Journal of Nanoparticle Research

, Volume 13, Issue 1, pp 283–291

Continuous optical coherence tomography monitoring of nanoparticles accumulation in biological tissues

Authors

    • Nizhny Novgorod State Medical Academy
  • M. V. Shirmanova
    • Nizhny Novgorod State Medical Academy
  • M. L. Bugrova
    • Nizhny Novgorod State Medical Academy
  • V. V. Elagin
    • Nizhny Novgorod State Medical Academy
  • P. A. Agrba
    • Institute of Applied Physics of RAS
  • M. Yu. Kirillin
    • Institute of Applied Physics of RAS
  • V. A. Kamensky
    • Institute of Applied Physics of RAS
  • E. V. Zagaynova
    • Nizhny Novgorod State Medical Academy
Research Paper

DOI: 10.1007/s11051-010-0028-x

Cite this article as:
Sirotkina, M.A., Shirmanova, M.V., Bugrova, M.L. et al. J Nanopart Res (2011) 13: 283. doi:10.1007/s11051-010-0028-x

Abstract

In this study, dynamics of nanoparticles penetrating and accumulating in biotissue (healthy skin) was investigated in vivo by the noninvasive method of optical coherence tomography (OCT). Gold nanoshells and titanium dioxide nanoparticles were studied. The processes of the nanoparticles penetration and accumulation in biotissue are accompanied by the changes in optical properties of skin which affect the OCT images. The continuous OCT monitoring of the process of the nanoparticles penetration into skin showed that these changes appeared in 30 min after application of nanoparticles on the surface; the time of accumulation of maximal nanoparticles concentration in skin was observed in period of 1.5–3 h after application. Numerical processing of the OCT signal exhibited the increase in contrast between upper and lower parts of dermis and contrast decay of the hair follicle border during 60–150 min. The transmission electron microscopy technique confirmed accumulation of the both types of nanoparticles in biotissue. The novelty of this study is presentation of OCT ability to in vivo monitor dynamics of nanoparticles penetration and their re-distribution within living tissues.

Keywords

NanoparticlesContinuous OCT monitoringNanoparticles accumulationIn vivo studyNanomedicineHealth and safety

Introduction

Nanoparticles with their unique physical, chemical, and biological properties open tremendous opportunities for biomedical applications. Carbon nanotubes, fullerenes, quantum dots, various metal, and metal oxide nanoparticles are currently widely examined (Parashar et al. 2008). Of special interest among metal nanoparticles are the so-called plasmon-resonant nanoparticles, for example, gold nanoshells. They may be used for directed drug delivery, as biosensors, as well as for optical bioimaging (Parashar et al. 2008). The titanium dioxide nanoparticles are primarily employed as UV-screening agents in cosmetic industry (Edlich et al. 2004; Innes et al. 2002), hence, it is important to know their penetration abilities into skin. Nanosize particles of titanium dioxide have strong backscattering in the visible spectral region and low absorption in the near-infrared range (McNeil et al. 2001). This feature makes them useful for optical visualization methods.

In spite of numerous studies of nanoparticles in biomedicine, the mechanisms of the interaction of nanosize particles with biological tissues, as well as the mechanisms of penetration into superficial tissue and biodistribution of particles in the organism are still to be further clarified. Today, these issues are addressed using the methods of qualitative assessment such as transmission electron microscopy, light microscopy (Gobin et al. 2007), confocal microscopy (Alvarez-Roma′n et al. 2004), two-photon luminescence microscopy (Park et al. 2008), and the methods of quantitative assessment of nanoparticle accumulation in biological tissues such as atomic absorption spectrometry (Liu et al. 2008) and neutron activation analysis (James et al. 2007). However, nearly all these methods are invasive or destructive and hard for implementation.

Rapid advance in new optical techniques in different fields of biology and medicine enable fast noninvasive studies of nanoparticle penetration and accumulation in biotissues of living objects. For example, diffusion optical spectroscopy allows one to determine quantitative nanoparticle distribution in tissues (Zaman et al. 2007), and optical coherence tomography (OCT) is a good tool for imaging the process of nanoparticle–biotissue interaction by changes in optical characteristics (Zagaynova et al. 2008a, b; Kirillin et al. 2009, Lee et al. 2003). Enhancement of OCT images contrast by utilizing of plasmon-resonant gold nanoparticles has been demonstrated on tissue phantoms (Troutman et al. 2007; Cang et al. 2005) and in vivo (Gobin et al. 2007; Kah et al. 2009). As optical visualization methods are based on detecting light scattering and absorption by medium components, the unique optical and physicochemical properties of nanoparticles may allow detection of individual structures, layers of biotissue or visualization of a pathological area containing these agents. By changes in the OCT images one can conclude about penetration and accumulation of nanoparticles in biotissue and trace the dynamics of these processes (Kim et al. 2009). It is worthy of notice that OCT studies are carried out in vivo, noninvasively, and in real time.

In our earlier studies, we showed a possibility of using gold nanoparticles and nanosize titanium dioxide as contrasting agents for OCT studies of skin (Zagaynova et al. 2008a, b; Kirillin et al. 2009). It was found that nanoparticles penetrate into different skin structures, thus increasing informativity of OCT images.

The goal of the current study is continuous OCT monitoring of alterations of optical properties of skin associated with penetration of gold and titanium dioxide nanoparticles.

Materials and methods

Optical coherence tomography system

The OCT system produced at the Institute of Applied Physics RAS (Nizhny Novgorod, Russia), equipped with a flexible probe was used in the present study (Gelikonov et al. 2003). The system has the following characteristics: outer diameter of the probe 2.7 mm, probing wavelength 900 nm, probing radiation power 2 mW, spatial resolution in air about 15 μm, and average time for acquiring a 2D image of 200 × 200 pixels 1.5 s.

Nanoparticles

In our study, we used aqueous suspension of gold nanoshells with silica core 130 nm in diameter and shell of 15 nm (Fig. 1a). The structure of these particles was studied in the article by Khlebtsov and Dykman (2010). Size distribution of nanoshells was minimal. The particles were stabilized by PEG molecules covalently attached to the surface. We used PEG of low molecular weight (5,000 Da). The nanoparticles were synthesized by the method described in detail in Khlebtsov et al. (2007), Zagaynova et al. (2008b) with minor modifications in the reagent concentrations. The concentration of particles in the suspension was 22 μg/ml (5 × 1010 nanoshells/ml). The extinction maximum of the gold nanoshells was at the wavelength of 900 nm (Tkachuk et al. 2005), which is optimal for the OCT technique.
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Fig. 1

Electron microphotographs of nanoparticles: a gold nanoshells with core/shell thickness of 130/15 nm, b titanium dioxide nanoparticles

We studied high purity (99.999%) nanosize titanium dioxide particles (PROMCHIM Group, Russia). According to the manufacturer’s information, the TiO2 nanoparticles are obtained by hydrolysis of titanium alcoholates. The X-ray structure analysis has shown that the nanoparticles are in rutile form and have tetragonal lattice. Other phases or free titanium are absent in the powder. The particles have spherical form with average diameter of 54 nm. The size distribution of the particles based on transmission electron microscopy (TEM) study is shown in Fig. 2.
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Fig. 2

The titanium dioxide nanoparticles size distribution based on TEM

Nanoparticles of titanium dioxide of such size have a large backscattering coefficient in the visible region of the spectrum (Lademann et al. 2000). It is supposed that titanium dioxide particles may be used for contrasting OCT images (Kirillin et al. 2009; Popov et al. 2005). In the current study, we employed nanoparticle suspension prepared by dissolving nanosize titanium dioxide powder in distilled water. The obtained suspension had the concentration of 1 mg/ml (nanoparticle concentration was selected experimentally by means of OCT studies so that the intensity of the OCT signal of titanium dioxide nanoparticle suspension should be comparable with the intensity of the signal in the gold nanoparticle solution). Titanium dioxide nanoparticles are highly aggregable; hence, the suspension was dispersed in ultrasonic bath for 30–40 min prior to application. The optimum time needed for full breaking of the aggregates was found by the TEM (Fig. 1b).

Study of the nanoparticles penetration and accumulation in rabbit skin in vivo

The research was done on rabbits (mass of 3.5–4 kg) kept in standard vivarium conditions.

The solutions of nanoparticles were applied by 25 μl drops on healthy skin of rabbit thigh after depilation in vivo. The optical changes caused by the gold nanoshells and TiO2 nanoparticles were continuously monitored by OCT. A special OCT probe holder was fixed by an adhesive tape. The probe was placed in the holder normally to skin surface providing constant uniform pressure. OCT monitoring started 15 min after nanoparticles application. The duration of monitoring was 3–4 h after nanoparticles application. The OCT images were obtained every 30 s. The total number of OCT images obtained during the series of measurements was about 400. The OCT images of the skin before application of solutions were used as a control.

Skin layers on the OCT images were identified by comparison with histological data. The identification of nanoparticles in skin was performed by TEM. Knowing the anatomic skin structure, we identified epidermis and upper dermal layer in the images as a single layer containing hair follicles and a lower layer of dermis. Due to the morphological features of dermal layer structure, a more or less distinct border was formed between the layers. Hair follicles in the OCT images looked like diagonal inclusions with low signal level and robust edges.

Results

OCT images of healthy skin showed no pronounced deep dermis layer; different structural skin formations were almost indistinguishable (Fig. 3).
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Fig. 3

Dynamics of nanoparticles penetration into skin on an example of gold nanoshells: a control, c in 30 min, d in 60 min, e 90 min, and f 150 min. The arrows show contrasting of the border between the upper and lower layers of dermis, and dotted lines show contrasting of inclusions. The averaging region by which A-scan was plotted is shown by the dashed curve in Fig. 3a, c, f. Histology image (b) and OCT image of rabbit skin before nanoparticles application (a): 1 epidermis, 2 superficial layer of the dermis, 3 deep layer of the dermis, 4 hair bulb. Histology slice was stained by hematoxiline–eosine, 200 magnification. The size of the OCT images is 2 by 1 mm. The light areas in OCT images feature high signal level, and dark areas low signal level

Our in vivo OCT study enabled continuous observation of the dynamics of nanoparticle penetration and accumulation in skin.

In the case of gold nanoparticles, the first changes in OCT images were observed 30 min after application of nanoparticles. The OCT signal level slightly increased in the upper dermal layer, resulting in visualization of the border between the upper and lower dermal layers (Fig. 3c) in contrast to the control OCT image (Fig. 3a) where the layers were not differentiated. Gradual increase of the signal in the upper layers of dermis was accompanied by gradual enhancement of the contrast between the dermal layers. Consequently, the contrasting dynamics fully corresponded to the dynamics of changes in the upper dermal layers.

This phenomenon is illustrated in Fig. 4 where a contrast plot for the border between the upper and lower dermal layers induced by gold nanoshells is given. The contrast plot demonstrates the difference in the signal intensities of the upper and lower layers of dermis. Values of the signal intensities were obtained by averaging the OCT signal over a manually selected region attributed with dermis layers in each fifth OCT image.
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Fig. 4

Contrast induced by gold nanoshells plotted versus time in the case of gold naoparticles: border between the upper and lower layers of dermis and border of inclusions

Simultaneously with signal increase in upper dermis, diagonally oriented inclusions with low signal level were visualized in the upper dermis. Comparison of OCT image with histological specimen leads to the conclusion that these inclusions are hair follicles. Hair follicles were not visualized in the control OCT image. At the beginning of monitoring (30 min after the start), the follicles were characterized by a low signal level. During the observation period from 30 to 45 min, contrast at the border of inclusions and dermis (Fig. 3c) increased due to signal enhancement in the upper dermal layer and low diffusion rate in the hair follicles. Therefore, the inclusions were the most contrasted at this time. Signal enhancement inside hair follicles led to contrast decay at the border between the follicle and the surrounding tissue. The signal of the inclusions gradually increased in the interval from 55 to 135 min due to partial penetration of the particles (Fig. 3d,e). The follicles were visualized during all the observation time.

The imaging depth started to increase 60 min after application of gold nanoparticles. This was accompanied by visualization of inclusions of different size and shape with low signal level in the middle and deep layers of dermis (Fig. 3e). The image depth was maximum in the period of observation from 1 to 2 h (Fig. 3d, e), and average from 2.5 to 3 h (Fig. 3f).

Analogous study was performed for titanium dioxide particles. The dynamics of changes in the OCT images was comparable with that for the case of gold nanoshells, but the changes described above were less pronounced for titanium dioxide particles. In particular, the border between the layers of dermis was less contrasted compared to the case of gold nanoshells. This is well seen in the A-scans (in-depth distribution of backscattering signal obtained by OCT at a fixed point) in Fig. 5. The A-scans were obtained by averaging a chosen region in the OCT images at different moments of time after application of nanoparticles. The averaging region for gold nanoparticles is shown in Fig. 3c, f, for TiO2 in Fig. 6b, d and for control in Fig. 3a and Fig. 6a. As was mentioned above, border contrast depends on the signal level in the upper layers of dermis. Apparently, the signal level with gold nanoshells is higher than that with titanium dioxide nanoparticles. The arrow points to the OCT signal drop, which corresponds to contrasting the border between the dermal layers. The OCT signal drop is less in the case of titanium dioxide particles (Fig. 5), hence, the border contrast is less too.
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Fig. 5

A-scans of rabbit skin: a gold nanoparticles; b nanosize TiO2 particles. The dashed line shows averaged A-scans of the control image (prior to application of agents). The arrows show the border between the layers of dermis

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Fig. 6

Accumulation of TiO2 nanoparticles in skin appendages: a control, b in 30 min, c 120 min, d 150 min. The arrows point to inclusions. The averaging region by which A-scan was plotted is shown by the dashed curve in Fig. 6a, b, d

Continuous OCT monitoring revealed accumulation of TiO2 particles in skin appendages (hair follicles according to skin histology) that are visualized in OCT images as inclusions with low signal intensity (Fig. 6b). TiO2 penetration into these structures resulted in signal intensity enhancement at the periphery of the inclusions (Fig. 6c). Gradually, due to TiO2 particles accumulation the OCT signal intensity of the inclusions became like that of the surrounding tissue (Fig. 6d) and the inclusions became less noticeable.

Using the electron microscopy technique, we detected both, gold nanoshells and titanium dioxide nanoparticles in skin structures (Fig. 7). These two types of nanoparticles were detected in all skin layers and structures: in epidermis, in connective tissue, inside cells, and in intercellular substance. Gold nanoshells were encountered in deeper layers of dermis, whereas titanium dioxide particles were primarily observed in epithelial cells and in the middle layers of dermis.
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Fig. 7

Electron microphotographs of rabbit skin: a gold nanoshells in dermis (36, 000 magnification), b titanium dioxide nanoparticles in epithelium (36,000 magnification). Nanoparticles are shown by arrows

Discussion

In the present study, we performed in vivo OCT monitoring of two types of nanoparticles penetrating into different structures of healthy skin with surface application. The proposed technique when the OCT probe is constantly attached to the skin gives information about the process of nanoparticle penetration with high accuracy. The acquiring of the images at fixed position is very important for their correct interpretation. It was demonstrated that there is no significant difference between dynamics of penetration of gold nanoparticles and titanium dioxide nanoparticles, although gold nanoparticles give rise to more pronounced alterations in optical properties such as contrasting of the border between the upper and lower dermal layers and OCT signal level enhancement. Penetrating into biotissue the particles change the intensity of backscattering, which is detected in OCT images.

There are many factors influencing the nanoparticles penetration to healthy skin. Nanoparticles, shape, superficial charge, composition, hydrodynamic diameter, and physicochemical properties of solvent are expected to affect penetration into skin (Elder et al. 2009). In our study, we used water suspensions of spherically shaped uncharged gold nanoshells and titanium dioxide nanoparticles. A majority of recent literature data shows that rigid particles unlike the elastic ones do not penetrate entire stratum corneum but penetrate the hair follicles, and the nanoparticles permeation in skin is strongly size-dependent (Baroli 2010). Particle composition and surface charges appear to be less significant. Based on the studies (Alvarez-Román et al. 2004; Borm et al. 2006; Langer 2004; Moranti et al. 2001; Ryman-Rasmussen et al. 2006; Teichmann et al. 2005; Toll et al. 2004) one can conclude that the passive penetration through the trans-epidermal, trans-follicular routes, or aqueous pores is attributed to particles of size below 36 nm, whereas larger particles penetrate through the trans-follicular route preferably due to anatomical features of skin. In contrast to above-mentioned studies, penetration of 0.5–1 μm beryllium particles through the stratum corneum of human skin being under flexing motion was shown in (Tinkle et al. 2003). Similar to Tinkle et al., we observed penetration of quite large rigid nanoparticles deeply into skin. Although the mechanism by which nanoparticles penetrate the intact skin is unclear both types of nanoparticles used in our study do cross the stratum corneum which was confirmed by TEM. The nanoparticles were detected in intercellular space and within epithelial cells, which indicate their capability to penetrate through cellular membrane, as well as in collagen fibers of dermis. This result may be explained by thickness of the stratum corneum which essentially varies in different objects or body regions. A human abdominal or porcine skin is commonly investigated. It is worth noting that the stratum corneum thickness of these skins is 10–20 μm which is at least two times larger than that of the rabbit skin in our estimation.

The methods currently exploited in nanoparticles researches lack the capability to investigate nanoparticles penetration in tissues of living organism in dynamics. While OCT provide such marvelous possibility as it has been demonstrated by Kim et al. (Kim et al. 2009) in dysplastic area of hamster cheek.

Alterations caused by nanoparticles in OCT images allow studying dynamics of their penetration in skin. We suppose that 30 min after topical application on skin surface nanoparticles start to diffuse from epidermis to the upper layers of dermis. This is accompanied by the following changes in the OCT images: signal enhancement in dermis (Fig. 3c) and contrasted border and low signal level in hair follicles as the diffusion rate in dermis is higher than through a follicle wall. Based on the contrast plot of the inclusion border (Fig. 4), we can conjecture that 20 more minutes after nanoparticles penetration into the upper dermal layers they will start to diffuse into hair follicles. Contrasting of the border between the dermal layers may be attributed to the difference in diffusion rates. Morphologically, the lower layer of dermis has a more dense structure and nanoparticle diffusion rate slows down at the border of these layers. OCT-detected nanoparticles penetration into skin appendages accompanied by signal enhancement within appendages and by simultaneous contrast decay of their border.

Analysis of the results obtained demonstrates that contrasting of the border between dermal layers and contrast decay of the hair follicle border are important indicators of nanoparticle penetration. According to our data, this occurs 60–150 min after nanoparticle application. It is the most effective period for visualizing the studied nanoparticles in skin structures.

Successful continuous study of nanoparticle accumulation in skin with surface application is promising for using this technique to monitor passive accumulation of nanoparticles in tumorous tissue localized immediately under skin. Knowing optical changes associated with the presence of nanoparticles in biotissue one can assess the degree of nanoparticle accumulation in tumor in real time. This will allow one to determine with high accuracy the time of nanoparticle accumulation in tumor. OCT monitoring may become an indispensable tool for controlling local laser hyperthermia with the use of plasmon-resonant nanoparticles.

Conclusion

The present study is concerned with in vivo study of accumulation of different types of nanoparticles in healthy tissue (skin) by the method of continuous OCT monitoring. The OCT technique revealed that nanoparticles penetrate into skin, accumulate in different layers and structures, thus changing optical characteristics of biotissue. Continuous OCT monitoring of these changes enables maximum accurate tracing of dynamics of the occurring processes, including nanoparticle penetration into skin appendages. Nanoparticle accumulation in skin was confirmed by the electron microscopy. Thus, results of our studies demonstrate that OCT is an objective technique for controlling nanoparticle accumulation in biotissues and may be a useful tool for assessing maximum time of nanoparticle accumulation in tumor for further efficient therapeutic action.

Acknowledgments

This study was supported in part by the Science and Innovations Federal Russian Agency (projects ## 02.512.11.2244, MD-3018.2009.7), RFBR grants (09-02-97072, 09-02-12215, 09-02-00539, 09-02-97040, 10-02-00744). The authors are grateful to L.B. Snopova (Nizhny Novgorod State Medical Academy) for help in performing the microscopic analysis. Also, the authors thank the Institute of Biochemistry and Physiology of Plants for providing gold–silica nanoshells and the group of companies PROMCHIM (Perm’, Russia) for providing titanium dioxide nanoparticles.

Copyright information

© Springer Science+Business Media B.V. 2010