Fabrication of Biocompatible Temperature-and pH-Responsive

Aug 4, 2010 - Sweccha Joshi , Indrajit Ghosh , Suman Pokhrel , Lutz Mädler , and Werner M. Nau. ACS Nano 2012 6 (6), 5668-5679. Abstract | Full Text ...
0 downloads 0 Views 2MB Size
Download PDF
8518

Ind. Eng. Chem. Res. 2010, 49, 8518–8525

Fabrication of Biocompatible Temperature- and pH-Responsive Magnetic Nanoparticles and Their Reversible Agglomeration in Aqueous Milieu Liangrong Yang, Chen Guo,* Lianwei Jia, Keng Xie, Qinghui Shou, and Huizhou Liu* State Key Laboratory of Biochemical Engineering, Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, P.R. China

A novel kind of biocompatible pH- and temperature-responsive magnetic nanoparticle consisting of iron oxide nanoparticles coated with pH-responsive chitosan oligosaccharide and temperature-responsive poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer was developed. The particles were characterized by TEM, DLS, VSM, FTIR spectroscopy, and TGA. The results indicated that the self-aggregation of the prepared nanoparticles was not only caused by the thermoinduced self-assembly of the immobilized block copolymers, but also affected by the pH-induced charge property change of the particle surface. The self-assembled behaviors can be readily reversed by adjusting the pH or temperature value. Thus, the attractive properties of reversible and controllable dual-responsive self-assembly might endow the biocompatible magnetic nanoparticles with potential applications in biomedical fields such as DNA delivery, drug targeting, and tissue engineering. 1. Introduction During the past decade, a variety of inorganic nanoparticles such as magnetic nanoparticles, metal nanoparticles, and metal oxide nanoparticles have gained increasing interest in the field of material science because of their specific optical, electronic, and magnetic properties.1,2 Among these particles, superparamagnetic iron oxide nanoparticles have been widely investigated because of their easy synthesis and simple control through an external magnetic field. Various functional polymers have been used to modify the surface of the magnetic nanoparticles. Such modifications can provide magnetic nanoparticles with stability, biocompatibility, and conjugation with ligands.3-5 The resulting particles can thus be used for many applications such as biomolecular separations, drug targeting and delivery, and magnetic resonance imaging (MRI).6-9 The current research trend in the modification of magnetic nanoparticles is focused on controlling their shape and size by changing their assembly behaviors. In recent years, the directed self-assembly of nanoparticles has been found to provide new functions for a wide range of potential applications.10-12 Selfassembly can be changed by employing an external stimulus such as temperature, pH, light, and so on.13,14 In particular, temperature-responsive magnetic particles have been widely studied because of the easily controlled property for temperature and the mature applications of hyperthermia.15-20 For example, Dong et al.15 synthesized thermally responsive PM(EO)2MA magnetic microgels by the electron-transfer atom-transfer radical polymerization method. These magnetic microgels are stable at 20 °C, but quickly precipitate from solution at 40 °C. Isojima et al.16 synthesized temperature-responsive Janus magnetic nanoparticles based on PAA [poly(acrylic acid)] and PNIPAM [poly(N-isopropylacrylamide)]. These Janus nanoparticles can disperse stably as individual particles at low temperature and self-assemble at high temperature (>31 °C) to form stable dispersions of clusters with hydrodynamic diameters of about 80-100 nm. pH-responsive polymers have also been widely investigated for many applications such as drug delivery systems, biosepa* E-mail: [email protected]; [email protected].

rations, and so on.21,22 Recently, a few researchers developed dual-responsive magnetic particles by introducing pH-responsive blocks into a temperature-responsive magnetic system.23,24 For example, Bhattacharya et al.23 explored temperature- and pHresponsive magnetic microgels based on the copolymerization of VCL (N-vinylcaprolactam), AAEM (acetoacetoxyethyl methacrylate), and Vim (vinylimidazole). Their results showed that magnetic particles in the gel could decrease its swelling degree and shift the volume phase transition temperature to higher values. Zhang and Wang24 prepared dual-responsive magnetic particles by assembling amino-modified Fe3O4@SiO2 particles on the surface of PSt/P(NIPAM-co-AA) core/shell microspheres (PSt ) polystyrene). The pH- and temperature-responsive shell provides the particles with tunable permeability. To date, there are only limited reports on temperature and pH dual-responsive magnetic particles. In most cases, thermosensitive polymers based on poly(N-isopropylacrylamide) (PNIPAAm) or poly(Nvinylcaprolactam) (PVCL) have been employed in dualresponsive magnetic systems. Pluronic copolymers, which consist of hydrophilic poly(ethylene oxide) (PEO) segments and hydrophobic poly(propylene oxide) (PPO) segments, are a type of temperature-responsive polymers.25,26 Pluronic polymers can assemble into a micelle structure with a PPO hydrophobic core surrounded by a PEO hydrophilic shell at body temperature.27 This self-assembly behavior is due to the thermoinduced dehydration of the ether backbone of Pluronics. Because of their biocompatibility, Pluronic copolymers have several advantages as drug delivery carriers in vivo.28-31 Chitosan, which is a representative pHresponsive biopolymer, has many unique advantages such as biocompatibility,32 antimicrobial activity,33 mucoadhesivity,34 and biodegradability.35 If Pluronic copolymers could be combined with pH-responsive building blocks such as chitosan and further modified with magnetic nanoparticles, the formed magnetic nanoparticles might have good temperature- and pHresponsive properties and, as a result, more potential for use in biomedical applications. Park et al.36 synthesized shell crosslinked magnetic capsules by first encapsulating hydrophobic magnetic nanoparticles with Pluronic F127 and then using chitosan to cross-link the outer shell of the nanocapsules. These particles are well-dispersed without significant aggregation in

10.1021/ie100587e  2010 American Chemical Society Published on Web 08/04/2010

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

aqueous solutions because of the charge repulsion of chitosan. However, the encapsulated structure, which restricts the Pluronic copolymer chains in the coiled conformation, limits the temperature-responsive self-assembly of the copolymer. This limitation might lead to the absence of reversible self-aggregation between encapsulated particles under the stimulus of temperature. In this study, Pluronic P103 was first grafted onto chains of chitosan oligosaccharide (CSO) (3000 < Mw < 5000) by Dess-Martin periodinane activation and sodium borohydride reduction, yielding a CSO-g-Pluronic copolymer. Then, the CSO-g-Pluronic copolymer was introduced onto the surface of hydrophilic magnetic nanoparticles. The formed nanoparticles had a spherical structure with Fe3O4 as the core, chitosan as the inner shell, and Pluronic P103 as the outer corona. The core-shell-corona (CSC) structure provided the nanoparticles with reversible pH- and temperature-responsive self-assembly properties. Thus, the corresponding volume transition property might endow the biocompatible dual-responsive magnetic nanoparticles with potential applications in biomedical fields such as DNA delivery, drug targeting, and tissue engineering. 2. Experimental Section 2.1. Materials. Randomly 80% deacetylated CSO (Mw ) 4.0 kDa) was supplied by Yuhuan Marine Biochemistry Co., Ltd. (Zhejiang, China). Dess-Martin periodinane, pyrene, and Eosin Y were purchased from Sigma-Aldrich (St. Louis, MO). The PEO-PPO-PEO block copolymer P103 [(EO)17-(PO)60(EO)17, Mw ) 4950] was obtained as a gift from BASF. Membra-Cel MD-34-3.5 and Membra-Cel MD-34-7 were purchased from Shanghai Green Bird Science & Technology Development Co., Ltd. All other chemicals used were purchased from Beijing Chemical Reagent Co. and were of analytical grade. 2.2. Preparation of CSO-g-Pluronic. Bhattarai and coworkers used acetic anhydride to oxidize poly(ethylene glycol) (PEG) into PEG-aldehyde and then conjugated PEG-aldehyde onto the chitosan backbone to synthesize PEG-g-chitosan copolymer.37 Yang et al. employed Dess-Martin periodinane to convert the terminal alcohols of Pluronic into aldehydes (Pluronic-CHO).38 Here, we used a protocol that first produced P103-CHO by oxidizing the terminal -OH groups of P103 with Dess-Martin periodinane and then conjugated the aldehyde groups of P103-CHO with the amino groups of CSO. Afterward, sodium borohydride was used to reduce the CdN groups (Schiff base) to produce the more stable CSO-g-Pluronic copolymer. Typically, 3.96 g of P103 (0.8 mmol) was dissolved in 200 mL of methylene chloride, and then, 84.8 mg of Dess-Martin periodinane (0.2 mmol) was added. After reaction for 24 h at room temperature, P103-CHO was isolated simply by filtration of the organic extracts through hexane and evaporation of the solvent at reduced pressure. Next, 0.4 mmol of activated Pluronic copolymer was added to 60 mL of CSO solution (3.33 mM), and the pH value was carefully adjusted to 4.5. Then, 250 mg of NaBH4 was added, and the resulting solution was stirred at room temperature. After 24 h, the product was dialyzed three times against distilled water using a MembraCel MD-34-7 membrane and finally lyophilized. 2.3. Conjugation of CSO-g-Pluronic with Magnetite Particles. An aqueous solution (100 mL) containing 2.344 g of FeCl3 · 6H2O (8.67 mmol), 0.862 g of FeCl2 · 4H2O (4.33 mmol), and 0.26 mmol of citrate acid trisodium salt [2% (mol/ mol) citrate/metallic species] was stirred for 30 min under a nitrogen gas atmosphere at 80 °C, and then 20 mL of concentrated ammonium hydroxide (25%) was poured into the

8519

mixture. After the solution turned black, the magnetic particles were separated by magnet, washed several times with distilled water, and redispersed in 50 mL of distilled water. Then, 50 mL of CSO-g-Pluronic (40 mg/mL) was added to the solution of magnetic particles with continuous stirring for 2 h. Afterward, the particles were separated by magnet, washed several times with distilled water, and suspended in distilled water for subsequent use. 2.4. Characterization of MCP (Magnetic CSO-gPluronic) Nanoparticles. 2.4.1. Fourier Transform Infrared (FTIR) Spectroscopic Measurements. FTIR spectra of bare Fe3O4 nanoparticles, P103, CSO, CSO-g-P103, and MCP nanoparticles were recorded on a Bruker Vector 22 FTIR spectrometer using KBr pellets. Temperature-dependent FTIR spectra of MCP aqueous samples were recorded with a thermocouple inserted into a stainless steel block that hosted a sample cell. The equilibration time for each temperature was 2 min. 2.4.2. Transmission Electron Microscopy (TEM) Measurements. MCP nanoparticles were characterized with a transmission electron microscope operated at 100 keV (FEI TECNAI 20). Samples were prepared by placing a drop of MCP nanoparticle aqueous solution onto a Formvar-covered copper grid. 2.4.3. Thermogravimetric Analysis (TGA). The number of CSO-g-Pluronic molecules bonded to magnetite particles was determined by thermogravimetric analysis (Netzsch STA 449C). Samples were heated from 30 to 800 °C at a heating rate of 5 °C/min in N2 gas atmosphere. 2.4.4. Magnetization Curve Measurements. Measurements of the magnetization of MCP and bare Fe3O4 nanoparticles were carried out with a vibrating sample magnetometer (VSM) at room temperature (LakeShore 7307). 2.4.5. Dynamic Light Scattering (DLS) and Zeta Potential Measurements. The hydrodynamic diameters of MCP nanoparticles were monitored by dynamic light scattering using a Delsa Nano Particle Analyzer (Beckman Coulter, Inc., Fullerton, CA) in the temperature range of 6-45 °C. The equilibrium time for each temperature was 20 min. The zeta potential of the nanoparticles was determined using a Delsa Nano Zeta Potential analyzer (Beckman Coulter, Inc.). The pH value of the MCP nanoparticle solution was adjusted using HCl and NaOH solutions. 3. Results and Discussion 3.1. Design of Stimuli-Responsive Magnetic Nanoparticles. The goal of this work was to synthesize dualresponsive magnetic nanoparticles that can self-assemble to aggregate through the application of an appropriate stimulus and can redisperse to form stable, singly dispersed nanoparticles upon removal of this stimulus. The dual-responsive magnetic structures synthesized in this work consisted of superparamagnetic iron oxide nanoparticles that were coated evenly with citrate ligands and were functionalized further by the attachment of the dual-responsive polymer CSO-g-Pluronic to the surface coating, as shown in Figure 1. The aldehyde groups formed at the end of Pluronic P103 were determined by 1H NMR and FTIR spectroscopies (Supporting Information). Citrate was selected as the base coating because its numerous carboxyl groups provide (i) strong anchoring of the citrate to the nanoparticles through ligand exchange with the hydroxyl groups on the particle surface39 and (ii) functional groups and excess negative charge on the surface. Pluronic can become hydrophilic (hydrated) or hydrophobic (dehydrated) depending

8520

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

Figure 1. Schematic illustration of the fabrication strategy for MCP nanoparticles.

Figure 2. FTIR spectra of (A) pure Pluronic P103, (B) CSO, (C) CSO-gP103, (D) bare Fe3O4 nanoparticles, and (E) MCP nanoparticles.

on the temperature. Thus, the temperature-responsive polymer Pluronic P103 [(EO)17-(PO)60-(EO)17, Mw ) 4950] was selected as a representative example of the Pluronic family. [Its critical micelle concentration (cmc) at 25 °C is 0.07% (w/v), and the critical micellar temperature (cmt) of a 0.05% w/v aqueous solution is about 26 °C.25] First, Pluronic P103 was covalently modified by the cationic biopolymer CSO. Then, the CSO-modified P103 polymer was conjugated to the anionic surface of magnetite nanoparticles through strong ionic interactions. In these particles, the excess amino groups of CSO are confined at the inner layer and provide the pH responsivity that can affect the aggregation behavior of the nanoparticles. 3.2. Characterization of MCP Nanoparticles. Evidence for the modification of Pluronic and attachment of CSO-g-Pluronic to the citrate-coated magnetic nanoparticles was obtained by FTIR spectroscopy. Figure 2 shows the FTIR spectra of pure Pluronic P103 copolymer, CSO, CSO-g-P103, bare Fe3O4 nanoparticles, and MCP nanoparticles. The broad absorption peak between 1200 and 1000 cm-1 is due to the C-O stretching vibration, which is a feature of the Pluronic copolymer (Figure 2A). The bands around 3200 cm-1 are attributed to the stretching vibration of N-H bonds of CSO (Figure 2B), and the bands at 1650 and 1581 cm-1 are assigned to the amine band that covalently links CSO and P103 (Figure 2C). The peak at around 600 cm-1 corresponds to iron oxide nanoparticles (Figure 2D). The FTIR results confirm the successful attachment of the CSOg-Pluronic to the magnetic nanoparticles (Figure 2E). Further confirmation was obtained by thermogravimeric analysis (TGA).

Figure 3. TG analysis of (A) bare Fe3O4 nanoparticles, (B) MCP nanoparticles, (C) CSO, and (D) P103.

As shown in Figure 3, MCP shows a mass loss of about 17 wt % after heating to 400 °C (Figure 3B), at which temperature the P103 copolymers decomposed (Figure 3D). In contrast, nearly no mass loss was observed for the bare Fe3O4 nanoparticles (Figure 3A). The content of CSO-g-P103 in MCP was determined to be about 17 wt % with respect to the total formula weight. The magnetic properties of MCP nanoparticles were investigated with a vibrating sample magnetometer (VSM). The

Figure 4. Room-temperature magnetization curves of (A) bare Fe3O4 nanoparticles and (B) MCP nanoparticles.

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

8521

Figure 5. TEM images of (A) MCP nanoparticles and (B) chitosan-coated magnetic nanoparticles.

Figure 6. DLS profiles of (A) aqueous MCP nanoparticles solution at 20 °C (pH 6.5) and (B) aqueous chitosan-coated magnetic nanoparticles solution at 20 °C (pH 6.5).

saturation magnetization value of MCP was found to be 54.29 emu/g at 25 °C (Figure 4B). No obvious loss of saturation magnetization was observed after conjugating the CSO-gPluronic with the magnetic nanoparticles (Figure 4A). Compared with the other dual-responsive magnetic particles, which have saturation magnetization values of 15 and 6 emu/g,23,24 the MCP nanoparticles have a much higher saturation magnetization value. This indicates that the MCP nanoparticles have good magnetic properties and are easy to control with an external magnetic field. Moreover, neither magnetic remanence nor coercivity was observed, which indicates that the MCP nanoparticles are superparamagnetic. Superparamagnetic particles no longer exhibit magnetic interactions upon removal of the magnetic field, so the aggregation between the particles can be reduced.40 Figures 5 and 6 show TEM micrographs and DLS profiles, respectively, of aqueous MCP nanoparticles. It was found that a typical particle of MCP had a ∼40-nm spherical magnetite core on average (Figure 5A) and a ∼75-nm hydrodynamic diameter (Figure 6A) in water at 20 °C. From the inset of Figure 6A, one can see that about 90% of the MCP particles were in the narrow size range of 50-200 nm. As shown in Figure 5B, chitosan-coated magnetic nanoparticles exhibit obvious aggregation, with a ∼1200-nm hydrodynamic diameter at 20 °C (Figure 6B). However, the magnetic nanoparticles coated with CSO-g-Pluronic copolymers show good monodispersity (Figure

5A). This is due to the hydrophilicity of the PEO segments of the Pluronic copolymers. Compared with the other dualresponsive magnetic particles, which have average diameters larger than 245 nm,23,24 the CSO-g-Pluronic micelles have a much smaller size, with an average diameter of about 75 nm. The size distribution can play an important role in determining the applied effect of a drug carrier.41 It has been reported that

Figure 7. Zeta potentials and hydrodynamic diameters of aqueous MCP nanoparticle solution (20 °C).

8522

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

Figure 8. DLS profiles and volume-average hydrodynamic diameter change of aqueous MCP nanoparticle solution over the temperature range of 5-40 °C at (A,C) pH 6.5 and (B,D) pH 7.93.

drug vehicles with diameters larger than 200 nm are readily scavenged nonspecifically by monocytes and the reticuloendothelial system (RES).30 In contrast, smaller particles tended to accumulate in the tumor sites because of the enhanced permeability and retention (EPR) effect, and a greater internalization was also observed. This indicates that the MCP nanoparticles are good potential candidates for biomedical applications. 3.3. pH- and Temperature-Responsive Self-Assembly of MCP Nanoparticles. The ability to control the self-assembly and aggregation behavior of the MCP nanoparticles was demonstrated by measuring their effective hydrodynamic diameters under different conditions using dynamic light scattering (DLS). The pH-responsive hydrodynamic diameters and zeta potentials of MCP nanoparticles are shown in Figure 7. The volumeaverage hydrodynamic diameter of MCP first increased and then decreased when the pH changed from 4.5 to 10.2. At low pH values (pH < 6.5), the MCP nanoparticles were electrically charged (35-50 mV), so a stable dispersion of particles of approximately 75 nm was obtained. With an increase in pH to achieve the isoeletric point (pI ) 7.93), the MCP nanoparticles began to aggregate, and the growth of the aggregates was limited by the hydrophilic PEO groups of Pluronic copolymers, which, at 20 °C, stabilized the clusters at a hydrodynamic diameter of

about 240 nm. With a further increase in pH to a higher value (pH 10.2), the clustered MCP nanoparticles began to disaggregate to form particles of approximately 80 nm, as a result of

Figure 9. Temperature-dependent relative peak intensities of the dehydrated PO (propylene oxide) groups for an MCP nanoparticle solution in FTIR spectra.

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

8523

Figure 10. Mechanism for the pH-responsive self-assembly of MCP nanoparticles.

the electrostatic repulsions between nanoparticles. This selfassembly of the MCP nanoparticles at pH 7.93 could be reversed readily by decreasing the pH back to about 4.5 to obtain, once again, stably dispersed nanoparticles. The temperature-responsive self-assembly of MCP nanoparticles in aqueous solution was characterized by DLS, as shown in Figure 8. The volume-average hydrodynamic diameter of the MCP nanoparticles exhibited contrary trends when the temperature was increased from 5 to 40 °C. Interestingly, the MCP nanoparticles showed contrary temperature-responsive behaviors in water over the range of 6-50 °C at pH 6.5 and pH 7.93. At pH 6.5, where the MCP nanoparticles were electrically charged (about 40 mV), the volume-average hydrodynamic diameter of the MCP particles underwent a sharp decrease from 80 to 40 nm when the temperature was increased from 10 to 35 °C (Figure 8A). At pH 7.93, where the MCP nanoparticles were nearly uncharged, the volume-average hydrodynamic diameter of the MCP particles underwent a sharp increase from 100 to 1100 nm when the temperature was increased from 10 to 35 °C (Figure 8B). The volume changes as a function of temperature were found to be reversible. This phenomenon can be attributed to the thermoinduced self-assembly of the PEOPPO-PEO block copolymers on the surfaces of the Fe3O4 nanoparticles. FTIR spectroscopy is a very effective technique for investigating the self-assembly of amphiphilic polymers and the conformation state of polymer chains. Figure 9 shows the temperature-dependent relative peak intensities around 1112 cm-1 bands that are assigned to dehydrated PO (propylene oxide) groups of Pluronic copolymers for MCP nanoparticle solution in FTIR spectra.42 It can be seen that the relative peak intensity associated with dehydrated PO groups of Pluronic copolymers increased when the temperature was increased from 10 to 30 °C. This indicates that dehydration of the ether backbone of P103 was due to the broken hydrogen bonds between ether oxygen atoms and water hydrogen atoms.43,44 The removal of water molecules from the backbone of P103 copolymers would result in the effective clustering of both PPO and PEO blocks.

Figure 11. Mechanism for temperature-responsive self-assembly of MCP nanoparticles (pH 6.5).

Figure 12. Mechanism for the temperature-responsive self-assembly of MCP nanoparticles (pH ) pI ) 7.93).

3.4. Mechanism of pH- and Temperature-Responsive Self-Assembly of MCP Nanoparticles. Taking the above experimental results into consideration, the following mechanism for pH- and temperature-responsive self-assembly of MCP nanoparticles is proposed, as illustrated in Figures 10-12. Changes in the surface charge properties of particles play a dominating role for pH-responsive behaviors. Compared with most of the relevant reports based on pH-responsive magnetic particles, which showed monotonic trends in the pH-responsive hydrodynamic diameters,16,24 the MCP nanoparticles exhibit a maximum hydrodynamic diameter at pH 7.93. When the pH is high (pH 9) or low (pH 6.5), the MCP nanoparticles are individually dispersed because of the electrostatic repulsions between nanoparticles, and when the pH changes to 7.93 (pI), where the zeta potential of the MCP nanoparticles is nearly 0, the MCP nanoparticles aggregate to form clusters because of

8524

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010

the disappearance of electrostatic repulsions. In contrast, the surface charge properties of most pH-responsive magnetic particles change simply from an electrically charged state to an uncharged state when the pH monotonically changed.16,24 This results in the monotonic trends in their pH-responsive properties. In addition, the clusters were stabilized at a specific size without unbridled growth because of the hydrophilicity of the PEO groups in the Pluronic copolymers. The MCP nanoparticles exhibited completely opposite temperature-responsive behaviors in water at pH 6.5 and pH 7.93, which is very different from most other temperature-responsive magnetic particles.16,23,24 The dominating driving force for temperature-responsive self-assembly is the change in polymer conformation and amphiphilic properties on the surface of the MCP nanoparticles (Figures 11 and 12). This change is caused by the thermoinduced dehydration of the Pluronic chains. At pH 6.5 (Figure 11), the MCP nanoparticles do not aggregate because of electrostatic repulsions between them, and the volume-average hydrodynamic diameter of the MCP nanoparticles decreases as the temperature increases, because the polymeric shell of the MCP particles becomes much more compact at higher temperature, accompanied by a conformational change from fully extended to highly coiled according to our previous studies.43-46 In contrast, at pH 7.93 (Figure 12), the volume-average hydrodynamic diameter of the MCP nanoparticles increases markedly as the temperature increases, because the electrostatic repulsions between MCP nanoparticles disappear, and at the same time, the properties of the Pluronic copolymers change from hydrophilic to hydrophobic because of the dehydration of the Pluronic chains at higher temperature. Therefore, the controllable self-assembled properties of MCP nanoparticles can be significantly changed by adjusting the pH and temperature. 4. Conclusions We have successfully developed a novel kind of pH- and temperature-responsive magnetic nanoparticle consisting of iron oxide nanoparticles coated with pH-responsive chitosan oligosaccharide and temperature-responsive poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) block copolymer. The attachment of the polymers to the nanoparticles was confirmed by FTIR spectroscopy and TGA, and the morphology and diameter of the MCP particles were characterized by TEM and DLS. The nanoparticles exhibit high aqueous stability, monodispersivity, and superparamagnetism. The nanoparticles can be dispersed individually at higher or lower pH values (pH 9 or 6.5) at room temperature (20 °C) and can self-assemble to form small clusters at the isoeletric point (pH 7.93). Moreover, at pH 6.5, the volume-average hydrodynamic diameter of the MCP nanoparticles decreases with increasing temperature. However, at pH 7.93, the volume-average hydrodynamic diameter of the MCP nanoparticles increases considerably with increasing temperature. The self-assembly behaviors can be readily reversed by adjusting the pH or temperature back to its original value. This pH and temperature dual-responsive selfassembly is one of the most attractive features of this material. Thus, controllable self-assembly properties might endow the biocompatible and dual-responsive magnetic nanoparticles with potential applications in biomedical fields. Acknowledgment This work was financially supported by the State Basic Research Development Program of China (2009CB219904),

National Basic Research Program of China (2007CB613507), National Natural Science Key Fund (No. 20490200), Beijing Natural Science Foundation (No.2102041), and Innovative Research Group Science Fund (No. 20221603). Supporting Information Available: Aldehyde groups determined during the preparation of CSO-g-Pluronic by 1H NMR and FTIR spectroscopies. This information is available free of charge via the Internet at http://pubs.acs.org/. Literature Cited (1) Alivisatos, P. The use of nanocrystals in biological detection. Nat. Biotechnol. 2004, 22 (1), 47–52. (2) Gao, X. H.; Yang, L. L.; Petros, J. A.; Marshal, F. F.; Simons, J. W.; Nie, S. M. In vivo molecular and cellular imaging with quantum dots. Curr. Opin. Biotechnol. 2005, 16 (1), 63–72. (3) Hong, R.; Fischer, N. O.; Emrick, T.; Rotello, V. M. Surface PEGylation and ligand exchange chemistry of FePt nanoparticles for biological applications. Chem. Mater. 2005, 17 (18), 4617–4621. (4) Lee, H.; Lee, E.; Kim, D. K.; Jang, N. K.; Jeong, Y. Y.; Jon, S. Antibiofouling polymer-coated superparamagnetic iron oxide nanoparticles as potential magnetic resonance contrast agents for in vivo cancer imaging. J. Am. Chem. Soc. 2006, 128 (22), 7383–7389. (5) Kim, B. S.; Qiu, J. M.; Wang, J. P.; Taton, T. A. Magnetomicelles: Composite nanostructures from magnetic nanoparticles and cross-linked amphiphilic block copolymers. Nano Lett. 2005, 5 (10), 1987–1991. (6) Jeong, U.; Teng, X. W.; Wang, Y.; Yang, H.; Xia, Y. N. Superparamagnetic colloids: Controlled synthesis and niche applications. AdV. Mater. 2007, 19 (1), 33–60. (7) Liu, X. Q.; Kaminski, M. D.; Chen, H. T.; Torno, M.; Taylor, L.; Rosengart, A. J. Synthesis and characterization of highly-magnetic biodegradable poly(D,L-lactide-co-glycolide) nanospheres. J. Controlled Release 2007, 119 (1), 52–58. (8) Huth, S.; Lausier, J.; Gersting, S. W.; Rudolph, C.; Plank, C.; Welsch, U.; Rosenecker, J. Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer. J. Gene Med. 2004, 6 (8), 923– 936. (9) Rosi, N. L.; Mirkin, C. A. Nanostructures in biodiagnostics. Chem. ReV. 2005, 105 (4), 1547–1562. (10) Klajn, R.; Bishop, K. J. M.; Fialkowski, M.; Paszewski, M.; Campbell, C. J.; Gray, T. P.; Grzybowski, B. A. Plastic and moldable metals by self-assembly of sticky nanoparticle aggregates. Science 2007, 316 (5822), 261–264. (11) Ge, J. P.; Hu, Y. X.; Biasini, M.; Beyermann, W. P.; Yin, Y. D. Superparamagnetic magnetite colloidal nanocrystal clusters. Angew. Chem., Int. Ed. 2007, 46 (23), 4342–4345. (12) Ge, J. P.; Hu, Y. X.; Yin, Y. D. Highly tunable superparamagnetic colloidal photonic crystals. Angew. Chem., Int. Ed. 2007, 46 (39), 7428– 7431. (13) Lattuada, M.; Hatton, T. A. Functionalization of monodisperse magnetic nanoparticles. Langmuir 2007, 23 (4), 2158–2168. (14) White, M. A.; Johnson, J. A.; Koberstein, J. T.; Turro, N. J. Toward the syntheses of universal ligands for metal oxide surfaces: Controlling surface functionality through click chemistry. J. Am. Chem. Soc. 2006, 128 (35), 11356–11357. (15) Dong, H. C.; Mantha, V.; Matyjaszewski, K. Thermally Responsive PM(EO)2MA Magnetic Microgels via Activators Generated by Electron Transfer Atom Transfer Radical Polymerization in Miniemulsion. Chem. Mater. 2009, 21 (17), 3965–3972. (16) Isojima, T.; Lattuada, M.; Vander Sande, J. B.; Hatton, T. A. Reversible clustering of pH- and temperature-responsive Janus magnetic nanoparticles. ACS Nano 2008, 2 (9), 1799–1806. (17) Chen, S.; Li, Y.; Guo, C.; Wang, J.; Ma, J. H.; Liang, X. F.; Yang, L. R.; Liu, H. Z. Temperature-responsive magnetite/PEO-PPO-PEO block copolymer nanoparticles for controlled drug targeting delivery. Langmuir 2007, 23 (25), 12669–12676. (18) Aqil, A.; Vasseur, S.; Duguet, E.; Passirani, C.; Benoit, J. P.; Jerome, R.; Jerome, C. Magnetic nanoparticles coated by temperature responsive copolymers for hyperthermia. J. Mater. Chem. 2008, 18 (28), 3352–3360. (19) Lai, J. J.; Hoffman, J. M.; Ebara, M.; Hoffman, A. S.; Estournes, C.; Wattiaux, A.; Stayton, P. S. Dual magnetic-/temperature-responsive nanoparticles for microfluidic separations and assays. Langmuir 2007, 23 (13), 7385–7391. (20) Zhu, Y. F.; Kaskel, S.; Ikoma, T.; Hanagata, N. Magnetic SBA15/poly(N-isopropylacrylamide) composite: Preparation, characterization and

Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010 temperature-responsive drug release property. Microporous Mesoporous Mater. 2009, 123 (1-3), 107–112. (21) Yang, L. R.; Guo, C.; Chen, S.; Wang, F.; Wang, J.; An, Z. T.; Liu, C. Z.; Liu, H. Z. pH-Sensitive Magnetic Ion Exchanger for Protein Separation. Ind. Eng. Chem. Res. 2009, 48 (2), 944–950. (22) Lai, J. J.; Nelson, K. E.; Nash, M. A.; Hoffman, A. S.; Yager, P.; Stayton, P. S. Dynamic bioprocessing and microfluidic transport control with smart magnetic nanoparticles in laminar-flow devices. Lab Chip 2009, 9 (14), 1997–2002. (23) Bhattacharya, S.; Eckert, F.; Boyko, V.; Pich, A. Temperature-, pH-, and magnetic-field-sensitive hybrid microgels. Small 2007, 3 (4), 650– 657. (24) Zhang, F.; Wang, C. C. Preparation of P(NIPAM-co-AA) Microcontainers Surface-Anchored with Magnetic Nanoparticles. Langmuir 2009, 25 (14), 8255–8262. (25) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Micellization of Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide) Triblock Copolymers in Aqueous SolutionssThermodynamics of Copolymer Association. Macromolecules 1994, 27 (9), 2414–2425. (26) Wanka, G.; Hoffmann, H.; Ulbricht, W. Phase Diagrams and Aggregation Behavior of Poly(oxyethylene)-Poly(oxypropylene)Poly(oxyethylene) Triblock Copolymers in Aqueous Solutions. Macromolecules 1994, 27 (15), 4145–4159. (27) Nakashima, K.; Bahadur, P. Aggregation of water-soluble block copolymers in aqueous solutions: Recent trends. AdV. Colloid Interface Sci. 2006, 123, 75–96. (28) Bhadra, D.; Bhadra, S.; Jain, P.; Jain, N. K. Pegnology: A review of PEG-ylated systems. Pharmazie 2002, 57 (1), 5–29. (29) Kohler, N.; Fryxell, G. E.; Zhang, M. Q. A bifunctional poly(ethylene glycol) silane immobilized on metallic oxide-based nanoparticles for conjugation with cell targeting agents. J. Am. Chem. Soc. 2004, 126 (23), 7206–7211. (30) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J. Controlled Release 2002, 82 (2-3), 189–212. (31) Kabanov, A. V.; Batrakova, E. V.; Sriadlbhatla, S.; Yang, Z. H.; Kelly, D. L.; Alakov, V. Y. Polymer genomics: Shifting the gene and drug delivery paradigms. J. Controlled Release 2004, 101 (1-3), 259–271. (32) Song, J. S.; Suh, C. H.; Park, Y. B.; Lee, S. H.; Yoo, N. C.; Lee, J. D.; Kim, K. H.; Lee, S. K. A phase I/IIa study on intra-articular injection of holmium-166-chitosan complex for the treatment of knee synovitis of rheumatoid arthritis. Eur. J. Nucl. Med. 2001, 28 (4), 489–497. (33) Liu, X. F.; Guan, Y. L.; Yang, D. Z.; Li, Z.; De Yao, K. Antibacterial action of chitosan and carboxymethylated chitosan. J. Appl. Polym. Sci. 2001, 79 (7), 1324–1335. (34) He, P.; Davis, S. S.; Illum, L. In vitro evaluation of the mucoadhesive properties of chitosan microspheres. Int. J. Pharm. 1998, 166 (1), 75–88.

8525

(35) Muzzarelli, R. A. A. Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cell. Mol. Life Sci. 1997, 53 (2), 131–140. (36) Bae, K. H.; Ha, Y. J.; Kim, C.; Lee, K. R.; Park, T. G. Pluronic/ chitosan shell cross-linked nanocapsules encapsulating magnetic nanoparticles. J. Biomater. Sci., Polym. Ed. 2008, 19 (12), 1571–1583. (37) Bhattarai, N.; Ramay, H. R.; Gunn, J.; Matsen, F. A.; Zhang, M. Q. PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release. J. Controlled Release 2005, 103 (3), 609–624. (38) Yang, T. F.; Chen, C. N.; Chen, M. C.; Lai, C. H.; Liang, H. F.; Sung, H. W. Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle. Biomaterials 2007, 28 (4), 725–734. (39) Bee, A.; Massart, R.; Neveu, S. Synthesis of Very Fine Maghemite Particles. J. Magn. Magn. Mater. 1995, 149 (1-2), 6–9. (40) Neuberger, T.; Schopf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B. Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system. J. Magn. Magn. Mater. 2005, 293 (1), 483–496. (41) Dong, Y. C.; Feng, S. S. Methoxy poly(ethylene glycol)poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials 2004, 25 (14), 2843–2849. (42) Chen, S.; Guo, C.; Liu, H. Z.; Wang, J.; Liang, X. F.; Zheng, L.; Ma, J. H. Thermodynamic analysis of micellization in PEO-PPO-PEO block copolymer solutions from the hydrogen bonding point of view. Mol. Simul. 2006, 32 (5), 409–418. (43) Guo, C.; Liu, H. Z.; Chen, J. Y. A Fourier transform infrared study of the phase transition in aqueous solutions of ethylene oxide-propylene oxide triblock copolymer. Colloid Polym. Sci. 1999, 277 (4), 376–381. (44) Su, Y. L.; Wang, J.; Liu, H. Z. FTIR spectroscopic investigation of effects of temperature and concentration on PEO-PPO-PEO block copolymer properties in aqueous solutions. Macromolecules 2002, 35 (16), 6426–6431. (45) Guo, C.; Wang, J.; Liu, H. Z.; Chen, J. Y. Hydration and conformation of temperature-dependent micellization of PEO-PPO-PEO block copolymers in aqueous solutions by FT-Raman. Langmuir 1999, 15 (8), 2703–2708. (46) Su, Y. L.; Wei, X. F.; Liu, H. Z. Influence of 1-pentanol on the micellization of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers in aqueous solutions. Langmuir 2003, 19 (7), 2995– 3000.

ReceiVed for reView March 11, 2010 ReVised manuscript receiVed July 21, 2010 Accepted July 25, 2010 IE100587E