Research Article www.acsami.org
Long-Term Tracking of the Osteogenic Differentiation of Mouse BMSCs by Aggregation-Induced Emission Nanoparticles Meng Gao,† Junjian Chen,‡ Gengwei Lin,† Shiwu Li,† Lin Wang,‡ Anjun Qin,† Zujin Zhao,*,† Li Ren,*,‡ Yingjun Wang,*,‡ and Ben Zhong Tang*,†,§,⊥ †
State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510640, China § Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China ⊥ Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Hong Kong, China ‡
S Supporting Information *
ABSTRACT: Bone marrow-derived mesenchymal stem cells (BMSCs) have shown great potential for bone repair due to their strong proliferation ability and osteogenic capacity. To evaluate and improve the stem cell-based therapy, long-term tracking of stem cell differentiation into bone-forming osteoblasts is required. However, conventional fluorescent trackers such as fluorescent proteins, quantum dots, and fluorophores with aggregation-caused quenching (ACQ) characteristics have intrinsic limitations of possible interference with stem cell differentiation, heavy metal cytotoxicity, and self-quenching at a high labeling intensity. Herein, we developed aggregation-induced emission nanoparticles decorated with the Tat peptide (AIE-Tat NPs) for long-term tracking of the osteogenic differentiation of mouse BMSCs without interference of cell viability and differentiation ability. Compared with the ability of the commercial Qtracker 655 for tracking of only 6 passages of mouse BMSCs, AIE-Tat NPs have shown a much superior performance in long-term tracking for over 12 passages. Moreover, long-term tracking of the osteogenic differentiation process of mouse BMSCs was successfully conducted on the biocompatible hydroxyapatite scaffold, which is widely used in bone tissue engineering. Thus, AIE-Tat NPs have promising applications in tracking stem cell fate for bone repair. KEYWORDS: aggregation-induced emission, long-term cell tracking, bone repair, stem cell, osteogenic differentiation
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INTRODUCTION Bone defects caused by injuries or diseases are commonly encountered in clinics and traditionally treated by autologous bone transplantation. However, this therapy is limited by bone sources, complex operations, and postoperative syndrome.1 Recently, bone marrow-derived mesenchymal stem cells (BMSCs) have shown great potential for bone repair due to their strong ability to proliferate and differentiate into bone-forming osteoblasts.2,3 This stem cell-based therapy requires long-term tracking of the distribution and differentiation process of delivered stem cells in the bone healing process without compromising effects on cell survival and differentiation ability. Compared with magnetic resonance imaging (MRI)4,5 and positron emission tomography (PET) imaging techniques,6 fluorescent imaging has advantages in terms of high resolution, easy operation, and low cost. Recently, various fluorescent agents have been used for long-term tracking of the differentiation of stem cells, including fluorescent proteins or luciferases,7−9 quantum dots,10 organic fluorophores,11 and nanoparticles.12,13 © 2016 American Chemical Society
Although much progress has been achieved to improve their performance in photostability and biocompatibility, they still suffer from intrinsic limitations.14,15 Fluorescent proteins or luciferases require high gene transfection efficiency and may interrupt normal cell activities.16 The heavy metal-containing quantum dots are cytotoxic after biodegradation of the surface ligands and can inhibit the differentiation of stem cells.17,18 The conventional organic fluorophores with aggregation-caused quenching (ACQ) properties can easily undergo self-quenching at high labeling intensity or strong fluorophore loading in NPs.19 Moreover, they also suffer from the drawbacks of poor photostability, easy diffusion out of cells, and small Stokes shifts.20 Aggregation-induced emission (AIE) fluorogens have recently emerged as a new generation of fluorescent materials with Received: May 8, 2016 Accepted: June 30, 2016 Published: July 11, 2016 17878
DOI: 10.1021/acsami.6b05471 ACS Appl. Mater. Interfaces 2016, 8, 17878−17884
Research Article
ACS Applied Materials & Interfaces Scheme 1. Fabrication of AIE-Tat NPs Based on PITBT-TPE Fluorogen
Figure 1. (A) Particle size distribution of AIE-Tat NPs measured by dynamic light scattering with a mean diameter of 121.2 nm and a PDI of 0.121. Inset: TEM images of AIE-Tat NPs. (B) Normalized UV−vis absorption (blue line) and PL spectra (red line) of AIE-Tat NPs in aqueous solution. λex = 483 nm.
various advantages such as high brightness, strong antiphotobleaching ability, large Stokes shifts, and low cytotoxicity.21−24 Due to the restriction of intramolecular motion, AIE fluorogens are highly emissive in the aggregated state and can thus be easily fabricated into highly bright NPs via
physical encapsulation with amphiphilic polymers or silica, covalent binding with block copolymer, and self-assembly.25,26 AIE NPs have broad applications in tumor imaging, vascular imaging, image-guided therapy, and long-term cell tracking.27−34 As biocompatible and fluorescent tracking agents 17879
DOI: 10.1021/acsami.6b05471 ACS Appl. Mater. Interfaces 2016, 8, 17878−17884
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ACS Applied Materials & Interfaces
Figure 2. Confocal laser scanning microscopy (CLSM) images of mouse BMSCs costained with AIE-Tat NPs and Hoechst 33342: (A) bright field image, (B) fluorescent image from AIE-Tat NPs, (C) fluorescent image from Hoechst 33342, and (D) merged image. Scale bar = 20 μm. (E) Metabolic viability of mouse BMSCs after incubation with 0, 12.5, 25, 50, 100, 200, 300, 400, and 500 pM AIE-Tat NPs for 48 h. (F) Photostability of internalized AIE-Tat NPs under continuous light irradiation (130 W).
Figure 3. Flow cytometry histograms of mouse BMSCs after incubation with (A) 50 pM AIE-Tat NPs and (B) 2 nM Qtracker 655 at 37 °C for 4 h and then subculture for the designated passages. The untreated mouse BMSCs were used as the control. The fluorescent images of mouse BMSCs for (C) AIE-Tat NPs and (D) Qtracker 655 on designated passages were recorded under excitation at 488 nm with a 550−780 nm bandpass filter.
are highly desirable for stem cell-based bone repair, we developed red-emissive AIE-Tat nanoparticles for long-term
tracking of the osteogenic differentiation process of mouse BMSCs. 17880
DOI: 10.1021/acsami.6b05471 ACS Appl. Mater. Interfaces 2016, 8, 17878−17884
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ACS Applied Materials & Interfaces
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RESULTS AND DISCUSSION The AIE fluorogen of PITBT-TPE was synthesized according to our previous report.35 The AIE nanoparticles with maleimide groups on the surface were synthesized according to a modified nanoprecipitation method (Scheme 1).30 A mixture of DSPEPEG2000 and DSPE-PEG2000-maleimide was used as an encapsulation matrix for encapsulating the fluorogen of PITBT-TPE under sonication to yield AIE nanoparticles with maleimide groups on the surface. The obtained AIE nanoparticles further reacted with a cysteine-modified cell penetrating Tat peptide (RKKRRQRRRC) to yield AIE-Tat NPs. The average diameter of AIE-Tat NPs in aqueous solution was measured to be 121.2 nm by a dynamic light scattering (DLS) experiment with a low polydispersity index (PDI) of 0.121 (Figure 1A). A smaller diameter of about 100 nm for AIE-Tat NPs was observed under TEM, possibly due to the shrinking effect during the drying process. The photophysical properties of AIE-Tat NPs were then investigated, and a maximum absorption
at 483 nm and an intense emission peak at 645 nm were observed (Figure 1B). The fluorescence quantum yield and lifetime of AIE-Tat NPs were measured to be 23.5% and 5.37 ns, respectively. The highly efficient red emission and large Stokes shift (162 nm) of AIE-Tat NPs will greatly favor their bioimaging applications with a high signal-to-noise ratio, as the possible interference of biological autofluorescence can be efficiently reduced. After incubation of mouse BMSCs with 50 pM AIE-Tat NPs at 37 °C for 4 h, the AIE-Tat NPs were distributed in the cytoplasm of mouse BMSCs and emitted intense red fluorescence which was verified by costaining with nuclear dye Hoechst 33342 under a confocal microscope (Figures 2A−D and Figure S1). An excellent 100% labeling efficiency for mouse BMSCs by AIE-Tat NPs was achieved and verified by a flow cytometry experiment (Figure S2). As determined by the CCK8 assays, the cell viability of mouse BMSCs was not affected by different concentrations of AIE-Tat NPs (Figure 2E), which indicates that
Figure 4. (A−C) Fluorescent images of mouse BMSCs grown on an HA scaffold. (D−F) RT-PCR results of OPN, BMP2, Col I, and ALP gene expression in AIE-Tat NP-labeled and unlabeled mouse BMSCs after osteogenic induction for 3, 7, and 14 days. 17881
DOI: 10.1021/acsami.6b05471 ACS Appl. Mater. Interfaces 2016, 8, 17878−17884
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AIE-Tat NPs have shown a superior tracking ability for over 12 passages. Moreover, long-term tracking of the osteogenic differentiation process was successfully conducted on the HA scaffold for bone tissue engineering, while the cell viability and differentiating ability were not compromised. AIE-Tat NPs are thus promising for the tracking of stem cell fate in the bone repair process.
AIE-Tat NPs have good biocompatibility. The photostability of AIE-Tat NPs after mouse BMSCs were stained was also measured, and the signal loss of AIE-Tat NPs was less than 20% after 5 min of continuous irradiation, which reveals their excellent photostability (Figure 2F). The significant advantages of AIE-Tat NPs in terms of high labeling efficiency, good biocompatibility, and excellent photostability make them especially suitable for long-term cell tracking applications. With the commercial Qtracker 655 labeling kit as a reference, the in vitro tracking ability of AIE-Tat NPs for mouse BMSCs was then evaluated. The mouse BMSCs labeled with AIE-Tat NPs and Qtracker 655 were subcultured continuously for different passages, and the fluorescence signals were quantitatively analyzed by flow cytometry (Figures 3A and B and Figure S3). After 1, 6, and 12 passages of subculture, the labeling efficiencies of mouse BMSCs by AIE-Tat NPs were 100, 96.7, and 35.3%, respectively. Comparatively, only 90.2, 5.36, and 0.35% of mouse BMSCs were labeled by Qtracker 655 after 1, 6, and 12 passages, respectively. These results clearly prove that AIE-Tat NPs have tracking ability superior to that of Qtracker 655. This is also verified by fluorescent imaging experiments (Figures 3C and D). AIE-Tat NPs-labeled cells showed fluorescence much brighter than that of Qtracker 655-labeled ones under the same irradiation conditions. After subculture for 6 passages, the cells labeled with AIE-Tat NPs still emitted strong fluorescence. On the contrary, almost no fluorescence was observed for cells labeled with Qtracker 655. The excellent cell tracking ability of AIE-Tat NPs can be ascribed to their efficient cell uptake and long-term intracellular retaining abilities. Hydroxyapatite (HA) has a similar composition to natural bone minerals and has been widely used as a scaffold to support cell adhesion and growth at bone defects.36 We previously developed an efficient method for preparing HA scaffolds with excellent biocompatibility and osteoinduction ability.37 As cultured osteoblast cells on the HA scaffold can be defined as regenerative bone tissue,38 long-term fluorescent tracking of the osteogenic differentiation process was then conducted by culturing mouse BMSCs on the HA scaffold under osteogenic conditions. The AIE-Tat NPs-treated mouse BMSCs showed bright fluorescence during the osteogenic differentiation process for over 14 days, which reveals that AIE-Tat NPs can be efficiently internalized and intracellularly retained long-term (Figures 4A−C). It was then examined whether the internalized AIE-Tat NPs would interfere with the osteogenic differentiation process. Osteopontin (OPN), bone morphogenetic protein 2 (BMP-2), collage 1 (Col I), and alkaline phosphatase (ALP) are well-accepted gene markers for osteogenic differentiation. These gene markers were measured by reverse transcription polymerase chain reaction (RT-PCR) after osteogenesis induction of mouse BMSCs for 3, 7, and 14 days, and their increasing expression clearly verified the successful osteogenic differentiation of mouse BMSCs (Figure S4). Moreover, no significant difference was observed for their expression in AIE-Tat NP-labeled and unlabeled mouse BMSCs (Figures 4D−F), which indicates that the internalization of AIE-Tat NPs did not give rise to adverse effects on osteogenic differentiation.
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EXPERIMENTAL SECTION
Materials and Chemicals. PITBT-TPE was synthesized according to our previous report.30 DSPE-PEG2000 and DSPE-PEG2000-Mal were purchased from Laysan Bio, Inc. DMEM (low glucose); DMEM/F12, FBS, penicillin−streptomycin, and Qtracker 655 were purchased from Thermo Fisher Scientific. Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), β-glycerophosphate, dexamethasone, and ascorbic acid were purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK8) was purchased from Dojindo, Japan. The cell penetrating peptide HIV-1 Tat with a cysteine-modified terminus (RKKRRQRRRC) was purchased from GenicBio. Equipment and Methods. UV−vis absorption spectra were measured on a Shimadzu UV-2600 spectrophotometer with a medium scanning rate and quartz cuvettes of a 1 cm path length. Photoluminescence spectra were recorded on a Horiba Fluoromax-4 spectrofluorometer. The absolute fluorescence quantum yield was measured using a Hamamatsu quantum yield spectrometer C11347 Quantaurus_QY. The fluorescence lifetime was measured using a Hamamatsu Compact Fluorescence Lifetime spectrometer C11367. Particle size measurements were performed using a Malvern ZetaSizer based on DLS. Fluorescent images were obtained by a CoolSnap HQ2 camera (Photometrics, Tuscon, AZ) under excitation at 488 nm with a 550−780 nm bandpass filter and analyzed by NIS Elements software (Version 3.0, Nikon Instruments, Melville, NY). TEM samples were prepared by drying a drop of aqueous solution of AIE-Tat NPs onto a carbon-coated copper grid. TEM measurements were performed on a JEM2010F instrument (JEOL Ltd., Akishima-Tokyo, Japan). Flow cytometry was measured on a Guava EasyCyte HT system by counting 7000 gated cells (excitation at 488 nm). Synthesis of AIE-Tat NPs. A THF solution containing PITBT-TPE, DSPE-PEG2000, and DSPE-PEG2000-Mal (1.0 mg each) was added to distilled water (9 mL) under sonification for 1 min (SCIENTZ-II D with 80% output). THF was then removed under stirring and nitrogen flow at 25 °C. After being filtered with a 0.2 μm filter, the AIE-active nanoparticle suspension was further reacted with HIV-1 Tat peptide (3 μM) for 12 h at 25 °C. Finally, the solution containing AIE-Tat NPs was dialyzed against distilled water for 2 days to remove excess peptide. The AIE-Tat NPs were collected for the following experiments. Cell Culture. Mouse BMSCs were purchased from ATCC (CRL12424) and cultured in DMEM with 1% penicillin-streptomycin and 10% FBS at 37 °C in a humidified incubator with 5% CO2. The culture medium was changed every other day, and the cells were collected by being treated with a 0.25% trypsin/1 mM EDTA solution. The cells of passages 3 and 4 were used in the following experiments of cell tracking, proliferation, and differentiation. The chemical mixture for inducing osteogenic differentiation includes β-glycerophosphate (10 mM), ascorbic acid (50 mM), and dexamethasone (100 nM). Cytotoxicity of AIE-Tat NPs. Cytotoxicity of AIE-Tat NPs was measured by the CCK-8 assay according to the manufacturer’s method (n = 5). A suspension of mouse BMSCs (density of 1 × 104 cells) was added onto the samples. After 24 h of culture, cell/sample constructs were transferred to a new plate followed by addition of 300 μL of the CCK-8 working solution into each well. After incubation at 37 °C for 1 h, 100 μL of the solution was pipetted into a 96-well plate, and the absorption was measured at 450 nm with a microplate reader (Thermo3001). Photostability of AIE-Tat NPs. Fluorescent images were obtained using the CoolSnap HQ2 camera under excitation at 488 nm at 130 W for a prolonged time (5 min) and statistically analyzed using image processing software (ImageJ, National Institutes of Health, United States). In Vitro Cell Tracking. After mouse BMSCs were cultured to achieve 80% confluence, the culture medium was removed, and the cells were washed with PBS. Then, 50 pM AIE-Tat NPs or 2 nM Qtracker
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CONCLUSION In summary, we developed a new kind of AIE-Tat NPs with the significant advantages of highly efficient red emission, easy preparation, high cell labeling efficiency, low cytotoxicity, and strong anti-photobleaching ability. Compared with commercial Qtracker 655 for the tracking of only 6 passages of mouse BMSCs, 17882
DOI: 10.1021/acsami.6b05471 ACS Appl. Mater. Interfaces 2016, 8, 17878−17884
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ACS Applied Materials & Interfaces 655 in DMEM was added, and the solution was further incubated at 37 °C for 4 h. The cells were then subcultured in 6-well plates for 1−12 passages. At designated time intervals, the cells were washed twice with PBS and detached for suspension in a DMEM medium. The fluorescence intensities of cells were analyzed by flow cytometry on a Guava EasyCyte HT system, and the histogram for each sample was obtained by counting 7000 gated cells. The fluorescence images were obtained by a CoolSnap HQ2 camera and analyzed by NIS Elements software. Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction. Mouse BMSCs were cultured with a density of 1 × 104 cells on the hydroxyapatite scaffold (Φ 9 mm × 3 mm, prepared with our previously reported method30) for a certain time (3, 7, and 14 days) in an osteogenic differentiation medium. RNA of different samples (n = 4) was isolated using HiPure Total RNA Kits (Magentec, China), and RNA concentrations were measured by a NanoDrop2000 spectrophotometer (Thermo Scientific, United States). The obtained cDNA was then subjected to PCR, and the gene expression was examined by an SYBR Green System (Invitrogen, United States). The relative quantification of target genes was determined using GAPDH as a reference. The expression levels of target genes were calculated by the 2-ΔΔCt method using primer sequences shown in Table 1.
Central Universities (Grants 2015ZY013 and 2015ZZ104), the Guangdong Natural Science Funds for Distinguished Young Scholar (Grant 2014A030306035), ITC−CNERC14S01, and the Guangdong Innovative Research Team Program (Grant 201101C0105067115).
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Table 1. Primer Sequences Used for RT-PCR Gene Expression Analysis gene
5′−3′
primers
OPN
forward reverse forward reverse forward reverse forward reverse
5′-TGCAAACACCGTTGTAACCAAAAGC-3′ 5′-TGCAGTGGCCGTTTGCATTTCT-3′ 5′-TGAGGATTAGCAGGTCTTTG-3′ 5′-CACAACCATGTCCTGATAAT-3′ 5′-CAGCCGCTTCACCTAGC-3′ 5′-TTTTGTATTCAATCACTGTCTTGCC-3′ 5′-TGCCTACTTGTGTGGCGTGAA-3′ 5′-TCACCCGAGTGGTAGTCACAATG-3′
BMP2 Col I ALP
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b05471. Three-dimensional sectional CLSM image, flow cytometry, cell labeling efficiency, relative gene expression, and calculation of AIE-Tat NP concentration (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
M.G. and J.C. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Key Project of the Ministry of Science and Technology of China (Grants 2013CB834702 and 2012CB619100), the National Science Foundation of China (Grants 21490571, 51273053, and 51232002), the Natural Science Foundation of Guangdong Province (Grants 2016A030313852 and 2016A030312002), the China Postdoctoral Science Foundation Grant (2015M580716 and 2016T90778), the Fundamental Research Funds for the 17883
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