Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb

Beta-casein and its complexes with chitosan as nanovehicles for delivery of a platinum anticancer drug
Mahdieh Razmi a , Adeleh Divsalar a,b,∗ , Ali Akbar Saboury b , Zhila Izadi a , Thomas Haertlé c , Hassan Mansuri-Torshizi d
a

Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran UR 1268 Biopolymères Interactions Assemblages, Fonctions et Interactions des Protéines, Institut National de la Recherche Agronomique, Nantes, France d Department of Chemistry, University of Sistan & Baluchestan, Zahedan, Iran
b c

a r t i c l e

i n f o

a b s t r a c t
The clinical application of platinum-based anticancer drugs is greatly limited by severe toxicity. Drugdelivery systems are much sought after to improve the efficacy and applicability of these drugs. Here, we describe a new drug-delivery system comprising a novel platinum complex (bipyridine morpholine dithiocarbamate Pt(II) nitrate) within nanoparticles composed of ␤-casein (␤-CN) and chitosan (CS). The influence of pH on the formation of a colloidally-stable nanocarrier system composed of Pt complex-loaded ␤-CN and chitosan nanoparticles was investigated using UV–vis spectrometry, dynamic light scattering (DLS) and scanning electron microscopy (SEM). The particles of Pt complex-loaded betacasein–chitosan formed were stable and soluble in the pH range 5.7–6.2. Hence, the optimal pH for complex formation is between the pI of casein (5.3) and the pKa of chitosan (6.5). DLS data showed that, at both pH values of 5.7 and 6.2, the particles formed had sizes between 200 and 300 nm. However, the optimum pH for particle formation was pH 5.7. At this pH, the zeta-potential values of nanoparticles were positive and the particles were stable. SEM analysis confirmed the formation of nanoparticles with good colloidal stability and an average particle size of 200 nm. The cytotoxicity of both free and encapsulated Pt complex was evaluated on colorectal carcinoma HCT116 cells. The results obtained indicated that both the cytotoxicity and cellular uptake of platinum were enhanced by its entrapment in ␤-CN–CS nanovehicles. These findings suggest that this novel drug-delivery system enables drugs to be thermodynamically stable in aqueous solutions and is potentially useful for targeted oral-delivery applications. © 2013 Elsevier B.V. All rights reserved.

Article history: Received 18 June 2013 Received in revised form 15 August 2013 Accepted 18 August 2013 Available online 28 August 2013 Keywords: Beta-casein Platinum complex Chitosan Nanovehicles Oral drug-delivery system

1. Introduction Platinum-based chemotherapy is effective in the treatment of various tumors. However, the clinical use of platinum compounds is limited by their severe systemic toxicity, induced tumor drug resistance and short half-life in the blood circulation system [1,2]. The most valuable progress in platinum-based chemotherapies will probably come from the invention of controlled and targeted drugdelivery nanovehicles [3]. In this context, nanoencapsulation of medicinal drugs inside biodegradable nanoparticles may provide many benefits [4]. It significantly increases the stability, solubility, efficacy, bioavailability, specificity and therapeutic index of the corresponding drugs [5].

∗ Corresponding author at: Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. Tel.: +98 2634579600; fax: +98 2634579600. E-mail addresses: divsalar@tmu.ac.ir, divsalar@khu.ac.ir (A. Divsalar). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.08.022

Biopolymer particles can be used to protect and deliver bioactive compounds in well-formulated delivery systems [6]. Hence, there is considerable interest in the use of natural biopolymers, such as proteins and polysaccharides, to build particulate delivery systems. Under controlled pH conditions, the attractive and repulsive forces between polysaccharides and proteins may lead to either biopolymer incompatibility or the formation of a nanocomplex [7]. In the latter case, the associative interactions may occur through electrostatic attraction between proteins and polysaccharides with opposite electrical charges. However, such particles may undergo dissociation when the conditions are changed, e.g. a change in pH or an increase in ionic strength [8,9]. Nanocomplex formation between (oppositely) charged macromolecules in solution has been widely investigated [9,10]. The present study describes an assay of an alternative strategy for the delivery of a new synthesized Pt (II) complex (bipyridine morpholine dithiocarbamate Pt(II) nitrate; Fig. 1) by encapsulating it in the nanoparticles formed from two oppositely-charged biopolymers such as beta-casein–chitosan.

Chitosan (CS) is a cationic polysaccharide (pKa 6. Particle size distribution was studied by DLS at 25 ◦ C using 0. However.1. Cytotoxicity studies 2.4. . micellar aggregations [16]. This is due to its abundance.89 cp for the viscosity of the medium. Based on previous studies. Mean diameter was only measured in solutions that showed no sedimentation after overnight equilibration.24]. The Pt (II) complex (bipyridine morpholine dithiocarbamate Pt (II) nitrate (Fig. according to fluorescent intensity data. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367 363 2. Molecular structure of the synthesized Pt(II) complex (bipyridine morpholine dithiocarbamate Pt(II) nitrate). biodegradability. albeit reversible. sample morphology was observed by microscope. Cell culture The human colon tumor cell line (HCT-116) was selected to assay the cytotoxicity in vitro of the new synthesized Pt(II) complex. transiently opening the tight junctions between epithelial cells [22]. Maillard reaction) or physical (e.M. DLS and zeta potential study The average size and size distribution of the drug-loaded NPs were determined using DLS/zeta potential analysis (Brookhaven Instruments Corporation analyzer). a fixed angle = 90◦ for the avalanche photo diode (APD) detector and the wavelength of 678 nm for the 90 mW laser. These authors suggested that hydrophobic interactions are responsible for the binding of lipid-soluble molecules to ␤-CN. ␤-CN was dissolved in a 0.1 M sodium phosphate buffer (PBS). 6. After preparation.05%. 1. w/v) to prepared drug-␤CN solution.g. 2.5. investigated using dynamic light scattering (DLS)/zeta potential analysis and SEM. most of the ␤-CN net charge is situated in the first 21 N terminal residues of the molecule.2.5. Nanoparticle solutions (NP) were prepared by adding stock chitosan solution (0. Britain).2 and 7) using DLS after 24 h of storage. 5. and other beneficial biological properties such as biocompatibility.01 M acetate buffer solution (pH 5. At neutral pH. Amphiphilic block copolymers such as ␤-CN micelles may be efficient as drug vehicles targeting neoplastic cells.7. chitosan nanoparticles have recently attracted interest because of their capacity to coat mucosal surfaces.5) with an increasing number of pharmaceutical and biomedical applications. capable of stable. The samples were diluted with distilled water and placed in the electrophoretic cell for zeta potential measurements. while the net charge of the remainder of the molecule is close to zero (C terminal). then stirred [24]. The final solutions were titrated using HCl and NaOH (Sigma–Aldrich) to the desired pH after mixing the polymers. Synthesis of nanoparticles A 2 mM platinum solution was added dropwise with constant stirring to 0. chitosan adopts the structure of a linear and polycationic biopolymer [21].18]. 2. 5. These attractive hydrophobic interactions are mostly responsible for this reversible micellar selfassociation [15].5) [15. pH 7. model: Leo UK. In this study. The intermacromolecular complex formation between chitosan and Pt complex-loaded ␤-CN in aqueous solutions was studied as a function of pH (3. was used for SEM study and the morphology of the surfaces of its nanoparticles was observed by scanning electron microscopy (SEM. All solutions were prepared with double distilled water. SEM study The best sample. A stock solution of 2 mM Pt (II) complex in distilled water was freshly prepared. inherent pharmacological properties. It has been suggested that ␤-CN is analogous to an amphiphilic diblock copolymer.12]. unique mucoadhesion. The bioefficacy and cytotoxicity of a platinum drug (bipyridine morpholine dithiocarbamate Pt(II) nitrate) loaded in ␤-CN–CS nanocarrier nanoparticles were evaluated on colorectal carcinoma HCT116 cells and compared with the efficacy and cytotoxicity of the free Pt complex. one of the most abundant proteins in bovine milk. chitosan can interact with whole casein to form either soluble or insoluble complexes depending on the pH [24]. Materials and methods 2. Other chemicals were of analytical grade.17]. Samples were also sonicated for 30 s before measurement to ensure that the particles were well dispersed and the dispersion was homogeneous [15. Previous studies have shown that chitosan can interact with proteins forming either soluble or insoluble complexes [23]. 2. This structure enables ␤-CN to self-organize in aqueous solutions into stable micelles [14].05%.1. w/v) was prepared by dispersing weighed amounts of powdered chitosan into 0. including four out of a total of five ␤-CN phosphoserines [PSer]).0. one of the problems of using ␤-CN nanoparticles as potential nanocarriers is that drug molecules may interact with their surfaces. electrostatic interactions). either free or encapsulated in CS–␤-CN micelles. 1) was synthesized in our laboratory using procedures reported previously [25]. based on DLS results.05 mM NaH2 PO4 . Chitosan solution (0.g. These interactions may be either chemical (e.5 mg/ml ␤-CN stock solution in PBS to reach. When solubilized in dilute acids. The stub was then coated with a platinum layer by the Auto Fine Platinum Coater before imaging. Cells were Fig. In addition. we used chitosan as a secondary coating for the controlled and targeted release of the drug and the protection of water-soluble health-promoting compounds [19]. non-toxicity and low immunogenicity [20]. Materials and preparation of solutions The Medium molecular weight chitosan (CS) and ␤-casein (␤CN) from bovine milk were purchased from Sigma–Aldrich. Razmi et al. The zeta potential of nanoparticles was measured using a zeta potential analyzer at 25 ◦ C. A drop of the suspension of nanoparticles was dripped onto the aluminum stubs placed on the surface of the sample stub and dried.65 mM Na2 HPO4 and 3.000 M−1 cm−1 at 280 nm. The results presented here are from a study of the influence of pH on the formation of a colloidally-stable nanocarrier system of Pt complex-loaded ␤-CN and chitosan. 2. Casein concentration was determined spectrophotometrically using the extinction coefficient of 11.3. Beta-casein (␤-CN). ␤-CN has a strongly amphiphilic structure due to the particular nature of its primary structure with hydrophilic N-terminal and hydrophobic C-terminal regions [13]. a final drug/␤-CN molar ratio of 3:1 (data not shown). is a single phosphorylated chain of 209 amino acid residues (molecular mass of ∼24 kDa) [11. The PBS used was composed of 80 mM NaCl. Recent reports suggest that ␤-CN nanoparticles can be used for the entrapment and oral delivery of anti-neoplastic agents [17.

The values are the mean (±S.2 without any evidence of phase separation or precipitation. The presence of hydrophilic CS molecules on the outside of the nanoparticles formed by the ␤-CN-bipyridine morpholine dithiocarbamate Pt (II) nitrate complex may be sufficient for steric stabilization of these nanoparticles preventing their self-aggregation. In vitro cytotoxicity study The cytotoxicity of bipyridine morpholine dithiocarbamate Pt (II) nitrate.01 Precipitation Zeta potential (mV) – +30 ± 0.2. However. 2) showed that the particles formed at these pHs measured between 200 and 300 nm with a mean diameter of 250 and 270 nm at pH 5. The isoelectric pH (pI) of ␤-CN is 5.11 ± 0. pH 3 5. indicating the formation of CS–␤-CN particles [17.17 ± 0. the optimal loading molar ratio was 3:1 Pt/␤-CN. chitosan and ␤-CN are positively charged and negatively charged. respectively. free and in nanoparticles. the polydispersity index ranged from 0. In the pH range 5. Table 1 Effect of pH on the particle size. At this pH.7 6.24].7.6 +29 ± 1 – . used as a carrier in the described proportions. further studies are required to verify this hypothesis [28].7 and 6. The systems containing beta-casein–chitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex were stable and soluble between pH 5. The mean diameter was only measured in systems that did not sediment after overnight equilibration.05 wt%. Effects of different pH on the mean nanoparticle diameter for systems containing 0. the ␤-CN molecules have a tendency for small-size aggregation before largescale aggregation and precipitation at their pI (pH 5.2. the mixture of CS and ␤-CN changed from crystal clear to somewhat opalescent. 3. The cleavage and conversion of the soluble yellowish MTT to the insoluble purple formazan by the active mitochondrial dehydrogenase of living cells has been used to develop an alternative assay system for measuring cell proliferation.33. zeta potential and polydispersity index of CS-␤-CN nanoparticles loaded with the bipyridine morpholine dithiocarbamate Pt (II) nitrate complex. respectively.2. With the decrease in pH of the system from pH 7.2. Razmi et al.) of three independent experiments. the interactions between these two oppositely-charged biopolymers.24]. 2. In this pH range.24]. 50 ␮L of MTT solution (5 mg/mL in PBS) was added to each well containing fresh and cultured medium. Results and discussion Their instability and formation of visually detectable aggregates in solution is one of the greatest limitations of the efficient dosage and use of anticancer platinum drugs. respectively. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367 grown in DMEM medium. was studied using the MTT assay.5. It should be mentioned that the nanoparticles aggregated at pH 3. At low pH (pH 3). bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at a 3:1 molar ratio and chitosan at 0. bipyridine morpholine dithiocarbamate Pt (II) nitrate cannot bind to ␤-CN nanoparticles either since both are positively charged at pH 3. which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ◦ C in a 5% CO2 /95% air atmosphere. one of the problems of ␤CN nanoparticles is that drug molecules may interact with their surface. Effect of pH on the size of ˇ-casein–chitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex The mean diameter and polydispersity of the nanocomplexes were determined at different pH by DLS and are shown in Fig. For controlled drug release and the protection of healthpromoting compounds. Asys Hitchech. indicating a pH-dependence of particle size and size distribution. According to fluorescence intensity measurements (not shown).7 and 6.7–6.5 mg/mL ␤-CN.7 to 6. Precipitation of nanoparticles was also observed at pH 7 caused by the insolubility and precipitation of chitosan [23. 2 and Table 1. Fluorescence measurements of the ␤-CN micelle–platinum complex formation revealed that Pt complex molecules bind to ␤-CN micelles. the solubility and stability of bipyridine morpholine dithiocarbamate Pt (II) nitrate is increased significantly. 2. and its solutions are completely transparent and lacking any Pt drug crystals. where Atreated and Acontrol are the absorbance of the treated and untreated cells. Upon addition of CS to the ␤-CN nanoparticle solution.2 7 Mean diameter (nm) – 250 ± 5 270 ± 4 – da (Polydispersity) Drug aggregation 0.3). in the presence of ␤-CN. The 50% cytotoxic concentration (Cc50 ) was measured as the drug concentration at which 50% of cells were viable compared with that of the control [26. However. both CS and ␤-CN are positively charged and strong dissociation disintegrates their nanoparticles.1. The mean diameter and polydispersity of the nanoparticles of beta-casein–chitosan loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex increased with increasing pH from 5. Harvested colorectal carcinoma HCT116 cells were seeded in a 24-well plate (1 × 105 cell/ml) with different amounts of free Pt complex (0–80 ␮M) and CS–␤-CN:Pt complex nanoparticles (0–30 ␮M) for 24 and 48 h.27]. However. chitosan was used as a secondary coating.17.11 to 0. Thus.D. which indicates a unimodal and homogeneous distribution of the nanoparticle suspension [23]. However. 3.01 0. probably due to the deprotonation of chitosan and the formation of larger particles at pH 6. Austria) at a wavelength of 570 nm. At the end. DLS data (Fig. yield nanocomplexes and nanoparticles [23. The cell viability was calculated using the following equation: Cell viability (%) = Atreated × 100 Acontrol Fig. In this case. Four hours before the end of incubations. the CS molecules may still bind to the outside of these small-size aggregates in the early stages of aggregation through Coulombic polyionic interactions between negatively-charged ␤-CN and positively-charged chitosan molecules. It can be proposed that the formation of nanoparticles of beta-casein–chitosan loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at constant ␤-CN and chitosan (polyanion and polycation) concentrations and different pH also depends on the self-aggregation of ␤-CN. certainly Coulombic in nature. the insoluble formazan generated was dissolved in a solution containing 1 ml of isopropanol 4% HCl 1 N (left for 24 h at room temperature in dark conditions) and the optical density (OD) was read against a reagent control with a multi-well scanning spectrophotometer (ELISA reader.2. the optimum pH for nanoparticle formation was pH 5.364 M.

pure chitosan solution was positively charged with an approximate z value of +60 mV. well-separated and roughly spherical.2 during titration with 0. Zeta potential as a function of pH of CS–␤-CN mixtures at 25 ◦ C (B).7) (mixtures of 0. HCT116 colorectal carcinoma cells were incubated with a number of equivalent concentrations of free and 3. the optimal pH based on DLS results.2. As can be seen in Fig. 4.3. 3B also shows for comparison the zeta potentials of pure ␤-CN and pure chitosan stock solutions used in these experiments. Consequently. preventing aggregation and stabilizing NPs in buffer solution.5 mg ␤-CN and Pt complex in a molar ratio of 3:1) ( ) at pH 5. Pt complex at a 3:1 molar ratio and chitosan at 0.05 wt% chitosan and 0.5 mg/ml ␤-CN) which agrees well with particle size measurements. No significant changes in the zeta potential were observed when the pH of the suspension increased from 5. 3 shows the results of studying the pH-dependence of the zeta potential of these nanocomplexes at pH 5.2. The values are the mean (±S. 3. Sedimentation was observed at pH 3 and 7 hence the zeta potential of these systems was not measured.5 mg/mL loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at a molar ratio of 1:3 and with chitosan at 0. Hence.) of three independent experiments.7 to 6.7. Fig. It describes strong repellent forces among particles.5 mg/ml ␤-CN). indicating that the formation of an electrostatic complex was responsible for this result.7. The zeta potential of beta-casein–chitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex decreased till approximately +30 mV. the NP population was homogeneous. indicating neutralization of anionic surface charges of ␤-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate by CS.1 mol/ml NaOH. Zeta potential of 0. cellular uptake and adsorption to cellular membranes in vivo. Cell culture and cytotoxicity assay To determine the anticancer activity of the free and encapsulated new synthesized bipyridine morpholine dithiocarbamate Pt (II) nitrate complex in CS–␤-CN NPs (mixtures of 0.05 wt% chitosan and 0. The NPs were intact.7.05 wt% chitosan ( ).M. The zeta potential of the nanoparticles (NPs) formed was measured only in systems that did not sediment after overnight equilibration. 0.05 wt% at pH 5. the zeta potential of the mixed solution was more positive than that of the pure ␤-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex.5 mg/mL beta-casein ( ) and chitosan–␤-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex (mixtures of 0.5 mg ␤-CN and bipyridine morpholine dithiocarbamate Pt (II) nitrate complex in a molar ratio of 3:1) at pH 5. with values of approximately −23 mV due to the net electrostatic charge of the ␤-casein surface.D. 3. The ␤-CN concentration was . were examined using SEM.05 wt%.2 and 5. 0. Fig. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367 365 Fig.5 mg/mL ␤-CN. 3. SEM micrograph of beta-casein–chitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex (mixtures of 0.5 mg/ml ␤-CN loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate was found to be negative at pH 6. as can be seen in Table 1.7 (A). Razmi et al. SEM measurement also indicated that NPs were approximately 200 nm at optimal pH (pH 5. Scanning electron microscopy (SEM) The morphological characteristics of the chitosan–␤-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex containing 0.7 and 6.7 and 6. A high absolute zeta potential value indicates a high electric charge on the surface of the drug-loaded NPs.4. 4. The zeta potential of 0. Fig.05 wt% chitosan and 0.05 wt% chitosan and 0. At these pH values.2 of nanocomplex solutions indicated their good stability and the presence of free NH3+ groups on the surface of the polymer [28]. These charges can greatly influence particle distribution. a zeta potential of +30 mV at pH 5. Effect of pH on the zeta potential of beta-casein–chitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex Zeta potential is a parameter used in the study of the surface charges and stability of NPs.

both untreated and encapsulated Pt complex show clear dose. Cc50 (after 24 h) (␮M) Pt complex ␤-casein:Pt complex:chitosan Cis-Pt (control) 60 ± 3 16 ± 1 154 ± 5 Cc50 (after 48 h) (␮M) 70 ± 5 21 ± 1.The values are the mean (±S. M.) of three independent experiments. At certain pH values (pH 5. 5.W. Chae. particularly those concerning different fragments of GIT (Gastro Intestinal Tract). The tumor cells were incubated with varying concentrations of the free Pt(II) complex ranging from 0 to 80 ␮M (A) and encapsulated in CS–␤-CN nanoparticles (bipyridine morpholine dithiocarbamate Pt (II) nitrate) complex ranging from 0 to 30 ␮M (B) for 24 h ( ) and 48 h ( ). Kataoka. Free ␤-casein and chitosan did not show any toxic effect on the cells studied (data not shown). Advances in polymeric micelles for drug delivery and tumor targeting. [2] V. possibly because of the adhesion of CS NPs to the mucosal surfaces and the transient opening of the tight junction between epithelial cells. nanoparticle-entrapped bipyridine morpholine dithiocarbamate Pt (II) nitrate complex for 24 and 48 h. [7] P. Kumari. 4. Food protein-based materials as nutraceutical delivery systems. are presented.D. Jun. Guo. [8] B.H.1 320 ± 4 .S.7 and 6. Ch. Shidhaye. M. SEM analysis provided additional proof of nanocomplex formation at the optimal pH. Thus.366 M. Funct. 71 (2011) 227–234. the Cc50 values of free and encapsulated bipyridine morpholine dithiocarbamate Pt (II) nitrate complex were calculated as 70 and 21 ␮M for 24 h and 60 and 16 ␮M for 48 h. However. Q. Biol. Norde. their aggregates dissociate while free drug (bipyridine morpholine dithiocarbamate Pt (II) nitrate) aggregates and precipitates. respectively.J. the studied solutions have a zeta potential of +30 mV. The 50% cytotoxic concentrations (Cc50 ) of both free and encapsulated complex were determined from Fig. Yadav. Wang. S. Biodegradable polymeric nanoparticles based drug delivery systems.J. J. Zhang. Technol. the optimal formation of nanoparticles was observed at pH 5.L. The results obtained also show that the Pt complex remains active after encapsulation in CS–␤-CN NPs. Voragen. Xing. S. References [1] R. Sohn. Control. 23 (2009) 1120–1126.K.E. depending on the pH. M. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367 has a strong influence on the suppression of HCT116 cell growth. 103 (2009) 1039–1044. A. These values both decreased significantly after longer incubation times.7 and 6. Park. A novel micelle-encapsulated platinum (II) anticancer agent. Fig. Influence of the overall charge and local charge density of pectin on the complex formation between pectin and ␤-lactoglobulin. Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for ␻-3 polyunsaturated fatty acids. Kim. Polymeric micelles for nano-scale drug delivery. It can be concluded that the newly-designed beta-casein–chitosan nanocarrier system loaded with the bipyridine morpholine dithiocarbamate Pt (II) nitrate complex could be a promising candidate for clinical trials for the treatment of different carcinomas. Cell growth after different incubation times was significantly reduced by various concentrations of complex. P. The cytotoxicity data also show that CS–␤-CN NP-encapsulated drug is more active than free bipyridine morpholine dithiocarbamate Pt (II) nitrate complex. potentially useful as a vehicle for cancer therapy. Nanomed. V. and aggregated at pH 3. Kadam. it could be more efficient in vivo [29]. because of the insolubility of chitosan at this pH.K. Song. Growth suppression activity of the free and encapsulated bipyridine morpholine dithiocarbamate Pt (II) nitrate complex on a colorectal carcinoma HCT116 cell line was assessed using MTT assay. R. S. Phutane. H. nanocomplexes with diameters between 200 and 300 nm were formed. Zimet. J. I.2. at which ␤-CN and chitosan had opposite charges. It is therefore evident that the bipyridine morpholine dithiocarbamate Pt (II) nitrate complex is more available when entrapped in CS–␤-CN NPs. B 75 (2010) 1–18. J. Zhang. measured by the cell uptake and cytotoxicity of the Pt complex by colorectal carcinoma HCT-116 cells. Trends Food Sci. Food Hydrocol. At pH 5. In addition.H. React. Chen. Yadav. Hyun Oh. These complexes were stable and soluble in the above-mentioned pH range. H.C. 5 and Table 2.A. Lee. Wang.and timeresponse suppression during growth of HCT116 cells.D. In the presence of CS–␤-CN NPs. Subirade. Nanotechnol. K. Y. the results of the study of novel biodegradable CS–␤-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex. Food Hydrocol.J. [5] K. 23 (2009) 765–772. It was shown that.G. Jadhav. Choi. Acknowledgement The authors acknowledge the financial support of the Research Council of Kharazmi University and express their gratitude. Inorg. 6 (2010) 714–729. S. 5. Y.J. Schols. The studied nanoparticles precipitated at pH 7. The results are presented in Fig. Livney. Stuart. the drug efficacy. G.7. Y. Sperber.M. Yang. It should be highlighted that the presence of the morpholine moiety in the structure of the Pt (II) complex Table 2 Cc50 values of free and encapsulated bipyridine morpholine dithiocarbamate Pt (II) nitrate complex after different incubation times. X. Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin. For example. indicating that they are stable at these optimal pHs. Conclusions In the present report. W. Z. Release 147 (2010) 144–150.C. Remondetto. [6] L.2). was improved. soluble or insoluble chitosan–␤-CN-loaded nanocomplexes containing a novel platinum-based drug can be formed. S. Miyata. J. [3] U. Med. [4] A. Y.B. Z. Biochem. Razmi et al. when chitosan and ␤-CN are positively charged. 17 (2006) 272–283. At pH 3. Polym.A. Colloids Surf. Kedar. Christie.

Paliwal. 20 (2010) 686–693.R. B. 6 (2010) 119–126. Gangnard. Shapira.G. Y. D. Zhao. I. Ron. Chitosan nanoconstructs for improved oral delivery of low molecular weight heparin: in vitro and in vivo evaluation.V. Chobert. A.K. J. G.S. 24 (2010) 239–248.A. W. J. E. Choiset. Adv. Biol. Zuev. M. Struct. Y. Tobiassen. Saboury.A. A. S. Esmaili. The adsorptioninduced secondary structure of ␤-casein and of distinct parts of its sequence in relation to foam and emulsion properties. Res. Z. Y. M. Li. Yousefi. A. Gruppen. Biol. Food Hydrocol. B 64 (2008) 104–110. Paliwal. Y. Res. J. [12] J. Dickinson.P. A. R. J. K. Beta-casein nanovehicles for oral delivery of chemotherapeutic drugs. Liu. [18] M. Farhadi. Biomaterials 26 (2005) 6041–6053. Y. 1430 (1999) 73–83. G. [17] A. Yousefi. Pan. J. H.D. Food Hydrocol. H. Axelos. Atri. [19] A. Norde. Kim. 17 (1997) 1053–1065. Beta-lactoglobulin–polysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in non-fat foods and clear beverages. Singh. J. I. J.G. A. Shapira. Pharm. Technol. Zheng. J. Dairy J. Singh.C. Diimine Platinum(II). 21 (2007) 180–190. Colloids Surf. [10] O. Beta-casein nanoparticles as an oral delivery system for chemotherapeutic drugs: impact of drug structure and properties on Co-assembly. Cholesterol-lowering. R. A. Guo. Haertlé. Drug Deliv. Thermal analysis of ␤-lactoglobulin complexes with pectins or carrageenan for production of stable biopolymer particles. J. Divsalar. A. Krishna. 249 (2002) 139–147. Y. Chobert. Hao. G. Saravanakumar. Gaudin. Razmi et al. Rev. Divsalar. S. J. M. Shapira. A. Livney. Caessens. Wei. [11] S. 62 (2010) 28–41. Food Sci. McClements. Livney. M. S. Bargarum. Assaraf. Nanotechnol. Anal. Agrawal.A. Aggregation in a concentrated model protein system: a mesoscopic simulation of ␤-casein self-assembly. Nanotechnol. Decker. Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo.J. based on self-assembled ␤-casein micelles. Trautwein. Choiset.J. H. H. Med. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362–367 [9] N. Epstein.A. Targeted delivery of low molecular drugs using chitosan and its derivatives. Bachar. Haertlé.D. A. Gaudin. Castrec.M. Int. Zimet. Xu. M. E. Mandelbaum. Ropers. Chem. Beta caseinmicelle as a nano vehicle for solubility enhancement of curcumin: food industry application. S. Pharm. H. Biotechnol. 422 (2012) 179–184. Assaraf. Int. Markman. Shchutskaya. J. [13] E. [15] A. Danino. U. 136 (2008) 67–73. and detail DNAbinding studies. MansooriTorshizi. Cui.W. [16] Y. Vyas.D. G. pH-dependent structures and properties of casein micelles. Ye.A. Moosavi-Movahedi.I. Pharm. Y. cytotoxicity.F. 27 (2010) 2175–2186. Release 160 (2012) 164–171. 27 (2009) 1124–1131. Muronetz.A. A. Livney. ␤-casein-based nanovehicles for oral delivery of chemotherapeutic drugs: drug-protein interactions and 367 [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] mitoxantrone loading capacity. V. Dyn. Chen. Y. . Food Hydrocol. Nutr. A.I. Perlstein. S. Biomol. [14] P.-LEB 44 (2011) 2166–2172. Modifications of the charges at the N-terminus of bovine ␤-casein: consequences on its structure and its micellisation. Spectroscopic and cytotoxic studies of the novel designed palladium(II) complexes: ␤-lactoglobulin and K562 as the targets. Parc. gallstone-preventing action of chitosans with different degrees of deacetylation in hamsters fed cholesterol-rich diets. Control. Engineering of caseins and modulation of their structures and interactions. Nanomed.R. H. Nanomed. Preparation and characterization of nanoparticles formed by chitosan–caseinate interactions.L.D. A. Y. Flanagan.J. J. Kwon. Even-Chen.M. Biol. Biophys. D. L.A. Int. Assaraf.H. Park. Portnaya. 6 (2010) 547–555. D. Adv. R. 15 (2001) 107–115. Y. Yousefi. Saboury. T. F. Flanagan. Biopolymers 82 (2006) 121–133. Haertlé. Development and characterization of a novel drug nanocarrier for oral delivery. Zuev. Mansouri-Torshizi. V. M. 26 (2009) 575–586. H. Formation of stable nanoparticles via electrostatic complexation between sodium caseinate and gum Arabic. Fedotov. Ghaffari. P. Erbersdobler. Subirade. T. Int. Chitosan/␤-lactoglobulin core–shell nanoparticles as nutraceutical carriers. characterization. Y. J. Y. 40 (2007) 381–386. Macromol. Y.P. Palladium(II) complexes of dithiocarbamate derivative as potential antitumor agents: synthesis. R. H. J. BBA-Protein Struct. J. Chobert. Moosavi-Movahedi. Moosavi-Movahedi. J. M. Livney. Moghaddam. Y. H. H. T. Sharifizadeh. M.G.H.D. Med. Barenholz. Jongh. R. Jürgensen. Jones.