Designed Glucose-Responsive Microgels with Selective Shrinking

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Designed Glucose-Responsive Microgels with Selective Shrinking Behavior Christophe Ancla,† Veronique Lapeyre,† Isabelle Gosse,† Bogdan Catargi,‡ and Valerie Ravaine*,† † ‡

Institut des Sciences Moleculaires, ENSCBP, Universite Bordeaux, 16 Av. Pey Berland, 33607 Pessac Cedex, France CBMN UMR 5248, Universite Bordeaux, Allee de Saint-Hilaire, 33600 Pessac, France

bS Supporting Information ABSTRACT: We report on the synthesis of various glucoseresponsive microgels based on N-alkylacrylamide derivatives and phenylboronic acid (PBA) as a glucose sensing moiety. Depending on their chemical composition, the microgels exhibit opposite behaviors in response to glucose concentration increase: they can either swell or shrink, using two different mechanisms for glucose recognition. Both behaviors may be suitable for glucose sensing and insulin delivery. When glucose binds a single boronate receptor, the microgel swells as glucose concentration increases. This mechanism can be used to deliver a drug by diffusion through the network. In other cases, glucose binds specifically to two boronates, which creates additional crosslinks within the network and provokes shrinkage. Such systems are promising for the development of sensors with improved selectivity and also as potential “intelligent” valves in microfabricated delivery systems. By a rational choice of the constituting units of the network structure, we show how to favor one or the other type of response to glucose variation. Therefore, glucose-swelling microgels operating under physiological conditions have been obtained by copolymerization with an appropriate choice of alkylacrylamide monomer and boronate derivative. At a pH above the pKa of the boronic acid derivative, the same structures shrink in response to glucose concentration. The nature of the cross-linker is a key parameter to enable this dual behavior. In other microgels, an amine group is introduced in the vicinity of the boronic acid, which lowers its pKa and favors microgel contraction at physiological pH. This work has allowed us to give some general rules to control the swelling/shrinking behavior of glucoseresponsive microgels.

’ INTRODUCTION The development of responsive (nano)structures is currently a major area of interest in nanotechnology. Bioresponsive materials focus particular attention because they can mimic natural systems, by changing their structure and function in response to a fine change in their environment. Glucose-responsive hydrogels, which can swell or shrink in response to glucose concentration variations, have pioneered this area1
4 while promising challenging application in the field of diabetes, as potential self-regulated insulin delivery systems.5 For a few years, the size of such systems has been scaled down to nano- and microgels,6
13 that can provide the suitable size for in vivo applications through blood circulation and exhibit much faster kinetics than their macrogel analogues.14,15 Both natural and synthetic ligands for glucose have been used. In the early studies, glucose oxidase was very popular and already proved many significant interests, but it presented certain severe disadvantages such as potential instability during use or sterilization. Conversely, phenylboronic acids (PBA) are highly stable and can be used as substitutes for natural receptors. These Lewis acids can reversibly bind to the cis-1,2- or cis-1,3-diols of saccharides to form a five- or six-membered boronic cyclic ester in aqueous media (Figure 1a). Most of the hydrogels functionalized r 2011 American Chemical Society

with PBA undergo this type of complexation and swell when glucose concentration increases. Indeed, the formation of a glucose
boronate complex,16 more stable than the glucose
boronic acid one,17 induces a shift in the ionization equilibrium and increases the fraction of charged phenylboronate. The charge density of the polymer increases simultaneously. The complexation also changes the polymer
solvent affinity: before complexation, in the neutral state, PBA has a hydrophobic character which becomes more hydrophilic upon complexation. The better polymer/solvent affinity and the increase of the Donnan potential are both parameters favoring the hydrogel swelling. However, a major issue for this class of ligands concerns their selectivity. Supramolecular chemists have already accomplished an important work to improve the selective recognition of selected sugars. Interestingly, while most physiological relevant sugars bind to boronic acid as monobidentate complexes, glucose has the unique property to be involved in a bis-bidentate glucose
boronic acid complex18 through its furanose form19,20 (Figure 1b). Therefore, this complexation is a key to improve the Received: July 27, 2011 Revised: September 4, 2011 Published: September 05, 2011 12693

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Figure 1. Equilibrium between a phenylboronic acid derivative and glucose in aqueous solution: (a) Conversion of the trigonal form of the boronic acid into the tetragonal form in the presence of OH
and the bidentate glucose
boronic acid complexation; (b) bis-bidentate glucose
boronate complexation with the furanose form of glucose in basic aqueous solution according to ref 19.

selectivity of polymeric systems for sugar detection. In this case, glucose
bis(boronate) complexes create additional cross-links that cause the gel to shrink. Asher’s team was the first to develop a glucose sensor based on this technology.21 Colorimetric sensors were obtained by embedding a crystalline colloidal array of nanoparticles within a glucose-sensitive hydrogel matrix. The lattice parameter of the colloidal crystal changed as the hydrogel swells or shrinks, thus provoking a shift in the diffraction wavelength. A blue shift related to the hydrogel shrinkage was thus observed upon glucose addition.21
23 Similarly, other colorimetric sensors were developed: instead of a colloidal crystal, holographic grating made of silver fringes was incorporated into a hydrogel matrix.24
27 These sensors also displayed a blue-shift in the diffraction wavelength due to the shrinkage of the hydrogel when glucose crosslinked two neighboring boronic acids.26,27 More recently, two other types of sensors used these supramolecular cross-links to detect glucose. The team of Stokke fabricated optical fibers modified at their end by a hydrogel. A change in the hydrogel swelling induced a change in their optical length which could be monitored by an interferometric fringe technique.28 Such sensors would be interesting for continuous in vivo monitoring of glucose and have shown minimal effect from interfering molecules.29,30 Li et al. used linear polymers modified with PBA moieties, that could undergo cross-linking upon bis-bidentate complex formation with glucose.31 In this case, the complexation induced a change in the solution viscosity, which could be monitored via a microelectromechanical system. Besides glucose monitoring application, glucose-responsive hydrogels are already attractive for their potential use as drug delivery device by two means. Thanks to their porous structure, hydrogels can entrap a drug and release it at a rate which is dependent on the diffusion across the network. Therefore, it is expected to be tuned by the hydrogel swelling state. To this end, hydrogels that can swell upon glucose addition are required because insulin will diffuse more rapidly across the network when this latter is extended.10,32 In a different strategy, hydrogels might be used as chemical valves incorporated in a silicon-based microdevice. In this case, the valving action requires a hydrogel

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that collapses and opens the circuit relating a pressurized insulin solution.33 This principle has been demonstrated using a glucoseshrinking hydrogel under nonphysiological conditions.34 The response kinetics of the sensitive hydrogels is a key issue concerning continuous in vivo monitoring as well as for insulin delivery application. Ben-Moshe et al.22 clearly showed that the kinetics was governed by hydrogel characteristics themselves, and neither by the formation of the bis-bidentate complex nor by the glucose diffusion within the hydrogel. Despite efforts to improve the gel formulation,22 the best results remained in the order of a few hundreds of seconds.22,29 The major parameter controlling the kinetics of hydrogel swelling/shrinking is its characteristic size.35 Reducing the hydrogel size is thus expected to decrease the response time. In particular, hydrogel particles of 200
350 nm diameter exhibit response times around 100 ns.14,15 Few microgels have already demonstrated their interesting potential for glucose sensing via fluorescence detection.13,32,36
38 These microgels swell as a function of glucose due to monobidentate formation. In this paper, we present the development of new glucoseresponsive microgels bearing phenylboronic acid that can either swell or shrink selectively according to glucose concentration. To the best of our knowledge, this is the first example of microgels cross-linked with bis-boronate complexes. Our intention is to show how to choose monomer composition to simply design microgels with a swelling/shrinking given behavior under physiological conditions. The interest of shrinkage over swelling will be compared in terms of selectivity.

’ EXPERIMENTAL SECTION Materials. All the reagents were purchased from Sigma-Aldrich unless otherwise noted. N-Isopropylacrylamide (NIPAM), N-isopropylmethacrylamide (NIPMAM), and N-ethylmethacrylamide (NEMAM) were recrystallized from hexane (ICS) and dried under vacuum prior to use. N,N0 -Methylenebis(acrylamide) (BIS), ethylene glycol dimethacrylate (EGDMA), allylamine (ALA), sodium dodecyl sulfate (SDS), and potassium persulfate (KPS) were used as received. 4-(1,6-Dioxo-2,5diaza-7-oxamyl)phenylboronic acid (DDOPBA) was synthesized according to the procedure described previously by Matsumoto et al.39 Deionized water, obtained with a Milli-Q system, was used for all synthesis reactions, purification, and solution preparation. Microgel Synthesis. The microgels were obtained by a method similar to an aqueous free-radical precipitation polymerization classically employed for the synthesis of thermoresponsive microgels and especially p-NIPAM microgels.40 The incorporation of the PBA derivative was performed by the copolymerization of DDOPBA with an alkylacrylamide (NIPAM, NIPMAM, or NEMAM) and a cross-linker agent (BIS or EGDMA). Polymerization was performed in a 200 mL three-neck round-bottom flask, equipped with a magnetic stir bar, a reflux condenser, a thermometer, and an argon inlet. The initial total monomer concentration was held constant at 70 mM, and the comonomer ratio ((100
x
y):x:y) (alkylacrylamide/cross-linker/ PBA derivative) was varied according to the desired receptor concentration. The alkylacrylamide, the cross-linker, and the surfactant (1 mM) were introduced into 47.5 mL of water. DDOPBA was dissolved in 1.5 mL of methanol and added to the previous solution. The whole solution was heated to 70 °C (to 80 °C in the case of NEMAM) and thoroughly purged with argon during at least 1 h prior to initiation. Free radical polymerization was then initiated with KPS (1.5 mM) dissolved in 2.5 mL and degassed during 10 min. The successful initiation was indicated by the occurrence of turbidity. The solution was allowed to react for a period of 6 h under argon. Note that EGDMA is poorly 12694

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Figure 2. Chemical structure of the different microgels.

Table 1. Summary of the Microgel Composition (x, y, and z Indicate the Molar Composition in the Feed) name

R1

R2

R3

R4

A1 A2 A3 B1 B11 B12 B2 B3 C1 C2 C3

H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

isopropyl isopropyl isopropyl isopropyl isopropyl isopropyl isopropyl isopropyl ethyl ethyl ethyl

H H H H H H H CH3 CH3 CH3 CH3

NH
CH2
NH NH
CH2
NH NH
CH2
NH NH
CH2
NH NH
CH2
NH NH
CH2
NH NH
CH2
NH O
CH2
CH2
O O
CH2
CH2
O O
CH2
CH2
O O
CH2
CH2
O

x y z (mol %) (mol %) (mol %) 2.5 2.5 2.5 2.5 2.5 2.5 5 10 2.5 10 10

5 10 15 10 10 10 10 10 10 10 15

separate size measurements, which themselves consist of 14 measurements with approximately 15 s integration time. Only one measurement per degree was carried out when recording a temperature program. The sample was allowed to equilibrate for 10 min between each temperature. Electrophoretic Mobilities. Experiments were carried out using the Zetasizer NanoZS (from Malvern Instruments, UK) instrument at the appropriate temperature. Particle electrophoretic mobilities were obtained after diluting the microgel suspension in the buffered solution (PBS or Tris buffer, 2 mM, pH adjusted with NaOH 1 M or HCl 1M) and after allowing the mixture to equilibrate for 10 min. Each value results from at least three cycles of 15 measurements. Elemental Analysis. The boron content in dilute suspensions of microgels was determined by atomic emission spectroscopy equipped with a He plasma and inductive coupling (ICPAES, Varian Liberty 220). The molar percentage of boron was calculated knowing the dried mass of polymer in the solution. Transmission Electron Microscopy. The core
shell particle morphology was visualized by transmission electron microscopy (TEM). One drop of the dilute suspension was deposited on a copper grid coated with a carbon membrane. A staining procedure using uranyl acetate was used to enhance the contrast after the microgels were deposited on the TEM grid. The grid was observed with a FEI Tecnai biotwin (120 kV) instrument.

’ RESULTS AND DISCUSSION 10 20

water-soluble. In this case, the method cannot be fully described as a precipitation polymerization procedure in which all monomers are water-soluble. Following synthesis, the microgels were purified by dialysis (Dialysis membrane, MCWO 10 000, Orange Scientific) against water (two changes per day for 2 weeks at 5 °C for NIPAM series and room temperature for other series). The microgels were then purified three times by centrifugation (21 255g) followed by redispersion in pure water, in order to remove water-soluble oligomers. They were finally lyophilized overnight. The same method was used to synthesize microgels containing an amino group. In this case, the comonomer ratio was the following: ((100
x
y
z):x:y:z) (alkylacrylamide/cross-linker/PBA derivative/ allylamine), with z being equal to 0, 10, and 20% mol. The chemical structure of the microgels is shown in Figure 2, and Table 1 summarizes the various compositions which have been synthesized. Characterization. Photon Correlation Spectroscopy. Particle sizes and polydispersities were determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano S90 Malvern Instruments apparatus operating with a HeNe laser at 90°. The hydrodynamic diameters dH were calculated from diffusion coefficients using the Stokes
Einstein equation. All correlogram analyses were performed with software supplied by the manufacturer. The polydispersity index (PDI) is given by the cumulant analysis method. Both material characterization and swelling behavior upon glucose recognition were investigated by this technique. Typically, a droplet of the initial particle suspension (10 μL) was dispersed in a buffered glucose solution, which had been prepared at least 1 day before. This procedure aimed at stabilizing glucose mutarotations.22 Before each data collection, the sample was allowed to equilibrate for 10 min at the appropriate temperature. Each data point reported is an average of five

1. Design of Glucose-Responsive Microgels Operating under Physiological Conditions. Glucose-responsive micro-

gels are obtained by the copolymerization of an alkylacrylamide monomer, a cross-linker, and a phenylboronate derivative as glucose sensing monomer. The introduction of an alkylacrylamide derivative helps to carry out simple precipitation polymerization, which usually yields monodisperse particles. A few years ago, we made the choice of copolymerization against postgrafting6 for two reasons. First, their synthesis is easier, and thus, hydrogel particles can be obtained in one step. Second, the grafting is not perfectly well-controlled: it requires the accessibility of the reactant to the functionalizable groups, which depends on their localization in the initial platform.8 The copolymerization method was further used to build up more advanced structure such as core
shell microgels10 or hollow microgels made of concentric hydrogel layers.11 In our first study, we synthesized microgels that could sense glucose under physiological salinity but not physiological pH or body temperature. In the present work, we chose selected building blocks to build up microgels with glucose sensing properties under physiological conditions. The copolymerization process requires taking into account their relative reactivity. The previously reported glucose-responsive microgels were sensitive to glucose through the equilibrium between phenylboronic acid and glucose depicted in Figure 1a. As shown, PBA derivatives exist in both charged and neutral forms in aqueous solution. However, upon glucose addition, the charged state makes a stable complex with glucose through reversible covalent binding16 whereas the neutral form is highly susceptible to hydrolysis.17 If the pH is close to the pKa of PBA, the acid (neutral) and the base (charged) are in equilibrium but the addition of glucose shifts the equilibrium toward the charged form. When used in combination with a thermosensitive polymer such as poly-N-isopropylacrylamide (pNIPAM), characterized by a lower critical solution temperature (LCST), the addition of glucose shifts the LCST of the polymer toward higher 12695

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Table 2. Summary of the Microgel Characterization for Various Microgel Compositionsa name

N-alkyl(meth)-

cross-linker

glucose receptor

boron content

WSP

maximal operating

dH (nm) (PDI) at

dH (nm) (PDI) at

acrylamide

(mol %)

(mol %)

(mol %)b

(wt %)v

temperature (°C)

T = 15 °C, pH 7.4

T > VPTT, pH 7.4

A1

NIPAM

BIS (2.5%)

DDOPBA (5%)

3.0

17

27

353 (0.014)

198 (0.043)

A2

NIPAM

BIS (2.5%)

DDOPBA (10%)

9.8

10

23

326 (0.041)

205 (0.031)

A3

NIPAM

BIS (2.5%)

DDOPBA (15%)

17.3

5

18

265 (0.05)

203 (0.013)

B1

NIPMAM

BIS (2.5%)

DDOPBA (10%)

8.1

51

35

502 (0.023)

251 (0.117)

B3

NIPMAM

EGDMA (10%)

DDOPBA (10%)

8.6

49

35

515 (0.054)

309 (0.002)

C1

NEMAM

EGDMA (2.5%)

DDOPBA (10%)

n.a.

62

n.a.

n.a.

C2 C3

NEMAM NEMAM

EGDMA (10%) EGDMA (10%)

DDOPBA (10%) DDOPBA (15%)

7.6 10.6

59 57

410 (0.017) 361 (0.124)

234 (0.167) 224 (0.068)

n.a. 55 50

a Percent of bore content in the microgel, amount of water-soluble polymers (WSP), hydrodynamic diameter at 15°C, i.e., in the most swollen state and the corresponding polydispersity index (PDI), hydrodynamic diameter at T > LCST and the corresponding PDI (both measured in phosphate buffer 2 mM, pH = 7.4). n.a.: Not available. b The boron content in the final microgel was measured by elemental analysis. v The amount of water-soluble polymer was obtained gravimetrically after ultracentrifugation (50 000 rpm for 30 min).

temperature, because the fraction of charged (and also hydrophilic) states increases over the fraction of uncharged (and relatively hydrophobic) ones.41,42 If this polymer is cross-linked, the corresponding hydrogel presents a volume phase transition temperature (VPTT) from swollen to shrunken state, which depends on the concentration of glucose. Since this phenomenon occurs at a pH close to the pKa of the PBA, the choice of the PBA monomer is fundamental to achieve glucose sensitivity under physiological pH conditions. The chosen PBA derivative, DDOPBA, has a pKa of 7.8, and its synthesis has been described elsewhere.39 Compared to other monomers that derived from 4-aminophenylboronic acid, DDOPBA has a lower pKa because of the presence of the para-positioned carbamoyl group, an electron-withdrawing group which decreases the electron density on the boron atom and renders the boron more acidic.39 Regarding the working temperature, we showed previously that the glucose-swelling response depended on temperature.6 It was necessary that the initial state of the microgel was partially swollen, meaning that no swelling occurred above the VPTT. To achieve glucose-responsiveness at body temperature, it is necessary to find a microgel composition with a VPTT above 37 °C. Various microgels were synthesized by copolymerization of an alkylacrylamide derivative, a cross-linker, and DDOPBA as glucose sensing monomer. The results of their synthesis and their main properties in aqueous solution are reported in Table 2. Whatever the DDOPBA content below 15 mol % of the total monomers, monodisperse particles were obtained, as verified by TEM (Figure 3 and Supporting Information Figure S1) and indicated by their PDI, measured by dynamic light scattering. A composition bearing more than 15 mol % yielded polydispersed particles and aggregates. At first, NIPAM was chosen as a main monomer and various amounts of DDOPBA were copolymerized (A1, A2, A3). The amount of PBA moieties had a significant effect on the VPTT of the microgels. Indeed, the PBA moieties are hydrophobic and decrease the VPTT of the microgels, compared to that of the pure pNIPAM microgels. The more PBA content, the lower the VPTT (Table 2 and Supporting Information Figure S3). In order to increase the VPTT, two other alkylacrylamide derivatives were chosen: NIPMAM and NEMAM. Their corresponding homopolymers have a LCST of 45 °C43,44 and 65 °C,45 respectively. The cross-linker classically employed with NIPAM

Figure 3. TEM view of the microgels in the dried state. Example of NEMAM-EGDMA10%-PBA10%. Scale bar is 100 nm.

is N,N0 -methylenebisacrylamide (BIS). It was suitable to crosslink pNIPMAM-based compositions, with a ratio below 10 mol % (samples B1 and B2). At this point, particles became polydisperse. In the case of pNEMAM particles, BIS was not suitable because its reactivity is too high and it yielded polydisperse particles. Furthermore, if the cross-linker is consumed too rapidly, the end of the polymerization yields poorly cross-linked polymers which are water-soluble (water-soluble polymers abbreviated as WSP). Therefore, BIS had to be replaced by another cross-linker, EGDMA, which had a poor solubility in water, thus limiting its availability and its polymerization rate.46 The first trials were carried out with 2.5 mol % EGDMA (sample C1) and yielded about 60 wt % WSP. Moreover, the synthesized microgels showed a very weak response to glucose, thus indicating a poor PBA incorporation. Therefore, in order to limit the amount of WSP and promote PBA copolymerization, a higher amount of EGDMA (10 mol %) was introduced. Finally, we studied two new microgel compositions with NEMAM (C2 and C3). The influence of the cross-linker (ratio and chemical structure) was studied by comparing B1 and B2 (NIPMAM crosslinked with BIS) with B3 (NIPMAM cross-linked with EGDMA). For all the compositions with NIPMAM or NEMAM, 12696

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Figure 4. Evolution of the swelling ratio (hydrodynamic volume at given temperature over hydrodynamic volume in the collapsed state) with temperature (phosphate buffer, 2 mM, pH 7.4): NIPAM-BIS-PBA10% (A2), NIPMAM-BIS-PBA10% (B1), and NEMAM-EGDMA-PBA10% (C2).

Figure 5. Relative swelling ratio (hydrodynamic volume at given glucose concentration over hydrodynamic volume without glucose) as a function of glucose concentration (T = 37 °C, PBS buffer, pH 7.4): NEMAM-EGDMA-PBA10% (C2) and NEMAM-EGDMA-PBA15% (C3).

the amount of water-soluble polymers was rather high, in spite of the efforts made to select the appropriate nature and amount of cross-linker. The amount of incorporated boronic acid in the microgels was also lower than the feed. This suggests that PBA moieties were more concentrated within water-soluble polymers than in the microgels. Of importance, all the microgels were thermoresponsive, with a VPTT depending upon the choice of the alkylacrylamide monomer and the amount of phenylboronic moieties. Figure 4 plots the evolution of the swelling ratio (hydrodynamic volume at considered temperature over hydrodynamic volume in the fully collapsed state above the VPTT; the hydrodynamic volumes being extracted from DLS measurements) as a function of temperature. At constant PBA content, the VPTT increases in agreement with the expected order NIPAM < NIPMAM