Size control of magnetic carbon nanoparticles for drug delivery

Size control of magnetic carbon nanoparticles for drug deliveryW.-K.Oh,H.Yoon,J.Jang *School of Chemical and Biological Engineering,Seoul National University,599Gwanangro,Sillim-dong,Gwanak-gu,Seoul 151-742,Republic of Koreaa r t i c l e i n f oArticle history:Received 22September 2009Accepted 8October 2009Available online 29October 2009Keywords:Drug delivery Nanoparticle Magnetism PorosityCell viablilitya b s t r a c tCarbonized polypyrrole nanoparticles with controlled diameters were readily fabricated by the pyrolysis of polypyrrole nanoparticles.The carbonized polypyrrole nanoparticles showed narrow size distribution,large micropore volume,and high surface area.Magnetic phases were introduced into the carbon nanoparticles during the pyrolysis without sophisticated process,which resulted in useful magnetic properties for selective nanoparticle separation.Field emission scanning electron microscopy,Raman spectrometer,N 2adsorption/desorption,X-ray diffraction,and superconducting interference device were employed for characterizing the carbonized polypyrrole nanoparticles.Hydrophobic guest molecules were incorporated into the carbonized polypyrrole nanoparticles by surface adsorption,pore filling,and surface covalent coupling.The carbonized polypyrrole nanoparticles exhibited embedding capability using pyrene as a typical hydrophobic fluorescent molecule.In addition,ibuprofen was incorporated into the carbon nanoparticles,and drug-loaded carbon nanoparticles sustained release property.In addition,the carbonized polypyrrole nanoparticles revealed low toxicity at concentrations below 100m g mL À1via cell viability test and were uptaken inside the cells.These results suggest a new platform for the drug delivery using carbonized polypyrrole nanoparticles.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionRecent progress in synthesis of nanomaterials has made it possible to fabricate nanometer-sized materials with controlled structures and functionalities [1–3].In particular,versatile porous materials with nanometer feature sizes have emerged as promising candidates for applications in the fields of catalysis [4–6],energy conversion and storage [7,8],separation [9],and biomedical science [10,11].For example,as a typical microporous material,zeolite has been extensively utilized for molecular sieves,scaffolds,and templates for microporous replicas [12].In addition,mesoporous carbons have been obtained using sacrificial templates including colloidal silica nanoparticles and mesoporous silicas.Various precursors,such as sucrose,phenol resin,polypyrrole,poly-acrylonitrile,and poly(furfuryl alcohol),were employed for obtaining porous carbon structures [13,14].Carbon nanomaterials are of great interest in applications for biological fields [15,16].Typically,carbon nanotubes (CNTs)have a feature of endohedral filling of 2–10nm in diameter leading to encapsulation of small Ts can be heterogeneously surface-functionalized and stained cytochemically with non-quenching and non-photobleaching.Accordingly,CNTs may be suitable for bio–applications in biorecognition and drug delivery systems [17–19].However the biocompatibility of CNTs is still controversial and the tedious functionalization process of CNT surface remains major obstacles to practical applications [20,21].The synthetic strategies towards carbon nanoparticles involve:i)pyrolysis of organic precursors under inert atmosphere [22]and ii)physical and chemical vapor deposition techniques [23].While the pyrolysis approach is applicable to large-scale production,it offers very limited control on the carbon nanostructure.It has been well-known that carbon black is a mass-product manufactured by thermal decomposition or incomplete combustion of carbon hydrogen compounds [24].However,carbon black consists of aggregates of spherical particles with the diameter of individual particles commonly ranging from 10nm to 200nm.Although the vapor deposition method allows precise control on the carbon nanostructure,it has also significant drawbacks such as limited yield,high cost,and complex equipment.In general,carbon encapsulated magnetic nanoparticles were obtained by deposition of carbon onto Fe,Co,and Ni nanocatalysts using arc-discharge techniques [25].However,it is known that the arc-discharge technique often gives an irreproducible and low yield due to the by-products such as graphitic flakes,carbon nanotubes,and carbides.Recently,we have explored the fabrication of several types of carbon nanoparticles from polymer nanoparticles as carbon*Corresponding author.Fax:þ8228881604.E-mail address:jsjang@plaza.snu.ac.kr (J.Jang).Contents lists available at ScienceDirectBiomaterialsjournal homepage:/locate/biomaterials0142-9612/$–see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.biomaterials.2009.10.018Biomaterials 31(2010)1342–1348precursors[26,27].Direct carbonization of tailored polymer nano-particles is favorable to the formation of carbon nanoparticles with the desired structure and composition.Furthermore,carbonization in mild temperature such as from700to900C remained their own biocompatibility of polymers,resulting in applying for biological fields.Notably,magnetic carbon nanospheres with controlled size and shape were fabricated via carbonization of iron-doped polymer nanomaterials[28,29].Magnetic carbon nanostructures have aroused a great deal of interest due to their possible biological applications,including drug/gene delivery,thermal tumor therapy, in–vitro cell separation,and magnetic resonance imaging[30,31]. The geometry(size and shape)of the nanostructures is a key factor determining cellular uptake rate and mechanism.It has been found that an optimum geometry for endocytotic uptake is ca.50nm and spherical shape[32,33].Here we report the fabrication of carbonized polypyrrole nanoparticles(CPyNs)with controlled diameters and their textural properties.Furthermore,we investigate the potential capability of CPyNs as imaging probes and drug carriers based on their porosity, magnetic property and biocompatibility.The guest molecule-loading of CPyNs was conducted with pyrene as a typical hydro-phobic dye and the guest molecule-releasing test was performed with ibuprofen as a typical hydrophobic drug.2.Materials and methods2.1.MaterialsDodecyltrimethylammonium bromide(DTAB,99.0%),decyl alcohol(99.0%), pyrrole(98.0%),and ferric chloride(97.0%)were purchased from Aldrich.Ethanol (99.9%),tetrahydrofuran(THF,99.9%),methanol(99.9%),and phosphate buffered solution(PBS:0.1M,pH7.4)were also obtained from Aldrich.Pyrene(99%)and ibuprofen(98%)were purchased from Aldrich.A humanfibroblast adherent cell line, which was derived from fetal lung tissue IMR90(CCL-186),was purchased from American Type Culture Collection(ATCC).2.2.Fabrication of CPyNsPolypyrrole(PPy)nanoparticles as the carbon precursor were prepared by micelle templating method and went through carbonization process in order to generate CPyNs.First,to prepare60nm–diameter PPy nanoparticles,DTAB(8g)was dissolved in distilled water(160mL)containing decyl alcohol(4.8g)at3C.Pyrrole (1g)was added dropwise into the DTAB/decyl alcohol solution and then ferric chloride(11.2g)was added into the above solution.The chemical oxidation poly-merization of pyrrole monomer was carried out for1h at3C.To prepare80nm–/ 105nm–diameter PPy nanoparticles,DTAB(8g/8.4g)was introduced into distilled water(160mL)containing decyl alcohol(4.4g/3.2g).Pyrrole(1.6g/2g)was added dropwise into the surfactant solution,and ferric chloride(11.2g)was added into the pyrrole/surfactant solution.The polymerization proceeded for1h at3C.The resulting products were washed with ethanol to remove the surfactant and other residual reagents and subsequently dried in a vacuum oven at room temperature. Carbonization of the PPy nanoparticles was conducted in a quartz tubular furnace under nitrogen atmosphere.The nanoparticles were heated to800C at a heating rate of1C minÀ1,held for3h,and cooled to room temperature.CPyNs with controlled diameters could be obtained from the PPy precursors at approximately char yields of ca.50%.Field emission scanning electron microscopy(FE-SEM)was performed with a JEOL6330F at an acceleration voltage of10kV.The size distribution of CPyNs was calculated by software based on more than100particles in the images.Raman spectra were obtained in the range of800–1800cmÀ1using a Jobin–Yvon T64000spectrometer.The Brunauer–Emmett–Teller(BET)nitrogen sorption experiments were conducted to calculate pore size distributions and cumulative pore volume with a Micromeritics ASAP2020at77K X-ray diffraction (XRD)measurement was performed using a M18XHF-SRA diffractometer(MAC Science Co.)equipped with a CuK a radiation source(l¼1.5406Å)at40kV and 300mA(12kW).The magnetic property of CPyNs was measured using a super-conducting interference device(SQUID)magnetometer(Quantum Design MPMS5).2.3.Pyrene loading into CPyNsPyrene was dissolved in THF solution at a concentration range of5Â10À2to 25m M and then mixed with an aqueous solution containing CPyNs for12h.The steady-statefluorescence spectrum of each solution(l exc¼334nm)was obtained and were scanned in the range of350–450nm using a Quanta Master Fluorescence Steady-State Spectrometer.2.4.Surface modification with amino groups using plasma treatmentThe plasma reactor(Korea Vacuum Co.)is the parallel–electrode type with a13.56MHz radio–frequency generator.The diameter of the powered electrode on which the sample is placed is35cm and the distance between the two electrodes is 8cm.The carbon nanoparticles were added into the plasma chamber,and the chamber was evacuated.When the chamber was evacuated below10À3Torr,the NH3carrier gas was introduced into the chamber at rate of30cm3minÀ1(operating pressure:160mTorr).The plasma output power wasfixed at80W and the plasma treatment was conducted in2min.This plasma treatment method and character-ization were previously reported in the literature[34].2.5.Release profile of ibuprofen from CPyNsIbuprofen(2mg)was dissolved in methanol(2mL)and then mixed into an aqueous solution(20mL)containing CPyN-1(10mg).The CPyN-1solution was stirred for12h,and subsequently it was separated by an external magnet and washed three times with water to remove residual drug.The drug-loaded CPyN-1 was dried in a vacuum oven at room temperature.To carry out time-dependent release tests,the following steps were employed:drug-loaded CPyN-1was introduced into0.1M PBS(50mL,pH)solution and incubated at37C.The samples(0.2mL)were extracted at a constant time interval and dissolved in methanol(1.8mL).The samples were quantitatively analyzed by HPLC(WatersÒ).Separation of the analytes was achieved with an acetonitrile/water(1:1)containing0.1% phosphate buffer,flow1mL minÀ1,detection228nm and270nm,pressure 10,35,000–1047000psi,injected volume50m L,and C18column.Linear regression coefficients from six-point linear calibration curves(concentration range 0.5–10m g mLÀ1)were between0.9982and0.9999for all compounds.2.6.Cell viability assayIMR90cells were cultured in Eagle’s Minimum Essential Medium(EMEM) containing sodium pyruvate(1m M),L–glutamine(2m M),10%heat–inactivated Fetal Bovine Serum(FBS),penicillin(10,000IU mLÀ1),and streptomycin(10mg mLÀ1). The cells were grown in a humidified incubator at37C(95%air,5%CO2).Cell viability of the CPyN-treated cells was investigated using CellTiter glow luminescent cell viability assay(Promega,Madison,WI).For estimating viable cells,10,000cells per well were plated and treated with different concentrations of CPyNs(10,25,100, 250,and500m g mLÀ1)for24h.2.7.Transmission electron microscopy(TEM)of CPyNs-treated cellsThe ultrastructural alterations of IMR90cell lines induced by the CPyNs were observed with TEM(JEM-2000EXII,JEOL).IMR90cells were incubated in Lab-Tek II chamber slides until80%confluence.After a24h exposure of CPyNs,IMR90was harvested by a cell scraper and prefixed with2%paraformaldehyde and2%glutar-aldehyde at4C for4h.After being washed with0.05M cacodylate buffer,IMR90was postfixed with1%osmiumtetraoxide at4C for2h and washed with distilled water and then stained with0.5%uranyl acetate at4C.The cells were dehydrated through a series of ethyl alcohol concentrations(30%,50%,70%,80%,90%,100%,100%,and dry alcohol).Then,the cells were treated with propylene oxide followed by1:1 propylene oxide:spurr’s resin for2h.The cells were infiltrated in spurr’s resin at 70 C for24h and ultramicrotome was performed with diamond knife,collected on carbon grids.Then,samples were observed with the TEM at120kV.3.Results and discussionPolypyrrole(PPy)nanoparticles with controlled diameters were prepared by micelle templating in oil/water emulsions and used for carbon precursors in order to fabricate CPyNs.PPy consists offive-membered heterocyclic rings with cross-linking structures(a,a-and a,b-links)and includes iron cations as the dopant from the oxidizingagent(ferric chloride),which can act as the catalyst to facilitate the formation graphite structure[35].During heat-treatment process under an inert atmosphere,the well-defined PPy nanoparticles can be readily converted into carbon nanoparticles and the doped iron cations are transformed into magnetic phases.As previously reported, spherical micelles were formed with DTAB in aqueous solution,and decyl alcohol as a cosurfactant was used to minimize the destabili-zation phenomenon such as Ostwald ripening effect to fabricate PPy nanoparticles with controlled diameters[2,25].The size of micelles strongly depends on the concentrations of surfactant.In general,the size of nanoparticle decreases with increasing the surfactant concentration[35].Under our experimental conditions,the diameterW.-K.Oh et al./Biomaterials31(2010)1342–13481343of PPy nanoparticle was effectively controlled with varying concen-trations of DTAB and decyl alcohol.DTAB was employed to form micelles as a nanoreactor,and decyl alcohol was used as a cosurfac-tant.Decyl alcohol,a water-insoluble long chain alcohol,can retard or hinder the diffusion of pyrrole through the aqueous phase.Therefore,the size of the nanoparticles was controlled by the ratio between pyrrole and DTAB/decyl alcohol.When the monomer/surfactant ratio decreases,the diameter of PPy nanoparticles decreases.PPy nano-particles were prepared with three different diameters (60,80,and 105nm)and were successfully transformed to CPyNs with uniform sizes by the carbonization procedure (char yield:ca .50%).Fig.1represents FE–SEM images and size distribution histograms of CPyNs.The FE–SEM images exhibited spherical nanoparticles with reason-ably uniform sizes and their diameters were 56Æ6nm (CPyN–1),76Æ9nm (CPyN–2),and 99Æ8nm (CPyN–3),respectively.The successful carbonization of CPyNs and the impregnation of iron oxides were confirmed by Raman spectroscopy.In Fig.2a,the Raman spectrum of the CPyNs represented two distinct peaks at 1595cm À1and 1350cm À1.The band at 1595cm À1(G band)was assigned to the E 2g vibration of graphitic carbon with sp 2configu-ration.On the other hand,the peak at 1350cm À1(D band)was attributable to the A 1g mode of an imperfection in the crystal structure of graphite (e.g.,iron)with sp 3configuration.Although CPyNs were carbonized at mild temperature (800C),the CPyNs represented the crystal structure and could be categorized as a glassy carbon structure.In addition,the XRD pattern of CPyNs confirmed the existence of graphite in the nanoparticles,origi-nating the characteristic 002and 100Bragg reflections of graphe-nes (Fig.2b).Furthermore,the XRD pattern showed the presence of g -Fe 2O 3and a -Fe (labeled F and A,respectively)as magneticphasesFig.1.FE-SEM images and size distribution histograms of CPyNs with different diameters:CPyN-1(56Æ6nm)(a),CPyN-2(76Æ9nm)(b),and CPyN-3(99Æ8nm)(c).W.-K.Oh et al./Biomaterials 31(2010)1342–13481344in the CPyNs.It is considered that iron-based complexes encapsu-lated in PPy nanoparticles formed iron oxides at 800C.Nitrogen sorption experiments were performed to characterize the textural properties of CPyNs.The nitrogen adsorption–desorption isotherms and the cumulative pore volume curves of CPyNs are displayed in Fig.3.The isotherms represent an increase in the volume adsorbed up to the relative pressure in the range of 0.01–0.02,depending on the particular sample,and then leveled off.At low relative pressures,micropore filling mainly occurs due to the strong adsorbent–adsorbate interactions and the amount adsorbed in the mesopores is negligible.Accordingly,this behavior is associated with adsorption in micropores and may also originate from adsorption in mesopores with sizes close to the micropore range.The CPyNs displayed a steep increase in the volume adsorbed at pressures close to the saturation vapor pressure.This phenomenon is due to the capillary condensation of nitrogen in interparticle pores with some contribution of multilayer adsorption on the surface of these pores [36].BET surface areas were ca .408m 2g À1,235m 2g À1,and 164m 2g À1for CPyN–1,CPyN–2,and CPyN–3,respectively,which was in inverse proportion to the diameter of CPyNs.The pore size distributions of CPyNs derived from nitrogen adsorption isotherms was close to the micropore range of 0.0–2.5nm in size.The total pore volumes of CPyNs were calculated to be ca .0.62cm 3g À1,0.39cm 3g À1,and 0.22cm 3g À1for CPyN–1,CPyN–2,and CPyN–3,respectively.The micropore volumescalculated from the Horvath–Kawazoe (HK)formalism were ca .0.17cm 3g À1,0.13cm 3g À1,and 0.10cm 3g À1,respectively (Fig.3insets).It is considered that the micropore volume increased in inverse proportion to the size of CPyNs because the heat conduc-tion and mass transfer of CPyNs were varied with respect to the size of CPyNs,where the heat conduction proceeded from outside to inside of CPyNs and the mass transfer proceeded from inside to outside of CPyNs during carbonization process.Interestingly,theFig.2.Raman spectrum of CPyN-1measured between 800and 1800cm-1a),powder XRD pattern of CPyN-1(G:graphite,F:g –Fe 2O 3,A:a –Fe)b).Fig.3.Nitrogen adsorption/desorption isotherms of CPyNs (insets:cumulative pore volumes of CPyNs at a pore diameter of less than 3nm calculated by HK formalism):CPyN-1(a),CPyN-2(b),and CPyN-3(c).W.-K.Oh et al./Biomaterials 31(2010)1342–13481345micropore volume of CPyNs was comparable to that of ZSM–5zeolite (micropore volume 0.14cm 3g À1,mesopore volume 0.02cm 3g À1),as a well–known microporous material [37].Magnetic properties of the CPyNs were investigated with an SQUID magnetometer.In Fig.4,the hysteresis loop of the CPyNs at 300K represented typical ferromagnetic behavior.Moreover,saturation of the magnetization from the hysteresis loop was found to be 12.5emu g À1.The coercivity (H c )was observed at a field of pared to the value of Hc for bulk iron (z 1Oe),a signif-icant increase of coercivity was observed in the CPyNs.The magnetic property of CPyNs is useful for efficient targeting and separating of drug carriers.In general,carbon nanoparticles could load functional guest molecules by surface adsorption,pore filling,and surface covalent coupling [38].As the diameter of a nanoparticle becomes smaller,it has more populations to contact with guest species due to the enhanced surface area.When the guest molecules reach to the nanoparticle surface,they can be incorporated into the nano-particle by physical interactions such as van der Waals force and hydrophobic interaction.In order to gain a better insight into the adsorption behavior of guest molecules in CPyNs,pyrene was loaded into CPyNs by a phase separation method.Emission spec-trum of pyrene shows characteristic the intensity ratio between the first (369.5nm)and the third (380nm)monomeric peaks (I m1/I m3).The I m1/I m3value is susceptible to the micro-environmental polarity of the solubilized pyrene molecules [39,40].In general,the I m1/I m3value increases as the solvent polarity increases.Accord-ingly,the location of pyrene in CPyNs could be traced on the molecular level by monitoring the I m1/I m3value.Fig.5a exhibits representative fluorescence spectrum of pyrene/CPyN-1/water solution.The I m1/I m3ratio was systematically investigated and normalized by dividing it by the initial value (the concentration of pyrene ¼5Â10À2m M).Fig.5b displays changes in the normalized I m1/I m3value for pyrene/CPyN/water systems as a function of pyrene concentration.The normalized I m1/I m3value did not change up to a critical concentration (C crit ),which was dependent on the size of CPyNs,and then gradually increased with further increasing pyrene concentration.At low concentrations,most pyrene mole-cules can exist in the hydrophobic pore or surface of CPyNs.The C crit increased in the order of CPyN-3<CPyN-2<CPyN-1.Because the pyrene amount in CPyN was determined by the pore volume and surface area of CPyNs,the C crit was strongly affected by the size of CPyNs.The increase in I m1/I m3value at higher concentrations reflects that pyrene locates a more hydrophilic microenvironment.In other words,it is considered that an excess of pyrene molecules over the C crit exists in aqueous phase.These results confirm the fact that CPyNs have high affinity with hydrophobic molecules and can readily load a certain amount of guest molecules.Potential capability of CPyNs to load guest molecules provides the application to a drug carrier.Ibuprofen is a well known nonsteroidal antiinflammatory drug and was investigated to releasing molecules of CPyNs in aqueous medium.Furthermore,a surface-modified CPyN-1(termed CPyN-1-NH 2)was prepared by further functionalization with ammonia plasma treatment for preparing the CPyN-1with amino groups.Fig.6exhibits the in-vitro release profiles of CPyNs for ibuprofen.CPyN-2and CPyN-3released over 70%of naproxen during 10h,and more than 90%of naproxen was released within 50h.In the case of CPyN-1,about 53%of ibuprofen released within 10h,and CPyN-1was sustained ca .70%of ibuprofen during 50h.CPyN-1exhibits the relatively slow release because of higher surface area and pore volume.Notably,CPyN-1-NH 2delayed the release of ibuprofen such as about 60%within 50h,indicating that the ionic interaction between the amino group of the CPyN-1-NH 2and the carboxyl group of ibuprofen plays a crucial role of sustaining release property [41].The biocompatibility of CPyNs is a key factor in their biological applications.The CPyNs were incubated with human lung fibro-blast cell line (IMR90),and ATP assay was used for the study on the cell viability of CPyNs (Fig.7a).The ATP assay is ahomogeneousFig.4.Magnetic hysteresis loop of CPyN-1measured at 300K between À15and þ15kOe (inset:magnified area between À50and þ50Oe).Fig.5.Adsoption behavior of pyrene-loaded CPyN-1;Fluorescence spectrum of pyr-ene/CPyN-1/aqueous solution (a),and relative hydrophobic index of pyrene-loaded CPyN-1as a function of pyrene concentration (b)(I m1/I m3value:micro-environmental polarity of the solubilized pyrene molecules).W.-K.Oh et al./Biomaterials 31(2010)1342–13481346technique for determining the number of viable cells based on quantification of the ATP concentration.After 24h treatments of CPyNs,the viability was declined to ca .52%,63%,and 72%for CPyN–1,CPyN–2,and CPyN–3at the highest concentration of CPyNs (500m g mL À1).Below 100m g mL À1,no significant decrease was observed.However,ATP content of the cells decreaseddrastically beyond 100m g mL À1.The cell viability of carbon nano-particles were concentration–dependent.Furthermore,cell viabil-ities decreased as the size of CPyNs decreased.As previously reported by Yen et al.for gold and silver nanoparticles,the smallest carbon nanoparticles exhibited most cytotoxic due to high surface area and large numbers [42].The TEM images of IMR90treated with CPyN-1at 25m g mL À1for 24h are shown in Fig.7V.CPyN-1was taken by endocytosis inside the vesicles in the cytoplasm and morphology of cells remained normal.The CPyN-1inside the vesicle formed a cluster.Magrez et al.reported carbon black nanoparticles showed hazardous effect compared to multiwalled carbon nanotubes and carbon nanofibers [20].In contrast,CPyNs represent relatively less cytotoxicity.It is thought that CPyNs are originated from non-cytotoxic precursor,polypyrrole nanoparticles.4.ConclusionPPy nanoparticles with controlled diameters were prepared by micelle templating in oil/water emulsions,and CPyNs with three different sizes (55,76,and 99nm)were successfully obtained by carbonization of the polymer precursors.CPyNs showed highly microporous compared to zeolite,resulting in loading guest molecules into CPyNs using phase separation.In addition,the magnetic property of CPyNs provided the selective separation and targeting.CPyNs sustained in-vitro drug release properties.Importantly,smaller size and amine surface modification of CPyNs provide an improved sustained property.Due to their superiorities such as microporous structure,monodispersity,magnetism,and biocompatibility,it is believed that the CPyNs open the way to use in fields such as biomaterials science,including bioimaging and magnetic induced drug carriers.AcknowledgementsThis work was supported by the Center for Advanced Materials Processing under the 21C Frontier Programs of the Ministry of Knowledge Economy,and WCU (World Class University)program through the Korea Science and Engineering Foundation funded by the Ministry of Education,Science and Technology (400-2008-0230),Republic of Korea.AppendixFigures with essential color discrimination.Figs.5and 6in this article are difficult to interpret in black and white.The full color images can be found in the on-line version,at doi:10.1016/j.biomaterials.2009.10.018.References[1]Lu A–H,Schu¨th F.Nanocasting:a versatile strategy for creating nanostructured porous materials.Adv Mater 2006;18:1793–805.[2]Jang J.Conducting polymer nanomaterials and their applications.Adv PolymSci 2006;199:189–259.[3]Lee KJ,Min SH,Jang J.Mesoporous nanofibers from dual structure-directingagents in AAO:mesostructural control and their catalytic applications.Chem Eur J 2009;15:2491–5.[4]Ko S,Jang J.A highly efficient palladium nanocatalyst anchored on a 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