Preparation of Aggregation-Resistant Biocompatible

Aug 29, 2012 - Novel biocompatible pH-stimuli responsive superparamagnetic hybrid hollow microspheres as tumor-specific drug delivery system. Xiaorui ...
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Preparation of Aggregation-Resistant Biocompatible Superparamagnetic Noncovalent Hybrid Multilayer Hollow Microspheres for Controlled Drug Release Xubo Zhao, Pengcheng Du, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Institute of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: Biocompatible superparamagnetic polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3-CS-NHCH2-PEG) were successfully prepared by PEGylation of multilayered polyelectrolyte hybrid shell encapsulated polystyrene sulfonate (PSS) microsphere templates fabricated by the layer-by-layer self-assembly of chitosan (CS) and citrate modified ferroferric oxide magnetic nanoparticles (Fe3O4-CA), after etching the templates by dialysis. Their hollow structure with diameter of about 200 nm was confirmed by TEM analysis. The pH and ionic strength responsive properties were retained after the PEGylation of the hollow microspheres. Furthermore, their biocompatibility and stability against aggregation and fusion in media with high ionic strength were distinctly improved. A typical anti-inflammatory drug, ibuprofen, was used for drug loading, and the release behaviors of ibuprofen in a simulated body fluid (SBF) were studied. The results indicate that the biocompatible superparamagnetic polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG) have a high drug loading capacity and favorable release property for ibuprofen; thus, they are very promising for application in drug delivery. KEYWORDS: multilayered hybrid hollow microspheres, layer-by-layer self-assembly, aggregation-resistant, biocompatible, superparamagnetic, controlled drug release

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INTRODUCTION During the past decade, hollow microspheres have attracted much attention due to their wide applications in biomedical fields. Polyelectrolyte multilayer hybrid hollow microspheres prepared by the layer-by-layer (LbL) self-assembly technique on various templates were demonstrated as convenient tools for biomedical fields and life science applications,1 especially for the delivery and controlled release of life-active compounds2−4 both in vitro5 and in vivo.6 However, a problem of salt-resistance of the hollow microspheres remains vital for their applications, in particular, non-salt-resistance of the hollow microspheres leads to aggregation and disintegration of the polyelectrolyte microcapsules. In addition, the flocculation phenomenon is a serious constraint in the simulation of human body fluid in vivo. In order to solve the critical problem, functional polymers and some small molecules were introduced. Covalent linkage of poly(ethylene glycol) (PEG) or other hydrophilic polymers to the surface of several carrier systems has been shown to reduce their nonspecific uptake by the cells (including cells from the mononuclear phagocyte system) due to their enhanced low fouling properties7,8 and the PEG chain having a shielding effect which can maintain stability to suppress the increase of the aggregation in a high salt concentration environment.9 In order to solve aggregation of such microcapsules in the solution of strong pH and/or higher salt concentration, Usov et al. used dextran to modify the layer-by-layer self-assembled polyelectrolyte microcapsules to provide stability against © 2012 American Chemical Society

aggregation in 0.75 M aqueous solutions of mono- and bivalent ions.10 Our groups successfully prepared the polymeric hollow microspheres with pH/ionic strength/temperature multiresponsive shell via the combination of layer-by-layer self-assembly techniques and grafting polymerization of N-isopropylacrylamide (NIPAm) with cerium ammonium nitrate as a redox initiator.11 The introduction of the poly(N-isopropylacrylamide) (PNIPAm) brushes onto the polyelectrolyte shells not only achieved the controlled release of drug molecules that could be dually controlled by solution pH and temperature, but also prevented flocculation among the obtained multiresponsive hollow microspheres in the solution with higher salt concentration. Poly(ethylene glycol) (PEG) is one of the most commonly used hydrophilic polymers, which possesses high biocompatibility, and can be rapidly and spontaneously cleared from human bodies as excellent candidates of biomedical materials.12 It is also believed to impart low fouling properties to surfaces due to the screening of interfacial charges, repulsion (entropic and osmotic), and excluded volume effects.13 Szczepanowicz reported a novel method to prepare nanosized capsules based on liquid core encapsulation by biocompatible polyelectrolyte (PE) multilayer adsorption, with a PEGylated outermost layer Received: Revised: Accepted: Published: 3330

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Scheme 1. Schematic Illustration of the Preparation of the Superparamagnetic Polyelectrolyte Hybrid Hollow Microspheres

to enhance biocompatibility of nanocapsules.14 Qian et al. developed a three-component copolymer containing PEG for drug release. PEG were designed as carriers for the sustained release of ketoprofen, naproxen, and ibuprofen.15 Zheng et al. showed that the chemical modification of the aginate/chitosan/ aginate hydrogel microcapsule with methoxy poly(ethylene glycol) would help to reduce the nonspecific protein adsorption and improve the biocompatibility in vivo.16 Dong et al. reported a uniform and biocompatible nanocapsule prepared via a novel “self-templating” strategy, the results indicating that the PEG chains on the surface of obtained nanocapsules would provide a shielding effect to prevent PICmicelles forming a vesicular structure with a smaller curvature.9 At present, it is well-recognized that the surfaces of biomaterials are immediately covered by biomolecules when they come in contact with a biological medium. So protein− nanoparticle interaction phenomena are possible impacts on the nanoparticles' fate and behavior in vivo.17−20 Mahmoudi reported a novel approach, which was used to verify the influence of surface roughness of nanoparticles on the composition of the corresponding protein corona.21 Therefore, to reduce the protein adsorption is a key element in resistance to protein adsorption. Among the uncharged and water-soluble polymers, PEG has been a successful route for the improved resistance of drug carriers to protein adsorption22 and the PEG chain has a shielding effect which can maintain stability to suppress the increase of the aggregation in a high salt concentration environment. Magnetite (Fe3O4) nanoparticles were extensively applied in drug targeting, bioseparation (cell sorting), magnetic resonance imaging (MRI), and magnetic transfection in nanomedicine because of their superparamagnetic properties and small size.23−26 However, these nanosized magnetite particles tend to aggregate because of their high specific area and strong interparticle interaction, which limits their utilization. Therefore, citrate was used to modify ferroferric oxide (Fe3O4) in order to enhance chemical stabilization of the naked magnetic nanoparticles against aggregation over a long period in our paper. In addition, nanoparticles also have toxicity in living organisms. Sharifi has discussed the biophysicochemical properties of various nanomaterials with emphasis on currently available toxicology data and methodologies for evaluating nanoparticle toxicity.27 Hussain also designed reliable methods for assessing novel nanomaterial internalization and their kinetics in living organisms.28 So it is imperative to assess potential toxicity of superparamagnetic noncovalent hybrid multilayer hollow microspheres. In the present paper, the biocompatible polyelectrolyte hybrid hollow microspheres were designed to be used as drug

delivery vehicles via LbL self-assembly with natural chitosan (CS) as polycation, and citrate modified ferroferric oxide (Fe3O4-CA) magnetic nanoparticles as hybrid anion on the polystyrene sulfonate microsphere (PSS) templates, respectively. Then the single-ended aldehyde group polyethylene glycol (APEG) was grafted onto the surface of the above magnetic hybrid polyelectrolyte coated microsphere via the nucleophilic addition between aldehyde groups of the APEG and the amino groups of CS. Finally, APEG grafted onto the surface of the above magnetic hybrid polyelectrolytes was treated with NaBH4 before the templates were removed by dialysis (Scheme 1). Dynamic light scattering (DLS) was employed to investigate the stability in the solution with higher mono- and bivalent ion concentration of the PEG modified magnetic hybrid polyelectrolyte hollow microspheres. The results indicated that the introduction of the PEG moiety improves efficiently water solubility and biocompatibility of the hollow microspheres, and enhances the salt-resistance and stability in solution with strong pH media and higher salt concentration. Their pH responsive controlled release of a model drug (ibuprofen) was also investigated in a simulated body fluid (SBF).

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EXPERIMENTAL SECTION Materials. Chitosan (CS, viscosity-average molecular weight, 6.0 × 105; degree of deacetylation, 90%) was purchased from the Zhejiang Yuhuan Marine Biotechnology Company. Polyethylene glycol 4000 (PEG 4000) was purchased from the Shanghai Wulian Chemical Factory. Sodium borohydride (NaBH4) was analytical reagent grade purchased from Shanghai Sapu Chemical Co. Ltd. Styrene (St), methacrylic acid (MAA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetic anhydride, ether, trichloromethane (CHCl3), dichloromethane (CH2Cl2), ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl 2 ·4H 2 O), sodium citrate (CA), ammonia−water (NH3·H2O, 28 wt %), ammonium persulfate (APS), sulfuric acid (H2SO4, 98%), and sodium carbonate (Na2CO3) were analytical reagent grade purchased from Tianjing Chemical Company in China. Deionized water was used throughout the experiments. Citrate Modified Ferroferric Oxide (Fe3O4-CA). The coprecipitation method was used to prepare the citrate modified ferroferric oxide. The FeCl3·6H2O (26.0 g) and FeCl2·4H2O (9.56 g) in a 1:2 molar ratio were dissolved in deionized water (400 mL) under nitrogen atmosphere with vigorous stirring. As the solution was heated to 70 °C, NH3·H2O (28 wt %, 50 mL) was added dropwise to the above mixture under vigorous stirring, then 48 mL of 2 M sodium 3331

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The CS and Fe3O4-CA were alternately deposited twice more onto the PSS templates to obtain the CS/Fe3O4-CA multilayer encapsulated PSS microspheres (PSS@(CS/Fe3O4-CA)3). At last, CS was adsorbed on the surface of the PSS@(CS/Fe3O4CA)3 again to obtain the CS/Fe3O4-CA multilayer encapsulated PSS microspheres with the outermost CS layer (PSS@(CS/Fe3O4-CA)3-CS) to be modified with APEG (Scheme 1). PEG Modified CS/Fe3O4-CA Multilayer Encapsulated PSS Microspheres (PSS@(CS/Fe3O4-CA)3-CS-NH-CH2PEG). The PSS@(CS/Fe3O4-CA)3-CS was dispersed in 250 mL of deionized water. The excess of APEG was added to the suspension of the PSS@(CS/Fe3O4-CA)3-CS after the pH of the mixture was adjusted to 5. The reaction mixture was stirred at 30 °C for 24 h, and the product was washed with deionized water for several times to remove the dissociated APEG by using the centrifugal separation technology to obtain the PEG modified PSS@(CS/Fe3O4-CA)3-CS microspheres via Schiff’s base structure (PSS@(CS/Fe3O4-CA)3-CS-NCH-PEG). In order to improve the stability of the grafted PEG, the Schiff base (−CN−) linking bond was transformed into −C−N− by the following procedure: 0.02 g of sodium borohydride (NaBH4) was dissolved in 20 mL of deionized water to get a 1 mg/mL solution. Then the sodium borohydride (NaBH4) solution was added dropwise to the suspension of the PSS@(CS/Fe3O4-CA)3-CS-NCH-PEG at 40 °C for 24 h by magnetic stirring and washed with deionized water several times to get the stable PEG modified CS/Fe3O4CA multilayer encapsulated PSS microspheres (PSS@(CS/ Fe3O4-CA)3-CS-NH-CH2-PEG).10 PEG Modified Polyelectrolyte Hybrid Hollow Microspheres. To obtain the hollow microspheres, the sacrificial templates (PSS) were removed by dialysis as follows: the aqueous suspension of the PSS@(CS/Fe3O4-CA)3-CS-NHCH2-PEG was dialyzed against DMF and deionized water using a dialysis membrane (MWCO = 14000) for 3 days with several changes of DMF and deionized water, respectively. The absence of the templates was confirmed by mixing the final dialysate with three times volume of water ensuring the absence of any precipitate. The resultant solution was centrifuged, washed with ethanol, and dried under vacuum at 40 °C for 24 h to obtain the ultimate PEG modified polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG). Furthermore, the PSS@(CS/Fe 3 O 4 -CA) 3 -CS and the PSS@(CS/Fe3O4-CA)3-CS-NCH-PEG microspheres were also dialyzed to etch the templates to obtain the magnetic polyelectrolyte hollow microspheres ((CS/Fe3O4-CA)3-CS and (CS/Fe3O4-CA)3-CS-NCH-PEG) in order to compare with the (CS/Fe3O4-CA)3-CS-NH-CH2-PEG hollow microspheres. Cell Toxicity Assays. Sulforhodamine-B (SRB) assay was performed to evaluate the cytocompatibility of the magnetic polyelectrolyte hybrid hollow microspheres with HepG2 cells. The cells were seeded into 96-well plates at densities of 1 × 105 cells per well for 24 h. Then, different concentrations of the magnetic polyelectrolyte hybrid hollow microspheres ((CS/ Fe3O4-CA)3-CS or (CS/Fe3O4-CA)3-CS-NH-CH2-PEG) were added to the cells and incubated for 48 h. Thereafter, the cells were washed three times with phosphate buffered saline (PBS) and processed for the SRB assay to determine the cell viability. For this, cells were fixed with a solution of 50% trichloroacetic acid and stained with 0.4% SRB dissolved in 1% acetic acid. Cell bound dye was extracted with 10 mM unbuffered Tris buffer

citrate was quickly added, the reaction was allowed to proceed for 1 h at 70 °C, and then the temperature was increased to 85 °C to vaporize the residual NH3. The ultimate citrate modified Fe3O4 nanoparticles (Fe3O4-CA) were washed more than three times with deionized water to discard the excessive sodium citrate by the magnetic separation procedure. Finally, the Fe3O4-CA nanoparticles were dispersed and stored in 200 mL of deionized water.29 Single-Ended Aldehyde Group Polyethylene Glycol (APEG). 10 g of PEG was dissolved in the mixture containing 25 mL of DMSO and 10 mL of trichloromethane in a 100 mL flask, and then 3.5 mL of acetic anhydride was added dropwise slowly to the flask and magnetically stirred at 35 °C for 10 h. The solution was precipitated with excess ether and filtered to obtain the white precipitate. Afterward the white precipitate was dissolved in the dichloromethane and precipitated with excess ether again. The white precipitate of single-ended aldehyde group polyethylene glycol (APEG) was filtered and dried at 35 °C in the vacuum oven.30 Preparation of Polystyrene Sulfonate Microsphere (PSS). The polystyrene (PS) latex was prepared by the emulsifier-free emulsion polymerization of styrene (St) with methacrylic acid (MAA) as the surfmer according to the procedure reported previously.31 Typically, 10 mL of styrene (St), 2 mL of methacrylic acid (MAA), and 95 mL of deionized water were added into a three-necked round-bottom flask fitted with a condenser and a magnetic stirrer and purged with nitrogen. A solution of ammonium persulfate (APS, 0.054 g) predissolved in deionized water (5 mL) was added dropwise slowly to the reaction vessel with vigorous stirring, bubbling with nitrogen. The polymerization was conducted for 24 h at 72 °C. The product was purified by repeated centrifugation and washing with ethanol. The white polystyrene nanospheres (PS) were finally obtained after being dried in a vacuum oven at 50 °C. The uniform PS particles (2.0 g) synthesized as presented above were dispersed in sulfuric acid (60 mL, 98%) with the aid of ultrasonic irradiation. The sulfonation was conducted at 45 °C under magnetic stirring for 8 h. After being cooled to room temperature, the product was separated by being centrifuged and washed with a large excess of deionized water after being diluted with deionized water until neutral pH. The obtained microspheres were finally dispersed and stored in 100 mL of deionized water.32 Chitosan/Ferroferric Oxide Multilayer Hybrid Encapsulated Polystyrene Sulfonate (PSS) Templates. The layer-by-layer assembly technique was used to prepare the magnetic hybrid coated polystyrene sulfonate templates with polyelectrolyte cation (CS) and the citrate modified ferroferric oxide (Fe3O4-CA) hybrid anion by the electrostatic interaction between the amino groups of CS and the carboxyl groups of the Fe3O4-CA, starting with CS. The adsorption of CS was completed in a solution of 250 mL of deionized water containing 0.5 g of sulfonate polystyrene (PSS) templates and 0.5 g of chitosan, at pH around 4.8 or 5.1 followed by centrifugation and washing twice in deionized water to obtain PSS-CS1. Next, the PSS-CS1 was dispersed into 250 mL of deionized water and added to the aqueous solution containing the citrate modified ferroferric oxide (Fe3O4-CA) in batches until to reach the adsorption balance at pH around 5.0 under magnetic stirring combined with ultrasonic. Then it was centrifuged and washed twice in water to obtain the hybrid shell encapsulated PSS templates (PSS@(CS/Fe3O4-CA)1). 3332

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The XRD patterns of the Fe3O4-CA, (CS/Fe3O4-CA)3-CS, and (CS/Fe3O4-CA)3-CS-NH-CH2-PEG were recorded in the range of 2θ = 20−70° by step scanning with a Shimadzu XRD6000 X-ray diffractometer using Cu Kα radiation. The behavior of drug loading and controlled release of the pH/ionic strength dual-responsive hollow microspheres (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG and hollow microspheres (CS/ Fe3O4-CA)3-CS were detected by a Perkin-Elmer Lambda 35 UV/vis spectrometer (Perkin-Elmer Instruments, USA) at room temperature. Each value represents the mean (SD < 5%) (n = 3).

solution (pH 10.5), and then the absorbance was measured at 550 nm using a plate reader. Drug Loading and Controlled Release. The ibuprofen solution (1 mg/mL) was prepared with deionized water. About 18.5 mg of the magnetic multilayer hybrid hollow microspheres (CS/Fe3O4-CA)3-CS or (CS/Fe3O4-CA)3-CS-NH-CH2-PEG was added into 5.0 mL of ibuprofen solution for the drugloading, respectively. After being swung by table concentrator for 48 h, the ibuprofen-loaded magnetic multilayer hybrid hollow microspheres were centrifuged to remove the excess free ibuprofen. The drug concentration in the supernatant solution was analyzed using an ultraviolet (UV) spectrophotometer by monitoring the loading capacities at a wavelength of maximum absorbance (264 nm). The drug-loading capacities of the magnetic multilayer hybrid hollow microspheres were calculated from the drug concentrations in solution before and after adsorption. The ibuprofen-loaded magnetic multilayer hybrid hollow microspheres (CS/Fe3O4-CA)3-CS, (CS/Fe3O4-CA)3-CS-NHCH2-PEG) were redispersed into 10 mL of phosphate buffer, transferred into dialysis tubes with a molecular weight cutoff of 14,000, and immersed into 100 mL of phosphate buffer at 37 °C under a pH value of 7.4 or 1.8, respectively. Aliquots (5.0 mL) of the solutions were taken at time intervals, and then the drug concentrations in the dialysates were analyzed for monitoring the 264 nm absorbance peak of ibuprofen using UV−vis spectrometry to detect the rate of drug release. 5.0 mL of fresh different phosphate buffer solutions was added after each sampling to keep the total volume of the solution constant. The cumulative release is expressed as the total percentage of drug molecules released through the dialysis membrane over time. Characterization. The magnetic hybrid polyelectrolyte coated PSS microspheres (PSS@(CS/Fe3O4-CA)3) and PEG modified magnetic hybrid polyelectrolyte hollow microspheres ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG) were characterized with a JEM1200 EX/S transmission electron microscope (TEM). The samples were dispersed in water and then deposited on a copper grid covered with a perforated carbon film. A Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany) was used for the Fourier transform infrared (FT-IR) spectroscopy analysis in the range of 400− 4000 cm−1 with a resolution of 4 cm−1. The KBr pellet technique was adopted to prepare the sample for recording the IR spectra. The mean particle size and size distributions of the obtained hollow microspheres were determined by the dynamical mode (dynamic light scattering (DLS)) on a light scattering system BI-200SM, Brookhaven Instruments, device equipped with a BI-200SM goniometer, a BI-9000AT correlator, a temperature controller, and a Coherent INOVA 70C argon ion laser at 20 °C. DLS measurements are performed using 135 mW intense laser excitation at 514.5 nm and at a detection angle of 90° using the emulsion directly at 25 °C. Each value represents the mean (SD < 5%) (n = 3). Magnetic properties of the citrate modified ferroferric oxide (Fe3O4-CA) and the magnetic polyelectrolyte hybrid hollow microspheres (CS/Fe3O4-CA)3-CS and (CS/Fe3O4-CA)3-CSNH-CH2-PEG were detected by vibrating sample magnetometer (Lakeshore 7304). The hysteresis of the magnetization was obtained by changing H between +11,000 and −11,000 Oe at room temperature.

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RESULTS AND DISCUSSION Preparation of PEG Modified Polyelectrolyte Hybrid Hollow Microspheres ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG). The PS latex was prepared by the emulsifier-free emulsion polymerization, and then the sulfonic acid groups were introduced onto the surface of the PS spheres to adsorb the polycation (chitosan (CS)). A typical TEM image of the polystyrene sulfonate microspheres (PSS) microspheres is spherical in shape and monodisperse in size, with a diameter of around 180 nm (Figure 1a). The layer-by-layer assembly technique was used to prepare the polyelectrolyte multilayer encapsulated PSS microspheres with chitosan (CS) and citrate modified magnetic nanoparticles (Fe3O4-CA) alternately adsorbed onto the PSS templates by the electrostatic interaction between the amino groups of chitosan and the carboxyl groups of citrate modified magnetic nanoparticles. The TEM image of the CS/Fe3O4-CA multilayer encapsulated PSS microspheres with the outermost CS layer (PSS@(CS/Fe3O4CA)3-CS) is shown as Figure 1b. It can be found that the diameter is about 240 nm. Furthermore, the diameter of the stable PEG modified CS/Fe3O4-CA multilayer encapsulated PSS microspheres (PSS@(CS/Fe3O4-CA)3-CS-NH-CH2-PEG) is approximately 320 nm in Figure 1d. It inferred that the PEG was successfully grafted on the magnetic polyelectrolyte hybrid multilayer encapsulated PSS microspheres (PSS@(CS/Fe3O4CA)3). The IR spectrum of the PSS@(CS/Fe3O4-CA)3-CS as shown in Figure 2 revealed the well-defined characteristic absorbance bands of polystyrene at 3060, 3022, 2920, 2845 cm−1 (C−H, stretching vibration), 1495, 1457 cm−1 (CC, stretching vibration), and 700 cm−1 (out-of-plane blending vibration, δring). The two characteristic peaks at 1702 and 1120 cm−1 are attributed to the carbonyl stretching of the carboxyl groups of MAA units and sulfonic acid groups, respectively. Furthermore, the representative absorbance band of Fe−O stretching vibration at 585 cm−1 from the IR spectrum of the PSS@(CS/Fe3O4-CA)3-CS can also be found, indicating the incorporation of the magnetic nanoparticles. Then polyethylene glycol (PEG) was grafted onto the magnetic hybrid polyelectrolyte shells via the nucleophilic addition between the aldehyde groups of APEG and the amino groups of CS for the sake of ameliorating the dispersion and biocompatibility of the magnetic hybrid polyelectrolyte hollow microspheres in media with higher salt concentrations. The Schiff base (−CN−) linking bond was transformed into −C−N− by being treated with sodium borohydride (NaBH4). A new peak is found in the IR spectrum of the PSS@(CS/ Fe3O4-CA)3-CS-NH-CH2-PEG at 1655 cm−1 compared with the PSS@(CS/Fe3O4-CA)3-CS. It indicated that PEG had been successfully grafted onto the surfaces of the PSS@(CS/Fe3O4CA)3-CS. 3333

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Figure 2. The FTIR spectra of (CS/Fe3O4-CA)3-CS-CH2-NH-PEG) (a), PSS@(CS/Fe3O4-CA)3-CS (b), PSS@(CS/Fe3O4-CA)3-CS-N CH-PEG (c), and PSS@(CS/Fe3O4-CA)3-CS-NH-CH2-PEG (d).

nm from the TEM analysis as revealed in Figure 1f. The reductive treatment did not result in collapse of the hollow microspheres. The magnetic hysteresis loops for the CA modified Fe3O4 nanoparticles (Fe3O4-CA) and the magnetic polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3-CS and (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG) are shown in Figure 3. Neither

Figure 3. The magnetic hysteresis loops of the Fe3O4-CA (a), (CS/ Fe3O4-CA)3-CS (b), and (CS/Fe3O4-CA)3-CS-NH-CH2-PEG (c).

remanence nor coercivity was observed, indicating the superparamagnetic property.29 The saturation magnetization of the Fe3O4-CA, the (CS/Fe3O4-CA)3-CS, and the (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG hollow microspheres was 63.32 emu/g, 37.45 emu/g, and 35.16 emu/g, respectively. The decrease in the saturation magnetization of the magnetic polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3CS and (CS/Fe3O4-CA)3-CS-NH-CH2-PEG) compared with the Fe3O4-CA nanoparticles was ascribed to the chitosan ingredients and PEG chains. The magnetite contents calculated from the ratio of the original saturation magnetization of the Fe3O4-CA nanoparticles and the magnetic polyelectrolyte hybrid hollow microspheres ((CS/Fe3O4-CA)3-CS and (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG) were 59.14% ((CS/Fe3O4CA)3-CS) and 55.53% ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG), respectively. The decrease of the magnetite content of the (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG compared with that of the (CS/Fe3O4-CA)3-CS resulted from the grafting of the PEG chains. The high magnetization properties were obtained while leaving sufficient polymer to retain drugs making these functional hollow microspheres suitable as a potential platform

Figure 1. The TEM of (a) PSS, (b) PSS@(CS/Fe3O4-CA)3-CS, (c) PSS@(CS/Fe3O4-CA)3-CS-NCH-PEG, (d) PSS@(CS/Fe3O4CA)3-CS-NH-CH2-PEG, (e) (CS/Fe3O4-CA)3-CS-NCH-PEG, and (f) (CS/Fe3O4-CA)3-CS-NH-CH2-PEG.

Then the PSS templates were removed by dialysis with DMF to get the biocompatible magnetic multilayer hybrid hollow microspheres (CS/Fe3O4-CA)3-CS-NH-CH2-PEG). The characteristic absorbance bands of the templates disappeared after the templates were etched by dialysis as shown in Figure 2, indicating that a large proportion of PSS templates were removed. The hollow structure of the biocompatible magnetic multilayer hybrid hollow microspheres (CS/Fe3O4-CA)3-CSNH-CH2-PEG) could be observed with diameter of about 200 3334

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for the design of magnetically guided drug delivery and other in vivo biomagnetic applications.33,34 And the grafting percentage of PEG (mass ratio of the PEG grafted and the (CS/Fe3O4CA)3-CS) could be calculated to be 3.63%. The X-ray diffraction analysis (XRD) patterns of the citratemodified Fe3O4 nanoparticles (Fe3O4-CA), the magnetic multilayer hybrid polyelectrolyte encapsulated PSS microspheres (PSS@(CS/Fe3O4-CA)3-CS-NH-CH2-PEG), and the magnetic hybrid hollow microspheres ((CS/Fe3O4-CA)3-CSNH-CH2-PEG) are shown in Figure 4. It can be seen that their

Figure 5. The typical average hydrodynamic diameter distributions of the (CS/Fe3O4-CA)3-CS (a), PSS@(CS/Fe3O4-CA)3-CS-NCHPEG (b), PSS@(CS/Fe3O4-CA)3-CS-NH-CH2-PEG (c), and (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG (d).

diameters of the polyelectrolyte hybrid hollow microspheres before and after the grafting of PEM by DLS. It could be seen from Figure 6 that the average hydrodynamic diameter (Dh) of

Figure 4. The XRD patterns of the citrate modified Fe 3 O 4 nanoparticles (Fe3O4-CA), the magnetic multilayer hybrid polyelectrolyte encapsulated PSS microspheres (PSS@(CS/Fe3O4-CA)3-CSNH-CH2-PEG), and the magnetic multilayer hybrid polyelectrolyte hollow microspheres ((CS/Fe3O4-CA)3-CS-NH-CH2-PEG).

peaks are identical although the baseline in the range of low 2θ was raised because of the existence of CS in the XRD patterns of the magnetic multilayer hybrid polyelectrolyte encapsulated PSS microspheres (PSS@(CS/Fe3O4-CA)3-CS-NH-CH2-PEG) and the magnetic polyelectrolyte hybrid (CS/Fe3O4-CA)3-CSNH-CH2-PEG hollow microspheres, indicating that the structure of magnetic nanoparticles keeps essentially unchanged during the preparation of the samples.31 All the diffraction peaks are in good agreement with that of the standard Fe3O4 with diffraction peaks of 311, 400, 511, and 440, which indicates a highly crystalline cubic spinel structure.35 pH and Ionic Strength Stimulus-Responsive Properties. The particle sizes of the immediate products and the final hollow microspheres in deionized water were tracked by the dynamic light scattering (DLS) technique. As shown in Figure 5, the average diameter of the PEG modified PSS@(CS/Fe3O4CA)3-CS microspheres via Schiff’s base structure (PSS@(CS/ Fe3O4-CA)3-CS-NCH-PEG) was found to be 347 nm. After reduction the Schiff’s base structure, the average hydrodynamic diameter (Dh) of the stable PEG modified CS/Fe3O4-CA multilayer encapsulated PSS microspheres (PSS@(CS/Fe3O4CA)3-CS-NH-CH2-PEG) decreased slightly to 331 nm. It might be due to the disappearance of the rigid Schiff’s base groups. After removal of the PSS templates, the biocompatible magnetic multilayer hybrid hollow microspheres (CS/Fe3O4CA)3-CS-NH-CH2-PEG) exhibited a Dh of 256 nm. It was higher than that of the magnetic polyelectrolyte hollow microspheres ((CS/Fe3O4-CA)3-CS) of 242 nm, resulted from the grafting of the PEG layer. The effect of the modification with PEG on the pHdependent behavior of the polyelectrolyte hybrid hollow microspheres was investigated by comparing the mean

Figure 6. pH strength dependence of the average hydrodynamic diameter (Dh) and polydispersity index (μ2/Γ2) of the magnetic multilayer polyelectrolyte hybrid hollow microspheres (CS/Fe3O4CA)3 -CS (a, c) and the PEG-modified magnetic multilayer polyelectrolyte hybrid hollow microspheres (CS/Fe3O4-CA)3-CSNH-CH2-PEG (b, d).

the (CS/Fe3O4-CA)3-CS hollow microspheres and the (CS/ Fe3O4-CA)3-CS-NH-CH2-PEG hollow microspheres decreased from 597 and 453 nm to186 and 196 nm with increasing of pH values in the range of 2−11, respectively. In most of the pH range (except for pH 11), the sizes of the (CS/Fe3O4-CA)3-CS hollow microspheres were higher than the corresponding sizes of the (CS/Fe3O4-CA)3-CS-NH-CH2-PEG hollow microspheres, although both of them exhibited the same trend. It indicated that the modification with PEG decreased the swelling ratio and the shrinking of the polyelectrolyte hybrid hollow microspheres in the electrolyte solution. It is well-known that the pKa values of citric acid as a weak acid are 3.13, 4.76, and 6.40, respectively.36 Chitosan is a weak polybase with the pKa value of about 6.5. The charge densities and the citric acid and chitosan are mainly controlled by the pH of solution. At low pH (