Photosensitizer encapsulated organically modified silica nanoparticles for direct two-photon photodynamic therapy and In Vivo functional imaging

Biomaterials 33 (2012) 4851e4860

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Photosensitizer encapsulated organically modified silica nanoparticles for direct two-photon photodynamic therapy and In Vivo functional imaging Jun Qian a, b, d, Dan Wang a, b, Fuhong Cai a, b, d, Qiuqiang Zhan a, b, Yalun Wang a, b, Sailing He a, b, c, * a

Centre for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, Zhejiang University (ZJU), Hangzhou, Zhejiang 310058, PR China JORCEP [Joint Research Center of Photonics of the Royal Institute of Technology (Sweden), Lund University (Sweden) and Zhejiang University (ZJU)], Hangzhou, Zhejiang 310058, China c Department of Electromagnetic Engineering, Royal Institute of Technology, 100 44 Stockholm, Sweden d Joint Research Laboratory of Optics of Zhejiang Normal University and Zhejiang University, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 February 2012 Accepted 27 February 2012 Available online 6 April 2012

Nanoparticle-assisted two-photon imaging and near infrared (NIR) imaging are two important technologies in biophotonics research. In the present paper, organically modified silica (ORMOSIL) nanoparticles encapsulated with either PpIX (protoporphyrin IX) photosensitizers or IR-820 NIR fluorophores were synthesized and optically characterized. Using the former ORMOSIL nanoparticles, we showed: (i) direct excitation of the fluorescence of PpIX through its efficient two-photon absorption in the intracellular environment of tumor cells, and (ii) cytotoxicity towards tumor cells by PpIX under two-photon irradiation. The latter ORMOSIL nanoparticles can be used as efficient NIR fluorescent contrast agents for various types in vivo animal imaging. We applied IR-820 doped ORMOSIL nanoparticles in in vivo brain imaging of mice. We also demonstrated the applications of them to sentinel lymph node (SLN) mapping of mice. Finally, we showed that the nanoprobes could target the subcutaneously xenografted tumor of a mouse for long time observations. ORMOSIL nanoparticles have great potentials for disease diagnosis and clinical therapies. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: ORMOSIL nanoparticles Two-photon Photodynamic therapy NIR Fluorescence In vivo imaging

1. Introduction During past years, organically modified silica (ORMOSIL) nanoparticles [1] have shown their great potentials as an ideal nanoplatform for various multimodal bio-imaging and “theranostic” (diagnosis-therapy) applications [2e5]. ORMOSIL nanoparticles are mesoporous with big pores in their matrix, which can facilitate some controlled release of encapsulated biomolecules like drugs, proteins and reactive oxygen species, etc. ORMOSIL nanoparticles can be loaded with either hydrophilic or hydrophobic drugs/dyes, protecting them against denaturation by the extreme bio-environment: by changing the dye type, the fluorescent ORMOSIL nanoparticles can achieve good fluorescence quantum yield and tunable photoluminescence that spans the entire visible and IR spectrum; by changing the drug type (such as genetic materials, chemotherapeutic and photodynamic drugs), the fluorescent ORMOSIL nanoparticles can be used to treat different kinds of diseases. ORMOSIL nanoparticles can be surface-functionalized with various chemical * Corresponding author. Centre for Optical and Electromagnetic Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, Zhejiang University (ZJU), Hangzhou, Zhejiang 310058, PR China. Tel.: þ86 571 88206525; fax: þ86 571 88206512. E-mail address: [email protected] (S. He). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.02.053

groups (e.g., carboxyl/thiol/amino/hydroxyl), and can be further conjugated with different targeting biomolecules (such as proteins and antibodies) and additional functionalities (such as probes for MR/radio imaging). Chemically inert ORMOSIL nanoparticles are transparent to visible/NIR light, and possess good biocompatibility. Due to the aforementioned features, ORMOSIL nanoparticles have been widely applied in photodynamic therapy (PDT). Previously, Roy et al. [6] and Ohulchanskyy et al. [7] have used HPPH doped ORMOSIL nanoparticles for in vitro PDT of tumor cells. Our group have utilized ORMOSIL nanoparticles to encapsulate protoporphyrin IX (PpIX), and applied them in PDT of HeLa cells [8,9]. Furthermore, ORMOSIL nanoparticles have also been used in many in vitro bioimaging examples [10e12]. Two-photon excitation induced bio-imaging has many unique advantages [13e16]. Due to a quadratic dependence of two-photon absorption on laser intensity [17,18], the sample region outside the beam focus cannot be excited, and it could reduce the possibility of photobleaching. The nonlinear excitation mode is also helpful to improve the spatial resolution of imaging, since only the site where the laser beam is focused can be efficiently excited. These two advantages of two-photon excitation are very important to long-term and selective imaging/PDT of biological specimen.

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Kim et al. used to co-encapsulate HPPH and a special type of twophoton absorbing fluorophores into ORMOSIL nanoparticles, and used them for indirect two-photon PDT of in vitro cells, which was based on the Foster resonance energy transfer (FRET) effect inside nanoparticles [19]. Furthermore, two-photon excitation has great potentials for deep-range tissue imaging by utilizing laser scanning microscopy. For one-photon bio-imaging, photosensitizers usually absorb light in the visible spectral region below 700 nm, where light penetration into the skin is only a few millimeters. On the other hand, the laser wavelength for two-photon excitation is usually in the range of 700e900 nm, which is typically considered as the transparent window of light for tissues [20], and thus the penetration of excitation light and the generation of deep-tissue signals can be improved. Another promising approach towards deep-range imaging is to use near infrared (NIR) excitation and emission [21,22]. Compared with visible light, NIR light (700e900 nm) is less absorbed and scattered by biological tissue and thus NIR excitation can greatly increase the penetration depth and emission. Furthermore, NIR light has lower energy than ultraviolet- and visible light, and less fluorophores in tissue can be stimulated if NIR excitation is adopted. Thus it can efficiently restrain the generation of autofluorescence and improve the contrast of the image. Different from two-photon excitation, NIR deep-tissue imaging is usually performed on a macro in vivo imaging system. Although ORMOSIL nanoparticles have been used in various types of in vitro bioimaging, the reports about their in vivo applications are relatively rare. Recently, Kumar et al. [23] have utilized DY776-doped ORMOSIL nanoparticles for NIR in vivo animal imaging. In their work, NIR signals from ORMOSIL nanoparticles were helpful to investigate the biodistribution and clearance process of ORMOSIL nanopaticles in animal bodies. Histological analysis of the dissected organs illustrated that ORMOSIL nanopaticles produced no cytotoxic effects in animal tissues. However, no further applications of NIR fluorescent ORMOSIL nanoparticles have been demonstrated in their research. In this paper, we report the synthesis of ORMOSIL nanoparticles, which are encapsulated with photosensitizer PpIX and NIR fluorophores IR-820 [24]. PpIX photosensitizers have been officially approved for use in clinical treatments and are commercially available [25,26]. Here we measure the one- and two-photon absorption/fluorescence properties of PpIX doped ORMOSIL nanoparticles, and investigate their feasibility in fluorescence imaging and PDT towards tumor cells under two-photon excitations. We

then verify the deep-range imaging capacity of IR-820 encapsulated ORMOSIL nanoparticles in simulated tissue, and investigate their applications in in vivo functional imaging of mice [e.g., brain imaging, sentinel lymph node (SLN) mapping and tumor targeting] 2. Experimental section 2.1. Materials Aerosol OT (98%), VTES (97%), APTES (98%), PpIX, IR-820 and O,O0 -Bis[2-(N-Succinimidyl-succinylamino)ethyl] polyethylene glycol 3000 (NHS-PEG-NHS, 3000) were purchased from Sigma Aldrich. DMSO, 1-butanol (99.8%), acetone and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co., Ltd, China. Cell-culture products, unless otherwise mentioned, were purchased from GIBCO. All the above chemicals were used without any additional purification, and DI water was used in all the experimental steps. 2.2. Synthesis of PpIX/IR-820 doped ORMOSIL nanoparticles ORMOSIL nanoparticles with PpIX or IR-820 were synthesized in the nonpolar core of Aerosol-OT/DMSO/water micelles (shown in Fig. 1) [12]. Typically, the micelles were prepared by dissolving a certain amount of Aerosol-OT and 1-butanol in 10 ml of DI water by vigorous magnetic stirring. 400 ml of PpIX/IR-820 in DMSO (1 mM) was then added to the solution under magnetic stirring. Half an hour later, 100 ml of neat VTES was added to the micellar system, and the resulting solution was stirred for about 1 h. Next, ORMOSIL nanoparticles were precipitated by adding 15 ml of APTES and stirred for another 20 h at room temperature. After the formation of the nanoparticles, surfactant Aerosol-OT, cosurfactant 1-butanol, residual VTES and APTES were removed by dialyzing the solution against DI water in a 12e14 kDa cutoff cellulose membrane for 50 h. The dialyzed solution was then filtered through a 0.45 mm cutoff membrane filter to be used in later experiments. 2.3. Conjugating polyethylene glycol (PEG) with IR-820 doped ORMOSIL nanoparticles As illustrated in Fig. 1B, 18 mg NHS-PEG-NHS (MW: 3000) was added to 2.5 ml aqueous dispersion of IR-820 doped ORMOSIL nanoparticles, and NaOH solution was added drop by drop to keep the pH value of the solution around 8. Since amino groups were grafted on the surfaces of ORMOSIL nanoparticles, PEG molecules could conjugate with nanoparticles through specific NHS-NH2 bonds in the weak alkali solution. 3 h later, the reaction solution was dialyzed against DI water for 24 h to remove the unreacted excess NHS-PEG-NHS molecules. 2.4. Characterization The structures of two kinds of ORMOSIL nanoparticles were taken by a JEOL JEM1200EX transmission electron microscope (TEM) operated at 160 kV in bright-field mode. The absorption/transmission spectra of nanoparticles within the wavelength region of 300 nme900 nm were recorded by a Shimadzu 2550 UVevis scanning spectrophotometer. One-photon excited fluorescence spectrum of PpIX doped ORMOSIL nanoparticles was obtained by a Fluorescence Spectrophotometer (F-2500, HITACHI, Japan). Two-photon fluorescence of PpIX doped ORMOSIL

Fig. 1. Synthesis illustration of PpIX doped ORMOSIL nanoparticles (A) and PEG modified IR-820 doped ORMOSIL nanoparticles (B).

J. Qian et al. / Biomaterials 33 (2012) 4851e4860 nanoparticles was excited by a fs laser (Spectra Physics, wavelength: 800 nm), and the spectrum was recorded with an optical fiber spectrometer (Ocean Optics). NIR fluorescence spectrum of IR-820 doped ORMOSIL nanoparticles was measured with a Maestro in vivo imaging system (CRI, Inc. Woburn, MA) consisting of an optical head, an optical coupler, a cooled scientific-grade monochrome CCD camera and an image acquisition/analysis software. The excitation source was deep-red broadband light with a peak wavelength at 704 nm, and a long-pass filter was used to extract the fluorescence signal and block the residual excitation light. A liquid-crystal tunable filter was automatically tuned with 10-nm increments from 740 to 950 nm, while the cooled monochrome CCD camera recorded the fluorescence intensity at each wavelength.

2.5. In Vitro studies HeLa cells (human cancer cell lines) were cultivated in Dulbecco minimum essential media (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin, and 1% amphotericin B. One day before the treatment, the cells were seeded in 35 mm cultivation dishes at a confluence of 70e80%. During the treatment, 200 ml stock dispersion of PpIX doped ORMOSIL nanoparticles was added into the HeLa cell plates. HeLa cell plates without any treatment were used for control experiment. The cell incubation process lasted for 2 h at 37  C with 5% CO2. Then the cells were washed thrice with PBS (phosphate buffered saline, 1x) and imaged with an upright two-photon fluorescence confocal microscope (FV1000, Olympus, Inc) under an 800 nm fs laser excitation. To confirm the in vitro two-photon PDT effect of PpIX doped ORMOSIL nanoparticles, HeLa cells with and without nanoparticle treatment were irritated by the 800 nm fs laser for 2 min. Cell morphologies at various times post laser-irritation were then recorded with the confocal microscope for further analysis.

2.6. In Vivo studies All in vivo experiments were performed in compliance with Zhejiang University Animal Study Committee’s requirements for the care and use of laboratory animals in research. 18e21 g male black mice (C57 line) and nude mice were used for animal imaging studies. The animal housing area (located in Animal Experimentation Center of Zhejiang University) was maintained at 24  C with a 12 h light/ dark cycle, and animals were fed with water and standard laboratory chow. Concerning NIR imaging of IR-820 doped ORMOSIL nanoparticles in the brain, the skull of a black mouse was open up through microsurgery, and four 0.5 ml aqueous dispersions of IR-820 doped ORMOSIL nanoparticles were microinjected into different depths of its brain (1 mm, 2 mm, 3 mm, 4 mm). The black mouse was anesthetized with pentobarbital, and then imaged using the aforementioned Maestro in vivo optical imaging system. To investigate the SLN mapping of ORMOSIL nanoparticles in mice, we intradermally injected 0.1 ml nanoparticles (in 5% glucose) into the right forepaw pad of a nude mouse, and anesthetized it with pentobarbital at various times after the injection. The sedated animals were then imaged using the in vivo optical imaging system. The xenografted mouse models were generated by subcutaneously injecting 3 ?106 HeLa cells/mouse (in 0.3 ml 1  BS, pH ¼ 7.4) in the left scapular region of 18e21 g male nude mice. Tumor growth was monitored every 2 days until a tumor size of approximately 1 cm in diameter was observed. The tumor-bearing mice were then intravenously injected with 0.3 ml IR-820 doped ORMOSIL nanoparticles (in 5% glucose) and imaged at different time points post-injection to study the targeting of the nanoparticles in the tumor.

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3. Results and discussion 3.1. Synthesis of ORMOSIL nanoparticles Fig. 2 shows TEM images of as-synthesized PpIX (A) and IR-820 (B) doped ORMOSIL nanoparticles. The former nanoparticles (A) have an average diameter of about 25 nm, and some mesoporous matrix could also be observed on their surfaces. The latter nanoparticles (B) have an average diameter of about 42 nm. Both kinds of nanoparticles have a spherical morphology with a narrow distribution of sizes.

3.2. Optical characterization of PpIX/IR-820 doped ORMOSIL nanoparticles The inset in Fig. 3A shows the normalized linear absorption spectrum of aqueous dispersion of PpIX doped ORMOSIL nanoparticles. Our two-photon fluorescence and PDT experiment was carried out under an upright confocal microscope, and a fs laser beam would pass through a certain length of water environment before it could interact with nanoparticles, and thus the influence of water towards the laser beam should be considered accordingly. As we can see in the figure, the strongest linear absorption band of PpIX doped ORMOSIL nanoparticles was located around the wavelength of 404 nm, and extended its decaying tail into 600e650 nm visible spectral range. Fig. 3A shows the linear transmission spectra of 1-cm-thick layer of water and aqueous dispersion of PpIX doped ORMOSIL nanoparticles. Both of them had negligible one-photon attenuation at 700e850 nm, indicating that this spectral range is very suitable as an optical window for twophoton excitation. One-photon (400 nm CW laser excited) and two-photon (800 nm fs laser excited) fluorescence spectra of PpIX doped ORMOSIL nanoparticles were shown in Fig. 3B, and their main envelopes were almost the same. That means in both one-photon and two-photon processes, the excited PpIX molecules were finally relaxed to the same lowest excited electronic-vibrational state(s), from which the fluorescence emission occurred. Thus, when the PpIX molecules are stimulated by 800 nm laser pulses, it needs to absorb at least two photons at the same time to get excited. However, some slight changes could still be observed under different excitation modes. Two-photon excited fluorescence showed a secondary peak around 675 nm, which has a 4 nm-redshift compared with that of one-photon excited fluorescence. Furthermore, the intensity of two-photon excited fluorescence in

Fig. 2. Representative TEM images of PpIX doped ORMOSIL nanoparticles (A) and IR-820 doped ORMOSIL nanoparticles (B).

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Fig. 3. Optical characterizations of PpIX doped ORMOSIL nanoparticles and IR-820 doped ORMOSIL nanoparticles. (A) Linear transmission spectra of a 1-cm-thick water sample and a 1-cm-thick aqueous solution of PpIX doped ORMOSIL nanoparticles, (Inset) normalized linear absorption of PpIX doped ORMOSIL nanoparticles; (B) One- and two-photon excited fluorescence spectra of PpIX doped ORMOSIL nanoparticles; (C) Quadratic dependence of two-photon induced fluorescence on the excitation intensity of PpIX doped ORMOSIL nanoparticles; (D) Normalized absorption and fluorescence spectra of IR-820 doped ORMOSIL nanoparticles, (Inset) Dependence of the absorption coefficient of light in tissue on the wavelength of light (Data taken from Ref. [28]).

the regions of 580e620 nm and 640e700 nm was weaker than that of one-photon excited fluorescence. To verify that the observed fluorescence was the result of twophoton excitation at 800 nm, we measured the emission intensity as a function of the power of the excitation laser, and the results are shown in Fig. 3C. The basic theory of two-photon absorption predicts a quadratic dependence of the emission intensity on the excitation intensity under our experimental conditions. Therefore, on logarithmic scales, there should be a straight line connecting the measured data with a slope of factor 2 for two-photon excitation. In Fig. 3C, the slope of the best-fitting straight line is 2.036, which confirms the two-photon mechanism for 800 nm-fs laser excitation [27]. The absorption and fluorescence spectra of IR-820 doped ORMOSIL nanoparticles were shown in Fig. 3D. They are both located in the "optical transparent windows" of tissues, which ranges from 700 nm to 900 nm (as shown in the inset) [28], and their peak wavelengths are 836 nm and 850 nm, respectively. Hence, NIR excitation and emission light of IR-820 doped ORMOSIL nanoparticles will be less absorbed and scattered by tissues, which can greatly increase the depth of in vivo imaging.

3.3. Two-photon imaging of PpIX doped ORMOSIL nanoparticles in HeLa cells PpIX doped ORMOSIL nanoparticles were used to treat HeLa cells, and a two-photon upright confocal microscope was utilized to confirm their uptake in tumor cells. The 800 nm fs laser in the confocal microscope was used as a direct two-photon excitation source, since PpIX molecules have a distinct one-photon absorption efficiency at 400 nm, while water has a negligible one-photon attenuation at 800 nm (as shown in Fig. 3A). From the results of two-photon fluorescence imaging (shown in Fig. 4B), one can see that bright red fluorescence covered the HeLa cells. The twophoton fluorescence spectrum was further acquired from HeLa cells (inset), and its envelope (with a main emission peak at 632 nm) accorded well with that shown in Fig. 3B, which verified that the red fluorescence was emitted by intracellular PpIX. The cell targeting of nanoparticles was clear and uniform, and no obvious aggregation could be observed. Fig. 4A showed the two-photon fluorescence imaging of control cells (without the treatment of ORMOSIL nanoparticles), and only some weak autofluorescence from cells could be observed. The two-photon imaging results

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Fig. 4. Two-photon excited fluorescence images of HeLa cells treated (A) without and (B) with PpIX doped ORMOSIL nanoparticles. (Inset) Localized two-photon fluorescence spectrum from the stained cells. The excitation wavelength was 800 nm.

illustrated that PpIX molecules could be effectively transferred into the HeLa cells by using ORMOSIL nanoparticles as carriers, which accorded well with what we reported previously [17]. 3.4. Two-photon PDT towards HeLa cells by using PpIX doped ORMOSIL nanoparticles The 800 nm fs laser in the confocal microscope was also used as a direct irradiation source of PDT, and 2 minute irradiation was carried out on both HeLa cell samples treated with and without nanoparticles. For the HeLa cells treated with nanoparticles, no

morphology changes could be observed immediately after the 2 minute irradiation finished (Fig. 5A). However, 8 min after the irradiation, one can see that the HeLa cells became round and some bubbles or the like appeared on their surfaces, indicating that cells were gradually becoming unhealthy (Fig. 5B). The morphology changes of the HeLa cells became more obvious as time went by, and 15 min after the irradiation more bubbles could be observed and some necrosis even appeared around the cells (Fig. 5C). For the nontreated HeLa cells, no obvious changes in the morphology of the HeLa cells could be observed after the light irradiation, as shown in Fig. 5DeF, indicating the excitation light itself was safe for these cells

Fig. 5. Transmission images of HeLa cells, which were treated with (A, B and C) and without (D, E and F) PpIX doped ORMOSIL nanoparticles. (A, D): immediately after the 2 minute irradiation finished; (B, E): 8 min after the 2 minute irradiation; (C, F): 15 min after the 2 minute irradiation. The irradiation light was from a 800 nm fs laser.

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and did not produce direct toxicity. Combining the above experimental results, we can conclude that the destruction of HeLa cells was induced by the reactive oxygen species, which were generated by two-photon excited PpIX. Due to the meso-porosity property of ORMOSIL nanoparticles, oxygen could contact the encapsulated (and two-photon excited) PpIX molecules well and further be stimulated to a reactive state. The reactive oxygen species could then be released from the meso-pores of ORMOSIL nanoparticles to destroy HeLa cells. Furthermore, compared to indirect two-photon PDT [19], fs laser could excite PDT drugs more efficiently in direct two-photon excitations, and thus avoid unnecessary loss of light power during energy transfer processes (e.g., FRET). In the future, we will try to introduce fs pulsed laser to deep tissues without distinct pulse-broadening, utilizing special optical fibers (e.g., photonic crystal fiber), and extend the application of PpIX doped ORMOSIL nanoparticles to in vivo direct two-photon PDT. 3.5. Assessment of NIR deep-range imaging capacity of IR-820 doped ORMOSIL nanoparticles in phantom To demonstrate the deep-range imaging capacity of NIR excited IR-820 doped ORMOSIL nanoparticles, we did some phantom experiments. We prepared a liquid phantom, which was composed of intralipid and black ink, to simulate typical human tissue as they have similar optical properties (especially in the NIR spectra). A glass tube filled with aqueous dispersion of as-synthesized IR-820 doped ORMOSIL nanoparticles was buried in the phantom solution

at three different depths (0.5 cm, 1 cm and 1.5 cm). The wellprepared samples were then imaged with an in vivo imaging system via reflection imaging mode (illumination light and CCD were on the same side of the phantom). Fig. 6AeC show the NIR images of the glass tube at different depths. With the increasing depth of the samples, the NIR signal intensity decreased, as both the excitation and emission lights experience more scattering and absorption for deeper position imaging. The typical NIR spectrum from samples was also recorded (inset of Fig. 6D), and it matched well with that of IR-820 molecules, illustrating that the photoluminescence was indeed emitted from the buried ORMOSIL nanoparticles. The average intensities (at 850 nm) of the central position of the tubes at various depths were shown in Fig. 6D. As one can see, even at 1.5 cm depth, the NIR fluorescence was still strong (280 units) and the NIR image had a high contrast (Fig. 6D), indicating that the excitation and emission lights (both located within a wavelength range from 700 nm to 900 nm) of the IR-820 doped ORMOSIL nanoparticles were not distinctly absorbed in the phantom. According to Fig. 6AeC, the shape of the tube at 1.5 cm depth did not broaden too much compared to that at 0.5 cm depth, indicating that the emission light of the nanoparticles was not distinctly scattered in the phantom. Considering the NIR imaging results in the “simulated human tissue”, we could anticipate the deep-range imaging ability of the IR-820 doped ORMOSIL nanoparticles in some small animals (e.g., nude mice), as their body dimension is usually less than 3 cm (the central position of the body is then no deeper than 1.5 cm).

Fig. 6. (AeC) NIR fluorescence imaging (exposure time: 2000 ms, peak wavelength of deep-red broadband excitation light: 704 nm) of a liquid phantom with a glass tube of IR-820 doped ORMOSIL nanoparticles buried at various depths (A: 0.5 cm, B: 1 cm and C: 1.5 cm). Insets illustrate “reflection imaging mode”; (D) Average intensities (at 850 nm) of the central position of the tubes at various depths, (Inset) Typical NIR fluorescence spectrum from imaged tubes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.6. NIR imaging of IR-820 doped ORMOSIL nanoparticles in mice brain We first applied PEG modified IR-820 doped ORMOSIL nanoparticles to mouse brain imaging. PEG can improve the chemical stability and long circulation of ORMOSIL nanoparticles during in vivo experiment. By counting the scale on the microinjection instrument, we could know the relatively accurate XY-Z (depth) location of the ORMOSIL nanoparticles. In our experiment, the depths of four injected ORMOSIL nanoparticles samples were about 1 mm, 2 mm, 3 mm and 4 mm. As shown in Fig. 7A, four distinct NIR fluorescent points inside the black mouse brain could be clearly discriminated, and the typical fluorescence spectrum accorded well with that of IR-820 doped ORMOSIL nanoparticles. As the injected sites of the nanoparticles got deeper, the brightness of the NIR fluorescence signal decreased accordingly. During microsurgery, the skin covering the skull of the mouse was stripped off, but the skull was still kept. Furthermore, the amount of microinjected ORMOSIL nanoparticles was very small (0.5 ml). Even in this condition, the NIR fluorescence from 4 mm depth ORMOSIL nanoparticles could still be observed very easily. Considering deeprange imaging and drug delivery capacities of ORMOSIL nanoparticles, we anticipate they will have great application potentials of disease diagnosis/functional therapy in animal brain, such as blood brain barrier penetration for drug supply, etc. 3.7. IR-820 doped ORMOSIL nanoparticles for SLN mapping of mice We then applied PEG modified IR-820 doped ORMOSIL nanoparticles in NIR mice sentinel lymph node (SLN) mapping. SLN is the first group of lymph nodes receiving metastatic cancer cells by direct lymphatic drainage from a primary tumor. Accurate

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identification and biopsy of SLN can enable clinicians to focus on certain lymph nodes and perform more detailed tracking of cancer cell diffusion. Hence, SLN mapping has attracted great attention as a key process in SLN biopsy for cancer staging and surgery [29]. In recent years, many nanomaterials [30e32] (e.g., quantum dots and gold nanoparticles) have been successfully applied in SLN mapping. The diffusion and accumulation process of PEG modified IR-820 doped ORMOSIL nanoparticles in the mouse SLN over time is shown in Fig. 8. When nanoparticles were injected intradermally into the forepaw pad of a mouse, they diffused rapidly from the injection site into the lymphatics. Four minutes later, some NIR fluorescence signals could already be observed at an axillary node of the mouse (Fig. 8A). As time went by, the NIR signal became stronger and more obvious in the SLN site, and 20 min later, its fluorescence intensity reached maximum (Fig. 8B). After that, nanoparticles gradually migrated from the SLN, and the NIR fluorescence signal intensity in the SLN decreased accordingly (Fig. 8C). Furthermore, the mouse was put down and its SLN was taken out and imaged accordingly. As shown in Fig. 8D, the dissected SLN emitted NIR fluorescence, and its spectrum has a peak wavelength of 850 nm (which is consistent with that of IR-820 doped ORMOSIL nanoparticles). The ex vivo imaging confirmed the in vivo experimental results yet again, illustrating that IR-820 doped ORMOSIL nanoparticles could be used as NIR optical probes for SLN mapping of live animals. 3.8. IR-820 doped ORMOSIL nanoparticles for tumor targeting of mice We intravenously injected (via the tail vein) PEG modified IR820 doped ORMOSIL nanoparticles into a nude mouse that bears a subcutaneously xenografted tumor to verify the applicability of

Fig. 7. (A) NIR imaging (exposure time: 400 ms, peak wavelength of deep-red broadband excitation light: 704 nm) of a black mouse with PEG modified IR-820 doped ORMOSIL nanoparticles microinjected into different depths of its brain, The NIR fluorescence signal was colored in red and the background autofluorescence was colored in green, (Inset) Typical fluorescence spectrum from NIR signal; (B) Bright field imaging of the treated black mouse. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Imaging of a nude mouse with PEG modified IR-820 doped ORMOSIL nanoparticles intradermally injected into the left forepaw pad at various times of post-injection. (AeC) NIR fluorescence imaging (exposure time: 5000 ms, peak wavelength of deep-red broadband excitation light: 704 nm) of the mouse: 4, 20, and 60 min after the injection. The NIR fluorescence signal was colored in red and the background autofluorescence was colored in green. (D) NIR fluorescence spectrum from dissected SLN, (Inset) real picture and NIR imaging of the dissected SLN. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the nanoparticles as NIR optical probes for in vivo tumor targeting. Another tumor-bearing mouse without any nanoparticle treatment acted as control. Fig. 9A and B show the in vivo imaging results 5 h post sample treatment. For the control mouse, only autofluorescence, the peak wavelength of which was located at 760 nm, could be detected. However, for the treated mouse, distinct NIR

signals could be observed in its liver, tumor and tail. The NIR optical signal had a peak wavelength at 850 nm, and accorded well with the fluorescence spectrum of IR-820 doped ORMOSIL nanoparticles, indicating (1) that most nanoparticles has accumulated in the liver of the treated mouse through blood circulation, and (2) that ORMOSIL nanoparticles could avoid capture/degradation by the

Fig. 9. NIR imaging (exposure time: 5000 ms, peak wavelength of deep-red broadband excitation light: 704 nm) of tumor-bearing mice treated with and without PEG modified IR820 doped ORMOSIL nanoparticles. (A) NIR fluorescence images of a treated mouse (right) and a non-treated mouse (left). The optical signals were pseudo-colored in red and the background was colored in green. (B) NIR fluorescence spectra of the signal and background from mice. Fluorescence peak wavelengths of the signal and background were 850 nm and 760 nm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 10. Imaging of a tumor-bearing mouse with PEG modified IR-820 doped ORMOSIL nanoparticles intravenously injected at various times (5 h, 24 h, 10 days and 30 days) postinjection. The NIR fluorescence signal was colored in red and the background autofluorescence in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

reticuloendothelial system (RES) due to the existence of PEG molecules on their surfaces, and they could effectively take up the tumor through the “enhanced permeability and retention” (EPR) effect [33]. The peak wavelength (850 nm) of the NIR signals was very far from that (760 nm) of autofluorescence, and they could easily be discriminated by the software in our in vivo imaging system, which is very helpful to increase the imaging contrast between signal and background. Furthermore, the liver is deep in the mouse body, and the distinct and clear NIR signals from the liver also confirmed the deep-tissue imaging capacity of IR-820 doped ORMOSIL nanoparticles. For the mouse treated with nanoparticles, long-term imaging and observation were carried out. 24 h post sample treatment, NIR fluorescence was observed in more parts of the tumor (Fig. 10B), indicating that more nanoparticles were accumulating in the tumor of the mouse. Meanwhile, the NIR fluorescence in its liver was still clear and distinct. 10 days later, there was no NIR fluorescence in the liver of the mouse (Fig. 10C), and we attributed this phenomenon to the metabolism and excretion process of the mouse. As Kumar et al. reported [23], ORMOSIL nanoparticles could effectively be cleared by the mouse itself over a long time through its hepatobiliary excretion. However, clear NIR fluorescence still existed in the tumor, which illustrated that (1) it was relatively more difficult for the mouse to clear out the ORMOSIL nanoparticles in the tumor compared to those in the liver, and (2) the fluorescence of IR-820 maintained stable and bright in a mouse tumor for quite a long time due to the protection of ORMOSIL nanoparticles against various bio-environments. This property is very helpful for the long time observation of various in vivo biological dynamic informations in the future. 30 days later, all the nanoparticles in the tumor and liver of the mouse were cleared, and no characteristic NIR signals could be observed (Fig. 10D). The mouse still stayed alive and no changes in its weight, shape, eating, drinking, exploratory behavior

or activity were observed, since ORMOSIL nanoparticles would not produce cytotoxicities and damages to the organs/tissues of the mouse [23]. 4. Conclusions We have reported on the synthesis of PpIX/IR-820 doped ORMOSIL nanoparticles. The one- and two-photon optical properties of PpIX doped ORMOSIL nanoparticles have been systematically investigated, and their applications in two-photon fluorescence imaging and PDT of tumor cells have been demonstrated. Furthermore, we have applied IR-820 doped ORMOSIL nanoparticles in various in vivo animal imaging applications. Our study indicated that microinjected ORMOSIL nanoparticles, which located beneath 4 mm-depth in the brain of a mouse, could be easily discriminated by utilizing NIR fluorescence imaging. We also demonstrated that the NIR fluorescent nanoparticles can effectively target the sentinel lymph node (SLN) of mice. We have found that intravenously injected NIR nanoparticles can target the subcutaneously xenografted tumor of a mouse through blood circulation and the “enhanced permeability and retention” (EPR) effect. ORMOSIL nanoparticles have bright prospects in future biomedical applications, such as disease diagnosis and clinical therapies. Acknowledgements This work was partially supported by the National Basic Research Program (973) of China (2011CB503700), the Special Financial Grant from the China Postdoctoral Science Foundation (No. 201104741), the National Natural Science Foundation of China (61008052 and 60990322), Science and Technology Department of Zhejiang Province (2010R50007), and the Fundamental Research Funds for the Central Universities. Jun Qian is grateful to Mr. Jiaqiang Li of

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Olympus Company (China) for his help in terms of two-photon laser scanning confocal microscopy. We also want to express our deepest gratitude towards Prof. Ying Mu and Dr. Wang Xi for their help in in vivo imaging.

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