Cellular response to chirality and amplified chirality

Journal ofMaterials Chemistry B

PAPER

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aDepartment of Biochemistry, University of

Kolkata-700019, India. E-mail: adgcal@gm

33 24614849bCancer Biology and Inammatory Disord

Industrial Research-Indian Institute of Chem

Jadavpur, Kolkata-700032, India

† Electronic supplementary information (SNP, cellular toxicity assay (PI, Annexin,given in this section. See DOI: 10.1039/c3

Cite this: J. Mater. Chem. B, 2013, 1,6634

Received 24th September 2013Accepted 11th October 2013

DOI: 10.1039/c3tb21322f

www.rsc.org/MaterialsB

6634 | J. Mater. Chem. B, 2013, 1, 6634

Cellular response to chirality and amplified chirality†

Sarita Roy,a Kaushik Bhattacharya,b Chitra Mandalb and Anjan Kr. Dasgupta*a

In this paper we have explored how cancer cell line U87MG responds to drug penicillamine (PA) with

reciprocal enantiomeric identities (i.e. L, D-forms). As nano-conjugation leads to amplification of chirality,

cellular response to respective chiral forms is studied in the presence and absence of nano-conjugation.

The L, D-forms of the drug (penicillamine) and their silver nanoparticle conjugated forms are used and

characterized by circular dichroism and fourier transform infrared spectroscopy. We report that cells

discriminate between the chiral forms through various mechanisms e.g. by altering mitochondrial

membrane potential and selected elements of the caspase pathway. The striking feature which we

would like to report is that chirality and the amplified chirality induced reciprocal responses may be

dissimilar and even reciprocal. The work shows that the cellular response to geometrical chirality is an

evolved concept and an amplified chirality by processes like nano-conjugation may be translated into an

altered message in the cell.

Introduction

The topic of chirality has always attracted researchers sinceancient times, starting from the days of Pasteur when he rstobserved the existence of molecular chirality and successivelyshowed how pairs of enantiomeric molecules (tartaric acid) arephysically identical and form non-superimposable mirrorimages with opposite optical rotation. The main questionsregarding the importance of chirality are twofold. Firstly, howdo they induce differential pharmaceutical response andsecondly, what is the mechanism that explains such a differ-ence? Differential interactions with DNA or protein are expectedas there might be steric requirements at the drug binding site.Chiral specicity is in fact seen in most of the enzymatic reac-tions and also at the receptor level (e.g. D-glucose has receptorsthat facilitate its entry, such receptors being absent forL-glucose). In addition the membrane may retain the chiraldrugs with reciprocal enantiomeric properties to a differentextent and the functionality or energetics of cells may bedifferently affected as a result.

The question regarding symmetry breaking, namely thechoice of only one of the two possible enantiomeric forms ofbuilding blocks (L-amino acids or D-sugars), in any functionalbiological system still remains unexplained, whereas racemic

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ESI) available: Details of FTIR study ofCFSE, Cell cycle) of conjugated SNP istb21322f

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mixtures with equal proportions are found in simple reactions.Chirality has signicant implications in pharmaceutical scienceor drug research. A chosen enantiomer (a molecule with hand-edness) may have enhanced biological activity as a drugcompared to its mirror image, which might even have an adverseeffect. Some classical examples of enantiomeric drug moleculesinclude thalidomide,1 one of whose enantiomers is useful inmorning sickness during pregnancy, while the other enantiomercauses birth defects. Other enantiomeric drugs include etham-butol, naproxen, Dopa, an essential drug for Parkinson's diseaseand many more.2 One of the examples of a chiral drug moleculethat is difficult to separate is the two enantiomeric forms ofWarfarin, whose S-form is more potent than the R-form and ismetabolized by the body by different pathways.3

While chirality and its evolutionary origin have been givensome attention in the literature,4 how the cell responds tochirality (like the differential actions of the enantiomeric formsof the drugs referred to above) is hardly understood. To explorethis problem we have inducted a simple approach. We exploredcell response to amplication of chirality. The amplication ofchirality is a well-known concept in organic chemistry.5

Recently, nano-conjugation has been employed for chiralamplication.6 A way of monitoring the amplication of chiralsignal is by recording the amplication of the circular dichroicresponse.

While difference in enantiomeric forms creating differentialcellular response is well studied, the issue of how a cellresponds to chiral amplication remains more or less unex-plored. Comparative study of cellular response to chiralityversus amplied chirality is likely to reveal whether the opticallydetermined metric of this symmetry breaking is translated inthe cellular response in equal proportions or not.

This journal is ª The Royal Society of Chemistry 2013

http://dx.doi.org/10.1039/c3tb21322f

http://pubs.rsc.org/en/journals/journal/TB

http://pubs.rsc.org/en/journals/journal/TB?issueid=TB001048

Paper Journal of Materials Chemistry B

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The potential of nanoparticles in inducing altered targetedcellular response and bio-sensing7–10 is well known in theliterature but the fate of chiral nanoparticles in induction ofcellular response towards cancer cells is not studied yet. In thispaper we probe into the chiral response of cancer cells. We try tond whether a cell can respond differently towards chiralnanoparticles, in a manner similar to chiral macromolecules.

To probe this question we synthesized penicillamine (PA)templated (both enantiomers, D-/L-) chiral silver nanoparticleswhose L and D-forms are known to induce differential cellularresponse. We checked for their cytotoxic activity towardshuman glioblastoma cell line U87MG in the presence andabsence of amplied chirality.

Experimental sectionSynthesis and conjugation of SNP with PA

The silver nanoparticles were synthesized by reducing a cooledsolution of silver nitrate (AgNO3), (Merck, Germany) by sodiumborohydride (NaBH4) (Merck, Germany) in a ratio of 1 : 5(AgNO3 : NaBH4) followed by uniform mixing of the reactionmixture.11 A yellow solution appeared aer the addition ofNaBH4 and the particles were stabilized by trisodium citrate(Merck, Germany).

For chiral silver nanoparticle (SNP) synthesis, the enantio-meric form of both D, L-Penicillamine (PA) was purchased fromSigma of highest purity grade and a stock solution (100 mM) ofD-penicillamine (DPA) and L-penicillamine (LPA) was preparedin milli Q water. Then the 1 : 1 mixture of DPA and LPA (1 mM)was added to pre-synthesized SNP and the mixture was incu-bated for a minimum of 3 hours for self-assembly of amino acidon the SNP surface.12

Characterization of PA conjugated SNP by UV-visiblespectroscopy and circular dichroism

Plasmonic properties of SNP and PA conjugate with SNP (i.e.DPA–SNP and LPA–SNP) were characterized by a Thermo-VisionSpectrophotometer in the wavelength region 300–800 nm. Theoptical activities of DPA, LPA and DPA–SNP or LPA–SNP wereveried by circular dichroism (Jasco-J810) from 190–420 nmwavelengths with a rectangular 1 mm path length cuvette.

FT-IR based measurement for verication of conjugation ofPA with SNP

Fourier transform IR spectra of trisodium citrate stabilized SNPand SNP conjugated PA i.e. DPA–SNP and LPA–SNP wererecorded by the Attenuated Total Reection (ATR) mode of aFTIR spectrophotometer (Thermo Scientic, Nicolet-6700). Thelms were prepared by air drying the concentrated samples ofnano-conjugates on the ATR plate to avoid water interference.FT-IR spectra of enantiomeric forms of PA (DPA, LPA) and tri-sodium citrate were recorded in powdered form by the samemethod. Each spectrum represents averaged data aer 256scans and acquired at 4 cm�1 wavenumber resolution at roomtemperature.


Morphology of nanoparticles

Study of size and shape of SNP and conjugated SNP with DPAand LPA has been done by Transmission Electron Microscopy(FEI, Model STWIN) with an accelerating potential of 200 kV.The data obtained were further analysed for size determinationby Gatan Microscopy Suit version 1.5.

Cell lines and culture condition

Human grade-IV glioblastoma multiforme cell line U87MG waspurchased from ATCC (USA) and grown in IMDM (Iscove'sModied Dulbecco's Medium) supplemented with 10% heatinactivated FCS (Fetal calf serum) and 1% antibiotic-anti-mycotic solution in a humidied atmosphere at 37 �C and with5% CO2.

Cell viability analysis by MTT assay

Cells (5 � 103) were treated with different concentrations (50 to400 mM at an interval of 50 mMw.r.t. PA) of DPA, LPA, SNP, DPA–SNP and LPA–SNP (stock concentration of silver nanoparticlesi.e. 48 mM being the same as that of PA) for 72 h and subse-quently subjected to MTT (100 mg ml�1) in fresh culturemedium for 3 h further incubation. Formazan crystals weredissolved in DMSO and absorbance was taken at 550 nm(Thermo Scientic).13

Cytotoxicity assay

Apoptotic cell surfaces have exposed phosphatidyl serine,which translocates from the inner to the outer leaet of theplasma membrane and is quantiable by Annexin-V conju-gated with uorescent probe FITC by ow cytometry.14 Cells(1 � 105) were exposed to DPA, LPA, DPA–SNP and LPA–SNP(150 and 250 mM) for 72 h. Evaluation of apoptosis was per-formed using Annexin V–FITC (5 mg ml�1) according to themanufacturer's instructions. At least 1 � 104 cells were exam-ined and the data were analyzed by Cell Quest Pro soware (BDFACS Calibur). Flow cytometric detection of cell death wasperformed by Propidium Iodide (PI) staining. For this, cells(1 � 105) were exposed to DPA, LPA, SNP, DPA–SNP and LPA–SNP (150 and 250 mM) for 72 h. Evaluation of cell death wasperformed using Propidium Iodide (5 mg ml�1).14 Briey,treated and control cells were washed in PBS and then incu-bated in PI solution for 15 min at 4 �C. Then cells were washedagain and ow cytometry was performed on a BD FACS Cal-ibur. At least 1 � 104 cells were examined and the data wereanalyzed by Cell Quest Pro.

Single Cell Gel Electrophoresis (SCGE) assay

Single Cell Gel Electrophoresis (SCGE), or Comet assay, is a wellknown and established method by which the damage of DNA incells is detected by proper staining with a uorescent dye.15 Forthis, cells (1 � 105) were treated with 250 mM of DPA, LPA, SNP,DPA–SNP and LPA–SNP along with cell only for 72 h. Aerwards,cells were washed with PBS and reconstituted to 1 � 103–1 �104 cells per ml in PBS. Treated cells were mixed with 1% lowmelting agarose (LMA) and layered over pre-coated glass slides

J. Mater. Chem. B, 2013, 1, 6634–6643 | 6635


Fig. 1 (A) Chemical structure of penicillamine. (B) Plasmon characterization ofSNP, DPA–SNP and LPA–SNP. (C) Verification of induction and amplification ofchirality of nano-conjugated drug.

Journal of Materials Chemistry B Paper

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and incubated at 0 �C for 10 min. Then a third layer of 1% LMAwas applied and further incubated at 0 �C for 10 min. Coatedcells on slides were immersed in lysis buffer (2.5 M NaCl, 100mM EDTA, 10 mM Trizma base, 1% Triton X-100) and lysis wascarried out overnight at 4 �C in the dark. Aer lysis, slides werekept in alkaline electrophoresis buffer (300 mM NaOH, 1 mMEDTA, pH > 13) for 20 min and then run at a voltage of 24V/300mA for 30 min. Aerwards, the slides were neutralized thricewith neutralizing buffer (0.4 M Tris, pH 6.5) for 5 min at roomtemperature. The slides were stained with 2 mg ml�1 of EtBr(ethidium bromide) and incubated for 5 min and then washedwith chilled milli Q water and covered with a glass cover slip foruorescence microscopy study. Images of comets were capturedwith 10� objective and analyzed by the freely available cometprogram COMET SCORE.

Mitochondrial transmembrane potential assay

To check the effects on mitochondrial membrane potential,polarization–depolarization assay was performed with treatedand control sets.16 In brief, cells (1 � 105) were incubated withDPA, LPA, DPA–SNP and LPA–SNP (150 and 250 mM) for 72 h,washed with PBS and stained with JC-1 (a potentiometric probefor mitochondria, 25 mM) in the dark for 30 min at 37 �C. Cells(1 � 104) were then analyzed by a ow cytometer to determinethe mitochondrial membrane depolarization. The dataobtained were analyzed by both Cell Quest Pro and Flow Josoware.

Identication of the cell proliferation index by CFSE assay

For the proliferation study, initially U87MG cells were loadedwith 2.5 mM Carboxy uorescein Diacetate Succinimidyl Ester(CFSE) (Molecular Probes, Invitrogen),17 at 37 �C for 15 min andsubsequently the reaction was stopped with the addition of FCS(5% v/v). Then labeled U87MG (1 � 105) cells were treated withDPA, LPA, SNP, DPA–SNP and LPA–SNP (250 mM) for 3 consec-utive days in appropriate cell culture condition. Aer the indi-cated time point, cells were analyzed by ow cytometry for thereduction of CFSE uorescence which gave a metric for cellproliferation. Day 0 uorescence of CFSE labeled U87MG cellsserved as the reference frame for the analysis. Proliferationindex was calculated by taking the ratio of 0th day uorescenceintensity and 3rd day uorescence intensity for a particulartreatment set.

Cell cycle analysis

Cells (1 � 105) were incubated with free PA, SNP and also PAconjugated SNP (250 mM) for 72 h, washed with PBS and theprotocol followed for cell cycle analysis according to the man-ufacturer's protocol from BD cycle test plus DNA analysis kit.Cells (1 � 104) were then analyzed by a ow cytometer todetermine the cell cycle distribution.18

Immunoblot assay

Proteins were extracted from treated and untreated whole cellsand quantied by the Bradford method. An equivalent amount

6636 | J. Mater. Chem. B, 2013, 1, 6634–6643

of protein (80 mg) was resolved by SDS-PAGE (10%) and electrotransferred to nitrocellulose membrane. The membrane wasnonspecically blocked by TBS–BSA, probed with primaryantibody (caspase-8, caspase-3, caspase-9, PARP, b-actin,1 : 1000 dilution) overnight at 4 �C. The membrane was washedwith TBS containing 0.1% Tween-20 and incubated with theappropriate HRP-conjugated secondary antibody (1 : 1000dilutions). Subsequently, the membrane was washed andimmune reactive complex was identied by the West Picochemiluminescence detector system.19

Results and discussionOptical characterization of SNP and its conjugates with PA

Differential response is obtained in many disease conditions(including cancer treatment) towards the enantiomeric forms ofchiral drug molecules,2 but whether such a response is seen inthe case of chiral nanoparticles is an interesting subject to bestudied. To full the aim, rst of all we synthesized silvernanoparticles and conjugated them with enantiomeric forms ofPA (Fig. 1A). The conjugation was done by addition of drugmolecules to a solution containing nanoparticles and allowingthe molecules to adsorb on the surfaces of SNP (mainly throughelectrostatic and hydrogen bonding interactions). The plas-monic nature of the particles was characterized by monitoringthe absorption spectra of SNP, DPA–SNP and LPA–SNP from300–800 nm (Fig. 1B). Bare SNP showed plasmon maxima (lmax)at �392 nm characteristic of typical SNP whereas conjugatednanoparticles showed red shied lmax at 408 nm and 406 nmwith reduced intensity for DPA–SNP and LPA–SNP respectively,which further conrmed the conjugation of nanoparticles withDPA or LPA.




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Chiral signatory patterns of SNP, DPA–SNP and LPA–SNPwere detected by circular dichroism in the wavelength span 190to 420 nm covering both far-UV and plasmonic regions of PAand SNP. Fig. 1C shows that free PA (both D- and L-enantiomericforms) absorbs near the far-UV range 190–250 nm which mightoriginate due to n / p* and p / p* transitions of thecarboxylic group and thiol group. Bare SNP does not show anyCD signal as it passes through zero which again conrms theachiral nature of SNP but SNP conjugates, i.e. DPA–SNP or LPA–SNP, showed enhanced CD signal w.r.t. DPA/LPA both in the UVand visible region (190–420 nm) which results due to the asso-ciation of drug and SNP through extensive hydrogen bondingfurther supporting the chiral nature of conjugated SNP. Hencenanoparticle conjugation of DPA or LPA gave rise to an ampli-ed CD signal compared to DPA or LPA alone. Generation ofplasmonic CD signal conrms the induction of chirality inconjugated SNP (DPA–SNP and LPA–SNP). The amplication ofCD signal can be explained by enhancement of the magnitudeof the magnetic and the electrical diploes, or by the re-align-ment of the two that leads to increase in the CD signal. As theamplication of an existing CD signal is primarily a property ofthe chiral template, it can be said that the amplication part isdue to the molecular re-arrangement that reects in the CDsignal. Generation of new plasmonic CD signal, on the otherhand can be associated to the template induced induction of anew dot product of electrical and magnetic dipole vectors.Interaction of nanosurfaces with the chiral template imposesasymmetric connement that led to emergence of novelchirality in the nano-material.

Verication of conjugation of PA with SNP by FT-IR and itsmorphology study by TEM

Conjugation of SNP with DPA or LPA is further veried by FT-IRstudies.20 For this, rst of all citrate stabilized SNP was sub-jected to IR measurement (ESI, Fig. S1A,† le panel). Then, pre-synthesized nanoparticle solution was mixed with a particularconcentration of conjugant molecule in a particular ratio.Fig. 2A and B respectively show FT-IR spectra for DPA–SNP andLPA–SNP. A marked changed in thiol (–SH group present in PA)stretching frequency in the range 2550–2600 cm�1 (mainly 2570cm�1) indicates that the major group responsible for conjuga-tion of SNP with PA (both DPA and LPA) is thiol. As the thiolstretching frequency of PA (D-/L-) vanishes completely uponconjugation with SNP, hence we predict that SNP conjugation ismediated through thiol bonding as also discussed by otherauthors in detail.21–23 Due to the presence of SNP in the vicinityof other functional groups of the PA molecule, we also ndchanges in the IR-vibrational mode of the stretching frequencyin the adjacent group. A broad IR band, ranging from 2500–3300 cm�1 characteristic of the O–H group of carboxylic acidwas also changed due to conjugation. IR stretching frequency ofpeaks near 1583 and 1390 cm�1 (DPA) or 1587 and 1390 cm�1

(LPA) also changed as a consequence of nano-conjugationwhich further conrms the participation of –COOH group. Ashi in the peaks from 1640–1550 cm�1 also indicates the roleof amine as the said region is characteristic of the N–H bending


mode of vibration which is also altered both in DPA–SNP andLPA–SNP compared to DPA or LPA alone, shown by the dottedlines. The environment of methyl group is also changed as aresult of interaction between SNP and the enantiomeric formsof drug (PA) molecule which is marked by the change in IRfrequency (region 1350–750 cm�1) for both DPA–SNP and LPA–SNP (shown by red and blue solid lines, respectively) whencompared with only DPA or LPA (shown by red and blue dottedlines). As we have seen from the IR study, only the thiol vibra-tional frequency disappears completely and alteration in otherfunctional groups takes place due to the presence of SNP in theneighborhood of the PA molecule, and we predict the structureof SNP conjugated PA as shown in the insets of Fig. 2A and B. Asthe major binding site is thiol the chiral centre of PA stillremains exposed to the environment and hence by this conju-gationmethod we successfully obtain chiral silver nanoparticlesconjugated with PA molecules.

Themorphology study by TEM indicates the spherical natureof SNP (ESI, Fig. S1B,† right panel) and the size of the SNP werefound to be 8 � 1.8 nm in diameter. Conjugation of SNP withDPA/LPA leads to a slight increase in the diameter of the particlewhich is also corroborated with the red shiing of the plasmonpeak (Fig. 1B). The diameters of the DPA–SNP and LPA–SNPwere found to be 12.9 � 1.6 and 10.7 � 0.7 nm, respectively(Fig. 3A and B, respectively).

Enhanced cytotoxic activity of DPA upon nano-conjugation,compared to conjugated form of LPA

To identify the potential of chiral nanoparticles with respect totheir naked components, initial cell viability assay was done byMTT assay and identied that chiral nanoparticles (DPA–SNPand LPA–SNP) were more effective towards cytotoxic activityover U87MG cell line in comparison to naked DPA or LPA.

More importantly, from Fig. 4A, it is seen that the DPAshowed reduced toxicity compared to LPA in naked form,whereas a changed toxicity pattern was observed upon conju-gation with SNP. DPA–SNP showed higher activity than LPA–SNP and IC50 values were documented as 180 mM and 220 mM,respectively, aer 72 h treatment. The IC50 value for DPA wasabout 500 mM and for LPA it was 344 mM. A bar diagramrepresentation of IC50 values is shown in Fig. 4B and the changein IC50 value from DPA to DPA–SNP is more signicant than thechange from LPA to LPA–SNP, as determined by the p valuestatistical analysis. The naked forms of DPA and LPA showedonly different activity at the higher concentrations ($300 mM) ofdrug. However the lower concentration range of drug showedsimilar cytotoxic activity. Upon nano-conjugation of DPA andLPA, the lower concentration range (#300 mM) becomes sensi-tive to change in cytotoxicity activity. Even there is reversal inIC50 value for bare drug and nano-conjugated drug. Whilecomparing the cytotoxic activity of DPA–SNP and LPA–SNP withthat of bare SNP, we observed the superior cytotoxicity of DPA–SNP and LPA–SNP as the IC50 value of SNP was evaluated to be275 mM in an equivalent period of treatment which was higherthan drug conjugated chiral nanoparticles.

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Fig. 2 (A) FT-IR study of DPA (red dotted line) and DPA–SNP (red solid line). (B) FT-IR study of LPA (blue dotted line) and LPA–SNP (blue solid line).

Fig. 3 (A) Morphology of DPA–SNP by TEM. (B) Morphology of LPA–SNP by TEM.The scale bar for TEM measurement is 20 nm in each case.


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Flow cytometry evaluation of enhanced apoptosis andsuppressed cell division by DPA–SNP than LPA–SNP

The mechanism of cytotoxicity of DPA, LPA, SNP, DPA–SNPand LPA–SNP was further explored to identify whether theobserved cell death was mediated by apoptosis or necrosis. Weperformed FACS based different cytotoxic assays like annexinV staining and PI uptake. Fig. S2 of ESI† section representshistogram plot of annexin V staining assay for twodifferent doses of DPA, LPA and its nano-conjugated forms toevaluate annexin V positive cells and also to assess thedifferent response of chiral drug vs. chiral nanoparticle

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conjugated with chiral drug. At 150 mM doses, it was foundthat there was not much difference in the annexin V positivecell counts for DPA or LPA and also between DPA–SNP or LPA–SNP but the annexin V positive cells were more than comparedto only DPA or LPA (lower le panel of Fig. S2†). In the case of250 mM dose it was found that the drug only samples do notshow difference in their annexin V positive cell counts butnano-conjugation leads to higher annexin V positive cells forDPA–SNP when compared to LPA–SNP (as shown in the insetof Fig. S2,† lower right panel for nano conjugate sets). Aquantitative measurement of annexin V positive cells has beendone by dening annexin V index (in arbitrary units) which isthe product of the annexin V positive cell and mean uores-cence intensity (MFI) of annexin V positive cells. From Fig. 5Ait is seen that at 150 mM dose the annexin V index value isgreater for nano-conjugate than that of only DPA or LPA butthe effect is similar for both conjugated nanoparticles.Whereas the 250 mM doses shows different responses forDPA–SNP and LPA–SNP and the index value is high for chiralDPA–SNP.

Further PI uptake assay, which detects the membraneintegrity, was assayed as a result of challenging cancer cell lineU87MG by chiral drug, bare SNP and chiral nanoparticles. Theassay indicated that dead cell membrane disintegrationoccurred more efficiently by DPA–SNP than LPA–SNP (Fig. S3,



Fig. 4 (A) Identification of enhanced cytotoxicity of DPA–SNP and LPA–SNP thantheir naked forms by MTT assay. U87MG cells were treated with different agentswithin the concentration range of 0–400 mM for 72 h. (B) IC50 values were eval-uated for each treatment set from the MTT assay and identified that DPA washighly sensitized after conjugation with SNP as IC50 was decreased significantlyfor DPA–SNP. Each value is the mean� SD of three independent experiments. **P< 0.01 and *P < 0.05, significant difference between two test groups.

Fig. 5 (A) Graphical representation of annexin V index value for different sets ofexperiment for two different doses i.e. 150 and 250 mM. (B) Graphical represen-tation of PI incorporation represented as PI index value in each set of experiments.Each value is the mean� SD of three independent experiments. **P < 0.01 and *P< 0.05, significant difference between two test groups.


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ESI† section). Here, we also further conrmed that both theconjugated nanoforms were more effective towards cellmembrane disintegration than naked DPA and LPA. At theconcentration of 250 mM DPA–SNP triggered �17% cellmembrane alteration and LPA–SNP triggered �13%. However,more importantly the difference between DPA–SNP vs. DPA was�12% where as LPA–SNP vs. LPA difference was �6% whichindicated higher efficacy of DPA–SNP compared to LPA–SNPtowards cytotoxicy of U87MG cells as revealed from the lowerright panel of Fig. S3, ESI.† Now comparing the annexin Vstaining and PI uptake assay data for bare SNP at 150 mM, weobserved almost similar result to that of DPA or LPA alone andslightly lower or the same when compared with DPA–SNP andLPA–SNP. Whereas for the 250 mM dose, the annexin V and PIindex value for SNP showed a lower index value whencompared with nano-conjugates. Reversal in cytotoxicityactivity is again observed in annexin V and PI assay. Initiallythe annexin V index value was the same for both DPA and LPAbut in case of chiral nanoparticle, DPA–SNP showed higherindex value in comparison to LPA–SNP. The reversalphenomenon is more prominent in PI uptake assay. In thecase of only drug, LPA showed a higher PI index value whilenano-conjugated DPA, i.e. DPA–SNP, showed a higher indexvalue.

Furthermore, we wanted to address whether the nano-conjugate drug has any effect over cell division. Accordingly we


performed cell based CFSE assay and identied signicantdecrease of cell division upon the treatment of DPA–SNP(250 mM) than DPA for 3 day prolonged study as shown inFig. S4 (ESI†). However, nano-conjugation did not sensitizeLPA for enhanced blockage in cell division. Even the bare SNPshows a similar extent of cell cycle blockade to that of DPA orLPA. This result indicated that nano-conjugated drug inducesarrest somewhere in the cell cycle progression. To identify theactual site of blockage in cell cycle pathway, PI stainingmediated cell cycle proling (ESI, Fig. S5A and B†) was donefor all the treated and control cells. This study indicated thatboth the naked drugs (DPA/LPA) were capable to inducesignicant G2/M phase cell cycle arrest and enhancement orthe % gated cell in G2/M phase was about 13% and 11%respectively for DPA and LPA w.r.t. cell only. Interestingly,upon nano-conjugation G2/M phase cell cycle arrest wasenhanced for both the chiral nano forms to the extent ofapproximately 27% and 23% respectively for DPA–SNP andLPA–SNP w.r.t. cell only. Whereas bare SNP (16%) showsslightly more arrest in G2/M phase than DPA/LPA but less thanDPA–SNP/LPA–SNP. Also, the difference of cell cycle arrest inG2/M phase between DPA–SNP and DPA was more statisticallysignicant than the difference between its other enantiomericforms (ESI, Fig. S5B†). All of the above cell based studiesindicated the higher efficacy of cellular cytotoxicity of DPA–SNPthan LPA–SNP.

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DPA–SNP was a greater potential DNA damaging agent thanLPA–SNP

SGCE or comet assay is a very sensitive method of detectingDNA damage. By performing this assay we have measured theextent of DNA damage induced by chiral drug and chiralnanoparticles by viewing the uorescence image of the DNAmigration pattern like a comet and further analyzing thestatistical data derived from those images by freely availablesoware COMET SCORE. For this assay we have chosen 250 mMdoses as this dose shows the differential response for both DPA/LPA and DPA–SNP/LPA–SNP. As is evident from Fig. 6A, it isseen that in the case of cell only image data the nucleus is intactand hence a comet like pattern is not seen. In between DPA andLPA a slight increase in tail of the comet is seen from themicroscopic image and also from Fig. 6B, in which a bardiagram representation of comet length and comet tail isshown. By comparing DPA and LPA, the length of both cometand tail is slightly more in the case of LPA.

Further, comparing the microscopic data between DPA–SNPand LPA–SNP, it is seen that the extent of DNA damagedmeasured by comet length is higher for the former case. Fromthe bar diagram representation of Fig. 6B it is also clear thatcomet length and tail length are both higher in the case of DPA–SNP than LPA–SNP. We further analyzed the microscopic dataand calculated the tail moment, which is expressed as theproduct of the length of the “tail” of DNA trailing the nucleusand the percent of total DNA in the tail and found that tailmoment is signicantly much higher for DPA–SNP than LPA–SNP (Fig. 6C). Both the change in length (comet and tail) and

Fig. 6 (A) Microscopic images of U87MG cells, treated and untreated (cell only)and stained with ethidium bromide and the length of the scale bar in each figureis equivalent to 10 mm. (B) Representation of comet length and comet tail as aresult of treatment with DPA/LPA, SNP and DPA–SNP/LPA–SNP. (C) A bar diagramrepresentation of tail moment (in arbitrary units) showing higher efficacy of DPA–SNP over other treatment sets. ***P < 0.001 and **P < 0.01, significant differencebetween two test groups.

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tail moment from DPA to DPA–SNP and LPA to LPA–SNP arestatistically signicant. Bare SNP also forms the comet likestructure but the length of the comet is less than that of nano-conjugates, as seen from both Fig. 6A and B, and the tailmoment (Fig. 6C) is also less than the chiral nano-forms.

Reversal of mitochondrial membrane potential as aconsequence of nano chirality

Next, we wanted to know whether mitochondrial stress wasgenerated and if the stress was different or not in the presenceof all co-factors. To address this question JC-1 staining assaywas done for the identication of mitochondrial stress inducedmembrane depolarization for two different doses (150 and 250mM) of drug (DPA/LPA) and chiral nano conjugated drug (DPA–SNP/LPA–SNP). As we have seen in all the above conditions thatbare SNP does not show signicant difference in cytotoxicitycompared to DPA–SNP and LPA–SNP and we were more inter-ested to probe the effect of drug conjugated chiral SNP,henceforth we have excluded the SNP data for further consid-eration. From the ow cytometric analysis of JC-1 (Fig. 7A), wehave critically identied that only chiral drugs were not effectiveto induce mitochondrial membrane depolarization irrespectiveof lower or higher dose. Calculations of percentage gated cellindicated that only about 4% increment of depolarized cell wasobserved in both the lower doses of DPA and LPA (150 mM)whereas higher dose (250 mM) of LPA triggered little enhance-ment of mitochondrial depolarized cell. On the contrary, DPA–SNP induces a signicant enhancement of mitochondrialdepolarization of about 4% and 11% respectively for 150 and250 mM doses w.r.t. naked DPA. Whereas, LPA–SNP did notshow any enhancement of mitochondrial depolarization whencompared to LPA for both lower and higher doses. By criticallyobserving the data, we can see that there was a reversal inmitochondrial membrane depolarization upon nano-conjuga-tion. Initially the % gated cell for LPA was higher (9.78% and12% for 150 and 250 mM, respectively) than DPA (9.18% and9.57% for 150 and 250 mM, respectively). Upon nano-conjuga-tion and with induction of chirality on nanosurfaces, themitochondrial depolarization status of a cell is reversed. Thepercent depolarized cell for DPA–SNP (13.28% and 19.81% for150 and 250 mM, respectively) was found to be enhancedcompared to LPA–SNP (8.2% and 11.67% for 150 and 250 mM,respectively).

To substantiate the superior role of DPA–SNP, JC-1 inducedgreen (Fig. 7B) and red uorescence (Fig. 7C) were analyzedseparately. In the green uorescence study we have interestinglyidentied a signicant reversal of mitochondrial depolarizingeffect of naked drug and chiral nano-conjugated drug at higherdose (250 mM). Here we observed that 250 mM LPA was morepotent than DPA to induce mitochondrial depolarization iden-tied by green uorescence intensity peak. On the contrary, thehigher efficacy of LPA shows an opposite depolarization effectupon nano-conjugation as green uorescence peak was higherin the case of chiral DPA–SNP than LPA–SNP. However wedid not found any signicant differential activity at lower dose(150 mM).



Fig. 7 (A) DPA–SNP was responsible for higher mitochondrial damage than its chiral counterpart at higher dose (250 mM) identified by the JC-1 mediated flowcytometric analysis. (B) Histogram overlay of JC-1 green fluorescence represented that reciprocal shifting occurred at 250 mM concentration of SNP conjugated chiralmolecules compared to the naked chiral molecules. (C) Overlay histogram analysis of JC-1 red fluorescence remains unchanged.


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We also did not observe any change of red uorescenceintensity in JC-1 staining assay. These results suggested thatDPA–SNP might be a potential inducer of mitochondrial deathcascade activation.

To evaluate the downstream pathway activation of mito-chondrial damage, protein proling of different caspases andPARP was done. Western blot data revealed the enhancedcaspase-9 and caspase-3 activation and deactivation of PARP inDPA–SNP treated U87MG cells. Activation of caspase andinactivation of PARP were identied by the reduction of proform protein level. As DPA–SNP previously showed its betterefficacy towards mitochondrial depolarization, direct mito-chondria dependent caspase-9 should be more pronouncedlyaltered and this was also veried by western blot analysis(Fig. 8). However, we did not nd any signicant change inprotein level in pro-form of caspase-8 which is mainly associ-ated with cell surface death receptor mediated apoptosis. Thisresult also suggested that nano-conjugated drug did not haveany effect in death receptor pathway activation at least for250 mM dose.


To understand the possible mechanisms for enhanced cyto-toxicity by DPA–SNP over U87MG cells, we did different cytotoxicevaluation assays and also the protein level identication assay.In the context of the mechanism, we could postulate that mito-chondrial dysfunction was the major event towards enhancedactivity of DPA–SNP. Mitochondrial transmembrane depolariza-tion is a hallmark of the initiation of the mitochondria mediatedintrinsic pathway of apoptosis. Several mitochondria targetingdrugs altered the mitochondrial membrane polarization andhence induced the cell death reported in different types ofcancer.24 Depolarization of mitochondrial membrane indicatesmainly the alteration or damage of inner mitochondrialmembrane induced by the enhanced generation of reactiveoxygen species (ROS) in the mitochondrial lumen due to theleakage of free electrons from the electron transport chain (ETC).Because of the excessive reactivity, ROS then can trigger proteinaggregation, lipid peroxidation, DNA damage etc., factorsresponsible for the mitochondrial dysfunction. As SNP is theknown inducer of ROS reported earlier,7,25 here we couldhypothesize that SNP conjugated with DPAmight have the higher

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Fig. 8 Western blot analysis at 250 mM dose of DPA–SNP activated caspase-9and caspase-3 and PARP was reduced notably compared to its chiral counterpartand naked drug in U87MG cell at 72 h treatment. Caspase-8 did not regulatepositively and showed almost equivalent expression levels in all the sets of theexperiment and B-actin was used as loading control.


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potential to induce an elevated level of mitochondrial ROS whichin turn induces mitochondrial dysfunction and triggers theintrinsic apoptotic pathway. Enhanced activation of the caspase-9/caspase-3 was also a concrete evidence for the intrinsic pathwayfor apoptosis which we have identied in the case of DPA–SNPtreated U87MG cells (Fig. 8). However, constant level expressionof caspase-8 prompted us to the fact that death receptor medi-ated apoptosis was not working under these circumstances. So,the overall enhanced cytotoxicity might be mediated through theintrinsic pathway of apoptosis in the presence of DPA–SNPmediated by mitochondrial dysfunction.

The reversal of the normal D and L drug function ascompared to their nano-conjugated form may be given specialattention. The cellular response may be triggered by topologicalchirality (an aspect rarely exhibited by chemical molecules, butcommon among biomolecules with non-planarity in theirgraph).26 Such non-geometrical chiral forms can emerge intopological structures like the nanosurface, the details of whichneed further exploration.

In brief, if we take the cell as a black box, we can see that itresponds differently when we challenge it with the enantiomers.The relative dominance of the L and D enantiomers is alsoreversed (in terms of cellular function) and therefore theamplied chirality effect which we observe from biophysicalexperiments is felt differently by the cell. The only mechanistichint we got in that ‘black box’ is that membrane potential isdifferently altered in response to the two chiral forms. Theamplied chirality is reported earlier (upon nano-conjugation)by several authors, but hardly anyone has demonstrated howthe cell responds to it.

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Conclusions

We chose nano-probes to amplify the chirality as in this casewe can avoid any additional asymmetric synthesis. Asymmetryin a nanostructure material can be template driven.27 Inci-dentally, synthesis and characterization of chiral nanoparticleshave been well explored in the literature and their versatileapplications in optical or electrochemical sensing or chiralrecognition,28,29 chiral catalysis,30 chiral enrichment,31 are wellcited. The origin of chirality at the nanoscale may be attributedin many cases due to transfer of ligand chirality to metalsurfaces by the “Cotton Effect”, enhancement of intrinsicchirality of metal by the ligand or due to the congurational orconformational chirality of nanoparticles.32–34 PA is used fortemplating and conjugation of chiral silver nanoparticles as itis well studied in the literature.12,20 Among the two enantio-meric forms of PA (i.e. D and L-forms), DPA is mainly used as adrug in copper chelating agent for the treatment of Wilson'sdisease as well as in the treatment of cystinuria, rheumatoidarthritis and heavy metal intoxication.35–38 Whereas, severalreports suggest LPA's toxic nature and hence the use of LPA asdrug is restricted. An interaction of cancer cell and DPA is wellreported in literature and the mode of action of anti-cancerousproperty exhibited by DPA is by the chelation of copper orheavy metal, by scavenging ROS (DPA as an antioxidant) andalso by activating apoptosis via mitochondrial dependentpathway.39

Let us now compare this existing knowledge with the datawe have obtained. The least we can say is that amplication ofchirality does not necessarily induce an amplied chiralresponse in the cell. Remarkably enough, the ampliedchirality may induce a reciprocal response. Thus we found inmitochondrial polarization de-polarization assay, the LPAchirality when amplied by SNP induces a cellularresponse akin to DPA and vice versa. Similar reciprocity is seenin some important pathways, e.g.caspase 9, where chiraland amplied chiral responses are not felt. On the contrary,the amplied chiral form plays the role of the mirrorimage molecule. Similar reversal of chiral forms and ampliedchiral forms are seen in the case of DNA damage assay (cometassay).

The insights gained from the study may provide uswith important insights in chiral drug research. Novel formu-lations of drugs40 have become fashionable in pharmaceuticalresearch particularly to improve bioavailability of drugs. Ifany such formulation leads to altered chirality the cellularresponse to the newly formulated drug may mimic anenantiomeric form which in turn may be toxic to the cell.Though further studies with various cell lines and various drugchoices are required to test the universality of the nding, itmay be apparent from this simple model study that thechirality we nd in nature using optical spectrometricmeasurements is realized in a much more complex fashion bythe cell. Understanding the cellular response to chirality maybe important for understanding the evolution of chiralselection and also for designing effective formulations of chiraldrugs.




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Acknowledgements

The authors SR and KB would like to thank CSIR, India forproviding the fellowship. We thank Mr Pulak Ray of SahaInstitute of Nuclear Physics (Electron Microscopy Facility),Kolkata for assistance in performing TEM studies of nano-particles. We are thankful to Jyoti Shaw (Department ofBiochemistry, CU) for her help. We acknowledge ICMR (grantnumber: 35/24/2010/BMS-NANO) for providing funds. Wefurther sincerely acknowledge CSIR-IICB, CSIR and DBT, Govt.of India. CM is grateful to nancial support by J.C. BoseFellowship, DST and a mutual grant from ICMR and theGerman Cancer Research Centre and a mutual grant from CSIRand Charite-Universitatsmedizin Berlin.

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Cellular response to chirality and amplified chirality