Enhanced cytotoxicity and apoptosis-induced anticancer effect of silibinin-loaded nanoparticles in oral carcinoma (KB) cells

Materials Science and Engineering C 41 (2014) 274–282

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Enhanced cytotoxicity and apoptosis-induced anticancer effect of silibinin-loaded nanoparticles in oral carcinoma (KB) cells M. Gohulkumar a, K. Gurushankar a, N. Rajendra Prasad b, N. Krishnakumar a,⁎ a b

Department of Physics, Annamalai University, Annamalainagar 608 002, Tamilnadu, India Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, 608 002, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 2 December 2013 Received in revised form 9 March 2014 Accepted 23 April 2014 Available online 2 May 2014 Keywords: Silibinin Nanoparticles KB cells Cytotoxicity Apoptosis

a b s t r a c t Silibinin (SIL) is a plant derived flavonoid isolated from the fruits and seeds of the milk thistle (Silybum marianum). Silibinin possesses a wide variety of biological applications including anticancer activities but poor aqueous solubility and poor bioavailability limit its potential and efficacy at the tumor sites. In the present study, silibinin was encapsulated in Eudragit® E (EE) nanoparticles in the presence of stabilizing agent polyvinyl alcohol (PVA) and its anticancer efficacy in oral carcinoma (KB) cells was studied. Silibinin loaded nanoparticles (SILNPs) were prepared by nanoprecipitation technique and characterized in terms of size distribution, morphology, surface charge, encapsulation efficiency and in vitro drug release. MTT assay revealed higher cytotoxic efficacy of SILNPs than free SIL in KB cells. Meanwhile, reactive oxygen species (ROS) determination revealed the significantly higher intracellular ROS levels in SILNPs treated cells compared to free SIL treated cells. Therefore, the differential cytotoxicity between SILNPs and SIL may be mediated by the discrepancy of intracellular ROS levels. Moreover, acridine orange (AO) and ethidium bromide (EB) dual staining and reduced mitochondrial membrane potential (MMP) confirmed the induction of apoptosis with nanoparticle treatment. Further, the extent of DNA damage (evaluated by comet assay) was significantly increased in SILNPs than free SIL in KB cells. Taken together, the present study suggests that silibinin-loaded nanoparticles can be used as an effective drug delivery system to produce a better chemopreventive response for the treatment of cancer. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cancer is a leading cause of death worldwide, reaching 7.6 million deaths (13% of all deaths) in 2008. Further, deaths from cancer worldwide are projected to continue rising to over 11 million in 2030 [1]. Currently, there are broad categories of cancer treatment, the most common of which are chemotherapy, radiotherapy and surgery or a combination of these methods. Because of systemic toxicity of chemotherapeutic drugs or radiotherapy, their effectiveness in cancer treatment has been a limitation [2]. Chemoprevention or chemotherapy via non-toxic agents could be one approach for decreasing the incidence of cancers. Many naturally occurring agents have shown chemopreventive and chemotherapeutic potential in a variety of bioassay systems and animal models [3]. This fact further stresses on the importance of developing new effective alternative chemopreventive therapies, with minimal adverse effects. Prevention and therapeutic intervention by developing new phytochemicals which are non-toxic, cost-effective, and physiologically bioavailable is an emerging field in cancer management [4]. Most of these phytochemicals are constituents of the human diet or are taken as dietary supplements. Bioflavonoids are a ubiquitous ⁎ Corresponding author. E-mail address: [email protected] (N. Krishnakumar).

http://dx.doi.org/10.1016/j.msec.2014.04.056 0928-4931/© 2014 Elsevier B.V. All rights reserved.

group of polyphenolic substances that are present in most plants. Silibinin is one such plant derived flavonoid present in silymarin isolated from the fruits and seeds of the milk thistle (Silybum marianum) [5]. One of the most prominent effects seen in preclinical studies of silibinin is G1 arrest and apoptosis. Silibinin increases the efficacy of several chemotherapy agents both in vitro [6] and in vivo [7]. Therefore, numerous formulations of silibinin have recently been developed, to increase its solubility and there by bioavailability, for examples, β-cyclodextrin inclusion complexes, phospholipid complex, and polymeric nanoparticles [8–10]. Among them, the colloidal delivery system of polymeric nanoparticles has attracted much attention as one of the new nanosuspensions, owing to excellent properties such as high biocompatibility, non-toxicity, long-term stability, good physiological options, and controlled release. Polymeric nanoparticles with small particle size (b200 nm) are reported to have increased drug accumulation in tumor cells via the enhanced permeability and retention (EPR) effects [11,12]. Therefore nanoparticles could act as an effective delivery system for improving phytochemical bioavailability and its anticancer effect. Eudragit® E is a cationic copolymer that has been widely used to improve the solubility of poorly water-soluble drugs [13]. It has a basic site containing tertiary amine groups which are ionized in gastric fluid and therefore it is easy to dissolve in the gastric environment [14]. This

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polymer composition enabled the fine tuning of the particle size and the release profile (especially the burst effect) with respect to a control of SILNPs [15]. Hence in the present study, silibinin-loaded eudragit nanoparticles were prepared by nanoprecipitation technique using PVA as a stabilizer. The prepared SILNPs were characterized by various analytical techniques such as dynamic light scattering (DLS), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimeter (DSC) and X-ray diffraction (XRD). Furthermore, encapsulation efficiency and in vitro drug release pattern was studied using UV spectrophotometer. In addition, the antitumor mechanism of SILNPs were investigated by studying the effect on cell viability, cell morphology, mitochondrial membrane potential (MMP), apoptotic morphological changes and DNA damage in oral carcinoma (KB) cells. 2. Materials and methods 2.1. Chemicals Silibinin, polyvinyl alcohol (M.W 25,000), 3-(4,5-dimethylthiazol2yl)-2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-dichlorofluoresein diacetate (DCFH-DA), heat-inactivated fetal calf serum (FCS), Dulbecco's modified Eagle's medium (DMEM) and antibiotics pencillin–streptomycin were procured from Sigma-Aldrich Chemical Pvt. Ltd, Bangalore, India and used as received. Acridine orange (AO), ethidium bromide (EB), rhodamine 123 (Rh-123) and phosphate buffered saline (PBS) were purchased from Himedia, Mumbai, India. Aminoalkyl methacrylate copolymer Eudragit® E 100 (EE 100) was a kind gift sample supplied by Evonik Industries (Mumbai, India). All other chemicals and solvents used were of analytical grade.

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was measured in aqueous dispersion. Measurements were realized in triplicate at 25 °C. 2.4.3. Transmission electron microscopy (TEM) analysis The internal morphology of nanoparticles was examined by JEOL2100 transmission electron microscopy (TEM). Precisely, ~ 1 mg of SILNPs was suspended in double distilled water and sonicated for 30 s. One drop of 1 mg ml−1 SILNP solution was placed on a carbon coated copper TEM grid, negatively stained with 1% uranyl acetate (w/v) for 10 min and allowed to air dry. The image was visualized at 100 kV under microscope. 2.4.4. Fourier transform infrared spectroscopy (FT-IR) study FT-IR spectral analysis was performed to investigate the possible chemical interactions between silibinin and the polymer matrix. Different samples (Blank nanoparticles, free SIL and SILNPs) were individually mixed with KBr (at a ratio of 1: 10) and ground into a fine powder using an agate mortar. The mixture was pressed into a pellet using a hydraulic press. FT-IR spectra were recorded in Nicolet Avatar 330 in the region of 4000–400 cm −1 with a resolution of 4 cm −1. 2.4.5. Differential scanning calorimeter (DSC) analysis The physical state of silibinin encapsulated in NPs was characterized using a differential scanning calorimeter (DSC) thermogram analysis (DSC Q 20V 24.2 Build 107). Each sample of ~ 10 mg (free SIL and SILNPs) was sealed separately in a standard aluminium pan and the samples were purged in DSC with pure dry nitrogen gas set at a flow rate of 20 ml min −1 . The temperature ramp speed was set at 10 °C min −1 and the heat flow was recorded from 30 °C to 275 °C.

2.2. Cell lines and culture conditions Human oral carcinoma (KB) cell lines were obtained from the National Center for Cell Science (NCCS, Pune, India) and were cultured in DMEM with 10% FCS, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37 °C in a humidified 95% air and 5% CO2 incubator.

2.4.6. X-ray diffraction (XRD) study XRD analysis was carried out to determine the crystallinity of the SILNP formulation. X-ray diffraction patterns were measured using X-ray diffractometer (X’PERT PRO-PANalytical Philips). The measurements were carried out at a voltage of 40 kV and 30 mA. The scanned angle was set from 10° to 80° at a scan speed of 2° per minute.

2.3. Synthesis of silibinin-loaded nanoparticles (SILNPs) The SILNP system was prepared in the ratio of SIL: EE: PVA (1:10:10; w/w/w) by the nanoprecipitation technique [16]. Briefly, 50 mg of SIL powder and 500 mg of EE 100 polymer were completely dissolved together in 25 ml of ethanol in a sealed glass vial. The internal organic phase solutions were quickly injected into the 75 ml external aqueous solution containing 500 mg of PVA, and then the solutions were added slowly with magnetic stirring. Then the solutions were homogenized at 18,000 rpm for 40 min. The ethanol was completely removed by vacuum rotary evaporation using a rotary evaporator and the remaining fraction was then lyophilized with a freeze dryer. The SILNPs freeze-dried powders were then collected for further experiments. 2.4. Characterization of SILNPs 2.4.1. Particle size analysis Particle size and size distribution measurements were carried out using nanotrac (Microtrac, Inc, USA) based on dynamic light scattering (DLS). Briefly, ~1 mg of SILNPs was suspended in 3 ml of double distilled water and sonicated for 30 s and measured immediately. The measurement was performed in triplicate. 2.4.2. Zeta potential measurement Zeta potential of nanoparticle dispersions was measured by Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK) to determine the surface charge of the nanosystem. Zeta potential of nanoparticles

2.4.7. Determination of % encapsulation efficiency (% EE) The amount of the drug entrapped within the nanoparticle was determined by measuring the non-entrapped (un-encapsulated) drug amount in the supernatant. The supernatant was collected after centrifugation and recovery of nanoparticles. The encapsulation efficiency was analyzed by UV spectrometer (UV-1800 Shimadzu) at 286 nm for SILNPs. Encapsulation efficiency was determined by using the following equation: EE ð%Þ ¼

Total weight of SIL in SILNPs−Free SIL released from SILNPs x100 Total weight of SIL in SILNPs

2.4.8. In vitro drug release studies Five milligrams of SILNPs was dispersed in 100 ml of phosphatebuffered saline (PBS, pH 7.4) at 37 °C and placed in an incubated shaking water bath with a paddle speed of 120 rpm. At predetermined time intervals, dispersion of 3 ml of the aliquots was withdrawn and the same amount of fresh PBS was added to maintain the constant volume under sink conditions. The concentration of the released drug was determined by UV spectrometer [UV-1800 Shimadzu] at 286 nm. The percentage of silibinin released was determined from the following equation:

Drug release ð%Þ ¼

Released silibinin from SILNPs  100: Total amount of silibinin in SILNPs

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2.5. Cytotoxicity assay The effect of the free SIL and SILNPs on cell proliferation/viability was determined by using MTT assay. The MTT assay is a quantitative and rapid colorimetric method, based on the cleavage of a yellow tetrazolium salt to insoluble purple formazan crystals by the mitochondrial dehydrogenase of viable cells [17]. KB cells were seeded in 96-well plates at the density of 8000–10,000 viable cells per well and incubated for 24 h to allow cell attachment. Cells treated with blank vehicles were used as controls. Cells were treated with different concentrations of free SIL and SILNPs (5, 10, 20, 30, 40 and 50 μg/ml) and the cells were incubated in the presence of 5% CO2 and 95% O2 at 37 ºC for 48 h. Upon completion of the incubation, stock MTT dye solution (10 μl, 100 mg ml–1) was added to each well and the cells were incubated for another 4 h. The cells were centrifuged for 10 min and the supernatant was removed. The formed MTT-formazan crystals were dissolved in 100 μl of DMSO and absorbance was recorded at 540 nm using a microplate reader. IC50 values were calculated and the optimum dose was used for further study.

Fluorescence was determined at 485/530 nm by spectrofluorimeter. Fluorescence microscopic images were taken using blue filter (450– 490 nm) (Nikon, Eclipse TS100, Japan). The stained cells were observed by a fluorescence microscope at 40× magnification.

2.7. Analysis of mitochondrial membrane potential (MMP, Δψm) The study of mitochondria and the changes in the mitochondrial transmembrane potential has become a focus of apoptotic analysis. KB cells were treated with IC 50 values of free SIL and SILNPs for 48 h, and then stained with 10 μg/ml rhodamine 123 for 30 min, which is easily sequestered by the mitochondrial membrane. After completion of incubation period, the cells were washed with PBS and analyzed by a fluorescence microscope using a blue filter. Rhodamine 123 is a lipophilic cationic dye, highly specific for mitochondria. Polarized mitochondria were marked by orange–red fluorescence, and depolarized mitochondria were marked by green fluorescence [19].

2.6. Measurement of intracellular reactive oxygen species (ROS) 2.8. Apoptotic morphological changes by AO/EB dual staining method The intracellular ROS level was measured by using a non-fluorescent probe 2′,7′-diacetate dichlorofluoresein (DCFH-DA), that can penetrate into the intracellular matrix of cells where it is oxidized by ROS to highly fluorescent dichlorofluoresein (DCF) [18]. IC50 values of Free SIL and SILNPs treated KB-cells were seeded in 6 well plates (2 × 106 cells/ well) and incubated with 10 μM DCFH-DA for 30 min at 37 °C. Cells were washed twice with PBS to remove the excess amount of dye.

a

AO and EB staining with DNA allowed visualization of the condensed chromatin of dead apoptotic cells. Viable cells had green fluorescent nuclei with organized structure, early apoptotic cells had yellow condensed chromatin in nuclei that were highly condensed or fragmented. Apoptotic cells also exhibited membrane blebbing. Late apoptotic cells had orange chromatin with nuclei that were highly condensed and fragmented; necrotic cells had bright orange chromatin in round nuclei [20]. After incubating the cells with 37 °C for 48 h in a CO2 incubator, the cells were stained with 2 μl of AO and EB solution (100 μg/ml, each) and then examined immediately by fluorescent microscope.

2.9. Analysis of DNA damage (comet assay)

b

DNA damage was estimated by alkaline single-cell gel electrophoresis (comet assay) [21]. A layer of 1% normal melting agarose was prepared on microscope slides. After treatment, KB cells (50 μl) were mixed with 120 μl of 0.5% low melting agarose. The suspension was pipetted onto the precoated slides. Slides were immersed in cold lysis solution at pH 10 (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, 1% Triton X-100, 10% DMSO) and kept at 4 °C for 60 min. To allow denaturation of DNA, the slides were placed in alkaline electrophoresis buffer at pH 13 and left for 25 min. Subsequently, slides were transferred to an electrophoresis tank with fresh alkaline electrophoresis buffer and electrophoresis was performed at field strength of 25 V and 300 mA for 25 min at 4 °C. Slides were neutralized in 0.4 M Tris, pH 7.5 for 5 min and stained with 20 μg/ml ethidium bromide. 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 four parameters were estimated to indicate DNA damage: % tail length (distance from the head center to the end of the tail), tail moment, tail DNA and olive tail moment were quantified.

2.10. Statistical analysis

Fig. 1. (a) Mean particle size of silibinin-loaded nanoparticles (SILNPs) determined using the dynamic light scattering, and (b) transmission electron microscopic (TEM) image of SILNPs (bar = 100 nm).

Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) by using statistical package of social science (SPSS) version 11.0 for windows. The values are ± S.D for six samples in each group. p values b 0.05 were considered as level of significance.

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3. Results 3.1. Characterization of silibinin-loaded nanoparticles (SILNPs) 3.1.1. Particle size, morphology and zeta potential of silibinin-loaded nanoparticles Fig. 1a shows the size distribution of the SILNPs in aqueous medium by means of dynamic light scattering (DLS). The average particle size of the prepared SILNPs was found to be around 120 nm. TEM studies were performed to further confirm the size and shape of the SILNPs. The SILNPs image obtained by TEM is shown in Fig. 1b. The TEM images revealed that the NPs are dispersed as individual NPs with a well-defined spherical shape and homogeneously distributed around 60–70 nm. The observed particle size was lower than that determined by DLS. In fact, DLS measures the hydrodynamic diameter of the nanoparticles, whereas the diameter observed by TEM indicates that of dried NPs. Zeta potential reveals information regarding the surface charge and stability of the nanoparticulate formulation. The positive charge of NPs is thought to be easily absorbed to negatively charged cellular membrane and then contributes to efficient intracellular trafficking [22]. As shown in Fig. 2, zeta potential values of the SILNPs possessed slight positive potential of + 4.61 mV. The positive charge of the prepared SILNPs would be helpful for its further application in vitro studies. 3.1.2. Fourier transform infrared spectroscopy (FT-IR) studies The FT-IR spectral analysis was conducted to identify any possibility of chemical interaction that might have occurred between the drug and the polymer-coated nanoparticle. Fig. 3 shows the FT-IR spectra of blank NPs, free SIL and SILNPs. FT-IR spectra showed characteristic bands due to the presence of different functional groups. The FT-IR spectrum of free SIL showed an absorption band at 3453 cm −1, 1632 cm −1, 1514 cm −1, 1277 cm −1, 1162 cm −1, 1082 cm −1 and 834 cm −1 corresponding to O\H stretch, CONH amide band I, N\H bending of amide band, aromatic C\O stretching, C\O stretching, benzopyran ring vibrations and C\O\C stretching respectively. However, more or less similar vibrational bands were observed in SILNPs at 3437 cm −1, 1639 cm −1, 1452 cm −1, 1256 cm −1 and 842 cm −1 and attributed to the vibrations of O\H stretching, CONH amide I, CH2 bending, C\N and C\C stretching, respectively. In SILNPs, O\H stretch is shifted from 3453 cm −1 to 3437 cm −1 and becomes broader, which indicates an enhanced hydrogen bonding due to some minor chemical interaction between the free silibinin and Eudragit matrix. 3.1.3. Physical state of silibinin-loaded nanoparticles (SILNPs) Differential scanning calorimeter (DSC) analysis was performed to find out the physical state of the drug entrapped in the nanoparticles.

Fig. 3. The FT-IR spectra of blank NPs, free SIL and SILNPs.

As shown in Fig. 4a DSC thermograms demonstrated that only free SIL had an endothermic peak of melting point at ~ 178 °C, whereas silibinin-loaded NPs had no such peak in that range, which showed encapsulated silibinin to be either in amorphous form or in disordered crystalline phase. Further to confirm the crystallinity, X-ray diffraction patterns of free SIL and SILNPs were recorded and presented in Fig. 4b. As seen from Fig. 4b, free SIL has displayed the characteristic crystalline peaks of 2θ at 12.54°, 13.85°, 16.50°, 17.70°, 20.94°, 27.30°, 33.31° and 35.10°. On the other hand, SILNPs have not shown any such crystalline peaks. This result clearly indicates that the XRD pattern of SILNPs is an amorphous or decreased drug crystallinity or solubilization of drug in Eudragit during the preparation process. 3.1.4. Drug encapsulation efficiency Encapsulation efficiency (EE) of NPs was determined indirectly through measurement of the free drug remaining unloaded in the reaction medium by UV spectral analysis. The encapsulation efficiency of SILNPs was found to be 79.0 ± 2.4%. 3.1.5. In vitro drug release The release profile of silibinin from the nanoparticles was studied in vitro using PBS (pH 7.4). As seen from Fig. 5. A typical two phase release profile was observed, indicating a relatively rapid release in the early time point (~ 24.1% of the entrapped silibinin release in 6 h) followed by the sustained and slow release over a prolonged time period (~79.2% of the drug released in 24 h). The observed initial burst release may be due to the diffusion of drug present at the surface of the SILNPs, which was followed by a slower sustained release of drug from the SILNPs [23].

Zeta Potential Distribution 400000

Total Counts

300000

200000

100000

0 -100

0

Zeta Potential (mV) Fig. 2. Zeta potential distribution of SILNPs.

100

200

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Fig. 6. Dose-dependent cytotoxicity study of free SIL and SILNPs (5–50 μg/ml) was observed by MTT assay in KB cells. Values are given as means ± S.D (n = 6) in each group (one-way analysis of variance [ANOVA] followed by Duncan's multiple range test [DMRT]).

3.3. Measurement of intracellular ROS levels in SILNPs Intracellular ROS levels were detected by H2DCF- DA dye in free SIL and SILNPs. As shown in Fig. 7a, fluorescence microscopic images confirmed bright DCF fluorescence in SILNPs treated cells than free SIL treated cells. Spectrofluorimetric analysis also confirm the increased intracellular ROS generation during SILNPs treatment than free SIL treatment (Fig. 7b).

3.4. Effect of SILNPs on mitochondrial membrane potential (MMP, ΔΨm) Fig. 4. (a) Differential scanning calorimetry (DSC) thermogram curves of free SIL and SILNPs. (b) X-ray diffraction (XRD) patterns of free SIL and SILNPs.

3.2. Effect of SILNPs on cell viability To investigate the therapeutic efficacy of the nano formulation, KB cells were treated with free SIL and SILNPs at different concentrations (5, 10, 20, 30, 40 and 50 μg/ml) for 48 h, and cell proliferation was measured by a standard MTT assay. As shown in Fig. 6, KB cells exposed to SIL and SILNPs exhibited significant cytotoxicity in the dose dependent manner after 48 h treatment. The estimated half maximal inhibitory concentration (IC50) value for free SIL and SILNPs was 38 μg/ml and 15 μg/ml respectively. This enhanced cytotoxicity of SILNPs in KB cell lines may be due to their efficient targeted binding and eventual uptake by the cells.

The loss of mitochondrial membrane potential is one of the important features to illustrate the initiation of the intrinsic apoptotic pathway [24]. Loss of membrane potential was detected using Rh-123 fluorescent dye. Fig. 8a shows the alteration in mitochondrial membrane potential (ΔΨm) in free SIL and SILNPs treated cells. After 48 h of treatment with SILNPs to KB cells, the fluorescence microscopic analysis resulted in the depolarization of the MMP as revealed by changes in Rh-123 fluorescence dye from orange red to green compared to free SIL and control. Further, SILNPs treatment showed elevated fluorescence intensity for Rh-123 absorption (Fig. 8b) indicating increased mitochondrial depolarization compared to free SIL.

3.5. Detection of apoptotic nuclei by EB/AO staining Fig. 9a shows the apoptotic morphological changes in the control, free SIL and SILNPs treated KB cells after staining with acridine orange/ethidium bromide. Cells with condensed or fragmented chromatin, indicative of apoptosis, were observed in SILNPs treated cells as compared to the control cells, which showed evenly, distributed green fluorescent chromatin. Fig. 9b shows the quantitative result of apoptosis in the control, free SIL and SILNPs treatment. These results together with membrane potential alterations in the SILNPs treatment indicate that apoptosis might be the mode of cell death.

3.6. Effect of SILNPs on DNA damage (comet assay)

Fig. 5. Cumulative in vitro release profile of free SIL from silibinin-loaded nanoparticles (SILNPs). Each data point represented as mean ± S.D (n = 3).

The induction of DNA single strand break is often used to predict oxidative damage of tumor cells. In the present work, as shown in Fig. 10a, free SIL and SILNPs treatment changes the levels of DNA damage in KB cells, whereas the control cells showed largely non-fragmented DNA (Fig. 10a). The extent of DNA damage was calculated by % tail length, tail DNA, tail moment and olive tail moment in the control, free SIL and SILNPs. SILNPs treatment shows a significant increase in % tail length, tail DNA, tail moment and olive tail moment in KB cells when compared to the free SIL (Fig. 10b).

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a

Control

% of Fluorescence Intensity

b

SILNPs

SIL

90

c

80 70 60 50 40

b

30

a

20 10 0

SIL

Control

SILNPs

Treatments Fig. 7. (a) Fluorescence microscopic showing the generation of intracellular reactive oxygen species (ROS) using DCFH-DA dye in KB-cells. Arrow mark (→) represents clearly visible high DCF fluorescence in SILNPs. (b) Intracellular ROS measurement by spectrofluorimeter. The values are given as mean ± SD of six experiments in each group (one-way analysis of variance [ANOVA] followed by Duncan's multiple range test [DMRT]). Values not sharing the common superscripts differ significantly (p b 0.05).

a

Control

% of Fluorescence Intensity

b

90

SIL

SILNPs

a

80 70 60

b

50 40

c

30 20 10 0 Control

SIL

SILNPs

Treatments Fig. 8. (a) Fluorescence microscopic images of mitochondrial membrane potential by Rh-123 staining in KB-cells. Arrow marks (→) represents emit green fluorescence in SILNPs. (b) Quantification of the spectrofluorimeter values is given as means ± SD of six experiments in each group (one-way analysis of variance [ANOVA] followed by Duncan's multiple range test [DMRT]). Values not sharing the common superscripts differ significantly (p b 0.05).

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a

Control

b

SIL

SILNPs c

90

% of apoptotic cells

80 70 60 50

b

40 30 20 10

a

0 Control

SIL

SILNPs

Treatments Fig. 9. (a) Photomicrograph effect of apoptotic morphological changes in KB- cells by dual staining. Arrow mark (→) represents orange colored cells which are late apoptotic cells. (b) Percentage of apoptosis. The values are given as mean ± SD of six experiments in each group (one-way analysis of variance [ANOVA] followed by Duncan's multiple range test [DMRT]). Values not sharing the common superscripts differ significantly (p b 0.05).

4. Discussion The search for new anticancer agents is necessary to increase the number of available therapeutic options and identify less toxic and more effective drug that can induced apoptosis and growth arrest of cancer cells without affecting normal cells. Plant-derived compounds played an important role in the development of several clinically useful anti-cancer agents. Phytochemicals have an advantage in drug discovery because they are potential sources of anticancer compounds with low toxicity to normal tissues, minimal side effect, safety and efficiency [25]. Despite, promising results in preclinical settings, the applicability of phytochemicals to human has met with only limited success, largely due to inefficient systemic delivery and bioavailability of promising agents [26]. Silibinin is one of the potent dietary phytochemicals and possesses various pharmacological activities, but the bioavailability of silibinin is quite low owing to degradation by gastric fluid and its poor aqueous solubility [27]. Pharmacokinetic studies showed that only 23– 47% of silibinin was absorbed from the gastrointestinal tract after being administered orally [28,29]. Therefore, nanoparticulate systems were developed to overcome major obstacles associated with silibinin bioavailability. The use of nanoparticles as drug delivery systems is gaining popularity because of a number of advantages such as placing nano-objects at the desired position, increasing the bioavailability of drugs, enhancing solubility and controlling the drug release rate. The particle size is an important parameter as it can directly affect physical stability, cellular uptake, and drug release from the nanoparticles and can affect its bio-distribution [30]. Incidentally, the fate of the drug loaded polymeric nanoparticles primarily depends on its physicochemical character. NPs with small particle size (b 200) are reported to have increased drug accumulation in tumor cell via the enhanced permeability and retention (EPR) effects. In the present study, the prepared SILNPs maintain a size of ~120 nm and would be expected to have a satisfactory

drug accumulation in the targeted cells. TEM analysis revealed that the prepared SILNPs possess much smaller particle size than those detected by DLS. Measurement based on DLS are based on the hydrodynamic diameter of the particles and provides an intensity weighted average particle size, whereas measurement obtained from microscope techniques are based on the diameter of dry particle and give average particle size [31]. Further, the zeta potential of the SIL nanoparticles is found to be slightly positive (Fig. 2). The positive charges on the surface of the complexes can help the nanoparticles bind tightly to the negatively charged cellular membrane, facilitating their entry into the cells by endocytosis [32]. Furthermore, FT-IR, DSC and XRD studies have demonstrated the chemical structure of silibinin and a lack of interaction with the polymer, confirming that the encapsulated silibinin retained its structural integrity inside the NPs. Moreover, it was observed that silibinin was loaded in NPs, reaching a high encapsulation efficiency of 79 ± 2.4%. High encapsulation efficiency is advantageous, since it transports enough drugs at the target site and increases the residence time of the drug. The therapeutic efficacy of the drug would largely depend on the dose and duration of their availability on the intracellular site of action. A carrier that could slowly release the drug at the site of action in the intracellular compartment would enhance the curative efficacy of the drug as well as could sustain its prolonged therapeutic effects. Sustained release of silibinin, as shown by the in vitro drug release kinetics study, indicates diffusion of the drug out of the polymeric matrix of the NPs, which is an essential requirement for effective antiproliferative activity [23]. The sustained release activity of the drugs from the SILNPs is might be due to the amorphous or disordered-crystalline state of the drug inside the formulation. The pharmacological activity of free SIL and SILNPs was further evaluated for theirin vitro cellular viability assay on KB cell line by MTT assay. SILNPs showed effective anti-proliferation activity with lower IC50 value of 15 μg/ml than free SIL, IC50 value around28 μg/ml. This

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a

Control

SIL

SILNPs

b

Fig. 10. (a) Photomicrographs of comet length in the control, free SIL and SILNPs, (b) Effect of comet parameters (% tail length, % tail DNA, tailmoment and olive tail moment). The values are given as mean ± SD of six experiments in each group (one-way analysis of variance [ANOVA] followed by Duncan's multiple range test [DMRT]). Values not sharing the common superscripts differ significantly (p b 0.05).

could be attributed due to the higher internalization of SILNPs inside the cells and escaping the multiple drug resistance barriers [33,34]. The higher cytotoxic efficacy of silibinin entrapped in NPs than the free SIL is also due to the continuous exposure and sustained release of the drug for a prolonged period of time at the site of action. One major biochemical change in cancer cells after treatments with anti-cancer agents is the increase in ROS generation, which is frequently considered as a cancer-promoting factor [35]. Silibinin is known to induce apoptosis by increasing ROS generation in cancer cells [36]. In the present study, SILNPs-treated cells showed 2.5 fold rapid increases in ROS generation compared with SIL-treated cells in KB cancer cells (Fig. 7b). These results indicate that SIL delivery of nanoparticles enables more accumulation in cells and consequently generates more intracellular ROS. The loss of mitochondrial membrane potential is one of the end-point features of apoptosis [37]. Mitochondria, which play a pivotal role in apoptosis, are major sites of ROS generation. Excessive ROS generation can lead to the opening of the mitochondrial permeability transition pore with consequent release of cytochrome c from the intermembrane space into the cytosol culminating in the activation of the caspase cascade and apoptotic cell death [38]. In the present study, the changes in the mitochondrial membrane potential in free SIL and SILNPs treated cells are observed. The changes from orange red to green fluorescence exhibit the loss of mitochondrial membrane potential in SILNPs treated KB cancer cells [39]. The increased MMP alteration in SILNPs treated cells than free SIL treatment indicates the direct and controlled release of SIL intracellularly by NPs. Apoptosis is a normal physiologic process and it plays an important role in homeostasis and development of the tissues in organism, morphological changes including cytoplasm shrinkage, chromatin condensation, plasma membrane blebs, DNA fragmentation and apoptotic

body formation which can be observed [40,41]. Silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways [42]. SILNPs treated KB cancer cells demonstrated significant apoptosis-related morphological alterations, such as apoptotic body formation and chromatin condensation (Fig. 9a). This could be due to the higher internalization of the drug-loaded NPs inside the cells, which leads to the effect of apoptosis. The increased ROS levels and subsequent loss of mitochondria membrane potential might also be the reason for the increased apoptotic morphological changes. The comet assay was further used to detect the induction of DNA damage in KB cells after 48 h treatment with free SIL and SILNPs. Damage DNA migrates during electrophoresis from the nucleus towards the anode, forming a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (released or broken DNA). In the present study, SILNPs treatment caused extensive DNA damage which is evident by the formation of comet. The reason for increased DNA damage in SILNPs might be due to increased generation of ROS. 5. Conclusion In this current study, silibinin-loaded eudragit NPs were prepared successfully using the nanoprecipitation approach in the presence of PVA as a stabilizer. DLS and TEM studies confirmed that the average particle size of SILNPs was below 120 nm with an entrapped efficiency of ~ 79%. The synthesized nanoparticles exhibited an approximate spherical structure with a smooth surface and FT-IR, DSC and XRD results confirmed that the encapsulated SIL retained its properties even inside the nanoparticles. The developed NPs have an excellent encapsulation efficiency of the anticancer drug SIL, and the in vitro drug study indicate that they may be useful for sustained drug

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