comparative study of the physical, chemical, and...

ISSN 1995�0780, Nanotechnologies in Russia, 2014, Vol. 9, Nos. 9–10, pp. 559–570. © Pleiades Publishing, Ltd., 2014.Original Russian Text © E.A. Genina, G.S. Terentyuk, A.N. Bashkatov, N.A. Mikheeva, E.A. Kolesnikova, M.V. Basko, B.N. Khlebtsov, N.G. Khlebtsov, V.V. Tuchin, 2014,published in Rossiiskie Nanotekhnologii, 2014, Vol. 9, Nos. 9–10.

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INTRODUCTION

In recent years, nanosize materials are being usedwith increasingly frequency in biomedical research.Gold nanoparticles occupy a specific place amongsuch materials for diagnostic and therapeutic pur�poses. In particular, gold nanoparticles are used as car�riers for delivering therapeutic agents, genetic mate�rial, and antigens, as well as a mediator in laser photo�thermolysis or as a diagnostic marker in tumor therapy[1, 2]. Much attention has been directed at studyingthe behavior of nanoparticles in contact with the sur�face of healthy and damaged skin and their interactionwith different skin layers, which, in the long run, canlead to an “ideal” deliverer or diagnostic tool from theviewpoint of the physicochemical parameters of nano�particles [3].

Numerous investigations [3–5] have demonstratedthat healthy skin is a barrier to penetration of sub�stances with a high molecular mass, including metalnanoparticles. Protective properties of the skin areprovided by the firm and dense stratum corneum, themacrophage system of the epidermis, and the dermis,

which includes Langerhans cells and histiocytes. Itwas established that nanoparticle�dependent deliveryto the epidermis and dermis is ineffective withoutinjury of the skin [5]. After injury to the skin barrier,e.g., in native or pathologically altered skin, the degreeof dermal absorption increases [6].

Permeability of the skin to nanoparticle�contain�ing preparations can be increased both by chemicalmethods [7–9] and physical actions, e.g., UV radia�tion [10], hyperthermia [11], iontophoresis [12], der�moporation [13], sonophoresis [14], etc. A combi�nation of different physical of physicochemicalapproaches favors more effective penetration of nano�particles through the skin barrier [15, 16].

It is well known that UV radiation has a negativeeffect on the barrier function of the skin by causingbiophysical and morphological changes in epidermallipids. However, according to [10], penetration ofnanoparticles into the skin was detected mainly in theregion of hair follicles, and the authors of [10] clas�sify this as one more possible mechanism of particletransfer.

Comparative Study of the Physical, Chemical, and Multimodal Approaches to Enhancing Nanoparticle Transport

in the Skin with Model DermatitisE. A. Geninaa, G. S. Terentyuka, b, c, A. N. Bashkatova, N. A. Mikheevab,

E. A. Kolesnikovaa, M. V. Baskoc, B. N. Khlebtsovd, N. G. Khlebtsova, d, and V. V. Tuchina, e, f

aChernyshevsky Saratov State University, ul. Astrakhanskaya 83, Saratov, 410012 RussiabUl’yanovsk State University, ul. L. Tolstogo 42, Ul’yanovsk, 432000 Russia

cRazumovskii Saratov State Medical University, ul. Bol’shaya Kazach’ya 112, Saratov, 410012 RussiadInstitute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences,

pr. Entuziastov 13, Saratov, 410049 RussiaeInstitute of Precision Mechanics and Control, Russian Academy of Sciences,

ul. Rabochaya 24, Saratov, 410028 RussiafUniversity of Oulu, Box 4500, Oulu, 90014 Finland

e�mail: [email protected] February 19, 2014; in final form, June 11, 2014

Abstract—This paper presents a comparative analysis of the combined and separate influence of ultrasoundand DMSO on the transport of a gold nanoshell suspension in intact and injured skin from data on opticalcoherence tomography and histochemical analysis. Experimental allergic contact dermatitis was used tomodel injury to the stratum corneum during various pathological changes in the skin. The studies were per�formed on outbred laboratory rats. It is shown that the best method for enhancing transdermal transport ofan immersion liquid is multimodal physical and chemical impact (a combination of DMSO and ultrasono�phoresis); the effectiveness of optical clearing of the dermis both in the presence and absence of the stratumcorneum is approximately the same. To enhance the transport of nanoparticles into the skin when it under�goes pathological changes related to injuries of the protective barrier, exposure to ultrasound is sufficient.

DOI: 10.1134/S1995078014050048

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GENINA et al.

Hyperthermia as a method of nanoparticle transferremains understudied. The results prevent a completeevaluation of the effect of thermal action that does notbreak the epidermis but only accelerates chemicalreactions [11].

Iontophoresis is a mechanism favoring the penetra�tion of hydrophilic and charged molecules through theskin with the application of direct current. A system oftransdermal delivery was applied for citrate�stabilizednegatively charged gold nanoparticles through humanskin [12]. At zero voltage, gold nanoparticles werelocalized on the surface epidermis and they passedalong an intercellular path after application of a lowvoltage. Unfortunately, further transfer to the dermiswas not shown [12].

Dermoporation is a new transdermal technique oftherapeutic agent delivery involving a pulsed electro�magnetic field. Experiments on increasing the pene�tration of 10 nm gold nanoparticles into the humanepidermis has demonstrated that the formation oftransition pores through which particles can diffuse is

a potential mechanism for improving transport [13].In addition, recent experiments on gold nanoparticlepenetration through the skin depending on the chargeshowed that negatively charged particles penetratebetter than positively charged ones [17].

Using low�frequency ultrasound makes it possibleto improve address delivery of micro� and nanoparti�cles to biological tissues [18]. However, overcomingthe epidermal barrier requires additional actions, suchas fractional laser microablation [15] or chemical epi�dermal diffusion enhancers [16].

Experiments on the permeability of intact andinjured skin to gold nanoparticles on model objects bysimple diffusion showed that penetration is possible;however, they did not reveal any quantitative differ�ence [6].

In a study of the influence of toluene as a dispersivemedium on gold nanoparticle penetration throughhuman skin, no radical changes that could favor thepenetration of nanoparticles through the skin wererevealed in the intercellular lipid composition [7].

At present, among the known compounds enhanc�ing transdermal diffusion, including low�molecularones, dimethyl sulfoxide (DMSO) is the best knownsulfur�containing compound. The mode of DMSOaction is caused by its ability to dissolve stratum cor�neum lipids and, consequently, to increase the watercontent in the stratum corneum [8, 9]. Owing to thisproperty, DMSO increases the rate of transdermalpenetration of both water�soluble and liposoluble sub�stances [8, 9].

The goal of this work is a study of gold�nanoshellpermeability of intact and injured skin, as well as acomparative analysis of the physical (ultrasound),chemical (DMSO), and multimodal approaches toenhancing nanoparticle transport in the skin with andwithout the protective epidermal barrier.

MATERIALS AND METHODS

A suspension of gold nanoshells with a silicate corediameter of 120 nm and shell thickness of 20 nm wassynthesized as described earlier [19]. The workinvolved a concentrated colloid of nanoshells with anoptical density of 20 in the wavelength range of 800–1000 nm (which corresponds to 1011 particles in1 mL). To increase biocompatibility and prevent theformation of particle conglomerates, the particle sur�face was modified by thiolated polyethylene glycol.Figure 1 shows the experimental extinction spectrumand an electron�microscope image of a gold nanoshellsample.

The gold nanoparticle colloid suspension for out�ward application was prepared as follows. A mixture of95% aqueous glycerol solution (ZAO Baza 1 Khim�reaktivov, Russian Federation) (refractive index n =1.467) and polyethylene glycol�400 (PEG�400, molec�ular weight 400, Aldrich, United States) (n = 1.463) in

(a)

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1.2

400 600 800 1000

Wavelength, nm

Diameter of the SiO2 nucleus is 120 nmShell thickness is 15–20 nm

Optical density, arb. units

200 nm

(b)

Fig. 1. (a) Extinction spectra and (b) electron�microscopyimages of gold nanoshells.


COMPARATIVE STUDY OF THE PHYSICAL, CHEMICAL, AND MULTIMODAL 561

equal proportions was prepared. The total volume ofthe solution was 920 μL (n = 1.465). The obtainedsolution was added dropwise with concentrated nano�particles (1 mL of the colloid). To 1 mL of the pre�pared colloid, 100 μL of DMSO (99%, Sigma, UnitedStates) (n = 1.477) were added. Then, the whole sys�tem was homogenized in an ultrasound bath (Elma�sonic One, Germany) for 10 min. The refractive indi�ces were measured on an IRF�454B2M refractometer(LOMO, Russia) at room temperature (~20°C) by thestandard technique.

Injuries of the stratum corneum of the epidermisare observed for different pathological states, includ�ing dermatitis of different etiology, malignant skinneoplasms, diabetes, etc. As a model of skin injury, onecan use experimental allergic contact dermatitis(ACD) in rats sensitized according to P.M. Zalkan’stechnique [20] with 2,4�dinitrochlorobenzene(DNCB).

Experiments were carried out in 24 male albinooutbred laboratory rats weighing 150–200 g on aver�age. The skin of white laboratory rats is close to humanskin in structure [21], and different dermatologicaldiseases can be modeled in these animals.

To model ACD, three droplets of 5% DNCB oilsolution were applied singly on a sensitized focus (dor�sum surface, preliminarily depilated skin) with addi�tional application of one droplet of the 1% solution toseven other skin areas. On the eighth day, an inflam�matory reaction with total necrosis of the epidermisand subepidermal vesicles formed.

The experiments were carried out on the ninth daysince the beginning of ACD modeling. The animalswere divided into eight groups (three animals pergroup): animals with ACD constituted groups I to IV;animals with uninjured skin, groups V to VIII. Theuninjured skin was preliminarily depilated. In everyanimal, two areas on the back were isolated. To studyskin permeability to gold nanoparticles, a suspensioncontaining gold nanoparticles combined with DMSOwas applied on both skin areas of the experimental ani�mals from groups I and V; a suspension containinggold nanoparticles without DMSO, from groups IIand VI. The exposure time at one of the areas was 20min. The second area, to enhance the transdermalgold nanoparticle transfer, was subjected to ultrasoundaction from a Dinatron 125 radiator (Dinatronics Co.,United States) with a frequency of 1 MHz, power of1.1 W, and total duration of 4 min (two times, by 2 minapiece). Groups III, IV, VII, and VIII, for which thepermeability of injured and healthy skin was studiedfor the immersion solution (the base of the suspen�sion) without gold nanoparticles, served as controlgroups. The solution composition for groups III andVII included DMSO, in contrast to groups IV andVIII. The studied skin areas for these groups were pro�cessed the same way as described above.

For noninvasive determination of gold nanoparti�cle accumulation zones in the skin and estimation ofthe probing light attenuation coefficient, opticalcoherent tomography was used for animals of allexperimental groups by means of an OCP930SR 022spectral optical coherent tomograph (OCT) (ThorlabsInc., United States) with a working wavelength of930 ± 5 nm and a full width at half�maximum of 100 ±5 nm. The optical power of the probing illuminationwas 2 mW and the scanning area was 6 mm. The axialand lateral resolutions of the device were 6.2 and9.6 μm, respectively. Every area was scanned beforeapplying the studied agent (gold nanoparticle suspen�sion or immersion solution without gold nanoparti�cles) and after processing: 20 min exposure of theagent or ultrasound action.

After termination of the experiments, animals fromgroups I and V were decapitated for further his�tochemical analysis.

The total coefficient of light attenuation μt at anarea of biological tissue is the sum of the absorption μa

and scattering μs coefficients. It can be obtained by fit�ting of the parameters of the approximating curve cal�culated using a corresponding model at the studiedarea near the slope of the A�scan of the OCT signal[22–24].

The single scattering model is based on theassumption that only the light undergoing single scat�tering preserves coherent properties and contributes tothe formation of an OCT signal. In this case, the OCTsignal is defined as [22–24]

(1)

where i(z) is the OCT signal and z is the distance in tis�sue depth from which the reflected signal arrives.

The single scattering model is valid both for weaklyscattering biological tissues and in the region of sur�face biological tissue layers where the single scatteringmode prevails. Biological tissues with an optical thick�

ness of can be treated as weakly scatter�

ing [22]. In this case, the calculation results for μs (orμt) are close to the values obtained from the results ofspectrophotometry optical measurements [24, 25].Thus, the value μt for surface skin layers (up to 100 μm),in our opinion, can be adequately estimated using thesingle scattering model.

It is well known that an OCT study results in mea�surement of the intensity of an OCT signal from thestudied tissue as a function of depth z,

. The intensity of an OCT signaldepends on the reflectivity α(z) of the biological tissueat the given depth and total attenuation coefficient

of the biological tissue. In accordancewith the single scattering model, the reflected power is

( ) ( ) ( )1/21/2 1/22 2

0( ) exp( 2 ) ,ti z i z≈ − μ

04

s

tdsτ = μ <∫

( )1/22( ) ( )R z i z∝

t a sµ = µ + µ

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proportional to exp(–μtz) [22], i.e., it can be approxi�mated by the expression

, (2)

where A is the proportionality factor equal to P0α(z),P0 is the optical power in the beam incident on the bio�logical tissue surface, α(z) is determined by the localrefractive index and local biological tissue’s ability toscatter the light backward, and B is the backgroundsignal.

Figure 2 presents the analyzed area in the OCTimage, the averaged A�scan of the OCT signal of thedermal skin layer of an in vivo rat, and an approximat�ing curve constructed using the single scatteringmodel. A region in the B�scan with a flat surface (iso�lated area in Fig. 2a) was chosen for the analysis. Thewidth of the studied region was 51 А�scans (approxi�mately 150 μm). The region of interest included thelayer of the upper part of the dermis with an opticaldepth of about 140 μm, which corresponds to a physi�cal depth of ~100 μm if the average index of the dermisis believed to be 1.4 [26]. This is the depth at which theOCT signal intensity decreases by е times as comparedto a signal from the surface (Fig. 2b). The fitting coef�ficients in Eq. (2) for the approximation of the exper�imental curve makes it possible to estimate the totalcoefficient of light attenuation by the dermis [27].

Biopsies of animal skins from groups I and V weretaken after decapitation. All experiments, care, andhusbandry were performed according to Directive2010/63/EU of the European Parliament and of theCouncil of September 22, 2010, on the protection ofanimals used for scientific purposes [28], and sanitaryregulations on organization, equipment, and mainte�nance of experimental�biological clinics of April 6,1973 [29].

The experimental material was fixed in 10% neutralformalin and poured into paraffin. Histological sec�tions were produced using an MPS�2 microtome

µ t( ) exp( )R z A z B= − +

(Tochmedpribor, Ukraine) and hematoxylin and eosinstain. To visualize zones of gold nanoparticle accumu�lation in skin tissue structures, the sections were his�tochemically stained with 0.2% aqueous silver nitratesolution in a mixture with 0.5% hydroquinone solu�tion in citrate buffer (pH = 3.8) [30]. The sectionswere additionally stained with toluidine blue. Descrip�tion and comparative morphological analysis of theskin structure were performed using a Motic B3microscope (Motic, People’s Republic of China).

RESULTS OF THE OCT INVESTIGATION

Figure 3 shows OCT images of the healthy skin ofan experimental animal and skin with ACD. In theOCT image of intact skin (Fig. 3a), one can see theepidermis (marked by the letter E) on the surface ofwhich the stratum corneum is observed (SC). Theboundary between epidermis and dermis (marked bythe letter D) is clearly differentiated.

In contrast to healthy skin, the injured SC is wellseen in the image of the skin with modeled ACD. Ininjured areas, either the absence of the epidermal layerand uncovering of the dermis (see Fig. 3b) or an eleva�tion of epidermal areas were observed (see Fig. 3c).The presence of elevated epidermal parts on the sur�face of skin with ACD attenuated the intensity of lightpenetrating into the skin due to multiple light reflec�tion from the air–epidermal tissue interfaces. As isseen in Fig. 3c, additional scattering created by theloose structure of the epidermal tissue almost impededpenetration of the probing radiation into the dermis.For this reason, the attenuation coefficient for the der�mis with the modeled ACD was calculated using areaswith uncovered dermis.

The values of the coefficient of light attenuation bythe dermis are presented in the table for different waysof introducing the gold nanoparticle suspension andimmersion solution.

Region of interest

Distinguished area

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600

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0

0 200 400 600 800 1000 1200z, µm

OCT signal, arb. units

Region

of interest

Averaged A�scan of OCT signalApproximating curve

Aexp(�µτz) + B

(a)

(b)

Fig. 2. (a) OCT image of area of the dermis in vivo (B�scan) and (b) averaged A�scan of OCT signal and approximating curveconstructed using single scattering model: .µ t( ) exp( )R z A z B= − +



After application of the studied suspension ontothe surface of the skin with the modeled ACD and20 min exposure for groups I and II of the experimen�tal animals, a decrease in μt values was observed onaverage by 3 and 2%, respectively. A 20 min exposureof the gold nanoparticle suspension on uninjured skin(groups V and VI) also caused a small decrease in μt

values. Such a change in the optical parameters can beassociated with the appearance of a reflecting screen ofthe skin surface; the gold nanoparticle suspensionlayer acts as the screen.

A more considerable decrease in μt values wasobserved in the control groups: approximately 20% ingroups III and IV and 6% in group VII. Only in groupVIII was the decrease in μt within the accuracy limit(1%).

Even more significant differences between groupstreated by the gold nanoparticle suspension andimmersion solution were observed after single anddouble application of particular agent and ultrasoundaction. For the skin with modeled ACD, an increase inthe attenuation coefficient by 4–7% (groups I and II)was observed when the gold nanoparticle suspensionwas the studied agent, which indicated an increase in

light scattering in the derma. For intact skin, the valueof the attenuation coefficient either decreased by 10%(group V) or remained almost constant (group VI). Inall control groups, in contrast, the value of μt signifi�cantly decreased. Namely, for group III after singleand double ultrasound treatment, the decreaseamounted to 37% and 48%; for group IV, 28% and42%; for group VII, 27% and 45%; and for group VIII,15% and 23%, respectively.

RESULTS OF THE HISTOCHEMICAL ANALYSIS

Figures 4–7 show a series of micrographs of histo�logical rat skin sections after introduction of the goldnanoparticle suspension by various means. Figure 4shows micrographs of ACD skin sections after 20 minexposure of the gold nanoparticle suspension withDMSO (group I of the experimental animals). InFig. 4a, one can clearly see the absence of epidermisthrough a large part of the section. The basementmembrane is absent in the remaining epidermis. Thestratum corneum of the epidermis is broken and con�tains nuclear cells. Basement layer cells form a stock�ade. Acantholysis is observed—degenerative changes

(a) (b) (c)

SC

D ED

D

SC E

500 µ

m

Fig. 3. OCT images of (a) healthy skin and (b, c) skin with allergic contact dermatitis: epidermis stratum corneum (SC), epider�mis (E), and dermis (D).

Attenuation coefficient of dermis (µt, cm–1) for different ways of introducing gold particle suspension and immersion solu�tion

Type of action

Experimental group

Skin with ACD Healthy skin

suspension with gold nanoparticle immersion solution suspension with gold

nanoparticle immersion solution

with DMSO

without DMSO

with DMSO

without DMSO

with DMSO

without DMSO

with DMSO

without DMSO

I II III IV V VI VII VIII

Without action 93 ± 12 93 ± 6 94 ± 10 91 ± 6 93 ± 5 101 ± 18 95 ± 2 97 ± 9

20 min exposure without ultrasound

90 ± 9 91 ± 8 78 ± 2 76 ± 4 91 ± 7 99 ± 14 90 ± 9 96 ± 8

2 min exposure with ultrasound

98 ± 13 97 ± 13 68 ± 4 71 ± 7 92 ± 11 104 ± 11 75 ± 2 84 ± 9

4 min exposure with ultrasound

102 ± 22 98 ± 10 63 ± 2 64 ± 9 84 ± 9 102 ± 11 66 ± 4 79 ± 9

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in cells of the spinous layer. The changes manifestthemselves as liquefying intercellular cytoplasmicbonds.

The dermis is characterized by sharp dystrophicchanges in collagen fibers in the form of mucoiddegeneration. In some areas, mucoid necrosis of der�mis fibers is pronounced. The vessels are full�bloodedwith determinable erythrostasis. In the subdermalmusculature, an interstitial edema is observed.

As a result of staining of the histological specimenwith silver nitrate, gold nanoparticle clusters werevisualized on the skin surface (marked in Fig. 4b by thelabel 1 and arrows) and, in insignificant quantities, inthe derma, mostly in the region of hair follicles (label2 and arrows).

Figure 5 shows micrographs of skin sections withmodel ACD after a single application of the gold

nanoparticle suspension with DMSO and low�fre�quency ultrasound action for 2 min (group I). It is seenin Fig. 5a that the epidermis is absent due to itsdesquamation and the wound surface is uncovered.The dermis is characterized by sharp dystrophicchanges in collagen fibers in the form of mucoiddegeneration. In some areas, mucoid necrosis of der�mis fibers is pronounced. There are hemorrhage focirepresented by lysed erythrocytes. The vessels are full�blooded with determinable erythrostasis. A distinctionis the neutrophilic infiltration of the dermis papillarylayer. It follows from Fig. 5b that gold nanoparticlesare revealed in the sarcoplasm of myosymplasts (label1 and arrows).

Figure 6 shows micrographs of histological sectionsof rat skin with ACD after double sequential applica�tion of the gold nanoparticle suspension with DMSO

2

1

(a) (b)

Fig. 4. Micrographs of histological sections of rat skin with allergic contact dermatitis after 20 min exposure of gold nanoparticlesuspension: (a) hematoxylin and eosin stained, magnification ×160, and (b) silver nitrate stained, magnification ×1000. The labelsand arrows mark clusters of gold nanoparticles.

1

(a) (b)

Fig. 5. Micrographs of histological sections of rat skin with allergic contact dermatitis after single application of gold nanoparticlesuspension and low�frequency ultrasound action for 2 min: (a) hematoxylin and eosin stained, magnification ×160; and (b) silvernitrate stained, additionally stained with toluidine blue, magnification ×600. Label 1 and arrows denote clusters of gold nanopar�ticles.



and ultrasound action (group I). Figure 6a shows localthickenings of the epithelium and epithelium damagein most areas. The stratum corneum is absent. Leuco�cytic infiltration of the dermis papillary layer is consid�erable. Collagen fibers are thickened, and deep dermislayers are edematous. The venules are full�blooded.Infiltrates by neutrophils and lymphocytes often occuralong vessels.

Staining of the histological specimen with silvernitrate revealed good skin permeability to gold nano�

particles, which were almost uniformly distributed inthe dermis reticular layer and somewhat concentratedin the region of oil glands (in Fig. 6b, they are denotedby label 1 and arrows). In addition, gold nanoparticleswere determined in considerable amounts in connec�tive�tissue interlayers of the subdermal musculature(denoted in Fig. 6c by label 1 and arrows).

Figure 7 shows micrographs of histological sectionsof intact rat skin after double sequential application ofgold nanoparticle suspension with DMSO and ultra�

(a)

(b)

(c)

1

1

Fig. 6. Micrographs of histological sections of rat skin with allergic contact dermatitis after double sequential application of goldnanoparticle suspension and low�frequency ultrasound action during 2 min: (a) hematoxylin and eosin stained, magnification×160; (b) silver nitrate stained, additionally stained with toluidine blue, magnification ×160; and (c) silver nitrate stained, mag�nification ×600. Label 1 and arrows denote clusters of gold nanoparticles.

(a) (b)

1

Fig. 7. Micrographs of histological sections of intact rat skin after double sequential application of gold nanoparticle suspensionand low�frequency ultrasound action during 2 min: (a) hematoxylin and eosin stained, magnification ×160; and (b) silver nitratestained, magnification ×1000. The labels and arrows denote clusters of gold nanoparticles.

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sound action (group V). The histology corresponds tohealthy skin. The stratum corneum in some areas isbroken. The dermis is insignificantly edematous; inthe papillary layer, loose leucocytic infiltration takesplace. Perivascular infiltration is typical for surfaceand deep vessels of the dermis (Fig. 7a).

It follows from Fig. 7b that trace gold nanoparticleswere revealed in the boundary zone between the der�mis and epidermis, rarely in the dermis. Label 1 andarrows mark gold nanoparticle clusters on the skin sur�face and in a hair follicle.

DISCUSSION

Among numerous methods for studying nanoparti�cle transport in different biological tissues, OCT occu�pies a prominent place. OCT is a noninvasive methodintended for visualizing the internal structure of opti�cally inhomogeneous objects and based on principlesof low�coherent interferometry using the light of thenear IR range (0.75–1.3 μm) [31]. The method allowsone to study the internal microstructure of biologicaltissues at the depth of up to 3 mm with spatial resolu�tion of 5–20 μm without penetration into the biologi�cal tissues [22]. In some works [16, 32, 33], it wasshown that OCT allows one to monitor the transportof metal nanoparticles introduced in a biological tissuebecause they increase the contrast in OCT images ofregions in which the nanoparticles are localized in thebiological tissue.

In addition to visualization of optical inhomogene�ities in a biological tissue, OCT allows one to measureoptical parameters of biological tissues such as therefractive index, coefficients of scattering and attenu�ation, and scattering anisotropy factor [23, 24, 34]. Aquantitative estimate of changes in optical parametersof the biological tissue due to the presence of nanopar�ticles in it can yield additional information about thestate of biological tissue, e.g., for diagnostic purposes.It is evident, however, that correspondence betweenthe probing radiation wavelength and plasmon reso�nance maximum of the nanoparticles is necessarywhen studying gold nanoparticle penetration into theskin by the OCT method [32]. Among the entire vari�ety of nanoparticles with tuned plasmon resonance,gold nanoshells possess a series of advantages (e.g., ascompared to gold nanorods) owing to low toxicity andhigh coefficients of absorption and scattering of a par�ticle in the IR region of the transparency window ofbiological tissues. A wide extinction band, which istypical of nanoshells (Fig. 1a),makes it possible to usethem for increasing the contrast of OCT images even ifthere is no exact coincidence between the probingradiation wavelength and plasmon resonance maxi�mum. In this case, in the spectral range of the tomo�graph radiation source (~930 ± 50 nm), the opticaldensity of the nanoshell suspension decreased by only~20% on average. Thus, the possibility of visualizing

nanoparticles in the skin and of estimating its opticalparameters was maintained.

In this study, the joint use of OCT and histochemi�cal analysis made it possible to reveal the features ofACD manifestation in OCT images and gold nanopar�ticle distribution in intact and injured skin for differentmeans of enhancing its permeability.

As is well known, ACD advances as a kind ofdelayed type of hypersensitivity. It manifests itself inthe appearance of inflammation foci on the immunebase with the involvement of microvasculature andcomplex morphological tissue and cell processesdeveloping in the injured area [35]. The ACD patho�morphological process involves the epidermis and der�mis, which undergo destructive changes.

A macroscopic study of the animals' skin with ACDrevealed the following: sustained hyperemia, desqua�mation, excoriation, and serous crusts. The patho�morphological pattern of ACD included zones of pro�nounced destruction of all epidermis layers; areas ofepidermis desquamation with an uncovered woundsurface, which could be observed in OCT images ofthe skin (see Figs. 3b and 3c). Microscopic morpho�logical changes also included acanthosis (epidermalhyperplasia)—an increase in the thickness of thespinous layer of the epidermis with elongation of inter�papillary crests. Results of individual investigationsshowed that ACD is accompanied by changes in thesynthesis of intercellular lipids of the stratum cor�neum, which is followed, first of all, by a discontinuityof barrier skin properties [36].

In connection with pronounced disfunctions in themicrovasculature, which are accompanied by discon�tinuities in the permeability of vessel walls, the dermisis characterized by oedemata and sharp dystrophicchanges in collagen fibers in the form of mucoiddegeneration. In OCT images, these phenomenamanifested themselves in a more homogeneous struc�ture of the dermis as compared to intact skin (seeFig. 3a).

The optical parameters of the skin with ACD andwithout pathology were also somewhat difference. Theaverage values of the attenuation coefficient of upperlayers of the dermis within an optical depth of 110 ±10 μm obtained as a result of calculations according tothe used model amounted to approximately 92 ±

1 cm–1 for the uncovered dermis (the skin with modelACD) and to 97 ± 3 cm�1 for the intact skin. In general,the values of the attenuation coefficient of upper layersof the dermis for intact skin agree well with spectro�photometry data [25].

Ultrasound as a physical method for enhancing dif�fusion is applied rather widely. It was shown in a num�ber of works that short�time acoustic cavitation andthermal and mechanical effects appearing in the near�surface layers of the skin under the action of ultra�sound improve the transdermal transport of different



medicinal preparations and chemical agents [37, 38],as well as of micro� and nanoparticles [14, 15, 33].

Comparative analysis of physical (ultrasound),chemical (DMSO), and multimodal physicochemicalmethods for enhancing the diffusion of a nanoparticlesuspension in an immersion solution and, individually,an immersion solution in the skin deprived of the pro�tective epidermal barrier and in the presence of thisbarrier may prove useful for developing these methods.

Figure 8 shows normalized values of the attenua�tion coefficient in the dermis of skin with an injuredepidermal layer and of healthy skin for different meansof introducing the gold nanoparticle suspension orimmersion solution as the base of the suspension.

It is well seen in the figure that 20 min exposure ofthe studied agent on the skin surface noticeablydecreased the attenuation coefficient of the skin der�

mis only in groups III and VII. Ultrasound action indifferent groups caused opposite changes in the opticalparameters of the dermis. In particular, in group I, anincrease in the μt value was observed after sequentialsingle and double application of the gold nanoparticlesuspension and ultrasound action on the dermis; ingroups II and VI, the μt value somewhat increased aftersingle ultrasound action and remained almostunchanged after additional introduction of the studiedsuspension into the dermis. In other groups, the atten�uation coefficient decreased to a different extent ofefficiency relative to its initial value.

These differences in the change dynamics of μt arerelated to the presence of gold nanoparticles in thestudied agents. Namely, in all groups of animals wherethe gold nanoparticle free suspension base was used,the attenuation coefficient noticeably decreased as

1.2

1.0

0.8

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0.4Without 20 min 2 min ultra� 4 min ultra�

No

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its

I group

II group

III group

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VIII group

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(a)

action exposure sound actionsound action

Without 20 min 2 min ultra� 4 min ultra�action exposure sound actionsound action

0.6

0.8

1.2

1.0

Fig. 8. Normalized values of the attenuation coefficient of the dermis in groups of experimental animals with (a) allergic contactdermatitis and (b) intact skin after different ways of introduction of the gold nanoparticle suspension and suspension base.

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compared to the initial values (see table), which isrelated to skin immersion as the skin interacts with thebase of the used suspension. Since the refractive indexof the studied solution is close to that of hydrated col�lagen and elastin fibers (n = 1.411) [39], the solutionequalized the refractive indices of main biological tis�sue components in the process of penetrating deepinto the dermis and replacing the interstitial fluid (n =1.345) [26]; this resulted in optical clearing of the der�mis [40]. Thus, a decrease in scattering of upper skinlayers favored the decrease in the attenuation coeffi�cient and, correspondingly, deeper penetration of theprobing radiation.

An insignificant (within the accuracy) decrease inμt observed after 20 min exposure in groups I and IIseems related to gold nanoparticle assembly on thedermis surface, as well as with penetration of the sus�pension base into the dermis tissue along hair follicles.Consequently, two processes occurred simultaneously:an increase in scattering due to gold nanoparticle pen�etration into surface layers of the dermis and adecrease in dermis scattering due to its immersionoptical clearing of the base suspension. This hypothe�sis is verified by the fact that rather efficient opticalclearing of the tissue was observed in the absence ofgold nanoparticles (groups III and IV).

Results of histological studies showed that applyingultrasound favored a deeper penetration of the goldnanoparticle suspension into the dermis and uniformdistribution in it, which caused an increase in the μt

values of the dermis in groups I and II. Ultrasoundaction also enhanced the efficiency of optical clearingof the dermis in groups III and IV.

In contrast to the skin with injured SC, the intactskin exhibited a decrease in the attenuation coefficientin all groups with the exception of the sixth group. Ingroup VI, changes in μt amounted only to 1–3%. Thisseems related to the presence of SC, which impededgold nanoparticle penetration into the dermis in theintact skin. In this case, the penetration of particlescould occur only along hair follicles and gland ducts.This is associated with the different behavior of theattenuation coefficient in groups V and VI. At thesame time, as follows from the analysis of histologicalsections, the nanoparticle concentration inside theepidermis and dermis remained insignificant for theintact skin. This indicates that the SC served as a filterfor nanoparticles. As a consequence, suspension com�ponents were separated: a dense gold nanoparticlelayer was formed on the skin surface and the immer�sion solution diffused into the dermis. In the fifthgroup, the diffusion occurred more efficiently than inthe sixth group due to the presence of DMSO in thesuspension composition.

Comparison of groups with similar physical actionsbut differing in the presence of the gold nanoparticlesuspension or immersion DMSO solution demon�strates that using DMSO as a diffusion enhancer had

no significant effect on the permeability of skindeprived of SC to nanoparticles. However, under thecombined physicochemical action on the skin, thedegree of its optical clearing was higher than under theaction of an individual chemical or physical enhancerof immersion agent transport. In addition, the degreeof optical clearing of the dermis in intact skin coin�cided approximately with the degree of dermis clarifi�cation in the absence of SC, which indicates an effi�cient overcoming of the epidermal skin barrier due tousing the multimodal approach.

CONCLUSIONS

We have presented the results of studies and a com�parative analysis of the combined and separate influ�ence of ultrasound and DMSO on the transport of ananoparticle suspension in an immersion solution andon the transport of a pure immersion solution in skinwith and without the protective epidermal barrier,based on data from optical coherent tomography andhistochemical analysis. It was shown that multimodalphysicochemical action (a combination of DMSOand ultrasonophoresis) is the optimum method forenhancing the transdermal transport of the immersionsolution; the efficiency of optical clearing of the der�mis is approximately equal both with and without thestratum corneum of the epidermis. To enhance nano�particle transport into a skin subjected to pathologicalchanges realted to injuries of the protective barrier,ultrasound action alone suffices.

ACKNOWLEDGMENTS

This work was supported by the Government of theRussian Federation (no. 14.Z50.31.0004) for a scientificresearch project under the supervision of a leading scien�tist, as well as partially supported by the Russian Foun�dation for Basic Research (project no. 13�02�91176�GFEN_a).

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Translated by A. Nikolskii

comparative study of the physical, chemical, and...

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