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Cite this: Chem. Commun., 2015, 51, 7203 Received 21st January 2015, Accepted 19th March 2015
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Combination drug release of smart cyclodextrin-gated mesoporous silica nanovehicles† Shengwang Zhou,a Huizi Sha,b Xiaokang Ke,a Baorui Liu,b Xizhang Wanga and Xuezhong Du*a
DOI: 10.1039/c5cc00585j www.rsc.org/chemcomm
An integrated c-cyclodextrin-gated mesoporous silica delivery system via dual dynamic covalent bonds was constructed with dual drug loading for simultaneous and cascade release in targeted combination drug therapy.
The combination of multiple drugs with different molecular targets can maximize therapeutic efficacy and is more likely to overcome drug resistance. Combination drug therapy has been adopted in clinics as a primary regimen of cancer treatment.1 There has been an ever increasing interest in developing stimuliresponsive drug delivery systems to improve therapeutic efficacy and minimize adverse effects of drugs. Mesoporous silica nanoparticles (MSNs) have attracted considerable attention as ideal drug carriers owing to their tunable pore size, large pore volume, good biocompatibility, and easy functionalization.2 To date, inorganic nanoparticles,3 macrocycles,4–7 and biomolecules8,9 have been used as MSN gatekeepers to show well-controlled release performances. The controlled release can be regulated by physical stimuli such as thermal9a–c and light irradiation,4f chemical stimuli such as pH3,4a,c,d,6a–c,7b,8d and redox,1,8c,9b and biochemical stimuli such as enzymes.4b,e,6d,9a–c However, simultaneous and cascade controlled release of two or more drugs for combination therapy is still difficult to achieve.9b,c Cyclodextrins (CDs) are linked by a-1,4-glycosidic bonds into a macrocycle, with the secondary O2 and O3 hydroxyls on one rim and the primary O6 hydroxyls on the other rim, and have been used as gatekeepers, such as nanovalves4a–d and nanogates,4e,f in the MSN-based drug delivery systems. Note that the hydrophobic cavities of CDs themselves can also accommodate drugs. It is a smart strategy to take advantage of the specific a
Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. E-mail: [email protected] b The Comprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China † Electronic supplementary information (ESI) available: Synthesis details, experimental details, supporting figures and illustrations. See DOI: 10.1039/c5cc00585j
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structures and properties of CDs for use in the MSN-based drug delivery systems not only as gatekeepers but also as drug carriers. Phenylboronic acid (PBA) and its derivatives are a class of Lewis acids and form boronate esters with 1,2- or 1,3-diols, such as saccharides and others containing diols, and their binding affinity depends on individual pKa and solution pH.10 Two methods have been used to reduce the pKa of PBA, so that stable boronate bonds are formed at physiological pH.11,12 One method is the introduction of electron-withdrawing groups to PBA at a meso-position with respect to boronic acid, and the other is the introduction of amino groups to decrease the apparent pKa of PBA because of the possible N–B coordination interaction.12–14 Herein, we report the simultaneous and cascade controlled release of two drugs from g-CD-gated MSN vehicles via dual dynamic covalent bonds (Scheme 1). MSNs were co-modified with disulfide-linked carbamoylphenylboronic acid moieties and amines on the surfaces. After a kind of drug was entrapped in the MSN pores, the secondary hydroxyls of g-CD on the wide rim effectively bound to the PBA moieties assisted by neighboring amino groups at physiological pH via the boronate linkages to encapsulate the drugs within the MSN pores. Afterward, the other kinds of drugs were included into the g-CD cavities. In addition, the dense-capped g-CD on the exterior surfaces played a role in bio-mimicking the saccharide-rich cell surfaces to overcome serum-susceptible drawbacks.15 The smart g-CDgated MSN delivery systems based on dual dynamic covalent bonds could well solve out the problem about simultaneous and cascade release of two drugs to meet diverse requirements. At acidic pH, the boronate bonds were hydrolyzed, and the drugs included inside the g-CD cavities could be protonated with a reduced binding affinity, which gave rise to simultaneous release of two drugs from the MSN pores and g-CD cavities. In the presence of monosaccharides, the drugs entrapped in MSN pores could be only released by the competitive binding of the monosaccharides to the PBA moieties, and then the drugs accommodated inside the g-CD cavities could be sequentially released upon decrease of pH. Similarly, the drugs entrapped in the MSN pores could be first released by the
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Fig. 1 (A) Solid-state 13C NMR spectra of PBA-MSNs, PBA/NH2-MSNs, and g-CD-gated MSNs without drug loading. The resonances labelled with the symbols * and # resulted from 2,2-dimethyl-1,3-propanediol and uncondensed ethoxysilane, respectively. (B) Solid-state 11B NMR spectra of PBA-MSNs and PBA/NH2-MSNs.
Scheme 1 Illustration of construction of g-CD-gated MSN vehicles functionalized with disulfide-linked carbamoylphenylboronic acid moieties and amines and simultaneous and cascade release of two drugs entrapped in the MSN pores and included inside the g-CD cavities.
cleavage of the disulfide linkages with the reducing agents dithiothreitol (DTT) or glutathione (GSH), and the other drugs could be successively released from the g-CD cavities upon decrease of pH. The combination of simultaneous and cascade release of the two drugs could maximize the therapeutic efficacy to overcome drug resistance. MCM-41 nanoparticles were synthesized by the base-catalyzed sol–gel method followed by removal of surfactant templates. Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of MCM-41 showed that MSNs had the sizes of 100 12 nm in diameter and possessed perfect parallelly arranged hexagonal pores (Fig. S1 in the ESI†). Small-angle X-ray diffraction (XRD) patterns of MCM-41 displayed four reflection peaks (Fig. S2, ESI†), typical of a well-fined hexagonal mesoporous structure. Nitrogen adsorption–desorption isotherms of MCM-41 showed a typical curve of type IV with a surface area of 1133.5 m2 g 1 and a pore size of 2.2 nm (Fig. S3, ESI†). Solid-state 13C NMR spectroscopy was used to characterize the functionalization of the PBA/amino moieties and the binding of g-CD (Fig. 1A). The strong resonance at 160 ppm was assigned to the –NHCONH– linkages, and the weak resonance at 167 ppm to the carbamoyl groups conjugated to the PBA moieties. The resonances in the range of 128–137 ppm were due to the phenyl groups of the PBA moieties (Fig. S4, ESI†).16 These spectral features confirm that the disulfide-linked PBA moieties were covalently immobilized on the MSN surfaces. After subsequent modification of 1,8-octanediamine (ODA), two main peaks at 28 and 30 ppm were clearly observed due to the resonances of –(CH2)6– groups except for the Ca atoms. After capping of g-CD without drug loading, four groups of new peaks in the ranges of 101–103, 76–82, 71–73, and 60–63 ppm were clearly observed owing to the
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various 13C resonances of g-CD,17 which demonstrate that PBA/NH2MSNs were capped with g-CD (g-CD-gated MSNs). FTIR spectroscopy was also used to characterize the modification of the PBA moieties and ODA and the capping of g-CD (Fig. S5, ESI†). The surface densities of the PBA moieties and amino groups were determined to be 1.290 and 0.345 mmol g 1 MCM-41 by thermogravimetric analysis (Fig. S6, ESI†), and the amount of bound g-CD was determined to be 0.302 mmol g 1 MCM-41 (without drug loading). The chemical shift and lineshape of solid-state 11B NMR spectroscopy are directly correlated with the coordination environment of boron species. One peak at 15–16 ppm could be assigned to the 3-coordinated (trigonal) boron species, and the other at less than 0 ppm to the 4-coordinated (tetrahedral) boron species (Fig. 1B). The neighboring amino groups promoted the conversion of the trigonal boron species into the tetrahedral ones, related to the mole ratio of the amino groups to the PBA moieties. The tetrahedral boron species facilitated the formation of stable cyclic boronate esters between the PBA/amino moieties and secondary hydroxyl groups of g-CD. The introduction of the amino groups in the vicinity of the PBA moieties probably promoted the Lewis acid–base interactions between the PBA moieties and amines, which thus lowered the apparent pKa of PBA.11 The markedly lowered PBA acidity was due to the ion-pairing stabilization from the neighboring amino groups and the N–B coordination interactions in equilibrium.13b,14 The zeta potential of MCM-41 was 29.4 mV and decreased to 35.5 mV for PBA-MSNs, followed by an increase to 2.1 mV for PBA/NH2-MSNs (Fig. S7A, ESI†). The zeta potential was slightly decreased to 6.7 mV after g-CD binding (g-CD-gated MSNs) and then underwent a change for inclusion of the two kinds of anticancer drugs inside the g-CD cavities, 0.4 mV in the case of doxorubicin (DOX) and 14.6 mV in the case of camptothecin (CPT) (Fig. S7B, ESI†). The average hydrodynamic diameters of these nanoparticles ranged from 102 to 110 nm (Fig. S8, ESI†), which reflect that the these nanoparticles were well monodispersed in aqueous solutions. However, the TEM images of PBA/NH2-MSNs and the g-CD-gated MSNs without drug loading did not show a significant difference from that of MCM-41 (Fig. S9, ESI†). PBA/ NH2-MSNs nearly displayed the same XRD patterns as MCM-41 (Fig. S10, ESI†). The MSN surface area decreased from 1133.5 m2 g 1 before modification to 926.3 m2 g 1 after modification, but the average pore diameters (2.2 nm) remained almost unchanged
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(Fig. S11 and Table S1, ESI†). However, none of the four XRD reflection peaks could be detectable after successive rhodamine 6G (Rh6G) loading, g-CD capping, and CPT inclusion. Moreover the surface area was drastically reduced to 100.8 m2 g 1 because the MSN pores were filled and capped (Fig. S10 and S11 and Table S1, ESI†). These results indicate that the g-CD-gated MSN vehicles with dual drug loading were thus constructed. UV-vis spectroscopy was used to monitor simultaneous release of Rh6G from the MSN pores and CPT from the g-CD cavities (Fig. S12 and S13, ESI†). The maximum absorption bands of Rh6G and CPT were well resolved without interference for the monitoring of release of the two drugs. Fluorescence spectroscopy was used to monitor cascade release of calcein from the MSN pores and DOX from the g-CD cavities (Fig. S14 and S15, ESI†). It is worth noting that the fluorescence of calcein was almost quenched at acidic pH and the release of calcein had no influence on monitoring the subsequent release of DOX triggered by acidic pH. The loading capacities of Rh6G and CPT were determined to be 0.0947 and 0.0707 mmol g 1 PBA/NH2-MSN using UV-vis spectroscopy (Fig. S16, ESI†), respectively. The loading capacities of calcein and DOX were determined to be 0.0316 and 0.0367 mmol g 1 PBA/NH2-MSN using fluorescence spectroscopy (Fig. S17, ESI†), respectively. The g-CD-gated MSNs with dual drug loading showed good blocking performance before pH trigger (Fig. 2 and Fig. S18, ESI†). Upon decrease of pH, simultaneous release of Rh6G and CPT was observed. The boronate bonds formed between the PBA/amino moieties and the secondary hydroxyl groups of g-CD were hydrolyzed at the acidic pH, which resulted in the removal of g-CD from the MSN surface and the release of Rh6G from the MSN pores. Simultaneously, CPT inside the g-CD cavities was protonated at the acidic pH, which gave rise to the weakening of their binding affinity and the release of CPT from the g-CD cavities. In order to gain an insight into the effect of the modified amino groups on the PBA moieties for g-CD binding, PBA-MSNs were loaded with Rh6G followed by addition of g-CD for comparison. The counterparts showed no cargo release even at pH 3.0 (Fig. S19, ESI†), because PBA-MSNs could not be capped with g-CD in the absence of modified amino groups. The cargo was finally washed off even if loaded, so that no cargo could be released. This further demonstrates that the introduction of the neighboring amino groups to the PBA moieties facilitated the development of the intermolecular N–B coordination interactions for g-CD binding.
Fig. 2 pH-responsive simultaneous release profiles from g-CD-gated MSNs with dual drug loading: (A) Rh6G (Amax = 527 nm); (B) CPT (Amax = 355 nm).
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Fig. 3 Cascade release profiles from g-CD-gated MSNs with dual loading of Rh6G and CPT upon first trigger of fructose (10 mM) at pH 7.4 and subsequent trigger of pH 5.0: Rh6G (Amax = 527 nm); CPT (Amax = 355 nm).
Saccharides are able to bind PBA to form cyclic boronate esters. In the presence of fructose or galactose (10 mM), significant release of Rh6G was observed from the g-CD-gated MSNs with dual drug loading at pH 7.4 but no CPT release; however, in the presence of glucose (10 mM) neither Rh6G nor CPT could be released (Fig. S20, ESI†). D-Fructose and D-galactose generally show stronger binding affinity to all aryl monoboronic acids in comparison to D-glucose.18 The competitive binding of fructose or galactose to the PBA moieties of the g-CD-gated MSNs drove the removal of g-CD and the release of Rh6G, while CPT inside the g-CD cavities remained unchanged. Importantly, glucose at the normal blood glucose levels could not cause premature release of the g-CD-gated MSNs with dual drug loading under physiological conditions. After the trigger of fructose, Rh6G was only released from the MSN pores. Upon further decrease of pH, subsequent release of CPT from the g-CD cavities was obviously observed (Fig. 3 and Fig. S21, ESI†). It is clear that the cascade release of Rh6G and CPT from the g-CD-gated MSNs could be realized upon first trigger of fructose (or galatose) and subsequent trigger of acidic pH. DTT could trigger release of calcein from the g-CD-gated MSNs with dual loading of calcein and DOX (Fig. S22 and S23, ESI†). It is worth noting that DTT contains diols as well as two mercapto groups. Further control experiments demonstrate that the DTT-responsive controlled release of calcein resulted from the cleavage of the disulfide bonds (Fig. 4A and Fig. S24, ESI†). GSH also caused the release of calcein but with about 50% release efficiency compared to DTT.
Fig. 4 (A) Stimuli-responsive release profiles from g-CD-gated MSNs only with the loading of calcein in the MSN pores at pH 7.4. (B) Cascade release profiles from g-CD-gated MSNs with dual loading of calcein and DOX upon first trigger of DTT (5 mM) at pH 7.4 and subsequent trigger of pH 3.0: calcein (lex = 458 nm, lem = 510 nm); DOX (lex = 480 nm, lem = 555 nm).
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This work was supported by National Natural Science Foundation of China (21273112) and Natural Science Foundation of Jiangsu Province (BK2012719). S. Zhou was supported by the program B for outstanding PhD candidate of Nanjing University.
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Notes and references
Fig. 5 CLSM images of intracellular uptake and drug release behaviors of the g-CD-gated MSNs with dual loading of CPT and DOX (100 mg mL 1) after incubation with (A) HeLa cells and (B) A549 cells for different times: CPT (lex = 365 nm); DOX (lex = 488 nm).
Similarly, the cascade release of calcein and DOX from the g-CD-gated MSNs was also realized (Fig. 4B and Fig. S25, ESI†). After the first trigger of DTT, calcein was only released, and then upon subsequent decrease of pH, DOX was released from the g-CD cavities. Further comparative experiments demonstrate that the first release of calcein almost had no influence on monitoring the subsequent release of DOX (Fig. S26 and S27, ESI†). To investigate intracellular combination drug release of the g-CD-gated MSNs, CPT and DOX were loaded within the MSN pores and g-CD cavities, respectively. HeLa and A549 cells were incubated with the g-CD-gated MSNs with dual drug loading. There are high GSH levels within A549 cells besides the weak acidic environments of tumor cells. The g-CD-gated MSNs with dual drug loading showed lower cell viabilities in the case of A549 cells than those in the case of HeLa cells, (Fig. S28, ESI†), attributed to the simultaneous/cascade release and synergistic effect of the two drugs triggered by acidic pH and GSH. Confocal laser scanning microscopy (CLSM) was further used to observe intracellular release behaviors of the two drugs (Fig. 5). The fluorescence images were acquired in different optical windows for CPT (blue color) and DOX (red color). Both blue and red fluorescence became stronger with incubation time. Their merged fluorescence images demonstrate that CPT and DOX were simultaneously released intracellularly in comparison to the bright-field images. In the case of A549 cells, the CPT fluorescence was enhanced to be comparable to that of DOX at the initial stages, owing to the co-trigger of acidic pH and GSH. It is clear that the smart g-CD-gated MSN delivery system has promising biological applications in combination drug therapy to maximize therapeutic efficacy and to overcome drug resistance. The g-CD-gated MSN vehicles functionalized with disulfidelinked PBA moieties and amines were constructed for combination drug release. The intermolecular N–B coordination interactions between the PBA moieties and neighboring amino groups facilitated the binding of g-CD via the boronate ester bonds at physiological pH. The integrated g-CD-gated MSN delivery system with dual drug loading provided a smart platform for combination drug release (both simultaneous release and cascade release), in addition to resistance to serum and normal blood glucose levels, and have promising practical biological applications in targeted combination drug therapy.
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1 C. Wang, Z. Li, D. Cao, Y.-L. Zhao, J. W. Gaines, O. A. Bozdemir, M. W. Ambrogio, M. Frasconi, Y. Y. Botros, J. I. Zink and J. F. Stoddart, Angew. Chem., Int. Ed., 2012, 51, 5460. 2 (a) K. K. Coti, M. E. Belowich, M. Liong, M. W. Ambrogio, Y. A. Lau, H. A. Khatib, J. I. Zink, N. M. Khashab and J. F. Stoddart, Nanoscale, 2009, 1, 16; (b) A. Popat, S. B. Hartono, F. Stahr, J. Liu, S. Z. Qiao and G. Q. Lu, Nanoscale, 2011, 3, 2801; (c) Y.-W. Yang, Y.-L. Sun and N. Song, Acc. Chem. Res., 2014, 47, 1950; (d) P. Yang, S. Gai and J. Lin, Chem. Soc. Rev., 2012, 41, 3679; (e) C. Coll, A. Bernardos, ´n ˜ez and F. Sancen ´on, Acc. Chem. Res., 2013, 46, 339. R. Martı´nez-Ma ´n ˜ez, F. Sanceno ´n, J. Soto, 3 E. Aznar, M. D. Marcos, R. Martı´nez-Ma ´s and C. Guillem, J. Am. Chem. Soc., 2009, 131, 6833. ´s, P. Amoro R. Casasu 4 (a) C. Park, K. Oh, S. C. Lee and C. Kim, Angew. Chem., Int. Ed., 2007, 46, 1455; (b) K. Patel, S. Angelos, W. R. Dichtel, A. Coskun, Y.-W. Yang, J. I. Zink and J. F. Stoddart, J. Am. Chem. Soc., 2008, 130, 2382; (c) H. Meng, M. Xue, T. Xia, Y.-L. Zhao, F. Tamanoi, J. F. Stoddart, J. I. Zink and A. E. Nel, J. Am. Chem. Soc., 2010, 132, 12690; (d) P. Dı´ez, ´nchez, M. Gamella, P. Martı´nez-Ruı´z, E. Aznar, C. de la Torre, A. Sa ´n ˜ez, R. Villalonga and J. M. Pingarro ´n, J. R. Murguı´a, R. Martı´nez-Ma J. Am. Chem. Soc., 2014, 136, 9116; (e) C. Park, H. Kim, S. Kim and C. Kim, J. Am. Chem. Soc., 2009, 131, 16614; ( f ) C. Park, K. Lee and C. Kim, Angew. Chem., Int. Ed., 2009, 48, 1275. 5 Y. L. Sun, Y. Zhou, Q. L. Li and Y. W. Yang, Chem. Commun., 2013, 49, 9033. 6 (a) S. Angelos, Y. Yang, K. Patel, J. F. Stoddart and J. I. Zink, Angew. Chem., Int. Ed., 2008, 47, 2222; (b) S. Angelos, N. M. Khashab, Y. Yang, A. Trabolsi, H. A. Khatib, J. F. Stoddart and J. I. Zink, J. Am. Chem. Soc., 2009, 131, 12912; (c) T. Chen, N. Yang and J. Fu, Chem. Commun., 2013, 49, 6555; (d) J. Liu, X. Du and X. Zhang, Chem. – Eur. J., 2011, 17, 810. 7 (a) Y.-L. Sun, Y.-W. Yang, D.-X. Chen, G. Wang, Y. Zhou, C.-Y. Wang and J. F. Stoddart, Small, 2013, 9, 3224; (b) X. Huang and X. Du, ACS Appl. Mater. Interfaces, 2014, 6, 20430. 8 (a) A. Schlossbauer, J. Kecht and T. Bein, Angew. Chem., Int. Ed., ´n ˜ez, 2009, 48, 3092; (b) E. Climent, A. Bernardos, R. Martı´nez-Ma A. Maquieira, M. D. Marcos, N. Pastor-Navarro, R. Puchades, ´n, J. Soto and P. Amoro ´s, J. Am. Chem. Soc., 2009, F. Sanceno 131, 14075; (c) Z. Luo, K. Cai, Y. Hu, L. Zhao, P. Liu, L. Duan and W. Yang, Angew. Chem., Int. Ed., 2011, 50, 640; (d) S. Wu, X. Huang and X. Du, Angew. Chem., Int. Ed., 2013, 52, 5580. 9 (a) C. Chen, J. Geng, F. Pu, X. Yang, J. Ren and X. Qu, Angew. Chem., Int. Ed., 2011, 50, 882; (b) S. Zhou, X. Du, F. Cui and X. Zhang, Small, 2014, 10, 980; (c) S. Zhou, H. Sha, B. Liu and X. Du, Chem. Sci., 2014, 5, 4424. 10 J. Yan, G. Springsteen, S. Deeter and B. Wang, Tetrahedron, 2004, 60, 11205. 11 A. Matsumoto, T. Ishii, J. Nishida, H. Matsumoto, K. Kataoka and Y. Miyahara, Angew. Chem., Int. Ed., 2012, 51, 2124. 12 R. Ma and L. Shi, Polym. Chem., 2014, 5, 1503. 13 (a) G. Wulff, Pure Appl. Chem., 1982, 54, 2093; (b) D. Shiino, Y. Murata, A. Kubo, Y. J. Kim, K. Kataoka, Y. Koyama, A. Kikuchi, M. Yokoyama, Y. Sakurai and T. Okano, J. Controlled Release, 1995, 37, 269; (c) K. T. Kim, J. J. L. M. Cornelissen, R. J. M. Nolte and J. C. M. van Hest, J. Am. Chem. Soc., 2009, 131, 13908. 14 L. Zhu, S. H. Shabbir, M. Gray, V. M. Lynch, S. Sorey and E. V. Anslyn, J. Am. Chem. Soc., 2006, 128, 1222. 15 B. Yang, H. Jia, X. Wang, S. Chen, X. Zhang, R. Zhuo and J. Feng, Adv. Healthcare Mater., 2014, 3, 596. ´rube ´ and D. G. Hall, J. Org. 16 M. Gravel, K. A. Thompson, M. Zak, C. Be Chem., 2002, 67, 3. 17 F. Adrian, T. Budtova, E. Tarabukina, M. Pinteala, S. Mariana, C. Peptu, V. Harabagiu and B. C. Simionescu, J. Inclusion Phenom. Macrocyclic Chem., 2009, 64, 83. 18 J. S. Hansena, J. B. Christensena, J. F. Petersena, T. Hoeg-Jensenb and J. C. Norrild, Sens. Actuators, B, 2012, 161, 45.
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Combination drug release of smart cyclodextrin-gated mesoporous silica nanovehicles† Shengwang Zhou,a Huizi Sha,b Xiaokang Ke,a Baorui Liu,b Xizhang Wanga and Xuezhong Du*a
DOI: 10.1039/c5cc00585j www.rsc.org/chemcomm
An integrated c-cyclodextrin-gated mesoporous silica delivery system via dual dynamic covalent bonds was constructed with dual drug loading for simultaneous and cascade release in targeted combination drug therapy.
The combination of multiple drugs with different molecular targets can maximize therapeutic efficacy and is more likely to overcome drug resistance. Combination drug therapy has been adopted in clinics as a primary regimen of cancer treatment.1 There has been an ever increasing interest in developing stimuliresponsive drug delivery systems to improve therapeutic efficacy and minimize adverse effects of drugs. Mesoporous silica nanoparticles (MSNs) have attracted considerable attention as ideal drug carriers owing to their tunable pore size, large pore volume, good biocompatibility, and easy functionalization.2 To date, inorganic nanoparticles,3 macrocycles,4–7 and biomolecules8,9 have been used as MSN gatekeepers to show well-controlled release performances. The controlled release can be regulated by physical stimuli such as thermal9a–c and light irradiation,4f chemical stimuli such as pH3,4a,c,d,6a–c,7b,8d and redox,1,8c,9b and biochemical stimuli such as enzymes.4b,e,6d,9a–c However, simultaneous and cascade controlled release of two or more drugs for combination therapy is still difficult to achieve.9b,c Cyclodextrins (CDs) are linked by a-1,4-glycosidic bonds into a macrocycle, with the secondary O2 and O3 hydroxyls on one rim and the primary O6 hydroxyls on the other rim, and have been used as gatekeepers, such as nanovalves4a–d and nanogates,4e,f in the MSN-based drug delivery systems. Note that the hydrophobic cavities of CDs themselves can also accommodate drugs. It is a smart strategy to take advantage of the specific a
Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. E-mail: [email protected] b The Comprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China † Electronic supplementary information (ESI) available: Synthesis details, experimental details, supporting figures and illustrations. See DOI: 10.1039/c5cc00585j
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structures and properties of CDs for use in the MSN-based drug delivery systems not only as gatekeepers but also as drug carriers. Phenylboronic acid (PBA) and its derivatives are a class of Lewis acids and form boronate esters with 1,2- or 1,3-diols, such as saccharides and others containing diols, and their binding affinity depends on individual pKa and solution pH.10 Two methods have been used to reduce the pKa of PBA, so that stable boronate bonds are formed at physiological pH.11,12 One method is the introduction of electron-withdrawing groups to PBA at a meso-position with respect to boronic acid, and the other is the introduction of amino groups to decrease the apparent pKa of PBA because of the possible N–B coordination interaction.12–14 Herein, we report the simultaneous and cascade controlled release of two drugs from g-CD-gated MSN vehicles via dual dynamic covalent bonds (Scheme 1). MSNs were co-modified with disulfide-linked carbamoylphenylboronic acid moieties and amines on the surfaces. After a kind of drug was entrapped in the MSN pores, the secondary hydroxyls of g-CD on the wide rim effectively bound to the PBA moieties assisted by neighboring amino groups at physiological pH via the boronate linkages to encapsulate the drugs within the MSN pores. Afterward, the other kinds of drugs were included into the g-CD cavities. In addition, the dense-capped g-CD on the exterior surfaces played a role in bio-mimicking the saccharide-rich cell surfaces to overcome serum-susceptible drawbacks.15 The smart g-CDgated MSN delivery systems based on dual dynamic covalent bonds could well solve out the problem about simultaneous and cascade release of two drugs to meet diverse requirements. At acidic pH, the boronate bonds were hydrolyzed, and the drugs included inside the g-CD cavities could be protonated with a reduced binding affinity, which gave rise to simultaneous release of two drugs from the MSN pores and g-CD cavities. In the presence of monosaccharides, the drugs entrapped in MSN pores could be only released by the competitive binding of the monosaccharides to the PBA moieties, and then the drugs accommodated inside the g-CD cavities could be sequentially released upon decrease of pH. Similarly, the drugs entrapped in the MSN pores could be first released by the
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Fig. 1 (A) Solid-state 13C NMR spectra of PBA-MSNs, PBA/NH2-MSNs, and g-CD-gated MSNs without drug loading. The resonances labelled with the symbols * and # resulted from 2,2-dimethyl-1,3-propanediol and uncondensed ethoxysilane, respectively. (B) Solid-state 11B NMR spectra of PBA-MSNs and PBA/NH2-MSNs.
Scheme 1 Illustration of construction of g-CD-gated MSN vehicles functionalized with disulfide-linked carbamoylphenylboronic acid moieties and amines and simultaneous and cascade release of two drugs entrapped in the MSN pores and included inside the g-CD cavities.
cleavage of the disulfide linkages with the reducing agents dithiothreitol (DTT) or glutathione (GSH), and the other drugs could be successively released from the g-CD cavities upon decrease of pH. The combination of simultaneous and cascade release of the two drugs could maximize the therapeutic efficacy to overcome drug resistance. MCM-41 nanoparticles were synthesized by the base-catalyzed sol–gel method followed by removal of surfactant templates. Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of MCM-41 showed that MSNs had the sizes of 100 12 nm in diameter and possessed perfect parallelly arranged hexagonal pores (Fig. S1 in the ESI†). Small-angle X-ray diffraction (XRD) patterns of MCM-41 displayed four reflection peaks (Fig. S2, ESI†), typical of a well-fined hexagonal mesoporous structure. Nitrogen adsorption–desorption isotherms of MCM-41 showed a typical curve of type IV with a surface area of 1133.5 m2 g 1 and a pore size of 2.2 nm (Fig. S3, ESI†). Solid-state 13C NMR spectroscopy was used to characterize the functionalization of the PBA/amino moieties and the binding of g-CD (Fig. 1A). The strong resonance at 160 ppm was assigned to the –NHCONH– linkages, and the weak resonance at 167 ppm to the carbamoyl groups conjugated to the PBA moieties. The resonances in the range of 128–137 ppm were due to the phenyl groups of the PBA moieties (Fig. S4, ESI†).16 These spectral features confirm that the disulfide-linked PBA moieties were covalently immobilized on the MSN surfaces. After subsequent modification of 1,8-octanediamine (ODA), two main peaks at 28 and 30 ppm were clearly observed due to the resonances of –(CH2)6– groups except for the Ca atoms. After capping of g-CD without drug loading, four groups of new peaks in the ranges of 101–103, 76–82, 71–73, and 60–63 ppm were clearly observed owing to the
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various 13C resonances of g-CD,17 which demonstrate that PBA/NH2MSNs were capped with g-CD (g-CD-gated MSNs). FTIR spectroscopy was also used to characterize the modification of the PBA moieties and ODA and the capping of g-CD (Fig. S5, ESI†). The surface densities of the PBA moieties and amino groups were determined to be 1.290 and 0.345 mmol g 1 MCM-41 by thermogravimetric analysis (Fig. S6, ESI†), and the amount of bound g-CD was determined to be 0.302 mmol g 1 MCM-41 (without drug loading). The chemical shift and lineshape of solid-state 11B NMR spectroscopy are directly correlated with the coordination environment of boron species. One peak at 15–16 ppm could be assigned to the 3-coordinated (trigonal) boron species, and the other at less than 0 ppm to the 4-coordinated (tetrahedral) boron species (Fig. 1B). The neighboring amino groups promoted the conversion of the trigonal boron species into the tetrahedral ones, related to the mole ratio of the amino groups to the PBA moieties. The tetrahedral boron species facilitated the formation of stable cyclic boronate esters between the PBA/amino moieties and secondary hydroxyl groups of g-CD. The introduction of the amino groups in the vicinity of the PBA moieties probably promoted the Lewis acid–base interactions between the PBA moieties and amines, which thus lowered the apparent pKa of PBA.11 The markedly lowered PBA acidity was due to the ion-pairing stabilization from the neighboring amino groups and the N–B coordination interactions in equilibrium.13b,14 The zeta potential of MCM-41 was 29.4 mV and decreased to 35.5 mV for PBA-MSNs, followed by an increase to 2.1 mV for PBA/NH2-MSNs (Fig. S7A, ESI†). The zeta potential was slightly decreased to 6.7 mV after g-CD binding (g-CD-gated MSNs) and then underwent a change for inclusion of the two kinds of anticancer drugs inside the g-CD cavities, 0.4 mV in the case of doxorubicin (DOX) and 14.6 mV in the case of camptothecin (CPT) (Fig. S7B, ESI†). The average hydrodynamic diameters of these nanoparticles ranged from 102 to 110 nm (Fig. S8, ESI†), which reflect that the these nanoparticles were well monodispersed in aqueous solutions. However, the TEM images of PBA/NH2-MSNs and the g-CD-gated MSNs without drug loading did not show a significant difference from that of MCM-41 (Fig. S9, ESI†). PBA/ NH2-MSNs nearly displayed the same XRD patterns as MCM-41 (Fig. S10, ESI†). The MSN surface area decreased from 1133.5 m2 g 1 before modification to 926.3 m2 g 1 after modification, but the average pore diameters (2.2 nm) remained almost unchanged
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(Fig. S11 and Table S1, ESI†). However, none of the four XRD reflection peaks could be detectable after successive rhodamine 6G (Rh6G) loading, g-CD capping, and CPT inclusion. Moreover the surface area was drastically reduced to 100.8 m2 g 1 because the MSN pores were filled and capped (Fig. S10 and S11 and Table S1, ESI†). These results indicate that the g-CD-gated MSN vehicles with dual drug loading were thus constructed. UV-vis spectroscopy was used to monitor simultaneous release of Rh6G from the MSN pores and CPT from the g-CD cavities (Fig. S12 and S13, ESI†). The maximum absorption bands of Rh6G and CPT were well resolved without interference for the monitoring of release of the two drugs. Fluorescence spectroscopy was used to monitor cascade release of calcein from the MSN pores and DOX from the g-CD cavities (Fig. S14 and S15, ESI†). It is worth noting that the fluorescence of calcein was almost quenched at acidic pH and the release of calcein had no influence on monitoring the subsequent release of DOX triggered by acidic pH. The loading capacities of Rh6G and CPT were determined to be 0.0947 and 0.0707 mmol g 1 PBA/NH2-MSN using UV-vis spectroscopy (Fig. S16, ESI†), respectively. The loading capacities of calcein and DOX were determined to be 0.0316 and 0.0367 mmol g 1 PBA/NH2-MSN using fluorescence spectroscopy (Fig. S17, ESI†), respectively. The g-CD-gated MSNs with dual drug loading showed good blocking performance before pH trigger (Fig. 2 and Fig. S18, ESI†). Upon decrease of pH, simultaneous release of Rh6G and CPT was observed. The boronate bonds formed between the PBA/amino moieties and the secondary hydroxyl groups of g-CD were hydrolyzed at the acidic pH, which resulted in the removal of g-CD from the MSN surface and the release of Rh6G from the MSN pores. Simultaneously, CPT inside the g-CD cavities was protonated at the acidic pH, which gave rise to the weakening of their binding affinity and the release of CPT from the g-CD cavities. In order to gain an insight into the effect of the modified amino groups on the PBA moieties for g-CD binding, PBA-MSNs were loaded with Rh6G followed by addition of g-CD for comparison. The counterparts showed no cargo release even at pH 3.0 (Fig. S19, ESI†), because PBA-MSNs could not be capped with g-CD in the absence of modified amino groups. The cargo was finally washed off even if loaded, so that no cargo could be released. This further demonstrates that the introduction of the neighboring amino groups to the PBA moieties facilitated the development of the intermolecular N–B coordination interactions for g-CD binding.
Fig. 2 pH-responsive simultaneous release profiles from g-CD-gated MSNs with dual drug loading: (A) Rh6G (Amax = 527 nm); (B) CPT (Amax = 355 nm).
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Fig. 3 Cascade release profiles from g-CD-gated MSNs with dual loading of Rh6G and CPT upon first trigger of fructose (10 mM) at pH 7.4 and subsequent trigger of pH 5.0: Rh6G (Amax = 527 nm); CPT (Amax = 355 nm).
Saccharides are able to bind PBA to form cyclic boronate esters. In the presence of fructose or galactose (10 mM), significant release of Rh6G was observed from the g-CD-gated MSNs with dual drug loading at pH 7.4 but no CPT release; however, in the presence of glucose (10 mM) neither Rh6G nor CPT could be released (Fig. S20, ESI†). D-Fructose and D-galactose generally show stronger binding affinity to all aryl monoboronic acids in comparison to D-glucose.18 The competitive binding of fructose or galactose to the PBA moieties of the g-CD-gated MSNs drove the removal of g-CD and the release of Rh6G, while CPT inside the g-CD cavities remained unchanged. Importantly, glucose at the normal blood glucose levels could not cause premature release of the g-CD-gated MSNs with dual drug loading under physiological conditions. After the trigger of fructose, Rh6G was only released from the MSN pores. Upon further decrease of pH, subsequent release of CPT from the g-CD cavities was obviously observed (Fig. 3 and Fig. S21, ESI†). It is clear that the cascade release of Rh6G and CPT from the g-CD-gated MSNs could be realized upon first trigger of fructose (or galatose) and subsequent trigger of acidic pH. DTT could trigger release of calcein from the g-CD-gated MSNs with dual loading of calcein and DOX (Fig. S22 and S23, ESI†). It is worth noting that DTT contains diols as well as two mercapto groups. Further control experiments demonstrate that the DTT-responsive controlled release of calcein resulted from the cleavage of the disulfide bonds (Fig. 4A and Fig. S24, ESI†). GSH also caused the release of calcein but with about 50% release efficiency compared to DTT.
Fig. 4 (A) Stimuli-responsive release profiles from g-CD-gated MSNs only with the loading of calcein in the MSN pores at pH 7.4. (B) Cascade release profiles from g-CD-gated MSNs with dual loading of calcein and DOX upon first trigger of DTT (5 mM) at pH 7.4 and subsequent trigger of pH 3.0: calcein (lex = 458 nm, lem = 510 nm); DOX (lex = 480 nm, lem = 555 nm).
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This work was supported by National Natural Science Foundation of China (21273112) and Natural Science Foundation of Jiangsu Province (BK2012719). S. Zhou was supported by the program B for outstanding PhD candidate of Nanjing University.
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Notes and references
Fig. 5 CLSM images of intracellular uptake and drug release behaviors of the g-CD-gated MSNs with dual loading of CPT and DOX (100 mg mL 1) after incubation with (A) HeLa cells and (B) A549 cells for different times: CPT (lex = 365 nm); DOX (lex = 488 nm).
Similarly, the cascade release of calcein and DOX from the g-CD-gated MSNs was also realized (Fig. 4B and Fig. S25, ESI†). After the first trigger of DTT, calcein was only released, and then upon subsequent decrease of pH, DOX was released from the g-CD cavities. Further comparative experiments demonstrate that the first release of calcein almost had no influence on monitoring the subsequent release of DOX (Fig. S26 and S27, ESI†). To investigate intracellular combination drug release of the g-CD-gated MSNs, CPT and DOX were loaded within the MSN pores and g-CD cavities, respectively. HeLa and A549 cells were incubated with the g-CD-gated MSNs with dual drug loading. There are high GSH levels within A549 cells besides the weak acidic environments of tumor cells. The g-CD-gated MSNs with dual drug loading showed lower cell viabilities in the case of A549 cells than those in the case of HeLa cells, (Fig. S28, ESI†), attributed to the simultaneous/cascade release and synergistic effect of the two drugs triggered by acidic pH and GSH. Confocal laser scanning microscopy (CLSM) was further used to observe intracellular release behaviors of the two drugs (Fig. 5). The fluorescence images were acquired in different optical windows for CPT (blue color) and DOX (red color). Both blue and red fluorescence became stronger with incubation time. Their merged fluorescence images demonstrate that CPT and DOX were simultaneously released intracellularly in comparison to the bright-field images. In the case of A549 cells, the CPT fluorescence was enhanced to be comparable to that of DOX at the initial stages, owing to the co-trigger of acidic pH and GSH. It is clear that the smart g-CD-gated MSN delivery system has promising biological applications in combination drug therapy to maximize therapeutic efficacy and to overcome drug resistance. The g-CD-gated MSN vehicles functionalized with disulfidelinked PBA moieties and amines were constructed for combination drug release. The intermolecular N–B coordination interactions between the PBA moieties and neighboring amino groups facilitated the binding of g-CD via the boronate ester bonds at physiological pH. The integrated g-CD-gated MSN delivery system with dual drug loading provided a smart platform for combination drug release (both simultaneous release and cascade release), in addition to resistance to serum and normal blood glucose levels, and have promising practical biological applications in targeted combination drug therapy.
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