Biocompatible Magnetic and Molecular Dual-Targeting Polyelectrolyte

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Biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres for controlled drug release Pengcheng Du, Jin Zeng, Bin Mu, and Peng Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300534a • Publication Date (Web): 18 Mar 2013 Downloaded from http://pubs.acs.org on March 25, 2013

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Molecular Pharmaceutics

Biocompatible magnetic and molecular dualtargeting polyelectrolyte hybrid hollow microspheres for controlled drug release Pengcheng Dua, Jin Zenga, Bin Mub, and Peng Liua,* a

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. b

Center of Eco-material and Green Chemistry, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China *Fax: 86-931-8912582; Tel: 86-931-8912582; E-mail: [email protected]

Abstract: Well-defined biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres have been accomplished via the layer-by-layer (LbL) self-assembly technique. The hybrid shell was fabricated by the electrostatic interaction between the polyelectrolyte cation, chitosan (CS), and the hybrid anion, citrate modified ferroferric oxide nanoparticles (Fe3O4-CA), onto the uniform polystyrene sulfonate microsphere templates. Then the magnetic hybrid core/shell composite particles were modified with a linear, functional

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poly(ethylene glycol) (PEG) mono-terminated with a biotargeting molecule (folic acid (FA)). Afterwards the dual targeting hybrid hollow microspheres were obtained after etching the templates by dialysis. The dual targeting hybrid hollow microspheres exhibit exciting pH response and stability in high salt-concentration media. Their pH-dependent controlled releasing of the drug molecule (anti-cancer drug, doxorubicin (DOX)) was also investigated in different human body fluids. As expected, cell viability of the HepG2 cells decreased more rapidly was treated by the FA modified hybrid hollow microspheres than the unmodified one in vitro study. The dual targeting hybrid hollow microspheres own selective killing of the tumor cells. The precise magnetic and molecular targeting properties and pH-dependent controlled release offers promise for cancer treatment.

Keywords: biocompatible polyelectrolyte hybrid hollow microspheres; magnetic and molecular dual-targeting; pH and ionic strength dual-responsive; layer-by-layer assembly; aggregationresistant

1. Introduction The stimuli-responsive hollow polymeric microspheres have attracted more and more attention due to their variety of potential applications in the encapsulation and drug delivery or controlled release systems, gene delivery, protective shells for cells and enzymes over the last decades.1-4 Various strategies have been developed for the fabrication of the stimuli-responsive hollow polymeric microspheres. Basically, the strategies for fabricating the hollow microspheres mainly included the colloid-templated layer-by-layer (LbL) self-assembly,5,6 core-gel-shell method,7 emulsion interfacial polymerization,8 template-free method,9 and self-assembly of block copolymer,10 and so on. Among them, the LbL self-assembly technique, based on the alternating


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adsorption of the charged species onto an oppositely charged substrate, was more attractive among the strategies abovementioned because of its unique advantages, such as mild preparation conditions and ability to readily tailor their properties (e.g., size, thickness, composition assembly involves the sequential alternate adsorption of charged species onto a sacrificial core via the electrostatic interaction,11 hydrogen-bonding12,13 or covalent bonding14,15 as the driving force). Beside the simple preparation technique, also the choice of the shell components plays a pivotal role in the LbL assembly since it directly influences the biocompatibility and degradability of the hollow microspheres inside living organisms.16 Up to now, many multifunctional synthetic polyelectrolytes, such as poly(styrene sulfonate, sodium salt) (PSS), poly(allylamine hydrochloride) (PAH), poly(methyl methacrylate) (PMAA) and poly(2diisopropylaminoethyl methacrylate) (PDPA), were used for fabricating the multi-response polyelectrolytes capsules.17-19 In comparison, the intracellular biodegradable polymer shows special advantage in the fabrication of the hollow spheres because of the biocompatibility and degradability inside living organisms. Hollow microspheres, made of intracellular biodegradable shell components such as poly(lactic-co-glycolic acid) (PLGA), poly-L-lysine, chitosan (CS) and sodium alginate (SAL), exhibit the most potential biomedical applications related to the delivery of active compounds such as genes, proteins or drugs inside living organisms.20,21 However, especially in drug-delivery systems, many special requirements have to be fulfilled to complete the transfer and controlled release of objects at required speed, at the right moment, in the right place, and at an adequate concentration guided by exterior stimuli, including temperature, ion strength, pH, magnetic field, and so on. Therefore, a considerable effort has been devoted to the development of the multifunctional hollow microspheres.7


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The hollow microspheres can be functionalized to impart optical, magnetic and photothermal properties by introducing the charged inorganic nanoparticles during the LbL self-assembly process via the electrostatic interactions.22-25 The modification of the shells with different types of nanoparticles allows for addressing important functions such as the targeting, the labeling, imaging and the controlled opening of the hollow microspheres. Compared with the nanoparticles, the hollow microspheres modified with the magnetic nanoparticles (such as Fe3O4) show more advantages including magnetic drug targeting, magnetic resonance imaging (MRI), magnetic hyperthermia therapy, targeted gene therapy and biosensors.26-28 Using the LbL selfassembly technique with the magnetic nanoparticle (Fe3O4) as the hybrid shell material, the magnetic composite particles were fabricated by alternate absorbing Fe3O4 nanoparticles and polyelectrolyte, and the multilayer hybrid hollow microspheres were prepared after calcined5 or treated with solvent.29 By incorporating magnetic nanoparticles to fluorescent capsules, capsules with dual-imaging functionalities, magnetic resonance imaging (MRI) and luminescent properties, can be produced as biomarkers in vitro and in vivo.30,31 Additionally, the hollow microspheres modified with magnetic nanoparticles can be externally manipulated using magnetic fields for directing and accumulating hollow spheres to the target region before delivering the chemotherapeutic drugs.32 In a different approach, high-frequency magnetic field (HFMF) has been proven to trigger the release of drugs from the hollow microspheres prepared by loading Fe3O4 nanoparticles into the walls.33 Covalent linkage of poly(ethylene glycol) (PEG) or other hydrophilic polymers to the surface of the drug delivery systems (DDS) have displayed to reduce their non-specific uptake by the cells (including cells from the mononuclear phagocyte system) due to their enhanced low fouling properties.34,35 And the PEG chains own the shield effect which can maintain stability to suppress the increase of the aggregation in high salt concentration environment.36 Meanwhile, the


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biotargeting molecules (folic acid (FA) or antibodies, etc) functionalized hollow microspheres bind specifically to cancer cells expressing the complementary antigen in vitro.37,38 The folate receptor (FR) has been identified as a tumor marker,39 which is expressed at elevated levels on epithelial malignancies, such as ovarian, colorectal, and breast cancer, relative to normal tissue.40-42 Interestingly, folic acid has been found to be a high affinity ligand for the FRs (Kd~ 10-10 mol L-1).40-42 It is also known that the folic acid linked cargos are efficiently bound and internalized by the FR-expressing cells, presumably via receptor-mediated endocytosis.43 In an attempt to develop novel cancer cell specific delivery vehicles, folic acid conjugated nanoparticles is a better choice. In the present work, the well-defined biocompatible polyelectrolyte hybrid hollow microspheres with magnetic and molecular targeting functional were designed and fabricated by the layer-by-layer self-assembly technique with the chitosan and the Fe3O4 nanoparticles as the assembling materials and surface-modification with PEG and targeting molecules (FA) after etching the templates (polystyrene sulfonate (PSS) microspheres) by dialysis with DMF (Scheme 1). The controlled releasing behavior of the obtained dual-targeting polyelectrolyte hybrid hollow microspheres was investigated in vitro by adjusting the different pH value simulation body fluids of the drug-loaded dual-targeting polyelectrolyte hybrid hollow microspheres, with Doxorubicin (DOX) as the model hydrophobic drug.


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LBL assembly of CS and SAL

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Adsorption Fe3O4-CA

PSS Repeating adsorption CS and Fe3O4

GA, NH2-PEG-NH2

Core removal

FA

CS

SAL

PEG

FA

Fe3O4-CA

Scheme 1. Schematic illustration of the fabrication of the biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres. 2. EXPERIMENTAL Materials. CS was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Its degree of deacetylation and molecular weight were determined to be 96 % and 6.0 ×105, respectively. Sodium alginate (viscosity 350 cps for a 1 mg/mL solution) was purchased from Xudong Chem. Co. Ltd. (Beijing, China). Bifunctional PEG (NH2-PEG-NH2) was provided by Beijing Kaizheng Biological Engineering Development Co., Ltd. (Beijing, China). Styrene (St) and methyl acrylic acid (MAA) (Tianjin Chemical Co. Tianjin, China) were distilled under vacuum before use. Ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Folic acid (FA), ammonium persulfate (APS), glutaraldehyde, N,N-Dimethylformade


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(DMF) and other reagents were all of analytical reagent grade from Tianjin Chemical Co. (Tianjin, China) and used without further purification. Deionized was used throughout. Preparation of the biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres. Uniform PS particles. The uniform polystyrene (PS) latices were prepared by the emulsifierfree emulsion polymerization according to the procedure reported previously.44 Styrene (St, 10.0 mL) and methacrylic acid (MAA, 2.0 mL) were added into 95 mL distilled water in a threenecked round-bottom flask equipped with a mechanical stirrer, a condenser, and a gas inlet. After the mixture was deoxygenated by bubbling with nitrogen gas at room temperature for 30 min, the flask was placed in a 72 °C oil bath and stirred mechanically at 300 rpm. A solution of ammonium persulfate (APS, 0.054 g) predissolved in water (5.0 mL) was added to the reaction vessel with vigorous stirring, bubbling with nitrogen (N2). The polymerization was continued for 24 h at 72 °C. After being cooled to room temperature, the product was isolated by being centrifuged and washed with ethanol. A white fine powder (PS latices) was finally obtained after being dried in a vacuum oven at 50 °C. Polystyrene sulfonate microspheres. The uniform PS particles (4.0 g) synthesized as presented above were dispersed in sulfuric acid (80 mL, 98%) with the aid of ultrasonic irradiation. The sulfonation was allowed to take place at 45 °C under magnetic stirring for 6 h. After being cooled to the room temperature, the product was separated by being centrifuged and washed with a large excess of water after being diluted with distilled water. The transformation of the sulfonated PS into sodium polystyrene sulfonate was performed by adding an excess of sodium bicarbonate after being resuspended in water, and then separated by being centrifuged and thoroughly rinsed with water until the neutral pH. The obtained microspheres were finally dispersed and stored in 50 mL of distilled water.


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Citrate modified ferroferric oxide (Fe3O4-CA). Fe3O4 nanoparticles were prepared by the chemical co-precipitation of Fe3+ and Fe2+ ions under a basic condition.45 FeCl3•6H2O (26.0 g) and FeCl2•4H2O (9.56 g) (nFe3+:nFe2+=2:1) were dissolved in 400 mL of deionized water with a mechanical stirring at 75 °C, then 50 mL of ammonia was added. The dispersion was stirred for 30 min upon addition of 50 mL trisodium citrate solution (2.0 M). The dispersion was heated to 85 °C and kept for 1.5 h. N2 was bubbled throughout the reaction. Subsequently, deionized water was added to wash and redisperse the functional ultrafine magnetic particles. Magnetic hybrid core/shell composite particles. The layer-by-layer assembly technique was used to prepare the magnetic multilayer hybrid encapsulated polystyrene sulfonate templates (PSS@PE). The first step in the coating of the PSS particles involved depositing a precursor polyelectrolyte multilayer film (CS/SAL/CS) (PE3) on the PSS templates. The outermost surface layer was cationic polyelectrolyte CS, making the particle surfaces positively charged in all cases. Electrostatic interactions between the negatively charged nanoparticles (citrate modified ferroferric oxide (Fe3O4-CA)) and the polycation CS were utilized to build up the hybrid multilayers. 1.0 g PSS@PE3 particles were dispersed into 400 mL deionized water and added to the aqueous solution containing a fixed amount the Fe3O4-CA at pH 5 under mechanical stirring combining with ultrasonic irradiation for 8 h. Then it was centrifuged and washed three times with water to obtain the multilayer hybrid shell covered PSS templates (PSS@(PE3/Fe3O4-CA)). The composite particles were then coated with CS in 400 mL deionized water at pH around 5 for 8 h with 0.4 g CS added. After being treated by centrifugation and washing three times with water, the Fe3O4-CA and CS alternately deposited four times onto the PSS templates to obtain magnetic hybrid core/shell composite particles. Folate-conjugated and PEG modified magnetic hybrid core/shell composite particles. Bifunctional PEG (NH2-PEG-NH2) has been used to modify the surface of the hybrid core/shell


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composite particles.46 About 0.2 g magnetic hybrid core/shell composite particles were dispersed in 25 mL deionized water, and 1.5 mL 1% glutaraldehyde was added to introduce the functional groups, after the mixture was centrifuged and washed three times with water, 0.050 g NH2-PEGNH2 was used to bring in the amino end-groups after being stirred for 24 h at room temperature, then 5 mL sodium borohydride (2.0 mg/mL) was added to the suspension to reduce the C=N bonds in order to form the stable C-N bonds. About 65 mg (0.15 mmol) FA was dissolved in 2.5 mL dimethylsulfoxide (DMSO). Then, 38 mg N-hydroxysuccinimide (NHS) (0.33 mmol) and 30 mg N-(3-dimethylaminopropyl)-N'ethylcarbodiimide hydrochloride (EDC⋅HCl) (0.17 mmol) were added into the solution to activate the COOH group of FA. The final molar ratio of FA/NHS/EDC was 1:2.2:1.1. Then the solution was added to the suspension. After 24 h, the product was treated by centrifugation and washing several times to remove the free FA. Biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres. After the dual-targeting functional core/shell hybrid microspheres were fabricated, the product was etched with DMF several times, followed centrifugation and washing to obtain the ultimate products, the dual-targeting polyelectrolyte hybrid hollow microspheres. For comparison, the polyelectrolyte hybrid hollow microspheres with neither PEG nor FA were also prepared by the similar process. Cell toxicity assays. Sulforhodamine-B (SRB) assay was performed to evaluate the cytocompatibility of the dual-targeting 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 dual-targeting polyelectrolyte hybrid hollow microspheres, drugloaded hybrid hollow microspheres or drug DOX were added to the cells and incubated for 48 h. Thereafter, the cells were washed three times with phosphate buffered saline (PBS) and


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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 solution (pH 10.5) and then the absorbance was measured at 550 nm using a plate reader. Drug loading. The obtained dual-targeting polyelectrolyte hybrid hollow microspheres (33 mg) were added into the DOX solution (5 mL, 1.0 mg/mL, pH 7). After 48 h, the DOX-loaded hollow microspheres were centrifuged to remove the free excess DOX molecules. Then the drug concentration in the supernatant solution was analyzed using a UV spectrophotometer at a wavelength of maximum absorbance (233 nm) after being diluted. The drug loading capacity of the hollow microspheres was calculated from the drug concentrations in the solutions before and after adsorption. Controlled release of DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres. A 10 mL aqueous solution containing the DOX-loaded hybrid hollow microspheres was transferred into dialysis tubes with a molecular weight cutoff of 14000 and immersed into 140 mL of buffer solution at the four different conditions: pH 1.8 at 25 °C, pH 5.0 at 25 °C and pH 7.4 at 25 °C, respectively. Aliquots (5.0 mL) of the solution were taken at certain time intervals. The solution was further diluted, and then the drug molecule concentration in the dialysate was analyzed for monitoring the 233 nm absorption peak of DOX using UV-vis spectrometry in order to detect the rate of drug releasing. Furthermore, 5.0 mL of fresh solution with the same pH value 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 molecule released through the dialysis membrane over time. Characterizations. Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany) was used for the Fourier transform infrared (FTIR) spectroscopy analysis in the range of 400 cm-1 to


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4000 cm-1 with the resolution of 4 cm-1. The KBr pellet technique was adopted to prepare the sample for recording the IR spectra. The morphologies of the hollow microspheres were characterized with a JEM-1200 EX/S transmission electron microscope. They were dispersed in water in an ultrasonic bath for 5 min, and then deposited on a copper grid covered with a perforated carbon film. The XRD patterns were taken from 20 ° to 80 °by step scanning with a Shimadzu XRD-6000 X-ray diffractometer (Shimadzu Corp., Kyoto, Japan). Nickel-filter Cu Ka radiation (l = 0.15418 nm) was used with a generator voltage of 40 kV and a current of 30 mA. The hysteresis of the magnetization was obtained by changing H between +11,000 and -11,000 Oe at room temperature with vibrating sample magnetometer (Lakeshore 7304). The Zeta potentials of the CS/SAL and CS/Fe3O4-CA multilayer coated PSS microspheres were determined with Zetasizer Nano ZS (Malvern Instruments Ltd, UK). The mean particle size and size distributions of the multilayer hybrid hollow microspheres were determined by the dynamical mode (dynamic light scattering (DLS)) on the ‘Light Scattering System BI-200SM, Brookhaven Instruments’ device equipped with the BI-200SM goniometer, the BI-9000AT correlator, temperature controller and the Coherent INOVA 70C argon-ion laser at 20 ºC. DLS measurements are performed using 135 mW intense laser excitation at 514.5 nm and at a detection angle of 90º using the emulsion directly at 25 ºC. Particle size distribution is calculated using the Brookhaven Instruments Particle sizing software. The loading of DOX onto the magnetic multilayer hybrid hollow microspheres and their controlled releasing behavior as well as the UV-vis spectra of the magnetic multilayer hybrid hollow microspheres were detected by TU-1901 UV/vis Spectrometer (Beijing Purkinje General Instrument Co. Ltd, Beijing, China) at room temperature. 3. RESULTS AND DISCUSSION


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The coprecipitation method was used to prepare the citrate (CA) modified Fe3O4 nanoparticles (Fe3O4-CA). The citrate surface modification of the magnetic nanoparticles could improve their dispersion and stability in water. Most importantly, it renders the negative charge on the nanoparticles surface, which was used as the hybrid anion in the LbL self-assembly process. The transmission electron microscope (TEM) image of the Fe3O4-CA nanoparticles is shown in Figure 1 (a). It shows that most of the Fe3O4-CA nanoparticles are quasi-spherical with an average diameter near 10 nm.

(a)

(d)

(b)

(e)


(c)

(f) 12

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(g)

(h)

Figure 1. TEM micrographs of (a) the citrate modified ferroferric oxide (Fe3O4-CA), (b) the PSS templates, (c) the PSS templates coated with PE3, (d) the magnetic hybrid core/shell composite microspheres, (e) the magnetic polyelectrolyte hybrid hollow microspheres, (f and g) the dualtargeting polyelectrolyte hybrid hollow microspheres and SEM micrograph of (h) the dualtargeting polyelectrolyte hybrid hollow microspheres. The PS template was prepared by the emulsifier-free emulsion polymerization of Styrene (St) with 2% surfmer methacrylic acid (MAA) added, and then treated with concentrated sulfuric acid. The sulfonic acid groups were introduced onto the surface of the PS templates, and then the surface anion richer PSS templates were used to adsorb the polycation chitosan (CS). The morphology of the PSS particles is described in Figure 1 (b). The microspheres are spherical in shape and monodisperse in size, with the diameter of 427.6 nm. The layer-by-layer self-assembly technique was used to fabricate the magnetic core/shell multilayer hybrid microspheres encapsulated via the alternately adsorption of the cationic polyelectrolyte (CS), the anionic polyelectrolyte alginate (SAL), and the hybrid anionic Fe3O4CA nanoparticles onto the PSS templates via the electrostatic interaction. The PSS templates


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were encapsulated by altering adsorption of cationic polyelectrolyte (CS) and anion polyelectrolyte (SAL) to prepare the precursor three-layer polyelectrolyte shells (PE3) with an outermost CS layer. After these steps, the Fe3O4-CA nanoparticles were adsorbed onto the modified PSS particles, by the electrostatic interaction between the amino groups of CS and the carboxyl groups of the hybrid Fe3O4-CA nanoparticles. Repeating this cycle for four times, the magnetic hybrid core/shell composite particles were obtained with the outermost CS layer for further modifications. The zeta potentials of the magnetic hybrid core/shell composite particles were conducted to track the polyelectrolyte multilayer and hybrid nanoparticles growth as shown in Figure 2. The odd layer numbers correspond to the CS deposition and the even layer numbers to the deposition of the Fe3O4-CA, except the second layer (SAL). The zeta potential was approximately +9 mv when the first layer of chitosan was deposited on the PSS template and -10.6 mv for the second layer of SAL. However, the zeta potential became negative charge of -4.36 when the last Fe3O4CA layer was completed. The zeta potential can demonstrate the altering adsorption of the polyelectrolyte and the hybrid Fe3O4-CA nanoparticles onto the surface of the PSS templates.

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CS

10

Zeta potential(mv)

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CS

CS

CS

CS

CS

5 0 PEG-Hybrid

-5

Microspheres Fe O -CA 3 4 Fe O -CA 3 4 Fe O -CA Fe O -CA 3 4 3 4

-10 -15 -20

SAL

Folate-conjugated Hybrid Microspheres GA-Hybrid Microspheres

PSS

-25 0

2

4

6

8

10

12

14

Layer number


14

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Figure 2. Zeta potentials during the fabrication of the magnetic hybrid core/shell composite particles. In order to improve the biocompatibility and molecular target function of the polyelectrolyte hybrid hollow microspheres, the functional NH2-PEG-NH2 was conjugated on the surfaces of the obtained magnetic hybrid core/shell composite particles. After the functional target molecule (FA) was successful coupled to the surface of the magnetic hybrid core/shell composite particles, the dual-targeting polyelectrolyte hybrid particles were treated with DMF to etch the inner PS template cores, then the product became yellow compared to the original color black. Finally the biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres were obtained. The IR spectrum of the functional PEG modified magnetic hybrid core/shell composite particles, FA and PEG modified magnetic hybrid core/shell composite particles, and the dualtargeting polyelectrolyte hybrid hollow microspheres (as shown in Figure 3) reveal that the welldefined characteristic absorbance band of PEG at 1112 cm-1 (νC-O-C). The absorbance peaks at 1712 cm-1 and 1606 cm-1 assigned to the vibration of the -COOH group and the benzene ring of FA, respectively. Therefore, the FTIR results identify that PEG and FA had been immobilized on the hollow microspheres successfully. However, the characteristic signals at 700 cm-1 of the phenyl group remained partly (Figure 3 d), might be ascribed to the sulfonated copolymer of styrene and AA adsorbed onto the surface of the Fe3O4 nanoparticles via the coordination bonds. The immobilized amount of FA was further quantified by UV-vis absorption spectroscopy (Figure 4). By subtracting the UV absorbance of the PEG modified hybrid polyelectrolyte hollow microspheres from the dual-targeting polyelectrolyte hybrid hollow microspheres, the grafting density of FA on the hollow microspheres surface were found to be 4.74 mmol/g.


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Transmittance(%)

120 100 80 60 40 20 0 4000 3500 3000 2500 2000 1500 1000

500

Wavenumber(cm-1)

Figure 3. The FT-IR spectra (up to down) of the magnetic hybrid core/shell composite particles, the PEG modified magnetic hybrid core/shell composite particles, the FA-terminated PEG modified magnetic hybrid core/shell composite particles, and the dual-targeting polyelectrolyte hybrid hollow microspheres.

1.2 1.0

Absorbance

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0.8 0.6 0.4

d c b a

0.2 0.0 200

300

400

500

600

700

800

Wavelength(nm)

Figure 4. UV-vis spectra of the polyelectrolyte hybrid hollow microspheres (a), the PEG modified polyelectrolyte hybrid hollow microspheres (b), the dual-targeting polyelectrolyte hybrid hollow microspheres (c) and folic acid (FA) (d).


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Additionally, the zeta-potential shown in Figure 2 decreased after the magnetic hybrid core/shell composite microspheres treated with glutaraldehyde (GA) due to the amino groups reacted with the GA. Afterwards, the zeta-potential increased after the modification with the amino functional PEG. At last, the zeta-potential decreased following the folic acid conjugation due to the reaction of folic acid with the amino groups (–NH2) of the magnetic hybrid core/shell composite microspheres thereby reducing the positive surface charge. The change of the zetapotential also can prove the PEG and FA groups immobilized on the microspheres successfully TEM was employed to follow the assembly of the Fe3O4 nanoparticle/polyelectrolyte multilayers on the PS templates. The uncoated template particles had smooth surface, and the templates with three layer polyelectrolyte were also smooth but their color changed to lighter in the edge shown in Figure 1 (c), which might be resulted from the polyelectrolyte three-layers. However, the surface of the magnetic hybrid particles became rough with the uniform Fe3O4 nanoparticles adsorbed onto their surface (Figure 1 (d)). The typical absorbance band of the FeO stretching vibration of the magnetic hybrid core/shell particles at 580 cm-1 can also confirm that the Fe3O4-CA nanoparticles had been coated successfully (Figure 3). Figure 1 (f, g) shows TEM images of the dual-targeting polyelectrolyte hybrid hollow microspheres obtained after etching the templates. The hollow structure of the dual-targeting polyelectrolyte hybrid hollow microspheres was uniform and well-defined with the diameter of 457.5 nm. However, the inner diameter of the functional hybrid microcapsules was 353.6 nm, which was obvious smaller than the corresponding magnetic hybrid core/shell composite particles before the removal of the template cores. It was resulted from the shrinking of the hybrid polyelectrolyte multilayer shells in some degree during the gradual drying process.47 The SEM image (Figure 1 h) confirmed that the dual-targeting polyelectrolyte hybrid hollow microspheres were uniform spheres and remained well-formed and unbroken. However, some


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hollow microspheres collapsed and creased due to the drying process. The TEM image of the polyelectrolyte hybrid hollow microspheres without PEG and FA is shown in Figure 1 e. Their morphology was also sphere not as regular as the dual-targeting polyelectrolyte hybrid hollow microspheres (Figure 1 f), which could be ascribed to the fact that glutaraldehyde played the role of crosslinking agent and the hollow mcirospheres were crosslinked partially. Otherwise the FAterminated PEG chains immobilized on the outermost layer can also protect the polyelectrolyte hybrid hollow microspheres from aggregation.36

(311) (440) (220)

(511) (422)

(400)

a

b

30

40

50

60

70

80

2θ(degree)

Figure 5. The XRD patterns of (a) the citrate modified Fe3O4 nanoparticles (Fe3O4-CA) and (b) the dual-targeting hybrid hollow microspheres. The X-ray diffraction analysis (XRD) patterns of the Fe3O4-CA and the dual-targeting functional polyelectrolyte hybrid hollow microspheres are presented in Figure 5. The diffraction patterns of Fe3O4 within the dual-targeting polyelectrolyte hybrid hollow microspheres were similar as those of the Fe3O4-CA with spinel structure, which also had six diffraction peaks assignable to (220), (311), (400), (422), (511), and (440).48,49 It is to say that the spinel structure Fe3O4 nanocrystals did not change throughout the preparation process.


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80

15

60

a b

10

c

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M(emu/g)

20 0

0

-20

-5

-40 -10

-60 -80 -15000 -10000 -5000

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H(mT)

10

d

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0

-5

-10 -15000 -10000 -5000

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H(mT)

Figure 6. The magnetic hysteresis loops of the (a) the Fe3O4-CA, (b) the magnetic hybrid core/shell composite particles, (c) the PEG modified magnetic hybrid core/shell composite particles, and (d) the dual-targeting polyelectrolyte hybrid hollow microspheres. Magnetic measurements were performed to confirm the magnetic nature of the magnetic Fe3O4 nanoparticles in the shells of the dual-targeting polyelectrolyte hybrid hollow microspheres as well as to determine the saturation magnetization of the phase created. The magnetic hysteresis loops for the CA modified Fe3O4, the magnetic hybrid core/shell composite particles, the PEG modified magnetic hybrid core/shell composite particles, and the dual-targeting polyelectrolyte


19


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hybrid hollow microspheres are shown in Figure 6. A superparamagnetic behavior with zero remanence and coercivity was observed.50,51 As expected, the saturation magnetization of the magnetic hybrid core/shell composite particles, the PEG modified magnetic hybrid core/shell composite particles, and the dual-targeting polyelectrolyte hybrid hollow microspheres was 9.60, 6.07 and 9.46 emu/g respectively, due to the nonmagnetic PSS templates, polyelectrolytes, and PEG. It should be noted that the saturation magnetization measured for the dual-targeting polyelectrolyte hybrid hollow microspheres had a magnetic content of 55.85%, larger than that of the PEG modified magnetic hybrid core/shell composite particles, ascribing to the etching of the PSS templates from the core/shell composite particles with DMF. And the different steps, the decrease in the saturation magnetization of the PEG modified magnetic hybrid particles compared with the magnetic hybrid core/shell composite particles implied that the PEG outmost layer was successfully coated onto the surface of the magnetic hybrid core/shell composite particles. The weight content of the Fe3O4 in the final hollow microspheres is about 14.76%, calculated by the VSM shown in Figure 6.

(a)

(b)

Figure 7. Photographic images of (a) the magnetic polyelectrolyte hybrid hollow microspheres and (b) the dual-targeting polyelectrolyte hybrid hollow microspheres.


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Both the magnetic polyelectrolyte hybrid hollow microspheres (Figure 7 a) and the dualtargeting polyelectrolyte hybrid hollow microspheres (Figure 7 b) show the magnetic-separation and redispersion in distilled water. In the absence of an external magnetic field, a dark yellow and homogeneous dispersion exists. When an external magnetic field was applied, both the magnetic polyelectrolyte hybrid hollow microspheres and the dual-targeting polyelectrolyte hybrid hollow microspheres were enriched and the dispersion became clear and transparent. The influence of the medium pH values on the average hydrodynamic diameters of the magnetic polyelectrolyte hybrid hollow microspheres and the dual-targeting polyelectrolyte hybrid hollow microspheres was investigated by dynamic light scattering (DLS) after the hollow microspheres had been immersed into the aqueous solutions with different pH values for 24 h. As shown in Figure 8, the hydrodynamic diameter of the magnetic polyelectrolyte hybrid hollow microspheres decreased with the pH value increased from 2 to 9, and the trend of change in size showed a parabola shape with pH variation and a small increase from 10 to 11. At low pH, the ionization of carboxyl groups was normally depressed; it is to say that less than one negative charge was carried by one citrate. At pH 2, CS was fully ionized and became -NH3+ groups,52 and the ionization of the amine groups in CS decreased greatly when the medium pH increased. The size of the polyelectrolyte hybrid hollow microspheres decreased with increasing the pH values because of the shrinkage of the polyelectrolytes hybrid shells. However, a marked increase in the hydrodynamic diameter was observed when the medium pH was in the range of 9-11, which could be attributed to the disintegration of the polyelectrolytes hybrid shells. At high pH (>9), the free CS chains entangled each other, which led to the increase in the size of the hollow microspheres.53


21


560

a 520

Diameter(nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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480

b

440

400

2

4

6

8

10

12

pH

Figure 8. pH dependence of average hydrodynamic diameter of (a) the magnetic polyelectrolyte hybrid hollow microspheres and (b) the dual-targeting polyelectrolyte hybrid hollow microspheres in various pH media. Compared to the magnetic polyelectrolyte hybrid hollow microspheres, the dual-targeting polyelectrolyte hybrid hollow microspheres present the similar pH responsive properties with the range of pH value from 2 to 11. However, the obvious decrease in the hydrodynamic diameter was observed when the medium pH was 2.0, the hydrodynamic diameter was investigated to be only 500 nm, which was obvious smaller than the magnetic polyelectrolyte hybrid hollow microspheres, at the same time the hydrodynamic diameter was 400 nm at pH 9, obviously larger than the magnetic polyelectrolyte hybrid hollow microspheres. In the preparation process, the functional PEG, which was introduced onto the surface of the dual-targeting polyelectrolyte hybrid hollow microspheres, enlarged the hydrodynamic diameter of the hollow spheres. Meanwhile, the PEG outmost layer maintains a stable state and avoids the aggregation of the hollow microspheres in high ionic strength media.36 On the other hand, the polyelectrolytes CS shell might also slightly be crosslinked by glutaraldehyde and the crosslinked shell partly limited the shrinking of the hollow microspheres.


22

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The influence of ionic strength on the average hydrodynamic diameters of the magnetic polyelectrolyte hybrid hollow microspheres and the dual-targeting polyelectrolyte hybrid hollow microspheres was also studied by DLS technique (Figure 9). It is found that introducing small molecule electrolytes (e.g., NaCl) has significant influence on the size of the obtained magnetic polyelectrolyte hybrid hollow microspheres, and the diameter of the hollow microspheres increases from 408 to 629 nm with increasing of the ionic strength from the range of 0 ~ 0.20 mol/L NaCl. In the range of 0 ~ 0.20 mol/L NaCl, the scattered intensity exhibits no appreciable changes. It means that the hollow microspheres are stable in the media with NaCl concentration in the range of 0 ~ 0.20 mol/L. This further confirms that the PEG layer can prevent the hollow microspheres from aggregation and fusion in high salt concentration media. 700

350

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50

0.20

CNaCl(mol/L)

Figure 9. Ionic strength dependence of average hydrodynamic diameter (a) and scattered intensity (b) of the magnetic polyelectrolyte hybrid hollow microspheres (a1, b1) and the dualtargeting polyelectrolyte hybrid hollow microspheres (a2, b2) obtained in various aqueous at 25 ºC.


23


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It is well known that the ionic strength of solution depends on the concentrations of polyelectrolyte and small electrolyte molecules. The small molecule electrolytes can weaken the electrostatic repulsion and the salt bond between the hybrid anion Fe3O4-CA and CS among the shells of the magnetic polyelectrolyte hybrid hollow microspheres. Hence, the chain of the polyelectrolytes could be stretched, and the size of the magnetic polyelectrolyte hybrid hollow microspheres would be increased because that salt usually had a shielding effect on the electrostatic force.29,54 On the contrary, the covalent linkage of PEG on the surface of the hollow microspheres can contract and wind to the skin of the hollow microspheres, the PEG outermost layer can remain stable and reduce expansion of the hollow microspheres. Furthermore the PEG layer also can prevent the hollow microspheres from aggregation and fusion in high salt concentration media.36 The in vitro toxicity of the dual-targeting polyelectrolyte hybrid hollow microspheres was evaluated in HepG2 cells using SRB assays. The cells were incubated with the dual-targeting polyelectrolyte hybrid hollow microsphres for 48 h at varying concentrations from 0 ~ 200 µg/mL. The results revealed that the dual-targeting polyelectrolyte hybrid hollow microspheres were practically non-toxic (cell viability = 99.7 ~ 102.9 %) up to a tested concentration of 100 µg/mL (Figure 10), indicating that the dual-targeting polyelectrolyte hybrid hollow microspheres have excellent biocompatibility on HepG2 cells in these concentration. However, when the concentration of the hollow microspheres was increased to 200 µg/mL, the viability after 48 h of incubation decreased to 46.5%.


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120 a b

Cell viability(%)

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Concentration( µg/mL)

Figure 10. Cell viability data of (a) the dual-targeting polyelectrolyte hybrid hollow microspheres and (b) the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres.

100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40

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Dox dosage (µg/mL)

Figure 11. Anti-tumor activity of the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres as a function of the DOX concentrations. HepG2 cells were incubated with the DOX-loaded hybrid hollow microspheres modified with PEG (a), the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres (b), or free DOX (c) for 48 h. The in vitro cytotoxicity of the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres was also investigated in HepG2 cells by SRB assays. The results showed that the


25


DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres showed pronounced cytotoxic effects (Figure 10). To evaluate the folate-mediated targeting function of the dualtargeting polyelectrolyte hybrid hollow microspheres, the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres and the DOX-loaded hybrid hollow microspheres modified with PEG were used for the in vitro study with HepG2 cells. Introduction of the DOX causes a reduction in cell viability with increasing the DOX concentration, significantly reduced cell viabilities were observed as shown in Figure 11, the order of efficacy as a killing agent is the free DOX, then the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres and finally the DOX-loaded hybrid hollow microspheres modified with PEG. At the same time, the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres show obvious cell inhibition than the DOX-loaded hybrid hollow microspheres modified with PEG which are known as folate receptor positive tumor cells. The result from the cell viabilities in the targeting study indicates that the cell inhibition is mainly caused by cell internalization of the hollow microspheres through folate-mediated targeting.55,56 Furthermore, it also proved that the surface modification with PEG decreased the non-specific cell adhesion.

Drug release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 12. In vitro drug release from the DOX-loaded dual-targeting polyelectrolyte hybrid microcapsules at pH 7.4, pH 5.0 and pH 1.8 at 25 ºC, respectively. The in vitro drug release from the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres was performed at 25 ºC under three different simulated body fluid conditions, i.e. pH 7.4, pH 5.0 and pH 1.8. According to the UV-vis result, the drug-loaded capacity of the dualtargeting polyelectrolyte hybrid hollow microspheres was calculated to be 21.9%. The results showed the sustained release of DOX over a period of 48 h (Figure 12). No burst release was observed at these three media. It is interesting to note that the release of DOX from the DOXloaded dual-targeting polyelectrolyte hybrid hollow microspheres was significantly faster at pH 1.8 and 5.0 than at pH 7.4. For example, approximately 69.8 %, 60.8% and 35.8% DOX was released in 12 h, while 99.3%, 81.6% and 46.6% in 48 h from the DOX-loaded dual-targeting polyelectrolyte hybrid hollow microspheres at pH 1.8, 5.0 and 7.4, respectively. The phenomenon of pH-dependent release of DOX was observed that the releasing ratio and the cumulative release of DOX from the drug carriers at pH 1.8 and pH 5.0 were quicker and higher than at pH 7.4. The results of the DOX cumulative release at different pH media revealed that the release at low pH could partly be attributed to the fact that the polyelectrolyte hybrid shell was highly permeable at low pH but not at high pH, because that the decreasing of pH weakened the ionic cross-linking salt bonds between CS and Fe3O4-CA due to the decrease in the charge density of citric acid and the cross-linking density. Furthermore, the polyelectrolyte hybrid shells were positively charged at low pH, the shells and DOX molecules likely charged the same sign because of the decrease in charge density of citric acid and the protonation of CS, which might make such a configuration unstable so that the water-soluble DOX molecules were impelled across the polyelectrolyte shells. On the other hand, the hollow microspheres shrank in


27


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Page 28 of 37

acid media, and the loading space of the hollow microspheres will be compressed. The pHdependent release of DOX from the dual-targeting polyelectrolyte hybrid hollow microspheres has a great advantage in curing the cancer cells with an acid medium about pH at 5.57,58 4. CONCLUSIONS Biocompatible polyelectrolyte hybrid hollow microspheres with magnetic and molecular dualtargeting were successfully prepared via the combination of layer-by-layer assembly techniques and modification with functional PEG and targeting molecules (FA) on their surfaces. The dualtargeting polyelectrolyte hybrid hollow microspheres present excellent pH responsiveness and salt concentration responsiveness. The PEG outmost layer maintains a stable state and avoids the aggregation of the hollow microspheres in high ionic strength media. The DOX-loaded dualtargeting polyelectrolyte hybrid hollow microspheres showed pronounced cytotoxic effects and pH-dependent

releasing

performance.

Their

magnetic

and

molecular

dual-targeting

characteristics were expected to use in targeted drug treatment for cancer treatment as excellent and smart drug carriers. Acknowledgments. This project was granted financial support from the National Nature Science Foundation of China (Grant No. 20904017), the Program for New Century Excellent Talents in University (Grant No. NCET-09-0441), and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2012-k12). References (1) Wang, Y. J.; Bansal, V.; Zelikin, A. N.; Caruso, F. Templated synthesis of singlecomponent polymer capsules and their application in drug delivery. Nano. Lett. 2008, 8, 1741.


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For Table of Contents Use Only

Biocompatible magnetic and molecular dual-targeting polyelectrolyte hybrid hollow microspheres for controlled drug release Pengcheng Dua, Jin Zenga, Bin Mub, and Peng Liua,*


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Biocompatible Magnetic and Molecular Dual-Targeting Polyelectrolyte

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