Fluorescence Resonance Energy Transfer Mediated Large Stokes

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Fluorescence Resonance Energy Transfer Mediated Large Stokes Shifting Near-Infrared Fluorescent Silica Nanoparticles for in Vivo Small-Animal Imaging Xiaoxiao He, Yushuang Wang, Kemin Wang,* Mian Chen, and Suye Chen State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, P.R. China S Supporting Information *

ABSTRACT: Fluorescent dye-doped silica nanoparticles are increasingly used for in vivo imaging due to their unique biocompatibility and easy surface modification. However, the utility of existing fluorescent dye-doped silica nanoparticles for in vivo imaging is still limited because most studies are focused on doping single near-infrared (NIR) dyes in the silica matrix, which would cause background and crosstalk between the excitation light and the emitting signals due to the small Stokes shift of the traditional NIR dyes. To address this issue, we present a novel large Stokes shifting NIR fluorescent silica nanoparticles (LSS-NFSiNPs) based on the principle of fluorescence resonance energy transfer. Two highly water-soluble dyes, tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy) and methylene blue (MB), were chose as the model donor−acceptor pair. The LSS-NFSiNPs were prepared by synchronously doping RuBpy and MB in the silica nanoparticles. By optimizing the molar ratio of RuBpy and MB for doping in the silica nanoparticles, the energy transfer from RuBpy to MB occurred in the silica matrix, resulting in a near-infrared fluorescent silica nanoparticles with strong fluorescence and large Stokes shift (>200 nm). As a result, it can effectively help to increase the discrimination of fluorescence signal of interest over other background signals. With a combination of excellent stability, large Stokes shift, and near-infrared spectral properties, this novel LSS-NFSiNPs provides real-time, deep-tissue fluorescent imaging of live animals. More importantly, the LSS-NFSiNPs can also be gradually cleared from the body through the urinary clearance system. We anticipate this design concept can lay a foundation for further development of in vivo optical nanoparticulate contrast toward clinical applications. n vivo fluorescence imaging has become a promising technique in preclinical studies of human disease detection and treatment because of its high sensitivity, lower cost, avoidance of using ionizing radiation, and multiplex detection abilities.1−3 The fluorescence signals can provide the molecular information of biological tissues in its intact and native physiological state. One of the key components for fluorescence imaging in vivo is the fluorescent contrast agents.3 Fluorescent dyes based on small organic molecules that excite and emit photons at the near-infrared (NIR) region (650−900 nm) are of great current interest in bioimaging and diagnosis. They allow for imaging with minimal autofluorescence from biological samples, reduced light scattering, and high tissue penetration.4−8 However, the direct employment of NIR fluorophores in bioimaging is still challenging for the following significant limitations. First, most of NIR fluorophores are hydrophobic, which induces self-aggregation in aqueous solution.9,10 Second, when biological targeting moieties, such as antibodies, peptides, nucleic acids are conjugated with fluorophores, the chemical modifications process is relative sophisticated and there is usually only a small number of fluorophores presented per targeting moiety, which dramati-

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© 2012 American Chemical Society

cally affect the detection sensitivity.11 Third, the small organic fluorophores generally exhibit poor circulation half-lives and low photobleaching thresholds which constrain their effectiveness in long-term tracking of physiological behaviors.12,13 Finally, most NIR dyes, such as polymethine cyanine dyes, have a small Stokes shift, which can cause self-quenching and measurement error due to overlap between the excitation and emission spectra.14 The recent advances in functionalized nanoparticles have produced significant contributions to tackle these challenges. A variety of nanoparticle-based NIR nanoprobes have been prepared, evaluated, and applied for in vivo imaging, including NIR-emitting semiconductor quantum dots (QDs), lanthanide doped up-converting nanoparticles, and NIR dye-containing nanoparticles. The QDs are known to exhibit vastly superior optical characteristics in term of high quantum yields, large effective Stokes shifts, deeper penetration, and high photostability when compared to traditional NIR organic fluoroReceived: May 28, 2012 Accepted: September 27, 2012 Published: September 27, 2012 9056

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Figure 1. Schematic illustration of the LSS-NFSiNPs based on the principle of FRET from RuBpy to MB and the chemical structures of RuBpy and MB.

phores.15−18 However, the major hurdle in the medical application of QDs is their unsure long-term toxicities, as their composite materials comprise intrinsically toxic elements such as cadmium or other heavy metals that are liable to release and are toxic to live organisms.19,20 Therefore, various synthesis techniques have been continuously developed to prepare low toxic QDs suitable for in vivo bioimaging. One technique is encapsulation of QDs within protective, biocompatible external shells, such as silica,21−23 polymers,24,25 phospholipid,26,27 etc., after the synthesized QDs in organic phase were transferred to aqueous solution. The other technique is the direct synthesizing and functionalizing of QDs in aqueous media, which can drastically decrease the QDs toxicity.28 On the basis of the improvement, the QDs have been successively applied for bioimaging.29,30 In addition to QDs, lanthanide doped upconverting nanoparticles, which can convert a longer wavelength radiation to shorter wavelength fluorescence, are emerging as a new class of fluorescent biolabels for in vivo imaging.31 For both of NIR QDs and lanthanide doped up-converting nanoparticles, appropriate chemical compositions and synthesis routes should be first selected for preparing the chromophore group parts. Usually, the synthesis process is relatively complex, and the fluorescence performance of these luminescent nanoparticles is greatly related to the chemical compositions. By comparison with NIR QDs and lanthanide doped upconverting nanoparticles, NIR dye-containing nanoparticles, a new generation of fluorescent probes for in vivo imaging, are easier to be obtained through direct encapsulation of NIR dyes in the organic or inorganic matrix, such as liposomes,32 polymeric nanomicelles,33 calcium phosphate,13,34 or silica.35,36 The nanoparticles encapsulation enhanced the photostability and biocompatibility of NIR dyes due to the matrix protection of the NIR dyes from outside quenching and degrading factors. The NIR dye-encapsulated nanoparticles can also be easily modified with targeting molecules through the functional groups on the matrix materials.37,38 Moreover, the nanoparticles encapsulation can improve fluorescence signal from high payloading dye molecules per nanoparticle. Among the reported nanoparticles matrix, silica nanoparticles appears to be an ideal matrix for entrapping NIR dyes because of their extraordinary properties, such as straightforward synthesis,

relatively low cost, sufficient water dispersibility, good biocompatibility, and easy bioconjugation.35,38 The incorporation of NIR dyes, such as ICG,39 Cy5,35 methylene blue,40 NIR persistent dyes,41 into the silica nanoparticles has been reported and used for cancer in vivo imaging. We recently reported the intravenous injected silica nanoparticles with a size of ∼45 nm could be partly excreted through the renal excretion route, which further endows the NIR dyes doped silica nanoparticles to be used as an appropriate fluorescent contrast agent for in vivo imaging.37 Unfortunately, current NIR dye doped silica nanoparticles prepared by trapping single NIR dyes cannot overcome the inherent fatal disadvantage of the small Stokes shift. Designing and preparing NIR dyes doped silica nanoparticles with large Stokes shift are very promising for NIR fluorescence in vivo imaging. Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.42−44 By comparison with a single fluorophore, FRET has a relatively larger gap between the excitation and emission and thus can significantly reduce crosstalk between the excitation light and the resulting fluorescence signals while imaging. Recently, the FRET mechanism has been used in the preparation of dye-containing nanoparticles by a synchronous encapsulation donor and receptor in one nanoparticle. For example, Law et al. have prepared polymeric nanoparticles doped with a FRET pair (DiD and DiR) for in vivo NIR imaging.45 Besides, Tan et al. have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stöber synthesis method.46,47 The prominence of these works lay in the construction of barcoding silica NPs for multiplexed signaling by encapsulating different ratio of three dyes. Subsequently, Chen et al. have reported coupled-dye-doped silica nanoparticles and used them as fluorescent labels for achieving super-resolution in a conventional scanning fluorescence microscope imaging.48 However, to the best of our knowledge, the dyes-doped FRET silica nanoparticles for in vivo NIR imaging has not yet been reported. In this work, we report the development of a kind of large Stokes shifting NIR fluorescent silica nanoparticles (LSS9057

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The stability of the LSS-NFSiNPs is very important for their applications as fluorescence contrast agents for in vivo smallanimal imaging. Thus, the fluorescence stability and leakage of LSS-NFSiNPs in full mouse serum at room temperature were first measured, respectively. The leakage of dyes from the LSSNFSiNPs particles in full mouse serum for up to 7 h has been investigated by measuring the fluorescence intensity of supernatants, which were obtained by centrifugation of the LSS-NFSiNPs at 14 000 r/min for 10 min. The fluorescence intensity of the filtrates was determined with the Maestro in vivo imaging system and analyzed with Image J software, symbolized as Fsupernatant. While the fluorescence intensity of full mouse serum without incubation of LSS-NFSiNPs at the same detection conditions was regarded as blank, Fblank. The total fluorescence intensity of LSS-NFSiNPs, Ftotal, was obtained by suspending nanoparticles in full mouse serum as above. The dyes leakage from the particles were calculated by using the relation (Fsupernatant − Fblank)/(Ftotal − Fblank) × 100%. The fluorescence stability of LSS-NFSiNPs was also measured in full mouse serum for up to 7 h at room temperature. At scheduled intervals, the fluorescence intensity of LSS-NFSiNPs suspension was obtained by the Maestro in vivo imaging system and analyzed with Image J software. The initial fluorescence intensity of LSS-NFSiNPs in the solution signs as Foriginal. The fluorescence intensity of full mouse serum at the same detection conditions was regarded as the blank, Fblank. Then the fluorescence intensity of LSS-NFSiNPs at other times was measured as Ftime. The stability of the LSS-NFSiNPs was calculated by using the relation (Ftime − Fblank)/(Foriginal − Fblank) × 100%. In Vivo Imaging of LSS-NFSiNPs. Male athymic BALB/c (Balb/C-nu) mice were obtained from the Hunan Slaccas Jingda Laboratory Animal Co., Ltd., (BALB/c). They were 4−5 weeks old at the start of each experiment and weighed 20−25 g. All animal operations were in accord with institutional animal use and care regulations, according to protocol no. SYXK (Xiang) 2008-0001, approved by the Laboratory Animal Center of Hunan. We first demonstrated whether the LSS-NFSiNPs can be used for small animal imaging by discriminating against background fluorescence. Briefly, the 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 the LSS-NFSiNPs suspended in deionized water (14.25 mg/mL) were dipped on the tail of the mice as the imaging signal and were subsequently exposed to the different excitation light sources. The emitted light was collected using the corresponding band-pass filters for RuBpy (515−600 nm) and MB (680−800 nm). RuBpy-SiNPs and MB-SiNPs were, respectively, used as the control and the images on the mice were acquired under the same condition as those of LSS-NFSiNPs. To demonstrate whether the LSS-NFSiNPs can be used as a potential tool for in-depth imaging, the penetration of the LSSNFSiNPs signal in the tissue has also been investigated through imaging the LSS-NFSiNPs soaked filter paper in different locations of the mice. The procedure was followed. First, the clean filter paper square (4 mm × 4 mm) was soaked in the LSS-NFSiNPs solution (14.25 mg/mL) for 1 min. After drying at room temperature, the LSS-NFSiNPs treated filter paper was then placed into the skin, muscle, and abdominal cavity of the anesthetized mice through the mini-incision, respectively. Following repairing the incision, the images of the mice with placement of LSS-NFSiNPs treated filter paper were then

NFSiNPs) for NIR in vivo imaging by taking advantage of the highly efficient energy transfer between the donor and receptor dually doped in the silica nanoparticles. As illustrated in Figure 1, the highly hydrophilic dyes of tris(2,2′-bipyridyl)dichloro ruthenium(II) (RuBpy) and methylene blue (MB) were selected as the model FRET donor−acceptor pair and were synchronously housed inside a silica nanoparticles by hydrolysis and polycondensation of tetraethyl orthosilicate (TEOS) in water-in-oil microemulsion. Energy transfer from RuBpy to MB occurred in the silica nanoparticles, resulting in near-infrared fluorescent silica nanoparticles with strong fluorescence and large Stokes shift (>200 nm). The tremendous potential of these nanoparticles for in vivo imaging has been well investigated. It was demonstrated these particles were promising for medical imaging uses.

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EXPERIMENTAL SECTION Chemicals and Materials. Methylene blue (MB), tris(2,2bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy) and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were obtained from Reagent & Glass Apparatus Corporation of Changsha and were used without further purification (Changhsa, P. R. China). All solutions were prepared and diluted using ultrapure water (18.2 MΩ cm) from the Millipore Milli-Q system (Barnstead/Thermolyne NANOpure, Dubuque, IA). The athymic BALB/c(Balb/C-nu) mice were obtained from Hunan Slaccas Jingda Laboratory Animal Co., Ltd. (BALB/c). Preparation of Large Stokes Shifting NIR Fluorescent Silica Nanoparticles. To prepare large Stokes shifting NIR fluorescent silica nanoparticles (LSS-NFSiNPs), the MB and RuBpy were synchronously doped in the silica matrix by using hydrolysis of TEOS in a water-in-oil microemulsion. Briefly, 1.8 mL of Triton X-100, 7.5 mL of cyclohexane, and 1.6 mL of nhexanol were mixed, and an appropriate amount of ultrapure water was added to form a transparent microemulsion. The two dyes mixture solution of MB and RuBpy was then added into the microemulsion and stirred for 30 min. TEOS was added as a precursor for silica formation and hydrolyzed under the catalysis of ammonia (volume ratio of TEOS to ammonia was 1.7). The reaction proceeded over a period of 24 h at room temperature. After the reaction was completed, the nanoparticles were precipitated by addition of ethanol and were washed with ethanol and water, respectively, for several times to remove the surfactant and excess dye molecules from the particles. In order to obtain LSS-NFSiNPs with high FRET efficiency, the amount of the two dyes for the LSS-NFSiNPs preparation was optimized. We had synthesized a series of particles by changing the molar ratio of RuBpy and MB (4:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5). The intensity of NIR emission of the prepared LSS-NFSiNPs was measured by the Maestro in vivo imaging system. For the control experiments, the RuBpy-doped silica nanoparticles (RuBpy-SiNPs) and MB-doped silica nanoparticles (MB-SiNPs) were also prepared, respectively. Nanoparticles Characterization. The surface morphology and size of the LSS-NFSiNPs were characterized using transmission electron microscopy (JEM-1230). Fluorescence excitation and emission spectra of free MB and RuBpy dyes were recorded with the F-7000 fluorescence spectrophotometer (Hitachi High Technologies America, Inc.). Fluorescence emission spectra of LSS-NFSiNPs, RSiNPs, and MSiNPs were measured by the Maestro in vivo imaging system (CRI, Inc., excitation, 465−495 nm; emission, 680 nm long-pass). 9058

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Figure 2. (A) Excitation and emission spectra of RuBpy and MB dyes recorded with the F-7000 fluorescence spectrophotometer. (B) Normalized emission spectra of the LSS-NFSiNPs (black line) and RuBpy-doped SiNPs (red line) obtained by the Maestro in vivo optical imaging system under the excitation of 465−495 nm.

dual-dye-doped SiNPs, the fluorescence intensity at the wavelength of 620 nm related to the emission peak of RuBpy-SiNPs was decreased obviously. However, as expected, a strong emission peak at 710 nm was detected. This emission peak of the MB and RuBpy dual-dye-doped SiNPs is corresponding to that of the MB-SiNPs under excitation of 615−665 nm. In addition, the MB-SiNPs showed no emission spectra from 550 to 800 nm under the excitation of 465−495 nm. Therefore, our results indicated the FRET effectively occurred in the silica nanoparticles. Importantly, the results also demonstrated that MB and RuBpy dual-dye-doped SiNPs exhibited a large Stokes shift (>200 nm), denoted as LSSNFSiNPs. It has been reported that the FRET efficiency is related to the ratio of donor and acceptor (D−A ratio).47,49 To obtain the FRET mediated LSS-NFSiNPs with high luminescence intensity, the effect of the molar ratios of RuBpy (donor) to MB (acceptor) on the fluorescence intensity was further investigated. Figure 3 shows the fluorescence images and relative fluorescence intensity of the resulting LSS-NFSiNPs which was obtained by the Maestro in vivo optical imaging system. As one can see from the fluorescence images and fluorescence intensity of the series of prepared LSS-NFSiNPs, the fluorescence of the LSS-NFSiNPs increased with increasing D−A molar ratio. As the D−A molar ratio was increased to 3, the emission intensity reached a maximum value. Further increasing of the D−A molar ratio would eventually result in a decrease of the emission intensity. These results supported that the fluorescence intensity of the FRET mediated LSS-NFSiNPs varied depending on the D−A molar ratio. Under the optimized D−A ratio, the prepared LSS-NFSiNPs were spherical in shape with a homogeneous size distribution around 40 nm in diameter as characterized by TEM (Figure 3C). MB and RuBpy are highly water-soluble dyes, thus the dye leaking from the silica matrix should not be ignored when the nanoparticles are used in the liquid environment of the biological system. By using full mouse serum as a demonstration, the dye leaking tests of the LSS-NFSiNPs were performed. As shown in Figure 4A, the percentage of dyes leakage from LSS-NFSiNPs was about 8.6% after incubation for 7 h in full mouse serum at 37 °C, revealing that using LSSNFSiNPs could effectively help to avoid dyes leakage. Additionally, the LSS-NFSiNPs prepared in this work also have excellent stability. With reference to Figure 4B, the LSS-

acquired at the FRET channel (excited at 465−495 nm and recorded at 680−800 nm). Furthermore, the in vivo real-time FRET imaging of LSS-NFSiNPs in mice was investigated. The LSS-NFSiNPs suspended in deionized water were first injected from the tail vein of the mice (0.1 mg of LSS-NFSiNPs/g of animal weight), then the FRET fluorescence images were acquired at many time points post injection (up to 24 h). The Maestro in vivo imaging 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 (Maestro2.4.3). For FRET imaging, the tunable filter was automatically stepped in 10-nm increments from 680 to 800 nm while the camera captured images at each wavelength with constant exposure. Total acquisition time was about 13 s. The 13 resulting TIFF images were loaded into a single data structure in memory, forming a spectral stack with a spectrum at every pixel h.

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RESULTS AND DISCUSSION Preparation and Characteristics of LSS-NFSiNPs. To construct large Stokes shifting near-infrared fluorescent nanoparticles based on the principle of FRET, two fluorescence dyes, which were a FRET donor−acceptor pair, were housed inside a silica matrix by hydrolysis and polycondensation of TEOS in water-in-oil microemulsion. Here, we chose two highly water-soluble dyes, RuBpy and MB, as the donor− acceptor pair. The fluorescent spectra of these two dyes were shown in Figure 2A. These two dyes have two distinct maximum emission wavelengths, with RuBpy at 596 nm and MB at 676 nm. The emission spectrum of RuBpy shares a broad overlapping with the excitation spectrum of MB, so they could be used as a FRET donor−acceptor pair. After silica nanoparticles preparation, the fluorescent emission spectra of the MB and RuBpy dual-dye-doped silica nanoparticles were measured by using the Maestro in vivo imaging system to investigate whether the FRET could effectively happen in the silica matrix. The fluorescence spectra was acquired with excitation of 465−495 nm and recorded from 550−800 nm. Figure 2B represents the normalized fluorescence emission spectra of MB and RuBpy dual-dye-doped SiNPs and RuBpySiNPs, respectively. The results demonstrated that the RuBpySiNPs showed emission peak at 620 nm. For MB and RuBpy 9059

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fluorescence, the LSS-NFSiNPs, RuBpy-SiNPs, and MB-SiNPs were carefully dipped on the tail of different nude mice. The background fluorescence imaging of the nude mice and nanoparticles imaging were directly obtained without use of spectral unmixing techniques. The resulting images were shown in Figure 5. As is demonstrated in the Figure 5A1−A4, with excitation at 465−495 nm and recorded at RuBpy channel (515−600 nm), both LSS-NFSiNPs and RuBpy-SiNPs showed fluorescence signal. However, whole animals also exhibited prominent autofluorescence (Figure 5A2, A4). Moreover, no fluorescence signal was detected for MB-SiNPs (Figure 5A3), because the MB dyes can not be excited at 465−495 nm. When the nude mice were excited at 615−665 nm and recorded at the NIR region (680−800 nm), strong background fluorescence can be visualized around the abdomen region for all of the nude mice imaging, which are mostly intestinal background that came from the mouse chow (Figure 5B1−B4).50 There was no detected fluorescence signal for RuBpy-SiNPs (Figure 5B4). By comparison with LSS-NFSiNPs (Figure 5B2), the NIRF image of the MB-SiNPs displayed an obvious bright signal (Figure 5B3). However, the signal of MB-SiNPs cannot be easily discriminated against the background fluorescence of the regular mouse chow. In the present study, the background fluorescence of the mice has been greatly minimized with excitation at 465−495 nm and recording at NIR region (680− 800 nm) (Figure 5C1−C4, FRET involved image.). By comparison with that of RuBpy-SiNPs, the FRET image of LSS-NFSiNPs showed a stronger brightness (Figure 5C2,C4). In addition, no fluorescence signal was detected for MB-SiNPs (Figure 5C3). Our results indicated that the LSS-NFSiNPs with a large Stokes shift demonstrated unique advantage over the RuBpy-SiNPs and MB-SiNPs in terms of increasing the contrast between the target signal and background fluorescence without the use of time-resolved measurements or spectral unmixing techniques, which is very promising for small animals imaging. Imaging Penetration of LSS-NFSiNPs in Animal. To demonstrate the effectiveness of the LSS-NFSiNPs for in-depth imaging in animals, the depth of image acquisition using LSSNFSiNPs has been qualitatively analyzed by placing the LSSNFSiNPs soaked filter paper into the skin, muscle, and abdominal cavity of the anesthetized mice, respectively. The resulting FRET images demonstrated that the bright fluorescence of LSS-NFSiNPs soaked filter paper can all be obviously visualized in the three localizations of skin (nearly 1 mm), muscle (nearly 3 mm), and abdominal cavity (nearly 6 mm) (Figure 6), which indicated that the LSS-NFSiNPs emission showed an efficient penetration through the animal body to be used for in vivo bioimaging. In Vivo Whole Animal Imaging with LSS-NFSiNPs. To further demonstrate the utility of LSS-NFSiNPs for in vivo small animal imaging, whole animal fluorescent imaging of LSSNFSiNPs injected Balb/c nude mice was performed up to 24 h postinjection (pi) to demonstrate both the distribution of the LSS-NFSiNPs as well as the clearance of the LSS-NFSiNPs over time. Figure 7A displays time-dependent in vivo fluorescence images of the mice after tail vein intravenous injection LSS-NFSiNPs (0.1 mg of LSS-NFSiNPs/g of animal weight). Under the excitation at 465−495 nm and recorded at 680−800 nm, there was almost no background fluorescence in the “background” control mice. Following the immediate intravenous injection of LSS-NFSiNPs, clear and bright fluorescence emitted from LSS-NFSiNPs can be easily

Figure 3. Fluorescence intensities (A) and images (B) of LSSNFSiNPs prepared with different molar ratios of RuBpy and MB (obtained with Maestro in vivo optical imaging system; excitation, 465−495 nm; emission, 680 nm long-pass). (C) Transmission electron microscope (TEM) images of prepared RuBpy and MBdoped LSS-NFSiNPs (doping D−A molar ratio = 3:1).

NFSiNPs were highly fluorescent and were stable after continuous incubation in full mouse serum for 7 h, which are very important for the LSS-NFSiNPs as a novel near-infrared fluorescent contrast agent for in vivo imaging over relative long periods of time. Discriminating Against Background Fluorescence Effect of LSS-NFSiNPs in Animal. Reliable discrimination of desired signal from background fluorescence in biological matrix in vivo has previously been limited to the traditional near-infrared fluorescent contrast agents, such as cyanine dyes and phthalocyanines. The background fluorescence mainly includes the biochemical autofluorescence came from living tissues and the intestinal fluorescence of the mouse chow.50 Recently, time-resolved measurements or spectral unmixing techniques have been integrated into systems allowing correction for this unwanted background fluorescence.51 With the aim of assessing whether the LSS-NFSiNPs can be used for small animals imaging by discriminating against background 9060

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Figure 4. (A) Cumulative percent leakage of dyes from LSS-NFSiNPs in full mouse serum at 37 °C. (B) Fluorescence stability of LSS-NFSiNPs in full mouse serum at 37 °C. Insets: The fluorescence images of the supernatant and LSS-NFSiNPs in the 96-wells plates were obtained with the Maestro in vivo optical imaging system (excitation, 465−495 nm; emission, 680 nm long-pass). All the fluorescence images were presented after processing by Image J software.

Figure 5. In vivo imaging of nude mice after dipping with different nanoparticles on the tails. (A) Excited at 465−495 nm and recorded at RuBpy channel (515−600 nm). (B) Excited at 615−665 nm and recorded at NIR region (680−800 nm). (C) Excited at 465−495 nm and recorded at NIR region (680−800 nm). The red circle in every image locates the site of the dipping nanoparticles. A1, B1, and C1 were the images of the nude mice without dipping nanoparticles. A2, B2, and C2 represented the images of the nude mice dipping with LSS-NFSiNPs. A3, B3, and C3 represented the images of the nude mice dipping with MB-SiNPs. A4, B4, and C4 represented the images of the nude mice dipping with RuBpy-SiNPs. Time of exposure for every fluorescence image was 200 ms.

literature for very small particles like the ones used here.37 For the mice with injection of RuBpy-SiNPs (0.1 mg of RuBpySiNPs/g of animal weight), there was only weak fluorescence signal that could be visualized through the FRET imaging. In addition, there was no fluorescence signal detected for MBSiNPs injected mice (0.1 mg of MB-SiNPs/g of animal weight) during the whole imaging process (Figure S1 in the Supporting Information). It was further confirmed that the fluorescence signal of LSS-NFSiNPs injected mice indeed came from the FRET effect. A dissected organs analysis of nude mice without injection of LSS-NFSiNPs, 1 h after LSS-NFSiNPs injection, and 24 h after LSS-NFSiNPs injection revealed accumulation of the LSS-NFSiNPs primarily in the liver, kidneys, and gall

visualized in the whole animal. As blood circulated, the fluorescence decreased gradually. From 0.5 to 3 h pi, two bright spots arose from the liver and urinary bladder and were observed through the abdomen imaging, which indicated that the LSS-NFSiNPs were seen to accumulate in the liver and urinary bladder. By 24 h pi, the fluorescence in many parts of the whole animal decreased to nearly background levels except for the liver and urinary bladder. Meanwhile, the fluorescence signals of LSS-NFSiNPs in the liver and gall bladder had decreased noticeably. The results provided preliminary evidence that the LSS-NFSiNPs can also be gradually cleared from the body through the urinary clearance system, an excretion pathway that had been widely reported in the 9061

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Figure 6. Penetration of LSS-NFSiNPs. (A) Image of LSS-NFSiNPs soaked filter paper in the skin. (B) Image of LSS-NFSiNPs soaked filter paper in the muscle. (C) Image of LSS-NFSiNPs soaked filter paper in the abdominal cavity. Time of exposure for every fluorescence image was 200 ms.

luminescent properties of LSS-NFSiNPs in terms of the detection sensitivity and the ability to increase the contrast between the tested sample and the animal background fluorescence allow their use for medical fluorescent imaging in vivo. The results of this study will also have a large impact on extending this design concept to other NIR fluorescent contrast agents for FRET imaging. As demonstrated above, by choosing RuBpy and MB as the model donor−acceptor pair, the energy transfer from RuBpy to MB indeed occurred in the silica nanoparticles, resulting in near-infrared fluorescent silica nanoparticles with strong fluorescence and a large Stokes shift (>200 nm). The in vivo imaging results confirmed that this nanoparticles probe could still work for in vivo imaging and effectively help to increase the discrimination of fluorescence signals of interest over other background signals, which made it be potentially employed as fluorescence contrast agents for FRET medical imaging. Of course, the insufficiency of this work is the selection of the donor with a visible wavelength range of excitation (465−500 nm). On the basis of the design of FRET mediated nanoparticles probes, a new donor−acceptor pair, NIR excitation with NIR emission would be a more promising solution for efficient imaging in future work.

Figure 7. (A) Real-time in vivo abdomen FRET imaging of nude mice intravenously injected with the LSS-NFSiNPs. (B) Ex vivo FRET imaging of dissected organs from the nude mice without injection of LSS-NFSiNPs, 1 h after LSS-NFSiNPs injection, and 24 h after LSSNFSiNPs injection. Arrows from left to right represented the organs of lung, muscle, spleen, bladder, skin, kidney, heart, large intestine, and liver, respectively. Time of exposure for every fluorescence image was 1000 ms.

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bladder at 1 h pi. The liver accumulation decreased obviously, and the kidneys accumulation greatly increased by 24 h pi, which is consistent with the urine excretion pathway. No lung, spleen, heart, or large intestine accumulation has been noted for both 1 and 24 h pi (Figure 7B). The results demonstrated that major sites of fluorescence seen by ex vivo optical imaging were almost consistent with those seen by in vivo imaging. Interestingly, the dissected organs imaging also confirmed that the intestinal background due to mouse chow could be completely avoided through the FRET imaging. Furthermore, the urine samples were collected directly from the urinary bladder of mice intravenously injected with LSS-NFSiNPs through bladder puncture at 1 h pi. The fluorescence imaging results of the collected urine specimens recorded using the Maestro optical in vivo imaging system further confirmed that the LSS-NFSiNPs of intravenously injected (iv-injected) are, in fact, partly excreted into urine (Figure S2 in the Supporting Information). Thus, our results indicated that the unique

CONCLUSIONS In conclusion, by doping the FRET donor−acceptor pair into the silica matrix, we proposed a facile method for preparation of large Stokes shifting near-infrared fluorescent contrast agents for FRET imaging. Here, we chose two highly water-soluble dyes, RuBpy and MB, as the model donor−acceptor pair. Because of the fact that the emission spectrum of RuBpy shares a broad overlapping with the excitation spectrum of MB, the energy transfer from RuBpy to MB occurred in the silica matrix. By optimizing the molar ratio of RuBpy and MB for doping in the silica nanoparticles, a near-infrared fluorescent silica nanoparticles with strong fluorescence and a large Stokes shift (>200 nm) have been obtained, leading to an ability to discriminate designed signal from background signals. The effectiveness of the LSS-NFSiNPs for in-depth imaging in animals has been realized. Furthermore, in vivo real time 9062

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imaging measurements confirmed that the fluorescence signal of LSS-NFSiNPs injected mice indeed came from the FRET effect. More importantly, the LSS-NFSiNPs can also be gradually cleared from the body through the urinary clearance system. Along with demonstrated enhanced discrimination against background fluorescence, deeper tissue imaging capability and gradual clearance efficiency, the LSS-NFSiNPs could be potentially employed as fluorescence contrast agents for FRET medical imaging.

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(14) Peng, X. J.; Song, F. L.; Lu, E.; Wang, Y. N.; Zhou, W.; Fan, J. L.; Gao, Y. L. J. Am. Chem. Soc. 2005, 127, 4170−4171. (15) So, M. K.; Xu, C. J.; Loening, A. M.; Gambhir, S. S.; Rao, J. H. Nat. Biotechnol. 2006, 24, 339−343. (16) Shuhendler, A. J.; Prasad, P.; Chan, H. K. C.; Gordijo, C. R.; Soroushian, B.; Kolios, M.; Yu, K.; OBrien, P. J.; Rauth, A. M.; Wu, X. Y. ACS Nano 2011, 3, 1958−1966. (17) Zhang, Y.; Li, Y.; Yan, X. P. Small 2009, 5, 185−189. (18) Weng, K. C.; Noble, C. O.; Sternberg, B. P.; Chen, F. F.; Drummond, D. C.; Kirpotin, D. B.; Wang, D. H.; Hom, Y. K.; Hann, B.; Park, J. W. Nano Lett. 2008, 8, 2851−2857. (19) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11−18. (20) Chang, E.; Thekkek, N.; Yu, W. W.; Colvin, V. L.; Drezek, R. Small 2006, 2, 1412−1417. (21) Erogbogbo, F.; Yong, K. T.; Roy, I.; Hu, R.; Law, W. C.; Zhao, W. W.; Ding, H.; Wu, F.; Kumar, R.; Swihart, T. M.; Prasad, P. N. ACS Nano 2011, 5, 413−423. (22) Gao, X. H.; Hu, X. G. ACS Nano 2010, 4, 6080−6086. (23) Wang, C.; Ma, Q.; Dou, W. C.; Kanwal, S.; Wang, G. N.; Yuan, P. F.; Su, X. G. Talanta 2009, 77, 1358−1364. (24) Ballou, B.; Lagerholm, B. C.; Ernst, A. L.; Bruchez, P. M.; Waggoner, S. A. Bioconjugate Chem. 2004, 15, 79−86. (25) Mancin, M. C.; Kairdolf, B. A.; Smith, A. M.; Nie, S. M. J. Am. Chem. Soc. 2008, 130, 10836−10837. (26) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759−1762. (27) Yong, K. T.; Hu, R.; Roy, I.; Ding, H.; Vathy, L. A.; Bergey, E. J.; Mizuma, M.; Maitra, A.; Prasad, P. N. Appl. Mater. Interfaces 2009, 1, 710−719. (28) Law, W. C.; Yong, K. T.; Roy, I.; Ding, H.; Hu, R.; Zhao, W. W.; Prasad, P. N. Small 2009, 5, 1302−1310. (29) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538−544. (30) Yu, W. W.; Chang, E.; Drezek, R.; Colvin, V. L. Biochem. Bioph. Res. Commun. 2006, 348, 781−786. (31) Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. Nano Lett. 2008, 8, 3834−3838. (32) Li, H.; Zhang, Z. H.; Blessington, D.; Nelson, D. S.; Zhou, R.; Katz, S. L.; Chance, B.; Glickson, J. D.; Zheng, G. Acad. Radiol. 2004, 11, 669−677. (33) Kumar, R.; Ohulchanskyy, T. Y.; Roy, I.; Gupta, S. K.; Borek, C.; Thompson, M. E.; Prasad, P. N. Appl. Mater. Intrfaces 2009, 1, 1474− 1481. (34) Altınoglu, E. I.; Russin, T. J.; Kaiser, J. M.; Barth, B. M.; Eklund, P. C.; Kester, M.; Adair, J. H. ACS Nano 2008, 2, 2075−2084. (35) Burns, A. A.; Vider, J.; Ow, H.; Herz, E.; Medina, O. P.; Baumgart, M.; Larson, S. M.; Wiesner, U.; Bradbury, M. Nano Lett. 2009, 9, 442−448. (36) Kumar, R.; Roy, I.; Ohulchanskyy, T. Y.; Vathy, L. A.; Bergey, E. J.; Sajjad, M.; Prasad, P. N. ACS Nano 2010, 4, 699−708. (37) He, X. X.; Nie, H. L.; Wang, K. M.; Tang, W. H.; Wu, X.; Zhang, P. F. Anal. Chem. 2008, 80, 9597−9603. (38) Smith, J. E.; Wang, L.; Tan, W. H. Trends Anal. Chem. 2006, 25, 848−855. (39) Lee, C. H.; Cheng, S. H.; Wang, Y. J.; Chen, Y. C.; Chen, N. T.; Souris, J.; Chen, C. T.; Mou, C. Y.; Yang, C. S.; Lo, L. W. Adv. Funct. Mater. 2009, 19, 215−222. (40) He, X. X.; Wu, X.; Wang, K. M.; Shi, B. H.; Hai, L. Biomaterials 2009, 30, 5601−5609. (41) Chermont, Q. M.; Chaneac, C.; Seguin, J.; Pelle, F.; Maîtrejean, S.; Jolivet, J. P.; Gourier, D.; Bessodes, M.; Scherman, D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9266−9271. (42) Wallrabe, H.; Periasamy, A. Curr. Opin. Biotechnol. 2005, 16, 19−27. (43) Förster, T. Ann. Phys. 1948, 2, 55. (44) Wu, C. F.; Zheng, Y. L.; Szymanski, C.; McNeill, J. J. Phys. Chem. C 2008, 112, 1772−1781.

ASSOCIATED CONTENT

S Supporting Information *

Real-time in vivo abdomen FRET imaging of nude mice intravenously injected with RuBpy-SiNPs and MB-SiNPs, respectively, and white light and fluorescence images of the urine samples collected from the urinary bladder of mice. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-731-88821566. Fax: 86-731-88821566. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Key Project of Natural Science Foundation of China (Grants 21175039 and 21190044), International Science & Technology Cooperation Program of China (Grant 2010DFB30300), Key Technologies Research and Development Program of China (Grant 2011AA02a114), Research Fund for the Doctoral Program of Higher Education of China (Grant 20110161110016) and the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (Grants 10JJ7002, 2011FJ2001, and 2012TT1003).

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REFERENCES

(1) John, V. F. Curr. Opin. Chem. Biol. 2003, 7, 626−634. (2) Ghoroghchian, P. P.; Therien, M. J.; Hammer, D. A. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2009, 1, 156−167. (3) Hickson, J. Urol. Oncol.: Semin. Orig. Invest. 2009, 27, 295−297. (4) Achilefu, S. Angew. Chem., Int. Ed. 2010, 49, 9816−9818. (5) He, X. X.; Wang, K. M.; Cheng, Z. WIREs Nanomed. Nanobiotechnol. 2010, 2, 349−367. (6) Hintersteiner, M.; Enz, A.; Frey, P.; Lise, A.; Kinzy, W.; Kneuer, R.; Neumann, U.; Rudin, M.; Staufenbiel, M.; Stoeck, M.; Wiederhold, K. H.; Gremlich, H. U. Nat. Biotechnol. 2005, 5, 577−583. (7) Urano, Y.; Asanuma, D.; Hama, Y.; Koyama, Y.; Barrett, T.; Kamiya, M.; Nagano, T.; Watanabe, T.; Hasegawa, A.; Choyke, P. L.; Kobayashi, H. Nat. Med. 2009, 15, 104−109. (8) Ho, N. H.; Weissleder, R.; Tung, C. H. Tetrahedron 2006, 62, 578−585. (9) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781−789. (10) Demeule, B.; Gurny, R.; Arvinte, T. Int. J. Pharm. 2007, 329, 37−45. (11) Kobayashi, S.; Makino, A. Chem. Rev. 2009, 109, 5288−5353. (12) Lichtman, J. W.; Conchello, J. A. Nat. Methods 2005, 2, 910− 919. (13) Muddana, H. S.; Morgan, T. T.; Adair, J. H.; Butler, P. J. Nano Lett. 2009, 9, 1559−1566. 9063

dx.doi.org/10.1021/ac301461s | Anal. Chem. 2012, 84, 9056−9064

Analytical Chemistry

Article

(45) Wagh, A.; Qian, S. Y.; Law, B. Bioconjugate Chem. 2012, 23, 981−992. (46) Wang, L.; Tan, W. H. Nano Lett. 2006, 6, 84−88. (47) Chen, X. L.; Estevez, M. C.; Zhu, Z.; Huang, Y. F.; Chen, Y.; Wang, L.; Tan, W. H. Anal. Chem. 2009, 81, 7009−7014. (48) Chen, J. F.; Cheng, Y. Opt. Lett. 2009, 34, 1831−1833. (49) Yakovlev, A. V.; Zhang, F.; Zulqurnain, A.; Zahoor, A. Z.; Luccardini, C.; Gaillard, S.; Mallet, J. M.; Tauc, P.; Brochon, J. C.; Parak, W. J.; Feltz, A.; Oheim, M. Langmuir 2009, 25, 3232−3239. (50) Leblond, F.; Davis, S. C.; Valdés, P. A.; Pogue, B. W. J. Photochem. Photobiol. B 2010, 98, 77−94. (51) Davis, S. C.; Dehghani, H.; Wang, J.; Jiang, S. D.; Pogue, B. W.; Paulsen, K. D. Opt. Express 2007, 15, 4066−4082.

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