Biomacromolecules 2008, 9, 733–740
733
Charge-Switching, Amphoteric Glucose-Responsive Microgels with Physiological Swelling Activity Todd Hoare* and Robert Pelton* Department of Chemical Engineering, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada L8S 4L7 Received October 31, 2007; Revised Manuscript Received November 29, 2007
Amphoteric, poly(N-isopropylacrylamide)-based microgels are functionalized with aminophenylboronic acid (PBA) functional groups to produce colloidally stable, glucose-responsive gel nanoparticles that exhibit glucose-dependent swelling responses at physiological temperature, pH, and ionic strength. Up to 2-fold volumetric swelling responses are observed in response to physiological glucose concentrations, the first such physiological response reported for a colloidally stable microgel. Amphoteric microgels can also be designed to both swell and deswell in response to glucose according to the pH of the medium, the concentration of PBA groups grafted to the microgel, and the relative concentrations of the cationic and anionic functional groups in the platform microgel. The increasing anionic charge density on the microgels observed at higher glucose binding fractions can be applied to switch the net charge of the microgels from cationic to anionic as the glucose concentration increases. Preliminary experiments suggest that such amphoteric PBA-microgels have a high capacity for insulin uptake and can selectively release more insulin at higher glucose concentrations under physiological conditions via glucose-induced, “on-off” switching of electrostatic attractions between insulin and the microgel.
Introduction Glucose-responsive materials have received considerable attention due to their potential to both sense changes in blood glucose levels and respond to these changes by regulating insulin release.1–4 Thermosensitive materials based on poly(N-isopropylacrylamide) (PNIPAM) or related N-alkylacrylamide derivatives have been of particular interest given their potential for facilitating environmentally triggerable swelling-deswelling or soluble-insoluble phase transitions. Furthermore, our previous work illustrated how the thermal phase transition of PNIPAMbased microgels can be applied to enhance or suppress charge-driven swelling transitions in glucose-responsive PNIPAM-based microgels, enabling the temperature to be used as a control switch for a targeted secondary phase transition.5 Glucose responsiveness in such materials is often achieved by incorporating phenylboronic acid (PBA) functional groups into the material.6–13 Ionized PBA groups bind to the cis-diol functional groups on glucose in equilibrium with the glucose solution concentration, according to the mechanism shown in Scheme 1. As more glucose is added to the microgel environment, the driving force for PBA-glucose complexation increases to generate more of the boronate-glucose complex (c). To maintain the chemical equilibrium at a given pH value, the boronic acid ionization equilibrium shifts to increase the concentration of the charged phenylboronate (b). When the PBA groups are anchored to a hydrogel network, this equilibrium shift increases the anionic charge density within the gel to drive a gel swelling response. A range of PNIPAM-based, PBA-functionalized linear polymers8,9 and hydrogels3,14 has been synthesized via the copolymerization of a PBA-containing comonomer or the postpolymerization grafting of a functional PBA derivative. * To whom correspondence should be addressed. E-mail: hoaretr@ mcmaster.ca (T.H.);
[email protected] (R.P.). Telephone: (905) 5259140, ext. 27045. Fax: (905) 528-5114.
Scheme 1. Reversible Binding of Glucose to (Alkylamido)phenylboronic Acid
However, linear polymers have only a limited capacity for reversible drug delivery responses, while hydrogels typically require relatively lengthy equilibration times that limit their practical use, particularly in blood glucose control applications in which large, rapid swings in glucose concentration can be observed. Microgels may therefore be attractive intermediates given their typically faster responses to environmental stimuli and the potential for controlled release through their hydrogel network microstructure. Glucose-responsive thermosensitive microgels have recently been explored in the literature both by us5 and others.7,12,13,15,16 However, the physiological utility of these microgels is significantly limited by two factors. First, given that the pKa of PBA moieties is ∼8.9,17 systems in which PBA alone (i.e., PBA with no electron-withdrawing substituents) is grafted to the polymer backbone are practically useful only at pH > 8, above physiological pH (∼7.4). In addition, most reported glucoseresponsive microgels have moderate to poor colloidal stability at the high ionic strength (∼0.15 M) of the physiological environment, particularly when heated to physiological temperature (37 °C). Indeed, while successful microgel responses under physiological temperature5 and ionic strength18 and have been reported, no microgel has yet been developed with both adequate colloidal stability and glucose swelling responses for physiological use.
10.1021/bm701203r CCC: $40.75 2008 American Chemical Society Published on Web 01/17/2008
734 Biomacromolecules, Vol. 9, No. 2, 2008
Hoare and Pelton
Table 1. Amphoteric Microgel Preparation Recipes moles in feed microgel
NIPAM
AA
DMAEA
CTAB
MBA
AMPA
APS
17–4(+) 22–5(+) 27–7(+) 17–5(-) 23–6(-) 28–8(-)
1.24 × 10-2 1.24 × 10-2 1.24 × 10-2 1.24 × 10-2 1.24 × 10-2 1.24 × 10-2
2.6 × 10-3 3.4 × 10-3 4.3 × 10-3 3.4 × 10-3 4.2 × 10-3 5.6 × 10-3
6.4 × 10-4 6.4 × 10-4 8.3 × 10-4 6.4 × 10-4 1.1 × 10-3 1.4 × 10-3
0.3 × 10-4 0.3 × 10-4 0.3 × 10-4 0.7 × 10-4 0.7 × 10-4 2.5 × 10-4
6.5 × 10-4 6.5 × 10-4 6.5 × 10-4 2.6 × 10-4 2.6 × 10-4 2.6 × 10-4
3.7 × 10-4 3.7 × 10-4 3.7 × 10-4 0 0 0
0 0 0 4.4 × 10-4 4.4 × 10-4 4.4 × 10-4
Amphoteric PNIPAM-based microgels containing both cationic and anionic functional groups have previously been prepared by Kokufuta’s group18,19 and investigated for their unique pH and temperature-responsive behaviors. Amphoteric microgels are useful for facilitating physiological glucose responses in PBA-functionalized amphoteric microgels in two ways. First, for polyelectrolyte gels containing only negative or positive charges, increasing the salt concentration lowers the Donnan equilibrium contribution to swelling and screens the electrostatic repulsions between fixed charges, driving gel collapse and (in the case of microgels) particle aggregation. In amphoteric gels, ionic cross-links formed within the gel at low salt concentrations are broken by charge screening at high salt concentrations. Consequently, amphoteric gels deswell much less than anionic gels in the presence of electrolyte; indeed, swelling and increased colloidal stability are often observed as the salt concentration increases.24 Second, physiological pH activity can concurrently be promoted via Lewis acid–base interactions between the electron-poor boron atom and the electron-rich nitrogen atom in the cationic monomer. This interaction increases the Lewis acidity of the boron center, lowers the pKa of the PBA residues, and protects the PBA-glucose complex from hydrolysis to increase the halflife of the charged boronate group at lower pH values.20 Amine groups have previously been incorporated into PBA-functionalized hydrogels and linear polymers for this purpose via copolymerization with N,N-(dimethylamino) propylacrylamide (DMPA),26 2-(dimethylamino) ethylmethacrylate (DMAEMA), or 2-(dimethylamino) ethylacrylate (DMAEA)21,22 or via covalent grafting of poly(L-lysine).23 In this paper, we illustrate how these properties of amphoteric polymers can be applied not only to induce targeted physiological glucose swelling responses in PBA-functionalized microgels but also to introduce the capacity for selectively switching the net charge of the microgel as a function of the glucose concentration. This charge-switching mechanism may provide a new option for facilitating the glucose concentration-controlled release of insulin in the physiological environment.
Experimental Section Materials. N-Isopropylacrylamide (NIPAM, 99%, Acros Organics) was purified by recrystallization from a 60:40 toluene:hexane mixture. Acrylic acid (AA, 99%, Aldrich) and N,N-dimethylamine ethylacrylate (DMAEA, 98%, Aldrich) were purified by vacuum distillation. N,NMethylene-bis-acrylamide (MBA, 99+%, Aldrich), cetyltrimethylammonium bromide (CTAB, 95%, Aldrich), 2,2′-azobis(2-methylpropionamindine) dihydrochloride (AMPA, 97%, Aldrich), ammonium persulfate (APS, 99%, BDH), N-3-dimethyl(aminopropyl)-N′-ethylcarbodiimide (EDC, commercial grade, Aldrich), 3-aminophenylboronic acid (APBA, 98%, Aldrich), human insulin (Aldrich), and D-glucose (Aldrich) were all used as received. The bicinchoninic acid (BCA) protein assays were performed using the BCA protein assay kit from Sigma-Aldrich using the recommended procedure. All water used in the synthesis and characterization was of Millipore Milli-Q grade.
Microgel Synthesis. A series of amphoteric microgels was synthesized using acrylic acid (AA) as the anionic monomer and N,Ndimethylamino ethylacrylate (DMAEA) as the cationic monomer. These two monomers were selected given their similar kinetic reactivities, promoting their colocalization within the microgel networks as per kinetic modeling work previously reported.24 Colocalization is particularly important in this application given that direct coordination between the amine and boronic acid groups is required to effectively lower the boronic acid pKa via Lewis acid interactions. The microgel recipes are given in Table 1. Each PBA-conjugated gel is labeled as “x-y(+/-)” where (+/-) is the charge of the initiator used in the synthesis, x is the mole percentage of AA in the feed, and y is the mole percentage of DMAEA in the feed. All reagents except the initiator are dissolved in 150 mL of distilled–deionized water and heated to 70 °C under nitrogen purge and 200 RPM mechanical mixing. After 30 min of preconditioning, the initiator is dissolved in 10 mL of water and added to the reactor. The reaction is allowed to proceed overnight. The product microgels are ultracentrifuged over five cycles (decanting the supernatant and resuspending the microgels in water between each cycle) to remove unreacted monomer and oligomers. The purified suspension is then lyophilized and stored dry. APBA Conjugation. Phenylboronic acid functional groups were incorporated into the microgel via the “graft to” approach outlined previously.5 Briefly, 36 mg of lyophilized microgel was suspended in 3.6 mL of MES buffer (pH 4.8, 20 mM ionic strength) in the presence of a specific APBA concentration (ranging from 1 mg/mL up to 10 mg/mL) under gentle magnetic stirring at 15 °C. After ∼30 min, 1.0 mL of a freshly prepared EDC solution (varying in concentration between 5 and 50 mg/mL) was immediately added to the vials. Each conjugation reaction was allowed to proceed for 2 h, at which point the microgel conjugates were isolated by four successive ultracentrifugation runs. All of the product microgel conjugates are assigned the code Ap Eq, where p is the concentration of the APBA and q is the concentration of EDC present in the conjugation solution (both in mg/ mL). Practically, these codes are representative of the relative phenylboronic acid graft densities in the microgels; the more APBA or EDC added to the conjugation reaction, the higher the PBA graft yield is achieved. The graft-modified microgels are stored in suspension at 4 °C prior to use. Measurement of PBA Functionalization. Prompt neutron activation analysis (PNAA) was used to determine the amount of boron present in the samples and, by extension, the graft yield of PBA functional groups to the microgels. Between 3 and 7 mg of a dried microgel sample was added at the midpoint of a 1 mL polyethylene cuvette filled to the top with fine sand. The samples were then placed in a thermalized beam of neutrons produced at the McMaster University nuclear research reactor. Samples are measured for the Doppler broadened prompt γ ray at 478 KeV using a high purity GE detector. Samples are compared to certified reference materials used to calibrate the system. Gamma radiation was counted by ActLabs (Ancaster, Ontario, Canada) with a detection limit of 1 µg. One sample was split to determine the reproducibility of the method; the relative error in the result was
