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Cite this: DOI: 10.1039/c4nr04796f
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Sugar and pH dual-responsive mesoporous silica nanocontainers based on competitive binding mechanisms† M. Deniz Yilmaz,‡a Min Xue,‡b Michael W. Ambrogio,c Onur Buyukcakir,a Yilei Wu,a Marco Frasconi,a Xinqi Chen,c Majed S. Nassar,d J. Fraser Stoddart*a and Jeffrey I. Zink*b A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs’ nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars,
Received 20th August 2014, Accepted 7th November 2014 DOI: 10.1039/c4nr04796f www.rsc.org/nanoscale
enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation. Acidification also induces the release of cargo molecules. Further investigations show that the presence of both a low pH and sugar molecules provides cooperative effects which together control the rate of release.
Introduction Mesoporous silica nanoparticles (MSNs) have exhibited considerable potential in controlled release and drug delivery.1–7 Numerous studies have focused on developing stimuli-responsive platforms8–14 by modifying the nanopore entrances, where a variety of complex molecules have been designed to separate the nanopores’ interiors from the outside environment. These molecular and supramolecular structures usually incorporate a bulky moiety, such as cyclodextrins,15 cucubiturils,16 transition metal complexes,17 polymers18 and quantum dots,19 to provide
a Center for the Chemistry of Integrated Systems, Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA. E-mail: [email protected] b Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095, USA. E-mail: [email protected] c Northwestern University Atomic and Nanoscale Characterization Experimental (NUANCE) Center, 2220 Campus Drive, Evanston, IL 60908, USA d National Center for Nano Technology Research, King Abdulaziz City of Science and Technology (KACST), P.O. Box 6086, Riyadh 11442, Kingdom of Saudi Arabia † Electronic supplementary information (ESI) available: Synthetic schemes, electron microscopy images and nitrogen adsorption/desorption isotherms of the nanoparticles, FT-IR spectra, isothermal titration calorimetry, X-ray photoelectron spectra and time-of-flight secondary ion mass spectra. DLS results for nanoparticle stability. See DOI: 10.1039/c4nr04796f ‡ These authors contributed equally to this work.
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steric hindrance to nanopore access. With well-chosen stimuli, these moieties cleave or disassociate from the surface, or undergo large amplitude motions to unblock the nanopores and hence release the stored cargo molecules. These integrated systems can be designed to be highly sensitive to stimuli such as pH change15 or redox process;20 indeed, there are many acids, bases, oxidants and reductants which produce the desired generic stimulus. One feature that is usually lacking in MSNs is their ability to respond to specific molecules. An example of moving towards higher specificity is provided by enzyme-responsive systems.21 Because of their well-defined nanopore structures and ease of functionalization, MSNs serve as good platforms for many physicochemical studies, such as the operation of supramolecular nanomachines,22 or to provide information about the status of molecules inside confined nanopores.23–25 Many complex molecular structures can be easily attached to the surface of mesoporous silica and their properties have become the focus of further investigations. In the case of stimuliresponsive binding, because the effective concentrations of the components on the surface are much higher than those in free solution, the behaviour of the binding event under certain stimuli can be altered dramatically. In this paper, we use MSNs as a scaffold (i) to investigate the effects of binding between different sugar molecules and boronic acids attached to the surface on the rates of cargo
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release,26–28 and (ii) to determine whether or not this relatively weak interaction can be employed for controlled delivery purposes. Because the complex can disassociate by either competitive binding processes or by protonation, we also investigate the behaviour of the binding in the presence of both sugar molecules and changes in pH.
Experimental Materials and general methods All reagents were purchased from commercial suppliers (Aldrich or VWR) and used without further purification. Reactions were carried out in anhydrous solvents under an inert nitrogen or argon atmosphere, unless otherwise stated. Thinlayer chromatography (TLC) was performed on silica gel 60 F254 TLC plates (Merck). Column chromatography was carried out on silica gel 60F (Merck 9385; 0.040–0.063 mm). UV/Vis Absorbance spectra were recorded using a UV-3600 Shimadzu spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 spectrometer, with a working frequency of 500 MHz for 1H. The chemical shifts in 1H NMR spectra are listed in ppm on the δ scale relative to the signals corresponding to the residual non-deuterated solvents, and the coupling constants are recorded in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; b, broad peaks; m, multiplet or overlapping peaks. High-resolution mass spectra (HRMS) were measured on an Agilent 6210 Time of Flight (TOF) LC-MS, employing an electrospray ionization (ESI) source, coupled with an Agilent 1100 HPLC stack, using direct infusion (0.6 mL min–1). The transmission electron microscope (TEM) images of the silica nanoparticles were collected on a CM 120 (Philips Electron Optics, Eindhoven, The Netherlands) instrument in the California NanoSystem Institute (CNSI). Microfilms for TEM imaging were made by placing a drop of the particle suspension in methanol onto a S3 200-mesh copper TEM grid (Ted Pella, Inc., Redding, CA) and drying at room temperature. Isothermal Titration Microcalorimetry (ITC) was carried out using a Microcal VP-ITC titration microcalorimeter. Software provided by Microcal LLC was used to compute the thermodynamic parameters of the titration (ΔG°, Ka, ΔS°, ΔH°) based on the one-site binding model. The controlled release profiles were obtained by recording luminescence spectra using an Acton SpectraPro 2300i CCD detector and either a Coherent Cube 375-16C laser or a Coherent Cube 445-40C laser. X-Ray photoelectron spectroscopy (XPS) was carried out using a Thermo Scientific ESCALAB 250 Xi, and all XPS spectra were calibrated by setting the peak corresponding to aliphatic carbon to 285 eV. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was carried out on a Physical Electronics PHI TRIFT III spectrometer. Samples for both XPS and TOF-SIMS were prepared by affixing doublesided copper tape to a silicon wafer, and subsequently spreading the sample of interest (which was always in powder form) onto the exposed side of the copper tape.
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C-βCD The synthetic scheme (Scheme S1†) is shown in the ESI.† Mono(6-(2-aminoethyl)amino-6-deoxy)-β-cyclodextrin (1, 500 mg, 0.425 mmol) and anhydrous MgSO4 (100 mg, 0.8 mmol) were suspended in anhydrous DMF (10 mL). Compound 2 (211 mg, 0.467 mmol) was added slowly into the solution. The mixture was stirred for 24 h under N2 atmosphere. After cooling to 0 °C in an ice-bath, NaBH4 (161 mg, 4.25 mmol) was added in small portions. The reaction mixture was allowed to warm up to room temperature and then stirred for 2 h under a N2 atmosphere. The mixture was poured into H2O (200 mL) to give a white precipitate (480 mg, 70%). The resulting precipitate was filtered, washed with Me2CO and used without further purification in the next step. This white solid (200 mg, 0.124 mmol) was dissolved in THF (10 mL)– MeOH (10 mL) solvent mixture. Tetrabutylammonium fluoride (130 mg, 0.496 mmol) was added to the solution and the mixture was stirred for 24 h under a N2 atmosphere at room temperature. Next, the reaction mixture was evaporated under reduced pressure. The resulting solid was dissolved in a minimum amount of H2O and poured into Me2CO (200 mL) to give C-βCD as an off-white solid (137 mg, 85%). 1H NMR (500 MHz, D2O, 298 K): δ = 6.95 (d, J = 7.5 Hz, 1H), 6.89 (s, 1H), 6.75 (d, J = 7.5 Hz, 1H), 5.10–4.90 (m, 7H), 4.20–3.20 (m, 44H), 3.10–2.60 (m, 4H). HR MS (EI): Calcd for C49H86O39 m/z = 1298.4745 [M]+, found m/z = 1299.4821 [M + H]+. AP-MSN Bare MCM-41 nanoparticles (200 mg) were synthesized according to an established procedure.15 The nanoparticles with surfactant were suspended in anhydrous PhMe (10 mL) and 3-aminopropyltriethoxysilane (30 μL) was added to the suspension which was heated under reflux in an atmosphere of argon for 15 h. After cooling to room temperature, the precipitate was collected by centrifugation. The nanoparticles were then suspended in 100 mL of MeOH and 80 mg of NH4NO3 was added to the suspension. The mixture was refluxed under argon for 40 min and the nanoparticles were collected and washed with MeOH. PBA-MSN 4-Carboxy-3-fluorophenylboronic acid (41 mg, 0.225 mmol) was reacted with N-hydroxysuccinimide (25 mg, 0.21 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (50 mg, 0.26 mmol) in Me2SO (5 mL) at room temperature for 30 min. This solution was added to the AP-MSN (100 mg) suspension in Me2SO (10 mL) and final mixture was stirred at room temperature for 24 h. The nanoparticles were collected through centrifugation, washed with Me2SO and MeOH, and then dried under vacuum. The synthetic scheme for PBA-MSN is shown (Scheme S2†) in the ESI.† Loading and capping of PBA-MSN PBA-MSN (20 mg) was suspended in an aqueous solution of propidium iodide (PI, 1 mL, 1 mM) and the mixture was
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stirred in the dark for 3 days. C-βCD (10 mg) was added into the suspension and the mixture was stirred for another 24 h. The nanoparticles were then collected by centrifugation and washed with H2O to remove the unloaded propidium iodide. After washing, the nanoparticles were dried under vacuum. The loading capacity was determined to be 3 wt% by absorption spectroscopy. Assessment of stimuli-responsive release The C-βCD capped, propidium iodide-loaded nanoparticles were placed in a cuvette and H2O was added without disturbing the nanoparticles. A laser beam (448 nm, 20 mW) was used to excite the cargo molecules released into the supernatant and the corresponding fluorescence was recorded by a CCD camera. In order to induce the cargo release, a small amount of HCl or sugar solution was added to the cuvette to reach the desired pH or sugar concentration. Alternatively, the solution was carefully removed and a new solution of the desired sugar concentration was added without disturbing the nanoparticles. The fluorescence spectra of propidium iodide (PI) were continuously monitored over the course of the experiment to generate release profiles. The standard of 100% release is obtained by soaking nanoparticles in a pH 2.0 solution for 24 h and then quantifying the amount of PI in the supernatant via UV/Vis absorption spectroscopy. The release profiles are subsequently normalized according to the 100% release standards.
Results and discussion C-βCD was prepared by condensation between the appropriate aldehyde and primary amine, followed by NaBH4 reduction and deprotection of the catechol hydroxy groups. Functionalized MSNs were synthesized according to previously reported procedures15 with some modifications. The assembly of the hybrid system is illustrated in Fig. 1. The nanoparticle size, surface morphology and chemical composition of PBA-MSNs and C-βCD capped PBA-MSNs (CD-PBA-MSNs) were characterized by TEM, N2 sorption, FTIR, XPS, and TOF-SIMS measurements (ESI†). TEM Images of PBA-MSNs (Fig. S1a†) and propidium iodide (PI) dye loaded CD-PBA-MSNs (Fig. S1b†) show the formation of uniform mesoporous nanoparticles with an average diameter of around 100 nm. Specific surface areas of PBA-MSNs and CD-PBA-MSNs were calculated from the N2 sorption data and found to be 727 m2 g−1 for PBA-MSNs and 601 m2 g−1 for CD-PBA-MSNs. A decrease in the specific surface area for CD-PBA-MSNs indicates the introduction of C-βCDs onto the surface of the nanoparticles. FTIR Spectra (Fig. S3†) of MSNs taken after each synthetic step show their own characteristics and confirm the successful functionalization. The boronate ester formation between 4-carboxy-3-fluorophenylboronic acid and C-βCD in H2O was studied first of all by isothermal titration calorimetry (ITC) measurements. An ITC titration of 10 mM 4-carboxy-3-fluorophenylboronic acid to
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Fig. 1 Schematic representations of the sugar and pH dual-responsive cargo release process and of the compounds used in this investigation.
1 mM C-βCD revealed (Fig. S4a†) a typical 1 : 1 binding event. Fitting to a 1 : 1 binding model yielded a Ka value of 6 × 103 M−1. In a control experiment (Fig. S4b†), an ITC titration of 10 mM 4-carboxy-3-fluorophenylboronic acid to 1 mM bare β-CD led to a weak binding with a Ka value of 1 × 102 M−1. The results prove that the higher binding constant stems from the catechol subunit and that the contribution from β-CD is negligible.29 The organic functionalization on the surface of MSNs was characterized using two analytical techniques. Firstly, X-ray photoelectron spectroscopy (XPS) was used to investigate the surface of the MSNs.30 The high resolution XPS spectra of the C1s region for AP-MSN, PBA-MSN, and CD-PBA-MSN are depicted in Fig. S6a–c,† respectively. For the CD-PBA-MSN sample, the large peak at 286.5 eV can be ascribed to the presence of β-CD on the surfaces of the nanoparticles. The increasing C : Si ratios of the three samples that were analyzed confirmed the presence of additional organic molecules being attached to the silica nanoparticles after each synthetic step, with C : Si ratios of 0.56, 1.01, and 1.54 being obtained for AP-MSN, PBA-MSN, and CD-PBA-MSN, respectively. Furthermore, XPS data confirm that boron is present in PBA-MSN and CD-PBA-MSN samples, as expected, based on the peak at around 191.5 eV. A second analytical technique, namely timeof-flight secondary ion mass spectrometry (TOF-SIMS) was also used to confirm (Fig. S7–S9†) the presence of boron and fluorine in the PBA-MSN and CD-PBA-MSN samples. On the other hand, neither boron nor fluorine are present in the AP-MSN precursor. These results show that the grafting of PBA onto MSN is successful and the C-βCD can indeed bind to the PBA and form boronate esters. The binding between PBA and catechol is fairly stable in aqueous solution. In order to ascertain whether the binding is strong enough to maintain the boronate ester linkages even
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Fig. 2 (a) Release of PI from CD-PBA-MSNs after treatment with different concentrations of glucose. (b) Release of PI from CD-PBA-MSNs with the different sugars (5 mM each) (fructose = red, glucose = black, maltose = blue, and sucrose = pink). Here, the original solution was removed carefully after taking baselines and new solutions of different sugars were added carefully to initiate the release of PI. Inset: bar graphs of percent released cargo upon treatment with sugars (5 mM each) after 16 h.
under diluted conditions, we loaded the PBA-MSNs with PI before capping with C-βCD and investigating their release behaviour. We use a continuous-monitoring fluorescence spectroscopic method to study the binding. If the PBA-C-βCD complex disassociates in diluted aqueous solution, the C-βCD will not be able to block the nanopores and PI will diffuse into the solution, causing an increase in the fluorescence. On the contrary, there is no obvious release (Fig. 2a) of PI during the first hour of the experiment. Only when a glucose solution was added, was a gradual increase in fluorescence intensity observed, indicating that the C-βCD-PBA complex was displaced by the glucose-PBA complex. In fact, because the size of glucose is much smaller than that of the nanopores’ orifices, the entrapped PI molecules are now able to diffuse out from
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the nanopores, causing the increase in the fluorescence intensity. Moreover, the release rate of PI varies as a function of the glucose concentration. At a low glucose concentration (1 mM), MSNs release 5% of the encapsulated cargo after 14 h of treatment, whereas almost 30% of the cargo molecules are released after the same period of time at a higher concentration (25 mM, Fig. 2) of glucose. This observation is in agreement with the proposed competitive binding mechanism. In order to probe the binding further, different sugar solutions were employed to initiate the cargo release. The release profiles are shown in Fig. 2b. Fructose and glucose trigger the release of the cargo from the nanopores faster than the other sugars, an observation which is consistent with the corresponding sugar-PBA-MSNs complex binding constant. The ratedifference among sugars seems to have a correlation with the accessibility of their furanose forms in solution.31 These results clearly demonstrate the binding-constant dependent responses and the competitive binding nature of the CD-PBA-MSNs system. It is well known that the binding between catechol and boronic acid is much weaker in an acidic environment. In order to demonstrate this pH-dependent feature, the release of PI entrapped in CD-PBA-MSNs was evaluated at different pH values. The resulting release profiles are shown in Fig. 3. As expected, the release rate of PI correlated with the final solution pH. In the case of pH 6.0, 30% of PI was released in about 6 h, while at pH 4.0, about 85% of PI was released in the same period of time. The rates of all the acid-triggered releases are much faster than those of the sugar-induced releases, as a consequence of the fact that the dye molecules are positively charged. Acidification changes the charge environment inside the nanopores, and assists the diffusion of positively charged cargo molecules from the nanopores.23 In the case of the sugar-induced stimulus, the release of the dye molecules relies mainly on their passive diffusion, which is driven by concentration gradients. After confirming that both sugar and acid can disassociate C-βCD and PBA, we turned our attention to the investigation of their binding behaviour in the presence of both stimuli. Fig. 4
Fig. 3
Release of PI from CD-PBA-MSNs after acidification from pH 7.2.
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ally, because of the acidic environment, the diffusion of the dye molecules is faster than that observed in the sugarinduced release, which also contributes to the overall synergistic effect caused by the combination of the two stimuli. When more acidic conditions ( pH 5.0) are introduced along with the glucose solution, the result (Fig. 4b) of the acid-sugar combination is similar to that arising from the accumulated effect of the stimuli. This observation is in agreement with our aforementioned explanation. Because of the higher proton concentration, the equilibrium already largely favours the disassociation of C-βCD, and so the addition of sugar molecules provides a minor influence on the equilibrium and results in a much less prominent synergistic effect.
Conclusions
Fig. 4 Release profile traces of PI from CD-PBA-MSNs at pH 6 (a) and pH 5 (b) in the presence of 5 mM glucose.
In conclusion, we have employed mesoporous silica nanoparticles as platforms for studying the binding properties of catechol-boronate esters on silica surfaces. The assembly of C-βCD and PBA serves as a model for catechol-boronate ester binding, as well as a controlled-release platform. We have demonstrated that sugar molecules can cause the dissociation of the C-βCD-PBA complex by means of a competitive binding mechanism, where the disassociation rate correlates with both the binding affinity and the concentration of sugar. We have shown that this binding is pH-responsive, to the extent that the lower the pH the faster the disassociation. We have also demonstrated that the combination of sugar and acid provides a synergistic effect on the disassociation process. We believe that this sugar and pH dual responsive hybrid system will inspire the development of new drug delivery protocols with advanced imaging and therapeutic goals.
Acknowledgements shows the release profiles resulting from these dual-responsive experiments. When mild acidic conditions ( pH 6.0) were employed, we observe that (Fig. 4a) the combination of sugar and acid induces a faster release rate that goes beyond a simple additive effect. Glucose resulted in around 7% release in 6 h, while pH 6.0 resulted in 30% PI release. If the effects of sugar and acid are simply additive, a 37% release after 6 h is expected. When both sugar and acid were introduced, however, a 70% release was detected in the same amount of time. This rate is nearly twice that based on a simple accumulative assumption. One possible explanation is that under these mild acidic conditions, the disassociation of the boronate ester caused by protonation is relatively slow, implying that the equilibrium only slightly favours the disassociation over the rebinding process. With the addition of a glucose solution, the glucose molecules compete with the C-βCD and therefore decrease significantly the probability of C-βCD rebinding with PBA. This protocol causes a shift of the equilibrium and therefore enhances the disassociation of the C-βCD, which translates into a faster release process. Addition-
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This research was supported by the NIH RO1 CA133697, by the Defense Threat Reduction Agency HDTRA1-13-1-0046, and is part of the Joint Center of Excellence in Integrated Nanosystems (JCIN) at King Abdul-Aziz City for Science and Technology (KACST) and Northwestern University (NU) (Project 34-941). The authors would like to thank both KACST and NU for their continued support of this research. Y. W. would like to thank the Fulbright Scholar Program for a Research Fellowship.
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15 (a) 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, 12690–12697; (b) M. Xue and J. I. Zink, J. Am. Chem. Soc., 2013, 135, 17659–17662. 16 (a) S. Angelos, Y. W. Yang, K. Patel, J. F. Stoddart and J. I. Zink, Angew. Chem., Int. Ed., 2008, 47, 2222–2226; (b) C. R. Thomas, D. P. Ferris, J. H. Lee, E. Choi, M. H. Cho, E. S. Kim, J. F. Stoddart, J. S. Shin, J. Cheon and J. I. Zink, J. Am. Chem. Soc., 2010, 132, 10623–10625; (c) J. S. Liu and X. Z. Du, J. Mater. Chem., 2010, 20, 3642–3649; (d) Y. L. Sun, B. J. Yang, S. X. A. Zhang and Y. W. Yang, Chem. – Eur J., 2012, 18, 9212–9216. 17 (a) M. Frasconi, Z. Liu, J. Lei, Y. Wu, E. Strekalova, D. Malin, M. W. Ambrogio, X. Chen, Y. Y. Botros, V. L. Cryns, J.-P. Sauvage and J. F. Stoddart, J. Am. Chem. Soc., 2013, 135, 11603–11613; (b) L. Z. He, Y. Y. Huang, H. L. Zhu, G. H. Pang, W. J. Zheng, Y. S. Wong and T. F. Chen, Adv. Funct. Mater., 2014, 24, 2754–2763. 18 A. Popat, J. Liu, G. Q. Lu and S. Z. Qiao, J. Mater. Chem., 2012, 22, 11173–11178. 19 T. Chem, H. Yu, N. Yang, M. D. Wang, C. Ding and J. Fu, J. Mater. Chem. B, 2014, 2, 4979–4982. 20 Z. Luo, K. Cai, Y. Hu, L. Zhao, P. Liu, L. Duan and W. Yang, Angew. Chem., Int. Ed., 2010, 50, 640–643. 21 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, 2382–2383. 22 T. D. Nguyen, H. R. Tseng, P. C. Celestre, A. H. Flood, Y. Liu, J. F. Stoddart and J. I. Zink, Proc. Natl. Acad. Sci. U. S. A., 2005, 29, 10029–10034. 23 M. Xue and J. I. Zink, J. Phys. Chem. Lett., 2014, 5, 839–842. 24 A. Zürner, J. Kirstein, M. Döblinger, C. Bräuchle and T. Bein, Nature, 2007, 450, 705–708. 25 M. Huang, B. S. Dunn and J. I. Zink, J. Am. Chem. Soc., 2000, 122, 3739–3745. 26 G. Springsteen and B. H. Wang, Tetrahedron, 2002, 58, 5291–5300. 27 Y. J. Huang, Y. B. Jiang, J. S. Fossey, T. D. James and F. Marken, J. Mater. Chem., 2010, 20, 8305–8310. 28 Y. Li, W. Xiao, K. Xiao, L. Berti, J. Luo, H. P. Tseng, G. Fung and K. S. Lam, Angew. Chem., Int. Ed., 2012, 51, 2864–2869. 29 Fluoride substituents on the phenyl ring reduce the pKa of phenylboronic acid and increase the stability of boronate ester at physiological pH, see: A. Matsumoto, T. Ishii, J. Nishida, H. Matsumoto, K. Kataoka and Y. Miyahara, Angew. Chem., Int. Ed., 2012, 51, 2124–2128. 30 M. W. Ambrogio, M. Frasconi, M. D. Yilmaz and X. Chen, Langmuir, 2013, 29, 15386–15393. 31 (a) J. C. Norrild and H. Eggert, J. Chem. Soc. Perkin Trans. 2, 1996, 2583–2588; (b) J. P. Lorand and J. O. Edwards, J. Org. Chem., 1959, 24, 769–774.
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Cite this: DOI: 10.1039/c4nr04796f
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Sugar and pH dual-responsive mesoporous silica nanocontainers based on competitive binding mechanisms† M. Deniz Yilmaz,‡a Min Xue,‡b Michael W. Ambrogio,c Onur Buyukcakir,a Yilei Wu,a Marco Frasconi,a Xinqi Chen,c Majed S. Nassar,d J. Fraser Stoddart*a and Jeffrey I. Zink*b A sugar and pH dual-responsive controlled release system, which is highly specific towards molecular stimuli, has been developed based on the binding between catechol and boronic acid on a platform of mesoporous silica nanoparticles (MSNs). By grafting phenylboronic acid stalks onto the silica surface, catechol-containing β-cyclodextrins can be attached to the orifices of the MSNs’ nanopores through formation of boronate esters which block access to the nanopores. These esters are stable enough to prevent cargo molecules from escaping. The boronate esters disassociate in the presence of sugars,
Received 20th August 2014, Accepted 7th November 2014 DOI: 10.1039/c4nr04796f www.rsc.org/nanoscale
enabling the molecule-specific controlled-release feature of this hybrid system. The rate of release has been found to be tunable by varying both the structures and the concentrations of sugars, as a result of the competitive binding nature associated with the mechanism of its operation. Acidification also induces the release of cargo molecules. Further investigations show that the presence of both a low pH and sugar molecules provides cooperative effects which together control the rate of release.
Introduction Mesoporous silica nanoparticles (MSNs) have exhibited considerable potential in controlled release and drug delivery.1–7 Numerous studies have focused on developing stimuli-responsive platforms8–14 by modifying the nanopore entrances, where a variety of complex molecules have been designed to separate the nanopores’ interiors from the outside environment. These molecular and supramolecular structures usually incorporate a bulky moiety, such as cyclodextrins,15 cucubiturils,16 transition metal complexes,17 polymers18 and quantum dots,19 to provide
a Center for the Chemistry of Integrated Systems, Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA. E-mail: [email protected] b Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095, USA. E-mail: [email protected] c Northwestern University Atomic and Nanoscale Characterization Experimental (NUANCE) Center, 2220 Campus Drive, Evanston, IL 60908, USA d National Center for Nano Technology Research, King Abdulaziz City of Science and Technology (KACST), P.O. Box 6086, Riyadh 11442, Kingdom of Saudi Arabia † Electronic supplementary information (ESI) available: Synthetic schemes, electron microscopy images and nitrogen adsorption/desorption isotherms of the nanoparticles, FT-IR spectra, isothermal titration calorimetry, X-ray photoelectron spectra and time-of-flight secondary ion mass spectra. DLS results for nanoparticle stability. See DOI: 10.1039/c4nr04796f ‡ These authors contributed equally to this work.
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steric hindrance to nanopore access. With well-chosen stimuli, these moieties cleave or disassociate from the surface, or undergo large amplitude motions to unblock the nanopores and hence release the stored cargo molecules. These integrated systems can be designed to be highly sensitive to stimuli such as pH change15 or redox process;20 indeed, there are many acids, bases, oxidants and reductants which produce the desired generic stimulus. One feature that is usually lacking in MSNs is their ability to respond to specific molecules. An example of moving towards higher specificity is provided by enzyme-responsive systems.21 Because of their well-defined nanopore structures and ease of functionalization, MSNs serve as good platforms for many physicochemical studies, such as the operation of supramolecular nanomachines,22 or to provide information about the status of molecules inside confined nanopores.23–25 Many complex molecular structures can be easily attached to the surface of mesoporous silica and their properties have become the focus of further investigations. In the case of stimuliresponsive binding, because the effective concentrations of the components on the surface are much higher than those in free solution, the behaviour of the binding event under certain stimuli can be altered dramatically. In this paper, we use MSNs as a scaffold (i) to investigate the effects of binding between different sugar molecules and boronic acids attached to the surface on the rates of cargo
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release,26–28 and (ii) to determine whether or not this relatively weak interaction can be employed for controlled delivery purposes. Because the complex can disassociate by either competitive binding processes or by protonation, we also investigate the behaviour of the binding in the presence of both sugar molecules and changes in pH.
Experimental Materials and general methods All reagents were purchased from commercial suppliers (Aldrich or VWR) and used without further purification. Reactions were carried out in anhydrous solvents under an inert nitrogen or argon atmosphere, unless otherwise stated. Thinlayer chromatography (TLC) was performed on silica gel 60 F254 TLC plates (Merck). Column chromatography was carried out on silica gel 60F (Merck 9385; 0.040–0.063 mm). UV/Vis Absorbance spectra were recorded using a UV-3600 Shimadzu spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 spectrometer, with a working frequency of 500 MHz for 1H. The chemical shifts in 1H NMR spectra are listed in ppm on the δ scale relative to the signals corresponding to the residual non-deuterated solvents, and the coupling constants are recorded in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; b, broad peaks; m, multiplet or overlapping peaks. High-resolution mass spectra (HRMS) were measured on an Agilent 6210 Time of Flight (TOF) LC-MS, employing an electrospray ionization (ESI) source, coupled with an Agilent 1100 HPLC stack, using direct infusion (0.6 mL min–1). The transmission electron microscope (TEM) images of the silica nanoparticles were collected on a CM 120 (Philips Electron Optics, Eindhoven, The Netherlands) instrument in the California NanoSystem Institute (CNSI). Microfilms for TEM imaging were made by placing a drop of the particle suspension in methanol onto a S3 200-mesh copper TEM grid (Ted Pella, Inc., Redding, CA) and drying at room temperature. Isothermal Titration Microcalorimetry (ITC) was carried out using a Microcal VP-ITC titration microcalorimeter. Software provided by Microcal LLC was used to compute the thermodynamic parameters of the titration (ΔG°, Ka, ΔS°, ΔH°) based on the one-site binding model. The controlled release profiles were obtained by recording luminescence spectra using an Acton SpectraPro 2300i CCD detector and either a Coherent Cube 375-16C laser or a Coherent Cube 445-40C laser. X-Ray photoelectron spectroscopy (XPS) was carried out using a Thermo Scientific ESCALAB 250 Xi, and all XPS spectra were calibrated by setting the peak corresponding to aliphatic carbon to 285 eV. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was carried out on a Physical Electronics PHI TRIFT III spectrometer. Samples for both XPS and TOF-SIMS were prepared by affixing doublesided copper tape to a silicon wafer, and subsequently spreading the sample of interest (which was always in powder form) onto the exposed side of the copper tape.
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C-βCD The synthetic scheme (Scheme S1†) is shown in the ESI.† Mono(6-(2-aminoethyl)amino-6-deoxy)-β-cyclodextrin (1, 500 mg, 0.425 mmol) and anhydrous MgSO4 (100 mg, 0.8 mmol) were suspended in anhydrous DMF (10 mL). Compound 2 (211 mg, 0.467 mmol) was added slowly into the solution. The mixture was stirred for 24 h under N2 atmosphere. After cooling to 0 °C in an ice-bath, NaBH4 (161 mg, 4.25 mmol) was added in small portions. The reaction mixture was allowed to warm up to room temperature and then stirred for 2 h under a N2 atmosphere. The mixture was poured into H2O (200 mL) to give a white precipitate (480 mg, 70%). The resulting precipitate was filtered, washed with Me2CO and used without further purification in the next step. This white solid (200 mg, 0.124 mmol) was dissolved in THF (10 mL)– MeOH (10 mL) solvent mixture. Tetrabutylammonium fluoride (130 mg, 0.496 mmol) was added to the solution and the mixture was stirred for 24 h under a N2 atmosphere at room temperature. Next, the reaction mixture was evaporated under reduced pressure. The resulting solid was dissolved in a minimum amount of H2O and poured into Me2CO (200 mL) to give C-βCD as an off-white solid (137 mg, 85%). 1H NMR (500 MHz, D2O, 298 K): δ = 6.95 (d, J = 7.5 Hz, 1H), 6.89 (s, 1H), 6.75 (d, J = 7.5 Hz, 1H), 5.10–4.90 (m, 7H), 4.20–3.20 (m, 44H), 3.10–2.60 (m, 4H). HR MS (EI): Calcd for C49H86O39 m/z = 1298.4745 [M]+, found m/z = 1299.4821 [M + H]+. AP-MSN Bare MCM-41 nanoparticles (200 mg) were synthesized according to an established procedure.15 The nanoparticles with surfactant were suspended in anhydrous PhMe (10 mL) and 3-aminopropyltriethoxysilane (30 μL) was added to the suspension which was heated under reflux in an atmosphere of argon for 15 h. After cooling to room temperature, the precipitate was collected by centrifugation. The nanoparticles were then suspended in 100 mL of MeOH and 80 mg of NH4NO3 was added to the suspension. The mixture was refluxed under argon for 40 min and the nanoparticles were collected and washed with MeOH. PBA-MSN 4-Carboxy-3-fluorophenylboronic acid (41 mg, 0.225 mmol) was reacted with N-hydroxysuccinimide (25 mg, 0.21 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (50 mg, 0.26 mmol) in Me2SO (5 mL) at room temperature for 30 min. This solution was added to the AP-MSN (100 mg) suspension in Me2SO (10 mL) and final mixture was stirred at room temperature for 24 h. The nanoparticles were collected through centrifugation, washed with Me2SO and MeOH, and then dried under vacuum. The synthetic scheme for PBA-MSN is shown (Scheme S2†) in the ESI.† Loading and capping of PBA-MSN PBA-MSN (20 mg) was suspended in an aqueous solution of propidium iodide (PI, 1 mL, 1 mM) and the mixture was
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stirred in the dark for 3 days. C-βCD (10 mg) was added into the suspension and the mixture was stirred for another 24 h. The nanoparticles were then collected by centrifugation and washed with H2O to remove the unloaded propidium iodide. After washing, the nanoparticles were dried under vacuum. The loading capacity was determined to be 3 wt% by absorption spectroscopy. Assessment of stimuli-responsive release The C-βCD capped, propidium iodide-loaded nanoparticles were placed in a cuvette and H2O was added without disturbing the nanoparticles. A laser beam (448 nm, 20 mW) was used to excite the cargo molecules released into the supernatant and the corresponding fluorescence was recorded by a CCD camera. In order to induce the cargo release, a small amount of HCl or sugar solution was added to the cuvette to reach the desired pH or sugar concentration. Alternatively, the solution was carefully removed and a new solution of the desired sugar concentration was added without disturbing the nanoparticles. The fluorescence spectra of propidium iodide (PI) were continuously monitored over the course of the experiment to generate release profiles. The standard of 100% release is obtained by soaking nanoparticles in a pH 2.0 solution for 24 h and then quantifying the amount of PI in the supernatant via UV/Vis absorption spectroscopy. The release profiles are subsequently normalized according to the 100% release standards.
Results and discussion C-βCD was prepared by condensation between the appropriate aldehyde and primary amine, followed by NaBH4 reduction and deprotection of the catechol hydroxy groups. Functionalized MSNs were synthesized according to previously reported procedures15 with some modifications. The assembly of the hybrid system is illustrated in Fig. 1. The nanoparticle size, surface morphology and chemical composition of PBA-MSNs and C-βCD capped PBA-MSNs (CD-PBA-MSNs) were characterized by TEM, N2 sorption, FTIR, XPS, and TOF-SIMS measurements (ESI†). TEM Images of PBA-MSNs (Fig. S1a†) and propidium iodide (PI) dye loaded CD-PBA-MSNs (Fig. S1b†) show the formation of uniform mesoporous nanoparticles with an average diameter of around 100 nm. Specific surface areas of PBA-MSNs and CD-PBA-MSNs were calculated from the N2 sorption data and found to be 727 m2 g−1 for PBA-MSNs and 601 m2 g−1 for CD-PBA-MSNs. A decrease in the specific surface area for CD-PBA-MSNs indicates the introduction of C-βCDs onto the surface of the nanoparticles. FTIR Spectra (Fig. S3†) of MSNs taken after each synthetic step show their own characteristics and confirm the successful functionalization. The boronate ester formation between 4-carboxy-3-fluorophenylboronic acid and C-βCD in H2O was studied first of all by isothermal titration calorimetry (ITC) measurements. An ITC titration of 10 mM 4-carboxy-3-fluorophenylboronic acid to
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Fig. 1 Schematic representations of the sugar and pH dual-responsive cargo release process and of the compounds used in this investigation.
1 mM C-βCD revealed (Fig. S4a†) a typical 1 : 1 binding event. Fitting to a 1 : 1 binding model yielded a Ka value of 6 × 103 M−1. In a control experiment (Fig. S4b†), an ITC titration of 10 mM 4-carboxy-3-fluorophenylboronic acid to 1 mM bare β-CD led to a weak binding with a Ka value of 1 × 102 M−1. The results prove that the higher binding constant stems from the catechol subunit and that the contribution from β-CD is negligible.29 The organic functionalization on the surface of MSNs was characterized using two analytical techniques. Firstly, X-ray photoelectron spectroscopy (XPS) was used to investigate the surface of the MSNs.30 The high resolution XPS spectra of the C1s region for AP-MSN, PBA-MSN, and CD-PBA-MSN are depicted in Fig. S6a–c,† respectively. For the CD-PBA-MSN sample, the large peak at 286.5 eV can be ascribed to the presence of β-CD on the surfaces of the nanoparticles. The increasing C : Si ratios of the three samples that were analyzed confirmed the presence of additional organic molecules being attached to the silica nanoparticles after each synthetic step, with C : Si ratios of 0.56, 1.01, and 1.54 being obtained for AP-MSN, PBA-MSN, and CD-PBA-MSN, respectively. Furthermore, XPS data confirm that boron is present in PBA-MSN and CD-PBA-MSN samples, as expected, based on the peak at around 191.5 eV. A second analytical technique, namely timeof-flight secondary ion mass spectrometry (TOF-SIMS) was also used to confirm (Fig. S7–S9†) the presence of boron and fluorine in the PBA-MSN and CD-PBA-MSN samples. On the other hand, neither boron nor fluorine are present in the AP-MSN precursor. These results show that the grafting of PBA onto MSN is successful and the C-βCD can indeed bind to the PBA and form boronate esters. The binding between PBA and catechol is fairly stable in aqueous solution. In order to ascertain whether the binding is strong enough to maintain the boronate ester linkages even
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Fig. 2 (a) Release of PI from CD-PBA-MSNs after treatment with different concentrations of glucose. (b) Release of PI from CD-PBA-MSNs with the different sugars (5 mM each) (fructose = red, glucose = black, maltose = blue, and sucrose = pink). Here, the original solution was removed carefully after taking baselines and new solutions of different sugars were added carefully to initiate the release of PI. Inset: bar graphs of percent released cargo upon treatment with sugars (5 mM each) after 16 h.
under diluted conditions, we loaded the PBA-MSNs with PI before capping with C-βCD and investigating their release behaviour. We use a continuous-monitoring fluorescence spectroscopic method to study the binding. If the PBA-C-βCD complex disassociates in diluted aqueous solution, the C-βCD will not be able to block the nanopores and PI will diffuse into the solution, causing an increase in the fluorescence. On the contrary, there is no obvious release (Fig. 2a) of PI during the first hour of the experiment. Only when a glucose solution was added, was a gradual increase in fluorescence intensity observed, indicating that the C-βCD-PBA complex was displaced by the glucose-PBA complex. In fact, because the size of glucose is much smaller than that of the nanopores’ orifices, the entrapped PI molecules are now able to diffuse out from
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the nanopores, causing the increase in the fluorescence intensity. Moreover, the release rate of PI varies as a function of the glucose concentration. At a low glucose concentration (1 mM), MSNs release 5% of the encapsulated cargo after 14 h of treatment, whereas almost 30% of the cargo molecules are released after the same period of time at a higher concentration (25 mM, Fig. 2) of glucose. This observation is in agreement with the proposed competitive binding mechanism. In order to probe the binding further, different sugar solutions were employed to initiate the cargo release. The release profiles are shown in Fig. 2b. Fructose and glucose trigger the release of the cargo from the nanopores faster than the other sugars, an observation which is consistent with the corresponding sugar-PBA-MSNs complex binding constant. The ratedifference among sugars seems to have a correlation with the accessibility of their furanose forms in solution.31 These results clearly demonstrate the binding-constant dependent responses and the competitive binding nature of the CD-PBA-MSNs system. It is well known that the binding between catechol and boronic acid is much weaker in an acidic environment. In order to demonstrate this pH-dependent feature, the release of PI entrapped in CD-PBA-MSNs was evaluated at different pH values. The resulting release profiles are shown in Fig. 3. As expected, the release rate of PI correlated with the final solution pH. In the case of pH 6.0, 30% of PI was released in about 6 h, while at pH 4.0, about 85% of PI was released in the same period of time. The rates of all the acid-triggered releases are much faster than those of the sugar-induced releases, as a consequence of the fact that the dye molecules are positively charged. Acidification changes the charge environment inside the nanopores, and assists the diffusion of positively charged cargo molecules from the nanopores.23 In the case of the sugar-induced stimulus, the release of the dye molecules relies mainly on their passive diffusion, which is driven by concentration gradients. After confirming that both sugar and acid can disassociate C-βCD and PBA, we turned our attention to the investigation of their binding behaviour in the presence of both stimuli. Fig. 4
Fig. 3
Release of PI from CD-PBA-MSNs after acidification from pH 7.2.
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ally, because of the acidic environment, the diffusion of the dye molecules is faster than that observed in the sugarinduced release, which also contributes to the overall synergistic effect caused by the combination of the two stimuli. When more acidic conditions ( pH 5.0) are introduced along with the glucose solution, the result (Fig. 4b) of the acid-sugar combination is similar to that arising from the accumulated effect of the stimuli. This observation is in agreement with our aforementioned explanation. Because of the higher proton concentration, the equilibrium already largely favours the disassociation of C-βCD, and so the addition of sugar molecules provides a minor influence on the equilibrium and results in a much less prominent synergistic effect.
Conclusions
Fig. 4 Release profile traces of PI from CD-PBA-MSNs at pH 6 (a) and pH 5 (b) in the presence of 5 mM glucose.
In conclusion, we have employed mesoporous silica nanoparticles as platforms for studying the binding properties of catechol-boronate esters on silica surfaces. The assembly of C-βCD and PBA serves as a model for catechol-boronate ester binding, as well as a controlled-release platform. We have demonstrated that sugar molecules can cause the dissociation of the C-βCD-PBA complex by means of a competitive binding mechanism, where the disassociation rate correlates with both the binding affinity and the concentration of sugar. We have shown that this binding is pH-responsive, to the extent that the lower the pH the faster the disassociation. We have also demonstrated that the combination of sugar and acid provides a synergistic effect on the disassociation process. We believe that this sugar and pH dual responsive hybrid system will inspire the development of new drug delivery protocols with advanced imaging and therapeutic goals.
Acknowledgements shows the release profiles resulting from these dual-responsive experiments. When mild acidic conditions ( pH 6.0) were employed, we observe that (Fig. 4a) the combination of sugar and acid induces a faster release rate that goes beyond a simple additive effect. Glucose resulted in around 7% release in 6 h, while pH 6.0 resulted in 30% PI release. If the effects of sugar and acid are simply additive, a 37% release after 6 h is expected. When both sugar and acid were introduced, however, a 70% release was detected in the same amount of time. This rate is nearly twice that based on a simple accumulative assumption. One possible explanation is that under these mild acidic conditions, the disassociation of the boronate ester caused by protonation is relatively slow, implying that the equilibrium only slightly favours the disassociation over the rebinding process. With the addition of a glucose solution, the glucose molecules compete with the C-βCD and therefore decrease significantly the probability of C-βCD rebinding with PBA. This protocol causes a shift of the equilibrium and therefore enhances the disassociation of the C-βCD, which translates into a faster release process. Addition-
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This research was supported by the NIH RO1 CA133697, by the Defense Threat Reduction Agency HDTRA1-13-1-0046, and is part of the Joint Center of Excellence in Integrated Nanosystems (JCIN) at King Abdul-Aziz City for Science and Technology (KACST) and Northwestern University (NU) (Project 34-941). The authors would like to thank both KACST and NU for their continued support of this research. Y. W. would like to thank the Fulbright Scholar Program for a Research Fellowship.
Notes and references 1 (a) Y. Lu, R. Ganguli, C. A. Drewien, M. T. Anderson, C. J. Brinker, W. Gong, Y. Guo, H. Soyez, B. Dunn, M. H. Huang and J. I. Zink, Nature, 1997, 389, 364–368; (b) B. G. Trewyn, I. I. Slowing, S. Giri, H.-T. Chen and V. S. Y. Lin, Acc. Chem. Res., 2007, 40, 846–853; (c) M. W. Ambrogio, C. R. Thomas, Y.-L. Zhao, J. I. Zink and J. F. Stoddart, Acc. Chem. Res., 2011, 44, 903–913;
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