Dual-Responsive Controlled Drug Delivery Based on Ionically

May 24, 2012 - The potential practical applications as drug delivery carriers were demonstrated using doxorubicin (DOX) as a model drug. With a lower ...
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Dual-Responsive Controlled Drug Delivery Based on Ionically Assembled Nanoparticles Wei Cui,† Xuemin Lu,† Kun Cui,† Lvye Niu,† Yen Wei,‡ and Qinghua Lu*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, P.R. China ‡ Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China S Supporting Information *

ABSTRACT: Ionically assembled nanoparticles (INPs) have been formed from poly(ionic liquid-co-N-isopropylacrylamide) with deoxycholic acid through electrostatic interaction. The structure and properties of the INPs were investigated by using 1H NMR, Fourier transform infrared (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), and so on. Due to pH-responsive deoxycholic acid (pKa = 6.2) and thermo responsive N-isopropylacrylamide included in the ionic complex, the INPs exhibit highly pH and thermal dual-responsive properties. The potential practical applications as drug delivery carriers were demonstrated using doxorubicin (DOX) as a model drug. With a lower pH (pH 5.2) and higher temperature (above 37 °C), structural collapse of the INPs occurred as well as release of DOX owing to protonated DA departure from the INPs and a lower LCST (lower critical solution temperature) at the pathological conditions. The result shows that 80% of DOX molecules were released from INPs within 48 h at pH 5.2, 43 °C, but only 30% of the drug was released within 48 h at 37 °C and pH 7.4. Moreover, drug-loaded INPs exhibit an inhibitory effect on cell growth. (caprolactone) nanoparticles for anticancer drug delivery.6 It was found that these nanoparticles deformed at body temperature under conditions of slightly acidic pH (pH 6.9), which triggered the release of the loaded anticancer drug. Similarly, Liu et al. developed poly (N,N-diethylacrylamide-comethacrylic acid) microspheres with well-defined temperaturesensitive cores and pH-sensitive shells for site-specific drug delivery in the intestine.9In this research, an alkaline pH of the medium led to swelling of the microspheres and an accelerated drug release rate. However, for most of the present dualresponsive systems, pH response resulted from the acidic pH environment reducing the lower critical solution temperature (LCST) of copolymers. However, acidic pH did not directly cause dissociation of nanoparticles. On the other hand, a complicated procedure was also included in preparation of such system. For further improving therapeutic efficacy of controlled

1. INTRODUCTION In the past decade, stimuli-responsive materials (nanoparticles, micelles, vesicles, gels, etc.) have received much attention as drug nanocarriers because they can offer the advantages of improving the therapeutic activity of the drug, reducing general drug toxicity, and decreasing drug dosage.1−4 Rapid growth of malignancy cells can cause a temperature increase and a pH decrease in its microenvironment, providing a handle for pathological cells treatment with controlled drug release. Such a concept has fueled research toward the development of thermo-, pH-, or dual-responsive polymeric nanocarriers.5−8 Among them, dual responsive polymeric nanoparticle is a much more attractive topic and received wide and intensive interest due to its potential advantage in intelligently distinguishing normal and pathological cells, so achieving much better release and control compared to monoresponsive nanocarriers. A series of smart nanoparticles were prepared based on the copolymer of thermo responsive poly(N-isopropylacrylamide) (PNIPAAM) and pH sensitive moiety. For example, Zhang et al. synthesized poly(N-isopropylacrylamide-co-acrylic acid)−poly© 2012 American Chemical Society

Received: April 23, 2012 Revised: May 19, 2012 Published: May 24, 2012 9413

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calculated from the ratio of the integrals of the peak at δ = 4.56 and 3.83 ppm. Preparation of DA−PILNPM20 (INPs). In a typical procedure for preparing DA−PILNPM20 complex, various volumes of aqueous DA solution (2.0 mg/mL in 0.1 M NaOH) were added dropwise to an aqueous solution of PILNPM20 (5.0 mL, 1.0 mg/mL). At the same time, the DA−PILNPM20 complex self-assembled to form INPs due to hydrophobic interactions between the DA molecules and PILNPM20. After stirring for 2 h at room temperature, a series of INP solutions was obtained with molar ratios of DA to PILNPM20 ranging from 0.05 to 1.0. Unless otherwise noted, the INP solution mentioned in this work refers to that with a DA to PILNPM20 charge ratio (Z) of 0.5. Measurement of Critical Aggregation Concentration (CAC). In this experiment, the INPs solution prepared as described above was lyophilized to obtain the solid sample. Then by diluting the lyophilized solid sample with water, various concentrations of INPs solution were obtained. Ten microliters of 3.0 × 10−6 M pyrene in acetone was added into an empty vial and evaporated to dry. Then 5.0 mL of various concentrations of complex of INPs solution were added. The final concentration of pyrene in the solution was 6.0 × 10−6 M. The INPs solutions containing pyrene were equilibrated at room temperature for 24 h before measurement. Fluorescence emission spectra of the solutions were excited at 333 nm and recorded in the range from 360 to 444 nm on a fluorescence spectrometer. Nile Red Loading and Release. The 0.1 mL of Nile red solution (20 mg/mL) in acetone was added to 10.0 mL of INPs solution. After stirring for 2 h, the solution was exposed to the air, allowing the acetone to evaporate. The excess of unloaded Nile red was removed by membrane filtration (0.8 μm, Millipore). A solution of Nile red-loaded INPs was thereby obtained. To study the reversible pH-induced release of Nile red from the INPs, fluorescence changes were monitored over three cycles from pH 7.4 to 5.2. A solution of INPs with encapsulated Nile red is clear purple. At pH 5.2, Nile red released from the dissociated INPs is precipitated at the bottom of the bottle. The supernatant in the bottle was withdrawn to measure the fluorescence intensity of residual Nile red in the INPs. This supernatant was returned to the bottle after measurement. After adjusting the pH to 7.4, Nile red molecules were encapsulated in the INPs once more, and the solution reverted to clear purple after shaking. Fluorescence emission spectra of Nile red were obtained by excitation at 550 nm and recorded in the range from 580 to 700 nm on a fluorescence spectrometer. The fluorescence intensity of Nile red was recorded at a wavelength of 660 nm. DOX Loading and Release. DOX as a model drug was loaded in INPs (DOX-INPs). A solution of DOX in DMSO (10 mg/mL, 0.2 mL) was added dropwise to INPs solution (10.0 mL). After stirring for 2 h, the solution was dialyzed (MW cutoff 7000) against distilled water (500 mL) at room temperature for 24 h, and the distilled water was replaced after 12 h to remove the unloaded free DOX. The drug loading efficiency (DL) is defined as the ratio of the loaded DOX weight to the total weight of loaded DOX and INPs, and entrapment efficiency (EE) is defined as the ratio of the loaded DOX weight to the initial weight of DOX feeding.11 In order to determine the DL and EE, the solution of DOX-INPs was lyophilized, the residue was dissolved in DMSO, and the UV absorbance at 485 nm was measured. The DL was determined to be 13.7% loading efficiency and 42.8% entrapment efficiency. For drug release experiments in vitro, 10 mL of DOX-INPs solution was placed in a dialysis membrane (MW cutoff 7000) and dialyzed against distilled water (250 mL) at pH 7.4 or pH 5.2. Aliquots of 3.0 mL were withdrawn from the dialysate periodically. The volume of dialysate was maintained constant by adding 3.0 mL of distilled water after each sampling. The amount of DOX released from the DOXINPs was determined by measuring the UV absorbance at 485 nm. Cell Viability. In vitro cell viability test, Hela cells were incubated in the presence of INPs (100 μg/mL), DOX-INPs (100 μg/mL) and free DOX (14 μg/mL) in 96-well plate, which was put into a 5% CO2, 37 °C incubator for 24 h and 48 h. The concentration of free DOX was the same as that of the DOX-INPs. The cell viability was evaluated

drug release, a kind of novel dual-stimuli-responsive nanoparticles prepared using a convenient approach, for which the both stimuli can directly cause the change in its shape and size, has been anticipated. Herein, we present a facile method for preparing pH and thermo dual-responsive nanoparticles based on ionic selfassembly of complexes of poly(ionic liquid-co-N-isopropylacrylamide) (PILNPM20) with deoxycholic acid (DA). Cationic copolymer PILNPM20 based on poly(ionic liquid) was chose as a polyelectrolyte backbone due to the plenty of charged cations along its backbone, and an anionic molecule DA was chosen to be the pH-sensitive tectonic unit. The complex of DA− PILNPM20 was self-assembled to form ionically assembled nanoparticles (INPs) in aqueous solution for hydrophobic/ hydrophilic interaction between the PILNPM20 and DA units. The formed INPs are sensitive to dual stimuli of pH and temperature due to the deprotonation/protonation process of DA and the highly thermo-responsive poly(N-isopropylacrylamide), and applied to explore the controlled guest release; doxorubicin (DOX) was elected as the model drug.

2. MATERIALS AND METHODS Materials. 4-Vinylpyridine was purchased from Alfa Aesar. NIsopropyl acrylamide, DA, DOX, and Nile red were purchased from Aldrich. Bromoethane, azodiisobutyronitrile, hydrochloric acid, and organic solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. The water used was doubly distilled. Instruments and Characterization. Samples for Fourier-transform infrared (FTIR) and nuclear magnetic resonance (1H NMR) were prepared by lyophilization of solutions of INPs. FTIR spectra were recorded on a Perkin-Elmer Paragon 1000 FTIR spectrometer using pressed thin transparent disks of the samples mixed with potassium bromide. Spectra were obtained by collecting and averaging 64 scans. 1H NMR studies were carried out on a Varian Mercury Plus 400 MHz spectrometer using solutions of the samples in D2O or deuterated dimethyl sulfoxide (DMSO-d6). Optical transmittance of INPs solutions at different pH values and temperatures was measured at 500 nm with a UV/vis spectrophotometer (Perkin-Elmer Lambda 20), and the heating rate was set at 1 °C/min. The LCST values of the INPs solutions were determined as the temperatures, showing an optical transmittance of 50%. Fluorescence emission spectra were measured on a Photon Technology International QM-4 luminescence spectrometer. The morphologies of the INPs were investigated by transmission electron microscopy (TEM) (JEOL JEM-2100). Samples for TEM were prepared by casting a drop of solution on a carboncoated copper grid, followed by drying in air. Dynamic light scattering (DLS) and zeta-potential measurements were performed using a Malvern Instruments Zetasizer Nano ZS. Preparation of PILNPM20. 1-Ethylvinyl pyridinium bromide was prepared according to a previously reported procedure.10 NIsopropylacrylamide (11.5 mmol, 1.3 g) was dissolved in ethanol (15 mL), and 1-ethylvinylpyridinium bromide (0.5 mmol, 0.11 g) and azodiisobutyronitrile (0.43 mmol, 0.07 g) were added sequentially. The reaction mixture was stirred at 80 °C for 24 h under the protection of a nitrogen atmosphere. After completion of the reaction, the mixture was added dropwise to cold ether to precipitate the copolymer. The final product was washed twice with ether and dried under vacuum. The average molecular weight (Mn) was about 16200 g/mol and Mw/Mn = 1.8 by gel permeation chromatography. 1H NMR (DMSO-d6): δ = 8.98 (2H, pyridine-H), 8.04 (2H, pyridine-H adjacent to main chain), 4.56 (2H, −CH2− adjacent to pyridine), 3.83 (1H, −CH− adjacent to amine), 1.14−2.13 ppm (−CH2−CH− in main chain, C (CH3), and C (CH3)2) are shown in Figure S1. FTIR: 3436 cm−1 (N−H stretching of amide), 1645, and 1545 cm−1 (CO and N−H stretching of CONH groups, respectively), 1367 and 1387 cm−1 (C−H bending of isopropyl groups) are shown in Figure S2. The ratio of poly(ionic liquid) to poly(N-isopropylacrylamide) was 1:20, as 9414

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Figure 1. Preparation of INPs and their pH and thermal responsive behavior. by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.12

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of INPs. The pH and thermal dual-responsive INPs were based on the self-

Figure 3. 1H NMR spectra of (A) PILNPM20 dissolved in D2O, (B) INPs dispersed in D2O, (C) INPs dissolved in DMSO-d6.

The formation of DA−PILNPM20 complex was confirmed by FTIR spectra, as shown in Figure 2, and the characteristic peaks at 1725, 1647, and 1550 cm−1 of the complexes are assigned to the stretching vibration of the carbonyl band from the DA units and amide bands I and II from the PILNPM20 units. A significant shift from 1715 to 1725 cm−1 of the carbonyl band of DA units suggested that ionic complexes were formed between the carboxyl group of DA and the pyridine group of PILNPM20.13,14 Moreover, 1H NMR spectra further confirmed the formation of INPs as shown in Figure 3. The samples for 1H NMR characterization were obtained by lyophilization of the INPs solution. Figure 3B is the 1H NMR spectrum of INPs dispersed in D2O, which features only the proton signals of PILNPM20 at 1.85 and 3.73 ppm. However, the signals corresponding to the protons of methylene adjacent to pyridine at 4.35 ppm

Figure 2. FTIR spectra of (a) INPs, (b) PILNPM20, and (c) DA.

assembly of complexes of PILNPM20 with DA. The preparation of INPs is illustrated in Figure1. As shown in Figure 1, the PILNPM20 matrix not only plays an important role in providing plenty of charged cationic centers along its backbone for the construction of various functional materials through Coulombic interactions with oppositely charged components, but also possesses significant thermo-responsive properties, while DA is responsible for providing a hydrophobic force and pHresponsive properties. 9415

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Figure 4. (a) TEM image, and (b) diameter and PDI of INPs by varying the Z value. Inset: the size distribution of INPs at Z = 0.5. Z: the molar ratio of DA to PILNPM20.

Figure 5. (A) Fluorescence emission spectra of pyrene (λex = 333 nm) in IPNs solutions by varying concentration. (B) Change of fluorescence intensity ratio (I383/I372) for pyrene as a function of the concentration of INPs. (a) 0.001 mg/mL; (b)0.01 mg/mL; (c) 0.1 mg/mL; (d) 0.6 mg/mL; (e) 1.0 mg/mL.

Figure 6. TEM image of INPs at (a) pH 5.2 and (b) pH 7.4 after five cycles. (c) The size distribution of INPs at pH 7.4, pH 5.2, and pH 7.4 after five cycles.

and the PILNPM20 chains. DA molecules are highly hydrophobic, and PILNPM20 chains possess plenty of hydrophilic groups. In order to minimize the system energy of the complex of DA and PILNPM20, DA molecules lie in the inner part of the complex, and spontaneously form INPs. By gradually increasing Z values from 0.13 to 0.73, it was found that the diameter of the formed INPs decreased from ca. 400 to ca. 130 nm due to increased hydrophobic interaction intensity of the ionic complexes cores.17 Moreover, the polydispersity index (PDI) of IPNs gradually decreased from 0.77 to 0.21, indicating relatively more uniform IPNs formation with increasing hydrophobic force. In this work, the Z value was fixed at 0.5 for obtaining INPs with monodisperse distribution in aqueous media, as shown in the inset of Figure 4b.

disappeared by comparison with spectrum of PILNPM20 dissolved in D2O, as shown in Figure 3A.15,16 It is because hydrophobic methylene adjacent to pyridine groups in INPs is confined within the core structure, thus causing shielding of proton signals. This shielding effect is further evidenced by the fact that the signals adjacent to pyridine at 4.35 ppm became once more pronounced when the INPs were dissolved in DMSO-d6, as shown in Figure 3C. The assembling behavior of DA and PILNPM20 in aqueous media at 0.5 of charge ratio (Z) assembly was investigated using TEM and DLS. As shown in Figure 4a, solid spherical INPs are clearly observable in the TEM image, and the size is 100−150 nm. The formation of INPs could be attributed to the difference in hydrophobic properties between DA molecules 9416

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Figure 7. (A) Fluorescence emission spectra of Nile red encapsulated in INPs on varying the pH between 7.4 and 5.2. (B) Photograph of solutions of INPs with encapsulated Nile red at (a) pH 7.4, (b) pH 5.2, and (c) pH 7.4 after three cycles.

Figure 8. (a) Optical transmittance of INPs solutions as a function of temperature at different pH values. (b) LCST of INPs solutions at different pH values.

the INPs at the turning point is 0.06 mg/mL, indicating that such a low concentration of complex of DA and PILNPM20 can form INPs and maintain the stability in dilute conditions. 3.2. pH-Responsive Property of INPs. The morphology of the INPs was first observed by TEM to investigate their pHdependent phase transition properties. These spherical INPs underwent a dramatic structural collapse when the pH was lowered from pH 7.4 to 5.2, as shown in Figure 6a. This was because protonation of the DA molecules at pH 5.2 (the pKa of DA is 6.2) resulted in their departure from the complexes, and hence the INPs decomposed.20 When the pH was increased to 7.4, whereupon the DA was deprotonated, DA-PILNPM20 complexes were reformed. After five cycles between pH 7.4 and pH 5.2, spherical INPs were also observed in TEM image of Figure 6b. However, the diameter of INPs after 5 cycles is slightly larger than initial observation probably because a small fraction of DA molecules could not be reassembled in complex. The DLS results of INPs at different pH are shown in Figure 6c. The size distribution of INPs changes from monodisperse to bimodal distribution as pH decreases from 7.4 to 5.2, suggesting that decomposed INPs with several different sizes exist in solution. Additionally, the average diameter of INP changes with five cycles between 7.4 and 5.2 is shown in Figure S3. Due to aggregation of decomposed INP segments at pH 5.2, the diameter of INPs increased to ∼1000 nm. However, after pH was increased to 7.4 after five cycles between 7.4 and 5.2, the size distribution of INPs almost recovered to the initial distribution, which agrees well with the results of TEM observation. Furthermore, Nile red molecules as a fluorescence probe were encapsulated in the INPs to study their pH-induced

Figure 9. UV−vis absorbance spectra of DOX and DOX-INPs.

The assembly behavior of DA and PILNPM20 in aqueous media was further studied using fluorescence probe technique. Pyrene as a fluorescence probe has very low fluorescence intensity in an aqueous solution due to its hydrophobic nature, and the fluorescent intensity ratio (I383 nm/I372 nm) increases significantly once it is transferred into a hydrophobic environment.18,19 Figure 5 shows the change in the fluorescent intensity ratio (I383 nm/I372 nm) of pyrene as a function of the concentration of INPs solutions. The fluorescence intensity ratio of pyrene is almost constant at low concentrations of INPs solutions, and above a certain concentration, the I383 nm/I372 nm increases dramatically, indicating the transfer of pyrene into a hydrophobic environment. Such a sudden change of the fluorescent intensity indicated the formation of INPs from complex of DA and PILNPM20 at this concentration. This concentration can be defined as the CAC. The CAC value of 9417

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Figure 10. (a) Drug release from DOX-INPs in response to pH and temperature; (b) viabilities of cells treated with INPs, DOX-INPs, and free DOX for 24 and 48 h.

copolymer chains, due to their amphiphilicity, raises the LCST (beyond 37 °C), and therefore the solution is clear.22,23 Further evidence supporting the above interpretation was provided by analysis of the change in the LCST of PILNPM20 solution without DA at different pH values (Figure S5). The LCST of a PILNPM20 solution was found to be almost the same at pH 7.4 and pH 5.2, which verified the solubilization of DA molecules at pH 7.4. One may envisage that such INPs could be stably dispersed in normal physiological blood at pH 7.4 and 37 °C (below LCST). Since these INPs nanocarriers reach a microenvironment of pathological cells with lower pH (6.8− 7.2 for pH of extracellular, 4.5−5.5 for pH of endosomes) and higher temperature, they could aggregate there and release drug cumulatively because of their lower LCST in those conditions. Therefore, the INPs represent a promising smart material with remarkable pH and thermal dual-stimuli-responsive properties for biomedical applications. 3.4. Drug Loading and Release Test. To explore the feasibility of using INPs as a controlled drug-delivery vehicle, the anticancer drug DOX was loaded into INPs (DOX-INPs), and then its release profile was investigated by dialysis in vitro. UV−vis spectra showed that DOX had been successfully loaded in the INPs since its characteristic absorbance appeared at 253 and 485 nm in Figure 9. FTIR characterization provided further proof of drug loading (Figure S6). Characteristic peaks at 1284−990 cm−1 can be assigned to O−H···H and C−CO deformation vibrations of DOX.24 Figure 10 shows cumulative drug release profiles of DOXINPs under different release conditions. It can clearly be observed that only 30% of the drug was released from DOXINPs within 48 h at 37 °C and pH 7.4 (at the stage b1 in Figure 1b). In contrast, approximately 60% of the drug was released when the pH was decreased from 7.4 to 5.2 at 37 °C, i.e., more than 30% of the enclosed drug was released because of dissociation of the INPs in weakly acidic medium (at the stage b2 in Figure 1b). In addition, due to the hydrophobic deformation of PILNPM20 copolymer and formation of aggregates caused by the temperature over its LCST (36.3 °C), some of the drug molecules were probably still entrapped in the interior of loose aggregates of PILNPM20 copolymer. However, when heating to 43 °C, PILNPM20 copolymer aggregates become more hydrophobic and compact due to more hydrogen bonds between copolymer and water being destroyed, which squeezes entrapped drug molecules, releasing them from interior as from the stage of b2 to b3 illustrated in Figure 1b.8 The cumulative drug release further increased to

reversible formation and dissociation. Nile red displays a strong fluorescence emission in hydrophobic environments, such as the core of INPs, but its emission decreases significantly once it is transferred into an aqueous solution due to its hydrophobic nature.21 Figure 7A shows fluorescence emission spectra of Nile red encapsulated in INPs with changing pH values between pH 7.4 and pH 5.2. As shown in Figure 7A, the fluorescence emission intensity of Nile red decreased around 60% at the center wavelength of 660 nm when the pH was lowered from 7.4 to 5.2 in the first cycle, indicating that most of the dye was transferred from within the INPs to the aqueous solution. When the pH was returned to 7.4, the pH at which the deprotonation of DA occurs, the fluorescence emission intensity of Nile red recovered upon stirring due to reloading of the core of the newly formed INPs. After three cycles, about 90% of the Nile red could be reloaded into the INPs, suggesting highly reversible pH-responsive behavior. The corresponding fluorescence intensity is shown in Figure S4 (Supporting Information). Figure 7B shows the conversion of Nile red encapsulated INPs solution from clear purple solution at pH 7.4 to the appearance of Nile red precipitate at pH 5.2, then it turns back to clear purple solution after three cycles with shaking. 3.3. Temperature-Responsive Property of INPs. Figure 8a shows optical transmittance changes of solutions of INPs as a function of temperature. At low temperature, the INPs solutions were transparent, and the transmittance of light was high. When the temperature was increased to a certain level, the INPs started to aggregate and phase separation occurred. The solutions became cloudy, and the transmittance decreased rapidly. It is worth noting that the INPs solutions also had a pH-dependent transition temperature. Figure 8b shows that the LCSTs of the INPs solutions gradually increased from 36.3 °C (pH 5.2) to 38.5 °C (pH 7.4), as obtained from the temperature corresponding to 50% transmittance of the solution. The inset photograph in Figure 8b shows that how the INPs solution changed from clear at pH 7.4 to opaque at pH 5.2 at 37 °C because of aggregation. This behavior of the INPs solution can be explained as follows: at pH 5.2, protonated DA departs from the INPs, and mixture solution of DA and PILNPM20 exhibits an LCST lower than 37 °C; aggregation of the copolymer chains along with the poor solubility of DA render the aqueous solution opaque. At pH 7.4, however, deprotonated DA anions become attached to PILNPM20 chains and are involved in the assembly of the hydrophobic core, and the solubilization of the DA to the 9418

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80% within 48 h at pH 5.2, 43 °C. The results proved that pH and thermal stimuli could dissociate and deform the INPs to release the drug, thereby achieving controlled drug delivery. Finally, Hela cervical cancer cells were incubated with INPs, DOX-INPs, and free DOX for 24 and 48 h to evaluate the cancer cell inhibition by DOX-INPs. As shown in Figure 10b, minor cytotoxicity was observed in the cells treated with INPs, indicating that the INPs were biocompatible. However, when cells were incubated with DOX-INPs, their viability decreased to ∼70% after 24 h, and further decreased to ∼40% when the incubation time was extended to 48 h. Compared with free DOX, through a sustained release of DOX from the INPs, DOX-INPs displayed 75% of the cytotoxic activity of the free DOX after 48 h, indicating a inhibitory effect on cell growth.

(3) Bagalkot, V.; Zhang, L. F.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Quantum dot−aptamer conjugates for synchronous cancer imaging, therapy and sensing of drug delivery based on bi-fluorescence resonance energy transfer. Nano Lett. 2007, 7, 3065−3070. (4) Choi, H. S.; Huh, K. M.; Ooya, T.; Yui, N. pH- and thermosensitive supramolecular assembling system: Rapidly responsive properties of β-cyclodextrin-conjugated poly(ε-lysine). J. Am. Chem. Soc. 2003, 125, 6350−6351. (5) Chang, B. S.; Sha, X. Y.; Guo, J.; Jiao, Y. F.; Wang, C. C.; Yang, W. L. Thermo and pH dual responsive, polymer shell coated, magnetic mesoporous silica nanoparticles for controlled drug release. J. Mater. Chem. 2011, 21, 9239−9247. (6) Zhang, L. Y.; Guo, R.; Yang, M.; Jiang, X. Q.; Liu, B. R. Thermo and pH dual-responsive nanoparticles for anti-cancer drug delivery. Adv. Mater. 2007, 19, 2988−2992. (7) Garbern, J. C.; Hoffman, A. S.; Stayton, P. S. Injectable pH- and temperature- responsive poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers for delivery of angiogenic growth Factors. Biomacromolecules 2010, 11, 1833−1839. (8) Soppimath, K. S.; Tan, D. C.-W.; Yang, Y.-Y. pH-triggered thermally responsive polymer core−shell nanoparticles for drug delivery. Adv. Mater. 2005, 17, 318−323. (9) Ma, L. W.; Liu, M. Z.; Liu, H. L.; Chen, J.; Cui, D. P. In vitro cytotoxicity and drug release properties of pH- and temperaturesensitive core-shell hydrogel microspheres. Int. J. Pharm. 2010, 385, 86−91. (10) Xiao, S. F.; Lu, X. M.; Lu, Q. H. Photosensitive polymer from ionic self-assembly of azobenzene dye and poly(ionic liquid) and its alignment characteristic toward liquid crystal molecules. Macromolecules 2007, 40, 7944−7950. (11) Wu, D. Q.; Lu, B.; Chang, C.; Chen, C. S.; Wang, T.; Zhang, Y. Y.; Cheng, S. X.; Jiang, X. J.; Zhang, X. Z.; Zhuo, R. X. Galactosylated fluorescent labeled micelles as a liver targeting drug carrier. Biomaterials 2009, 30, 1363−1371. (12) Zhang, X. K.; Meng, L. J.; Lu, Q. H.; Fei, Z. F.; Dyson, P. J. Targeted delivery and controlled release of doxorubicin to cancer cells using modified single wall carbon nanotubes. Biomaterials 2009, 30, 6041−6047. (13) Thünemann, A. F. Complexes of polyethyleneimine with perfluorinated carboxylic acids: wettability of lamellar structured mesophases. Langmuir 2000, 16, 824−828. (14) Ozer, B. H.; Smarsly, B.; Antonietti, M.; Faul, C. F. J. DNAanalogous structures from deoxynucleophosphates and polylysine by ionic self-assembly. Soft Matter 2006, 2, 329−336. (15) Cui, F. Y.; Qian, F.; Zhao, Z. M.; Yin, L. C.; Tang, C.; Yin, C. H. Nanoparticles incorporated in bilaminated films: A smart drug delivery system for oral formulations. Biomacromolecules 2009, 10, 1253−1258. (16) Kim, K.; Kown, S.; Park, J. H.; Chung, H.; Jeong, S. Y.; Kwon, I. C.; Kim, I.-S. Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan−deoxycholic acid conjugates. Biomacromolecles 2005, 6, 1154−1158. (17) Kown, S.; Park, J. H.; Chung, H.; Kwon, I. C.; Jeong, S. Y.; Kim, I.-S. Physicochemical characteristics of self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid. Langmuir 2003, 19, 10188−10193. (18) Jiang, G. B.; Quan, D. P.; Liao, K. R.; Wang, H. H. Novel polymer micelles prepared from chitosan grafted hydrophobic palmitoyl groups for drug delivery. Mol. Pharmaceutics 2005, 3, 152−160. (19) Li, X.; Wu, Q.; Chen, Z. C.; Gong, X. G.; Lin, X. F. Preparation, characterization and controlled release of liver-targeting nanoparticles from the amphiphilic random copolymer. Polymer 2008, 49, 4769− 4775. (20) Duret, G.; Delcour, A. H. Deoxycholic acid blocks vibrio cholerae OmpT but not OmpU porin. J. Biol. Chem. 2006, 281, 19899−19905.

4. CONCLUSIONS A new strategy for preparing pH and thermal dual-responsive INPs has been presented. The INPs reported here are distinct from previously reported nanoparticles derived from covalent copolymers, in that they are assembled from ionic PILNPM20 with hydrophobic groups of DA through electrostatic interaction. The INPs can dissociate and deform to release drug in response to both pH and temperature. This makes them promising for application in controlled drug delivery to pathological cells. Moreover, the drug-loaded INPs exhibit comparable cytotoxicity to the free drug DOX. Thus, this work may provide useful information for the design of functional nanoparticles in the fields of biology and pharmacology.

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ASSOCIATED CONTENT

S Supporting Information *

The 1H NMR and FTIR spectra of PILNPM20; average diameter of INPs at pH 5.2 and pH7.4 after 5 cycles between pH 7.4 and 5.2.; fluorescent intensity of Nile red in INPs at pH 5.2 and pH7.4 after 5 cycles between pH 7.4 and 5.2; optical transmittance of PILNPM20 solutions as a function of temperature at different pH values; FTIR spectra of DOX and DOX-INPs. This information 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: (021)54747535; Fax: (021)54747535. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Science Fund for Distinguished Young Scholars (50925310), the National Science Foundation of China (50902094,51173103), the 973 project (2009CB93043, 2012CB933803), and excellent academic leaders of Shanghai (11XD1403000).

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REFERENCES

(1) Cayre, O. J.; Chagneuxb, N.; Biggs, S. Stimulus responsive core− shell nanoparticles: Synthesis and applications of polymer based aqueous systems. Soft Matter 2011, 7, 2211−2234. (2) Zhu, X. L.; Liu, Y. X.; Huang, J. Y.; Li, G. X. A pH-responsive gate fabricated with nano-channels and nano-particles. Chem.Eur. J. 2010, 16, 1441−1444. 9419

dx.doi.org/10.1021/la3016436 | Langmuir 2012, 28, 9413−9420

Langmuir

Article

(21) Goodwin, A. P.; Mynar, J. L.; Ma, Y. Z.; Fleming, G. R.; Fréchet, J. M. J. Synthetic micelle sensitive to IR light via a two-photon process. J. Am. Chem. Soc. 2005, 127, 9952−9953. (22) Schild, H. G.; Tirrell, D. A. Interaction of poly(Nisopropylacrylamide) with sodium n-alkyl sulfates in aqueous solution. Langmuir 1991, 7, 665−671. (23) Benrebouh, A.; Avoce, D.; Zhu, X. X. Thermo- and pH-sensitive polymers containing cholic acid derivatives. Polymer 2001, 42, 4031− 4038. (24) Das, G.; Nicastri, A.; Coluccio, M. L.; Gentile, F.; Candeloro, P.; Cojoc, G.; Liberale, C.; DeAngelis, F.; Di Fabrizio, E. FT-IR, Raman, RRS measurements and DFT calculation for doxorubicin. Microsc. Res. Tech. 2010, 73, 991−995.

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