Disulfide-Containing Molecular Sticker Assists Cellular Delivery of DNA Nanoassemblies by Bypassing Endocytosis

Open AccessCCS ChemistryCOMMUNICATION1 Mar 2021Disulfide-Containing Molecular Sticker Assists Cellular Delivery of DNA Nanoassemblies by Bypassing Endocytosis Wen Yang, Xiaochen Liu, Haofei Li, Jie Zhou, Shan Chen, Pengfei Wang, Juan Li and Huanghao Yang Wen Yang MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 , Xiaochen Liu MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 , Haofei Li Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240. , Jie Zhou MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 , Shan Chen MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 , Pengfei Wang Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240. , Juan Li *Corresponding author: E-mail Address: [email protected] MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 and Huanghao Yang MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108 https://doi.org/10.31635/ccschem.020.202000250 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail The delivery efficiency of DNA nanoassemblies to the cytosol remains unsatisfactory due to endo- and lysosomal entrapment and degradation, which restricts their bioanalytical and biomedical applications. Herein, the authors developed a disulfide-containing molecular sticker (DSMS) assisted approach to achieve efficient cytosolic delivery of DNA nanoassemblies. DSMS is designed to consist of a disulfide unit for thiol-mediated uptake and a guanidinium cation unit for strongly adhering to DNA nanoassemblies. After attaching with DSMS, a set of DNA nanoassemblies maintained their original three-dimensional features but had a different cellular internalization pathway. These results demonstrated that DSMS-DNA nanoassemblies complex did not enter the cells via endocytosis but instead entered through thiol-mediated direct uptake. Taking advantages of this facile assembly, universal applicability, and direct cytosolic delivery, DSMS might serve as a potent agent to facilitate the applications of DNA nanoassemblies in a variety of fields, such as bioimaging, drug delivery, and gene therapy. Download figure Download PowerPoint Introduction The development of DNA nanotechnology leads to the generation of different DNA nanoassemblies with precise programmability, high biocompatibility, and versatile functionality,1,2 such as DNA origami,3 DNA hydrogels,4 and DNA micelles.5 Furthermore, DNA has been used as a powerful tool to biofunctionalized nanoparticles for morphology control,6,7 surface modification,8,9 spatial addressing,10,11 and dynamic assembly.12,13 Owing to the unique features of DNA, these DNA hybrid nanoparticles are endowed with targeted function, diversified controllability, and multifunctional properties. These features enable DNA nanoassemblies to be used widely in bioanalytical and biomedical applications.14,15 However, there remain several bottlenecks that impede their practical applications. The major one is that DNA nanoassemblies are internalized via endocytotic pathways, leading to subsequent endo- and lysosomal trapping and degradation.16,17 In such a way, the three-dimensional (3D) nanostructure of DNA nanoassemblies is broken down, resulting in false detection signals or low therapeutic efficiency. In attempts to address these limitations, many efforts have been put into facilitating endo/lysosomal escape of DNA nanoassemblies. For instance, cationic polymers or cationic albumin have been employed for the release of DNA nanoassemblies from endosomes and lysosomes by taking advantage of the "proton sponge effect."18 Unfortunately, high cytotoxicity of cationic carriers and low cytosolic release efficiency have limited their application.19,20 As an alternative solution, the strategies via endocytosis-independent pathways have attracted considerable attention. Membrane fusion and thiol-mediated uptake have been used for the direct cytosolic delivery of biomolecules.21,22 In particular, thiol-mediated uptake is a natural mechanism that bypasses the endocytotic internalization of disulfide-containing cargoes. Cells express thiols on their surface to protect them against the oxidative environment.23 Disulfide exchange reaction between disulfide-containing cargoes and exofacial thiols mediates translocation of the cargoes into the cytosol; then, the disulfide undergoes rapid cleavage, catalyzed by endogenous glutathione (GSH) to free cargoes.24 Taking advantage of cell-penetrating poly(disulfide)s (CPDs), which were first reported by Matile's group,25,26 thiol-mediated uptake has been applied in the delivery of diverse artificial cargoes, including proteins,27 nucleic acids,28 and small-molecule drugs.29 Therein, Yao et al.30,31 did pioneer work for applying CPDs to the cytosolic delivery of nucleic acids. They used mesoporous silica nanoparticles (MSNs) as nanocarriers and CPDs as gatekeepers to deliver small-molecule inhibitors and antisense oligonucleotides for target detection, imaging, and gene therapy.30,31 Recently, our group has reported a DNA template-assisted polymerization method to create size-controllable DNA nanospheres for antisense gene therapy.32 While substantial progress has been made, there are still some shortcomings. Most of these methods require time-consuming polymerization procedures to covalently or noncovalently conjugated with cargoes, which might increase the risk of damage to the DNA nanostructure and biological activities. Therefore, the development of a facile and universal method for direct cytosolic delivery of DNA nanoassemblies without structural deformation remains a desirable goal. Herein, we develop a facile and universal approach by using a disulfide-containing molecular sticker (DSMS) to assist DNA nanoassemblies with bypassing endocytosis. In this approach, the DSMS was designed to consist of two functional units (Scheme 1a): a disulfide unit (colored orange) for thiol-mediated uptake and a guanidinium cation unit (Gu+, colored green) for strong adherence on DNA nanoassemblies. The Gu+ cations have a strong affinity for phosphate groups of DNAs via the formation of multiple salt bridges.33 Due to the high rigidity of DNA nanoassemblies, the DSMS could adhere firmly to them and maintain their original 3D nanostructures. To illustrate the generality of the molecular sticker, three different DNA nanoassemblies with different nanostructures and components were selected as models (Scheme 1b). Their cellular uptake ability and mechanism of the nanoassemblies with or without molecular stickers were well demonstrated. Taking advantage of the facile assembly, universal applicability, and direct intracellular delivery, DSMS provides an efficient avenue for DNA nanoassemblies in broad applications, such as bioimaging, drug delivery, and gene therapy. Scheme 1 | (a) Schematic illustration of DSMS adhering to DNA nanoassemblies via multiple salt bridges. (b) Intracellular uptake of DNA nanoassemblies via endocytosis-dependent pathways (left) and by the DSMS-DNA nanoassemblies complex system, bypassing endocytotic internalization delivery via thiol-mediated direct uptake (right). Download figure Download PowerPoint Results and Discussion First, as a proof of concept, we employed DNA tetrahedron nanostructure as a model of DNA nanoassemblies with a small size. It is a classical and readily synthesized DNA nanostructure with excellent mechanical rigidity and structural stability, which has been widely applied in bioimaging and drug delivery in living cells.34 The DNA tetrahedron nanostructure was self-assembled with four designed strands (A, B, C, and D; Supporting Information Table S1 for detailed sequences) through a simple annealing process.35 A polyacrylamide gel electrophoresis (PAGE) analysis suggested that DNA tetrahedron was formed as designed ( Supporting Information Figure S1). The DSMS was prepared according to previously reported protocol25 and then characterized by 1H NMR ( Supporting Information Figure S2), 13C NMR ( Supporting Information Figure S3), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) ( Supporting Information Figure S4a). To obtain the DSMS-DNA tetrahedron complex, we mixed the DSMS and DNA tetrahedron with different N/P ratios (N/P = [Gu+]/[PO4−]). Dynamic light scattering (DLS) was used to determine the average size. With the rise of the proportion of DSMS, the hydrodynamic size of the DSMS-DNA tetrahedron complex was increased. DNA tetrahedron was 5.67 ± 0.55 nm, and the hydrodynamic sizes of DSMS-DNA tetrahedron complex increased to 10.81 ± 0.60 nm (N/P = 5/1), 11.89 ± 0.26 nm (N/P = 8/1), and 352.07 ± 29.83 nm (N/P = 15/1) (Figure 1a). The hydrodynamic size increased dramatically at N/P ratio 15/1, which might be attributable to the formation of assemblies of multiple DSMS-DNA tetrahedron complexes. Besides, the zeta potentials of the DSMS-DNA tetrahedron complexes were about −8.31 mV (N/P = 5/1), −8.44 mV (N/P = 8/1), and −7.91 mV (N/P = 15/1), which were more positive than that of DNA tetrahedron alone (−12.1 mV) and more negative than that of the DSMS (+28.3 mV) ( Supporting Information Figure S5). To further study the morphology of the complex, an atomic force microscopy (AFM) was conducted. The DSMS-DNA tetrahedron complex and the DNA tetrahedron had similar well-defined 3D nanostructures while the thickness of DSMS-DNA tetrahedron complex was slightly increased with DSMS addition (Figures 1b and 1c). The increased hydrodynamic size, AFM thickness, and neutralization of zeta potential indicated that DSMS was assembled successfully with DNA tetrahedron. Figure 1 | (a) Hydrodynamic size of DSMS-DNA tetrahedron complex with different N/P ratio. AFM images of (b) DNA tetrahedron and (c) DSMS-DNA tetrahedron complex. Scale bars: 100 nm. (d) Assessing the integrity of the DSMS-DNA tetrahedron complex by fluorescence studies. Download figure Download PowerPoint To investigate whether the DSMS could influence on the structural integrity of the DNA tetrahedron, we examined the efficiency of fluorescence resonance energy transfer (FRET) of dual fluorescence dyes (Cy3 and Cy5)-labeled DNA tetrahedron (Figure 1d). With the gradual assembly of DNA tetrahedron, Cy3 fluorophore came close to Cy5 fluorophore, and FRET efficiency was enhanced. The FRET efficiency of DSMS-DNA tetrahedron assembly was consistent with that of DNA tetrahedron alone, demonstrating that assembly with DSMS did not impact on the integrity of DNA tetrahedron 3D configuration. To further unveil the potential formation mechanism of the DSMS-DNA tetrahedron complex, MALDI-TOF MS measurement was performed. Only an ion peak corresponding to monomers was observed after mixing DNA tetrahedron with DSMS for 72 h, suggesting no polymerization reaction occurred under this condition ( Supporting Information Figure S4b), which might be attributable to the low local effective concentration of DSMS, not enough to reach the critical concentration for ring-opening disulfide-exchange polymerization. On the other hand, the structural rigidity of the DNA tetrahedron is much higher than that of single-stranded DNA, making it hard to bring DSMS into proximity. Therefore, DNA tetrahedron might serve as a framework surrounded by several DSMS. To test the structural stability, the DSMS-DNA tetrahedron complexes were incubated in 10% fetal bovine serum (FBS) for 0–24 h. Then native PAGE analysis was performed to analyze digestion products ( Supporting Information Figure S6). The DSMS-DNA tetrahedron band and DNA tetrahedron remained almost unchanged within 8-h incubation, indicating high structural stability. The biocompatibility of the DSMS-DNA tetrahedron complex was investigated in HeLa cells before their intracellular applications, which showed negligible cytotoxicity on HeLa cells up to 2 µM, suggesting good biocompatibility ( Supporting Information Figure S7). Next, we investigated cellular uptake and intracellular structural integrity of the DSMS-DNA tetrahedron complex. First, HeLa cells were treated with Cy5-labeled DSMS-DNA tetrahedron complex of varying concentrations at a fixed incubation time or a fixed DSMS-DNA tetrahedron complex concentration, but at varying incubation times. Upon increasing the incubation times and concentrations, the cytosolic red fluorescence intensity increased with either increased DSMS-DNA tetrahedron complex concentration or increased incubation time, indicating that DSMS-DNA was taken up in a dose- and time-dependent manner ( Supporting Information Figures S8 and S9). Furthermore, the structural integrity of DSMS-DNA tetrahedron was tested by FRET experiments in living cells ( Supporting Information Figure S10). Hela cells were incubated with the dual fluorescence dyes (Cy3 and Cy5)-labeled DSMS-DNA tetrahedron for 2 h and analyzed by confocal laser scanning microscopy (CLSM). The CLSM images showed that the two dyes were highly colocalized and gave a FRET signal inside the living cells. To exclude the FRET signal generated by the fluorescence of the DNA degradation product,36 we further assessed the integrity of DSMS-DNA tetrahedron in cell lysate for 0–4 h at 37 °C. The almost unchanged bands showed that DSMS-DNA tetrahedron remained intact within 4 h incubation ( Supporting Information Figure S11). Collectedly, DSMS-DNA tetrahedron remained intact in the cytosol. We investigated whether the DSMS could influence the subcellular localization of DNA tetrahedron by tracking their subcellular distribution using CLSM. The lysosomes and nucleus were stained with Lysotracker red (LysoTracker; Invitrogen, Eugene, OR, USA)) and Hoechst33342 as references. Most of the red fluorescence from the DNA tetrahedron overlapped with the green fluorescence of the lysosomes, implying the high occurrence of lysosomal trapping (Figure 2a, line i). This result was in good agreement with previous reports.37 In contrast, the DSMS-DNA tetrahedron complex was mostly separated from the lysosomes and localized in the cytoplasm (Figure 2a, line ii). We quantitatively analyzed the colocalization values between DSMS-DNA tetrahedron complex and lysosomes via Pearson's correlation coefficients (PCCs; Figure 2b). The value of the complex was 0.37 ± 0.03, which was below the threshold of >0.5 required for correlation and significantly different from DNA tetrahedron (PCCs = 0.61 ± 0.03). Collectively, after attaching with DSMS, DNA tetrahedron nanostructures could be directly delivered into the cytosol through endocytosis-independent pathways. Figure 2 | (a) Confocal images of HeLa cells incubated with Cy5-labeled DNA tetrahedron or DSMS-DNA tetrahedron complex (red), Lysotracker Red (green), and Hoechst33342 (blue). Scale bars: 20 μm. Enlarge images are magnified boxed regions from merge images. Scale bars: 5 μm. (b) PCCs of DNA tetrahedron and DSMS-DNA tetrahedron complex (***p < 0.001). (c) CLSM relative fluorescence of DNA tetrahedron and DSMS-DNA tetrahedron assembly complex in HeLa cells with treatment of different inhibitors, including methyl-β-cyclodextrin (M-β-CD; 50 μM), wortmannin (50 nM), CPZ (10 μg/mL), and DTNB (4.8 mM), were normalized to no inhibitor (n = 3). (d) Flow cytometry analysis of HeLa cells treated with DNA tetrahedron or DSMS-DNA tetrahedron assembly complexes in the presence of different inhibitors. CLSM, confocal laser scanning microscopy; PCCs, Pearson's correlation coefficients; CPZ, chlorpromazine; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid. Download figure Download PowerPoint To demonstrate whether the DSMS-DNA tetrahedron complex underwent a thiol-mediated uptake, HeLa cells were pretreated with different inhibitors (methyl-β-cyclodextrin, wortmannin, and chlorpromazine [CPZ]). As expected, the endocytosis inhibitors had a significant influence on the cellular uptake efficiency of the DNA tetrahedron alone, while only slight influence was observed with the DSMS-DNA tetrahedron complex (Figure 2c and Supporting Information Figure S12). However, the internalization of the complex was obviously suppressed by treatment with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), exofacial thiols blocking reagent (Figure 2c and Supporting Information Figure S12). The results of CLSM imaging were consistent with those of flow cytometry (Figure 2d). In addition, intracellular uptake efficiency of both DNA tetrahedron and DSMS-DNA tetrahedron complex significantly decreased with pretreatment at 4 °C, which matched with endocytosis and the thiol-mediated process is a temperature-dependent process (Figures 2c and 2d and Supporting Information Figure S9).38,39 Collectively, these results suggested strongly that thiol-mediated uptake was the dominant mechanism for DSMS-DNA tetrahedron complex and endocytosis was that for DNA tetrahedron. Having demonstrated the direct intracellular delivery of DNA nanoassemblies with small size, we next evaluated the potential of DSMS for that with large sizes, such as DNA origami. We used DNA origami nanorod as a second model. The DNA nanorod was designed and constructed from M13 bacteriophage genome DNA (p7560; Supporting Information Table S2) according to the reported protocol.40 The DNA nanorod was 127 nm in length, with a cross section of 8 nm by 8 nm. The successful preparation of the designed nanorod was examined by native agarose gel electrophoresis ( Supporting Information Figure S13). AFM images showed that both DNA nanorod and DSMS-DNA nanorod (N/P = 1/10) were solid rod shape. The thickness of the DSMS-DNA nanorod was higher than that of DNA nanorod alone (Figures 3a and 3b). Importantly, the zeta potential of DSMS-DNA nanorod (about −4.09 mV) was more positive than that of DNA nanorod alone (about −12.4 mV), suggesting that the DSMS was attached on the DNA nanorod ( Supporting Information Figure S14). Next, we investigated the cellular uptake and subcellular localization of DSMS-DNA nanorod by using CLSM imaging. As expected, most of DSMS-DNA nanorods were distributed throughout the cytosol with high cellular internalization efficiencies (Figure 3c). The PCCs were calculated to analyze the association between DSMS-DNA nanorod and lysosomes. The value of DNA nanorod was 0.69 ± 0.05, while that of DSMS-DNA nanorod was 0.43 ± 0.03, confirming that DSMS-DNA nanorod underwent intracellular uptake through an endocytosis-independent pathway (Figure 3d). Figure 3 | AFM images of (a) DNA nanorod and (b) DSMS-DNA nanorod. Scale bars: 200 nm. (c) Confocal images of HeLa cells incubated with Cy5-labeled DNA nanorod or DSMS-DNA nanorod (red), Lysotracker Red (green), and Hoechst33342 (blue). Scale bars: 20 μm. Enlarge images are magnified boxed regions from merge images. Scale bars: 5 μm. (d) PCCs of DNA nanorod and DSMS-DNA nanorod (***p < 0.001). AFM, atomic force microscopy; PCCs, Pearson's correlation coefficients. Download figure Download PowerPoint Apart from DNA nanoassemblies that were generated by pure DNA molecules as building blocks, we next investigated whether DSMS could assist DNA functionalized nanoparticles via bypassing endocytosis. Herein, we used the DNA tetrahedron-upconversion nanoparticles (TH-UCNPs) as the other model. UCNPs with the size of ∼25 nm were synthesized, as previously described,41 and TH-UCNPs were prepared according to our previously published protocol.42 After assembled with DSMS, a lager hydrodynamic size and more positive zeta potential of DSMS-TH-UCNPs were observed ( Supporting Information Figure S15). Besides, there was negligible change in morphology and optical features (Figures 4a–4c and Supporting Information Figure S16). Conventionally, the subcellular localization of TH-UCNPs and DSMS-TH-UCNPs was analyzed by CLSM imaging. As expected, DSMS-TH-UCNPs showed minimal lysosomal trapping (Figure 4d). The PCCs were calculated to analyze the association between DSMS-TH-UCNPs and lysosomes. The values of the PCCs were 0.62 ± 0.01 and 0.33 ± 0.03 for TH-UCNPs and DSMS-TH-UCNPs, respectively (Figure 4e). These results revealed that the DSMS-TH-UCNPs could be delivered directly into the cytosol through endocytosis-independent pathways. Overall, DSMS could serve as a general molecular sticker to assist not only pure DNA nanoassemblies but also DNA functionalized nanoparticles for bypassing endocytosis. Figure 4 | TEM images of (a) oleic acid-UCNPs, (b) TH-UCNPs, and (c) DSMS-TH-UCNPs. Scale bars: 50 nm. (d) Confocal images of HeLa cells incubated with Cy5-labeled TH-UCNPs or DSMS-TH-UCNPs (red), Lysotracker Red (green), and Hoechst33342 (blue). Scale bars: 20 μm. Enlarge images are magnified boxed regions from merge images. Scale bars: 5 μm. (e) PCCs of TH-UCNPs and DSMS-TH-UCNPs (***p < 0.001). TEM, transmission electron microscopy. Download figure Download PowerPoint Conclusion We proposed the DSMS as a general molecular sticker that assists DNA nanoassemblies in bypassing endocytosis. After conjugating DNA with DSMS, both pure DNA nanoassemblies (DNA tetrahedron and DNA nanorod, in this case) and DNA modified nanoparticles were able to direct the delivery of the DNA nanomaterials into the cytosol through thiol-mediated uptake. This proposed approach presents several advantageous features, including facile assembly, retainment of the DNA 3D nanostructures, high biocompatibility, minimal lysosomal trapping, and efficient cell uptake. Benefiting from these excellent advantages, DSMS provides a potential tool for enhancing and broadening DNA nanoassemblies in intracellular biosensing, drug delivery, and gene regulation therapy in vivo. Supporting Information Supporting Information is available. Conflicts of Interest The authors declare no conflict of interest. Acknowledgments This study was supported by the Natural Science Foundation of China (grant nos. 91959102, 21635002, and 61805041), the Natural Science Foundation of Fujian Province of China (grant no. 2017J06004), the Shanghai Rising-Star Program (grant no. 19QA1405400), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT15R11). References 1. Seeman N. C.; Sleiman H. F.DNA Nanotechnology.Nat. Rev. Mater.2017, 3, 1–23. Google Scholar 2. Chen Y. J.; Groves B.; Muscat R. A.; Seelig G.DNA Nanotechnology from the Test Tube to the Cell.Nat. Nanotech.2015, 10, 748–760. Google Scholar 3. Jiang Q.; Liu S.; Liu J.; Wang Z. G.; Ding B.Rationally Designed DNA-Origami Nanomaterials for Drug Delivery In Vivo.Adv. Mater.2019, 31, e18047851. Google Scholar 4. Li J.; Zheng C.; Cansiz S.; Wu C.; Xu J.; Cui C.; Liu Y.; Hou W.; Wang Y.; Zhang L.; Teng I. T.; Yang H. 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Google Scholar Previous articleNext article FiguresReferencesRelatedDetails Issue AssignmentVolume 3Issue 3Page: 1178-1186Supporting Information Copyright & Permissions© 2020 Chinese Chemical SocietyKeywordsendocytosis-independent pathwayself-assemblylysosomal trappingDNA nanoassembliesthiol-mediated uptakeAcknowledgmentsThis study was supported by the Natural Science Foundation of China (grant nos. 91959102, 21635002, and 61805041), the Natural Science Foundation of Fujian Province of China (grant no. 2017J06004), the Shanghai Rising-Star Program (grant no. 19QA1405400), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT15R11). Downloaded 1,351 times Loading ...