701A. Prokop (ed.), Intracellular Delivery: Fundamentals and Applications, Fundamental Biomedical Technologies 5, DOI 10.1007/978-94-007-1248-5_22, © Springer Science+Business Media B.V. 2011

Abstract In this paper, the principle of terahertz molecular imaging (TMI) used for the visualization of nanoparticle delivery is presented. In addition, a brief review of terahertz (THz) technology in terms of the generation, detection, and characteristics of THz waves is given. The TMI technique entails the sensitive measurement of the change in the THz optical coefficients of water induced by the surface plasmon resonance of nanoparticles. It is shown that the measured THz response is linearly proportional to the concentration of the nanoparticles. Nanoparticle delivery to a cancerous tumor and the organs in a mouse is imaged by employing the TMI technique. This technique enables target-specific sensing of cancers as well as molecular diagnosis of drug delivery.

Keywords Cancer diagnosis • Drug delivery • Optical coefficients • Molecular imaging • Terahertz waves

1 Introduction

A number of nanoparticle probes and drugs have recently been developed for use in the areas of therapeutics and diagnostics (Lee et al. 2007, 2008; McCarthy and Weissleder 2008). It is imperative that nanoparticles be delivered to the targeted cells or organs for obtaining maximum diagnostic and therapeutic results; further, accurate monitoring of nanoparticle delivery to the target cells is important in

J.-H. Son (*) Department of Physics, University of Seoul, Seoul 130-743, Korea e-mail: joohiuk@uos.ac.kr

S.J. Oh, J.-S. Suh, and Y.-M. Huh Department of Radiology, College of Medicine, Yonsei University, Seoul 120-752, Korea

J. Choi and S. Haam Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-752, Korea

Imaging of Nanoparticle Delivery Using Terahertz Waves

Joo-Hiuk Son, Seung Jae Oh, Jihye Choi, Jin-Suck Suh, Yong-Min Huh, and Seungjoo Haam

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theranostics. A number of molecular imaging techniques have been developed to visualize such biological processes at the cellular or molecular level. Among these techniques are positron emission spectroscopy (PET) (Massoud and Gambhir 2003), magnetic resonance imaging (MRI) (Weissleder et al. 2000), and optical imaging using bioluminescence or fluorescence (Moore et al. 1948, Weissleder and Ntziachristos 2003), all of which have their pros and cons. For example, PET is a highly sensitive technique but has low spatial resolution and requires the use of radioactive isotopes. MRI offers better spatial resolution than does PET but has lower sensitivity despite the fact that iron oxide or gadolinium derivatives, which are expected to increase the sensitivity of the technique, are used as contrast agents. Optical imaging techniques are relatively simple and inexpensive, but the pene-tration depth and spatial resolution that can be achieved are very low because of the Rayleigh scattering of optical waves by cells.

In this review, a novel molecular imaging technique based on the use of terahertz (THz) electromagnetic waves and nanoparticle probes (Oh et al. 2009) is introduced. The basics of THz technology, including the generation and detection of THz waves, are explained in Sect. 2. In addition, the THz characteristics of water are reviewed because they form the basis of TMI. In Sect. 3, the principle of TMI is presented. An example of nanoparticle delivery imaging of a cancerous tumor and the organs in a mouse is shown in Sect. 4. A brief summary is presented in Sect. 5.

2 Review of Terahertz Technology

THz waves, ranging from 0.1 to 10 THz, occupy the electromagnetic spectrum between microwave and infrared bands. This region is scientifically rich because of the rotational and vibrational energies of the materials that lie in that frequency range (see Fig. 1), but it has gained importance only in recent times because of the difficulty involved in the generation and detection of THz signals. In this section, THz technology is reviewed with respect to generation and detection techniques. In addition, the THz characteristics of water are discussed because they form the basis of the THz molecular imaging technique.

2.1 Generation and Detection of Terahertz Waves

The generation of electromagnetic waves in the THz frequency range is difficult because transport-type devices such as transistors cannot move electrons fast enough to generate THz signals, and transit-type devices such as lasers do not have natural gain media with a small energy separation of a only a few meV. However, two decades ago when femtosecond lasers were developed, researchers worked to combine optical pulses with semiconductors and thus generate THz electromagnetic pulses. When 800-nm laser pulses from a mode-locked Ti:sapphire laser (Spence

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et al. 1991, Son et al. 1992) illuminate a semiconductor, such as semi-insulating GaAs (SI-GaAs) having a band gap of 1.45 eV, electron–hole pairs are generated and drift to the electrodes fabricated on the semiconductor wafer (see Fig. 2). The drifted charges are collected by the opposite electrodes biased with a dc voltage, which, in turn, generate a photocurrent surge in the coplanar waveguide. The increase in the photocurrent is proportional to the integration of the optical pulse, and the decrease is dependent on the semiconductor carrier lifetime. The time-varying photocurrent gives rise to the free-space radiation of electromagnetic waves with an equivalent Lienard-Wiechert potential (Schwartz 1972). This generation scheme is known as the photoconductive switching technique. Using this technique, Fattinger et al. achieved a frequency spectrum of over 1 THz by Fourier trans-formation of the subpicosecond electromagnetic pulse in the free space. They referred to this radiation as the THz beams (Fattinger and Grischkowsky 1988). Other generation methods using femtosecond laser interaction include techniques using a built-in surface field in semiconductors, the photo-Dember effect, and optical rectification. These techniques do not require the use of external voltage bias (Zhang and Xu 2010).

Variations in THz pulses can occur in less than a picosecond, making them impossible to measure with conventional electronic equipment such as oscillo-scopes or network analyzers. The first technique to measure such high-frequency

Fig. 1 Characteristic energies in electromagnetic spectrum

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signals was a photoconductive sampling technique that is the reverse of photocon-ductive switching. Free-space THz waves are captured by a waveguide antenna and the induced electric fields are switched on a semiconductor such as low-temperature-grown GaAs (LT-GaAs) by a femtosecond laser. The switching occurs in less than a picosecond to give a dc voltage to a lock-in amplifier because the carrier lifetime of LT-GaAs is approximately 0.5 ps (Gupta et al. 1991). The full THz waveform can be reconstructed by varying the optical delay between the generation and detection femtosecond optical pulses. The detection can also be accomplished with the electro-optic (EO) sampling technique using Pockels’ effect (Valdmanis and Mourou 1986). Some examples of EO crystals that can be used for freespace detection are ZnTe, GaP, GaSe, etc. A bandwidth of over 30 THz was realized using this technique (Wu and Zhang 1997).

One example of a THz spectroscopic system is shown in Fig. 3. This system generates THz pulses from an InAs wafer by the photo-Dember effect and detects them using a photoconductive switch of LT-GaAs. It operates in a reflection mode to measure signals from an absorptive sample such as water or certain biological materials. This system can obtain spectroscopic information from 0.1 to several THz from a specific measurement point, and can also construct a spectroscopic image of a sample by raster scanning. This experimental setup was utilized to produce the measurement results shown in Sects. 3 and 4.

Because THz technology has tremendous potential for applications in scientific study, security, and medicine, many generators and detectors have been developed. Among the more notable generators are quantum cascade lasers (Kohler et al. 2002), THz parametric generators and oscillators (Kawase et al. 1996, 2001), and THz photomixers using two diode lasers (McIntosh et al. 1995). A detector fabricated with a microbolometer array can take live images of THz signals (Behnken et al. 2008). See the work by Son (2009) for a detailed review of generators and detectors.

Fig. 2 Schematic of THz pulse generation. (a) Generation of electron–hole pairs in photoconduc-tive switching antenna. (b) Waveforms of excitation laser pulse, induced photocurrent, and THz pulse in free space

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2.2 THz Characteristics of Water

Water is the most abundant material in living cells and organs, and its characteristics play an important role in diagnosis based on the use of THz waves. THz waves are sensitive to the dynamics of water molecules because their rotational and vibrational energies and hydrogen bond stretching energy lie in the THz region. Another impor-tant feature is that the low energy of the THz waves prevents ionization of the water molecules. Therefore, THz waves have been utilized to study the microscopic and macroscopic characteristics of water. The microscopic dynamics between water and a solute resulted in a larger absorption of THz waves due to the coherent oscil-lation between the hydration water and the solute and caused the hydrogen bond rearrangement process to slow down (Heugen et al. 2006). For THz molecular imaging for nanoparticle delivery, the macroscopic characteristics are more important because the TMI technique is based on a principle that relies on the temperature-dependent property of bulk water, as will be explained in the next section. As can be seen in Fig. 4, the power absorption and the refractive index of liquid water are highly dependent on temperature because it affects bonding strength. In addition,

Fig. 3 Schematic of reflection-mode THz imaging setup with infrared (IR) laser for the induction of surface plasma polaritons (From Oh et al. (2009). Copyright © 2009 by Optical Society of America. Reprinted by permission of Optical Society of America)

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the molecule’s motions are active in the THz frequency range (Son 2009). The power absorption becomes greater as the temperature increases, and reaches a local maximum around 6 THz due to the hydrogen-bond resonance (Zelsmann 1995). THz measurement with a Debye model fit has shown that bulk water has two relaxation dynamics with fast and slow times. The slow time of approximately 8 ps is related to the loosening of hydrogen bonds, and the fast time of 170 fs arises from the realignment of molecules under an external electric field (Oh et al. 2007).

3 Principle of Terahertz Molecular Imaging

THz molecular imaging (TMI) is achieved by monitoring the changes in water’s THz optical property such as absorption and index caused by temperature variation. Cells and organs contain an abundant amount of water, and the water temperature can be controlled by nanoparticle probes (nanoprobes) delivered to the cells and organs. The gold nanorods (GNRs) shown by the transmission electron microscopy (TEM) images in Fig. 5 (Oh et al. 2009) are an example of nanoprobes. The GNRs have resonant features around 800 nm, as shown in Fig. 5d, and their resonant wavelength can be engineered by adjusting the dimension. GNRs can be targeted to specific cells or tumors by antibody conjugation, and they can be used to generate heat in the targeted location by surface-plasmon resonance under infrared (IR) irra-diation at the resonant wavelength. To simulate GNRs in cells, the GNRs were immersed in water and the THz response was measured by the reflection-mode THz imaging setup shown in Fig. 3. The increased heat induced by the surface plasmon elevated the temperature of the water, and THz reflectivity increased, as shown in Fig. 6. Similar results were obtained with an A431 skin cancer cell endocytosed with GNRs. Fig. 7 shows the cancer cell images with and without GNRs. Figs 7b, c are the THz images with and without IR irradiation, respectively. The cell with GNRs subjected to IR irradiation was the only one to display higher THz reflectivity

Fig. 4 Temperature-dependent power absorptions and refractive indices of water (From Son (2009). Copyright © 2009 by American Institute of Physics. Reprinted by permission of American Institute of Physics)

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Fig. 5 TEM images of gold nanorods (GNRs) with aspect ratios of (a) 3.2, (b) 4.0, and (c) 4.2; (d) UV-visible absorption spectra of the GNRs of (a), (b), and (c) (From Oh et al. (2009). Copyright © 2009 by Optical Society of America. Reprinted by permission of Optical Society of America)

Fig. 6 Reflected THz time-domain waveforms from water with GNRs before (black line) and 5 minutes after their irradiation with an IR laser (red line). (From Oh et al. (2009). Copyright © 2009 by Optical Society of America. Reprinted by permission of Optical Society of America)

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resulting from the temperature rise caused by the surface-plasmon resonance. The cell without GNRs did not show any change regardless of IR irradiation. Although the increase in reflectivity was small (less than 10%), the contrast became evident when the difference was measured between Figs. 7b, c to give Fig. 7e. The differential THz image from the cell without GNRs was negligible, but that from the cell with GNRs showed clear evidence of nanoparticle distribution. The ratio between the amplitudes with and without GNRs was approximately 30. Therefore, very-high-contrast THz molecular imaging can be accomplished for the sensitive monitoring of nanoprobe delivery.

Fig. 7 Cancer cell images with and without GNRs. (a) Visible image; (b) THz image without IR irradiation; (c) THz image with IR irradiation; (d) amplitudes along the lines in (b) (black) and (c) (red); (e) differential image between (b) and (c); and (f) amplitude along the line in (e) (From Oh et al. (2009). Copyright © 2009 by Optical Society of America. Reprinted by permission of Optical Society of America)

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4 Imaging of Nanoparticle Delivery

Nanoparticle delivery to a cancerous tumor and the organs in a mouse was measured using the technique of THz molecular imaging with a differential modu-lation of the IR irradiation beam. As shown in Figs. 8a, b a male nude mouse bearing an A431 epidermoid carcinoma tumor in the proximal thigh region was prepared, and the nanoprobes were injected through the tail-vein; a similar mouse without nanoprobe injection served as a control. The nanoprobe was the GNR composite conjugated with Cetuximab (Cetuximab PEGylated gold nanorods: CET-PGNRs) for epidermal growth factor receptor (EGFR) specific tumor-cell targeting. The nanoprobes were absorbed by the liver and the spleen on their way to being delivered to the tumor. The organs were surgically removed to measure the concentration of nanoprobes delivered to specific locations by the TMI technique. As can be seen in Fig. 9a, most of the nanoprobes were captured by the liver, a small amount was captured by the spleen, and a fair amount was targeted to the cancerous tumor as the CET-PGNR was designed. Almost no nanoprobes reached the brain. Fig. 9b shows the conventional THz imaging without nanoprobe injection. It is clear that detailed quantitative imaging of nanoparticle delivery can be achieved using THz waves.

Fig. 8 Preparation of mice with A431 skin cancer tumors. (a) A mouse with the injection of nanoprobes; (b) a mouse without the nanoprobe injection to serve as a control

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5 Conclusion

The principle of TMI is presented along with a brief review of the method of genera-tion and detection of THz waves. The use of the TMI technique enables the quantitative measurement of nanoparticle distributions and allows for the high-contrast imaging of these distributions. The effectiveness of this technique is demonstrated by consi dering nanoprobe delivery to a cancerous tumor and the organs in a mouse. The images show that the signal amplitudes are linearly proportional to the nanoprobe concentration (Oh et al. 2011). The TMI technique has promising applications in the target-specific sensing of cancers as well as in the molecular diagnosis of drug delivery.

Acknowledgments The authors thank Mr. Heejun Shin and Mr. Dong-Gyu Lee for their assistance in the preparation of this paper. This study was supported by a grant from the Korean Health Technology R&D Project of the Ministry for Health, Welfare and Family Affairs, Republic of Korea (A101954), and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) under Grant Nos. 20100020647, 20100001979, 20100015989, 20100011934, and 20090054519.

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