Colloids and Surfaces B: Biointerfaces 112 (2013) 362367

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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 Haertl 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 Biopolymres Interactions Assemblages, Fonctions et Interactions des Protines, 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 efcacy 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 inuence of pH on the formation of a colloidally-stable nanocarrier system composed of Pt complex-loaded -CN and chitosan nanoparticles was investigated using UVvis spectrometry, dynamic light scattering (DLS) and scanning electron microscopy (SEM). The particles of Pt complex-loaded betacaseinchitosan formed were stable and soluble in the pH range 5.76.2. 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 conrmed 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 -CNCS nanovehicles. These ndings 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 benets [4]. It signicantly increases the stability, solubility, efcacy, bioavailability, specicity 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-caseinchitosan.

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2. Materials and methods 2.1. Materials and preparation of solutions The Medium molecular weight chitosan (CS) and -casein (CN) from bovine milk were purchased from SigmaAldrich. The Pt (II) complex (bipyridine morpholine dithiocarbamate Pt (II) nitrate (Fig. 1) was synthesized in our laboratory using procedures reported previously [25]. Other chemicals were of analytical grade. All solutions were prepared with double distilled water. A stock solution of 2 mM Pt (II) complex in distilled water was freshly prepared. -CN was dissolved in a 0.1 M sodium phosphate buffer (PBS), pH 7.0. The PBS used was composed of 80 mM NaCl, 5.65 mM Na2 HPO4 and 3.05 mM NaH2 PO4 . Casein concentration was determined spectrophotometrically using the extinction coefcient of 11,000 M1 cm1 at 280 nm. Chitosan solution (0.05%, w/v) was prepared by dispersing weighed amounts of powdered chitosan into 0.01 M acetate buffer solution (pH 5.5) [15,24]. 2.2. Synthesis of nanoparticles A 2 mM platinum solution was added dropwise with constant stirring to 0.5 mg/ml -CN stock solution in PBS to reach, according to uorescent intensity data, a nal drug/-CN molar ratio of 3:1 (data not shown). The intermacromolecular complex formation between chitosan and Pt complex-loaded -CN in aqueous solutions was studied as a function of pH (3, 5.7, 6.2 and 7) using DLS after 24 h of storage. Nanoparticle solutions (NP) were prepared by adding stock chitosan solution (0.05%, w/v) to prepared drug-CN solution. The nal solutions were titrated using HCl and NaOH (SigmaAldrich) to the desired pH after mixing the polymers, then stirred [24]. 2.3. 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). Particle size distribution was studied by DLS at 25 C using 0.89 cp for the viscosity of the medium, a xed angle = 90 for the avalanche photo diode (APD) detector and the wavelength of 678 nm for the 90 mW laser. The zeta potential of nanoparticles was measured using a zeta potential analyzer at 25 C. The samples were diluted with distilled water and placed in the electrophoretic cell for zeta potential measurements. Mean diameter was only measured in solutions that showed no sedimentation after overnight equilibration. Samples were also sonicated for 30 s before measurement to ensure that the particles were well dispersed and the dispersion was homogeneous [15,17]. 2.4. SEM study The best sample, based on DLS results, was used for SEM study and the morphology of the surfaces of its nanoparticles was observed by scanning electron microscopy (SEM; model: Leo UK, Britain). A drop of the suspension of nanoparticles was dripped onto the aluminum stubs placed on the surface of the sample stub and dried. The stub was then coated with a platinum layer by the Auto Fine Platinum Coater before imaging. After preparation, sample morphology was observed by microscope. 2.5. Cytotoxicity studies 2.5.1. 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, either free or encapsulated in CS-CN micelles. Cells were

Fig. 1. Molecular structure of the synthesized Pt(II) complex (bipyridine morpholine dithiocarbamate Pt(II) nitrate).

Beta-casein (-CN), one of the most abundant proteins in bovine milk, is a single phosphorylated chain of 209 amino acid residues (molecular mass of 24 kDa) [11,12]. -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]. At neutral pH, most of the -CN net charge is situated in the rst 21 N terminal residues of the molecule, including four out of a total of ve -CN phosphoserines [PSer]), while the net charge of the remainder of the molecule is close to zero (C terminal). This structure enables -CN to self-organize in aqueous solutions into stable micelles [14]. These attractive hydrophobic interactions are mostly responsible for this reversible micellar selfassociation [15]. It has been suggested that -CN is analogous to an amphiphilic diblock copolymer, capable of stable, albeit reversible, micellar aggregations [16]. Amphiphilic block copolymers such as -CN micelles may be efcient as drug vehicles targeting neoplastic cells. Recent reports suggest that -CN nanoparticles can be used for the entrapment and oral delivery of anti-neoplastic agents [17,18]. These authors suggested that hydrophobic interactions are responsible for the binding of lipid-soluble molecules to -CN. However, one of the problems of using -CN nanoparticles as potential nanocarriers is that drug molecules may interact with their surfaces. In this study, 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]. Chitosan (CS) is a cationic polysaccharide (pKa 6.5) with an increasing number of pharmaceutical and biomedical applications. This is due to its abundance, unique mucoadhesion, inherent pharmacological properties, and other benecial biological properties such as biocompatibility, biodegradability, non-toxicity and low immunogenicity [20]. When solubilized in dilute acids, chitosan adopts the structure of a linear and polycationic biopolymer [21]. In addition, chitosan nanoparticles have recently attracted interest because of their capacity to coat mucosal surfaces, transiently opening the tight junctions between epithelial cells [22]. Previous studies have shown that chitosan can interact with proteins forming either soluble or insoluble complexes [23]. These interactions may be either chemical (e.g. Maillard reaction) or physical (e.g. electrostatic interactions). Based on previous studies, chitosan can interact with whole casein to form either soluble or insoluble complexes depending on the pH [24]. The results presented here are from a study of the inuence of pH on the formation of a colloidally-stable nanocarrier system of Pt complex-loaded -CN and chitosan, investigated using dynamic light scattering (DLS)/zeta potential analysis and SEM. The bioefcacy and cytotoxicity of a platinum drug (bipyridine morpholine dithiocarbamate Pt(II) nitrate) loaded in -CNCS nanocarrier nanoparticles were evaluated on colorectal carcinoma HCT116 cells and compared with the efcacy and cytotoxicity of the free Pt complex.

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grown in DMEM medium, which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 C in a 5% CO2 /95% air atmosphere. 2.5.2. In vitro cytotoxicity study The cytotoxicity of bipyridine morpholine dithiocarbamate Pt (II) nitrate, free and in nanoparticles, was studied using the MTT assay. 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. Harvested colorectal carcinoma HCT116 cells were seeded in a 24-well plate (1 105 cell/ml) with different amounts of free Pt complex (080 M) and CS-CN:Pt complex nanoparticles (030 M) for 24 and 48 h. Four hours before the end of incubations, 50 L of MTT solution (5 mg/mL in PBS) was added to each well containing fresh and cultured medium. At the end, 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, Asys Hitchech, Austria) at a wavelength of 570 nm. The cell viability was calculated using the following equation: Cell viability (%) = Atreated 100 Acontrol

Fig. 2. Effects of different pH on the mean nanoparticle diameter for systems containing 0.5 mg/mL -CN, bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at a 3:1 molar ratio and chitosan at 0.05 wt%. The values are the mean (S.D.) of three independent experiments.

where Atreated and Acontrol are the absorbance of the treated and untreated cells, respectively. The 50% cytotoxic concentration (Cc50 ) was measured as the drug concentration at which 50% of cells were viable compared with that of the control [26,27]. 3. Results and discussion Their instability and formation of visually detectable aggregates in solution is one of the greatest limitations of the efcient dosage and use of anticancer platinum drugs. However, the solubility and stability of bipyridine morpholine dithiocarbamate Pt (II) nitrate is increased signicantly, and its solutions are completely transparent and lacking any Pt drug crystals, in the presence of -CN, used as a carrier in the described proportions. Fluorescence measurements of the -CN micelleplatinum complex formation revealed that Pt complex molecules bind to -CN micelles. According to uorescence intensity measurements (not shown), the optimal loading molar ratio was 3:1 Pt/-CN. However, one of the problems of CN nanoparticles is that drug molecules may interact with their surface. For controlled drug release and the protection of healthpromoting compounds, chitosan was used as a secondary coating. Upon addition of CS to the -CN nanoparticle solution, the mixture of CS and -CN changed from crystal clear to somewhat opalescent, indicating the formation of CS-CN particles [17,24]. 3.1. Effect of pH on the size of -caseinchitosan 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. 2 and Table 1. The mean diameter was only measured in systems that did not sediment after overnight equilibration. The systems containing beta-caseinchitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex were stable and soluble between pH 5.7 and 6.2 without any evidence of phase separation or precipitation. DLS data (Fig. 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 5.7 and 6.2, respectively. However, the optimum pH for nanoparticle formation was pH 5.7,

probably due to the deprotonation of chitosan and the formation of larger particles at pH 6.2. In this pH range, chitosan and -CN are positively charged and negatively charged, respectively. Thus, the interactions between these two oppositely-charged biopolymers, certainly Coulombic in nature, yield nanocomplexes and nanoparticles [23,24]. In the pH range 5.76.2, the polydispersity index ranged from 0.11 to 0.17, which indicates a unimodal and homogeneous distribution of the nanoparticle suspension [23]. The mean diameter and polydispersity of the nanoparticles of beta-caseinchitosan loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex increased with increasing pH from 5.7 to 6.2, indicating a pH-dependence of particle size and size distribution. It should be mentioned that the nanoparticles aggregated at pH 3. The isoelectric pH (pI) of -CN is 5.33. At low pH (pH 3), both CS and -CN are positively charged and strong dissociation disintegrates their nanoparticles. At this pH, bipyridine morpholine dithiocarbamate Pt (II) nitrate cannot bind to -CN nanoparticles either since both are positively charged at pH 3. Precipitation of nanoparticles was also observed at pH 7 caused by the insolubility and precipitation of chitosan [23,24]. It can be proposed that the formation of nanoparticles of beta-caseinchitosan 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. With the decrease in pH of the system from pH 7, the -CN molecules have a tendency for small-size aggregation before largescale aggregation and precipitation at their pI (pH 5.3). In this case, 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. 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 sufcient for steric stabilization of these nanoparticles preventing their self-aggregation. However, further studies are required to verify this hypothesis [28].

Table 1 Effect of pH on the particle size, zeta potential and polydispersity index of CS--CN nanoparticles loaded with the bipyridine morpholine dithiocarbamate Pt (II) nitrate complex. pH 3 5.7 6.2 7 Mean diameter (nm) 250 5 270 4 da (Polydispersity) Drug aggregation 0.11 0.01 0.17 0.01 Precipitation Zeta potential (mV) +30 0.6 +29 1

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Fig. 4. SEM micrograph of beta-caseinchitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex (mixtures of 0.05 wt% chitosan and 0.5 mg -CN and bipyridine morpholine dithiocarbamate Pt (II) nitrate complex in a molar ratio of 3:1) at pH 5.7.

Fig. 3. Zeta potential of 0.05 wt% chitosan ( ), 0.5 mg/mL beta-casein ( ) and chitosan-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex (mixtures of 0.05 wt% chitosan and 0.5 mg -CN and Pt complex in a molar ratio of 3:1) ( ) at pH 5.7 (A). Zeta potential as a function of pH of CS-CN mixtures at 25 C (B). The values are the mean (S.D.) of three independent experiments.

0.5 mg/mL loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at a molar ratio of 1:3 and with chitosan at 0.05 wt%. Fig. 3B also shows for comparison the zeta potentials of pure -CN and pure chitosan stock solutions used in these experiments. The zeta potential of 0.5 mg/ml -CN loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate was found to be negative at pH 6.2 and 5.7, with values of approximately 23 mV due to the net electrostatic charge of the -casein surface. At these pH values, pure chitosan solution was positively charged with an approximate z value of +60 mV. The zeta potential of beta-caseinchitosan nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex decreased till approximately +30 mV, indicating neutralization of anionic surface charges of -CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate by CS. Hence, 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, indicating that the formation of an electrostatic complex was responsible for this result. No signicant changes in the zeta potential were observed when the pH of the suspension increased from 5.7 to 6.2 during titration with 0.1 mol/ml NaOH, as can be seen in Table 1. Consequently, a zeta potential of +30 mV at pH 5.7 and 6.2 of nanocomplex solutions indicated their good stability and the presence of free NH3+ groups on the surface of the polymer [28]. 3.3. Scanning electron microscopy (SEM) The morphological characteristics of the chitosan-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex containing 0.5 mg/mL -CN, Pt complex at a 3:1 molar ratio and chitosan at 0.05 wt% at pH 5.7, the optimal pH based on DLS results, were examined using SEM. As can be seen in Fig. 4, the NP population was homogeneous. The NPs were intact, well-separated and roughly spherical. SEM measurement also indicated that NPs were approximately 200 nm at optimal pH (pH 5.7) (mixtures of 0.05 wt% chitosan and 0.5 mg/ml -CN) which agrees well with particle size measurements. 3.4. 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 0.5 mg/ml -CN), HCT116 colorectal carcinoma cells were incubated with a number of equivalent concentrations of free and

3.2. Effect of pH on the zeta potential of beta-caseinchitosan 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. These charges can greatly inuence particle distribution, cellular uptake and adsorption to cellular membranes in vivo. A high absolute zeta potential value indicates a high electric charge on the surface of the drug-loaded NPs. It describes strong repellent forces among particles, preventing aggregation and stabilizing NPs in buffer solution. The zeta potential of the nanoparticles (NPs) formed was measured only in systems that did not sediment after overnight equilibration. Fig. 3 shows the results of studying the pH-dependence of the zeta potential of these nanocomplexes at pH 5.7 and 6.2. Sedimentation was observed at pH 3 and 7 hence the zeta potential of these systems was not measured. The -CN concentration was

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has a strong inuence on the suppression of HCT116 cell growth. The cytotoxicity data also show that CS-CN NP-encapsulated drug is more active than free bipyridine morpholine dithiocarbamate Pt (II) nitrate complex. It is therefore evident that the bipyridine morpholine dithiocarbamate Pt (II) nitrate complex is more available when entrapped in CS-CN NPs. In addition, possibly because of the adhesion of CS NPs to the mucosal surfaces and the transient opening of the tight junction between epithelial cells, it could be more efcient in vivo [29]. Free -casein and chitosan did not show any toxic effect on the cells studied (data not shown). 4. Conclusions In the present report, the results of the study of novel biodegradable CS-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate Pt (II) nitrate complex, potentially useful as a vehicle for cancer therapy, are presented. It was shown that, depending on the pH, soluble or insoluble chitosan-CN-loaded nanocomplexes containing a novel platinum-based drug can be formed. At certain pH values (pH 5.7 and 6.2), at which -CN and chitosan had opposite charges, nanocomplexes with diameters between 200 and 300 nm were formed. These complexes were stable and soluble in the above-mentioned pH range. However, the optimal formation of nanoparticles was observed at pH 5.7. The studied nanoparticles precipitated at pH 7, because of the insolubility of chitosan at this pH, and aggregated at pH 3. At pH 3, when chitosan and -CN are positively charged, their aggregates dissociate while free drug (bipyridine morpholine dithiocarbamate Pt (II) nitrate) aggregates and precipitates. At pH 5.7 and 6.2, the studied solutions have a zeta potential of +30 mV, indicating that they are stable at these optimal pHs. SEM analysis provided additional proof of nanocomplex formation at the optimal pH. In the presence of CS-CN NPs, the drug efcacy, measured by the cell uptake and cytotoxicity of the Pt complex by colorectal carcinoma HCT-116 cells, was improved. It can be concluded that the newly-designed beta-caseinchitosan 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, particularly those concerning different fragments of GIT (Gastro Intestinal Tract). Acknowledgement The authors acknowledge the nancial support of the Research Council of Kharazmi University and express their gratitude. References
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Fig. 5. 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. 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 ( ).The values are the mean (S.D.) of three independent experiments.

nanoparticle-entrapped bipyridine morpholine dithiocarbamate Pt (II) nitrate complex for 24 and 48 h. The results are presented in Fig. 5 and Table 2. The 50% cytotoxic concentrations (Cc50 ) of both free and encapsulated complex were determined from Fig. 5. These values both decreased signicantly after longer incubation times. For example, 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, respectively. Thus, both untreated and encapsulated Pt complex show clear dose- and timeresponse suppression during growth of HCT116 cells. The results obtained also show that the Pt complex remains active after encapsulation in CS-CN NPs. Cell growth after different incubation times was signicantly reduced by various concentrations of complex. 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. 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 1.1 320 4

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