International Journal of Biological Macromolecules 69 (2014) 546–553

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Docetaxel loaded chitosan nanoparticles: Formulation, characterization and cytotoxicity studies Ankit Jain a,∗ , Kanika Thakur b , Preeti Kush a , Upendra K. Jain a a

Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali 140 110, India Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125 001, India b

a r t i c l e

i n f o

Article history: Received 19 April 2014 Received in revised form 26 May 2014 Accepted 13 June 2014 Available online 24 June 2014 Keywords: Chitosan Docetaxel Nanoparticles Nanoemulsion

a b s t r a c t The primary objective of the present investigation was to explore biodegradable chitosan as a polymeric material for formulating docetaxel nanoparticles (DTX-NPs) to be used as a delivery system for breast cancer treatment. Docetaxel loaded chitosan nanoparticles were formulated by water-in-oil nanoemulsion system and characterized in terms of particle size, zeta potential, polydispersity index, drug entrapment efficiency (EE), loading capacity (LC), scanning electron microscopy (SEM), in vitro release study and drug release kinetics. Further, to evaluate the potential anticancer efficacy of docetaxel loaded chitosan nanoparticulate system, in vitro cytotoxicity studies on human breast cancer cell line (MDA-MB-231) were carried out. The morphological studies revealed the spherical shape of docetaxel loaded chitosan nanoparticles having an average size of 170.1 ± 5.42–227.6 ± 7.87 nm, polydispersity index in the range of 0.215 ± 0.041–0.378 ± 0.059 and zeta potential between 28.3 and 31.4 mV. Nanoparticles exhibited 65–76% of drug entrapment and 8–12% loading capacity releasing about 68–83% of the drug within 12 h following Higuchi’s square-root kinetics. An increase of 20% MDA-MB-231 cell line growth inhibition was determined by docetaxel loaded chitosan nanoparticles with respect to the free drug after 72 h incubation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Chitosan, derived from chitin by deacetylation, is the second most abundant naturally occurring biopolymer and a major structural polysaccharide found in the exoskeleton of crustaceans such as crab and shrimp [1]. It consists of ␤-(1, 4)-2-acetamido-2deoxy-d-glucose and ␤-(1, 4)-2-amino-2-deoxy-d-glucose units. Thus, it comprises of copolymers of glucosamine and N-acetyl glucosamine [2]. Chitosan is considered to be the most widespread polycationic biopolymer having non-toxic, biocompatible and biodegradable characteristics [3]. Till date, chitosan has been extensively employed in food processing, agriculture, biomedicine, biochemistry and pharmaceutical applications [4–7]. Chitosan, due to its biodegradable behavior has a strong potential for application as drug carrier. The amino and carboxyl groups in the chitosan molecule can be combined with glycoprotein in mucus to form a hydrogen bond leading to an adhesive effect which prolongs the retention time of drugs as well as improves drug bioavailability

∗ Corresponding author. Tel.: +91 9466831003. E-mail address: [email protected] (A. Jain). http://dx.doi.org/10.1016/j.ijbiomac.2014.06.029 0141-8130/© 2014 Elsevier B.V. All rights reserved.

[8–11]. Chitosan can act on tumor cells to interfere directly with the cell metabolism and inhibit cell growth. Previously done studies clearly suggest that chitosan had antitumor effects in vitro and in vivo, leading to good prospects for its application as a supplementary antitumor drug and drug carrier [12–15]. Studies have also indicated the selectivity of chitosan nanoparticles for tumor cells [16]. Chitosan nanoparticles have attracted great attention in pharmaceutical applications including being targeted for colon or mucosal delivery, cancer therapy, delivery of vaccines, genes and antioxidants etc. Docetaxel (DTX), a semisynthetic derivative of taxoid family has emerged as one of the most important chemotherapeutic agent against cancer over the past several decades. It is an analog of paclitaxel which is extracted from the needles of the European yew tree (Taxus baccata L.) [17]. DTX has been effective against breast, ovarian, lung, head and neck cancer. Being a microtubule stabilizing agent, it inhibits microtubule assembly and consequently inhibits cell proliferation [18]. Due to the higher lipophilicity and poor solubility of DTX, its main marketed product used clinically i.e., Taxotere is formulated using polysorbate 80 and ethanol (50:50, v/v) which causes hypersensitivity reaction, reduced uptake by tumor tissue and increased exposure to other body compartments [19]. It is

A. Jain et al. / International Journal of Biological Macromolecules 69 (2014) 546–553

mentioned in the instructions of Taxotere that there may be foam on top of the solution in the presence of polysorbate 80 or precipitation due to the high lipophilicity of DTX, indicating that its clinical use is not completely safe [20]. This leads to the need of developing alternative formulations of DTX so as to induce desirable clinical response and reduce toxicity associated with the adjuvants used. Nanoparticle based drug delivery system holds great potential to overcome the side effects associated with the use of DTX. In the present study docetaxel loaded chitosan nanoparticles (DTX-NP) were prepared by water-in-oil nanoemulsion system. Triton X-100 was used as a surfactant, cyclohexane as the oil phase, n-hexanol as a cosurfactant and dilute acetic acid solution containing chitosan as the aqueous phase. The effect of number of variables on the physicochemical properties of nanoparticles was studied. Nanoparticles were characterized in terms of particle size, zeta potential, polydispersity index, drug entrapment efficiency (EE), loading capacity (LC), scanning electron microscopy (SEM), in vitro release study, in vitro cytotoxicity screening, stability studies and drug release kinetics.

2.4. Characterization of docetaxel loaded chitosan nanoparticles (DTX-NP) 2.4.1. Particle size, size distribution and zeta potential of nanoparticles The mean particle size, polydispersity index and zeta potential of DTX-NP was measured at 25 ◦ C by photon correlation spectroscopy using the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The nanoparticle suspension was diluted 10 times with deionized water and the analysis was performed at a scattering angle of 90◦ . 2.4.2. Encapsulation efficiency and loading capacity The percentage encapsulation of DTX in chitosan nanoparticles was determined by separating the unentraped drug from nanoparticles by centrifugation at 14,000 rpm for 30 min by Cooling centrifuge C-24BL, Remi Instruments, Mumbai, India. The clear supernatant was analyzed for the contents of DTX by measuring absorbance in a UV-Visible spectrophotometer (Shimadzu UV spectrophotometer, Japan) at 230 nm. The percentage encapsulation efficiency and loading capacity were calculated as follows:

2. Experimental

EE (%) =

2.1. Materials

LC (%) =

Docetaxel was purchased from Sigma–Aldrich (USA). Chitosan (degree of deacetylation – 80%) was procured from Himedia Pvt. Ltd (Mumbai, India). Glutaraldehyde (GLU), cyclohexane, n-hexanol and glacial acetic acid were obtained from Loba chemie Pvt. Ltd (Mumbai, India). All other chemicals used were of reagent grade. 2.2. Preparation of water-in-oil (w/o) nanoemulsion Blank chitosan nanoparticles were prepared by the w/o nanoemulsion method. Briefly, an aqueous dispersion of chitosan (1–6%, w/v) was prepared in 100 ml of glacial acetic acid solution (1%, v/v). Further cyclohexane, hexanol and chitosan solution were stirred together continuosly in a flask at the volume ratio of 11:6:6 [21]. Triton X-100 was added as a surfactant into the mixture while stirring until the mixture became transparent or semitransparent, indicating the formation of nanoemulsion. The w/o nanoemulsion containing glutaraldehyde (10–60%, w/v) as a cross linking agent, but without chitosan was prepared with the same procedure. 2.3. Preparation of docetaxel loaded chitosan nanoparticles (DTX-NP) For the preparation of DTX-NP, various concentrations of DTX (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) were prepared in cyclohexane. The w/o nanoemulsion containing glutaraldehyde was added dropwise into the w/o chitosan nanoemulsion under stirring and kept the mixed nanoemulsion at 40 ◦ C for 6 h to allow the water pools containing glutaraldehyde to collide with chitosan. Fig. 1 depicts the procedure for the preparation of DTX-NP by w/o nanoemulsion method. Chitosan was cross-linked and chitosan nanoparticles were formed. The nanoparticles were then precipitated with centrifugation at 10,000 rpm for 30 min (Cooling centrifuge C-24BL, Remi Instruments, Mumbai, India) at room temperature and rinsed with acetone. The pellet obtained was further redispersed in 10 ml of Phosphate buffer saline (pH 7.4). Mannitol (2%, w/v) was added as a cryoprotectant and freezed at −80 ◦ C for 4 h followed by lyophilization in laboratory model freeze dryer (Alpha 2–4 LD Plus, Martin Christ, Germany) for 24 h at −48 ◦ C and 0.0010 mbar.

547

DTXt − DTXf DTXt

× 100

DTXt − DTXf Weight of nanoparticles

(1) × 100

(2)

where DTXt is the total amount of docetaxel used in the preparation of nanoparticles and DTXf is the unentraped docetaxel present in the supernatant. 2.4.3. Morphological characterization of nanoparticles The surface morphology of DTX-NP was observed by scanning electron microscopy (Jeol, JSM-6100, Japan). The lyophilized samples were carefully mounted on an aluminum stub using a double adhesive carbon tape. Samples were then introduced into an automated sputter coater and coated with a very thin film of gold. The photomicrographs were recorded at an accelerating voltage of 10 kV at different magnifications. 2.4.4. Fourier transform infrared spectroscopy (FT-IR) The interaction between the drug and polymer was studied by subjecting the samples to FT-IR spectroscopy in FTIR spectrophotometer (IR Affinity, Shimadzu, Japan) using the KBr pellet method. 2.4.5. In vitro drug release The in vitro release profile of various batches of DTX-NP was evaluated by the dialysis sac method. For the in vitro release studies, about 20 mg of nanoparticles were suspended in 3.0 ml of release medium (phosphate buffer saline solution pH 7.4). The mixture was then introduced into a cellophane membrane dialysis bag. The dialysis bag was suspended into 250 ml of release medium by tying to the paddle of USP type II dissolution apparatus (TDT-08L, Electrolab, Mumbai, India). The release media was kept at 37 ± 0.5 ◦ C and 50 rpm. A 5 ml of sample was withdrawn at various time intervals and replaced with an equal volume of dissolution medium. The contents of DTX in the samples were determined spectrophotometrically by measuring absorbance at 230 nm in a UV-Visible Spectrophotometer (Shimadzu UV spectrophotometer, Japan). 2.4.6. In vitro cytotoxicity screening Docetaxel loaded chitosan nanoparticles (Formulation DTXNP2) which showed an optimum particle size, polydispersity index and maximum in vitro drug release was selected for the in vitro cytotoxicity studies and evaluated comparatively against pure drug (DTX) and a batch of blank nanoparticles (Blank-NPs).

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Fig. 1. Preparation of docetaxel loaded chitosan nanoparticles by water-in-oil nanoemulsion method.

Cancer cell viability of the drug-loaded nanoparticles was screened employing MDA-MB-231 cell lines using the MTT assay method. Briefly, MDA-MB-231 cell lines were seeded in 96-well plates at the density of 5000 viable cells/well and incubated for 24 h at 37 ◦ C in 5% CO2 humidified incubator [22]. The cells were incubated with pure DTX, DTX-NP2 at 0.05, 0.5, 5.0 ␮g/ml equivalent DTX concentrations, and blank nanoparticles for 24, 48 and 72 h, respectively. These concentrations correspond to plasma levels of the drug which can be achieved in humans [23]. At specified time intervals, the medium was removed, and the wells were washed twice with phosphate buffer solution and 10 microliters of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well of the plate and incubated for another 1–4 h. After 4 h the plate was observed at 560 nm in a microplate reader. % Cell viability was calculated by the following equation: Cell viability (%) =

Abs sample × 100 Abs control

(3)

where Abs sample is the absorbance of the cells incubated with the nanoparticles, Abs control is the absorbance of the cells incubated with the culture medium only (positive control).

2.4.7. Stability studies DTX-NP were evaluated for the change in particle size and entrapment efficiency i.e. aggregation over the period of 3 months. The formulation (DTX-NP2) was kept at a temperature of 4 ± 2 ◦ C for a period of 3 months.

2.4.8. Statistical analysis The results were presented as mean ± standard deviation. Statistical analysis performed with one-way ANOVA and statistical significance was designated as p < 0.05.

3. Results and discussion 3.1. Particle size, size distribution and zeta potential of nanoparticles DTX-NP were prepared by water in oil nanoemulsion system. The particle size and zeta potential of chitosan nanoparticles are influenced by a number of factors such as pH, molecular weight of chitosan, concentration of chitosan and glutaraldehyde. Among these parameters, concentration of chitosan and glutaraldehyde are the most important factors that control the size of the prepared nanoparticles. The effect of these parameters on the properties of formulated nanoparticles were investigated by changing one parameter while keeping the others constant. On the basis of preliminary trials, the varying parameters were selected which include concentration of glutaraldehyde (10–60%, v/v), concentration of chitosan (1–6%, w/v) and drug concentration (0–1 mg/ml). 3.2. Effect of glutaraldehyde (cross linker) concentration Glutaraldehyde is mainly used as a cross linker in nanoemulsion method. It is a dialdehyde and the most commonly used cross linker with chitosan [24–26]. Glutaraldehyde is a dipolar anionic linear molecule and reacts with free amine groups present in chitosan. Chitosan cross links with glutaraldehyde by nucleophilic addition reaction between amine group of chitosan and aldehyde group of glutaraldehyde [27]. The aldehyde groups of glutaraldehyde forms covalent imine bonds with the amino groups of chitosan, due to the resonance established with adjacent double ethylenic bonds [28,29] via a Schiff reaction. Thus, cross linking is greatly attributed to the formation of covalent imine bond between chitosan and glutaraldehyde. Chitosan concentration was fixed at (1%, w/v) for all formulations and glutaraldehyde concentration was varied from (10–60%, v/v) as shown in Table 1. Fig. 2(a) represents a comparative effect of glutaraldehyde concentration on particle size, polydispersity index and zeta potential. It is observed that an initial increase

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Fig. 2. Bar graph showing comparative effect of Glutaraldehyde concentration (a), chitosan concentration (b), drug concentration (c) on particle size, polydispersity index and zeta potential of docetaxel loaded chitosan nanoparticles.

in concentration of the glutaraldehyde decreases the particle size up to a concentration of 40%. This may be attributed to the fact that at higher concentration of cross linking agent, pore networks get partially filled and size of particles appears to be smaller [30]. The higher concentration of glutaraldehyde (>40%) has no significant influence on particle size. Zeta potential influences the stability of the nanoparticles through electrostatic repulsion. The results showed no significant effect of cross linking agent on zeta potential. A small decrease of zeta potential can be observed due to the decrease of residual amine group into the solution. The polydispersity index is below 0.5 indicating uniformity of particle size.

size, polydispersity index and zeta potential. An increase in concentration of chitosan increases the particle size due to the increase in amine group of chitosan. Glutaraldehyde interacts with two amino groups of chitosan [31]. Further, values of polydispersity index are less than 0.5 indicating uniformity of particle size. 3.4. Effect of drug concentration

Chitosan undergoes nucleophilic interaction with the cross linking agent leading to the formation of nanoparticles. Glutaraldehyde concentration was kept constant at 40% (v/v) and chitosan concentration was varied from 1 to 6% (w/v) as shown in Table 2. Fig. 2(b) shows a comparative effect of chitosan concentration on particle

DTX-NP was prepared upon the addition of DTX (0.2–1 mg/ml) in 40% v/v glutaraldehyde into 2% w/v chitosan solution. Fig. 2(c) represents a comparative effect of drug concentration on particle size, polydispersity index and zeta potential. It can be observed that the size of nanoparticles did not grow significantly at concentrations up to 0.4 mg/ml. A significant increase in size was observed at concentration 0.6 mg/ml and the size remains constant at concentrations above 0.6 mg/ml of docetaxel. Table 3 shows the effect of different concentrations of DTX on the %EE and %LC of docetaxel loaded chitosan nanoparticles. The observed results did not show a significant change in EE of

Table 1 Effect of glutaraldehyde concentration on particle size and zeta potential of docetaxel loaded chitosan nanoparticles.

Table 2 Effect of chitosan concentration on particle size and zeta potential of docetaxel loaded chitosan nanoparticles.

3.3. Effect of chitosan concentration

GLU concn. (%, v/v)

Particle size (nm)

10 20 30 40 50 60

290.5 241.1 207.2 170.6 176.7 183.4

± ± ± ± ± ±

3.98 4.01 5.81 3.01 5.37 4.34

Polydispersity Index (PI) 0.367 0.298 0.321 0.208 0.278 0.345

± ± ± ± ± ±

0.03 0.03 0.02 0.02 0.03 0.03

Zeta potential (mV) 28.1 30.9 30.3 31.6 27.7 29.1

± ± ± ± ± ±

1.01 1.19 0.98 1.21 1.45 1.34

Mean ± S.D., n = 3. Note: Concentration of Chitosan: (2%, w/v). GLU concn: Concentration of Glutaraldehyde.

Chitosan concn. (%, w/v)

Particle size (nm)

1 2 3 4 5 6

173.5 197.3 249.6 292.5 331.9 349.8

± ± ± ± ± ±

3.07 4.87 5.98 7.12 7.34 7.23

Polydispersity Index (PI) 0.208 0.301 0.291 0.411 0.434 0.455

± ± ± ± ± ±

0.02 0.05 0.02 0.03 0.02 0.02

Zeta potential (mV) 31.9 33.7 35.1 35.9 37.6 38.4

± ± ± ± ± ±

0.94 1.45 1.95 1.65 1.34 1.54

Mean ± S.D., n = 3. Note: Concentration of Glutaraldehyde: (40%, v/v). Chitosan concn: Concentration of chitosan.

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Table 3 Characterization of docetaxel loaded chitosan nanoparticles by varying drug concentration. Formulation code

DTX concentration (mg/mL)

Particle size (nm)

DTX-NP DTX-NP1 DTX-NP2 DTX-NP3 DTX-NP4 DTX-NP5

– 0.2 0.4 0.6 0.8 1

165.4 170.1 176.9 193.3 201.1 208.6

± ± ± ± ± ±

3.21 5.42 4.23 7.59 7.02 7.87

Polydispersity Index (PI) 0.220 0.215 0.227 0.297 0.321 0.378

± ± ± ± ± ±

0.02 0.05 0.02 0.05 0.04 0.06

Zeta potential (mV) 30.9 31.2 31.4 29.6 28.3 29.9

± ± ± ± ± ±

1.23 1.02 1.47 2.01 1.94 1.78

Encapsulation efficiency (%)

Loading capacity (%)

– 65.4 75.3 70.4 67.4 66.1

– ± ± ± ± ±

0.94 1.12 1.52 1.75 1.80

8.9 10.2 12.0 9.5 9.1

± ± ± ± ±

0.71 0.89 0.95 0.97 0.91

Mean ± S.D., n = 3. Note: DTX concentration: Concentration of Docetaxel.

nanoparticles, but maximum EE (75%) was achieved at 0.4 mg/ml and maximum LC was observed at 0.6 mg/ml of drug concentration. 3.5. Morphological characterization of nanoparticles Fig. 3(a and b) displays the scanning electron micrographs of DTX-NP showing their shape and surface morphology. The nanoparticles were found to be spherical in shape with smoother surfaces. 3.6. Fourier transform infrared spectroscopy (FT-IR)

nanoparticle ingredients and the drug leading to frequency shifts. The polymer may be able to interact with the above mentioned groups of DTX. However, FT-IR spectra of nanoparticles indicated the characteristics peaks of similar functional groups of drug, confirming the successful formulation of nanoparticles without any chemical interaction Thus, providing evidence that such hydrogen bond interactions are not present in the prepared nanoparticle and that the drug remained intact during the preparation procedure of nanoparticles Thus, FT-IR analysis indicated absence of any drug excipient interaction between the drug and polymer during the formulation of nanoparticles.

Fourier transform infrared (FT-IR) spectroscopy is a well known technique to study the interaction between two functional groups. FT-IR spectra of the drug, drug-polymer mixture and drug loaded nanoparticles was used to study the interaction between the drug and the polymer. Fig. 4 shows the FT-IR spectra of DTX, DTXChitosan physical mixture (50:50) and DTX-NP. The spectra of DTX shows characteristic peaks at 3481.84 cm−1 and 3337.96 cm−1 which may be ascribed to N H stretching and O H stretching of alkanes, respectively. Peaks at 1740 cm−1 and 1711 cm−1 can be attributed to C O stretching and at 1631.97 cm−1 due to N H plane bending, respectively. During the preparation of nanoparticles, there is always a possibility of any kind of physico-chemical interaction such as the formation of hydrogen bonds between

Fig. 3. Scanning electron micrographs showing shape (a) and surface morphology (b) of docetaxel loaded chitosan nanoparticles.

Fig. 4. Comparative FT-IR spectra of docetaxel, docetaxel-chitosan physical mixture and docetaxel loaded chitosan nanoparticles.

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Table 4 Release kinetics of different batches of docetaxel loaded chitosan nanoparticles. Release kinetics Formulation code

Zero order 2

DTX-NP1 DTX-NP2 DTX-NP3 DTX-NP4 DTX-NP5

First order −1

R

k (% h

0.8123 0.8533 0.7798 0.7951 0.8817

0.0695 0.0896 0.0811 0.0783 0.0816

)

2

Higuchi’s square-root −1

R

k (h

)

0.9091 0.9609 0.8907 0.9283 0.9589

0.0011 0.0018 0.0016 0.0016 0.0013

2

−0.5

R

k (%h

0.9582 0.9813 0.9478 0.9496 0.9823

2.1451 2.7324 2.5406 2.4334 2.4484

Korsmeyer Peppas )

R2

k (%h−n )

n

0.8772 0.9115 0.9134 0.8598 0.9237

0.3375 0.3023 0.2931 0.3748 0.2691

0.5566 0.5971 0.5954 0.5708 0.5831

3.7. In vitro drug release The various batches of nanoparticles showed a sustained release of about 68–83% of drug within 24 h. Fig. 5 displays the in vitro release profile of DTX-NP prepared by w/o nanoemulsion method. This can be attributed to the drug release occurring in three phases. An initial burst release due to the release of drug from the surface of nanoparticles followed by the second phase which shows a sustained release due to the release of drug from matrix and lastly, slow release of drug due to polymer degradation [32]. To determine the release kinetics, the release data was fitted into various kinetic models. Table 4 represents the release kinetics of various batches of DTX-NP. The drug was released following Higuchi’s square-root kinetics. Further, the value of ‘n’ the release exponent of korsmeyer–Peppas (0.45 ≤ n ≤ 0.89) indicates that nanoparticles released the drug by combination of both diffusion of drug through the polymer and dissolution of the polymer [33–35]. Fig. 5. In vitro release profile of docetaxel from various batches of docetaxel loaded chitosan nanoparticles.

3.8. In vitro cytotoxicity screening The in vitro cytotoxicity screening of DTX as a free drug and drug loaded chitosan nanoparticles, at the same drug equivalent

Fig. 6. % Cell viability of MDA-MB-231 cancer cells after incubation for 24 h (a), 48 h (b) and 72 h(c).

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Table 5 Stability data. Docetaxel loaded chitosan nanoparticles

Initial

Encapsulation efficiency (%) Size (nm)

75.3 ± 1.1 176.6 ± 4.2

4 ± 2 ◦C 1 month

2 months

3 months

73.8 ± 1.3 179.1 ± 3.9

71.5 ± 1.4 183.2 ± 4.7

71.0 ± 1.9 187.7 ± 4.9

Mean ± S.D., n = 3.

concentration of 0.05, 0.5 and 5.0 ␮g/ml was evaluated by the MTT assay using MDA-MB-231 as a model breast cancer cell line. Untreated MDA-MB-231 cells as well as cells treated with empty chitosan nanoparticles, with the same polymer content of that of the drug-loaded nanosystems, were used for comparison [36]. Fig. 6 (a–c) shows the viability of MDA-MB-231 cancer cells, cultured with blank chitosan nanoparticles (Blank-NPs) and docetaxel-loaded chitosan nanoparticles [DTX-NP2] after incubation for 24 h (a), 48 h(b) and 72 h(c), in comparison with that of pure drug [DTX] at 0.05, 0.5 and 5.0 ␮g/ml equivalent docetaxel concentrations. Blank nanoparticles appeared nearly non-toxic to MDA-MB-231 cells, even at a higher concentration, thus confirming the good biocompatibility of chitosan. After 24 h incubation Fig. 6(a), the MDA-MB-231 cell viability was decreased to about 65.11, 54.23 and 43.31% for DTX-NP2 at 0.05, 0.5 and 5.0 ␮g/ml drug concentrations respectively, corresponding to an increase in cytotoxicity of 22% compared with that DTX. After 48 h incubation Fig. 6(b), the cytotoxicity was increased to about 46.09, 37.41 and 26.29% for the DTX-NP2 and 69.98, 60.78 and 49.59% for the DTX, respectively. The more marked inhibition of cell growth was obtained for longer incubation period (72 h). With an approximately 80% cell viability reduction, the strongest cytotoxic effect was achieved with nanoparticles at 5.0 ␮g/ml drug concentration. On the whole, the results clearly demonstrate that docetaxel loaded chitosan nanoparticles are more effective against cancer cells than free docetaxel drug. The higher cytotoxicity of the drug loaded into nanoparticles can be attributed to synergistic combination of different but exclusive mechanisms. Docetaxel loaded chitosan nanoparticles were adsorbed onto the cell surface leading to an increase in drug concentration near the cell membrane, thus generating a concentration gradient that promotes the drug influx into the cell [37]. Cancerous cells exhibit enhanced endocytic activity and internalize the polymeric nanoparticles in the interior of the cell leading to an increase in drug concentration. Besides, free DTX molecules, are transported out by P-glycoprotein (P-gp) pumps, drug loaded nanoparticles are taken up by cells through an endocytosis pathway. Thus, it results in a higher cellular uptake of the entrapped drug, thereby enabling them to escape from the effect of P-gp pumps [38]. Moreover, intracellular delivery of DTX-NP allows enhanced drug concentration near the site of action [39].

3.9. Stability studies The long term storage stability of the drug chitosan nanoparticles is an important parameter. Nanoparticles formulations increase the surface area by many folds and this may lead to very high aggregation after long periods of storage [40]. This poor long term stability may be due to different physical and chemical factors that may destabilize the system. Table 5 represents the effect of storage time on particle size and % encapsulation efficiency of docetaxel loaded chitosan nanoparticles (DTX-NP2). After 3 months of storage at 4 ± 2 ◦ C, the nanoparticles appeared to be stable without any collapse or aggregation. There were no major changes in nanoparticles besides a slight increase in particle size and a slight decrease in encapsulation efficiency and drug loading. Therefore,

formulated docetaxel loaded chitosan nanoparticles were found to be stable for a long period of time. 4. Conclusion The present work is an effort to target Docetaxel using nanoparticulate drug delivery system in order to increase its therapeutic efficacy and minimize the toxicity associated with its use. Chitosan was successfully employed as a polymeric material for the encapsulation of docetaxel by water-in-oil nanoemulsion method. The nanoparticles exhibited particle size in the range of 170–227 nm and showed a sustained drug release behavior. The in vitro cytotoxicity studies proved an increased efficacy of nanoparticles compared with the free drug against MDA-MB-231 cell line. However, nanoparticles of desired particle size and release behavior can be prepared by optimizing the concentration of chitosan and glutaraldehyde. In conclusion, chitosan can be considered as a promising biocompatible polymer to be used for the development of nanoparticulate drug delivery system for cancer chemotherapy. Acknowledgements The authors are grateful to Central Instrumentation Laboratory, Panjab University, Chandigarh for particle size and SEM analysis and also gratified to the Management of Chandigarh College of Pharmacy, Mohali (Punjab), India for financial support and providing the facilities to carry out the research work References [1] X. Juan, M. Lili, L. Yang, X. Fei, N. Jun, M. Guiping, Int. J. Biol. Macromol. 50 (2012) 438–443. [2] M. Kaloti, H.B. Bohidar, Colloids Surf. B 81 (2010) 165–173. [3] C. Muzzarelli, V. Stanic, L. Gobbi, G. Tosi, R.A.A. Muzzarelli, Carbohydr. Polym. 57 (2004) 73–82. [4] M.L. Tsai, R.H. Chen, S.W. Bai, W.Y. Chen, Carbohydr. Polym. 84 (2011) 756–761. [5] J.S. Chang, K.L.B. Chang, M.L. Tsai, J. Appl. Polym. Sci. 105 (2007) 2670–2675. [6] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632. [7] M.L. Tsai, S.W. Bai, R.H. Chen, Carbohydr. Polym. 71 (2008) 448–457. [8] M.X. Jin, Q.H. Hu, Centr. South Pharm. 6 (003) (2008) 324–327. [9] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, J. Controlled Release 100 (2004) 5–28. [10] A. Vila, A. Sanchez, K. Janes, I. Behrens, T. Kissel, J.L. Vila Jato, M.J. Alonso, Eur. J. Pharm. Biopharm. 57 (2004) 123–131. [11] X.B. Yuan, H. Li, Y.B. Yuan, Carbohydr. Polym. 65 (2006) 337–345. [12] J. Cao, N.J. Zhou, Chin. J. Biochem. Pharm. 26 (2) (2005) 127–128. [13] Y. Maeda, Y. Kimura, J. Nutr. 134 (4) (2004) 945–950. [14] S.H. Zhou, Y. Hong, G.J. Fang, J. Clin. Rehab. Tiss. Eng. Res. 11 (48) (2007) 9688–9691. [15] G.J. Fang, Y. Hong, Y.Y. Jiang, J. Clin. Rehab. Tiss. Eng. Res. 11 (48) (2007) 9696–9699. [16] T. Torzsas, C. Kendall, M. Sugano, Y. Iwamoto, A. Rao, Food Chem. Toxicol. 34 (1) (1996) 73–77. [17] Y. Afrouz, E. Farnaz, R. Sima, A. Fatemeh, D. Rassoul, Sci. Pharm. 77 (2009) 453–464. [18] S.B. Horwitz, Trends Pharmacol. Sci. 13 (1992) 134–136. [19] E. Lee, H. Kim, I.H. Lee, S. Jon, J. Controlled Release 140 (2009) 79–85. [20] M. Zhao, M. Su, X. Lin, Y. Luo, H. He, C. Cai, X. Tang, Pharm. Res. 27 (2010) 1687–1702. [21] J. Zhi, Y. Wang, G. Luo, React. Funct. Polym. 65 (2005) 249–257. [22] V. Sanna, A.M. Roggio, A.M. Posadino, A. Cosssu, S. Marceddu, A. Mariani, V. Alzari, S. Uzzau, G. Pintus, M. Sechi, Nanoscale Res. Lett. 61 (2011) 1–9. [23] C. Fonseca, S. Simoes, R. Gaspar, J. Controlled Release 83 (2002) 273–286. [24] A.S. Aly, Angew. Makromol. Chem. 259 (1998) 13–18.

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Docetaxel loaded chitosan nanoparticles: formulation, characterization and cytotoxicity studies.

The primary objective of the present investigation was to explore biodegradable chitosan as a polymeric material for formulating docetaxel nanoparticl...
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