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Terahertz (THz) imaging is a nondestructive, label-free, rapid imaging technique which gives the possibility of a real-time tracing of drugs within the skin. We evaluated the feasibility of THz dynamic imaging for visualizing serial changes in the distribution and penetration of a topical agent, dimethyl sulfoxide (DMSO) containing ketoprofen, using excised mouse skins. THz imaging was performed for 6 h after drug application to the skin and was compared with the results obtained using the Franz cell diffusion test, a standard in vitro skin absorption test. THz dynamic reflection imaging showed that the reflection signals decreased rapidly during the early time period, and remained constant through the late time period. The area of drug permeation within the skin layer on THz imaging increased with time. The dynamic pattern of THz reflection signal decrease was similar to that of DMSO absorption analyzed by the Franz cell diffusion test, which indicates that THz imaging mainly reflects the DMSO component. This study demonstrates that THz imaging technique can be used for imaging the spatial distribution and penetration of drug-applied sites.
©2012 Optical Society of America
1. Introduction
Terahertz (THz) imaging is a rapidly developing novel imaging technique that has broadened its biomedical application into the detection of skin and breast cancer and molecular imaging [1–5]. THz imaging using spectroscopy techniques has been actively studied in the pharmaceutical and chemical industries because of its spectroscopic ability [6,7]. However, only one study has attempted to use THz technology for drug imaging in biological tissues [8]. Based on its imaging and spectroscopic abilities, THz imaging has the potential to be a good modality for imaging drug distribution and diffusion.
Transdermal drug delivery systems have been increasingly used in the past several decades because of advantages such as avoidance of the hepatic first-pass metabolic effect, noninvasive drug delivery, and better patient compliance [9]. To understand and optimize the skin absorption of topical drugs, exact and reproducible data of the local distribution or diffusion kinetics of the topical drug are necessary. From this perspective, guidance notes on skin absorption studies were developed by the Organization for Economic Co-operation and Development (OECD) Expert Group on Dermal Absorption [10]. Currently, a well-established method that has been adopted by the “OECD guideline 428” is the in vitro skin absorption test, which uses diffusion cells that measure the diffusion of chemicals across the skin to a receptor fluid reservoir. In vitro methods can provide a great deal of valuable information such as penetration of chemicals expressed as a percentage of the dose or as a rate; however, they cannot provide the information in real time and require many steps such as diffusion cell experiments and high-performance liquid chromatography (HPLC) analysis. Therefore, there is a great demand for real-time imaging methods to provide information on drug permeation mechanisms.
In this work, we used THz dynamic imaging, to follow the penetration of a topical drug, ketoprofen dissolved in dimethyl sulfoxide (DMSO), to evaluate serial changes in the distribution and penetration of the topical drug. We compared the results of THz dynamic imaging with those of in vitro skin absorption test.
2. Materials and methods
2.1 Terahertz imaging system
THz reflection imaging of ex vivo skin was performed using a conventional THz time-domain spectroscopy (TDS) system (Fig. 1 ). A mode-locked Ti:sapphire laser, which provided 40-fs pulses at a wavelength of 800 nm, was divided into two beams. One of the beams was illuminated on a p-InAs wafer to generate THz pulses, and the other was focused on a photoconductive antenna fabricated on GaAs to detect the THz pulses. THz pulse waveforms were measured by obtaining the cross-correlated signal of the THz pulse at the detector and the optical gating pulse. The generated THz pulses were focused on a sample with two parabolic mirrors (f/number 1), and the reflected THz pulses from the sample were guided to the detector. The THz beam was focused down to 200 μm on the reflected sample surface, quartz plate-dermis interface, to obtain good spatial resolution images of the skin dermis. The sampled signal was amplified by a preamplifier, and the time-domain waveform was acquired using an optical delay [11]. The sample was moved by an x-y axis stepper in increments of 0.25 mm during scanning to form a pixel-by-pixel two-dimensional image. It took 8 min to scan a 10 × 10-mm2 area with a 0.25 × 0.25-mm2 pixel size.
A THz pulse is reflected back when it reaches an interface between media with different refractive indices. Reflections from different depths result in different optical time delays that can be used to estimate depth information [2]. We generated THz B-scan images by plotting the time-domain profile as a function of position across a slice of the skin, such that the grayscale represents the THz amplitude and the vertical axis represents the optical time delays.
2.2 Topical drug formulation
A simple form of the topical agent comprising the active drug component (ketoprofen) and the drug media (DMSO) was used to reduce the mixed effects of various components of topical agents. Pure ketoprofen (Sigma Aldrich, St Louis, USA) which is an analgesic drug, was dissolved in DMSO (Sigma Aldrich), a well-known transdermal penetration enhancer [12]. In this study, a percent-by-weight (%Wt) mixture of 20% ketoprofen and 80% DMSO was used.
2.3 Terahertz dynamic imaging of drug-applied sites
The study was approved by the institutional animal care and use committee of Seoul National University Hospital. The in vitro experimental setting with excised skin was adopted to compare the THz dynamic imaging and the in vitro skin absorption test in similar experimental geometry. The abdominal skin of hairless mice (SKH1 mice; Oriental-bio, Seoul, Korea) was excised immediately after euthanasia. The excised skin was placed onto a crystalline quartz plate with the stratum corneum side facing upward, and the dermis side in close contact with the quartz plate, as shown in Fig. 1. The test formulation (5 μL) was applied to a circular test area (5-mm radius, 0.19 cm2). To evaluate serial changes in the drug application site, THz reflection images and reconstructed B-scan images were obtained at 0, 8, 16, 30, and 45 min, and at 1, 1.5, 2, 3, 4, and 6 h after drug application. Four experimental sessions were carried out. Among these, 2 sessions ended at 2 h. The skin was secured with a sealed cap to prevent dehydration of the skin and evaporation of the topical drug. These experiments were performed in our laboratory space, where temperature and humidity are controlled at 20–24 °C and 30–60%, respectively.
2.4 In vitro skin absorption test
Franz diffusion cells were used as an in vitro device for assessing percutaneous absorption [13,14]. The Franz diffusion cell system (Hanson Research, Chatsworth, CA) is illustrated in Fig. 2 . Briefly, the diffusion cell was composed of a donor chamber and a receptor chamber between which the skin was positioned. The diffusion cells had a diffusion area of 2.0 cm2 (round shape with 16-mm diameter). The receptor chamber contained 7 mL of isotonic phosphate-buffered saline (PBS; Gibco Invitrogen, Grand Island, NY), and was stirred with a stirring magnet.
Fig. 2 Schematic drawing of excised skin mounted in the Franz cell diffusion system. The donor chamber (1) above the skin contains the applied topical agent. The chamber below the skin is the receptor chamber (2) from which samples are taken through the sampling port. The receptor chamber is surrounded by a water jacket (3) maintained at 32 °C. A magnetic stirrer and stirring helix are magnetically rotated at the bottom of the receptor chamber. The topical drug, which is applied to the stratum corneum side of the skin, permeates into the dermis side and then crosses the skin.
Freshly excised full-thickness skin of hairless mice was placed on the receptor chamber with the stratum corneum side facing upward and the dermis side facing downward. The prepared Franz cells were kept at 32 °C for 1 h with a circulating water bath. The same temperature was maintained throughout the experiment. After 1 h, 2 different formulations with finite doses of 50 μL and 100 μL were applied to 2 donor chambers in contact with the skin. The donor chambers were covered with a Franz cell cap. To evaluate penetration kinetics, aliquots (0.5 mL) of receptor fluid were sampled at predefined times (0, 7.5, 15, 30, and 45 min, and 1, 1.5, 2, 3, 4, 6, 8, 10, 18, and 24 h) and immediately replaced with 0.5 mL of fresh PBS. The sampled receptor fluid was assayed by HPLC to determine the concentration of DMSO and ketoprofen that had permeated through the skin.
2.5 HPLC analysis
HPLC analysis was performed on an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an ultraviolet diode array detector and a rapid scan in-line fluorescence detector. The column was YMC-Pack ODS-A (250 mm × 4.6 mm, id 5 μm) (YMC Co., Ltd., Kyoto, Japan). The flow rate of the mobile phase was 1 mL/min, and the detection wavelength was set to 210 nm for DMSO and 255 nm for ketoprofen. The mobile phase was a mixture of acetonitrile and 0.1% phosphate buffer (90:10 V/V for DMSO and 40:60 V/V for ketoprofen). All procedures were performed at ambient temperature.
3. Results
THz dynamic reflection images of the drug-applied site are presented in Fig. 3 . On the serial THz reflection images, the drug-applied sites were not visualized on the initial images scanned 0–8 min after drug application, but appeared on the second images scanned 8–16 min after drug application. The drug-applied sites showed a lower reflection signal than that of the skin area on which the drug was not applied. The THz reflection signal of the drug-applied sites gradually decreased until 60 min after application, and then remained constant until the end of THz imaging (6 h in 2 sessions; 2 h in 2 sessions).
Fig. 3 Serial THz reflection images of the drug-applied site. On the image obtained 8 min after drug application, the drug-applied site appeared as a dark shaded area with a low reflection signal; the intensity of darkness gradually increased for 1 h. The area of the dark shade increased with time, which may indicate diffusion and distribution changes of the topical drug in the skin. The scan time was 8 min for each image series.
Serial time-domain waveforms [Fig. 4(a) ] obtained at the drug-applied site also showed that the maximal amplitude (MA) of the first main peak gradually decreased at the early time period after drug application. The first main peak indicates the reflection from a boundary between the quartz plate and dermis. The permeated DMSO reduced the difference of the refractive index between the skin dermis and quartz plate, thus decreased the reflection of the THz pulse at the quartz plate-dermis interface, which is the main factor to determine THz reflection images. The signal of the second small peak (black curved arrows in Fig. 4(a) increased as time progressed, which corresponded with the layer with a high signal (white curved arrows in Fig. 4(b) just above the quartz plate–dermis interface. This small peak may indicate that the signals were reflected from the interface between the lower part of the skin dermis and the drug-permeated layer in the skin.
Fig. 4 Time-domain waveforms and B-scan images of a drug-applied site. (a) Serial time-domain waveforms obtained before (Ref), and 15 min, 30 min, and 60 min after the drug application show the MA of the main peak (arrowheads) at the drug-applied sites (at the red dot in the THz reflection image on the upper right) gradually decreased with time. (b) THz B-scan images of the drug-applied site show the reconstructed perpendicular images (at the red dotted line in the THz reflection image on the upper right) as the vertical axis represents the optical delay, and the gray-scale represents the THz amplitude. These data indicate that the signal and optical time width of the quartz-dermis interface (arrows) decreased with time, which corresponds to the main peak on the time-domain waveforms [arrowheads in (a)]. The signal of the layer (white curved-arrows) just above the quartz plate-dermis interface, which corresponds to the small peak on the time-domain waveforms [black curved-arrows in (a)], increased with time.
In this experimental setting, the main factor that altered the THz reflection signal over time was drug permeation across the skin. The THz reflectivity of the drug-applied site is determined by the mixed THz response of skin components such as water, protein, and fat, and diffused drug component [8]. Within few hours in these experiments, changes in the skin components might be minimal; hence, the permeated drug is the main factor that lowers THz reflectivity.
We assumed that the difference in the measured MA of the first main peak between scanning before and after drug application was directly related to the amount of permeated drug. To adjust the variable range of MA between experimental sessions, the percentage of MA difference [MAD (%)] was calculated by dividing the MA difference by MA measured before drug application [MA (ref)] as in Eq. (1). When the MAD (%) was plotted as a function of time, it increased rapidly at the early time period after drug application until approximately 1 h, and then reached a plateau in the plotted curve (Fig. 5 ):
Fig. 5 A diagram showing the time course of the MAD (%) difference on THz reflection imaging. In all experimental sessions of serial THz reflection imaging of the drug-applied site (2 sessions, followed up for 6 h; 2 sessions, followed up for 2 h), the measured MAD (%) increased rapidly in the early time period until approximately 1 h, and then, remained constant.
The cumulative amount of drug that permeated through the skin per unit of area in Franz cells was calculated from the concentration of each substance in the sampled fluid and plotted as a function of time (Fig. 6 ). The permeated amount of DMSO was much larger than that of ketoprofen (e.g., 187- to 351-fold at 60 min). The pattern of the absorption curve of DMSO was similar with that of the MAD (%) plot of THz reflection imaging, in that both showed rapid absorption of the drug in the early period and reached a plateau after a certain time. The pattern of the absorption curve of ketoprofen was different from that of the DMSO or MAD (%) plot, in that the penetration of ketoprofen started at a delayed time period, approximately 1 h after drug application, and increased continuously. These drug permeation profiles support that THz reflection imaging mainly reflects the DMSO component.
Fig. 6 Profiles of the cumulative permeated drug amount obtained using the Franz cell diffusion test. Two conditions were tested: applying 50-μL and 100-μL doses of the topical agent. (a) Permeation profiles of all drug components for 24 h. (b) Permeation profiles of all drug components for 6 h.
4. Discussion
Our study results demonstrate that THz dynamic imaging can be utilized for following up topical drug application by imaging the distribution and diffusion of the drug in the skin. THz dynamic imaging mainly reflects the rapidly permeating DMSO component. The main reason for THz imaging to reflect the distribution of DMSO is its sensitivity to polar solvents such as water, because THz radiation is strongly absorbed in polar molecules [15]. DMSO is a polar, hydrophilic solvent [16] that has an absorption level similar to that of water in the THz region [8,17]. In addition, the absolute amount of ketoprofen permeating through the skin is much smaller than that of DMSO, as shown in Fig. 6. Among the 3 skin layers, including the stratum corneum, epidermis, and dermis (outermost to innermost), the dermis layer is a highly aqueous tissue that has little resistance to the diffusion of water-soluble molecules and acts as a permeability barrier to lipophilic compounds [18]. DMSO is water soluble, permeating easily into the dermis layer, while ketoprofen is not soluble in water. These data imply that most of ketoprofen applied to the skin remained at the skin surface. Our THz dynamic imaging system, in which the THz beam was focused down to approximately 200 μm on the quartz plate–dermis interface, mainly reflected the drug component in the dermis layer of the skin, DMSO.
The Franz cell diffusion test is a well-established in vitro approach for evaluation of skin drug absorption, providing valuable information for penetration kinetics. There were several differences in the experimental conditions between the THz imaging set up and the Franz cell diffusion test. First, THz imaging was performed in the natural test environment, while the Franz diffusion cell was performed under a strictly controlled environment, including temperature and humidity. In addition, the receptor fluid in the Franz cell was stirred continuously, which may enhance the drug absorption across skin throughout the experiment. Second, THz imaging reflects the permeated drug into the dermis layer, while the Franz cell diffusion test measures the permeated drug across the skin into the receptor fluid. Therefore, THz imaging may reflect earlier events than Franz cell diffusion. Despite these differences, the pattern of the MAD (%) plot was similar with that of the DMSO absorption curve, which suggests that THz imaging can be used as an alternative analysis method of skin drug absorption.
THz dynamic imaging for skin drug absorption has several potential advantages. Conventional standard methods for analyzing skin drug absorption are mostly in vitro studies such as the Franz cell diffusion method or in vivo studies that require special labeling such as fluorescence or radioisotopes [19,20]. THz imaging is a noninvasive, label-free imaging method to observe skin changes under natural conditions. THz imaging may enable real-time tracking of drugs in the skin. Currently, real-time THz imaging is under development [21,22]. In this study, it took 8 min to scan a 1 × 1-cm2 area. The scan time of the current THz imaging system is enough for analysis of penetration kinetics because sampling is usually done on an hourly basis in the Franz cell diffusion method. The Franz cell diffusion method requires HPLC analysis or liquid scintillation counting for quantification of permeated drug, which may hamper immediate analysis. THz dynamic imaging that enables immediate analysis of skin drug absorption without special preparation can be used as a tool complementary to the currently used methods.
In this study, THz imaging mainly reflected the distribution of the penetration enhancer DMSO, because of its polarity and greater permeation into the skin. Current biophysical imaging modalities for skin drug absorption such as fluorescence imaging or autoradiography focus on the distribution of active drug components [23]. However, in transdermal drug delivery research, it is essential to study the penetration kinetics of many types of penetration enhancers. Reproducible data and an exact knowledge of the local distribution of penetration enhancers are decisive prerequisites to understand and optimize the mode of action of active drug components. As such, the characteristics of THz imaging useful in the evaluation of the distribution of penetration enhancers are important. If drug media that are relatively inert to THz radiation were used, THz imaging would show the distribution of the active drug components. We evaluated the THz imaging of only one topical agent, namely, ketoprofen in DMSO. Various kinds of topical agents might induce variable responses to THz radiation, which requires further investigation.
5. Conclusion
This study showed for the first time that THz reflection imaging could be used to visualize the distribution and diffusion of topically applied drugs, especially DMSO, which is a penetration enhancer. We expect that this technique will be useful to characterize the effects of various transdermal penetration enhancers and to provide more insight into the dynamic penetration of drugs across the skin.
Acknowledgments
This work was supported by the Basic Science Research Program through the Korean Health Technology R&D Project of the Ministry for Health, Welfare & Family Affairs (A101954) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (Nos. 2007-2004047, 2009-0093432, 2009-0076933, 2011-0013169, 2011-0001303 and 2012-0000936).
References and links
1. J.-H. Son, “Terahertz electromagnetic interactions with biological matter and their applications,” J. Appl. Phys. 105(10), 102033 (2009). [CrossRef]
2. R. M. Woodward, B. E. Cole, V. P. Wallace, R. J. Pye, D. D. Arnone, E. H. Linfield, and M. Pepper, “Terahertz pulse imaging in reflection geometry of human skin cancer and skin tissue,” Phys. Med. Biol. 47(21), 3853–3863 (2002). [CrossRef] [PubMed]
3. A. J. Fitzgerald, V. P. Wallace, M. Jimenez-Linan, L. Bobrow, R. J. Pye, A. D. Purushotham, and D. D. Arnone, “Terahertz pulsed imaging of human breast tumors,” Radiology 239(2), 533–540 (2006). [CrossRef] [PubMed]
4. S. J. Oh, J. Kang, I. Maeng, J. S. Suh, Y. M. Huh, S. Haam, and J.-H. Son, “Nanoparticle-enabled terahertz imaging for cancer diagnosis,” Opt. Express 17(5), 3469–3475 (2009). [CrossRef] [PubMed]
5. S. J. Oh, J. Choi, I. Maeng, J. Y. Park, K. Lee, Y. M. Huh, J. S. Suh, S. Haam, and J.-H. Son, “Molecular imaging with terahertz waves,” Opt. Express 19(5), 4009–4016 (2011). [CrossRef] [PubMed]
6. D. F. Plusquellic, K. Siegrist, E. J. Heilweil, and O. Esenturk, “Applications of terahertz spectroscopy in biosystems,” ChemPhysChem 8(17), 2412–2431 (2007). [CrossRef] [PubMed]
7. Y. Ueno and K. Ajito, “Analytical terahertz spectroscopy,” Anal. Sci. 24(2), 185–192 (2008). [CrossRef] [PubMed]
8. K. W. Kim, H. Kim, J. Park, J. K. Han, and J.-H. Son, ““Terahertz tomographic imaging of transdermal drug delivery,” IEEE Trans. THz,” Sci. Tech. (Paris) 2, 99–106 (2012).
9. A. Tfayli, O. Piot, F. Pitre, and M. Manfait, “Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy,” Eur. Biophys. J. 36(8), 1049–1058 (2007). [CrossRef] [PubMed]
10. OECD, “Test Guideline 428. Skin absorption: in vitro method” (OECD, Paris, 2004).
11. J. Y. Park, H. J. Choi, K.-S. Cho, K.-R. Kim, and J.-H. Son, “Terahertz spectroscopic imaging of a rabbit VX2 hepatoma model,” J. Appl. Phys. 109(6), 064704 (2011). [CrossRef]
12. M. Rizwan, M. Aqil, S. Talegaonkar, A. Azeem, Y. Sultana, and A. Ali, “Enhanced transdermal drug delivery techniques: an extensive review of patents,” Recent Pat. Drug Deliv. Formul. 3(2), 105–124 (2009). [CrossRef] [PubMed]
13. T. J. Franz, “Percutaneous absorption on the relevance of in vitro data,” J. Invest. Dermatol. 64(3), 190–195 (1975). [CrossRef] [PubMed]
14. T. J. Franz, “The finite dose technique as a valid in vitro model for the study of percutaneous absorption in man,” Curr. Probl. Dermatol. 7, 58–68 (1978). [PubMed]
15. J. Y. Suen, P. Tewari, Z. D. Taylor, W. S. Grundfest, H. Lee, E. R. Brown, M. O. Culjat, and R. S. Singh, “Towards medical terahertz sensing of skin hydration,” Stud. Health Technol. Inform. 142, 364–368 (2009). [PubMed]
16. M. S. Skaf, “Static dielectric properties of a model for liquid DMSO,” Mol. Phys. 90(1), 25–34 (1997). [CrossRef]
17. Y. B. Ji, E. S. Lee, S. H. Kim, J. H. Son, and T. I. Jeon, “A miniaturized fiber-coupled terahertz endoscope system,” Opt. Express 17(19), 17082–17087 (2009). [CrossRef] [PubMed]
18. S. C. Wilkinson, W. J. M. Maas, J. B. Nielsen, L. C. Greaves, J. J. M. van de Sandt, and F. M. Williams, “Interactions of skin thickness and physicochemical properties of test compounds in percutaneous penetration studies,” Int. Arch. Occup. Environ. Health 79(5), 405–413 (2006). [CrossRef] [PubMed]
19. K. König, A. Ehlers, F. Stracke, and I. Riemann, “In vivo drug screening in human skin using femtosecond laser multiphoton tomography,” Skin Pharmacol. Physiol. 19(2), 78–88 (2006). [CrossRef] [PubMed]
20. E. Touitou, V. M. Meidan, and E. Horwitz, “Methods for quantitative determination of drug localized in the skin,” J. Control. Release 56(1-3), 7–21 (1998). [CrossRef] [PubMed]
21. T. Hattori and M. Sakamoto, “Deformation corrected real-time terahertz imaging,” Appl. Phys. Lett. 90(26), 261106 (2007). [CrossRef]
22. S. H. Cho, S. H. Lee, C. Nam-Gung, S. J. Oh, J.-H. Son, H. Park, and C. B. Ahn, “Fast terahertz reflection tomography using block-based compressed sensing,” Opt. Express 19(17), 16401–16409 (2011). [CrossRef] [PubMed]
23. K. Cal, J. Stefanowska, and D. Zakowiecki, “Current tools for skin imaging and analysis,” Int. J. Dermatol. 48(12), 1283–1289 (2009). [CrossRef] [PubMed]
