International Journal of Biological Macromolecules 69 (2014) 267–273

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Fabrication, characterization and bioevaluation of silibinin loaded chitosan nanoparticles Deep Pooja a , Dileep J. Babu Bikkina a , Hitesh Kulhari a,b,∗∗ , Nalla Nikhila a , Srinivas Chinde c , Y.M. Raghavendra d , B. Sreedhar e , Ashok K. Tiwari a,∗ a

Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India c Toxicology Unit, Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India d Crop Protection Chemical Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India e Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, India b

a r t i c l e

i n f o

Article history: Received 19 April 2014 Accepted 16 May 2014 Available online 23 May 2014 Keywords: Silibinin Chitosan nanoparticles Characterization Dissolution Cytotoxicity

a b s t r a c t Silibinin is reported to possess multiple biological activities. However, its hydrophobic nature limits its bioavailability compromising in vivo biological activities. Nanoparticles-based delivery of such molecules has emerged as new technique to resolve these issues. Bio-degradable, compatible and adhesive nature of chitosan has recently attracted its suitability as a carrier for biologically active molecules. This study presents fabrication and characterization of chitosan-tripolyphosphate based encapsulation of silibinin. Various preparations of silibinin encapsulated chitosan-tripolyphosphate nanoparticles were studied for particle size, morphology, zeta-potential, and encapsulation efficiencies. Preparations were also evaluated for cytotoxic activities in vitro. The optimized silibinin loaded chitosan nanoparticles were of 263.7 ± 4.1 nm in particle size with zeta potential 37.4 ± 1.57 mV. Nanoparticles showed high silibinin encapsulation efficiencies (82.94 ± 1.82%). No chemical interactions between silibinin and chitosan were observed in FTIR analysis. Powder X-ray diffraction analysis revealed transformed physical state of silibinin after encapsulation. Surface morphology and thermal behaviour were determined using TEM and DSC analysis. Encapsulated silibinin displayed increased dissolution and better cytotoxicity against human prostate cancer cells (DU145) than silibinin alone. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Silibinin (SLB) is a polyphenolic flavonoligan and is a major biologically active component in silymarin which is derived from seeds of the milk thistle plant, Silybum marianum [1,2]. It is widely used for treatment of various acute and chronic liver toxicities, inflammation, fibrosis, and oxidative stress [3–5]. Various studies indicate that SLB is also active against different carcinomas like breast [6], lung [7], colon [8,9], brain [10] and prostate cancer [11]. SLB has shown anti proliferation activities through cell cycle regulation, apoptosis induction, chemosensitization, growth inhibition, antiinflammation, inhibition of angiogenesis, reversal of multi-drug resistance and inhibition of invasion and metastasis [12–14]. Bulky

∗ Corresponding author. Tel.: +91 40 2719 1875. ∗∗ Corresponding author at: IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India. Tel.: +91 9494751080. E-mail addresses: [email protected] (H. Kulhari), [email protected] (A.K. Tiwari). http://dx.doi.org/10.1016/j.ijbiomac.2014.05.035 0141-8130/© 2014 Elsevier B.V. All rights reserved.

multi-ring structure and poor oral bioavailability (23–47%) of SLBs leads to its low aqueous solubility and hence its clinical role is limited [3,15,16]. Nano-drug delivery systems such as nanoparticles, nanomicelles, nanosuspension and nanoemulsion have great potential to deliver the hydrophobic drugs in improved manner [17,18]. Many approaches have been employed to improve the efficacy and bioavailability of SLB, such as silybin-phospholipid complex [19], dendrimers [15], nanosuspension [20], silybin nano-structured lipid carriers [6,3], silibinin hydrogel [16] and self-emulsifying drug delivery system [21]. Biodegradable polymeric nanoparticles have attracted much attention to overcome drug associated problems [22]. Chitosan is a naturally occurring cationic polysaccharide derived by deacetylation of chitin. Chitosan based formulations have been used for the delivery of pharmaceutically active ingredients, nucleic acids [23], protein therapeutics and antigens [24]. Due to the biocompatible, biodegradable, non-toxic, non-immunogenic and noncarcinogenic properties, chitosan is widely used for the preparation of nanoparticles [25,26]. Because of the mucoadhesive

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and permeation-enhancing properties of chitosan, its nanoparticles are very promising carriers for oral drug delivery. ChitosanTPP nanoparticles have been previously used to encapsulate various natural compounds like rutin [27], quercetin [28], tea catechins [29], curcumin [30] and ascorbic acid [31]. Chitosan nanoparticles are generally prepared by using cross-linking agents like glutaraldehyde, tripolyphosphate and polyaspartic acid [32,33]. The objective of the present study was to fabricate the silibinin encapsulated chitosan-TPP nanoparticles for oral delivery and to evaluate the potential of encapsulated SLB for anticancer activity. The nanoparticles were characterized for various physicochemical properties using dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and powdered X-ray diffraction (XRD) techniques. 2. Materials and methods Silibinin, chitosan and sodium tripolyphosphate (TPP) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM), trypsin–EDTA, antibiotic antimycotic solution, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], dimethyl sulfoxide (DMSO) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Foetal bovine serum (FBS) was purchased from Gibco, USA. Cell culture 96 well plates and plastic wares were obtained from Techno plastic products (CH-8219 Trasadingen, Switzerland). All other chemicals were of analytical grade. 2.1. Preparation of silibinin loaded chitosan nanoparticles (SCN) Chitosan nanoparticles were prepared by ionic gelation method with different ratios of chitosan and TPP (Table 1). Chitosan was dissolved in 1% acetic acid solution and pH was adjusted to pH 4.8. SLB was dissolved in acetone and was added dropwise to chitosan solution with continuous stirring and then sonicated for 2 min at 65% amplitude using probe sonicator (Vibra Sonics, Biotech-North America). TPP solution was added dropwise in above solution and further stirred at 500 rpm for 3 h. Nanoparticle suspension was centrifuged at 15,000 rpm for 30 min. Supernatant was removed and nanoparticles were washed twice with distilled water. Nanoparticles were lyophilized using trehalose (5%, w/v) as cryoprotectant using (Modulyod-230, Thermo electron freeze dryer, USA). The instrument was set at −50 ◦ C with vacuum pressure less than 1 mbar. Blank chitosan nanoparticles (BCN) were prepared without SLB. 2.2. Physicochemical characterizations of nanoparticles 2.2.1. Particle size and zeta potential Blank and SLB loaded nanoparticles were characterized for particle diameter, polydispersity index (PDI) and zeta potential by using Zetasizer, Nano-ZS (Malvern Instruments, UK). Before the analysis, nanoparticles dispersions were diluted with distilled water. 2.2.2. Transmission electron microscopy (TEM) The surface morphology of SCN was examined by TEM analysis. Nanoparticles were dispersed in distilled water and a drop of the suspension was casted onto a copper grid. Extra solution was removed by filter paper, air-dried and directly observed under the transmission electron microscope (FEI Tecnai, G112, Philips, USA) at room temperature without staining.

2.2.3. Fourier transform infrared (FTIR) analysis FTIR analysis was carried out for SLB, chitosan and SCN by using potassium bromide pellet method. Samples were mixed with potassium bromide, compressed into pellets and scanned for percent transmittance in the range of 4000–450 cm−1 using FTIR spectrophotometer (Perkin Elmer, Spectrum One, UK). 2.2.4. Differential scanning calorimetry (DSC) Thermal behaviour of SLB, BCN, and SCN was performed by using DSC (EXSTAR DSC7020, Japan). The samples were heated in hermetically sealed aluminium pans at a scanning rate of 10 ◦ C/min from 50 to 250 ◦ C under inert nitrogen atmosphere. An empty aluminium pan was used as a reference. 2.2.5. Powder X-ray diffraction study (PXRD) The X-ray diffraction patterns of SLB, BCN and SCN were obtained by using Siemens D-5000 X-ray diffractometer (Germany) with Cu K␣ radiation generated at 45 kV and 20 mA. The diffraction patterns run over the range of 2 from 2◦ to 60◦ , at steps of 0.013◦ with continuing time 13.6 s. 2.3. Silibinin entrapment efficiency The entrapment efficiency (EE) was calculated by indirect method. SCN was centrifuged at 15,000 rpm for 30 min. Supernatant was collected and analyzed for silibinin content at 287 nm by using UV/VIS spectrophotometer (Perkin Elmer, Lambda 25, UK). The % EE was calculated as follows: % EE =

Amount of SLB added − Amount of SLB in supernatant Amount of SLB added × 100

2.4. Dissolution study Dissolution studies of pure SLB and SCN were carried out by using Dissolution test Station USP apparatus-2 (paddle type) (SR8 plus, Hanson Research, USA). The studies were carried out in two buffer mediums namely simulated gastric fluid (pH 1.2) and phosphate buffer (pH 6.8). A volume of 900 ml of buffer solution was placed in dissolution flasks and maintained at a constant temperature of 37 ± 0.5 ◦ C. The apparatus was set at a speed of 100 rpm and 10 mg SLB or SCN (equivalent to 10 mg SLB) was added to individual dissolution flasks. An aliquot of 5 ml of sample was withdrawn at predetermined time intervals and equal volume of fresh buffer was added to maintain the sink conditions. Silibinin concentration in the samples was determined by UV/VIS spectrophotometer at 287 nm against the buffer solution. 2.5. Cell culture and preparation of test samples Human prostate cancer cell line (DU-145) was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were grown in DMEM medium supplemented with 10% FBS, 0.3% sodium bicarbonate, 10 ml/L antibiotic antimycotic solution (10,000 U/ml penicillin, 10 mg/ml streptomycin and 25 ␮g/ml amphotericin B), 1 ml of 2 mM l-glutamine and 1 ml of 100 mM sodium pyruvate. The culture was maintained in CO2 incubator at 37 ◦ C with a 90% humidified atmosphere and 5% CO2 . For the preparation of stock solutions of test compounds, pure SLB was dissolved in DMSO (1 mg/ml) whereas SCN was dispersed in sterile PBS. Various further dilutions were made with sterile PBS to get desired concentrations. All formulations were filtered with 0.22 ␮m sterile filter and were kept under UV radiation for 20 min before using.

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Table 1 Optimization and physicochemical characterization of blank and silibinin loaded chitosan nanoparticles (mean ± SD, n = 3). Formulation

CS:TPP:SLB (w/w/w)

Particle size (nm)

F1 F2 F3 F4 F5 SCN 1 SCN 2 SCN 3 SCN 4 SCN 5

2:1:0 3:1:0 4:1:0 5:1:0 6:1:0 4:1:0.25 4:1:0.5 4:1:1 4:1:1.5 4:1:2

410.2 368.3 237.1 276.3 284.1 225.9 217.7 263.7 374.6 437.1

± ± ± ± ± ± ± ± ± ±

3.5 2.8 3.9 4.2 1.5 3.2 2.4 4.1 5.1 3.4

PDI

Zeta potential (mV)

0.659 0.437 0.290 0.304 0.300 0.266 0.347 0.243 0.333 0.358

± ± ± ± ± ± ± ± ± ±

0.022 0.03 0.016 0.024 0.019 0.027 0.011 0.028 0.015 0.026

23.8 35.9 39.4 44.7 36.5 27.7 42.2 37.4 43.1 36.6

± ± ± ± ± ± ± ± ± ±

1.23 2.16 1.89 1.2 1.69 2.21 1.76 1.57 0.96 2.45

% EE – – – – – 99.52 ± 95.17 ± 82.94 ± 73.54 ± 65.43 ±

0.43 0.67 1.82 2.72 1.39

CS: chitosan; SLB: silibinin; TPP: sodium tripolyphosphate; PDI: polydispersity index.

2.6. Cytotoxicity studies (MTT assay) Cytotoxicity of different SLB samples was assessed using MTT assay. The assay is based on the reduction of MTT by the mitochondrial dehydrogenase of viable cells into purple formazan crystals which are dissolved in DMSO and absorbance is measured at 570 nm. Briefly, 1 × 104 exponentially growing cells (counted by Trypan blue exclusion dye method) were seeded into each 96 well plate and allowed to grow. At 70% confluency, the cells were treated with different formulations blank nanoparticles, pure silibinin, and SCN at concentrations ranging from 5 to 200 ␮M along with controls (negative and positive). After 48 h, the media of the wells was replaced with 90 ␮l of fresh serum free media and 10 ␮l of MTT (5 mg/ml in PBS) and plates were incubated at 37 ◦ C for another 2 h. Thereafter, the media was discarded, plate was dried for 30 min and 100 ␮l of DMSO was added in each well to dissolve purple formazan crystals. The absorbance of samples was read immediately at 570 nm using Spectra Max plus 384 UV-Visible plate reader (Molecular Devices, Sunnyvale, CA, USA). IC-50 values were determined by probit analysis software package of MS-excel. 2.7. Stability studies For the determination of physical and chemical stability of SCN, formulation was stored at 4 ◦ C up to 2 months. The stability samples were analyzed for particle size, zeta potential, polydispersity index and drug content.

ratio was increased from 2:1 to 4:1, the mean particle diameter was decreased from 410.6 nm to 237.1 nm. Further increase in chitosan/TPP ratio resulted in increase in particle size due to precipitation. Data suggested that optimum interaction (inter and intramolecular) between chitosan and TPP occurred at 4:1 ratio. The interaction between chitosan and TPP was also supported by the change in surface charge of nanoparticles. All the formulations (F1–F6) showed the positive zeta potential indicating the presence of unconjugated NH3 + groups of chitosan. With the increase in chitosan/TPP ratio, the surface charge has also increased from 23.8 to 44.7 mV (F1–F4). Increase in the surface charge is due to presence of more free, positive amine groups in chitosan-TPP complex. Surprisingly, as the chitosan/TPP ratio was increased (7:1, F5), the zeta potential was decreased to 36.5 mV which can be attributed to clustering or precipitation of chitosan molecules at high mass level. Overall, high zeta potential values indicated the high physical stability of nanoparticles. The entrapment efficiency of SLB was indirectly measured by determining SLB content in supernatant. The EE was found to be depending on polymer content. More than 95% of SLB was encapsulated at high polymer content (SCN1 and SCN2). As the SLB content was increased in formulation, the EE was decreased and particle size was increased dramatically. The decrease in entrapment efficiency can be explained by the presence of insufficient amount of polymer to encapsulate SLB. Formulation of SCN3 was considered as finally optimal because of good EE (82.94 ± 1.82%) with optimum particle size (263.7 ± 4.1 nm), low polydispersity (0.243) and high zeta potential (37.4 ± 1.57 mV).

2.8. Statistical analysis All the studies were performed in triplicate and data are presented as mean ± SD (standard deviation). Statistical significance was analyzed using Student’s t-test for two groups and one way ANOVA for multiple groups. The p value

Fabrication, characterization and bioevaluation of silibinin loaded chitosan nanoparticles.

Silibinin is reported to possess multiple biological activities. However, its hydrophobic nature limits its bioavailability compromising in vivo biolo...
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