Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging

Biomaterials 32 (2011) 5880e5888

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Aggregation-enhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging Dan Wang a, b,1, Jun Qian a,1, Sailing He a, b, *, Jin Sun Park c, Kwang-Sup Lee c, Sihai Han d, Ying Mu d a

Centre for Optical and Electromagnetic Research, State Key Laboratory of Modern Optical Instrumentations, JORCEP (KTH-ZJU Joint Center of Photonics), Zhejiang University, Hangzhou 310058, PR China b ZJU-SCNU Joint Research Center of Photonics, South China Normal University (SCNU), 510006 Guangzhou, PR China c Department of Advanced Materials, Hannam University, Daejeon 305-811, Republic of Korea d Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Lab. of Industrial Control Technology, Zhejiang University (ZJU), Hangzhou 310058, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 April 2011 Accepted 26 April 2011 Available online 20 May 2011

We report polymeric nanomicelles doped with organic fluorophores (StCN, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]-acrylonitrile), which have the property of aggregation-enhanced fluorescence. The fluorescent nanomicelles have two unique features: (1) They give much brighter fluorescence emission than mono-fluorophores. (2) The nanomicelles with amphiphilic copolymers [e.g., phospholipids-PEG (polyethylene glycol)] make the encapsulated fluorophores more stable in various bio-environments and easy for further conjugation with bio-molecules. After chemical and optical characterization, these fluorescent nanomicelles are utilized as efficient optical probes for in vivo sentinel lymph node (SLN) mapping of mice. The StCN-encapsulated nanomicelles, as well as their bioconjugates with arginine-glycine-aspartic acid (RGD) peptides, are used to target subcutaneously xenografted tumors in mice, and in vivo fluorescence images demonstrate the potential to use PEGylated phospholipid nanomicelles with aggregation-enhanced fluorescence as bright nanoprobes for in vivo diagnosis of tumors. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Aggregation-enhanced fluorescence Phospholipid-PEG nanomicelles Bright nanoprobes In vivo imaging

1. Introduction In recent years, photoluminescence based bioimaging forms a major thrust of bio-photonics [1]. Much focus has been given to the development of efficient, inexpensive, stable, and tunable exogenous optical agents in biological systems, such as quantum dots/rods [2e6], silica nanoparticles [7e9], metal nanoparticles [10,11], carbon nanomaterials [12,13] and up-converting nanophosphores [14e17]. Although organic fluorophores exhibit remarkably high photoluminescence quantum efficiencies, their applications in the area of biological imaging are still limited due to their intrinsic hydrophobic property and instability in bio-environments. To overcome these problems, some approaches (e.g., phospholipid nanomicelles encapsulation) have been adopted.

* Corresponding author. Centre for Optical and Electromagnetic Research, Zhejiang University, PR China. Department of Electromagnetic Engineering, Royal Institute of Technology (KTH), Sweden. Tel.: þ86 46 8 7908465; fax: þ86 571 88206512. E-mail address: [email protected] (S. He). 1 D. Wang and J. Qian contributed equally to this work. 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.04.080

Phospholipid-PEG nanomicelles can be utilized to encapsulate various fluorophores and drugs, and have many advantages in bioapplications: (1) They possess no cytotoxicity; (2) The preparation process of phospholipid-PEG nanomicelles is much simpler than that of other nanocarriers, such as silica nanoparticles [7e9] and gold nanoparticles [10,11]; (3) A large hydrophobic core in the nanomicelle, which arises due to the presence of long acyl chains of phospholipids, can facilitate the loading of high concentrations of hydrophobic molecules per micelle; (4) The long PEG chains in nanomicelles can improve the long-time circulation of nanoparticles in an animal body and help to avoid capture/degradation by reticuloendothelial systems (RES), and this is very important for in vivo animal experiments [18e20]. Self-assembled constructions of phospholipid-PEG nanomicelles have been widely utilized in the applications of drug delivery [21,22] and in vivo animal imaging [23] in the past several years. However, there is still an obstacle for fluorophore doped nanomicelles. Most commonly used organic fluorophores suffer from an aggregation-induced fluorescence quenching phenomenon and their fluorescence emission decreases when they are highly loaded in phospholipid-PEG nanomicelles. Thus, it would be very useful if

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certain fluorescent materials, which have aggregation-induced enhanced emission properties, could be encapsulated in phospholipid-PEG nanomicelles and applied in fluorescence bioimaging. Recently, some groups have successfully synthesized these “special” fluorescent materials and demonstrated that they could exhibit enhanced emission when aggregated in certain solvents, or fabricated into solid films [24,25]. Furthermore, some other groups also began to use these fluorescent materials for various in vitro cell imaging experiments [7,26e28]. However, to the best of our knowledge, aggregation-enhanced emission dyes have still not been applied in in vivo animal imaging. In this paper, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl] acrylonitrile (StCN), a DeAeD type stilbene derivative with cyano group (as fluorescence acceptor group) in the center and vinyl and triphenylamine (as fluorescence donor group) in both ends, was synthesized as previously reported [29]. The intermolecular orientation is interrupted by the bulky cyano group during the formation of aggregation, which prevents the parallel overlap of fluorophores from becoming face-to-face oriented H-aggregations, but facilitates the formation of head-to-tail oriented J-aggregations [30,31]. Subsequently, StCN-encapsulated phospholipid-PEG (StCN@PEG) nanomicelles with a diameter of w20 nm were prepared, and excellent chemical stability of the nanomicelles was demonstrated. The absorption and emission properties of StCN@PEG nanomicelles with various loading densities were characterized with an absorbance spectrophotometer and photoluminescence spectroscopy, and no noticeable aggregationinduced fluorescence quenching or emission peak-wavelength shift was observed. We then used the StCN@PEG nanomicelles for in vitro stain of HeLa cells, and their biological uptake by tumor cells was confirmed with fluorescence microscopy. To demonstrate the potential of StCN@PEG nanomicelles as bright fluorescent probes for in vivo animal imaging, we first used them for sentinel lymph node (SLN) mapping, which is a key process in SLN biopsy (SLNB) for cancer staging and surgery [32]. Subsequently, we developed a fluorescence imaging protocol based on StCN@PEG nanomicelles as contrast probes for in vivo diagnosis of tumors of mice. As is well known, nanoparticles have a property to be preferentially taken up by malignant tissues (e.g., tumors) due to the “enhanced permeability and retention” (EPR) effect [33,34]. We demonstrated the passive targeting of tumors using StCN@PEG nanomicelles as fluorescent agents in nude mice, which bear subcutaneous lung tumor xenografts. Furthermore, StCN@PEG nanomicelles bioconjugated with arginine-glycine-aspartic acid (RGD) peptides were used for in vivo tumor targeting. RGD peptides, which have high binding affinity to the avb3 integrin receptor [16,35e39], are promising new tools for imaging of tumors. The integrin avb3 is a type of cell-surface receptors that are overexpressed at the endothelium of growing blood vessels (vasculature) associated with tumor growth (angiogenesis) [40,41]. It plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new diagnosis and therapeutic treatment of tumors in vivo [35,36]. Our experimental results revealed that RGD peptide-conjugated StCN@PEG nanomicelles provided higher targeting efficiency to the subcutaneous lung tumor xenografts of mice than StCN@PEG nanomicelles without RGD peptides.

First, maleimide-functionalized StCN@PEG nanomicelles were prepared by mixing 1.5 mL StCN solutions (1 mg/mL in chloroform), 80 mL mPEG-DSPE solutions (10 mg/mL in chloroform) and 10 mL DSPE-PEG-maleimide solutions (10 mg/mL in chloroform) together. After evaporated and dried under vacuum, 2.5 mL PBS (pH ¼ 7.4, 10 mM) solutions were added into the obtained lipidic film, and the solution was sonicated for 2 min to obtain StCN@PEG-maleimide nanomicelles. The StCN@PEG-maleimide nanomicelles were then conjugated with thiolated RGD through specific thiolemaleimide reactions. Briefly, 1 mL PBS solution of StCN@PEG-maleimide nanomicelles was mixed with 0.3 mL thiolated RGD peptide solution (24 mg/mL) and incubated for 2 h in room temperature. The resulting dispersion was further centrifugated at 12,000 rpm in a 0.2 mm membrane filter for 15 min to remove the excess unreacted RGD molecules and the pellets blocked on the membrane (mainly containing bioconjugates) were redispersed in 1 mL of PBS (pH ¼ 7.4, 10 mM) and kept at 4  C for further use.

2. Experimental section

2.4. Release kinetics and chemical stability analyses

2.1. Materials and instruments

For release kinetics studies, 1 mg StCN@PEG (60 wt% of StCN loading) nanomicelles were incubated with 1 mL Tween-20 solutions (1% in DI water) at 40  C. After a certain time, the sample was spin-filtered using microfuge membrane-filter (NANOSEP 100K OMEGA, Pall Corporation, USA) at 12,000 rpm for 15 min (spinfiltration). The filtrated solution, which passed through the

(Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]-acrylonitrile (StCN) was synthesized by the Department of Advanced Materials, Hannam University, Daejeon, Korea. The detailed information, including preparation and spectroscopic characterizations, were

described in Ref. [29]. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (mPEGDSPE-5000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide[poly(ethylene glycol)] (DSPE-PEG-Maleimide-3400) were purchased from Creative PEGWorks, Inc. Chloroform, 3,30 -diethylthiadicarbocyanine iodide (DTDC), Nile Red, hydrochloric acid and sodium hydroxide were obtained from the Chemical Reagent Department of Zhejiang University. RGD peptides (PCI-3686-PI) were purchased from Peptides International, Inc. Cell-culture products, unless otherwise mentioned, were purchased from Gibco. All the reagents, which were not specially pointed out, were analytical grade, and deionized (DI) water was used in all the experimental procedures. Transmission electron microscopy (TEM) images were taken by a JEOL JEM-1230 transmission electron microscope operating at 160 kV in bright-field mode. A Shimadzu 2550 UVevis scanning spectrophotometer and a HITACHI F-2500 fluorescence spectrophotometer were used to measure the absorption and photoluminescence (PL) spectra of samples. 2.2. Preparation of StCN@PEG nanomicelles Typically, 1.5 mL of StCN solutions in chloroform (1 mg/mL) were injected into 100 mL mPEG-DSPE solutions in chloroform (10 mg/mL). The mixture solution was then evaporated and dried under vacuum in a rotary evaporator at 70  C. Next, 2.5 mL of DI water was added into the solid lipidic mass obtained, and the solution was sonicated for 2 min. After that, an optically clear suspension containing StCN@PEG was prepared. As excess mPEG-DSPE molecules were used to encapsulate StCN molecules, the small loss of StCN in the experimental process could be negligible. The loading density (StCN/ [StCN þ mPEG-DSPE] in wt%) of StCN in StCN@PEG nanomicelles was calculated by the weight ratio of the materials put into the reaction. By varying the added quantities of StCN and mPEG-DSPE solution, nanomicelles with different StCN loading densities (20 wt%, 40 wt%, 60 wt%) were prepared by the same method (Table S1). 2.3. Preparation of RGD-conjugated StCN@PEG nanomicelles

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membrane, was collected. Its extinction spectrum, as well as that of the original aqueous solution of StCN@PEG nanomicelles, was measured. The ratio of the two extinction peak intensities could be used to characterize the release percentage of StCN from StCN@PEG nanomicells. For chemical stability tests, the PL spectra of 100 mg StCN@PEG nanomicelles incubated in 1 mL solutions (pH values from 4e10) of PBS and serum were monitored. The chemical stabilities were evaluated according to the changes of PL intensity. 2.5. In vitro studies with tumor cells HeLa cells were cultured in a Dulbecco’s minimum essential media (DMEM/f12) with 10% fetal bovine serum (FBS), 1% penicillin, and 1% amphotericinB. For fluorescence imaging, cells were treated separatedly with nothing, 200 mL solutions of PEG (400 mg/mL in water), 200 mL solutions of StCN@PEG nanomicelles (60 wt% of StCN loading, 1 mg/mL in water), and then incubated at 37  C with 5% CO2 for 2 h. Thereafter, all the cell samples were gently washed thrice with PBS (pH ¼ 7.4, 10 mM) and directly imaged with a fluorescence microscope underblue broadband light excitation. 2.6. In vivo imaging 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 nude mice from the Animal Experimentation Center of Zhejiang University were used for animal imaging studies. Every time before imaging, the mice were anaesthetized with intraperitoneal injection of 0.5% pentobarbital sodium. To investigate the SLN mapping of StCN@PEG nanomicelles in mice, samples (60 wt% of StCN@PEG, 1 mg/mL in 10 mM PBS, 10 mL solutions per mouse) were intradermally injected into the right forepaw pad of a group of nude mice. As a control experiment, the other group of nude mice were intradermally injected with DSPE-mPEG solutions (0.4 mg/mL in 10 mM PBS, 10 mL solutions per mouse) on their right forepaw pads. The in vivo fluorescence imaging of the experimental group and the control group was performed immediately after sample injection, by utilizing a Maestro in vivo optical imaging system (CRI, Inc. Woburn, MA). The in vivo imaging system consists of an optical head, an optical coupler, a cooled scientific-grade monochrome CCD camera, and an image acquisition/analysis software. A liquid crystal tunable filter was automatically tuned with 10 nm increments from 500 nm to 750 nm while the camera captured the image at each wavelength with a constant exposure time (500 ms). The resulting images were then used to create the unmixed images of the mouse with both auto-fluorescence and fluorescence signals. To set up tumor models, A549 cells (human lung cancer cell lines, 3  106 cells in 0.3 mL 10 mM PBS, pH ¼ 7.4) were implanted subcutaneously in the scapular region of 18e21 g male nude mice.

Fig. 1. Synthesis and characterization of StCN@PEG nanomicelles. (a) A schematic illustration for the preparation of StCN@PEG nanomicelles. (b) A representative TEM image of StCN@PEG nanomicelles. (c) UVevis absorption spectra of StCN in chloroform and StCN@PEG nanomicelles in water solution. (d) PL spectra of StCN in chloroform and StCN@PEG nanomicelles in water solution. Inset: Pictures of StCN in chloroform (left) and StCN@PEG nanomicelles in water (right) under visible light and ultraviolet light excitation (325 nm), respectively. The StCN in both vials are with the same masses of 0.2 mg.

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Tumor growth was monitored until a palpable tumor size was observed. Tumor-bearing mice were intravenously injected with StCN@PEG nanomicelles (with 60 wt% loading density, 1 mg/mL in PBS, 100 mL solutions per mouse) and RGD peptide-conjugated StCN@PEG nanomicelles (with 60 wt% loading density, 1 mg/mL in PBS, 100 mL solutions per mouse). mPEG-DSPE solutions (0.4 mg/ mL in PBS, 100 mL solutions per mouse) were also intravenously injected to tumor-bearing mice, acting as control. All the mice were imaged with the in vivo imaging system, and the imaging results were analyzed with the software package provided by CRI Inc. 3. Results and discussion 3.1. Synthesis and characterization of StCN@PEG nanomicelles Hydrophobic StCN molecules were encapsulated in amphiphilic PEGylated phospholipid based on a well-established method [23]. The major steps of StCN@PEG nanomicelles synthesis and the chemical structures of StCN and mPEG-DSPE are shown in Fig. 1a. mPEG-DSPE is a type of well-established

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surfactant with stable properties in aqueous solutions, which has been widely used in bio-applications [18e23]. The presence of PEG chains not only enabled the water solubility of StCN@PEG nanomicelles, but also provided a feasible bioconjugation method due to the existence of functional groups (e.g., COOHe, NH2e, or SHe) on its molecule end. Fig. 1b shows a representative TEM image of the StCN@PEG nanomicelles with 60 wt% loading density, and the nanomicelles exhibited roughly spherical shapes and were well dispersed. The StCN@PEG nanomicelles also appeared uniform in size and had an average diameter of less than 30 nm, which is small enough to minimize any disturbance of normal cellular physiology [42]. A digital camera was used to observe the solutions of StCN in chloroform and StCN@PEG nanomicelles in DI water under visible light and ultraviolet light (325 nm) excitation in a darkroom. The photographs were presented in the insets of Fig. 1c and d. As the StCN in both vials is of the same masses (0.2 mg), both solutions exhibited similar yellow colors under visible light (shown in Fig. 1c). After the excitation of ultraviolet light (325 nm), StCN chloroform solution emitted orange fluorescence while aqueous solution of StCN@PEG nanomicelles

Fig. 2. Stability studies of StCN@PEG nanomicelles. (a) Release kinetics studies of StCN@PEG nanomicelles (60 wt% of StCN loading) in 1% Tween-20 suspension at 40  C. The ratios of the two absorbance peak intensities of the filtrate solution containing Tween-20 nanomicelles and the original aqueous solution of StCN@PEG nanomicelles exhibit nearly no changes at various time points (0e12 h), indicating that the release of StCN from StCN@PEG nanomicelles is not severe and independent of incubation time. (b) Stability comparison of the StCN@PEG nanomicelles treated with PBS, serum, and pH 4 to 10 solutions. The PL intensities of mPEG-DSPE solutions in water were also measured as control. The panels inset show the corresponding fluorescence images of StCN@PEG nanomicelles dispersed in various solutions and the control solutions. The excitation source was a blue broadband light with a peak wavelength of 455 nm.

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emitted reddish fluorescence (shown in Fig. 1d). The two solutions were then characterized using a UVevis scanning spectrophotometer and a fluorescence spectrophotometer, respectively. As both the absorbance and PL intensities of mPEG-DSPE in water were almost “none” (Fig. S1), we can conclude that the absorbance and emission of StCN@PEG nanomicelles in water were mainly contributed from the encapsulated StCN molecules. As indicated by the results shown in Fig. 1c and d, the absorbance spectrum of StCN@PEG nanomicelles in DI water was similar to that of StCN in chloroform, with a maximum absorbance wavelength at 435 nm. However, compared to StCN chloroform solution, the fluorescence peak-wavelength of the nanomicelles solution (in water) redshifted from 554 nm to 577 nm (the excitation wavelength was 435 nm). We attributed the red-shift of the fluorescence peakwavelength to the nanomicelle-encapsulation with PEGylated phospholipid and/or the difference of the solvents, since StCN@PEG nanomicelles were “dispersed” in water while StCN molecules were “dissolved” in chloroform.

3.2. Stability analyses of StCN@PEG nanomicelles Since the stability of nanomicelles is of high importance for optical imaging, we tested the leakage of the encapsulated StCN from StCN@PEG nanomicelles in Tween-20 solution. As StCN dye can’t dissolve (or disperse) in water, if it leaked from nanomicelles and formed aggregates, the aqueous solutions would turn turbid. In our experiments, the Tween-20 solutions containing StCN@PEG nanomicelles were always optically clear, and thus the possibility that aggregates were formed by leaked dye could be excluded. According to the release kinetics study, if the nanomicelles structure is not stable, Tween-20 solution would break phospholipid-PEG nanomicelles and extract StCN molecules from them, forming smaller StCN@Tween-20 nanomicelles in the aqueous solution [23]. Under high speed centrifugation, microfuge membrane-filters would allow Tween-20 nanomicelles to pass through the membrane with the phospholipid-PEG nanomicelles arrested by the membrane. According to Beer’s Law, the optical

Fig. 3. Aggregation-enhanced fluorescence analysis of StCN@PEG nanomicelles. (a) UVevis absorption and PL spectra of the StCN@PEG nanomicelles with various StCN loading densities (20 wt%, 40 wt%, 60 wt%). (b) Normalized fluorescence intensities of hydrophobic dyes (StCN, DTDC and Nile Red) doped in phospholipid-PEG nanomicelles with various loading densities (20 wt%, 40 wt%, 60 wt%), respectively.

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absorbance of a chromophore in a transparent solvent varies linearly with the concentration of the chromophore, if the pathlength of light in the chromophore sample is fixed. In our experiment, the filtrated solutions containing StCN@Tween-20 micelles were collected and dissolved in a certain volume of DI water for absorbance measurement by a UVevis scanning spectrophotometer. Measurements were carried out at various time points and the absorbance intensity at 435 nm was used to calibrate the StCN concentration. The release percentage of StCN from StCN@PEG nanomicelles was calculated by the concentration ratio of StCN in the filtrated solution and StCN in the original solution of StCN@PEG nanomicelles. As shown in Fig. 2a, the release percentages of StCN from StCN@PEG nanomicelles were less than 5% after incubated with 1% of the Tween-20 surfactant at 40  C during the entire time of the experiment (even after 12 h), indicating that no severe release of StCN from StCN@PEG nanomicelles occurred in aqueous solutions. Furthermore, as a kind of efficient fluorescent probes for biological applications, StCN@PEG nanomicelles should be stable in various bio-environments including a wide range of pH values. Otherwise, monodispersed nanoparticles may be transformed into clusters composed of large numbers of particles, which are too big to be taken up by cells, and cannot circulate smoothly in the vessels for in vivo experimental applications. To confirm the biological and chemical stabilities of StCN@PEG nanomicelles, we systematically studied the fluorescence intensity changes of these nanomicelles under different treatments (e.g., PBS, serum, and pH 4 to 10 solutions) for 12 h. The solutions are selected with consideration to the most basic environments in the human body: PBS is a buffer solution commonly used in biological research; serum is used to simulate the bio-environments of in vivo experiments; pH ¼ 4 and pH ¼ 10 are almost the limit values of pH in the human body. As shown in Fig. 2b, the fluorescence peak intensities of StCN@PEG nanomicelles changed by less than 10% in all experimental conditions (normalized by the fluorescence peak intensity at pH ¼ 7), indicating that the StCN@PEG nanomicelles was chemically stable in those solutions, which is very positive for various bio-applications. 3.3. Aggregation-enhanced fluorescence properties of StCN@PEG nanomicelles Nanomicelles solutions with various StCN loading densities (20 wt%, 40 wt%, 60 wt%) were optically characterized in order to investigate the aggregation-induced properties of StCN@PEG nanomicelles. For an accurate quantitative comparison of photoluminescence emission, absorption intensities of the excitation wavelength for all the samples were kept the same, by utilizing different amounts of nanomicelles to encapsulate the same amount of StCN molecules (Table S2). The total PL intensity increased significantly without any distinct wavelength shift in emission peak (Fig. 3a), as the StCN loading density increased. Furthermore, two common fluorophores (DTTC and Nile Red, with absorbance and PL spectra shown in Fig. S2) were selected, and the PL intensities of DTDC and Nile Red encapsulated phospholipid-PEG nanomicelles with various loading densities were monitored for comparison with those of StCN@PEG nanomicelles. As shown in Fig. 3b, the PL intensities (normalized by the PL intensity in the condition of loading density ¼ 20 wt%) of DTDC/Nile Red doped nanomicelles decreased significantly as the loading density of DTDC/Nile Red in nanomicelles increased, which was due to the aggregation-induced fluorescence quenching. However, the behavior of StCN doped nanomicelles was quite opposite to those of the two common dyedoped nanomicelles. It can be concluded that StCN@PEG nanomicelles exhibit an aggregation-enhanced fluorescence emission effect, due to the unique chemical structure of StCN molecules.

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3.4. StCN@PEG nanomicelles for in vitro cell imaging Due to the properties of ultra small size, highly monodispersity, excellent chemical stability and aggregation-enhanced fluorescence emission, StCN@PEG nanomicelles were utilized for in vitro optical bioimaging of cells. Fig. 4c shows the in vitro images of HeLa cells treated with StCN@PEG nanomicelles. The bright-field images showed that the morphologies of all the cells (treated with StCN@PEG nanomicelles) kept very well, and they were viable after the sample treatment, indicating StCN@PEG nanomicelles did not cause any toxicity to the cells. The fluorescence images illustrated that the fluorescence of StCN could be clearly observed from the HeLa cells, indicating that the StCN@PEG nanomicelles were effectively taken up by cells. However, control cells A (without any treatment, Fig. 4a) and control cells B (treated only with mPEG-DSPE, Fig. 4b) showed no fluorescence. 3.5. StCN@PEG nanomicelles for in vivo SLN mapping and tumor targeting of mice To assess the potential of StCN@PEG nanomicelles for bioimaging in living animals, we first investigated whether they can be used for fluorescence mapping of SLN (sentinel lymph node) in a mouse model. SLN is the first group of lymph nodes receiving metastatic cancer cells by direct lymphatic drainage from a primary tumor. Accurate identification and biopsy of SLN can enable clinicians to focus on certain lymph nodes and perform more detailed tracking of cancer cell diffusion. SLN imaging utilizing fluorophores as labeling agents has become a research area with intense interest. The aggregation-enhanced fluorescence emission of StCN, as well as the excellent chemical stability of StCN@PEG nanomicelles, motivate us to investigate their applicability in in vivo SLN mapping. In our experiment, StCN@PEG nanomicelles were intradermally injected into the right forepaw pads of nude mice. A Maestro optical imaging system was then used to record the in vivo fluorescence imaging immediately after sample injection. The diffusion and accumulation process of nanomicelles at the SLN over time was shown in Fig. 5b. After injected, nanomicelles diffused rapidly from

Fig. 4. In vitro images of HeLa cells treated with nothing (a), mPEG-DSPE (b), StCN@PEG nanomicelles (c) for 2 h at 37  C. The scale bar is 50 mm.

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Fig. 5. Pseudo-color fluorescence images of mice with mPEG-DSPE (a) and StCN@PEG nanomicelles (b) injected into its right paw at various time points (5, 20, 40 and 60 min). Arrows indicate the SLN sites. (c) Fluorescence spectra obtained from the SLN (red) and skin (green) of the mice. (d) Variations of fluorescence peak intensities in the SLN sites of the mice, which were injected with StCN@PEG nanomicelles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the injection site into the lymphatics. 5 min later, fluorescence signals were detected at an axillary node, giving a clear SLN mapping of the mouse (shown in Fig. 5a). In contrast, mice injected with mPEG-DSPE solutions (Fig. 5a) showed no signal in the area of SLN. Fluorescence spectra acquired from the SLN and the skin of the mouse were shown in Fig. 5c. As time went by, nanomicelles gradually migrated from SLN, and the fluorescence signal intensity at the SLN decreased (as shown in Fig. 5d). Furthermore, a mouse was put down and its SLN was taken out and dissected, thereafter the in vivo SLN mapping of StCN@PEG nanomicelles was confirmed according to ex vivo imaging result (Fig. S3). 3.6. StCN@PEG nanomicelles for tumor targeting of mice To verify the applicability of StCN@PEG nanomicelles for in vivo tumor targeting, we injected them intravenously into nude mice bearing subcutaneous lung tumor xenografts. PEG molecules can improve the long-time circulation of nanomicelles in the animal body, and due to the EPR effect in the tumor tissues, nanomicelles could be preferentially taken up by tumors. Hence, we could anticipate that StCN@PEG nanomicelles could passively target tumors of mice. Furthermore, in order to obtain high efficiency of tumor targeting by using nanomicelles, we conjugated StCN@PEG

nanomicelles with a kind of triplet peptide called RGD (arginineglycine-aspartic acid peptides). Triplet peptide RGD can specifically target (bind to) avb3 integrins overexpressed on the tumor endothelium, which can potentially play a critical role for the diagnosis of developing tumors in vivo via noninvasive optical imaging. In our experiment, mice bearing subcutaneous lung tumor xenografts were intravenously injected separately with mPEG-DSPE (as control), StCN@PEG nanomicelles and RGD peptide-conjugated StCN@PEG nanomicelles. In vivo fluorescence imaging of tumorbearing nude mice was carried out at various time points postinjection and spectrally unmixed using the Maestro imaging software. Fig. 6 shows the representative whole body in vivo optical imaging results of mice injected with StCN@PEG nanomicelles (top of the images) and RGD peptide-conjugated StCN@PEG nanomicelles (bottom of the images). Spectral signatures from the tumor sites and the auto-fluorescence of skin sites were also respectively presented in Fig. 6. It is obvious that the fluorescence spectra of signals were consistent with the fluorescence spectra of StCN and it could easily be differentiated from the auto-fluorescence of skin sites. For control mice, no fluorescence contrast could be observed from tumors and surrounding skin after injection of mPEG-DSPE (Fig. S4). For the mice injected with StCN@PEG nanomicelles, there were fluorescence signals from the tumors at 48 h post-

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Fig. 6. In vivo imaging of mice bearing subcutaneous lung tumor xenografts, injected with StCN@PEG nanomicelles (top) and RGD peptides-conjugated StCN@PEG nanomicelles (bottom). The concentrations of two StCN@PEG nanomicelles samples were the same (with 60 wt% loading density, 1 mg/mL in PBS, 100 mL solutions per mouse). Spectral profiles in (dei) were used to unmix images.

injection (Fig. 6f and l top), while the images taken 1 h and 24 h post-injection only showed bright signals at the injection sites of tails (Fig. 6d, e, j, k, top). These results showed that the accumulation of the StCN@PEG nanomicelles in the tumor sites needs long time (e.g., 48 h post-injection) to take place, which could be attributed to the slow process of the EPR effect [33,34]. For the mice injected with RGD peptide-conjugated StCN@PEG nanomicelles, there were no signals in the tumor sites 1 h post-injection (Fig. 6d and j bottom), but intense fluorescence signals could be observed in the tumor sites at 24 h post-injection (Fig. 6e and k bottom), as well as at 48 h post-injection (Fig. 6f and l bottom). The accumulation of the RGD peptide-conjugated StCN@PEG nanomicelles in the tumor sites was much faster than StCN@PEG nanomicelles, and one possible explanation is: the high binding affinity of RGD peptides to the avb3 integrin receptor contributed more than the EPR effect to tumor targeting of nanomicelles, making the RGD peptideconjugated StCN@PEG nanomicelles exhibit higher efficiency of targeting to the subcutaneous lung tumor xenografts in mice. Our experiment results have suggested that bioconjugated/nonbioconjugated StCN@PEG nanomicelles of aggregation-enhanced fluorescence can be used as bright nanoprobes for potential applications in in vivo tumor targeting and diagnoses. 4. Conclusions We have synthesized phospholipid-PEG nanomicelles (<30 nm) loaded with (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]acrylonitrile (StCN). The as-prepared StCN@PEG nanomicelles were

characterized and exhibited ultra small size, high monodispersity, excellent chemical stability and aggregation-enhanced fluorescence emission. In vitro fluorescence microscopy and in vivo SLN mapping using StCN@PEG nanomicelles (as bright luminescent labels) were carried out. Moreover, we have demonstrated that StCN@PEG nanomicelles (with and without RGD-conjugated) could be used as an excellent tool for tumor targeting in live animals. Acknowledgments This work is partially supported by the National Natural Science Foundation of China (No. 61008052 and 60688401), the Swedish Foundation for Strategic Research (SSF), AOARD, the Fundamental Research Funds for the Central Universities and the China Postdoctoral Science Foundation (No. 20090461394). We are also grateful to National Basic Research Program of China 2007CB714503 and Innovation Method Fund of China 2008IM040800. Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2011.04.080. References [1] Prasad PN. Introduction to biophotonics. New York: Wiley-Interscience; 2004. [2] Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science 1998;281(5385):2013e6.

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