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Biomedicine & Preventive Nutrition 3 (2013) 64–73

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Original article

Anticancer activity of resveratrol-loaded gelatin nanoparticles on NCI-H460

non-small cell lung cancer cells

S. Karthikeyan a, N. Rajendra Prasad a,∗, A. Ganamanib, E. Balamurugan a

a Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608002, Chidambaram, Indiab Microbiology Division, Central Leather Research Institute, Adyar, Chennai 600020, Tamilnadu, India

a r t i c l e i n f o

Article history:

Received 27 August 2012Accepted 23 October 2012

Keywords:

Resveratrol

Coacervation

Anticancer

Gelatin nanoparticles

Lung cancer

Controlled release

a b s t r a c t

Resveratrol (RSV), a grape phytochemical, has drawn greater attention because of its beneficial effects

against cancer. However, RSV has some drawbacks such as unstabilization,poor water solubility and short

biological half time, which limit the utilization of RSV in medicine, food and pharmaceutical industries. In

this study, we have encapsulated RSV in gelatin nanoparticles (GNPs) and studied its anticancer efficacy

in NCI-H460 lung cancer cells. SEM and DLS studies have revealed that the prepared RSV-GNPs possess

spherical shape with a mean diameter of 294 nm. The successful encapsulation of RSV in GNPs has been

achieved by the cross-linker glutaraldehyde probably through Schiff base reaction and hydrogen bond

interaction. Spectrophotometric analysis revealed that the maximum of 93.6% of RSV has been entrapped

in GNPs. In vitro drug release kinetics indicated that there was an initial burst release followed bya slow

and sustained release of RSV from GNPs. The prepared RSV-GNPs exhibited very rapid and more efficient

cellular uptake than free RSV. Further, RSV-GNPs treatment showed greater antiproliferative efficacy than

free RSV treatment in NCI-H460 cells. It has been found that greater ROS generation, DNA damage and

apoptotic incidence in RSV-GNPs treated cells than free RSV treatment. Erythrocyte aggregation assay

showed that the prepared RSV-GNPs formulation elicit no toxic response. HPLC analysis revealed that

RSV-GNPs was more bioavailable and had a longer half-life than free RSV. Hence, GNPs carrier system

might be a promising mode for controlled delivery and for improved therapeutic index of poorly water

soluble RSV.© 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Non-small cell lung cancer (NSCLC) constitutes 75–80% of all

lung cancers and is a most frequent tumor in the elderly [1]. The

long-term survival rate of lung cancer patients treated by con-

ventional modalities such as surgery, radiation and chemotherapy

remains far from satisfactory [2]. RSV is a naturally occurring

polyphenolic phytoalexin, synthesized by a wide variety of plant

species such as grapes, berries, peanuts, and a variety of food

sources in response to injury or fungal attack [3]. Recently, RSVhas drawn greater attention because of its beneficialeffects against

many diseases such as cancer [4], inflammatory disease, etc. [5].

However, RSV has somedrawbacks such as unstabilization [4], poor

watersolubility [6] and shorter biological half time [7]. Allof which

limit the utilization of RSV in medicine, food and pharmaceutical

industries. Many researchers have attempted to improve its solu-

bility by using dimethylsulfoxide (DMSO) [8]. However, the safety

∗ Correspondingauthor. Tel.: +91 9842 305384, fax: +91 4144 239141.

E-mail address: [email protected](N. Rajendra Prasad).

of DMSO is questionable due to its risk on vasoconstriction and

neurological toxicity [9]. Biodegradable polymers have attracted

greater interest in the recent years for clinical administration of

anticancer drugs. Incorporation of bioactive agents into polymer

matrices for extending their shelf life, protecting against oxidation

and increasing bioavailability have been growing rapidly [10].

Gelatin is a naturally occurring protein based biopolymer with

relatively low antigenicity. It has been used for decades in par-

enteral formulations as an approved plasma expander [11]. Its

biodegradability, biocompatibility, chemical modification poten-tial and cross-linking possibility make gelatin-based nanoparticles

a promising carrier system fordrug delivery [12]. Numberof inves-

tigations showed glutaraldehyde as an effective cross-linker that

gives stability, shape and an enhanced circulation time to GNPs

[12,13]. The aldehyde groups of glutaraldehyde and the amino

groups of gelatin undergo Schiff base reaction and form a net-

work structure [12], which will favour stabilization and controlled

release of RSV. The objective of this work was to prepare RSV-GNPs

usingglutaraldehydeas a cross-linker for the controlledrelease and

improved anticancer efficacy in non-small cell lung cancer (NSCLC)

cell line.

2210-5239/$ – seefrontmatter © 2012 Elsevier Masson SAS. All rights reserved.

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S. Karthikeyan et al. / Biomedicine & Preventive Nutrition 3 (2013) 64–73 65

2. Materials and methods

2.1. Chemicals

Resveratrol (RSV), bovine skin gelatin (type B), glutaraldehyde

(25% v/v aqueous solution), span 80, Hoechst 33258, ace-

tonitrile, HPLC water, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT), 2-7’ dichlorodihydrofluorescein diac-

etate (DCFH-DA), rhodamine 123 (Rh-123), ethidium bromide

(EBr), acridine orange (AO), heat inactivated fetal calf serum (FCS),

RPMI-1640 medium, glutamine-penicillin-streptomycin solution

and trypsin-EDTA were purchased from Sigma Chemicals Co., St.-

Louis, USA. HPLC grade methonal, analytical grade ethanol and

dimethyl sulfoxide (DMSO) were purchased from SRL, India.

2.2. Preparation and characterization of RSV-GNPs

2.2.1. Preparation of resveratrol-loaded GNPs

Gelatin nanoparticles were prepared by coacervation-phase

separation technique with slight modification [14]. Briefly, 200mg

of gelatin-Bwas dissolvedin distilledwater(20 mL) underconstant

heating at 40±1 ◦C. RSV (10mg dissolved in 500l of DMSO) was

added in aqueous polymer phase, followed by a drop wise addi-

tion of span 80 (30mL) to form GNPs. At the end of the process,

glutaraldehyde solution (25% v/v aqueous solution) was added as a

cross-linking agent,and thesolutionwas stirred for12 h at 700rpm

(Remi, Mumbai, India). DMSO was removed with repeated mild

washing with distilled water. The prepared RSV-GNPs were stored

at 25◦C under vacuum (2mmHg) for further investigations.

2.2.2. Particle size, size distribution and zeta potential

DLS (ZetasizerNano, Malvern Instruments Ltd.United Kingdom)

was used to measure the average size and size distribution of the

prepared nanoparticles. Three different batches were analyzed to

give an average value and standard deviation for the particle diam-

eter and zeta potential.

2.2.3. Scanning electron microscopy (SEM)The morphological features of RSV-GNPs were examined by

scanning electron microscopy (Quanta 200F, FEI, Hillsboro, OR,

USA). The samples were sprinkled onto a double-sided tape and

sputter-coated with a 5 nm thick gold layer. The inner-structure of

nanoparticles was observed after fracturing by a razor blade.

2.2.4. Fourier transform-infrared spectroscopy (FT-IR)

FT-IR spectra were recorded using a Perkin Elmer 1700 FT-IR

spectrometer, USA. About 5 mg of sample were mixed with 100 mg

of KBr and compressed into pellet using a hydraulic press.

2.2.5. Differential scanning calorimetry (DSC) and

thermogravimetry analysis (TGA)

Thermal properties of polymers and particles were measured byDSC and TGA. Nitrogen was used as the purge gas. Top pierced alu-

minum pans were used throughout the study with sample weights

varied between 5 and 10mg. The DSC system was controlled by

DSC-Q-200-TA instruments USA, Built 79 software (version 23.10).

Samples were characterized by DSC in the range of 25–360◦C a t a

heating rate of 10 ◦C per minute. The TGA system was controlled by

TGA-Q-50-TA instruments USA, Built 31 software (version 20.6).

2.2.6. Determination of drug encapsulation efficiency (EE), yield

and actual drug loading

RSV-GNPs (10 mg) were dispersed in an aqueous solution

(10 mL; NaCl, 0.9%, w/v and 5% v/v DMSO) containing trypsin

(200g/mL) at the ratio of 1:5 (w/w). The dispersion was kept

for 5 h at 37±1◦

C in the dark under magnetic agitation. The clear

solution obtained was measured by UV spectrophotometer (Elico

SL159, India) at the wavelength 270 nm. The EE percent can be

determined by the following equation (1):

Encapsulation efficiency (%) =(Drug)tot − (Drug)free

(Drug)tot

× 100 (1)

The purified nanosuspension was ultracentrifuged (Centrifuge

5415R, eppendorf, Germany) at 13,000 g for 1 h at 4±1 ◦C. The

supernatant was discarded and the pellet was freeze-dried. Theyields of RSV-GNPs were calculated using Eq. (2). The actual drug

content of GNPs was calculated using Eq. (3);

Nanoparticles yield (%w/w)

=Mass of recovered GNPs

Totalmass ofpolymer anddrugadded × 100 (2)

Actual drugloading (%w/w) =MassofdruginGNPS

Massof GNPsrecovered × 100 (3)

2.2.7. In vitro drug release studies

The in vitro drug release tests were carried out on all formu-

lations (RSV and RSV-GNPs). Fifty milligrams of each sample was

suspendedin 100 mL ofPBS bufferat various pHat 37◦C andplacedin an incubated shaker at 120rpm. At predetermined time inter-

vals, 3 mL of aliquots waswithdrawnand the concentration of drug

released was monitored by UV spectrophotometer (Elico SL159,

India) at 270 nm. The dissolution medium was replaced with fresh

buffer to maintain the total volume. The drug release percent can

be determined by the following equation (4):

Drug release (%) =C(t)

C(0)× 100 (4)

where C(0) = amount of drug loaded, C(t) = amount of drug released

at a specified time. All studies were carried out in triplicate.

2.2.8. Analysis of resveratrol uptake by fluorescence microscopyThecellular uptakeof RSV-GNPs in NCI-H460 cells wasanalyzed

by the fluorescence microscopy. In brief, cells were incubated with

Hoechst 33258 dye (50 ng/mL; blue fluorescence) for 30min, and

then washed two times with PBS. The washed cells were resus-

pended in media and then incubated with RSV for 3h. Cells were

then examined under a fluorescence microscope (BX51; Olympus,

Tokyo, Japan) andimages werecapturedusing a Photometrics Cool-

snap SHC-745 color camera (Samsung, Korea).

2.3. Anticancer efficacy of RSV-GNPs

2.3.1. Cell culture

The present work was carried out in NCI-H460 non-small cell

lung cancer (NSCLC) cell line. NCI-H460 cells were obtained fromNational Centre for Cell Science (NCCS), Pune, India. Cells were cul-

turedas monolayerin RPMI-1640 medium,supplemented with10%

fetal bovine serum (FBS), penicillin and streptomycin in a humidi-

fied atmosphere of 95% air and 5% CO2 at 37 ◦C. Cells were grown

in 75 cm2 tissue culture flasks and used for experiments when in

exponential growth phase. Cells were treated with different con-

centration of RSV and RSV-GNPs (1, 5, 10, 15, 20, 25, 30, 35, 40and

50g) and cytotoxicity was observed after 24h incubation by MTT

assay [15]. IC50 values were calculated and the optimum dose was

used for further study.

The NCI-H460 cells were divided into four experimentalgroups.

Group 1: untreated control cells, Group 2: RSV alone (20g/mL)

treated cells, Group 3: RSV-GNPs (5g/mL) treated cells, Group 4:

Cisplatin (5g/mL) treated cells.



66 S. Karthikeyan et al. / Biomedicine & Preventive Nutrition 3 (2013) 64–73

2.3.2. Determination of intracellular ROS levels

Intracellular ROS level was measured using a non-fluorescent

probe, 2,7-diacetyl dichlorofluorescein (DCFH-DA), that can pen-

etrate into the intracellular matrix of cells where it is oxidized

by ROS to fluorescent dichlorofluorescein (DCF) [16]. RSV-GNPs

and free RSV treated NCI-H460 cells were seeded in 6 well plates

(2×106cells/well) and incubated with 10M DCFH-DA for 30min

at 37 ◦C. Fluorescent measurements were made with excitation

and emission filters set at 485±10nm and 530±12.5 nm, respec-

tively (Shimadzu RF-5301 PC spectroflurometer). The cells were

alsoobservedunder fluorescencemicroscope usingblue filter(450-

490nm) (Nikon, Eclipse TS100, Japan).

2.3.3. Changes in mitochondrial transmembrane potential ()The changes in mitochondrial membrane potential during RSV-

GNPs and RSV treatment condition were analyzed using Rh-123

staining. RSV and RSV-GNPs treated cells were mixed with 1L

of Rh-123 (5mmol/L) and kept incubation for 15min [17]. Then,

the cells were washed with PBS and observed under fluores-

cence microscope using blue filter (450–490nm). The fluorescence

intensity in treated cells were also recorded using spectrofluro-

metric with excitation and emission filters set at 485±10nm and

530±12.5nm, respectively.

2.3.4. Measurement of oxidative DNA damage

DNA damage was estimated by alkaline single cell gel elec-

trophoresis (comet assay). The extent of DNA damage was

estimated by fluorescence microscopy using a digital camera and

analyzed by image analysis software, CASP. For each sample, 100

comet images were analyzed and tail moment, tail length and olive

tail moment were quantified [18].

2.3.5. Apoptotic morphological changes

Acridine orange (AO) and ethidium bromide (EBr) dual staining

method was adopted to differentiate condensed apoptotic nuclei

from normal cells [16]. The control, RSV andRSV-GNPs treated cells

were seeded in 6 well plate (3×104/well) and incubated at CO2

incubator for 24h. The cells were stained with 1:1 ratio of AO/EBr,

and viewed under a fluorescence microscope with a magnification

of 40×. The number of cells showing features of apoptosis was

counted as a function of the total number of cells present in the

field.

2.3.6. Erythrocyte aggregation assay

Fresh blood was isolated andaddedto an equal volumeof buffer

containing PBS and 10mM EDTA. Erythrocytes were pelleted by

centrifugation, washed multiple times with PBS. To one volume of

fresh erythrocytes one volume of RSV or RSV-GNPs were added.

Solutions were incubated for 1 h at 37◦C and placed into a 96well

plate for phase contrast imaging.

2.4. Pharmacokinetics study

2.4.1. Animals care and handling

Healthy maleSwiss albino miceweighing20–22 g obtainedfrom

the Central Animal House, Raja Muthiah Medical College, Anna-

malai University, Annamalainagar, India were grouped and housed

in poly acrylic cages (38cm×23cm×10cm) with not more than

six animals per cage and maintained under standard laboratory

conditions (temperature 25±2 ◦C and dark/light cycle 14/10h).

They were allowed free access to standard dry pellet diet (Hin-

dustan Lever, Kolkata, India) and water ad libitum. All procedures

described were reviewed and approved by the University Animal

Ethical Committee (proposal No: 864; 160/1999/CPCSEA).

2.4.2. RSV-GNPs Extraction and quantification

The mice were divided in to two groups (6 mice in each group),

group 1 was given RSV and group 2 was given RSV-GNPs. RSV

and RSV-GNPs were administered intravenously (10 mg/kg) and

the blood was collected at different time intervals. Serum was

separated and the RSV levels were determined by HPLC analy-

sis. The HPLC analysis of RSV was performed using A Nova-pak

C18 (150×3.9mm, 5m) column fromElite AnalyticalInstruments

(DalianCity, China). The mobile phase consisted of solvent A: 10%

methanol in water, solvent B: 90% methanol in water. The mobile

phase was delivered at a flow-rate of 0.60mL/min with a linear

gradient from 0% to 90% B in 25min, the detection wavelength was

303nm, the attenuation was 0.001, and the injection volume was

25L.

2.5. Statistical analysis

Statistical analysis was performed using one way analysis

of variance (ANOVA) followed by Duncan’s Multiple Range Test

(DMRT) by using Statistical Package of Social Science (SPSS) ver-

sion 11.5 for windows. The values of anticancer study are given as

means±S.D. for six samples in each group. The value of in vitro

drug release and cellular uptake study are given as means± S.D.

for triplicates in each group. P ≤0.05 were considered as level of

significance.

3. Results

3.1. Physicochemical characterization of RSV-Loaded GNPs

It has been noticed that the prepared RSV-GNPs nanoparticles

possess average size of 294nm and polydispersity index (PI) of

0.295 (Fig. 1i and ii). Further, the prepared RSV-GNPs had zeta

potential of −18.6mV. Ithas been found that93.6% RSV was encap-

sulated in GNPs (Table 1). SEM images of the RSV-GNPs are shown

in Fig. 2A. The prepared RSV-GNPs had smooth surface but with

some irregular small particles (Fig. 2A-ii). The major character-

istic peaks of RSV (1100–1500cm−1) and -OH phenolic bending

(1200–1600cm−1) are present in free and GNPs encapsulated RSV

(Fig. 2B-i). These RSV peaks has not been present in GNPs alone

(Fig. 2B-ii). Further, GNPs characteristic peaks like–C= O stretch-

ing (1634.4 cm−1) and C= H stretching (1089.4cm 1) are present

in the RSV-GNPs (Fig. 2B-ii and iii). Further, there was no drug-

melting peak observed in the DSC curve for RSV-GNPs particles

indicating that RSV was present in a non-crystalline state (Fig. 3A).

Thermal gravitometric analysis clearly indicates RSV-GNPs combi-

nation degrades in a slower rate, whereas GNP was degraded in a

faster manner (Fig. 3B).

3.2. In vitro drug release

Fig. 4A shows release behaviors of RSV-GNPs in PBS at various

pH levels, respectively. Upon 12h incubation, RSV-GNPs released

28.1%, 32.5% and 40.6% RSV at pH 1.4, 10.5 and 7.4, respectively.

After 48h incubation, we observed 63.7%, 69.9% and 80.2% RSV

release at pH 1.4, 10.5 and 7.4, respectively.

3.3. Uptake of RSV-loaded nanoparticles by NCI-H460 cells

Visual evidence of RSV-GNPs uptake by the NCI-H460 cells

during 30min incubation time was analyzed by fluorescence

microscopy using Hoechst 33258 dye. Free RSV treated cells show

diminished fluorescence due to the minimum uptake of RSV

(Fig. 4B-i); whereas RSV-GNPs treated cells show brighter fluores-

cence dueto enhanced intracellular uptakeof RSV-GNPs(Fig. 4B-ii).




Fig. 1. Size distribution of:i: RSVand ii: RSV-GNPs by DLS.

3.4. Anticancer efficacy of RSV-GNP nanoparticles

3.4.1. In vitro cytotoxicity assay

Fig.5 showsthe percentagecytotoxicityof RSVand RSV-GNPs(1,

5,10,15,20,25,30,35,40,45 and 50g/ml) in NCI-H460 cells.RSV-

GNPs treatment (for 24h) showed cytotoxicity on NCI-H460 cells

in a concentration dependent manner. There was a 100% cell death

at 20g/ml concentration of RSV. Conversely, RSV-loaded GNPs

showed 100% cell death at much lower 10g/ml concentration in

NCI-H460 cells. Hence, the inhibitory concentration 50 (IC50) was

fixed as 10g/ml for RSV and 5g/ml for RSV-GNPs in NCI-H460

cells.

Table 1

Physicochemical characterizations of RSV-GNPs. Values are given as means±S.D. of six experiments in each group.

Formulations Size (nm) PI EE (% w/w) Actual d rug l oading ( % w/w) Nanoparticles y ield ( % w/w) Zeta potential ( mV)

GNPs 636.3 0.381 – – 68.29 ± 3.24 −8.1 ± 0.62

RSV-GNPs 294±30 0.295 93.6 1.96±0.39 65.14 ± 3.91 −18.6 ± 0.51




Fig. 2. A. Morphology of RSV-GNPs by SEM: i: surface morphology of RSV-GNPs before freeze-drying and ii: surface morphology of RSV-GNPs after freeze-drying. B. FT-IR

spectrumof: i: free RSV; ii: gelatin and iii: RSV-GNPs.

3.4.2. Effect of RSV-GNPs on intracellular ROS generation in

NCI-H460 cells

RSV-GNPs showed maximum generation of ROS in NCI-H460

cells when compared with RSV treatment alone (Fig. 6A-i). We

observed weak ROS generation in the untreated NCI-H460 cells.

RSV treatment showed significant ROS production and a further

enhanced ROS generation was found in RSV-GNPs treated NCI-

H460 cells (Fig. 6B-i).

3.4.3. RSV-GNPsmodulates mitochondrialmembrane potential in

NCI-H460 cells

Mitochondrial membrane potential has been found to be

reduced in the RSV-GNPs treated cells when compared with RSV

alone treated cells (Fig. 6A-ii). Fluorescence microscopic images

(Fig. 6B-ii) showed accumulation of Rh-123 dye in the control

group. No Rh-123 accumulation was found in RSV-GNPs treated

cells as the membrane potential decreased.

3.4.4. RSV-GNPs induced DNA damage

RSV-GNPs treated cells showed significantly increased percent-

agetaillength (58%),tailmoment(50%)and olive tail moment(54%)

in NCI-H460 cells when compared to free RSV treatment alone

(Fig. 6A-iii). The control cellsshowed largely non-fragmentedintact

nucleoid. We observed enhanced percentage tail DNAin RSV-GNPs

treated cells than RSV treatment alone (Fig. 6B-iii).

3.4.5. Effect of RSV-GNPs on apoptotic morphological changes

Apoptotic features with condensed or fragmented chromatin,

indicativeof apoptosis,were observed in RSV andRSV-GNPstreatedNCI-H460 cells. Control cells (Fig. 6B-iv) showedevenly distributed

acridine orange stain (green fluorescence) with no morphological

changes whereas RSV andRSV-GNPstreatedcells showedapoptotic

morphological features and showed ethidium bromide fluores-

cence dueto membrane damage. RSV-GNPs treatmentshowed98%

of apoptotic cells whereas free RSV treatment showed only 42%

apoptotic cells (Fig. 6A-iv).

3.4.6. Erythrocyte aggregation assay

The RSV treatment alone caused a significant aggregation of

erythrocytes upon 1 h incubation at room temperature, whereas

RSV-GNPshave notshowedany effectwiththe erythrocytes(Fig.7).




Fig. 3. A. Thermal gravitometric curve of GNP andRSV-GNPs. B. DSC curve on weight reduction of i: GNP and ii: RSV-GNPs.

3.5. Pharmacokinetic study

3.5.1. Bioavailability of RSV-GNPs in mice blood

The mice were divided into two groups (6 mice in each group),

group 1 was given RSV and group 2 was given RSV-GNPs. RSV

and RSV-GNPs were administered intravenously (10 mg/kg) and

the blood was collected at different time intervals. Present results

clearly show that serum levels of RSV were almost twice as high in

the case of RSV-GNPs administration when compared to free RSVadministration alone (Fig. 8).

4. Discussion

In this study, RSV-GNPs were prepared by coacervation process

andstudied itsanticancer efficacy in NCI-H460 cells. GNPs have the

potential to be an efficient, viable, safe and cost-effective system

for administration of anticancer agents due to its biodegradabil-

ity, biocompatibility, suitability for intravenous applications and

low immunogenicity [12]. In this study,we cross-linked GNPs with

RSV using glutaraldehyde in situ. Glutaraldehyde is a non-zero

length cross-linker, which induces poly- or bifunctional cross-links

into the network structure of GNP by bridging free amino groups

of lysine or hydroxyl lysine. We have noticed no aggregation or

precipitation of particles during the addition of glutaraldehyde

(25% w/w).The prepared RSV-GNPs possess average size of 294 nm

(Fig. 1i and ii), polydispersity index (PI) of 0.295 and zeta poten-

tial of −18.6mV. Entrapment efficiency (EE) of RSV in GNPs was

found to be 93.6% (Table 1). Previous studies showed that the EE

of RSV in liposomes [19] and mPEG-PCL nanoparticles [20] were

only 76.00% and 91.00% respectively, indicating GNPs might be an

efficient system for the delivery of RSV. SEM images of the RSV-

GNPs are shown in Fig. 2A. The prepared RSV-GNPs had smoothsurface but with some irregular small particles (Fig. 2A-ii), which

were attributed to the results of the mechanical stress during the

stirring process or the movement of the moisture during the dry-

ing period. The internal structureof RSV-GNPsshowed a compacted

and continuous network.

FT-IR analysis is one of the important tools for the quick and

efficient identification of encapsulated chemical molecule. The

presence of both RSVand GNP characteristic peaks in theRSV-GNPs

was a direct conformation of RSV encapsulation on GNPs (Fig. 2Bi-

iii). This RSV encapsulation might be achieved by the cross-linker

glutaraldehyde probably through Schiff base reaction and hydro-

gen bond interaction. This improved thermal stability of RSV-GNPs

compared to GNPs alone might be due to the presence of RSV

(Fig. 3A and B). Previous reports indicate that the incorporation of




Fig.4. A.In vitro RSV releasekineticsfromGNP nanoparticlesat variouspH levels(1.4, 10.5 and 7.4) in PBS. Values are givenas means±S.D.of six experiments in eachgroup.

B. Cellular uptake of RSV-GNPs by NCI-H460 cells. Microscopic images (Hoechst staining) of NCI-H460 cells incubated with: i: free RSV and ii: RSV-GNPs for 3h. Enhanced

staining indicates improved cellular uptake of RSV-GNPs.

Fig. 5. Viability of NCI-H460 cells upon RSV and RSV-GNPs treatment. Cells were

treated with different concentration of RSV, RSV-GNPs and cisplatin (1-50g/ml)

and cell viability was observed by MTT assay. IC50 value for RSV, RSV-GNPs and

cisplatin was found to be 10, 5 and 5g/ml, respectively. Values are given as

means±S.D. of six experiments in each group.

RSV into chitosan microspheres using vanillin as the cross-linker

protected RSV from heat effects and increased the thermostability

[21].

In PBS, RSV-GNPs released 28.1%, 32.5% and 40.6% of RSV at pH

1.4, 10.5 and7.4,respectivelyupon 12h incubation. After 48h incu-

bation, 63.7%, 69.9% and 80.2% RSV has been released at pH 1.4,

10.5 and 7.4, respectively (Fig. 4A). The present results demon-

strate that there was slower release kinetics of RSV at higher pH

conditions. Moreover, the drug release plots revealed that there

were two stages for the release of RSV from the GNP nanoparticles.

The first stage of release was initially rapid, which might be due to

burst release of RSV from GNPs. Following rapid release, RSV wasreleased in a sustained manner from the GNPs (controlled release).

The burst release may help to reach the effective concentration of

RSV rapidly in PBS, whereas the controlled release would maintain

the effective concentration of RSV in PBS for a long time. Similar

to this finding, GNPs released amphotericin-B through initial burst

followed by a controlled release in PBS at pH 7.4 [13].

RSV-GNPs/Hoechst 33258 entered the cells during incubation

period and the fluorescence was found inside the nuclei, which

indicate that RSV-GNPs might prove to be useful in site-specific

delivery of drugs whose site of pharmacological activity might

be the cell nucleus. Free RSV has not been effectively uptake

by the cells, which has been evidenced by diminished fluores-

cence observed in RSV alone treated cells (Fig. 4B-i and ii). The

DLS results proved that the prepared RSV-GNPs possess 294 nm




Fig.6. A. Effect of RSV-GNPs on: i: intracellularROS generation; ii: mitochondrialmembrane potential; iii:% taillength,tail moment andolive tailmoment andiv: percentage

of apoptosis in NCI-H460 cells. Values are given as means±S.D. of six experiments in each group. Bars not sharing the common superscripts differ significantly at P ≤0.05

vs. control (DMRT). B. Photomicrographs show the effect of RSV-GNPs on: i: intracellular ROS generation; ii: mitochondrial membrane potential; iii: oxidative DNA damage

(comet assay) and iv: apoptotic morphology changes in NCI-H460 cells.




Fig. 7. Brightfield microphotograph of RBCaggregation: i: control, ii: RSV andiii: RSV-GNPs.

whereas the RSV possesses 636.3 nm. Particles with size in the

10–400 nm range can accumulate preferentially in tumor cells due

to the enhanced permeability and retention (EPR) effect [22]. This

might be thereason forenhancedintracellular accumulationof RSV

when given as RSV-GNPs.It hasbeen previously demonstratedthat

FITC-D-labeled GNP nanoparticles were taken up by Caco-2 cells

whereas free FITC-D (not attached to the nanoparticles) has not

been detected inside the cell [23].

Both RSV and RSV-GNPs treatment (24h) showed cytotoxic-

ity on NCI-H460 cells in a concentration dependent manner. TheIC50 value was found to be 10g/ml for RSV and 5g/ml for

RSV-GNPs.Further, RSV-GNPs showedsignificantly enhanced cyto-

toxicity when compared to RSV treatment alone. Interestingly, the

anticancer efficacy of RSV-GNPs was comparable to standard anti-

cancer drug, cisplatin (Fig. 5). This superior anticancer efficacy of

RSV-GNPs in NCI-H460cells at relatively lower doses maybedueto

enhanced intracellular RSV accumulation. It has been established

that, RSV kills cancerous cells at least in part through the genera-

tion of elevated amounts of intracellular ROS [24,25]. Elevated ROS

levels can induce DNA damage, thereby activating p53 depend-

ent apoptotic cascade [26]. The intracellularly accumulated RSV

possibly interacts with peroxidase-H2O2 system and significantly

generates ROS in NCI-H460 cells (Fig. 6A and B-i). Thus, RSV acts

as pro-oxidant, disrupting intracellular redox balance and leadingto cancer cell apoptosis [25,27]. Loss of mitochondrial potential ()

is an early stage of apoptosis. Mitochondrion is one of the most

important organelles in regulating apoptosis [28]. It has been well

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0

10

20

30

40

50

60

70

80

90

2 12 24 48 72 96

Time (h)

S e r u m c

o n c e n

t r a t i o n ( µ g / m g )

RSV-GNPs RSV

Fig. 8. Bioavailability of RSVand RSV-GNPs. Themice weredivided intotwo groups

(s ix mice in e ach g ro up) , gro up 1 was give n RS V and gr ou p 2 w as given RSV-

GNPs.RSV andRSV-GNPswereadministeredintravenously(10mg/kg)and theblood

was collected at different time intervals. Serum was separated and the concentra-

tion of RSV and RSV-GNPs were determined by HPLC analysis. Values are given as

means±S.D. of six determinations.

established that RSV alters mitochondrial membrane potential [29]

and triggers apoptotic signaling cascades in a numberof cancer cell

lines [26,30]. The increased MMP alteration in RSV-GNPs treated

cells than RSV treatment alone indicates the direct and controlled

release of RSV intracellularly by GNPs (Fig. 6A and B-ii). DNA is an

importantmolecular target for tumor cellkilling [31]. The induction

of DNA single strand breaks is often used to predict oxidative dam-

ageof tumor cells.Since,cancer cells possess centrallyacidic region

RSV could not able to act as antioxidant and act as prooxidant in

cancercells [20] and mightinducedsignificant DNAdamage (Fig.6Aand B-iii). Apoptotic features with condensed or fragmented chro-

matin,indicative of apoptosis, were observed in RSVand RSV-GNPs

treated NCI-H460 cells (Fig. 6A and B-iv). It has been previously

demonstrated that RSV induced apoptosis in EC-9706 cells with

typical apoptotic characteristics includes chromatin condensation,

nucleus fragmentation and apoptotic body formation [32]. The

increased apoptotic incidence during RSV-GNPs treatments clearly

indicates the enhanced anticancer potential of RSV-GNPs combi-

nation. The RSV treatment alone caused a significant aggregation

of erythrocytes upon 1 h incubation at room temperature, whereas

RSV-GNPs (average size 294) have not showed any effect with the

erythrocytes (Fig. 7). Similar to these findings a 10-times reduction

in hemolysis of erythrocytes during GNP-amphotericin-B treat-

ment when compared with plain amphotericin-B [13].Present results clearly show that serum levels of RSV were

almost twice in the case of RSV-GNPs administration when com-

pared to free RSV administration alone (Fig. 8). In addition,

half-life of RSV-GNPs was substantially longer than that of RSV

alone treatment. Similarly, resveratrol-loaded Ca-pectinate beads

and Zn-pectinate microparticles have been shown to increase its

half-life in rats in the upper gastro-intestinal tract [33]. Further,

resveratrol formulated with polymeric lipid-core nanocapsules,

when given to animals, had a higher plasma levels than the unfor-

mulated resveratrol. It has also been observed that improved

biodistribution and decreased metabolism rate in experimen-

tal animals [34]. Recently, in vivo, oral administration of the

liposome-encapsulated curcumin-resveratrol showed an increase

in resveratrol andcurcuminlevels in theserumand prostate tissue;and synergistically improves their bioavailability and enhances

their antitumor effect against prostate cancer [35].

In conclusion, the RSV-GNPs were prepared by a modified

coacervation method. The obtained RSV-GNPs have a spherical

morphology; average size of 294 nm; and 93.6% of RSV was encap-

sulated in GNPs. Further, RSV-GNPs showed enhanced anticancer

activity than free RSV by decreasing cell viability, mitochondrial

membrane potential, and increasing cytotoxicity, intracellular ROS

levels and DNA damage in NCI-H160 cells. Moreover, the prepared

RSV-GNPs elicited no hemolytic property in erythrocyte aggrega-

tion assay in vitro. Pharmacokinetic assays revealed that there was

more bioavailability of RSV when it has given as RSV-GNPs combi-

nation than RSV treatment alone. Based on these results, it can be

concluded that the GNPs is an ideal way to deliver RSV because of




its high loading efficiency and superior efficacy in cancer cell line

and animal model.

Disclosure of interest

The authors declare that they have no conflicts of interest con-

cerning this article.

Acknowledgements

The financial assistance in the form of Senior Research Fel-

lowship to Mr. S. Karthikeyan, by the Indian Council of Medical

Research (ICMR), Government of India, New Delhi, is gratefully

acknowledged. Further, we acknowledge Mr. N. Radhakrishnan for

his assistance in RSV-GNPs preparation.

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