RETRACTED: Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells

Acta Biomaterialia xxx (2016) xxx–xxx

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Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells Yang Wu a,b,1, Bo Zhou b,c,1, Fuben Xu b,c, Xiaoyong Wang d, Gang Liu d, Li Zheng b,c,e,⇑, Jinmin Zhao a,b,e,f,⇑, Xingdong Zhang b,c,g a

Department of Orthopaedics Trauma and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, Guangxi Medical University, Nanning, China Collaborative Innovation Center of Guangxi Biological Medicine, Guangxi Medical University, Nanning, China d State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, China e The Medical and Scientific Research Center, Guangxi Medical University, Nanning, China f Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Nanning, China g National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China b c

a r t i c l e

i n f o

Article history: Received 10 May 2016 Received in revised form 2 September 2016 Accepted 7 September 2016 Available online xxxx Keywords: Quantum dot RNA interference Chondrogenesis Mesenchymal stem cell

a b s t r a c t SOX9 plays an important role in mesenchymal condensations during the early development of embryonic skeletons. However, its function in the chondrogenic differentiation of adult mesenchymal stem cells (MSCs) has not been fully investigated because SOX9 RNA interference in adult MSCs has seldom been studied. This study used SOX9 gene as the target gene and the quantum dot (QD)-based nanomaterial QD-NH2 (ZnS shell and poly-ethylene glycol (PEG) coating) with a fluorescent tracer function as the gene carrier to transfect siSOX9 into MSCs after sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1carboxylate (sulfo-SMCC) activation in vitro and in vivo. The results showed that QD-SMCC could effectively bind and deliver siRNAs into the MSCs, followed by efficient siRNA escape from the endosomes. The siRNAs released from QD-SMCC retained their structural integrity and could effectively inhibit the targeted gene expression, leading to reduced chondrogenic differentiation of MSCs and delayed cartilage repair. QDs were excreted from living cells instead of dead cells, and the ZnS shell and PEG coating layer greatly reduced the cytotoxicity of the QDs. The transfection efficiency of QD-SMCC was superior to that of polyethylenimine (PEI). In addition, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection. Statement of Significance In this study, we developed functional quantum dot (QD) nanoplexes by sulfosuccinimidyl-4-(N-maleimi domethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation of PEG-coated CdSe/ZnS QDs as the gene carrier of siRNA to study the effect of SOX9 RNA interference on the chondrogenic differentiation of MSCs. This study confirmed the importance of SOX9 in chondrogenesis, as evidenced by the findings that SOX9 knockdown significantly inhibited the expression of cartilage-specific markers including acan and col2a1 in MSCs and further delayed cartilage repair. Moreover, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction ⇑ Corresponding authors at: Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, Guangxi Medical University, Nanning, China. E-mail addresses: [email protected] (L. Zheng), [email protected] (J. Zhao). 1 Yang Wu and Bo Zhou contributed equally to this work.

The chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by a series of signaling molecules, among which SOX9 plays an important role. This has been verified via

http://dx.doi.org/10.1016/j.actbio.2016.09.008 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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an analysis of SOX9 knockout chimeras and conditional knockout mice. In chimeric mice, SOX9 / cells are excluded from cartilage primordia throughout embryonic development [1]. During embryogenesis, SOX9 is required for mesenchymal condensation, a prerequisite for limb bud formation, and for the inhibition of precocious hypertrophic conversion of proliferating chondrocytes in the growth plate [1]. SOX9 can bind to type II collagen (col2a1) and aggrecan (acan) enhancer elements[2]. The overexpression of SOX9 promotes the expression of col2a1 and acan and accelerates the differentiation of adult MSCs toward chondrocytes [3]. However, few studies have reported the effect of SOX9 interference on the chondrogenesis of adult MSC. One of the major challenges in RNA interference is the lack of a safe and efficient gene delivery method. Carriers for RNA include viral and non-viral carriers [4]. Viral carriers enclose RNA sequences in the viral capsid and achieve efficient and stable transfection by taking advantage of the viruses’ infection properties [5]. However, using viruses as RNA carriers poses potential risks such as cell mutation and immune responses [6]. Another limitation is the lack of an intrinsic signal for long-term and real-time imaging

of siRNA transport and release [7]. Non-viral carriers include liposomes, cationic polymers and nanomaterials, which can bind with RNA molecules by electrostatic adsorption or covalent coupling [8]. Many non-viral carriers are easy to synthesize and modify. In addition, they are less immunogenic and have been successfully used for delivery of genes to stem cells [9,10]. Overall, there is a critical need for an effective and low-toxic nanocarrier that can not only deliver RNA but also monitor the transport process in real time. Water-soluble quantum dots (QDs), which are often applied to label biological molecules, may be an excellent choice. QDs exhibit strong fluorescence and can pass through the cell membranes and blood-brain barrier [11]. The toxic effect of CdSe QDs can be improved by modification such as the incorporation of a ZnS shell and poly-ethylene glycol (PEG) coating [12,13]. Previously, QDbased siRNA delivery was usually achieved by mixing QDs with transfection agents such as polyethyleneimine (PEI) or combined with another class of nanomaterial [7,14]. However, the addition of other agents may lead to vulnerability to intracellular degradation or increased cytotoxicity. An alternative is the use of non-selective or selective bioconjugation techniques. Selective

Fig. 1. Schematic diagram of the QD-SMCC-si conjugation process and its in vitro and in vivo inhibitory effects on the chondrogenic differentiation of MSCs. (A) Aminomodified QDs were conjugated with thiol-modified biomolecules via SMCC. (B) The transfection process and inhibitory effects of QD-SMCC-si in vitro. (C) The inhibitory effects of QD-SMCC-si in vitro and in vivo.

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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bioconjugation can occur without a preceding reduction reaction, thus retaining the integrity of the siRNA [15]. The use of a heterobifunctional crosslinker such as sulfosuccinimidyl-4-(N-maleimido methyl)cyclohexane-1-carboxylate (sulfo-SMCC) results in selective bioconjugation toward specific sites on the protein to form a stable thioether bond with a sulfhydryl-exposed antibody [16]. QD-SMCC has been applied in immunohistochemistry only for antibody bioconjugation. The potential for gene delivery, particularly in the study of MSC differentiation, has yet to be sufficiently investigated. In this study, we applied PEG-coated CdSe/ZnS QDs that were bioconjugated with SMCC, which are commonly used to label antibodies, for gene transfer to study the effect of SOX9 RNA interference on the chondrogenic differentiation of adult MSCs (Fig. 1). As a control, PEI was also investigated. The effect of SOX9 knockdown on cartilage regeneration in vivo was further studied in a cartilagedefective model. This study suggests that QD-SMCC is a promising vector for real-time tracking of transfection. 2. Materials and methods 2.1. Materials Polyethyleneimine (PEI, 25 kD) was purchased from Sigma (Shanghai, China). Water-soluble PEG-amino QDs (CdSe/ZnS, 625 nm peak emission) were purchased from Jiayuan (Wuhan, China), whereas thiol-modified FAM-labeled siRNAs (SOX9-siRNA sequence: GGAGAGCGAGGAAGATAAA) were purchased from Genechem (Shanghai, China). Fifty microliters of QDs was diluted with borate buffer to a concentration of 4 lM, which was then added to 10 lL of the required SMCC (Life Technologies, USA) solution and reacted by stirring at room temperature for 1 h. A desalting column was prepared by equilibrating it with MES buffer. The activated QD-SMCCs were centrifuged to remove aggregates, passed through the equilibrated desalting column, and eluted with MES, followed by collection of the fluorescent fraction. The thiol-modified siRNAs were added to the purified, active QD-SMCCs and reacted by stirring at 4 °C for 20 min. The final product was centrifuged to remove aggregates and stored at 20 °C. The binding effect was determined by gel electrophoresis. 2.2. Gel electrophoresis To determine the optimal concentration of QD-SMCC for siRNA loading, the siRNAs (10 pmol) and QD-SMCCs (1, 2, 10, 50 or 100 pmol) were diluted in borate buffer to molar ratios of 10:1, 5:1, 1:1, 1:5 or 1:10, respectively. A 1% agarose gel was prepared. Next, the sample was mixed with loading buffer and then loaded. After electrophoresis at 90 V for 20 min, the results were observed with a gel imager. 2.3. Isolation and culture of MSCs

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2.5. Transfection experiment The MSCs were seeded at a density of 1  105 cells/well on 6-well plates and incubated for 24 h before transfection. The MSCs were then washed with a-MEM. The various QD-SMCC-si complexes were prepared in a 1:5 ratios and added to each well. The PEI-siSOX9 complex was prepared, and 3 lL of PEI was added to the well as a reference. The MSCs were incubated with each complex for 12 h. The MSCs were then washed with PBS, and fresh cell culture medium was added. 2.6. Cytotoxicity measurement The MTT assay was used to assess the toxicity of the QD-SMCCs. The cells were seeded in 96-well plates at 5000 cells per well, and then 100 lL of a-MEM and varying concentrations of QD-SMCCs were added to each well and cultured for 72 h. The MTT stock solution was then added. After the MTT was removed, the cells were treated with 150 lL of dimethyl sulfoxide (DMSO, Gibco, USA) to dissolve the formazan product. The absorbance was determined at 570 nm using an ELISA plate reader (Thermo Fisher Scientific, UK). 2.7. Flow cytometry The ability of QD-SMCC nanoparticles to deliver siSOX9 into the cells was detected by flow cytometry. The MSCs were seeded in 24-well plates at 7  104 cells per well and cultured overnight in a 37 °C incubator. The QD-SMCC-si complex was added to the 24-well plates seeded with MSCs, and then the cells were cultured in a 37 °C incubator for 24 h. After trypsinization, the cells were collected, and the intracellular FAM fluorescence signals were detected by flow cytometry in a fluorescence activated cell sorting (FACS) Canto apparatus. 2.8. Imaging and observations of the cells by confocal laser scanning microscopy (CLSM) The intracellular distribution of QD-SMCC-si was studied by CLSM (Nikon A1, Japan). Two milliliters of the MSC suspension were seeded in a confocal dish at a density of 2.5  105 cells/well. After culturing at 37 °C and 5% CO2 for 24 h, the old medium was removed, and 2.25 mL of a-MEM medium (FBS-free) was added to each well, along with 250 lL of the solution containing the QD-SMCC-si complex (5:1 ratio of the complex). After an additional 24-h incubation, the cells were rinsed three times with sterile PBS solution to remove the residual complex. Subsequently, the cells were stained with LysoTracker Yellow for 30 min, followed by with Hoechst 33342 for 5 min, and then the samples were observed and photographed by CLSM. The untransfected MSCs were used as the control. 2.9. Observation of cell ultrastructure with transmission electron microscope (TEM)

The MSCs from the femoral marrow cavity were flushed out with a syringe containing a-MEM medium (Gibco, USA). Then, the extracted cells were seeded in 24-well plates and incubated at 37 °C and 5% CO2.

The cells were washed three times with PBS and centrifuged at 1000 rpm for 5 min. The cells were fixed and dehydrated. The samples were cut into 60 nm ultrathin sections, mounted onto copper mesh, and observed under TEM.

2.4. Nanoparticle characterization

2.10. Animal experiment

The mean hydrodynamic diameter was examined by dynamic light scattering (DLS) analysis. The z-potential of QD-SMCC-si complex was measured by Zeta Sizer (Malvern, UK) according to the manufacturer’s instruction.

Three-week-old SD rats were anesthetized with 30 mg/kg of 2.5% sodium pentobarbital and fixed on the operating table in the supine position. After routine skin preparation, a medial patellar incision was made, the soft tissues were separated, and the

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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patella was dislocated laterally. The knee joint was flexed to completely expose the chondral surface of the femur. At the center of the femoral surface, a 3-mm diameter drill was used to produce a full-thickness defect (approximately 3 mm in depth). Approximately 0.4 mL of the QD-SMCC-si-transfected cell clusters (1  106 cells/mL) were injected into the defect site; the surface of the defect site was slightly lower than the surrounding cartilage surface. Then, the patella was carefully repositioned, and the incisions were tightly sutured layer by layer. After 2 weeks and 4 weeks, the rats were sacrificed, and their bones were harvested and immediately frozen in liquid nitrogen for the HE, IHC, WB and PCR tests. All in vivo animal procedures were approved by the Committee on the Ethics of Animal Experiments of Guangxi Medical University (2015-07-11).

prepared, and each reaction tube contained 10 lL of Mastermix (Life Technologies, USA), 1.4 lL of forward primer, 1.4 lL of reverse primer (Life Technologies, USA), 1 lL of cDNA and 6.2 lL of DNase/ RNase-free H2O. The PCR cycling conditions were as follows: 95 °C for 10 min, 94 °C for 15 s, and 60 °C for 1 min for 40 cycles. Then, the dissolution curves were detected. The experiment was repeated three times, and three replicate wells were included in each experiment. The results were expressed as the mean Ct values. An American ABI7500 real-time PCR system was used to detect the products. The following primers were designed: SOX9 Forward-TCCAGCAAGAACAAGCCACA and Reverse-CGAAGGGTCTC TTCTCGCTC; collagen type II Forward-CTGGTCCTTCCGGCCCTAGA and Reverse-GGATCGGGGCCCTTCTCTCT; and aggrecan Forward-CCG CTGGTCTGATGGACACT and Reverse-AGGTGTTGGGGTCTGTGCAA.

2.11. In vivo imaging studies

2.15. Inductively coupled plasma-mass spectrometry (ICP-MS) quantification of cadmium and selenium

Before imaging, the rats were anesthetized with 30 mg/kg of 2.5% sodium pentobarbital, and the transfected MSCs were injected into the rats’ articular cavities. The rats were imaged with a Maestro in vivo optical imaging system (KODAK, USA). Bioluminescence imaging was performed 3 days after the injection. The signal intensity was quantified as the sum of all detected photon counts within a region of interest prescribed over the ankle site. 2.12. HE and immunohistochemistry Paraffin sections were deparaffinized and hydrated, and the antigen was retrieved. Each section was incubated with 3% hydrogen peroxide (H2O2) at room temperature for 10 min and incubated with an anti-SOX9 antibody (1:100 dilution, Boster, China) overnight at 4 °C. Then, a biotin-labeled secondary antibody was added to the sections drop-wise and incubated for 10 min at room temperature. Horseradish peroxidase (HRP)-labeled streptavidin was added to the sections and incubated for 10 min at room temperature. Next, a freshly prepared DAB (Boster, China) chromogenic solution was added to the sections drop-wise and incubated for 5 min at room temperature. The sections were then washed with PBS and counterstained with hematoxylin. Finally, the sections were dehydrated with a gradient of ethanol, cleared in xylene and mounted with neutral gum.

ICP-MS (Thermo Electron Corp, Franklin, MA) was used to detect the tissue distribution of QDs in vivo. Tissues were weighted and ground into homogenates. One milliliter of HNO3 (67%) and the homogenates were combined in a microwave digestion vessel and digested at 200 °C, 250 psi, and 300 W for 40 min. Recovery was accomplished by adding Cd and Se standards at 10 ng/ml in 1 ml 3% HNO3 prior to homogenization. The ICP-MS instrument was optimized using the manufacturer’s recommendations. 2.16. FDA/PI assay Live/dead cell viability assay was carried out with a FDA/PI Staining Kit (Invitrogen) according to the manufacturer’s instructions. 2.17. Statistical analysis SPSS 19.0 was used for data analysis. Comparison among groups was performed by one-way ANOVA, whereas relative expression levels of different genes and proteins were analyzed by independent samples t-test. P < 0.05 was considered statistically significant.

2.13. Western blot

3. Results

The cells or tissues were washed twice with cold PBS, and 1 mL of RIPA and 10 lL of PMSF were added to lyse the cells by shaking in water bath for 1 h. After centrifugation for 10 min, the supernatant was withdrawn and stored at 80 °C. The protein concentration was determined using the BCA assay reagent. After SDSpolyacrylamide gel electrophoretic separation, 50 lg of the total protein was electro-transferred to a PVDF membrane. The membrane was incubated with 5% BSA at room temperature, followed by successive incubations with an anti-SOX9 antibody and an HRP-labeled secondary antibody. A murine monoclonal anti-bactin antibody was similarly incubated with the membrane and served as a control. The AB fluorogenic substrate was then added at a 1:1 ratio to the HRP-conjugated secondary antibody, followed by developing, fixing and analyzing the grayscale images.

3.1. Characterization of QD-SMCC-siRNA

2.14. RT-PCR The RNA was extracted according to the instructions provided with the RNA isolation kit (Tiangen Biotechnology, China) and reverse transcribed in a 20-lL system using a reverse transcription kit (Fermentas Company, USA) to obtain the corresponding cDNA. A 20-lL real-time fluorescent quantitative PCR reaction was

A transmission electron microscope (TEM) was used to detect the micromorphology of QDs and QD-SMCC-si (Fig. 2A and B). The DLS measurements showed that the size of the QD particles was 9.6 nm (Fig. 2C) and that of the QD-SMCC-si complex was 16.7 nm (Fig. 2D). Zeta potential measurements revealed that the QD-SMCC complex had a zeta potential of 16.5 ± 1.6 mV (Fig. 2E), with a negatively charged siRNA surface ( 17.2 ± 2.1 mV). After the siRNA was conjugated to QD-SMCC in a 1:5 ratio, the complex became positively charged (11.4 ± 1.3 mV). 3.2. Assessment of the QD-loads of siRNAs To determine the appropriate QD-loads of the siRNAs, the siRNAs were mixed with several concentrations of QD-SMCC at molar ratios of 10:1, 5:1, 1:1, 1:5 and 1:10 (Fig. 2F). As the ratio of the siRNA/QD-SMCC complex decreased, the fluorescence intensity of the siRNA gradually decreased. When the molar ratio was higher than 1:5, free siRNA was detected on the agarose gel, indicating that the siRNA molecules were not completely complexed with QD-SMCC. When the ratio of the complex was 1:5 or less, the

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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Fig. 2. TEM, DLS, MTT, and zeta-potential analyses of the QD-SMCCs, as well as a gel retardation assay to evaluate the binding between the siRNAs and QD-SMCCs. (A) TEM image of the QDs. (B) TEM image of QD-SMCC-si. (C) DLS image of the QDs. (D) DLS image of QD-SMCC-si. (E) Changes in the surface charge. (F) Effective loading of siRNA on the QD-SMCC, as determined by agarose gel electrophoresis. Lanes 3–7 correspond to siRNA/QD-SMCC ratios of 10:1, 5:1, 1:1, 1:5 and 1:10, respectively. Lanes 1 and 2 correspond to siSOX9 and QD-SMCC. (G) The cytotoxicities of QD-SMCC and PEI were assessed with an MTT viability assay.

RNA was fully bound to QD-SMCC, and there was no RNA in the complex that would migrate toward the anode.

cells. The FDA/PI assay indicated more than 97% of cells were labeled by green fluorescence, demonstrating that QDs were excreted by living cells instead of dead cells.

3.3. Cell viability 3.5. Cellular ultrastructural response to the QD-SMCC-siRNAs As shown in Fig. 2G, cell viability was maintained at approximately 95% for a QD-SMCC concentration range of 20–80 pmol. When the concentration of QD-SMCC was higher than 200 pmol, the cell viability was 63% (Fig. 2G). In comparison, 25–35 pmol/L of PEI maintained the best cell viability. The cytotoxicity of QDSMCC cytotoxicity was lower than that of PEI within a range of 20–120 pmol/L (Fig. 2G, P < 0.05). The time-dependent cytotoxicity at a QD concentration of 40 pmol/L and a PEI concentration of 35 pmol/L (optimal transfection concentration) was also measured (Fig. S1). Compared with that of the control group, the OD value of the QD-SMCC group did not result in a significant change. Fourteen days after incubation, the MTT assay showed an increase in OD value (0.58 ± 0.04 in control group, 0.57 ± 0.03 in QD-SMCC group and 0.47 ± 0.04 in PEI group). The OD value of the PEI group was lower than that of the QD-SMCC group (P < 0.05). 3.4. The elimination of QDs Fig. S2 shows the time course for the cellular elimination of QDs. The intracellular concentration of QDs decreased as the elimination rate decreased with time. Many QDs still accumulated in MSCs after 48 h (Fig. S2 A). Following the internalizing effects on cells, a proportion of Se was reserved in cells, whereas little Cd was eliminated by MSCs (Fig. S2 C and D). Moreover, nearly 85% of Cd accumulated in cells even after 48 h. To investigate whether the excreted QDs came from dead or intact cells, the supernatants of transfected MSCs were collected, and red fluorescence was then observed under a confocal laser scanning microscope (CLSM) (Fig. S2 B). The results clearly indicate that QDs leaked out of the

To study the ultrastructure of MSCs transfected with QD-SMCCsi, the cells were fixed at different time points and evaluated by TEM. After the QD-SMCC-si were transfected into the MSCs, a large amount of QD-SMCC-si was aggregated onto the surface of the cells (Fig. 3A). Three hours after transfection, the MSCs extended a large number of filopodia toward a cluster of QD-SMCC-si and enveloped them (Fig. 3B). Six hours after transfection, the phagocytized QDSMCC-si gradually moved away from the cell membrane into endosomes wrapped by lipid membranes (Fig. 3C). Twelve hours later, after some of the endosomes had ruptured, the QD-SMCC-si were released into the cytoplasm (Fig. 3D). 3.6. The QD-SMCC-si transfection process We further observed the QD-SMCC-si transfection process by CLSM. Within the MSCs, green fluorescence represented the FAM-siRNAs, red fluorescence represented the QD-SMCCs, and blue fluorescence represented the Hoechst 33342-labeled nuclei. Furthermore, acidic organelles (including endosomes and lysosomes) were labeled in yellow with LysoTrackerTM Yellow. After 3 h of incubation, the red QD-SMCCs and the green FAM-siRNAs gathered around the cell membranes (Fig. 3E). After 6 h of incubation, the green FAM-siRNAs and the red QD-SMCCs were mainly distributed in the yellow-labeled organelles, suggesting that the nanoparticles were retained in the endosomes or early lysosomes. Twelve hours later, the green FAM-siRNAs and the red QD-SMCCs were no longer distributed in the yellow-labeled organelles, which escaped from the endosomes. This finding indicates that the QD-SMCCs could effectively deliver the siRNAs into MSCs and

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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Fig. 3. TEM and confocal laser-scanning microscopy images of MSCs following treatment with the QD-SMCC-siRNA. (A) TEM showed that the MSCs extended many filopodia toward a cluster of QD-SMCC-si particles after 2 h of transfection. (B) Three hours after transfection, the QD-SMCC-si clusters were being phagocytized. (C) Six hours after transfection, the early endosomes contained QD-SMCC-si clusters. (D) Twelve hours later, the late endosomes resembled transparent droplets containing the QD-SMCCs and cell debris and were dispersed in the cytoplasm. (E) CLSM showed the transfection process of QD-SMCC-si in MSCs.

enable their escape from endosomes to silence genes in the cytoplasm. 3.7. Quantification of the transfection efficiency of QD-SMCC-si To compare the effects of different transfection times on transfection efficiency, we determined the number of FAM-siSOX9positive transfected MSCs using fluorescence-activated cell sorting (FACS). The FACS analysis showed that 0.58% of the untreated MSCs were positive, whereas 52.5%, 65.4% and 61.6% of the MSCs were positive following incubation with the QD-SMCC-si complex for 6 h, 12 h, and 24 h, respectively (Fig. 4A). The results indicated that 12 h of transfection could efficiently deliver the siRNA into the cells. The effects of different concentrations on transfection efficiency were further examined by FACS, which showed that QDSMCC had the highest transfection efficiency (62.6%) at a concentration of 40 pmol/L (Fig. 4B). Finally, we determined the efficiency

of different transfection reagents. The FACS analysis showed that 68.6% of the MSCs were positive for QD-SMCC-siSOX9 (Fig. 4C), whereas 56.4% of the cells were positive for PEI-si (P < 0.05). The results indicate that the transfection efficiency of QD-SMCC was higher than that of PEI. 3.8. Gene and protein silencing efficiency of QD-SMCC-si in vitro We examined the effect of the QD-SMCC transfected siRNA on the SOX9 silencing efficiency by quantitative RT-PCR. After transfection, there were no significant differences between the control, free siRNA, QD-SMCC and QD-SMCC-siNC groups. As expected, the level of SOX9 was significantly decreased in the QD-SMCC-si and PEI-si groups compared with that of the control (0.34 ± 0.04 and 0.58 ± 0.07, Fig. 4D). The decrease was time-dependent, with the best silencing effect observed at day 2 after transfection. Between the QD-SMCC-si and PEI-si groups, QD-SMCC-si had a relatively higher

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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Fig. 4. FACS, RT-PCR and Western blot analyses of SOX9 expression in MSCs following treatment with the QD-SMCC-si particles for various times. RT-PCR analysis of col2a1 and acan in the MSCs following treatment with the QD-SMCC-si particles in vitro for various times. (A) Number of siSOX9-positive MSCs following treatment with the QDSMCC-si particles for various times. (B) Number of siSOX9-positive MSCs following treatment with various concentrations of the QD-SMCC-si particles. (C) Number of siSOX9positive MSCs following treatment with the different transfection reagents. (D) RT-PCR analysis of SOX9 mRNA levels in the MSCs following treatment with the different transfection reagents for 1 day, 2 days, and 3 days. (E) The levels of the SOX9 mRNA changed from day 1 to day 21. (F) Western blot analysis of the SOX9 protein levels in the MSCs following treatment with the different transfection reagents for 1 day, 2 days, and 3 days. (G) The levels of the SOX9 protein changed from day 1 to day 21. (H) RT-PCR analysis of the col2a1 and acan mRNA levels in the MSCs following treatment with the different transfection reagents for 7 days, 14 days, and 21 days. The bars with different letters are significantly different from each other at P < 0.05.

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inhibition efficiency (0.30 ± 0.06) than PEI-si (0.52 ± 0.08) at day 3 (P < 0.05, Fig. 4D). After the cells were treated with a chondrogenic supplement, the reduced SOX9 expression in the QD-SMCC-si and PEI-si groups gradually increased in a time-dependent manner. After 21 days of induction, the level of SOX9 was close to the control (Fig. 4E), suggesting that QD-SMCC-si inhibited the early stages of chondrogenic differentiation. During chondrogenesis, QD-SMCC-si showed higher transfection efficiency than PEI. WB showed that SOX9 protein level was the lowest on the third day after transfection, the expression of SOX9 at the protein level was reduced to 0.16 ± 0.07 by QD-SMCC-si and to 0.36 ± 0.08 by PEI-siSOX9 (Fig. 4F), indicating that the QDS-transfected siSOX9 played an interference effect within cells. During chondrogenic differentiation, QD-SMCC-si-downregulated SOX9 levels were gradually elevated (Fig. 4G). Comparatively, QD-SMCC was slightly superior to PEI. 3.9. Suppression of SOX9 affected cartilage-specific markers in vitro

drogenesis, downregulation of the col2a1 and acan genes (0.58 ± 0.05 and 0.63 ± 0.08) was also observed in the PEI-si group. During culture, the expression of cartilage-specific markers, including col2a1 and acan, increased in the control group over time after TGF-b stimulation, demonstrating that chondrogenesis was induced (Fig. 4H). 3.10. In vivo fluorescence imaging In vivo fluorescence imaging was performed to investigate the potential of QD-SMCC as a promising tool for real-time gene tracking. The significant bioluminescence of the transfected cells was also confirmed in vivo, whereas little to no signal above background was found in the rats that were injected with the unlabeled control cells (Fig. 5A and B, P < 0.05). The results suggest that the QD-SMCC exhibited strong fluorescence and offer great potential for imaging and related biomedical applications because deep imaging within tissues has become possible by the introduction of advanced fluorescence microscopy. 3.11. ICP-MS quantification of cadmium and selenium

The inhibition of SOX9 clearly downregulated the expression of the col2a1 and acan genes (0.35 ± 0.07 and 0.40 ± 0.06) in the MSCs transfected with QD-SMCC (Fig. 4H). During the early stage of chon-

Liver taken from control rats at 1 d contained 4.2 ± 1.6 ng of cadmium (Fig. S3). The levels of cadmium in the kidney, spleen,

Fig. 5. Macroscopic assessment, in vivo fluorescence images and HE staining of the articular cartilage defects in rat models. (A) In vivo optical images and (B) quantitative analysis of the transfected cells. (C) Macroscopic assessment of the repaired tissue at the articular cartilage defects in rat models. (D) HE staining of the repaired tissue at the articular cartilage defect in rat models. * indicates P < 0.05.

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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blood, muscle, heart and bone marrow were maintained at 3.4 ± 0.6 ng, 2.3 ± 0.5 ng, 1.5 ± 0.3 ng, 2.5 ± 0.5 ng, 2.3 ± 0.8 ng and 2.7 ± 1.1 ng from 1 d to 14 d. For transfected rats, the levels of cadmium in the liver, kidney, spleen, blood, muscle, heart and bone marrow were 4.7 ± 1.3 ng, 3.8 ± 1.1 ng, 2.6 ± 0.8 ng, 3.1 ± 1.4 ng, 2.9 ± 0.9 ng, 2.8 ± 1.1 ng and 4.2 ± 2.1 ng. No significant difference was found between transfected rats and control rats. Seven days after transfection, the levels of cadmium in the liver, kidney, spleen, blood and bone marrow increased to 147.8 ± 16.4 ng 83.5 ± 13.7 ng, 55.6 ± 10.4 ng, 24.3 ± 9.1 ng and 92.7 ± 12.1 ng (P < 0.05). No significant changes were detected in the muscle and heart (3.3 ± 1.2 ng, 3.9 ± 1.6 ng). Fourteen days after transfec-

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tion, liver taken from transfected rats contained 124.6 ± 18.9 ng of cadmium. The level of cadmium in the kidneys increased to 94 ± 15.7 ng. The levels of cadmium in the spleen, blood and bone marrow were 48.5 ± 10.7 ng, 27.6 ± 6.4 ng, 67.3 ± 18.2 ng. The levels of cadmium in the muscle and heart did not change during the 14 d of the study. 3.12. Macroscopic observation of the cartilage defects To investigate the effect of SOX9 knockdown on cartilage formation, we transplanted transfected or untreated MSCs into a cartilage defect. Based on macroscopic observations after the

Fig. 6. Sox9, col2a1 and acan expression in the rat cartilage defect sites. (A) Immunohistochemical staining for SOX9 in rat cartilage. (B) RT-PCR and Western blot analysis of the SOX9 levels in the MSCs following treatment with the different transfection reagents for 2 weeks and 4 weeks. (C) COL2A1 and ACAN protein levels in the MSCs following treatment with different transfection reagents for 2 weeks and 4 weeks. The bars with different letters are significantly different from each other at P < 0.05.

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samples were harvested, severe synovitis, osteophyte formation, or infection was not observed (Fig. 5C). The defect was still visible in every knee examined, but the repair process appeared to evolve slowly. Two weeks after the operation, defects in all groups were filled with fibrin-like tissues with an irregular surface, and the boundary between the newly formed tissue and original cartilage was obvious in all the groups. At 4 weeks, although the repaired surface appeared fibrillated and less mature than the natural cartilage, the newly formed tissue appeared to be integrated with the surrounding tissue in the control and QD-SMCC groups. There was a slight decrease in the defects of the QD-SMCC-si and PEI-si groups. 3.13. Histological evaluation HE staining images of the repaired tissue in cartilage defects are shown in Fig. 5D. Two weeks after the operation, the defects in all groups showed poor cartilage repair. Imaged sections showed that the defects were unfavorably repaired in a minority of tissue samples, appearing more fibrous than fibrocartilaginous and loosely bound to the surrounding tissue in the QD-SMCC-si and PEI-si groups. In contrast, the control and QD-SMCC groups showed relatively tightly bound tissue in the defect. Four weeks after the operation, the interface between the newborn tissue and the original cartilage became integrated, and the fibrocartilaginous tissue was present in the defect. In control knees, articular contours were restored, but a significant gap was noted in the border area stretching to subchondral bone. The differences between the control, QDSMCC, QD-SMCC-si and PEI-si groups were not as significant as those observed at 2 weeks after surgery. In the QD-SMCC-si group, the surface was irregular and covered with thin, fibrin-like tissue. 3.14. Silencing efficiency of QD-SMCC-si in vivo To investigate the expression of SOX9 in the cartilage defect model, we used immunohistochemical, RT-PCR and WB analyses. Two weeks after the operation, there was very little SOX9 staining in the QD-SMCC-si and PEI-si groups. In contrast, there was SOX9positive staining in the control and QD-SMCC groups (Fig. 6A). Four weeks after the operation, the specimens in the QD-SMCC-si and PEI-si groups showed positive staining for SOX9 compared with the control and QD-SMCC groups. The RT-PCR and WB results further verified that the expression of SOX9 was decreased in the QD-SMCC-si and PEI-si groups compared with that observed for the control and QD-SMCC groups (Fig. 6B). The mRNA level of SOX9 decreased (0.49 ± 0.07) for QD-SMCC transfected cells 2 w after operation (Fig. 6B), and the WB result (0.55 ± 0.08) was also consistent with the PCR results. The level of SOX9 mRNA and protein expression in MSCs was significantly lower following transfection with QD-SMCC compared with that of PEI. The expression level of SOX9 increased significantly from week 2 to week 4 for the QD-SMCC and PEI groups during culture. 3.15. Suppression of SOX9 affected neocartilage formation in vivo To examine the role of SOX9 interference in the chondrogenic differentiation of MSCs in vivo, we injected transfected cell clusters into the defect sites in rat cartilage. Two weeks postoperatively, the inhibition of SOX9 clearly downregulated the expression of COL2A1 and ACAN protein (0.46 ± 0.09 and 0.56 ± 0.08) in the QD-SMCC-si group (Fig. 6C). Four weeks postoperatively, QDSMCC-si and PEI-si did not affect the protein level of COL2A1 and ACAN significantly. The result indicated that the impact of siSOX9 on the expression of the cartilage-specific genes was reduced during the process of cartilage repair.

4. Discussion This study demonstrated the effect of SOX9 interference on the chondrogenic differentiation of adult MSCs, in which the delivery of siRNA was facilitated by bioconjugated QD-SMCC. The observed interference was superior to that of PEI, and QD-SMCC contains an intrinsic signal for long-term and real-time imaging of siRNA transport, demonstrating the potential advantage of using QD-SMCC as a delivery platform. In contrast to other silencing reports using nanoparticle delivery, the interference was recorded at several moments during chondrogenesis. The RNA and protein levels of SOX9 were detected when the cells were treated with siRNA both in in vitro and in vivo studies. When targeting siRNA against a specific gene, it is known that associated genes related to chondrogenic differentiation may also be affected. We have shown that cartilage-specific markers are influenced when SOX9 is silenced, demonstrating that SOX9 may be a potential therapeutic target for cartilage therapy. As siRNA carriers, QDs have attracted attention regarding the treatment of diseases [17–19]. Most QDs have been modified with other siRNA delivery agents, resulting in greatly increased QD sizes. Li modified QDs with PEG, and the QDs diameter was 84.2 nm after modification. PEI-modified QD605 had a diameter of up to 150 nm [20,21]. Comparatively, QD-peptide conjugates are relatively smaller. In our study, the diameter of the QDSMCC-si complex increased from 9.6 nm to 16.7 nm after bioconjugation (Fig. 2C and D). We know that small nanoparticles are clearly advantageous in gene transfection because they can easily pass through capillaries and the tissue space to be ingested by the cells [22]. Our study is unique in that it relies on simple bioconjugation between QD-SMCC and siRNA, leading to effective interference of the MSCs. For the QD-NH2 platform to function as a gene carrier, it must be highly soluble in aqueous solutions and stable under physiological conditions and should not form aggregates. Sulfo-SMCC can form stable amide bonds with QD-NH2. PEG-amino modified on the QDs have a strong proton-absorbing capability inside acidic cellular organelles such as endosomes. The co-existence of PEG-amino and QD is expected to facilitate siRNA release inside cells. Our study also showed that the QD-SMCC nanocomposites were positively charged, with zeta potentials in the range of 11.4 ± 0.9 mV (Fig. 2E). The positive charge of QD-SMCC-si may also interact with the negative charge on the surface of MSCs to promote QD-SMCC-si entry into the cells. The TEM results verified that the particles were uniformly distributed after they were covalently bioconjugated to the siRNA. Covalent bioconjugation to the siRNA permits a higher degree of protection against nucleases and ensures that the siRNA is delivered to the cytoplasm, which contributes to the improved gene transfection. The transfection efficiency of the QD-SMCC-si complexes was 68.6%, which was higher than that of PEI (56.4%). We observed the cell entry process of the QD-SMCC-si by CLSM and found that the cells extended many filopodia toward a cluster of QD-SMCC and simultaneously enveloped them (Fig. 3). After the endosomes ruptured, QD-SMCC-si were released into the cytoplasm. Some studies have shown that the QDs were eventually deposited within the cells and caused cytotoxicity due to nonspecific uptake and peroxidation [23,24], but we found that QDs are excreted from living cells rather than dead cells. The elimination rate of QDs decreased over time; in accord with the survey performed by ICP-MS, a portion of the initial QDs were retained in the cells after 48 h. Previous reports indicated that elimination of nanoparticles slowed over time and not all internalized nanoparticles were excreted [25–27]. It is recognized that many types of stem cells with membrane transporters are capable of

Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008

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expelling toxic reagents from the cytoplasm for self-protection [28]. However, the mechanism of QD excretion should be studied. Because the toxicity of QDs is related to the release of toxic elements, Seon evaluated the toxicity of Se and Cd. Cadmium is recognized as a toxic material that leads to side effects such as mitochondrial dysfunction, the induction of oxidative stress, the disruption of intracellular calcium signaling and apoptosis [29]. After evaluating the toxicity of Cd, Peng found that the toxicity of Cd was apparent at concentrations reaching 8.9 mM [30]. In contrast, Selenium is an essential element that is toxic when the concentration becomes excessive [31]. We found that QD-SMCC had the highest transfection efficiency at a concentration of 40 pmol. The dose of QDs is far lower than 8.9 mM; therefore, no significant cytotoxic effects were observed in the study. The low cytotoxicity of QD-SMCC may result from the ZnS and PEG coating layer that protects the QDs from being exposed to the intracellular environment, thereby preventing Cd2+ release. We further verified the interference effects of the QDtransfected siSOX9 at the gene and protein levels in vitro. We showed that siSOX9 knocked down the expression at both the RNA and protein levels and was superior to PEI (Fig. 4). The gene silencing effect of SOX9 can significantly reduce the expression of chondrogenic-specific markers, including col2a1 and acan. The results indicated that QD-SMCC did not have detectable adverse effects that limit the differentiation. Using this delivery system, we demonstrated that SOX9 knockdown reduced the expression of col2a1 and acan and delayed the differentiation of adult MSCs into chondrocytes (Fig. 4). Similarly to chondrogenesis during embryonic development, SOX9 plays key roles in the early stage of chondrogenesis in adult MSCs [10]. We further studied the effect of SOX9 interference on neocartilage formation after implanting the MSCs into a cartilage defect model. Articular cartilage is an avascular tissue with very low cell density; it is therefore difficult for the QDs to be translocated to other organs. At first, no significant changes were detected in the tissues. However, QDs accumulated in the liver, kidney, spleen, blood, and bone marrow through time-dependent redistribution. Many researchers are convinced that QDs will never be used in clinical practice because of their potential toxicity [32]. The perception that QDs are toxic is derived from in vitro studies indicating that cadmium can induce oxidative stress, DNA damage and apoptosis. However, such a deduction is inappropriate because few toxicity tests based on cells can be adapted to more complex biosystems [32]. ZnS-capped CdSe QDs do not produce ROS, and the ZnS shell is effective in inhibiting the reactivity of QDs [33]. Hauck showed that ZnS-capped CdSe QDs did no short-term (less than a week) or long-term harm (more than 80 days) on SD rats after implementing a pioneering and comprehensive in vivo toxicity test. Moreover, they detected no changes in the weight or behavior of the animals or hematological markers, in contrast to controls. Based on biochemical and histological analyses, no organic damage or inflammation was identified even though QDs or materials degraded from them were found in the kidney, spleen and liver [34]. Because the amount and structure of nanoparticles are in constant flux in vivo, only a small portion of injected nanoparticles will interact with cells. Parameters such as size and surface chemistry will be altered as nanoparticles travel through the human body. The histological findings showed that the QD-SMCCsi-transfected MSCs exhibited delayed cartilage regeneration compared with the control, as demonstrated by the presence of more fibrous tissue in the newly formed tissue that loosely interfaced with the surrounding tissue in the early stage (Fig. 5). The PCR and WB results also agreed with the histological staining and showed a relative decrease in the expression of the cartilagespecific markers. We proved that the silencing of SOX9 delayed

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cartilage repair. Gene overexpression and knockdown provide useful tools for genetic functional analysis. Insights gained from experimental RNAi use may be useful in identifying potential therapeutic targets. SOX9 plays an important role in the chondrogenesis of MSCs. The results also showed that transfection with QDSMCC was superior to that with PEI at 2 weeks after the operation in an in vivo study (Fig. 6). The silencing effect of QD-SMCC-si was confirmed with a single QD-SMCC to preclude the toxicity caused by the inhibition of cartilage repair. However, the delayed neocartilage formation was not as significant for the MSCs transfected with the QD-SMCC and PEI vehicles compared with the control after 4 weeks. The results indicated that nanoparticle-based transfection plays a role in the early stage of chondrogenesis. Unlike a virus vector, QD and PEI are transfection reagents for transient gene expression. In transient transfection, the introduced nucleic acid is inserted into the nucleus of the cell for a certain period and is not integrated into the genome. Transiently transfected siSOX9 is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division. Over time, the interference is weakened and eventually eliminated. The signal in the joint can be tracked using the bioconjugated QD-SMCC (Fig. 5). The cationic surface of the QD-SMCC effectively binds the gene via bioconjugation, leading to efficient gene delivery in vitro and in vivo while maintaining the fluorescence properties and high biocompatibility of the QD-SMCC. 5. Conclusion In this study, we developed the nanomaterial QD-NH2 with a fluorescent tracer function as a gene carrier to transfect siSOX9 into MSCs after SMCC activation. Our results suggest that SOX9 is crucial for chondrogenic differentiation of MSCs, which may be a potential target for cartilage therapy. QD-SMCC is promising in gene delivery both in vitro and in vivo. We envision the application of QD-SMCC as imaging-trackable nanocarriers for safe and efficient gene delivery. Acknowledgments This work has been financially supported by National key research and development program of China (2016YFB0700800), Guangxi scientific Research and Technological Development Foundation (Grant No. Guikegong 1598013-15), Guangxi Science Fund for Distinguished Young Scholars (Grant No. 2014GXNSFGA1 18006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2016.09. 008. References [1] W. Bi, J.M. Deng, Z. Zhang, R.R. Behringer, B. de Crombrugghe, Sox9 is required for cartilage formation, Nat. Genet. 22 (1) (1999) 85–89. [2] I. Sekiya, K. Tsuji, P. Koopman, H. Watanabe, Y. Yamada, K. Shinomiya, A. Nifuji, M. Noda, SOX9 enhances aggrecan gene promoter/enhancer activity and is upregulated by retinoic acid in a cartilage-derived cell line, TC6, J. Biol. Chem. 275 (15) (2000) 10738–10744. [3] L.J. Ng, S. Wheatley, G.E. Muscat, J. Conway-Campbell, J. Bowles, E. Wright, D.M. Bell, P.P. Tam, K.S. Cheah, P. Koopman, SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse, Dev. Biol. 183 (1) (1997) 108–121. [4] W.B. Tan, S. Jiang, Y. Zhang, Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference, Biomaterials 28 (8) (2007) 1565–1571.

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Please cite this article in press as: Y. Wu et al., Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.09.008