research article synthesis, characterization, and dna...

Research ArticleSynthesis Characterization and DNA BindingStudies of Nanoplumbagin

Sheik Dawood Shahida Parveen1 Abdullah Affrose1 Basuvaraj Suresh Kumar1

Jamespandi Annaraj1 and Kasi Pitchumani12

1School of Chemistry Madurai Kamaraj University Madurai 625021 India2Centre for Green Chemistry Processes School of Chemistry Madurai Kamaraj University Madurai 625021 India

Correspondence should be addressed to Kasi Pitchumani pit12399yahoocom

Received 14 August 2014 Revised 8 November 2014 Accepted 10 November 2014 Published 17 December 2014

Academic Editor Zhi Li Xiao

Copyright copy 2014 Sheik Dawood Shahida Parveen et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The traditional anticancer medicine plumbagin (PLN) was prepared as nanostructured material (nanoplumbagin NPn1) from itscommercial counterparts simultaneously coencapsulating with cetyltrimethylammonium bromide or cyclodextrin as stabilizersusing ultrasonication technique Surface morphology of NPn analysed from atomic force microscopy (AFM) indicates that NPnhas tunable size between 75 nm and 100 nm with narrow particle size distribution Its binding efficiency with herring sperm DNAwas studied using spectral and electrochemical techniques and its efficiency was found to be more compared to the commercialmicrocrystalline plumbagin (PLN) DNA cleavage was also studied by gel electrophoresis The observed results indicate that NPn1has better solubility in aqueous medium and hence showed better bioavailability compared to its commercial counterparts

1 Introduction

Though traditional medicines are generally claimed to be safeand efficacious in most cases neither the chemical entity northe molecular mechanism of action is well defined Plumba-gin (5-hydroxy-2-methyl-14-naphthoquinone) a quinonoidconstituent isolated from the root of Plumbago zeylanica Lhas been shown to exert anticarcinogenic antiatheroscleroticand antimicrobial effects [1ndash3] Oral administration of drugsis the most popular route for the majority of drugs Howeverwhen drugs are poorly soluble they have low bioavailabilitydue to their inherent low solubility and slow dissolutionrate primarily in the gastrointestinal tract which necessitateshighly concentrated administration of drugs than normallyneeded Many synthesized anticancer drugs exert their cyto-toxicity by inhibiting DNA synthesis and cell replication[4] The side effects of these drugs such as bone marrowsuppression and gastrointestinal toxicity are difficult to avoidIn this context nanoencapsulation of drug particles is a usefultechnique in controlled and targeted delivery [5ndash7] During

the past few decades a large variety of biocompatible drugdelivery systems such as liposomes micelles emulsions andpolymeric micro- and nanoparticles have been developedPolyelectrolyte capsules were also identified as promisingdrug delivery vehicles [8 9] In this context nanoparticlescan be considered as useful platforms as they have uniqueadvantages such as high surface area to volume ratio andallow many functional groups to be attached to a nanopar-ticle which can seek out and bind to selected tumor cellsAdditionally the small size of nanoparticles allows them topreferentially accumulate at tumor sites Dissolution ratesmay increase by reducing the particle size to increase theinterfacial surface area [10]

To overcome the problems of using bulk materials asdrugs nanoparticle based drug delivery approaches weredeveloped [11] in which curcumin is encapsulated in lipo-somes [12] solid lipid microparticles such as bovine serumalbumin [13] and chitosan [14] or complexed with phos-pholipids [15] and cyclodextrin [16ndash19] Currently the sono-chemical method has been used extensively to generate novel

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2014 Article ID 179149 9 pageshttpdxdoiorg1011552014179149

2 Journal of Nanomaterials

materials with unusual properties since they form particlesof a much smaller size and higher surface area than thosereported by other methods [20 21]

Cetyltrimethylammonium bromide (CTAB) is a well-known surfactant and has been widely used in the synthesisof many types of nanoparticles with controlled morphologies[22 23] Using this cationic surfactant a rich variety ofaggregate microstructures can be synthesised even at highdilution and this can serve as novel organic templatesfor mesostructured materials with various remarkable mor-phologies

Recently cyclodextrins (CDs) were found to play impor-tant roles in self-assembly systems of amphiphiles or surfac-tants As an amphiphilic building unit CDs can be chemicallyor physically attached to a hydrophobicmoiety where the CDouter surface acts as a hydrophilic moiety and the resultantcompound as a classic amphiphile The amphiphilic CDs canself-assemble in a classic way similar to that of surfactantwhere the CD portion acts like the hydrophilic head groupof a surfactant [24] Many amphiphilic CDs were developedand were found to be able to form micelles monolayers atairwater surface vesicles and fibers Compared to surfactantaggregates the amphiphilic CD-based aggregates can providemacrocyclic hosting sites on the surface creating new possi-bilities for host-guest interaction and recognition

The principle of ultrasonication is based on formationgrowth and implosion of acoustic cavity and is consideredas a green simple fast popular and reproducible method Inthis study we have used low frequency ultrasonication pro-cess for the synthesis ofNPn usingCTABor120573-CD as a surfac-tantstabilizers It was characterized using FT-IR and UV-Visspectroscopy AFM and scanning electronmicroscopy (SEM)and DLS techniques Their interaction with DNA was alsoinvestigated through spectral and electrochemical studies

2 Experimental Techniques

21 Materials and Methods Plumbagin cetyltrimethylam-monium bromide (CTAB) and 120573-cyclodextrin (120573-CD) werepurchased from Sigma Aldrich pUC18 DNA from SRLHerring sperm DNA (hs-DNA) from GENEI and sodiumchloride and Tris hydrochloride (Tris-HCl) were purchasedfrom Merck All other chemicals were used without furtherpurification Double distilled water was used throughout theexperimental studies All the experimental studies involvingthe interaction of the plumbagin (PLN) and nanoplumbagin(NPn) with hs-DNA were carried out in aerated buffer (Tris-HCl pH 71) containing NaCl Gel electrophoresis experi-ments were performed using pUC18-DNA and 1 agarosegels together with a TAE (Tris-acetic acid-EDTA) runningbuffer solution DNA samples were mixed with loading dye(bromophenol blue) and loaded into the slots of the sub-merged gel and resolved at 50VThe gels were photographedunder UV light

22 Physical Measurements UV-Vis absorption spectra wererecorded using JASCO-spectra manager (V-550) in 1 cm pathlength quartz cuvette with a volume of 2mL at 25∘C Infrared

spectra of PLN and NPn were recorded on a JASCO FT-IR-410 (4000ndash400 cmminus1) spectrophotometer The SEM imageswere obtained using a JEOL JSM-6390 scanning electronmicroscope The powder samples were placed over carbontape and a thin layer of platinumwas coated over the samplesto avoid charging Cyclic voltammetric measurements werecarried out on a Bioanalytical System (BAS) model CV-50Welectrochemical analyzer The three-electrode cell compriseda reference AgAgCl platinum wire as counter electrode andworking glassy carbon (GC) electrodes with surface area of007 cm2TheGCwas polishedwith 03 and 005mmaluminabefore each experiment and if necessary the electrode wassonicated in distilled water for 10min Dissolved oxygenwas removed by purging the solution with pure nitrogengas for about 15min before each experiment A bare cyclicvoltammogram has been carried out for a blank solution tocheck the purity of the supporting electrolyte and the solventNuclear magnetic resonance spectroscopic measurementswere recorded using Bruker 300MHz spectrometer Circulardichroism spectra were recorded on a JASCO J-810 (200ndash900 nm) spectropolarimeter The spectral changes of PLNand NPn during the addition of increasing concentrationof DNA were monitored at 25∘C using a quartz cuvette of1 cm optical path length with a volume of 1mL at roomtemperature Gel electrophoretograms of the samples wereinvestigated by BIORAD Model XI computer controlledelectrophoresis power supply

23 Preparation of Nanoplumbagin (NPn) Commerciallyavailable microcrystalline plumbagin (0094 g 05mM) wasdissolved in 5mL of ethanol and poured into HCl (005M20mL) An aqueous solution of NaOH (005M 20mL) wasprepared separately in presence of CTAB or 120573-cyclodextrinThen twodifferent concentrations of 02 and 05wtofCTABand 120573-cyclodextrin were prepared the dropwise additionof plumbagin solution into CTAB120573-cyclodextrin solutionin NaOH is effected separately under ultrasonic conditionsusing an ultrasonic power of 250w and a frequency of59Hz After sonication for 40min yellow precipitate wasobtained which was washed with ethanol several times toremove unbounded surfactants (CTAB or 120573-CD) to give purenanoplumbagin It was characterized by UV-Vis absorptionFT-IR and 1H NMR spectroscopy techniques The absorp-tion data clearly showed the enhanced solubility of NPn1in water compared to an equivalent concentration of PnFurther these spectral data also confirm that NPn does notundergo any structural change during its synthesis fromits commercial counterparts It was characterised by UV-Vis 1H amp 13C-NMR and FT-IR spectra IR (] cmminus1 KBr)] (OndashH) = 3412 nm ] (C=O) = 1664 nm 1643 nm ] (ndashCH3) = 2922 nm UV-Vis (DMSO) 120582max = 416 nm 1H NMR

(300MHz CDCl3) 120575 ppm 12 (1H) 78 (2H) 73 (1H) 72

(1H) 68 (1H) 22 (3H)13C NMR (CDCl3 75MHz) 120575 ppm

190 184 161 149 136 133 124 119 and 16 (Figures S1 S2and S3 in SI) (see Supplementary Material available onlineat httpdxdoiorg1011552014179149) The schematic rep-resentation of the synthesis is shown in Scheme 1

Journal of Nanomaterials 3

CTAB

Plumbagin

SonicationWashed

with EtOH

Washedwith EtOH

NPn1 and NPn2

120573-CDSonication

NPn3 and NPn4

Scheme 1 Schematic representation of the synthesis of NPn

24 Preparation of Stock Solution of Nanoplumbagin All themeasurements were carried out in Tris-hydrochloride bufferat pH 71 and double distilled water was used to preparebuffersThe stock solution of nanoplumbagin (1times 10minus2M)wasprepared by dissolving 0094 g of nanoplumbagin (NPn1) in50mL Tris-hydrochloride buffer at pH 71

3 Results and Discussion

31 Characterization of Nanoplumbagin (NPn)

311 DLS Data Analysis The analysis of dynamic lightscattering data of NPn1 given in supporting information(Figure S4) showed that Npn1 has the particle size rangingfrom 7 nm to 1200 nm with most of the particles lying in therange of 75 nmndash110 nm

312 Atomic Force Microscopy (AFM) The effect and theamount of stabilizers were taken into account during thesynthesis of NPn namely NPn1ndashNPn4 AFM images andsize distribution of the NPn prepared in presence of 02 and05 wt CTAB namely NPn1 and NPn2 respectively areshown in Figure 1 From this data it is clear that in NPn1 thenanoparticles are in the range of 84ndash110 nm (Figure 1(a)) withthe height profile of the three-dimensional (3D) AFM image(Figure 1(b)) of NPn1 found to be about 109 nm whereasin NPn2 the microparticles are in the range of 110ndash350 nm(Figure 1(c)) with a height of 15 nm (Figure 1(d)) due to thefact that CTAB as stabilizer can be absorbed at the surface tocontrol crystal growth and coagulation

Similarly AFM images and size distribution of the NPnprepared in the presence of 02 and 05 wt 120573-CD namelyNPn3 and NPn4 respectively are also shown in Figure 2AFM images of NPn3 andNPn4 show that the particles are inthe range of 110ndash160 nmwith the height of 26 nm (Figures 2(a)and 2(b)) and 530ndash700 nmwith the height of 140 nm (Figures2(c) and 2(d)) respectively

From these observations it can be concluded that 02 wtof CTAB that is NPn1 is appropriate for the preparation of

NPn which is taken into account for further studies likeDNAbinding and cleavage studies

313 SEM Analysis The morphology of the nanocrys-talline plumbagin (NPn1) was studied by scanning electronmicroscopic (SEM) technique This material consisted ofnanospheres with sim84ndash110 nm grain size (Figure 3) whichis consistent with the data obtained from AFM images andsome larger sized particles present in the system are also seenin these images

314 XRD Diffraction Pattern of NPn1 The powder X-raydiffraction pattern of NPn1 is as shown in the SupportingInformation (Figure S5) A definite line broadening of theXRD peaks indicates that the prepared material consists ofparticles which are in the nanoscale range The diffractionpeaks are observed at 2120579 values 1161∘ 1346∘ 2672∘ 3194∘4562∘ 5663∘ and 754∘ from the calculated lattice constants119886 = 119887 = 3254 A and 119888 = 5202 A The observed pattern indi-cates that these NPn1 nanoparticles are present as hexagonalwurtzite phase and the average crystalite size is 1498 nm

32 DNA Binding Studies

321 Electronic Absorption Studies The interaction of syn-thesized nanocrystalline plumbagin with herring spermDNA was investigated by monitoring the changes observedin the UV-Vis absorption bands (120587 rarr 120587lowast and 119899 rarr 120587lowasttransitions) When increasing the concentration of herringsperm DNA to a constant concentration of nanodrug causeda hypochromism in theirUV-Vis absorption bands alongwitha slight red shift (2ndash5 nm) (Figure 4 and Table 1 in SI) whichindicates the possibility of deep penetration of NPn1 particlesinto the core of DNA In contrast PLN showed no change inits absorption measurements (Figure 5) due to its poor or nosolubility in aqueous medium These results show that themain action mode of NPn1 binding to DNA is intercalation[25 26]


109

(nm)

00

(a)

10900 (n

m)

y 18 120583mx18120583m

(b)

(nm)

50

0

(c)

150 (n

m)

y25120583mx 25 120583m

(d)

Figure 1 (a and c) Two-dimensional (2D) AFM image of NPn1 and NPn2 (b and d) Three-dimensional (3D) images of NPn1 and NPn2

The intrinsic binding constant119870119887values were determined

from a plot of [DNA](120576119886minus 120576119891) versus [DNA] using the fol-

lowing equation

[DNA](120576119886minus 120576119891)=[DNA](120576119887minus 120576119891)+1

119870119887

(120576119887minus 120576119891) (1)

where [DNA] is the concentration of DNA in base pairs 120576119886

is the apparent extinction coefficient obtained by calculating119860obs[complex] and 120576

119891and 120576

119887correspond to extinction

coefficients of the complex in its free and bound formsrespectively [27] From the plots of [DNA](120576

119886minus 120576119891) versus

[DNA] 119870119887is measured by the ratio of slope to intercept as

shown in Figures 4 and 5The intrinsic binding constants (119870

119887) for NPn1 and PLN

with DNA are 29 times 104Mminus1 and 54 times 103Mminus1 The observed119870119887value for PLN closely matches with the previous report

[27] and that of NPn1 showed much higher value thanPLN and its copper complexes due to its better solubility inaqueous medium than its counterpartsThe binding constantvalues observed from electronic spectral results indicate thatNPn1 strongly intercalates into the core DNA compared to itscommercial counterparts

322 Circular Dichroism Studies The changes whichoccurred in the CD signals of the DNA upon interactions

with drugs may often be assigned to the correspondingchanges in the DNA structure [28] The groove bindingand electrostatic interaction of a molecules with DNA showless or no perturbations on the base stacking and helicitybands because these two binding modes do not influence thesecondary structure of DNA while the observed intensitychange can be attributed to an intercalation mode whensmall planar molecules interact with DNA [29]

The observed ICD spectrum (Figure 6) of herring spermDNA (hs-DNA) consists of a positive band at 275 nm due tobase stacking and a negative band at 245 nmdue to its helicitywhich are characteristic of right-handed B form DNA [30]Upon addition of PLN to DNA the CD spectrum exhibitsa slight alteration on both negative and positive absorptionintensities without much change in its wavelength indicatinga relatively weak intercalation of plumbagin to hs-DNA Inthe presence of NPn1 a significant decrease in the peakintensities of both bands was observed which indicates thatNPn1 can effectively intercalate the neighbouring base pairsof hs-DNA to reduce the energy of the 120587-120587 electronictransition and also due to strong conformational changes byNPn1 and its binding to DNA mainly by intercalation mode[31]

323 Electrochemical Studies The electrochemical methodis extremely useful in probing the nature and mode of


163

00

(nm

)

(a)

16

0 (nm

)

y 120 120583m x120120583m

(b)

142

0

(nm

)

(c)

014000

y 20120583m x20120583m

(120583m

)

(d)


Figure 3 SEM images of nanoplumbagin (NPn1)

DNA binding with metal complexes Cyclic voltammetricbehaviour of NPn1 and PLN in the absence and presenceof DNA is shown in Figures 7(a) and 7(b) The separationof the cathodic and anodic peak potentials Δ119864

119901 and the

ratio of cathodic to anodic peak current 119894119901119886119894119901119888

(Table 2in SI) indicate that the quasi-reversible redox process wasobserved for bothNPn1 andPLNThe formal potential11986401015840 (orvoltammetric119864

12) taken as the average of119864

119901119888and119864

119901119886 in the

absence of DNA for NPn1 and PLN is minus0311 and minus0289mV

respectively which upon the addition of DNA undergo aslight positive shift indicating significant association of NPn1and PLN with DNA [32]

It is reported by Bard and Faulkner [33] that if the peakpotential is shifted negatively when the molecule interactedwith DNA the interaction will be electrostatic in nature Onthe contrary if the peak potential is shifted positively theinteraction will be through intercalation mode If there is nochange of peak potential then the interaction of the reduced


250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]

(120576aminus120576 b)times10

10

Kb = 29 times 104 M minus1Nanoplumbagin

(b)

Figure 4 UV-Vis spectrum of NPn1 (1 times 10minus4M) in 5mM Tris-HCl buffer at pH 71 with increasing amounts of DNA (a) R = 0 (b) R = 02(c) R = 04 (d) R = 06 (e) R = 08 and (f) R = 1 R = [DNA][compound]

250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9

Kb = 54 times 103 M minus1Pn

(b)

Figure 5 UV-Vis spectrum of PLN (1 times 10minus4M) in 5mM Tris-HCl buffer at pH 71 with increasing amounts of DNA (a) R = 0 (b) R = 02(c) R = 04 (d) R = 06 (e) R = 08 and (f) R = 1 R = [DNA][compound]

form of the molecule will be the same as that of its oxidizedform In the present study it is clear that the potential shiftoccurs in both NPn1 and PLN during the addition of DNAand hence the interactions of the reduced and oxidized formsare not similarThepeak potentials ofNPn1 andPLNundergoa slight positive shift with decrease in peak current whichwas well distinguished for the NPn1 than PLN These resultsindicate that as expected NPn1 were interacted with DNAmuch efficiently than PLN due to its smaller size whichenhanced its solubility This decrease in current is due todiffusion of an equilibriummixture of free DNA-bound drugto electrode surface [34]

The Nernst equation for the reversible redox reactions ofthe free and bound species and the corresponding equilib-rium constants for binding of each oxidation state to DNAfor an electron process is given as follows

11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891

are the formal potentials of oxidisedreduced couple in the bound and free forms respectivelyand 119870ox and 119870red are the binding constants of oxidized andreduced forms to DNA respectively The conclusions could


240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)

Figure 6 Circular dichroism spectra of HS-DNA (24 times 10minus4M) in 5mM Tris-HCl buffer at pH 71 in the absence and presence of PLN andNPn1 at a concentration of (5 times 10minus4M)

00 minus01 minus02 minus03 minus04 minus05 minus06 minus07

E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)

Figure 7 (a) Cyclic voltammogram of NPn1 (1 times 10minus4M) in Tris-HCl buffer pH = 71 with increasing amounts of DNA (a) R = 0 (b) R = 02(c) R = 04 (d) R = 06 (e) R = 08 and (f) R = 1 at 100mVsminus1 scan rate (b) Cyclic voltammogram of PLN (1 times 10minus4M)with increasing amountsof DNA (a) R = 0 (b) R = 02 (c) R = 04 (d) R = 06 (e) R = 08 and (f) R = 1 at 100mVsminus1 scan rate where R = [DNA][compound]

be drawn in the light of Bardrsquos report that bothNPn1 and PLNinteracted with DNA relatively through weak intercalationmode

33 DNA Cleavage Study Gel electrophoresis was used tostudy cleavage of supercoiled pUC18 DNA by the synthesizedNPn1 and commercially available PLN Gels were run inTris-HClNaCl buffer (pH = 71) and analyzed after ethidiumbromide staining in the absence of any external reagent orlight

When circular plasmid DNA is subjected to gel elec-trophoresis relatively fast migration is observed for the intactsupercoil form (form I) If scission occurs on one strand(nicked circular) the supercoil will relax to generate a slower-moving open circular form (form II) If both strands arecleaved a linear form that migrates between forms I and IIwill be generated [35ndash37]

From gel electrophoresis studies plumbagin nanoparti-cles show the change of DNA form I to form II (Figure 8) dueto its nanosize which increases the surface area and solubility


Lanes 1 2 3 4 5

Form II

Form I

Figure 8 Cleavage of plasmid pUC18-DNA treated with increasingconcentrations of NPn1 after incubation for 2 h at 37∘C in Tris-HClNaCl buffer pH = 71 Lane 1 control DNA lanes 2ndash5 DNA+1020 50 and 100120583MNPn1 respectively

Lanes 1 2 3 4 5

Form II

Form I

Figure 9 Cleavage of plasmid pUC18 DNA treated with increasingconcentrations of PLN after incubation for 2 h at 37∘C in Tris-HClNaCl buffer pH = 71 Lane 1 control DNA lanes 2ndash5 DNA+1020 50 and 100120583M PLN respectively

in incubation of DNA with plumbagin the supercoiledform of plasmid pUC18 DNA being lengthened withoutany obvious unwinding but the DNA mobility for PLNwas slightly reduced (Figure 9) [27] These observed resultsare also in accordance with the previously studied spectraland electrochemical investigations All these results clearlyindicate that the present NPn1 interacts more significantlythan its original counterparts of PLN due its smaller size andenhanced solubility

4 Conclusions

In summary ultrafine nanoparticles of plumbagin were syn-thesised successfully by sonochemical methods via control-ling the surfactant concentrationThey were characterised byUV-Vis FT-IR NMR DLS AFM SEM and XRD techniquesand the observed spectral and electrochemical studies indi-cate that this PLN and Npn1 were partially intercalated to theDNA in that Npn1 intercalated with DNAmore significantlycompared to the commercial PLN due to its small size whichenhanced its solubility in aqueous medium

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Financial assistance from University Grants Commission(for UPE programme to Madurai Kamaraj University) isgratefully acknowledged

References

[1] S B Padhye and B A Kulakarni ldquoStudies on the root constitu-ents of Plumbago zeylanicardquo Journal of the University of PoonaScience and Technology vol 44 pp 27ndash29 1973

[2] J C Tilak S Adhikari and T P A Devasagayam ldquoAntioxidantproperties of Plumbago zeylanica an Indian medicinal plantand its active ingredient plumbaginrdquo Redox Report vol 9 no4 pp 219ndash227 2004

[3] J-M Shieh T-A Chiang W-T Chang et al ldquoPlumbagininhibits TPA-induced MMP-2 and u-PA expressions by reduc-ing binding activities of NF-120581B and AP-1 via ERK signalingpathway in A549 human lung cancer cellsrdquo Molecular andCellular Biochemistry vol 335 no 1-2 pp 181ndash193 2010

[4] Y Chen X Lin H Park and R Greever ldquoStudy of artemisininnanocapsules as anticancer drug delivery systemsrdquo Nanomed-icine Nanotechnology Biology and Medicine vol 5 no 3 pp316ndash322 2009

[5] C P Reis R J Neufeld A J Ribeiro and F Veiga ldquoNanoencap-sulation I Methods for preparation of drug-loaded polymericnanoparticlesrdquo Nanomedicine Nanotechnology Biology andMedicine vol 2 no 1 pp 8ndash21 2006

[6] K NThakkar SM Snehit andY P Rasesh ldquoBiological synthe-sis of metallic nanoparticlesrdquo Nanomedicine NanotechnologyBiology and Medicine vol 6 no 2 pp 257ndash262 2010

[7] E Tomlinson and S S Davis Eds Site-Specific Drug Deliveryvol 7 John Wiley amp Sons Chichester UK 1986

[8] F Caruso R A Caruso andHMohwald ldquoNanoengineering ofinorganic and hybrid hollow spheres by colloidal templatingrdquoScience vol 282 no 5391 pp 1111ndash1114 1998

[9] J H Bang and K S Suslick ldquoApplications of ultrasound to thesynthesis of nanostructuredmaterialsrdquoAdvancedMaterials vol22 no 10 pp 1039ndash1059 2010

[10] Z Zhao D Huang Z Yin X Chi X Wang and J Gao ldquoMag-netite nanoparticles as smart carriers tomanipulate the cytotox-icity of anticancer drugs magnetic control and pH-responsivereleaserdquo Journal ofMaterials Chemistry vol 22 no 31 pp 15717ndash15725 2012

[11] P Anand H B Nair B Sung et al ldquoDesign of curcumin-loaded PLGAnanoparticles formulationwith enhanced cellularuptake and increased bioactivity in vitro and superior bioavail-ability in vivordquo Biochemical Pharmacology vol 79 no 3 pp330ndash338 2010

[12] D Wang M S Veena K Stevenson et al ldquoLiposome-encap-sulated curcumin suppresses growth of head and neck squa-mous cell carcinoma in vitro and in xenografts through the inhi-bition of nuclear factor 120581B by an AKT-independent pathwayrdquoClinical Cancer Research vol 14 no 19 pp 6228ndash6236 2008

[13] V Gupta A Aseh C N Rıos B B Aggarwal and A B MathurldquoFabrication and characterization of silk fibroin-derived cur-cumin nanoparticles for cancer therapyrdquo International journalof nanomedicine vol 4 pp 115ndash122 2009

[14] R K Das N Kasoju and U Bora ldquoEncapsulation of cur-cumin in alginate-chitosan-pluronic composite nanoparticlesfor delivery to cancer cellsrdquo Nanomedicine NanotechnologyBiology and Medicine vol 6 no 1 pp 153ndash160 2010

[15] KMaiti KMukherjee A Gantait B P Saha and P KMukher-jee ldquoCurcumin-phospholipid complex preparation therapeu-tic evaluation and pharmacokinetic study in ratsrdquo InternationalJournal of Pharmaceutics vol 330 no 1-2 pp 155ndash163 2007

[16] M M Yallapu M Jaggi and S C Chauhan ldquoPoly(120573-cyclodex-trin)curcumin self-assembly a novel approach to improve


curcumin delivery and its therapeutic efficacy in prostate cancercellsrdquo Macromolecular Bioscience vol 10 no 10 pp 1141ndash11512010

[17] S Bisht M Mizuma G Feldmann et al ldquoSystemic admin-istration of polymeric nanoparticle-encapsulated curcumin(NanoCurc) blocks tumor growth and metastases in preclinicalmodels of pancreatic cancerrdquo Molecular Cancer Therapeuticsvol 9 no 8 pp 2255ndash2264 2010

[18] L Jiang Y Yan and J Huang ldquoVersatility of cyclodextrins inself-assembly systems of amphiphilesrdquo Advances in Colloid andInterface Science vol 169 no 1 pp 13ndash25 2011

[19] C W Lim O Crespo-Biel M C A Stuart D N ReinhoudtJ Huskens and B J Ravoo ldquoIntravesicular and intervesicularinteraction by orthogonal multivalent host-guest and metal-ligand complexationrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 104 no 17 pp 6986ndash6991 2007

[20] J Zhu S Liu O Palchik Y Koltypin and A Gedanken ldquoAnovel sonochemical method for the preparation of nanophasicsulfides synthesis of HgS and PbS nanoparticlesrdquo Journal ofSolid State Chemistry vol 153 no 2 pp 342ndash348 2000

[21] K S Suslick Ultrasound Its Chemical Physical and BiologicalEffects VCH Weinheim Germany 1988

[22] S-HWu and D-H Chen ldquoSynthesis of high-concentration Cunanoparticles in aqueous CTAB solutionsrdquo Journal of Colloidand Interface Science vol 273 no 1 pp 165ndash169 2004

[23] X Li J Shen A Du et al ldquoFacile synthesis of silver nanopar-ticles with high concentration via a CTAB-induced silvermirror reactionrdquo Colloids and Surfaces A Physicochemical andEngineering Aspects vol 400 pp 73ndash79 2012

[24] B Aswathy G S Avadhani S Suji andG Sony ldquoSynthesis of 120573-cyclodextrin functionalized gold nanoparticles for the selectivedetection of Pb2+ ions from aqueous solutionrdquo Frontiers ofMaterials Science vol 6 no 2 pp 168ndash175 2012

[25] P F Shi Q Jiang Y M Zhao et al ldquoDNA binding properties ofnovel cytotoxic gold(III) complexes of terpyridine ligands theimpact of steric and electrostatic effectsrdquo Journal of BiologicalInorganic Chemistry vol 11 no 6 pp 745ndash752 2006

[26] H L Chan D L Ma M Yang and C M Che ldquoSynthesis andbiological activity of a platinum(II) 6-Phenyl-221015840-bipyridinecomplex and its dimeric analoguerdquo ChemBioChem vol 4 no1 pp 62ndash68 2003

[27] Z-F Chen M-X Tan L-M Liu et al ldquoCytotoxicity of the tra-ditional chinese medicine (TCM) plumbagin in its copperchemistryrdquo Dalton Transactions no 48 pp 10824ndash10833 2009

[28] V I Ivanov L E Minchenkova A K Schyolkina and A IPoletayev ldquoDifferent conformations of double-stranded nucleicacid in solution as revealed by circular dichroismrdquo Biopolymersvol 12 no 1 pp 89ndash110 1973

[29] B A Jackson V Y Alekseyev and J K Barton ldquoA versatile mis-match recognition agent specific cleavage of a plasmid DNA ata single base mispairrdquo Biochemistry vol 38 no 15 pp 4655ndash4662 1999

[30] A K Patra M Nethaji and A R Chakravarty ldquoSynthesiscrystal structure DNA binding and photo-induced DNA cleav-age activity of (S-methyl-l-cysteine)copper(II) complexes ofheterocyclic basesrdquo Journal of Inorganic Biochemistry vol 101no 2 pp 233ndash244 2007

[31] HWangM Tan J Zhu et al ldquoSynthesis cytotoxic activity andDNA binding properties of copper (II) complexes with hes-peretin naringenin and apigeninrdquo Bioinorganic Chemistry andApplications vol 2009 Article ID 347872 9 pages 2009

[32] J Sun and D K Y Solaiman ldquoThe cyclic voltammetric study ofiron-tallysomycin in the absence and presence of DNArdquo Journalof Inorganic Biochemistry vol 40 pp 271ndash277 1990

[33] A J Bard and L R Faulkner Electrochemical Methods WileyNew York NY USA 1980

[34] M T Carter M Rodriguez and A J Bard ldquoVoltammetricstudies of the interaction of metal chelates with DNA 2Tris-chelated complexes of cobalt(III) and iron(II) with 110-phenanthroline and 22rsquo-bipyridinerdquo Journal of the AmericanChemical Society vol 111 no 24 pp 8901ndash8911 1989

[35] J A Cowan ldquoMetal activation of enzymes in nucleic acid bio-chemistryrdquoChemical Reviews vol 98 no 3 pp 1067ndash1088 1998

[36] A Sridhar and J A Cowan ldquoCatalytic hydrolysis of DNA bymetal ions and complexesrdquo JBIC Journal of Biological InorganicChemistry vol 6 no 4 pp 337ndash347 2001

[37] E L Hegg and J N Burstyn ldquoToward the development ofmetal-based synthetic nucleases and proteases a rationale andprogress report in applying the principles of coordinationchemistryrdquo Coordination Chemistry Reviews vol 173 no 1 pp133ndash165 1998

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


materials with unusual properties since they form particlesof a much smaller size and higher surface area than thosereported by other methods [20 21]

Cetyltrimethylammonium bromide (CTAB) is a well-known surfactant and has been widely used in the synthesisof many types of nanoparticles with controlled morphologies[22 23] Using this cationic surfactant a rich variety ofaggregate microstructures can be synthesised even at highdilution and this can serve as novel organic templatesfor mesostructured materials with various remarkable mor-phologies

Recently cyclodextrins (CDs) were found to play impor-tant roles in self-assembly systems of amphiphiles or surfac-tants As an amphiphilic building unit CDs can be chemicallyor physically attached to a hydrophobicmoiety where the CDouter surface acts as a hydrophilic moiety and the resultantcompound as a classic amphiphile The amphiphilic CDs canself-assemble in a classic way similar to that of surfactantwhere the CD portion acts like the hydrophilic head groupof a surfactant [24] Many amphiphilic CDs were developedand were found to be able to form micelles monolayers atairwater surface vesicles and fibers Compared to surfactantaggregates the amphiphilic CD-based aggregates can providemacrocyclic hosting sites on the surface creating new possi-bilities for host-guest interaction and recognition

The principle of ultrasonication is based on formationgrowth and implosion of acoustic cavity and is consideredas a green simple fast popular and reproducible method Inthis study we have used low frequency ultrasonication pro-cess for the synthesis ofNPn usingCTABor120573-CD as a surfac-tantstabilizers It was characterized using FT-IR and UV-Visspectroscopy AFM and scanning electronmicroscopy (SEM)and DLS techniques Their interaction with DNA was alsoinvestigated through spectral and electrochemical studies

2 Experimental Techniques

21 Materials and Methods Plumbagin cetyltrimethylam-monium bromide (CTAB) and 120573-cyclodextrin (120573-CD) werepurchased from Sigma Aldrich pUC18 DNA from SRLHerring sperm DNA (hs-DNA) from GENEI and sodiumchloride and Tris hydrochloride (Tris-HCl) were purchasedfrom Merck All other chemicals were used without furtherpurification Double distilled water was used throughout theexperimental studies All the experimental studies involvingthe interaction of the plumbagin (PLN) and nanoplumbagin(NPn) with hs-DNA were carried out in aerated buffer (Tris-HCl pH 71) containing NaCl Gel electrophoresis experi-ments were performed using pUC18-DNA and 1 agarosegels together with a TAE (Tris-acetic acid-EDTA) runningbuffer solution DNA samples were mixed with loading dye(bromophenol blue) and loaded into the slots of the sub-merged gel and resolved at 50VThe gels were photographedunder UV light

22 Physical Measurements UV-Vis absorption spectra wererecorded using JASCO-spectra manager (V-550) in 1 cm pathlength quartz cuvette with a volume of 2mL at 25∘C Infrared

spectra of PLN and NPn were recorded on a JASCO FT-IR-410 (4000ndash400 cmminus1) spectrophotometer The SEM imageswere obtained using a JEOL JSM-6390 scanning electronmicroscope The powder samples were placed over carbontape and a thin layer of platinumwas coated over the samplesto avoid charging Cyclic voltammetric measurements werecarried out on a Bioanalytical System (BAS) model CV-50Welectrochemical analyzer The three-electrode cell compriseda reference AgAgCl platinum wire as counter electrode andworking glassy carbon (GC) electrodes with surface area of007 cm2TheGCwas polishedwith 03 and 005mmaluminabefore each experiment and if necessary the electrode wassonicated in distilled water for 10min Dissolved oxygenwas removed by purging the solution with pure nitrogengas for about 15min before each experiment A bare cyclicvoltammogram has been carried out for a blank solution tocheck the purity of the supporting electrolyte and the solventNuclear magnetic resonance spectroscopic measurementswere recorded using Bruker 300MHz spectrometer Circulardichroism spectra were recorded on a JASCO J-810 (200ndash900 nm) spectropolarimeter The spectral changes of PLNand NPn during the addition of increasing concentrationof DNA were monitored at 25∘C using a quartz cuvette of1 cm optical path length with a volume of 1mL at roomtemperature Gel electrophoretograms of the samples wereinvestigated by BIORAD Model XI computer controlledelectrophoresis power supply

23 Preparation of Nanoplumbagin (NPn) Commerciallyavailable microcrystalline plumbagin (0094 g 05mM) wasdissolved in 5mL of ethanol and poured into HCl (005M20mL) An aqueous solution of NaOH (005M 20mL) wasprepared separately in presence of CTAB or 120573-cyclodextrinThen twodifferent concentrations of 02 and 05wtofCTABand 120573-cyclodextrin were prepared the dropwise additionof plumbagin solution into CTAB120573-cyclodextrin solutionin NaOH is effected separately under ultrasonic conditionsusing an ultrasonic power of 250w and a frequency of59Hz After sonication for 40min yellow precipitate wasobtained which was washed with ethanol several times toremove unbounded surfactants (CTAB or 120573-CD) to give purenanoplumbagin It was characterized by UV-Vis absorptionFT-IR and 1H NMR spectroscopy techniques The absorp-tion data clearly showed the enhanced solubility of NPn1in water compared to an equivalent concentration of PnFurther these spectral data also confirm that NPn does notundergo any structural change during its synthesis fromits commercial counterparts It was characterised by UV-Vis 1H amp 13C-NMR and FT-IR spectra IR (] cmminus1 KBr)] (OndashH) = 3412 nm ] (C=O) = 1664 nm 1643 nm ] (ndashCH3) = 2922 nm UV-Vis (DMSO) 120582max = 416 nm 1H NMR

(300MHz CDCl3) 120575 ppm 12 (1H) 78 (2H) 73 (1H) 72

(1H) 68 (1H) 22 (3H)13C NMR (CDCl3 75MHz) 120575 ppm

190 184 161 149 136 133 124 119 and 16 (Figures S1 S2and S3 in SI) (see Supplementary Material available onlineat httpdxdoiorg1011552014179149) The schematic rep-resentation of the synthesis is shown in Scheme 1


CTAB

Plumbagin

SonicationWashed

with EtOH

Washedwith EtOH

NPn1 and NPn2

120573-CDSonication

NPn3 and NPn4















109

(nm)

00

(a)

10900 (n

m)

y 18 120583mx18120583m

(b)

(nm)

50

0

(c)

150 (n

m)

y25120583mx 25 120583m

(d)




lowing equation


119870119887

(120576119887minus 120576119891) (1)



119891and 120576



119886minus 120576119891) versus











163

00

(nm

)

(a)

16

0 (nm

)

y 120 120583m x120120583m

(b)

142

0

(nm

)

(c)

014000

y 20120583m x20120583m

(120583m

)

(d)




119901 and the




119901119888and119864

119901119886 in the





250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]


10


(b)


250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9


(b)




11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891



240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




CTAB

Plumbagin

SonicationWashed

with EtOH

Washedwith EtOH

NPn1 and NPn2

120573-CDSonication

NPn3 and NPn4















109

(nm)

00

(a)

10900 (n

m)

y 18 120583mx18120583m

(b)

(nm)

50

0

(c)

150 (n

m)

y25120583mx 25 120583m

(d)




lowing equation


119870119887

(120576119887minus 120576119891) (1)



119891and 120576



119886minus 120576119891) versus











163

00

(nm

)

(a)

16

0 (nm

)

y 120 120583m x120120583m

(b)

142

0

(nm

)

(c)

014000

y 20120583m x20120583m

(120583m

)

(d)




119901 and the




119901119888and119864

119901119886 in the





250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]


10


(b)


250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9


(b)




11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891



240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




109

(nm)

00

(a)

10900 (n

m)

y 18 120583mx18120583m

(b)

(nm)

50

0

(c)

150 (n

m)

y25120583mx 25 120583m

(d)




lowing equation


119870119887

(120576119887minus 120576119891) (1)



119891and 120576



119886minus 120576119891) versus











163

00

(nm

)

(a)

16

0 (nm

)

y 120 120583m x120120583m

(b)

142

0

(nm

)

(c)

014000

y 20120583m x20120583m

(120583m

)

(d)




119901 and the




119901119888and119864

119901119886 in the





250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]


10


(b)


250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9


(b)




11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891



240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




163

00

(nm

)

(a)

16

0 (nm

)

y 120 120583m x120120583m

(b)

142

0

(nm

)

(c)

014000

y 20120583m x20120583m

(120583m

)

(d)




119901 and the




119901119888and119864

119901119886 in the





250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]


10


(b)


250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9


(b)




11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891



240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




250 300 350 400 450 50000

05

10

15

20

25

30

Abs

Wavelength (nm)

a

f

(a)

[DNA] times 106 (M)

05

15

25

35

45

0 2 4 6 8 10 12

[DN

A]


10


(b)


250 300 350 400 450 500

Abs

Wavelength (nm)

00

05

10

15

a

f

(a)

[DNA] times 106 (M)

05

0

1

15

0 2 4 6 8 10

[DN

A]


9


(b)




11986401015840119887

minus 11986401015840119891

= 00591 log(119870red119870ox) (2)

where 11986401015840119887

and 11986401015840119891



240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




240 260 280 300

0

CD (m

deg)

DNA PLNNPn1

Wavelength (nm)



E (V)

a

f

minus002

000

002

004

006

008

010

012

014

I(A)times10

minus4

(a)


000

002

004

006

E (V)

a

f

I(A)times10

minus4

(b)







Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Lanes 1 2 3 4 5

Form II

Form I


Lanes 1 2 3 4 5

Form II

Form I



4 Conclusions




Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article synthesis, characterization, and dna...

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