1.

Introduction

Lung cancer is the leading cause of cancer deaths in men and the second leading cause of cancer deaths in women, with approximately 1.6 million new cases of lung cancer diagnosed and 1.4 million deaths each year throughout the world.^1–3 Nonsmall cell lung cancer, which includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and bronchioloalveolar carcinoma, accounts for nearly 85% of all cases of lung cancer.^2,3 Cigarette smoking is the most common etiological factor, accounting for about 80% of global lung cancer deaths in men and 50% of the deaths in women.^4,5 In addition, environmental exposures other than smoking may contribute to the incidence of lung cancer.⁶

With the development of nanotechnology, there has been a tremendous growth in the application of nanoparticles (NPs) for drug delivery systems, antibacterial materials, cosmetics, sunscreens, and electronics.^7,8 ${\mathrm{TiO}}_2$ NPs, with the property of absorption and reflection of ultraviolet lights, are used extensively in a variety of cosmetics. ${\mathrm{TiO}}_2$ fine particles, traditionally considered as poorly soluble, low toxicity particles, have been used as a “negative control” in many in vitro and in vivo particle toxicological studies.⁹ However, this view was challenged after Lee et al.¹⁰ revealed that rats suffered from lung tumors after 2 years of exposure to high concentrations of fine ${\mathrm{TiO}}_2$ particles. In recent years, ${\mathrm{TiO}}_2$ NPs have been widely used in industrial and consumer products due to their stronger catalytic activity when compared with ${\mathrm{TiO}}_2$ fine particles, which are due to their smaller sizes and larger surface area per unit mass. Nevertheless, studies have revealed that ${\mathrm{TiO}}_2$ NPs are more toxic than ${\mathrm{TiO}}_2$ fine particles.^9,11,12 Oberdorster et al.¹³ have reported that ${\mathrm{TiO}}_2$ NPs caused a greater pulmonary inflammatory response than ${\mathrm{TiO}}_2$ fine particles at the same mass burden, with greater amounts of ${\mathrm{TiO}}_2$ NPs entering the alveolar interstitium in the lungs. Li et al.¹⁴ have shown that instilled ${\mathrm{TiO}}_2$ NPs could induce lung damage and change the permeability of the alveolar-capillary barrier. Wide application of ${\mathrm{TiO}}_2$ NPs confers substantial potential for human exposure and environmental release, which inevitably allows for a potential health risk to humans, livestock, and the ecosystem, especially the possible induction of lung cancer.^13–15 The potential adverse effects of ${\mathrm{TiO}}_2$ NPs on lung tissue and their challenge to human health have raised particular concerns, therefore, it is very necessary to study the effects of ${\mathrm{TiO}}_2$ NPs penetrating and accumulating in the human body, especially in human lung tissue.

Although there have been numerous studies of ${\mathrm{TiO}}_2$ NPs in biomedicine, most of them have focused on the study of toxicological information of ${\mathrm{TiO}}_2$ NPs within different organs^16,17 while some studies focused on the research of ${\mathrm{TiO}}_2$ NPs accumulation in human or animal skin.^18,19 Our group has reported the effects of NPs accumulation in ex vivo human colon tissue on tissue optical properties.²⁰ However, there are few studies that investigate the effects of ${\mathrm{TiO}}_2$ NPs penetrating and accumulating in human normal lung (NL) and lung squamous cell carcinoma (LSCC) tissue on tissue optical properties. Herein, the issues are addressed using the methods of optical coherence tomography (OCT) imaging and diffuse reflectance (DR) spectra. OCT is a noninvasive technique for imaging biotissues at a depth of up to 2 mm with axial spatial resolution down to units of micrometers based on low-coherent interferometry in the near-infrared range of wavelengths (0.75 to $1.3\mu \mathrm{m}$). In recent years, OCT has proved to be an efficient tool for the imaging of the superficial tissues of skin and mucous membranes.^21,22 DR spectra measurement is also a noninvasive method widely used in monitoring analyses concentrations in bulk tissue and investigating tissue optical parameters.^23–27 It is one of the simplest and most cost effective methods for understanding biological tissue characteristics. This technique involves detection and analysis of a portion of the incident light that undergoes multiple elastic scattering caused by inhomogeneities in the refractive index of the tissue.

With the development of medical laser techniques, lasers have been shown to have great advantages in enhancing transdermal drug delivery of not only small molecules but also of large and hydrophilic molecules. Lasers are different from ultrasound and are an established technology for drug delivery.²⁸ The displacement caused by ultrasound oscillates equally between positive and negative pressures, whereas the laser is a unipolar compression wave (positive pressure).²⁹ Reduction of light scattering has the potential to improve many current and novel diagnostic and therapeutic applications of lasers in medicine. A plethora of current and novel diagnostically and therapeutically useful applications of lasers in medicine will benefit from precise control over tissue optical properties and our ability to temporarily influence them. Studies have reported that after QS-Nd:YAG laser irradiation, both the concentration and penetration depth of methylene blue into biofilms were enhanced,³⁰ and the penetration of 5-fluorouracil into irradiated rabbit ear skin was increased.³¹ In addition, Genina et al.¹⁸ have reported that fractional laser microablation with Er:YAG laser allowed for efficient administering of NPs into the skin. Stumpp et al.³² have revealed that using an inexpensive 980 nm diode laser can significantly enhance the delivery of topically applied glycerol for optical skin clearing. However, there are few studies to investigate laser irradiation for improving NPs penetration into biological tissues. As a result, we try to use 650-nm diode laser irradiation to enhance ${\mathrm{TiO}}_2$ NPs penetration into lung tissue and then investigate the induced changes of tissue optical properties.

In this study, in order to assess the effects of ${\mathrm{TiO}}_2$ NPs on optical properties of human lung tissue for further understanding the interactions between ${\mathrm{TiO}}_2$ NPs and lung tissue, we monitored the continuous process of two different sized ${\mathrm{TiO}}_2$ NPs penetrating and accumulating in normal and cancerous lung tissues with OCT and DR spectra. In addition, 650-nm diode lasers were used to irradiate the lung tissues to enhance the amounts of ${\mathrm{TiO}}_2$ NPs penetrating in the tissues and then evaluate the induced changes of tissue optical properties.

2.

Materials and Methods

2.1.

${\mathsf{TiO}}_{\mathsf{2}}$ Nanoparticles Preparation and Transmission Electron Microscopy Measurements

In this study, 60 and 100 nm commercially available high purity (99.8%) rutile ${\mathrm{TiO}}_2$ NPs (Aladdin Chemistry Co. Ltd, Shanghai, China) were used, which are the primary particle sizes of food grade ${\mathrm{TiO}}_2$ and are also the main ${\mathrm{TiO}}_2$ NPs sizes existing in personal care products.³³ An ${\mathrm{TiO}}_2$ NPs suspension was prepared by dissolving ${\mathrm{TiO}}_2$ powder in distilled water to a concentration of $1\mathrm{mg}/\mathrm{ml}$. ${\mathrm{TiO}}_2$ NPs are highly aggregable; hence, the suspension was dispersed in an ultrasonic bath (the power of 35 W and the frequency of 43 to 45 kHz) for 30 to 40 min for thorough mixing of the content both in the process of preparation and immediately prior to use. High-resolution images of two different sized ${\mathrm{TiO}}_2$ NPs were obtained via transmission electron microscopy (TEM). Samples were dropped to 400-mesh carbon-coated copper grids and dried for measurement. Without further preparation, the samples were evaluated in TEM (JEM-2100HR, JEOL, Japan). The TEM images are shown in Fig. 1.

Fig. 1

Transmission electron microscopy (TEM) images of ${\mathrm{TiO}}_2$ NPs with two different sizes: (a) 60-nm ${\mathrm{TiO}}_2$ NPs; and (b) 100-nm ${\mathrm{TiO}}_2$ NPs.

2.5.

Data Processing

In order to characterize the changes in optical properties of lung tissues during the penetration and accumulation of ${\mathrm{TiO}}_2$ NPs, the attenuation coefficients of each sample were calculated from the 2-D OCT image. The total OCT attenuation coefficient of the tissue is the sum of the absorption coefficient ${\mu }_{\mathrm{a}}$ and scattering coefficient ${\mu }_{\mathrm{s}}:{\mu }_{\mathrm{t}}={\mu }_{\mathrm{a}}+{\mu }_{\mathrm{s}}$, and can be determined from the slope of the A-scan of the OCT signal measured for the region of interest.^34–37 In the superficial layers of many tissues, a single-scattering model can be adequately used.^36,37 In accordance with the single-scattering model, the measured signal in the OCT system is defined as^34–37

Eq. (1)

$${[\langle i^2(z)\rangle ]}^{\frac{1}{2}}\approx {(\langle i^2{\rangle }_0)}^{\frac{1}{2}}[{\exp (-2{\mu }_{t}z)]}^{\frac{1}{2}},$$

where the $\langle i^2(z)\rangle$ is the photodetector heterodyne signal current received by the OCT system from the probing depth $z$ and ${\langle i^2\rangle }_0$ is the mean square heterodyne signal. In OCT, the intensity of the optical reflection, $R(z)\propto {[\langle i^2(z)\rangle ]}^{\frac{1}{2}}$, via the tissue probing depth $z$ is measured. The reflectance depends on the optical properties of tissue, i.e., the total attenuation coefficient ${\mu }_{\mathrm{t}}$. Thus, in the single-scattering model, the reflected power can be approximately proportional to $-{\mu }_{\mathrm{t}}z$, i.e., ³⁴

Eq. (2)

$$R(z)=I_{0}a(z)\times \exp (-{\mu }_{\mathrm{t}}z),$$

where $I_0$ is the incident light intensity launched into the tissue sample, and $a(z)$ is the reflectivity of the tissue at depth $z$. $a(z)$ is linked to the local refractive index and backscattering property of the tissue.³⁴ However, the reflectivity $a(z)$ is considered as weakly dependent on depth for a homogeneous tissue layer. Therefore, ${\mu }_{\mathrm{t}}$ can be obtained theoretically from the reflectance measurements at two different depths, $z_1$ and $z_2$:^34–37

Eq. (3)

$${\mu }_{\mathrm{t}}=\frac{1}{\mathrm{\Delta }z}\ln \left[\frac{R(z_1)}{R(z_2)}\right],$$

where $\mathrm{\Delta }z=|z_1-z_2|$. As noise is inevitable in the measurement, a final result should be obtained by the use of a best-fit exponential curve technique in order to improve the accuracy of the determined value of ${\mu }_{\mathrm{t}}$. An averaged optical intensity profile that represents the reflected light intensity distribution in depth is obtained by averaging the 2-D image laterally over 1 mm, which is wide enough for speckle noise suppression.

Figure 2 shows the example for calculating the attenuation coefficients of lung tissues. For calculation, we selected the region [white rectangle in Fig. 2(a)] from which OCT depth intensity profiles with a corresponding exponential best-fit curve were obtained. The region of interest [red rectangle in Fig. 2(a)] was selected from a depth of $130\mu \mathrm{m}$ to a depth of $287\mu \mathrm{m}$, where the OCT signal distribution is relatively smooth. The fitted curve [red curve in Fig. 2(b)] in the region of interest allows evaluating the attenuation coefficient.

Fig. 2

Typical B-scan of lung tissue with (a) marked selection region and region of interest and (b) plot of the averaged A-scan and the exponential best-fit curve for calculating attenuation coefficient.

In the spectroscopic measurements, the changes of reflectance induced by the NPs solution were quantitatively evaluated by calculating the increase in DR after 60-, 120-, 180-, and 210-min treatment. The increase in DR by the solutions at the time intervals of treatment was calculated as Eq. (4):^38,39

Eq. (4)

$$\mathrm{\Delta }R=\frac{R_{x\min }-R_{\text{control}}}{R_{\text{control}}}\times 100\%,$$

where $R_{\text{control}}$ and $R_{x\min }$ are the measured diffuse reflectances at 543 and 578 nm of each group of samples before and after $x\min$ treatment in different ways. The reason for selecting 543 and 578 nm to assess reflectance is that these wavelengths represent visible wavelength with skin oxyhemoglobin absorption.^40–42

3.

Results and Discussion

Figures 3(a)–3(d) illustrate the dynamic changes of DR as a function of time elapsed after the NL tissue samples were topically treated with 60-nm ${\mathrm{TiO}}_2$ NPs alone, 60-nm ${\mathrm{TiO}}_2$ NPs in combination with laser, 100-nm ${\mathrm{TiO}}_2$ NPs alone, and 100-nm ${\mathrm{TiO}}_2$ NPs in combination with laser, respectively. The lowest black lines in Figs. 3(a) and 3(c) represent spectra that were measured from the intact lung tissues without any treatment, whereas the lines in Figs. 3(b) and 3(d) represent spectra that were measured immediately from laser-treated tissues. There is a distinguishable difference between the spectra with and without laser irradiation. With the laser irradiation, the DR spectra of laser-treated tissues were relatively lower than the spectra of intact tissues. The increase in the intensity of radiation reflected from the sample was observed, which is related to a substantial increase of the scattering coefficient of the tissue, promoted by the NPs located in the lung tissues. In the spectroscopic measurements, the absorption bands of blood hemoglobin at the wavelengths 416 (Soret band), 543, and 578 nm are very pronounced. Figure 4 shows the enhancement of DR for NL tissues at the wavelengths 543 and 578 nm per time of penetration of 60- and 100-nm ${\mathrm{TiO}}_2$ NPs with and without laser irradiation. As with ${\mathrm{TiO}}_2$ NPs penetration into the tissues, the DR of the samples increases; however, after the tissues are irradiated by laser, the enhancement of DR is markedly larger than the tissues treated with the same type of NPs alone at the same time. Additionally, the enhancement of DR is higher for 100-nm ${\mathrm{TiO}}_2$ NPs than for 60-nm ${\mathrm{TiO}}_2$ NPs under the same conditions. This may be caused by the fact that 100-nm ${\mathrm{TiO}}_2$ NPs have a larger backscattering compared with 60-nm ${\mathrm{TiO}}_2$ NPs. Figs. 5(a)–5(d) illustrate the dynamic changes of DR per time of LSCC tissues treated with 60-nm ${\mathrm{TiO}}_2$ NPs alone, 60-nm ${\mathrm{TiO}}_2$ NPs in combination with laser, 100-nm ${\mathrm{TiO}}_2$ NPs alone, and 100-nm ${\mathrm{TiO}}_2$ NPs in combination with laser, respectively. By comparing Figs. 3 and 5, it is obvious that the spectra of LSCC tissues are very different from the spectra of NL tissues in the amplitudes. The amplitudes for the spectra of NL tissue are obviously higher as compared with the spectra of LSCC tissue under the same conditions. The wavelengths of the main peaks of both NL and LSCC tissues and the absorption bands of blood hemoglobin at the wavelengths 416, 543, and 578 nm are not changed.^42,43 Figure 6 shows the enhancement of DR for LSCC tissue at the wavelengths 543 and 578 nm per time of penetration of the 60- and 100-nm ${\mathrm{TiO}}_2$ NPs with and without laser irradiation. The results show that the enhancement of DR for LSCC tissue with laser irradiation is significantly larger than for the tissue without laser irradiation when treated with the same type of NPs, and the enhancement of DR is bigger for 100-nm ${\mathrm{TiO}}_2$ NPs than for 60-nm ${\mathrm{TiO}}_2$ NPs under the same conditions. The results are similar to the previous results for NL tissues. Moreover, the enhancement of DR for LSCC tissue is markedly larger than that for NL tissue when under the same treatment. The difference in optical properties between NL and LSCC tissue may be induced by the morphological and structural differences between the two types of tissues, such as larger nuclei, the higher nuclear-to-cytoplasmic ratio in tumor cells, and the higher regional tumor cell density of the tumor tissues.⁴³ As the result, LSCC tissue may be easier to penetrate compared with NL tissue, thus, more NPs would spread into the LSCC tissue and induce a higher enhancement of DR.

Fig. 3

Measured diffuse reflectance spectra over 400 to 1100 nm for in vitro human normal lung (NL) tissue before and after different applications at 60, 120, 180, and 210 min: (a) NL samples with 60-nm ${\mathrm{TiO}}_2$; (b) NL samples with 60-nm ${\mathrm{TiO}}_2/\text{laser}$; (c) NL samples with 100-nm ${\mathrm{TiO}}_2$; and (d) NL samples with 100-nm ${\mathrm{TiO}}_2/\text{laser}$.

Fig. 4

Enhancement of diffuse reflectance at wavelengths (a) 543 nm and (b) 578 nm for human NL tissue after different applications of ${\mathrm{TiO}}_2$ NPs solution on the tissues at 60, 120, 180, and 210 min, respectively.

Fig. 5

Measured diffuse reflectance spectra over 400 to 1100 nm for in vitro human lung squamous cell carcinoma (LSCC) tissue before and after different applications at 60, 120, 180, and 210 min: (a) LSCC samples with 60-nm ${\mathrm{TiO}}_2$; (b) LSCC samples with 60-nm ${\mathrm{TiO}}_2/\text{laser}$; (c) LSCC samples with 100-nm ${\mathrm{TiO}}_2$; and (d) LSCC samples with 100-nm ${\mathrm{TiO}}_2/\text{laser}$.

Fig. 6

Enhancement of diffuse reflectance at wavelengths (a) 543 nm and (b) 578 nm for human LSCC tissue after different applications of ${\mathrm{TiO}}_2$ NPs solution on the tissues at 60, 120, 180, and 210 min, respectively.

The attenuation coefficients of each group at different times were calculated from the data of the best exponential fit curve corresponding to the averaged intensity profiles. All the attenuation coefficients of NL and LSCC tissues are shown in Table 2. Figs. 7 and 8 present the changes in the attenuation coefficients of NL and LSCC treated with 60-nm ${\mathrm{TiO}}_2$ NPs, 60-nm ${\mathrm{TiO}}_2$ NPs/laser, 100-nm ${\mathrm{TiO}}_2$ NPs, and 100-nm ${\mathrm{TiO}}_2$ NPs/laser, respectively. In Figs. 7(a)–7(d), the attenuation coefficients of NL tissue are about $4.66\pm 0.21{\mathrm{mm}}^{-1}$ for 60-nm ${\mathrm{TiO}}_2$ NPs treatment and $4.83\pm 0.24{\mathrm{mm}}^{-1}$ for 100-nm ${\mathrm{TiO}}_2$ NPs treatment, then they changed to be $3.29\pm 0.12{\mathrm{mm}}^{-1}$ for 60-nm ${\mathrm{TiO}}_2$ NPs treatment at about 163 min and $3.42\pm 0.13{\mathrm{mm}}^{-1}$ for 100-nm ${\mathrm{TiO}}_2$ NPs treatment at about 175 min when the penetration process reached the stable state. As with laser pretreatment, the starting values of the attenuation coefficients of NL tissue are $4.46\pm 0.11{\mathrm{mm}}^{-1}$ for 60-nm ${\mathrm{TiO}}_2$ NPs treatment and $4.73\pm 0.11{\mathrm{mm}}^{-1}$ for 100-nm ${\mathrm{TiO}}_2$ NPs treatment, then it took about 151 min for the 60-nm ${\mathrm{TiO}}_2$ NPs treatment and 156 min for the 100-nm ${\mathrm{TiO}}_2$ NPs treatment to reach the stable state, and the values changed to $2.28\pm 0.08$ and $2.67\pm 0.07{\mathrm{mm}}^{-1}$, respectively. Similarly, as to LSCC tissue (Fig. 8), the attenuation coefficients were calculated to be $10.87\pm 0.58$, $10.28\pm 0.43$, $11.85\pm 0.61$, and $11.48\pm 0.59{\mathrm{mm}}^{-1}$ at 0 min for treatment of 60-nm ${\mathrm{TiO}}_2$ NPs, 60-nm ${\mathrm{TiO}}_2$ NPs/laser, 100-nm ${\mathrm{TiO}}_2$ NPs, and 100-nm ${\mathrm{TiO}}_2$ NPs/laser, respectively. Then, as with NPs penetrating into LSCC tissues, the penetration of ${\mathrm{TiO}}_2$ NPs at LSCC tissues took approximately 124, 108, 132, and 115 min for the treatment of 60-nm ${\mathrm{TiO}}_2$ NPs, 60-nm ${\mathrm{TiO}}_2$ NPs/laser, 100-nm ${\mathrm{TiO}}_2$ NPs, and 100-nm ${\mathrm{TiO}}_2$ NPs/laser, respectively, to reach the stable state. Their attenuation coefficients changed to $5.38\pm 0.31$, $4.55\pm 0.27$, $5.56\pm 0.24$, and $5.42\pm 0.33{\mathrm{mm}}^{-1}$, respectively.

Table 2

The attenuation coefficients of NL and LSCC tissues and the time required to reach the stable state.

Fig. 7

Average attenuation coefficients of NL tissue after different applications of two-sized ${\mathrm{TiO}}_2$ NPs solution: (a) NL samples with 60-nm ${\mathrm{TiO}}_2$; (b) NL samples with 60-nm ${\mathrm{TiO}}_2/\text{laser}$; (c) NL samples with 100-nm ${\mathrm{TiO}}_2$; and (d) NL samples with 100-nm ${\mathrm{TiO}}_2/\text{laser}$.

Fig. 8

Average attenuation coefficients of LSCC tissue after different applications of two-sized ${\mathrm{TiO}}_2$ NPs solution: (a) LSCC samples with 60-nm ${\mathrm{TiO}}_2$; (b) LSCC samples with 60-nm ${\mathrm{TiO}}_2/\text{laser}$; (c) LSCC samples with 100-nm ${\mathrm{TiO}}_2$; and (d) LSCC samples with 100-nm ${\mathrm{TiO}}_2/\text{laser}$.

As is seen from the results of all the data in Figs. 7 and 8, using a laser pretreatment, for both of NL and LSCC tissues the penetration of ${\mathrm{TiO}}_2$ NPs into the tissues was dramatically increased and accelerated as compared with the same kind of NPs treatment alone. Moreover, with the same treatment, the penetration of ${\mathrm{TiO}}_2$ NPs in LSCC tissue is much faster than that in NL tissue; whereas for the same type of tissue, the penetration of 100-nm ${\mathrm{TiO}}_2$ NPs in the tissue is slightly slower than 60-nm ${\mathrm{TiO}}_2$ NPs. This is very consistent with the former results of DR spectra. The morphological and structural differences of lung tissues significantly affect the penetration of ${\mathrm{TiO}}_2$ NPs. Different lung pathologies can affect the extracellular matrix.⁴⁴ Lung’s physiological behavior reflects both the mechanical properties of the individual tissue components and their complex structural organization and thus induces the differences of permeability of lung tissue. This is well illustrated in our previous study, in which Wei et al.⁴⁵ assessed the effects of ultrasound-mediated glucose on the permeability of normal, benign, and cancerous human lung tissues with OCT. During the laser irradiation, the temperature rise of the tissue surfaces was almost up to 8°C. Some studies have revealed that the transient photothermal effect could disrupt the stratum corneum and enhance transdermal drug delivery.^30,31,46,47 However, few studies revealed the physical and physiological mechanisms of diode laser irradiation for improving NPs penetration into lung tissues. As for this study, the temperature rise of the sample caused by laser irradiation may be the main cause of the increase in ${\mathrm{TiO}}_2$ NPs penetration. But it is still worth further investigation into the mechanisms of diode laser irradiation for improving NPs penetration into lung tissues.

4.

Conclusions

This study considered the effects of two-sized ${\mathrm{TiO}}_2$ NPs on the optical properties of human lung tissues, including NL and LSCC tissues. Our results show the penetration process of ${\mathrm{TiO}}_2$ NPs with both sizes (60 and 100 nm) in LSCC tissue is faster than that in NL tissue and that 60-nm ${\mathrm{TiO}}_2$ NPs penetrate and accumulate faster than 100-nm ${\mathrm{TiO}}_2$ NPs in the same type of lung tissue. In addition, the use of an inexpensive diode laser can significantly enhance the penetration of topically applied ${\mathrm{TiO}}_2$ NPs in the tissue. Moreover, results of our studies show that OCT and DR provide an ideal tool to perform qualitative and quantitative analyses of the ${\mathrm{TiO}}_2$ NPs penetration and accumulation in lung tissue to reveal the localization of NPs within the tissues and to investigate the changes in optical properties of biotissue induced by NPs. However, the findings in this study and the effects of laser irradiation should be further tested in the future.