Penetrating Peptide-Bioconjugated Persistent Nanophosphors for

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Penetrating peptide bioconjugated persistent nanophosphors for long-term tracking of adipose-derived stem cells with superior signal-to-noise ratio Shu-Qi Wu, Chongwei Chi, Cheng-Xiong Yang, and Xiu-Ping Yan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00449 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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Analytical Chemistry

Penetrating Peptide

Bioconjugated

Persistent

Nanophosphors

for

Long-term Tracking of Adipose-derived Stem Cells with Superior Signal-to-noise Ratio Shu-Qi Wu,† Chong-Wei Chi,§ Cheng-Xiong Yang † and Xiu-Ping Yan*,†,‡ †

College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal

Chemical Biology (Nankai University), Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071, China. ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071,

China §

Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, Institute of Automation,

Chinese Academy of Sciences, Beijing 100190, China

* Correspondence should be addressed to X.-P. Yan ([email protected])

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ABSTRACT Reliable long-term in vivo tracking of stem cells is of great importance in stem cell-based therapy and research. Fluorescence imaging with in situ excitation has significant autofluorescence background, which results in poor signal-to-noise ratio (SNR). Here we report TAT penetrating peptide-bioconjugated long persistent luminescence nanoparticles (LPLNPs-TAT) for long-term tracking of adipose-derived stem cells (ASCs) without constant external excitation. LPLNPs-TAT exhibits near-infrared emitting, red light renewable capability, and superior in vivo imaging depth and SNR compared with conventional organic dye and quantum dots. Our findings show that LPLNPs-TAT can successfully label ASCs without impairing their proliferation and differentiation, and can effectively track ASCs in skin regeneration and tumor homing models. We believe that LPLNPs-TAT represents a new generation of cell tracking probes and will open a broad application in diagnosis and therapy.

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Mesenchymal stem cells are multipotent cells that can differentiate into a variety of specialized cells.1 They are well known for natural high tumor affinity, which can be used as a biological drug or gene carrier for targeted cancer therapy.2,3 As stem cells possess significant self-renewability and exhibit low immunogenicity, stem cell-based therapy has played a very important role in regeneration medicine, such as wound healing, cartilage repair, and arthritis treatment.4,5 The treatment in these therapeutic applications usually takes a few days to weeks.2,5 So, it is of critical importance to track the long-term fate, migration, and regenerative capability of delivered stem cells in tissue regeneration or targeted homing studies. Reliable and non-invasive tracking of stem cells is urgently needed for scientists and clinicians to understand the natural behaviors of stem cells and to evaluate the treatment efficacy.6 There are two main strategies for cell labeling. One is genetic transfection of reporter genes and the other is direct labeling with nanoparticles or organic probes. Genetically encoded proteins such as green fluorescent protein (GFP) or luciferase are widely used for cell labeling to provide precise and quantitative information on the fate and distribution of administered stem cells.7,8 However, reporter gene labeling needs complicated gene modification and transfection steps which may cause concern of the safety in clinical application.9 In recent years, several imaging probes have been used for tracking stem cells, including Au nanoparticles,10 iron oxide nanoparticles,11,12 organic dyes and quantum dots (QDs)13-15 via various imaging techniques such as X-ray computed tomography imaging, magnetic resonance imaging, and optical imaging. Among those imaging techniques, optical imaging has much higher sensitivity, larger throughputs, cheaper and smaller equipment and multiple detection wavelengths.16,17 Except for the commercial fluorescent organic dyes and QDs, there are a few newly developed fluorescent probes such as organic nanodots,18 polymer nanodots19 and upconversion nanoparticles20,21 for reporting the long-term fate of stem cells. However, conventional fluorescent probes have very short lifetimes (several nanoseconds to microseconds22), thus make it prerequisite to image under in situ excitation. As a result, the so caused interference from significant autofluorescence background signals limits the usage in tracking small numbers of stem cells and imaging in deep tissue. Upconversion nanoparticles show minimal autofluorescence background, but their low quantum yield and thermal effect caused by continuous near-infrared (NIR) light excitation may limit extensive applications.23 Persistent luminescence nanoparticles can store excitation energy (e.g., UV light, visible light or X ray), and then slowly emit persistent luminescence for a long time after the stoppage of the excitation.24,25 With the development of NIR-emitted and small sized long-persistent luminescence nanoparticles (LPLNPs) in recent years, LPLNPs has been used as optical probes in biosensing and in vivo bioimaging without in situ excitation.26-28 LPLNPs-based in vivo imaging can be conveniently carried out on commercial imaging systems by acquiring 3



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persistent luminescence signals. More importantly, the persistent luminescence of several Cr3+-doped LPLNPs is renewable with tissue penetrable red light instead of UV light, which makes the LPLNPs-based imaging no longer limited by the persistent luminescence decay lifetime.29,30 These persistent luminescence nanoparticles have attracted increasing interest in chemistry and cell biology as a new generation of advanced optical materials.25,31 Herein, we report the first fabrication of TAT penetrating peptide functionalized NIR-emitting LPLNPs with composition of Zn1.1Ga1.8Ge0.1O4:Cr3+,Eu3+ (LPLNPs-TAT) for long-term tracking of stem cells. The prepared LPLNPs-TAT can successfully label the stem cells and shows excellent biocompatibility, high signal-to-noise ratio (SNR), and red LED light renewable capability. Our data demonstrate that labeling of LPLNPs-TAT not only has superior SNR for tracking adipose-derived mesenchymal stem cells (ASCs) in skin regeneration and tumor homing models, but also has no effect on both proliferation and multilineage differentiation of ASCs. The superlong persistent luminescence and red light renewability makes functional LPLNPs very promising for long-term in vivo stem cell tracking and provides us insights to explore the contribution and migration of ASCs.

EXPERIMENTAL SECTION Synthesis of LPLNPs. Zn1.1Ga1.8Ge0.1O4:0.5%Cr3+,0.5%Eu3+ was synthesized via a hydrothermal process in combination with sintering in air.25,32-34 Firstly, Ga2O3 was dissolved in dilute nitric acid solution under hydrothermal condition at 150 oC. GeO2 was dissolved in dilute ammonia solution (3%). Then, 4.59 mmol Zn2+, 7.5 mmol Ga3+, 0.0375 mmol Cr3+, 0.0375 mmol Eu3+, and 0.417 mmol Ge4+ were mixed together under vigorous stirring. The resulting solution was adjusted to pH 7.0 with concentrated ammonia solution (28%), stirred for 3 h at room temperature, and sealed in a Teflon-lined autoclave at 160 oC for 24 h. The resulting precipitate was washed with ultrapure water and ethanol sequentially and dried at 70 oC for 4 h. Finally, the dry powder was sintered in air at 900 o

C for 4 h. The resulting powder was wet ground with an agate mortar and pestle for 30 min, and then dispersed in 5

mM NaOH solution. After stirring overnight and centrifugation, hydroxylated LPLNPs (LPLNPs-OH) were obtained. Surface Modification of LPLNPs. LPLNPs-OH was coated with APTES according to a previously reported protocol.28 LPLNPs-NH2 was obtained by adding 400 µL of APTES to a suspension of 100 mg LPLNPs-OH in 40 mL DMF. The reaction mixture was sonicated and kept vigorous stirring for 6 h at room temperature. The resulting LPLNPs-NH2 was centrifuged and washed with DMF to remove unreacted APTES. LPLNPs-COOH was prepared by adding 120 mg of SC-PEG-COOH and 20 µL triethylamine to a suspension

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of 20 mg of LPLNPs-NH2 in 4 mL DMF. To ensure a maximum PEG density, the reaction mixture was stirred overnight at room temperature. The resulting LPLNPs-COOH was centrifuged and washed with DMF and ethanol. To conjugate TAT peptide to the LPLNPs-COOH, 20 mg of LPLNPs-COOH was pre-activated with 16 mg of EDC and 40 mg of NHS in 10 mM PBS (pH 6) for 2 h. Following activation, 70 mg of TAT peptide was added, and the mixture was adjusted to pH 8 with 1 M NaOH and stirred in dark at room temperature for 8 h. The unreacted peptide was removed by centrifugation and the final LPLNPs-TAT was washed with 10 mM PBS. Characterization. X-ray powder diffraction (XRD) pattern was recorded using a D/max-2500 diffractometer (Rigaku, Japan) using Cu Kα radiation (λ = 1.5418 Å). The transmission electron microscopic (TEM) images were obtained on a Tecnai G2 F20 transmission electron microscope (FEI, America). The photoluminescence spectra were obtained on an F-4500 spectrofluorometer (Hitachi, Japan). Fourier transform infrared (FT-IR) spectra (4000−400 cm-1) in KBr were recorded on a Magna-560 spectrometer (Nicolet, Madison, WI). Dynamic light scattering and Zeta potential analysis were performed on a Zetasizer Nano-ZS (Malvern, UK). Thermal gravimetric analyzer (TGA) experiments were performed on a TG209 thermal analyzer (Netzsch, Germany) under pure N2 in the range from 20 to 700 °C with a heating rate of 10 °C/min. The microscopic images were observed on an inverted fluorescence microscope (DMIL LED, Leica, Germany) equipped with an Electron-Multiplying Charge-coupled Device (EMCCD camera, Princeton Instruments, America). NIR persistent luminescence and fluorescence images were acquired on an IVIS Imaging System (Caliper Co., America). Persistent luminescence signals were captured under bioluminescence imaging mode, while fluorescence signals were captured under fluorescence imaging mode with various excitation and emission filters. MTT assay was performed on a plate reader (Synergy HT, BioTek, America). Persistent luminescence nanoparticles were pre-excited with a UV lamp (254 nm, 6W) or red LED light (650 nm, 5,000 lm). Cell Culture. GFP-expressed ASCs isolated from the abdominal and inguinal adipose tissue of C57BL/6 transgenic mice were purchased from Cyagen Bioscences (Guangzhou, China). The ASCs were cultured and expanded in 10-cm2 plate in complete growth medium (Cyagen Bioscences, China) at 37 oC in 5% CO2. Normal (GFP-unexpressed) ASCs isolated from the abdominal and inguinal adipose tissue of KM mice were cultured in α-Minimum Essential Medium (α-MEM) supplemented with 20% fetal bovine serum (FBS) and 100 U/mL of penicillin-streptomycin at 37 oC in 5% CO2.18 4T1 murine breast cancer cells (a gift from Prof. Jie Tian, Key Laboratory of Molecular Imaging, Institute of Automation, China) were cultured in Dulbecco’s modified Eagle’s high glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin-streptomycin at 37 oC in 5% CO2. Cells were washed with Phosphate Buffered Saline (PBS). 5



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In Vitro Tracking of ASCs. Upon 80% confluence, ASCs were trypsinized and seeded in a 6-well plate with 50,000 cells per well. LPLNPs-TAT was diluted in culture medium (50 µg/mL) and incubated with ASCs overnight. After incubation, unbound nanoparticles were removed by repetitive washing with PBS, followed by the addition of fresh culture medium. The cells were excited with a red LED light for 1 min before observing on an inverted microscope equipped with an EMCCD camera. NIR persistent luminescence decay images were acquired without any excitation. Animal Model. All animal procedures were performed isoflurane gas anaesthesia (3% isoflurane-air mixture) and all efforts were made to minimize suffering. All animal experiments were carried out in accordance with guidelines of the Tianjin Committee of Use and Care of Laboratory Animals, and all experimental protocols were approved by the Animal Ethics Committee of Nankai University. The seven- to eight-week-old Balb/c nude mice were obtained from Vital River Laboratories (Beijing, China). The subcutaneous 4T1 tumor mice model was established by subcutaneously injecting 5 × 106 tumor cells in nude mice. The tumor carrying mice were used on 14 d after injection. Biodistribution and In Vivo Toxicity Studies. LPLNPs-TAT (800 µg) diluted in 100 mM PBS were irradiated with a UV lamp for 5 min before intravenous injection into each healthy mouse (n = 3). The time-dependent biodistribution of the LPLNPs-TAT in mice was imaged on the IVIS Imaging System. On 6 h, 12 h, and 24 h after injection, the mice were excited with a red LED light for 1 min before acquiring the persistent luminescence signal. The mice were sacrificed 7 days later and the major organs were collected for ex vivo luminescence imaging. All organs were also excited with the red LED light for 1 min before imaging. The histological changes in the organs of the injected mice were observed on 15 d after injection. The organs (heart, liver, spleen, lung and kidney) were embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek), sliced at thickness of 8 µm on Leica CM1950, and stained with hematoxylin and eosin (H&E). Healthy mice without injecting LPLNPs-TAT were also conducted following the same procedures as a control. All H&E-stained slices were imaged on optical microscope. In Vivo Comparison of LPLNPs-TAT with Conventional Fluorescent Probes. LPLNPs-TAT (30 µg), CdTe QDs (700 nm emitting, BEIDA JUBANG SCIENCE & TECHNOLOGY Co. Ltd., Beijing, China) and the BODIPY-derivative dye (710 nm emitting, a gift from Prof. Li-Gong Chen at Tianjin University) were subcutaneously injected onto the flanks of three nude mice, respectively. The mice were placed on their back and abdomen for signal acquisition. For NIR persistent luminescence-based imaging, LPLNPs-TAT was pre-excited with a UV lamp for 5 min before injection. The luminescence images were acquired without any excitation/emission 6


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filters. The fluorescence acquisition filters were set as 500 ± 20 nm excitation/707 ± 20 nm emission for the QDs, and 696 ± 20 nm excitation/719 ± 20 nm emission for the dye. ASCs were incubated overnight with fresh culture medium containing 50 µg/mL of the LPLNPs-TAT, 50 ng/mL of the QDs and 10 µg/mL of the dye, respectively. The cells were collected by trypsin treatment and washed with PBS for three times. 1 × 106 of each labeled ASCs were injected onto the backs of three mice, and persistent luminescence or fluorescence images were then acquired. Mouse administrated with LPLNPs-TAT labeled ASCs was excited with a red LED light for 1 min before acquisition of NIR persistent luminescence images. In Vivo Tracking of ASCs. The mouse was anesthetized and two 1-cm full-thickness excisional skin wounds were created on each side of the midline. 1 × 106 of LPLNPs-TAT labeled and unlabeled ASCs in 50 µL of Matrigel were injected onto the wound bed on the left and right side, respectively. All luminescence images were taken using IVIS Imaging System on 0, 3, 7, 10, 14 and 21 d after injection. The mouse was excited with a red LED light for 1 min before luminescence acquisition. LPLNPs-TAT Labeled ASCs Homing to 4T1 Tumor Model. LPLNPs-TAT labeled ASCs (1 × 106) and LPLNPs-TAT (400 µg) in 200 µL of PBS were intravenously injected into 4T1 tumor bearing mice, respectively. The mice were irradiated with a red LED light for 1 min before acquiring NIR persistent luminescence signals. The mice were sacrificed on 2 d or 7 d after injection, and the major organs and tumors were collected for luminescence imaging. All tissues were irradiated with a red LED light for 1 min before imaging. The tumor and lung slices from mouse treated with LPLNPs-TAT labeled ASCs after 2 d were fixed, stained with DAPI and observed on the inverted microscope equipped with an EMCCD camera.

RESULTS AND DISCUSSION Functionalization and Characterization of LPLNPs. The NIR-emitting LPLNPs was synthesized via a hydrothermal method in combination with sintering in air.25,32-34 The synthesized LPLNPs has a diameter of 37.4 ± 7.0 nm (calculated from 100 randomly selected particles) (Figure 1a) with a pure spinel phase of zinc gallogermanates solid solution (Figure S1a, b). The LPLNPs can be excited by a broad wavelength range of light to give a NIR emission band at 694 nm (Figure 1b), which was attributed to the 2E → 4A2 transition of distorted Cr3+ ions in gallogermanate.28 The LPLNPs also exhibits excellent long lasting NIR luminescence after irradiation with a UV lamp for 5 min (Figure 1c), and can be repeatedly activated with a red LED light to restore the persistent luminescence signal (Figure 1d). Such persistent luminescence signal can also be easily captured on an IVIS Imaging

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system (Figure 1e). The LPLNPs in aqueous solution (1 mg/mL) exhibits similar optical properties (Figure 1f and Figure S2a, b).

Figure 1. Morphology and persistent luminescence properties of the LPLNPs. (a) TEM image of the LPLNPs. (b) Excitation and emission spectra of the LPLNPs powder (λem = 694 nm). (c) NIR-persistent luminescence decay curve of the LPLNPs monitored at 694 nm after irradiation with a UV lamp for 5 min. (d) LED light reactivated persistent luminescence decay curves monitored at 694 nm after reactivation every 1000 s with a red LED light for 1 min. (e) NIR persistent luminescence and LED reactivated persistent luminescence images of LPLNPs powder (200 mg). (f) NIR persistent luminescence and LED reactivated persistent luminescence images of aqueous solution of the LPLNPs (100 µL, 1 mg/mL). The sample was irradiated with a UV lamp for 5 min before imaging. Nature decay of persistent luminescence was captured to 72 h or 24 h. The solid sample was reactivated every two days with a red LED light for 1 min, and the aqueous sample was reactivated daily with the LED for 1 min. Images at 1 h after the reactivation were also captured. The as-prepared LPLNPs were finally functionalized with TAT peptide to improve the biocompatibility, solubility, and cellular uptake. Briefly, hydroxyl groups were firstly introduced on the surface of LPLNPs, followed by aminosilanization and PEGylation. TAT penetration peptide was finally conjugated via amide condensation 8


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reaction28,35,36 (Figure 2a). As a result, the hydrodynamic size of the LPLNPs increased from 60.1 nm to the final 104.4 nm (Figure S3a). The zeta potential changed from –36.0 to +28.1 mV after coating aminosilanes, whereas PEGylation caused an opposite shift to –6.5 mV. The final zeta potential of LPLNPs-TAT became +6.8 mV after TAT peptide grafting (Figure S3b), as TAT peptide is abundant in arginine. The surface modification of the LPLNPs was further confirmed by Fourier transform infrared (FT-IR) spectrometry and thermogravimetric analysis (TGA). LPLNPs-NH2 gave strong absorption bands of stretching vibration of O–Si–O at 1105 and 1045 cm-1, the asymmetric and symmetric –CH2– stretching bands at 2925 and 2858 cm-1, and the N–H stretching bands at 3412 and 3242 cm-1. After modification with PEG, the resulting LPLNPs-COOH had two absorption bands of stretching vibration of C–O and –CH2– at 1110 and 2874 cm-1. LPLNPs-TAT has an obvious absorption band at 1658 cm-1 for stretching vibration of –CO–NH–27,28 (Figure S3c). TGA shows increased weight loss with the gradual modification (Figure S3d). All the results indicate the successful modification of the LPLNPs. The time-dependent distribution and metabolization in the mice after intravenous injection of LPLNPs-TAT was investigated (Figure 2b). Although the persistent luminescence signal of LPLNPs-TAT was getting weak, it was recovered with 1-min red LED excitation (Figure 2c). The mouse was dissected and the main organs were collected at day 7. We observed a majority of LPLNPs-TAT in the liver and a low uptake by the spleen, indicating that the LPLNPs-TAT was accumulated in the reticuloendothlial system (RES) organs. The above results also show the possibility of the red LED light renewable persistent luminescence of LPLNPs-TAT for tracking ASCs in therapeutic applications, such as tissue regeneration and tumor homing.

Figure 2. Surface modification and biodistribution of LPLNPs-TAT. (a) Schematic representation of LPLNPs surface modification. (b) In vivo biodistribution of LPLNPs-TAT after intravenous injection (800 µg, irradiated with a UV

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lamp for 5 min before injection). (c) LED light reactivated images of post-injected mouse.

In Vitro Labeling and Imaging of ASCs with LPLNPs-TAT. To achieve good internalization and labeling efficiency, all cell lines were overnight incubated with 50 µg/mL of LPLNPs-TAT. The obtained LPLNPs-TAT labeled ASCs were imaged on a microscope equipped with an EMCCD camera. After 1-min excitation with a red LED light, obvious persistent luminescence signal was observed in the ASCs cytoplasm (Figure 3a). The luminescence decay images of LPLNPs-TAT labeled ASCs were captured after excitation with a red LED light for 1 min. It is worth noting that the cells were still detectable for more than 30 min without external excitation. Although the luminescence signal in the cells was getting very weak after 30 min, it could be repeatedly activated with a red LED light (Figure 3b). We used thin-section TEM images to study the stability and location of the LPLNPs-TAT inside the ASCs. LPLNPs-TAT was accumulated in the cell endosomes/lysosomes, and able to be carried over to the daughter cells (Figure S4a). The formation of endocytic vesicles indicated that the uptake of LPLNPs-TAT was mainly via endocytosis (Figure S4b). A common challenge with ASCs tracking is that the labeling probes leach out from the cell over time by exocytosis, leading to the possible uptake of these probes by other nearby cells and introducing false positive signals.37 To address if this problem appears for LPLNPs-TAT, we co-cultured LPLNPs-TAT labeled ASCs and unlabeled GFP-expressed ASCs in the same plate. In this way, we observed the persistent luminescence signals of the mixed ASCs on a microscope during the tested 1 and 3 days, but did not see obvious persistent luminescence signals in the unlabeled GFP-expressed ASCs (Figure S4c). These results suggest that LPLNPs-TAT could effectively label ASCs for at least three days without significant leakage from the labeled cells. The advantages of LED light renewable ability and no background interference render great potential to LPLNPs-TAT for long-term tracking of ASCs.

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Figure 3. Luminescence microscopic images of LPLNPs-TAT labeled ASCs. (a) Uptake of LPLNPs-TAT by GFP-expressed ASCs. The cellular nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, blue). The NIR persistent luminescence signals of the cells were captured after excitation with a red LED light for 1 min. (b) Luminescence decay images of the cells and repeatedly activated signals after LED excitation. The exposure time of all persistent luminescence images is 10 s. Scale bar is 50 µm. Toxicity Study of LPLNPs-TAT Labeled ASCs. The effect of LPLNPs-TAT on the differentiation capability of ASCs, the cytotoxicity and in vivo toxicity of LPLNPs-TAT were evaluated before tracking stem cells. To examine whether LPLNPs-TAT labeling interferes with the differentiation capacity of ASCs toward multiple cell lineages, we treated LPLNPs-TAT labeled and unlabeled ASCs with adipogenic, chondrogenic, and osteogenic supplemented medium, respectively. The results showed no significant stain difference between LPLNPs-TAT labeled and unlabeled ASCs in each kind of differentiation (Figure S5a). These data suggest that the LPLNPs-TAT labeling did not affect the differentiation of ASCs. The in vitro cytotoxicity of LPLNPs-TAT was studied on ASCs (Figure S5b) and 4T1 (Figure S5c) cell lines using MTT assay. For all the cell lines studied, the viability remains > 90% within 5-day study duration (up to 100 µg/mL), indicating low cytotoxicity of LPLNPs-TAT. The in vivo toxicity of LPLNPs-TAT was evaluated via histological studies of main organs including heart, liver, spleen, lung, and kidney from LPLNPs-TAT pre-injected mice (Figure S5d). The hematoxylin and eosin (H&E) stained slices showed that the LPLNPs-TAT hardly caused damage in any lesions to organs. Moreover, the mice viability, weight, and activity had no significant change 3 months after injection. All these data reveal the low toxicity of LPLNPs-TAT. In Vivo Comparison of LPLNPs-TAT with Conventional Fluorescent Probes. The performance of in vivo 11



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imaging of LPLNPs-TAT labeled ASCs was evaluated by subcutaneously injecting various numbers of ASCs onto the back of a nude mouse. Strong persistent luminescence signals were visualized after 1-min excitation with a red LED light (Figure 4a). The SNR for in vivo imaging of LPLNPs-TAT labeled ASCs significantly improved as the cell numbers increased (Figure 4b). Noteworthy is that as few as ~ ten LPLNPs-TAT labeled ASCs could be successfully detected and the SNR remained 2.9 with such low cell numbers. The crucial advantage of persistent luminescence for in vivo tracking of ASCs over other classical fluorescence techniques was revealed by comparing LPLNPs-TAT with commercial CdTe QDs (700 nm emission) and a BODIPY-derivative dye (710 nm emission). 30 µg of the LPLNPs-TAT, the QDs and the dye were subcutaneously injected onto the back flanks of three nude mice, respectively. In spite of the much higher photons generated by the QDs or the dye over the LPLNPs-TAT, the SNR of the LPLNPs-TAT was far better than that with the QDs and the dye (dorsal view). As shown in the ventral view of each mouse (Figure 4c), the fluorescence signal of the QDs or the dye was hard to see as the autofluorescence became obviously significant, but the persistent luminescence of LPLNPs-TAT was still clear with the SNR of 22.2. The high SNR of the LPLNPs-TAT resulted from the absence of autofluorescence background without in situ excitation. We next used these three probes to label ASCs, and injected the same number (1 × 106) of each labeled ASCs onto the middle backs of three mice, respectively. After 1-min excitation with a red LED light, manifest persistent luminescence signals of the LPLNPs-TAT labeled ASCs were visualized clearly. On the contrary, the fluorescence signals of the QDs or the dye labeled ASCs were covered by the overwhelming autofluorescence background, and the injected position of the labeled ASCs was invisible (Figure 4d). The comparison of these probes reveals a much better SNR of LPLNPs-TAT for in vivo tracking of ASCs.

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Figure 4. Detection depth and SNR study of LPLNPs-TAT-based imaging. (a) In vivo tracking of LPLNPs-TAT labeled ASCs. Mouse was subcutaneously injected with various numbers of LPLNPs-TAT labeled ASCs (10 to 1,000). (b) SNR for various numbers of LPLNPs-TAT labeled ASCs. (c) In vivo imaging depth comparison of LPLNPs-TAT, QDs and dye. (d) In vivo imaging comparison of LPLNPs-TAT, QDs and dye labeled ASCs.

Long-term Tracking and Engraftment Evaluation of LPLNPs-TAT Labeled ASCs in Skin Wound Bearing Mouse Model. ASCs have the ability to accelerate the repairing of damaged tissue.19 We next used a full-thickness wound mouse model to study the regeneration capability of LPLNPs-TAT labeled ASCs. Mice with the same size wound (~ 1 cm) on the dorsal skin were divided randomly into four groups (n = 6 in each group), followed by injecting PBS, Matrigel, LPLNPs-TAT labeled and unlabeled ASCs in Matrigel onto the wound sides, respectively. The appearance of in vivo wound closure was recorded by a digital camera and the wound sizes were measured over time (Figure S6a, b). Wounds treated with LPLNPs-TAT labeled and unlabeled ASCs exhibited faster healing capability compared to those treated with PBS or Matrigel. Histological evaluation of the wound sides from four groups shows that the ASCs treated wounds were healed after 7 days and newly formed skin and hair follicles were found within 21 days, while the PBS and Matrigel treated groups showed no hair follicles generation (Figure S6c).

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All these results indicate that ASCs promoted the wound healing, and the labeling of LPLNPs-TAT did not affect the wound repair efficacy of ASCs. We then studied the feasibility of the LPLNPs-TAT labeling for long-term tracking of ASCs in wound regeneration model. To this end, two wounds on both left and right sides of the nude mice (n = 4) were generated, followed by injecting the same amount (1 × 106) of LPLNPs-TAT labeled ASCs to the left wound side and unlabeled ASCs to the right wound side. The in vivo persistent luminescence images of ASCs transplanted mouse (pre-excited with red LED light for 1 min) at different time points were captured (Figure 5a). We observed obvious luminescence signals from the left wound side distinguished from the right wound side or any other places of the mouse body, due to the elimination of in situ excitation and the absence of autofluorescence background of mouse body. The SNR of persistent luminescence signals from the left wound side decreased during the skin regeneration. The persistent luminescence signals decreased obviously after the scars shedding at day 7 due to the loss of dead ASCs, but the SNR remained 10.4 after transplantation for 21 days. To make sure the persistent luminescence signals in the left wound side were truly from the LPLNPs-TAT labeled ASCs rather than free nanoparticles, the skin section on the left wound side was collected after transplantation with LPLNPs-TAT labeled ASCs for 3 days. Persistent luminescence image of LPLNPs-TAT and fluorescence image of GFP were captured and merged together. The LPLNPs-TAT and GFP signals were co-localized efficiently, indicating that the in vivo persistent luminescence signals resulted from the LPLNPs-TAT labeled ASCs (Figure 5b).

Figure 5. In vivo tracking of ASCs in skin wound bearing mouse model. (a) Representative time-dependent persistent luminescence images of the wound sites from mouse transplanted with LPLNPs-TAT labeled (1 × 106, left side wound) and unlabeled (1 × 106, right side wound) ASCs (n = 4). (b) Luminescence and fluorescence microscopy images of wound slice taken from the left side wound after transplantation for 3 days. Cell nuclei were stained by DAPI (blue). Green and red colors represent fluorescence signals of GFP and persistent luminescence signals of LPLNPs-TAT, respectively. Scale bar is 50 µm. 14


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Scars shed from wounds pre-treated with PBS, Matrigel, LPLNPs-TAT labeled or unlabeled ASCs in Matrigel after 7 days were collected and imaged to compare the imaging performance of persistent luminescence with conventional fluorescence. The scar from wound pre-treated with LPLNPs-TAT labeled ASCs showed obvious persistent luminescence signal compared with other three scars in the absence of in situ excitation. In contrast, we also imaged the scars under excitation at wavelengths for various common used fluorescent probes, and found overwhelming autofluorescence background due to the constant external excitation (Figure S7). This result further indicates that LPLNPs-TAT-based imaging has much better SNR than conventional fluorescent probes. Tracking of ASCs Homing to the Tumor. It is well-accepted that mesenchymal stem cells could target to tumors and retain in the tumors. This natural tumor affinity is possibly mediated with chemokines.4 To show the capability of LPLNPs-TAT labeled ASCs for tumor homing, 1 × 106 of the labeled ASCs were intravenously injected 14 days after 4T1 tumor cells subcutaneous injection. For comparison, 400 µg LPLNPs-TAT was intravenously injected as a control. In vivo persistent luminescence imaging was performed within 24 h after injection and the mice were pre-excited with LED for 1 min before imaging. The results show that most ASCs were trapped in the lung due to the rapid sequestration of phagocytes in the lung capillary bed,25 but a small amount of ASCs survived and homed to the 4T1 tumor,8 in contrast to the accumulation in RES organs of LPLNPs-TAT (Figure 6a).

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Figure 6. In vivo tracking of the LPLNPs-TAT labeled ASCs (1 × 106) in 4T1 tumor bearing mice. (a) Representative time-dependent biodistribution of LPLNPs-TAT and LPLNPs-TAT labeled ASCs after intravenous injection. (b) Representative ex vivo NIR-persistent luminescence images of isolated organs from 4T1 tumor bearing mice pre-injected with LPLNPs-TAT or LPLNPs-TAT labeled ASCs at day 2 and 7. (c) Relative intensity of organs in (b). The ex vivo biodistribution of LPLNPs-TAT labeled ASCs was assessed by imaging the persistent luminescence signals of main organs. Most persistent luminescence signals associated with LPLNPs-TAT labeled ASCs were found in lung and tumor, which is in good agreement with the in vivo luminescence images (Figure 6b). The LPLNPs-TAT labeled ASCs accumulated in tumor significantly increased with time after injection (Figure 6c). However, in the control group, most nanoparticles were accumulated in the liver and spleen instead of tumor in LPLNPs-TAT injected mice. Further microscopy images of lung and tumor sections show the persistent luminescence signals from LPLNPs-TAT labeled ASCs (Figure S8). These results confirm that labeling of LPLNPs-TAT has no adverse side effect on tumor homing ability of ASCs, and offers great potential for long-term tracking of ASCs during homing to the tumor process.

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CONCLUSIONS We have developed TAT-bioconjugated persistent luminescence nanoparticles as a novel cell tracker for noninvasive long-term tracking of ASCs. The prepared LPLNPs-TAT shows long lasting NIR-persistent luminescence, red LED light renewable property, and low toxicity. All these promising features enable LPLNPs-TAT for ultrasensitive bioimaging without causing any adverse side effect on ASCs. Compared with common fluorescent probes such as QDs or organic dyes for bioimaging under in situ excitation which suffers from pronounced autofluorescence background, LPLNPs-TAT-based imaging is easily achieved by acquiring persistent luminescence signals without in situ excitation. Owing to the absence of autofluorescence background from animal tissue, LPLNPs-TAT shows an outstanding SNR compared with other optical probes. Moreover, tracking with LPLNPs-TAT only needs simple incubation to label the cells, and thus is more stable and convenient than genetic transfected luciferase with the need for complicated genetic transfection and repeated injection of enzyme substrates. Although skin regeneration is a superficial model, and many probes have been used for tracking stem cells in this model,19,20 labeling of LPLNPs-TAT shows more details about long-term fate of transplanted ASCs. Our studies clearly demonstrate that some transplanted ASCs became part of the new tissue of wound side and promoted skin healing, while a few transplanted ASCs detached from the wound. Taking advantage of no autofluorescence interference, LPLNPs-TAT-based imaging shows great potential for clinical applications. Labeling of LPLNPs-TAT can track ASCs during tumor homing process both in vivo and ex vivo. As the NIR-emitting light has better penetration, LPLNPs will have a wider use in deep tissue tracking than common white-light emitted luciferase. This labeling strategy will also have universal applications in monitoring other significant cells, such as dendritic cells, metastatic cancer cells and transplanted cells. This approach of LPLNPs-TAT-based ASCs tracking will not only inspire tracking cells in important life processes, but also lead to broad application in stem cell-based research and therapy.

ASSOCIATED CONTENT Supporting Information Available: Materials and reagents, cytotoxicity assay, differentiation study, TEM images of LPLNPs-TAT labeled ASCs, and histochemical studies. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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Corresponding Author *E-mail: [email protected]. Fax: +86-22-23506075.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grants 21435001, 21275079, 81227901, 61501462) and the National Basic Research Program of China (grant 2011CB707703). We thank the support of optical multi-modal platform from Key Laboratory of Molecular Imaging of Chinese Academy of Sciences.

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