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Novel biocompatible pH-stimuli responsive superparamagnetic hybrid hollow microspheres as tumor-specific drug delivery system
Accepted Manuscript Title: Novel biocompatible pH-stimuli responsive superparamagnetic hybrid hollow microspheres as tumor-specific drug delivery system Author: Xiaorui Li Pengcheng Du Peng Liu PII: DOI: Reference:
S0927-7765(14)00347-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.06.054 COLSUB 6500
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
17-2-2014 22-6-2014 24-6-2014
Please cite this article as: X. Li, P. Du, P. Liu, Novel biocompatible pHstimuli responsive superparamagnetic hybrid hollow microspheres as tumorspecific drug delivery system, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.06.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Novel biocompatible pH-stimuli responsive superparamagnetic
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hybrid hollow microspheres as tumor-specific drug delivery system
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Xiaorui Li, Pengcheng Du, Peng Liu*
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State Key Laboratory of Applied Organic Chemistry and Key Laboratory of
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Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of
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Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
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Abstract
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Novel biocompatible pH-stimuli responsive superparamagnetic hybrid hollow
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microspheres have been designed via the layer-by-layer (LBL) self-assembly
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technique via the electrostatic interaction between the poly(ethylene glycol) grafted
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chitosan (CS-g-PEG) as polycation and the citrate modified ferroferric oxide
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nanoparticles (Fe3O4-CA) as hybrid anion onto the uniform polystyrene sulfonate
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(PSS) microsphere templates. The well-defined hybrid hollow microspheres
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((CS-g-PEG/Fe3O4-CA)4/CS-g-PEG) were obtained after etching the templates by
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washing with DMF. They possessed superparamagnetic characteristics with a saturation magnetization of 37.23 emu/g, and exhibited excellent stability in high ion-strength media and pH dependent DOX release. Their unique structure and outstanding performance make them potential platform for tumor-specific delivery in
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the tumor diagnostic and therapy.
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Keywords: Drug delivery system; pH dependent release; superparamagnetic; hollow
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microspheres; LBL self-assembly
*
Corresponding author. Fax: 86 0931 8912582; Tel: 86 0931 8912582; E-mail: [email protected]. 1
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1. Introduction
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The stimuli-responsive hollow microspheres have attracted more and more attention
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because of their variety of potential applications such as drug carriers, biomedicine
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and enzyme transplantation, gene therapy, and contrast agents in diagnostics [1-3]. By
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now, various physical and chemical methods have been developed to prepare the
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stimuli-responsive hollow microspheres. Among these methods, the layer-by-layer
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(LbL) self-assembly of the oppositely charged polyelectrolytes (PEs) onto the
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sacrificial templates has been widely used due to its unique advantages (e.g., size,
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thickness, composition, porosity, stability, surface functionality, tunable permeability)
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[4,5]. In most of the LbL engineered multilayer hollow microspheres, two kinds of the
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PEs of opposite charge have been used as the raw materials via the electrostatic
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interaction.
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Chitosan (CS), a non-toxic, biocompatible and biodegradable polysaccharide and
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cationic polyelectrolyte, has shown some favorable bioactivities, such as anti-bacterial,
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immunological and wound healing activity [6-8]. It has been widely used for biomedical applications, especially for the delivery and control release of drugs, genes, and vaccines [9-12]. However, its poor solubility in water and some organic solvents limits its wide application. Many chitosan derivatives have been prepared by chemical
40
modification to overcome this disadvantage and generate new biomaterials [13],
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among which chemical modification of chitosan with poly(ethylene glycol) (PEG) is
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considered to be a convenient way to improve its water solubility and
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biocompatibility [14,15]. PEG has been widely used in modification of a variety of 2
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materials because of its excellent physicochemical and biological properties,
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including hydrophilicity, flexibility, lack of toxicity, ease of chemical modification,
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absence of immunogenicity and antigenicity, biocompatibility and steric repulsion
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[16,17]. Especially for the multilayer hollow microspheres fabricated with the
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non-covalent bonds such as electrostatic interaction or hydrogen bond, the surface
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PEGylation could efficiently prevent them from aggregation or fusion in high
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ion-strength media [18]. Furthermore, many reports have been published on the water
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solubility and bioactivity of CS-g-PEG and the results show that the grafting of PEG
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onto chitosan not only improves water solubility and biocompatibility of chitosan, but
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also improve the bioavailability of drugs in vivo [19,20].
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The hollow microspheres modified with the magnetic nanoparticles show a lot of
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advantages including magnetic-targeting and magnetic hyperthermia therapy, or
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application in magnetic resonance imaging and biosensors [21]. The magnetic
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nanoparticles could be used as the hybrid anion to prepare the multilayer
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superparamagnetic hybrid hollow microspheres via the LbL self-assembly technique, after surface modification with citric acid [22-24]. The magnetic hollow microspheres can be widely used in magnetic fields for directing and accumulating hollow microspheres to the target sites before releasing the chemotherapeutic drugs,
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achieving the magnetic targeting function [25]. With such a magnetic-sensitive shell
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structure, active substances can be well-regulated in a manageable manner with a
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designable profile according to the time duration under high frequency magnetic field
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[26]. 3
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In the present work, the well-defined biocompatible pH-stimuli responsive
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superparamagnetic hybrid hollow microspheres have been accomplished via the
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layer-by-layer (LBL) self-assembly technique with the poly(ethylene glycol) grafted
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chitosan (CS-g-PEG) and the citrate modified ferroferric oxide nanoparticles
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(Fe3O4-CA) as the assembling materials, after etching the templates (polystyrene
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sulfonate microspheres (PSS)) by washing with DMF (Scheme 1). The controlled
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releasing behavior was investigated in vitro in simulated body fluids with different pH
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values, with doxorubicin (DOX) as model hydrophobic drug.
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2. Experimental
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2.1. Materials and reagents
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Chitosan (CS) was obtained from Golden-Shell Biochemical Co. Ltd., Zhejiang,
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China with degree of deacetylation and viscosity-average molecular weight of 96%
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and 6.0×105, respectively. Aldehyde-terminated poly(ethylene glycol)
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(CH3O-PEG-CHO, Mn=2000) was purchased from Taiyuan Rongyuan Co. Ltd., Taiyuan, China.
Styrene (St) and methacrylic acid (MAA) (Tianjin Chemical Co. Ltd., Tianjin, China)
were distilled under vacuum before use. Ferric chloride hexahydrate (FeCl3·6H2O)
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and ferrous chloride tetrahydrate (FeCl2·4H2O) were purchased from Sinopharm
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Chemical Reagent Co., Ltd. Shanghai, China. Sodium citrate (Na3C6H5O7·2H2O) was
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an analytical grade reagent from Shanghai Chemical Co. Ltd., Shanghai, China.
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Sodium cyanoborohydride (NaCNBH3) was obtained from the Sigma Chemical Co. 4
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(St. Louis, MO, USA). Ammonium persulfate (APS), N,N-dimethylformamide
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(DMF), and other reagents were all of analytical reagent grade from Tianjin Chemical
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Co. Ltd., Tianjin, China and used without further purification. Deionized water was
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used throughout.
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2.2. Uniform polystyrene (PS) particles
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The uniform polystyrene (PS) particles were prepared via the emulsifier-free emulsion
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polymerization according to the procedure as reported previously [22]. Styrene (St,
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10.0 mL) and methacrylic acid (MAA, 2.0 mL) were added to 95 mL distilled water
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in a three-necked round bottom flask fitted with a condenser and a magnetic stirred
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and purged with nitrogen. A solution of ammonium persulfate (APS, 0.054 g)
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pre-dissolved in water (5 mL) was added to the reaction vessel with vigorous stirring
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and bubbling with nitrogen. The polymerization was continued for 24 h at 72 °C.
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After cooling to room temperature, the product was purified by repeating
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centrifugation (10000 rpm for 10 min) and washing with ethanol. The white fine powder was finally obtained after being dried in a vacuum oven at 50 °C.
2.3. Polystyrene sulfonate (PSS) microspheres
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4.0 g PS particles were dispersed in 80 mL 98% sulfuric acid with ultrasonication and
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the dispersion was stirred at 45 °C for 8 h. After being cooled to the room temperature,
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the product was separated by repeating centrifugation and washing with a large excess
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of water after being diluted by distilled water. The transformation of the polystyrene 5
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sulfonic acid into the sodium polystyrene sulfonate was performed by adding an
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excess of sodium bicarbonate after being re-dispersed in water and then separated by
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repeating centrifugation (10000 rpm for 10 min) and thoroughly rinsing with water
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until neutral. The sulfonate percentage was measured to be about 0.8 mmol/g by the
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sulfur element analysis method. The obtained PSS microspheres were finally
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dispersed and stored in 100 mL distilled water.
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2.4. PEG grafted chitosan (CS-g-PEG)
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The poly(ethylene glycol) grafted chitosan (CS-g-PEG) was synthesized according to
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the method described by Harris [27]. 1.0 g CS was dissolved in a mixture of aqueous
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acetic acid (2.0 wt%, 20 mL) and methanol (10 mL), to which 10 ml aqueous solution
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of the aldehyde-terminated poly(ethylene glycol) (CH3O-PEG-CHO, Mn=2000)
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(0.745 g) was added subsequently. The mixture was stirred for 24 h at room
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temperature, and its pH was adjusted to 6.0-6.5 with aqueous 1mol/L NaOH solution.
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After stirring for another 2 h at room temperature, 10 mL of aqueous NaCNBH3 (0.2342 g) solution was added drop-by-drop in 30 min. To complete the reaction, the solution was stirred for another 24 h at room temperature under nitrogen protection. The mixture was dialyzed against distilled water for 1 week, with water-replacement
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every 5 h. The remained APEG was removed by freezing or air drying in combination
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with acetone (100 mL) washing process. The final copolymer CS-g-PEG was
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obtained after evaporating the residual solvent in vacuum.
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2.5. Citrate modified ferroferric oxide (Fe3O4-CA)
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The co-precipitation method was used to prepare the citrate modified ferroferric oxide
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[22]. The FeCl3·6H2O (26.0 g) and FeCl2·4H2O (9.56 g) (Fe3+ : Fe2+ = 2:1) were
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dissolved in distilled water (400 mL) under nitrogen atmosphere with mechanical
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stirring. As the solution was heated to 70 °C, NH3·H2O (28 wt%, 50 mL) was added
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dropwise to the mixture under vigorous stirring, then 48 mL of 2 mol/L sodium citrate
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was quickly added, and the mixture was heated to 85°C and kept for 1.5 h.
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Subsequently, the ultimate citrate modified Fe3O4 nanoparticles (Fe3O4-CA) were
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washed more than three times with distilled water to discard the excessive sodium
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citrate by the magnetic separation procedure. Finally, the Fe3O4-CA nanoparticles
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were dispersed and stored in 100 mL distilled water.
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2.6. PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres
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The LbL assembly technique was used to prepare the multilayer hybrid shell
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encapsulated polystyrene sulfonate templates (PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG) with the positively charged CS-g-PEG and the Fe3O4-CA hybrid anion via electrostatic interaction. The adsorption of the CS-g-PEG was completed in a solution of 400 mL deionized water containing 1.0 g
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sodium polystyrene sulfonate (PSS) templates and 0.25 g CS-g-PEG, at pH around 5
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for 8 h followed by centrifugation (10000 rpm for 10 min) and washing three times in
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water to obtain the PSS@CS-g-PEG. Then the PSS@CS-g-PEG was dispersed into
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400 mL deionized water and added to the solution containing the Fe3O4-CA in 7
Page 7 of 38
batches until reaching the adsorption balance at pH 8.5 under mechanical stirring
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combining with ultrasonic. Then the microspheres were centrifuged (10000 rpm for
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10 min) and washed three times in water to obtain the core-shell hybrid microspheres
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covered with one bilayer (PSS@(CS-g-PEG/Fe3O4-CA)1). The CS-g-PEG and
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Fe3O4-CA were alternately deposited for another four and three times onto the PSS
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templates to obtain the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
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microspheres.
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2.7. (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres
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The sacrificial templates (PSS) in the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4-
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/CS-g-PEG microspheres were removed by washing with DMF: 1.0 g core-shell
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PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres were dispersed into 20 mL
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DMF. After the dispersion was stirred for 4 h, the microspheres were magnetically
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separated and dispersed into 20 mL new DMF. The washing with DMF was repeated
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for 4 times, followed with excess water for three times and centrifugation (10000 rpm for 10 min) to obtain the ultimate products, the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
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2.8. Drug loading
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The drug loading was carried out by dispersing the
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(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres in DOX solution
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with certain concentration. A typical procedure was as follows: 10 mg of the 8
Page 8 of 38
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres were added into the
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DOX aqueous solution (3.0 mL, 1.0 mg/mL, pH=5). After being stirred for 3 days, the
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DOX-loaded hollow microspheres were centrifuged (10000 rpm for 10 min) to
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remove the free excess DOX molecules. Then the drug concentration in the
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supernatant solution was analyzed using a UV spectrophotometer at a wavelength of
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maximum absorbance (233 nm) after being diluted 100 times. The drug-loading
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capacities of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
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were calculated from the drug concentrations in solution before and after adsorption.
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2.9. Controlled release
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A 10 mL buffer solution containing the DOX-loaded
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(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres was transferred into dialysis
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tubes with a weight cutoff 14000 and immersed into 150 mL buffer solutions at 37 °C
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and three different pH values, 5.0, 6.5 and 7.4, respectively. Aliquots (5.0 mL) of the
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solution were taken at time intervals. The drug concentration in the dialysates was analyzed for monitoring the 233 nm absorption peak of DOX using UV-vis spectrometry in order to detect the rate of drug release. Furthermore, 5.0 mL fresh buffer solution with the same pH value was added after each sampling to keep the
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total volume of the solution constant. The cumulative release is expressed as the total
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percentage of drug molecule released through the dialysis membrane over time.
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Mi= Ci×160mL+∑j=1j=i-1 Cj×5mL
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MA= (1mg/mL-Cs) ×5mL 9
Page 9 of 38
The cumulative release (%) = (Mi/MA) ×100%
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where Mi is the total cumulative drug mass released from the hollow microsphere as
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of measurement i, Ci (mg/mL) is the drug concentration of sample i, ∑j=1j=i-1 Cj ×5mL
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is the total drug mass in previously extracted samples, Cs is the drug concentration in
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the supernatant after centrifugation (10000 rpm for 10 min), MA is the total drug mass
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of the hollow microspheres loaded.
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2.10. Cell compatibility assays
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Sulforhodamine-B (SRB) assay was applied to estimate the cell compatibility of the
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(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres with HepG2 cells. The
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cell were seeded into 96-well plates at densities of 1×105 cells per well for 24 h.
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Then, the different concentrations of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow
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microspheres and the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow
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microspheres were added to the cells and incubated for 48 h. Afterwards, the cells
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were washed three times with phosphate buffered saline and the SBR assay were used to test the cell viability. For this process, cells were fixed with the 50 % trichloroacetic acid solution and stained with 0.4 % SBR dissolved in 1 % acetic acid. Cell bound dye was extracted with 10 mL unbuffered Tris buffer solution, the absorbance were measured at 550 nm using a plate reader.
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2.11. Analysis and characterization
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The morphologies of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow 10
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microspheres and other samples were characterized with a JEM1200 EX/S
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transmission electron microscope (TEM) (JEOL Tokyo, Japan). The samples were
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dispersed in deionized water and stirred for 30 min, and then deposited on a copper
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grid covered with a perforated carbon film.
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The Zeta potentials of the core-shell and hybrid hollow microspheres in different pH
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media were determined with Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.).
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A Bruker IFS 66 v/s infrared spectrometer (Bruker, Germany) was used for the
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Fourier transform infrared (FT-IR) spectroscopy analysis in the range of 400-4000
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cm-1 with a resolution of 4 cm-1. The KBr pellet technique was adopted to prepare the
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sample for recording the IR spectra.
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To determine the amount of carboxylic acid groups on the surface of the Fe3O4-CA
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was carried out by the conductometric titration method in a flask equipped with a
232
conductivity meter (model DDS-11A) [27].
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The element analysis of the PSS templates was conducted with Elementar Vario EL
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instrument (Elementar Analysen systeme GmbH, Munich, Germany). 1
H NMR spectra were recorded on 400 MHz with Brucker ARX 400 spectrometer
(Bruker, Germany) using CDCl3 as solvent with internal TMS as the reference (0 ppm).
The mean particle size of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow
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microspheres were determined by dynamical mode (dynamic light scattering (DLS))
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on the “Light Scattering System BI-200SM, Brookhaven Instruments” device
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equipped with the BI-200SM Goniometer, the BI-9000AT Correlator, Temperature 11
Page 11 of 38
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Controller and the Coherent INOVA 70C argon ion laser at 20 °C. DLS measurements
243
are performed using 135 mW intense laser excitation at 514.5 nm and at a detection
244
angle of 90° using the emulsion directly at 25 °C.
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(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres were detected at a
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wavelength of maximum absorbance of 233 nm by TU-1901 UV/vis spectrometer
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(Beijing Purkinje General Co. Ltd., Beijing China) at room temperature.
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3. Results and discussion
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3.1. CS-g-PEG copolymer
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In the 1H NMR spectrum of the poly(ethylene glycol) grafted chitosan (CS-g-PEG)
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(Figure S1), the chemical shift at 3.58 ppm (-OCH3) and 3.847-3.939 ppm
254
(-OCH2CH2-) are due to the PEG brushes grafted. The chemical shift at 5.05 ppm
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(-O(C)CHO-) is attributed to the C-H of CS. The integrated areas of the -OCH3 and
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-O(C)CHO- are 3.00 and 26.67, respectively. So the PEG content of the CS-g-PEG copolymer could be calculated as 3.75% from the 1H-NMR spectrum. Furthermore, the absorbance peaks at 3435 cm-1 (O-H), 2875 cm-1 (C-H), 1651 cm-1 (amide I), 1386 cm-1 (amide III), and 1093 cm-1 (C-O-C) are attributed to the
260
characteristics of CS (Figure S2 (a)). By comparing the FT-IR spectra of CS and the
261
CS-g-PEG, it could be seen that the absorbance corresponding to the C-H stretching at
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2875 cm-1 was significantly strengthened after the modification. The C-O-C stretching
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shifted to 1110 cm-1 (stretching shift of ether C-O) in comparison with that of CS at 12
Page 12 of 38
1093 cm-1, and the absorbance of PEG also appeared at 955 cm-1 (wagging vibration
265
of ether C-H), 1247 cm-1 (twisting vibration of ether C-H), 1286 cm-1 (wagging
266
vibration of ether C-H), and 1464 cm-1 (formation vibration of ether C-H) [28]. All
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the analysis indicated that PEG had been successfully grafted onto chitosan to form
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the copolymer CS-g-PEG.
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3.2. Fe3O4-CA nanoparticles
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In the FT-IR spectrum of the citrate modified ferroferric oxide nanoparticles
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(Fe3O4-CA) (Figure S2 (b)), the absorbance peak at 571 cm-1 is attributed to the
273
characteristic of the Fe-O stretching vibration of the Fe3O4 nanoparticles [22].
274
Additionally, the absorbance of the symmetric C–O stretching, asymmetric C–O
275
stretching, C-H, and O-H of citrate at 1394, 1622, 2923, and 3423 cm-1 were found,
276
indicating that the Fe3O4 nanoparticles were successfully modified by citrate [29]. The
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six X-ray diffraction peaks at 30.1°, 35.5°, 42.4°, 52.0°, 57.6°, and 62.8° are
279 280 281
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assignable to the (220), (311), (400), (422), (511), and (440) planes of the spinel structure of Fe3O4 (Figure S3) [30]. The TEM image of the citrate modified ferroferric oxide (Fe3O4-CA) nanoparticles
is showed as Figure 1(a). Its average diameter was less than 10 nm. In the magnetic
282
hysteresis loop of the Fe3O4-CA nanoparticles (Figure S4), neither remanence nor
283
coercivity was observed, indicating the superparamagnetic property [31] with a high
284
saturation magnetization of 58.04 emu/g. Its carboxylic acid percentage was measured
285
to be about 1.3 mmol/g by the conductometric titration method [27].
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3.3. LbL assembly process
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The layer-by-layer (LbL) assembly technique was used to prepare the hybrid
289
multilayer coated PSS core-shell microspheres with the CS-g-PEG copolymer as
290
polycation and the Fe3O4-CA nanoparticles as hybrid anion. Initially, the copolymer
291
CS-g-PEG was adsorbed onto the PSS templates by the electrostatic interaction
292
between the amino groups of the CS-g-PEG and the carboxyl and sulfonic acid groups
293
of the PSS templates. Next, the hybrid anion Fe3O4-CA nanoparticles were added to
294
the aqueous solution containing the PSS@CS-g-PEG to achieve the layer-by-layer
295
assembly between the CS-g-PEG and the Fe3O4-CA. Repeating this cycle for four
296
times, the hybrid multilayer coated PSS core-shell microspheres
297
(PSS@(CS-g-PEG/Fe3O4-CA)n) (n=1, 2, 3, 4) were then obtained. Finally, the
298
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres were obtained with
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the CS-g-PEG copolymer as the outermost layer by adsorption it on the core-shell
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PSS@(CS-g-PEG/Fe3O4-CA)4 microspheres, as described in Scheme 1.
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The zeta potentials of the hybrid multilayer encapsulated PSS core-shell
microspheres were conducted to track the hybrid multilayer growth as shown in Figure 2. The odd layer numbers correspond to the CS-g-PEG deposition and the even layer numbers to the Fe3O4-CA adsorption. When the first layer of the CS-g-PEG was
305
deposited on the PSS templates, the zeta potential was about 38.0 mV, indicating that
306
the deposition of the CS-g-PEG could completely cover the PSS template surface.
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Then the zeta potential changed to about 23.1 mV after the adsorption of the hybrid
308
anion Fe3O4-CA nanoparticles over the CS-g-PEG coated PSS templates. The zeta 14
Page 14 of 38
potential of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)1/CS-g-PEG,
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PSS@(CS-g-PEG/Fe3O4-CA)2, PSS@(CS-g-PEG/Fe3O4-CA)2/CS-g-PEG,
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PSS@(CS-g-PEG/Fe3O4-CA)3, PSS@(CS-g-PEG/Fe3O4-CA)3/CS-g-PEG,
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PSS@(CS-g-PEG/Fe3O4-CA)4 and PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
313
microspheres were about 23.2 , 14.8 , 16.1 , 15.3 , 20.4 , 19.3 and 20.1 mV,
314
respectively. The regular increase or decrease in the zeta potentials of the core-shell
315
microspheres with different coated layers stated clearly the alternative deposition of
316
the polycation CS-g-PEG copolymer and the hybrid anion Fe3O4-CA nanoparticles on
317
the PSS templates to form the hybrid multilayer shells. For each of the intermediate
318
products and the final PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG core-shell
319
microspheres, their positively charged surface is favorable for their dispersion
320
stability due to the electrostatic repulsion.
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Different from the polyelectrolyte multilayers, the zeta potentials of the core-shell
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hybrid microspheres remained positive during the LbL assembly procedure in the
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present work. It is due to that the assembled anion used here is negative charged Fe3O4-CA nanoparticles with particle size of less than 10 nm, which could not cover completely the surface of the CS-PEG layers. The zeta potential of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres was 20.1 mV. It means that
327
the hybrid hollow microspheres obtained after the following template-removal
328
procedure might be positively charged. The positively charged surfaces show
329
pH-sensitive cell interactions, which enables specific drug delivery to cells in a
330
weakly acidic environment, such as the tumor tissues [32]. And their surface has been 15
Page 15 of 38
typically protected by PEG brushes to achieve the prolonged circulation and evasion
332
of immune clearance via reducing opsonization and phagocytic uptake by the
333
hydrophilic surface. So the final resultant hybrid hollow microspheres are expected to
334
be potential tumor-specific drug delivery system [32].
ip t
331
cr
335
3.4. Core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres
337
The TEM images of the PSS templates and the core-shell
338
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres are compared in Figure 1.
339
Their average diameters were about 436 nm and 526 nm, respectively. Obviously, as
340
the hybrid multilayer successfully coated onto the PSS templates, the average
341
diameter increased. Comparing the average diameters of the PSS templates and the
342
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, one can calculate
343
the thickness of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid shells of 45 nm.
345 346 347 348
an
M
d
te
Ac ce p
344
us
336
3.5. (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres The FT-IR spectra of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres are compared in Figure 3. The characteristic absorbance of benzene ring in polystyrene at 3059, 3025cm-1 (C-H,
349
stretching vibration), 1601, 1492 and 1452 cm-1(C=C, stretching vibration), 699 cm-1
350
(out of plane blending vibration) and the characteristic absorbance of carbonyl
351
stretching of the carboxyl groups in MAA and sulfonic acid groups at 1698 and 1074
352
cm-1, which were present in the FT-IR spectrum of the core-shell 16
Page 16 of 38
353
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, disappeared in that of the
354
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres, indicating that the PSS
355
templates had been completely removed by washing with DMF.
ip t
After the PSS templates encapsulated in the core-shell
356
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres had been etched with DMF,
358
the well-defined (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
359
were obtained with an average diameter of 510 nm (Figure 1(d)). Compared with that
360
of the core-shell microspheres, the average diameter hybrid hollow microspheres
361
decreased 16 nm, due to the shrinkage of the hybrid shells. The XRD patterns of the
362
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and the
363
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres are similar as those
364
of the Fe3O4-CA nanoparticles (Figure S3). It indicates that the structure of the
365
magnetic nanoparticles keeps essentially unchanged during the LbL assembly and
366
washing manipulations. After the removal of the PSS templates, the saturation
368 369 370
us
an
M
d
te
Ac ce p
367
cr
357
magnetization of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres increased to 37.23 emu/g from 25.98 emu/g of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres. They also exhibited the superparamagnetic property (Figure S4) [33]. Compared with the saturation
371
magnetization of the Fe3O4-CA nanoparticles, their magnetite content could be
372
calculated as 64.14%. Figure 4 shows the digital photographs of the aqueous dispersions of the core-shell
373 374
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and the 17
Page 17 of 38
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or
376
without (left) external magnetic field, which were well dispersed in water under
377
normal conditions. This result indicates that the core-shell microspheres and the
378
hybrid hollow microspheres can be easily manipulated by an external magnetic field.
379
The superparamagnetic characteristics with high saturation magnetization of the
380
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres predict their potential
381
application for magnetic resonance imaging and magnetic-target drug delivery [21].
us
cr
ip t
375
an
382
3.6. Stimuli-responsive properties
384
The influence of ionic strength on the average hydrodynamic diameter of the
385
superparamagnetic hybrid hollow microspheres ((CS-g-PEG/Fe3O4-CA)4/CS-g-PEG)
386
was studied by dynamic light scattering (DLS) technique. It is found that introducing
387
small molecule electrolytes (e.g., NaCl) had a significant influence on the size of the
388
obtained hybrid hollow microspheres, the average hydrodynamic diameter of the
390 391 392
d
te
Ac ce p
389
M
383
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres increased from 407.1 to 488.4 nm with increasing the ionic strength in the range of 0-0.20 mol/L NaCl (Figure 5a), due to the shielding effect of the small electrolytes on the electrostatic force [34]. However, it remained constant (488.4 nm) at higher salt concentration
393
(0.15-0.20 mol/L NaCl), covering the NaCl concentration in the physiological media
394
of 0.154 mol/L. It showed that neither aggregation nor fusion of the
395
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres will occur in the
396
physiological media due to the surface concealing effect of the PEG brushes [18]. 18
Page 18 of 38
The pH dependence of the average hydrodynamic diameter of the
397
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was also investigated
399
by DLS. As shown in Figure 5b, the average hydrodynamic diameter of the
400
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres decreased from 489.5
401
to 413.3nm with increasing pH value from 3 to 11, while the average hydrodynamic
402
diameter of the hybrid hollow microspheres remained the same in the near neutral
403
media (pH between 5 and 7). In the basic media, the hybrid hollow microspheres
404
shrank with the increasing of the media pH values, due to the deprotonation of the
405
amino groups in the CS-g-PEG copolymer [35]. At low pH values, the ionization of
406
the carboxyl groups of the Fe3O4-CA nanoparticles was normally depressed and the
407
amino groups in CS-g-PEG were protonated [22], resulting to the expansion of the
408
hybrid shells. It is favorable to the drug-release in the media with lower pH values
409
such as the acidic tumor mircoenvironmets [36].
411 412 413 414
cr
us
an
M
d
te
Ac ce p
410
ip t
398
3.7. Drug-loading and controlled release The drug-loading and pH-responsive controlled release performance of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was investigated with an anticancer drug doxorubicin (DOX) as model drug. The DOX-loading capacity
415
was calculated to be 10.44 %. It is lower than those data of the polyelectrolyte
416
multilayer hollow microspheres reported, due to the lower content of the carboxyl
417
groups in the hybrid hollow microspheres, as the Zeta potential analysis (Figure 2).
418
The in vitro drug releases from the DOX-loaded hybrid hollow microspheres were 19
Page 19 of 38
performed at 37°C under three different simulated body fluids (pH 5.0, 6.5 and 7.4).
420
The drug release was faster in the lower pH media and the cumulative release of the
421
DOX was 54.67 %, 34.59 %, and 29.78 % at pH 5.0, 6.5 and 7.4 at 37 °C within 50 h
422
(Figure 6). The pH-dependent release of DOX was observed that the releasing ratio
423
and cumulative release of DOX from the drug carriers was quicker and higher at pH
424
5.0 than those at pH 6.5 or pH 7.4.
us
cr
ip t
419
However, the common feature of these three curves is that the release curve does not
425
tend to be flat, and gradually upward trend as the growth of the time. The results of
427
the DOX cumulative release at different pH media revealed that the release at low pH
428
could partly be attributed to the fact that the permeability of the polyelectrolyte hybrid
429
shells was better at low pH values than at high ones, because of the decrease in pH
430
weakened the ionic crosslinking bonds between the CS-PEG copolymer and the
431
Fe3O4-CA nanoparticles due to the decrease in the charge density of citric acid and the
432
crosslinking density. Furthermore, the hybrid shells were positively charged at low
434 435 436
M
d
te
Ac ce p
433
an
426
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 the CS-PEG, which might make such configuration unstable so that the water soluble DOX molecules were impelled across the hybrid shells. The pH-dependent release of DOX from the
437
superparamagnetic multilayer hybrid hollow microspheres has a great advantage in
438
curing the cancer cells with an acid medium about pH at 5 [37,38].
439 440
3.8. Cytocompatibility 20
Page 20 of 38
The in vitro cytocompatibility of the superparamagnetic
442
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was studied with
443
HepG2 cells using SRB assays. The cells were incubated with the hollow
444
microspheres and the DOX-loaded hollow microspheres for 48 h at various
445
concentrations (0-40 μg/mL). The result indicated that the
446
(CS-g-APEG/Fe3O4-CA)4-CS-g-APEG hybrid hollow microspheres showed high
447
biological compatibility (cell viability = 88.86-70.19 %) up to a tested concentration
448
of 10-40 μg/mL (Figure 7).
an
us
cr
ip t
441
Moreover, the DOX-loaded hollow microspheres had higher toxicity (cell viability =
449
72.07-29.45 %) up to a tested concentration of 10-40 μg/mL. With increasing the
451
tested concentration of the hybrid hollow microspheres, the cell viability had small
452
change. So it could be concluded that the superparamagnetic hybrid hollow
453
microspheres (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG exhibit preferably biocompatibility.
454
Furthermore, the cytotoxicity was found to be reduced by using the
456 457 458
d
te
Ac ce p
455
M
450
superparamagnetic hybrid hollow microspheres as drug carriers with the same DOX dosage (Figure 8). And the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres possessed higher antitumor activity only with higher dosages.
459
4. Conclusions
460
In the present work, biocompatible superparamagnetic pH-stimuli responsive
461
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with magnetite
462
content of 64.14% and saturation magnetization of 37.23 emu/g were desiged with the 21
Page 21 of 38
Fe3O4-CA nanoparticles as hybrid anion via the LBL assembly technique. They
464
exhibit stable dispersibility in the physiological media. Their positively charged
465
surfaces showing the pH-sensitive cell interactions and the pH-stimuli responsive
466
property could enable the specific drug delivery to cells such as the tumor
467
microenvironments. All the features make them potential magnetic-targeting
468
tumor-specific drug delivery system, magnetic hyperthermia therapy, or magnetic
469
resonance imaging.
us
cr
ip t
463
an
470
Acknowledgments
472
This work was supported by the Fundamental Research Funds for the Central
473
Universities and the Open Project of Key Laboratory for Magnetism and Magnetic
474
Materials of the Ministry of Education, Lanzhou University.
475
477 478 479 480
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471
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te
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Ac ce p
542
M
538
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25
Page 25 of 38
544
Figure Captions
545 546
Scheme 1. Schematic illustration of the fabrication of the pH-stimuli responsive superparamagnetic (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow
548
microspheres.
Figure 1. TEM images of (a) the Fe3O4-CA nanoparticles, (b) the PSS templates, (c)
cr
549
ip t
547
the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, and (d)
551
the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres.
Figure 2. Zeta potentials of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)n
an
552
us
550
microspheres (n = 1, 2, 3, 4) (mean ± standard deviation, n=3).
554
M
553
Figure 3. The FT-IR spectra of the core-shell
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and
556
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
558 559 560 561 562
te
Figure 4. Photographic images of (a) the core-shell
Ac ce p
557
d
555
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and (b) the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or without (left) external magnetic field.
Figure 5. Ionic strength (a) and pH (b) dependences of average hydrodynamic diameter (D) of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres (mean ± standard deviation, n=3).
563 564
Figure 6. In vitro drug release from the DOX-loading superparamagnetic hybrid
565
hollow microspheres at pH 5.0, 6.5 and 7.4 at 37 °C, respectively (mean ±
566
standard deviation, n=3). 26
Page 26 of 38
567
Figure 7. Cell viability data of the CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
569
(mean ± standard deviation, n=3).
570
Figure 8. Antitumor activity of free DOX and the DOX-loaded
ip t
568
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres for 48h (mean ±
572
standard deviation, n=3).
cr
571
Ac ce p
te
d
M
an
us
573 574
27
Page 27 of 38
574 575
adsorption CS-g-PEG
repeating four times
dialyze
CS
PSS
PEG
us
576 577
cr
adsorption CS-g-PEG
DMF
ip t
adsorption Fe 3O4 -CA
Fe3O4 -CA
Scheme 1. Schematic illustration of the fabrication of the pH-stimuli responsive
an
superparamagnetic (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
578
M
579
Ac ce p
te
d
580
28
Page 28 of 38
580
cr
ip t
581
us
582
(a)
(b)
584
(c)
Figure 1. TEM images of (a) the Fe3O4-CA nanoparticles, (b) the PSS templates, (c)
588 589 590
Ac ce p
586 587
(d)
te
585
d
M
an
583
the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, and (d) the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres.
29
Page 29 of 38
590 591 592
ip t
35
cr
30 25 20 15 0
2
4
6
8
10
an
layer number (2n)
us
Zetal potential (mV)
40
593
Figure 2. Zeta potentials of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)n
595
microspheres (n = 1, 2, 3, 4) (mean ± standard deviation, n=3).
M
594
d
596
Ac ce p
te
597
30
Page 30 of 38
597 598
1698
591
80 2852 3422
1492 1452 1650
1601
60 50
cr
70
1073 1698
3025
us
Transmittance (%)
3059
ip t
1099
90
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG)
an
4000 3500 3000 2500 2000 1500 1000
592
500
Wavenumber ( cm ) -1
599
Figure 3. The FT-IR spectra of the core-shell
601
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
602
hybrid hollow microspheres.
te
d
M
600
604
Ac ce p
603
31
Page 31 of 38
604 605 606
608 609
us
cr
ip t
607
(a)
(b)
Figure 4. Photographic images of (a) the core-shell
an
610
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and (b) the
612
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or
613
without (left) external magnetic field.
d
M
611
te
614
Ac ce p
615
32
Page 32 of 38
615 616
500 500
(a)
(b)
480
460 440 420
ip t
D (nm)
460 440 420
400
400 0.00
0.05
0.10
0.15
0.20
2
4
CNaCl (mol/L)
pH
8
10
12
us
617
6
cr
D (nm)
480
Figure 5. Ionic strength (a) and pH (b) dependences of average hydrodynamic
619
diameter (D) of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
620
(mean ± standard deviation, n=3).
an
618
M
621
Ac ce p
te
d
622
33
Page 33 of 38
622 623
pH 5.0 pH 6.5 pH 7.4
ip t
50 40 30 20 10 0
500
1000
1500
2000
2500
t (min)
624
3000
3500
us
0
cr
Cumulative release (%)
60
Figure 6. In vitro drug release from the DOX-loading superparamagnetic hybrid
626
hollow microspheres at pH 5.0, 6.5 and 7.4 at 37 °C, respectively (mean ± standard
627
deviation, n=3).
M
an
625
628
Ac ce p
te
d
629
34
Page 34 of 38
629 630
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
cr
80 60 40 20 0 10
20
30
Concentration ( μg/mL)
40
an
0
us
Cell viability (%)
100
ip t
631
632
Figure 7. Cell viability data of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and the
634
DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres (mean
635
± standard deviation, n=3).
d
M
633
te
636
Ac ce p
637
35
Page 35 of 38
637 638
free DOX DOX-loaded ( CS-g-PEG/Fe3O4-CA) 4/CS-g-PEG
cr
80 60 40 20 0 0
2
4
6
8
10
an
DOX (ug/mL)
us
Cell viability (%)
100
ip t
639
640
Figure 8. Antitumor activity of free DOX and the DOX-loaded
642
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres for 48h (mean ±
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standard deviation, n=3).
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Highlights Biocompatible pH‐responsive superparamagnetic hollow microspheres were fabricated. They exhibited stable dispersibility in the physiological media. Their positively charged surfaces enabled the specific drug delivery to tumor. The pH‐responsive property enabled the selective release in the acidic media.
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Graphical Abstract The superparamagnetic characteristics, excellent stability and pH dependent DOX release make the well‐defined(CS‐g‐PEG/Fe3O4‐CA)4/CS‐g‐PEG hybrid hollow microspheres potential platform for tumor‐specific delivery.
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S0927-7765(14)00347-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.06.054 COLSUB 6500
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
17-2-2014 22-6-2014 24-6-2014
Please cite this article as: X. Li, P. Du, P. Liu, Novel biocompatible pHstimuli responsive superparamagnetic hybrid hollow microspheres as tumorspecific drug delivery system, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.06.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Novel biocompatible pH-stimuli responsive superparamagnetic
2
hybrid hollow microspheres as tumor-specific drug delivery system
3
Xiaorui Li, Pengcheng Du, Peng Liu*
4
State Key Laboratory of Applied Organic Chemistry and Key Laboratory of
5
Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of
6
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
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Abstract
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Novel biocompatible pH-stimuli responsive superparamagnetic hybrid hollow
9
microspheres have been designed via the layer-by-layer (LBL) self-assembly
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technique via the electrostatic interaction between the poly(ethylene glycol) grafted
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chitosan (CS-g-PEG) as polycation and the citrate modified ferroferric oxide
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nanoparticles (Fe3O4-CA) as hybrid anion onto the uniform polystyrene sulfonate
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(PSS) microsphere templates. The well-defined hybrid hollow microspheres
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((CS-g-PEG/Fe3O4-CA)4/CS-g-PEG) were obtained after etching the templates by
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washing with DMF. They possessed superparamagnetic characteristics with a saturation magnetization of 37.23 emu/g, and exhibited excellent stability in high ion-strength media and pH dependent DOX release. Their unique structure and outstanding performance make them potential platform for tumor-specific delivery in
19
the tumor diagnostic and therapy.
20
Keywords: Drug delivery system; pH dependent release; superparamagnetic; hollow
21
microspheres; LBL self-assembly
*
Corresponding author. Fax: 86 0931 8912582; Tel: 86 0931 8912582; E-mail: [email protected]. 1
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1. Introduction
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The stimuli-responsive hollow microspheres have attracted more and more attention
24
because of their variety of potential applications such as drug carriers, biomedicine
25
and enzyme transplantation, gene therapy, and contrast agents in diagnostics [1-3]. By
26
now, various physical and chemical methods have been developed to prepare the
27
stimuli-responsive hollow microspheres. Among these methods, the layer-by-layer
28
(LbL) self-assembly of the oppositely charged polyelectrolytes (PEs) onto the
29
sacrificial templates has been widely used due to its unique advantages (e.g., size,
30
thickness, composition, porosity, stability, surface functionality, tunable permeability)
31
[4,5]. In most of the LbL engineered multilayer hollow microspheres, two kinds of the
32
PEs of opposite charge have been used as the raw materials via the electrostatic
33
interaction.
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Chitosan (CS), a non-toxic, biocompatible and biodegradable polysaccharide and
34
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cationic polyelectrolyte, has shown some favorable bioactivities, such as anti-bacterial,
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immunological and wound healing activity [6-8]. It has been widely used for biomedical applications, especially for the delivery and control release of drugs, genes, and vaccines [9-12]. However, its poor solubility in water and some organic solvents limits its wide application. Many chitosan derivatives have been prepared by chemical
40
modification to overcome this disadvantage and generate new biomaterials [13],
41
among which chemical modification of chitosan with poly(ethylene glycol) (PEG) is
42
considered to be a convenient way to improve its water solubility and
43
biocompatibility [14,15]. PEG has been widely used in modification of a variety of 2
Page 2 of 38
materials because of its excellent physicochemical and biological properties,
45
including hydrophilicity, flexibility, lack of toxicity, ease of chemical modification,
46
absence of immunogenicity and antigenicity, biocompatibility and steric repulsion
47
[16,17]. Especially for the multilayer hollow microspheres fabricated with the
48
non-covalent bonds such as electrostatic interaction or hydrogen bond, the surface
49
PEGylation could efficiently prevent them from aggregation or fusion in high
50
ion-strength media [18]. Furthermore, many reports have been published on the water
51
solubility and bioactivity of CS-g-PEG and the results show that the grafting of PEG
52
onto chitosan not only improves water solubility and biocompatibility of chitosan, but
53
also improve the bioavailability of drugs in vivo [19,20].
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The hollow microspheres modified with the magnetic nanoparticles show a lot of
54
advantages including magnetic-targeting and magnetic hyperthermia therapy, or
56
application in magnetic resonance imaging and biosensors [21]. The magnetic
57
nanoparticles could be used as the hybrid anion to prepare the multilayer
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superparamagnetic hybrid hollow microspheres via the LbL self-assembly technique, after surface modification with citric acid [22-24]. The magnetic hollow microspheres can be widely used in magnetic fields for directing and accumulating hollow microspheres to the target sites before releasing the chemotherapeutic drugs,
62
achieving the magnetic targeting function [25]. With such a magnetic-sensitive shell
63
structure, active substances can be well-regulated in a manageable manner with a
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designable profile according to the time duration under high frequency magnetic field
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[26]. 3
Page 3 of 38
In the present work, the well-defined biocompatible pH-stimuli responsive
66
superparamagnetic hybrid hollow microspheres have been accomplished via the
68
layer-by-layer (LBL) self-assembly technique with the poly(ethylene glycol) grafted
69
chitosan (CS-g-PEG) and the citrate modified ferroferric oxide nanoparticles
70
(Fe3O4-CA) as the assembling materials, after etching the templates (polystyrene
71
sulfonate microspheres (PSS)) by washing with DMF (Scheme 1). The controlled
72
releasing behavior was investigated in vitro in simulated body fluids with different pH
73
values, with doxorubicin (DOX) as model hydrophobic drug.
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2. Experimental
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2.1. Materials and reagents
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Chitosan (CS) was obtained from Golden-Shell Biochemical Co. Ltd., Zhejiang,
78
China with degree of deacetylation and viscosity-average molecular weight of 96%
79
and 6.0×105, respectively. Aldehyde-terminated poly(ethylene glycol)
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(CH3O-PEG-CHO, Mn=2000) was purchased from Taiyuan Rongyuan Co. Ltd., Taiyuan, China.
Styrene (St) and methacrylic acid (MAA) (Tianjin Chemical Co. Ltd., Tianjin, China)
were distilled under vacuum before use. Ferric chloride hexahydrate (FeCl3·6H2O)
84
and ferrous chloride tetrahydrate (FeCl2·4H2O) were purchased from Sinopharm
85
Chemical Reagent Co., Ltd. Shanghai, China. Sodium citrate (Na3C6H5O7·2H2O) was
86
an analytical grade reagent from Shanghai Chemical Co. Ltd., Shanghai, China.
87
Sodium cyanoborohydride (NaCNBH3) was obtained from the Sigma Chemical Co. 4
Page 4 of 38
(St. Louis, MO, USA). Ammonium persulfate (APS), N,N-dimethylformamide
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(DMF), and other reagents were all of analytical reagent grade from Tianjin Chemical
90
Co. Ltd., Tianjin, China and used without further purification. Deionized water was
91
used throughout.
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2.2. Uniform polystyrene (PS) particles
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The uniform polystyrene (PS) particles were prepared via the emulsifier-free emulsion
95
polymerization according to the procedure as reported previously [22]. Styrene (St,
96
10.0 mL) and methacrylic acid (MAA, 2.0 mL) were added to 95 mL distilled water
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in a three-necked round bottom flask fitted with a condenser and a magnetic stirred
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and purged with nitrogen. A solution of ammonium persulfate (APS, 0.054 g)
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pre-dissolved in water (5 mL) was added to the reaction vessel with vigorous stirring
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and bubbling with nitrogen. The polymerization was continued for 24 h at 72 °C.
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After cooling to room temperature, the product was purified by repeating
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centrifugation (10000 rpm for 10 min) and washing with ethanol. The white fine powder was finally obtained after being dried in a vacuum oven at 50 °C.
2.3. Polystyrene sulfonate (PSS) microspheres
106
4.0 g PS particles were dispersed in 80 mL 98% sulfuric acid with ultrasonication and
107
the dispersion was stirred at 45 °C for 8 h. After being cooled to the room temperature,
108
the product was separated by repeating centrifugation and washing with a large excess
109
of water after being diluted by distilled water. The transformation of the polystyrene 5
Page 5 of 38
sulfonic acid into the sodium polystyrene sulfonate was performed by adding an
111
excess of sodium bicarbonate after being re-dispersed in water and then separated by
112
repeating centrifugation (10000 rpm for 10 min) and thoroughly rinsing with water
113
until neutral. The sulfonate percentage was measured to be about 0.8 mmol/g by the
114
sulfur element analysis method. The obtained PSS microspheres were finally
115
dispersed and stored in 100 mL distilled water.
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2.4. PEG grafted chitosan (CS-g-PEG)
118
The poly(ethylene glycol) grafted chitosan (CS-g-PEG) was synthesized according to
119
the method described by Harris [27]. 1.0 g CS was dissolved in a mixture of aqueous
120
acetic acid (2.0 wt%, 20 mL) and methanol (10 mL), to which 10 ml aqueous solution
121
of the aldehyde-terminated poly(ethylene glycol) (CH3O-PEG-CHO, Mn=2000)
122
(0.745 g) was added subsequently. The mixture was stirred for 24 h at room
123
temperature, and its pH was adjusted to 6.0-6.5 with aqueous 1mol/L NaOH solution.
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After stirring for another 2 h at room temperature, 10 mL of aqueous NaCNBH3 (0.2342 g) solution was added drop-by-drop in 30 min. To complete the reaction, the solution was stirred for another 24 h at room temperature under nitrogen protection. The mixture was dialyzed against distilled water for 1 week, with water-replacement
128
every 5 h. The remained APEG was removed by freezing or air drying in combination
129
with acetone (100 mL) washing process. The final copolymer CS-g-PEG was
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obtained after evaporating the residual solvent in vacuum.
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2.5. Citrate modified ferroferric oxide (Fe3O4-CA)
133
The co-precipitation method was used to prepare the citrate modified ferroferric oxide
134
[22]. The FeCl3·6H2O (26.0 g) and FeCl2·4H2O (9.56 g) (Fe3+ : Fe2+ = 2:1) were
135
dissolved in distilled water (400 mL) under nitrogen atmosphere with mechanical
136
stirring. As the solution was heated to 70 °C, NH3·H2O (28 wt%, 50 mL) was added
137
dropwise to the mixture under vigorous stirring, then 48 mL of 2 mol/L sodium citrate
138
was quickly added, and the mixture was heated to 85°C and kept for 1.5 h.
139
Subsequently, the ultimate citrate modified Fe3O4 nanoparticles (Fe3O4-CA) were
140
washed more than three times with distilled water to discard the excessive sodium
141
citrate by the magnetic separation procedure. Finally, the Fe3O4-CA nanoparticles
142
were dispersed and stored in 100 mL distilled water.
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2.6. PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres
145
The LbL assembly technique was used to prepare the multilayer hybrid shell
146 147 148 149
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encapsulated polystyrene sulfonate templates (PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG) with the positively charged CS-g-PEG and the Fe3O4-CA hybrid anion via electrostatic interaction. The adsorption of the CS-g-PEG was completed in a solution of 400 mL deionized water containing 1.0 g
150
sodium polystyrene sulfonate (PSS) templates and 0.25 g CS-g-PEG, at pH around 5
151
for 8 h followed by centrifugation (10000 rpm for 10 min) and washing three times in
152
water to obtain the PSS@CS-g-PEG. Then the PSS@CS-g-PEG was dispersed into
153
400 mL deionized water and added to the solution containing the Fe3O4-CA in 7
Page 7 of 38
batches until reaching the adsorption balance at pH 8.5 under mechanical stirring
155
combining with ultrasonic. Then the microspheres were centrifuged (10000 rpm for
156
10 min) and washed three times in water to obtain the core-shell hybrid microspheres
157
covered with one bilayer (PSS@(CS-g-PEG/Fe3O4-CA)1). The CS-g-PEG and
158
Fe3O4-CA were alternately deposited for another four and three times onto the PSS
159
templates to obtain the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
160
microspheres.
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2.7. (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres
163
The sacrificial templates (PSS) in the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4-
164
/CS-g-PEG microspheres were removed by washing with DMF: 1.0 g core-shell
165
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres were dispersed into 20 mL
166
DMF. After the dispersion was stirred for 4 h, the microspheres were magnetically
167
separated and dispersed into 20 mL new DMF. The washing with DMF was repeated
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for 4 times, followed with excess water for three times and centrifugation (10000 rpm for 10 min) to obtain the ultimate products, the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
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2.8. Drug loading
173
The drug loading was carried out by dispersing the
174
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres in DOX solution
175
with certain concentration. A typical procedure was as follows: 10 mg of the 8
Page 8 of 38
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres were added into the
177
DOX aqueous solution (3.0 mL, 1.0 mg/mL, pH=5). After being stirred for 3 days, the
178
DOX-loaded hollow microspheres were centrifuged (10000 rpm for 10 min) to
179
remove the free excess DOX molecules. Then the drug concentration in the
180
supernatant solution was analyzed using a UV spectrophotometer at a wavelength of
181
maximum absorbance (233 nm) after being diluted 100 times. The drug-loading
182
capacities of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
183
were calculated from the drug concentrations in solution before and after adsorption.
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2.9. Controlled release
186
A 10 mL buffer solution containing the DOX-loaded
187
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres was transferred into dialysis
188
tubes with a weight cutoff 14000 and immersed into 150 mL buffer solutions at 37 °C
189
and three different pH values, 5.0, 6.5 and 7.4, respectively. Aliquots (5.0 mL) of the
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solution were taken at time intervals. The drug concentration in the dialysates was analyzed for monitoring the 233 nm absorption peak of DOX using UV-vis spectrometry in order to detect the rate of drug release. Furthermore, 5.0 mL fresh buffer solution with the same pH value was added after each sampling to keep the
194
total volume of the solution constant. The cumulative release is expressed as the total
195
percentage of drug molecule released through the dialysis membrane over time.
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Mi= Ci×160mL+∑j=1j=i-1 Cj×5mL
197
MA= (1mg/mL-Cs) ×5mL 9
Page 9 of 38
The cumulative release (%) = (Mi/MA) ×100%
198
where Mi is the total cumulative drug mass released from the hollow microsphere as
200
of measurement i, Ci (mg/mL) is the drug concentration of sample i, ∑j=1j=i-1 Cj ×5mL
201
is the total drug mass in previously extracted samples, Cs is the drug concentration in
202
the supernatant after centrifugation (10000 rpm for 10 min), MA is the total drug mass
203
of the hollow microspheres loaded.
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2.10. Cell compatibility assays
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Sulforhodamine-B (SRB) assay was applied to estimate the cell compatibility of the
207
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres with HepG2 cells. The
208
cell were seeded into 96-well plates at densities of 1×105 cells per well for 24 h.
209
Then, the different concentrations of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow
210
microspheres and the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow
211
microspheres were added to the cells and incubated for 48 h. Afterwards, the cells
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were washed three times with phosphate buffered saline and the SBR assay were used to test the cell viability. For this process, cells were fixed with the 50 % trichloroacetic acid solution and stained with 0.4 % SBR dissolved in 1 % acetic acid. Cell bound dye was extracted with 10 mL unbuffered Tris buffer solution, the absorbance were measured at 550 nm using a plate reader.
217 218
2.11. Analysis and characterization
219
The morphologies of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow 10
Page 10 of 38
microspheres and other samples were characterized with a JEM1200 EX/S
221
transmission electron microscope (TEM) (JEOL Tokyo, Japan). The samples were
222
dispersed in deionized water and stirred for 30 min, and then deposited on a copper
223
grid covered with a perforated carbon film.
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The Zeta potentials of the core-shell and hybrid hollow microspheres in different pH
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media were determined with Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.).
226
A Bruker IFS 66 v/s infrared spectrometer (Bruker, Germany) was used for the
227
Fourier transform infrared (FT-IR) spectroscopy analysis in the range of 400-4000
228
cm-1 with a resolution of 4 cm-1. The KBr pellet technique was adopted to prepare the
229
sample for recording the IR spectra.
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To determine the amount of carboxylic acid groups on the surface of the Fe3O4-CA
230
was carried out by the conductometric titration method in a flask equipped with a
232
conductivity meter (model DDS-11A) [27].
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The element analysis of the PSS templates was conducted with Elementar Vario EL
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instrument (Elementar Analysen systeme GmbH, Munich, Germany). 1
H NMR spectra were recorded on 400 MHz with Brucker ARX 400 spectrometer
(Bruker, Germany) using CDCl3 as solvent with internal TMS as the reference (0 ppm).
The mean particle size of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow
238 239
microspheres were determined by dynamical mode (dynamic light scattering (DLS))
240
on the “Light Scattering System BI-200SM, Brookhaven Instruments” device
241
equipped with the BI-200SM Goniometer, the BI-9000AT Correlator, Temperature 11
Page 11 of 38
242
Controller and the Coherent INOVA 70C argon ion laser at 20 °C. DLS measurements
243
are performed using 135 mW intense laser excitation at 514.5 nm and at a detection
244
angle of 90° using the emulsion directly at 25 °C.
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The DOX-loading and controlled release behaviors of the
245
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres were detected at a
247
wavelength of maximum absorbance of 233 nm by TU-1901 UV/vis spectrometer
248
(Beijing Purkinje General Co. Ltd., Beijing China) at room temperature.
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3. Results and discussion
251
3.1. CS-g-PEG copolymer
252
In the 1H NMR spectrum of the poly(ethylene glycol) grafted chitosan (CS-g-PEG)
253
(Figure S1), the chemical shift at 3.58 ppm (-OCH3) and 3.847-3.939 ppm
254
(-OCH2CH2-) are due to the PEG brushes grafted. The chemical shift at 5.05 ppm
255
(-O(C)CHO-) is attributed to the C-H of CS. The integrated areas of the -OCH3 and
257 258 259
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-O(C)CHO- are 3.00 and 26.67, respectively. So the PEG content of the CS-g-PEG copolymer could be calculated as 3.75% from the 1H-NMR spectrum. Furthermore, the absorbance peaks at 3435 cm-1 (O-H), 2875 cm-1 (C-H), 1651 cm-1 (amide I), 1386 cm-1 (amide III), and 1093 cm-1 (C-O-C) are attributed to the
260
characteristics of CS (Figure S2 (a)). By comparing the FT-IR spectra of CS and the
261
CS-g-PEG, it could be seen that the absorbance corresponding to the C-H stretching at
262
2875 cm-1 was significantly strengthened after the modification. The C-O-C stretching
263
shifted to 1110 cm-1 (stretching shift of ether C-O) in comparison with that of CS at 12
Page 12 of 38
1093 cm-1, and the absorbance of PEG also appeared at 955 cm-1 (wagging vibration
265
of ether C-H), 1247 cm-1 (twisting vibration of ether C-H), 1286 cm-1 (wagging
266
vibration of ether C-H), and 1464 cm-1 (formation vibration of ether C-H) [28]. All
267
the analysis indicated that PEG had been successfully grafted onto chitosan to form
268
the copolymer CS-g-PEG.
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3.2. Fe3O4-CA nanoparticles
271
In the FT-IR spectrum of the citrate modified ferroferric oxide nanoparticles
272
(Fe3O4-CA) (Figure S2 (b)), the absorbance peak at 571 cm-1 is attributed to the
273
characteristic of the Fe-O stretching vibration of the Fe3O4 nanoparticles [22].
274
Additionally, the absorbance of the symmetric C–O stretching, asymmetric C–O
275
stretching, C-H, and O-H of citrate at 1394, 1622, 2923, and 3423 cm-1 were found,
276
indicating that the Fe3O4 nanoparticles were successfully modified by citrate [29]. The
277
six X-ray diffraction peaks at 30.1°, 35.5°, 42.4°, 52.0°, 57.6°, and 62.8° are
279 280 281
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assignable to the (220), (311), (400), (422), (511), and (440) planes of the spinel structure of Fe3O4 (Figure S3) [30]. The TEM image of the citrate modified ferroferric oxide (Fe3O4-CA) nanoparticles
is showed as Figure 1(a). Its average diameter was less than 10 nm. In the magnetic
282
hysteresis loop of the Fe3O4-CA nanoparticles (Figure S4), neither remanence nor
283
coercivity was observed, indicating the superparamagnetic property [31] with a high
284
saturation magnetization of 58.04 emu/g. Its carboxylic acid percentage was measured
285
to be about 1.3 mmol/g by the conductometric titration method [27].
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Page 13 of 38
3.3. LbL assembly process
288
The layer-by-layer (LbL) assembly technique was used to prepare the hybrid
289
multilayer coated PSS core-shell microspheres with the CS-g-PEG copolymer as
290
polycation and the Fe3O4-CA nanoparticles as hybrid anion. Initially, the copolymer
291
CS-g-PEG was adsorbed onto the PSS templates by the electrostatic interaction
292
between the amino groups of the CS-g-PEG and the carboxyl and sulfonic acid groups
293
of the PSS templates. Next, the hybrid anion Fe3O4-CA nanoparticles were added to
294
the aqueous solution containing the PSS@CS-g-PEG to achieve the layer-by-layer
295
assembly between the CS-g-PEG and the Fe3O4-CA. Repeating this cycle for four
296
times, the hybrid multilayer coated PSS core-shell microspheres
297
(PSS@(CS-g-PEG/Fe3O4-CA)n) (n=1, 2, 3, 4) were then obtained. Finally, the
298
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres were obtained with
299
the CS-g-PEG copolymer as the outermost layer by adsorption it on the core-shell
300
PSS@(CS-g-PEG/Fe3O4-CA)4 microspheres, as described in Scheme 1.
302 303 304
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The zeta potentials of the hybrid multilayer encapsulated PSS core-shell
microspheres were conducted to track the hybrid multilayer growth as shown in Figure 2. The odd layer numbers correspond to the CS-g-PEG deposition and the even layer numbers to the Fe3O4-CA adsorption. When the first layer of the CS-g-PEG was
305
deposited on the PSS templates, the zeta potential was about 38.0 mV, indicating that
306
the deposition of the CS-g-PEG could completely cover the PSS template surface.
307
Then the zeta potential changed to about 23.1 mV after the adsorption of the hybrid
308
anion Fe3O4-CA nanoparticles over the CS-g-PEG coated PSS templates. The zeta 14
Page 14 of 38
potential of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)1/CS-g-PEG,
310
PSS@(CS-g-PEG/Fe3O4-CA)2, PSS@(CS-g-PEG/Fe3O4-CA)2/CS-g-PEG,
311
PSS@(CS-g-PEG/Fe3O4-CA)3, PSS@(CS-g-PEG/Fe3O4-CA)3/CS-g-PEG,
312
PSS@(CS-g-PEG/Fe3O4-CA)4 and PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
313
microspheres were about 23.2 , 14.8 , 16.1 , 15.3 , 20.4 , 19.3 and 20.1 mV,
314
respectively. The regular increase or decrease in the zeta potentials of the core-shell
315
microspheres with different coated layers stated clearly the alternative deposition of
316
the polycation CS-g-PEG copolymer and the hybrid anion Fe3O4-CA nanoparticles on
317
the PSS templates to form the hybrid multilayer shells. For each of the intermediate
318
products and the final PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG core-shell
319
microspheres, their positively charged surface is favorable for their dispersion
320
stability due to the electrostatic repulsion.
te
d
M
an
us
cr
ip t
309
Different from the polyelectrolyte multilayers, the zeta potentials of the core-shell
321
323 324 325 326
hybrid microspheres remained positive during the LbL assembly procedure in the
Ac ce p
322
present work. It is due to that the assembled anion used here is negative charged Fe3O4-CA nanoparticles with particle size of less than 10 nm, which could not cover completely the surface of the CS-PEG layers. The zeta potential of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres was 20.1 mV. It means that
327
the hybrid hollow microspheres obtained after the following template-removal
328
procedure might be positively charged. The positively charged surfaces show
329
pH-sensitive cell interactions, which enables specific drug delivery to cells in a
330
weakly acidic environment, such as the tumor tissues [32]. And their surface has been 15
Page 15 of 38
typically protected by PEG brushes to achieve the prolonged circulation and evasion
332
of immune clearance via reducing opsonization and phagocytic uptake by the
333
hydrophilic surface. So the final resultant hybrid hollow microspheres are expected to
334
be potential tumor-specific drug delivery system [32].
ip t
331
cr
335
3.4. Core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres
337
The TEM images of the PSS templates and the core-shell
338
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres are compared in Figure 1.
339
Their average diameters were about 436 nm and 526 nm, respectively. Obviously, as
340
the hybrid multilayer successfully coated onto the PSS templates, the average
341
diameter increased. Comparing the average diameters of the PSS templates and the
342
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, one can calculate
343
the thickness of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid shells of 45 nm.
345 346 347 348
an
M
d
te
Ac ce p
344
us
336
3.5. (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres The FT-IR spectra of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres are compared in Figure 3. The characteristic absorbance of benzene ring in polystyrene at 3059, 3025cm-1 (C-H,
349
stretching vibration), 1601, 1492 and 1452 cm-1(C=C, stretching vibration), 699 cm-1
350
(out of plane blending vibration) and the characteristic absorbance of carbonyl
351
stretching of the carboxyl groups in MAA and sulfonic acid groups at 1698 and 1074
352
cm-1, which were present in the FT-IR spectrum of the core-shell 16
Page 16 of 38
353
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, disappeared in that of the
354
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres, indicating that the PSS
355
templates had been completely removed by washing with DMF.
ip t
After the PSS templates encapsulated in the core-shell
356
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres had been etched with DMF,
358
the well-defined (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
359
were obtained with an average diameter of 510 nm (Figure 1(d)). Compared with that
360
of the core-shell microspheres, the average diameter hybrid hollow microspheres
361
decreased 16 nm, due to the shrinkage of the hybrid shells. The XRD patterns of the
362
core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and the
363
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres are similar as those
364
of the Fe3O4-CA nanoparticles (Figure S3). It indicates that the structure of the
365
magnetic nanoparticles keeps essentially unchanged during the LbL assembly and
366
washing manipulations. After the removal of the PSS templates, the saturation
368 369 370
us
an
M
d
te
Ac ce p
367
cr
357
magnetization of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres increased to 37.23 emu/g from 25.98 emu/g of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres. They also exhibited the superparamagnetic property (Figure S4) [33]. Compared with the saturation
371
magnetization of the Fe3O4-CA nanoparticles, their magnetite content could be
372
calculated as 64.14%. Figure 4 shows the digital photographs of the aqueous dispersions of the core-shell
373 374
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and the 17
Page 17 of 38
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or
376
without (left) external magnetic field, which were well dispersed in water under
377
normal conditions. This result indicates that the core-shell microspheres and the
378
hybrid hollow microspheres can be easily manipulated by an external magnetic field.
379
The superparamagnetic characteristics with high saturation magnetization of the
380
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres predict their potential
381
application for magnetic resonance imaging and magnetic-target drug delivery [21].
us
cr
ip t
375
an
382
3.6. Stimuli-responsive properties
384
The influence of ionic strength on the average hydrodynamic diameter of the
385
superparamagnetic hybrid hollow microspheres ((CS-g-PEG/Fe3O4-CA)4/CS-g-PEG)
386
was studied by dynamic light scattering (DLS) technique. It is found that introducing
387
small molecule electrolytes (e.g., NaCl) had a significant influence on the size of the
388
obtained hybrid hollow microspheres, the average hydrodynamic diameter of the
390 391 392
d
te
Ac ce p
389
M
383
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres increased from 407.1 to 488.4 nm with increasing the ionic strength in the range of 0-0.20 mol/L NaCl (Figure 5a), due to the shielding effect of the small electrolytes on the electrostatic force [34]. However, it remained constant (488.4 nm) at higher salt concentration
393
(0.15-0.20 mol/L NaCl), covering the NaCl concentration in the physiological media
394
of 0.154 mol/L. It showed that neither aggregation nor fusion of the
395
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres will occur in the
396
physiological media due to the surface concealing effect of the PEG brushes [18]. 18
Page 18 of 38
The pH dependence of the average hydrodynamic diameter of the
397
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was also investigated
399
by DLS. As shown in Figure 5b, the average hydrodynamic diameter of the
400
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres decreased from 489.5
401
to 413.3nm with increasing pH value from 3 to 11, while the average hydrodynamic
402
diameter of the hybrid hollow microspheres remained the same in the near neutral
403
media (pH between 5 and 7). In the basic media, the hybrid hollow microspheres
404
shrank with the increasing of the media pH values, due to the deprotonation of the
405
amino groups in the CS-g-PEG copolymer [35]. At low pH values, the ionization of
406
the carboxyl groups of the Fe3O4-CA nanoparticles was normally depressed and the
407
amino groups in CS-g-PEG were protonated [22], resulting to the expansion of the
408
hybrid shells. It is favorable to the drug-release in the media with lower pH values
409
such as the acidic tumor mircoenvironmets [36].
411 412 413 414
cr
us
an
M
d
te
Ac ce p
410
ip t
398
3.7. Drug-loading and controlled release The drug-loading and pH-responsive controlled release performance of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was investigated with an anticancer drug doxorubicin (DOX) as model drug. The DOX-loading capacity
415
was calculated to be 10.44 %. It is lower than those data of the polyelectrolyte
416
multilayer hollow microspheres reported, due to the lower content of the carboxyl
417
groups in the hybrid hollow microspheres, as the Zeta potential analysis (Figure 2).
418
The in vitro drug releases from the DOX-loaded hybrid hollow microspheres were 19
Page 19 of 38
performed at 37°C under three different simulated body fluids (pH 5.0, 6.5 and 7.4).
420
The drug release was faster in the lower pH media and the cumulative release of the
421
DOX was 54.67 %, 34.59 %, and 29.78 % at pH 5.0, 6.5 and 7.4 at 37 °C within 50 h
422
(Figure 6). The pH-dependent release of DOX was observed that the releasing ratio
423
and cumulative release of DOX from the drug carriers was quicker and higher at pH
424
5.0 than those at pH 6.5 or pH 7.4.
us
cr
ip t
419
However, the common feature of these three curves is that the release curve does not
425
tend to be flat, and gradually upward trend as the growth of the time. The results of
427
the DOX cumulative release at different pH media revealed that the release at low pH
428
could partly be attributed to the fact that the permeability of the polyelectrolyte hybrid
429
shells was better at low pH values than at high ones, because of the decrease in pH
430
weakened the ionic crosslinking bonds between the CS-PEG copolymer and the
431
Fe3O4-CA nanoparticles due to the decrease in the charge density of citric acid and the
432
crosslinking density. Furthermore, the hybrid shells were positively charged at low
434 435 436
M
d
te
Ac ce p
433
an
426
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 the CS-PEG, which might make such configuration unstable so that the water soluble DOX molecules were impelled across the hybrid shells. The pH-dependent release of DOX from the
437
superparamagnetic multilayer hybrid hollow microspheres has a great advantage in
438
curing the cancer cells with an acid medium about pH at 5 [37,38].
439 440
3.8. Cytocompatibility 20
Page 20 of 38
The in vitro cytocompatibility of the superparamagnetic
442
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres was studied with
443
HepG2 cells using SRB assays. The cells were incubated with the hollow
444
microspheres and the DOX-loaded hollow microspheres for 48 h at various
445
concentrations (0-40 μg/mL). The result indicated that the
446
(CS-g-APEG/Fe3O4-CA)4-CS-g-APEG hybrid hollow microspheres showed high
447
biological compatibility (cell viability = 88.86-70.19 %) up to a tested concentration
448
of 10-40 μg/mL (Figure 7).
an
us
cr
ip t
441
Moreover, the DOX-loaded hollow microspheres had higher toxicity (cell viability =
449
72.07-29.45 %) up to a tested concentration of 10-40 μg/mL. With increasing the
451
tested concentration of the hybrid hollow microspheres, the cell viability had small
452
change. So it could be concluded that the superparamagnetic hybrid hollow
453
microspheres (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG exhibit preferably biocompatibility.
454
Furthermore, the cytotoxicity was found to be reduced by using the
456 457 458
d
te
Ac ce p
455
M
450
superparamagnetic hybrid hollow microspheres as drug carriers with the same DOX dosage (Figure 8). And the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres possessed higher antitumor activity only with higher dosages.
459
4. Conclusions
460
In the present work, biocompatible superparamagnetic pH-stimuli responsive
461
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with magnetite
462
content of 64.14% and saturation magnetization of 37.23 emu/g were desiged with the 21
Page 21 of 38
Fe3O4-CA nanoparticles as hybrid anion via the LBL assembly technique. They
464
exhibit stable dispersibility in the physiological media. Their positively charged
465
surfaces showing the pH-sensitive cell interactions and the pH-stimuli responsive
466
property could enable the specific drug delivery to cells such as the tumor
467
microenvironments. All the features make them potential magnetic-targeting
468
tumor-specific drug delivery system, magnetic hyperthermia therapy, or magnetic
469
resonance imaging.
us
cr
ip t
463
an
470
Acknowledgments
472
This work was supported by the Fundamental Research Funds for the Central
473
Universities and the Open Project of Key Laboratory for Magnetism and Magnetic
474
Materials of the Ministry of Education, Lanzhou University.
475
477 478 479 480
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Ac ce p
476
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471
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te
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Ac ce p
542
M
538
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25
Page 25 of 38
544
Figure Captions
545 546
Scheme 1. Schematic illustration of the fabrication of the pH-stimuli responsive superparamagnetic (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow
548
microspheres.
Figure 1. TEM images of (a) the Fe3O4-CA nanoparticles, (b) the PSS templates, (c)
cr
549
ip t
547
the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, and (d)
551
the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres.
Figure 2. Zeta potentials of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)n
an
552
us
550
microspheres (n = 1, 2, 3, 4) (mean ± standard deviation, n=3).
554
M
553
Figure 3. The FT-IR spectra of the core-shell
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and
556
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
558 559 560 561 562
te
Figure 4. Photographic images of (a) the core-shell
Ac ce p
557
d
555
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and (b) the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or without (left) external magnetic field.
Figure 5. Ionic strength (a) and pH (b) dependences of average hydrodynamic diameter (D) of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres (mean ± standard deviation, n=3).
563 564
Figure 6. In vitro drug release from the DOX-loading superparamagnetic hybrid
565
hollow microspheres at pH 5.0, 6.5 and 7.4 at 37 °C, respectively (mean ±
566
standard deviation, n=3). 26
Page 26 of 38
567
Figure 7. Cell viability data of the CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and the DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
569
(mean ± standard deviation, n=3).
570
Figure 8. Antitumor activity of free DOX and the DOX-loaded
ip t
568
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres for 48h (mean ±
572
standard deviation, n=3).
cr
571
Ac ce p
te
d
M
an
us
573 574
27
Page 27 of 38
574 575
adsorption CS-g-PEG
repeating four times
dialyze
CS
PSS
PEG
us
576 577
cr
adsorption CS-g-PEG
DMF
ip t
adsorption Fe 3O4 -CA
Fe3O4 -CA
Scheme 1. Schematic illustration of the fabrication of the pH-stimuli responsive
an
superparamagnetic (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres.
578
M
579
Ac ce p
te
d
580
28
Page 28 of 38
580
cr
ip t
581
us
582
(a)
(b)
584
(c)
Figure 1. TEM images of (a) the Fe3O4-CA nanoparticles, (b) the PSS templates, (c)
588 589 590
Ac ce p
586 587
(d)
te
585
d
M
an
583
the core-shell PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres, and (d) the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hollow microspheres.
29
Page 29 of 38
590 591 592
ip t
35
cr
30 25 20 15 0
2
4
6
8
10
an
layer number (2n)
us
Zetal potential (mV)
40
593
Figure 2. Zeta potentials of the core-shell PSS@(CS-g-PEG/Fe3O4-CA)n
595
microspheres (n = 1, 2, 3, 4) (mean ± standard deviation, n=3).
M
594
d
596
Ac ce p
te
597
30
Page 30 of 38
597 598
1698
591
80 2852 3422
1492 1452 1650
1601
60 50
cr
70
1073 1698
3025
us
Transmittance (%)
3059
ip t
1099
90
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG)
an
4000 3500 3000 2500 2000 1500 1000
592
500
Wavenumber ( cm ) -1
599
Figure 3. The FT-IR spectra of the core-shell
601
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
602
hybrid hollow microspheres.
te
d
M
600
604
Ac ce p
603
31
Page 31 of 38
604 605 606
608 609
us
cr
ip t
607
(a)
(b)
Figure 4. Photographic images of (a) the core-shell
an
610
PSS@(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG microspheres and (b) the
612
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres with (right) or
613
without (left) external magnetic field.
d
M
611
te
614
Ac ce p
615
32
Page 32 of 38
615 616
500 500
(a)
(b)
480
460 440 420
ip t
D (nm)
460 440 420
400
400 0.00
0.05
0.10
0.15
0.20
2
4
CNaCl (mol/L)
pH
8
10
12
us
617
6
cr
D (nm)
480
Figure 5. Ionic strength (a) and pH (b) dependences of average hydrodynamic
619
diameter (D) of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres
620
(mean ± standard deviation, n=3).
an
618
M
621
Ac ce p
te
d
622
33
Page 33 of 38
622 623
pH 5.0 pH 6.5 pH 7.4
ip t
50 40 30 20 10 0
500
1000
1500
2000
2500
t (min)
624
3000
3500
us
0
cr
Cumulative release (%)
60
Figure 6. In vitro drug release from the DOX-loading superparamagnetic hybrid
626
hollow microspheres at pH 5.0, 6.5 and 7.4 at 37 °C, respectively (mean ± standard
627
deviation, n=3).
M
an
625
628
Ac ce p
te
d
629
34
Page 34 of 38
629 630
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG
cr
80 60 40 20 0 10
20
30
Concentration ( μg/mL)
40
an
0
us
Cell viability (%)
100
ip t
631
632
Figure 7. Cell viability data of the (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG and the
634
DOX-loaded (CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres (mean
635
± standard deviation, n=3).
d
M
633
te
636
Ac ce p
637
35
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637 638
free DOX DOX-loaded ( CS-g-PEG/Fe3O4-CA) 4/CS-g-PEG
cr
80 60 40 20 0 0
2
4
6
8
10
an
DOX (ug/mL)
us
Cell viability (%)
100
ip t
639
640
Figure 8. Antitumor activity of free DOX and the DOX-loaded
642
(CS-g-PEG/Fe3O4-CA)4/CS-g-PEG hybrid hollow microspheres for 48h (mean ±
643
standard deviation, n=3).
d
M
641
Ac ce p
te
644
36
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644 645 646 647 648
Highlights Biocompatible pH‐responsive superparamagnetic hollow microspheres were fabricated. They exhibited stable dispersibility in the physiological media. Their positively charged surfaces enabled the specific drug delivery to tumor. The pH‐responsive property enabled the selective release in the acidic media.
Ac ce p
te
d
M
an
us
cr
ip t
649
37
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Graphical Abstract The superparamagnetic characteristics, excellent stability and pH dependent DOX release make the well‐defined(CS‐g‐PEG/Fe3O4‐CA)4/CS‐g‐PEG hybrid hollow microspheres potential platform for tumor‐specific delivery.
an
us
cr
ip t
649 650 651 652 653
654
M
655
Ac ce p
te
d
656
38
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