Dual Stimuli-Responsive Polymeric Hollow Nanogels Designed as

Oct 4, 2012 - Dual stimuli-responsive hollow nanogel spheres serving as an efficient intracellular drug delivery platform were obtained from the spont...
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Dual Stimuli-Responsive Polymeric Hollow Nanogels Designed as Carriers for Intracellular Triggered Drug Release Wen-Hsuan Chiang,†,⊥ Viet Thang Ho,†,⊥ Wen-Chia Huang,‡ Yi-Fong Huang,‡ 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: Dual stimuli-responsive hollow nanogel spheres serving as an efficient intracellular drug delivery platform were obtained from the spontaneous coassociation of two graft copolymers into the vesicle architecture in aqueous phase. Both copolymers comprise acrylic acid (AAc) and 2-methacryloylethyl acrylate (MEA) units as the backbone and either poly(Nisopropylacrylamide) (PNIPAAm) alone or both PNIPAAm and monomethoxypoly(ethylene glycol) (mPEG) chain segments as the grafts. The assemblies were then subjected to covalent stabilization within vesicle walls with ester-containing cross-links by radical polymerization of MEA moieties, thereby leading to hollow nanogel particles. Taking the advantage of retaining a low quantity of payload within polymer layer-enclosed aqueous chambers through the entire loading process, doxorubicin (DOX) in the external bulk phase can be effectively transported into the gel membrane and bound therein via electrostatic interactions with ionized AAc residues and hydrogen-bond pairings with PNIPAAm grafts at pH 7.4. With the environmental pH being reduced (e.g., from 7.4 to 5.0) at 37 °C, the extensive disruption of AAc/DOX complexes due to the reduced ionization of AAc residues within the gel layer and the pronounced shrinkage of nanogels enable the rapid release of DOX species from drug-loaded hollow nanogels. By contrast, the drug liberation at 4 °C was severally restricted, particularly at pH 7.4 at which the DOX molecules remain strongly bound with ionized AAc residues and PNIPAAm grafts. The in vitro characterizations suggest that the DOX-loaded hollow nanogel particles after being internalized by HeLa cells via endocytosis can rapidly release the payload within acidic endosomes or lysosomes. This will then lead to significant drug accumulation in nuclei (within 1 h) and a cytotoxic effect comparable to free drug. This work demonstrates that the novel DOX-loaded hollow nanogel particles show great promise of therapeutic efficacy for potential anticancer treatment.

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INTRODUCTION Development of stimuli-responsive nanogels serving as functional drug delivery vehicles has attracted considerable interests because of their enhanced stability and drug loading capacity and well-modulated drug release in response to biological stimuli such as difference in pH or temperature, redox reactions, and enzymes, etc.1−5 Representative approaches to the preparation of such smart colloidal assemblies usually composed of hydrophilic polymeric networks or core−shell architectures with either cross-linked cores or shells include inverse microemulsion polymerization,6,7 cross-linking of preformed polymeric micelles8−14 or vesicles (also being referred to as polymersomes),15−18 and one-step ring-opening polymerization.19 Zhang et al. prepared multifunctional and degradable zwitterionic nanogels capable of carrying both fluorescent-labeled dextran as a drug model and Fe3O4 nanoparticles as a magnetic resonance (MR) imaging agent by the inverse microemulsion polymerization of carboxybetaine methacrylate in the presence of L-cystine bis(acrylamide) acting as a disulfide (redox-sensitive) cross-linker.7 The cargo-loaded © 2012 American Chemical Society

nanogels showed a superior reduction-responsive drug release and MR imaging contrast, thereby rendering the assemblies rather promising as a deliverable theranostic device. As reported by Shuai and co-workers,13 via formation of disulfide cross-links within the intermediate layer of polymeric micelles from self-assembly of a triblock copolymer comprising poly(ethylene glycol) (PEG), 2-mercaptoethylamine-grafted poly(L-aspartic acid) (PAsp(MCEA)), and 2(diisopropylamino)ethylamine-grafted poly(L-aspartic acid) (PAsp(DIP)) in aqueous phase of pH 10.0, dual redox- and pH-sensitive cross-linked micelles were developed. While being internalized into cells and confined within glutathionecontaining acidic lysosomes (ca. pH 5.0), the resultant micelles displayed a burst release of loaded anticancer drugs. This was attributed to both cleavage of the disulfide cross-links of intermediate gel layers and disintegration of PAsp(DIP) cores. Received: July 18, 2012 Revised: October 3, 2012 Published: October 4, 2012 15056

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Figure 1. Chemical structures, compositions, and average molecular weights of copolymers A and B. The numbers associated with the chemical structures correspond to the proton signals of copolymers A and B in the 1H NMR spectra shown in Figure 2c.

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Distinct from the above stimuli-responsive nanogel-like asssemblies prepared mainly by cross-linking of the interfacial layer within polymeric micelles, stable pH-responsive micelles constituting PEG shells and PAsp cores were developed by core cross-linking of the target from the PEG/PAsp diblock copolymer using 1,6-hexanediamine as a cross-linker.14 By opposite charge interaction between Asp residues and doxorubicin (DOX) species, the device was characterized by not only a relatively high drug-loading capacity but also an accelerated drug release at pH 5.0 with respect to that at pH 7.4. This was due to the increased aqueous solubility of DOX species and reduced charge attraction in response to such a pH change. In this study, a novel strategy was proposed for development of dual stimuli-responsive hollow nanogels as an effective intracellular drug delivery system. Polymer assemblies in vesicle form were first obtained from coassociation of two graft copolymers both comprising AAc and MEA units as the backbone and either PNIPAAm alone or both PNIPAAm and mPEG as the grafts in aqueous phase of pH 3.0 at ambient temperature via hydrogen-bond pairings of un-ionized PAAc and PNIPAAm segments. Hollow nanogels were then attained by photoinitiated radical polymerization of the MEA moieties within vesicle walls. Hollow nanogels developed in this work show several superior characteristics, as described below. First, the nanoparticles can be readily prepared in aqueous phase without resort to undesirable organic solvents, thus making their potential biomedical applications more attractive. High DOX loading can be achieved at pH 7.4 through a facile diffusion process driven by an extremely low drug concentration within inner aqueous chambers as compared to the drug concentration in the outer bulk phase. Such a concentration gradient is established by the complementary complexation of DOX with ionized AAc residues along with cooperative hydrogen-bond pairings with PNIPAAm segments. While the hollow nanogel particles are preferentially accumulated in the tumor microenvironment via the enhanced permeability and retention effect, drug liberation can be largely promoted in acidic endosomal/lysosomal compartments after cellular endocytic internalization by virtue of extensive detachment of DOX species from nanogel membranes and combined transport process involving concentration-dependent diffusion and water elution pertinent to the pH-evolved morphologic transition of hollow nanogel particles.

EXPERIMENTAL SECTION

Materials. Synthesis and characterization of two graft copolymers both comprising AAc and MEA units as the backbone and either PNIPAAm alone (referred to hereinafter as copolymer A) or both PNIPAAm and mPEG as grafts (B) employed in this work were performed according to our previous studies.11,12 The chemical structures, compositions, and average molecular weights of the graft copolymers are illustrated in Figure 1. The apparent pKa values of both copolymers herein referred to as the pH of the aqueous medium at which half (50%) of the AAc residues were ionized were determined by the potentiometric titration of graft copolymers in water (25 °C) with an aqueous NaOH solution (0.025 M), using a Mettler Toledo DL53 autotitration system equipped with a DG 101-SC pH electrode. Based on the similar potentiometric titration profiles of copolymers A and B, the apparent pKa values were obtained to be ca. 7.0 for both copolymers. In addition, the dissociation degrees of the AAc residues were ca. 75% at pH 7.4 and 25 °C and only 11% at pH 5.0. 2,2Diethoxyacetophenone (DEAP) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma and DOX (in the hydrochloride salt form) was obtained from Seedchem. Dulbecco’s modified Eagle medium (DMEM), Hoechst 33258, and fetal bovine serum (FBS) were purchased from Invitrogen. Deuterium solvents used in 1H NMR measurements were obtained from Cambridge Isotope (Andover, MA). Deionized water was produced from Milli-Q Synthesis (18 MΩ, Millipore). All other chemicals were reagent grade and used as received. Hollow Nanogel Preparation. Copolymers A and B at a weight ratio of 3/1 (w/w) were added together into aqueous NaCl solution (0.01 M) to a total concentration of 1.0 mg/mL. The copolymer solution was adjusted to pH 7.4 with 0.1 N NaOH and then stirred at 4 °C for 30 min. The solution was passed through a 0.45 μm filter. Polymersome suspension was attained by reducing the medium pH from 7.4 to 3.0 with 0.1 N HCl at 25 °C under stirring and equilibrated under such conditions for an additional 24 h. After being purged with N2 for 10 min, the aqueous vesicle suspension containing DEAP (10 wt %) as the photoinitiator was placed into ultraviolet cross-linkers (UVP CL-1000) equipped with five 8 W 254 nm UV tubes. Covalent cross-linking of vesicle membranes was achieved by radical polymerization of MEA residues within vesicle membranes under UV light of 80 mW/cm2 for 30 min. The pristine hollow nanogel particles thus obtained were thoroughly purified by ultrafiltration (Amicon 8200 with a Millipore PBMK membrane, MWCO 300 000) against aqueous NaCl solution (0.01 M) at ambient temperature to remove copolymers that were not cross-linked. 1 H NMR Characterization. Aqueous solutions containing copolymers A and B at pD 7.4 and 3.0 in D2O were prepared as stated above for 1H NMR measurements while the medium pD was adjusted by either NaOD or DCl. The 1H NMR characterization of the sample at pD 7.4 or 3.0 was conducted on a Varian Unity Inova-500 NMR spectrometer at 500 MHz. The pulse width of 4.9 μs with a 15057

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Figure 2. (a) DLS particle size distribution profiles of mixed copolymers A and B, polymeric assemblies, and hollow nanogel particles in aqueous phases at 25 °C. (b) Photograph of aqueous solutions of mixed copolymers A and B at pH 7.4 and 3.0 at 25 °C. (c) 1H NMR spectra of mixed copolymers A and B in D2O at pD 7.4 and 3.0 at 25 °C. relaxation delay of 2.0 s was utilized. The sample was equilibrated at 25 °C for 30 min prior to measurement. Dynamic and Static Light Scattering (DLS/SLS) Measurements. The mean hydrodynamic particle diameter (Dh) and particle size distribution of polymer vesicles in aqueous media were determined by a Brookhaven BI-200SM goniometer equipped with a BI-9000 AT digital correlator using a solid-state laser (35 mW, λ = 637 nm) detected at a scattering angle of 90° based on the cumulant method. The experimental results reported herein represent an average of at least triplicate measurements. In addition, the angular dependence of the autocorrelation functions was measured using the same instrument as described above. Correlation functions were also analyzed by the cumulant method at varying angles. To assess the morphology of polymeric assemblies in terms of the ratio of the gyration radius to the hydrodynamic radius of assemblies, the mean hydrodynamic radius (Rh) was obtained at a scattering angle of 90° based on the CONTIN method. The root-mean-square radius of gyration (Rg) of polymeric assemblies was determined by the angular dependent measurements of the light scattering intensity. The partial Zimm plot of the scattering intensity (Iex−1) versus the square of the scattering vector (q2) was used for the quantitative determination of Rg. Transmission Electron Microscopy (TEM) Examination. TEM images were obtained from a JEOL JEM-1200 CXII microscope operating at an accelerating voltage of 120 kV. Samples were prepared by placing a few drops of the polymer colloid on a 300-mesh copper grid covered with carbon and then negatively stained with uranyl acetate (5.0 wt %) for 20 s and dried at 25 °C for 2 days before measurements. DOX Loading and Stimuli-Triggered Drug Release. DOX originally in salt form was dissolved in the aqueous phase and added directly into the aqueous hollow nanogel suspension (pH 7.4) to a final concentration of 0.5 mM, and subsequently, the pH was adjusted back to 7.4. The DOX-containing suspension was then equilibrated under stirring at 25 °C for 24 h, followed by thorough dialysis (Cellu Sep MWCO 12 000−14 000) against the phosphate buffer of pH 7.4 (ionic strength 0.01 M) for 3 days to remove unloaded DOX. To

determine the drug loading level, a small portion of DOX-loaded hollow nanogel particles was withdrawn and diluted with DMF to a volume ratio of DMF/H2O = 9/1. The amount of DOX encapsulated was quantitatively determined by a fluorescence spectrophotometer (Hitachi F-7500). The excitation was performed at 480 nm, and the emission spectrum was recorded in the range 500−700 nm. The calibration curve used for drug loading characterization was established by the fluorescence intensity of DOX with different concentrations in DMF/H2O (9/1 (v/v)) solutions. Drug loading efficiency (DLE) and drug loading content (DLC) were calculated according to the formulas

DLE (%) = (weight of loaded DOX/weight of DOX in feed) × 100% DLC (%) = (weight of loaded DOX/weight of copolymer from lyophilization of nanogel suspension of a prescribed volume after purification) × 100% The drug release profiles at different pH values were determined by the dialysis technique. The DOX-loaded hollow nanogel dispersion (1.0 mL) was placed within a dialysis tube (Cellu Sep MWCO 12 000−14 000), followed by dialysis against different buffer solutions including succinic acid buffers of pH 5.0 and phosphate buffer saline of pH 7.4 (50 mL, ionic strength 0.15 M) at 37 or 4 °C. At prescribed time intervals, 1.0 mL of the external buffer solution (pH 5.0 or 7.4) was withdrawn and replaced with an equivalent volume of fresh medium. The concentration of DOX was determined by the fluorescence technique using the pertinent calibration curve of DOX with various concentrations in aqueous solution of either pH 5.0 or 7.4. The experimental results presented herein represent an average of at least triplicate measurements. Cellular Uptake. HeLa cells (1 × 105 cells/well) were treated with free DOX species and DOX-loaded hollow nanogel particles, 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. Drug 15058

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cellular uptake was then analyzed on a FACSCalibur flow cytometer (BD Biosciences). A minimum of 1 × 104 cells were analyzed from each batch with fluorescence intensity displayed on a three-decade log scale. For confocal laser scanning microscopy (CLSM) studies, HeLa cells (2 × 105 cells/mL) were seeded onto 22 mm round glass coverslips, placed in a 6-well plate, and cultured overnight. The cells were then incubated with free DOX and DOX-loaded hollow nanogels at a DOX concentration of 10 μM for 1 h. Cells were washed twice with PBS and fixed with 4% formaldehyde. Finally, cells were stained with Hoechst 33258 for 10 min, and the slides were rinsed with PBS three times. Coverslips were placed onto the glass microscope slides, and cellular uptake of DOX was visualized at the excitation and emission wavelength of 488 and 590 nm, respectively, by a ZESS LSM 510 META. Cytotoxicity Analysis. HeLa cells were seeded in a 96-well plate at a density of 1 × 104 cells/well in DMEM (100 μL) 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 either free DOX or DOX-loaded hollow nanogel particles at varying DOX concentrations or DOX-free nanogels, and cells were incubated for 24 h. Thereafter, 5 μL of MTT (5.0 mg/mL) was added into each well, followed by incubation at 37 °C for 4 h. After discarding the culture medium, DMSO was added to dissolve the precipitate, and the resulting solution was measured for absorbance at 570 nm with a reference wavelength of 690 nm using a SpectraMax M5 microplate reader.

segments largely impairs the segmental mobility of complexed PNIPAAm grafts. The spin−lattice relaxation time (T1) of the methyl protons of PNIPAAm segments is thus significantly reduced, rendering the corresponding protons undetectable by 1 H NMR. Nevertheless, this feature proton signal at pD 3.0 does not disappear completely, indicative of incomplete hydrogen-bond pairings due to in part the excess NIPAAm residues on molar basis with respect to AAc moieties from mixed copolymers. Distinct from the hydrogen-bond paired PNIPAAm segments with the severely restricted segmental flexibility, free PNIPAAm grafts maintain excellent solubility and segmental mobility in the aqueous phase at temperature far below their LCST. On the other hand, just like the sample at pD 7.4, the feature signal of ethylene protons at δ 3.75 ppm from mPEG grafts of copolymer B within assemblies remains fully detectable by 1H NMR at pD 3.0. This strongly suggests that mPEG grafts be capable of interacting with water molecules extensively to retain the prominent segmental mobility, thereby effectively preventing colloidal particles from aggregation via its steric repulsion effect. Furthermore, a linear relationship between the relaxation frequency (Γ) and the square of the scattering vector (q2) was observed in the angle-dependent DLS measurements at pH 3.0 (Figure 3). This indicates the target polymeric assemblies in a

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RESULTS AND DISCUSSION Development and Characterization of Pristine Hollow Nanogel Platform. Pristine hollow nanogel particles were prepared first by spontaneous coassociation of copolymers A and B in the continuous aqueous phase into assemblies in polymersome form. This was achieved by reducing the medium pH from 7.4 to 3.0 at 25 °C far below the LCST of PNIPAAm. The DLS data illustrate that mixed copolymers A and B (at a weight ratio of copolymer A/copolymer B = 3/1, total concentration = 1.0 mg/mL) underwent self-assembly, as shown by dramatic changes in hydrodynamic particle diameter and size distribution and the corresponding light scattering intensity in Figure 2a. Obviously, assemblies with very poorly defined, loose structure at pH 7.4 were developed. Once experiencing the pH change to 3.0, mixed copolymers A and B underwent coassociation into stable assemblies with Dh equal to ca. 170 nm and a very narrow particle size distribution (polydispersity index (PDI): ca. 0.08). In supporting the DLS data, the aqueous solution containing both copolymers A and B instantly turned from the clear to turbid state upon the pH change (Figure 2b) by virtue of the hydrophobic coassociation of mixed copolymers in aqueous solution. This is primarily due to the increased protonation of AAc moieties of mixed copolymers at pH 3.0 that promotes facile complementary pairings of un-ionized AAc moieties with NIPAAm residues in a consecutive manner via hydrogen bonds. Remarkable hydrophobic association between PAAc and PNIPAAm segments thus evolved further induces the formation of colloidal assembly. Similar observations can be found in the literature.11,20−22 Figure 2c illustrates 1H NMR spectra of mixed copolymers A and B in D2O at pD 7.4 and 3.0, respectively, at 25 °C. As compared to that at pD 7.4, the signal intensity of the methyl protons (at δ 1.25 ppm) of PNIPAAm segments was appreciably reduced at pD 3.0, in agreement with the above postulation that PNIPAAm grafts are involved extensively in the development of hydrophobic dehydrated regions within polymeric assemblies. Apparently, such hydrophobic association of un-ionized PAAc with PNIPAAm chain

Figure 3. Angle-dependent DLS/SLS measurements for polymeric assemblies before and after cross-linking at pH 3.0 and 25 °C.

spherical form.23,24 The Rg value was also obtained from the partial Zimm plot of Iex−1 versus q2 by angle-dependent SLS measurements (Figure 3). The Rg/Rh ratio of ca. 0.99 is essentially identical to the theoretical value (1.0), which serves as a supporting evidence for thin shell hollow spheres (note that an Rg/Rh ratio of 0.77 implies the formation of solid sphere-like particles).23−26 The TEM photograph (Figure S1 in the Supporting Information) also confirms the formation of spherical vesicles, as evidenced by the appearance of the negatively stained peripheral walls with uranyl acetate. Similar observations were reported elsewhere.27,28 There is no doubt that polymeric vesicles formed with an internal hollow chamber enclosed by a hydrophobic PAAc/PNIPAAm membrane that was further elaborated by highly hydrated mPEG and unpaired PNIPAAm graft segments extending toward the continuous aqueous phase (Scheme 1). It is noteworthy that the mPEG content of mixed copolymers A and B, similar to the appropriate hydrophilic mass fractions of amphiphilic copolymers prone to selfassembly into vesicles in aqueous phase as described in the 15059

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Scheme 1. Schematic Illustration of Development of Hollow Nanogel Particles

Figure 4. TEM images of pristine hollow nanogels at pH (a) 3.0, (b) 5.0, and (c) 7.4.

literatures,24,29,30 plays a profound role in the morphology of self-assembled polymeric particles and their colloidal stability. For example, at a constant total copolymer concentration (1.0 mg/mL), while a weight ratio of copolymer A to B was 1/3 (instead of 3/1 chosen for this study), the mixed copolymers at pH 3.0 tended to coassociate into micelles comprising hydrophobic PAAc/PNIPAAm cores surrounded by mPEG coronas due to the relatively high mPEG content. By contrast, at a weight ratio of copolymer A/copolymer B = 4/1, huge colloidal particles formed due to the interparticle aggregation in the absence of sufficient steric stabilization provided by mPEG chain segments (data not shown). These results illustrate that an appropriate mPEG content (e.g., at a weight ratio of 3/1 (A/ B) in this study) is required to acquire well-defined polymeric vesicles from the coassociation of mixed copolymers A and B. It is crucial that the polymeric vesicles are imparted the desirable structural integrity at pH 7.4 in combination with facile pathways for effective drug loading and release via the stimuli-induced structural evolution in drug delivery applications. To achieve this goal, the polymeric assemblies were further elaborated by covalent cross-links within vesicle membranes. As a result of the cross-linking process, nanogels in hollow particle form were produced. The reaction took place in the aqueous phase (at pH 3.0 and 25 °C) by the photoinitiated radical polymerization of MEA residues confined within the vesicle membrane. It is worthy to note that each individual MEA residue carries two ester linkages capable of undergoing slow hydrolysis and subsequent assembly disintegration under physiologic conditions. Figures 2a and 3 demonstrate that the resultant hollow nanogel particles have very similar particle size and Rg/Rh value to the polymeric vesicles at pH 3.0 prior to cross-linking. While experiencing a pH increase from 3.0 to 7.4, these nanogel particles are apparently enlarged yet retain very narrow particle size distribution (Figure 2a). Being consistent with the DLS data, TEM images (Figure 4) indicate the very good dispersion of nanogels as individual spherical hollow particles at all pH (3.0, 5.0, and 7.4) by virtue of effective polymerization of MEA residues occurring within the assembly membrane. The result

of TEM images at pH 3.0 and 7.4 is qualitatively consistent with the DLS data shown in Figure 2a. Stimuli-Evolved Structural Transformation for Facile Drug Loading and Release. In view of the hollow nanogel particles comprising both pH-responsive PAAc and thermosensitive PNIPAAm chain segments, they are expected capable of exhibiting a dual stimuli-responsive property. As shown in Figure 5, substantial changes in both Dh and Rg/Rh ratio of

Figure 5. Dh and Rg/Rh data for hollow nanogels in aqueous solutions of 25 °C as a function of pH: (□) (Dh), (●) (Rg/Rh).

hollow nanogels occur between high (i.e., 7.4) and low pH (5.0) of the external media at 25 °C. The AAc residues undergo significant deprotonation at pH 7.4, which results in not only extensive disruption of hydrogen bonds between AAc and NIPAAm units but also development of ionic osmotic pressure gradient induced by the immobilized ionized AAc residues within nanogel membranes, as described by the Donnan equilibrium theory.11,12 This inevitably enhances water influx into both the peripheral gel layer and the inner aqueous chamber. At pH 7.4 and 25 °C, a more remarkable increase in Rg in comparison with Rh (ca. 1.4-fold higher than the increase of Rh) was observed (Figure 5). It should be noted that, by definition, Rg is closely related to the mean distance of 15060

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Figure 6. Dh data for hollow nanogels in aqueous solutions of pH 5.0 and 7.4 at 4 and 37 °C, respectively.

individual atoms (groups) constructing the target assembly to its mass center. This confirms that the gel layer becomes loose and swollen in addition to a spatial enlargement of the internal aqueous chamber at pH 7.4.23,25 Ionized AAc units residing within such a loose and hydrated layer make themselves available as binding sites for electrostatic interactions with the positively charged anticancer agent (DOX) chosen for this study. Extensive binding of DOX molecules with ionized AAc residues can thus induce the development of hydrophobic and π−π stacking association of DOX species within the gel layer,14,31,32 thereby preventing DOX molecules from being transported in the dissolved state into the inner aqueous chamber. In addition, hydrogen bonds developed between the amide groups of PNIPAAm grafts with DOX species undoubtedly enhance the stability of AAc/DOX complexes.33 As a consequence, a very high drug loading capacity (DLE: ca. 84% and DLC: ca. 23 wt %) can be achieved by retaining a very low concentration of soluble DOX within the gel-enclosed aqueous chamber as compared to that in the external bulk phase during the essentially entire drug encapsulation process. With the temperature being raised to 37 °C, an appreciable reduction in the particle volume (ca. 42%, estimated from the change of Dh shown in the right half of Figure 6) of hollow nanogel particles occurs in response to a change in the medium pH from 7.4 to 5.0. Such a pH change (around 37 °C) stimulates the physiologic pH variation before and after cellular uptake of particles into endosomes/lysosomes via endocytosis. This suggests a considerable outflow of water molecules from the hollow nanogels as primarily a result of the enhanced AAc protonation in collaboration with hydrophobic association with thermally induced dehydrated PNIPAAm grafts. Taking advantage of the concomitant interruption of electrostatic pairings of drug species with AAc residues within gel layers owing to the pH-evolved increase of AAc protonation at 37 °C, this water efflux may essentially facilitate the payload elution in addition to the conventional diffusion pathway. Functionalizing this unique drug delivery hollow nanogel device with capability of undergoing temperature/pH-controllable morphologic tuning provides a facile, yet effective, route to rapidly liberate the target payload intracellularly upon pH changes along with their cellular uptake via endocytosis. Figure 7 illustrates the pH-controlled drug release profiles obtained from DOX-loaded hollow nanogel particles in aqueous solution (ionic strength 0.15 M) at 37 or 4 °C. In agreement with the above postulation, the cumulative drug release performed at pH 5.0 and 37 °C over a period of 3 h (50%) is much higher than that (20%) in the milieu of pH 7.4 at the same temperature. In addition to an increase in the

Figure 7. Cumulative drug release profiles of DOX-loaded hollow nanogels in buffer solutions of different pH at 37 and 4 °C.

aqueous solubility of DOX with the medium pH being lowered from 7.4 to 5.0, the dramatically reduced AAc ionization also induces the disruption of the electrostatic binding of AAc residues with DOX species. Considering extensive detachment of drug species from mixed copolymers A and B that constitute nanogel walls, the promotion in payload release at pH 5.0 and 37 °C was therefore driven by virtue of concentrationdependent diffusion and gel shrinkage-induced water elution as well. However, because a part of the AAc residues within nanogels at pH 5.0 remain deprotonated, thus retaining the AAc/DOX complexes to some extent and stabilizing the DOX π−π electron stacking closely associated via hydrogen-bond pairings with the PNIPAAm grafts. This leads to the incomplete liberation of the originally loaded DOX species from nanogels. The water efflux effect was demonstrated in part by an appreciably higher drug unloading, particularly within the first 3 h period, at 37 °C than that at 4 °C (both at the same pH of 5.0 as shown in Figure 7) at which temperature the particle volume contraction and thus water outflow was somewhat retarded (Figure 6). This is primarily attributed to the lack of phase transition and hydrophobic association of PNIPAAm segments from nanogels at 4 °C (far below the LCST of PNIPAAm grafts). Under the circumstances, the drug was released primarily by a diffusion-controlled mechanism. A similar observation has been reported previously.34−36 The results illustrated in Figure 7 also suggest that DOX-loaded hollow nanogels can fully retain the structural integrity at 4 °C for storage with negligible drug elution prior to the in-vivo therapeutic applications, particularly at pH 7.4, the most frequently used milieu pH that meets the physiologic requirement. In conclusion, the hollow nanogel system 15061

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developed herein preserves the structural stability while allowing drug loading and retention in an effective manner at pH 7.4 via ester cross-links within polymeric vesicle membranes. Furthermore, this delivery platform exhibits prominent payload release at pH 5.0 (at 37 °C), thereby demonstrating itself great promise in intracellular drug release and transport within acidic endosomal or lysosomal compartments. Cellular Uptake and in Vitro Cytotoxicity. The cellular uptake of DOX-loaded hollow nanogel particles by HeLa cells was evaluated by flow cytometry and CLSM. Figure 8 shows

intensity within HeLa cells incubated with DOX-loaded nanogel assemblies for 1 h is rather profound, which is only slightly lower than that of cells treated with free DOX species. In agreement with the in vitro release study, DOX molecules were rapidly released from endocytosed hollow nanogel vehicles most probably due to prominent stimuli-triggered responses, thereby leading to localization in a large measure in the nucleus regions comparable to free drug used in the control experiment. The in vitro cytotoxicity of DOX-loaded hollow nanogel particles against HeLa cells in comparison with free DOX species determined by MTT assay is shown in Figure 10. As an

Figure 8. Flow cytometric histogram profiles of HeLa cells incubated with free DOX and DOX-loaded hollow nanogels at 37 °C for 1 h (DOX concentration = 10 μM).

Figure 10. Viability of HeLa cells incubated with free DOX molecules and DOX-loaded hollow nanogels for 24 h. Data are presented as mean ± SD (n = 3).

flow cytometric histograms of HeLa cells incubated with free DOX and DOX-loaded hollow nanogel particles over a period of 1 h. Cells without any DOX treatment were used as a negative control. Compared to the control, HeLa cells incubated with either free DOX or DOX-loaded hollow nanogels exhibit much enhanced DOX fluorescence intensity, which is indicative of the cellular uptake of free DOX or DOXloaded hollow nanogels. In comparison with DOX-loaded nanogels, a slightly higher extent of free DOX uptaken by HeLa cells was observed. This is primarily due to distinct routes of cellular internalization. It is well-known that free DOX molecules are transported into cells by passive diffusion pathway,37,38 whereas DOX-loaded hollow nanogel particles are internalized by cells via endocytosis. The CLSM images (Figure 9) clearly demonstrate that the DOX fluorescence

important control, high viability of HeLa cells exposed to pristine nanovehicles in the concentration range 0.25−32 μg/ mL for 24 h was observed (Figure S2). This demonstrates that the drug-free hollow nanogels are virtually nontoxic to HeLa cells. When HeLa cells were incubated with free DOX molecules or DOX-loaded hollow nanogels in the drug concentration range 0.16−20 μM, DOX-loaded nanogels exhibited comparable cytotoxic effect to free DOX species. The observed drug doses required for 50% cellular growth inhibition (IC50) of the free DOX and the DOX-loaded hollow nanogels are ca. 3.6 and 4.3 μM, respectively. As compared to other delivery systems reported elsewhere,37−40 such an amazing similarity in cytotoxicity to free DOX shows the superiority of hollow nanogels developed in this work. Thus, this novel hollow nanogel system may serve as an effective

Figure 9. CLSM images of HeLa cells incubated with free DOX molecules and DOX-loaded hollow nanogels at 37 °C for 1 h (DOX concentration = 10 μM). Cell nuclei were stained with Hoechst 33358. 15062

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ACKNOWLEDGMENTS This work is supported by the National Science Council (NSC99-2627-M007-009 and NSC99-2221-E-007-006-MY3) and National Tsing Hua University (99N2913E1 and 99N82309E1), Taiwan.

intracellular pH-triggered drug release carrier. Upon the cellular uptake of the DOX-loaded hollow nanogels via endocytosis, it is expected that a high intracellular drug concentration can be achieved via the pH-induced rapid DOX release from drugloaded hollow nanogels within acidic endosomes and lysosomes, thereby not only maximizing the therapeutic efficacy of solid tumor chemotherapeutic treatment but also possibly further overcoming the multidrug resistance of cancer cells.41,42

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ASSOCIATED CONTENT

S Supporting Information *

TEM image of polymeric assemblies before cross-linking at pH 3.0 and 25 °C and the viability of HeLa cells after being coincubated with pristine hollow nanogels for 24 h. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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CONCLUSION Hollow nanogel particles were developed to serve as a dual stimuli-responsive drug delivery carrier from spontaneous coassociation of two graft copolymers into polymersome structure. Both copolymers comprise AAc and MEA units as the backbone and either PNIPAAm alone or both PNIPAAm and mPEG chain segments as the grafts. Polymeric vesicles were produced via hydrogen-bond pairings of un-ionized AAc and NIPAAm residues at pH 3.0 and 25 °C. The vesicle wall was then covalently stabilized with ester-containing cross-links by photoinitiated radical polymerization of the carbon−carbon double bond containing MEA units therein, thereby leading to hollow nanogel particles. Taking the advantage of a low quantity of payload retention within the gel-enclosed aqueous chamber through the entire drug loading process, DOX species can be effectively bound via the electrostatic interaction with ionized AAc residues and hydrogen-bond pairings with PNIPAAm grafts within the gel membrane at pH 7.4. Accompanied with the dissociation of DOX species from mixed copolymers within the gel layer in response to the decreased medium pH from 7.4 to 5.0 at 37 °C, an accelerated drug release from DOX-loaded hollow nanogels was achieved via the operative mechanism combining concentration-dependent diffusion with water elution action resulting from pHinduced gel volume contraction. The in vitro characterization of drug-loaded hollow nanogels suggested that the delivery system be capable of rapidly releasing the payload within intracellular endosomes or lysosomes of HeLa cells after being internalized via endocytosis, leading to significant drug accumulation within nuclei. As a consequence, DOX-loaded hollow nanogels displayed a cytotoxic effect against HeLa cells comparable to free DOX. The results obtained from this work demonstrated that hollow nanogel particles showed great promise of therapeutic efficacy for the potential anticancer treatment.

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*Fax 886-35718649; Tel 886-35750829; e-mail hscchiu@mx. nthu.edu.tw. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 15063

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