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Cite this: Chem. Commun., 2015, 51, 6544 Received 20th January 2015, Accepted 2nd March 2015 DOI: 10.1039/c5cc00557d
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DNA-templated in situ growth of silver nanoparticles on mesoporous silica nanospheres for smart intracellular GSH-controlled release† Changhui Liu,ab Zhihe Qing,c Jing Zheng,a Li Deng,a Cheng Ma,a Jishan Li,a Yinhui Li,a Sheng Yang,ac Jinfeng Yang,d Jing Wang,d Weihong Tanae and Ronghua Yang*ac
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In this work, we propose a method for constructing silver nanoparticles (AgNPs) on mesoporous silica nanospheres (MSNs) by a DNA-templated process. By in situ formation, the gatekeeper can be easily modulated to meet different degrees of glutathione (GSH) stimuli for location-specific drug release.
In the past decade, MSNs have emerged as efficient nanovectors for drug delivery and biosensing because of their remarkable biocompatibility and high enveloping efficiency.1 For facilitating drug uptake and controlled release, various kinds of gatekeepers have been fabricated, allowing the release of loaded drug molecules into a specific environment in response to external or internal stimuli, such as pH, redox reaction, temperature, and enzymatic activity.2 Among these developed gatekeepers, metal nanomaterials have intensely attracted researchers’ interest, and have been widely used as gatekeepers for MSNs.3 To the best of our knowledge, the preparation of metal gatekeepers and their modification on MSNs are usually carried out seperately, resulting in time-consumption, use of intensive technology, and/or biological instability.4 Therefore, it is necessary to develop a simple method to construct metal gatekeepers for a stable MSN-based drug delivery system.
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, 410082, P. R. China. E-mail: [email protected]; Fax: +86-731-88822523; Tel: +86-731-88822523 b Department of Chemistry and Environmental Engineering, Hunan City University, Yiyang, 413000, P. R. China c School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, P. R. China d The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410011, P. R. China e Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, 32611-7200, USA † Electronic supplementary information (ESI) available: Chemicals and materials, experimental conditions, and supporting figures, and additional figures as noted in the text. See DOI: 10.1039/c5cc00557d
6544 | Chem. Commun., 2015, 51, 6544--6547
Recently, DNA has been widely employed as a scaffold for metal ions, which can be reduced to form metal nanoparticles that spontaneously follow the contour of DNA templates.5 Typically, cytosine-rich DNA is used for preparing AgNPs via a DNA-templated process.6 In addition, the interaction between template-synthesized AgNPs and DNA, which turns on sizedependent binding affinity, is also much weaker than that of thiol groups and silver.7 GSH, as the most predominant endogenous thiol species, has important physiological functions in fetal metabolism.8 Especially, the intracellular GSH concentration is much higher than extracellular levels,9 providing a unique opportunity for preferential intracellular release. Inspired by these findings, we propose here a previously undescribed route for the simultaneous synthesis of the gatekeeper and their modification on MSNs, making use of DNA-guided in situ formation of AgNPs on MSNs, and subsequently GSH-induced dismantle of AgNPs. Importantly, by in situ formation, the size and amount of AgNPs capped on MSNs are easily modulated so that the gated systems are tunable to meet different degrees of external stimuli, which is a practical demand in clinical applications for defined-location drug release. As proof-of-concept, the design scheme of our proposed approach is shown in Scheme 1. We demonstrate that the DNA strands conjugated on the MSN surface act as ‘‘universal molecular glues’’ for the formation of AgNPs to close the pores, leading to a little drug leakage. Additionally, the GSH-triggered drug release mechanism, which is different from existing methods that rely on chemical cleavage of a S–S linkage connecting the cap and MSNs,3a,c,10 is governed by GSH-mediated dismantle of AgNPs through the ligand exchange process, that is, a special Ag–S interaction.5b,7 The direct interaction of the trigger with the gatekeeper allows rapid drug release without the formation of cellular toxic –SH components.11 Therefore, this delivery system would provide multiple advantages, including cost-effective fabrication and facile modulation of the gatekeepers, and selective intracellular release. MSNs were synthesized using a base-catalyzed sol–gel process.12 Scanning electron microscopy (SEM) showed that the particles
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Scheme 1 MSN gatekeepers designed for drug delivery, showing in situ growth and capping of AgNPs on the MSN surface via reduction of Ag+ with a DNA template and removal of the capped AgNPs by intracellular GSH to release the loaded drug.
Fig. 1 SEM images (a), TEM images (b) and small-angle XRD patterns (c) of MSNs. The concentration ratio of Ag+ to P1 = 40 : 1.
were spherical in shape with a diameter of about 90 nm (Fig. 1a), while a hexagonally ordered porous array of uniform channels was shown by transmission electron microscopy (TEM) (Fig. 1b), which was further confirmed by the powder X-ray diffraction pattern (XRD) (Fig. 1c). To graft cytosine-rich DNA strands (denoted as P1), the MSNs were firstly functionalized with isocyanatopropyl groups (MSN-ICP), which was confirmed using Fourier transform infrared spectroscopy (FT-IR) (Fig. S1, ESI†). After conjugation with the DNA scaffolds, the strong and sharp absorption bands of the NQCQO moiety at 2275 cm1 disappeared,13 along with the decrease of zeta potential (Fig. S2, ESI†), indicating that P1 was covalently linked to the MSNs (MSN-P1). After the model drug, such as rhodamine 6G (Rh6G), was loaded into the nanopores of MSN-P1, AgNPs were formed by reducing the MSN-P1/Ag+ mixture (nAg+ : nP1 = 40 : 1) with HEPES buffer having a mild reducing ability, which is a popular pH buffer used extensively in chemical and biochemical experiments.14 Fig. 2b shows distinctive dark spots on the MSN surface, indicating that AgNPs were formed and capped on MSNs (Rh6G loaded AgNP-2@MSNs) in concomitant with a
Fig. 2 TEM images of AgNP@MSNs with concentration ratios of Ag+ to P1 of 20 : 1 (a), 40 : 1 (b), and 80 : 1 (c), respectively.
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remarkable increase of zeta potential (Fig. S2, ESI†), and a significant decrease of the surface area and pore size during the functionalized process (Fig. S3 and Table S1, ESI†). Additionally, EDX spectroscopy analysis showed the coexistence of Si, O, N, P and Ag elements of Rh6G loaded AgNP-2@MSNs (Fig. S4, ESI†). By contrast, the reduction products of (MSNs + Ag+) or (MSNs + P1 + Ag+) are far away from the MSN surface (Fig. S5, ESI†). Therefore, this DNA-directed simultaneous synthesis of AgNPs and capping of the MSN pores provide a facile and efficient route for the fabrication of MSN-based delivery systems. Since the AgNPs capped on MSNs are formed by in situ reduction of the bound Ag+ ions, the size and amount of AgNPs are easily modulated. To demonstrate this advantage, another two types of AgNP-capped MSNs with the concentration ratios of Ag+ to P1 of 20 : 1 (AgNP-1@MSNs) and 80 : 1 (AgNP-3@MSNs) were prepared, respectively. When the Ag+ to P1 ratio was 20 : 1, the dark spots present approximately 3.0 nm size and small amounts of distribution on the surface of MSNs, while the size and distribution are obviously increased with the concentration ratio of Ag+ to P1 from 20 : 1 to 80 : 1 (Fig. 2), demonstrating that the capped AgNPs could be adjusted by manipulating the concentration of Ag+ ions. To demonstrate GSH-triggered uncapping of AgNPs and the resultant cargo release in response to GSH at different concentrations in the biological environment, both the absorbance of AgNPs and the fluorescence of Rh6G for the three systems were examined. As shown in Fig. 3a, for AgNP-2@MSNs, in phosphate buffer saline solution (PBS, 10 mM, pH 7.4) containing 10% fetal bovine serum (FBS, v/v), the absorbance is scarcely influenced, indicating that Rh6G loaded AgNP-2@MSN is highly stable in biological systems. After addition of 5.0 mM GSH, however, a dramatic decrease of AgNPs’ absorbance was observed, together with the restoration of hexagonally packed nanopores of the MSNs (Fig. S6, ESI†), and the significant increase in fluorescence emission of Rh6G (Fig. S7, ESI†), suggesting that AgNPs were detached from the MSN surface. This utility is further supported by evaluating the effects of substrates containing different functional groups on the release of Rh6G. Fig. S8 (ESI†) shows that the sulfhydryl group is the key factor for the detachment of AgNPs from MSNs.
Fig. 3 Effects of the different concentration ratios of Ag+ to P1 on GSHtriggered uncapping of AgNPs from AgNP@MSNs. (a) Absorbance changes of Rh6G loaded three types of AgNP@MSNs. A0 and A denote the absorbance of AgNP@MSNs at 410 nm in PBS and in 10% FBS, respectively. (b) The corresponding cumulative Rh6G-release triggered by different concentrations of GSH for 10 h. Error bars indicate s.d. (n = 3).
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Furthermore, when the Ag+ to P1 ratio was 20 : 1, the absorbance was markedly decreased in 10% FBS, indicating an undesirable dismantle of the AgNPs in the biological environment. However, a further increase in the amount of Ag+ to 80 : 1 did not give better response to GSH than that of 40 : 1 under the same conditions (Fig. 3a). These results indicate that the gatekeeper AgNPs present good GSH-responsive capability, which can be easily modulated by adjusting the concentration of Ag+ ions. Due to the detachment of AgNPs from MSNs is integrated with the uncapping event, in vitro release of Rh6G with corresponding GSH concentrations was performed (Fig. 3b). For AgNP-1@MSNs, only at a blood plasma concentration of GSH of 1.0–10 mM,9 appreciable release (420%) of the entrapped Rh6G was observed within 5 h (Fig. S9a, ESI†), suggesting that the system presents free elution of the loaded cargo and cannot realize selective intracellular release. However, for AgNP-2@MSNs, upon addition of 10 mM GSH, physiologically corresponding to maximal extracellular GSH concentration,9,15 less than 5% of Rh6G is released over a period of 10 h, signifying efficient confinement of the cargo in the pores of MSNs by virtue of capping with AgNPs; while for 5.0 mM of GSH, the intracellular GSH concentration level,15 leads to a faster release of Rh6G, reaching a maximal cumulative release of B90% within 5 h (Fig. S9b, ESI†). And for AgNP-3@MSNs, much higher concentrations of GSH would be needed to remove the capped AgNPs from the MSN surface with the cost of reducing release efficiency (Fig. S9c, ESI†). These results suggest that the difference in accumulative release and release rate is attributed to the size of the AgNPs, which provides a tunable gatekeeper for stimuli-based cargo release and displays the potential for selective release at a defined time and location. Furthermore, we proceeded to evaluate the ability of our system with respect to cellular uptake and intracellular release. Firstly, the in vitro cytotoxicity of the nanoparticles against cancer cells was evaluated by MTT assay. As shown in Fig. S10 (ESI†), neither the blank nanoparticles (AgNP-2@MSNs) nor their released products (i.e., AgNPs) did show an inhibitory effect toward HeLa cells, respectively, suggesting that the system had good biocompatiblity and could serve as a promising nanovector for intracellular release. Then, confocal laser scanning microscopy (CLSM) was performed by determining the fluorescence of the released Rh6G. Because of self-quenching and/or quenching by AgNPs, faint fluorescence is observed in the capped and loaded particles, while the fluorescence signal is further enhanced as the incubation time increased (Fig. S11, ESI†), indicating cell uptake of the nanocarriers and cargo release from the nanopores. Additionally, to further test whether intracellular GSH-triggered Rh6G release, N-methylmaleimide (NMM, a GSH scavenger)16 and GSH-reduced ethyl ester (GSH-OEt, a GSH synthesis enhancer)17 were used to regulate the intracellular GSH levels. Compared to the control cells without additional treatment, obviously decreased fluorescence is observed by treating with NMM, but a significantly enhanced signal by treating with GSH-OEt (Fig. S12, ESI†). These results demonstrate that the cargo release depends on intracellular GSH concentration.
6546 | Chem. Commun., 2015, 51, 6544--6547
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Fig. 4 CLSM of the three Rh6G loaded AgNP@MSNs in HeLa (top) and HepG2 (bottom) cells: (a and a 0 ) AgNP-1@MSNs, (b and b 0 ) AgNP-2@MSNs, and (c and c 0 ) AgNP-3@MSNs. Scale bars: 40 mm.
Finally, to further assess the facile modulation of cellselective release, fluorescence imaging experiments were carried out in other biological models. Due to much higher concentration of GSH in HepG2 cells than that in HeLa cells,17 GSH-induced fluorescence enhancement within the two cells was investigated. Both Rh6G loaded AgNP-1@MSNs and AgNP-2@MSNs caused high fluorescence signals in either HeLa or HepG2 cells (Fig. 4 and Fig. S13, ESI†), yet a distinct increase of fluorescence in HepG2 cells is observed compared to HeLa cells. Especially, upon incubation with Rh6G loaded AgNP-3@MSNs, HeLa cells show the lowest fluorescence intensity among other groups, while HepG2 cells present a relatively strong signal, which is well matched with the results described in Fig. 3b. These results indicate that such flexibility allows us to manipulate the cargo release in a controlled and cell-selective manner by tuning the gatekeeper AgNPs. In summary, we have successfully fabricated MSN-based systems with tunable gatekeepers, which offer capabilities unavailable in conventional systems. Firstly, the decoration of MSNs with AgNPs via a DNA-templated process is different from conventional covalent or noncovalent strategies, providing a less laborious but more cost-effective and robust approach to construct nanocarriers. Secondly, by in situ formation, the size and amount of the gatekeeper AgNPs are easily modulated by manipulating the amounts of Ag+ ions to meet different degrees of GSH stimuli. Also, the simultaneous synthesis of gatekeeper AgNPs and their capping on MSNs achieved by our approach can be easily applied to produce different types of gatekeepers, including Au-, Cu-, Pt- and Au/ Cu/Pt-coated MSNs.18 Finally, loaded cargo release is determined by specific Ag–S interaction, which may reduce the toxicity without the formation of toxic –SH components. We thus envisioned that the approach would provide a unique methodology for diverse applications in diagnostics, imaging, and drug delivery. The work was supported by National Natural Science Foundation of China (21135001, 21305036, 21405038 and J1103312) and the ‘‘973’’ National Key Basic Research Program (2011CB91100-0).
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Notes and references 1 (a) D. Tran, C. E. Ashley, M. Xue, E. C. Carnes, J. I. Zink and C. J. Brinker, Acc. Chem. Res., 2013, 46, 792–801; (b) I. I. Slowing, B. G. Trewyn, S. Giri and V. S.-Y. Lin, Adv. Funct. Mater., 2007, 17, 1225–1236. 2 (a) Z. X. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink, Chem. Soc. Rev., 2012, 41, 2590–2605; (b) M. W. Ambrogio, C. R. Thomas, Y.-L. Zhao, J. I. Zink and J. F. Stoddart, Acc. Chem. Res., 2011, 44, 903–913. 3 (a) C. Y. Lai, B. G. Trewyn, D. M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija and V. S.-Y. Lin, J. Am. Chem. Soc., 2003, 125, 4451–4459; (b) C.-L. Zhu, C.-H. Lu, X.-Y. Song, H.-H. Yang and X.-R. Wang, J. Am. Chem. Soc., 2011, 133, 1278–1281; (c) S. Giri, B. G. Trewyn, M. P. Stellmaker and V. S.-Y. Lin, Angew. Chem., Int. Ed., 2005, 44, 5038–5044; (d) Y. Wang, S. Song, J. Liu, D. Liu and H. Zhang, Angew. Chem., Int. Ed., 2015, 54, 536–540; (e) M. Li, P. Shi, C. Xu, J. Ren and X. Qu, Chem. Sci., 2013, 4, 2536–2542. 4 N. Singh, A. Karambelkar, L. Gu, K. Lin, J. S. Miller, C. S. Chen, M. J. Sailor and S. N. Bhatia, J. Am. Chem. Soc., 2011, 133, 19582–19585. 5 (a) H. A. Becerril and A. T. Woolley, Chem. Soc. Rev., 2009, 38, 329–337; (b) Y. Tao, E. Ju, J. Ren and X. Qu, Chem. Commun., 2013, 49, 9791–9793. 6 (a) J. T. Petty, J. Zheng, N. V. Hud and R. M. Dickson, J. Am. Chem. Soc., 2004, 126, 5207–5212; (b) A. A. Zinchenko, K. Yoshikawa and D. Baigl, Adv. Mater., 2005, 17, 2820–2823. 7 (a) J. Jin, X. Ouyang, J. Li, J. Jiang, H. Wang, Y. Wang and R. Yang, Sci. China: Chem., 2011, 54, 1266–1272; (b) L. Gao and N. Ma, ACS Nano, 2012, 6, 689–695; (c) X. Ran, H. Sun, F. Pu, J. g. Ren and X. Qu, Chem. Commun., 2013, 49, 1079–1081.
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ChemComm 8 B. K. McMahon and T. Gunnlaugsson, J. Am. Chem. Soc., 2012, 134, 10725–10728. 9 A. Verma, J. M. Simard, J. W. E. Worrall and V. M. Rotello, J. Am. Chem. Soc., 2004, 126, 13987–13991. 10 (a) Z. Luo, K. Cai, Y. Hu, L. Zhao, P. Liu, L. Duan and W. Yang, Angew. Chem., Int. Ed., 2011, 50, 640–643; (b) R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen and Z. Zhong, J. Controlled Release, 2011, 152, 2–12. 11 L. Poon, W. Zandberg, D. Hsiao, Z. Erno, D. Sen, B. D. Gates and N. R. Branda, ACS Nano, 2010, 4, 6395–6403. 12 D. R. Radu, C.-Y. Lai, K. Jeftinija, E. W. Rowe, S. Jeftinija and V. S. Y. Lin, J. Am. Chem. Soc., 2004, 126, 13216–13217. 13 A. R. McDonald, H. P. Dijkstra, B. M. J. M. Suijkerbuijk, G. P. M. V. Klink and G. V. Koten, Organometallics, 2009, 28, 4689–4699. 14 (a) R. W. Y. Sun, R. Chen, N. P. Y. Chung, C. M. Ho, C. L. S. Lin and C. M. Che, Chem. Commun., 2005, 5059–5061; (b) J. Xie, J. Y. Lee and D. I. C. Wang, Chem. Mater., 2007, 19, 2823–2830. 15 R. Deng, X. Xie, M. Vendrell, Y. T. Chang and X. Liu, J. Am. Chem. Soc., 2011, 133, 20168–20171. 16 Y.-H. Ahn, J.-S. Lee and Y.-T. Chang, J. Am. Chem. Soc., 2007, 129, 4510–4511. ´ndez, B. J. Kim, N. S. Forbes and 17 R. Hong, G. Han, J. M. Ferna V. M. Rotello, J. Am. Chem. Soc., 2006, 128, 1078–1079. 18 (a) Z. Qing, X. He, D. He, K. Wang, F. Xu, T. Qing and X. Yang, Angew. Chem., Int. Ed., 2013, 52, 9719–9722; (b) I. Ocsoy, B. Gulbakan, T. Chen, G. Zhu, Z. Chen, M. M. Sari, L. Peng, X. Xiong, X. Fang and W. Tan, Adv. Mater., 2013, 25, 2319–2325.
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Cite this: Chem. Commun., 2015, 51, 6544 Received 20th January 2015, Accepted 2nd March 2015 DOI: 10.1039/c5cc00557d
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DNA-templated in situ growth of silver nanoparticles on mesoporous silica nanospheres for smart intracellular GSH-controlled release† Changhui Liu,ab Zhihe Qing,c Jing Zheng,a Li Deng,a Cheng Ma,a Jishan Li,a Yinhui Li,a Sheng Yang,ac Jinfeng Yang,d Jing Wang,d Weihong Tanae and Ronghua Yang*ac
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In this work, we propose a method for constructing silver nanoparticles (AgNPs) on mesoporous silica nanospheres (MSNs) by a DNA-templated process. By in situ formation, the gatekeeper can be easily modulated to meet different degrees of glutathione (GSH) stimuli for location-specific drug release.
In the past decade, MSNs have emerged as efficient nanovectors for drug delivery and biosensing because of their remarkable biocompatibility and high enveloping efficiency.1 For facilitating drug uptake and controlled release, various kinds of gatekeepers have been fabricated, allowing the release of loaded drug molecules into a specific environment in response to external or internal stimuli, such as pH, redox reaction, temperature, and enzymatic activity.2 Among these developed gatekeepers, metal nanomaterials have intensely attracted researchers’ interest, and have been widely used as gatekeepers for MSNs.3 To the best of our knowledge, the preparation of metal gatekeepers and their modification on MSNs are usually carried out seperately, resulting in time-consumption, use of intensive technology, and/or biological instability.4 Therefore, it is necessary to develop a simple method to construct metal gatekeepers for a stable MSN-based drug delivery system.
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, 410082, P. R. China. E-mail: [email protected]; Fax: +86-731-88822523; Tel: +86-731-88822523 b Department of Chemistry and Environmental Engineering, Hunan City University, Yiyang, 413000, P. R. China c School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, P. R. China d The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410011, P. R. China e Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, 32611-7200, USA † Electronic supplementary information (ESI) available: Chemicals and materials, experimental conditions, and supporting figures, and additional figures as noted in the text. See DOI: 10.1039/c5cc00557d
6544 | Chem. Commun., 2015, 51, 6544--6547
Recently, DNA has been widely employed as a scaffold for metal ions, which can be reduced to form metal nanoparticles that spontaneously follow the contour of DNA templates.5 Typically, cytosine-rich DNA is used for preparing AgNPs via a DNA-templated process.6 In addition, the interaction between template-synthesized AgNPs and DNA, which turns on sizedependent binding affinity, is also much weaker than that of thiol groups and silver.7 GSH, as the most predominant endogenous thiol species, has important physiological functions in fetal metabolism.8 Especially, the intracellular GSH concentration is much higher than extracellular levels,9 providing a unique opportunity for preferential intracellular release. Inspired by these findings, we propose here a previously undescribed route for the simultaneous synthesis of the gatekeeper and their modification on MSNs, making use of DNA-guided in situ formation of AgNPs on MSNs, and subsequently GSH-induced dismantle of AgNPs. Importantly, by in situ formation, the size and amount of AgNPs capped on MSNs are easily modulated so that the gated systems are tunable to meet different degrees of external stimuli, which is a practical demand in clinical applications for defined-location drug release. As proof-of-concept, the design scheme of our proposed approach is shown in Scheme 1. We demonstrate that the DNA strands conjugated on the MSN surface act as ‘‘universal molecular glues’’ for the formation of AgNPs to close the pores, leading to a little drug leakage. Additionally, the GSH-triggered drug release mechanism, which is different from existing methods that rely on chemical cleavage of a S–S linkage connecting the cap and MSNs,3a,c,10 is governed by GSH-mediated dismantle of AgNPs through the ligand exchange process, that is, a special Ag–S interaction.5b,7 The direct interaction of the trigger with the gatekeeper allows rapid drug release without the formation of cellular toxic –SH components.11 Therefore, this delivery system would provide multiple advantages, including cost-effective fabrication and facile modulation of the gatekeepers, and selective intracellular release. MSNs were synthesized using a base-catalyzed sol–gel process.12 Scanning electron microscopy (SEM) showed that the particles
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Scheme 1 MSN gatekeepers designed for drug delivery, showing in situ growth and capping of AgNPs on the MSN surface via reduction of Ag+ with a DNA template and removal of the capped AgNPs by intracellular GSH to release the loaded drug.
Fig. 1 SEM images (a), TEM images (b) and small-angle XRD patterns (c) of MSNs. The concentration ratio of Ag+ to P1 = 40 : 1.
were spherical in shape with a diameter of about 90 nm (Fig. 1a), while a hexagonally ordered porous array of uniform channels was shown by transmission electron microscopy (TEM) (Fig. 1b), which was further confirmed by the powder X-ray diffraction pattern (XRD) (Fig. 1c). To graft cytosine-rich DNA strands (denoted as P1), the MSNs were firstly functionalized with isocyanatopropyl groups (MSN-ICP), which was confirmed using Fourier transform infrared spectroscopy (FT-IR) (Fig. S1, ESI†). After conjugation with the DNA scaffolds, the strong and sharp absorption bands of the NQCQO moiety at 2275 cm1 disappeared,13 along with the decrease of zeta potential (Fig. S2, ESI†), indicating that P1 was covalently linked to the MSNs (MSN-P1). After the model drug, such as rhodamine 6G (Rh6G), was loaded into the nanopores of MSN-P1, AgNPs were formed by reducing the MSN-P1/Ag+ mixture (nAg+ : nP1 = 40 : 1) with HEPES buffer having a mild reducing ability, which is a popular pH buffer used extensively in chemical and biochemical experiments.14 Fig. 2b shows distinctive dark spots on the MSN surface, indicating that AgNPs were formed and capped on MSNs (Rh6G loaded AgNP-2@MSNs) in concomitant with a
Fig. 2 TEM images of AgNP@MSNs with concentration ratios of Ag+ to P1 of 20 : 1 (a), 40 : 1 (b), and 80 : 1 (c), respectively.
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remarkable increase of zeta potential (Fig. S2, ESI†), and a significant decrease of the surface area and pore size during the functionalized process (Fig. S3 and Table S1, ESI†). Additionally, EDX spectroscopy analysis showed the coexistence of Si, O, N, P and Ag elements of Rh6G loaded AgNP-2@MSNs (Fig. S4, ESI†). By contrast, the reduction products of (MSNs + Ag+) or (MSNs + P1 + Ag+) are far away from the MSN surface (Fig. S5, ESI†). Therefore, this DNA-directed simultaneous synthesis of AgNPs and capping of the MSN pores provide a facile and efficient route for the fabrication of MSN-based delivery systems. Since the AgNPs capped on MSNs are formed by in situ reduction of the bound Ag+ ions, the size and amount of AgNPs are easily modulated. To demonstrate this advantage, another two types of AgNP-capped MSNs with the concentration ratios of Ag+ to P1 of 20 : 1 (AgNP-1@MSNs) and 80 : 1 (AgNP-3@MSNs) were prepared, respectively. When the Ag+ to P1 ratio was 20 : 1, the dark spots present approximately 3.0 nm size and small amounts of distribution on the surface of MSNs, while the size and distribution are obviously increased with the concentration ratio of Ag+ to P1 from 20 : 1 to 80 : 1 (Fig. 2), demonstrating that the capped AgNPs could be adjusted by manipulating the concentration of Ag+ ions. To demonstrate GSH-triggered uncapping of AgNPs and the resultant cargo release in response to GSH at different concentrations in the biological environment, both the absorbance of AgNPs and the fluorescence of Rh6G for the three systems were examined. As shown in Fig. 3a, for AgNP-2@MSNs, in phosphate buffer saline solution (PBS, 10 mM, pH 7.4) containing 10% fetal bovine serum (FBS, v/v), the absorbance is scarcely influenced, indicating that Rh6G loaded AgNP-2@MSN is highly stable in biological systems. After addition of 5.0 mM GSH, however, a dramatic decrease of AgNPs’ absorbance was observed, together with the restoration of hexagonally packed nanopores of the MSNs (Fig. S6, ESI†), and the significant increase in fluorescence emission of Rh6G (Fig. S7, ESI†), suggesting that AgNPs were detached from the MSN surface. This utility is further supported by evaluating the effects of substrates containing different functional groups on the release of Rh6G. Fig. S8 (ESI†) shows that the sulfhydryl group is the key factor for the detachment of AgNPs from MSNs.
Fig. 3 Effects of the different concentration ratios of Ag+ to P1 on GSHtriggered uncapping of AgNPs from AgNP@MSNs. (a) Absorbance changes of Rh6G loaded three types of AgNP@MSNs. A0 and A denote the absorbance of AgNP@MSNs at 410 nm in PBS and in 10% FBS, respectively. (b) The corresponding cumulative Rh6G-release triggered by different concentrations of GSH for 10 h. Error bars indicate s.d. (n = 3).
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Furthermore, when the Ag+ to P1 ratio was 20 : 1, the absorbance was markedly decreased in 10% FBS, indicating an undesirable dismantle of the AgNPs in the biological environment. However, a further increase in the amount of Ag+ to 80 : 1 did not give better response to GSH than that of 40 : 1 under the same conditions (Fig. 3a). These results indicate that the gatekeeper AgNPs present good GSH-responsive capability, which can be easily modulated by adjusting the concentration of Ag+ ions. Due to the detachment of AgNPs from MSNs is integrated with the uncapping event, in vitro release of Rh6G with corresponding GSH concentrations was performed (Fig. 3b). For AgNP-1@MSNs, only at a blood plasma concentration of GSH of 1.0–10 mM,9 appreciable release (420%) of the entrapped Rh6G was observed within 5 h (Fig. S9a, ESI†), suggesting that the system presents free elution of the loaded cargo and cannot realize selective intracellular release. However, for AgNP-2@MSNs, upon addition of 10 mM GSH, physiologically corresponding to maximal extracellular GSH concentration,9,15 less than 5% of Rh6G is released over a period of 10 h, signifying efficient confinement of the cargo in the pores of MSNs by virtue of capping with AgNPs; while for 5.0 mM of GSH, the intracellular GSH concentration level,15 leads to a faster release of Rh6G, reaching a maximal cumulative release of B90% within 5 h (Fig. S9b, ESI†). And for AgNP-3@MSNs, much higher concentrations of GSH would be needed to remove the capped AgNPs from the MSN surface with the cost of reducing release efficiency (Fig. S9c, ESI†). These results suggest that the difference in accumulative release and release rate is attributed to the size of the AgNPs, which provides a tunable gatekeeper for stimuli-based cargo release and displays the potential for selective release at a defined time and location. Furthermore, we proceeded to evaluate the ability of our system with respect to cellular uptake and intracellular release. Firstly, the in vitro cytotoxicity of the nanoparticles against cancer cells was evaluated by MTT assay. As shown in Fig. S10 (ESI†), neither the blank nanoparticles (AgNP-2@MSNs) nor their released products (i.e., AgNPs) did show an inhibitory effect toward HeLa cells, respectively, suggesting that the system had good biocompatiblity and could serve as a promising nanovector for intracellular release. Then, confocal laser scanning microscopy (CLSM) was performed by determining the fluorescence of the released Rh6G. Because of self-quenching and/or quenching by AgNPs, faint fluorescence is observed in the capped and loaded particles, while the fluorescence signal is further enhanced as the incubation time increased (Fig. S11, ESI†), indicating cell uptake of the nanocarriers and cargo release from the nanopores. Additionally, to further test whether intracellular GSH-triggered Rh6G release, N-methylmaleimide (NMM, a GSH scavenger)16 and GSH-reduced ethyl ester (GSH-OEt, a GSH synthesis enhancer)17 were used to regulate the intracellular GSH levels. Compared to the control cells without additional treatment, obviously decreased fluorescence is observed by treating with NMM, but a significantly enhanced signal by treating with GSH-OEt (Fig. S12, ESI†). These results demonstrate that the cargo release depends on intracellular GSH concentration.
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Fig. 4 CLSM of the three Rh6G loaded AgNP@MSNs in HeLa (top) and HepG2 (bottom) cells: (a and a 0 ) AgNP-1@MSNs, (b and b 0 ) AgNP-2@MSNs, and (c and c 0 ) AgNP-3@MSNs. Scale bars: 40 mm.
Finally, to further assess the facile modulation of cellselective release, fluorescence imaging experiments were carried out in other biological models. Due to much higher concentration of GSH in HepG2 cells than that in HeLa cells,17 GSH-induced fluorescence enhancement within the two cells was investigated. Both Rh6G loaded AgNP-1@MSNs and AgNP-2@MSNs caused high fluorescence signals in either HeLa or HepG2 cells (Fig. 4 and Fig. S13, ESI†), yet a distinct increase of fluorescence in HepG2 cells is observed compared to HeLa cells. Especially, upon incubation with Rh6G loaded AgNP-3@MSNs, HeLa cells show the lowest fluorescence intensity among other groups, while HepG2 cells present a relatively strong signal, which is well matched with the results described in Fig. 3b. These results indicate that such flexibility allows us to manipulate the cargo release in a controlled and cell-selective manner by tuning the gatekeeper AgNPs. In summary, we have successfully fabricated MSN-based systems with tunable gatekeepers, which offer capabilities unavailable in conventional systems. Firstly, the decoration of MSNs with AgNPs via a DNA-templated process is different from conventional covalent or noncovalent strategies, providing a less laborious but more cost-effective and robust approach to construct nanocarriers. Secondly, by in situ formation, the size and amount of the gatekeeper AgNPs are easily modulated by manipulating the amounts of Ag+ ions to meet different degrees of GSH stimuli. Also, the simultaneous synthesis of gatekeeper AgNPs and their capping on MSNs achieved by our approach can be easily applied to produce different types of gatekeepers, including Au-, Cu-, Pt- and Au/ Cu/Pt-coated MSNs.18 Finally, loaded cargo release is determined by specific Ag–S interaction, which may reduce the toxicity without the formation of toxic –SH components. We thus envisioned that the approach would provide a unique methodology for diverse applications in diagnostics, imaging, and drug delivery. The work was supported by National Natural Science Foundation of China (21135001, 21305036, 21405038 and J1103312) and the ‘‘973’’ National Key Basic Research Program (2011CB91100-0).
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