8/14/2019 1-s2.0-S

1/6

Colloids and Surfaces B: Biointerfaces 112 (2013) 362367

Contents lists available atScienceDirect

Colloids and Surfaces B: Biointerfaces

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

Beta-casein and its complexes with chitosan as nanovehicles fordelivery 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, Iranb Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iranc UR 1268 Biopolymres Interactions Assemblages, Fonctions et Interactions des Protines, Institut National de la Recherche Agronomique, Nantes, Franced Department of Chemistry, University of Sistan & Baluchestan, Zahedan, Iran

a r t i c l e i n f o

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

a b s t r a c t

The clinical application of platinum-based anticancer drugs is greatly limited by severe toxicity. Drug-

delivery 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 morpho-

line 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 UVvis spectrometry, dynamiclight scattering (DLS) and scanning electron microscopy (SEM). The particles of Pt complex-loaded beta-

caseinchitosan formed were stable and soluble in the pH range 5.76.2. Hence, the optimal pH for

complex formation is between the pIof 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 forparticle formationwas pH 5.7. At this pH,the zeta-potential valuesof nanoparticleswere

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 wasevaluatedon colorectal carcinoma HCT116cells. Theresults obtained indicatedthat boththecytotoxicity andcellular uptake of platinum were enhanced by its entrapment in-CNCS nanovehi-cles. These findings suggest thatthis 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.

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 mostvaluableprogressin platinum-based chemotherapies will

probably come from the inventionof controlled and targeted drug-delivery nanovehicles [3]. In this context, nanoencapsulation of

medicinal drugs inside biodegradable nanoparticles may provide

many benefits[4]. It significantly increases the stability, solubil-

ity, 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).

Biopolymer particlescan be usedto protect anddeliverbioactive

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 sys-

tems. 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 throughelectrostatic 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 macro-

molecules 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 morpho-

line dithiocarbamate Pt(II) nitrate; Fig. 1) by encapsulating it in the

nanoparticles formed from two oppositely-charged biopolymers

such as beta-caseinchitosan.

0927-7765/$ see front matter 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.colsurfb.2013.08.022

http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.colsurfb.2013.08.022http://www.sciencedirect.com/science/journal/09277765http://www.elsevier.com/locate/colsurfbmailto:divsalar@tmu.ac.irmailto:divsalar@tmu.ac.irmailto:divsalar@khu.ac.irhttp://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.colsurfb.2013.08.022http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.colsurfb.2013.08.022mailto:divsalar@khu.ac.irmailto:divsalar@tmu.ac.irhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.colsurfb.2013.08.022&domain=pdfhttp://www.elsevier.com/locate/colsurfbhttp://www.sciencedirect.com/science/journal/09277765http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.colsurfb.2013.08.022

8/14/2019 1-s2.0-S

2/6

M. Razmi et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362367 363

Fig.1. Molecularstructure ofthe synthesizedPt(II)complex(bipyridinemorpholine

dithiocarbamate Pt(II) nitrate).

Beta-casein (-CN), one of the most abundant proteins inbovine milk, is a single phosphorylated chain of 209 amino acid

residues (molecular mass of24 kDa)[11,12]. -CN has a stronglyamphiphilic structure due to the particular nature of its primary

structurewith hydrophilic N-terminal and hydrophobic C-terminal

regions [13]. At neutral pH, most of the-CN net charge is situatedin the first 21 N terminal residues of the molecule, including four

out of a total of five -CN phosphoserines [PSer]), while the net

charge of the remainder of the molecule is close to zero (C ter-minal). This structure enables -CN to self-organize in aqueoussolutions into stable micelles [14].These attractive hydrophobic

interactions are mostly responsible for this reversible micellar self-

association [15]. It hasbeen suggested that-CN is analogous to anamphiphilic diblock copolymer, capable of stable, albeit reversible,

micellar aggregations[16].Amphiphilic block copolymers such as

-CN micelles may be efficient as drug vehicles targeting neo-plastic cells. Recent reports suggest that -CN nanoparticles canbe used for the entrapment and oral delivery of anti-neoplastic

agents [17,18]. These authors suggested that hydrophobic inter-

actions are responsible for the binding of lipid-soluble molecules

to-CN. However, one of the problems of using -CN nanoparti-cles as potential nanocarriers is that drug molecules may interact

with their surfaces. In this study, we used chitosan as a sec-ondary 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 phar-

macological properties, and other beneficial 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 inter-

est because of their capacity to coat mucosal surfaces, transiently

opening the tight junctions between epithelial cells [22]. Previ-

ous studies have shown that chitosan can interact with proteinsforming either soluble or insoluble complexes [23]. These inter-

actions 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 influence

of pH on the formation of a colloidally-stable nanocarrier sys-

tem of Pt complex-loaded -CN and chitosan, investigated usingdynamiclight scattering (DLS)/zeta potential analysis and SEM. The

bioefficacy andcytotoxicity of a platinumdrug (bipyridine morpho-

line dithiocarbamate Pt(II) nitrate) loaded in -CNCS nanocarriernanoparticleswere evaluatedon colorectal carcinoma HCT116 cells

and compared with the efficacy and cytotoxicity of the free Pt

complex.

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 pre-

pared. -CN was dissolved in a 0.1M sodium phosphate buffer(PBS),pH 7.0.The PBS usedwas composed of 80mM NaCl, 5.65mM

Na2HPO4and 3.05mM NaH2PO4. Casein concentration was deter-

mined spectrophotometrically using the extinction coefficient of

11,000 M1 cm1 at 280nm. 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, accord-ing to fluorescent intensity data, a final drug/-CN molar ratio of3:1 (data not shown). The intermacromolecular complex formation

between chitosan and Pt complex-loaded -CN in aqueous solu-tions was studied as a function of pH (3, 5.7, 6.2 and 7) using DLS

after 24h of storage. Nanoparticle solutions (NP) were prepared by

adding stock chitosan solution (0.05%, w/v) to prepared drug--CN solution. The final 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 wasstudiedbyDLSat25 Cusing0.89cpfortheviscosityofthemedium,

a fixed angle= 90 for the avalanche photo diode (APD) detectorand the wavelengthof 678 nmfor the 90mW laser. The zeta poten-

tial 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 wasonly measured in solutions that showedno sedimen-

tation after overnight equilibration. Samples were also sonicated

for 30s 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 SEMstudy and the morphology of the surfaces of its nanoparticles was

observed by scanning electron microscopy (SEM; model: Leo UK,

Britain).A dropof thesuspension of nanoparticleswas 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) com-

plex, either free or encapsulated in CS-CN micelles. Cells were

8/14/2019 1-s2.0-S

3/6

364 M. Razmi et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362367

grown in DMEM medium, whichwas supplemented with 10% fetal

bovine serum (FBS) and 1% penicillin-streptomycin at 37C i n 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 alter-

native assay system for measuring cell proliferation. Harvested

colorectal carcinoma HCT116 cells were seeded in a 24-well

plate (1105 cell/ml) with different amounts of free Pt complex

(080M) and CS-CN:Pt complex nanoparticles (030M) for24 and 48 h. Four hours before the end of incubations, 50L ofMTT solution (5 mg/mL in PBS) was added to each well containing

freshand culturedmedium.At the end,the insolubleformazangen-

erated 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 570nm. The cell viability was

calculated using the following equation:

Cellviability (%) =AtreatedAcontrol

100

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 formationof visually detectable aggregates

in solution is one of the greatest limitations of the efficient dosage

and use of anticancer platinum drugs. However, the solubility and

stability of bipyridine morpholine dithiocarbamate Pt (II) nitrate is

increased significantly, and its solutions are completely transpar-ent and lacking any Pt drug crystals, in the presence of-CN, usedas a carrier in the described proportions. Fluorescence measure-

ments of the -CN micelleplatinum complex formation revealedthatPt complex moleculesbind to-CN micelles.According to fluo-rescence 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 health-

promoting compounds, chitosan was used as a secondary coating.

Upon addition of CS to the -CN nanoparticle solution, the mixtureofCS and-CN changed from crystal clear to somewhatopalescent,indicating the formation of CS-CN particles[17,24].

3.1. Effect of pH on the size of-caseinchitosan nanoparticlesloaded 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. Themean diameter was only measured in systems that

did not sediment after overnight equilibration. The systems con-

taining beta-caseinchitosan nanoparticles loaded with bipyridine

morpholine dithiocarbamate Pt (II)nitrate complex werestable and

soluble between pH 5.7and 6.2without any evidence of phase sep-

aration 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 270nm at pH 5.7 and 6.2, respectively.

However, the optimum pH for nanoparticle formation was pH 5.7,

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.

probably due to the deprotonation of chitosan and the formation

of larger particles at pH 6.2. In this pH range, chitosan and -CNare positively charged and negatively charged, respectively. Thus,

the interactions between these two oppositely-charged biopoly-

mers, certainly Coulombic in nature, yield nanocomplexes and

nanoparticles [23,24]. In the pH range 5.76.2, the polydisper-sity 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 dithio-

carbamate Pt (II) nitrate complex increased with increasing pH

from 5.7to 6.2, indicating a pH-dependence of particle size and size

distribution. It should be mentioned that the nanoparticles aggre-

gated at pH 3. The isoelectric pH (pI) of-CN is5.33.At low pH(pH3), bothCS and-CN are positivelycharged and strong dissociationdisintegratestheir nanoparticles.At thispH, bipyridine morpholine

dithiocarbamate Pt (II) nitrate cannot bind to-CN nanoparticleseither since both are positively charged at pH 3. Precipitation of

nanoparticles was also observed at pH 7 caused by the insolubil-

ity and precipitation of chitosan [23,24].It can be proposed thatthe formationof nanoparticlesof beta-caseinchitosan loaded with

bipyridine morpholine dithiocarbamate Pt (II) nitrate complex at

constant -CN and chitosan (polyanion and polycation) concen-trations and different pH also depends on the self-aggregation of

-CN. With the decrease in pH of the system from pH 7, the -CNmolecules have a tendency for small-size aggregation before large-

scale 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 Coulom-

bic polyionic interactions between negatively-charged -CN andpositively-chargedchitosan molecules.The presenceof hydrophilic

CS molecules on the outside of the nanoparticles formed by the

-CN-bipyridine morpholine dithiocarbamate Pt (II) nitrate com-

plex maybe sufficient for steric stabilization of these nanoparticlespreventing 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 withthe bipyridinemorpholine dithiocarbamatePt (II)nitrate

complex.

pH Mean diameter

(nm)

da (Polydispersity) Zeta potential

(mV)

3 Drug aggregation

5.7 2505 0.110.01 +300.6

6.2 2704 0.170.01 +291

7 Precipitation

8/14/2019 1-s2.0-S

4/6

M. Razmi et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362367 365

Fig. 3. Zeta potential of 0.05wt% chitosan ( ), 0.5 mg/mL beta-casein ( ) and

chitosan-CN nanoparticles loaded with bipyridine morpholine dithiocarbamate

Pt (II) nitrate complex (mixtures of 0.05wt% chitosan and 0.5 mg-CN and Pt com-

plex in a molar ratio of 3:1) () at pH 5.7 (A). Zeta potential as a function of pH of

CS-CNmixtures at25 C (B). Thevaluesarethe mean (S.D.)of three independent

experiments.

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 influence

particle distribution, cellular uptake and adsorption to cellular

membranes in vivo. A high absolute zeta potential value indi-

cates a high electric charge on the surface of the drug-loaded

NPs. It describes strong repellent forces among particles, preven-

ting 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. Sedi-

mentation was observed at pH 3 and 7 hence the zeta potential

of these systems was not measured. The -CN concentration was

Fig. 4. SEM micrograph of beta-caseinchitosan nanoparticles loaded with bipyri-

dine morpholine dithiocarbamate Pt (II) nitrate complex (mixtures of 0.05wt%

chitosanand 0.5mg-CN and bipyridinemorpholine dithiocarbamatePt (II)nitratecomplex in a molar ratio of 3:1) at pH 5.7.

0.5 mg/mL loaded with bipyridine morpholine dithiocarbamate Pt

(II) nitrate complex at a molar ratio of 1:3 and with chitosan at

0.05wt%.Fig. 3B also shows for comparison the zeta potentials

of pure -CN and pure chitosan stock solutions used in theseexperiments. The zeta potential of 0.5mg/ml -CN loaded withbipyridine 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 sur-face. At these pH values, pure chitosan solution was positively

charged with an approximate zvalue of +60 mV. The zeta poten-

tial of beta-caseinchitosan nanoparticles loaded with bipyridine

morpholine dithiocarbamate Pt (II) nitrate complex decreased till

approximately +30mV, indicating neutralization of anionic surface

charges of-CN nanoparticles loaded with bipyridine morpholinedithiocarbamate Pt (II) nitrate by CS. Hence, the zeta potential of

the mixed solution was more positive than that of the pure -CNnanoparticles loaded with bipyridine morpholine dithiocarbamate

Pt (II) nitrate complex, indicating that the formation of an elec-

trostatic complex was responsible for this result. No significantchanges inthe zeta potentialwereobservedwhenthepH ofthe sus-

pension increased from 5.7 to 6.2 during titration with 0.1 mol/ml

NaOH, as can be seen inTable 1.Consequently, a zeta potential of

+30mV at pH 5.7 and 6.2 of nanocomplex solutions indicated their

good stability and the presence of free NH 3+ groups on the surface

of the polymer[28].

3.3. Scanning electron microscopy (SEM)

The morphological characteristics of the chitosan-CNnanoparticles loaded with bipyridine morpholine dithiocarbamate

Pt (II) nitrate complex containing 0.5 mg/mL-CN, Pt complex at a3:1 molar ratio and chitosan at 0.05wt% 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 indi-

cated that NPs were approximately 200 nm at optimal pH (pH 5.7)

(mixtures of 0.05wt% chitosan and 0.5 mg/ml -CN) which agreeswell with particle size measurements.

3.4. Cell culture and cytotoxicity assay

To determine the anticancer activity of the free and encapsu-

lated new synthesized bipyridine morpholine dithiocarbamate Pt

(II) nitrate complex in CS-CN NPs(mixtures of 0.05 wt%chitosanand0.5mg/ml -CN), HCT116 colorectal carcinoma cells wereincu-

bated with a number of equivalent concentrations of free and

8/14/2019 1-s2.0-S

5/6

366 M. Razmi et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362367

Fig. 5. Growth suppression activity of the free and encapsulated bipyridine mor-

pholine dithiocarbamate Pt (II) nitrate complex on a colorectal carcinoma HCT116

cell line wasassessedusingMTT assay.The tumorcellswereincubated with varying

concentrations of the free Pt(II) complex ranging from 0 to 80M (A) and encap-

sulated in CS-CN nanoparticles (bipyridine morpholine dithiocarbamate Pt (II)nitrate) complex ranging from 0 to 30 M (B) for 24h () and 48h ().The valuesare the mean (S.D.) of three independent experiments.

nanoparticle-entrapped bipyridine morpholinedithiocarbamate Pt

(II) nitrate complex for 24 and 48h.

The results are presented inFig. 5andTable 2.

The 50% cytotoxic concentrations (Cc50) of both free and encap-

sulated complex were determined fromFig. 5.These values both

decreased significantly after longer incubation times. For exam-

ple, the Cc50values of free and encapsulated bipyridine morpholine

dithiocarbamate Pt (II) nitrate complex were calculated as 70 and

21M for 24 h and 60 and 16M for 48 h, respectively. Thus, bothuntreatedand encapsulated Pt complex showclear dose-and time-

response 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 afterdifferent incubation times was significantly reduced by various

concentrations of complex. It should be highlighted that the pres-

ence ofthe morpholine moietyin the structureof thePt (II) complex

Table 2

Cc50 values of free and encapsulated bipyridine morpholine dithiocarbamate Pt (II)

nitrate complex after different incubation times.

Cc50 (after 24h)

(M)

Cc50 (after 48h)

(M)

Pt complex 60 3 70 5

-casein:Pt complex:chitosan 16 1 21 1.1

Cis-Pt (control) 154 5 320 4

has a strong influence on the suppression of HCT116 cell growth.

The cytotoxicity data also show that CS-CN NP-encapsulateddrug is more active than free bipyridine morpholine dithiocar-

bamate 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, pos-sibly because of the adhesion of CS NPsto themucosal surfaces and

the transientopening of the tight junction between epithelial cells,

it could be more efficient in vivo [29].Free

-casein and chitosan

did not show any toxic effect on the cells studied (data not shown).

4. Conclusions

Inthe present report, theresults of thestudy of novelbiodegrad-

able CS-CN nanoparticles loaded with bipyridine morpholinedithiocarbamate Pt (II)nitrate complex, potentially useful as a vehi-

cle for cancertherapy,are presented. It was shown that, depending

on the pH, soluble or insoluble chitosan-CN-loaded nanocom-plexes containing a novel platinum-based drug can be formed. At

certain pH values (pH 5.7 and 6.2), at which-CN andchitosan hadopposite charges,nanocomplexes withdiameters between 200and

300 nm were formed. These complexes were stable and soluble in

the above-mentioned pH range. However, the optimal formationof nanoparticles was observed at pH 5.7. The studied nanoparti-

cles precipitated at pH 7, because of the insolubility of chitosan at

this pH, and aggregated at pH 3. At pH 3, when chitosan and -CNare positively charged, their aggregates dissociate while free drug

(bipyridine morpholine dithiocarbamate Pt (II) nitrate) aggregates

andprecipitates.At pH 5.7and6.2, thestudiedsolutions have a zeta

potential of +30 mV, indicating that they are stable at these opti-

mal pHs. SEM analysis provided additional proof of nanocomplex

formation at the optimal pH. In the presence of CS-CN NPs, thedrug efficacy, 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 dithio-

carbamate Pt (II) nitrate complex could be a promising candidatefor clinical trials for the treatment of different carcinomas, particu-

larly those concerning different fragments of GIT (Gastro Intestinal

Tract).

Acknowledgement

The authors acknowledge the financial support of the Research

Council of Kharazmi University and express their gratitude.

References

[1] R.Xing,X. Wang,Ch. Zhang,Y. Zhang,Q. Wang,Z. Yang,Z. Guo, Characterizationand cellular uptake of platinum anticancer drugs encapsulated in apoferritin,

J. Inorg. Biochem. 103 (2009) 10391044.[2] V.B. Jadhav, Y.J. Jun, J.H. Song, M.K. Park, J. Hyun Oh, S.W. Chae, I. Kim, S. Choi,

H.J.Lee, Y.S.Sohn, A novel micelle-encapsulatedplatinum (II)anticancer agent,J. Control. Release 147 (2010) 144150.

[3] U.Kedar, P.Phutane,S. Shidhaye, V.Kadam, Advancesin polymericmicellesfordrug delivery and tumortargeting, Nanomed. Nanotechnol. Biol.Med. 6 (2010)714729.

[4] A.Kumari,S.K. Yadav,S.C. Yadav,Biodegradablepolymeric nanoparticlesbaseddrug delivery systems, Colloids Surf. B 75 (2010) 118.

[5] K. Miyata, R.J.Christie, K. Kataoka,Polymeric micellesfor nano-scaledrug deliv-ery, React. Funct. Polym. 71 (2011) 227234.

[6] L. Chen, G.E. Remondetto, M. Subirade, Food protein-based materials asnutraceutical delivery systems, Trends Food Sci. Technol. 17 (2006) 272283.

[7] P. Zimet, Y.D. Livney, Beta-lactoglobulin and its nanocomplexes with pectinas vehicles for -3 polyunsaturated fatty acids, Food Hydrocol. 23 (2009)11201126.

[8] B.L.H.M. Sperber, H.A. Schols, M.A.C.Stuart, W. Norde, A.G.J. Voragen, Influenceof the overall charge and local charge density of pectin on the complex forma-tion between pectin and -lactoglobulin, Food Hydrocol. 23 (2009) 765772.

8/14/2019 1-s2.0-S

6/6

M. Razmi et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 362367 367

[9] N. Ron, P. Zimet, J. Bargarum, Y.D. Livney, Beta-lactoglobulinpolysaccharidecomplexesas nanovehiclesfor hydrophobic nutraceuticalsin non-fatfoods andclear beverages, Int. Dairy J. 20 (2010) 686693.

[10] O. Jones, E.A. Decker, D.J. McClements, Thermal analysis of-lactoglobulincomplexes with pectins or carrageenan for production of stable biopolymerparticles, Food Hydrocol. 24 (2010) 239248.

[11] S. Gangnard,Y. Zuev, J. Gaudin, V. Fedotov, Y. Choiset, M.A.V.Axelos, J. Chobert,T. Haertl, Modifications of the charges at the N-terminus of bovine -casein:consequences on its structure and its micellisation, Food Hydrocol. 21 (2007)180190.

[12] J. Gaudin, A.L. Parc, B. Castrec, M. Ropers, Y. Choiset, J. Shchutskaya, R. Yousefi,

V.I.Muronetz,Y. Zuev,J. Chobert,T. Haertl,Engineeringof caseinsand modula-tionof theirstructuresand interactions,Biotechnol.Adv. 27 (2009)11241131.

[13] E. Dickinson, S. Krishna, Aggregation in a concentrated model protein system:a mesoscopic simulation of-casein self-assembly, Food Hydrocol. 15 (2001)107115.

[14] P.W.J.R. Caessens, H.H.J.D. Jongh, W. Norde, H. Gruppen, The adsorption-induced secondary structureof-casein andof distinct partsof itssequence inrelation to foam and emulsion properties, BBA-Protein Struct. M. 1430 (1999)7383.

[15] A. Shapira, Y.G. Assaraf, Y.D. Livney, Beta-casein nanovehicles for oral deliv-ery of chemotherapeutic drugs, Nanomed. Nanotechnol. Biol. Med. 6 (2010)119126.

[16] Y. Liu, R. Guo, pH-dependent structures and properties of casein micelles, Bio-phys. Chem. 136 (2008) 6773.

[17] A. Shapira, Y.G. Assaraf, D. Epstein, Y.D. Livney, Beta-casein nanoparticles as anoraldeliverysystem for chemotherapeutic drugs: impactof drug structureandproperties on Co-assembly, Pharm. Res. 27 (2010) 21752186.

[18] M. Bachar, A. Mandelbaum,I. Portnaya,H. Perlstein, S. Even-Chen, Y. Barenholz,D. Danino, Development and characterization of a novel drug nanocarrier for

oraldelivery, basedon self-assembled-casein micelles,J. Control.Release160(2012) 164171.

[19] A. Shapira, G. Markman, Y.G. Assaraf, Y.D. Livney, -casein-based nanovehi-clesfor oraldeliveryof chemotherapeutic drugs: drug-protein interactions and

mitoxantrone loading capacity, Nanomed. Nanotechnol. Biol. Med. 6 (2010)547555.

[20] J.H. Park, G. Saravanakumar, K. Kim, I.C. Kwon, Targeted delivery of lowmolec-ular drugs using chitosan and its derivatives, Adv. Drug Deliv. Rev. 62 (2010)2841.

[21] E.A. Trautwein, U. Jrgensen, H.F. Erbersdobler, Cholesterol-lowering,gallstone-preventing action of chitosans with different degrees of deacetyla-tion in hamsters fed cholesterol-rich diets, Nutr. Res. 17 (1997) 10531065.

[22] Y. Pan, Y. Li, H. Zhao, J. Zheng, H. Xu, G. Wei, J. Hao, F. Cui, Bioadhesivepolysaccharide in protein delivery system: chitosan nanoparticles improve theintestinal absorption of insulin in vivo, Int. J. Pharm. 249 (2002) 139147.

[23] L. Chen, M. Subirade, Chitosan/-lactoglobulin coreshell nanoparticles asnutraceutical carriers, Biomaterials 26 (2005) 60416053.

[24] A.K. Anal, A. Tobiassen, J. Flanagan, H. Singh, Preparation and characterizationof nanoparticles formed by chitosancaseinate interactions, Colloids Surf.B 64(2008) 104110.

[25] H. Mansouri-Torshizi, M.I.Moghaddam, A. Divsalar, A.A.Saboury,Diimine Plat-inum(II), Palladium(II) complexes of dithiocarbamate derivative as potentialantitumor agents: synthesis, characterization, cytotoxicity, and detail DNA-binding studies, J. Biomol. Struct. Dyn. 26 (2009) 575586.

[26] A. Divsalar, A.A. Saboury, R. Yousefi, A.A. Moosavi-Movahedi, H. Mansoori-Torshizi, Spectroscopic and cytotoxic studies of the novel designedpalladium(II) complexes:-lactoglobulin and K562 as the targets, Int. J. Biol.Macromol. 40 (2007) 381386.

[27] M. Esmaili, S.M. Ghaffari, Z. Moosavi-Movahedi, M.S. Atri, A. Sharifizadeh, M.Farhadi, R. Yousefi, J. Chobert, T. Haertl, A.A. Moosavi-Movahedi, Beta casein-micelleas a nanovehiclefor solubilityenhancement ofcurcumin:foodindustryapplication, Food Sci. Technol.-LEB 44 (2011) 21662172.

[28] A. Ye, J. Flanagan, H. Singh, Formation of stable nanoparticles via electrostaticcomplexation between sodium caseinate and gum Arabic, Biopolymers 82

(2006) 121133.[29] R. Paliwal, S.R. Paliwal, G.P. Agrawal, S.P. Vyas, Chitosan nanoconstructs for

improved oral delivery of low molecular weight heparin: in vitro and in vivoevaluation, Int. J. Pharm. 422 (2012) 179184.