characteristics of micro- and nano-emulsions Micro-emulsions as well as nano-emulsions are composed of water, oil, and a surfactant. Surfactants are amphiphilic molecules, which are usually used in conjunction with a co-surfactant (Callender et al., 2017). Micro-emulsions are thermodynamically stable and form spontaneously. In contrast to nano- emulsions, micro-emulsions usually require a higher percentage of surfactant or emulsifier (i.e., 15–30% w/w of the oil phase) for formation. Additionally, the co-surfactant required is usually of a shorter carbon chain length than that required in nano-emulsions (Rosen and Kunjappu, 2012). Nano-emulsions are kinetically resistant and require energy input for their formation. They require less emulsifier than microemulsions 1–3% of the volume of the oil phase (Rosen and Kunjappu, 2012). Ultrasonic agitation and use of high-pressure homogenization with microfluidic devices are used for the nano-emulsion preparation (Mason et al., 2006). Applications of micro- and nano-emulsions in drug delivery Composition of microemulsions improve the solubility, chemical stability, and oral bioavailability of many poorly water-soluble drugs, moreover they can encapsulate and deliver both hydrophilic and hydrophobic compounds. Furthermore, they have low interfacial tension, large interfacial area and capacity to solubilize both aqueous and oil soluble compounds (Callender et al., 2017). Microemulsions also have slow degradation, controlled drug release rate, and target specificity (Jadhav et al., 2006). Thus, microemulsions are very competitive candidates as drug delivery vehicles. Nano-emulsions also have utility in drug delivery research, precisely a study of Zulli and his team revealed that encapsulation of the cosmetic substance coenzyme Q10 in nano-emulsions results in significantly enhanced bioavailability (Zulli, 2006). Additionally, nano-emulsions prepared according to a patented process, allow the administration of several incompatible substances at the same time, for example Vitamin E and coenzyme Q10, which are poorly water-soluble substances, form a 44 “double” nano-emulsion that can be successfully used for cosmetic purposes (Guglielmini, 2008; Merisko et al., 2003). STATE-OF-THE ART The 3D reconstructed human dermal and epidermal models are considered a breakthrough in cosmetic industry since they are the best alternatives to animal testing (Sheasgreen et al., 2009). Consequently, they increasingly became an indispensable part for pharmaceutical investigation and development as well (Mathes et al., 2014). In addition, 3D models for rare and common skin diseases are valuable tools in studies aiming to decipher the molecular mechanisms underlying a variety of skin diseases such as melanoma progression (Li et al., 2011), recessive dystrophic epidermolysis bullosa (Mittapalli et al., 2016), atopic dermatitis (Pendaries et al., 2014) etc. In other words, 3D RHS models provide novel experimental systems that recapitulate adequately the in vivo state and could be used for a wealth of reasons. The 3D reconstructed skin developed here, is a model of a full-thickness normal skin, which mimics native human skin both structurally and biochemically. More precisely, it contains a distinct dermis and a well-structured epidermis consisting of a cornified layer (SC), a granular layer (SG), a spinous layer (SS) and a basal layer (SB). Moreover, it expresses the typical skin differentiation markers (involucrin, loricrin, keratin 5), proliferation markers (Ki67), cell-adhesion molecules (E-cadherin), structural proteins (DSC1, DSG1, CDSN) and proteases (KLK5, KLK6, KLK7) with key functions in skin physiology. Our in-house 3D skin substitute was generated to be exploited in the evaluation of the cosmetic properties and skin irritation of a variety of essential oils (EOs) and cosmetic micro-and nano- emulsion formulations with encapsulated natural EOs as bioactive compounds. This novel formulation could provide safety given that it contains only natural ingredients, controlled release of the bioactive compound as well as skin moisturizing action. To evaluate safety, preliminary studies of cytotoxicity were performed in the frame of the current study prior to examination of cosmetic properties, such as the moisturizing action of these formulations, and the extent of potential skin irritation on the 3D RHS. SPECIFIC AIMS OF THE STUDY The main purpose of this study was the generation of a 3D skin model that simulates the structural and biochemical characteristics of the native human skin. For the achievement of this objective, a 3D reconstructed full-thickness human skin was developed. More accurately, immortalized human dermal fibroblasts (iNHFs) mixed with collagen were used for the development of the reconstructed dermis and immortalized human epidermal keratinocytes (iNHKs) were used for the development of the reconstructed epidermis. In addition, the key objective was to validate whether the keratinocytes seeded in the dermis substrate had the ability to multiply and differentiate to form a differentiated epithelium that mimics perfectly the in vivo state. Histology was used to show that the in-house model resembles native skin remarkably, which was one of the main aims of the study. The investigation of the expression of proteins that exist principally in native human skin was the other basic goal to achieve for the proof-of-concept that our in-house 3D model mimics biochemically the native skin. Therefore, the expression of the proliferation marker Ki67, of the cell adhesion molecule E-cadherin, of the differentiation markers (involucrin, loririn, keratin 5) and structural proteins (DSC1, DSG1, CDSN) as well as of kallikrein-related peptidases (KLK5, KLK6, KLK7) was the key to validate the success of our 3D skin model. Finally, this study’s purpose was to examine the cytotoxicity of EOs and novel cosmetic micro-and nano- emulsion formulations. Cytotoxicity of these cosmetic products was evaluated firstly on iNHKs. Studies of cytotoxicity on iNHKs were essential as a preliminary step before their evaluation on the in-house 3D RHS. The developed products will be examined on the 3D RHS for cosmetic properties, such as their moisturizing action. Skin irritation and corrosion should also be tested on the 3D skin model after treatment with the specific cosmetic products. MATERIALS, METHODS AND INSTUMENTATION Instruments Equipment Supplier Camera Olympus stylus Cold plate Leica Centrifuge, Rotofix 32 Hettich CO2 water jacketed incubator series II Forma Scientific, Inc Diaphragm vacuum KNF Hemocytometer Neubauer Incubator MMM medcenter Inverted light microscope Hund wetzlar Microtiter plate reader - Infinite F50 Tecan Microflow biological safety cabinet Astec Microtome LEICA Orbital shaker Thermo Scinetific Upright light microscope with Axiocam ERc5s ZEISS Tissue embedder Leica Water bath Memmert Plastic or glass laboratory consumables Material Company 6-well deep plate Falcon 96-well cell culture plate Corning Cell culture inserts Falcon Cryovials SDL Life Sciences Embedding cassettes Fisher Scientific Microtome blades Leica Microscope slides Thermo scientific Pasteur pipettes Kisher-Biotech G Serological pipettes SDL Life Sciences Sterile petri-dishes 100 x15 mm, 60 x16 mm Falcon Syringe filters 0.22 μm Sigma Aldrich 50 ml and 15 ml conical centrifuge tubes Falcon T25 and T75 tissue culture flask Falcon Media and Reagents Adenine, Sigma Aldrich, A2786-25G Agar, Invitrogen, 30391-023 Ascorbic acid, Sigma Aldrich, A1300000 Aquaguard-1 Solution, Promocell, PK-CC01-867-1B Bovine serum albumin BSA, Sigma Aldrich, A7906-50G Calcium chloride CaCl2, Honeywell, C1016-100G Citrate sodium, Sigma Aldrich, C8532-500G Collagen, Corning, 354236 Dulbecco’s Modified Eagle Medium DMEM, Gibco, 41965-039 Dimethyl Sulfoxide DMSO, Sigma Aldrich, D2650 Ethanol, Sigma Aldrich, 24194 Eosin, Sigma Aldrich Eukitt Quick-Hardening mounting medium, Sigma Aldrich, 03989 Fetal Bovine Serum FBS, Gibco 12676029 Formaldehyde, Merck, K41839403 Hanks’ Balanced Salt Solution HBSS, Lonza, 10-527F Hematoxylin, Sigma Aldrich, MHS16 Hydrochloric acid HCl, Sigma Aldrich, 30721 Hydrocortisone, Millipore Hydrogen peroxide H2O2, Sigma Aldrich, 31642 Insulin, Millipore, I-5500 Isopropanol, Sigma Aldrich, 33539 Glacial acetic acid, Sigma Aldrich, 33209 Keratinocyte SFM, Gibco, 17005-034 Keratinocytes supplements, Gibco, 37000-015 L-Glutamine 200mM, Gibco, 25030-081 Neutral red NR, Sigma Aldrich, N4638-1G MTT, Sigma Aldrich, M5655 Penicillin/streptomycin, Gibco, 15140-122 Recombinant human epidermal growth factor rhEGF, Millipore Sodium dodecyl sulfate SDS, Sigma Aldrich, L3771-100G Sodium hydroxide, SO04211000 Triton® X-100, Sigma Aldrich, X114 Trypan Blue, Sigma, T8154 Trypsin- Ethylenediaminetetracetic acid EDTA 0.05%, Gibco 25300-054 Tween® 20, Sigma Aldrich, P1379 Xylene, Sigma Aldrich, 16446 Antibodies ? Anti-KLK5, R&D Systems AF1108 (working dilution: IHC- P 1/100) ? Anti-KLK6 IgY (Sotiropoulou et al., 2012) (working dilution: IHC- P 1/200) ? Anti- KLK7, R&D Systems AF2624 (working dilution: IHC-P 1/ 100) ? Anti- Cytokeratin 5, Abcam ab 24647 (working dilution: IHP 1/1000) ? Anti- CDSN, Flarebio CSB-PA005124 (working dilution: IHC-P 1/500) ? Anti-Loricrin, Abcam ab24722 (working dilution: IHC- P 1/1000) ? DSC1 (L-15), Santa Cruz SC- 20114 (working dilution: IHC- P 1/250) ? DSG1 (H-290), Santa Cruz SC- 15230 (working dilution: IHC- P 1/300) ? Anti-E-cadherin (G10), Santa Cruz SC8426 (working dilution: IHC- P 1/ 1/200) ? Anti-Ki67 Abcam 15580 (working dilution: IHC-P 1/ 1/1000) ? Involucrin (M-15), Santa Cruz SC-15230 (working dilution: IHC- P 1/100) ? Anti-chicken IgY HRP, Sigma Aldrich A9046 (working dilution: IHC-P 1/300) ? Anti-mouse IgG HRP, Sigma Aldrich A9044 (working dilution: IHC-P 1/200) ? Anti-rabbit HRP, Millipore AP132P (working dilution: IHC- P 1/ 200) ? Donkey anti-goat IgG-HRP, Santa Cruz sc-2020 (working dilution: IHC-P 1/200) Native human skin Native human skin was kindly provided by Manthoula Valari, MD, PhD, i.e. ear skin tissue excised by plastic surgery on healthy donors for cosmetic purposes. Biopsy samples were embedded in paraffin for further analysis including microscopic observation of the skin structure and immunohistochemical analysis. Biopsies were taken with full informed consent and the study was approved by the Ethics Committee. Cell lines Human epidermal keratinocytes and dermal fibroblasts cell lines used in this study, were kindly offered by Dr. Med. Dimitra Kiritsi, Department of

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Dermatology, Faculty of Medicine, Medical Center-University of Freiburg, Freiburg, Germany. Skin specimens were obtained from healthy donors after written informed consent and was approved by the Ethics Committee of Freiburg University (#521/13). Primary keratinocytes and fibroblasts were then isolated from human skin samples and cultured in serum-free keratinocyte growth medium and DMEM respectively, in the presence of 5% CO2 at 37oC. Stable cell lines were generated upon immortalization of both epidermal keratinocytes and dermal fibroblasts with standard protocols. Micro- and nano-emulsions, oils and surfactants Micro- and nano-emulsions, EOs and surfactants used in terms of this study, were kindly offered by Dr. Vassiliki Papadimitriou, Department of Biomimetics and Nanobiotechnology, Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece. METHODS Cell culture Normal human fibroblast cell lines were cultured at 37°C in 5% CO2 in DMEM medium containing 10% FBS, 2% L-glutamine and 1% penicillin/streptomycin. Normal human keratinocyte cell lines were grown in KSFM, supplemented with growth factors and hormones necessary for cell growth including hEGF and pituitary bovine extract. Cells were passaged when more than ~70-80% confluence was observed. For subculture, both fibroblasts and keratinocytes had to be detached from the bottom of the culture flask. Firstly, the culture medium was discarded and 1 or 2 ml trypsin/EDTA solution was added into 25 cm2 or 75 cm2 flask, respectively, following by 3 min incubation at 37oC. Action of trypsin was stopped when cells detached by addition of two volumes of phosphate-buffered saline (PBS), 10% FBS, then cell suspension was centrifuged at 1000 x rpm for 5 min, following by resuspension of cell pellet in fresh medium and finally cell suspension was transferred to culture flasks for cell growth. Photographs of iNHFs and iNHKs were taken with an OLYMPUS stylus SP-100EE camera focused in 35 mm, optical microscope’s total magnification was 100x (10x objective lenses * 10x eyepieces). Cell cryopreservation Cells were observed under an inverted light microscope to assess % confluence, which should be close to 80-90% and to confirm the absence of contamination by bacteria and/ or fungi. Then, the cells were detached with trypsin/EDTA, as described previously in “cell culture’’, centrifuged and finally, 1-2 x 106 cells were resuspended in 1ml freezing medium containing 80% DMEM, 10% FBS and 10% DMSO, which is the most commonly used cryoprotectant. Afterwards, ~0.5 -1 x 106 cells were transferred into each cryovial, which was labeled and stored at -800C for 1-3 days and finally, transferred in liquid nitrogen for long-term storage. Cell viability Number and viability of the cells were estimated using a glass hemocytometer and trypan blue exclusion test according to Current Protocols in Immunology (Strober., 2001). Cells were detached from the bottom of the flask by trypsinization, centrifuged at 1000 x rpm for 5 min and then, resuspend in the required prewarmed medium depending on the cell type. Once the cells were fully resuspended, a small amount of cell suspension was mixed with 0.4% trypan-blue at a ratio of 1:1 (i.e. dilution factor, DF= + ) and counting of both dead and alive cells was performed (Figure 9). Trypan blue exclusion test is based on the principle that live cells possess intact cell membranes that exclude trypan blue dye while dead cells do not. Viable cells have clear cytoplasm while a dead cell’ s cytoplasm is blue. The percentage of cells’ viability was calculated using the following general equation: 100. + In addition, considering that the depth of a chamber is 0.1 mm and each large square’s surface is 1 mm2 (i.e. volume of one large square is 0.1 mm3 =10-4 ml) (Figure 9), the density of cells was estimated using the following equation: DF = 1 * 104 * DF cells/ml In conclusion, to calculate the total concentration of viable cells, the following equation was used: cells cell density ( ml ) total volume of cell suspension A B C Figure 9. Cell number calculation and cell viability estimation. A, chemical structure of the trypan blue dye. B, schematic representation of a hemocytometer used for cell number calculation and cell viability estimation; an enlarged area is shown on the right. C, microscopic appearance of cells stained with trypan blue. Viable cells have clear cytoplasm, while in dead cells the cytoplasm is stained blue, since their membranes are not intact allowing penetration of the trypan blue dye. Development of the 3D RHS Development of the 3D RHS was described previously (Mittapalli et al., 2016). Briefly, fibroblasts were detached from the bottom of the flask via trypsinization and then cells were measured using a glass hemocytometer and finally resuspended in FBS to a final concentration of 3,000,000 cells/ml. Τrypan blue exclusion assay confirmed that iNHFs had viability over 90%. The ice-cold rat tail collagen I solution (80% of total volume) was mixed with HBSS 10x (10% of total volume) to a final concentration of 3 .5 mg/ml and then, pH was adjusted to 7.4 with 5 M NaOH under gently stirring on ice to avoid formation of air bubbles and premature gelation, following by addition of 750,000 fibroblasts/ml collagen gel. Using pre-cooled pipettes, 2.5 ml of the collagen I-fibroblasts mixture was transferred to each 1 μm pore size filter insert and then incubated at 37oC, 5% CO2 for gelation. Thereafter, a glass ring with 12 mm inner diameter and 10 mm height were placed on each gel and gently pushed down to confine the area for the epithelial cell growth. Gels, were placed for 1 hour at 370C in a humidified incubator and then, excess liquid was carefully aspirated and finally, the gels were submerged in 15 ml DMEM containing 2% FBS, 2% L-glutamine, 1% penicillin/streptomycin per well and incubated for 24 hours in an incubator (37°C, 5% CO2). After 1 day, medium was carefully removed and 1-2 * 106 keratinocytes were seeded on each gel. Four days later, the culture at the air–liquid interface is initiated therefore, the culture medium was replaced by 10 ml of organotypic culture -organotypic cultures (OTC) medium (2% L-glutamine, 5 ng/ml rhEGF, 1 μg/ml hydrocortisone, 5 μg/ml insulin, 0.18 mM adenine, 5% FBS and 1.8 mM Ca2+) containing 10 μl of 50 mg/ml ascorbic acid. Culture medium was renewed every 2- 3 days and the 3D RHS equivalents were harvested four weeks after initiating the culture at the air-liquid interface. Embedding tissue into paraffin blocks Inserts with the reconstructed tissue were placed in a glass plate and cut out in pieces using sterilized surgical knifes, with caution not to disturb the epidermis. Tissue was placed in 3.7% formaldehyde in PBS and then embedded in 2% agar in PBS. Afterwards, reconstructed tissues were left 24 hours in PBS and then were embedded in paraffin. More specifically, tissue was placed in embedding cassette and dehydrated through a series of ethanol dilutions (70, 85, 95, and 100%) and finally cleared with two washes with xylene, 45 min each at room temperature (RT). Subsequently, tissue was left to incorporate paraffin for approximately 2 hours. Then, liquid warmed paraffin was poured in stainless-steel base mold and the agar-tissue block was placed in the middle of the mold, which was then filled with paraffin and was left to cool and solidify on a cold plate. Hematoxylin and eosin staining Tissue paraffin sections of 7 μm were cut using a microtome and placed on a microscope slide. Sections were then air-dried for 1 hour and incubated in 600C for paraffin melting. Subsequently, sections were deparaffinized in xylene (20 dips), rehydrated in decreasing concentrations of ethanol (100, 75, and 50%) and finally in water (10 dips in each solution). Finally, sections were stained with hematoxylin, which stains the nucleus of the cells for approximately 3 minutes and then with eosin, which stains the cytoplasm. Afterwards, sections were dehydrated in increasing dilutions of ethanol (50, 75, and 100%), cleared in xylene (20 dips) and mounted with Eukitt. Photographs of skin tissues were taken with Axiocam ERc5s under a light microscope using Zeiss Zen software. Total magnification was 200x (10x objective lenses * 20x eyepieces) and 400x (10x objective lenses * 40x eyepieces). Immunohistochemistry Paraffin sections of 7 μm were cut, air- dried and then paraffin was melted in a preheated incubator at 60oC. Then, sections were deparaffinized in xylene (20 dips) and rehydrated in a serial dilution of ethanol (100% to 0%). Afterwards, sections were boiled 20 min in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween pH 6.0). The sections were washed in PBS for 5 min and endogenous peroxidase activity was quenched by incubation in 3% H2O2 in PBS for 10 min. In addition, sections were immersed in blocking buffer (0.3% bovine serum albumin or BSA in PBS- Triton 0.1%) for 5 min. Consequently, the primary antibody was placed on sections at the appropriate concentration and time. Sections were washed twice with 0.3% BSA in PBS and the secondary antibody conjugated with a peroxide enzyme was added on sections for 45 min to bind to the 1st antibody. Sections were washed again, twice with 0.3% BSA in PBS and once with PBS. Then, 3,3-diaminobenzidine (DAB) substrate was placed upon sections. As already mentioned, 2nd antibody is conjugated with a peroxidase enzyme, which in the presence of hydrogen peroxide, oxidizes DAB to a brown product. Finally, sections were counterstained with hematoxylin, dehydrated and mounted with Eukitt. Axiocam ERc5s was used under a light microscope using Zeiss Zen software for capturing photographs under 400x (10x objective lenses*40x eyepieces) magnification. Neutral red uptake assay The NRU assay was carried out according to the experimental protocol reported by Repetto et al. (2008) . The basic principle of THE NRU assay is that viable cells are able to incorporate and bind the supravital dye NR in the lysosomes. The chemical structure of NR is shown in Figure 10. Specifically, NHK were grown in keratinocyte serum-free medium (KSFM) and when reached 80% confluence, cells were detached via trypsinization and were counted using a hemocytometer. Subsequently, cell viability was checked using trypan blue exclusion test as detailed in Material and Methods. Finally, cells were diluted in order to form a suspension of 75 x 103 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate, which was incubated at 37±1°C, 5±1 % CO2, 95 % relative humidity (RH) for 24 hours. On the next day, cells were exposed to increasing concentrations of testing products in KSFM and incubated at 37±1°C, 5±1 % CO2, 95 % RH for the appropriate time. Alongside, a NR solution 40 μg/ml in water was prepared and incubated for an hour at 370C. The testing medium was aspirated from the cells and the cells were washed twice with 150 μl of PBS. Subsequently 100 μl of NR medium was added in each well plate and incubated for two hours at 37±1°C, 5±1 % CO2, 95 % RH. Then, microscopic evaluation was conducted and cells were washed with 150 μl water. In addition, each well was filled with destaining solution (50% ethanol-49% deionized water and 1% glacial acetic acid) and plate was left under stirring for 15 min. Finally, the OD was measured at 490 nm in a microtiter plate spectrophotometer. Figure 10. Chemical structure of the NR dye. Viable cells incorporate and bind the supravital dye NR in the lysosomes and are stained red. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was conducted following the recommended protocol from Abcam. The basic principle of MTT assay is that water soluble yellow MTT, is reduced to an insoluble purple formazan product by active mitochondrial dehydrogenases of viable cells (Figure 11). Briefly, iNHKs were grown in KSFM to 80% confluence, then, they were used in MTT assay. Cells were collected and measured as detailed in NRU assay. Approximately 75 x 10 cells/ml were dispersed per well in 96-well plates. Plates were then incubated at 37± 1 °C, 5± 1 % CO2, 95 % RH for 24 hours. Cells were exposed to different concentrations of testing products in KSFM and incubated at 37±1°C, 5±1 % CO2, 95 % RH for the appropriate time. Then, 50 μl of fresh KSFM and 50 μl of MTT (5 mg/ml in PBS) were added into each well and the plate was incubated for 3 hours at 37oC, 5% CO2, followed by addition of 150 μl of MTT solvent (0.1 Ν ΗCl in isopropanol) into each well. Then, the plate was wrapped in foil and placed in an orbital shaker for 15 min and finally, the OD was measured at 590 nm in a microtiter plate spectrophotometer. Figure 11. Structure of both MTT and formazan. A yellow tetrazole, is reduced to purple formazan in living cells. RESULTS CHAPTER 1: Generation of the 3D RHS Development of 3D RHS could be summarized in 4 steps including, establishment of fibroblast and keratinocyte cell cultures, preparation of cellularized collagen with the appropriate concentration of dermal fibroblasts, seeding of keratinocytes and finally tissue harvesting and precipitation for further analysis (Carlson et al., 2008). Morphology of immortalized normal human dermal fibroblasts Cell lines of iNHFs were cultured in flasks with surface area 25 cm2 (T25) and 75 cm2 (T75) in feeding medium DMEM supplemented with 10% FBS, 2% L-glutamine and 1% penicillin/streptomycin and when they had reached ~80% confluence, they were trypsinized, counted and, finally, used for the formation of normal 3D RHS (Figure 12). Figure 12. Immortalized normal human dermal fibroblasts. The 2D iNHF cultures were routinely observed under an optical microscope until 80% confluence, then cells were used for the 3D RHS, as described in Figure 14. Morphology of immortalized normal human epidermal keratinocytes Cell lines of iNHKs were firstly cultured in KSFM in T25 flasks, with keratinocytes supplements and consequently they were used for the formation of normal 3D RHS (Figure 13). Figure 13. Immortalized normal human epidermal keratinocytes. The 2D iNHK cultures were routinely observed under an optical microscope until 80% confluence, then cells were used for the 3D RHS as described in Figure 14. Preparation of cellularized collagen gel Firstly, iNHFs viability was estimated higher than 95% using trypan blue exclusion test as detailed in Materials and Methods. The rat tail collagen 1 was mixed with iNHFs using chilled pipets on ice to prevent premature gelation. It is important to neutralize the acidic collagen with 5M NaOH to avoid cell damaging. The final concentration of iNHFs was 3 x 105 fibroblasts/ml gel. Figure 14. Preparation of 3D RHS. Day 0, collagen gel mixed with iNHFs was placed above the culture insets. Day 5, iNHKs were seeded onto the surface of the matrix, attached to the collagen- fibroblast gel and allowed to be fully adhered. Day 10, tissues were exposed to the air-liquid interface in order to complete stratification and full morphological and biochemical differentiation, and the medium was changed from DMEM to OTC. Day 37, tissues were ready for paraffin embedding and sectioning followed by histological characterization. Seeding of keratinocytes During the second day of 3D’s reconstructed skin development, fibroblasts mixed with rat tail collagen I have begun to form a massive gel (the dermis). On day five, 2,000,000 iNHKs with viability of ~94% were seeded onto the collagen- fibroblast gel. Keratinocyte differentiation began a few days later and on day 10, tissues were exposed to an air liquid interface to complete stratification and full morphological and biochemical differentiation (Figure 14 and 15). Furthermore, medium was changed from DMEM containing, 2% FBS, 2% L-glutamine, 1% penicillin/streptomycin to OTC containing, 2% L-glutamine, 5 ng/ml rhEGF, 1 μg/ml hydrocortisone, 5 μg/ml insulin, 0.18 mM adenine, 5% FBS and 1.8 mM Ca+2. Harvesting of 3D reconstructed tissues for analysis Around day 37, reconstructed tissues were ready to be harvested and precipitated for further analysis including histological and immunohistochemical characterization. Using sterile scissors and forceps, tissues were carefully harvested and then embedded in paraffin as detailed in Materials and Methods. Briefly, they were placed in embedding cassettes, dehydrated, cleared with xylene and finally embedded in paraffin. Figure 15. Macroscopic appearance of 3D RHS at air-liquid interface. Keratinocytes were seeded on the collagen -fibroblasts gel, after 28 days of growth at air-liquid interface, keratinocytes were completely differentiated and the squamous stratified epidermal epithelium was fully formed by day 37. CHAPTER 2: Characterization of 3D RHS The 3D RHS displays a microstructure very similar to normal skin tissue The microstructure of 3D RHS was evaluated by eosin-hematoxylin (Η&Ε) staining. Eosin is an anionic and acidic stain that stains the cytoplasm while hematoxylin is a basic and cationic stain that stains the nucleus of the cells (Chan, 2014), as a result, cytoplasm appears pink while nucleus appears blue. Histological examination of skin paraffin sections revealed that skin microstructure was normal in 3D RHS collected upon 28 days culture at the air-liquid interface compared to native human skin (Figure 16A). Epidermis, the outermost layer of the skin, as well as dermis, the inner skin layer, were distinct both in native skin and 3D RHS paraffin sections. All epidermal layers, including from superficial to deep SC, SG,

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SS, and SB, were clearly observed (Figure 16A) suggesting that iNHKs were differentiated and formed the characteristic stratified squamous epithelium of the epidermis. Overall, the skin microstructure of 3D RHS appears very similar to native human skin. Nevertheless, in accordance with previous reports (Mc Govern et al., 2016; Vicanova et al., 1996), it was observed that epidermis of 3D RHS was almost twice as thick as native’s skin epidermis. In addition, hyperkeratosis is another characteristic observed in 3D RHS (Figure 16B). Therefore, epidermal thickness varies according to ethnic origin, age, gender, body region and smoking habits (Elder, 2009; Lee et al., 2002; Hoffmann et al., 1994; Seidenari et al., 1994; Sandby Møller et al., 2003; Dao et al., 2007; Shuster et al., 1975; Southwood et al., 1946; Chopra et al., 2015; Gültekin et al., 2011), which may have affected the results obtained in this study. IHC assessment showed that 3D RHS mimics biochemically the native human skin The method of IHC was utilized to evaluate the biochemical functions of the 3D RHS. More specifically, 3D RHS was characterized for the proliferation activity of keratinocytes, for dermo-epidermal junction, for competence in differentiation markers, for the expression of structural proteins of desmosomes and corneodesmosomes and finally for the expression of KLKs. A B Epidermal Thickness, μm 300 Stratum Corneum 250 32.5 Cellular epidermis 200 150 15 100 240 50 150 0 Native skin 3D RHS Figure 16. The 3D RHS forms a stratified squamous epithelium. Α, representative paraffin sections of 7 μm stained with H&E show that the structure of 3D RHS appears similar to native human skin. Epidermal and dermal layers of 3D RHS and native skin are distinct. The SC, SS, SG and SB were also visible in the epidermis. The blue dashed line represents the epidermis-dermis junction. B, quantifications of epidermal thickness. The graph shows the thickness of the SC and of the viable layers of the epidermis both in 3D RHS and native skin. Native skin had smaller total epidermal thickness than the 3D RHS. Data are shown as median ± SEM, n>14, n; measured in randomly selected fields. A Β 50 %Ki67 positive nuclei 40 30 20 10 0 23 10.33 Native skin 3D RHS Figure 17. Epidermis of 3D RHS is hyperproliferative. A, Ki67 IHC staining in 3D RHS and native skin biopsies. The blue dashed line represents the epidermis-dermis junction. B, percentage of Ki67-positive cells was increased in epidermis of 3D RHS. Data are shown as median ± SEM, n> 7, n =ratio of Ki67 positive cells/ total cells, calculated for randomly chosen fields. 40x Figure 18. Immunohistochemical analysis of E- cadherin. E-cadherin was expressed throughout the epidermis both in native skin and 3D RHS. Immunostained areas in the blue panel are enlarged and indicated by blue arrows. The blue dashed line represents the epidermis-dermis junction. The 3D RHS shows increased proliferation index of keratinocytes in the epidermis Ki67 is a proliferation marker, which is expressed in the nuclei of all keratinocytes that are not in the G0 resting phase of the cell cycle (Noszczyk et al., 2001). Immunohistochemical analysis of biopsy sections revealed that Ki67 immunoreactivity was restricted mostly to the basal keratinocytes both in native skin and 3D RHS (Figure 17A). Quantification of Ki67 positive cells showed that 3D RHS sustained a higher epidermal proliferative index than that observed in native human skin. Specifically, approximately 23% of keratinocytes were hyperproliferative in 3D RHS, while 10% were hyperproliferative in native human skin (Figure 17B). Generally, keratinocyte proliferation is important for the formation of the SC. Thus, hyperkeratosis observed in 3D RHS is compatible with its hyperproliferation. The 3D RHS maintains structural integrity similar to normal skin tissue To investigate skin integrity in 3D RHS, we next investigated the expression levels of E-cadherin, a calcium-dependent cell adhesion molecule whose function is a main contributor of epidermal epithelial polarity and skin structural integrity. It was observed that E-Cadherin was expressed in both native skin and 3D RHS and staining was localized to membrane of keratinocytes (Figure 18) indicating that cell-cell adhesions potentially exist in epidermis of 3D RHS. Cell differentiation is normal in the 3D RHS Epidermal differentiation process was investigated by immunohistochemical analysis of several differentiation markers since abnormal keratinocyte differentiation is linked with several epidermal abnormalities. Immunostaining of paraffin sections obtained from 3D RHS and native skin revealed that loricrin, a terminal differentiation marker, localized in the outer epidermal layers of native skin and 3D RHS. Involucrin, a differentiation marker, which appears in the early stages of keratinocyte terminal differentiation process, was expressed in the outer epidermal layers of the native skin and 3D RHS (Figure 19). Keratin 5, an intermediate filament protein, was expressed predominantly in the basal keratinocytes of the native human epidermis in the SB but was also slightly expressed in the other layers of the epidermis except for the SC, at the RHS (Figure 19). Normal expression pattern of structural (corneo)desmosomal proteins in 3D RHS The SC, the outer layer of the epidermis, contains DSG1, DSC1, which are the structural proteins of desmosomes and corneodesmosomes. Another structural protein is CDSN, which is only found in corneodesmosomes. Expression profile of all structural proteins (DSG1, DSC1, CDSN) was similar to both 3D RHS and native skin (Figure 20). Precisely, DSG1 was expressed mostly in the outer epidermal layers of native skin and 3D RHS while DSC1 and CDSN were predominately expressed in SC of both native human epidermis and 3D RHS (Figure 20). The skin proteases KLK5, KLK6, and KLK7 are comparably expressed in 3D RHS and native skin Generally, KLKs are related to the regulation of skin’s exfoliation and desquamation process. KLK5 and KLK7 are expressed by keratinocytes at the SG and their activity is confined at the SC (McGovern et al., 2006). Moreover, these KLKs cleave some of the structural proteins of desmosomes and corneodesmosomes, precisely, KLK5 cleaves DSC1, DSG1, CDSN, while KLK7 cleaves DSC1 and CDSN (Caubet et al., 2004; Descargues et al., 2006). The expression of KLK5 and KLK7 in native human skin was detected mostly in the SC. In addition, these proteins were weakly expressed in the other upper layers of the epidermis (Figure 21). KLK5 and KLK7 were expressed primarily in SC of 3D RHS and slightly in the other epidermal layers. KLK6, which is generally a protein that participates in the differentiation process of keratinocytes (Lin et al., 2002), localized predominantly in SC and SG epidermal layers of native human skin. In the epidermis of 3D RHS, KLK6 staining was strongly evident in the upper layers of keratinocytes (Figure 21). Figure 19. Immunohistochemical analyses of skin differentiation markers. Immunoreactivity of differentiation markers loricrin, involucrin and keratin 5. Loricrin and involucrin were primarily expressed in the SC of both native human skin and 3D RHS however, keratin 5 was expressed predominantly in SB of native human epidermis and 3D RHS. Areas of loricrin and keratin 5 staining are indicated by arrows. The blue dashed line represents the epidermis-dermis junction. Figure 20. Immunohistochemical analyses of the structural proteins of desmosomes and corneodesmosomes. Expression profiles of DSG1, DSC1 and CDSN was similar to both native epidermis and 3D RHS. Areas of intense staining are indicated by arrows. The blue dashed line represents the epidermis-dermis junction. Figure 21. Immunohistochemical analyses of human KLK proteases in skin. KLK5 and KLK7 were expressed mostly in the SC of native human skin, while KLK6 appeared equally at the SC and the SG. KLK5, KLK6 and KLK7 appeared mostly at the cornified layer of the 3D RHS. The blue dashed line represents the epidermis- dermis junction. CHAPTER 3: Toxicity tests of micro- and nano-emulsions, EOs and surfactants, on iNHKs Micro- and nano-emulsions are promising drug delivery vehicles/systems, as detailed in the introduction, since they have a dual role: [1] to protect the bioactive ingredient from metabolism and physical barriers, and [2] to improve the delivery of bioactive compound to the skin. In addition, micro- and nano-emulsions have been developed for cosmetic purposes, offering unique advantages including improvement of product efficiency and enhancement of stability of the bioactive compounds. Generally, the surfactants/co -surfactants used in the composition of micro- and nano- emulsions, are excipients that decrease the interfacial tension and expand the flexibility of the interfacial film, respectively (Boonme, 2007). Many different EOs were tested here as potential bioactive cosmetic compounds encapsulated in o/w micro- and nano-emulsions. In this study, we present toxicity data from a number of different EOs, different types of surfactants and cosurfactants, which could be candidate compounds in o/w micro- emulsion cosmetic formulations (Table 6). Moreover, cytotoxicity of eight different micro-emulsions as well as one nano-emulsion, was examined on iNHKs (Table 6) using NRU and/or the MTT assay. Their exact formulation cannot be disclosed in this Master thesis. Table 6. Different micro-emulsions and their potential substances tested for in vitro cytotoxicity on iNHKs. The EOs, surfactants and cosurfactants, which were tested as candidate substances for micro- and nano-emulsions. In addition, the micro- and nano-emulsions tested are shown. Essential oils Achillea millefolium-Lavandin Achillea millefolium Citrepel Geraniol Isopropyl myristate Lavandin Lavender Melissa officinalis Mentha spicata Oregano Surfactants: Cosurfactants Diethylene glycol monoethyl ether Span 85 Labrasol Tween 80 Micro- or Nano-emulsions 6% isopropyl palmitate encapsulated in micro- emulsion 10% citrepel encapsulated in micro-emulsion 20% citrepel encapsulated in micro-emulsion 30% citrepel encapsulated in micro-emulsion 10% geraniol encapsulated in micro-emulsion 20% geraniol encapsulated in micro-emulsion 30% geraniol encapsulated in micro-emulsion without encapsulated essential oil in micro-emulsion 1% geraniol encapsulated in nano-emulsion Commercially available product with 15% citrodiol encapsulated The NRU and MTT assays revealed that EOs at concentrations 0.1 %, 0.01% are not cytotoxic Generally, EOs could be encapsulated in the micro-emulsions, creating a promising cosmetic micro-emulsion formulation as detailed previously. The in vitro EOs cytotoxicity effects were examined on iNHKs using the NRU and MTT assay for determination of cell viability. The iNHKs were grown in KSFM until they reached a culture density of approximately 80% confluence. Cells were trypsinized and counted using a hemocytometer to estimate their exact number following by confirmation of a higher than 90% cellular viability using the trypan blue exclusion test, prior to treatment with EOs. Afterwards, cells were diluted to 7 .5 x 104 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate resulting in a density of 1. 5 x 103 cells /well. Finally, the plate was covered and incubated at 37±1°C, 5±1 % CO2, 95 % RH for 24 hours. Next day, cell morphology and confluence in the wells of the 96-well tissue plate was evaluated under a phase contrast light microscope (Figure 22), cells were then exposed for 1 hour to the testing EOs in a working dilution of 0.1% EO in KSFM containing DMSO as solvent for EOs with the limitation that DMSO should not exceed 0.5% of the total volume since it is cytotoxic in higher concentrations. Figure 22. Indicative photographs of iNHKs before treatment. The iNHKs were > 80% confluent. Normal cellular morphology before treatment, no cellular stress observed. It should be pinpointed that each plate encompasses control wells containing cells that were not treated with EOs but with KSFM (negative control), cells treated with cytotoxic 1% SDS (positive control) and wells containing growth medium without cells for use as assay blanks. At the end of the treatment period, examination of iNHKs under a phase contrast light microscope, revealed no changes in cell morphology indicative of no cytotoxic effects. Afterwards, cell viability was estimated by the NRU and/or MTT assay. The NRU assay showed that viability of iNHKs upon treatment with 0.1% Achillea millefolium, citrepel, geraniol, lavandin and mentha spicata oil was between 70-80%, only viability of iNHKs treated with Melissa officinalis oil was around 60% while iNHKs treated with lavander and isopropyl myristate (IPM) oil reached viability around 85% (Figure 23B; Table 7). Viability results obtained after comparing untreated cells with iNHKs upon treatment with EOs. Finally, iNHKs treated with 0.5% DMSO showed viability higher than 95% assuring that 0.5% DMSO was not cytotoxic and can be definitely used as a solvent for EOs, while iNHKs exposed to 1% SDS showed viability equal to zero as expected. In addition, a lower concentration of EOs was tested to determine a range of concentrations where EOs were not cytotoxic to iNHKs. Similarly, iNHKs were exposed for 1 hour to the testing EOs in a working dilution of 0.01% EO in KSFM containing 0.1% DMSO as solvent for EOs. Each plate contained control wells, as detailed previously. Microscopic examination after treatment with EOs and staining with NR showed absence of cellular stress and successful incorporation of NR in the lysosomes indicative of cell viability (Figure 24A). Αs evident in Figure 24B and Table 8, the tested EOs at concentration 0.01% were not cytotoxic and cell viability was higher than 90%. A Achillea millefolium Citrepel Geraniol Isopropyl myristate Lavandin Lavander Mellissa officinalis Mentha spicata Oregano B Viability, % 120 100 80 60 40 20 0 Figure 23. Viability testing of iNHKs following treatment with 0.1% EOs by NRU assay. A, microscopic observation of iNHKs following treatment with 0.1% EOs for 1 hour. Representative photographs showed no cellular stress response. B, cytotoxic effects of 0.1% EOs on iNHKs. The % viability compared to control cells treated only with KSFM. Data are shown as median ± SEM, n=2 independent experiments. Table 7. Viability percentage values of iNHKs treated with 0.1% EOs determined by NRU assay. The highest viability was observed in iNHKs treated with 0.1% lavander or IPM, while the lowest viability was seen in iNHKs treated with 0.1% Melissa officinalis. Essential oils, 0.1% Viability, % Achillea millefolium 73.9 Citrepel 70.0 Geraniol 71.2 Isopropyl myristate 87.1 Lavandin 77.0 Lavander 84.9 Melissa officinalis 59.6 Mentha spicata 76.6 Oregano 71.9 Table 8. Viability percentage values of iNHKs treated with 0.01% EOs determined by NRU assay. Observed viability in iNHKs treated with 0.01% EOs was higher than 90%, indicating that 0.01% EOs were not cytotoxic. Essential oils, 0.01% Viability, % Achillea millefolium-Lavandin 99.1 Achillea millefolium 96.1 Citrepel 97.8 Geraniol 94.5 Lavandin 96.1 A Achillea millefolium-Lavandin Achillea millefolium Citrepel Geraniol Lavandin 0.1% DMSO Β 120 Viabiliy, % 100 80 60 40 20 0 Figure 24. Viability testing of iNHKs following treatment with 0.01% EOs by NRU assay. A, visualization of iNHKs under the light microscope following treatment with 0.01% EOs for 1 hour and staining with NR. Cells treated with 0.01% EOs showed no cellular stress. B, the % viability of iNHKs treated with EOs compared to untreated control cells. Data are shown as median ± SEM, n=3 independent experiments. Viability of iNHKs treated with 0.01% EOs was also determined by the MTT assay to assess the potential cytotoxicity of EOs used in the developed formulations. Morphology of iNHKs before treatment was analyzed under a phase contrast light microscope to assure that cells had normal morphology and higher than 80% confluence (Figure 25). Microscopic evaluation revealed that iNHKs treated with 0.01% EOs for 1 hour showed normal morphology resembling that of control- untreated iNHKs (Figure 26A). In accordance with the % viability values estimated by NRU assay, which were presented previously, it was found by MTT assay that 0.01% EOs were not cytotoxic (Figure 26B; Table 9). Τhis, may be correlated with the potential ability of EOs to enhance cellular proliferation, but this remains further investigation. Statistical analysis by Student’s t- tests revealed that differences between % viability values of control-untreated cells and those of treated iNHKs with 0.01% are not statistically significant, showing p values > 0.5. Consequently, both NRU and MTT assays confirmed that EOs at low concentrations examined, do not have a negative impact on viability of iNHKs in 2D cell cultures. Thus, EOs tested could be encapsulated in micro- and nano-emulsions to enhance their properties such as moisturizing action. Figure 25. Morphology of iNHKs before treatment with EOs. Cultured iNHKs were observed under the light microscope until they reached 80% confluence, then cells were used for the NRU and MTT cytotoxicity assays. A Achillea millefolium-Lavandin Achillea millefolium Citrepel Geraniol Lavandin B 140 Viability, % 120 100 80 60 40 20 0 Figure 26. Viability testing of iNHKs following treatment with 0.01% EOs by MTT assay. A, microscopic observation of iNHKs upon treatment with EOs was assessed by the MTT assay. MTT was successfully reduced in mitochondria of viable

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cells, thus insoluble blue formazan crystals are visible under a phase contrast light microscope. No cellular stress was observed. B, graph showing % viability of iNHKs upon treatment with 0.01% EOs. The % viability was estimated compared to % viability of control-untreated cells, which was 100%. Results are shown as median ± SEM n=3 independent experiments. Table 9. Viability percentage values of iNHKs treated with 0.01% EOs determined by MTT assay. The EOs were not cytotoxic. Essential oils, 0.01% Viability, % Achillea millefolium-Lavandin 109.0 Achillea millefolium 100.8 Citrepel 109.7 Geraniol 109.0 Lavandin 102.9 Toxicity tests of surfactants on iNHKs assessed by the NRU and MTT assays, showed that the surfactants do not reduce cell viability at the concentrations tested Commonly, micro-emulsions in cosmetic formulations compromised of oil, water, a surfactant, a cosurfactant and/or an active ingredient. It was considered necessary to study the viability of iNHKs after treatment with some different substances that could be used for the development of cosmetic micro-emulsion formulations. Thus, candidate surfactants-cosurfactants have been tested for cytotoxicity at different concentrations, range between 0.01-0.04% in KSFM. Precisely, iNHKs were grown in KSFM, trypsinized, counted and cellular viability was always estimated higher than 95%, prior to 1 hour treatment with surfactants- cosurfactants. Subsequently, cells were diluted to 7 .5 x 104 cells/ml and then 200 μl of the cell suspension was dispersed into each well of a 96-well tissue plate resulting in a density of 1. 5 x 103 cells /well. Finally, the plate was covered and incubated at 37±1°C, 5±1 % CO2, 95 % RH for 24 hours. Morphology of iNHKs before treatment with EOs was examined under a phase contrast light microscope (Figure 25). The iNHKs were grown in KSFM until they reached a culture density of approximately 80%. Subsequently, NRU assay was conducted, and microscopic evaluation of the iNHKs after treatment showed that cells did not face cellular stress and they successfully incorporated NR inside their lysosomes (Figure 27A). The % viability of iNHKs treated with surfactants was compared with the viability of cells treated only with KSFM while 1% SDS was used as an indicative cytotoxic substance. The viability of iNHKs was higher than 90%, showing that surfactants were not cytotoxic at the examined concentrations (Figure 27B; Table 10). A Diethylene glycol Labrasol Span 85 monoethyl ether Tween 80 Untreated 1% SDS B viability, % 140 120 100 80 60 40 20 0 Figure 27. Toxicity testing of surfactants on iNHKs by NRU assay. A, microscopic examination showed that iNHKs had successfully incorporated NR and were confluent. B, viability of iNHKs treated with surfactants compared to untreated cells, showed that surfactants were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments. Table 10. The % viability values of the surfactants tested for cytotoxicity on iNHKs determined by NRU assay. Tested samples were not cytotoxic. Surfactants Viability, % Diethylene glycol monoethyl ether 0.04% 102.0 Labrasol 0.03% 90.7 Span 85 0.01% 102.7 Tween 80 0.01% 95.1 Evaluating the toxicity of the surfactants by the MTT assay was conducted to confirm the results obtained with the NRU assay. Visualization of iNHKs under the light microscope before treatment is shown in Figure 25, indicating that iNHKs were confluent. After treatment with surfactants, iNHKs where metabolically active as yellow tetrazole was successfully reduced to visible purple formazan crystals (Figure 28A). In addition, viability percentage values were higher than 95% for all of the surfactants (diethylene glycol monoethyl ether, labrasol, span 85, tween 80) examined (Figure 28B; Table 11). In conclusion, this study indicates that constituents added to micro- and nano- emulsions, such as the diethylene glycol monoethyl ether, labrasol, tween 80 and span 85 at low concentrations do not affect the viability of iNHKs in 2D cell cultures, as shown in Table 11. These results were dually confirmed by the NRU and MTT assay. Table 11. Viability percentage values of iNHKs treated with surfactants determined by MTT assay. Most of the samples had viability higher than 90%. Surfactants Viability, % Diethylene glycol monoethyl ether 0.04% 101.7 Labrasol 0.03% 95.2 Span 85 0.01% 98.1 Tween 80 0.01% 95.5 A Diethylene glycol Labrasol Span 85 monoethyl ether Tween 80 Untreated 1% SDS B 120 viability, % 100 80 60 40 20 0 Figure 28. Toxicity testing of surfactants on iNHKs by MTT assay. A, microscopic examination showed no cellular stress response. B, cellular viability was higher than 90% for iNHKs treated with surfactants indicating that samples were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments. Treatment with micro-emulsion at concentration 10% and 1% could significantly reduce cell viability according to NRU assay but treatment with micro-emulsions at concentration 0.1% does not reduce cellular viability Whilst it may be easier to characterize separately each ingredient of emulsions, the actual properties and the characteristics may be modified into the emulsion, during application or residence in the skin or affected by other environmental parameters. Consequently, it is crucial to examine whether micro- and nano-emulsions produced here, are cytototoxic. The toxicity of micro- and nano-emulsion, listed in Table 6 was assessed though different in vitro assays including NRU and MTT assay as detailed previously. Microscopic evaluation of cells before the cytotoxicity assay was always necessary to evaluate their condition (Figure 25). Initially, 10, 1 and 0.1 % of micro- and nano- emulsions diluted in KSFM were examined for cytotoxicity with NRU. Microscopic evaluation after treatment with 10 and 1% of micro-emulsions for 1 hour, in comparison with microscopic evaluation before treatment (Figure 25) showed that micro-emulsions provoked cellular stress response. The viability percentage values were estimated compared to % viability of untreated cells, which was 100% while 1% SDS used as indicative cytotoxic substance (positive control). Especially, viability of iNHKs was estimated under 50% for 10% micro-emulsion having 6% isopropyl palmitate encapsulated, 10% micro-emulsion having 10, 20, 30% citrepel encapsulated, 10% micro-emulsiom having 10, 20, 30% geraniol encapsulated (Figure 29) and 10% of control micro-emulsion having no EO encapsulated, indicating that 10% micro- emulsions were cytotoxic. Therefore, viability of iNHKs after 1 hour treatment with the same micro-emulsions was around 50-60% (Figure 30). Precisely, micro-emulsion having no EO encapsulated recorded the greatest viability (63%, micro-emulsion 8), while micro- emulsions with 10 and 20% geraniol encapsulated, do not reduce further the cell viability compared to micro-emulsion 8 (Table 12). A Isopropyl palmitate 10% 20% 30% Citrepel 10% 20% 30% Without EO Geraniol Untreated 1% SDS B 120 100 80 60 40 20 0 Citrepel Geraniol Figure 29. Viability testing of iNHKs following treatment with 10% o/w micro- emulsions by the NRU assay. A, representative photographs of iNHKs after treatment with 10% micro-emulsions. Intense cellular stress response was observed. B, viability of iNHKs after treatment with micro- emulsions was estimated under 50% indicating that micro-emulsions were cytotoxic. Data are shown as median ± SEM n=2 independent experiments. A Isopropyl palmitate 10% 20% Citrepel 30% 10% 20% Geraniol 30% Without EO Commercial product Untreated 1% SDS B Citrepel Geraniol Figure 30. Viability testing of iNHKs following treatment with 1% o/w micro- emulsions by the NRU assay. A, microscopic observation of iNHKs after treatment with 1% micro-emulsions. Changes in cells’ morphology were observed. B, viability of iNHKs after treatment with micro-emulsions was estimated around 50- 60% for most of the micro-emulsions. Data are shown as median. Contrary, the NRU assay revealed that treatment for 1 hour with 0.1% of micro- emulsion having 10% citrepel encapsulated, 0.1% of micro-emulsion having 10% geraniol encapsulated and 0.1% nano-emulsion having 1% geraniol encapsulated, showed absence of cellular stress in iNHKs and viability of iNHKs was higher than 95%, showing that emulsions were not cytotoxic at low concentrations (Figure 31). In addition, the viability of iNHKs exposed to the commercially available product having 15% citrodiol encapsulated, was almost 100% and no cellular stress response was observed. Viability values are also represented at Table 12. The cell viability to cytotoxicity ratio of iNHKs treated with 0.1% micro and nano- emulsions was also assessed by the MTT assay. Morphology of iNHKs before treatment was observed under a light microscope to confirm that cells had normal morphology and were confluent, a representative example is shown in Figure 25. Observation after treatment revealed that iNHKs were not stressed and they efficiently reduced MTT in their mitochondria (Figure 31A). In addition, viability of iNHKs treated with 0.1% micro-emulsion having 10% citrepel encapsulated as well as 0.1% micro-emulsion containing 1% geraniol encapsulated showed viability greater than 90%, while iNHKs treated with 0.1% micro-emulsion having 10% geraniol encapsulated showed viability 75%, however the difference is not statistically significant (t-test between micro-emulsion with 10% geraniol encapsulated and untreated cells) (Figure 31B; Table 13). Across the novel emulsions tested here, the factor of concentration led to iNHKs toxicity with a concentration of 1% being less toxic than 10%, which was cytotoxic. Micro- and nano-emulsions with a concentration of 0.1% were not cytotoxic showing that concentration can have a profound effect on cells’ viability. As a result, a range of concentrations where o/w micro- and nano-emulsions are not cytotoxic to iNHKs was determined. A 10% Citrepel 10% Geraniol 1% Geraniol Commercial product Untreated 1% SDS B 140 120 Viability, % 100 80 60 40 20 0 Figure 31. Toxicity testing of 0.1% o/w micro and nano-emulsions on iNHKs by the NRU assay. A, microscopic observation showed that iNHKs were successfully incorporated neutral red inside their lysosomes and were confluent. B, micro-emulsions’ and nano-emulsion’s viability was higher than 95%, indicating that samples were not cytotoxic. Data are shown as median ± SEM n=3 independent experiments. Table 12. Viability percentage values of iNHKs following treatment with 10%, 1% and 0.1% micro-emulsions determined by the NRU assay. The viability of cells treated with 10% micro-emulsions was determined to be below 50%, showing that micro-emulsions were cytotoxic. The viability of cells treated with 1% micro- emulsions was estimated between 50-60%. The viability of cells treated with 0.1% micro-emulsions and nano-emulsion were higher than 95%, thus those emulsions were not cytotoxic. Sample Viability, % 1. 10% of 6% isopropyl palmitate encapsulated in micro-emulsion 48.1 2. 10% of 10% citrepel encapsulated in micro-emulsion 40.7 3. 10% of 20% citrepel encapsulated in micro-emulsion 42.6 4. 10% of 30% citrepel encapsulated in micro-emulsion 48.7 5. 10% of 10% geraniol encapsulated in micro-emulsion 51.7 6. 10% of 20% geraniol encapsulated in micro-emulsion 49.1 7. 10% of 30% geraniol encapsulated in micro-emulsion 48.6 8. 10% without encapsulated essential oil in micro-emulsion 53.1 1. 1% of 6% isopropyl palmitate encapsulated in micro-emulsion 58.8 2. 1% of 10% citrepel encapsulated in micro- emulsion 52.8 3. 1% of 20% citrepel encapsulated in micro-emulsion 51.8 4. 1% of 30% citrepel encapsulated in micro-emulsion 53.8 5. 1% of 10% geraniol encapsulated in micro-emulsion 60.0 6. 1% of 20% geraniol encapsulated in micro-emulsion 63.8 7. 1% of 30% geraniol encapsulated in micro-emulsion 53.8 8. 1% without encapsulated essential oil in micro-emulsion 63.8 0.1% of 10% citrepel encapsulated in micro-emulsion 104.8 0.1% of 10% geraniol encapsulated in micro-emulsion 100.9 0.1% of 1% geraniol encapsulated in nano-emulsion 98.9 Commercially available product with 15% citrodiol encapsulated* 97.1 *median; Figure 30D; Figure 31B. Table 13. Viability percentage values of iNHKs treated with 0.1% micro- and nano-emulsions determined by MTT assay. Viability of iNHKs treated with 0.1% micro- emulsion with 10% citrepel encapsulated and nano-emulsion with 1% geraniol encapsulated were higher than 90% while iNHKs treated with 0.1% micro-emulsion with 10% geraniol encapsulated were 75.6%. Emulsions Viability, % 0.1% of 10% citrepel encapsulated in micro-emulsion 97.6 0.1% of 10% geraniol encapsulated in micro-emulsion 75.6 0.1% of 1% geraniol encapsulated in nano-emulsion 91.2 Commercially available product with 15% citrodiol encapsulated 101.1 A 10 % Citrepel 10 % Geraniol 1% Geraniol Commercial product Untreated 1% SDS B 120 Viability, % 100 80 60 40 20 0 Figure 32. Toxicity testing of 0.1% o/w micro- and nano-emulsions on iNHKs by the MTT assay. A, microscopic evaluation showed that iNHKs were not affected by the treatment of micro-emulsions. B, viability was higher than 90%. Therefore, the samples were not cytotoxic. Data are shown as medians ± SEM n=3 independent experiments. DISCUSSION Overall, 3D RHS models are quite useful tools for the development and evaluation of pharmaceuticals and cosmetic products (Suhail et al., 2019) and for the study of skin diseases especially rare skin diseases (Carlson et al., 2008). Finally, RHS models are valid in vitro alternatives to animal testing as they mimic perfectly the in vivo state. Therefore, this kind of models play a pivotal role in cosmetic industry as animal welfare concerns are increasing globally while animal experimentation for cosmetic products in the EU has already be banned. The main aim of this study was the generation of a 3D skin model that resembles native skin both structurally and biochemically. The iNHFs were mixed with collagen to form the dermis of the RHS and iNHKs were then seeded above the reconstructed “dermis” for the construction of the epidermis. Keratinocytes begun to differentiate until they formed a stratified squamous epithelium and the 3D skin model was ready to be characterized with histology and IHC. Histology was the method used for studying the structure of the 3D RHS. Staining with eosin and hematoxylin (H&E) was enough to show that the generated 3D skin model, resembles excellently the native skin in structure. The layers of the reconstructed epidermis (SC, SG, SS, SB) as well as the reconstructed dermis were distinct and successfully developed. In addition, immunohistochemical analysis was used for determining the existence of skin proteins in the 3D RHS model. Proliferation marker (Ki67), protein related with epidermal junction (E-cadherin), differentiation markers (keratin 5, involucrin, loricrin), structural proteins of desmosomes and corneo desmosomes (DSG1, DSC1, CDSN) that are important for preserving normal skin structure and physiology, were well expressed throughout the epidermis. Even the activity of kallikrein-related peptidases has been evaluated in the epidermis of the in-house 3D RHS, showing that epidermal proteolytic enzymes like KLK5, KLK6, KLK7 that are key to normal skin function and to the desquamation process are expressed equally in 3D RHS and native skin. In conclusion, the developed 3D skin model resembles normal human skin in terms of structure and biochemistry/physiology and can be used as a reliable human skin substitute for the examination of toxicity and moisturizing action of cosmetic micro- and nano- emulsion formulations. Generally, in vitro studies using 3D RHS in contrast to animal experimentation, offer higher speed, considering that animal testing can take months while in vitro testing in a skin substitute is a matter of days or weeks, and greater accuracy. Additionally, testing on 3D RHS helps gathering information in order to highlight the advantages and disadvantages of cosmetic products. 3D RHS has several advantages compared to 2D cultures in efficacy examination of a cosmetic product, because it gives the possibility of long term application of the testing product, it has cellular heterogeneity, structural complexity, and it resembles better the human skin considering the interactions between dermis and epidermis (Niehues et al., 2018; Schlotmann et al., 2001). Nevertheless, 2D cultures could be a valuable tool for the preliminary evaluation of skin care cosmetic products and topically applied drugs before their examination on 3D RHS substitutes. 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J Dermatol Sci 95: 28-35. https://www.abcam.com/kits/mtt-assay-protocol https://www.episkin.com https://www.mattek.com/products/epiderm/ APPENDIX Immortalization of keratinocytes Primary keratinocytes were isolated from the skin of healthy donors and cultured in serum-free keratinocyte growth medium (Gibco) in the presence of 5% CO2 at 370C. After immortalization with standard protocol stable cell lines with normal keratinocytes were generated by BIOSS Center for Biological Signaling Studies (University of Freiburg, Freiburg, Germany) with lentiviral infection, and infected keratinocytes were selected with puromycin. Immortalization of fibroblasts Primary fibroblasts were isolated from the skin of healthy donors and cultured in serum-free keratinocyte growth medium (Gibco) in the presence of 5% CO2 at 370C. Following immortalization by an established protocol (Zingkou et al., 2019) the stable cell line of normal fibroblasts was generated by BIOSS Center for Biological Signaling Studies (University of Freiburg, Freiburg, Germany) with lentiviral infection, and infected fibroblasts were selected with puromycin. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 35 36 40 41 42 43 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 104 105 106 107 109 110 111

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