PHYSICAL AND CHEMICAL METHODS OF SKIN DRUG DELIVERY ENHANCEMENT: COMPARATIVE STUDY OF HEALTHY SKIN AND SKIN WITH DERMATITIS Ekaterina A. Kolesnikova, Elina A. Genina, Georgy S. Terentyuk, Natalya A. Tsyganova, Alexey N. Bashkatov, Daniil S. Chumakov, Marina V. Basko, Valery V. Tuchin Saratov State University, Saratov State Medical University, Ulianovsk State University, Institute of Precise Mechanics and Control of RAS, University of Oulu Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

The main advantages of transcutaneous administration of preparations are: 1) minimal invasiveness or even noninvasiveness; 2) improved drug pharmacokinetics; and 3) targeted drug delivery However, living epidermis and its upper layer stratum corneum (SC) represent a major barrier making delivery of drugs deep into the skin a rather difficult problem Goal of the study is to investigate the effect of low-frequency US, DMSO, TSO and their combination on transdermal permeation of gold nanoparticles suspension through intact skin and skin with the partly removed epidermis 2 Motivation Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

Materials and methods  For this study laboratory rats with a healthy skin and experimental allergic contact dermatitis were used  As a potential drug carrier, a suspension of gold nanoshells (GNSs) in immersion solution of glycerol (50%) and PEG-400 (50%) was used  To enhance drug delivery into the skin dermis the suspension was mixed with dimethyl sulfoxide (DMSO) or thiophane sulfoxide (TSO)  For physical enhancement of the transdermal transport, the skin sites were treated with low-frequency ultrasound (US)  Monitoring of skin optical properties was implemented using an optical coherence tomography (OCT)  All measurements were performed at room temperature (about 20°C) 3 Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

 Experimental setup  OCT system 4 Materials and methods Commercial spectral optical coherence tomography system OCP930SR (Thorlabs, USA) (fig.1) working at the central wavelength 930 ± 5 nm with 100 ± 5 nm full width at half maximum spectrum, an optical power of 2 mW, a maximum image depth of 1.6 mm, and a length of scanned area 6 mm Axial and lateral resolutions were 6.2 and 9.6 µm in air, respectively Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

 Experimental setup  OCT system 5 Materials and methods Fig.1. A general view of the spectral optical coherent tomography (left image) and tomography probe with the sample stage (right image) Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

 Experimental setup  US system 6 Materials and methods Sonicator “Dynatron 125” (Dynatronics, USA) (fig.2) equipped with a 2.2- cm diameter probe The US frequency was 1 MHz, the power was 1.1 W in the continuous mode, and the time of sonication was 4 min. During sonication, the US probe was immersed into applied solutions. Fig.2. US system “Dynatron 125” Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

 Immersion agents 7 Materials and methods As an OCA the mixture of dehydrated glycerol and polyethylene glycol with the MW 400 (PEG-400) in equal proportion was prepared. Refractive index of the prepared mixture was evaluated the mean wavelength of the used OCT system as The mixture was divided into 3 parts. Dimethyl sulfoxide (DMSO, 99%, Sigma, USA) and thiophane sulfoxide were added to the 1 st and to the 2 nd part, respectively, to obtain 9%-DMSO-OCA and 9%-TSO-OCA solutions.  Test objects Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013 In vivo experiments were carried out with white outbred laboratory rats. The age of the animals was nearly 12 months. Weight was g. Before the experiment they were subjected to general anesthesia by intramuscular injection of Zoletil 50 (Virbac, France). The dose was 0.18±0.02 mL. The hair on the chosen skin areas was depilated previously using the depilatory cream Nair.

 Test objects 8 Materials and methods In dependence on exposure type laboratory rats were divided into 8 investigated groups: I group - 20 minutes of mixture OCA - DMSO exposure II group - 20 minutes of mixture OCA - TSO exposure III group - mixture OCA - DMSO application and single ultrasonic exposure IV group - mixture OCA - TSO application and single ultrasonic exposure V group - 20 minutes of mixture OCA - DMSO exposure VI group - 20 minutes of mixture OCA - TSO exposure VII group - mixture OCA - DMSO application and single ultrasonic exposure VIII group - mixture OCA - TSO application and single ultrasonic exposure Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013 Healthy skin Skin with dermatitis

9 Results a b c d e Fig.3. OCT images of healthy skin before the treatment (a), after 20 minutes of mixture OCA - DMSO application (b), after 20 minutes of mixture OCA - TSO application (c), after mixture OCA - DMSO application and single ultrasonic exposure (d), and after mixture OCA - TSO application and single ultrasonic exposure (e). SC- stratum corneum, E - epidermis, D - dermis, OCA – optical clearing agent, F – hair follicle. The vertical label corresponds to 500 microns. SC E D OCA F Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

10 Results a b c d e Fig.4. OCT images of skin with dermatitis before the treatment (a), after 20 minutes of mixture OCA - DMSO application (b), after 20 minutes of mixture OCA - TSO application (c), after mixture OCA - DMSO application and single ultrasonic exposure (d), and after mixture OCA - TSO application and single ultrasonic exposure (e). SC- stratum corneum, E - epidermis, D - dermis, OCA – optical clearing agent. The vertical label corresponds to 500 microns. SC D OCA Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

11 Results a b c d e Fig. 5. OCT images of healthy skin before the treatment (a), after 20 minutes of mixture GNSs - DMSO application (b), after 20 minutes of mixture GNSs - TSO application (c), after mixture GNSs - DMSO application and single ultrasonic exposure (d), and after mixture GNSs - TSO application and single ultrasonic exposure (e). SC- stratum corneum, E - epidermis, D - dermis, GNSs - gold nanoshells. The vertical label corresponds to 500 microns. SC E D GNSs Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

12 Results a b c d e Fig.6. OCT images of skin with dermatitis before the treatment (a), after 20 minutes of mixture GNSs - DMSO application (b), after 20 minutes of mixture GNSs - TSO application (c), after mixture GNSs - DMSO application and single ultrasonic exposure (d), and after mixture GNSs - TSO application and single ultrasonic exposure (e). SC- stratum corneum, E - epidermis, D - dermis, GNSs - gold nanoshells. The vertical label corresponds to 500 microns. GNSs SC D GNSs Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

13 Results Fig.7. Microphotographs of the rat skin with dermatitis after 20 minutes of mixture GNSs - DMSO application. (a) – dyeing by hematoxylin-eosin, ×160; (b) – dyeing by silver nitrate on Hacker G.W. method, × 1000, 1 - clusters of GNSs on the skin surface, 2 - clusters of GNSs in the hair follicle. GNSs a b Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

14 Results Fig.8. Microphotographs of the rat skin with dermatitis after 20 minutes of mixture GNSs - TSO application. (a) – dyeing by hematoxylin-eosin, ×160; (b) – dyeing by silver nitrate on Hacker G.W. method, × 600, 1 - clusters of GNSs on the boundary between the epidermis and dermis and in the hair follicle area. GNSs a b Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

15 Results Fig.9. Microphotographs of the rat skin with dermatitis after mixture GNSs - DMSO application and single ultrasonic exposure, dyeing by hematoxylin-eosin, ×160 (a); (b) –subcutaneous musculature, dyeing by silver nitrate on Hacker G.W. method with toluidine blue addutional dyeing, × 600, 1 - clusters of GNSs in the myosymplasts sarcoplasm. GNSs a b Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

16 Results Fig.10. Microphotographs of the rat skin with dermatitis after mixture GNSs - TSO application and single ultrasonic exposure. (a) – dyeing by hematoxylin-eosin, ×160; (b) – dyeing by silver nitrate on Hacker G.W. method, × 600, 1 - clusters of GNSs in the sebaceous glands. GNSs a b Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

Table 1. The characteristic optical probing depth l t of the dermis at different methods of OCA suspension delivery Results 17 Exposure type l t, mkm Intact skinSkin with dermatitis Without exposure119±5123±4 20 min after application DMSO127±3154±4 TSO132±3153±3 Single US exposure DMSO142±6175±6 TSO152±3174±11 Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

Table 2. The characteristic optical probing depth l t of the dermis at different methods of GNSs suspension delivery Results 18 Exposure type l t, mkm Intact skinSkin with dermatitis Without exposure119±5123±4 20 min after application DMSO115±6132±4 TSO118±7137±7 Single US exposure DMSO112±5150±6 TSO108±6162±7 Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

 The analysis of the results has shown that OCA usage in both cases of intact skin and skin with dermatitis led to the characteristic optical probing depth (OPD) increase because of the matching of refractive indices of collagen and elastin fibers and of interstitial fluid  The effectiveness of DMSO and TCO as amplifiers of diffusion through the SC is about the same  OPD of the skin with dermatitis is higher than that of intact skin for all exposure types. It can be explained by damage of SC - natural skin barrier  GNSs delivery into the skin led to OPD decrease regardless of delivery method because of the formation reflecting and scattering screen on the skin surface that prevented deep light penetration into the dermis.  Our results can be used for the development of new methods and optimization of the existing ones of drug delivery into the skin 19 Summary Saratov State University Department of Optics & Biophotonics Saratov Fall Meeting 2013

Acknowledgements 20 This research was supported by:  Grant of President of RF NSH  FiDiPro, TEKES Program (40111/11), Finland  RF State contracts № and 14.B

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