Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized...

8
Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle Composite Inspired by Mussel Thread Coating and Squid Beak Interfacial Chemistry Ondrej Zvarec, ,Sreekanth Purushotham, § Admir Masic, Raju V. Ramanujan, and Ali Miserez ,,, * School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798; Centre for Biomimetic Sensor Science, Nanyang Technological University, Singapore 637553; § Energy Research Institute at NTU, Nanyang Technological University, Singapore 637553; Max-Planck Institute of Colloids and Interfaces, Department of Biomaterials, Science Campus Golm, 14424 Potsdam, Germany; School of Biological Sciences, Nanyang Technological University, Singapore 639798. * S Supporting Information ABSTRACT: Biological materials oer a wide range of multifunctional and structural properties that are currently not achieved in synthetic materials. Herein we report on the synthesis and preparation of bioinspired organic/inorganic composites that mimic the key physicochemical features associated with the mechanical strengthening of both squid beaks and mussel thread coatings using chitosan as an initial template. While chitosan is a well-known biocompatible material, it suers from key drawbacks that have limited its usage in a wider range of structural biomedical applications. First, its load-bearing capability in hydrated conditions remains poor, and second it completely dissolves at pH < 6, preventing its use in mild acidic microenvironments. In order to overcome these intrinsic limitations, a chitosan-based organic/inorganic biocomposite is prepared that mimics the interfacial chemistry of squid beaks and mussel thread coating. Chitosan was functionalized with catechol moieties in a highly controlled fashion and combined with superparamagnetic iron oxide (γ-Fe 2 O 3 ) nanoparticles to give composites that represent a signicant improvement in functionality of chitosan-based biomaterials. The inorganic/organic (γ-Fe 2 O 3 /catechol) interfaces are stabilized and strengthened by coordination bonding, resulting in hybrid composites with improved stability at high temperatures, physiological pH conditions, and acid/base conditions. The inclusion of superparamagnetic particles also makes the composites stimuli-responsive. INTRODUCTION Synthetic materials that mimic the chemical and structural complexity of biological systems are the holy grailof bioinspired composite formulation. With increasing under- standing of structureproperty relationships of many natural materials at various length scales, from the molecular level up to the micro- and macro-scale, an increasing number of bioinspired synthetic strategies are emerging. 1 Among the various length scales probed in biological materials, the molecular scale, which particularly includes the elucidation of primary sequence in protein-based materials, their post- translational modications, or their interfacial chemistry, perhaps remains the most elusive. However, there are notable exceptions of biological materials whose molecular-scale designs have been elucidated, and those represent unique source of bioinspired synthesis strategies. Two such materials include squid beaks and mussel thread cuticle coatings, whose strengthening mechanisms at the molecular scale have recently been revealed. Squid beaks are highly sclerotized chitin-protein- water composites with graded structural properties that correlate with molecular gradients. 2 The compliant region attached to the jaw muscular tissue is mostly composed of hydrated chitin (80 wt. pct water) with a small amount of hydrophilic proteins. Moving toward the hard rostrum, the water is gradually exchanged by Gly- and His-rich proteins. These proteins impregnate the chitinous nanobrillar network, and are subsequently stabilized by covalent cross-linking between His and catecholic residues, the latter originating either from post-translational modication of Tyr into dihydroxylphenylalanine (DOPA), or from secretion of low- molecular weight catecholic compounds. This curing mecha- nism has a dual role in squid beak maturation. First, it results in a dense cross-linked network of proteins that enclose the chitin nanobrous network. Second, it drives water molecules out of the chitin network, stiening the chitin ber by localized dehydration. 3 Hence, a key physicochemical characteristic is the controlled desolvation of molecular-bound water from the chitin polymer. In mussel thread coatings, a central and unusual Received: May 20, 2013 Revised: July 16, 2013 Published: July 18, 2013 Article pubs.acs.org/Langmuir © 2013 American Chemical Society 10899 dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 1089910906

Transcript of Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized...

Page 1: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

Catechol-Functionalized Chitosan/Iron Oxide NanoparticleComposite Inspired by Mussel Thread Coating and Squid BeakInterfacial ChemistryOndrej Zvarec,†,‡ Sreekanth Purushotham,§ Admir Masic,∥ Raju V. Ramanujan,† and Ali Miserez†,‡,⊥,*†School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798;‡Centre for Biomimetic Sensor Science, Nanyang Technological University, Singapore 637553;§Energy Research Institute at NTU, Nanyang Technological University, Singapore 637553;∥Max-Planck Institute of Colloids and Interfaces, Department of Biomaterials, Science Campus Golm, 14424 Potsdam, Germany;⊥School of Biological Sciences, Nanyang Technological University, Singapore 639798.

*S Supporting Information

ABSTRACT: Biological materials offer a wide range ofmultifunctional and structural properties that are currentlynot achieved in synthetic materials. Herein we report on thesynthesis and preparation of bioinspired organic/inorganiccomposites that mimic the key physicochemical featuresassociated with the mechanical strengthening of both squidbeaks and mussel thread coatings using chitosan as an initialtemplate. While chitosan is a well-known biocompatiblematerial, it suffers from key drawbacks that have limited itsusage in a wider range of structural biomedical applications. First, its load-bearing capability in hydrated conditions remains poor,and second it completely dissolves at pH < 6, preventing its use in mild acidic microenvironments. In order to overcome theseintrinsic limitations, a chitosan-based organic/inorganic biocomposite is prepared that mimics the interfacial chemistry of squidbeaks and mussel thread coating. Chitosan was functionalized with catechol moieties in a highly controlled fashion and combinedwith superparamagnetic iron oxide (γ-Fe2O3) nanoparticles to give composites that represent a significant improvement infunctionality of chitosan-based biomaterials. The inorganic/organic (γ-Fe2O3/catechol) interfaces are stabilized and strengthenedby coordination bonding, resulting in hybrid composites with improved stability at high temperatures, physiological pHconditions, and acid/base conditions. The inclusion of superparamagnetic particles also makes the composites stimuli-responsive.

■ INTRODUCTION

Synthetic materials that mimic the chemical and structuralcomplexity of biological systems are the “holy grail” ofbioinspired composite formulation. With increasing under-standing of structure−property relationships of many naturalmaterials at various length scales, from the molecular level up tothe micro- and macro-scale, an increasing number ofbioinspired synthetic strategies are emerging.1 Among thevarious length scales probed in biological materials, themolecular scale, which particularly includes the elucidation ofprimary sequence in protein-based materials, their post-translational modifications, or their interfacial chemistry,perhaps remains the most elusive. However, there are notableexceptions of biological materials whose molecular-scaledesigns have been elucidated, and those represent uniquesource of bioinspired synthesis strategies. Two such materialsinclude squid beaks and mussel thread cuticle coatings, whosestrengthening mechanisms at the molecular scale have recentlybeen revealed. Squid beaks are highly sclerotized chitin-protein-water composites with graded structural properties thatcorrelate with molecular gradients.2 The compliant region

attached to the jaw muscular tissue is mostly composed ofhydrated chitin (∼80 wt. pct water) with a small amount ofhydrophilic proteins. Moving toward the hard rostrum, thewater is gradually exchanged by Gly- and His-rich proteins.These proteins impregnate the chitinous nanofibrillar network,and are subsequently stabilized by covalent cross-linkingbetween His and catecholic residues, the latter originatingeither from post-translational modification of Tyr intodihydroxylphenylalanine (DOPA), or from secretion of low-molecular weight catecholic compounds. This curing mecha-nism has a dual role in squid beak maturation. First, it results ina dense cross-linked network of proteins that enclose the chitinnanofibrous network. Second, it drives water molecules out ofthe chitin network, stiffening the chitin fiber by localizeddehydration.3 Hence, a key physicochemical characteristic is thecontrolled desolvation of molecular-bound water from thechitin polymer. In mussel thread coatings, a central and unusual

Received: May 20, 2013Revised: July 16, 2013Published: July 18, 2013

Article

pubs.acs.org/Langmuir

© 2013 American Chemical Society 10899 dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−10906

Page 2: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

characteristic that hardens the protective layer (known as thecuticle) is the presence of DOPA-Fe3+ coordination bonds,which cross-link the cuticle proteins into dense micro- andnanoscale globular particles.4,5 Under mechanical stress, theload is transferred onto the dense DOPA-Fe3+ globular regions,circumventing cuticle failure through formation of micro-fractures, highlighting the role of the DOPA-Fe3+ coordinationas a sacrificial bond.6

In the first stage of our composite strategy, we functionalizedchitosan fibers (CS) with catecholic moieties (CAT) in acontrolled manner, with the dual goals of (i) reducing thenumber of hydrophilic amine motifs along the backbone, and(ii) providing the polymer with side-chain functionality thattriggers subsequent cross-linking (Scheme 1). Catecholic

functional groups were chosen because of their versatilecharacteristics,7 including covalent cross-linking8 and coordina-tion bonding.6,9 This is followed by induced coordination offunctionalized chitosan with superparamagnetic iron oxide (γ-Fe2O3) nanoparticles, allowing catecholic side groups to form arobust coordinated-bonded network with the surface of ironoxide nanoparticles, similar to the catechol-Fe3+ coordinationcross-linked network observed in mussel thread coatings.6 Theaddition of superparamagnetic γ-Fe2O3 nanoparticles (MNP)enables magnetic functionality,10 making these hybrid compo-sites multifunctional, robust, and stimuli-active materials thatcould be employed in various biomedical devices.

■ EXPERIMENTAL SECTIONSynthesis of Iron(III) Oxide (Fe2O3) Nanoparticles. FeCl3·

6H2O (27g, 0.1 M) and FeCl2·4H2O (10g, 0.05M) were dissolved inultrapure Milli-Q water (200 mL) and heated to 80 °C under nitrogen.To the solution was added 0.14 M NaOH dropwise over 20 min andthe solution was stirred at 80 °C for 30 min. The solution was thencooled, centrifuged at 8000 rpm for 15 min, and the precipitate wascollected. The precipitate was washed with water (5 × 200 mL) andmagnetically separated to give magnetite nanoparticles (Fe3O4), whichwere stored under an inert atmosphere. Magnetite nanoparticles(Fe3O4) were converted to maghemite (γ-Fe2O3) by oxidation in air at150 °C for 1 h, yielding particles with an average size of 11.6 nm (SD= 3).

Synthesis of Catechol-Functionalized Chitosan (CATCS). To asolution of 92% deacetylated chitosan (1 equiv) in water (∼500−800mL) was added at ambient temperature 1-hydroxybenzotriazolehydrate (HOBT) (2 equiv) and stirred for 1 h. Respective Catechol(0.25−3 equiv) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDCI) (2 equiv) in ethanol (100 mL) was added andthe solution stirred for 7 days. The solution was dialyzed with wateruntil the dialysate tested negative to the Arnow assay (typically 6 × 4ltr changes). The resulting solution was then lyophilized to give therequired functionalized chitosan polymer. L-DOPA ; 1H NMR (400MHz; D6-DMSO-d6, δ) 12.47 (s, 1H), 8.71 (s, 2H), 6.94 (d, 1H, J =6.0 Hz), 6.92 (t, 1H, J = 4 Hz), 6.47 (d, 1H, J = 4 Hz), 4.01−3.85 (m,1H), 2.80−2.63 (m, 2H), 1.34 (s, 9H).11 Chitosan; 1H NMR (400MHz; D2O, δ) 3.83 (s, 1H) 3.65 (s, 2H), 3.09 (3H).12 DOPACS (2),1H NMR (400 MHz; D2O, δ) 7.79−7.50 (m, 3H, DOPA) 3.82 (s, 1H,chitosan), 3.63 (s, 2H, chitosan). 3.08 (s, 3H, chitosan), 2.81−2.79 (m,2H, DOPA). DHACS (3),

1H NMR (400 MHz; D2O, δ) 7.79−7.49(m, 3H, DHA), 3.81 (s, 1H, chitosan), 3.63 (s, 2H, Chitosan), 3.07 (s,3H, chitosan), 2.81−2.78 (m, 2H, DHA), 2.71−2.69 (m, 1H, DHA),2.53−2.56 (m, 1H, DHA).

Chitosan Polymer-Fe2O3 Nanoparticle Incorporation Prep-aration (mnp-CATCS). To a solution of requisite chitosan polymer(100 mg) in 15% acetic acid (20 mL) was added Fe2O3 nanoparticle(1 equiv or 0.1 equiv respective to CATCS monomer unit) in 1 mL in asingle portion and allowed to stir for 72 h. The solution was dialyzedwith water (3 × 4 ltr changes), and the solution decanted fromunreacted nanoparticles into glass molds (∼5 cm diameter) and curedunder atmospheric conditions. The resulting cured film was washedwith water to give the desired composites.

Surface Chemistry Analysis. 1H NMR spectra were obtainedusing a Bruker AVANCE I 400 MHz spectrometer in either deuterateddimethyl sulfoxide, chloroform, or water. Dimethyl sulfoxide (1H =2.50 ppm, 13C = 39.5 ppm), chloroform (1H = 7.26 ppm, 13C = 77.0ppm) and water (1H = 4.79 ppm) were used as internal standards forNMR analysis. Arnows assay was performed as per literature for eachpolymer to identify the presence of catechol.13,14 UV−vis spectra wereobtained using a Thermo Scientific NanoDrop 2000c spectropho-tometer for all modified chitosan polymers. Fourier transform infrared(FTIR) spectra were recorded for all polymers using a Bruker Vertex70. For Raman spectroscopy, a continuous laser beam was focused onthe sample through a confocal Raman microscope (CRM200, WITec)equipped with a piezo-scanner (P-500, Physik Instrumente). Thediode-pumped 785 nm laser excitation (Toptica Photonics AG) wasused in combination with a 20× objective lens (Nikon, NA = 0.4). Thespectra were acquired using a CCD camera (DU401A-DR-DD,Andor) behind a grating (300 g mm−1) spectrograph (Acton,Princeton Instruments Inc.) (for each spectrum a total of 30 sintegration time was applied). The ScanCtrlSpectroscopyPlus software(version 2.08, Witec) was used for spectra processing.

Physical and Magnetic Properties Analysis. Transmissionelectron microscopy samples of the nanoparticles were prepared bydrying a drop of the aqueous colloidal suspension on a copper grid. AJEOL 2010F transmission electron microscope operating at 200 kVwas used for bright field imaging and particle size measurements weremade using ImageJ software (see Figure S1a of the SupportingInformation, SI). X-ray diffraction scans were performed on the ironoxide nanoparticle powder using a Shimadzu 6000 X-ray diffrac-tometer with Cu Kα radiation of wavelength 1.54 Å at a scan rate of0.5° min−1 through the 2Θ range of 10°−90°. The Intensity vs 2Θ plotwas matched to reference JCPDS files to indentify the phase and indexthe peaks (see Figure S1b of the SI). Magnetic measurements of theiron oxide nanoparticles as well as composite films were performed atroom temperature up to an applied field strength of 20 kOe with avibrating sample magnetometer attached to a Quantum Design PPMS-6500 physical property measurement system.

Mechanical Properties Analysis. Nanoindentation studies ofcomposites were completed using a Hysitron TI950 triboindenter.Samples were mounted in epoxy resin before analysis. Dehydratedconditions: Using a 20 μm conical diamond probe with a load of 300μN a series ∼20−25 indentations across a 200 μm grid were

Scheme 1. Synthetic Strategy of Chitosan Derived Bio-Inspired Organic/Inorganic Composites

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610900

Page 3: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

performed for each composite. Hydrated conditions: Samples werepresoaked before analysis in ultrapure milli-q water. With a 20 μmconical diamond probe a series of ∼20−25 indentations at a controlledload of 100 μN across a 200 μm grid were acquired for eachcomposite. Thermal gravimetric analysis of the composite films wasperformed using a TA Instruments Q500 TGA system. Weight loss ofbare iron oxide nanoparticles as well as pure chitosan was measured forreference. Samples (∼5 mg) were heated in the temperature range30−700 °C at a ramp rate of 10 °C·min−1 in air and N2.

■ RESULTS AND DISCUSSION

Using water-soluble EDCI/HOBT carbodiimide chemistry, aseries of CATCS polymers were prepared with a catecholiccontent in the range of 20−82%, representing the highestreported example of covalently bonded catechol into apolysaccharide backbone (see Table S1 of the SI). In tandem,recent developments in catechol functionalization of thechitosan backbone have emerged.15 A similar level of catecholicfunctionalization was recently reported on polysiloxaneelastomers by Heo et al.,16 with the goal of providing highaffinity toward oxides. By limiting the addition of catechol,amidation could be achieved with highly controlled monomericratios. Typically, 1D HNMR (see Figures S2−S5 of the SI)spectrum showed resonances in the region of 6.64−6.81 ppm,attributed to the aryl motif of the catechol with reduction of theamine peak at 3.08 ppm, all of which confirmed the amidationof chitosan’s backbone. The “active” phenolic motifs wereestablished through Arnow’s staining, an effective assay inidentifying the presence of catecholic motifs.17 Iron oxidenanoparticles were incorporated into CATCS polymers, and theimpregnation process was controlled such that molar ratios of1:1 and 1:0.1 (CATCS: MNP) were obtained and cast into thinfilms. With ∼82% catechol functionalization (4a), thecomposites yielded a clustered microstructure, while with∼47% functionalization, the composites (4b/d) were brittle.

During thin film deposition, appropriate films of uniformthickness (35−100 μm) were only obtained when thecomposites catecholic content was below 50% (4c/e),presumably due to the intrinsic hydrophobic interactions withthe high catecholic content. These composites were sub-sequently characterized in more detail.In order to probe the nature of the functionalized chitosan/

Fe2O3 interfaces, the composites were investigated by FTIRand Raman spectroscopy at various stages of processing. FTIRspectroscopy (Figure 1a) of CATCS polymers showed theexpected signals associated with the polysaccharide back-bone18−21 and the presence of the catecholic motif includingthe aryl6 (1405 cm−1) and phenolic alcohol bands (1267cm−1).22 Physical attachment of the catechol was confirmedwith the presence of the amide I/II and carbonyl bands in theregion 1532−1706 cm−1. For unmodified chitosan, iron oxideaddition resulted in a sharpening of the amine band with a shiftof the CO alcohol band, suggesting close proximity of the ironoxide to these motifs.23 Solubility tests (see Table S2 of the SI)revealed that these observations are most likely a result ofphysical entrapment and not coordination for unmodifiedchiton-based composites. The FTIR spectral changes observedfor mnp-CATCS composites were more pronounced, confirm-ing the formation of the γ-Fe2O3-catechol coordination bondthrough loss of the phenolic band at 1260 cm−1. Furtherindicative of the new coordination bonds, the aryl vibrationalbands broadened and split into two distinct signals, which,commonly occurs when aryl groups are in close proximity tometals.4,24 The amide I/II bands associated with the amidebond grew in intensity, which is also attributed to the closeproximity of these motifs to iron oxide.23 These changes inFTIR spectra signal highlighted the successful coordination ofiron oxide with our catechol-chitosan composites.

Figure 1. FTIR analysis (A) of functional chitosan and composites, pH 3 and Raman spectra (B) of (1:1) mnp-DOPACS and mnp-DHACScomposites.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610901

Page 4: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

Raman spectroscopy of mnp-CATCS (Figure 1b) showed thepresence of key [M−O] vibrational frequencies in the range500 to 700 cm−1, which also supports the presence of γ-Fe2O3-catechol coordination bonds. These vibrational frequencieshave been previously outlined as key features for oxygen−metalchelation, with the 591 and 635 cm−1 indicative of coordinationof the metal center by the oxygens on C3 and C4 of thecatechol ring, respectively.6,25,26 The 540 cm−1 band arisesupon charge transfer that proceeds only when both phenolicmotifs are chelated to a transitional metal as is the case with γ-Fe2O3-catechol chelation.26 Upon wetting the samples, thesignals associated with chelation remained unchanged evenwith applied strains 50% that of the strain to failure. Theseresults suggest that the γ-Fe2O3-catechol coordinated networkis stabilized by a relatively strong binding energy and that thecoordinated bonds do not undergo reversible bindinginteractions, as has been documented for Fe3+-DOPA richmfp-1 protein27,28 but in contrast to functionalized PEG single-molecules.29 Further to this TGA measurement indicated thatthese prevailing coordination bonds are thermally stable up to276 °C (see Figure S6 and Table S3 of the SI).With higher catecholic (DOPA/DHA) content in the

composites, water uptake decreased leading to a dehydrationeffect due to the increasing hydrophobic motifs expelling water.In tandem, this modification led to improved stability (Figure2a), with all of the modified films shown to be resistant to awider pH range from basic (∼10) to acidic (∼3) conditions. Ithas been well-documented30−33 that chitosan is soluble in mildacidic conditions with its exposed hydrophilic amine groupsprotonated at low pH. With our process, the newly formed

amide bond decreases the charged state of chitosan at low pH,thus improving the stability of the CATCS composites at pH 3.Addition of iron oxide into neat chitosan composites gavematerials that were still susceptible to mild acidic environments,with the composites completely degrading within 24hrs. Thiswas not seen for the mnp-CATCS composites, highlighting theimportance of γ-Fe2O3-catechol coordination in maintainingphysical integrity and opening the pathway for use underphysiological conditions, which is a key feature for biomedicalapplications. In summary, the vibrational spectroscopy dataindicate that our composites successfully mimic key interfacialchemistry features from both squid beaks and mussel threadcuticles. First they mimic through catechol-assisted dehydrationthe controlled dehydration of chitin as in the squid beak; andsecond the functionalized chitin/inorganic interfaces arestabilized through the formation of γ-Fe2O3-catechol coordina-tion bonds that mimic DOPA-Fe3+ bonds detected in cuticlemussel threads.6

The magnetic property measurements (Figure 2b; see TableS4 of the SI) showed the expected magnetization properties for1:1 and 0.1:1 mnp-CATCS composites, with a 10- and 100-folddecrease in magnetization compared to bare γ-Fe2O3 (66.1emu·g−1). This demonstrates the first addition of super-paramagnetic iron chitosan-based scaffolds. The appropriatedecrease in magnetic saturation with mass fractions(CATCS:MNP; 10:1 and 100:1) indicates that: (i) iron oxidenanoparticles retain their magnetic properties once coordinate-bonded, and (ii) no unbound iron oxide is present within thecomposite.

Figure 2. Physical properties of CATCS and mnp-CATCS composites. (A) Polymers hydrated in 5% acetic acid pH 3. (B) Magnetization curves formnp-CATCS films. Control measurements (inset) show an anhysteric loop typical of superparamagnetic nanoparticles, with saturation magnetizationof 66.1 emu·g−1. (C) Representative cat-MNPCS film magnetized with neodymium magnet.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610902

Page 5: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

The mechanical properties of the composites were probed bynanoindentation in both dry and hydrated states, using asphero-conical tip (20 μm nominal radius) and Hertziancontact mechanics. Data summary (see Table S5 of the SI) andrepresentative curves are presented in Figure 3. Nano-mechanical values of the elastic modulus of chitosan andother polysaccharides are usually obtained using the classicalOliver-Pharr analysis.34 However, we found that chitosan-basedmaterials in this work systematically exhibited moduliapproximately 3-fold higher when using the Oliver-Pharrmethod instead of the loading Hertzian portion of anindentation cycle, and attribute this phenomenon to poroelasticbehavior.35 Chitosan film contain a certain degree of porosity,hence it is likely that the network is axially packed duringindentation, leading to higher modulus values at the peak load.The Hertzian contact solution is thus deemed more accurate inorder to obtain elastic moduli of our materials. The indentationresponse of functionalized chitosan in the dry state highlighteda 25% stiffer network with addition of the catechol motif. Alongwith the hydrogen bonding of chitosan, addition of thephenolic motif can induce additional aryl interactions, as well asincrease dehydration and π−π organized packing. With MNPaddition, the elastic modulus of mnp-DOPACS increased, goingfrom 1.45 GPa (±0.06) for unreinforced DOPACS, to 1.65 GPa(±0.17) with 0.1 M iron oxide, up to 1.88 GPa (±0.19) with 1M iron oxide. The increase in modulus was more pronounced

for mnp-DHACS composites, with elastic modulus values of1.84 GPa (±0.21) and 2.37 GPa (±0.29) for 0.1 and 1 M ironoxide, respectively. We speculate that with 0.1 molar iron oxide,the moderate increase in elastic modulus is a result perhaps dueto γ-Fe2O3-DOPA coordination bonds being masked by theinherently high stiffness of dry chitosan fibers. Increasing ironoxide content to 1 molar led to high increases in elasticmodulus, with mnp-DHACS demonstrating nearly a 2-foldincrease while in the case of mnp-DOPACS, the elastic modulusonly increased slightly. We speculate that this observation is afunction of the amine side arm of DOPA imparting “sites” ofirregular organizational packing in the composites, whichdecreases H-bonding and aryl π−π interactions.In the hydrated state, differences between unmodified

chitosan and the hybrid composites become more obvious,with the addition of MNPs mitigating the loss in stiffness uponhydration. Expressed in terms of the ratio Ewet/Edry (Figure 3b)the relative decrease was lower for functionalized chitosan, andeven less pronounced for iron oxide reinforced composites. TheEwet/Edry ratio was 0.24 for raw chitosan, increasing to 0.37−0.48 for functionalized chitosan, and maximal values in therange 0.54−0.58 for MNPs-reinforced composites. Representa-tive nanoindentation curves obtained under hydrated con-ditions (Figure 3c) demonstrate the clear effect of addingMNPs on the indentation response of mnp-CATCS composites.It should also be mentioned that in the wet state, no significant

Figure 3. Nanoindentation analysis of composites. (A) Young’s modulus (E) of dehydrated and hydrated composites. (B) Relative change inYoung’s modulus (E) expressed in terms of the ratio Ewet/Edry. (C) Representative load−displacement curves for each state, left-dehydrated; right-hydrated.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610903

Page 6: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

differences were noticed for the two types of composites (mnp-DOPACS versus mnp-DHACS), with similar elastic modulivalues obtained in both cases. Overall, the best-performingcomposite (mnp-DHACS 1:1 molar ratio) displayed a morethan 3-fold increase in elastic modulus in comparison to rawchitosan in hydrated conditions with a value of 1.3 GPa, whichis higher than currently available chitosan-based materials.36 Inthe hydrated state, our composites exhibit higher modulus(∼50%) and hardness (2-fold) compared to the native musclethread cuticle (M. galloprovincialis),37 while significantly lessthan the squid beak.2 γ-Fe2O3 coordination improves themechanical properties of the functionalized chitosan, though weanticipate that further covalent cross-linking of the catecholmotif would yield further improvements in modulus andhardness as observed in the squid beak.2

Hybrid composites exhibit slight increases in mechanicalproperties in the dry state. Differences in mechanical responseare much more dramatic in the hydrated state, where the elasticmodulus of the hybrid composites is as much as 10-fold higherthan the unmodified chitosan. Previous studies have underlinedthe key role of controlled water desolvation and cross-linking inincreasing the strength and stiffness of sclerotized/chitosancomposites, including squid beaks.3 The present data can beexplained on the basis of equivalent physicochemicalmechanisms resulting from the chemical design inspired byour model systems. First, by mimicking the squid beak model(chitinuous network dehydration), controlled functionalizationof DOPA/DHA moieties imparts the chitosan network withhydrophobic aryl motifs, thereby preventing excessive wateruptake, which has a direct role in plasticizing the chitosannetwork. Second, the γ-Fe2O3−catechol coordinated networkacts as a mimic of the mussel thread coating chemistry (DOPA-Fe3+ coordination), providing sites on the surface of thepolysaccharide fibrils for efficient load transfer to the iron oxideparticles. Finally, a dual role of these two mechanisms is alsoplausible. It is reasonable to assume that the dense network ofγ-Fe2O3-catechol coordination bonds further prevents adsorp-tion of water molecules on the surface of the chitosan chains,thereby mitigating the plasticization effect of water associatedwith interchain shearing during deformation. Overall, thecombined mechanical and chemical degradation results high-light that the γ-Fe2O3−catechol coordination bond is anefficient means to prevent extreme losses in structural integrity(elastic modulus/hardness).

■ CONCLUSIONSOne major challenge in bioinspired materials synthesis andprocessing resides in preparing materials that mimic multiplelength scales of the biological system. Here we have establisheda route toward γ-Fe2O3-catechol clad composites that fulfillsthis goal, whereby chitosan was functionalized with catecholicmotifs up to 82% and coordinated bonded with γ-Fe2O3nanoparticles. Raman spectroscopy confirmed bidentate γ-Fe2O3-catechol chelation, while nanoindentation measurementsdemonstrated that this form of cross-linking is an efficientmeans to prevent loss in structural integrity in the hydratedcondition. This bioinspired strategy also allows us to overcometwo key drawbacks of chitosan, namely its poor load-bearingcapability in hydrated conditions and its solubility in mild acidicmicroenvironments, which chitosan or iron oxide dopedchitosan cannot achieve. The composites exhibit multifunc-tional properties after coordination bonding, displaying super-paramagnetic behavior provided by the iron oxide nano-particles. Biocompatible and magnetically responsive polymer-magnetic nanoparticle composites38,39 have been typicallylimited by their low stiffness in the physiological milieu, thuslimiting the actuation forces that can be generated. The presentcoordinated-bonded hybrid composites, with an elasticmodulus exceeding 1 GPa in hydrated conditions, overcomesthis key limitation, opening the pathway for their use as stimuli-responsive materials in various regenerative applications.

■ ASSOCIATED CONTENT*S Supporting InformationExperimental details; 1H NMR, TGA, TEM, XRD; nano-indentation and magnetization data for chitosan, catechol-chitosan and mnp-CATCS. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSA.Mi. acknowledges the support of the Singapore NationalResearch Foundation (NRF) through a NRF Fellowship. A.Ma.

Figure 4. Hardness (left) and Young’s Modulus E (right) of composites DOPACS, DHACS and native materials (hydrated conditions); muscle threadcuticle (M. galloprovincialis)37 and Humboldt squid beak (tip).2.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610904

Page 7: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

is grateful for support by the Alexander von HumboldtFoundation and the Max Planck Society in the framework ofthe Max Planck Research Award funded by the FederalMinistry of Education and Research. R.V.R. acknowledges thesupport of the U.S. AOARD, Tokyo.

■ REFERENCES(1) Studart, A. Towards High-Performance Bioinspired Composites.Adv. Mat. 2012, 24 (37), 5024−5044.(2) Miserez, A.; Li, Y.; Waite, J. H.; Zok, F. Jumbo Squid beaks:Inspiration for design of robust organic composites. Acta Biomater.2007, 3 (1), 139−149.(3) Miserez, A.; Schneberk, T.; Sun, C.; Zok, F. W.; Waite, J. H. Thetransition from stiff to compliant materials in squid beaks. Science2008, 319 (5871), 1816−1819.(4) Sever, M. J.; Weisser, J. T.; Monahan, J.; Srinivasan, S.; Wilker, J.J. Metal-mediated cross-linking in the generation of a marine-musseladhesive. Angew. Chem., Int. Ed. 2004, 43 (4), 448−450.(5) Holten-Andersen, N.; Mates, T. E.; Toprak, M. S.; Stucky, G. D.;Zok, F. W.; Waite, J. H. Metals and the integrity of a biologicalcoating: The cuticle of Mussel Byssus. Langmuir 2008, 25 (6), 3323−3326.(6) Harrington, M. J.; Masic, A.; Holten-Andersen, N.; Waite, J. H.;Fratzl, P. Iron-clad fibers: A metal-based biological strategy for hardflexible coatings. Science 2010, 328 (5975), 216−220.(7) Sedo, J.; Saiz-Poseu, J.; Busque, F.; Ruiz-Molina, D. Catechol-based biomimetic functional materials. Adv. Mater. 2013, 25 (5), 653−701.(8) Miserez, A.; Rubin, D.; Waite, J. H. Cross-linking chemistry ofsquid beak. J. Biol. Chem. 2010, 285 (49), 38115−38124.(9) Taylor, S. W.; Luther, G. W.; Waite, J. H. Polarographic andspectrophotometric investigation of iron(III) complexation to 3,4-dihydroxyphenylalanine-containing peptides and proteins from Mytilusedulis. Inorg. Chem. 1994, 33 (25), 5819−5824.(10) Amstad, E.; Gillich, T.; Bilecka, I.; Textor, M.; Reimhult, E.Ultrastable iron oxide nanoparticle colloidal suspensions usingdispersants with catechol-derived anchor groups. Nano Lett. 2009, 9(12), 4042−4048.(11) Sever, M. J.; Wilker, J. J. Synthesis of peptides containing DOPA(3,4-dihydroxyphenylalanine). Tetrahedron 2001, 57 (29), 6139−6146.(12) Lai, W.-F.; Lin, M. C.-M. Nucleic acid delivery with chitosan andits derivatives. J. Controlled Release 2009, 134 (3), 158−168.(13) Arnow, L. E. Colorimetric determination of the components of3,4-dihydroxyphenylalaninetyrosine mixtures. J. Biol. Chem. 1937, 118(2), 531−537.(14) Barnum, D. W. Spectrophotometric determination of catechol,epinephrine, dopa, dopamine and other aromatic vic-diols. Anal. Chim.Acta 1977, 89 (1), 157−167.(15) Kim, K.; Ryu, J. H.; Lee, D. Y.; Lee, H. Bio-inspired catecholconjugation converts water-insoluble chitosan into a highly water-soluble, adhesive chitosan derivative for hydrogels and LbL assembly.Biomater. Sci. 2013, 1 (7), 783−790.(16) Heo, J.; Kang, T.; Jang, S. G.; Hwang, D. S.; Spruell, J. M.;Killops, K. L.; Waite, J. H.; Hawker, C. J. Improved performance ofprotected catecholic polysiloxanes for bioinspired wet adhesion tosurface oxides. J. Am. Chem. Soc. 2012, 134 (49), 20139−20145.(17) Waite, J. H.; Tanzer, M. L. Specific colorimetric detection of o-diphenols and 3,4-dihydroxyphenylalanine-containing peptides. Anal.Biochem. 1981, 111 (1), 131−6.(18) Chen, F.; Wang, Z. C.; Lin, C. J. Preparation andcharacterization of nano-sized hydroxyapatite particles and hydrox-yapatite/chitosan nano-composite for use in biomedical materials.Mater. Lett. 2002, 57 (4), 858−861.(19) Jenjob, S.; Sunintaboon, P.; Inprakhon, P.; Anantachoke, N.;Reutrakul, V. Chitosan-functionalized poly(methyl methacrylate)particles by spinning disk processing for lipase immobilization.Carbohydr. Polym. 2012, 89 (3), 842−848.

(20) Zhao, D.-L.; Wang, X.-X.; Zeng, X.-W.; Xia, Q.-S.; Tang, J.-T.Preparation and inductive heating property of Fe3O4−chitosancomposite nanoparticles in an AC magnetic field for localizedhyperthermia. J. Alloys Compd. 2009, 477 (1−2), 739−743.(21) Zhu, H. Y.; Jiang, R.; Xiao, L.; Li, W. A novel magneticallyseparable gamma-Fe2O3/crosslinked chitosan adsorbent: Preparation,characterization and adsorption application for removal of hazardousazo dye. J. Hazard. Mater. 2010, 179 (1−3), 251−257.(22) Bilinska, B. On the structure of human hair melanins from aninfrared spectroscopy analysis of their interactions with Cu2+ ions.Spectrochim. Acta A-Mol. Biomol. Spectrosc. 2001, 57 (12), 2525−2533.(23) Hernandez, R.; Zamora-Mora, V.; Sibaja-Ballestero, M.; Vega-Baudrit, J.; Lopez, D.; Mijangos, C. Influence of iron oxidenanoparticles on the rheological properties of hybrid chitosanferrogels. J. Colloid Interface Sci. 2009, 339 (1), 53−59.(24) Gulley-Stahl, H.; Hogan, P. A.; Schmidt, W. L.; Wall, S. J.;Buhrlage, A.; Bullen, H. A. Surface Complexation of catechol to metaloxides: An ATR-FTIR, adsorption, and dissolution study. Environ. Sci.Technol. 2010, 44 (11), 4116−4121.(25) Farquhar, E.; Emerson, J.; Koehntop, K.; Reynolds, M.; Trmcic,M.; Que, L., Jr. In vivo self-hydroxylation of an iron-substitutedmanganese-dependent extradiol cleaving catechol dioxygenase. JBIC J.Biol. Inorg. Chem. 2011, 16 (4), 589−597.(26) Hwang, D. S.; Harrington, M. J.; Lu, Q.; Masic, A.; Zeng, H.;Waite, J. H. Mussel foot protein-1 (mcfp-1) interaction with titaniasurfaces. J. Mater. Chem. 2012, 22 (31), 15530−15533.(27) Holten-Andersen, N.; Fantner, G. E.; Hohlbauch, S.; Waite, J.H.; Zok, F. W. Protective coatings on extensible biofibres. Nat. Mater.2007, 6 (9), 669−672.(28) Zeng, H.; Hwang, D. S.; Israelachvili, J. N.; Waite, J. H. Strongreversible Fe3+-mediated bridging between dopa-containing proteinfilms in water. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (29), 12850−12853.(29) Lee, H.; Scherer, N. F.; Messersmith, P. B. Single-moleculemechanics of mussel adhesion. Proc. Natl. Acad. Sci. 2006, 103 (35),12999−13003.(30) Cho, Y.-W.; Jang, J.; Park, C. R.; Ko, S.-W. Preparation andsolubility in acid and water of partially deacetylated chitins.Biomacromolecules 2000, 1 (4), 609−614.(31) Pillai, C. K. S.; Paul, W.; Sharma, C. P. Chitin and chitosanpolymers: Chemistry, solubility and fiber formation. Prog. Polym. Sci.2009, 34 (7), 641−678.(32) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Influence of acetic acidconcentration on the solubilization of chitosan. Polymer 1999, 40 (25),7029−7032.(33) Sogias, I. A.; Khutoryanskiy, V. V.; Williams, A. C. Exploring thefactors affecting the solubility of chitosan in water. Macromol. Chem.Phys. 2010, 211 (4), 426−433.(34) Aryaei, A.; Jayatissa, A. H.; Jayasuriya, A. C. Nano and micromechanical properties of uncross-linked and cross-linked chitosanfilms. J. Mech. Behavior Biomed. Mater. 2012, 5 (1), 82−89.(35) Hu, Y.; Chan, E. P.; Vlassak, J. J.; Suo, Z. Poroelastic relaxationindentation of thin layers of gels. J. Appl. Phys. 2011, 110 (8), 086103−086103.(36) Gray, K. M.; Kim, E.; Wu, L.-Q.; Liu, Y.; Bentley, W. E.; Payne,G. F. Biomimetic fabrication of information-rich phenolic-chitosanfilms. Soft Matter 2011, 7 (20), 9601−9615.(37) Holten-Andersen, N.; Zhao, H.; Waite, J. H. Stiff coatings oncompliant biofibers: The cuticle of Mytilus californianus ByssalThreads. Biochemistry 2009, 48 (12), 2752−2759.(38) Nguyen, V. Q.; Ahmed, A. S.; Ramanujan, R. V. Morphing softmagnetic composites. Adv. Mater. 2012, 24 (30), 4041−4054.(39) Purushotham, S.; Ramanujan, R. V. Thermoresponsive magneticcomposite nanomaterials for multimodal cancer therapy. ActaBiomater. 2010, 6 (2), 502−510.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610905

Page 8: Catechol-Functionalized Chitosan/Iron Oxide Nanoparticle ... · Catechol-Functionalized Chitosan/Iron Oxide ... usage in a wider range of structural biomedical applications. ... Squid

■ NOTE ADDED AFTER ASAP PUBLICATIONThis paper was published on the Web on August 7, 2013, withan error in Figure 4. The corrected version was reposted onAugust 8, 2013.

Langmuir Article

dx.doi.org/10.1021/la401858s | Langmuir 2013, 29, 10899−1090610906