Letter pubs.acs.org/Langmuir
Glucose- and pH-Responsive Charge-Reversal Surfaces B. V. V. S. Pavan Kumar, Krishnachary Salikolimi, and M. Eswaramoorthy* Nanomaterials and Catalysis Lab, Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India S Supporting Information *
ABSTRACT: We have shown a pH- and glucose-responsive charge reversal on silica surface through heterogeneous functionalization utilizing amines and boronic acid moieties. The dual responsiveness of the charge reversal has been unambiguously demonstrated through the desorption of charged chromophores. Interestingly, we observed a concentration-dependent desorption response to glucose at physiologically relevant levels.
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INTRODUCTION Systems capable of having charge reversal in response to external stimuli such as pH,1−4 electrical potential,5 and light6,7 have attracted a lot of attention in recent years by virtue of their applications in switchable assemblies,6,8 permselective transport,9−11 controlled cell adhesion,5 drug/gene delivery,12−14 switchable electrode interfaces,15,16 and bacterial targeting.17−19 The charge-reversal systems designed so far often respond to only one type of stimulus. To our knowledge, there are only a few systems which are capable of responding to multiple stimuli, therefore widening the scope of their application in various environments. Systems responding to both pH and biomolecule stimuli are well suited for application in living systems and more specifically glucose-responsive systems20,21 owing to the role of glucose as a ubiquitous fuel for biological systems. Furthermore, if the system responds to the stimulus in a quantitative way, then it would help to regulate its response in a controlled fashion. Herein, we report a heterogeneously functionalized mesoporous silica system to achieve charge-reversal surfaces which respond to both pH and glucose. We chose mesoporous silica (MCM-41) as a scaffold to study charge reversal due to its large and easily modifiable surface and its wide range of applications in biology. The surface was covalently functionalized with propylamine and phenylboronic acid groups to impart pHresponsive charge reversal (MCM-B). At low pH (8), the amine groups are sparsely protonated while the boronic acid binds with hydroxyl ion to form a negatively charged tetrahedral boronate ion, leaving the overall surface charge negative (Figure 1 and S1, Supporting Information - SI). Additionally, the shifting of equilibrium from the neutral trigonal form of phenylboronic acid to its negatively charged tetrahedral form on binding to glucose22 at pH 7.4 was exploited for glucose-responsive charge © 2014 American Chemical Society
Figure 1. Schematic illustrating the dual-mode charge reversal of a functionalized mesoporous silica surface (MCM-B).
Received: January 30, 2014 Revised: April 9, 2014 Published: April 9, 2014 4540
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Figure 2. (a) Variation of zeta potential with the change in pH for MCM-N, MCM-B, and MCM-B bound to glucose (100 mM). Desorption of brilliant yellow (negatively charged) at pH 7.4 showing (b) the concentration-dependent response and (c) the stepwise response on increasing glucose concentration at fixed intervals. (d) Desorption profile of a positively and negatively charged chromophore on addition of glucose (100 mM) at pH 7.4. centrifuged after 24 h, and the supernatant was analyzed to monitor cumulative dye desorption. To monitor the time-dependent desorption, aliquots (0.2 mL) were taken at predetermined time intervals to monitor the dye desorption, and the volume of the sample was adjusted with buffer (containing the same concentration of glucose as being used in the respective desorption study). Stepwise Dye Desorption. The BY-loaded MCM-B (5 mg) sample was dispersed in 5 mL of HEPES buffer (10 mM, pH 7.4) at room temperature. To this solution increasing amounts of glucose were added at intervals of 3 h. Aliquots (0.2 mL) were taken every hour to monitor the dye desorption, and the volume of the sample was adjusted with buffer (containing glucose at the respective concentration in the desorption study). The amount of dye released was monitored by UV−visible absorption spectroscopy. Saccharide-induced desorption: The procedure followed was the same as for the time-dependent desorption, with the only difference being that the concentration of the saccharides (mannose or fructose) added was 0.1 mM. Stepwise desorption: The procedure followed was the same as in the case of glucose. pH-Induced Desorption. BY-loaded MCM-B (5 mg) was dispersed in 5 mL of pH 4 solution at room temperature. The pH of the solution was gradually increased in small steps to pH 8.5. Aliquots (0.2 mL) were taken at regular intervals of 15 min to monitor the dye desorption, and the volume of the sample was adjusted with Millipore water Crystal violet-loaded MCM-B (5 mg) was dispersed in 5 mL of pH 8.5 solution at room temperature. The pH of the solution was gradually lowered in small steps to pH 4. Aliquots (0.2 mL) were taken at regular intervals of 15 min to monitor the dye desorption, and the volume of the sample was adjusted with Millipore water.
reversal (Figure 1 and S1, SI). The glucose-driven chargereversal was a controlled process and exhibited a quantitative response to the concentration of glucose.
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EXPERIMENTAL SECTION
Materials. N-Cetyltrimethylammonium bromide, tetraethyl orthosilicate, (3-aminopropyl)triethoxysilane, 4-carboxyphenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, Nhydroxysuccinimide, brilliant yellow (BY), alizarin red S, crystal violet, 1,3,6,8-pyrenetetrasulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and D-glucose were purchased from SigmaAldrich. All solvents were purchased from Spectrochem. Synthesis of MCM-B. Mesoporous silica nanoparticles (MCM-41) were synthesized according to a reported procedure.23 Surfactant-free MCM-41 (1.0 g) and 1.0 mL (5.67 mmol) of (3-aminopropyl)triethoxysilane (APTES) were added to 80 mL of anhydrous toluene and then refluxed under stirring for 24 h. The reaction mixture was centrifuged and washed extensively with toluene, hexane, and ethanol to get MCM-N. N-hydroxysuccinimide (0.2 g, 1.74 mmol) and 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.4 g, 2.08 mmol) were dissolved in 10 mL of anhydrous DMF, and then 4carboxyphenylboronic acid (0.3 g, 1.8 mmol) was added. This reaction mixture was stirred at room temperature under an inert atmosphere for 15 min before adding it to a dispersion of 0.8 g of MCM-N in 40 mL of anhydrous DMF. The mixture was stirred for another 24 h, followed by centrifugation and washing with DMF, water, and finally methanol. Dye Loading. MCM-B (5 mg) was dispersed in 1 mL of 1 mM dye solution at pH 7.4 for glucose-responsive desorption and at either pH 4 or 8.5 for pH-responsive desorption. The dispersion was stirred for 12 h, centrifuged, and washed extensively to remove physisorbed dye. The supernatant and washings were analyzed to quantify the amount of dye loading. Glucose-Induced Dye Desorption. Single-Step (Cumulative) and Time-Dependent Desorption. The BY-loaded MCM-B (3 mg) was dispersed in 4 mL of HEPES buffer (10 mM, pH 7.4) with different concentrations of glucose at room temperature. The suspensions were
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RESULTS AND DISCUSSION
MCM-41 nanoparticles with an average size of 100 nm (Figure S2, SI) were prepared following a well-known procedure,23 and the surface was functionalized with (3-aminopropyl)triethoxysilane to form MCM-N. The amine groups were 4541
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then covalently linked to the −COOH group of carboxyphenylboronic acid using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling to form MCM-B (Scheme S3, SI). Thermogravimmetric analysis and the ninhydrin test24 show that 40% of the amine groups are coupled to carboxyphenylboronic acid through an amide bond (Figures S4 and S5, SI). Nitrogen adsorption−desorption isotherms were recorded at 77 K to follow the progress of functionalization inside the pores (Figure S6a, SI). The Barret−Joyner−Halenda (BJH) pore size distribution showed a gradual decrease in pore size from 2.4 nm (MCM) through 2.2 nm (MCM-N) to 1.8 nm (MCM-B), indicating progressive functionalization inside the pores (Figure S6b, SI). The presence of a low-angle peak in the powder X-ray diffraction pattern of MCM-B confirms the retention of mesostructural ordering on functionalization (Figure S7, SI), which is further supported by the transmission electron microscopy (TEM) analysis (Figure S2c,d, SI). The number of active boronic acid groups available for binding to cis diols was estimated to be ∼0.5 mmol/g using an Alizarin red S (ARS) binding assay25 (Figures S8 and S9, SI). The binding of ARS to boronic acid groups turns on its fluorescence while its displacement from boronic acid by glucose quenches the fluorescence. This allowed us to follow the binding of boronic acid groups in MCM-B with glucose through emission spectra (Figure S10, SI). To probe the surface charge we used zeta potential measurements. The variation of zeta potential was monitored with the change in pH in the range of 3−9. Aminopropylfunctionalized MCM-N exhibited a zeta potential of +45 mV at pH 3, which decreased to +5 mV at pH 9 without showing any sign of charge reversal (Figure 2a). It should be noted that even at pH 3 only less than 50% of the amine groups in MCM-N would be protonated due to charge crowding.26 On the other hand, MCM-B, which has both amine and phenylboronic acid groups, shows charge reversal on increasing the pH from 3 to 9 (Figure 2a). At pH 3, MCM-B is positively charged at +47 mV, similar to that of MCM-N which can be understood by considering the fact that still a significant number of amine groups are available for protonation in MCM-B. The zeta potential of MCM-B shows a slow but gradual decrease up to pH 6, beyond which there is a steep drop in the surface charge. The surface charge drops to zero at around pH 7.6 and goes to the negative side as the pH is increased. This suggests that the pKa of the carboxyphenylboronic acid (pKa = 8.027) is shifted to a lower value in the presence of amine groups.28,29 As the pH is increased further, the zeta potential goes on decreasing due to the increased formation of negatively charged boronate ions and the neutralization of amines. The zeta potential decreases to about −30 mV at around pH 9. The isoelectric point of MCM-B falls at pH 7.6, whereas for MCMB bound to glucose (MCM-B-glu) it is at pH 7.2 (Figure 2a). At pH 7.4, MCM-B is still positively charged and MCM-B-glu is negatively charged. Therefore, the binding of glucose has caused a charge reversal of the system at lower pH (Figure S11, SI). The reversal of charge at pH 7.4 by glucose was further verified by studying the desorption behavior of a negatively charged chromophore, brilliant yellow (BY) with increasing concentration of glucose (Figure 2b). It was observed that the desorption of BY showed a concentration-dependent response. This was further illustrated by monitoring the desorption profile of BY with time at different glucose levels at pH 7.4 (Figure S12, SI). The desorption profile in the absence of
glucose showed marginal release with time in comparison to the desorption in the presence of glucose. The steplike desorption profile for incremental doses of glucose clearly shows the versatility of the system to respond to the sequential addition of glucose (Figure 2c). The crystal violet (positively charged chromophore)-loaded MCM-B showed no desorption on addition of glucose, in contrast to the BY-loaded MCM-B which showed a strong desorption response, confirming the reversal of charge induced by the binding of glucose (Figure 2d). To further prove the glucose-induced charge reversal we studied the desorption of another negatively charged dye, 1,3,6,8-pyrenetetrasulphonic acid, which showed a similar desorption profile to that of BY (Figure S13, SI). The interference by fructose and mannose was studied to check the selectivity of our system. At their maximum concentration in blood (0.1 mM), no significant interference was found (Figure S14, SI), but at equal concentrations, fructose exhibited a 40% greater response than glucose while mannose showed a response similar to that of glucose (Figure S15, SI). The pH-induced charge reversal was studied by monitoring the desorption behavior of BY adsorbed on MCM-B at pH 4 (Figure 3a). As the pH was gradually increased, we see that there is not much release of BY until about pH 7, which can be attributed to the fact that MCM-B is still highly positively charged. Moreover, the two negative charges on BY lead to a strong electrostatic interaction with MCM-B, giving a weak
Figure 3. Stepwise desorption profiles of (a) negatively charged brilliant yellow with increasing pH and (b) positively charged crystal violet dye with decreasing pH from MCM-B. 4542
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(3) Mikhail, M.; Tsz Kin, T.; Marcos, P.; Ihor, T.; Evgeny, K.; Sergiy, M. Switchable Selectivity for Gating Ion Transport with Mixed Polyelectrolyte Brushes: Approaching ‘Smart’ Drug Delivery Systems. Nanotechnology 2009, 20, 434006. (4) Tam, T. K.; Pita, M.; Motornov, M.; Tokarev, I.; Minko, S.; Katz, E. Modified Electrodes with Switchable Selectivity for Cationic and Anionic Redox Species. Electroanalysis 2010, 22, 35−40. (5) Ng, C. C. A.; Magenau, A.; Ngalim, S. H.; Ciampi, S.; Chockalingham, M.; Harper, J. B.; Gaus, K.; Gooding, J. J. Using an Electrical Potential to Reversibly Switch Surfaces between Two States for Dynamically Controlling Cell Adhesion. Angew. Chem., Int. Ed. 2012, 51, 7706−7710. (6) Hu, L.-C.; Yonamine, Y.; Lee, S.-H.; van der Veer, W. E.; Shea, K. J. Light-Triggered Charge Reversal of Organic−Silica Hybrid Nanoparticles. J. Am. Chem. Soc. 2012, 134, 11072−11075. (7) Han, G.; You, C.-C.; Kim, B.-j.; Turingan, R. S.; Forbes, N. S.; Martin, C. T.; Rotello, V. M. Light-Regulated Release of DNA and Its Delivery to Nuclei by Means of Photolabile Gold Nanoparticles. Angew. Chem., Int. Ed. 2006, 45, 3165−3169. (8) Baram, J.; Shirman, E.; Ben-Shitrit, N.; Ustinov, A.; Weissman, H.; Pinkas, I.; Wolf, S. G.; Rybtchinski, B. Control over Self-Assembly through Reversible Charging of the Aromatic Building Blocks in Photofunctional Supramolecular Fibers. J. Am. Chem. Soc. 2008, 130, 14966−14967. (9) Yasui, M.; Hazama, A.; Kwon, T.-H.; Nielsen, S.; Guggino, W. B.; Agre, P. Rapid Gating and Anion Permeability of an Intracellular Aquaporin. Nature (London) 1999, 402, 184−187. (10) Tagliazucchi, M.; Azzaroni, O.; Szleifer, I. Responsive Polymers End-Tethered in Solid-State Nanochannels: When Nanoconfinement Really Matters. J. Am. Chem. Soc. 2010, 132, 12404−12411. (11) Yameen, B.; Ali, M.; Neumann, R.; Ensinger, W.; Knoll, W.; Azzaroni, O. Single Conical Nanopores Displaying pH-Tunable Rectifying Characteristics. Manipulating Ionic Transport with Zwitterionic Polymer Brushes. J. Am. Chem. Soc. 2009, 131, 2070− 2071. (12) LaManna, C. M.; Lusic, H.; Camplo, M.; McIntosh, T. J.; Barthélémy, P.; Grinstaff, M. W. Charge-Reversal Lipids, PeptideBased Lipids, and Nucleoside-Based Lipids for Gene Delivery. Acc. Chem. Res. 2012, 45, 1026−1038. (13) Han, L.; Zhao, J.; Zhang, X.; Cao, W.; Hu, X.; Zou, G.; Duan, X.; Liang, X.-J. Enhanced siRNA Delivery and Silencing Gold− Chitosan Nanosystem with Surface Charge-Reversal Polymer Assembly and Good Biocompatibility. ACS Nano 2012, 6, 7340−7351. (14) Xu, P.; Van Kirk, E. A.; Zhan, Y.; Murdoch, W. J.; Radosz, M.; Shen, Y. Targeted Charge-Reversal Nanoparticles for Nuclear Drug Delivery. Angew. Chem., Int. Ed. 2007, 46, 4999−5002. (15) Bocharova, V.; Katz, E. Switchable Electrode Interfaces Controlled by Physical, Chemical and Biological Signals. Chem. Rec. 2012, 12, 114−130 and references therein. (16) Katz, E.; Bocharova, V.; Privman, M. Electronic Interfaces Switchable by Logically Processed Multiple Biochemical and Physiological Signals. J. Mater. Chem. 2012, 22, 8171−8178 and references therein. (17) Pranzetti, A.; Mieszkin, S.; Iqbal, P.; Rawson, F. J.; Callow, M. E.; Callow, J. A.; Koelsch, P.; Preece, J. A.; Mendes, P. M. An Electrically Reversible Switchable Surface to Control and Study Early Bacterial Adhesion Dynamics in Real-Time. Adv. Mater. 2013, 25, 2181−2185. (18) Liu, Y.; Mu, L.; Liu, B.; Zhang, S.; Yang, P.; Kong, J. Controlled Protein Assembly on a Switchable Surface. Chem. Commun. 2004, 1194−1195. (19) Radovic-Moreno, A. F.; Lu, T. K.; Puscasu, V. A.; Yoon, C. J.; Langer, R.; Farokhzad, O. C. Surface Charge-Switching Polymeric Nanoparticles for Bacterial Cell Wall-Targeted Delivery of Antibiotics. ACS Nano 2012, 6, 4279−4287. (20) Hoare, T.; Pelton, R. Charge-Switching, Amphoteric GlucoseResponsive Microgels with Physiological Swelling Activity. Biomacromolecules 2008, 9, 733−740.
desorption response of 7% at pH 7. At pH 8 and 9, the desorption was 20 and 48%, respectively. From the zeta potential data, it can be seen that MCM-B is negatively charged above pH 7.6, so it is to be expected that desorption should be complete at pH 8. Instead, the desorption at pH 9 (48%) is greater than at pH 8 (20%). We can be reconciled to this fact by taking into account that MCM-B is a system with heterogeneous functionalization, and the zeta potential gives us an idea of the overall charge of the system but not its spatial distribution. From the zeta potential data of MCM-N at pH 8, it can be understood that the amine groups would still be protonated in MCM-B. Hence even though the surface as a whole has gained a negative charge, the presence of localized positive charges on the amine groups enable the negatively charged chromophore to bind to the surface. On further increasing the pH to 9, there is an increased desorption of brilliant yellow due to the neutralization of amines and negative charging of boronic acid groups. To further verify the pHinduced charge reversal, the desorption of the positively charged chromophore (crystal violet - CV) was also monitored with respect to pH (Figure 3b). The desorption profile shows its maximum response in the pH range of 6 to 7. The presence of amine groups adjacent to boronic acid is known to shift its pKa to smaller values,28,29 which in turn increases the desorption response in the pH range of 6 to 7. The desorption is not complete even after reaching pH 4, where 65% of CV is still adsorbed on MCM-B. It should be noted that unlike BY, CV is monopositive and the positive charge is highly delocalized, which in turn weakens the electrostatic repulsions. In summary, we have shown a simple system capable of charge reversal showing a response to dual stimuli, pH as well as glucose. The charge reversal was carefully studied using zeta potential measurements as well as the desorption of charged chromophores to reveal the heterogeneity in surface charge. The uneven distribution of boronic acid groups on the silica surface gave rise to a range of binding sites for glucose, allowing for multiple levels of response.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Detailed account of experimental methods, supporting figures and instrumentation. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*E-mail:
[email protected] Author Contributions
B.V.V.S.P.K. and K.S. contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS B.V.V.S.P.K. thanks CSIR for a fellowship. REFERENCES
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