Ekaterina A. Kolesnikova, Elina A. Genina, Georgy S. Terentyuk,

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

e-mail: [email protected]

Saratov State UniversityDepartment 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

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MotivationSaratov State University

Department of Optics & Biophotonics


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)

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Experimental setup OCT system

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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


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Experimental setup OCT system

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Fig.1. A general view of the spectral optical coherent tomography (left image) and tomography probe with the sample stage (right image)


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Experimental setup US system

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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”


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Immersion agents

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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 1.421. The

mixture was divided into 3 parts. Dimethyl sulfoxide (DMSO, 99%, Sigma, USA) and

thiophane sulfoxide were added to the 1st and to the 2nd part, respectively, to obtain

9%-DMSO-OCA and 9%-TSO-OCA solutions. Test objects


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In vivo experiments were carried out with white outbred laboratory rats. The age of

the animals was nearly 12 months. Weight was 150-200 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

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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


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He

alth

y sk

inS

kin

with

d

erm

atit

is

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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 OCA

F


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Results

a b c

d eFig.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

OCA


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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 GNSs

GNSsGNSs


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Results

a b c

d eFig.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

GNSsGNSs

SC

D

GNSs GNSs


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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.

GNSsGNSs GNSs

a b


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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.

GNSsGNSs GNSs

a b


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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.

GNSsGNSs GNSs

a b


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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.

GNSsGNSs GNSs

a b


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Table 1. The characteristic optical probing depth lt of the dermis at different methods of OCA

suspension delivery

Results

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Exposure typelt, mkm

Intact skin Skin with dermatitis

Without exposure 119±5 123±4

20 min after application

DMSO 127±3 154±4

TSO 132±3 153±3

Single US exposure

DMSO 142±6 175±6

TSO 152±3 174±11


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Table 2. The characteristic optical probing depth lt of the dermis at different methods of GNSs

suspension delivery

Results

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Exposure typelt, mkm

Intact skin Skin with dermatitis

Without exposure 119±5 123±4

20 min after application

DMSO 115±6 132±4

TSO 118±7 137±7

Single US exposure

DMSO 112±5 150±6

TSO 108±6 162±7


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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

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SummarySaratov State University

Department of Optics & Biophotonics


Acknowledgements

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This research was supported by:

Grant of President of RF NSH-1177.2012.2

FiDiPro, TEKES Program (40111/11), Finland

RF State contracts № 14.512.11.0022 and 14.B37.21.0728

Thanks for your attention!

Ekaterina A. Kolesnikova, Elina A. Genina, Georgy S. Terentyuk,

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