Nanomaterials for Cancer Diagnosis and TherapyView this Special Issue
Development of Near Infrared-Fluorescent Nanophosphors and Applications for Cancer Diagnosis and Therapy
The use of near infrared (NIR) light for biomedical photonics in the wavelength region between 800 and 2000 nm, which is called “biological window”, has received particular attention since water and biological tissues have minimal optical loss due to scattering and absorption as well as autofluorescence in this region. Recent development of InGaAs CCD enables observations in this wavelength region. In the present paper, we report development of Yb and Er-doped yttrium oxide nanoparticles (:YbEr-NP) which show strong NIR emission under NIR excitation (NIR-NIR emission). We also demonstrate that NIR emission can be observed through swine colon wall. Based on these results, we propose a possible application of :YbEr-NP for cancer diagnosis and therapy using NIR-NIR imaging system. Our results also suggest potential applications of :YbEr-NP for noninvasive detection of various diseases.
Bioimaging technique has received particular attention as an essential tool in the field of biomedical research through the observation of biological phenomena both in vivo and in vitro. The use of near infrared (NIR) light in the wavelength region between 800 and 2000 nm for biomedical photonics attracts great interest because this region is a so-called “biological window”, where water and biological tissues have minimal absorbance and autofluorescence. As shown in the loss spectrum of human skin  (see Figure of Supplementary Material available online at http://dx.doi.org/10.1155/2010/491471.), one can expect the lowest loss of the spectrum within the above region.
Recently, upconverting (UC) phosphors (UCPs) have been used for bioimaging (Figure 1) [2–8]. UCPs are ceramic materials containing rare earth ions. The materials can absorb IR radiation and upconvert it to emit visible light by stepwise excitation among discrete energy levels of the rare earth ions (NIR-VIS imaging) . For example, yttrium oxide (Y2O3) matrix containing several atomic % of erbium (Er) exhibits upconversion emission at 550 nm (green) and 660 nm (red) following excitation at 980 nm. The advantage of NIR-VIS bioimaging is that NIR light can penetrate deeper into tissues due its lower scattering.
The wavelength for biomedical photonics has been limited due to the use of the silicon-based CCD. The observation wavelength is limited to at most 1100 nm due to the band gap of silicon. In recent years, however, the InGaAs CCD which can cover wavelength between 800 and 2200 nm has become available. Considering various advantages of the NIR window, the time is ideal for the development of phosphors to emit fluorescence in this region.
Rare-earth doped ceramics can be a good candidate, since these are known to emit efficient fluorescence in the NIR wavelength region by NIR excitation. For example, the most representative solid state laser material Nd : YAG (Nd-doped yttrium aluminum garnet) can emit light with a wavelength of 1064-nm with 800-nm excitation . Er-doped silicate glass fibers are used to amplify the signal of long-distance fiber optical communication by emitting 1550-nm fluorescence with 980-nm excitation . The authors have previously reported that Er-doped yttrium oxide nanoparticles (Y2O3:Er-NP) showed NIR fluorescence (1550 nm) with NIR excitation . The advantage of this NIR-NIR imaging is that both excitation and emission light can penetrate deep into/from tissues, which enables imaging of the target inside the tissues (Figure 1).
In this study, we report a development of Yb and Er-doped yttrium oxide nanoparticles (Y2O3:YbEr-NP), which possess higher NIR emission than Y2O3:Er-NP. Yb3+ was added as a so-called “sensitizer” to increase the NIR emission. Since Yb3+ has much larger absorption efficiency and the excitation energy can be efficiently transferred to Er3+ in case of upconversion phosphors , we added the Yb3+ codopant as a sensitizer expecting the same effect for the 1550 nm NIR emission of Er3+. We also demonstrated for the first time that NIR emission could be observed even through the swine colon wall. Based on this observation, we propose possible new NIR-NIR biophotonics applications for cancer diagnosis and therapy using Y2O3:YbEr-NP, especially for resection surgery of colon cancer.
2. Material and Methods
Y(NO3)36H2O (99.99% purity) and Urea (99.0% purity) were purchased from Kanto Chemicals (Tokyo, Japan). Er(NO3)35H2O (>99% purity), Yb(NO3)35H2O (99.9% purity) and Na2CO3 (99% purity) were obtained from Kojundo Chemical Laboratory (Saitama, Japan).
2.2. Preparation of NIR Biophotonic Nanoparticles
Y2O3:YbEr-NP were prepared by the homogeneous precipitation method as used for preparation of upconversion nanoparticles . Twenty mmol/L Y(NO3)3, 0.2 mmol/L Yb(NO3)3, and 0.2 mmol/L Er(NO3)3 were dissolved in 200 mL purified water, mixed with 100 mL of 4 mol/L Urea solution, and stirred for 1 hour at 100°C. The obtained precipitates were separated by centrifugation, and dried at 80°C for 12 hours. The hydroxide or hydroxyl carbonated precursors were calcinated at 1200°C for 60 minutes in an electric furnace to convert them into anhydrous crystalline Y2O3 nanoparticles doped with Yb and Er.
2.3. Characterization of NIR Biophotonic Nanoparticles
The prepared Y2O3:YbEr-NP were provided for characterization using FE-SEM (S-4200, Hitachi Ltd., Tokyo, Japan) and XRD (XRD-6100, Shimadzu, Kyoto, Japan) with CuKα radiation.
Optical absorption spectra were observed using a spectrometer (U-4000, Hitachi Ltd., Tokyo, Japan) equipped with an integrating sphere. The loss spectrum of the swine colon was also observed using the same equipment and sandwiching a slice of the colon (thickness: 250–330 m) between two glass slides. The loss spectra were measured in a normal mode without using the integrating sphere.
Fluorescence spectra of Y2O3:YbEr-NP and Y2O3:Er-NP were recorded using a spectrometer (AvaSpec-NIR256-1.7, Avantes, Eerbeek, Netherlands) under an excitation of 980-nm and a laser diode (LD, SLI-CW-9MM-C1-980-1M-PD, Semiconductor Laser International Corp., USA).
2.4. NIR Imaging System
NIR-NIR imaging was carried out using the NIR imaging system, consisting of a fiber pigtail laser diode at 980 nm with 2 W power (LU0975T050, Lumics, Berlin, Germany), a laser scanner (VM500+, GSI Group, Massachusetts, USA) for planer irradiation of the excitation light, and InGaAs CCD camera (NIR-300PGE, VDS Vosskühler, Osnabrück, Germany) for detection of the NIR fluorescence between 1100 and 1600 nm.
2.5. NIR Imaging Inside Swine Colon
In order to demonstrate that NIR light under NIR excitation can be observed through the colon wall, a tablet of Y2O3:YbEr-NP with a diameter of 3 mm and a length of 6 mm was formed by mixing Y2O3:YbEr-NP with a conventional dental composite resin (Fuji I, GC, Tokyo, Japan). An endoscopic clip  (Olympus, Tokyo, Japan) painted with Y2O3:YbEr-NP-containing paint (NIR clip) was also prepared in order to demonstrate that NIR light from the clip can also be observed through colon wall. After fixing the NIR clip in the mucosal side (inside) of a piece of the tubular swine colon, we observed the colon using the NIR-NIR imaging system. It is expected that the painted portion of these endoscopic clips will be observed through the colon wall from the serosal side (outside) of the colon.
3. Results and Discussion
3.1. Characterization of NIR Biophotonic Nanoparticles
Figure 2(a) shows FE-SEM images of Y2O3:YbEr-NP synthesized by homogeneous precipitation and calcination at 1200°C for 60 minutes. The particle size was approximately 130 ± 25 nm. Figure 2(b) shows the XRD pattern of Y2O3:YbEr-NP. The sample was confirmed to be single-phase Y2O3 since all of the peaks were identified as those of cubic Y2O3 (JCPDS 41-1105).
3.2. Absorption and Fluorescence Spectra
Absorption and fluorescence spectra of Y2O3:YbEr-NP are shown in Figure 3. Yb3+ was added as a so-called “sensitizer” for increasing the absorption efficiency of the excitation light at 980 nm in this study. In the absorption spectrum (Figure 3(a)), a strong absorption band of Yb3+ was observed. The absorbed excitation light at 980 nm was mainly absorbed by Yb3+ and the excitation energy transfers to Er3+ to emit the NIR fluorescence at 1550 nm, as shown in Figure 3(b). The absorption and florescence schemes are well known in the field of optical communication and the phenomenon has been well understood [10, 11]. Figure 3(c) shows that the NIR emission of Y2O3:YbEr-NP is much higher than that of Y2O3:Er-NP, indicating that codoping of Yb3+ is also effective to enhance NIR emission.
Figure 4 shows the loss spectrum of the slice of swine colon. The spectrum was obtained by deducting the spectrum due to a thickness of 250 m from that of 330 m to yield the net loss due to a swine colon thickness of 110 m. The spectrum is divided by the corresponding thickness to make it a coefficient spectrum. A water absorption spectrum as well as the emission spectrum of Y2O3:YbEr-NP, were also coplotted. There are absorption band peaks at 1420 nm, which are due to the second harmonic absorption of the O-H stretching vibration in water molecules. In the spectrum, the fluorescence spectrum is super imposed. Although the fluorescence and the absorption bands overlap, the tail of the fluorescence is still out of the absorption band and one can expect observation of the fluorescence through the colon wall. It appeared better to select phosphors which could emit fluorescence avoiding the water absorption at 1420 nm. The development of the phosphors that can emit NIR light at different wavelength by doping different rare-earth ions such as Nd, Pr or Tm is now in progress.
3.3. NIR Imaging
Figure 5(a) shows images of the Y2O3:YbEr-NP tablet set in a tubular swine colon. The tablet emission could be clearly observed even through the colon wall. This result indicates that the NIR excitation light and the NIR emission from Y2O3:YbEr-NP is strong enough to penetrate the colon wall.
In an effort to show the applicability of Y2O3:YbEr-NP in cancer therapy, NIR imaging of Y2O3:YbEr-NP-coated medical clips and Y2O3:YbEr-NP solution injected from the mucosal side (inside) of the colon were carried out. The clips used in this experiment are commercially available for endoscopic therapy and can be easily employed to mark the part of cancer using a conventional endoscopy system. The coating was applied onto the plastic part of the clip. Figure 5(b) shows the NIR imaging Y2O3:YbEr-NP-coated clips and those set inside of the swine colon under NIR excitation. Although the coating was as thin as several tens of m, the NIR fluorescence was clearly observed and was comparable to the case of the tablets.
NIR imaging of Y2O3:YbEr-NP solution injected inside the colon was also carried out. As shown in Figure 5(c), NIR emission from Y2O3:YbEr-NP injected in the other side of the colon wall was clearly observed. This result suggests that Y2O3:YbEr-NP can be used as a substitution for tattoo (black ink) solution which is usually used in cancer therapy as described below. Since tattoo solution is usually injected at both ends of tumor region before laparoscopic surgery, Y2O3:YbEr-NP solution was also injected at two points.
3.4. Possible Applications of NIR Photonic Nanomaterials for Cancer Diagnosis and Therapy
The spectroscopic properties of swine colon and the development and demonstrative work using Y2O3:YbEr-NP suggest a great potential of NIR-NIR photonic nanomaterials for cancer therapy. For example, this technology can be applied to the intraoperative recognition of the tumor site in laparoscopic surgery for the gastrointestinal cancer (Figures 6(a) and 6(b)). Tattooing into the submucosal layer of the colon is generally performed in laparoscopic surgery, which sometimes leads to difficulty in recognition of cancer site due to faint tattoo and diffused tattoo, which causes spread resection of the colon (Figure 6(b)) . Figure 6(a) shows our proposed procedure using NIR clips. After endoscopic detection of colorectal cancer, NIR clips are fixed to mark cancer site using endoscopy. Cancer site can be recognized through the serosa of the intestinal wall by NIR fluorescence from the NIR clips fixed inside the colon during cancer surgery using NIR-NIR imaging system. Using this new imaging system, we will be able to determine the proper resection margins (normally 10 cm from the cancer site) for curative resection during surgery, which is much more advantageous compared with the current procedure using tattoo (Figure 6(b)).
Y2O3:YbEr-NP can also be used for caner diagnostics. Previously we have demonstrated tumor cell-targeted upconversion imaging using Y2O3:Er-NP modified with cyclic arginine-glycine-aspartic acid (RGD) peptide as a specific probe for tumor cell detection . The RGD peptide strongly binds to integrin , whose expression is significantly upregulated in invasive tumor cells of certain cancer types (glioblastoma, melanoma, breast, ovarian, and prostate cancers, and in almost all tumor vasculature), but not in quiescent endothelium and normal tissues [16, 17]. Thus, modification of Y2O3:YbEr-NP with cyclic RGD peptide will also be useful for the development of a tumor cell-targeted NIR-NIR imaging probe. Successful observation of NIR emission from Y2O3:YbEr-NP solution injected inside the colon (Figure 5(c)) supports the idea that targeting and detection of cancer sites in colon using Y2O3:YbEr-NP are possible. Research along this line is currently in progress. Our results also suggest that probe-modified Y2O3:YbEr-NP could be used for noninvasive detection of various diseases.
Cell toxicity is another important issue when considering probes for use in bioimaging. Previous studies showed that Y2O3 and Er3+-doped Y2O3 nanoparticles were nontoxic to cultured cell [6, 18]. Since the chemical properties of Yb3+ are similar to those of Er3+ , it is plausible that Yb3+ and Er3+-doped Y2O3 nanoparticles also are nontoxic. However, further studies on biocompatibility such as inflammation assays and long-term toxicity assays using animal models are important for their medical application.
The use of near infrared (NIR) light in the wavelength region between 800 and 2000 nm for biomedical photonics attracts great interest. This region is a so-called “biological window”, where water and biological tissues have minimal absorbance and autofluorescence. In the present study, we report high NIR emission under NIR excitation (NIR-NIR emission) of Yb and Er-doped yttrium oxide nanoparticles (Y2O3:YbEr-NP), and propose a possible NIR-NIR biophotonic application using Y2O3:YbEr-NP for cancer diagnosis and therapy based on demonstrative experiments. Observations of NIR emission through swine colon wall support our idea that NIR-NIR biophotonic nanomaterials can be used for cancer diagnosis and therapy.
The authors thank Professor Atsuo Yasumori for the XRD measurement and Dr. Karin Sörgjerd for helpful comments. This work is financially supported by Industrial Technology Research Grant Program from New Energy and Industrial Technology Development Organization (NEDO) of Japan (Tamotsu Zako).
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