Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 421967 9 pageshttpdxdoiorg1011552013421967

Research ArticleAnticancer Activity of Ferulic Acid-InorganicNanohybrids Synthesized via Two Different HybridizationRoutes Reconstruction and Exfoliation-Reassembly

Hyoung-Jun Kim1 Kitae Ryu2 Joo-Hee Kang3 Ae-Jin Choi4 Tae-il Kim2 and Jae-Min Oh1

1 Department of Chemistry and Medical Chemistry College of Science and Technology Yonsei University WonjuGangwon-do 220-710 Republic of Korea

2Department of Biosystems and Biomaterials Science and Engineering College of Agriculture and Life SciencesSeoul National University Seoul 151-921 Republic of Korea

3 RampI Korea GBU SC Solvay Industry-University Cooperation Building Ewha Womans University 150 Bukahyun-roSeodaemun-gu Seoul 120-750 Republic of Korea

4National Institute of Horticultural amp Herbal Science (NIHHS) of RDA Eumseong-gunChungcheongbuk-do 369-873 Republic of Korea

Correspondence should be addressed to Tae-il Kim seal1004snuackr and Jae-Min Oh jaeminohyonseiackr

Received 31 August 2013 Accepted 26 September 2013

Academic Editors K Liu and H E Unalan

Copyright copy 2013 Hyoung-Jun Kim et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We have successfully prepared nanohybrids of biofunctional ferulic acid and layered double hydroxide nanomaterials throughreconstruction and exfoliation-reassembly routes From X-ray diffraction and infrared spectroscopy both nanohybrids weredetermined to incorporate ferulic acid molecules in anionic form Micrsocopic results showed that the nanohybrids had averageparticle size of 150 nm with plate-like morphology As the two nanohybridization routes involved crystal disorder and randomstacking of layers the nanohybrids showed slight alteration in z-axis crystallinity and particle size The zeta potential values ofpristine and nanohybrids in deionized water were determined to be positive while those in cell culture media shifted to negativevalues According to the in vitro anticancer activity test on human cervical cancer HeLa cells it was revealed that nanohybridsshowed twice anticancer activity compared with ferulic acid itselfTherefore we could conclude that the nanohybrids of ferulic acidand layered double hydroxide had cellular delivery property of intercalated molecules on cancer cell lines

1 Introduction

Organic-inorganic nanohybrids in which functional organicmolecules were homogeneously combined with inorganicmaterials in nanoscale have attracted great interests inmany research and industry fields [1 2] Among them theintercalation of biologically functionalized molecules intobiocompatible 2-dimensional inorganics has been extensivelystudied during last decades 2D layered materials such aslayered double hydroxides (LDHs) are known to have phys-ical strength [3] chemical inertness [4] highly anisotropicstructure [5] biocompatibility [6] pH-dependent solubility[7] and high cellular uptake efficiency [8] and therefore the

intercalation of biofunctional molecules into LDHs has beenknown to enhance their biological availability [8 9]

LDHs of which general chemical formula isM(II)

1minus119909M(III)

119909(OH)2(A119899minus)119909119899sdot 119898H2O (M metal A ani-

onic species 119898 and 119899 integer 0 lt 119909 lt 1) have electro-statically stacked layers with interlayer anions [10 11] Whena part of M(II) cations in M(II)(OH)

2structure is iso-

morphically substituted with M(III) ones there evolvespermanent positive charge on the framework In this waythe layer of LDHs acquires positive layer charge which iscompensated by the anions Various kinds of biomoleculessuch as deoxyribonucleotides oligonucleotides anticancerdrugs antibiotics vitamins and antioxidants have been

2 The Scientific World Journal

incorporated to LDHs for various biological applications [812 13] In this study we intercalated ferulic acid (FA) a kind ofantioxidant into MgAl-LDHs in order to enhance the intrin-sic anticancer activity of FA by increasing cellular uptake

FA is one of phytochemical phenolic acids and widelyknown for its anti-inflammatory antiviral immunoprotec-tive and antioxidant property [14ndash16] FA molecules caninhibit cancer development by either scavenging reactiveoxygen species or being involved in cell cycle upon cellularuptake [17] or they show antiproliferative and antimetastasiseffect by specifically inhibiting antiapoptotic protein like Bcl-XL and Bcl2 [18] The induction of detoxification by FAspecifically through the phase II metabolism is also reported[17] In spite of those advantageous properties instabilityagainst oxidation low solubility in water and small cellularuptake limit the clinical applications [19] Thus the nanohy-bridization of phenolic acids or flavonoids with LDHs can besuggested as a strategy to enhance their biological functional-ity through stabilizing them from external stress improvingwater solubility and increasing cellular internalization

Generally four kinds of reactions are utilized in theintercalation of functional organic molecules into LDHlayers coprecipitation ion-exchange reconstruction andexfoliation-reassembly In coprecipitation LDHrsquos ionic pre-cursors and organic molecules are dissolved in a solutionand they are simultaneously precipitated into nanohybridsupon base titration [20] On the other hand the ion-exchangeutilizes the already prepared LDH pristine and anionicorganic molecules topotactically replace the preexisting oneaccording to concentration gradient [21] In reconstructionthe pristine LDHs with carbonate anion are first thermallytreated atmild temperature (sim400∘C) and the resultingmetaloxide (often referred to as layered double oxide (LDO))recovers LDH structure under the existence of water andintended anions [22] For exfoliation-reassembly the LDHlayers are first delaminated into a few sheets by treating in anappropriate solvent and the obtained nanosheets are mixedwith organic anions to restack the LDH structure [23] Thelatter two methods are relatively less utilized conventionallybut have advantages the hybridization reaction occurs fastand the intercalation of large sized molecules is possible

The intercalation of functional organic molecules intoLDHs for biomedical applications has been extensivelystudied in various fields Nevertheless there have beennot enough researches to investigate the effect of differentintercalation methods on the physicochemical propertiesof organic-LDH hybrids and their biological activity Inthis paper we intercalated FA molecules into LDHs viareconstruction (REC) and exfoliation-reassembly (ER) routeThe physico-chemical properties of resulting nanohybridswere characterized in detail compared with pristine LDHsAnd the anticancer efficacy of nanohybrids was evaluated inthe human cervical adenocarcinoma epithelial cell lineHeLa

2 Materials and Methods

21 Materials The magnesium nitrate hexahydrate(Mg(NO

3)2sdot6H2O) aluminum nitrate nonahydrate

(Al(NO3)3sdot9H2O) sodium bicarbonate (NaHCO

3)

dimethylsulfoxide (DMSO) thiazolyl blue tetrazoliumbromide (MTT) and ferulic acid (C

10H10O4) were purchased

from sigma-aldrich Co Ltd (USA) Sodium hydroxidepellet (NaOH) formamide (CH

3NO) and sodium nitrate

(NaNO3) were obtained from Daejung Chemicals amp Metals

Co Ltd (Gyonggi-do Korea) Dulbeccorsquos Modified eaglesrsquoMedium (1X) + GlutaMax-1 (DMEM) Dulbeccorsquos PhosphateBuffered Saline (DPBS) and 025 trypsin-EDTA weregained from Invitrogen-Gibco (Carlsbad CA) All reagentswere used without purification

22 Preparation of the Pristine LDHs and Nanohybrids Forthe preparation of CO

3-LDH (Mg

2Al(OH)

6(CO3)05) pris-

tine themixedmetal solution (03MofMg(NO3)2sdot6H2Oand

015M Al(NO3)3sdot9H2O) was titrated with alkaline solution

(09M of NaOH and 0675M NaHCO3) until pH sim 95

NO3-LDH pristine (Mg

2Al(OH)

6(NO3)) was synthesized by

titrating mixed metal solution (03M of Mg(NO3)2sdot6H2O

and 015M Al(NO3)3sdot9H2O) with alkaline solution (09M of

NaOHand supersaturatedNaNO3) until pHsim 95withN

2gas

purging Bothmixtures were hydrothermally treated at 150∘Cfor 24 h The products were freeze-dried after centrifugationand washing with decarbonated water

In order to obtain FA-LDH (FL) nanohybrid via RECroute (REC-FL) CO

3-LDH was first calcined at 400∘C for

8 h and mixed with FA solution with FAAl ratio of 3and then aged for 24 h at dark The obtained precipitateswere centrifuged washed with decarbonated water and thenfreeze-dried To synthesize FL nanohybrid through ER (ER-FL) NO

3-LDH was dispersed into formamide (05 gL) and

stirred for 24 h in order to delaminate LDHs as previouslyreported [21] Thus obtained colloidal suspension was mixedwith 007M FA solution and then aged for 24 h at dark Finalproducts were collected washed with decarbonated waterseveral times and freeze-dried

23 Characterization Powder X-ray diffraction (XRD) pat-terns were obtained with a Bruker AXS D2 Phaser with Ni-filtered Cu-K120572 radiation (120582 = 15418 A) Scans were from 3∘ to80∘ with rate 002∘1 sec The Fourier transform infrared (FT-IR) spectroscopy was obtained using Perkin Elmer spectrumone Bv50 spectrometer with conventional KBr methodThescanning electron microscope (SEM) images were obtainedwith Quanta 250 FEG after PtPd sputtering The shape andthickness of pristine LDHs and nanohybrids were investi-gated using atomic forcemicroscope (AFM) fromNX10 (Parksystems) with tappingmode at room temperature Formicro-scopies all the samples were dispersed in deionized water(DW) gently deposited on a silicon wafer and dried in ambi-ent air All the AFM images and profiles were recorded with05MHz scan rate and obtained images were analyzed usingXEI software Zeta-potential of pristine and nanohybrids ineitherDWorDMEMweremeasured byELSZ-1000 (Otsuka)The hydrodynamic size of pristine and nanohybrids in DWwere obtained with dynamic light scattering (DLS) mode ofELSZ-1000 The samples were prepared in 1mgmL concen-tration and 05Tween 80 (Daejung) was used as dispersant

The Scientific World Journal 3

Table 1 Crystallographic information of pristine LDHs andnanohybrids

Sample Interlayerdistance (nm)

Crystallite sizealong z-axis (nm)

Lattice parametera (nm) c (nm)

CO3-LDH 028 186 0305 227NO3-LDH 041 145 0306 267REC-FL 123 124 0303 516ER-FL 128 104 0305 532

24 InVitro Anticancer Activity Test Theanticancer activitiesof pristine LDHs nanohybrids and FA only were evaluatedusing the MTT assay with human cervical adenocalcinomaepithelial cells line (HeLa) Cells were cultured in DMEM(10 FBS) at 37∘C with 5 CO

2 The 100 120583L cultured HeLa

cells (1 times 104 cellwell) were seeded in a 96-well plate After24 h of incubation the DMEM was removed and cells weretreated with different concentrations of samples (FA and FLhybrid in serum-free DMEM (5 DMSO) concentrationof FA to be 1 5 10 20 30 40 and 50 120583M and pristineLDH in serum-free DMEM (5 DMSO)) The dose of FLnanohybrids was determined to have equal amount withFA only and that of pristine was decided referring to FLnanohybrids The cells were incubated for 24 h and 25120583L ofMTT (2mgmL in DPBS) solution was added to each welland incubated for 2 h The unreacted MTT was removedand formazan crystals formed by proliferating cells were dis-solved in 150120583L DMSOThe absorbance was measured usingmicroplate reader (Synergy H1 BioTek USA) at 570 nm

3 Result and Discussion

The powder XRD patterns of pristine and nanohybridswere displayed in Figure 1 The (hkl) index was identifiedbased on JCPDS powder diffraction file 14-0191 and previousreport [24] As the LDHwas 2-dimensional layered inorganicmaterial of which interlayer ions existed through electrostaticinteractionwith layers the interlayer distance could be variedaccording to the size of interlayer anions The interlayerdistance values could be calculated from (00l) diffractionpeaks considering the layer thickness of LDH sim 048 nm andwere determined to be 028 041 123 and 128 nm for CO

3-

LDH NO3-LDH REC-FL and ER-FL respectively (Table 1)

As the molecular dimension of FA was sim101 nm which waslarger than CO

3

2minus or NO3

minus the interlayer distance of LDHsignificantly expanded upon the intercalation of FA Accord-ing to the previous reports on FL nanohybrids the interlayerdistance of 12sim13 nm stood for the zig-zag orientation ofFA molecules enhancing their interlayer stabilization via 120587-120587 interaction of aromatic groups [13 25]

The crystallite sizes of pristine and nanohybrids along 119911-axis were calculated by solving Scherrerrsquos equation (equationbelow where 120582 119861 and 120579 stood for wavelength of applied X-ray full width half maximum of each peak and the radianvalue of peak position) of (00l) peaks

119905 =09120582

119861 cos 120579(1)

10 20 30 40 50 60 70 80

(c)

(b)

(a)

(1 1

0)

(0 1

2)

(1 1

3)

(0 1

2)

(0 0

15)

(0 0

12)

(0 0

9)

(0 0

6)

(0 0

3)

(1 1

3)

(1 1

0)

(0 1

8)

(0 1

5)

(0 1

2)

(0 0

6)

Inte

nsity

(au

)

(0 0

3)

(1 1

3)

(1 1

0)

(0 1

8)

(0 1

5)

(0 1

2)

(0 0

6)

(0 0

3)

(0 0

12)

(0 0

9)

(0 0

6)

(0 0

3)

(d)

2120579 (deg)Figure 1 Powder X-ray diffraction patterns for pristine LDHs (a)CO3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL

1800 1600 1400 1200 1000 800 600

Tra

nsm

ittan

ce (a

u)

Wavenumbers (cmminus1)

(c)

(b)

(a)

(d)

(e)

(f)

Figure 2 Fourier transform infrared spectra for pristine LDHs (a)CO3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL FT-IR spectrum of (e) ferulic acid and (f) sodium ferulatesalt is displayed for comparison

The crystallite sizes of pristine LDHs were determinedto be 186 and 145 nm for CO

3-LDH and NO

3-LDH while

nanohybrids showed relatively lower values of 124 and104 nm for REC-FL and ER-FL respectively (Table 1) Thisdecrease in crystallinity along 119911-axis could be explained bythe chemical change during nanohybridization process Inthe reconstruction (REC) process the pristine CO

3-LDH

first underwent heat treatmentwhere sequential dehydrationdehydroxylation and decarbonation occurred The resultingmetal oxide (LDO) could then be reconstructed into LDHphase in the presence of water and appropriate anionicspecies [22 26] During this retrotopotactic transformationLDH went through a collapse in layered structure result-ing in the reduction of crystallinity in the layer stackingdirection (119911-axis) [27ndash29] Similarly exfoliation-reassembly(ER) process went with crystal disorder The delaminatedlayers spontaneously restacked with anions forming house-of-cards-like structure with low crystallinity [30 31] The

4 The Scientific World Journal

200nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)Figure 3 Scanning electron microscope images of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL

same reduced crystallinity and disorder of stacking in thenanohybrids were also visualized with SEM (Figure 3) andAFM (Figure 4) and will be further discussed in detail

It is worthy to note here that the (hkl) peaks of pristineand nanohybrids were observed at similar 2-theta valuesat around 35 40 and 62 degrees for (012) (015) and(113) respectively This showed that the crystallinity uponxy-plane direction was not significantly modified duringnanohybridization The calculated lattice parameters 119886 and 119888for pristine and nanohybrids were displayed in Table 1 The 119886values lay between 0303 and 0305 nm while 119888 values showedclear variationThis result verified that the FAmolecules weresuccessfully intercalated into LDH interlayer without alteringthe 2-dimensional layer structure of LDH nanomaterials

The successful intercalation of FA moiety into LDHswas also verified with infrared spectroscopy As shown inFigure 2 the FT-IR spectra of two nanohybrids REC-FL andER-FL exhibited characteristic peaks of FA Peaks at 1595and 1428 cmminus1 indicated with the dotted line in Figure 2were attributed to the aromatic nucleus in FA The peak at1269 cmminus1 marked with solid line was from the ]as(COC)stretching vibration It revealed that the intact structure of FAwas well preserved after hybridization reaction The stretch-ing vibration of carboxylic acid at 1692 cmminus1 in FA (998771) splitinto asymmetric (]as(COOminus) I in Figure 2) and symmetric(]s(COOminus) ∙ in Figure 2) stretching vibration at 1541 and

1384 cmminus1 respectively after hybridizationThe IR spectra ofNa+-ferulate salt also showed the same splitting Thereforethe carboxylic acid terminal of FA was thought to be depro-tonated and stabilized between LDH layers via strong electro-static interaction [13 32]When interpreting the IR spectra ofcarboxylate group the energy difference between symmetricand asymmetric stretching (Δ]a-s)was often utilized to deter-mine the geometry of carboxylate and its interaction withcounterion For example theΔ]a-s value was the largest whenthe carboxylate was unidentate to cation and it decreased inionic state or bidentate coordination [33 34] The calculatedΔ]a-s values for Na

+-ferulate REC-FL and ER-FL were fairlysimilar with 161 145 and 157 cmminus1 revealing that the FAmoiety was electrostatically stabilized like in ionic lattice

The size and morphology of inorganic nanoparticles arevery important when those materials were utilized as drugencapsulating and delivering carrier into intracellular systemas those properties have been reported to strongly affectthe interaction between nanomaterials and biosubstancesLDH nanoparticles with size between 100 and 300 nm werereported to be easily taken up by cells and retained efficientlycompared to smaller or larger ones [35] It was also sug-gested that the rod morphology LDHs could have differentsubcellular compartment compared to plate-like ones andenter into the nucleus [36] According to recent report

The Scientific World Journal 5

Hei

ght (

nm)

0 50 100 150(nm)

200nm

minus1491419

(a)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus1

4

9

14

(b)

0 50 100 150 200

Hei

ght(n

m)

(nm)

200nm

minus149141924

(c)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus14914192429

(d)Figure 4 Atomic force microscopic images and height profile (inset) of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL The AFM images were obtained with tapping mode

on nanoparticles-cell interaction the surface roughness ofnanoparticles played an important role in determining thecellular uptake pathway as well as amount [37]

We evaluated both particle size and morphology of pris-tine LDHs as well as nanohybrids utilizing SEM and AFMIt was determined that all the samples had average primaryparticle diameter around 100 to 200 nm with homogeneousdistribution and typical plate-likemorphology (Figures 3 and4)As previouslymentioned the hybridizationmethods RECand ER were different from the conventional ion-exchangereaction which was a kind of topotactic reaction and theinterlayer ions were simply exchanged with external ionsthrough concentration gradient However the LDH latticesundergo chemical or physical treatment during REC andER routes Therefore it is worthy to carefully evaluate thechange in particle size and morphology between pristine andnanohybrids The particle sizes calculated from randomlyselected 100 particles of microscopic image were sim75 145145 and 140 nm for CO

3-LDH NO

3-LDH REC-FL and ER-

FL respectively showing that the particle size of REC-FLslightly increased compared with its pristine CO

3-LDH It

suggested that there were partial destruction and aggregationof particles during reconstruction process Figure 4 showedthat the overall crystallinity and smoothness of nanohybrids

changed compared with pristine the surface of nanohybridswas rugged and rough compared with the relatively cleansurface of pristine LDHs As discussed in the XRD patterns(Figure 1) and Scherrerrsquos equation (Table 1) the LDH latticesunderwent disorder of stacking during the REC or ERprocessThe particle thickness was determined to lie between10 and 20 nm suggesting that one particle either pristine andnanohybrids has tens of sheets-stacking structure

Surface charge of nanomaterials was one of the majorfactors determining their interaction with cells Nanomate-rials due to its very small size had large portion of surfaceand therefore the surface properties took great part in deter-mining their biological behavior In particular surface chargewas the most important factor as the plasma membraneswere known to have negative charge [38] Many researchershave reported that the surface charge was strongly relatedto the biological behavior of nanomaterials such as plasmamembrane interaction [39 40] cellular uptake [41] blood-brain-barrier interaction [42] toxicity [43 44] aggregationwith proteins [45] and so forth It was reported that thepositively charged nanomaterials had active interaction withcellularmembrane giving rise to enhanced cellular uptake AsLDHs had permanent positive layer charge they have beenreported to effectively deliver the intercalated molecules intointracellular system [46 47]

6 The Scientific World Journal

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

1212 plusmn 202mV(A)

(B)

(C)

(D)

pH 812

3706 plusmn 125mVpH 861

636 plusmn 093mVpH 781

minus1084 plusmn 204mVpH 618

(a)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus974 plusmn 208mV

minus1009 plusmn 472mV

minus1218 plusmn 099mV

minus1853 plusmn 615mV

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

(D)

(C)

(B)

(A)

(b)

Figure 5 Zeta potential values of each sample in (a) deionized water and (b) DMEM media Pristine LDHs (A) CO3-LDH (B) NO

3-LDH

and nanohybrids (C) REC-FL and (D) ER-FL

Figure 5 showed the zeta potential of pristine LDHs andnanohybrids when they were dispersed in deionized water(DW) and cell culture medium DMEM respectivelyThe pHvalues of DW dispersion were also displayed in Figure 5(a)As the pristine LDHs had abundant hydroxyl groups on thesurface which deprotonated surrounding water moleculesthe pH of dispersion was slightly basic and the zeta potentialwas highly positive On the other hand nanohybrids suspen-sions (DW) showed relatively lower pH values and their zetapotentials slightly positive or negative values possibly due tothe adsorbed FAmoiety on the rough surface of nanohybridsDifferent from the DW dispersion all the samples showed

negative zeta potential values in DMEM environment Cellculture media DMEM contained various anionic moleculeslike amino acids chlorides carbonates phosphates sulfatesand so forth and they could reduce the positive zeta potentialof LDHs by adsorbing on the surface Similar zeta potentialdecrease from highly positive to negative direction in LDHsattributed to anionic species was reported previously [48 49]

We also evaluated the hydrodynamic size of pristineLDHs and nanohybrids in DW dispersion utilizing DLSAs shown in Figure 6 the pristine LDHs showed averagediameter of 80sim150 nm and nanohybrids exhibited slightlyhigher values of 100 to 200 nm which well correspond to the

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

The Scientific World Journal 3

Table 1 Crystallographic information of pristine LDHs andnanohybrids

Sample Interlayerdistance (nm)

Crystallite sizealong z-axis (nm)

Lattice parametera (nm) c (nm)

CO3-LDH 028 186 0305 227NO3-LDH 041 145 0306 267REC-FL 123 124 0303 516ER-FL 128 104 0305 532

24 InVitro Anticancer Activity Test Theanticancer activitiesof pristine LDHs nanohybrids and FA only were evaluatedusing the MTT assay with human cervical adenocalcinomaepithelial cells line (HeLa) Cells were cultured in DMEM(10 FBS) at 37∘C with 5 CO

2 The 100 120583L cultured HeLa

cells (1 times 104 cellwell) were seeded in a 96-well plate After24 h of incubation the DMEM was removed and cells weretreated with different concentrations of samples (FA and FLhybrid in serum-free DMEM (5 DMSO) concentrationof FA to be 1 5 10 20 30 40 and 50 120583M and pristineLDH in serum-free DMEM (5 DMSO)) The dose of FLnanohybrids was determined to have equal amount withFA only and that of pristine was decided referring to FLnanohybrids The cells were incubated for 24 h and 25120583L ofMTT (2mgmL in DPBS) solution was added to each welland incubated for 2 h The unreacted MTT was removedand formazan crystals formed by proliferating cells were dis-solved in 150120583L DMSOThe absorbance was measured usingmicroplate reader (Synergy H1 BioTek USA) at 570 nm

3 Result and Discussion

The powder XRD patterns of pristine and nanohybridswere displayed in Figure 1 The (hkl) index was identifiedbased on JCPDS powder diffraction file 14-0191 and previousreport [24] As the LDHwas 2-dimensional layered inorganicmaterial of which interlayer ions existed through electrostaticinteractionwith layers the interlayer distance could be variedaccording to the size of interlayer anions The interlayerdistance values could be calculated from (00l) diffractionpeaks considering the layer thickness of LDH sim 048 nm andwere determined to be 028 041 123 and 128 nm for CO

3-

LDH NO3-LDH REC-FL and ER-FL respectively (Table 1)

As the molecular dimension of FA was sim101 nm which waslarger than CO

3

2minus or NO3

minus the interlayer distance of LDHsignificantly expanded upon the intercalation of FA Accord-ing to the previous reports on FL nanohybrids the interlayerdistance of 12sim13 nm stood for the zig-zag orientation ofFA molecules enhancing their interlayer stabilization via 120587-120587 interaction of aromatic groups [13 25]

The crystallite sizes of pristine and nanohybrids along 119911-axis were calculated by solving Scherrerrsquos equation (equationbelow where 120582 119861 and 120579 stood for wavelength of applied X-ray full width half maximum of each peak and the radianvalue of peak position) of (00l) peaks

119905 =09120582

119861 cos 120579(1)

10 20 30 40 50 60 70 80

(c)

(b)

(a)

(1 1

0)

(0 1

2)

(1 1

3)

(0 1

2)

(0 0

15)

(0 0

12)

(0 0

9)

(0 0

6)

(0 0

3)

(1 1

3)

(1 1

0)

(0 1

8)

(0 1

5)

(0 1

2)

(0 0

6)

Inte

nsity

(au

)

(0 0

3)

(1 1

3)

(1 1

0)

(0 1

8)

(0 1

5)

(0 1

2)

(0 0

6)

(0 0

3)

(0 0

12)

(0 0

9)

(0 0

6)

(0 0

3)

(d)

2120579 (deg)Figure 1 Powder X-ray diffraction patterns for pristine LDHs (a)CO3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL

1800 1600 1400 1200 1000 800 600

Tra

nsm

ittan

ce (a

u)

Wavenumbers (cmminus1)

(c)

(b)

(a)

(d)

(e)

(f)

Figure 2 Fourier transform infrared spectra for pristine LDHs (a)CO3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL FT-IR spectrum of (e) ferulic acid and (f) sodium ferulatesalt is displayed for comparison

The crystallite sizes of pristine LDHs were determinedto be 186 and 145 nm for CO

3-LDH and NO

3-LDH while

nanohybrids showed relatively lower values of 124 and104 nm for REC-FL and ER-FL respectively (Table 1) Thisdecrease in crystallinity along 119911-axis could be explained bythe chemical change during nanohybridization process Inthe reconstruction (REC) process the pristine CO

3-LDH

first underwent heat treatmentwhere sequential dehydrationdehydroxylation and decarbonation occurred The resultingmetal oxide (LDO) could then be reconstructed into LDHphase in the presence of water and appropriate anionicspecies [22 26] During this retrotopotactic transformationLDH went through a collapse in layered structure result-ing in the reduction of crystallinity in the layer stackingdirection (119911-axis) [27ndash29] Similarly exfoliation-reassembly(ER) process went with crystal disorder The delaminatedlayers spontaneously restacked with anions forming house-of-cards-like structure with low crystallinity [30 31] The

4 The Scientific World Journal

200nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)Figure 3 Scanning electron microscope images of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL

same reduced crystallinity and disorder of stacking in thenanohybrids were also visualized with SEM (Figure 3) andAFM (Figure 4) and will be further discussed in detail

It is worthy to note here that the (hkl) peaks of pristineand nanohybrids were observed at similar 2-theta valuesat around 35 40 and 62 degrees for (012) (015) and(113) respectively This showed that the crystallinity uponxy-plane direction was not significantly modified duringnanohybridization The calculated lattice parameters 119886 and 119888for pristine and nanohybrids were displayed in Table 1 The 119886values lay between 0303 and 0305 nm while 119888 values showedclear variationThis result verified that the FAmolecules weresuccessfully intercalated into LDH interlayer without alteringthe 2-dimensional layer structure of LDH nanomaterials

The successful intercalation of FA moiety into LDHswas also verified with infrared spectroscopy As shown inFigure 2 the FT-IR spectra of two nanohybrids REC-FL andER-FL exhibited characteristic peaks of FA Peaks at 1595and 1428 cmminus1 indicated with the dotted line in Figure 2were attributed to the aromatic nucleus in FA The peak at1269 cmminus1 marked with solid line was from the ]as(COC)stretching vibration It revealed that the intact structure of FAwas well preserved after hybridization reaction The stretch-ing vibration of carboxylic acid at 1692 cmminus1 in FA (998771) splitinto asymmetric (]as(COOminus) I in Figure 2) and symmetric(]s(COOminus) ∙ in Figure 2) stretching vibration at 1541 and

1384 cmminus1 respectively after hybridizationThe IR spectra ofNa+-ferulate salt also showed the same splitting Thereforethe carboxylic acid terminal of FA was thought to be depro-tonated and stabilized between LDH layers via strong electro-static interaction [13 32]When interpreting the IR spectra ofcarboxylate group the energy difference between symmetricand asymmetric stretching (Δ]a-s)was often utilized to deter-mine the geometry of carboxylate and its interaction withcounterion For example theΔ]a-s value was the largest whenthe carboxylate was unidentate to cation and it decreased inionic state or bidentate coordination [33 34] The calculatedΔ]a-s values for Na

+-ferulate REC-FL and ER-FL were fairlysimilar with 161 145 and 157 cmminus1 revealing that the FAmoiety was electrostatically stabilized like in ionic lattice

The size and morphology of inorganic nanoparticles arevery important when those materials were utilized as drugencapsulating and delivering carrier into intracellular systemas those properties have been reported to strongly affectthe interaction between nanomaterials and biosubstancesLDH nanoparticles with size between 100 and 300 nm werereported to be easily taken up by cells and retained efficientlycompared to smaller or larger ones [35] It was also sug-gested that the rod morphology LDHs could have differentsubcellular compartment compared to plate-like ones andenter into the nucleus [36] According to recent report

The Scientific World Journal 5

Hei

ght (

nm)

0 50 100 150(nm)

200nm

minus1491419

(a)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus1

4

9

14

(b)

0 50 100 150 200

Hei

ght(n

m)

(nm)

200nm

minus149141924

(c)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus14914192429

(d)Figure 4 Atomic force microscopic images and height profile (inset) of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL The AFM images were obtained with tapping mode

on nanoparticles-cell interaction the surface roughness ofnanoparticles played an important role in determining thecellular uptake pathway as well as amount [37]

We evaluated both particle size and morphology of pris-tine LDHs as well as nanohybrids utilizing SEM and AFMIt was determined that all the samples had average primaryparticle diameter around 100 to 200 nm with homogeneousdistribution and typical plate-likemorphology (Figures 3 and4)As previouslymentioned the hybridizationmethods RECand ER were different from the conventional ion-exchangereaction which was a kind of topotactic reaction and theinterlayer ions were simply exchanged with external ionsthrough concentration gradient However the LDH latticesundergo chemical or physical treatment during REC andER routes Therefore it is worthy to carefully evaluate thechange in particle size and morphology between pristine andnanohybrids The particle sizes calculated from randomlyselected 100 particles of microscopic image were sim75 145145 and 140 nm for CO

3-LDH NO

3-LDH REC-FL and ER-

FL respectively showing that the particle size of REC-FLslightly increased compared with its pristine CO

3-LDH It

suggested that there were partial destruction and aggregationof particles during reconstruction process Figure 4 showedthat the overall crystallinity and smoothness of nanohybrids

changed compared with pristine the surface of nanohybridswas rugged and rough compared with the relatively cleansurface of pristine LDHs As discussed in the XRD patterns(Figure 1) and Scherrerrsquos equation (Table 1) the LDH latticesunderwent disorder of stacking during the REC or ERprocessThe particle thickness was determined to lie between10 and 20 nm suggesting that one particle either pristine andnanohybrids has tens of sheets-stacking structure

Surface charge of nanomaterials was one of the majorfactors determining their interaction with cells Nanomate-rials due to its very small size had large portion of surfaceand therefore the surface properties took great part in deter-mining their biological behavior In particular surface chargewas the most important factor as the plasma membraneswere known to have negative charge [38] Many researchershave reported that the surface charge was strongly relatedto the biological behavior of nanomaterials such as plasmamembrane interaction [39 40] cellular uptake [41] blood-brain-barrier interaction [42] toxicity [43 44] aggregationwith proteins [45] and so forth It was reported that thepositively charged nanomaterials had active interaction withcellularmembrane giving rise to enhanced cellular uptake AsLDHs had permanent positive layer charge they have beenreported to effectively deliver the intercalated molecules intointracellular system [46 47]

6 The Scientific World Journal

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

1212 plusmn 202mV(A)

(B)

(C)

(D)

pH 812

3706 plusmn 125mVpH 861

636 plusmn 093mVpH 781

minus1084 plusmn 204mVpH 618

(a)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus974 plusmn 208mV

minus1009 plusmn 472mV

minus1218 plusmn 099mV

minus1853 plusmn 615mV

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

(D)

(C)

(B)

(A)

(b)

Figure 5 Zeta potential values of each sample in (a) deionized water and (b) DMEM media Pristine LDHs (A) CO3-LDH (B) NO

3-LDH

and nanohybrids (C) REC-FL and (D) ER-FL

Figure 5 showed the zeta potential of pristine LDHs andnanohybrids when they were dispersed in deionized water(DW) and cell culture medium DMEM respectivelyThe pHvalues of DW dispersion were also displayed in Figure 5(a)As the pristine LDHs had abundant hydroxyl groups on thesurface which deprotonated surrounding water moleculesthe pH of dispersion was slightly basic and the zeta potentialwas highly positive On the other hand nanohybrids suspen-sions (DW) showed relatively lower pH values and their zetapotentials slightly positive or negative values possibly due tothe adsorbed FAmoiety on the rough surface of nanohybridsDifferent from the DW dispersion all the samples showed

negative zeta potential values in DMEM environment Cellculture media DMEM contained various anionic moleculeslike amino acids chlorides carbonates phosphates sulfatesand so forth and they could reduce the positive zeta potentialof LDHs by adsorbing on the surface Similar zeta potentialdecrease from highly positive to negative direction in LDHsattributed to anionic species was reported previously [48 49]

We also evaluated the hydrodynamic size of pristineLDHs and nanohybrids in DW dispersion utilizing DLSAs shown in Figure 6 the pristine LDHs showed averagediameter of 80sim150 nm and nanohybrids exhibited slightlyhigher values of 100 to 200 nm which well correspond to the

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

4 The Scientific World Journal

200nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)Figure 3 Scanning electron microscope images of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c) REC-FL and (d)

ER-FL

same reduced crystallinity and disorder of stacking in thenanohybrids were also visualized with SEM (Figure 3) andAFM (Figure 4) and will be further discussed in detail

It is worthy to note here that the (hkl) peaks of pristineand nanohybrids were observed at similar 2-theta valuesat around 35 40 and 62 degrees for (012) (015) and(113) respectively This showed that the crystallinity uponxy-plane direction was not significantly modified duringnanohybridization The calculated lattice parameters 119886 and 119888for pristine and nanohybrids were displayed in Table 1 The 119886values lay between 0303 and 0305 nm while 119888 values showedclear variationThis result verified that the FAmolecules weresuccessfully intercalated into LDH interlayer without alteringthe 2-dimensional layer structure of LDH nanomaterials

The successful intercalation of FA moiety into LDHswas also verified with infrared spectroscopy As shown inFigure 2 the FT-IR spectra of two nanohybrids REC-FL andER-FL exhibited characteristic peaks of FA Peaks at 1595and 1428 cmminus1 indicated with the dotted line in Figure 2were attributed to the aromatic nucleus in FA The peak at1269 cmminus1 marked with solid line was from the ]as(COC)stretching vibration It revealed that the intact structure of FAwas well preserved after hybridization reaction The stretch-ing vibration of carboxylic acid at 1692 cmminus1 in FA (998771) splitinto asymmetric (]as(COOminus) I in Figure 2) and symmetric(]s(COOminus) ∙ in Figure 2) stretching vibration at 1541 and

1384 cmminus1 respectively after hybridizationThe IR spectra ofNa+-ferulate salt also showed the same splitting Thereforethe carboxylic acid terminal of FA was thought to be depro-tonated and stabilized between LDH layers via strong electro-static interaction [13 32]When interpreting the IR spectra ofcarboxylate group the energy difference between symmetricand asymmetric stretching (Δ]a-s)was often utilized to deter-mine the geometry of carboxylate and its interaction withcounterion For example theΔ]a-s value was the largest whenthe carboxylate was unidentate to cation and it decreased inionic state or bidentate coordination [33 34] The calculatedΔ]a-s values for Na

+-ferulate REC-FL and ER-FL were fairlysimilar with 161 145 and 157 cmminus1 revealing that the FAmoiety was electrostatically stabilized like in ionic lattice

The size and morphology of inorganic nanoparticles arevery important when those materials were utilized as drugencapsulating and delivering carrier into intracellular systemas those properties have been reported to strongly affectthe interaction between nanomaterials and biosubstancesLDH nanoparticles with size between 100 and 300 nm werereported to be easily taken up by cells and retained efficientlycompared to smaller or larger ones [35] It was also sug-gested that the rod morphology LDHs could have differentsubcellular compartment compared to plate-like ones andenter into the nucleus [36] According to recent report

The Scientific World Journal 5

Hei

ght (

nm)

0 50 100 150(nm)

200nm

minus1491419

(a)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus1

4

9

14

(b)

0 50 100 150 200

Hei

ght(n

m)

(nm)

200nm

minus149141924

(c)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus14914192429

(d)Figure 4 Atomic force microscopic images and height profile (inset) of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL The AFM images were obtained with tapping mode

on nanoparticles-cell interaction the surface roughness ofnanoparticles played an important role in determining thecellular uptake pathway as well as amount [37]

We evaluated both particle size and morphology of pris-tine LDHs as well as nanohybrids utilizing SEM and AFMIt was determined that all the samples had average primaryparticle diameter around 100 to 200 nm with homogeneousdistribution and typical plate-likemorphology (Figures 3 and4)As previouslymentioned the hybridizationmethods RECand ER were different from the conventional ion-exchangereaction which was a kind of topotactic reaction and theinterlayer ions were simply exchanged with external ionsthrough concentration gradient However the LDH latticesundergo chemical or physical treatment during REC andER routes Therefore it is worthy to carefully evaluate thechange in particle size and morphology between pristine andnanohybrids The particle sizes calculated from randomlyselected 100 particles of microscopic image were sim75 145145 and 140 nm for CO

3-LDH NO

3-LDH REC-FL and ER-

FL respectively showing that the particle size of REC-FLslightly increased compared with its pristine CO

3-LDH It

suggested that there were partial destruction and aggregationof particles during reconstruction process Figure 4 showedthat the overall crystallinity and smoothness of nanohybrids

changed compared with pristine the surface of nanohybridswas rugged and rough compared with the relatively cleansurface of pristine LDHs As discussed in the XRD patterns(Figure 1) and Scherrerrsquos equation (Table 1) the LDH latticesunderwent disorder of stacking during the REC or ERprocessThe particle thickness was determined to lie between10 and 20 nm suggesting that one particle either pristine andnanohybrids has tens of sheets-stacking structure

Surface charge of nanomaterials was one of the majorfactors determining their interaction with cells Nanomate-rials due to its very small size had large portion of surfaceand therefore the surface properties took great part in deter-mining their biological behavior In particular surface chargewas the most important factor as the plasma membraneswere known to have negative charge [38] Many researchershave reported that the surface charge was strongly relatedto the biological behavior of nanomaterials such as plasmamembrane interaction [39 40] cellular uptake [41] blood-brain-barrier interaction [42] toxicity [43 44] aggregationwith proteins [45] and so forth It was reported that thepositively charged nanomaterials had active interaction withcellularmembrane giving rise to enhanced cellular uptake AsLDHs had permanent positive layer charge they have beenreported to effectively deliver the intercalated molecules intointracellular system [46 47]

6 The Scientific World Journal

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

1212 plusmn 202mV(A)

(B)

(C)

(D)

pH 812

3706 plusmn 125mVpH 861

636 plusmn 093mVpH 781

minus1084 plusmn 204mVpH 618

(a)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus974 plusmn 208mV

minus1009 plusmn 472mV

minus1218 plusmn 099mV

minus1853 plusmn 615mV

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

(D)

(C)

(B)

(A)

(b)

Figure 5 Zeta potential values of each sample in (a) deionized water and (b) DMEM media Pristine LDHs (A) CO3-LDH (B) NO

3-LDH

and nanohybrids (C) REC-FL and (D) ER-FL

Figure 5 showed the zeta potential of pristine LDHs andnanohybrids when they were dispersed in deionized water(DW) and cell culture medium DMEM respectivelyThe pHvalues of DW dispersion were also displayed in Figure 5(a)As the pristine LDHs had abundant hydroxyl groups on thesurface which deprotonated surrounding water moleculesthe pH of dispersion was slightly basic and the zeta potentialwas highly positive On the other hand nanohybrids suspen-sions (DW) showed relatively lower pH values and their zetapotentials slightly positive or negative values possibly due tothe adsorbed FAmoiety on the rough surface of nanohybridsDifferent from the DW dispersion all the samples showed

negative zeta potential values in DMEM environment Cellculture media DMEM contained various anionic moleculeslike amino acids chlorides carbonates phosphates sulfatesand so forth and they could reduce the positive zeta potentialof LDHs by adsorbing on the surface Similar zeta potentialdecrease from highly positive to negative direction in LDHsattributed to anionic species was reported previously [48 49]

We also evaluated the hydrodynamic size of pristineLDHs and nanohybrids in DW dispersion utilizing DLSAs shown in Figure 6 the pristine LDHs showed averagediameter of 80sim150 nm and nanohybrids exhibited slightlyhigher values of 100 to 200 nm which well correspond to the

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

The Scientific World Journal 5

Hei

ght (

nm)

0 50 100 150(nm)

200nm

minus1491419

(a)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus1

4

9

14

(b)

0 50 100 150 200

Hei

ght(n

m)

(nm)

200nm

minus149141924

(c)

0 50 100 150

Hei

ght(n

m)

(nm)

200nm

minus14914192429

(d)Figure 4 Atomic force microscopic images and height profile (inset) of pristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL The AFM images were obtained with tapping mode

on nanoparticles-cell interaction the surface roughness ofnanoparticles played an important role in determining thecellular uptake pathway as well as amount [37]

We evaluated both particle size and morphology of pris-tine LDHs as well as nanohybrids utilizing SEM and AFMIt was determined that all the samples had average primaryparticle diameter around 100 to 200 nm with homogeneousdistribution and typical plate-likemorphology (Figures 3 and4)As previouslymentioned the hybridizationmethods RECand ER were different from the conventional ion-exchangereaction which was a kind of topotactic reaction and theinterlayer ions were simply exchanged with external ionsthrough concentration gradient However the LDH latticesundergo chemical or physical treatment during REC andER routes Therefore it is worthy to carefully evaluate thechange in particle size and morphology between pristine andnanohybrids The particle sizes calculated from randomlyselected 100 particles of microscopic image were sim75 145145 and 140 nm for CO

3-LDH NO

3-LDH REC-FL and ER-

FL respectively showing that the particle size of REC-FLslightly increased compared with its pristine CO

3-LDH It

suggested that there were partial destruction and aggregationof particles during reconstruction process Figure 4 showedthat the overall crystallinity and smoothness of nanohybrids

changed compared with pristine the surface of nanohybridswas rugged and rough compared with the relatively cleansurface of pristine LDHs As discussed in the XRD patterns(Figure 1) and Scherrerrsquos equation (Table 1) the LDH latticesunderwent disorder of stacking during the REC or ERprocessThe particle thickness was determined to lie between10 and 20 nm suggesting that one particle either pristine andnanohybrids has tens of sheets-stacking structure

Surface charge of nanomaterials was one of the majorfactors determining their interaction with cells Nanomate-rials due to its very small size had large portion of surfaceand therefore the surface properties took great part in deter-mining their biological behavior In particular surface chargewas the most important factor as the plasma membraneswere known to have negative charge [38] Many researchershave reported that the surface charge was strongly relatedto the biological behavior of nanomaterials such as plasmamembrane interaction [39 40] cellular uptake [41] blood-brain-barrier interaction [42] toxicity [43 44] aggregationwith proteins [45] and so forth It was reported that thepositively charged nanomaterials had active interaction withcellularmembrane giving rise to enhanced cellular uptake AsLDHs had permanent positive layer charge they have beenreported to effectively deliver the intercalated molecules intointracellular system [46 47]

6 The Scientific World Journal

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

1212 plusmn 202mV(A)

(B)

(C)

(D)

pH 812

3706 plusmn 125mVpH 861

636 plusmn 093mVpH 781

minus1084 plusmn 204mVpH 618

(a)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus974 plusmn 208mV

minus1009 plusmn 472mV

minus1218 plusmn 099mV

minus1853 plusmn 615mV

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

(D)

(C)

(B)

(A)

(b)

Figure 5 Zeta potential values of each sample in (a) deionized water and (b) DMEM media Pristine LDHs (A) CO3-LDH (B) NO

3-LDH

and nanohybrids (C) REC-FL and (D) ER-FL

Figure 5 showed the zeta potential of pristine LDHs andnanohybrids when they were dispersed in deionized water(DW) and cell culture medium DMEM respectivelyThe pHvalues of DW dispersion were also displayed in Figure 5(a)As the pristine LDHs had abundant hydroxyl groups on thesurface which deprotonated surrounding water moleculesthe pH of dispersion was slightly basic and the zeta potentialwas highly positive On the other hand nanohybrids suspen-sions (DW) showed relatively lower pH values and their zetapotentials slightly positive or negative values possibly due tothe adsorbed FAmoiety on the rough surface of nanohybridsDifferent from the DW dispersion all the samples showed

negative zeta potential values in DMEM environment Cellculture media DMEM contained various anionic moleculeslike amino acids chlorides carbonates phosphates sulfatesand so forth and they could reduce the positive zeta potentialof LDHs by adsorbing on the surface Similar zeta potentialdecrease from highly positive to negative direction in LDHsattributed to anionic species was reported previously [48 49]

We also evaluated the hydrodynamic size of pristineLDHs and nanohybrids in DW dispersion utilizing DLSAs shown in Figure 6 the pristine LDHs showed averagediameter of 80sim150 nm and nanohybrids exhibited slightlyhigher values of 100 to 200 nm which well correspond to the

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

6 The Scientific World Journal

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

1212 plusmn 202mV(A)

(B)

(C)

(D)

pH 812

3706 plusmn 125mVpH 861

636 plusmn 093mVpH 781

minus1084 plusmn 204mVpH 618

(a)

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus100 minus80 minus60 minus40 minus20 0 20 40 60 80 100

minus974 plusmn 208mV

minus1009 plusmn 472mV

minus1218 plusmn 099mV

minus1853 plusmn 615mV

Inte

nsity

(au

)In

tens

ity (a

u)

Inte

nsity

(au

)In

tens

ity (a

u)

Zeta potential (mV)

(D)

(C)

(B)

(A)

(b)

Figure 5 Zeta potential values of each sample in (a) deionized water and (b) DMEM media Pristine LDHs (A) CO3-LDH (B) NO

3-LDH

and nanohybrids (C) REC-FL and (D) ER-FL

Figure 5 showed the zeta potential of pristine LDHs andnanohybrids when they were dispersed in deionized water(DW) and cell culture medium DMEM respectivelyThe pHvalues of DW dispersion were also displayed in Figure 5(a)As the pristine LDHs had abundant hydroxyl groups on thesurface which deprotonated surrounding water moleculesthe pH of dispersion was slightly basic and the zeta potentialwas highly positive On the other hand nanohybrids suspen-sions (DW) showed relatively lower pH values and their zetapotentials slightly positive or negative values possibly due tothe adsorbed FAmoiety on the rough surface of nanohybridsDifferent from the DW dispersion all the samples showed

negative zeta potential values in DMEM environment Cellculture media DMEM contained various anionic moleculeslike amino acids chlorides carbonates phosphates sulfatesand so forth and they could reduce the positive zeta potentialof LDHs by adsorbing on the surface Similar zeta potentialdecrease from highly positive to negative direction in LDHsattributed to anionic species was reported previously [48 49]

We also evaluated the hydrodynamic size of pristineLDHs and nanohybrids in DW dispersion utilizing DLSAs shown in Figure 6 the pristine LDHs showed averagediameter of 80sim150 nm and nanohybrids exhibited slightlyhigher values of 100 to 200 nm which well correspond to the

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

The Scientific World Journal 7

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

854 plusmn 212nm

(a)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1331 plusmn 315nm

(b)

100 200 300 400 500Diff

eren

tial n

umbe

r (au

)

1272 plusmn 284nm

(c)

100 200 300 400 500Diameter (nm)

Diff

eren

tial n

umbe

r (au

)

2033 plusmn 434nm

(d)

Figure 6 Hydrodynamic sizes of each sample in deionized waterPristine LDHs (a) CO

3-LDH (b) NO

3-LDH and nanohybrids (c)

REC-FL and (d) ER-FL

results ofmicroscopies (Figures 3 and 4) FAmolecules on thesurface of nanohybrids played as a kind of binder resultingin the slight aggregation in suspension Nevertheless thecurrent nanohybrids were thought to show good cellularuptake property as the particle size of LDHbelow 300 nmwasknown to be effectively internalized into cells via endocytosis

Therefore we evaluated the anticancer effect of nanohy-brids in cultured cell line Figure 7 displayed the concen-tration dependent anticancer efficacy of FL nanohybridscompared with pristine LDHs and FA itself when they aretreated to cervical cancer HeLa cells For precise comparisonthe dose of FL nanohybridswas set to have equivalent amountof FA considering the chemical formula of nanohybridsTheFA only treated HeLa cells showed sim30 of cell viability

0 10 20 30 40 500

20

40

60

80

100

Relat

ive c

ell v

iabi

lity

()

FA REC-FL

ER-FL

Concentration (ferulic acid 120583M)

NO3-LDHCO3-LDH

Figure 7 Concentration dependent anticancer activity of ferulicacid and nanohybrids RE-FL and ER-FL in HeLa cell lines

decrease at the administration concentration 5 120583M and didnot exhibit additional effect upon increasing concentrationOn the other hand both nanohybrids showed concentrationdependent cell viability suppression upto sim60 until 50120583MTaking into account that the pristine LDH did not havecytotoxic effect regardless of concentration the anticanceractivity of nanohybrids almost twice of FA only was con-sidered as the efficient cellular delivery ability of LDHs Inthe previous research it was revealed that the cellular uptakeof anionic drug methotrexate was enhanced more than 10times by intercalation into LDHs whichmodified the cellularuptake pathway of drug molecules [47] Similarly we couldsuggest in this study that the nanohybridization of FAswith LDHs modified the cellular internalization route andincreased the biological activity of FAs such as detoxificationinhibition of antiapoptotic proteins and so forth The twonanohybrids REC-FL and ER-FL showed almost similaranticancer efficacy although their crystallinity decreasedcompared with pristine We could thus conclude that thecrystallinity was not a critical factor in LDH nanohybridsin determining their cellular uptake Both nanohybrids hadgood anticancer activity in spite of negative zeta potentialsin DMEM dispersion Even though the zeta potential wasnegative the intrinsic positive charge of LDHs layer wasthought to maximize interaction between nanohybrids andcellular membrane increasing their cellular uptake

4 Conclusion

We have demonstrated the physico-chemical property andbiological activity of FA-LDH nanohybrids Two differ-ent hybridization routes reconstruction and exfoliation-reassembly were utilized to intercalate FA molecules intoLDH interlayer space FromXRDpatterns and FT-IR spectrait was revealed that the FA molecules were successfully

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

8 The Scientific World Journal

stabilized between LDH layers through electrostatic inter-action although the crystallinity was slightly altered duringintercalation route Microscopic and DLS results revealedthat the nanohybrids had less than 300 nmparticle size whichwas an optimumvalue for cellular uptakeThe anticancer effi-cacy of both nanohybrids showed concentration dependentmanner with maximum twice higher cancer proliferationsuppression effect compared to FA only It was thereforeconcluded that the nanohybrids of FA with LDHs obtainedby reconstruction or exfoliation-reassembly had beneficialphysico-chemical properties for cellular uptake increasingthe anticancer activity of the FA itself

Acknowledgments

This study was supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea govern-ment (MSIP) (2005-0049412) and the Herbal Crop ResearchProject (PJ006921) of RDA Republic of KoreaThis work wasalso supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Education Science and Technology (2011-0015045)

References

[1] M Z bin Hussein Z Zainal A H Yahaya and A B A AzizldquoSynthesis of layered organic-inorganic nanohybrid materialan organic dye naphthol blue black in magnesium-aluminumlayered double hydroxide inorganic lamellardquo Materials Scienceand Engineering B vol 88 no 1 pp 98ndash102 2002

[2] J-M Oh T T Biswick and J-H Choy ldquoLayered nanomaterialsfor green materialsrdquo Journal of Materials Chemistry vol 19 no17 pp 2553ndash2563 2009

[3] W Chen and B Qu ldquoLLDPEZnAl LDH-exfoliated nanocom-posites effects of nanolayers on thermal and mechanical prop-ertiesrdquo Journal of Materials Chemistry vol 14 no 11 pp 1705ndash1710 2004

[4] J-H Choy S-J Choi J-M Oh and T Park ldquoClay minerals andlayered double hydroxides for novel biological applicationsrdquoApplied Clay Science vol 36 no 1ndash3 pp 122ndash132 2007

[5] X Wang J Lu W Shi et al ldquoA thermochromic thin film basedon hostmdashguest interactions in a layered double hydroxiderdquoLangmuir vol 26 no 2 pp 1247ndash1253 2010

[6] S-J Choi J-M Oh and J-H Choy ldquoToxicological effects ofinorganic nanoparticles on human lung cancer A549 cellsrdquoJournal of Inorganic Biochemistry vol 103 no 3 pp 463ndash4712009

[7] J-H Choy M Park and J-M Oh ldquoBio-nanohybrids based onlayered double hydroxiderdquoCurrentNanoscience vol 2 no 3 pp275ndash281 2006

[8] J-M Oh M Park S-T Kim J-Y Jung Y-G Kang and J-H Choy ldquoEfficient delivery of anticancer drug MTX throughMTX-LDH nanohybrid systemrdquo Journal of Physics and Chem-istry of Solids vol 67 no 5-6 pp 1024ndash1027 2006

[9] E Lima J Flores A S Cruz G Leyva-Gomez and E KrotzschldquoControlled release of ferulic acid from a hybrid hydrotalciteand its application as an antioxidant for human fibroblastsrdquoMicroporous and Mesoporous Materials vol 181 pp 1ndash7 2013

[10] T H Kim H J Kim and J M Oh ldquoInterlayer structureof bioactive molecule 2-aminoethanesulfonate intercalatedinto calcium-containing layered double hydroxidesrdquo Journal ofNanomaterials vol 2012 Article ID 987938 2012

[11] H Abdolmohammad-Zadeh K Tavarid and Z Talleb ldquoDeter-mination of iodate in food environmental and biologicalsamples after solid-phase extraction with Ni-Al-Zr ternarylayered double hydroxide as a nanosorbentrdquoTheScientificWorldJournal vol 2012 Article ID 145482 8 pages 2012

[12] J M Oh J H Choy and S J Choi ldquoNanoceramics-biomolecular conjugates for gene and drug deliveryrdquo Advancesin Science and Technology vol 45 pp 769ndash778 2006

[13] T Biswick D-H Park Y-G Shul S-J Hwang and J-H ChoyldquoUV screening of ferulic acid-zinc basic salt nanohybrid withcontrolled release raterdquo Journal of Nanoscience and Nanotech-nology vol 11 no 1 pp 413ndash416 2011

[14] M Kampa V-I Alexaki G Notas et al ldquoAntiproliferative andapoptotic effects of selective phenolic acids on T47D humanbreast cancer cells potential mechanisms of actionrdquo BreastCancer Research vol 6 no 2 pp R63ndash74 2004

[15] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[16] L-C Lin Y-C Kuo and C-J Chou ldquoImmunomodulatoryprinciples of Dichrocephala bicolorrdquo Journal of Natural Prod-ucts vol 62 no 3 pp 405ndash408 1999

[17] MMMansonHW L BallM C Barrett et al ldquoMechanism ofaction of dietary chemoprotective agents in rat liver inductionof phase I and II drug metabolizing enzymes and aflatoxin B1metabolismrdquo Carcinogenesis vol 18 no 9 pp 1729ndash1738 1997

[18] S Karthikeyan G Kanimozhi N R Prasad and R Mahalak-shmi ldquoRadiosensitizing effect of ferulic acid on human cervicalcarcinoma cells in vitrordquo Toxicology in Vitro vol 25 no 7 pp1366ndash1375 2011

[19] M Kikugawa M Tsuchiyama K Kai and T SakamotoldquoSynthesis of highly water-soluble feruloyl diglycerols by ester-ification of an Aspergillus niger feruloyl esteraserdquo AppliedMicrobiology and Biotechnology vol 95 no 3 pp 615ndash622 2012

[20] J M Oh D H Park S J Choi and J H Choy ldquoLDHnanocontainers as bio-reservoirs and drug delivery carriersrdquoRecent Patents on Nanotechnology vol 6 no 3 pp 200ndash2172012

[21] S Tezuka R Chitrakar A Sonoda K Ooi and T TomidaldquoStudies on selective adsorbents for oxo-anions Nitrate ion-exchange properties of layered double hydroxides with differentmetal atomsrdquo Green Chemistry vol 6 no 2 pp 104ndash109 2004

[22] V Rives Layered Double Hydroxides Present and Future NovaPublishers New York NY USA 2001

[23] D-H Park J-E Kim J-M Oh Y-G Shul and J-H ChoyldquoDNA coreInorganic shellrdquo Journal of the American ChemicalSociety vol 132 no 47 pp 16735ndash16736 2010

[24] C Rossi A Schoubben M Ricci et al ldquoIntercalation of theradical scavenger ferulic acid in hydrotalcite-like anionic claysrdquoInternational Journal of Pharmaceutics vol 295 no 1-2 pp 47ndash55 2005

[25] V Ambrogi G Fardella G Grandolini L Perioli and M CTiralti ldquoIntercalation compounds of hydrotalcite-like anionicclays with anti-inflammatory agents II uptake of diclofenac fora controlled release formulationrdquo AAPS PharmSciTech vol 3no 3 pp 77ndash82 2002

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008

The Scientific World Journal 9

[26] D Pan H Zhang T Zhang and X Duan ldquoA novel organic-inorganic microhybrids containing anticancer agent doxifluri-dine and layered double hydroxides structure and controlledrelease propertiesrdquoChemical Engineering Science vol 65 no 12pp 3762ndash3771 2010

[27] D G Costa A B Rocha W F Souza S S X Chiaro and AA Leitao ldquoAb initio study of reaction pathways related to initialsteps of thermal decomposition of the layered double hydroxidecompoundsrdquo The Journal of Physical Chemistry C vol 116 no25 pp 13679ndash13687 2012

[28] J Perez-Ramırez S Abello and N M van der Pers ldquoMemoryeffect of activated Mg-Al hydrotalcite in situ XRD studies dur-ing decomposition and gas-phase reconstructionrdquo ChemistrymdashA European Journal vol 13 no 3 pp 870ndash878 2007

[29] W Yang Y Kim P K T Liu M Sahimi and T T TsotsisldquoA study by in situ techniques of the thermal evolution of thestructure of a Mg-Al-CO

3layered double hydroxiderdquo Chemical

Engineering Science vol 57 no 15 pp 2945ndash2953 2002[30] S-M Paek H Jung Y-J Lee M Park S-J Hwang and J-

H Choy ldquoExfoliation and reassembling route to mesoporoustitania nanohybridsrdquo Chemistry of Materials vol 18 no 5 pp1134ndash1140 2006

[31] S Vial V Prevot F Leroux and C Forano ldquoImmobilizationof urease in ZnAl layered double hydroxides by soft chemistryroutesrdquoMicroporous andMesoporous Materials vol 107 no 1-2pp 190ndash201 2008

[32] J Wang Y Cao B Sun and C Wang ldquoCharacterisation ofinclusion complex of trans-ferulic acid and hydroxypropyl-120573-cyclodextrinrdquo Food Chemistry vol 124 no 3 pp 1069ndash10752011

[33] M Nara H Torii and M Tasumi ldquoCorrelation between thevibrational frequencies of the carboxylate group and the typesof its coordination to a metal ion an ab initio molecular orbitalstudyrdquo Journal of Physical Chemistry vol 100 no 51 pp 19812ndash19817 1996

[34] J-H Yang Y-S Han M Park T Park S-J Hwang andJ-H Choy ldquoNew inorganic-based drug delivery system ofindole-3-acetic acid-layered metal hydroxide nanohybrids withcontrolled release raterdquo Chemistry of Materials vol 19 no 10pp 2679ndash2685 2007

[35] J-M Oh S-J Choi G-E Lee J-E Kim and J-H ChoyldquoInorganic metal hydroxide nanoparticles for targeted cellularuptake through clathrin-mediated endocytosisrdquo ChemistrymdashAn Asian Journal vol 4 no 1 pp 67ndash73 2009

[36] Z P XuMNiebert K Porazik et al ldquoSubcellular compartmenttargeting of layered double hydroxide nanoparticlesrdquo Journal ofControlled Release vol 130 no 1 pp 86ndash94 2008

[37] A Schrade V Mailander S Ritz K Landfester and U ZienerldquoSurface roughness and charge influence the uptake of nanopar-ticles fluorescently labeled pickering-type versus surfactant-stabilized nanoparticlesrdquoMacromolecular Bioscience vol 12 no11 pp 1459ndash1471 2012

[38] Y Zhang M Yang N G Portney et al ldquoZeta potentiala surface electrical characteristic to probe the interaction ofnanoparticles with normal and cancer human breast epithelialcellsrdquo Biomedical Microdevices vol 10 no 2 pp 321ndash328 2008

[39] L Chen J M Mccrate J C-M Lee and H Li ldquoThe role ofsurface charge on the uptake and biocompatibility of hydroxya-patite nanoparticles with osteoblast cellsrdquo Nanotechnology vol22 no 10 Article ID 105708 2011

[40] G Su H Zhou Q Mu et al ldquoEffective surface charge densitydetermines the electrostatic attraction between nanoparticles

and cellsrdquo Journal of Physical Chemistry C vol 116 no 8 pp4993ndash4998 2012

[41] M Merhi C Y Dombu A Brient et al ldquoStudy of seruminteraction with a cationic nanoparticle implications for invitro endocytosis cytotoxicity and genotoxicityrdquo InternationalJournal of Pharmaceutics vol 423 no 1 pp 37ndash44 2012

[42] P R Lockman J M Koziara R J Mumper and D DAllen ldquoNanoparticle surface charges alter blood-brain barrierintegrity and permeabilityrdquo Journal of Drug Targeting vol 12no 9-10 pp 635ndash641 2004

[43] B Chertok A E David and V C Yang ldquoPolyethyleneimine-modified iron oxide nanoparticles for brain tumor drug deliveryusing magnetic targeting and intra-carotid administrationrdquoBiomaterials vol 31 no 24 pp 6317ndash6324 2010

[44] B J Marquis Z Liu K L Braun and C L Haynes ldquoInvesti-gation of noble metal nanoparticle 120577-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelec-trode amperometryrdquo Analyst vol 136 no 17 pp 3478ndash34862011

[45] M Kendall P Ding and K Kendall ldquoParticle and nanoparticleinteractions with fibrinogen the importance of aggregation innanotoxicologyrdquo Nanotoxicology vol 5 no 1 pp 55ndash65 2011

[46] J H Choy S Y Kwak Y J Jeong and J S Park ldquoInorganiclayered double hydroxides as nonviral vectorsrdquo AngewandteChemiemdashInternational Edition vol 39 no 22 pp 4042ndash40452000

[47] J-M Oh S-J Choi S-T Kim and J-H Choy ldquoCellular uptakemechanismof an inorganic nanovehicle and its drug conjugatesenhanced efficacy due to clathrin-mediated endocytosisrdquo Bio-conjugate Chemistry vol 17 no 6 pp 1411ndash1417 2006

[48] P C Pavan E L Crepaldi and J B Valim ldquoSorption of anionicsurfactants on layered double hydroxidesrdquo Journal of Colloidand Interface Science vol 229 no 2 pp 346ndash352 2000

[49] Z P Xu Y Jin S Liu Z P Hao and G Q Lu ldquoSurface chargingof layered double hydroxides during dynamic interactions ofanions at the interfacesrdquo Journal of Colloid and Interface Sciencevol 326 no 2 pp 522ndash529 2008