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Fabrication, characterization and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles
Accepted Manuscript Title: Fabrication, characterization and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles Author: Ankit Jain Kanika Thakur Gajanand Sharma Preeti Kush Upendra K. Jain PII: DOI: Reference:
S0144-8617(15)00992-3 http://dx.doi.org/doi:10.1016/j.carbpol.2015.10.012 CARP 10429
To appear in: Received date: Revised date: Accepted date:
21-7-2015 1-10-2015 4-10-2015
Please cite this article as: Jain, A., Thakur, K., Sharma, G., Kush, P., and Jain, U. K.,Fabrication, characterization and cytotoxicity studies of ionically crosslinked docetaxel loaded chitosan nanoparticles, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.10.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Ionically cross-linked DTX-CH-NP prepared by Ionotropic gelation Influence of fabrication conditions on micromeritics investigated
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Spherically shaped nanoparticles with an average size of 159.2-220.7 nm
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Nanoparticles released 78-82% of drug following Korsmeyer-Peppas kinetics
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An increase of 25% MDA-MB-231 cell line growth inhibition by nanoparticles
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*Manuscript
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Fabrication, characterization and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles
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Ankit Jain1, Kanika Thakur 2, Gajanand Sharma2, Preeti Kush1, Upendra K. Jain1 1
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Department of Pharmaceutics, Chandigarh College of Pharmacy,
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University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh, India 160014
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Mohali-140110, India
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*Address for correspondence:
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Ankit Jain
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Department of Pharmaceutics
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Chandigarh College of Pharmacy, Mohali-140110, India
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E-mail address: [email protected]
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Tel: +91-09466831003
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Abstract
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The present investigation aimed at the fabrication and characterization of ionically cross-
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linked docetaxel (DTX) loaded chitosan nanoparticles (DTX-CH-NP) using ionic gelation
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technique with sodium tripolyphosphate (TPP) as the cross-linking agent. The formulated
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nanoparticles were characterized in terms of particle size, drug entrapment efficiency (EE),
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scanning electron microscopy (SEM), in vitro release and cytotoxicity studies. Formulation
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factors (chitosan, TPP and drug concentration) were examined systematically for their effects
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on size of the nanoparticles. The average size of the nanoparticles was observed to be in the
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range of 159.2 ± 3.31 to 220.7 ± 2.23 nm with 78-92% encapsulation efficiency (EE). The in
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vitro cytotoxicity studies on breast cancer cell lines (MDA-MB-231) revealed the advantages
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of DTX-CH-NP over pure DTX with approximately 85% cell viability reduction. The results
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indicate that systematic modulation of the surface charge and particle size of ionically cross-
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linked nanoparticles can be readily achieved with the right control of critical processing
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parameters. Thus, DTX-CH-NP presents a promising delivery alternative for breast cancer
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treatment.
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Keywords: Chitosan, Docetaxel, Tripolyphosphate, Nanoparticles, Cross-linking
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1. Introduction
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Cancer is a growing menace, with the number of patients increasing at an alarming rate.
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Chemotherapy as a treatment option has been successful, but to some extent in cancer
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treatment. The main drawbacks of chemotherapy include the limited accessibility of drugs to
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the tumor tissues, high toxicity, development of multiple drug resistance and non-specific
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targeting (Parveen & Sahoo, 2008). Nanoparticles (NPs), an evolution of nanotechnology,
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have demonstrated great potential in successfully addressing the problems related to
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chemotherapeutic drug delivery by providing high drug payload, improvement in the
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biodistribution of drugs and ability to target tumors with an enhanced accumulation (Naahidi
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et al., 2008; Danhier, Feron & Preat, 2010; Bertrand et al., 2014; Ferrari, 2005).
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In the recent decades, polymeric nanoparticles have witnessed an exceptional growth and
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usage in anti-cancer drug delivery. The high surface-to-volume ratio of polymeric
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nanoparticles improves the loading capacity of the selected molecule, while providing it
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therequired protection (Rodrigues, Rosa da Costa & Grenha, 2012; Peer et al., 2007; Prabhu,
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Patravale & Joshi, 2015). These particles also help in enhancement of the therapeutic efficacy
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of anticancer drugs by regulating their release, improvement in stability and prolonged
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circulation time. A number of studies have reported that nano-sized drug carriers composed
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of natural and synthetic polymers sustain in the body for prolonged periods by evading the
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reticulo-endothelial system (RES) (Hwang, Kim, Kwon & Kim, 2008). Polymeric
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nanoparticles accumulate in the tumor tissue, resulting in a disorganized vascular
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architecture, referred to as the enhanced permeability and retention (EPR) effect (Sultana,
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Khan, Kumar, Kumar, & Ali, 2013; Garcia-Fuentes & Alonso, 2012).
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Chitosan-based polymeric nanoparticles have received great attention in the recent times due
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to their biodegradability, biocompatibility, non-toxicity and low immunogenicity (Dash,
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Chiellini, Ottenbrite & Chiellini, 2011). Chitosan, the N-deacetylated form of chitin, mostly
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found in the exoskeleton of crustaceans, insects, and fungi, is a natural polysaccharide
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(Muzzarelli, Stanic, Gobbi, Tosi & Muzzarelli, 2004; Dutta, Dutta & Tripathi, 2004). It has
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been recognized as a promising polymer for drug delivery, possessing high density of
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positive charge, attributed to the presence of glucosamine group on its backbone (Cooney,
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Petermann, Lau & Minteer, 2009; Jee at al., 2012). Chitosan acts on tumor cells to interfere
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with the cell metabolism by inhibiting cell growth or inducing cell apoptosis (Cao & Zhou,
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2005). Previous studies have reported that low-molecular weight and modified chitosan could
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inhibit tumor growth leading to good prospects for their application in cancer therapy (Maeda
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& Kimura, 2004). The strong mucoadhesive interactions of chitosan with the mucous
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membranes associated with tumors, makes it capable for efficient anticancer drug delivery
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(Zhou, Hong & Fang, 2007). Chitosan contains abundant amino and hydroxyl groups, thus
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enabling nanoparticulate formulation via both physical and chemical cross-linking (Makhlof,
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Tozuka & Takeuchi, 2011). Ionic cross-linking of chitosan with negatively charged
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multivalent ions such as tripolyphosphate (TPP) is a typical non-covalent interaction (Tsai,
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Chen, Bai & Chen, 2011). Under acidic conditions, the amino group of chitosan molecule
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protonizes to –NH3+ which ultimately, interacts with an anion such as tripolyphosphate (TPP)
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to form nanoparticles (Tsai, Bai & Chen, 2008). The reversible physical cross-linking by
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electrostatic interaction is a very simple method and prevents possible toxicity of reagents
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and other undesirable side effects (Ringel & Horwtiz, 1991).
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Docetaxel (DTX), a second-generation semi-synthetic taxane derivative is effective against a
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variety of solid tumors including breast, ovarian, prostate and non-small cell lung cancer
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(Lyseng-Williamson & Fenton, 2005; Bissery, Nohynek, Sanderink & Lavelle, 1995). The
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clinical applications of DTX are limited due to its poor aqueous solubility, rapid phagocytic
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activity, renal clearance and non-selective distribution (Zhao et al., 2010). Since, DTX is
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highly lipophilic and practically insoluble in water, the main marketed product of DTX
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(Taxotere®) used clinically is formulated using Tween 80 (Zhang & Zhang, 2013). Tween 80
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tends to alter the membrane fluidity, resulting in an increase of membrane permeability
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associated with serious hypersensitivity reactions and cumulative-fluid retention (Dou,
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Zhang, Liu, Zhang & Zhai, 2014). A number of drug delivery systems have been reported for
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DTX such as micelles, liposomes, self-emulsified DTX formulations (Yang, Li, Wang, Dong
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& Qi, 2014), PEGylated immunoliposomes (Zhao et al., 2009) and PEG-liposomes-folic acid
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bioconjugates (Song et al., 2011), lipid emulsified nanoparticles (Zhang et al., 2015), folate
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decorated human serum albumin nanoparticles (Jiang, Gong, Zhag & Zu, 2015),
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hypersensitive and biodegradable nanoparticles (Wu et al., 2015; Liu et al., 2012), and
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diblock copolymer nanoparticles (Tao et al., 2013) . All these delivery systems are limited by
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the complicated preparative procedures, high cost and low stability of the formulations.
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In the present study, ionically cross-linked docetaxel loaded chitosan nanoparticles (DTX-
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CH-NP) were formulated by ionotropic gelation method. Chitosan was cross-linked with
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tripolyphosphate (TPP) and the influence of a number of formulation parameters like chitosan
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and TPP concentration, chitosan-TPP volume ratio, pH and temperature of chitosan solution
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on the fabrication process were investigated systematically. The nanoparticles were
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characterized in terms of particle size, zeta potential, polydispersity index, drug entrapment
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efficiency (EE), loading capacity (LC), transmission electron microscopy (TEM), scanning
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electron microscopy (SEM), in vitro release and cytotoxicity screening, stability studies and
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drug release kinetics.
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2. MATERIALS AND METHODS
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2.1. Materials
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Docetaxel was purchased from Sigma-Aldrich (USA). Chitosan (degree of deacetylation-
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80%, molecular weight 40-80 kDa) was procured from Himedia Pvt. Ltd (Mumbai, India).
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Sodium Tripolyphosphate (TPP) and glacial acetic acid were procured from Loba chemie
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Pvt. Ltd (Mumbai, India). All other chemicals and reagents used in the study were of
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analytical grade.
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2.2. Preparation of ionically cross-linked Docetaxel loaded Chitosan nanoparticles (DTX-
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CH-NP)
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Blank ionically cross-linked chitosan nanoparticles were prepared by the ionic gelation of
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chitosan with TPP anions as described by Calvo et al. (Calvo, Remu˜nán-López, Vila-Jato &
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Alonso, 1997; Vila et al., 2004). Briefly, chitosan was dissolved in 1% acetic acid solution
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(0.2-1%, w/v) at room temperature under sonication. The pH of the resulting solution was
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adjusted to 3-6 using 20% w/v sodium hydroxide solution and passed through a syringe filter
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(pore size 0.45 μm, Millipore, USA) to remove insoluble particles. Sodium TPP was
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dissolved in double distilled water at a concentration of (0.25-1.25% w/v) and also passed
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through a syringe filter (pore size 0.22 μm, Millipore, USA). Blank nanoparticles were
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prepared upon the dropwise addition of TPP solution (0.25-1.25%, w/v) to chitosan solution,
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kept under stirring at room temperature. Fig. 1 depicts the procedure for the preparation of
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DTX-CH-NP by ionotropic gelation method. For the preparation of DTX-CH-NP, various
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concentrations of DTX (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) in TPP solution (firstly drug was
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dissolved in 1ml of methanol) were prepared. Nanoparticles were formed by adding this
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solution dropwise into chitosan solution. The nanoparticle suspension was continuously
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stirred for 1 h and centrifuged at 16,000 rpm for 30 min. (Cooling centrifuge C-24BL, Remi
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Instruments, Mumbai, India). The pellet obtained was further redispersed in 10 ml of
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Phosphate buffer saline (pH 7.4). Mannitol (2%, w/v) was added as a cryoprotectant and
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freezed at −80ºC for 4 h followed by lyophilization in laboratory model freeze dryer (Alpha
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2–4 LD Plus, Martin Christ, Germany) for 24 h at −48ºC and 0.0010 mbar.
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2.3. Characterization of ionically cross-linked Docetaxel loaded Chitosan nanoparticles
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(DTX-CH-NP)
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2.3.1. Micromeritics and zeta potential
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The mean particle size, size distribution and zeta potential of DTX-CH-NP was analyzed by
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photon correlation spectroscopy using the Zetasizer Nano ZS (Malvern Instruments, Malvern,
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UK). All the measurements were carried out in triplicates at 25ºC. The nanoparticle
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suspension was diluted ten times with deionized water and the analysis was performed at a
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scattering angle of 90º.
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2.3.2. Entrapment efficiency and loading capacity
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The entrapment efficiency (%) of docetaxel in DTX-CH-NP was determined by separating
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DTX containing supernatant from nanoparticles by centrifugation at 15000 rpm for 40 min.
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(C-24BL, Remi Instruments, Mumbai, India). The clear supernatant was analyzed for the
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contents of docetaxel by measuring the absorbance in a UV-Visible spectrophotometer
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(Shimadzu UV spectrophotometer, Japan) at 230 nm. All the samples were measured in
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triplicates. The percentage entrapment efficiency and loading capacity were calculated as
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follows:
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×100………………………….. (1)
EE (%) LC (%)
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×100…………… (2)
Where DTXt is the total amount of docetaxel used in the preparation of DTX-CH-NP and
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DTXf is the free docetaxel present in the supernatant.
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2.3.3. Morphological characterization of nanoparticles
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The shape and surface morphology of DTX-CH-NP was determined by scanning electron
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microscopy (Jeol, JSM-6100, Japan). The lyophilized samples were mounted on an
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aluminium stub using a double adhesive carbon tape. The photomicrographs were recorded at
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an accelerating voltage of 10kV at different magnifications.
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2.3.4. Transmission electron microscopy (TEM)
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The topography of nanoparticles was determined using transmission electron microscopy
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(JEOL, JEM-1230, Japan Ltd.). The samples were negatively stained with freshly prepared
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phosphotungstic acid solution (2%, pH 6.8).
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2.3.5. Fourier transform infrared spectroscopy (FT-IR)
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DTX-CH-NP samples were subjected to FT-IR spectroscopy in a Fourier-transform infrared
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spectrophotometer (IR Affinity, Shimadzu, Japan) in the range of 4000–500 cm-1. The freeze-
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dried nanoparticles were mixed with KBr and pressed to a pellet to investigate the chemical
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interactions between the drug and polymer matrix.
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2.3.6. In vitro drug release
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The in vitro release of docetaxel from the various batches of DTX-CH-NP was carried out by
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dialysis sac method. An accurately weighed quantity of about 20 mg of nanoparticles was
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suspended in 3.0 ml of release medium (Phosphate buffer saline solution, pH 7.4) in the
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dialysis tubing (cut off 10,000 kDa). The dialysis tube was tied to the paddle of USP type II
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dissolution apparatus (TDL–08L, Electrolab, India) and immersed in 250 ml of dissolution
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medium (Phosphate buffer, pH 7.4). The dissolution media was maintained at 37ºC ± 0.5ºC
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with continuous stirring at 50 rpm. An aliquot of 5 ml sample was withdrawn at various time
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intervals and replenished with equal volumes of fresh media. The content of DTX in the
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samples was determined spectrophotometrically by measuring the absorbance at 230 nm in a
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UV-Visible Spectrophotometer (Shimadzu UV spectrophotometer, Japan).
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2.3.7. In vitro Cytotoxicity Screening
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MTT assay using breast cancer cell lines (MDA-MB-231) was employed to determine the
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cell viability. The cell lines were seeded in 96-well plates at the density of 150,000 viable
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cells per well and incubated for 24 hours to allow cell attachment at 37ºC in 5% CO2
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humidified incubator (Sanna et al., 2011). The cells were then incubated for another 24 hours
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with blank nanoparticles (NP), ionically cross-linked docetaxel loaded chitosan nanoparticles
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(DTX-CH-NP) and the pure drug (DTX) at 0.05, 0.5, 5.0 μg/ml equivalent concentrations for
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24, 48 and 72 h, respectively. These concentrations correspond to plasma levels of the drug
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which can be achieved in humans (Fonseca, Simoes & Gaspar, 2002). The medium was
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removed, at specific time intervals and the wells were washed twice with phosphate buffer
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solution and 10 μL of 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide
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(MTT) solution was added to each well of the plate and incubated for another 1 to 4 h.
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Following media removal, the MTT-formazan formed by metabolically viable cells was
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dissolved in 200 μL of Diemthyl sulphoxide (DMSO) and after 4 h, the plates were observed
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at 560 nm in a microplate reader. % Cell viability was calculated by the following equation:
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Cell viability (%)
×100…………… (3)
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Where Abs sample is the absorbance of the solution incubated with the ionically cross linked
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nanoparticles
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Abs control is the absorbance of the solution incubated with the culture medium only
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(positive control).
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2.3.8. Stability studies
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DTX-CH-NP were evaluated for the change in particle size and entrapment efficiency i.e.
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aggregation over the period of 3 months. All the formulations were kept at a temperature of
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4°C for a period of 3 months.
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2.3.9. Statistical analysis
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The results were presented as mean ± standard deviation. Statistical analysis was performed
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with one-way ANOVA and statistical significance was designated as p < 0.05.
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3. Results and discussion
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3.1. Particle size, size distribution and zeta potential of nanoparticles
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Based on an optimization procedure designed, the effect of formulation parameters (chitosan,
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TPP and drug concentration, chitosan to TPP volume ratio, temperature and pH of chitosan
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solution) was investigated systematically by changing one parameter while keeping the others
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constant. On the basis of preliminary trials, the parameters were varied as follows:
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concentration of chitosan (0.2–1%, w/v), concentration of TPP (0.25–1.25%, w/v), chitosan-
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TPP volume ratio (3:1-7:1), stirring speed (800-1600 rpm), temperature (35ºC - 65ºC) and pH
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(3.5-6) of chitosan solution, respectively. The effect of each of these parameters is discussed
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as follows:
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3.1.1. Effect of TPP (cross-linker) concentration
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TPP has five negative charge groups that interact with the positive amino groups of chitosan
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in acetic acid solution (Hosseinzadeh, Atyabi, Dinarvand & Ostad, 2012).The concentration
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of chitosan was fixed at (0.2%, w/v) and TPP concentration was varied from (0.25–1.25%,
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w/v). Fig. 2(a) represents a comparative effect of TPP concentration on particle size,
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polydispersity index and zeta potential. It was observed that the initial increase in TPP
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concentration (0.75%, w/v) led to a significant reduction in particle size. A high TPP
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concentration resulted in high degree of cross-linking of chitosan molecules, thus compact
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particle structures were formed. Further, increase in TPP concentration beyond (0.75%, w/v)
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led to a significant enhancement in the particle size. The increase in the number of anionic
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groups led to an increase in the electrostatic interaction with positive amino sites on chitosan
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molecule, thereby leading to reduction of the surface charge and increase in particle size. Zeta
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potential is an important parameter which influences the stability of the nanoparticles through
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electrostatic repulsion (Gang & Wang, 2007; Ajun, Yan, Li & Huili, 2009). The positive zeta
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potential is attributed to the presence of amino groups and further, neutralization of charged
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amino groups led to a decrease in the overall zeta potential. TPP concentration higher than
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(1.25%, w/v) formed aggregated solution, giving a gel-like consistency. The concentration of
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TPP had little effect on the polydispersity of particles since the polydispersity index (PDI)
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value was below 0.5 indicating uniformity of particle size.
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3.1.2. Effect of chitosan concentration
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Chitosan has the property of protonization under acidic conditions due to which it undergoes
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cross-linking with anions like Tripolyphosphate. TPP concentration was fixed at (0.75%,
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w/v) and chitosan concentration was varied from (0.2-1%, w/v). Fig. 2(b) represents a
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comparative effect of chitosan concentration on particle size, polydispersity index and zeta
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potential. The increase in size and zeta potential of the nanoparticles with increase in chitosan
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concentration from (0.2-1 mg/ml) may be attributed to the linear increase in viscosity of
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chitosan solution with increasing concentration. The high viscosity prevents effective ionic
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interaction between TPP and chitosan solution (Berger et al., 2004). The electrostatic
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repulsions and interchain hydrogen bonding interactions between chitosan molecules remain
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in equilibrium upto the concentration of 2 mg/ml leading to the formation of large particle
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sizes (Gana, Wang, Cochrane & McCarron, 2005).The zeta potential of the nanoparticles
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increased linearly due to a more protonized –NH3+ ion on the surface of nanoparticles formed
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with higher chitosan concentration. The PDI of the nanoparticles was found to be favorable
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i.e. below 0.5.
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3.1.3. Effect of chitosan-TPP volume ratio
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The effect of chitosan-TPP volume ratio on the particle size, polydispersity index and zeta
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potential was very significant. TPP concentration was fixed at (0.75%, w/v) and chitosan
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concentration was fixed at (0.2%, w/v). Fig. 2(c) represents a comparative effect of chitosan-
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TPP volume ratio on particle size, polydispersity index and zeta potential. The results
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indicate that, with increasing chitosan volume (3:1 to 5:1) in the solution, the particle size
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decreased from 295.3 nm to 159.4 nm and then increased to 502.1 nm at ratio 7:1. When
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chitosan-TPP volume ratio was below 3:1, the volume of chitosan solution was not sufficient
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for the formation of a cross-linked structure with TPP. With an increase in volume ratio from
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3:1 to 5:1, the particle size decreased due to increased cross-linking density between chitosan
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and TPP. As its volume ratio increased from 5:1 to 7:1, though the molecules were cross-
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linked, the excess of chitosan resulted in the formation of larger-sized nanoparticles. The zeta
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potential of chitosan–TPP nanoparticles showed a linear relationship to chitosan-TPP volume
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ratio from 3:1 to 5:1. The PDI values were all below 0.5 and least value of PDI (0.267) was
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seen at 5:1 volume ratio of chitosan-TPP.
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3.1.4. Effect of temperature of chitosan solution
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Fig. 2(d) shows the comparative effect of temperature of chitosan solution on the particle
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size, polydispersity index and zeta potential. The particle size decreased from 489.4 nm to
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160.1 nm with increase in temperature of the chitosan solution (35 ºC-65ºC) due to decrease
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in intrinsic viscosity. The increase in temperature reduced the water of hydration of chitosan
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molecules, resulting in an increase in chitosan chain flexibility (Chen & Tsaih, 1998). As a
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result, chitosan molecules approached each other with greater force resulting in the formation
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of compactly cross-linked nanoparticles. The decrease in particle size is negligible at a
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temperature above 65ºC. A decrease in zeta potential was also observed and the resulting PDI
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values were all below 0.5.
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3.1.5. Effect of pH of chitosan solution
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Chitosan is a weak base polyelectrolyte insoluble at neutral and alkaline pH (Mi, Sung &
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Shyu, 2003).The amine groups are positively charged in an acidic medium and hence, the
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surface charge density of chitosan molecules depends on the solution pH (Leong, Mao &
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Truong-Le, 1998; Ko, Park & Hwang, 2002). As the ionic cross-linking process for the
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formation of chitosan–TPP nanoparticles is pH-dependent, the effect of changing pH of
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chitosan solution on particle size, zeta potential and polydispersity index was investigated
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keeping the concentration of chitosan (0.2%, w/v), TPP (0.75%, w/v) and chitosan-TPP
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volume ratio (5:1) fixed. Fig.2 (e) represents a comparative effect of pH of chitosan solution
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on particle size, polydispersity index and zeta potential.
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At a low pH (3.5), the amino groups of chitosan and TPP molecule are protonated, leading to
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a lower charge density of the molecules. This causes an insufficient cross-linking and hence
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larger size of the particles. With an increase in pH, the deprotonation degree of TPP
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increases, while the protonation degree of chitosan is less affected. Hence, the particle size
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decreases significantly at pH 5.5. The sharp increase in size at pH 6.0 indicates that the
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degree of protonization at surface of the particles was reduced leading to particle aggregation.
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The same decreasing pattern was also shown in zeta potential at pH 5.5 and increase at pH 6.
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3.1.6. Effect of the stirring speed
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Fig. 2(f) represents a comparative effect of stirring speed on particle size, polydispersity
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index and zeta potential. The results indicate that the particle size was significantly reduced
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by increasing stirring speed from 800 rpm to 1400 rpm. There was slight decrease in zeta
319
potential and no significant effect on PDI was observed.
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3.1.7. Effect of the drug concentration
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DTX-CH-NPs were prepared by the addition of DTX in 0.75% w/v TPP into 0.2% w/v of
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chitosan solution. Fig. 2(g) represents a comparative effect of drug concentration on particle
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size, polydispersity index and zeta potential. The results indicate that the addition of DTX led
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to an increase in the size of chitosan nanoparticles. In general, the size of DTX-CH-NP did
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not grow significantly at concentration up to 0.6 mg/ml, but there was a change in size when
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the concentration of the drug was increased from 0.6 to 1 mg/ml. The drug concentration did
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not influence the zeta potential of the prepared nanoparticles significantly.
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Table 1 depicts the effect of different concentrations of DTX on the %EE and %LC of DTX-
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CH-NP. A maximum EE (78%) was achieved at 0.6 mg/ml of DTX concentration. Initially,
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%EE showed an increase with increase in DTX concentration up to 0.6 mg/ml. From 0.6- 1
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mg/ml, the %EE decreased from 78% to 73%. The %LC of DTX-CH-NP was in the range of
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9-12%. A high loading capacity was obtained due to the hydrophobicity of DTX. The %LC
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increased as a function of initial DTX content. The increase in %LC as a function of increase
334
in drug content corresponds to the previous literature reports such as loading of bovine serum
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albumin (Jiang, Gong, Zhag & Zu, 2015) and ammonium glycyrrhizinate (Wu et al., 2005)
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into chitosan–TPP nanoparticles. The results showed that 0.6 mg DTX in the formulation
337
resulted in the best % EE and %LC. Further increasing the DTX amount did not significantly
338
affect %LC.
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3.2. Morphological characterization of nanoparticles
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The morphological characteristics of DTX-CH-NP were imaged using SEM and TEM. Fig.
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3(a-b) displays the scanning electron micrographs of DTX-CH-NP showing their surface
342
morphology while Fig. 3(c) depicts the transmission electron micrograph. The nanoparticles
343
showed a dense and spherical structure with uniformity in size.
344
3.3. Fourier Transform infrared spectroscopy (FT-IR)
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FT-IR spectroscopy was used to study the drug–polymer interactions. Fig. 4 shows the FT-IR
346
spectra of DTX, DTX-Chitosan physical mixture (50:50), DTX-TPP physical mixture (50:50)
347
and DTX-CH-NP. The spectra of DTX shows characteristic peaks at 3481.84 cm-1 which may
348
be ascribed to N-H stretching of alkanes. Peaks at 1740 cm-1 and 1711 cm-1 may be due to C-
349
O stretching and at 1631.97 cm-1 can be due to N-H plane bending. The spectra of DTX-
350
Chitosan and DTX-TPP indicated the absence of any drug-excipient interaction between the
351
drug and polymer as the spectra of the drug remained intact without any changes in peaks.
352
The peak at 1651 cm-1 of DTX-chitosan spectra disappeared in the spectra of DTX-CH-NP
353
and two new peaks at 1643 cm-1 and 1543 cm-1 appeared. The disappearance of the peak
354
could be attributed to the linkage between the phosphoric and ammonium ions. The cross-
355
linked chitosan nanoparticles showed a peak for P = O at 1157 cm-1. All recorded spectra
356
show that the peaks of docetaxel remain unaffected indicating that the drug remained intact
357
during the preparation procedure of nanoparticles. Thus, FT-IR analysis indicated absence of
358
any drug excipient interaction between the drug and polymer during the formulation of
359
nanoparticles.
360
3.5. In vitro drug release
361
The cumulative percent drug release of DTX from different formulations of DTX-CH-NP
362
varied from 77.46 ± 1.17% and 73.79 ± 0.79% respectively over a period of 24 h as shown in
363
Fig. 5. The release profiles of the nanoparticles are similar, and exhibit an initial burst release
364
followed by a slow and sustained release due to the release of drug from the matrix. The
365
initial rapid release was mainly due to the drug present at the surface of the nanoparticles
366
which diffuses in the release medium in the initial time. To determine the release kinetics,
367
the release data was fitted into various kinetic models. Table 2 represents the release kinetics
368
of various batches of ionically cross-linked docetaxel loaded chitosan nanoparticles and the
369
co-efficient of correlation (R2) of zero order, first order, Korsmeyer–Peppas model and
370
Higuchi’s square root model was determined. Korsmeyer–Peppas model was observed to be
371
the best fit model indicating Fickian diffusion as the mechanism of drug release from
372
nanoparticles. Further, the value of ‘n’ the release exponent of Korsmeyer–Peppas (0.45≤ n ≤
373
0.89) indicates that nanoparticles released the drug by combination of both diffusion of drug
374
through the polymer and dissolution of the polymer (Costa & Lobo, 2001; Siepmann &
375
Peppas, 2001; Higuchi, 1963).
376
3.6. In vitro Cytotoxicity Screening
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The in vitro cytotoxicity screening of docetaxel as a free drug and ionically cross-linked
378
docetaxel loaded chitosan nanoparticles was determined by MTT assay, which is a
379
colorimetric test based on the selective ability of viable cells to reduce the tetrazolium
380
component of MTT into purple colored formazan crystals. MTT assay was performed for the
381
three different concentrations i.e., 0.05, 0.5 and 5.0 μg/ml using MDA-MB-231 as a model
382
breast cancer cell line.
383
Untreated MDA-MB-231 cells as well as cells treated with blank cross-linked chitosan
384
nanoparticles, with the same polymer content were used as control. Fig.6 (a-c) shows the
385
viability of MDA-MB-231 cancer cells, cultured with blank cross-linked chitosan
386
nanoparticles (Blank-NPs) and ionically cross-linked docetaxel loaded chitosan nanoparticles
387
[DTX-CH-NP3] after incubation for 24 h (a), 48 h (b) and 72 h (c), in comparison with that
388
of pure drug [DTX]. The biocompatibility of chitosan was confirmed by the negligible
389
toxicity of blank chitosan nanoparticles [NPs]. After 24 h incubation, as depicted in Fig. 6(a),
390
the cell viability decreased to about 59.22, 47.12 and 38.45% for DTX-CH-NP3 at 0.05, 0.5
391
and 5.0 μg/ml drug concentrations respectively, corresponding to an increase in cytotoxicity
392
of 25% compared with that of free drug. After 48 h incubation, portrayed in Fig. 6(b), the
393
cytotoxicity increased to about 41.34, 35.12 and 25.31% for the DTX-CH-NP3 and 65.88,
394
58.98 and 48.87% for the free drug, respectively. The more marked inhibition of cell growth
395
was obtained for longer incubation period (72 h). The strongest cytotoxic effect was achieved
396
with nanoparticles at 5.0 μg/ml drug concentration.
397
The storage stability of chitosan nanoparticles cross-linked with TPP is an important
398
parameter. Particles in the colloidal dispersion may adhere to one another and form
399
aggregates of increasing size. The physical stability was investigated at 4ºC over a period of 3
400
months. A special emphasis was placed on the changes in particle size and entrapment
401
efficiency which are the two important physicochemical parameters of nanoparticles. The
402
nanoparticles were found to be stable with no major changes except a slight increase in
403
particle size and a slight decrease in entrapment efficiency and drug loading.
404
4. Conclusion
405
The present work is an effort to explore ionically cross-linked docetaxel loaded chitosan
406
nanoparticles for breast cancer treatment in order to improve the therapeutic efficacy and
407
minimize severe toxicity associated with the clinical usage of docetaxel. TPP was
408
successfully employed as a cross-linking agent to formulate nanoparticles by ionotropic
409
gelation. The influence of a number of fabrication parameters was systematically investigated
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13 Page 14 of 32
and the concentrations of TPP, chitosan, chitosan-TPP volume ratio were found to have a
411
profound impact on the physicochemical characteristics of nanoparticles. The nanoparticles
412
exhibited particle size in the range of 159.2 ± 3.31 nm to 220.7 ± 223 nm with a sustained
413
drug release pattern. The in vitro cytotoxicity screening studies demonstrated a 25% increase
414
in cytotoxicity compared with the free drug against MDA-MB-231 cancer cell lines. Thus,
415
the present study unraveled the effect of fabrication parameters on the ionic cross-linking
416
process. The results of this investigation clearly demonstrate that the ionically cross-linked
417
docetaxel loaded nanoparticles seems to have a great potential as a drug carrier in cancer
418
chemotherapy. The present research work offers immense scope for further exploitation of
419
chitosan in future for the development of nanoparticulate drug delivery system for cancer
420
chemotherapy.
421
Acknowledgements
422
The authors are grateful to Central Instrumentation Laboratory, Panjab University,
423
Chandigarh for particle size and SEM analysis and also gratified to the Management of
424
Chandigarh College of Pharmacy, Mohali (Punjab), India for financial support and providing
425
the facilities to carry out the research work.
426
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Table(s)
List of Tables Table 1 Characterization of ionically cross-linked docetaxel loaded chitosan nanoparticles by varying drug concentration
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Table 2 Release kinetics and stability data of different formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles
Page 20 of 32
Characterization of ionically cross-linked docetaxel loaded chitosan nanoparticles by varying drug concentration Zeta potential (mV)
Blank
˗˗˗
159.2 ± 3.31
0.278 ± 0.029
33.2 ± 1.66
DTX-CH-NP1
0.2
163.2 ± 3.43
0.259 ± 0.034
32.7 ± 1.12
DTX-CH-NP2
0.4
170.9 ± 4.58
0.247 ± 0.041
31.9 ± 2.11
DTX-CH-NP3
0.6
175.2 ± 3.43
0.229 ± 0.027
DTX-CH-NP4
0.8
204.1 ± 6.09
0.287 ± 0.043
DTX-CH-NP5
1
220.7± 2.23
0.345± 0.036
Entrapment Efficiency (%)
Loading Capacity (%)
˗˗˗
˗˗˗
69.37 ± 0.74
9.94 ± 0.81
cr
(nm)
Polydispersity Index (PDI)
75.11 ± 1.02
11.26 ± 0.79
32.3 ± 1.67
78.28 ± 0.91
12.01 ± 0.92
29.8 ± 1.96
70.41 ± 1.35
10.56 ± 0.99
73.23± 1.42
10.21± 0.65
us
Particle size
an
DTX concentration (mg/ml)
Formulation code
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Table 1:
34.8± 3.40
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M
Mean ± S.D., n =3. Note: DTX concentration: Concentration of Docetaxel.
Page 21 of 32
Table 2: Release kinetics and stability data of different formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles Release kinetics
Zero order
First order
Higuchi’s square- root
Korsmeyer-Peppas
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Formulation Code
-1
k
R2
-1
(% h )
R2
(h )
k (%h
0.6452
0.0479
0.7859
0.0009
0.8946
DTX-CH-NP2
0.6277
0.0519
0.7767
0.0011
0.8849
DTX-CH-NP3
0.6962
0.0557
0.8639
0.0013
0.9254
DTX-CH-NP4
0.6206
0.0436
0.7862
0.0009
0.8779
DTX-CH-NP5
0.7025
0.0407
0.8342
0.0006
R2 )
2.1451
M
0.9188
k
(%h-n)
0.9439
n
0.2348
0.5880
2.3431
0.9210
0.2948
0.5781
2.4480
0.9114
0.3268
0.5982
2.2568
0.9598
0.3932
0.5437
1.9710
0.9475
0.2785
an
DTX-CH-NP1
-0.5
cr
k
us
R2
0.5165
Stability Data
Entrapment Efficiency (%)
d
4±2ºC
1 month
2 months
3 months
78.28 ± 0.91
77.4 ± 0.3
76.5 ± 1.8
76.0 ± 3.0
167.2 ± 3.43
169.6 ± 4.3
176.5± 3.2
181.5 ± 3.3
Ac ce p
Size (nm)
Initial
te
Ionically cross-linked Docetaxel loaded chitosan nanoparticles
Page 22 of 32
Figure(s)
Figure captions Fig. 1 Preparation of ionically cross-linked docetaxel loaded chitosan nanoparticles by Ionotropic gelation.
cr
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Fig. 2 Bar graph showing comparative effect of (a) TPP concentration (b) Chitosan concentration (c) Chitosan: TPP volume ratio (d) Temperature of chitosan solution (e) pH of chitosan solution (f) Stirring speed (g) Drug concentration on particle size, polydispersity index and zeta potential of ionically cross-linked docetaxel loaded chitosan nanoparticles.
us
Fig. 3 Scanning electron micrographs showing shape and surface morphology (a-b) and transmission electron micrograph (c) of ionically cross-linked docetaxel loaded chitosan nanoparticles.
an
Fig. 4 Comparative FT-IR spectra of docetaxel, docetaxel-chitosan physical mixture, docetaxelTPP physical mixture and ionically cross-linked docetaxel loaded chitosan nanoparticles.
M
Fig. 5 Release profile of docetaxel from various formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles.
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Fig. 6 % Cell viability of MDA-MB-231 cancer cells after incubation for 24 h (a), 48 h (b) and 72 h (c) with ionically cross-linked docetaxel loaded chitosan nanoparticles.
Page 23 of 32
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Fig. 1
Fig. 2(a)
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Fig. 2(b)
Fig. 2(c)
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d
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Fig. 2(d)
Fig. 2(e)
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d
Fig. 2(f)
Fig. 2(g)
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d
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Fig. 3(a)
Fig. 3(b)
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Fig. 3(c)
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d
Fig. 5
Fig. 6(a)
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Fig. 6(b)
Fig. 6(c)
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S0144-8617(15)00992-3 http://dx.doi.org/doi:10.1016/j.carbpol.2015.10.012 CARP 10429
To appear in: Received date: Revised date: Accepted date:
21-7-2015 1-10-2015 4-10-2015
Please cite this article as: Jain, A., Thakur, K., Sharma, G., Kush, P., and Jain, U. K.,Fabrication, characterization and cytotoxicity studies of ionically crosslinked docetaxel loaded chitosan nanoparticles, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.10.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Ionically cross-linked DTX-CH-NP prepared by Ionotropic gelation Influence of fabrication conditions on micromeritics investigated
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Spherically shaped nanoparticles with an average size of 159.2-220.7 nm
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Nanoparticles released 78-82% of drug following Korsmeyer-Peppas kinetics
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An increase of 25% MDA-MB-231 cell line growth inhibition by nanoparticles
Page 1 of 32
*Manuscript
1 2
Fabrication, characterization and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles
3
Ankit Jain1, Kanika Thakur 2, Gajanand Sharma2, Preeti Kush1, Upendra K. Jain1 1
4
Department of Pharmaceutics, Chandigarh College of Pharmacy,
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University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh, India 160014
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*Address for correspondence:
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Ankit Jain
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Department of Pharmaceutics
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Chandigarh College of Pharmacy, Mohali-140110, India
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E-mail address: [email protected]
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Tel: +91-09466831003
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Abstract
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The present investigation aimed at the fabrication and characterization of ionically cross-
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linked docetaxel (DTX) loaded chitosan nanoparticles (DTX-CH-NP) using ionic gelation
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technique with sodium tripolyphosphate (TPP) as the cross-linking agent. The formulated
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nanoparticles were characterized in terms of particle size, drug entrapment efficiency (EE),
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scanning electron microscopy (SEM), in vitro release and cytotoxicity studies. Formulation
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factors (chitosan, TPP and drug concentration) were examined systematically for their effects
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on size of the nanoparticles. The average size of the nanoparticles was observed to be in the
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range of 159.2 ± 3.31 to 220.7 ± 2.23 nm with 78-92% encapsulation efficiency (EE). The in
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vitro cytotoxicity studies on breast cancer cell lines (MDA-MB-231) revealed the advantages
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of DTX-CH-NP over pure DTX with approximately 85% cell viability reduction. The results
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indicate that systematic modulation of the surface charge and particle size of ionically cross-
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linked nanoparticles can be readily achieved with the right control of critical processing
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parameters. Thus, DTX-CH-NP presents a promising delivery alternative for breast cancer
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treatment.
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Keywords: Chitosan, Docetaxel, Tripolyphosphate, Nanoparticles, Cross-linking
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1. Introduction
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Cancer is a growing menace, with the number of patients increasing at an alarming rate.
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Chemotherapy as a treatment option has been successful, but to some extent in cancer
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treatment. The main drawbacks of chemotherapy include the limited accessibility of drugs to
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the tumor tissues, high toxicity, development of multiple drug resistance and non-specific
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targeting (Parveen & Sahoo, 2008). Nanoparticles (NPs), an evolution of nanotechnology,
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have demonstrated great potential in successfully addressing the problems related to
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chemotherapeutic drug delivery by providing high drug payload, improvement in the
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biodistribution of drugs and ability to target tumors with an enhanced accumulation (Naahidi
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et al., 2008; Danhier, Feron & Preat, 2010; Bertrand et al., 2014; Ferrari, 2005).
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In the recent decades, polymeric nanoparticles have witnessed an exceptional growth and
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usage in anti-cancer drug delivery. The high surface-to-volume ratio of polymeric
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nanoparticles improves the loading capacity of the selected molecule, while providing it
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therequired protection (Rodrigues, Rosa da Costa & Grenha, 2012; Peer et al., 2007; Prabhu,
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Patravale & Joshi, 2015). These particles also help in enhancement of the therapeutic efficacy
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of anticancer drugs by regulating their release, improvement in stability and prolonged
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circulation time. A number of studies have reported that nano-sized drug carriers composed
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of natural and synthetic polymers sustain in the body for prolonged periods by evading the
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reticulo-endothelial system (RES) (Hwang, Kim, Kwon & Kim, 2008). Polymeric
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nanoparticles accumulate in the tumor tissue, resulting in a disorganized vascular
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architecture, referred to as the enhanced permeability and retention (EPR) effect (Sultana,
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Khan, Kumar, Kumar, & Ali, 2013; Garcia-Fuentes & Alonso, 2012).
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Chitosan-based polymeric nanoparticles have received great attention in the recent times due
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to their biodegradability, biocompatibility, non-toxicity and low immunogenicity (Dash,
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Chiellini, Ottenbrite & Chiellini, 2011). Chitosan, the N-deacetylated form of chitin, mostly
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found in the exoskeleton of crustaceans, insects, and fungi, is a natural polysaccharide
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(Muzzarelli, Stanic, Gobbi, Tosi & Muzzarelli, 2004; Dutta, Dutta & Tripathi, 2004). It has
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been recognized as a promising polymer for drug delivery, possessing high density of
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positive charge, attributed to the presence of glucosamine group on its backbone (Cooney,
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Petermann, Lau & Minteer, 2009; Jee at al., 2012). Chitosan acts on tumor cells to interfere
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with the cell metabolism by inhibiting cell growth or inducing cell apoptosis (Cao & Zhou,
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2005). Previous studies have reported that low-molecular weight and modified chitosan could
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inhibit tumor growth leading to good prospects for their application in cancer therapy (Maeda
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& Kimura, 2004). The strong mucoadhesive interactions of chitosan with the mucous
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membranes associated with tumors, makes it capable for efficient anticancer drug delivery
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(Zhou, Hong & Fang, 2007). Chitosan contains abundant amino and hydroxyl groups, thus
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enabling nanoparticulate formulation via both physical and chemical cross-linking (Makhlof,
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Tozuka & Takeuchi, 2011). Ionic cross-linking of chitosan with negatively charged
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multivalent ions such as tripolyphosphate (TPP) is a typical non-covalent interaction (Tsai,
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Chen, Bai & Chen, 2011). Under acidic conditions, the amino group of chitosan molecule
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protonizes to –NH3+ which ultimately, interacts with an anion such as tripolyphosphate (TPP)
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to form nanoparticles (Tsai, Bai & Chen, 2008). The reversible physical cross-linking by
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electrostatic interaction is a very simple method and prevents possible toxicity of reagents
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and other undesirable side effects (Ringel & Horwtiz, 1991).
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Docetaxel (DTX), a second-generation semi-synthetic taxane derivative is effective against a
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variety of solid tumors including breast, ovarian, prostate and non-small cell lung cancer
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(Lyseng-Williamson & Fenton, 2005; Bissery, Nohynek, Sanderink & Lavelle, 1995). The
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clinical applications of DTX are limited due to its poor aqueous solubility, rapid phagocytic
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activity, renal clearance and non-selective distribution (Zhao et al., 2010). Since, DTX is
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highly lipophilic and practically insoluble in water, the main marketed product of DTX
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(Taxotere®) used clinically is formulated using Tween 80 (Zhang & Zhang, 2013). Tween 80
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tends to alter the membrane fluidity, resulting in an increase of membrane permeability
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associated with serious hypersensitivity reactions and cumulative-fluid retention (Dou,
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Zhang, Liu, Zhang & Zhai, 2014). A number of drug delivery systems have been reported for
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DTX such as micelles, liposomes, self-emulsified DTX formulations (Yang, Li, Wang, Dong
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& Qi, 2014), PEGylated immunoliposomes (Zhao et al., 2009) and PEG-liposomes-folic acid
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bioconjugates (Song et al., 2011), lipid emulsified nanoparticles (Zhang et al., 2015), folate
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decorated human serum albumin nanoparticles (Jiang, Gong, Zhag & Zu, 2015),
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hypersensitive and biodegradable nanoparticles (Wu et al., 2015; Liu et al., 2012), and
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diblock copolymer nanoparticles (Tao et al., 2013) . All these delivery systems are limited by
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the complicated preparative procedures, high cost and low stability of the formulations.
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In the present study, ionically cross-linked docetaxel loaded chitosan nanoparticles (DTX-
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CH-NP) were formulated by ionotropic gelation method. Chitosan was cross-linked with
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tripolyphosphate (TPP) and the influence of a number of formulation parameters like chitosan
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and TPP concentration, chitosan-TPP volume ratio, pH and temperature of chitosan solution
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on the fabrication process were investigated systematically. The nanoparticles were
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characterized in terms of particle size, zeta potential, polydispersity index, drug entrapment
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efficiency (EE), loading capacity (LC), transmission electron microscopy (TEM), scanning
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electron microscopy (SEM), in vitro release and cytotoxicity screening, stability studies and
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drug release kinetics.
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2. MATERIALS AND METHODS
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2.1. Materials
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Docetaxel was purchased from Sigma-Aldrich (USA). Chitosan (degree of deacetylation-
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80%, molecular weight 40-80 kDa) was procured from Himedia Pvt. Ltd (Mumbai, India).
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Sodium Tripolyphosphate (TPP) and glacial acetic acid were procured from Loba chemie
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Pvt. Ltd (Mumbai, India). All other chemicals and reagents used in the study were of
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analytical grade.
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2.2. Preparation of ionically cross-linked Docetaxel loaded Chitosan nanoparticles (DTX-
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CH-NP)
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Blank ionically cross-linked chitosan nanoparticles were prepared by the ionic gelation of
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chitosan with TPP anions as described by Calvo et al. (Calvo, Remu˜nán-López, Vila-Jato &
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Alonso, 1997; Vila et al., 2004). Briefly, chitosan was dissolved in 1% acetic acid solution
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(0.2-1%, w/v) at room temperature under sonication. The pH of the resulting solution was
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adjusted to 3-6 using 20% w/v sodium hydroxide solution and passed through a syringe filter
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(pore size 0.45 μm, Millipore, USA) to remove insoluble particles. Sodium TPP was
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dissolved in double distilled water at a concentration of (0.25-1.25% w/v) and also passed
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through a syringe filter (pore size 0.22 μm, Millipore, USA). Blank nanoparticles were
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prepared upon the dropwise addition of TPP solution (0.25-1.25%, w/v) to chitosan solution,
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kept under stirring at room temperature. Fig. 1 depicts the procedure for the preparation of
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DTX-CH-NP by ionotropic gelation method. For the preparation of DTX-CH-NP, various
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concentrations of DTX (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) in TPP solution (firstly drug was
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dissolved in 1ml of methanol) were prepared. Nanoparticles were formed by adding this
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solution dropwise into chitosan solution. The nanoparticle suspension was continuously
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stirred for 1 h and centrifuged at 16,000 rpm for 30 min. (Cooling centrifuge C-24BL, Remi
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Instruments, Mumbai, India). The pellet obtained was further redispersed in 10 ml of
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Phosphate buffer saline (pH 7.4). Mannitol (2%, w/v) was added as a cryoprotectant and
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freezed at −80ºC for 4 h followed by lyophilization in laboratory model freeze dryer (Alpha
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2–4 LD Plus, Martin Christ, Germany) for 24 h at −48ºC and 0.0010 mbar.
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2.3. Characterization of ionically cross-linked Docetaxel loaded Chitosan nanoparticles
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(DTX-CH-NP)
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2.3.1. Micromeritics and zeta potential
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The mean particle size, size distribution and zeta potential of DTX-CH-NP was analyzed by
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photon correlation spectroscopy using the Zetasizer Nano ZS (Malvern Instruments, Malvern,
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UK). All the measurements were carried out in triplicates at 25ºC. The nanoparticle
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suspension was diluted ten times with deionized water and the analysis was performed at a
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scattering angle of 90º.
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2.3.2. Entrapment efficiency and loading capacity
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The entrapment efficiency (%) of docetaxel in DTX-CH-NP was determined by separating
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DTX containing supernatant from nanoparticles by centrifugation at 15000 rpm for 40 min.
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(C-24BL, Remi Instruments, Mumbai, India). The clear supernatant was analyzed for the
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contents of docetaxel by measuring the absorbance in a UV-Visible spectrophotometer
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(Shimadzu UV spectrophotometer, Japan) at 230 nm. All the samples were measured in
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triplicates. The percentage entrapment efficiency and loading capacity were calculated as
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follows:
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×100………………………….. (1)
EE (%) LC (%)
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×100…………… (2)
Where DTXt is the total amount of docetaxel used in the preparation of DTX-CH-NP and
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DTXf is the free docetaxel present in the supernatant.
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2.3.3. Morphological characterization of nanoparticles
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The shape and surface morphology of DTX-CH-NP was determined by scanning electron
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microscopy (Jeol, JSM-6100, Japan). The lyophilized samples were mounted on an
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aluminium stub using a double adhesive carbon tape. The photomicrographs were recorded at
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an accelerating voltage of 10kV at different magnifications.
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2.3.4. Transmission electron microscopy (TEM)
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The topography of nanoparticles was determined using transmission electron microscopy
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(JEOL, JEM-1230, Japan Ltd.). The samples were negatively stained with freshly prepared
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phosphotungstic acid solution (2%, pH 6.8).
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2.3.5. Fourier transform infrared spectroscopy (FT-IR)
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DTX-CH-NP samples were subjected to FT-IR spectroscopy in a Fourier-transform infrared
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spectrophotometer (IR Affinity, Shimadzu, Japan) in the range of 4000–500 cm-1. The freeze-
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dried nanoparticles were mixed with KBr and pressed to a pellet to investigate the chemical
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interactions between the drug and polymer matrix.
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2.3.6. In vitro drug release
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The in vitro release of docetaxel from the various batches of DTX-CH-NP was carried out by
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dialysis sac method. An accurately weighed quantity of about 20 mg of nanoparticles was
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suspended in 3.0 ml of release medium (Phosphate buffer saline solution, pH 7.4) in the
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dialysis tubing (cut off 10,000 kDa). The dialysis tube was tied to the paddle of USP type II
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dissolution apparatus (TDL–08L, Electrolab, India) and immersed in 250 ml of dissolution
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medium (Phosphate buffer, pH 7.4). The dissolution media was maintained at 37ºC ± 0.5ºC
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with continuous stirring at 50 rpm. An aliquot of 5 ml sample was withdrawn at various time
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intervals and replenished with equal volumes of fresh media. The content of DTX in the
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samples was determined spectrophotometrically by measuring the absorbance at 230 nm in a
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UV-Visible Spectrophotometer (Shimadzu UV spectrophotometer, Japan).
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2.3.7. In vitro Cytotoxicity Screening
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MTT assay using breast cancer cell lines (MDA-MB-231) was employed to determine the
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cell viability. The cell lines were seeded in 96-well plates at the density of 150,000 viable
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cells per well and incubated for 24 hours to allow cell attachment at 37ºC in 5% CO2
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humidified incubator (Sanna et al., 2011). The cells were then incubated for another 24 hours
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with blank nanoparticles (NP), ionically cross-linked docetaxel loaded chitosan nanoparticles
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(DTX-CH-NP) and the pure drug (DTX) at 0.05, 0.5, 5.0 μg/ml equivalent concentrations for
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24, 48 and 72 h, respectively. These concentrations correspond to plasma levels of the drug
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which can be achieved in humans (Fonseca, Simoes & Gaspar, 2002). The medium was
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removed, at specific time intervals and the wells were washed twice with phosphate buffer
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solution and 10 μL of 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide
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(MTT) solution was added to each well of the plate and incubated for another 1 to 4 h.
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Following media removal, the MTT-formazan formed by metabolically viable cells was
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dissolved in 200 μL of Diemthyl sulphoxide (DMSO) and after 4 h, the plates were observed
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at 560 nm in a microplate reader. % Cell viability was calculated by the following equation:
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Cell viability (%)
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Where Abs sample is the absorbance of the solution incubated with the ionically cross linked
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nanoparticles
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Abs control is the absorbance of the solution incubated with the culture medium only
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(positive control).
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2.3.8. Stability studies
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DTX-CH-NP were evaluated for the change in particle size and entrapment efficiency i.e.
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aggregation over the period of 3 months. All the formulations were kept at a temperature of
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4°C for a period of 3 months.
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2.3.9. Statistical analysis
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The results were presented as mean ± standard deviation. Statistical analysis was performed
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with one-way ANOVA and statistical significance was designated as p < 0.05.
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3. Results and discussion
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3.1. Particle size, size distribution and zeta potential of nanoparticles
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Based on an optimization procedure designed, the effect of formulation parameters (chitosan,
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TPP and drug concentration, chitosan to TPP volume ratio, temperature and pH of chitosan
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solution) was investigated systematically by changing one parameter while keeping the others
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constant. On the basis of preliminary trials, the parameters were varied as follows:
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concentration of chitosan (0.2–1%, w/v), concentration of TPP (0.25–1.25%, w/v), chitosan-
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TPP volume ratio (3:1-7:1), stirring speed (800-1600 rpm), temperature (35ºC - 65ºC) and pH
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(3.5-6) of chitosan solution, respectively. The effect of each of these parameters is discussed
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as follows:
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3.1.1. Effect of TPP (cross-linker) concentration
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TPP has five negative charge groups that interact with the positive amino groups of chitosan
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in acetic acid solution (Hosseinzadeh, Atyabi, Dinarvand & Ostad, 2012).The concentration
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of chitosan was fixed at (0.2%, w/v) and TPP concentration was varied from (0.25–1.25%,
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w/v). Fig. 2(a) represents a comparative effect of TPP concentration on particle size,
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polydispersity index and zeta potential. It was observed that the initial increase in TPP
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concentration (0.75%, w/v) led to a significant reduction in particle size. A high TPP
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concentration resulted in high degree of cross-linking of chitosan molecules, thus compact
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particle structures were formed. Further, increase in TPP concentration beyond (0.75%, w/v)
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led to a significant enhancement in the particle size. The increase in the number of anionic
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groups led to an increase in the electrostatic interaction with positive amino sites on chitosan
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molecule, thereby leading to reduction of the surface charge and increase in particle size. Zeta
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potential is an important parameter which influences the stability of the nanoparticles through
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electrostatic repulsion (Gang & Wang, 2007; Ajun, Yan, Li & Huili, 2009). The positive zeta
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potential is attributed to the presence of amino groups and further, neutralization of charged
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amino groups led to a decrease in the overall zeta potential. TPP concentration higher than
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(1.25%, w/v) formed aggregated solution, giving a gel-like consistency. The concentration of
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TPP had little effect on the polydispersity of particles since the polydispersity index (PDI)
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value was below 0.5 indicating uniformity of particle size.
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3.1.2. Effect of chitosan concentration
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Chitosan has the property of protonization under acidic conditions due to which it undergoes
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cross-linking with anions like Tripolyphosphate. TPP concentration was fixed at (0.75%,
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w/v) and chitosan concentration was varied from (0.2-1%, w/v). Fig. 2(b) represents a
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comparative effect of chitosan concentration on particle size, polydispersity index and zeta
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potential. The increase in size and zeta potential of the nanoparticles with increase in chitosan
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concentration from (0.2-1 mg/ml) may be attributed to the linear increase in viscosity of
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chitosan solution with increasing concentration. The high viscosity prevents effective ionic
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interaction between TPP and chitosan solution (Berger et al., 2004). The electrostatic
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repulsions and interchain hydrogen bonding interactions between chitosan molecules remain
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in equilibrium upto the concentration of 2 mg/ml leading to the formation of large particle
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sizes (Gana, Wang, Cochrane & McCarron, 2005).The zeta potential of the nanoparticles
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increased linearly due to a more protonized –NH3+ ion on the surface of nanoparticles formed
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with higher chitosan concentration. The PDI of the nanoparticles was found to be favorable
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i.e. below 0.5.
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3.1.3. Effect of chitosan-TPP volume ratio
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The effect of chitosan-TPP volume ratio on the particle size, polydispersity index and zeta
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potential was very significant. TPP concentration was fixed at (0.75%, w/v) and chitosan
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concentration was fixed at (0.2%, w/v). Fig. 2(c) represents a comparative effect of chitosan-
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TPP volume ratio on particle size, polydispersity index and zeta potential. The results
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indicate that, with increasing chitosan volume (3:1 to 5:1) in the solution, the particle size
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decreased from 295.3 nm to 159.4 nm and then increased to 502.1 nm at ratio 7:1. When
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chitosan-TPP volume ratio was below 3:1, the volume of chitosan solution was not sufficient
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for the formation of a cross-linked structure with TPP. With an increase in volume ratio from
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3:1 to 5:1, the particle size decreased due to increased cross-linking density between chitosan
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and TPP. As its volume ratio increased from 5:1 to 7:1, though the molecules were cross-
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linked, the excess of chitosan resulted in the formation of larger-sized nanoparticles. The zeta
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potential of chitosan–TPP nanoparticles showed a linear relationship to chitosan-TPP volume
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ratio from 3:1 to 5:1. The PDI values were all below 0.5 and least value of PDI (0.267) was
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seen at 5:1 volume ratio of chitosan-TPP.
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3.1.4. Effect of temperature of chitosan solution
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Fig. 2(d) shows the comparative effect of temperature of chitosan solution on the particle
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size, polydispersity index and zeta potential. The particle size decreased from 489.4 nm to
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160.1 nm with increase in temperature of the chitosan solution (35 ºC-65ºC) due to decrease
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in intrinsic viscosity. The increase in temperature reduced the water of hydration of chitosan
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molecules, resulting in an increase in chitosan chain flexibility (Chen & Tsaih, 1998). As a
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result, chitosan molecules approached each other with greater force resulting in the formation
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of compactly cross-linked nanoparticles. The decrease in particle size is negligible at a
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temperature above 65ºC. A decrease in zeta potential was also observed and the resulting PDI
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values were all below 0.5.
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3.1.5. Effect of pH of chitosan solution
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Chitosan is a weak base polyelectrolyte insoluble at neutral and alkaline pH (Mi, Sung &
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Shyu, 2003).The amine groups are positively charged in an acidic medium and hence, the
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surface charge density of chitosan molecules depends on the solution pH (Leong, Mao &
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Truong-Le, 1998; Ko, Park & Hwang, 2002). As the ionic cross-linking process for the
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formation of chitosan–TPP nanoparticles is pH-dependent, the effect of changing pH of
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chitosan solution on particle size, zeta potential and polydispersity index was investigated
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keeping the concentration of chitosan (0.2%, w/v), TPP (0.75%, w/v) and chitosan-TPP
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volume ratio (5:1) fixed. Fig.2 (e) represents a comparative effect of pH of chitosan solution
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on particle size, polydispersity index and zeta potential.
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At a low pH (3.5), the amino groups of chitosan and TPP molecule are protonated, leading to
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a lower charge density of the molecules. This causes an insufficient cross-linking and hence
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larger size of the particles. With an increase in pH, the deprotonation degree of TPP
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increases, while the protonation degree of chitosan is less affected. Hence, the particle size
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decreases significantly at pH 5.5. The sharp increase in size at pH 6.0 indicates that the
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degree of protonization at surface of the particles was reduced leading to particle aggregation.
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The same decreasing pattern was also shown in zeta potential at pH 5.5 and increase at pH 6.
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3.1.6. Effect of the stirring speed
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Fig. 2(f) represents a comparative effect of stirring speed on particle size, polydispersity
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index and zeta potential. The results indicate that the particle size was significantly reduced
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by increasing stirring speed from 800 rpm to 1400 rpm. There was slight decrease in zeta
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potential and no significant effect on PDI was observed.
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3.1.7. Effect of the drug concentration
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DTX-CH-NPs were prepared by the addition of DTX in 0.75% w/v TPP into 0.2% w/v of
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chitosan solution. Fig. 2(g) represents a comparative effect of drug concentration on particle
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size, polydispersity index and zeta potential. The results indicate that the addition of DTX led
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to an increase in the size of chitosan nanoparticles. In general, the size of DTX-CH-NP did
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not grow significantly at concentration up to 0.6 mg/ml, but there was a change in size when
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the concentration of the drug was increased from 0.6 to 1 mg/ml. The drug concentration did
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not influence the zeta potential of the prepared nanoparticles significantly.
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Table 1 depicts the effect of different concentrations of DTX on the %EE and %LC of DTX-
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CH-NP. A maximum EE (78%) was achieved at 0.6 mg/ml of DTX concentration. Initially,
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%EE showed an increase with increase in DTX concentration up to 0.6 mg/ml. From 0.6- 1
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mg/ml, the %EE decreased from 78% to 73%. The %LC of DTX-CH-NP was in the range of
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9-12%. A high loading capacity was obtained due to the hydrophobicity of DTX. The %LC
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increased as a function of initial DTX content. The increase in %LC as a function of increase
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in drug content corresponds to the previous literature reports such as loading of bovine serum
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albumin (Jiang, Gong, Zhag & Zu, 2015) and ammonium glycyrrhizinate (Wu et al., 2005)
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into chitosan–TPP nanoparticles. The results showed that 0.6 mg DTX in the formulation
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resulted in the best % EE and %LC. Further increasing the DTX amount did not significantly
338
affect %LC.
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3.2. Morphological characterization of nanoparticles
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The morphological characteristics of DTX-CH-NP were imaged using SEM and TEM. Fig.
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3(a-b) displays the scanning electron micrographs of DTX-CH-NP showing their surface
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morphology while Fig. 3(c) depicts the transmission electron micrograph. The nanoparticles
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showed a dense and spherical structure with uniformity in size.
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3.3. Fourier Transform infrared spectroscopy (FT-IR)
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FT-IR spectroscopy was used to study the drug–polymer interactions. Fig. 4 shows the FT-IR
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spectra of DTX, DTX-Chitosan physical mixture (50:50), DTX-TPP physical mixture (50:50)
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and DTX-CH-NP. The spectra of DTX shows characteristic peaks at 3481.84 cm-1 which may
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be ascribed to N-H stretching of alkanes. Peaks at 1740 cm-1 and 1711 cm-1 may be due to C-
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O stretching and at 1631.97 cm-1 can be due to N-H plane bending. The spectra of DTX-
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Chitosan and DTX-TPP indicated the absence of any drug-excipient interaction between the
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drug and polymer as the spectra of the drug remained intact without any changes in peaks.
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The peak at 1651 cm-1 of DTX-chitosan spectra disappeared in the spectra of DTX-CH-NP
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and two new peaks at 1643 cm-1 and 1543 cm-1 appeared. The disappearance of the peak
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could be attributed to the linkage between the phosphoric and ammonium ions. The cross-
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linked chitosan nanoparticles showed a peak for P = O at 1157 cm-1. All recorded spectra
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show that the peaks of docetaxel remain unaffected indicating that the drug remained intact
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during the preparation procedure of nanoparticles. Thus, FT-IR analysis indicated absence of
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any drug excipient interaction between the drug and polymer during the formulation of
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nanoparticles.
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3.5. In vitro drug release
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The cumulative percent drug release of DTX from different formulations of DTX-CH-NP
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varied from 77.46 ± 1.17% and 73.79 ± 0.79% respectively over a period of 24 h as shown in
363
Fig. 5. The release profiles of the nanoparticles are similar, and exhibit an initial burst release
364
followed by a slow and sustained release due to the release of drug from the matrix. The
365
initial rapid release was mainly due to the drug present at the surface of the nanoparticles
366
which diffuses in the release medium in the initial time. To determine the release kinetics,
367
the release data was fitted into various kinetic models. Table 2 represents the release kinetics
368
of various batches of ionically cross-linked docetaxel loaded chitosan nanoparticles and the
369
co-efficient of correlation (R2) of zero order, first order, Korsmeyer–Peppas model and
370
Higuchi’s square root model was determined. Korsmeyer–Peppas model was observed to be
371
the best fit model indicating Fickian diffusion as the mechanism of drug release from
372
nanoparticles. Further, the value of ‘n’ the release exponent of Korsmeyer–Peppas (0.45≤ n ≤
373
0.89) indicates that nanoparticles released the drug by combination of both diffusion of drug
374
through the polymer and dissolution of the polymer (Costa & Lobo, 2001; Siepmann &
375
Peppas, 2001; Higuchi, 1963).
376
3.6. In vitro Cytotoxicity Screening
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The in vitro cytotoxicity screening of docetaxel as a free drug and ionically cross-linked
378
docetaxel loaded chitosan nanoparticles was determined by MTT assay, which is a
379
colorimetric test based on the selective ability of viable cells to reduce the tetrazolium
380
component of MTT into purple colored formazan crystals. MTT assay was performed for the
381
three different concentrations i.e., 0.05, 0.5 and 5.0 μg/ml using MDA-MB-231 as a model
382
breast cancer cell line.
383
Untreated MDA-MB-231 cells as well as cells treated with blank cross-linked chitosan
384
nanoparticles, with the same polymer content were used as control. Fig.6 (a-c) shows the
385
viability of MDA-MB-231 cancer cells, cultured with blank cross-linked chitosan
386
nanoparticles (Blank-NPs) and ionically cross-linked docetaxel loaded chitosan nanoparticles
387
[DTX-CH-NP3] after incubation for 24 h (a), 48 h (b) and 72 h (c), in comparison with that
388
of pure drug [DTX]. The biocompatibility of chitosan was confirmed by the negligible
389
toxicity of blank chitosan nanoparticles [NPs]. After 24 h incubation, as depicted in Fig. 6(a),
390
the cell viability decreased to about 59.22, 47.12 and 38.45% for DTX-CH-NP3 at 0.05, 0.5
391
and 5.0 μg/ml drug concentrations respectively, corresponding to an increase in cytotoxicity
392
of 25% compared with that of free drug. After 48 h incubation, portrayed in Fig. 6(b), the
393
cytotoxicity increased to about 41.34, 35.12 and 25.31% for the DTX-CH-NP3 and 65.88,
394
58.98 and 48.87% for the free drug, respectively. The more marked inhibition of cell growth
395
was obtained for longer incubation period (72 h). The strongest cytotoxic effect was achieved
396
with nanoparticles at 5.0 μg/ml drug concentration.
397
The storage stability of chitosan nanoparticles cross-linked with TPP is an important
398
parameter. Particles in the colloidal dispersion may adhere to one another and form
399
aggregates of increasing size. The physical stability was investigated at 4ºC over a period of 3
400
months. A special emphasis was placed on the changes in particle size and entrapment
401
efficiency which are the two important physicochemical parameters of nanoparticles. The
402
nanoparticles were found to be stable with no major changes except a slight increase in
403
particle size and a slight decrease in entrapment efficiency and drug loading.
404
4. Conclusion
405
The present work is an effort to explore ionically cross-linked docetaxel loaded chitosan
406
nanoparticles for breast cancer treatment in order to improve the therapeutic efficacy and
407
minimize severe toxicity associated with the clinical usage of docetaxel. TPP was
408
successfully employed as a cross-linking agent to formulate nanoparticles by ionotropic
409
gelation. The influence of a number of fabrication parameters was systematically investigated
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13 Page 14 of 32
and the concentrations of TPP, chitosan, chitosan-TPP volume ratio were found to have a
411
profound impact on the physicochemical characteristics of nanoparticles. The nanoparticles
412
exhibited particle size in the range of 159.2 ± 3.31 nm to 220.7 ± 223 nm with a sustained
413
drug release pattern. The in vitro cytotoxicity screening studies demonstrated a 25% increase
414
in cytotoxicity compared with the free drug against MDA-MB-231 cancer cell lines. Thus,
415
the present study unraveled the effect of fabrication parameters on the ionic cross-linking
416
process. The results of this investigation clearly demonstrate that the ionically cross-linked
417
docetaxel loaded nanoparticles seems to have a great potential as a drug carrier in cancer
418
chemotherapy. The present research work offers immense scope for further exploitation of
419
chitosan in future for the development of nanoparticulate drug delivery system for cancer
420
chemotherapy.
421
Acknowledgements
422
The authors are grateful to Central Instrumentation Laboratory, Panjab University,
423
Chandigarh for particle size and SEM analysis and also gratified to the Management of
424
Chandigarh College of Pharmacy, Mohali (Punjab), India for financial support and providing
425
the facilities to carry out the research work.
426
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Table(s)
List of Tables Table 1 Characterization of ionically cross-linked docetaxel loaded chitosan nanoparticles by varying drug concentration
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Table 2 Release kinetics and stability data of different formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles
Page 20 of 32
Characterization of ionically cross-linked docetaxel loaded chitosan nanoparticles by varying drug concentration Zeta potential (mV)
Blank
˗˗˗
159.2 ± 3.31
0.278 ± 0.029
33.2 ± 1.66
DTX-CH-NP1
0.2
163.2 ± 3.43
0.259 ± 0.034
32.7 ± 1.12
DTX-CH-NP2
0.4
170.9 ± 4.58
0.247 ± 0.041
31.9 ± 2.11
DTX-CH-NP3
0.6
175.2 ± 3.43
0.229 ± 0.027
DTX-CH-NP4
0.8
204.1 ± 6.09
0.287 ± 0.043
DTX-CH-NP5
1
220.7± 2.23
0.345± 0.036
Entrapment Efficiency (%)
Loading Capacity (%)
˗˗˗
˗˗˗
69.37 ± 0.74
9.94 ± 0.81
cr
(nm)
Polydispersity Index (PDI)
75.11 ± 1.02
11.26 ± 0.79
32.3 ± 1.67
78.28 ± 0.91
12.01 ± 0.92
29.8 ± 1.96
70.41 ± 1.35
10.56 ± 0.99
73.23± 1.42
10.21± 0.65
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Particle size
an
DTX concentration (mg/ml)
Formulation code
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Table 1:
34.8± 3.40
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Mean ± S.D., n =3. Note: DTX concentration: Concentration of Docetaxel.
Page 21 of 32
Table 2: Release kinetics and stability data of different formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles Release kinetics
Zero order
First order
Higuchi’s square- root
Korsmeyer-Peppas
ip t
Formulation Code
-1
k
R2
-1
(% h )
R2
(h )
k (%h
0.6452
0.0479
0.7859
0.0009
0.8946
DTX-CH-NP2
0.6277
0.0519
0.7767
0.0011
0.8849
DTX-CH-NP3
0.6962
0.0557
0.8639
0.0013
0.9254
DTX-CH-NP4
0.6206
0.0436
0.7862
0.0009
0.8779
DTX-CH-NP5
0.7025
0.0407
0.8342
0.0006
R2 )
2.1451
M
0.9188
k
(%h-n)
0.9439
n
0.2348
0.5880
2.3431
0.9210
0.2948
0.5781
2.4480
0.9114
0.3268
0.5982
2.2568
0.9598
0.3932
0.5437
1.9710
0.9475
0.2785
an
DTX-CH-NP1
-0.5
cr
k
us
R2
0.5165
Stability Data
Entrapment Efficiency (%)
d
4±2ºC
1 month
2 months
3 months
78.28 ± 0.91
77.4 ± 0.3
76.5 ± 1.8
76.0 ± 3.0
167.2 ± 3.43
169.6 ± 4.3
176.5± 3.2
181.5 ± 3.3
Ac ce p
Size (nm)
Initial
te
Ionically cross-linked Docetaxel loaded chitosan nanoparticles
Page 22 of 32
Figure(s)
Figure captions Fig. 1 Preparation of ionically cross-linked docetaxel loaded chitosan nanoparticles by Ionotropic gelation.
cr
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Fig. 2 Bar graph showing comparative effect of (a) TPP concentration (b) Chitosan concentration (c) Chitosan: TPP volume ratio (d) Temperature of chitosan solution (e) pH of chitosan solution (f) Stirring speed (g) Drug concentration on particle size, polydispersity index and zeta potential of ionically cross-linked docetaxel loaded chitosan nanoparticles.
us
Fig. 3 Scanning electron micrographs showing shape and surface morphology (a-b) and transmission electron micrograph (c) of ionically cross-linked docetaxel loaded chitosan nanoparticles.
an
Fig. 4 Comparative FT-IR spectra of docetaxel, docetaxel-chitosan physical mixture, docetaxelTPP physical mixture and ionically cross-linked docetaxel loaded chitosan nanoparticles.
M
Fig. 5 Release profile of docetaxel from various formulations of ionically cross-linked docetaxel loaded chitosan nanoparticles.
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Fig. 6 % Cell viability of MDA-MB-231 cancer cells after incubation for 24 h (a), 48 h (b) and 72 h (c) with ionically cross-linked docetaxel loaded chitosan nanoparticles.
Page 23 of 32
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Fig. 1
Fig. 2(a)
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Fig. 2(b)
Fig. 2(c)
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Fig. 2(d)
Fig. 2(e)
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d
Fig. 2(f)
Fig. 2(g)
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Fig. 3(a)
Fig. 3(b)
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Fig. 3(c)
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Fig. 5
Fig. 6(a)
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Fig. 6(b)
Fig. 6(c)
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