Superparamagnetic Hollow Hybrid Nanogels as a Potential

Deionized water was produced from Milli-Q synthesis (18 MΩ, Millipore). ... The magnetic hyperthermia characterization of the hollow hybrid nanogels ...
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Superparamagnetic Hollow Hybrid Nanogels as a Potential Guidable Vehicle System of Stimuli-Mediated MR Imaging and Multiple Cancer Therapeutics Wen-Hsuan Chiang,†,∥ Viet Thang Ho,†,∥ Hsin-Hung Chen,‡ Wen-Chia Huang,‡ Yi-Fong Huang,‡ Sung-Chyr Lin,‡ Chorng-Shyan Chern,§ and Hsin-Cheng Chiu*,† †

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan § Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan ‡

S Supporting Information *

ABSTRACT: Hollow hybrid nanogels were prepared first by the coassembly of the citric acid-coated superparamagnetic iron oxide nanoparticles (SPIONs, 44 wt %) with the graft copolymer (56 wt %) comprising acrylic acid and 2methacryloylethyl acrylate units as the backbone and poly(ethylene glycol) and poly(N-isopropylacrylamide) as the grafts in the aqueous phase of pH 3.0 in the hybrid vesicle structure, followed by in situ covalent stabilization via the photoinitiated polymerization of MEA residues within vesicles. The resultant hollow nanogels, though slightly swollen, satisfactorily retain their structural integrity while the medium pH is adjusted to 7.4. Confining SPION clusters to such a high level (44 wt %) within the pH-responsive thin gel layer remarkably enhances the transverse relaxivity (r2) and renders the MR imaging highly pH-tunable. For example, with the pH being adjusted from 4.0 to 7.4, the r2 value can be dramatically increased from 138.5 to 265.5 mM−1 s−1. The DOX-loaded hybrid nanogels also exhibit accelerated drug release in response to both pH reduction and temperature increase as a result of the substantial disruption of the interactions between drug molecules and copolymer components. With magnetic transport guidance toward the target and subsequent exposure to an alternating magnetic field, this DOX-loaded nanogel system possessing combined capabilities of hyperthermia and stimuli-triggered drug release showed superior in vitro cytotoxicity against HeLa cells as compared to the case with only free drug or hyperthermia alone. This work demonstrates that the hollow inorganic/organic hybrid nanogels hold great potential to serve as a multimodal theranostic vehicle functionalized with such desirable features as the guidable delivery of stimuli-mediated diagnostic imaging and hyperthermia/chemotherapies.

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copolymers with hydrophobic SPIONs into micelles.10,18,19 Gao and co-workers showed that hydrophobic SPIONs were efficiently encapsulated into micelles of amphiphilic diblock copolymers consisting of cRGD-bearing poly(ethylene glycol) (PEG) and poly(D,L-lactide) blocks.10 The clustering of SPIONs within the micelle core and a high SPION loading density (up to 50 wt %) significantly enhanced the sensitivity of MRI. Kim et al. utilized the diblock copolymer composed of polystyrene (PS) and poly(acrylic acid) (PAA) blocks to form hybrid micelles with oleic acid-coated SPIONs in the aqueous phase in which the loaded number of SPIONs per micelle was virtually controlled by the initial concentration ratio of SPIONs to copolymer.18 Recently, Hickey et al. observed that the morphology of the self-assemblies from PS-b-PAA with SPIONs in the aqueous phase was influenced by the level of iron oxide nanoparticles incorporated. With the increase in the

INTRODUCTION Supramolecular assemblies serving as powerful tools in biomedical applications such as theranostic delivery and nanobioreactors can be attained by the intermolecular packing of amphiphilic copolymers into well-defined hierarchical architectures in highly ordered functional states.1−4 Hybrid associations of copolymers with other functional building components (either organic or inorganic) apt to supplement assembly functions have also become an important paradigm of the nanotechnology, in particular, for medical applications.5−7 For example, the incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into various organic nanoassemblies, such as polymeric micelles, polymersomes, and liposomes, has been studied by virtue of their appealing biomedical applications including magnetic resonance imaging (MRI),8−10 magnetically guidable drug delivery,11,12 hyperthermia cancer therapy,12−14 and remotely controlled payload release.15−17 As a key prerequisite to achieving these goals, a high loading level of SPION within assemblies was usually attained by manipulating the coassembly of amphiphilic © XXXX American Chemical Society

Received: January 16, 2013 Revised: March 24, 2013

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Scheme 1. Development of the DOX-Loaded Hollow Hybrid Nanogels Serving as a Multifunctional Anticancer Theranostic Platform

(mPEG) chain segments as the grafts via hydrogen bond pairings. Being quite distinct from our previous work, this work contains a description of a facile approach to developing multifunctional nanovehicles capable of carrying very high levels of SPION and doxorubicin (DOX) and hence displays stimuli-triggered chemo/hyperthermia therapeutics and MRI contrast imaging, which serve as a potential transport-guidable cancer theranostic system. The hybrid assemblies were prepared first by the cooperative assembly of citric acid-coated SPION (44 wt %) with a graft copolymer (56 wt %) comprising AA and 2-methacryloylethyl acrylate (MEA) residues as the backbone and PNIPA and mPEG as the grafts in the aqueous phase of pH 3.0. This was followed by in situ covalent stabilization through the photoinitiated polymerization of MEA residues to form ester cross-links (Scheme 1). The resultant assemblies possess a hollow nanogel architecture with an inner aqueous chamber that is enclosed by the hybrid gel membrane composed of SPIONs and PNIPA/PAA chain segments elaborated by the surrounding mPEG chains as coronas (Scheme 1). In addition to the structural characterization, the feasibility of the organic/inorganic nanohybrids serving as a multifunctional delivery system was preliminarily evaluated with respect to their biological performance in vitro. Superior enhancement of the T2-type MRI contrast of SPIONs confined within hollow hybrids and their high sensitivity to the medium pH in fine tuning the image contrast were achieved in this work. Furthermore, the local hyperthermia reaction induced by the treatment of hollow hybrid nanogels under a high-frequency magnetic field (HFMF) and the dual pH/ thermally triggered drug release response corresponded quite well to the drastically promoted cytotoxicity of the hybrid nanogel assemblies under external magnetic guidance toward

encapsulated inorganic nanoparticle content to 35.8%, the relative volume ratio between the soft and hard segmental domains became more symmetric and thus beneficial to the formation of hybrid vesicles instead of the micelle structure.19 Owing to the presence of SPIONs hybridized with organic components within the stimuli-responsive assemblies, it becomes plausible to functionalize these assemblies with the desired tunable MRI contrast in response to the intracellular pH or redox potential.20−22 The reduction-sensitive nanogels carrying both fluorescently labeled dextran and waterdispersible SPIONs were developed by the inverse microemulsion polymerization of zwitterionic monomer, carboxybetaine methacrylate, with a disulfide-containing cross-linker.21 Being exposed to the dithiothreitol-rich environment, the nanogels were structurally disrupted via the cleavage of disulfide linkages, thereby leading to not only the release of drug but also the disassembly of clustered SPIONs that largely alters the MRI contrast. The enclosure of SPION clusters with a pHresponsive AA-rich hydrogel has provided an alternative to imparting to hybrid assemblies sensitivity in the MRI contrast to the medium pH.22 As quantitatively reflected by the proton transverse relaxivity (r2), the negative-type MRI contrast of SPIONs enclosed within nanogels was significantly enhanced by the increased hydration of the gel coating with the increase in pH. Recently, we reported stimuli-responsive hollow polymeric nanogels as carriers for efficient intracellular drug delivery.23 The functional hollow polymeric nanogels were prepared from the covalent cross-linking of the colloidal particles attained by the coassociation of two graft copolymers comprising AA units as the backbone and either poly(N-isopropylacrylamide) (PNIPA) alone or both PNIPA and monomethoxy-PEG B

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pH and of HeLa cells (2 × 105/well) after being incubated with the hybrid nanogels of varying doses for 1 h were acquired on a Bruker S300 Biospec/Medspec MRI at 7 T using the following parameters: TR = 3000 ms, TE = 15−150 ms, FOV = 60 × 60 mm2cm, matrix = 192 × 192, and slice thickness = 1 mm. Parameter r2 was evaluated via the linear least-squares best fitting of the 1/T2 relaxation time (1/s) versus the iron concentration (mM Fe). DOX Loading and in Vitro Release. An aqueous DOX solution (5.0 mM, pH 5.0) was added directly to the purified aqueous hybrid nanogel suspension to a final concentration of 0.5 mM, and the pH was adjusted to 7.4. The sample was then equilibrated under stirring at 25 °C for 24 h, followed by thorough dialysis (Cellu Sep MWCO 12 000−14 000) against an aqueous solution at pH 7.4 (I 0.01 M) for 3 days to remove unloaded DOX. To determine the drug-loading level, a small portion of the DOX-loaded hybrid nanoparticles was withdrawn and subjected to full swelling by the addition of DMF to a ratio of 9/1 v/v DMF/H2O. The amount of DOX loaded was quantitatively determined by fluorescence spectrophotometry (Hitachi F-7500) using a calibration curve previously established by the fluorescence intensity of DOX with different concentrations in 9/1 DMF/H2O. Excitation was performed at 480 nm, and the emission spectra were recorded in the range from 500 to 700 nm. The drug-loading efficiency (DLE) and drug-loading capacity (DLC) were calculated according to the following formulas, respectively:

HeLa cells in vitro as compared to only anticancer drugs or local heat effects.

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EXPERIMENTAL SECTION

Materials. The synthesis and characterization of the graft copolymer employed in this work are described in the Supporting Information (SI) and our previous work.23−25 The chemical structure, composition, and average molecular weight of the copolymer are illustrated in Scheme 1. The preparation of citric acid-coated SPIONs is described in detail in the Supporting Information. 2,2-Diethoxyacetophenone (DEAP) and 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma, and DOX (in the hydrochloride salt form) was obtained from Seedchem. Dulbecco’s modified Eagle’s medium (DMEM) and Hoechst 33258 were purchased from Invitrogen. Deionized water was produced from Milli-Q synthesis (18 MΩ, Millipore). All chemicals were reagent grade and used as received. Preparation of Hollow SPION/Copolymer Hybrid Nanogels. The graft copolymer (6.0 mg) was dissolved directly in the aqueous suspension of SPIONs (pH 7.4, 1.0 mg/mL, 4.0 mL). After adjustment of the pH to 3.0 under vigorous stirring, the dispersion was heated to 60 °C for 1 h. Immediately after the dispersion was cooled to room temperature, DEAP (10.0 wt %) as the photoinitiator was added to initiate the radical polymerization of MEA residues under UV light within ultraviolet cross-linkers (UVP CL-1000) equipped with five 8-W 254 nm UV tubes at 25 °C for 30 min. The suspension was then subjected to centrifugation (Hettich Universal 320R, 30 min, 10 000 rpm) and ultrafiltration (Amicon 8200 with a Millipore PBMK membrane, MWCO 300 000) to remove unloaded SPIONs and unreacted copolymer, respectively. Characterization of Hollow Hybrid Nanogels. The mean hydrodynamic diameter (Dh) and particle size distribution of the hybrid nanogels in aqueous solution were analyzed by dynamic light scattering (DLS) on a Malvern Zetasizer Nano-ZS instrument at an angle of 173° equipped with a 4 mW He−Ne laser operating at λ = 632.8 nm in combination with the cumulant analysis method. In addition, the dependence of the Dh of the hybrid nanogels in aqueous solution on the light-scattering angle was also measured in the range of 45−135° with a Brookhaven BI-200SM goniometer equipped with a BI-9000 AT digital correlator using a solid-state laser (35 mW, λ = 637 nm). The TEM images of the citric acid-coated SPIONs and the hollow SPION/copolymer hybrid nanogels with and without negative staining (uranyl acetate, 2.0 wt %) were obtained on a JEOL JEM1200 CXII microscope operating at an accelerating voltage of 120 V. The magnetite content of the hybrid nanogels was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). In brief, the lyophilized magnetic nanogels were weighed and then added to the HCl solution (1.0 N) to achieve the complete dissolution of SPIONs. The iron concentration was determined at the specific iron emission wavelength (259.9 nm) using a previously established calibration curve. The SPION content was then calculated as the ratio of the weight of the enclosed SPIONs to the total weight of the hybrid nanogels. Magnetic studies were carried out on an MPMS XL-7 Quantum Design SQUID magnetometer at 300 K. The applied magnetic field was varied from 4 × 104 to −4 × 104 Oe in order to attain the hysteresis loops. The magnetic hyperthermia characterization of the hollow hybrid nanogels in aqueous solution was conducted on an HFMF consisting of a power supply, functional generator, amplifier, and cooling water system. The coil (35 mm in diameter) was constructed in a seven-loop structure, the frequency was 37 kHz, and the strength of the magnetic field was 2.5 kA/m. The temperature of the HFMF generator was controlled by cycling cooling water at 25 °C. T2 Relaxivity Measurement. For the aqueous suspensions of citric acid-covered SPIONs and hollow hybrid nanoparticles at different pH, T2 relaxation time measurements were conducted on a Bruker Minispec mq20 at 20 MHz (0.47 T, which is comparable to the magnetic field strength of clinically used MR scanners) and 37 °C. The T2-weighted MR phantom images of the hybrid nanogels at different

DLE% =

wt of loaded DOX × 100% wt of DOX in feed

DLC% =

wt of loaded DOX wt of drug‐loaded magnetic nanogel after thorough lyophilization × 100%

For the release experiment, the DOX-loaded nanogel dispersions (1.0 mL) were dialyzed (Cellu Sep MWCO 12 000−14 000) against succinic acid buffers of pH 5.0 and 6.0 and phosphate buffer of pH 7.4 (50 mL, I = 0.15 M), respectively, at 4, 37, or 45 °C. At prescribed time intervals, the dialysate (1.0 mL) was withdrawn and replaced with an equivalent volume of fresh medium. The concentration of DOX was determined by fluorescence measurements using the pertinent calibration curve of DOX at various concentrations in aqueous buffer solution of pH 5.0, 6.0, or 7.4. To explore the HFMF-induced hyperthermia effect on the drug release, a glass tube containing the DOX-loaded magnetic nanogel suspension (pH 5.0, 1.0 mL) was positioned at the coil center of the HFMF for different durations (10, 20, and 30 min). Afterward, the above nanogel dispersion was dialyzed against succinic acid buffer of pH 5.0 at 37 °C over a period of 2 h (including the duration of HFMF treatment), and the cumulative drug release was determined as described above. The experimental results presented in this work represent an average of at least triplicate measurements. Cellular Uptake. HeLa cells (1 × 105 cells/well) were treated with free DOX and the DOX-loaded magnetic nanogels, respectively, at a DOX concentration of 10 μM at 37 °C for 1 h. After being washed twice with PBS, cells were detached by the trypsine−EDTA solution and then dispersed in 0.5 mL of PBS. The cellular uptake of drug was analyzed on a FACSCalibur flow cytometer (BD Biosciences). For the laser scanning confocal microscope (LSCM) studies, HeLa cells (2 × 105) were seeded onto 22 mm round glass coverslips, placed in a sixwell plate, and then cultured overnight. The cells were then incubated respectively with free DOX and the DOX-loaded magnetic nanogels at a DOX concentration of 10 μM for 1 h. Cells were washed twice with PBS and then fixed with 4% formaldehyde. To identify the location of nuclei inside cells, cells were stained with Hoechst 33258 for 10 min. The cellular uptake of DOX was directly visualized with a ZESS LSM 510 META. To examine the effect of magnetic guidance on the cellular uptake of the hybrid nanogels, a prescribed quantity of the DOX-loaded magnetic nanogels (DOX concentration of 10 μM) was added to a 60 mm Petri dish containing 5 × 105 HeLa cells in DMEM medium (4.0 C

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mL) supplemented with 10% FBS and 1% penicillin at 37 °C. A cylindrical N35 Nd−Fe−B magnet (dimensions: d = 10 mm, h = 10 mm; field strength: 0.6 T) was then placed under the Petri dish (on the right side) for 1 h. Afterward, cells were washed twice with PBS and then fixed with 4% formaldehyde. Prior to the LSCM examination, cells were stained with Hoechst 33258 for 10 min, and the slides were rinsed three times with PBS. The cellular uptake of DOX was visualized with a ZESS LSM 510 META. For the direct observation of intracellular SPIONs, HeLa cells were treated with Prussian blue solution (4.0 mL) containing 2% potassium ferrocyanide(II) trihydrate and 2% HCl for 20 min at 37 °C after being coincubated with the DOX-loaded magnetic nanogels under the external magnetic direction for 1 h as described above and then fixed with formaldehyde. Iron staining was observed with a transmission light microscope (Zeiss, Axiovert40 CFL) equipped with a fluorescence system and a digital camera (Axiocam MRC5). Cytotoxicity Analysis. HeLa cells were seeded in a 96-well culture plate at a density of 1 × 104 cells/well in DMEM containing 10% FBS and 1% penicillin and incubated at 37 °C for 24 h. The medium was then replaced with 100 μL of fresh medium containing free DOX, the DOX-loaded magnetic nanogels (with varying amounts of DOX), or the DOX-free hollow nanogels and incubated at 37 °C for 24 h. To evaluate the effect of the magnetic guidance of the drug-loaded magnetic nanoparticles toward the target cells on the cytotoxicity, a 96-well magnetic plate (Magou, Magtractor-96, field strength 3500 G) was applied to cells for the first 7 h during the course of cell incubation. Thereafter, 5 μL of MTT (5.0 mg/mL) was added to each well and incubated at 37 °C for 4 h. After the culture medium was discarded, DMSO was added to dissolve the precipitate. The absorbance of the resulting solution in each well at 570 nm was determined by using a SpectraMax M5 microplate reader. The combined effects of magnetic hyperthermia and drug release from the guided drug-loaded magnetic nanogels on the cytotoxicity were evaluated as follows. First, HeLa cells (2 × 104 cells/well) were incubated with free DOX, the DOX-loaded magnetic nanogels (with varying amounts of DOX), or the DOX-free magnetic nanogels and then incubated at 37 °C for 7 h in the presence of magnetic guidance. After being washed twice with PBS, cells were harvested from monolayers with the trypsine−EDTA solution and placed into a glass tube. The glass tube containing cells was then subjected to the HFMF treatment for 20 min. After DMEM (500 μL) was added to each well, the cells treated with HFMF were reseeded in each well and then incubated for an additional 17 h. The in vitro cytotoxicity was evaluated by MTT assay.

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Table 1. Dh, PDI, and Zeta Potentials of the Mixture of SPION and Copolymer, Un-Cross-Linked Hybrid Assemblies, Hollow Hybrid Nanogels, and DOX-Loaded Hybrid Nanogels in Aqueous Solutions at 25 °C Dh (nm)

sample mixture of SPION and copolymer at pH 7.4 un-cross-linked hybrid assemblies at pH 3.0 hollow hybrid nanogels at pH 3.0 hollow hybrid nanogels at pH 7.4 DOX-loaded hybrid nanogels at pH 7.4

PDI

zeta potential (mV)

66

0.13

−24.0

198

0.14

−0.5

196 208 190

0.15 0.17 0.13

−0.3 −18.5 −17.4

attributed to the development of the hybrid assemblies from the cooperative association of macromolecules with the citric acid-bearing SPIONs via massive hydrogen bonds between PNIPA grafts and un-ionized carboxyl groups of citric acid residues. Complementary hydrogen-bond pairings of PNIPA chain segments with the pairing targets containing carboxyl groups and the subsequent hydrophobic effect of supramolecular association thus induced have been extensively documented.23,24,26−28 The resultant nanohybrids are highly stabilized by the pronounced steric repulsion mechanism of the hydrated mPEG graft segments. The particle size distribution profiles of the hybrid assemblies determined by DLS are shown in Figure S3. To promote the potential applications in drug delivery by improving the colloidal stability of the hybrid assemblies, the structural covalent cross-linking reaction was further performed by the photoinitiated free radical polymerization of MEA residues using DEAP as the initiator at pH 3.0 and 25 °C. The resultant SPION-hybridized nanogels at pH 3.0 possess a Dh value of ca. 196 nm and a monomodal particle size distribution with the polydispersity index (PDI) equal to 0.15 (Table 1). Notably, as the pH was raised back to 7.4, the cross-linked hybrid assemblies fully retained their structural integrity although the particles were slightly enlarged in size (Dh = 208 nm) compared to those (196 nm) at pH 3.0 (Table 1). This is mainly because of the increased ionization of AA and citric acid residues within the hybrid nanogels at pH 7.4, as evidenced by a substantial change in the zeta potential from −0.3 mV (pH 3.0) to −18.5 mV (pH 7.4), which not only enhances their interaction with water molecules24,25 but also sabotages the hydrogen bond pairings originally formed between SPIONs and PNIPA chain segments. As a consequence, water influx and nanogel swelling are established. Upon large volume dilution with PBS (ionic strength 0.15 M), the colloidal particle morphology of the hybrid hollow nanogels remains intact (Figure S4). This implies the excellent structural and colloidal stability provided by the interior ester cross-links and the external PEG chain segments attached to the gel external surfaces, respectively. On the basis of the ICP-AES data, about 44.2 wt % of the citric acid-coated SPIONs were ultimately incorporated into the hybrid nanogels. The percentage (44 wt %) of SPION within the hybrid nanogels that is somewhat higher than that in the feed (40 wt %) in the coassembly process indicates the quite effective binding of SPION with copolymer via the SPION surface citric acid residues (at pH 3.0) prior to the cross-linking reaction. Such a level of SPION within the nanogels achieved in this work is substantially higher than those in the previously reported polymeric assemblies.15,29,30 Although the hybrid-

RESULTS AND DISCUSSION

Formation and Characterization of Hollow Hybrid Nanogels. Judging from the thermal gravimetric analysis (data not shown here) and TEM image (Figure S2 in the SI), the citric acid content of the citric acid-covered SPIONs and their particle size are ca. 25 wt % and 8−10 nm in diameter, respectively. While the pH of the aqueous SPION suspension (1.0 mg/mL) was lowered to 4.0 or below, severe particle aggregation and then precipitation occurred, obviously owing to the considerable reduction in ionization of the citric acid groups anchored on the magnetic particle surfaces that impaired the electrostatic stabilization. With the graft copolymer being added to the SPION dispersion at pH 7.4 and subsequently the pH adjusted to 3.0, the SPIONs remained well-dispersed. The DLS measurements (Table 1) demonstrate that the particle size (Dh = 66 nm) is essentially the same for the SPION suspensions in the presence and absence of the copolymer at pH 7.4. While the pH was being adjusted to 3.0, the particle size was dramatically increased from 66 to 198 nm. Note that the graft copolymer alone in aqueous solution at pH 3.0 exists in the form of unimers, as described in our previous work.24 Therefore, the increased particle size is primarily D

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Figure 1. (a) Field-dependent magnetic curves of (I) the citric acid-covered SPIONs and (II) the hollow hybrid nanogels. (b) Photographs of the hollow hybrid nanogel suspension in the presence (right) and absence (left) of an external magnet.

PNIPA grafts with those un-ionized carboxyl groups enables the transformation of the hybrid particles into those with a vesicle architecture (Scheme 1). In addition, the inclusion of SPION to such a high level (44 wt %) enlarges the effective hydrophobic volume occupied by PNIPA. As a result, the relative volume ratio between the polar segments and the nonpolar PNIPA and SPION regions becomes more symmetric, which is beneficial to vesicle structure formation. A similar viewpoint on the coassembly of magnetic nanoparticles with amphiphilic copolymers in vesicle architecture was described previously.19 Relaxivity Measurement. The hybrid nanogels over the entire pH range (4.0−7.4) exhibit much higher r2 values in comparison to those of the citric acid-coated SPIONs alone at pH 5.0 and 7.4 (r2 = ca. 84 mM−1 s−1) (Figure 3a) and the SPION/dextran particles (30−50 mM−1 s−1) as reported elsewhere.33 This is attributed to the increased packing density of clustered SPIONs within the gel layer that induces the synergistic effect by promoting magnetic coupling among these SPIONs.9,21 In addition, owing to its water-retaining capacity, the gel layer reduces the mobility of water molecules near the SPION surface and thus retards the exchange of water species with those outside the gel layer. This then prolongs the time period in which water molecules interact with the magnetic field generated by the hybrid particles.22 More importantly, with the pH being adjusted from 4.0 to 7.4, r2 increases significantly from 138.5 to 265.5 (mM−1 s−1). This is ascribed primarily to the scenario in which the pH-induced increase in the water content retained intensively within the swollen gel layer as described above enables more water protons to interact with the magnetic field at the SPION surfaces, thereby greatly enhancing the relaxivity (Figure 3a). A similar observation was reported in the literature.22 Being consistent with the pHevolved T2 relaxation (Figure 3a), in the iron concentration range of 0.15−0.60 mM the hollow hybrid nanogels show prominent differences in the T2-weighted MR images among pH 7.4, 6.0, and 5.0 (Figure 3b) and thus illustrate great potential in the noninvasive in vivo imaging of tumoral acid microenvironments (pH 7.4−6.0) for diagnosis. It is important to note that the hybrid nanogels after being internalized by HeLa cells still preserve satisfactory MR imaging contrast (Figure 3c). The increased cellular uptake was clearly reflected in the significant contrast enhancement observed in the T2weighted MR images of HeLa cells exposed to higher magnetic nanogel doses.

ization of SPION with copolymer inevitably dilutes the concentration of SPION, thereby leading to a lower saturation magnetization value (36 emu/g) of the hybrid nanogels than that (77 emu/g) of the citric acid-coated SPIONs alone, the cross-linked hybrid nanogels retain excellent superparamagnetic behavior with negligible hysteresis (Figure 1a). The high sensitivity to external magnetic field can be readily confirmed by the instant agglomeration and full attachment (within 1 min) of the gel particles to an external magnet (Figure 1b). The morphology of the hybrid nanogels was assessed first by the angular dependence of the Dh of the hybrid nanogels in the angle range of 45−135° at pH 3.0, at which the target hybrid nanogels were prepared. As shown in Figure S5, the particle size of hybrid nanogels is essentially unchanged irrespective of the light-scattering angle. This confirms that the translational diffusion coefficient of the hybrid nanogels remains essentially constant in response to changes in the light-scattering angle, strongly implying hybrid nanogels in spherical form.31,32 The TEM images of the hybrid nanogels nevertheless manifest a hollowlike (vesicle) architecture, as evidenced by the clear-cut contrast between the center and the negatively stained hybrid gel layer with uranyl acetate (Figure 2a). The vesicle wall

Figure 2. TEM images of hollow hybrid nanogels at pH 3.0 (a) with and (b) without negative staining using uranyl acetate.

thickness in the dry state observed by TEM is ca. 30 nm. On the basis of the lower electron scattering density of the organic constituent without staining as compared to that of the inorganic counterpart, Figure 2b further illustrates that these SPIONs cluster together primarily within the thin walls of the hollow particles. A similar TEM observation of polymeric vesicles hybridzed with SPION in hydrophobic membranes was reported elsewhere.15 This is in agreement with the hypothesis pertinent to the vesicle wall formation via the cooperative binding of SPIONs with the graft copolymer. It is postulated herein that the enhanced hydrophobic association by virtue of the pH-evolved (i.e., from pH 7.4 to 3.0) increase in the protonation of both PAA chain segments3,4 and citric acid residues on SPION surfaces and the hydrogen-bond pairings of E

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Figure 3. (a) Concentration-dependent relaxation rate (1/T2) of the citric acid-coated SPIONs and magnetic hybrid nanogels at different pH values. (b) T2-weighted MR images of the hybrid nanogels at 37 °C and various pH values. (c) T2-weighted MR images of HeLa cells incubated with hybrid nanogels at different concentrations for 1 h.

Figure 4. (a) pH-evolved drug release profiles of the DOX-loaded hybrid nanogels in buffer solutions of different pH at 37 °C. (b) Cumulative drug release of the DOX-loaded hybrid nanogels at pH 5.0 and 7.4 at various temperatures and at pH 5.0 and 37 °C over a period of 2 h after HFMF treatment of varying durations.

Drug Loading and Stimuli-Triggered Drug Release Behavior. In taking advantage of retaining the pronounced drug concentration gradient between the aqueous phases inside and outside the hollow nanoparticle at pH 7.4 essentially for the entire drug encapsulation process, it was found that positively charged DOX molecules tended to permeate the swollen gel layer and be deposited therein by binding to ionized AA and citric acid residues via electrostatic attraction and a stacking interaction.34,35 The hydrophobic DOX/AA and DOX/citric acid complexes could be further stabilized by hydrogen bonds between DOX and PNIPA.36 As a result, a rather high drug loading efficiency (88.3%) corresponding to a payload quantity of 9.6 wt % within the hollow hybrid nanogels was achieved. The in vitro release of encapsulated DOX from the hybrid nanoparticles was primarily governed by both pH and temperature (Figure 4a,b). The pH-evolved DOX release from the hybrid nanogels at 37 °C is demonstrated in Figure 4a. The cumulative drug release at pH 7.4 is severely limited. However, a remarkably enhanced drug release (>50%) at pH

5.0 can be achieved over a period of 24 h. With the temperature being raised from 4 to 37 or 45 °C far beyond the lower critical solution temperature (LCST, ca. 32 °C) of PNIPA, the hybrid nanogels at pH 5.0 exhibit greatly enhanced drug release compared to those at pH 7.4 (Figure 4b). In addition to an increase in the aqueous solubility of DOX at pH 5.0, the significantly reduced ionization of AA and citric acid moieties and the thermally induced coil-to-globule transition of PNIPA segments largely impair the original electrostatic attraction and hydrogen bonding between DOX species and copolymer components constituting the nanogel wall, thereby leading to enhanced drug liberation. Thus, the physically bound drug species can be liberated promptly in the intracellular acidic organelles (endosomes/lysosomes) after these vehicles are internalized into target cells via endocytosis compared to the physiological pH condition of the bloodstream. Besides, the very low drug leakage at pH 7.4 and 4 °C (Figure 4b) allows their long-term storage prior to the in vivo theranostic application. F

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Figure 5. (a) LSCM images (top) and optical micrographs (bottom) of HeLa cells exposed to the DOX-loaded magnetic nanogels in the presence (red circle) and absence (green circle) of external magnetic guidance, respectively, at 37 °C for 1 h. (b) Intracellular iron concentrations of HeLa cells treated with the hollow hybrid nanogels at 37 °C for 7 h with and without external magnetic guidance as determined by ICP-AES.

Figure 6. LSCM images of HeLa cells incubated with free DOX and DOX-loaded magnetic nanogels at 37 °C for 1 h (DOX concentration = 10 μM).

hybrid nanogels were internalized more profoundly by magnetic guidance compared to those in the absence of an external magnetic field, as shown by the dramatic difference in the intensities of both DOX fluorescence and Prussian blue associated with SPIONs (Figure 5a). This is in agreement with the quantitative ICP-AES measurements of intracellular iron concentrations with and without magnetic guidance (Figure 5b). These preliminary data positively illustrate that both the controlled delivery of anticancer drugs and MRI can be achieved by the precise magnetic guidance of the hybrid nanogels to target cells. With free DOX being used alone as a positive control, the internalization of the hybrid nanogels by HeLa cells in the absence of both magnetic guidance and HFMF-triggered drug release was demonstrated by LSCM (Figure 6). In comparison to free DOX that accumulates appreciably in the nuclei of HeLa cells, DOX species transported by the magnetic hollow nanogels still largely deposit in the cytosol region of the cell. Differences in both the intracellular quantity and localization of drug species essentially reflect an outcome of different cellular uptake mechanisms between free DOX (i.e., passive diffusion)40,41 and the magnetic hollow nanoparticles (i.e., endocytosis). In agreement with the LSCM observation, the flow cytometric histogram data (Figure S7) also indicate a slight difference in the intracellular DOX fluorescence intensity between the free drug form and the drugloaded magnetic hybrid system. The high viability of HeLa cells treated with drug-free magnetic nanogels of various concentrations in the presence or absence of an external magnetic field (>90%) was observed (Figure 7a). This indicates the nontoxic nature of the hybrid nanogels to the cells. Figure 7a also shows that, while being magnetically directed to the target cells and then subjected to the HFMF treatment for 20 min, the drug-free hollow hybrid

Notably, as the hybrid nanogels were exposed to an external HFMF, the suspension temperature was increased dramatically from 37 to 55 °C in a short time period (Figure S6) upon the oscillation of the SPION’s magnetic moment due to the Neel and Brownian relaxations.13,37 According to the literature,38,39 raising the temperature into the range 40−45 °C can impair cells directly in addition to rendering the cells more radiosensitive, whereas above 45 °C the thermal ablation (direct destruction) of some tumors occurs. This result implies the feasibility of using the hybrid nanogels to serve as promising candidates in dual-modal cancer therapy (i.e., both hyperthermia and thermoregulated drug release). Because it has been demonstrated above that the drug release from the hollow hybrid nanogels can be thermally triggered, the HFMF-induced local heat effect on the impairment of interactions between DOX and gel constituents and, consequently, the enhanced drug elution, particularly at pH 5.0, can be readily realized. For instance, the cumulative drug release over a period of 2 h was appreciably higher for the case with the HFMF treatment for 20 min than that in the absence of the HFMF trigger at pH 5.0 and 37 °C (Figure 4b). Furthermore, when the stimulus duration was prolonged from 20 to 30 min, the cumulative drug liberation was further increased from 27 to 36%. Cell Internalization and Cytotoxicity. The accumulation of the hollow hybrid particles at the target sites, particularly the tumor tissues, obviously becomes a crucial factor in the success of the multimodal cancer theranostics. In addition to the in vivo enhanced permeation and retention effects to concentrate nanoparticles primarily in the tumor area during circulation, the hybrid nanogels developed herein show great potential of being magnetically guided to a specific site. While being coincubated with HeLa cells in a Petri dish under which an external magnetic field was applied only on one side, the DOX-loaded G

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Figure 7. (a) Cell viability of HeLa cells incubated with the drug-free hybrid nanogels at 37 °C for 24 h with and without magnetic guidance (7 h) and of HeLa cells treated with drug-free hybrid nanogels at 37 °C under magnetic direction for 7 h, followed by HFMF stimulus of 20 min and additional incubation of 17 h. (b) Cell viability of HeLa cells incubated with free DOX and SPION/DOX-loaded nanogels with and without external magnetic guidance of 7 h at 37 °C for 24 h (n = 3). (c) Cell viability of HeLa cells treated with free DOX, DOX-free magnetic nanogels, and DOXloaded magnetic nanogels at 37 °C under magnetic direction for 7 h, followed by HFMF stimulus of 20 min and additional incubation of 17 h (n = 3).

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CONCLUSIONS The hollow hybrid nanogel system attained from the covalent stabilization of the coassembly of SPION with the copolymer, poly(AA-co-MEA)-g-mPEG/PNIPA, in the vesicle structure represents a promising versatile theranostic platform. Owing to the high level of SPION incorporated into coassociation with the pH/thermosensitive graft copolymer, the resultant hollow hybrid nanogels exhibit such advantageous features as stimulimediated MR imaging contrast, hyperthermia, and controlled drug elution and cellular uptake. All of these desirable characteristics lead to a prominent in vitro cytotoxic effect against tumor cells. Although the evaluation of the in vivo performance of the magnetic hybrid nanogels against tumor growth is obviously required, this work demonstrated the great potential of the multimodal theranostic system capable of combining hyperthermia and chemotherapy for cancer treatment in a magnetically guidable manner under MR imaging monitoring without resorting to an additional contrast agent.

nanogels show concentration-dependent cytotoxicity against HeLa cells. This demonstrates the prominent effect of guided hyperthermia on cell proliferation. With DOX being loaded, the effect of magnetic guidance on the promotion of the cellular uptake of the hollow hybrid nanogels also essentially reflects the in vitro drug cytotoxicity against HeLa cells. For example, while the cells remained ca. 45% viable from the coculture with the DOX-loaded hybrid nanogels (DOX concentration = 10 μM) alone, the survival rate was significantly reduced to ca. 27% with magnetic guidance (3500 G) and even slightly lower than that (34%) achieved by free DOX (Figure 7b). More importantly, the DOX-loaded magnetic nanogels experiencing the HFMF treatment after the magnetic direction to the target cells display the greatly enhanced ability to inhibit cell proliferation (for instance, as low as ca. 14% of the cell survival rate at a DOX concentration of 10 μM) in comparison to the drug-free magnetic nanogels (hyperthermia therapy only) and free DOX (chemotherapy alone, Figure 7c). Though rather preliminary, this work clearly demonstrated the feasibility of the proposed strategy: the payload was liberated within or in close proximity to cancer cells, and cooperatively enhanced cytotoxic effects were achieved via the simultaneous hyperthermia treatment.13 It was also corroborated that combining magnetothermo therapy and chemotherapy within a single platform functionalized with magnetic guidance as a nanoscopic multimodal theranostic system essentially promoted its cytotoxic performance against tumor cells.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of the graft copolymer. 1H NMR spectra of poly(NAS-co-MEA) in DMSO-d6 and poly(AA-coMEA)-g-mPEG/PNIPA in CDCl3 at 20 °C. TEM image of the citric acid-covered SPIONs. DLS particle size distribution profiles of the mixture of the SPION and graft copolymer, uncross-linked hybrid assemblies, hollow hybrid nanogels, and DOX-loaded hybrid nanogels in aqueous solutions at 25 °C. H

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DLS particle size distribution profiles of the hollow hybrid nanogels in aqueous solution (pH 7.4 and 25 °C) with different dilutions. Time-evolved temperature profile of the aqueous suspension of the hollow hybrid nanogels originally at 37 °C after being subjected to an external HFMF treatment for various durations. Flow cytometry histograms of HeLa cells incubated with free DOX and the DOX-loaded magnetic nanogels at 37 °C for 1 h (DOX concentration = 10 μM). 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]. Fax: 886-35718649. Tel: 886-35750829. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Science Council (NSC99-2627-M007-009 and NSC99-2221-E007-006-MY3) and National Tsing Hua University (102N2046E1), Taiwan. We thank the 7T Animal MRI Core Lab of the Neurobiology and Cognitive Science Center for technical and facility support and the Instrumentation Center for MRI experiments at National Taiwan University.

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