Chitosan Composite

Sep 20, 2008 - Fe3O4/poly(N-Isopropylacrylamide)/Chitosan Composite Microspheres with Multiresponsive Properties. Pei Li, Ai Mei Zhu, Qing Lin Liu* an...
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Ind. Eng. Chem. Res. 2008, 47, 7700–7706

Fe3O4/poly(N-Isopropylacrylamide)/Chitosan Composite Microspheres with Multiresponsive Properties Pei Li, Ai Mei Zhu, Qing Lin Liu,* and Qiu Gen Zhang Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen UniVersity, Xiamen 361005, China

Multiresponsive composite microspheres were fabricated via emulsion polymerization in two steps. Fe3O4 nanoparticles modified by oleic acid (about 13 nm in diameter) were first prepared, and then they were embedded in biocompatible chitosan (CS) and N-isopropylacrylamide (NIPAm). Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) examined the structure and morphology of the composite microspheres. Scanning electron microscopy (SEM) and transmission electronic microscopy (TEM) indicate the diameter of the composite microspheres to be about 400 nm. The magnetic, thermo, and pH-sensitive properties of the composite microspheres were investigated. Magnetic measurements with magnet and superconducting quantum interference device (SQUID) reveal that the composite microspheres are superparamagnetic. The electromagnetically induced heating shows that the composite microspheres could be heated up to 45 °C in an alternating electromagnetic field. The dynamic light scattering (DLS) results confirm the thermoresponsive and pHresponsive properties. It was found that the lower critical solution temperature (LCST) of the composite microspheres is 29 °C in water, and the LCST changed from 28 to 32 °C in the pH range from 4.7 to 7.4. These composite microspheres with the multiresponsive properties show great promise in biomedical applications. 1. Introduction Recently, there has been considerable interest in the preparation of materials (microgels or microspheres) that can respond to external stimuli, such as magnetic field, thermo, or pH, etc. The conformation of the stimuli-sensitive materials is able to be changed with external stimuli, and their properties would be changed in response. These characters made the materials especially suitable for application in biological and medical fields, such as separation and purification of proteins,1,2 magnetic targeting,3 cell separation and purification,4,5 drug targeting carriers for hyperthermia,6 and controlled drug release.7-9 Of the smart polymeric materials, N-isopropylacrylamide (NIPAm) exhibits a lower critical solution temperature (LCST) around 32 °C; theoretical efforts on its thermoresponsiveness behavior have been well-documented.10,11 The NIPAm involved dual thermoresponsive and pH-responsive systems have been commonly investigated.12 Such compounds are usually prepared by combining NIPAm with a pH-sensitive polymer such as acrylic acid or chitosan by copolymerization.13 Alvarez-Lorenzo et al.14 prepared thermosensitive and pH-sensitive interpenetrated polymer networks (IPNs) using chitosan and NIPAm. Lin et al.15 reported the synthesis of a core-shell copolymer latex, in which the core is the copolymer of NIPAm and chitosan and the shell is the copolymer of methacrylic acid and methyl methacrylate. Chitosan with high biocompatibility can undergo chemical modification attributed to the presence of free amino groups (-NH2) and hydroxyl groups (-OH), which increases the reactivity of the polymer.16 The NIPAm involved dual thermoresponsive magnetic system has also been researched widely.17,18 The magnetic particles generally exist in a core-shell configuration, in which polymeric particles are bound to the magnetic core through organic linkers. The * Corresponding author. E-mail: [email protected]. Tel.: 86-5922183751. Fax: 86-592-2184822.

biomolecules may be coupled with the organic shell by its organic groups.19 Chiu et al.20 and Cai et al.21 indicated that a responsive system can be synthesized using Fe3O4 nanoparticles and NIPAm microgels via layer-by-layer adsorption. Sun et al.22 and Lai et al.23 prepared magnetic Fe3O4 nanoparticles coated by polyNIPAm, which has a LCST of 31 °C. These particles have been used as magnetic resonance imaging (MRI) contrast agents and hyperthermia. A lot of investigations have been processed currently on dualresponsive systems; the research on multiresponsive systems has also received great attention. The interesting physical phenomena exhibited by multiresponsive polymer and its medical importance make it worth confronting the experimental challenges. Gohy et al.24 reported a pH, ionic strength, and temperature sensitive system based on a mixture of P2VP-b-PEO and PMAA-b-PEO copolymers. Wang et al.25 studied multifunctional microspheres that combined several advances of photoluminescence, magnetic, and temperature responses into one single entity. However, there have been no reports that focus on multiresponsive microspheres with thermal, pH, and magnetic responsive characteristics. In this study, novel multiresponsive composite microspheres with thermal, pH, and magnetic responsive characteristics were prepared. Fe3O4 nanoparticles modified by oleic acid were encapsulated together with chitosan and NIPAm. The effects of the weight ratio of chitosan/NIPAm, the reaction temperature, and the stirring speed on the morphology of the composite microspheres were discussed. The thermal, pH, and magnetic responsive characteristics of the synthesized microspheres were tested by dynamic light scattering (DLS) and magnetic analysis. The morphology and elemental composition of the composite microspheres were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electronic microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and elemental analysis.

10.1021/ie800824q CCC: $40.75  2008 American Chemical Society Published on Web 09/20/2008

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Figure 1. Schematic illustration of the composite microsphere growth on Fe3O4 nanoparticles.

2. Materials and Methods 2.1. Materials. Chitosan with deacetylation degree (DD) of 90% and molecular weight (Mw) of 200 kD was purchased from Yuhuan Ocean Biochemical Company, China. NIPAm (Tokyo Chemical industry Co. Ltd.) was recrystallized in hexane before use. The water used in the experiment was purified by a Milli-Q reagent-grade system. All other solvents and reagents were of analytical grade and used without further purification. 2.2. Preparation of Magnetic Particles. Fe3O4 particles were synthesized by chemical coprecipitation method under alkaline conditions.26 In a typical run, 0.208 g of FeCl2 · 4H2O and 0.298 g of FeCl3 · 6H2O (molar ratio of Fe2+/Fe3+ ) 2:3) were dissolved in 40 mL of water with 1000 rpm stirring in N2 atmosphere. NH3 · H2O solution (0.7 mL) was added as the water bath temperature reached 60 °C. Then oleic acid (2 mL) was added 30 min later, and the reaction was maintained for 2 h. The precipitate was separated by magnet and rinsed with water several times. The obtained magnetic particles were redispersed in water to form a magnetic fluid containing 5.0 wt % microspheres for further use. 2.3. Synthesis of the Composite Microspheres. The composite microspheres were synthesized by polymerization of NIPAm and chitosan onto the oleic acid modified Fe3O4 nanoparticles. The synthesis mechanism of the microspheres is shown in Figure 1.27 First, chitosan (0.1 g) in 100 mL of 2.0 wt % acetic acid solution, Tween-80 (0.05 mL), and sodium sulfate (3 g) were added into a three-necked flask. Second, a suitable amount of the freshly made magnetic fluid was added dropwise and dispersed into the solution with stirring of 500 rpm at room temperature for 2 h. The chitosan-Fe3O4 nanoparticles were then used as seed to polymerize with 0.05 g of NIPAm in the presence of the cross-linker N,N-methylene bisacrylamide (MBA, 0.01 g) and the initiator ammonium persulfate (APS, 0.02 g). The reaction proceeded with stirring of 150 rpm in nitrogen atmosphere at 60 °C for 24 h. At the end of polymerization, the obtained composite microspheres were enriched with a magnet and rinsed with water and ethanol several times. Then the composite microspheres were freezedried for further analysis. 2.4. Characterization of Composite Microspheres. 2.4.1. Structure and Morphology Analysis. FTIR (NICOLET FTIR-740SX, U.S.A.) spectra of the samples were recorded. XRD (Panalytical X’pert Philip, Holland) was used to determine the crystal structure of the samples. The freeze-dried samples were sputtered with a conductive gold layer. Then low voltage (3-5 kV) SEM (Leo1530, Germany) was used to observe morphologies of the samples. A drop of an aqueous dispersion of the microspheres was placed on a Formvar-coated copper TEM grid (300 mesh size and covered with Formvar/carbon) and allowed to air-dry for TEM (JEOL, F30, 100 kV FEI Company, Holland) characterization. 2.4.2. Thermal and pH-Sensitive Analysis. TGA (Netzsch DSC 204 Phoenix, Germany) was performed with intracooler. Samples were heated from room temperature to 600 °C at a heating rate of 10 °C · min-1 under constant nitrogen purging. Then elemental analysis (Vario EL III, Elementar, Germany)

Figure 2. TEM of the nanoparticles: (a) pristine Fe3O4 nanoparticles and (b) Fe3O4 nanoparticles modified by oleic acid.

was used to characterize the remains. The dependence of the change in hydrodynamic size of the composite microspheres on temperature was examined by dynamic light scattering analyzer (DLS, Malvern-Zeta3600 Sizer, U.K.) from 25 to 40 °C. The samples were prepared in Milli-Q water and standard phosphate buffer solution (PBS) under different pH’s for DLS analysis.

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Figure 3. FTIR spectra of (a) NIPAm, (b) chitosan polymer, (c) pristine Fe3O4 nanoparticles, (d) Fe3O4 nanoparticles modified by oleic acid, and (e) composite microspheres.

2.4.3. Magnetic Properties. The magnetic response of the composite microspheres was first observed using a magnet (the results are not shown in this article). Then superconducting quantum interference device (SQUID, MPMS XL-7, Quantum Design, U.S.A.) magnetometer was used to confirm it. The electromagnetically induced heating was also carried out by a generator-oscillator combination (Huayan Company, Shanghai, China) under a field amplitude of 20 kA · m-1 with a frequency of 360 kHz. 3. Results and Discussion 3.1. Structure and Morphology Analysis. Figure 2 shows TEM analysis of the pristine Fe3O4 and oleic acid-treated Fe3O4 nanoparticles. The pristine Fe3O4 nanoparticles (Figure 2a) have a spherical shape with a size of 10 nm, and the aggregation is obvious. After surface modification by oleic acid (Figure 2b), the particles maintained their original spherical shape with a size of 13 nm and a slight aggregation was observed. The increase of 3 nm in size estimated by the software Sigmascan on the basis of TEM picture approximately equals the thickness of double oleic acid layers. The results indicate that the oleic acid modified Fe3O4 nanoparticles have good dispersivity.28 The Fe3O4 nanoparticles used hereafter are all modified by oleic acid. The FTIR spectra of the samples are shown in Figure 3. In NIPAm spectrum (curve a), the peak at 1658 cm-1 is due to the OdCsNH, and the characteristic isopropyl groups give the doublet absorption peaks at 1386 and 1368 cm-1 for sCH(CH3)2.29 For the spectrum of chitosan (curve b), the characteristic absorption bands appeared at 1646 (amide I), 1559 (amide II), and 1380 cm-1 (amide III). In Fe3O4 nanoparticles spectrum (curve c), the characteristic adsorption of FesO bonds appeared at 571 cm-1. In oleic acid modified Fe3O4 nanoparticles spectrum (curve d), the FesO bonds appeared at 586 cm-1. Another sharp band can be observed at 1627 cm-1, which has been shifted left from 1710 cm-1 (CdO bond asymmetric vibration). This can be explained as follows: as the carboxyl groups of oleic acid combined with the Fe atoms on the surface of Fe3O4 nanoparticles and rendered a partial single bond character of the CdO bond to weaken the bond, there was a shift of the stretching frequency to a lower value.30 For the spectrum of the composite microspheres (curve e), it was originally assumed that both hydroxyl and amino groups in chitosan could have reacted with oleic acid, resulting in ester and amide linkage, respectively. However, curve e does not show any absorption peaks of CdO stretching from ester linkage, which is normally found in the range between 1720

Figure 4. XRD patterns of (a) Fe3O4 nanoparticles modified by oleic acid and (b) composite microspheres.

and 1740 cm-1. Instead, the original amide peak (amide I) increased in intensity after reaction and shifted to lower wavenumber 1637 cm-1 due to hydrogen bonding. The 1559 cm-1 peak of sNH2 bending vibration of chitosan also blueshifted to 1546 cm-1. These results indicate that there are CS chains in the composite microspheres. The characteristic bands of NIPAm at 2972 and 1387 cm-1 confirmed that the NIPAm chains existed in the composite microspheres. The characteristic adsorption of Fe3O4 appeared at 619 cm-1. These results of the FTIR suggest the integration of Fe3O4, NIPAm, and chitosan in the composite microspheres. Figure 4 shows XRD patterns of the Fe3O4 and composite microspheres samples. Six characteristic peaks for Fe3O4 (2θ ) 30.2, 35.5, 43.1, 53.4, 57.0, and 62.6°) were observed in both the samples. These peaks are consistent with the database in JCPDS file (PCPDFWIN v.2.02).5 The results confirm that Fe3O4 is embedded in the composite microspheres. The size (D) of Fe3O4 nanoparticles can be estimated by Scherrer’s formula on the basis of XRD data D ) 0.9

λ β cos θ

(1)

where λ is the wavelength of the radiation, θ is Bragg’s angle, and β is the full-width at half-maximum.31 The particle size was determined to be 13 nm, which is a common value for superparamagnetic iron oxide nanoparticles as reported.32 This is also in accordance with the average diameter (13 nm) of Fe3O4 nanoparticles observed previously by TEM. Figure 5 (parts a-f) shows SEM micrographs of the composite microspheres with different weight ratios of chitosan/ NIPAm. Significant differences can be noted in the structure of the samples. All of the samples have sphere structure morphology. The coagulation effect on surface area decreases with decreasing NIPAm content; thus, the microspheres show distinct boundaries, and the degree of dispersion of the samples gets better (Figure 5f). The diameter of the microspheres (Figure 5g) decreases with decreasing NIPAm loading, and the system exhibits high hydrophilicity and swelling properties at high NIPAm concentration; therefore, the composite microspheres have strong aggregation and the diameter of the microspheres is bigger.33 They display good morphological characteristics and spherical shapes with a diameter of 400 nm. When the ratio is >2.0, the thermoresponsive behavior of the composite microspheres would become less observable, so an optimal of 2.0 was obtained. Some other influence factors such as reaction temperature and stirring speed were investigated. However, the results show that they did not have as obvious an effect as the weight ratio of chitosan/NIPAm.

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Figure 5. (a-f) SEM of the composite microspheres with different chitosan/NIPAm weight ratios, (a) 0.1, (b) 0.2, (c) 0.5, (d) 1.0, (e) 1.5, and (f) 2.0; (g) the relationship between the size distribution of the composite microspheres and the weight ratios of chitosan/NIPAm.

Figure 6a shows the TEM micrographs of the composite microspheres with chitosan/NIPAm weight ratio of 2.0. The morphology of the microspheres is fairly spherical with a size of about 400 nm. The microspheres have the morphology of a homogeneous structure. The result of DLS (Figure 6b) confirms that the average diameter of the composite microspheres is about 400 nm; this result agrees with the TEM well. The composite microspheres used afterward are the samples with a chitosan/ NIPAm weight ratio of 2.0. 3.2. Thermal and pH-Sensitive Analysis. Figure 7 shows the TGA result of the composite microspheres. The line reveals two steps of weight loss. The first step between 40 and 180 °C

is due to the evaporation of residual water and decomposition of polymers of low molecular weights in the sample, and the second weight loss stage between 180 and 400 °C is due to the decomposition of the chains of chitosan and NIPAm bound on the surface.34 There is no significant weight change within the temperature range from 400 to 600 °C; the elemental analysis of the remains showed only 0.005% carbon residue. This indicates that the mass content of Fe3O4 in microspheres was about 25%. The thermoresponsiveness of the composite microspheres was confirmed by DLS (Figure 8) in solution at different pH’s within a temperature range from 25 to 40 °C. Each of the curves

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Figure 8. Relationship between the hydrodynamic diameter and the temperature of the composite microspheres.

Figure 6. (a) TEM images of the composite microspheres with chitosan/ NIPAm weight ratio of 2 and (b) size distribution of the composite microspheres by DLS at 25 °C.

Figure 7. Thermal degradation curves of the composite microspheres.

appears to show a similar behavior: an increase in hydrodynamic diameter (Dh) is noted when temperature is below the LCST, then the Dh decreases sharply at the LCST and finally remains approximately constant. At a temperature below the LCST, with increasing temperature, the chitosan and NIPAm chains are more easily protonated, leading to an increase in the hydrophilicity and swelling of the microspheres owing to the hydrogen bonding between water and the amide groups. Meanwhile, the aggregation of microspheres may be increased to a certain extent because the interaction among chitosan, water, and NIPAm chains increases with increasing temperature. Therefore, the Dh of the microspheres increases accordingly. When the system is above the LCST, the microspheres surface would turn hydrophobic; the hydration capability of the microspheres is suppressed due to the progressive replacement of the intermolecular interaction. The chain of NIPAm shrinks and the interaction chains collapse, which results in much smaller particles, so the Dh decreases sharply.35 At the same time, the results show that

the LCST of the microspheres is around 31 °C in Milli-Q water, which is lower than 32 °C (the LCST of pure NIPAm microgels).36 This is because the hydrophobic modification of NIPAm by oleic acid and chitosan results in the LCST shifting to a lower temperature.37 Figure 8 also shows the effect of pH on the LCST of the composite microspheres. The LCST in the PBS solution is 28 °C at pH ) 4.7, whereas it turns to 32 °C at pH ) 7.4. The change of LCST can be explained by the existence of a large amount of amino groups in chitosan.38 The LCST of composite microspheres decreases owing to inter- or intramolecular hydrogen bonding between the amide group of polyNIPAm and the amino group of CS below the pKa value of CS (the pKa value of amino group of CS was reported to be about 6.2-6.8). With decreasing pH, the amino groups in CS segments are more easily protonated; hence, the hydrophilicity of CS segments increases, which leads to a decrease in the LCST of the microspheres. The phase-separation temperature of the composite microspheres depends on the hydrophilic/hydrophobic balance in the polymer chains and on the hydrogen-bonding capabilities from its chemical structure. The hydrophilic/ hydrophobic balance depends mainly on the amount of comonomer, which affects the extent of ionization of the amine groups. The hydrophobicity relies mainly on the isopropyl units of NIPAm, and the hydrophilicity is caused by the amino group of CS. This effect increases with increasing the ionization degree of the amine, which depends on the pH of the solution: the lower the pH, the higher is the ionization degree of the amine groups; furthermore, it depends on the pKa of the CS segments. 3.3. Magnetic Properties. The microspheres have strong magnetic properties and can be quickly separated from the solution by a magnetic field (a magnet of 0.2 T in our experiments). Figure 9 shows the magnetic hysteresis curves of the Fe3O4 nanoparticles and the composite microspheres measured by SQUID at 300 K. Magnetization intensity increases greatly under the magnetic field, and the superparamagnetic phenomena appears. The inset curves show very low coercivity of 17 and 19 Oe, respectively, which confirms the superparamagnetic properties of both the microspheres.39 The saturation magnetization of Fe3O4 nanoparticles and the composite microspheres is 35.8 and 9.3 emu · g-1, respectively. These low saturation magnetization values are less than the literature value for the pure magnetite nanoparticles (64.3 emu · g-1). This can be explained by considering the diamagnetic contribution of the oleic acid shells surrounding the magnetite nanoparticles. The saturation magnetization of the latter is about 74% lower than that of the former. It is possible that surface magnetic anisotropy

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Figure 9. Magnetization curves of (a) Fe3O4 nanoparticles modified by oleic acid and (b) composite microspheres (sample weight of 4.4 mg).

Fe3O4 nanoparticles. The oleic acid modified Fe3O4 nanoparticles exhibit a good dispersion. The SEM and TEM results suggest that the composite microspheres are in a spherical shape with size of 400 nm when the ratio of chitosan/NIPAm is 2.0. The composite microspheres are thermoresponsive, and pHresponsive, and magnetic-sensitive. The incorporation of chitosan into the structure induced pronounced pH sensitivity. DLS studies indicate that the composite microspheres have a LCST of 31 °C in water; the LCST was observed to vary from 28 °C at acidic pH to 32 °C at basic pH. SQUID reveals that the composite microspheres are superparamagnetic. The electromagnetically induced heating shows that the composite microspheres could be heated to 45 °C in an alternating electromagnetic field. The multiresponsive properties of these microspheres suggest that they could be used more effectively than with a single stimulus in the application of biotechnology and magnetically guided drug delivery. Acknowledgment The support of National Nature Science Foundation of China (No. 50573063), the Program for New Century Excellent Talents in University, the research fund for the Doctoral Program of Higher Education (No. 2005038401), and Xiamen University (No. 0000X071C1) in the preparation of this work is gratefully acknowledged. Literature Cited

Figure 10. Heating test curve of the composite microspheres in alternating electromagnetic field.

was changed with the existence of thick carbon layers, the surface spins disorientation increased, and thus the magnetic moment decreased.25 Figure 10 describes the relation of temperature with time in an alternating electromagnetic field (0.3 T). The experiment was performed in a glass tube thermo-insulated with concentrations of 10 and 20 mg · mL-1 of the composite microspheres placed in the center of the coil. The results demonstrate that sufficient energy deposition leads to an increase of the solution temperature within a short time. The temperature of the solution ranged from 15 to 35 or 45 °C, i.e., the extent of temperature variation is 25 or 30 °C. The temperature is directly proportional to the time in the first hour, and then the heating speed slowed down because of the thermal losses and the hysteresis loss during the reorientation of the magnetization.40 The specific absorption rates (SARs) can be calculated by SAR ) C

∆T ∆t

(2)

Here, C is the sample specific heat capacity (water as the main part of the system), and ∆T /∆t is the slope of the temperature versus time.41 The SAR results lie between 10 and 15 W · g-1. At a temperature of 45 °C, the sample presented optimum behavior for the hyperthermia treatment, so the system should be much more promising to achieve the curative effect for the treatment of tumor. 4. Summary The composite microspheres were prepared via emulsion polymerization of NIPAm and chitosan with oleic acid modified

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ReceiVed for reView May 22, 2008 ReVised manuscript receiVed July 26, 2008 Accepted August 7, 2008 IE800824Q