Degradable Thermoresponsive Nanogels for Protein Encapsulation

Article pubs.acs.org/bc

Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release Neha Bhuchar,† Rajesh Sunasee,† Kazuhiko Ishihara,‡ Thomas Thundat,† and Ravin Narain*,† †

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, T6G 2G6, Canada Department of Materials Engineering and Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

‡

S Supporting Information *

ABSTRACT: Reversible addition−fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h.

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INTRODUCTION Nanogels are networks of swollen cross-linked polymers in the nanometer range. These systems are increasingly being studied for their attractive properties in drug delivery systems, biotechnology, and biomedical fields.1−6 Nanosized particles have the advantage of enhanced permeability and retention (EPR) effect7 owing to their small size. Apart from their small size, extra stability in aqueous medium, and high water retention, they can also be made to respond to changes in the external environment, such as temperature and pH. These so-called “smart” nanogels are increasingly being considered for drug delivery systems (DDS).1,8 For example, change in size of pH controlled systems helps in controlled release of encapsulated drugs.9,10 Similarly, temperature sensitive nanogels undergo a phase change from hydrophilic to hydrophobic at a transition temperature called the lower critical solution temperature (LCST). Poly(methoxydiethylene glycol methacrylate (poly(MeODEGM)) and poly(N-isopropylacrylamide) (poly(NIPAM)) are examples of thermoresponsive polymers. NIPAM has a LCST of 32 °C and can be easily copolymerized with other hydrophilic monomers to raise the LCST close to body temperature.9,11 Despite the extensive biological studies, PNIPAM based materials have not yet been approved by FDA © 2011 American Chemical Society

due to toxicity concerns. On the other hand, MeODEGM is widely used in medicine and pharmacology because of its excellent biocompatibility with an LCST of 24 °C.12−14 In order to be used as DDS, surface functionalization of nanogels with organ specific ligands can be done to target specific tissues or organs. Nanocarriers have shown very high drug loading capacity (sometimes as high as 800%, i.e., 1 mg of nanogel can load about 8.0 mg of BSA).15 The success of using such nanocarriers relies on their ability for a controlled release of encapsulated drugs. Nanogels have been synthesized using various approaches: physical self-assembly, covalent cross-linking of preformed polymers, and template supported nanofabrication.16 Emulsionfree or precipitation polymerization is an easy way to produce nanomicelles where ionic surfactants are used to stabilize these nanoparticles in water. While emulsion polymerization is carried out in an aqueous medium and the core consists of the hydrophobic monomer, the inverse mini-emulsion polymerization technique is used for hydrophilic monomers like N-vinyl Received: July 20, 2011 Revised: November 30, 2011 Published: December 15, 2011 75

dx.doi.org/10.1021/bc2003814 | Bioconjugate Chem. 2012, 23, 75−83

Bioconjugate Chemistry

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caprolactam.17,18 The presence of residual surfactants with the nanogels has been a major concern of toxicity and hence is not viable for in vivo studies. Also, removal of surfactants from the nanogel is not easy. Self-assembly of amphiphilic polymers is another approach which eliminates the need for surfactants in the synthesis. Wooley et al. synthesized triblock cross-linked micelles forming stabilized nanogels.19−22 Multiresponsive polymeric micelles, which respond to changes in both pH and temperature, have been synthesized by Kuckling et al.3 Various living polymerization techniques are used for the synthesis of nanogels to maintain control over molecular weights and polydispersity of the polymer units.7 These techniques include ring-opening polymerization, ring-opening metathesis polymerization, cyanoxyl-mediated free radical polymerization, atom transfer radical polymerization (ATRP), reversible addition−fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated polymerization (NMP).23−26 RAFT polymerization is a suitable technique and is very popular for biological applications. Micelles containing RAFT agents are easy to cross-link and thus form core cross-linked,27−29 shell cross-linked,30,31or nexus between both.32,33 Self-assembly of the amphiphilic copolymers in water leads to the formation of micelles. However, under special conditions, like low concentration or high ionic environment, these micellar structures may not exhibit a strong amphiphilic character and hence can be unstable in aqueous solution, existing as unimers.32,34 Cross-linking of polymers can play a significant role in imparting the required stability. This technique combines self-assembly and cross-linking and provides excellent control over the “spatial distribution of polymeric chains” in an aqueous solution.16 Cross-linkers that degrade or hydrolyze under special conditions, for example, low pH, reductive environment, or the presence of dithiothreitol (DTT), are conducive for drug delivery, as their degradation causes the release of encapsulated macromolecules. Encapsulation of a drug prevents its fast clearance from body.32 Responsive nanogel systems have been shown to control the activity of biopolymers and, at the same time, control the release of a therapeutic drug.22,35,36 Previous reports have shown that nanoparticle/nanogel formation increases the stability of trapped enzymes/proteins against degradation and denaturation and modulated the release of these biomolecules at specific or targeted sites.37−40 Protein encapsulation in nanogels, microgels, nanotubes, and nanoparticles is under intense study, and these systems have shown immense potential in drug delivery systems.37,41−43 Protein encapsulation in hydrogel systems and controlled release of insulin, lysozome, calcitonin, and interleukin-2 has been studied intensively.22,44−46 The present study entails the synthesis, characterization, and application of thermoresponsive, acid degradable core crosslinked nanogels with a hydrophobic core composed of MeODEGM and AEMA cross-linked with an acid degradable cross-linker and a hydrophilic shell of poly(methacryloyloxyethyl phosphorylcholine) poly(MPC). Solubility and stability of the nanogels are attributed to the hydrophilic and zwitterionic nature of the poly(MPC) shell. The phosphorylcholine group on the methacrylate monomer resembles the polar phospholipid group present on the cell membrane. As a result, MPC based polymers show very high biocompatibility, and various copolymers of MPC are popularly

used for biomedical applications.47−49 The thermoresponsive MeODEGM core swells and shrinks with the change in temperature, which helps the entrapment of controlled encapsulation and release of therapeutic molecules. A cationic monomer, AEMA, is incorporated in the core to enhance the encapsulation of charged protein. A range of pH domains is found in the human body. While physiological pH is 7.4, pH values in endosomes and lysosomes are 5.5−6 and 4−5, respectively. The cross-linker is acid degradable, and the release of encapsulated protein was evaluated as a function of pH. Size trends and polydispersities of various nanogels synthesized by RAFT polymerization have been analyzed. Protein encapsulation and release from such nanogels have been evaluated and compared for different crosslinker concentrations.

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EXPERIMENTAL SECTION Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was obtained from NOF Co (Tokyo, Japan), which was synthesized by a method reported previously.50 2Hydroxyethyl methacrylate (HEMA), 2,2-dimethoxypropane, p-toluene sulfonic acid (pTSA), triethyl amine (TEA), and 4,4′azobis-(cyanovaleric acid) (ACVA) were purchased from Sigma Aldrich (Canada) and were used as received. Methoxydiethylene glycol methacrylate (MeODEGM) was purchased from Sigma Aldrich (Canada) and was passed through a short pad of silica prior to use. Aminoethyl methacrylamide hydrochloride (AEMA)51 and 4-cyanopentanoic acid dithiobenzoate (CTP)52,53 was synthesized as described according to previous reports. 2-Propanol, HPLC grade water, acetone, hexane, and ethyl acetate were purchased from Caledon Laboratories (Canada). Micro BCA assay kit was obtained from Fisher Scientific. Gwiz β-galactosidase was purchased from Aldevron. Bovine serum albumin (BSA) was purchased from Promega Corporation. Insulin was obtained from Sigma Aldrich. The chemical structures of monomers, initiator, chain transfer agent and cross-linker used in this study are depicted in Figure 1.

Figure 1. Chemical structures of monomers (MPC, MeODEGM and AEMA), cross-linker (CL), chain transfer agent (CTP), and initiator (ACVA).

Synthesis of 2,2-Dimethacroyloxy-1-ethoxypropane (Cross-Linker). The synthesis of the cross-linker has been recently reported by Aspinwall and co-workers.54 However, their synthesis has been carried out under different conditions 76



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(refluxing in benzene for 20 h). A typical protocol for our synthesis is as follows. HEMA (2-hydroxyethyl methacrylate) (10 g, 76.8 mmol), 2,2-dimethoxypropane (4.0 g, 38.4 mmol), and p-TSA (66 mg, 0.34 mmol) were added to a 25 mL roundbottom flask and the mixture was stirred for 6 h at room temperature in the dark. The crude product was purified by flash column chromatography on silica gel using a mobile phase of 85:14:1 hexane/ethylacetate/TEA. Fractions containing the pure product were pooled, and the solvent was removed by rotary evaporation and dried overnight under vacuum to afford pure cross-linker as a pale yellow oil. The cross-linker was stored at 0 °C. 1 H NMR (400 MHz, CDCl3) δ 1.39 (6H, s, C(CH3)2), 1.96 (6H, s, (6H, s, (OCOCCH3CH2)2), 3.75 (4H, t, J = 10.1, 5.0 Hz, C(CH3)2(OCH2CH2O)2), 4.30 (4H, t, J = 10.0, 5.1 Hz, C(CH3)2(OCH2CH2O)2), 5.59 (2H, s, OCOCCH3CH2 − syn to methyl), 6.15 (2H, s, OCOCCH3CH2 − anti to methyl). 13C NMR (100 MHz, CDCl3) δ 17.9 (OCOCCH3CH2), 24.4 (C(CH 3 ) 2 ), 58.5 (C(CH 3 ) 2 (OCH 2 CH 2 O) 2 ), 63.6 (C(CH3)2(OCH2CH2O)2), 99.8 (C(CH3)2), 125.2 (OCOCCH 3 CH 2 ), 135.8 (OCOCCH 3 CH 2 ), 166.9 (OCOCCH3CH2). HRMS (M+Na+) for C15H24O6Na, m/z = 323.1464 observed, m/z = 323.1465 calculated. Synthesis of Poly(MPC) MacroCTA by RAFT Polymerization. MPC macroCTA was synthesized as previously reported.55 MPC (3 g, 10.2 mmol), CTP (2.8 × 10−2 g, 0.1 mmol), and ACVA (1.4 × 10−2 g, 0.051 mmol) were dissolved in 9 mL methanol in a 25 mL reactor. The solution was degassed by purging nitrogen for 30 min and then placed under stirring at a temperature of 60 °C for 6 h. The polymer was obtained by precipitating the mixture in acetone. Any remaining traces of monomer were removed by washing the solution in water/acetone (1:7). The precipitate was again washed with acetone for 3−4 times and the final powder was freeze−dried. The poly(MPC) macroCTA molecular weight and PDI were found to be 1.2 × 104 g/mol and 1.21, respectively, by GPC. General Procedure for the Synthesis of poly(MPC-b(MeODEGM-st-AEMA-st-CL)) Nanogel by RAFT Polymerization. The RAFT polymerizations were performed in water/ 2-propanol (4:1 by volume) using ACVA as initiator, Poly(MPC) as macroCTA, and 2,2-dimethacroyloxy-1-ethoxypropane as the cross-linker at 70 °C for 24 h. The molar ratio of the macroCTA and ACVA (2:1) was held constant and the molar ratio between the macroCTA and the monomers was varied in order to prepare nanogels with different chain lengths (Table 1) . A typical general procedure is as follows: Poly(MPC) macroCTA (3.9 × 10−2 g, 3.3 × 10−3 mmol), MeODEGM (0.2 g, 1.1 mmol), AEMA (4.4 × 10−2 g, 0.26 mmol), and CL (6.0 × 10−2 g, 0.19 mmol) (15 mol % with respect to total moles of MeODEGM and AEMA) were dissolved in 4 mL water and sonicated. ACVA (4.0 × 10−4 g, 1.6 × 10−3 mmol) was dissolved in 1 mL 2-propanol and added to the reaction mixture. The solution was degassed by purging with nitrogen for 30 min and the reaction was carried out at 70 °C for 24 h. Reaction was stopped by quenching in liquid nitrogen. Nanogel was centrifuged at 14 000 rpm and 40 °C. The supernatant was discarded and the precipitate was washed with distilled water 3−4 times. The final precipitate was dissolved in water and freeze−dried overnight. A white powder was recovered and stored in the refrigerator. Protein Encapsulation. Drug loading was done using incubation method. Aqueous solutions of nanogels were

Table 1. Comparison of Hydrodynamic Diameter of MPC-b(MeODEGM-st-AEMA-st CL) Nanogel with Varying CrossLinker Concentrations and MEODEGM-AEMA Chain Length nanogel

nanogel composition

NG1

MPC70-b-(MeODEGMst-AEMA-st-CL)500 MPC70-b-(MeODEGMst-AEMA-st-CL)300 MPC70-b-(MeODEGMst-AEMA-st-CL)400 MPC43-b-(MeODEGMst-AEMA-st-CL)400 MPC43-b-(MeODEGMst-AEMA-st-CL)400 MPC43-b-(MeODEGMst-AEMA-st-CL)400 MPC43-b-(MeODEGMst-AEMA-st-CL)400

NG2 NG3 NG4 NG5 NG6 NG7

crosslinker (mol %)

hydrodynamic diameter (nm)

polydispersity

7

45

0.09

10

60

0.29

10

114

0.07

15

140

0.09

20

150

0.14

25

282

0.08

35

-

-

prepared in 10 mg/mL concentrations. The nanogel solution was mixed with 1.5 × 102 μL solution of 1 mg/mL BSA solution and incubated for 24 h at 4 °C. The total amount of protein encapsulated was calculated after centrifuging the sample at 40 °C in Beckmann Coulter Centrifuge (Microfuge 22R) (14 000 rpm, 15 min). The solution was separated into a white precipitate and supernatant. The amount of protein encapsulated in the nanogel was measured by incubating with bicinchoninic acid (BCA assay) for 2 h at 37 °C and measuring the absorbance at 570 nm using TECAN Genios Pro microplate reader. The plate reader was precalibrated using varying concentrations of protein and data were analyzed using Boltzmann function to give the best sigmoidal fit. The feed amounts for encapsulation of insulin and β-galactosidase are 50 and 2 μg, respectively. The amount of protein encapsulated (D) was calculated as follows:

D=

total protein − free protein total protein

where free protein is the protein in supernatant. Release of Protein from Nanogels at Acidic pH. In order to study the release of encapsulated protein, the nanogel was precipitated and redispersed in a citrate buffer solution of pH 4.8 and 6.4. The solution was divided into 7 aliquots. At regular intervals, the nanogels aliquots were precipitated and total protein content in the supernatant (released protein) was determined using BCA assay. Activity of Protein. The activity of β-galactosidase protein encapsulated in nanogel was studied by β-galactosidase assay. The nanogel was precipitated and separated from the supernatant (solution 1). The precipitate was redispersed in 100 μL sodium phosphate buffer solution (solution 2). 150 μL O-nitrophenyl-β-D-galactopyranoside (ONPG) (4 mg/mL) was added to 100 μL solutions 1 and 2 in the presence of 4.5 μL 100× Mg solution (0.64 μL of β-mercaptoethanol in 100 μL of 0.1 M MgCl2) in a 96 well plate and was incubated for 4 h at 37 °C. The yellow color developed was detected using the TECAN Genios Pro microplate reader at 420 nm. Characterizations. Nuclear Magnetic Resonance. 1H and 13C NMR spectra of the cross-linker were recorded on a 77



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Scheme 1. Schematic Representation of Core Cross-Linked Micelles with Thermoresponsive and Degradable Cores

conditions, and the rate of degradation was dependent on both pH and cross-linker concentration. The acid degradable nanogels were then used to study the encapsulation efficiency and the release profile of proteins. Synthesis of 2,2-Dimethacroyloxy-1-ethoxypropane (acid degradable cross-linker). The pH in the human body varies from 7.4 (physiological pH) to 5.5−6 in endosomes and 4−5 in lysosomes. Acid degradable cross-linker was synthesized for this study, to facilitate release of encapsulated proteins at low pH. Aspinwall and co-workers54 recently reported the synthesis of the cross-linker, which involved the use of benzene as solvent under high temperature with a reaction time of 20 h. However, our synthesis proceeds under much milder conditions, namely, in the absence of organic solvent at room temperature for 6 h of reaction time. The reaction of HEMA with 2,2-dimethoxypropane in a molar ratio of 2:1 yields acid degradable cross-linker as shown in Figure S1 (Supporting Information). Excess HEMA was removed during flash column chromatography on silica gel using a small amount of triethylamine in the eluent system. The presence of triethylamine prevents the degradation of the acid sensitive crosslinker. It was observed that, if the reaction was continued for more than the specified time of 6 h, the product turned dark brown, which is possibly due to side reactions that started simultaneously. The cross-linker was stored at 0 °C to avoid these side reactions. The 1H and 13C NMR spectra of the purified cross-linker are shown in Figures S2 and S3 (Supporting Information). Synthesis of Nanogels. Core−shell nanogels were synthesized using acid degradable cross-linker, by RAFT polymerization technique. A one-pot synthesis method developed by Pan et al.,56 which involved the polymerization of cross-linker with the monomer to get stabilized core crosslinked micelles, was followed for synthesizing nanogels. Recently, our group has extended this powerful one-pot synthesis for the fabrication of fixed CCL micelles having primary amino groups on the outer coronas for further surface functionalization.58 Overall, in this RAFT polymerization process, cross-linking and polymerization happened simultaneously. The core shell nanogel comprising a temperature sensitive MeODEGM core along with a primary amine monomer AEMA, imparting an additional functionality to the core, was cross-linked by an acid degradable cross-linker (CL) and a hydrophilic shell of MPC polymer chains (Scheme 2). MPC macroCTA (Mn = 1.2 × 104 g/mol) was synthesized in methanol as discussed in previous protocol.55 The conversion

Varian 400 MHz instrument. The sample was prepared using CDCl3 as solvent. MeODEGM shows thermoresponsive behavior and undergoes coil to globule transition at a lower critical solution temperature (LCST) of 24 °C. This transition was also studied at a molecular level by NMR spectroscopy in D2O. The signal intensities of MeODEGM protons vs that of solvent protons were compared at various temperatures (below and above the LCST). To account for the chemical shift of signal intensities at elevated temperatures, 1% 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) solution was added as a reference at 0 ppm. Mass Spectroscopy. Mass spectroscopy was carried out in Agilent Technologies 6220 orthogonal acceleration TOF (oaTOF) (Santa Clara California, USA). Gel Permeation Chromatography (GPC). The number average molecular weight (Mn) and polydispersity (Mw/Mn) of the macro CTA was studied using Viscotek conventional GPC connected to two Waters Ultrahydrogel linear WAT011545 columns (pore size: blend; exclusion limit = 7.0 × 106) and has a Viscotek model 250 dual detector. An acidic buffer of 0.50 M sodium acetate/0.50 M acetic acid was used as eluent. Calibration of GPC was done by six monodispersed poly(ethylene oxide) (PEO) standards (Mp = (1.01 × 103)−(1.01 × 105) g mol−1). Dynamic Light Scattering (DLS). Size of MeODEGM nanogels was analyzed using Viscotek DLS 802 instrument, which is equipped with a He−Ne laser at a wavelength of 632 nm and a Peltier temperature controller. The aqueous nanogel solution was filtered through a 0.45 μm pore size Millipore membrane. Data were obtained at an angle of 90° within the temperature range 25−50 °C. Omnisize software was used to record the DLS size data. 0.5 mL aliquots were drawn at 0 min, 30 min, 1 h, 2 h, 5 h, 18 h, and 24 h of reaction time. Transmission Electron Microscopy (TEM). Size and morphology of nanogels at room temperature were analyzed on a Philips transmission electron microscope operated at 80 kV and fitted with a CCD camera. A droplet of a properly diluted nanogel solution was placed on the TEM carbon coated copper grid and allowed to air-dry before it was stained with phosphotungstic acid (2 wt % aqueous solution) for 1 min. The sample was then allowed to dry overnight prior to observation.

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RESULTS AND DISCUSSION

Nanogels (core cross-linked micelles) were successfully synthesized with a hydrophilic shell and a thermoresponsive core in a one-pot manner using the RAFT polymerization method (Scheme 1). These nanogels degraded under acidic 78



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amounts of cross-linker (Figure 2). It was observed that the nanogels attained a stable size within 5−6 h of reaction time

Scheme 2. RAFT Synthesis of poly(MPC-b-(MeODEGM-stAEMA-st-CL)) Core Cross-Linked Micelles with Thermoresponsive and Degradable Cores

Figure 2. Evolution of hydrodynamic diameter of reaction mixture of cross-linking nanogel and variation of size with varying cross-linker (CL) concentration.

and with increasing cross-linker concentration, the hydrodynamic size also increased (Table 1). However, for poorly degradable cross-linker concentrations (7−10 mol %), the sizes were small (45−60 nm) and increased with temperature. This may be due to the unstable micelle structures and formation of aggregates of MeODEGM polymers. On the other hand, for a high concentration of cross-linker (35 mol %), a very turbid solution was obtained. Particle precipitation was observed when the solution was left undisturbed for 10−15 min. Thus, the size of particles formed was in the micrometer range, and hence, particles were unstable in solution. Stable nanogels were obtained at an optimum cross-linker concentration of 15−20 mol % with particle sizes of 125−150 nm and low polydispersities (0.09−0.14) as determined by DLS. TEM images of these nanogels (NG4 and NG6) are depicted in Figure 3 and showed that they were

was kept low (60%) in order to prevent the formation of dead chains as a result of the termination reactions that set in toward the end of the reaction. The macroCTA was further used to copolymerize MeODEGM and AEMA in the presence of the cross-linker in water and 2-propanol using ACVA as initiator (Scheme 2). The use of ACVA as initiator is driven by two factors. First, it can be thermally decomposed and has a half-life of 10 h in aqueous solution at 69 °C, and second, it is slightly more hydrophilic than other thermally sensitive initiators like AIBN. Tao et al. observed that a hydrophobic initiator could interfere with the hydrophobic core and hence produce unstable micelles. On the contrary, ACVA, which is more hydrophilic, yields controlled and more stable nanogels.57 Micelle structures can easily dissociate after dilution, and in order to prevent dissociation of micelle structures of diblock copolymers, they are stabilized by cross-linking.58 The size and stability of nanogels depend on cross-linker concentration (molar ratio with respect to the total molar monomers concentration) (expressed in percentage). For the synthesis of uniform, fixed micelles, it is necessary to have a suitable amount of cross-linker. Branched copolymers and unstable nanostructures can be formed as a result of low cross-linker concentration.12 On the other hand, very high cross-linker concentration can result in the formation of microgel or hydrogel networks.59 To confirm this effect, cross-linker concentration was varied from 7, 10, 15, 20, 25, and 35 mol %. The particle size and their polydispersities were determined using DLS as shown in Table 1. The evolution of hydrodynamic size of the nanogel with time was studied for varying

Figure 3. TEM images of nanogels showing (A) NG4 (15 mol % CL) and (B) NG6 (25 mol % CL).

spherical in shape with a smooth surface. Both NG3 (10 mol % CL) and NG4 (15 mol % CL) (Figure S4, Supporting Information) displayed good dispersibility; however, for NG6 (25 mol % CL), the particles started to aggregate. More importantly, the TEM results indicated that the nanogel had a core−shell structure, which confirmed nanogel formation. MeODEGM is a thermoresponsive polymer and experiences a phase change from being hydrophilic at low temperatures (24 79



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°C) to hydrophobic at high temperatures. The MeODEGM core of the micelle, therefore, collapses at high temperatures, which is indicated by a notable decrease in size above 24 °C. Table 1 shows the variation in size of nanogels with change in MeODEGM chain length and cross-linker concentration. MeODEGM chain length has little effect on the size of nanogels, but it affects the LCST of the nanogel. Lutz et al. observed that an increase in MeODEGM levels in the nanogel raised LCST from 24 °C to as high as 84 °C.13,60 Temperature dependent DLS studies showed that nanoparticles shrunk from 147 nm at 15 °C to 115 nm at 50 °C (NG4) and 75 nm at 15 °C to 53 nm at 50 °C (NG2) as shown in Figure 4. The phase transition of MeODEGM from

Figure 5. Normalized ratio of 1H NMR signal intensity of MeODEGM protons to D2O protons vs temperature as observed for the methylene group protons of MeODEGM at 3.4 ppm and D2O for NG4.

(Figure 6). Also, the rate of degradation of the nanogels is more pronounced for low cross-linker concentration (10 mol %),

Figure 4. Decrease in mass-average hydrodynamic diameter with increase in temperature obtained for 0.5 mg/mL aqueous solution of nanogel, for varying cross-linker (CL) concentration.

hydrophilic to hydrophobic state could be seen as the solution immediately turned milky white from transparent as the temperature was raised (Figure S5, Supporting Information). The nanogels were found to be quite stable at 50 °C. The phase transition behavior of MeODEGM core was studied using VTNMR (Variable Temperature 1H NMR) as depicted in Figure S6 (Supporting Information). While disappearance of the peak for the thermoresponsive was expected at higher temperature, only a small reduction in peak intensity was observed. This shows that nanogels behave differently from homogeneous and macroscopic systems. Furthermore, Schönhoff et al. have attributed this reduction in NMR signal intensity of poly(NIPAM) to the decrease in polymer segmental mobility, which further decreases the relaxation time, T.61 The 1H NMR peak intensity of methylene group of poly(MeODEGM) at 3.4 ppm was compared with normalized solvent (D2O) peak intensity. The reduction in polymer signal intensity above phase transition is shown in Figure 5. The effect of pH on the nanogel was studied to find the rate of degradation of the cross-linker at low pH. The pH of distilled water is slightly acidic (pH 6.7). Nanogels in distilled water could be stored for 2−3 days without degradation. A comparative study of the rate of degradation at different pH (pH 4.5, 5.2, and 6.4) and varying cross-linker concentration (10 and 15 mol %) was carried out. As expected, the nanogels were found to be unstable and degrade under acidic conditions

Figure 6. Degradation profile of NG3 and NG4 at pH 4.5, 5.4, and 6.2.

which degraded within 2 h at pH 4.5 and 5.2. The nanogels were found to be very stable under neutral and alkaline pH. For higher cross-linker concentrations, however, the degradation curve shifted to the right, indicating higher stability at low pH for a longer time. Protein Encapsulation. An important application of stimuli-responsive nanogels is the encapsulation of macromolecules in their core or coronas. Cross-linking of polymers provides a stable micelle structure, which can be exploited to trap macromolecules of various sizes. However, the release of trapped drugs is only possible by the degradation of cross-linker under physiological conditions. We have used acid degradable cross-linker, which degrades at low pH, thereby releasing the encapsulated proteins. The thermoresponsive nature of the synthesized nanogels plays a vital role for protein encapsulation, since at low temperature (4 °C), the nanogel core and shell are both 80



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hydrophilic and, as temperature increases to 37 °C, the core of the nanogels become hydrophobic and therefore the nanogels can easily be separated out of solution by simple centrifugation. Thus, the nanogels were collapsed at 37 °C to trap the protein within the core and hence the amount of entrapped protein can easily be estimated. The protein was loaded using the incubation method. Insulin, BSA, and β-galactosidase were incubated overnight in aqueous nanogel solutions (5 mg/mL) at 4 °C. The three proteins vary largely in their sizes. Insulin is the smallest protein with size 5.81 kDa, while BSA has a molecular weight of 66.8 kDa and β-galactosidase is the largest protein of size 540 kDa. MeODEGM nanogels are hydrophobic at room temperature. The proteins were incubated at low temperature (7 °C) to ensure maximum encapsulation. The incubation method is reported to be less efficient than the encapsulation method, as per previous reports.62 However, these nanogels showed high loading probably due to the additional functionality of the core of the nanogels (presence of protonated amino groups). The cationic nature of the core has (PAEMA-pKa 8.8) has facilitated the encapsulation of negatively charged macromolecules. For example, BSA has a pI (isoelectric point or the point of zero charge) of 4.6 and therefore has a negative surface charge at neutral pH. The nanogel was precipitated at 14 000 rpm and 40 °C. Before precipitating, the gels were collapsed to trap the protein within the core. The amount of entrapped protein was estimated by calculating the difference between the amount of protein added and the amount of nonencapsulated protein in the aqueous phase of precipitated protein. It was observed that two factors that dictated the amount of protein encapsulated were the cross-linker concentration and the percentage of AEMA in the core. A comparative study of three samples is shown in Table 2. The analysis showed that 92% of insulin was encapsulated for

cross-linker decrease the pore size leading to reduced loading capacity. To study the effect of AEMA, nanogels with varying amount of cationic components were studied and compared. For the same amount of cross-linker, the AEMA component also affects the encapsulation efficiency. This may be due to the electrostatic interaction between the protein and cationic nanogel due to opposite charges of protein and MeODEGMAEMA nanogel at neutral pH. Table 2 indicates that NG4b has the optimum cross-linker concentration (15 mol %) and AEMA concentration (5 mol %) for the encapsulation of proteins. A careful analysis of data indicates that the size and properties of macromolecules also play an important role in determining the total encapsulation. Insulin showed highest percentage encapsulation in agreement with its smallest size and lowest molecular weight (5.81 kDa). β-Galactosidase, on the other hand, has a molecular weight of 540 kDa, and consequently the lowest encapsulation efficiency. The encapsulation efficiency decreased from 92% for insulin to 35% for β-galactosidase. Release Profile. The release profile of nanogels was studied at varying pH. Nanogels with 15% and 20% cross-linker concentration were incubated overnight with 50 μL insulin protein (1 mg/mL). The nanogels were precipitated and the amount of protein encapsulated was determined by BCA assay. The precipitated nanogel was redispersed in buffer solutions of pH 4.8 and 6.4. Aliquots from buffer solution were taken at regular intervals and the amount of protein released was calculated. Almost 90% protein was released over a period of 48 h at pH 4.8. Thus, the release profile of insulin from MeODEGM nanogels is slow, and no burst release was observed (Figure 7).

Table 2. Protein Encapsulation Efficiencies for Nanogels of Varying Cross-Linker Concentration and AEMA Contenta samples

protein

protein encapsulation (μg)

encapsulation (%)

NG4a

Insulin BSA β-Gal Insulin BSA β-Gal Insulin BSA β-Gal

46 54 46 90 0.7 40 40 -

92 36 92 60 35 80 27 -

NG4b

NG5

a

(A) NG4a: 15 mol % CL and 5% AEMA feed ratio. (B) NG4b: 15 mol % CL and 25% AEMA feed ratio. (C) NG 5: 20 mol % CL and 10% AEMA feed ratio. Figure 7. Cumulative release profile of insulin from NG4 and NG5 at pH 4.8 and 6.4.

nanogel with 15% feed cross-linker concentration and 25% feed ratio AEMA (NG4b). For higher cross-linker concentration, the encapsulation was reduced to 80%. A possible explanation for the strong dependence of encapsulation efficiency on the cross-linker concentration may be due to its effect on pore size of the cross-linked nanogel. A higher cross-linker concentration is expected to produce a tighter network, therefore providing a smaller pore size. Encapsulation of macromolecules is easier for a larger pore size network. While lower cross-linker concentration fails to cross-link stably, higher amounts of

To confirm the structure and activity of β-galactosidase after loading, β-galactosidase assay was conducted on the 400 μL of 5 mg/mL aqueous nanogel solution loaded with β-gal. The amount of encapsulation based on BCA assay calculation was found to be 35%. Figure S7 (Supporting Information) showed the change in color of nanogel when ONPG was hydrolyzed to o-nitrophenol (yellow) and galactose by β-galactosidase. The activity of protein in supernatant and nanogel was calculated to 81



Article

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be 1384 mU/mg and 4642 mU/mg, respectively. It was therefore found that the protein was present mostly in its native form after encapsulation and likely retained its catalytic activity.

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CONCLUSION In summary, we have successfully synthesized novel stable thermoresponsive and acid degradable poly(MeODEGMAEMA) core-cross-linked micelles via RAFT polymerization using poly(MPC) macro RAFT agent. Sizes of these nanogels can be tuned accordingly by varying the cross-linker concentration and MeODEGM chain length. AEMA provides cationic character to the nanogel core, which facilitates the encapsulation of oppositely charged proteins, for example, insulin, BSA, and β-galactosidase. The loading efficiency of these proteins largely depends on the pore size of nanogels, the cationic component, and the size of protein. The degradation profile of these proteins encapsulated in acid-degradable nanogels was studied at lower pH, and a controlled release profile of protein was observed. These MPC-b-(MeODEGM-stAEMA-st-CL) nanogels therefore have promising applications as smart carriers for targeted drug delivery systems and controlled release.

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis of acid degradable cross-linker, HNMR and CNMR spectra of cross-linker, TEM image of NG3, VT-NMR study of nanogel, digital photograph of aqueous solution of nanogel on heating above and cooling below LCST, digital photograph of β-galactosidase assay conducted on aqueous solution of nanogel. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Telephone: 1-780-492-1736, Fax: 1-780-492 2881.

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ACKNOWLEDGMENTS The authors acknowledge support from Natural Sciences and Engineering Research Council of Canada (NSERC) for the funding of this work.

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REFERENCES

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Degradable Thermoresponsive Nanogels for Protein Encapsulation

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