Development of NIR Bioimaging Systems

Journal of Physics: Conference Series 16 (28) 1223 doi:1.188/1742-6596/16/1/1223 Development of NIR Bioimaging Systems Kohei SOGA 1, Takashi TSUJI 1, Fumio TASHIRO 1, Joe CHIBA 1 Motoi OISHI 2, Keitaro YOSHIMOTO 2, Yukio NAGASAKI 2 Katsuhisa KITANO 3 and Satoshi HAMAGUCHI 3 1 Polyscale Technology Research Center, Tokyo University of Science 2 Tsukuba Research Center for Interdisciplinary Materials Science, University of Tsukuba 3 Center for Atomic and Molecular Technologies, Osaka University E-mail: mail@ksoga.com Abstract. Fluorescence bioimaging is one of the most important technologies in the biomedical field. The most serious issue concerning current fluorescence bioimaging systems is the use of short wavelength light, UV or VIS, for the excitation of phosphors such as fluorescent proteins or quantum dots. The authors propose a fluorescence bioimaging system excited by near infrared light using rare-earth doped ceramic nanophosphors. The requirements for the nanophosphors are a designed emission scheme under the near infrared excitation, a controlled size between 1 and 2 nm and surface modification of the particles with a biofunctional polymer, which prevents particle agglomeration and non-specific interaction to nontargeting substances and gives them a specific interaction for the targeted objects. The preparation of the bioimaging probe and demonstrative imaging work are reported. 1. Introduction Fluorescence bioimaging (FBI) is one of the key technologies for the biomedical sciences. It is used for the imaging of biological substances, and can also be utilized in techniques involving fluorescence immunoassay, photo dynamic therapy, and drug delivery systems. Fluorescent proteins and quantum dots are currently used for FBI under excitation by ultraviolet (UV) or short-wavelength visible light. In the case of fluorescent proteins, the probe proteins themselves and biological substances are damaged within a few minutes by the photo-toxicity due to the high quantum energy of the excitation light. Although light emitted from quantum dots can be utilized in various applications for periods of up to several tens of minutes, the potential toxicity associated with their use is a concern since quantum dots for the most part comprise toxic elements selected for their unique optical properties. For both of the probes, the strong scattering of the excitation light due to the short wavelength is a disadvantage for imaging with a deep observation depth. A solution to this problem involves a shift to a longer excitation wavelength. Rare-earth doped ceramic materials show efficient fluorescence under near infrared (NIR) excitation [1]. The most well known is a Nd:YAG laser, which emits light at 164 nm under 8-nm excitation [2]. One of the most important items currently used for long distance optical fiber communication is a fiber amplifier made of erbium-doped silica fiber, which emits c 28 Ltd 1

Journal of Physics: Conference Series 16 (28) 1223 doi:1.188/1742-6596/16/1/1223 fluorescence at 155 nm under a 98-nm excitation [3]. Rare-earth doped ceramic also shows infraredto-visible upconversion (UC) [4, 5], where the stepwise excitation by the NIR light excites the states to higher energy levels than the excitation energy to emit higher energy photons by an immediate transition to the ground state. Since most FBI systems currently use a charge-coupled device (CCD) for visible light observation, the UC process is useful for having the advantages of NIR excitation and visible observation with the currently used CCD. Our strategy for achieving FBI under NIR excitation (NIR-FBI) utilizes a rare-earth doped device with ceramic nanophosphors (RED-CNP). The requirements of the RED-CNP for achieving NIR-FBI are a particle size between 1 and 2 nm with a narrow size distribution, dispersion in an ionic solution, and a specific adsorption to the targeting substance, such as proteins. The lower limit of the particle size is determined by the minimum size for the long lifetime of the rare-earths in the ceramic particles. Ionic vibrations surrounding the particles tend to quench the emission by converting the excitation energy to vibration energies. An efficient emission is difficult to achieve if the particle size is less than 1 nm. If the particle size is too large, the particles settle down even with steric repulsion of surface polymers. The required particle size varied according to the targeting objects, though it is limited for the above reasons. The latter two requirements can be fulfilled by surface modification of the RED-CNP with a particular polymer. The dispersion of the particles is required in ionic solution with an ionic strength of.15 mol/l, which corresponds to that of physiological saline. Poly(ethylene glycol) (PEG) can modify the dispersion effects for inorganic particles [6, 7]. The PEG chains are also known to prevent non-specific adsorption to general substances in a living body. Acetal-PEG-block poly [2-(N,N-dimethylamino) ethylmethacrylate] (acetal-peg-b-pama) can be installed with ligand molecules, such as biotin, with specific adsorption to a targeting protein by an avidin-biotin interaction. The PAMA part has a positive charge to interact with the surface of the ionic solids. The objective of this paper is to describe the development of the NIR-FBI system. The technology developed in this study was used to prepare Y 2 O 3 particles doped with 4 mol%er (Y 2 O 3 :Er) nanoparticles as the UC emissive particles with an approximate size of 2 nm through so-called homogeneous precipitation, achieve surface modification of the particles with acetal-peg-b-pama, and present a demonstration of the NIR-FBI system using macrophage cells. 2. Experimental 2.1. Preparation of Y 2 O 3 :Er nanoparticles The Y 2 O 3 :Er particles were prepared by calcinating precursors at 9 o C for 3 hrs. The precursor, Y,Er(CO 3 )OH, was precipitated using homogeneous precipitation. A mixture of urea and nitrates of yttrium and erbium was dissolved into distilled water as a starting solution. By heating the solution up to 8 o C, the urea decomposed into CO 3 2- and NH 4 + as precipitants for the precursor. 2.2. Surface Modification using acetal-peg-b-pama The surface of Y 2 O 3 :Er particles is positively charged, as is the PAMA end of acetal-peg-b- PAMA. To allow an interaction between these two positively charged parts, we used poly (acrylic acid) (PAAc) as an interfacing agent. Using this method, acetal-peg-b-pama is known to be firmly adsorbed on the surface of Y2O3 particles even with three centrifugal washes [8]. The precise experimental conditions for the surface modification have been described [8]. 2.3. Demonstration of NIR-FBI using macrophages For a demonstration of NIR-FBI, Y 2 O 3 :Er particles with a 2-nm size were modified with acetal-peg-b-pama and introduced into macrophage cells. The cells were incubated for a total of 44 hrs with the particles and observed with a biological fluorescence microscope equipped with a 98-nm laser diode for the NIR excitation. 2

Journal of Physics: Conference Series 16 (28) 1223 doi:1.188/1742-6596/16/1/1223 3. Results and Discussion 3.1. Preparation of Y 2 O 3 :Er nanoparticles Figure 1 shows FE-SEM images of the precursors obtained by a conventional carbonate precipitation method and homogeneous precipitation methods. Using the homogeneous precipitation, we prepared spherical particles of the precursor with a size of 1-2 nm within a narrow size distribution. The size and shape were maintained even after the calcination. Figure 2 shows the particle size distribution of Y 2 O 3 particles based on the analysis of FE-SEM images of calcinated particles. We successfully prepared Y2O3:Er particles with a size of 1-2 nm for NIR-FBI. 3.2. Surface Modification using acetal-peg-b-pama and a Demonstration of NIR-FBI Figures 3 and 4 show the zeta potential of particles after PAAc and acetal-peg-b-pama modification, respectively. The potential once moved into the negative region by modification with PAAc and recovered to a neutral state by post-modification with acetal-peg-b-pama. The particles were successfully modified by the double layer of the PAAc and Acetal-PEG-b-PAMA polymers. The firm (a) (b) 1. μm 1. μm Figure 1 FE-SEM images of precursors obtained by a conventional carbonate precipitation method (a) and homogeneous precipitation methods (b). 2 2 Particle size(nm) 1 (a) Particle size(nm) 1 (b) 2 4 6 81 Number 1 2 3 4 Number Figure 2 Particle size distribution of the Y 2 O 3 particles obtained by analyzing the FE-SEM images of the particles obtained by a conventional carbonate precipitation method (a) and the homogeneous precipitation methods (b). 3

Journal of Physics: Conference Series 16 (28) 1223 doi:1.188/1742-6596/16/1/1223 modification was also confirmed using FT-IR spectroscopy. The modified particles were then introduced into macrophage cells for a total incubation period of 44 hrs. Figure 5 shows the bright-field image and UC image of the macrophages. We successfully obtained cell imaging using the NIR-FBI system. 4. Conclusion FBI under NIR excitation was achieved for the cellular bioimaging of macrophages. The requirements for the imaging were achieved by the design of rare-earth phosphors, fabrication of the ceramic particles, surface modification of the particles with a bio-functional polymer, micro-scale installation of the particles into cells, and design of the NIR microscope. The homogeneous precipitation of the precursors for 2-nm Y 2 O 3 :Er particles and the surface modification of particles with double layers of PAAc and acetal-peg-b-pama led to successful NIR imaging. Acknowledgement This work was supported by "Academic Frontier" Project for Private Universities: Matching Fund Subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 26-21. This work was also partly supported by Industrial Technology Research Grant Program in 5 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. Zeta potential (mv) 2 1-1 -2-3 -4 2 4 6 8 Number of PAAc to one Y 2 O 3 nanoparticle 1x1 3 Figure 3 Zeta potential of Y 2 O 3 nanoparticles as a function of the number of PAAc in the solution. Dashed line is drawn as a visual guide. Zeta potential (mv) 2 1-1 -2-3 -4 2 4 6 8 1 Number of Acetal-PEG-b-PAMA to one Y 2 O 3 nanoparticle Figure 4 Zeta potential of Y 2 O 3 nanoparticles as a function of the number of acetal-peg-b-pama added to the colloidal solution. Dashed line is drawn as a visual guide. (a) (b) (c) 1um Figure 5 Englobement of Y 2 O 3 :Er nanoparticles by macrophage cells. (a) Bright-field image, (b) UC image, and (c) merged image. 4

Journal of Physics: Conference Series 16 (28) 1223 doi:1.188/1742-6596/16/1/1223 References [1] K. Soga et al., J. Appl. Phys., 93 (23) 2946-2951. [2] R. C. Powell, Physics of Solid State Laser Materials, (Springer, 1998). [3] S. Sudo et al., Optical Fiber Amplifiers: Materials, Devices, and Applications (Artech House Publishers, 1997). [4] D. Matsuura et al., Appl. Phys. Lett., 81 (22) 4526-4528. [5] D. Matsuura et al., J. Electrochem. Soc., 152 (25) H39-42. [6] T. Ishii et al., Langmuir, 2 (24) 564-564. [7] Y. Nagasaki et al., Langmuir, 2 (24) 6396-64. [8] T. Konishi, J. Photopolymer Sci. Tech., 19 (26) 145-149. 5