Photocontrolled SiRNA Delivery and Biomarker-Triggered

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Photocontrolled SiRNA Delivery and Biomarker-Triggered Luminogens of Aggregation-Induced Emission by Up-Conversion NaYF4:Yb3+Tm3+@SiO2 Nanoparticles for Inducing and Monitoring Stem-Cell Differentiation Jinming Li,† Chris Wai Tung Leung,# Dexter Siu Hong Wong,† Jianbin Xu,† Rui Li,† Yueyue Zhao,# Chris Yu Yee Yung,# Engui Zhao,# Ben Zhong Tang,*,# and Liming Bian*,†,‡,§,∥,⊥ †

Division of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong, China Shun Hing Institute of Advanced Engineering, §Shenzhen Research Institute, and ∥Centre for Novel Biomaterials, The Chinese University of Hong Kong, Hangzhou, China ⊥ China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China # Department of Chemistry, Institute of Molecular Functional Materials, The Hong Kong University of Science and Technology (HKUST), Kowloon, Hong Kong, China ‡

S Supporting Information *

ABSTRACT: Controlling the differentiation of stem cells and monitoring cell differentiation has attracted much research interest since the discovery of stem cells. In this regard, a novel near-infrared (NIR) light-activated nanoplatform is obtained by encapsulating the photoactivatable caged compound (DMNPE/siRNA) and combining a MMP13 cleaved imaging peptide-tetrapheny-lethene (TPE) unit conjugated with the mesoporous silica-coated up-conversion nanoparticles (UCNPs) for the remote control of cell differentiation and, simultaneously, for the real-time monitoring of differentiation. Upon NIR light illumination, the photoactivated caged compound is activated, and the siRNA is released from UCNPs, allowing controlled differentiation of stem cells by light. More importantly, MMP13 enzyme triggered by osteogenic differentiation would effectively cleave the TPE probe peptide, thereby allowing the real-time monitoring of differentiation in living stem cells by aggregation-induced emission (AIE). KEYWORDS: stem cell, controlled differentiation, real-time monitoring cell differentiation, up-conversion nanoparticles, aggregation-induced emission

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INTRODUCTION Stem cells, especially the adult stem cells, such as human mesenchymal stem cells (hMSCs), are becoming increasingly attractive as a cell source for regenerative medicine because of their easy availability and multipotency.1 However, inducing the differentiation of hMSCs efficiently toward an intended lineage remains a challenge. For example, conventional osteogenic differentiation protocols require between 3 to 4 weeks for fully differentiating MSCs into the mature osteoblastic phenotype. In addition to the use of inductive agents, such as small molecules and growth factors, gene-silencing technologies, including RNA interference (RNAi), have also been demonstrated to promote the directed differentiation of stem cells by selectively knocking down the targeted genes that impede stem cell differentiation toward the desired lineages.2 However, safe and efficient delivery and subsequent control of the intracellular release of small interfering RNA (siRNA) remains a major challenge to controlling differentiations of stem cells, which are known for being difficult to transfect. Various means have been used to © XXXX American Chemical Society

spatially and temporally control the activity of biomolecules; light irradiation is one such method that has gained popularity in the past decade because of its safety and effectiveness.3 Several reports have described the “photoinduced RNAi” by the caged siRNA strategy.4,5 The activity of the caged siRNA is blocked by the caging groups (such as 4,5-dimethoxy-2nitroacetophenone, DMNPE) and can be completely recovered via photoactivation. These caging groups bind the nucleic acids through a covalent bond to from the photosensitive precursor group, thereby rendering the siRNA inert. UV exposure leads to the breakage of the covalent bond and, therefore, the activation and release of the bound siRNAs.6 These caging groups have Special Issue: Current Trends in Functional Surfaces and Interfaces for Biomedical Applications Received: January 17, 2017 Accepted: March 28, 2017 Published: March 28, 2017 A

DOI: 10.1021/acsami.7b00845 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. (A) Synthesized Process of UCNP@mSiO2, (B) Conjugation of RGD Peptide and MMP13−Peptide−AIE Unit to UCNP@mSiO2 to Form the UCNP@mSiO2−Peptide−AIEgen (UCNP−Peptide−AIE) and the Encapsulation of DMNPE/ siRNA to the UCNP−Peptide−AIE to form the DMNPE/siRNA−UCNP@mSiO2−Peptide−AIEgen (UCNP−Peptide−AIE− siRNA), and (C) Mechanism of Controlling and Detecting Differentiation of hMSCs by UCNP−Peptide−AIE−siRNAa

a

The NIR-triggered release of siRNA from UCNP−peptide−AIE−siRNA enhances the osteogenic differentiation of hMSCs by suppressing the expression of PPARγ. The up-regulated MMP13 enzyme in the differentiated hMSCs specifically cleaves the MMP13-sensitive peptide, resulting in the release and subsequent aggregation of AIEgen. The aggregated AIEgen in differentiated hMSCs switched on the luminescence signal by aggregation that indicated the differentiation of stem cells.

been used in a number of biological studies to study cell motility, muscle fiber physiology, active transport of proteins, and other intracellular responses.7−9 The major limitation of this strategy is that most photo-activated caging molecules respond to UV radiation rather than visible or near-infrared (NIR) light, which give rise to problems such as phototoxicity and limited tissue-penetration depth. In contrast, NIR light possesses deep tissue penetration and minimal photodamage to the exposed cells and tissue, and NIR light is believed to be the ideal light source for photoactivation. The up-conversion nanoparticles (UCNPs) are made of host lattices of ceramic materials embedded with trivalent lanthanide ions, and UCNPs are capable of converting the absorbed NIR light to UV or visible light through the unique ladder-like energy level structures of lanthanide ions.10 Therefore, UCNPs have become a powerful tool for biomedical applications,11 including controlled drug release,12 detection of biological molecules,13 photothermal therapy14 and photodynamic therapy,15 photon lithography,16 and in vivo imaging and cancer therapy.17

However, monitoring the progression of stem cell differentiation is crucial to uncovering the key cellular events involved in the stem cell differentiation and developing effective stem-cell-based therapies. Conventional methods, such as qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, allow the assessment of stem cell differentiation by examining the expression level of marker genes.18 These analytical methods are reliable. However, a large number of cell samples need to be sacrificed to isolate the target genes or proteins, and this necessity severely limits the application of these methods. Immunofluorescence and chemical staining are two other commonly used methods to examine the differentiation status of the fixed stem cells. For example, Alizarin Red staining (ARS) and Von Kossa staining (VKS) are widely used to assess the extent of mineralization by stem cells upon osteogenic induction. However, these staining methods are not sensitive enough to monitor the early differentiation stage of stem cells, and they also preclude realtime monitoring of the intracellular expression of biomarkers in cell cultures. Thus, developing a facile and nondestructive B


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Dimethoxy-2-nitroacetophenone (DMNPE) was purchased from Molecular Probes, Inc. MAL−(PEG)4−NHS ester linker was purchased from Quanta Biodesign, Ltd.. Propiolic acid− PLGVRGKGGC−COOH peptide was synthesized by Bankpeptide, Inc. (Hefei, China). The PPARγ siRNA was purchased from Ribobio., Ltd. (Guangzhou, China), and the sequence was sense (5′−3′): GAC AUU CCA UUC ACA AGA A and antisense (5′−3′): UUC UUG UGA AUG GAA UGU CTT. Phospha-e buffered saline (PBS), fetal bovine serum (FBS), a-MEM medium, and penicillin/streptomycin were purchased from Gibco (Thermo Fisher Scientific, Gaithersburg, MD). PCR relative primers and regents were from Sangon Biotech (Shanghai, China). Transmission electron microscopy (TEM) was performed on CM-200 (Philips) with an accelerating voltage of 100 kV. Dynamic light scattering (DLS) was measured by using photon correlation spectroscopy (Brookhaven Instruments Corporation). Cell viability of alamarblue was tested by Multiskan GO (Thermo Scientific). UV−vis absorption spectra were recorded on DU800 UV−visible spectrophotometer (Beckman Coulter, Inc., Fullerton, CA). Fourier transform infrared spectrometry (FTIR) was performed on an IRTracer-100 (Shimadzu, Kyoto, Japan). Confocal laser scanning microscopy (CLSM) was performed on a SP8 instrument (Leica, Wetzlar, Germany). Polymerase chain reaction (PCR) was performed on a MJ Mini PCR instrument (Bio-Rad, Hercules, CA). Photoirradiation experiments were carried out using a MDL-H-980 nm laser (0−2.6 W) and a UV lamp (Blak-Ray, B-100AP/R, 100 W). Human mesechymal stem cells are from Lonza Group Ltd. (Basel, Switzerland). Synthesis of UCNPs, UCNP@SiO2, and UCNP@mSiO2 Nanoparticles. The UCNPs were synthesized in accordance with a previously reported method.27 Briefly, RECl3 (0.2 M, RE = Y, Yb, and Tm) in methanol were added to a 50 mL flask containing 3 mL of oleic acid and 7 mL of 1-octadecene, and the solution was heated to 160 °C for 30 min and then cooled to room temperature. Thereafter, 5 mL of methanol solution of NH4F (1.6 mmol) and NaOH (1 mmol) were added, and the solution was stirred for 30 min. After the evaporation of methanol, the solution was heated up to 300 °C under argon for 1.5 h and then cooled to room temperature. The resulting nanoparticles were precipitated by the addition of ethanol, collected by centrifugation, washed with methanol and ethanol several times, and finally redispersed in cyclohexane. The UCNP@SiO2 were also synthesized with a previously reported method.27 UCNPs@SiO2: Briefly, UCNPs (4 mg) were dissolved in cyclohexane (16 mL) and followed by adding Triton X-100 (4 mL), 1hexnol (4 mL), and DI water (680 μL). Then, TEOS (tetraethyl orthosilicate) (10 μL) and (3-aminopropyl)triethoxysilane (APS, 2 μL) were added. After 6 h of stirring, NH4OH (200 μL) was added to form the UCNPs@SiO2. Finally, acetone was used to terminate the reaction and the resultant precipitate was washed in sequence with butanol, isopropyl alcohol, ethanol, and water to produce an aqueous suspension of the composite silica-coated UCNPs with amino groups on the surface (UCNPs@SiO2−NH2) for further characterization and peptide conjugation. The mesoporous silica-coated UCNPs (UCNP@mSiO2) were synthesized through a NaOH-based etching method.22 Briefly, 1 mM NaOH was added to 4 mL of solution of the prepared UCNP@ SiO2 (2 mg/mL), and the mixture solution was stirred at RT for 4 h. Upon the etching of silica, which was coated on UCNPs particles surface, the clear solutions became opaque. Then, the UCNP@mSiO2 nanoparticles were washed with ethanol and water three times. After repeated washing and centrifugation, the prepared colloidal spheres were generated, which were finally dispersed in polar solvents, such as deionized water or ethanol for further applications. Synthesis of AIEgen−PLGVRGKGGC Units. The AIEgen (Cy− Py−N3) was synthesized by previous report.28 The AIEgen− PLGVRGKGGC was synthesized by a “click” chemical reaction with previous report.24 Briefly, the propiolic acid−PLGVRGKGGC− COOH peptide (5 μmol) and AIEgen−N3 (6 μmol) were dissolved in 50 μL of dimethyl sulfoxide (DMSO). A mixture of DMSO/H2O solution (v/v = 1/1; 0.5 mL) was subsequently added, and the reaction was shaken for a few minutes to obtain a clear solution. The

method to monitor the differentiation process and to elucidate the differentiation status of living stem cells is highly desirable. Aggregation-induced emission (AIE) is a unique photophysical phenomenon that was first reported in 2001. Propellershaped fluorogens, such as tetraphenylethene (TPE), are nonemissive when molecularly dissolved in solution; they are, however, induced to emit efficiently by aggregate formation.19 Luminogens of AIE characteristics (AIEgen) have become a powerful tool for a variety of biological applications, such as chemical sensors,20,21 biological imaging,20 and optoelectronic devices.22 Notably, AIEgens have been utilized to monitor the activities of various enzymes.23,24 These assays rely on the weakly fluorescent AIEgens in a solution to interact with the oppositely charged chemical or protein substrates to yield fluorescent complexes, and these complexes then release the AIEgens back to the solution after enzyme digestion, thereby leading to the “turning on” of the fluorescence by the aggregation of AIEgens. In the present study, we developed a multifunctional UCNPs system to control and simultaneously monitor the differentiation of stem cells. In our design (Scheme 1), Tm/Yb codoped NaYF4 core−shell UCNPs have a reduced surfacequenching effect, and these UNCPs were chosen as the platform for the conjugation of the peptide−AIEgen unit and the encapsulation of caged siRNA (DMNPE/siRNA). The UCNPs possess a mesoporous silica-coated structure (Scheme 1A), which facilitates the conjugation of the RGD peptide and the peptide−AIEgen unit on the surface of the UCNPs to form the UCNP@SiO2−peptide−AIEgen (UCNP−peptide−AIE). The mesoporous silica structure of UCNP−peptide−AIE also harbors the DMNPE/siRNA compound via physical absorption to form the DMNPE/siRNA−UCNP@SiO2−peptide−AIEgen nanocomplexes (UCNP−peptide−AIE−siRNA) (Scheme 1B). The up-converted UV from UCNPs triggers the activation and release of the siRNA intracellularly upon NIR excitation (Scheme 1C). The activation and release lead to gene knockdown and effective induction of the stem cell differentiation. Thus, controlling the differentiation of stem cells in vivo through the tissue penetration of NIR is enabled (Scheme S1). Meanwhile, the surface of UCNPs is conjugated with peptide−AIEgen unit via a RGD peptide sequence to target the cells and an enzyme (Matrix metallopeptidase 13, MMP13)sensitive peptide sequence to detect the stem cell differentiation by the emission of AIEgens. The MMP13 enzyme is a marker expressed in osteoblastic cells,25,26 and this enzyme in the differentiating stem cells specifically cleaves the MMP13 peptide linker and releases the hydrophobic AIEgens. The released AIEgens tend to self-aggregate in the aqueous cytoplasmic environment due to their low water solubility, and this self-aggregation of the AIEgen leads to the “turning on” of AIEgen emission upon excitation by the up-converted UV emission from the UCNP nanocarrier under NIR excitation. Thus, detecting the differentiation in deep tissues is possible through the more-biocompatible NIR. Our findings demonstrate that the UCNP−peptide−AIE−siRNA affords efficient control over the directed stem cell differentiation and simultaneous monitoring of the differentiation process in stem cells.

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EXPERIMENTAL SECTION

Materials and Chemicals. All reagents were purchased from Aldrich. Anhydrous solvents for organic synthesis were purchased from Aldrich and stored over-activated molecular sieves (4 Å). 4,5C


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ACS Applied Materials & Interfaces “click” reaction was initiated by sequential addition of catalytic amounts of sodium ascorbate (2 μmol) and CuSO4 (1 μmol). The reaction was continued with shaking at room temperature for another 24 h. The final product was purified by HPLC and further characterized by UV. Conjugation of AIEgen−PLGVRGKGGC and RGD Peptides to UCNP@mSiO2 to Form the UCNP−Peptide−AIE Nanocarrier. MAL−(PEG)4−NHS linker was used to conjugate AIEgen− PLGVRGKGGC and RGD peptide (CRGD) to the surface of UCNP@mSiO2. Briefly, the UCNP@mSiO2 (10 mg) were dissolved in water (1 mL), and then MAL−(PEG)4−NHS ester (100 μL, 20 mM) and N,N-diisopropylethylamine (DIPEA: 2 μL) were added. After overnight stirring, the MAL−(PEG)4-functionalized UCNPs were collected by centrifugation at 8000 rpm. The supernatant was removed, and the nanoparticles were redispersed in DMF by ultrasound and then washed three times with DMF. Then, the collected MAL−(PEG)4 -functionalized UCNPs, the AIEgen− PLGVRGKGGC (2 mM in DMF, 400 μL) and RGD peptide (2 mM in DMF, 200 μL), and DIPEA (2 μL) were mixed together for the reaction. The reaction was continued for 24 h in the dark at RT. Finally, the UCNP−peptide−AIE nanocomplexes were obtained by centrifugation at 8000 rpm and washed with H2O/isopropanol (v/v = 1:5) three times. The nanocomplexes were redispersed by ultrasound in PBS to the desired concentrations. DMNPE-Caged siRNA (DMNPE/siRNA). The 4,5-dimethoxy-2nitroacetophenone hydrazone precursor (DMNPE, 5 mg) was oxidized by MnO2 (20 mg) in 1 mL of DMSO at room temperature under agitation for 20 min to activate the DMNPE. Then, the activated diazo-cage solution was mixed with siRNA aqueous solution to achieve a 2:1 acetonitrile/aqueous buffer for forming the DMNPE/ siRNA compound, and the resultant mixture was agitated overnight. The DMNPE/siRNA compound reaction solutions were lyophilized for purification, and the purified compound was stored at 4 °C for further application. Encapsulation of DMNPE/siRNA with UCNP−Peptide−AIE to Form the UCNP−Peptide−AIE−siRNA Nanocomplexes and NIR-Triggered Release of siRNA in PBS Buffer. Caged siRNA (DMNPE/siRNA) was loaded into the UCNP−peptide−AIE by stirring. The UCNP−peptide−AIE (1 mg/mL) were added drop-wise to 50 μg of DMNPE/siRNA solution (1 mL) and agitated gently at room temperature for 4 h. The solution was then centrifuged at 8000 rpm for 10 min, and the pellet containing the UCNPs loaded with caged siRNA was resuspended in deionized water or PBS for further experiments. The caged siRNA was triggered for release from UCNP− peptide−AIE−siRNA by NIR irradiation. The UCNP−peptide−AIE− siRNA (1 mg/mL, PBS) were exposed to a UV lamp (5 min, 2 W/ cm2) or NIR laser (20 min, 2 W/cm2), and the solution was centrifuged at 8000 rpm for 10 min. The supernatant was collected and tested by intensity spectrophotometry of UV−vis for measuring the release of siRNA and was found to be approximately 150 nM under UV irradiation and 100 nM under NIR irradiation. The release of siRNA was also checked by agarose gel electrophoresis, and tissue (pigskin, 1 mm) was used to test the tissue penetration of UV and NIR. After various treatments, the supernatant was collected and directly loaded to the agarose gel (2%, 120 mV, 20 min) for siRNA release testing. Detection of MMP13 Enzyme by UCNP−Peptide−AIE in PBS. To detect the activity of MMP13 enzyme in solution, the MMP13 was pretreated by the MMP buffer (SensoLytePlus MMP-13 Assay Kit, AnaSpec, Inc.) for 30 min at 37 °C. Next, the UCNP−peptide−AIE nanoprobes (1 mg/mL) mixed with MMP13 buffer solution (10 nM) for 1 h of incubation at 37 °C. After the reaction, the solution was centrifuged, and the supernatant was collected for PL measurement by fluorescence microplate reader. The solution was excited at 360 nm, and the emission was collected from 450 to 650 nm for AIEgen. To detect MMP13 by NIR, the NIR laser (980 nm) was put into the fluorescence microplate reader as the excitation light source and the UV lamp of fluorescence microplate reader was converted. Instead of UV lamp in the fluorescence microplate reader, the upconverted NIRto-UV from UCNPs was as the excitation light to excite the AIEgen

after MMP13 enzyme cleavage and AIE characteristics, and the emission was collected from 300 to 600 nm for the changed emission of UCNPs and AIEgen. Concentration-dependence experiments (concentration of MMP13: 0−10 nM) and different enzymedependence (enzyme specificity) experiments (MMP13, MMP7, Cathepsin B, Trypsin, and bovine serum albumin (BSA)) were also performed with a similar process with NIR or UV excitation. Silencing PPARγ by UCNP−Peptide−AIE−siRNA Nanocomplexes. Reverse-transcription polymerase chain reaction (RT-PCR) analysis was used to measure the gene expression after silencing the PPARγ by UCNP−peptide−AIE−siRNA. The traditional nucleic acid transfection reagent Lipofectamine2000 was used to deliver siRNA (silence PPARγ) into hMSCs first for observing the osteogenic differentiation relative gene expression (Runt-related transcription factor 2 (Runx2),38,39 a major transcription factor of osteogenesis, and MMP13). The Lipofectamine2000 (2 uL) was mixed with siRNA (100 nM) in the medium (no FBS) for 30 min at RT, and then the mixture was added to the hMSCs for 4 h of incubation. After transfection, the treated hMSCs were continued to culture in the osteogenic differentiation medium to induce differentiation for 7 days. Next, the treated hMSCs were collected to get the total amount of RNA, which then underwent reverse transcription to cDNA by PCR. For controlled silencing PPARγ by NIR, the hMSCs were incubated with UCNP−peptide−AIE−siRNA (1 mg/mL, 4 h incubation) first. The treated hMSCs were exposed to UV or NIR with or without tissue covered. After UV/NIR exposure, the hMSCs were continued to culture in osteogenic differentiation medium to induce differentiation for 7 days. Next, the hMSCs were collected to get the total amount of RNA, which underwent reverse transcription to cDNA by PCR. The PCR reverse transcription reaction was carried out at 37 °C for 15 min and 85 °C for 5 s and followed by PCR: 1 cycle, 95 °C, 5 min; 30 cycles, 95 °C, 30 s, 55 °C, 30 s, and 72 °C, 30 s; 1 cycle, 72 °C, 10 min. Primers for PCR: β-actin (sense, 5′-AAATCGTGCGTGACATTAA3′; antisense, 5′-CTCGTCATACTCCTGCTTG-3′), PPARγ (sense, 5′-CATAAAGTCCTTCCCGCTGA-3′; antisense, 5-GGGCTCCATAAAGTCACCAA-3), Runx2 (sense, 5′-AGATGGGACTGTGGTTACTG-3′; antisense, 5′-GTAGCTACTTGGGGAGGATT-3′), MMP13 (sense, 5′-TCTGGTCTGTTGGCTCACGCTT-3′, antisense, 5′-TCTCGGGTAGTCTTTATCCATCAC-3′). The PCR products were analyzed by electrophoresis on a 1% agarose gel (120 V, 30 min) with a gel imaging system (Gel Doc XR+, Bio-Rad). Controlling Differentiation of hMSCs by UCNP−Peptide− AIE−siRNA with Immunofluorescence and Histochemical Staining. hMSCs were incubated with UCNP−peptide−AIE− siRNA (1 mg/mL) for 4 h and washed with PBS three times to remove the untransfected UCNP−peptide−AIE−siRNA. Next, the treated hMSCs were exposed to UV or NIR with or without tissue cover. The light-exposed hMSCs were continued to culture with osteogenic differentiation medium to induce differentiation for 7 days. Light untreated hMSCs were used as the control. After 7 days of differentiation, the hMSCs were fixed with 4% paraformaldehyde at 4 °C for 1 h and then washed with PBS three times. The cell layer was pretreated with 0.1% Triton-X in PBS for 15 min at room temperature and then washed with PBS three times. Nonspecific antibody binding was blocked with 5% bovine serum albumin in PBS, followed by washing in PBS three times. For immunodetection, the primary antibody of Runx2 and MMP13 diluted at 1:200 were added for overnight incubation at 4 °C for specific antibody binding. The specific bindings were visualized by adding FITC-labeled secondary antibody 1:500 (Invitrogen) for 2 h at room temperature, and the samples were washed with PBS for 3 times. Finally, 1 mL of PBS was added, and the cells were visualized by using CLSM (SP8, Leica). The fluorescence images were taken using 20 × objective, and the laser was emitted at 488 nm to excite FITC-labeled secondary antibody. The fluorescent signals were collected on the basis of the emission filter set at 530 nm for FITC. For histological staining (ARS and VKS), the hMSCs were washed by PBS three times and were fixed by 4% formalin at 4 °C for 24 h. The fixed hMSCs were washed by PBS three times and stored at room temperature for further histological analysis. Alkaline phosphatase staining by VKS and extracellular matrix mineralization by ARS D


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ACS Applied Materials & Interfaces was performed as previously described.29 All differentiation studies were performed in triplicate to facilitate statistical analysis. Detection of Differentiation by UCNP−Peptide−AIEgen Nanoprobes in hMSCs. hMSCs were incubated with UCNP− peptide−AIE−siRNA for 4 h and washed with PBS three times to remove the untransfected nanocomplexes. Next, the treated hMSCs were exposed to NIR with or without tissue cover. The NIR-exposed hMSCs were continued to culture with osteogenic differentiation medium for 1, 3, and 7 days to induce differentiation. NIR-untreated hMSCs were the control. After 1, 3, and 7 days of differentiation, the hMSCs were fixed with 4% paraformaldehyde at 4 °C for 1 h, followed by washing with PBS three times. Next, the hMSCs were incubated with 50 μL of 4,6-diamidino-2-phenylindole (DAPI, 50 μg/mL, Invitrogen) at 37 °C for 1 h to stain the nuclei. The cells were then washed three times with PBS to remove excessive DAPI. Finally, 1 mL of PBS was added, and the cells were visualized by using CLSM (SP8, Leica, Germany). The fluorescence images were taken using a 63× oilimmersion objective lens, and the laser at wavelengths of 405 nm was emitted to excite DAPI and AIEgen. The fluorescent signals were collected on the basis of the emission filter sets at 450 nm for DAPI and 560 nm for AIEgen, respectively. For flow cytometry, the pretreatment of hMSCs was similar to the previous experiment as described above. After 7 days of differentiation, the hMSCs were collected by Trypsin and wash with PBS. For flow-cytometry analysis, the treated hMSCs were excited at 405 nm for AIEgen, and its emission was acquired at 560 nm. Statistical Analysis. All data are presented as the mean ± standard deviation. Statistica (Statsoft, Tulsa, OK) was used to perform statistical analyses by using two-way ANOVA and the Tukey HSD post hoc test of the means (n = 3) with culture duration and experimental groups as independent factors.

peptide−AIE (Scheme 1B). The TEM images show that the average size of UCNP−peptide−AIE is 35 ± 5.1 nm (Figure 1A), which is similar to the UCNP@mSiO2. The dynamic light

Figure 1. Characterization of UCNP−peptide−AIE. (A) TEM image of UCNP−peptide−AIE and (B) DLS of UCNP−peptide−AIE in PBS. The size of the UCNP−peptide−AIE in PBS is about 38 ± 2.7 nm by DLS. (C) UV−vis spectra of UCNP@mSiO2, the MMP13− peptide−AIEgen unit, and UCNP−peptide−AIE. (D) Luminescence of UCNP−peptide−AIE in PBS under NIR excitation. The inserted photograph shows the UCNP−peptide−AIE suspension under NIR laser excitation. UCNP−peptide−AIE: 1 mg/mL; NIR: 2 W/cm2.

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RESULTS AND DISCUSSION Synthesis and Characteristic. First, we synthesized the UCNPs according to a previous report.30 The obtained UCNPs were then treated with tetraethyl orthosilicate (TEOS) and (3aminopropyl)triethoxysilane to undergo surface modification with silica, and this process forms the silica-coated UCNPs (UCNP@SiO2) that bear the reactive terminal amino groups. Then, the mesoporous silica coating was generated by the NaOH etching to produce the mesoporous silica-coated UCNPs (UCNP@mSiO2, Scheme 1A).31 The N2 adsorption−desorption isotherms and the corresponding pore-size distributions of the UCNP@mSiO2 produced by this method is generally about 180 cm3/g and 3 nm, respectively, after 4 h of etching.31 The TEM images show that the average size of UCNPs, UCNP@SiO2, and UCNP@mSiO2 is approximately 17 ± 2.7, 35 ± 3.5, and 35 ± 1.8 nm, respectively (Figure S1). The FTIR analysis further demonstrates the transformation of UCNP@SiO2 to UCNP@mSiO2, as evidenced by the reduced intensity of Si−OH stretching vibration (956 cm−1, Figure S2). Next, we synthesized the MMP13 enzyme-sensitive probes, which consists of a hydrophilic MMP13-specific substrate peptide (propiolic acid−Pro−Leu−Gly−Val−Arg−Gly−Lys− Gly−Gly−Cys−COOH, propiolic acid−PLGVRGKGGC−Ac) and a hydrophobic AIEgen (Cy−Py−N3).29 The coupling between AIEgen and propiolic acid−PLGVRGKGGC−Ac was achieved through Cu(I)-catalyzed “click” reaction24 to form the AIEgen−PLGVRGKGGC−Ac unit. The UV−vis absorption spectra of AIEgen and AIEgen−PLGVRGKGGC−Ac show a similar absorption band in the 350−400 nm range, which confirms the conjugation of AIEgen to MMP13 peptide (Figure S3).24 AIEgen−PLGVRGKGGC−Ac unit and Arg−Gly−Asp peptide (CRGD) were further conjugated to UCNP@mSiO2 through a Mal−(PEG)4−NHS linker to yield UCNP−

scattering (DLS) analysis (Figure 1B) shows that the size of UCNP−peptide−AIE was 38 ± 2.7 nm in PBS solution. The UV−vis absorption spectra of AIEgen−PLGVRGKGGC−Ac and UCNP−peptide−AIE in DMSO/PBS both have similar absorption bands in the 350−400 nm range (Figure 1C); these similar absorption bands demonstrate the successful conjugation of AIEgen−PLGVRGKGGC−Ac to UCNPs by the Mal− (PEG)4−NHS linker. Furthermore, the UCNP−peptide−AIE showed strong photoluminescence under NIR excitation (Figure 1D). The strong photoluminescence (up-converted UV) suitably excites aggregated AIEgen to monitor the differentiation of stem cells; this monitoring is achieved because the osteogenic differentiation of hMSCs leads to upregulated MMP13 expression and induces the enzyme cleavage of AIEgen−PLGVRGKGGC. Cell Viability and Cell Uptake with the UCNP− Peptide−AIE. The up-converted UV emission from UCNPs is of low intensity and is largely restricted to within a few nanometers from the surface of UCNPs.32 The hMSCs were treated with 1 mg/mL of UCNP−peptide−AIE for 4 h and then irradiated with different dosages of NIR (980 nm), and the viability of these hMSCs was then evaluated. The alamarBlue assay indicates no apparent reduction in the viability of the hMSCs that were treated with an increasing concentration of the UCNP−peptide−AIE (0−5 mg/mL) (Figure S4A). Furthermore, all cells (not treated and treated with the UCNP−peptide−AIE (1 mg/mL)) did not show significant decreases in viability under increasing duration of NIR exposure (0 to 20 min, Figures S4B and S4C). Furthermore, the cell uptake of the UCNP−peptide−AIE was studied by Bio-TEM (The use of TEM to study biological structures), as shown in E


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Figure 2. NIR triggering of a release of siRNA from the UCNP−peptide−AIE−siRNA nanocomplexes. (A) The UV absorption of siRNA at 270 nm with various treatments. Red: the absorption spectrum of naked siRNA solution shows an absorption peak at 270 nm. Black: the spectrum of the supernatant of nanocomplexes without UV or NIR exposure shows no siRNA absorption peak because of the lack of siRNA release. Green: absorption spectrum of the supernatant of UCNP−peptide−AIE−siRNA + UV irradiation group shows an obvious peak at 270 nm because of the UV-triggered release of siRNA from the UCNP−peptide−AIE−siRNA. Blue: absorption spectrum of the supernatant of UCNP−peptide−AIE− siRNA + NIR irradiation group also shows the characteristic absorption peak of siRNA because the up-converted UV from UCNPs triggers the siRNA release from the UCNP−peptide−AIE−siRNA. (B) The release of siRNA−FAM from UCNP−peptide−AIE−siRNA with light irradiation (UV or NIR) by agarose gel electrophoresis. (a) DNA marker, (b) UCNP−peptide−AIE−siRNA irradiated with UV,( c) UCNP−peptide−AIE− siRNA irradiated with UV through a layer of pigskin, (d) UCNP−peptide−AIE−siRNA without UV or NIR irradiation, (e) UCNP−peptide−AIE− siRNA irradiated with NIR, and (f) UCNP−peptide−AIE−siRNA irradiated with NIR through a tissue (pigskin, 1 mm). UV: 5 min, 2 W/cm2; NIR: 20 min, 2 W/cm2.

Figure 3. Detection of MMP13 enzyme activity by UCNP−peptide−AIE nanoprobes with UV irradiation (A) and NIR irradiation (B) in PBS (PBS/DMSO, v/v = 199:1). The fluorescence was “turned on” after the peptide cleavage of MMP13 enzyme and the release and aggregation of AIE. (C) The photoluminescence spectra of UCNP−peptide−AIE (1 mg/mL) when incubated with MMP13 enzyme with different concentrations (0, 1, 2, 5, and 10 nM) under the NIR irradiation. (D) The fluorescence emission intensity of UCNP−peptide−AIE in the presence of various proteins. Incubation time: 1 h, 37 °C; UCNP−peptide−AIE: 1 mg/mL; MMP13: 10 nM; inhibitor: 10 μM. MMP13, MMP7, Cathepsin B, Trypsin, BSA: 10 nM.

siRNA.4,34 In a previous study, the UCNPs were shown to be capable of uncaging and activating the DMNPE-caged siRNA (DMNPE/siRNA).35 In the present study, we used UCNP− peptide−AIE to harbor the DMNPE/siRNA to form the UCNP−peptide−AIE−siRNA nanocomplexes for the NIRtriggered release of siRNA in hMSCs. We synthesized the DMNPE/siRNA and loaded the DMNPE/siRNA to the UCNP−peptide−AIE by using a previously reported proto-

Figure S5. In Figure S5, the Bio-TEM images showed that the RGD conjugated UCNPs have higher cell uptake efficiency compared to the UCNPs without RGD modification (control). This result indicated that the RGD peptide modification can enhance the cell uptake of the UNCPs.33 NIR-Trigged Release of siRNA from the UCNP− Peptide−AIE−siRNA Nanocomplexes in Vitro. The small molecule DMNPE was found to be an effective photocage of F


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Figure 4. (A) RT-PCR data of osteogenic marker gene in hMSCs. In the UCNPs-treated hMSCs, the activation and release of the photocaged PPARγ siRNA by UV or NIR led to the reduced expression of PPARγ and increased the expression of Runx2 and MMP13 in the hMSCs (*, p < 0.05; **, p < 0.01; n = 3). (B) Immunofluorescence staining against osteogenic markers after various treatments. After silencing of the PPARγ gene by UCNP−peptide−AIE−siRNA with UV or NIR irradiation, the expression of Runx2 protein and MMP13 protein presented a increase in the differentiated hMSCs. Osteogenic differentiation: 7 days. UV: 2 W/cm2, 5 min. NIR: 2 W/cm2, 20 min. Scale bar: 20 μm.

col.35 As shown in Figure S6, the UV−vis spectra of the DMNPE/siRNA complexes have two absorption peaks in the range of 250−300 and 350−400 nm that are found in the spectra of siRNA and DMNPE, respectively. These spectra indicate the successful formation of the DMNPE/siRNA complex. The obtained DMNPE/siRNA was encapsulated into the mesoporous of the UCNP−peptide−AIE by physical adsorption to generate the UCNP−siRNA−AIE−siRNA. Next, we examined the NIR-triggered siRNA release from UCNP−siRNA−AIE−siRNA by conducting the UV−vis analysis on the supernatant of the incubating buffer. The UV−vis data show that both UV and NIR irradiation are able to trigger the release of the siRNA, as evidenced by the characteristic absorption peak of the siRNA (Figure 2A). In addition, agarose gel electrophoresis data show new bands associated with a fluorescently labeled control siRNA (siRNA− FAM), which was preloaded to the UCNPs prior to the UV− NIR irradiation (Figure 2Bb and Be). This result further demonstrates the release of siRNA from the UCNP−siRNA− AIE−siRNA after UV−NIR irradiation. Furthermore, only NIR successfully induced the release of siRNA through the intervening layer of animal tissue (pigskin, 1 mm, Figure 2Bf), and UV irradiation was unable to induce the same. Thus, compared to UV, NIR possesses enhanced tissue penetration capability to trigger the release of cargo molecules from UCNPs-based nanoparticles in deep tissues; this capability affords the potential application to control the differentiation of stem cells in vivo. Detecting the MMP13 Enzyme by UCNP−Peptide− AIE Nanoprobes in Vitro. The conjugation of the AIEgen to

UCNPs with the MMP13 sensitive peptide (PLGVRGKGGC) allows the MMP13-triggered release of the AIEgen via the specific cleavage of the linker peptide. These released hydrophobic AIEgen molecules can form aggregates in the aqueous solution, leading to a typical fluorogen with AIE characteristics by UV or upconverted UV excitation.19 As shown in Figure 3A, upon treatment with MMP13 for 1 h at 37 °C, the PBS solution of UCNP−peptide−AIE showed a strong fluorescence signal at a wavelength of 560 nm; this result is consistent with the emission wavelength of the AIEgen29 conjugated to the UCNPs under UV excitation. In contrast, the addition of ethylenediaminetetraacetic acid (EDTA), a highly specific inhibitor of metalloproteinases,36,37 abrogated the MMP13-triggered AIEgen emission; this nullification indicates that the previously detected emission is specifically due to the MMP13-mediated cleavage of the peptide linker. Furthermore, NIR irradiation also produced a strong AIEgen emission (∼560 nm) from the PBS solution of UCNP−peptide−AIE upon treatment with MMP13 for 1 h at 37 °C (Figure 3B). Moreover, the diminished peaks at 360 and 475 nm were due to the absorption by the AIEgen.29 Similarly, the addition of EDTA also significantly reduced the AIEgen emission (∼560 nm) under NIR irradiation. To study the correlation between the concentration of the MMP13 enzyme and that of the UCNP−peptide−AIE nanoprobes, 1 mg/mL UCNP−peptide−AIE were incubated with different concentrations of MMP13 ranging from 0 to 10 nM for 1 h at 37 °C. As shown in Figure 3C, the increasing concentration of MMP13 enzyme under NIR excitation produced increasing intensity of the AIEgen emission (∼560 nm); this increased AIEgen emission is G


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Figure 5. Detecting osteogenic differentiation of hMSCs by UCNP−peptide−AIE−siRNA nanocomplexes. A) Confocla images of osteogenic differentiated hMSCs. After NIR irradiation, the UCNP-treated hMSCs presented prominent fluorescence signal from aggregated AIE, which indicated that the release of siRNA from UCNP−peptide−AIE−siRNA enhanced the differentiation of hMSCs and the differentiation can be detected by UCNPs nanocomplexes simultaneously in the differentiated hMSCs. B) Flow cytometry result of the differentiated hMSCs with various treatments. After NIR irradiation, the UCNPs-treated hMSCs showed strong fluorescence signal by the aggregation of AIEgen, which indicated that the distensible differentiation of hMSCs, which was promoted by the NIR-triggered release of siRNA, can be detected by the UCNPs nanocomplexes simultaneously. UCNP−peptide−AIE-siRNA: 1 mg/mL. Osteogenic differentiation: 7 days. NIR: 20 min, 2 W/cm2. Tissue: pigskin (1 mm).

33 ± 4.3% of the control level) because of its excellent tissue penetration. The knocking down of the PPARγ expression in the hMSCs treated with nanocomplexes and light irradiation led to the enhanced expression of the osteogenic-related genes, such as Runx2 (Figure 4A). The Runx2 level in the hMSCs treated with UV and NIR increased 5- and 4.5-fold compared to that of the control cells, respectively. When the cells were covered with the pigskin tissue and then irradiated by UV lamp, the Runx2 expression level only increased 0.5-fold, whereas the Runx2 expression level in the cells irradiated with NIR under the same conditions increased more than 2.5-fold. The expression level of MMP13 follows the same trend as that of the Runx2 expression under these conditions (Figure 4A). Controlling Differentiation of hMSCs by UCNP− Peptide−AIE−siRNA Nanocomplexes. Next, we used the UCNP−peptide−AIE−siRNA to control the osteogenic differentiation of hMSCs by light. As shown in Figure 4B, after the light irradiation (UV or NIR) followed by 7 days of osteogenic culture, the confocal microscopy images of the immunofluorescence staining against Runx2 and MMP13 indicated that only faint Runx2 and MMP13 staining in the control hMSCs (treated with the nanocomplexes but without light irradiation and with 7 days of osteogenic differentiation), and this faint staining was due to the short differentiation time. In contrast, substantial Runx2 expression (in the nucleus) and MMP13 (in the cytoplasm) in the hMSCs treated with the nanocomplexes and UV or NIR irradiation were observed, and this result indicates the accelerated osteogenic differentiation of the hMSCs because of the photoactivated PPARγ siRNA released from the nanocomplexes. The UV-induced Runx2 and MMP13 expression declined substantially when pigskin tissue was placed over the cells. In comparison, the NIR-triggered expression of Runx2 and MMP13 remained at a high level, owing to the effective penetration of NIR through the tissue. Furthermore, ARS and VKS were then applied to measure the mineralization from the osteogenic differentiation of the hMSCs treated with the UCNPs nanocomplexes after 7 days of osteogenic culture following the light treatment (Figure S8). In Figure S8A, the ARS result showed that compared to the control group (UCNPs-treated hMSCs with a relative ARS intensity of 100% ± 4.3), both the UV group (UCNPs-treated hMSCs with UV irradiation with a relative ARS intensity of 203% ± 7.8) and the

likely due to the increased AIEgen being released. This result indicates that the AIEgen emission is positively related to the concentration of MMP13 present in the buffer. To check the detection specificity, the UCNP−peptide−AIE nanoprobes were incubated with several proteins, such as MMP13, MMP7, Cathepsin B, trypsin, and BSA under identical conditions (1 h of incubation at 37 °C). As shown in Figure 3D, MMP13 induced significant AIEgen emission, whereas other proteins only produced weak emissions; these results suggest that our UCNPs nanoprobes have high specificity toward MMP13 detection to monitor differentiation. Silencing the PPARγ Gene by UCNP−Peptide−AIE− siRNA Nanocomplexes in hMSCs. The peroxisome proliferator-activated receptor γ (PPARγ) gene is a key regulator of the adipogenesis of hMSCs.28 The down-regulated expression of PPARγ promotes osteogenic differentiation by up-regulating the expression of Runx2. Furthermore, Runx2 is known to regulate the expression of MMP 13, and MMP13 is critical for bone remodeling, osteoblast differentiation, and bone development.40−42 We first used Lipofectamine 2000 to deliver siRNA (targeting PPARγ) to hMSCs; the RT-PCR result reveals that the down-regulated expression of PPARγ promotes the expression of Runx2 (Figure S7). Consistent with a previous report, the up-regulated expression of MMP13 was also observed with the increased expression of Runx2. To confirm the feasibility of the photoactivation of the caged siRNA in hMSCs, the hMSCs were treated with UCNP− peptide−AIE−siRNA before being further cultured in osteogenic induction media. The UCNPs-treated hMSCs underwent UV (2 W/cm2, 5 min) or NIR exposure (2 W/cm2, 20 min); the RT-PCR result (Figure 4A) shows that these hMSCs had significantly lower PPARγ gene expression because of the efficient activation of the photocaged siRNA (down to 12 ± 6.3% and 17 ± 2.5% of the control expression level, respectively), compared to the control (treated with the nanocomplexes but without light irradiation and with 7 days of osteogenic differentiation). Interestingly, when we covered the hMSCs with a layer of tissue (pigskin, 1 mm), the UV irradiation produced a poor PPARγ silencing effect (down to 90 ± 7.2% of the control level) because of its limited tissue penetration. However, NIR laser irradiation was still capable of significantly down-regulating the PPARγ expression (down to H



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CONCLUSIONS In summary, we developed multifunctional UCNPs nanocomplexes (UCNP−peptide−AIE-siRNA) to control and detect the differentiation of stem cells. Our findings demonstrate that the UCNPs nanocomplexes are capable of the special delivery and controlled release of siRNA against PPARγ in response to NIR to control the osteogenic differentiation of hMSCs. Meanwhile, the UCNPs nanocomplexes also can be used as a nanoprobe, which allows the detection of the osteogenic marker enzyme MMP13 in the differentiated hMSCs via the MMP13-triggered switching on the AIEgen after controlling differentiation by NIR. Thus, we believe that this multifunctional UCNPs nanosystem can be a powerful tool to assist the basic scientific research on stem cells, and this UCNPs system can also potentially be translated into clinical applications for targeted multimodal imaging and photodynamic therapy once the long-term safety of UNCPs is confirmed.

NIR group (UCNPs-treated hMSCs with UV irradiation and a relative ARS intensity of 185% ± 3.9) exhibited substantially more staining against mineralization. However, UV treatment through the pigskin tissue (UV+P group with a relative ARS intensity of 112% ± 2.6) showed less mineralization in the hMSC culture, whereas NIR treatment (NIR+P group with a relative ARS intensity of 153% ± 6.5) still produced robust mineralization despite the presence of the animal tissue layer. The VKS staining showed the same result as that of ARS staining (control: 100% ± 2.3, UV: 311% ± 8.3, NIR: 256% ± 6.2, UV+P: 123% ± 7.1, NIR+P: 212% ± 3.8). This finding indicates that NIR is capable of penetrating the animal tissue and photoactivating the caged siRNA in the UCNPs nanocomplexes to induce the osteogenesis and mineralization of the hMSCs. Detecting Differentiation of hMSCs by UCNP−Peptide−AIE−siRNA Nanocomplexes. Lastly, we evaluated the efficacy of the UCNP−peptide−AIE−siRNA to detect the MMP13 enzyme activity in differentiated hMSCs for detection of differentiation by confocal and flow cytometry (Figure 5A and B). The hMSCs were incubated with the UCNP−peptide− AIE−siRNA and then exposed to NIR (2 W/cm2, 20 min) for the photoactivation of the caged PPARγ siRNA. The treated hMSCs were further cultured in the osteogenic differentiation medium for 1, 3, and 7 days for assessments. The induced MMP13 expression in the differentiated hMSCs was expected to cleave the MMP13-sensitive peptide linker and release the conjugated hydrophobic AIEgen. The released hydrophobic AIEgen should then aggregate in the cell cytoplasm and generate the characteristic emission.24 The control hMSCs (treated with UCNP−peptide−AIE−siRNA but no NIR irradiation with osteogenic differentiation medium) were used as the control for comparison. After 7 days of osteogenic culture, the control hMSCs showed a slight AIEgen fluorescence signal, and this indicates the early stage of the osteogenic differentiation with low expression level of MMP13 (Figure 5A). In sharp contrast, the AIEgen fluorescence signal (red) can be detected in the NIR-treated hMSCs after only 1 day of culture; the signal intensifies over time and becomes highly prominent on day 7 of the culture. The results clearly demonstrate that the nanocomplexes significantly speed up the osteogenic differentiation of the hMSCs upon photoactivation of siRNA. Moreover, the nanocomplexes enable the tracking of the MMP13 activity level in the differentiating hMSCs. Furthermore, the NIR-triggered differentiation is almost not affected when the pigskin (1 mm) is used to cover the treated hMSCs, as shown by the slightly reduced AIEgen signal (Figure 5A, NIR (P)). This result is due to the deep tissue penetration of NIR light. In addition, the flow cytometry result (Figure 5B) also revealed a significantly stronger signal of the AIEgen in the NIR-treated hMSCs after 7 days of differentiation compared with the control hMSCs (control hMSCs show only faint emission, indicating slow osteogenic differentiation), and this result further confirms the induced MMP13 expression in the treated hMSCs. The application of NIR through the pigskin still induced increased MMP13, as shown in the result. Despite the successful qualitative detection of differentiation markers in stem cells, a limitation of our multifunctional nanosystem is the inability to quantitatively assess the expression of these biomarkers.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00845. Figures showing NIR tissue-penetration triggered release of siRNA; TEM images; FTIR and UV−vis absorption spectra; cell viability; and Bio-TEM, RT-PCR, ARS, and VKS imaging. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Liming Bian: 0000-0003-4739-0918 Author Contributions

J.M.L. and C.W.T.L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region (reference no.: 02133356). This research is supported by the Chow Yuk Ho Technology Centre for Innovative Medicine (The Chinese University of Hong Kong). This work is also partially supported by the National Basic Research Program of China (973 Program, 2013CB834701 and 2013C834702), the University Grants Committee of Hong Kong (AoE/P-03/08), the Research Grants Council of Hong Kong (16301614, 16305015 and N_HKUST604/14), and the Innovation and Technology Commission (ITC-CNERC14SC01 and RE:ITCPD/17-9). B.Z.T. is grateful for the support from the G u a ng d o n g I nn o v a t i v e Re s e a r c h T e a m Pr og r a m (201101C0105067115).

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