In Vivo Study of Biodistribution and Urinary Excretion of Surface

Nov 14, 2008 - These findings would have direct implications for the use of SiNPs as delivery systems and imaging tools in live animals. Furthermore, ...
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Anal. Chem. 2008, 80, 9597–9603

In Vivo Study of Biodistribution and Urinary Excretion of Surface-Modified Silica Nanoparticles Xiaoxiao He, Hailong Nie, Kemin Wang,* Weihong Tan, Xu Wu, and Pengfei Zhang Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, and Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China The biodistribution and urinary excretion of different surface-modified silica nanoparticles (SiNPs) in mice were investigated in situ using an in vivo optical imaging system. Three types of surface-modified SiNPs, including OH-SiNPs, COOH-SiNPs, and PEG-SiNPs with a size of ∼45 nm, have been prepared with RuBPY doped for imaging purposes. Intravenous (iv) injection of these SiNPs followed by fluorescence tracing in vivo using the Maestro in vivo imaging system indicated that OH-SiNPs, COOH-SiNPs, and PEG-SiNPs were all cleared from the systemic blood circulation, but that both the clearance time and subsequent biological organ deposition were dependent on the surface chemical modification of the SiNPs. Thus, for instance, the PEG-SiNPs exhibited relatively longer blood circulation times and lower uptake by the reticuloendothelial system organs than OH-SiNPs and COOH-SiNPs. More interestingly, in vivo real-time imaged dominant signal in bladder and urine excretion studies revealed that all three types of iv-injected SiNPs with a size of ∼45 nm were partly excreted through the renal excretion route. These conclusions were further confirmed through ex vivo organ optical imaging and TEM imaging and energy-dispersed X-ray spectrum analysis of urine samples. These findings would have direct implications for the use of SiNPs as delivery systems and imaging tools in live animals. Furthermore, our results demonstrate that the in vivo optical imaging method is helpful for in vivo sensing the biological effects of SiNPs by using luminescent dye doped in the silica matrix as a synchronous signal. Silica nanoparticles (SiNPs) possess extraordinary properties, including straightforward synthesis, relatively low cost, easy separation, high hydrophilicity, and facile surface modification. As such, SiNPs are being widely developed for a broad spectrum of biomedical and biotechnological applications,1-12 such as * To whom correspondence should be addressed. E-mail: kmwang@ hnu.cn. Tel: 0731-8821566. (1) Zhao, X. J.; Tapec-Dytioco, R.; Tan, W. H. J. Am. Chem. Soc. 2003, 125, 11474–11475. (2) Peng, J. F.; He, X. X.; Wang, K. M.; Tan, W. H.; Wang, Y.; Liu, Y. Anal. Bioanal. Chem. 2007, 388, 645–654. (3) Ye, Z. Q.; Tan, M. Q.; Wang, G. L.; Yuan, J. L. Anal. Chem. 2004, 76, 513–518. (4) Wang, J.; Liu, G. D.; Engelhard, M. H.; Lin, Y. H. Anal. Chem. 2006, 78, 6974–6979. 10.1021/ac801882g CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

biosensors for DNA,1 intracellular pH,2 human prostate-specific antigen,3 and tumor necrosis factor,4 biomarkers for tumor cells and bacteria recognition using optical microscopy imaging,5-7 cancer therapy,8 gene delivery,9,10 and drug delivery.11,12 Similar to other nanomaterials,13-22 the safety and overall bioeffects of SiNPs have emerged as a question that necessarily affects the use of SiNPs in most types of biomedical applications where compatibility with the biological milieu is required. For this reason, several groups, including ours, have explored the interaction and toxicity of SiNPs with biomolecules and cells.23-28 For instance, studies have demonstrated that SiO2 nanoparticles cause aberrant clusters of topoisomerase I in the nucleoplasm in cells;23 our group has previously demonstrated those positively charged aminomodified SiNPs could enrich plasmid DNA and protect it from (5) Santra, S. S.; Zhang, P.; Wang, K. M.; Tapec-Dytioco, R.; Tan, W. H. Anal. Chem. 2001, 73, 4988–4993. (6) Zhao, X. J.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R. P.; Jin, S.; Tan, W. H. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15027–15032. (7) Nakamura, M.; Shono, M.; Ishimura, K. Anal. Chem. 2007, 79, 6507–6514. (8) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13549–13554. (9) Bharali, D. J.; Klejbor, I.; Stachowiak, E. K.; Dutta, P.; Roy, I.; Kaur, N.; Bergey, E. J.; Prasad, P. N.; Stachowiak, M. K. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 11539–11544. (10) Roy, I.; Ohulchanskyy, T. Y.; Bharali, D. J.; Pudavar, H. E.; Mistretta, R. A.; Kaur, N.; Prasad, P. N. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 279–284. (11) Huo, Q. S.; Liu, J.; Wang, L. Q.; Jiang, Y. B.; Lambert, T. N.; Fang, E. J. Am. Chem. Soc. 2006, 128, 6447-6453. (12) Chen, J. F.; Ding, H. M.; Wang, J. X.; Shao, L. Biomaterials 2004, 25, 723–727. (13) Akerman, M. E.; Chan, W. C. W.; Laakkonen, P.; Bhatia, S. N.; Ruoslahti, E. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12617–12621. (14) Hoshino, A.; Fujioka, K.; Oka, T.; Suga, M.; Sasaki, Y. F.; Ohta, T.; Yasuhara, M.; Suzuki, K.; Yamamoto, K. Nano Lett. 2004, 4, 2163–2169. (15) Ballou, B.; Lagerholm, B. C.; Ernst, L. A.; Bruchez, M. P.; Waggoner, A. S. Bioconjugate Chem. 2004, 15, 79–86. (16) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stoelzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331–338. (17) Liu, W. H.; Choi, H. S.; Zimmer, J. P.; Tanaka, E.; Frangioni, J. V.; Bawendi, M. J. Am. Chem. Soc. 2007, 129, 14530–14531. (18) Choi, H. S.; Liu, W. H.; Mistra, P.; Tanaka, E.; Zimmer, J. P.; Ipe, B. I.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2007, 25, 1165–1170. (19) Kamps, J. A.; Morselt, H. W. M.; Swart, P. J.; Meijer, D. K. F.; Scherphof, G. L. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 11681–11685. (20) Krieger, M.; Herz, J. Annu. Rev. Biochem. 1994, 63, 601–637. (21) Singh, R.; Pantarotto, D.; Lacerda, L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3357– 3362. (22) Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen, X. Y.; Dai, H. J. Nat. Nanotechnol. 2007, 2, 47–52. (23) Chen, M.; Von Mikecz, A. Exp. Cell Res. 2005, 305, 51–62.

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enzymatic cleavage.24 More recently, Kong et al., Zhao et al., and our group have all reported that SiNPs were nontoxic to cells and molecules at low dosages.25-27 Yinfa Ma et al. have reported the exposure to 15- and 46-nm silica nanoparticles results in a dosedependent cytotoxicity in cultural human bronchoalveolar carcinoma-derived cells.28 These findings pave the way for the use of SiNPs as core nanomaterials for extracellular and intracellular applications. In view of potential in vivo applications, elucidating the in vivo pharmacokinetics of administered SiNPs, as an important indication of the in vivo behavior, is considered very important in the context of the underlying medical debate regarding the safety of novel nanomaterials. Previous studies suggested the inhalation of silica at microscale size by humans has been linked to the pulmonary disease silicosis.29 Chronic inhalation studies with crystalline silica in rats have induced pulmonary fibrosis and cancer.30 Size-dependent bioeffects of other particles have been well documented.31,32 It is likely that the unique properties of nanosized SiO2 particles maybe impose biological effects in vivo that are quite different from its microscale particles. Thus, the biological effects in vivo of nanosized SiO2 particles warrant further studies. However, in contrast to a significant number of studies that report the in vivo bioeffects of nanomaterials, such as quantum dots,13-18 liposomes,19,20 and carbon nanotubes,21,22 the pharmacokinetics of SiNPs has not been extensively reported yet, likely due to the lack of suitable tracking methods. Most of the toxicology and biodynamics of silica nanoparticle studies so far were focused on the pulmonary toxicity after inhalation, intratracheal instillation, and pharyngeal aspiration, as well as their effects on skin toxicity after exposure of skin to SiNPs, which were carried out by mainly using histopathology study, body weight analysis, and biochemical evaluation. Shimada et al. reported the acute and subacute lung toxicity of low dose of ultrafine colloidal SiNPs using bronchoalveolar lavage techniques and histopathological evaluations.33 Xue et al. showed that the silica nanoparticles are not toxic by detecting variations of pathology in organs.34 Cho et al. evaluated the biological distribution as well as the potential toxicity of silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate by using many in vitro tests.35 However, these methods cannot obtain the information of SiNPs (24) He, X. X.; Wang, K. M.; Tan, W. H.; Liu, B.; Lin, X.; He, C. M.; Li, D.; Huang, S. S.; Li., J. J. Am. Chem. Soc. 2003, 125, 7168–7169. (25) Chang, J. S.; Chang, K. L. B.; Hwang, D. F.; Kong, Z. L. Environ. Sci. Technol. 2007, 41, 2064–2068. (26) Jin, Y. H.; Kannan, S.; Wu, M.; Zhao, X. J. Chem. Res. Toxicol. 2007, 20, 1126–1133. (27) He, X. X.; Liu, F.; Wang, K. M.; Ge, J.; Qin, D. L.; Gong, P.; Tan, W. H. Chin. Sci. Bull. 2006, 51, 1939–1946. (28) Lin, W. S.; Huang, Y. W.; Zhou, X. D.; Ma, Y. F. Toxicol. Appl. Pharmacol. 2006, 217, 252–259. (29) Castranova, V.; Vallyathan, V. Environ. Health Perspect. 2000, 108, 675– 684. (30) Saffiotti, U. Prog. Clin. Biol. Res. 1992, 374, 51–69. (31) Brown, D. M.; Wilson, M. R.; MacNee, W.; Stone, V.; Donaldson, K. Toxicol. Appl. Pharmacol. 2001, 175, 191–199. (32) Donaldson, K.; Tran, C. L. Inhalation Toxicol. 2002, 14, 5–27. (33) Kaewamatawong, T.; Shimada, A.; Okajima, M.; Inoue, H.; Morita, T.; Inoue, K.; Takano, H. Toxicol. Pathol. 2006, 34 (7), 958–965. (34) Xue, Z. G.; Zhu, S. H.; Pan, Q.; Liang, D. S.; Li, Y. M.; Liu, X. H.; Xia, K.; Xia, J. H. Zhong Nan Da Xue Xue Bao, Yi Xue Ban 2006, 31, 6–8. (35) Kim, J. S.; Yoon, T. J.; Yu, K. N.; Kim, B. G.; Park, S. J.; Kim, H. W.; Lee, K. H.; Park, S. B.; Lee, J. K.; Cho, M. H. Toxicol. Sci. 2006, 89 (1), 338– 347.

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in vivo and real time and need many animals to be used for getting more samples. Recently, in vivo optical imaging using fluorescent probes is commonly used for tracking of quantum dots and other fluorescence probe functionalized nanomaterials in animals.14,15,18,36 Highly fluorescent core-shell silica nanoparticles made by luminescent materials doped in the silica shells are widely used for cellular and subcellular structure imaging,5-7 which will also be promising as tools for sensing and imaging in vivo if they can be delivered to the animals. Thus, for instance, Scherman et al. have recently developed persistent luminescent silica nanoparticle probes and determined the feasibility in real time, in vivo biodistribution imaging.37 However, the biological fate of SiNPs has never been visually revealed in vivo by using an optical imaging system. More importantly, critical pharmacological parameters such as blood circulation and clearance half-life and urinary excretion that are essential for the development of any pharmaceutical have yet to be determined. This need is of fundamental importance for the development of SiNP-based gene and drug delivery systems. Here, we used an in vivo optical imaging system to study the biological effects of intravenous (iv) injected SiNPs in vivo by using RuBPY dye doped in the silica matrix as an adoptable method to track SiNPs. Three types of chemically modified SiNPs with a size of ∼45 nm were investigated, giving us some interesting results and a more complete understanding of the related in vivo behaviors of SiNPs on surface characteristics. SiNPs in mice, such as their blood circulation and clearance half-life, their biodistribution in biological organs, and their partial renal excretion have been observed in real time. Ex vivo analysis, including resected organ optical imaging, transmission electron microscope (TEM) imaging, and energy-dispersed X-ray spectrum analysis of urine samples, were used to further confirm the in vivo visualization. EXPERIMENTAL SECTION Chemicals and Materials. N-[(3-Trimethoxysilyl)propyl]ethylenediamine triacetic acid (EDTAS) and 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (MPEGTMS) (6-9 PEG units) were obtained from Gelest Inc. and used as received. Tris(2,2bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy), Triton X-100, and 50% glutaraldehyde solution were purchased from Sigma-Aldrich. All the other chemicals were purchased from Reagent & Glass Apparatus Corp. of Changsha and were used without further purification. All solutions were prepared with deionized water. Preparation of Surface-Modified RuBPY-Doped SiNPs. RuBPY-doped SiNPs with free hydroxyl groups were first synthesized according to previously published procedures.5 Briefly, a water-in-oil microemulsion was prepared by mixing 1.77 mL of Triton X-100, 7.5 mL of cyclohexane, 1.8 mL of n-hexanol, 400 µL of water, and 50 µL of 0.1 M RuBPY solutions. After stirring for 1 h, 200 µL of tetraethyl orthosilicate (TEOS) was then added as a precursor for silica formation, followed by the addition of 100 µL of NH4OH to initiate the polymerization process. The reaction (36) Chen, H. Y.; Zhang, J.; Qian, Z. Y.; Liu, F.; Chen, X. Y.; Hu, Y. Z.; Gu, Y. Q. Nanotechnology. 2008, 19, 185707–185717. (37) Chermont, Q. L. M.; Chane´ac, C.; Seguin, J.; Pelle´, F.; Maıˆtrejean, S.; Jolivet, J. P.; Gourier, D.; Bessodes, M.; Scherman, D. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 9266–9271.

Figure 1. (A). Schematic drawings of three types of surface-modified SiNPs. (B). TEM imaging of prepared three types of surface-modified SiNPs: (a) OH-SiNPs; (b) COOH-SiNPs; (c) PEG-SiNPs. Scale bar, 50 nm. (C). The cumulative percent leakage of RuBPY from surfacemodified SiNPs incubated in full mouse serum at 37 °C. (D). The photobleaching of the three types surface-modified SiNPs excited for 7200 s by successive xenon lamp irradiation at 488 nm.

was allowed to continue for 24 h at room temperature to form SiNPs with free hydroxyl groups. Carboxyl-terminated SiNPs (COOH-SiNPs) and poly(ethylene glycol) (PEG)-terminated SiNPs (PEG-SiNPs) were then prepared from additional coating procedures in the water-in-oil microemulsion containing hydroxy-SiNPs (OH-SiNPs) through the synchronous hydrolysis of 50 µL of TEOS and 100 µL of N-[(3-trimethoxysilyl)propyl]ethylenediamine triacetic acid, and 50 µL of TEOS and 100 µL of MPEGTMS (6-9 PEG units), respectively (Figure 1A). The success of the modification procedure was assessed by zeta potential measurements (Malvern Zetasizer 3000HS). Characterization of Surface-Modified RuBPY-Doped SiNPs. The prepared surface-modified SiNPs were examined and characterized by high-resolution TEM (JEM-3010). Owing to the importance of stability of RuBPY-doped SiNPs for tracking the behavior of surface-modified SiNPs in live animals, the stability of the RuBPY doped in SiNPs was investigated by incubating RuBPY-doped, surface-modified SiNPs in full mouse serum at 37 °C for up to 8 h. At scheduled intervals, portions of RuBPY-doped, surface-modified SiNPs suspension were collected at different time

points and dialyzed through 3000-Da pore size membranes. The fluorescence intensity of RuBPY in the filtrates was analyzed using the fluorescence plate reader, sign as F supernatant. While the fluorescence intensity of full mouse serum at the same detection conditions was regarded as blank, F blank. And the total fluorescence intensity of RuBPY-doped, surface-modified SiNPs, F total, was measured by suspending nanoparticles in full mouse serum as above. The RuBPY leakage from surface-modified SiNPs was calculated by using the relation (F supernatant - F blank)/ (F total - F blank) × 100%. The photobleaching of the RuBPY doped in SiNPs has also been quantitatively tested. The SiNPs in PBS buffer were excited for 7200 s by successive xenon lamp irradiation at 488 nm. The average intensity was recorded and normalized to its intial value. Animal Models and SiNPs Injection. The athymic BALB/c (Balb/C-nu) mice were obtained from Beijing Vital River Laboratory Animal Co., Ltd. (BALB/c); 65 male nude mice were used at ages from 6 to 12 weeks. Adult female Sprague-Dawley (SD) rats (n ) 6), weighing 50-80 g were obtained from Hunan provincial center for disease prevention and control. All animal Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 2. In vivo imaging biodistribution of different iv injected surface-modified SiNPs at different time points, postinjection (A-C, (a), abdomen imaging; (b), back imaging.). (A) OH-SiNPs; (B) COOHSiNPs; (C) PEG-SiNPs. Arrows mark the location of the kidney (K), liver (L), and urinary bladder (Ub).

operations were in accord with institutional animal use and care regulations, according to protocol No. SYXK (Xiang) 2008-0001, approved by Laboratory Animal Center of Hunan. For the in vivo biodistribution imaging, mice were first anesthetized intraperitoneally by 2% pentobarbital (4 µL/g of animal weight) and 0.08% promethazine hydrochloride (2.5 µL /g of animal weight). Then, three types of surface-modified RuBpy dye doped SiNPs (3 mg/mL) in 200 µL of PBS buffer were injected, respectively, into the tail veins of mice (typical 20 g, thus 0.03 mg of SiNPs/g of animal weight). To further directly visualize the renal excretion of SiNPs, PEG-terminated SiNPs, as a representative, were injected to the SD rats from the abdominal arcus aorta by the following method. The SD rats were first anesthetized intraperitoneally as described above and anatomized with exposure of abdominal aortae. The abdominal aorta was ligated with a silk thread, and then the PEG-terminated SiNPs was injected into the abdominal arcus descendens of rats at a dose of 0.03 mg/g of animal weight. In Vivo Imaging and Analysis. We performed in vivo imaging experiments on nude (nu/nu) mice bearing different types of SiNPs. Whole body images of SiNPs intravenously injected mice were acquired and analyzed at many time points postinjection (pi) (up to 24 h) using the Maestro in vivo imaging system (CRI, Inc., excitation, 465-495 nm; emission, 515 nm long-pass). The Maestro optical system consists of an optical head that includes a liquid crystal tunable filter (with a bandwidth of 10 nm and a scanning wavelength range of 500-950 nm), an optical coupler, and a scientific-grade monochrome CCD camera, along with image acquisition and analysis software (Nuance 1.4.2). The 9600

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tunable filter was automatically stepped in 10-nm increments from 550 to 700 nm while the camera captured images at each wavelength with constant exposure. The 16 resulting TIFF images were loaded into a single data structure in memory, forming a spectral stack with a spectrum at every pixel. The total acquisition time was ∼16 s. With spectral imaging software, small but meaningful spectral differences could be rapidly detected and analyzed. The pure autofluorescence spectra and RuBpy dye doped SiNPs spectra were manually selected from the spectral image using the control mice to select appropriate regions. Spectral unmixing using the pure autofluorescence and RuBpy dye doped SiNPs spectra yielded the superimposed image, unmixed autofluorescence image and unmixed RuBpy dye doped SiNPs image (Figure S1, Supporting Information, SI). The mean fluorescence intensity was analyzed using MetaMorph 6.1 image analysis software. To obtain circulating lifetimes by whole mouse imaging, mice intravenously injected with different types of SiNPs were imaged respectively at several minute intervals posinjection. Lifetime data were then generated by selecting six 2 × 2 mm regions of interest per mouse, where the regions of interest were selected to accord with conspicuous surface blood vessels, and by tracking them through time. Mean fluorescence intensities from six regions of interest on each mouse were measured, normalized to maximum initial values, and plotted versus time after injection. The fluorescence change in the bladder of PEG-SiNPs intraarterially injected SD rats with skin and peritoneum removed was also in vivo imaged and analyzed at many time points pi as described above. Ex Vivo Imaging and Analysis. After in vivo imaging, we killed the mice bearing different types of SiNPs by cervical dislocation under narcosis at 4.5 h pi. The anatomized mice and dissected organs, including liver, kidney, spleen, lung, heart, spermary, bladder, brain, and muscle, were imaged with the Maestro in vivo imaging system as described above. The relative fluorescence intensity of resected organs at 4.5 h pi was also further compared using MetaMorph 6.1 image analysis software. Urine Sample Collection and Analysis. The urine samples were collected directly from the urinary bladder of mice intravenously injected with different types of SiNPs through bladder puncture. The fluorescence imaging and optical spectra of the collected urine samples were first recorded using the Maestro optical in vivo imaging system. Next, for TEM imaging and energy-dispersed X-ray spectrum (EDS) analysis of the urine samples, the collected urine samples were respectively centrifuged. Then the precipitates were incubated with 1 M HCl solution for 30 min and centrifuged again. The pretreated precipitates were washed three times with water, followed by suspension in water, and dropped onto a TEM grid for detection. The images of prepared surface-modified SiNPs and the presence of surface-modified SiNPs in urine samples were detected using high-resolution TEM (JEM-3010) and analyzed with EDS (Inca Oxford). RESULTS AND DISCUSSION Characterization of Surface-Modified SiNPs. In this work, we investigated the biodistribution and urinary excretion of three types of surface-modified silica nanoparticles in mice, including SiNPs with free hydroxyl groups, carboxyl-terminated SiNPs, and

Figure 3. Real-time in vivo imaging of fluorescence signal changes in the urinary bladder and urinary meatus of the mice injected with the OH-SiNPs at different time points, postinjection (A, superimposed image; B, unmixed RuBpy dye doped SiNPs image). (C) Real-time in vivo imaging of the fluorescence change process in the urinary bladder of the SD rat at different time points, postinjection after abdominal aorta injection of PEG-SiNP. Rings and arrows mark the location of the liver (L), urinary bladder (Ub), and urinary meatus (Um).

PEG-terminated SiNPs (hereinafter referred to as OH-SiNPs, COOH-SiNPs, and PEG-SiNPs). The surface-modified SiNPs were examined and characterized by Malvern zetasizer and TEM. Figure 1B shows images characteristics of the three types of surface-modified SiNPs obtained after dispersion of the sample onto a TEM grid and evaporation of the solvent. The different surface coating had a profound effect on the charge of SiNPs, possessing surface charges of -11.0, -34.5, and -18.1 mV, for OH-SiNPs, COOH-SiNPs, and PEG-SiNPs, respectively. However, the TEM images demonstrated that the OH-SiNPs, COOH-SiNPs, and PEG-SiNPs were quite uniform in size, having diameters of 45 ± 2, 47 ± 3, and 47 ± 4 nm, respectively, which indicated that the diameter of the SiNPs have not changed obviously after further surface modification. Additional experiments were carried out to investigate the stability of RuBPY doped in SiNPs to account for leakage of RuBPY from nanoparticles. As shown in Figure 1C, the percentage of RuBPY leakage from the OH-SiNPs, COOH-SiNPs, and PEGSiNPs was about 5.2, 4.4, and 3.3%, respectively, after incubation for 8 h in full mouse serum at 37 °C, suggesting that the three surface-modified RuBPY-doped SiNPs could effectively avoid RuBPY leakage. Additionally, the surface-modified RuBPY-doped SiNPs prepared in this paper have strong fluorescence intensity and excellent photostability. With reference to Figure 1D, over 7200-s successive scans, the relative intensity of the three types

of surface-modified RuBPY-doped SiNPs all still kept more than ∼85% of their initial value, which are very important for the RuBPY dye doped in the silica matrix as a synchronous fluorescent signal indicator to track SiNPs in mice. In Vivo Biodistribution and Urinary Excretion of Different Surface-Modified SiNPs. The biodistribution of the three different surface-modified SiNPs in mice was investigated by noninvasive in vivo imaging, and the resulting images were shown in Figure 2A-C and QuickTime movies in Supporting Information. Immediately after iv tail vein injection, fluorescence emitted from all the SiNPs with different surface modification was easily visualized in the superficial vasculature of the whole body. Subsequently, as blood circulated, the SiNPs were seen to gradually distribute and deposit inside different biological organs in a surface coating dependent manner. By 3-h pi, both OH-SiNPs and COOHSiNPs were cleared from the circulation and exhibited prominent distribution in the liver, urinary bladder, and kidney. By comparison, the PEG-SiNPs remained visible in the circulation at 3-h pi and presented lower liver uptake than OH-SiNPs and COOH-SiNPs, although the fluorescence signal could be imaged in the urinary bladder and kidney. As indicated by the fluorescence signal, images show that PEG-SiNPs remained visible in the circulation, even at 5-h pi. By 24-h pi., the fluorescence signals of OH-SiNPs and COOHSiNPs in the liver had decreased, but were still grossly visible, while the fluorescence of PEG-SiNPs was still not evident. The circulation Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 4. Ex vivo optical imaging of anatomized mice with injection of surface-modified SiNPs and some resected organs during necropsy at 4.5-h pi. (1), (2), (3), and (4) representing intravenously injected mice groups with OH-SiNPs, COOH-SiNPs, PEG-SiNPs and uninjected mice, respectively. (a) Superimposed images of anatomic mice; (b) superimposed images of some resected organs; (c) images of some resected organs that were analyzed using MetaMorph 6.1 image analysis software. Arrows represented the organs of liver, kidney, spleen, lung, heart, spermary, bladder, brain, and muscle, respectively, from left to right in (b).

Figure 5. Fluorescence images of collected urine samples from the iv-injected, surface-modified SiNPs mice (A) and the optical spectra of the collected urine samples and OH-SiNPs solutions (B) obtained using an in vivo imaging system. (a, OH-SiNPs solution; b, collected urine from uninjected mice; c-e, collected urine from mice iv injected with OH-SiNPs, COOH-SiNPs, and PEG-SiNPs, respectively.).

lifetime obtained by in vivo imaging revealed that PEG-SiNPs exhibit a much longer blood circulation time (t1/2 ) 180 ± 40 min) than OHSiNPs (t1/2 ) 80 ± 30 min) and COOH-SiNPs (t1/2 ) 35 ± 10 min) (Figure S2, SI). These results suggest that the PEG chemically modified SiNPs showed minimal liver uptake, but increased circulatory lifetime, as has been generally observed for other PEG-capped nanoparticles.15,22,37,38 Interestingly, the in vivo optical imaging results also demonstrated the fluorescence signal of surfaced-modified SiNPs was obviously presented in some organs involved in the formation and excretion of urine for all three types of SiNPs iv-injected mice. Specifically, the behavior and stability of SiNPs in full mouse serum enabled the utilization of in vivo real-time 9602

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imaging to view the fluorescence signal in some organs involved in the formation and excretion of urine. For example, parts A and B of Figure 3 show real-time images of fluorescence signal changes in the urinary bladder and urinary meatus of the mice injected with the OH-SiNPs. The observed fluorescence signals of SiNPs and their stability of SiNPs in serum enabled us to conceive that some iv-injected SiNPs were excreted through the renal route. To further demonstrate the renal excretion of SiNPs with a size of ∼45 nm, PEG-SiNPs was selected as representative and injected into SD rats from the arcus aorta. Then some of PEG(38) Moghimi, S. M.; Szebeini, J. Prog. Lipid Res. 2003, 42, 463–478.

SiNPs were flowed directly into the afferent arteriole. If some SiNPs can be excreted, the renal excretion could be visualized quickly and directly, as could transport down the ureters bilaterally and into the bladder. As shown in Figure 3C, with time postinjection increased, the fluorescence signal of PEG-SiNPs in the bladder increased. After 30-min postinjection, obvious red fluorescence of PEG-SiNPs was found in the bladder, indicating that the SiNPs with a size of ∼45 nm were indeed partially excreted from the renal route. Further Evidence for the Biodistribution and Urinary Excretion of Different Surface-Modified SiNPs. To confirm the in vivo imaging results, the ex vivo organ optical imaging and urine samples analysis have been performed. Figure 4 shows ex vivo optical images of anatomized mice with skin and peritoneum removed and some organs directly resected at 4.5-h pi. It was obvious that relatively intensive luminescence of OH-SiNPs and COOH-SiNPs remained mainly in the liver, kidney, and urinary bladder. However, intensive fluorescence could be detected in the whole body and all of the organs removed from those mice examined 4.5 h after PEG-SiNP administration, even though the signal intensity of the liver was much lower than that of either OH-SiNPs or COOH-SiNPs. However, uninjected mice showed no comparable fluorescence in any organs. The results demonstrated that major sites of fluorescence seen by ex vivo optical imaging were almost consistent with those seen by noninvasive imaging, which indicated that the biodistribution of surfacemodified SiNPs inside biological organs could be sensed and imaged in vivo by using RuBPY dye doped in the silica matrix as an adoptable method to track SiNPs. After the biodistribution validation, we carried out an excretion study to establish whether the excretion of SiNPs was through the renal route, as speculated above. The three modified SiNPs were respectively injected into different mice from the tail veins, and urine was collected directly from the urinary bladder through bladder puncture at 4.5 pi. The fluorescence imaging and optical spectra of the collected urine specimens were recorded by the in vivo imaging system and were correlated with those of OH-SiNPs solution (Figure 5). TEM and EDS analysis of urine samples confirmed the presence of intact OH-SiNPs, COOH-SiNPs, and PEG-SiNPs. (SI, Figure S3). The results from the excretion studies confirmed that the three types of iv-injected, chemically modified SiNPs with a size of ∼45 nm are, in fact, excreted into urine as

intact SiNPs through the renal route. Hence, the real-time, in vivo imaged fluorescence signal in some organs of the urinary system can reveal the in vivo excretion of iv-injected, chemically modified SiNPs. However, the fact that obvious fluorescence signal of SiNPs remains in mice at 24-h pi suggested relatively a little excretion of iv-injected SiNPs. CONCLUSION We have investigated the in vivo biodistribution and urinary excretion of three types of surface-modified silica nanoparticles (OH-SiNPs, COOH-SiNPs, and PEG-SiNPs) in animals using an optical imaging method, taking advantage of RuBPY dye doped in the silica matrix as a synchronous fluorescence signal. Results from the in vivo imaging studies show that the iv-injected OHSiNPs, COOH-SiNPs, and PEG-SiNPs, with a size of ∼45 nm, can all be cleared from the circulation and presented inside organs. The PEG-SiNPs exhibit relatively longer blood circulation times and lower uptake by the liver than OH-SiNPs and COOH-SiNPs. Even more interestingly, in vivo imaging reveals that all the ivinjected types of SiNPs are partly excreted through the renal route. Furthermore, the in vivo imaging results were well confirmed by ex vivo organ optical imaging, TEM imaging, and energydispersed X-ray spectrum analysis of urine samples. This work puts forward direct implications for the use of SiNPs as delivery systems and imaging tools in live animals. ACKNOWLEDGMENT This work was supported by the National Key Basic Research Program (2002CB513100-10), National Science Foundation of P.R.China (90606003, 20775021), Program for New Century Excellent Talents in University (NCET-06-0697), and Outstanding Youth Foundation of Hunan Province (06JJ10004). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review September 5, 2008. Accepted October 25, 2008. AC801882G

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