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Xanthan gum stabilized gold nanoparticles: Characterization, biocompatibility, stability and cytotoxicity
Accepted Manuscript Title: Xanthan gum stabilized gold nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity Author: Deep Pooja Sravani Panyaram Hitesh Kulhari Shyam S Rachamalla Ramakrishna Sistla PII: DOI: Reference:
S0144-8617(14)00273-2 http://dx.doi.org/doi:10.1016/j.carbpol.2014.03.041 CARP 8703
To appear in: Received date: Revised date: Accepted date:
8-1-2014 18-2-2014 5-3-2014
Please cite this article as: Pooja, D., Panyaram, S., Kulhari, H., Rachamalla, S. S., & Sistla, R.,Xanthan gum stabilized gold nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.03.041 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.
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Xanthan gum stabilized gold nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity
3 Deep Pooja,a Sravani Panyaram,a Hitesh Kulhari,a,b Shyam S Rachamalla,c Ramakrishna Sistlaa,* a
Medicinal Chemistry & Pharmacology Division, bIICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India.
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Faculty of Pharmacy, College of Technology, Osmania University, Hyderabad, India
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*Corresponding author:
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Sistla Ramakrishna, PhD
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Principal Scientist,
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Medicinal Chemistry & Pharmacology Division,
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CSIR-Indian Institute of Chemical Technology,
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Hyderabad-500007, India
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Phone: +91-40-27193753 (office)
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Email: [email protected]
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Highlights
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Gold nanoparticles were prepared using xanthan gum as reducing and capping agent.
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Different formulation and process variables were optimized. 1 Page 1 of 36
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XG stabilized nanoparticles were non-toxic and biocompatible.
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DOX loaded on GNP showed more therapeutic efficacy than free DOX in cancer cells.
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Formulations displayed colloidal stability against pH and ionic strength changes.
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Graphical Abstract
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Abstract
Xanthan gum (XG) has been widely used in food, pharmaceutical and cosmetic industries. In
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the present study, we explored the potential of XG in the synthesis of gold nanoparticle. XG
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was used as both reducing and stabilizing agent. The effect of various formulation and
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process variables such as temperature, reaction time, gum concentration, gum volume and
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gold concentration, in GNP preparation was determined. The XG stabilized, rubey-red XGNP
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were obtained with 5 ml of XG aqueous solution (1.5 mg/ml). The optimum temperature was
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80 ºC whereas the reaction time was 3 h. The optimized nanoparticles were also investigated
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as drug delivery carrier for doxorubicin hydrochloride. DOX loaded gold nanoparticles
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(DXGP) were characterized by dynamic light scattering, TEM, FTIR, and DSC analysis. The
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synthesized nanoparticle showed mean particle size of 15-20 nm and zeta potential -29.1 mV.
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The colloidal stability of DXGP was studied under different conditions of pH, electrolytes
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and serum. Nanoparticles were found to be stable at pH range between pH 5-9 and NaCl
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concentration up to 0.5 M. In serum, nanoparticles showed significant stability up to 24 h.
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During toxicity studies, nanoparticles were found biocompatible and non-toxic. Compared
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with free DOX, DXGP displayed 3 times more cytotoxicity in A549 cells. In conclusion, this
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study provided an insight to synthesize GNP without using harsh chemicals.
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Key words: Xanthan Gum; Gold nanoparticles; Reducing agent, Doxorubicin, Cytotoxicity, Stability
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1.
Introduction
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Xanthan gum (XG) is an anionic, high molecular weight, exo-polysaccharide
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produced by aerobic fermentation of sugars by Xanthomonas campestris. Its basic chain
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consists of a β-(1→4) linked glucose backbone with the substitution of a charged
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trisaccharide side-chains of [β-(1→3)-mannose-α-(1→2)-glucuronic acid-β-(1→4)-mannose]
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on alternate glucose residues (Jian, Zhu, Zhang, Sun, & Jiang, 2012). It is non-toxic,
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hydrophilic and biodegradable bio-polymer. XG is being used in food, cosmetic and
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pharmaceutical industries. The industrial applications of XG are based upon its exceptional
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rheological properties. XG is soluble in both cold and hot water, hydrates quickly and
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produces high viscosity at low concentration (Sereno, Hill, & Mitchell, 2007; Sharma,
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Naresh, Dhuldhoya, Merchant, Merchant, 2006). In pharmaceutical industries, XG has been
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reported for formulation of both solid and liquid dosage forms. In solid dosage formulation, it
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is used as controlled release agent (Jian et al., 2012; Phaechamud and Ritthidej, 2007; Santos,
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H., Veiga, F., Pina, M. E., & Sousa, 2005; Sinha, Mittal, Bhutani, & Kumaria, 2004) whereas
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in liquid formulations, it is used as thickening agent, suspending agent and emulsion
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stabilizer (Desplanques, Renou, Grisel, & Malhiac, 2012).
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Recently, XG has also been used in the preparation and stabilization of inorganic iron
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and palladium nanoparticles (Fan et al., 2013; Xue & Sethi, 2012; Comba , Dalmazzo,
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Santagata, & Sethi, 2011; Vecchia, Luna, & Sethi, 2009). Nanoparticles are solid structures
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with a size below 200 nm and have found their applications in sensing, imaging and in drug
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and gene delivery. Gold nanoparticles (GNP) are one of the most commonly explored and
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used nanoparticles in drug delivery because of controlled size, improved efficacy and
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targeted delivery (Almeida, Figueroa, & Drezek; 2013; Pissuwan, Niidome, & Cortie, 2011).
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As drug delivery carrier, GNP has been used for the delivery of both hydrophilic and
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hydrophobic drugs (Oliveira et al., 2013; Chen et al., 2007; Gibson, Khanal, & Zubarev,
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2007; Aryal, Grailer, Pilla, Steeber, & Gong, 2009; Prabaharan M, Grailer JJ, Pilla S, Steeber
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DA, & Gong, 2009). But, the conventional synthesis of GNP involves the use of chemicals
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like sodium borohydrate, tri-sodium citrate etc as reducing agent (Burygin et al., 2009;
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Duncan, Kim, & Rotello, 2010; Khan, Jung et al., 2013; Vishakante, & Siddaramaiah, 2013;
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Saha et al., 2007). These GNP have been found to be very unstable and form aggregates with
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the slight change in pH and electrolyte concentration (Mirza and Shamshad, 2011; Rouhana,
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Jaber, & Schlenoff, 2007). The GNP prepared with gellan gum showed the stability between
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pH 4 to 8 and addition of NaCl up to 0.1M (Dhar, Reddy, Shiras, Pokharkar, & Prasad,
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2008). Natural gums stabilize the inorganic nanoparticles by two mechanisms: first, by
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adsorbing to the surface of nanoparticles which creates steric repulsion among the particles.
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In second mechanism, they increase the viscosity of nanoparticle suspension, and therefore
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slow down the aggregation processes (Tiraferri, Chen, Sethi, & Elimelech, 2008; Xue &
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Sethi, 2012; Comba & Sethi, 2009). The aim of this investigation was to synthesize the
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environment friendly gold nanoparticles using XG as reducing agent. The nanoparticle
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preparation was optimized for gum concentration and volume, gold concentration,
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temperature and reaction time. Gum stabilized GNP were studied for its utility as drug
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delivery carrier using Doxorubicin hydrochloride as model drug. Doxorubicin hydrochloride
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is hydrophilic in nature and has been clinically used for the treatment of various cancers,
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haematological malignancies, soft tissue sarcomas and solid tumors (Carvalho et al., 2009;
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Laginha, Verwoert, Charrois, & Allen, 2005). Two measure limitations of DOX are non-
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specific cytotoxicity and multi drug resistance. Multi drug resistance to DOX is due to drug
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efflux by P-glycoproteins. Doxorubicin is substrate for P-glycoproteins which efflux DOX
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and decrease intracellular drug level. DOX loaded GNP have shown to overcome both the
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problems of non-specific toxicity and drug resistance (Gu, Cheng, Man, Wong, & Cheng,
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2012). The prepared nanoparticles were also investigated for biocompatibility, stability and
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cytotoxicity study in lung cancer cells.
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2.
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2.1.
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Materials and methods Materials
Xanthan gum and Tetrachloroauric acid (HAuCl4) were purchased from Sigma-
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Alderich (St Louis, MO, USA). Doxorubicin hydrochloride was received as gift sample from
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TherDose pharma pvt ltd (Hyderabad, India). The chemical used for buffer preparations were
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of analytical grade and were purchased from sd Fine-Chem Ltd. (Hyderabad, India). MTT (3-
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(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromide, Dulbecco’s modified eagle
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medium (DMEM), trypsin–EDTA, fetal bovine serum (FBS) and antibiotic solution (10,000
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U/ml penicillin, 10 mg/ml streptomycin) were purchased from Sigma-Aldrich (St Louis, MO, 6 Page 6 of 36
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USA). Cell culture plastic wares were obtained from Tarsons Products Pvt. Ltd. (Kolkata,
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India). All the formulations were prepared in MilliQ water.
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2.2.
Preparation of gum solution The stock solution of gum was prepared by dissolving 500 mg of the gum in 100 ml
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water and was stirred overnight at room temperature. The solution was centrifuged to remove
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the insoluble materials and supernatant was lyophilized. The lyophilized dry powder was
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dissolved in water to get desired concentration of xanthan gum.
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2.3.
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Synthesis of gold nanoparticles
Gold nanoparticles were prepared by reducing the aqueous solution of HAuCl4 by
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heating at 80 ºC in the presence of XG solution (1.5 mg/ml). The change in colour was
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obtained from colourless to purple to rubey-red after 2 h. The colloidal solution was cooled at
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room temperature and stored in amber colour vials at 4 ºC. The nanoparticle formulation
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showed the absorption maxima at 525 nm.
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2.4.
Optimization of formulation and process variables
The effect of formulation and process variables such as gum concentration, gold
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concentration, gold to gum volume ratio, reaction time and temperature were studied by
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changing one parameter at a time and keeping other constant.
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2.5.
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2.5.1. Hydrodynamic diameter and polydispersity
Characterizations of Xanthan gum stabilized gold nanoparticles(XGNP)
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The hydrodynamic diameter and polydispersity index of GNP were determined by
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dynamic light scattering using Zetasizer Nano-ZS (Malvern instrument Ltd., Malvern, UK). 7 Page 7 of 36
Before measurement, the samples were diluted appropriately to get particle count rate
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between 100-300 kcps and instrument was set up at 25 ºC with a backscattering angle of
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173°.
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2.5.2. Transmission electron microscopy (TEM)
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A drop of sample was placed on carbon coated copper grid, air dried at room
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temperature and stained with 2% uranyl acetate. The nanoparticle size measurement was
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done using transmission electron microscope (Hitachi, H-7500) and average of 10
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nanoparticle size was considered as the size of the sample.
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2.5.3. Surface charge
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The electrokinetic properties of the GNP were determined by measuring the zeta
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potential using Zetasizer Nano-ZS. The samples were diluted 10 times with MilliQ water
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before the measurement.
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2.5.4. Fourier transform infrared analysis
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compressed to form a pellet. The pellet was placed in pellet holder and scanned for the
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measurement of % transmittance in the wave number range of 4000 to 450 cm-1 using FTIR
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spectrophotometer (Perkin Elmer, USA). The spectra requisition was carried out using the
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software Spectrum One (Perkin Elmer, USA).
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2.6.
Doxorubicin loading to nanoparticles
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Blank gold nanoparticles were dispersed in phosphate buffer saline and were incubated
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with 1 mg of DOX solution (1 mg/ml) at room temperature overnight. The nanoparticles
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dispersion was centrifuged at 15000 rpm for 30 min. DOX loading was determined by
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measuring the free drug content in supernatant. Percent drug loading was calculated as
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follows: % DOX loading = (1-DS/DT) × 100
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Whereas, DS is amount of DOX present in supernatant, DT = Total amount of DOX
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loaded initially.
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2.7.
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In vitro drug release studies
The in vitro drug release studies were performed using dialysis method. In dialysis
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tubing, the nanoparticles equivalent to 1 mg of DOX were dispersed in 1 ml of distilled water
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and placed in 100 ml of release medium (phosphate buffer saline pH 7.4 and sodium acetate
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buffer pH 4.5) at 37 ºC in dark. Three millilitres of sample was withdrawn at different time
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intervals up to 12 hours and was replaced with same volume of fresh medium. The samples
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were analyzed for drug content using UV/VIS spectrophotometer (Perkin Elmer, Lambda
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25, USA) at 480 nm.
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2.8.
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The biocompatibility of prepared gold nanoparticles was determined by estimating the
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% hemolysis, caused by nanoparticles. The assay procedure reported by Kumar et al. 2011,
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was used with slight modification (Kumar, Paul, Sharma, 2011). Blank gold nanoparticles
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were dispersed in normal physiological saline (0.9 %w/v NaCl). Varying concentrations (50-
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200 µg/ml) of the dispersion were added into red blood cells suspension (2 %v/v), mixed well
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and incubated at 37 °C. After 1 h, the samples were centrifuged at 3000 rpm for 5 min and
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absorbance of supernatant was measured at 540 nm spectrophotometrically. The supernatant
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absorbance of RBC suspension treated with distilled water and normal saline were taken as
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standard and control; respectively. The % hemolysis was calculated as: % Hemolysis =
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(Asample – Acontrol) / (Astandard - Acontrol) × 100
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2.9.
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Cytotoxicity studies 9 Page 9 of 36
Cellular toxicity of pure DOX and DXNP was evaluated by MTT assay in A549 human
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lung cancer cells. The assay is based on the mitochondrial reduction of yellow MTT, a
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tetrazolium dye, to a highly coloured blue formazan product. About 10000 cells per well were
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cultured in 96 wells plate and allowed to attach overnight. Next day, cells were incubated
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with different concentration (0.1-10 µg/ml) of pure doxorubicin, blank gold nanoparticles
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(positive control) and doxorubicin loaded gold nanoparticles. Cells were incubated in CO2
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incubator at 37 °C for 48 h. The solution of each well was replaced with fresh serum free
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media and 10 µl of MTT reagent (5 mg/ml) was added. After 4 h, the media was removed,
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200 µl DMSO was added and incubated for 20 min. The absorbance was measured at 570 nm
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using Spectra Max plus 384 UV-Visible plate reader. The cell viability was determined as a
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percent based on the absorbance measured relative to the absorbance of untreated or control
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cells. The half maximum inhibitory concentration (IC50) values were determined by non-
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regression analysis using GraphPad Prism v. 5.03 (GraphPad Software Inc. CA).
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2.10. Stability studies
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2.10.1. Storage stability
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Doxorubicin loaded nanoparticles were stored at 4 °C and were studied for particle
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size, zeta potential and drug content up to 1 month. Particle size and zeta potential were
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determined by particle size analyzer and DOX content was determined using UV/VIS
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spectrophotometer.
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2.10.2. Effect of pH and ionic concentration of medium
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For the determination of effect of pH on the stability, DXGP were incubated in
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different pH medium (pH 3 to pH 11). Nanoparticles incubated in phosphate buffer saline pH
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7.4 were taken as control. Absorbance was measured after 24 h and compared with
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absorbance of control. 10 Page 10 of 36
The effect of ionic strength on the stability of DXNP was determined by incubating the
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nanoparticles in deionised water containing varying concentrations (0.1-2M) of sodium
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chloride (NaCl).
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2.10.3. Serum Stability of DXGP
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Serum stability of DXGP was evaluated at two different serum concentration
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levels (10%v/v and 100% serum). Nanoparticles were dispersed in serum and particles
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size, zeta potential and absorbance were measured at different time intervals up to 72 h.
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2.11. Statistical analysis
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All the experiments were performed in triplicates. Data are expressed as Mean ± SD
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(standard deviation) (n=3). The values of in vitro drug release, cytotoxicity and stability
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studies were statistically analyzed using student t-test. Statistical significance level was set at
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p < 0.05.
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3.
Result and discussion
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3.1.
Preparation and optimization of gold nanoparticles
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reducing agent. The role of gum concentration was studied by using gum solution in the
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range of 0.5-2 mg/ml containing 10 mM of HAuCl4 concentration (Fig. 1a). The absorbance
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in the UV spectrum which describes the reduction of GNP, was found to be directly
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related to gum concentration. The intensity of the absorption band gradually increased and
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the maximum intensity of absorption was attained at 1.5 mg/ml which indicated the
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maximum gold nanoparticle concentration.
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The effect of gold to gum volume ratio was evaluated by varying the gum volume in
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the range of 2.5-10 ml (Fig. 1b). At lower gum volume (2.5 ml) a peak shift was observed 11 Page 11 of 36
and the absorption band shifted to 550 nm which suggested that lower gum volume was not
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sufficient to reduce all Au ions. When the gum volume was changed from 5 to 7.5 ml
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followed by 10 ml, there was no significant shift in the characteristic absorption band but the
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absorbance was decreased. The maximum intensity of absorption was attained at 5 ml and
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was considered optimum gum volume for the synthesis of XGNP. The effect of gold
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concentration (Fig. 1c) and volume (Fig. 1d) was also studied and was found as per earlier
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reports. A volume of 100 µl of 10 mM gold solution showed better absorbance at 525 nm.
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Reaction temperature and time are two important process parameters in the synthesis
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on gold nanoparticles. In this study, the temperature was changed in the range of 60-90 °C
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with 10 mM HAuCl4 concentration and 1.5 mg/ml gum solution (Fig. 2a). The maximum
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intensity of the characteristic absorption band was attained at 80 ˚C which indicates the
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maximum gold nanoparticle concentration. For the optimization of reaction time,
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nanoparticle synthesis was studied up to 4 h. The absorbance was found to be increased up to
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2 h and after that there was no change in the absorbance (Fig. 2b).
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3.2.
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Characterization of Xanthan gum stabilized gold nanoparticles The hydrodynamic diameter and polydispersity index of optimized XGNP were
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determined using dynamic light scattering method. The observed mean hydrodynamic
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diameter was 41.2 ± 3.78 nm. The prepared nanoparticles showed high polydispersity (0.35-
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0.6) which can be attributed to presence of wide range of particles. The high negative surface
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potential (-47.2±2.59) indicated the high stability of nanoparticles and presence of gum
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molecules on nanoparticle surface. The presence of acetyl group on O-6 position and pyruvic
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acid linked with mannose sugar are responsible for the negative charge. After DOX loading,
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the surface charge was decreased to -29.1±2.78 mV indicating the interaction between
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positively charged amine group of DOX and negative acidic group of gum. The nanoparticle
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surface morphology and actual particle size were determined by transmission electron 12 Page 12 of 36
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microscopy. Figure 3 showed that nanoparticles were spherical in shape and 15-20 nm in
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size. The chemical interactions between DOX and nanoparticles were studied by FTIR
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analysis (Fig. 4). In XG spectra, the vibration peak of hydrogen bonded −OH and C–H
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stretching were observed at 3416 and 2902 cm−1, respectively. The vibration peak of the
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acetal was found at 1051.8 cm−1 whereas C=O stretching peak was found at 1728.4 cm−1. The
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FITR spectra of blank nanoparticles exhibited all the characteristic peaks of XG at 3398,
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1728 and 1042 cm−1. The peak at 1254.2 cm−1 presented the C-O-C group. The DXGP
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showed principal IR peaks of both XG and DOX showing the non-covalent binding of DOX
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to nanoparticles.
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DSC is a useful technique to explain the thermal stability and phase transitions. Figure
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5 illustrates the DSC thermograms of pure DOX, native XG and DXGP. DSC scan of XG
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exhibited endothermic transition at 125 °C. The endothermic peak of DOX appeared at its
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melting point 195 °C. This peak was disappeared in DXGP spectra indicating that DOX was
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not precipitated on nanoparticle surface but was present as molecular or amorphous
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dispersion.
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3.3.
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DOX loading
The observed doxorubicin loading was found to be 71.4 ± 2.53%. The high drug
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loading can be attributed to presence of XG on the nanoparticle surface. Xanthan gum
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contains negatively charged moieties such as pyruvic acid which interacts with positively
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charged amine group of doxorubicin.
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3.4.
In vitro drug release studies
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In vitro drug release from the nanoparticles was studied in PBS and SAB buffer (Fig.
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6). The nanoparticles showed complete drug release within 10 h. Up to 4 h, there was no
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significant difference (p > 0.05) in doxorubicin release in both the medium but at the end
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DOX release was higher in SAB (98.1%) as compared to PBS (83.6%). The faster release of
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DOX in SAB can be attributed to protonation of amine groups of DOX molecules which
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increases hydrophilicity and solubility at lower pH. The protonation of amine group also
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leads to decreased interaction between DOX and GNP (Minati et al., 2012). The PBS pH 7.4
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is a simulated condition for blood plasma whereas SAB pH 4.5 is for the internal
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environment of cancer cell. Thus the slower release of DOX in PBS and faster release in SAB
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would help to release the maximum amount of drug in tumour and hence to enhance the
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therapeutic efficacy of the formulation.
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3.5.
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Hemolytic toxicity
Nanoparticles are reported to cause oxidative-stress induced hemolysis (Teodora,
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2013). Therefore, the suitability for intravenous administration of nanoparticles is generally
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determined by hemolytic study. In this study, dose-dependent hemolytic toxicity of xanthan
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gum stabilized gold nanoparticle was carried out. The prepared nanoparticles did not show
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hemolysis up to 200 µg/ml.
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3.6.
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Cytotoxicity studies
Anti-proliferative activity of doxorubicin loaded xanthan gum stabilized gold
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nanoparticles (DXGP) and free DOX was studied in A549 human lung cancer cells. The cell
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viability was determined by MTT assay after 48 h of incubation with different concentrations
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(Fig. 7). Blank xanthan stabilized gold nanoparticles (BXNP) were taken as positive control
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and did not decrease the cell viability at any test concentration. It suggested that the prepared
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nanoparticles were non-toxic and biocompatible. Drug loaded nanoparticles showed 14 Page 14 of 36
concentration-dependent cytotoxicity. The cell viability was decreased with the increase in
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DOX concentration. The IC50 value for pure DOX and DXGP was 2.46 and 0.79 µg/ml,
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respectively. The lower IC50 value of DXGP indicated the more cytotoxicity which can be
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attributed to enhanced cellular uptake and hence increased intracellular drug concentration.
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However, the exact mechanism and role of carbohydrate moieties present on nanoparticle in
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cellular uptake require detailed investigation. One possible reason may be due to the
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presence of mannose sugars in the composition of xanthan gum which was used for the
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stabilization of GNP. It is documented that mannose receptors are up regulated in
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human lung cancer cells A549 (Basuki et al., 2014; El-Boubbou et al., 2010). Basuki et al
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reported the improvement in cell uptake of mannose functionalized iron nanoparticles
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in human lung cancer cells A549. Hence, the presence of mannose in xanthan gum
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might have contributed to increase in uptake of drug through receptor-mediated
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endocytosis pathway.
d Stability studies
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3.7.
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The colloidal stability of DXGP was studied for both physical and chemical
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stabilities. The change in particle size and zeta potential are shown in Fig. 8. No significant
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(p>0.05) changes in particle size and zeta potential were observed up to 15 days. But,
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chemical degradation of DXNP was faster than physical instability. The DOX content was
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less than 90% within 24 hours. However, the DOX degradation was slower with DXGP in
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comparison to pure DOX. The observed drug degradation rate constant (K) was 8.28 × 10-3
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and 5.07 × 10-3 day-1 for pure DOX and DXGP (Fig. 8c). Therefore, decease in drug content
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was due to light sensitivity of DOX itself not because of formulation.
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DXGP showed a wide range of pH stability (pH 5-pH 9). The observed absorbance of
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DXGP at 480 nm was 1.109, 0.899, 0.908, 0.927 and 0.776 for pH 3, 5, 7, 9 and pH 11
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respectively (Fig. 9a). The absorbance of control sample was 0.931. The results suggested
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that XG or carbohydrate capping of nanoparticles helped to increase the colloidal stability of
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gold nanoparticles. The decrease in absorbance at pH 11 may due to gel formation properties
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of XG at higher pH levels (pH > 10) (Sharma et al., 2006).
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In other study, DXGP were treated with different NaCl concentration to determine the
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stability of nanoparticles in varying ionic concentrations. Figure 9b showed the absorbance
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pattern of DXGP after 24 h of incubation with different NaCl concentrations. The control
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group was incubated in deionised water without NaCl. The DXGP were found to be stable up
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to 0.5M NaCl concentration.
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The stability of DXGP was further evaluated at two serum conditions-diluted
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(10% v/v serum) and undiluted or 100% serum. Figure 10a showed the particle
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diameter of DXGP exposed to serum, after different time period. In both the conditions,
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particle size was increased continuously up to 72 h. But, the increase in particle
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diameter was more in undiluted serum than diluted serum. After 2 h, particle size was
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increased by 7.1 nm in 10% serum wile it increased by 31.5 nm in 100% serum. From
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the figure 10a it is clear that DXGP were stable up to 24 h in both the conditions but
380
after that size of nanoparticle was increased tremendously. The increase in particle size
381
can be explained by the adsorption of serum proteins or other components on
382
nanoparticle surface. (Yang, Zhang, Wang, White, & Jiang, 2014; Zang & Zang, 2010).
383
The nanoparticle dispersion in distilled water had a high negative zeta potential of -30.2
384
mV and thus the electrostatic interactions between electronegative nanoparticles and
385
positive serum components can also not be ruled out. This possibility was well
386
supported by the zeta potential values observed after addition in to serum (Fig. 10b).
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Zeta potential of nanoparticle was increased (less negative values) dramatically due to
388
charge neutralization. After 24 h, surface charge of DXGP was -18.2 and -14.5 mV in
389
10% and 100% serum, respectively. Higher zeta potential in 100% serum indicated the
390
more interactions between nanoparticles and serum proteins leading to higher particle
391
size.
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The change in absorption pattern of DXGP with time was also observed over the
393
range of 350-750 nm. Interestingly, the absorption pattern in diluted serum (Fig. 10c)
394
was similar to that of spectra observed throughout the study but new absorption
395
spectrum was observed in 100% serum (Fig. 10d). It may be due to, as earlier
396
mentioned, adsorbed or electro-statically interacted serum components. However, no
397
change in absorption spectra was observed up to 24 h in both serum conditions. Overall,
398
DXGP were stable physically and chemically up to 24 h even under the extreme
399
conditions of serum.
400
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4.
Conclusions
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The applications of gold nanoparticles as multifunctional vehicle (drug delivery carrier,
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imaging) are increasing because of ease of synthesis, biocompatibility and opportunity for
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surface conjugation. In this investigation, gold nanoparticles were synthesized using natural
405
and biocompatible Xanthan gum as reducing and capping agent. Xanthan gum at very low
406
concentration of 1.5 mg/ml converted the ionic gold to neutral gold particles with a particle
407
size of 15-20 nm. Nanoparticles were found to be non-toxic and biocompatible in hemolysis
408
study. Doxorubicin was loaded to the nanoparticles through non-covalent interaction. The
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prepared carbohydrate-rich gold nanoparticles showed high drug loading, good colloidal
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stability and enhanced cytotoxicity in A549 lung cancer cells. High physical stability of gum 17 Page 17 of 36
stabilized nanoparticles can be attributed to steric repulsion among the particles provided by
412
same charge of gum moieties present on the nanoparticle surface. Receptor-mediated
413
endocytosis of nanoparticles could be a reason for better anticancer activity of DXGP than
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free drug. Presence of mannose in xanthan gum might have contributed this effect.
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Hence, natural gum based approach could be a better method for the synthesis of gold
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nanoparticles.
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Acknowledgements
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The research work was financially supported by Council of Scientific and Industrial
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Research (CSIR) grant under project Advanced Drug Delivery (ADD-CSC 0302).
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Doxorubicin hydrochloride was kindly provided by Therdose Pharma. HK thanks to the Head
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of IICT-RMIT research centre for providing the Junior Research Fellowship. DP is awarded
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Senior Research Fellowship by CSIR, New Delhi. Authors are also thankful to the Director,
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CSIR-Indian Institute of Chemical Technology, Hyderabad for providing the necessary
425
facilities.
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Figure legends
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Fig. 1. Optimization of different formulation variables for the synthesis of Xanthan gum stabilized gold nanoparticles. Effect of a) Xanthan gum concentration b) gum volume c) gold volume and d) gold concentration
560 561
Fig. 2. Effect of temperature a) and reaction time b) in the synthesis of XG stabilized gold nanoparticles
562 563
Fig. 3. Transmission electron microscopy (TEM) image of DOX loaded Xanthan gum stabilized gold nanoparticles (DXGP)
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Fig. 4. FTIR Spectra of a) pure Dox b) Native XG c) Blank GNP and d) Dox loaded GNP
565 566
Fig. 5. Differential scanning thermogram of a) pure DOX b) Dox loaded xanthan gum stabilized nanoparticles and c) Native xanthan gum
567 568
Fig. 6. In vitro drug release from DXNP in sodium acetate buffer pH 4.5 and phosphate buffer saline pH 7.4
569 570
Fig. 7. Percent cell viability of A549 human lung cancer cells after 48 h of treatment with free
571
Fig. 8. Storage stability of DXGP: a) particles diameter b) zeta potential and c) DOX content
572
Fig. 9. Effect of pH and ionic strength on the stability of DXGP
573 574
Fig. 10. Serum stability of DXGP a) particle size b) zeta potential c) absorption spectra in diluted (10%) serum and d) absorption spectra in undiluted (100%) serum
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DOX, BGNP and DXNP (Mean ± SD, n=3)
23 Page 23 of 36
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Figures
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a)
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616 617 618
b)
619 620 24 Page 24 of 36
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c)
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655 657 659 661 663
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666 667 668
Fig. 1. Optimization of different formulation variables for the synthesis of Xanthan gum stabilized gold nanoparticles. Effect of a) Xanthan gum concentration b) gum volume c) gold volume and d) gold concentration
669 25 Page 25 of 36
671 673 675 677
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a)
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702 704 706 708 710
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711 712
b)
713 714
Fig. 2. Effect of temperature a) and reaction time b) in the synthesis of XG stabilized gold nanoparticles
715 716 26 Page 26 of 36
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Fig. 3. Transmission electron microscopy (TEM) image of DOX loaded Xanthan gum stabilized gold nanoparticles (DXGP)
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a) 911.53 870.17 1523.92
3525.46
1729.37
1212.41 1235.55
2931.91 3336.70 1617.36 1582.51
1413.68 1384.31
b)
466.13 583.32
969.48 1114.22 1072.51 1005.97
1285.36
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2255.89 1984.06 2165.66 2079.45
789.59
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1403.97 1279.69 1614.79 1051.82
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728 729
3600
3200
1413.94
2800
2400
2000
734 735 736 737 738
1200
1000
800
600
450.0
Fig. 4. FTIR Spectra of a) pure Dox b) Native XG c) Blank GNP and d) Dox loaded GNP
d te
733
1400
Ac ce p
732
1600
cm-1
730 731
1800
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4000.0
2934.15
731.16 465.91 969.69 804.16 688.06 536.88 991.36 870.16 721.63 600.45 1005.37 761.60 583.10 1072.42 911.75 709.22 950.20 846.07 1114.33
1211.94 1235.28
739 740
28 Page 28 of 36
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Fig. 5. Differential scanning thermogram of a) pure DOX b) Dox loaded xanthan gum stabilized nanoparticles and c) Native xanthan gum
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750 751 752 753
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Fig. 6. In vitro drug release from DXNP in sodium acetate buffer pH 4.5 and phosphate buffer saline pH 7.4
an
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762 763 764 765 766 767
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768 769 770 771 772
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an
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Fig. 7. Percent cell viability of A549 human lung cancer cells after 48 h of treatment with free DOX, BGNP and DXNP (Mean ± SD, n=3)
M
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a)
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Ac ce p
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b)
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c)
795
Fig. 8. Storage stability of DXGP: a) particles diameter b) zeta potential and c) DOX content
M
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33 Page 33 of 36
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a)
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b)
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Fig. 9. a) Effect of pH and b) ionic strength on the stability of DXGP
814 815 816 34 Page 34 of 36
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a)
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b)
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c)
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Fig. 10. Serum stability of DXGP a) particle size b) zeta potential c) absorption spectra in diluted (10%) serum and d) absorption spectra in undiluted (100%) serum
835 836 837 838
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S0144-8617(14)00273-2 http://dx.doi.org/doi:10.1016/j.carbpol.2014.03.041 CARP 8703
To appear in: Received date: Revised date: Accepted date:
8-1-2014 18-2-2014 5-3-2014
Please cite this article as: Pooja, D., Panyaram, S., Kulhari, H., Rachamalla, S. S., & Sistla, R.,Xanthan gum stabilized gold nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.03.041 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.
1 2
Xanthan gum stabilized gold nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity
3 Deep Pooja,a Sravani Panyaram,a Hitesh Kulhari,a,b Shyam S Rachamalla,c Ramakrishna Sistlaa,* a
Medicinal Chemistry & Pharmacology Division, bIICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad, India.
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Faculty of Pharmacy, College of Technology, Osmania University, Hyderabad, India
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*Corresponding author:
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Sistla Ramakrishna, PhD
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Principal Scientist,
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Medicinal Chemistry & Pharmacology Division,
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CSIR-Indian Institute of Chemical Technology,
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Hyderabad-500007, India
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Phone: +91-40-27193753 (office)
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Email: [email protected]
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Highlights
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Gold nanoparticles were prepared using xanthan gum as reducing and capping agent.
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Different formulation and process variables were optimized. 1 Page 1 of 36
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XG stabilized nanoparticles were non-toxic and biocompatible.
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DOX loaded on GNP showed more therapeutic efficacy than free DOX in cancer cells.
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Formulations displayed colloidal stability against pH and ionic strength changes.
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Graphical Abstract
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Abstract
Xanthan gum (XG) has been widely used in food, pharmaceutical and cosmetic industries. In
64
the present study, we explored the potential of XG in the synthesis of gold nanoparticle. XG
65
was used as both reducing and stabilizing agent. The effect of various formulation and
66
process variables such as temperature, reaction time, gum concentration, gum volume and
67
gold concentration, in GNP preparation was determined. The XG stabilized, rubey-red XGNP
68
were obtained with 5 ml of XG aqueous solution (1.5 mg/ml). The optimum temperature was
69
80 ºC whereas the reaction time was 3 h. The optimized nanoparticles were also investigated
70
as drug delivery carrier for doxorubicin hydrochloride. DOX loaded gold nanoparticles
71
(DXGP) were characterized by dynamic light scattering, TEM, FTIR, and DSC analysis. The
72
synthesized nanoparticle showed mean particle size of 15-20 nm and zeta potential -29.1 mV.
73
The colloidal stability of DXGP was studied under different conditions of pH, electrolytes
74
and serum. Nanoparticles were found to be stable at pH range between pH 5-9 and NaCl
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concentration up to 0.5 M. In serum, nanoparticles showed significant stability up to 24 h.
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During toxicity studies, nanoparticles were found biocompatible and non-toxic. Compared
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with free DOX, DXGP displayed 3 times more cytotoxicity in A549 cells. In conclusion, this
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study provided an insight to synthesize GNP without using harsh chemicals.
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Key words: Xanthan Gum; Gold nanoparticles; Reducing agent, Doxorubicin, Cytotoxicity, Stability
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1.
Introduction
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Xanthan gum (XG) is an anionic, high molecular weight, exo-polysaccharide
94
produced by aerobic fermentation of sugars by Xanthomonas campestris. Its basic chain
95
consists of a β-(1→4) linked glucose backbone with the substitution of a charged
96
trisaccharide side-chains of [β-(1→3)-mannose-α-(1→2)-glucuronic acid-β-(1→4)-mannose]
97
on alternate glucose residues (Jian, Zhu, Zhang, Sun, & Jiang, 2012). It is non-toxic,
98
hydrophilic and biodegradable bio-polymer. XG is being used in food, cosmetic and
99
pharmaceutical industries. The industrial applications of XG are based upon its exceptional
100
rheological properties. XG is soluble in both cold and hot water, hydrates quickly and
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produces high viscosity at low concentration (Sereno, Hill, & Mitchell, 2007; Sharma,
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Naresh, Dhuldhoya, Merchant, Merchant, 2006). In pharmaceutical industries, XG has been
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reported for formulation of both solid and liquid dosage forms. In solid dosage formulation, it
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is used as controlled release agent (Jian et al., 2012; Phaechamud and Ritthidej, 2007; Santos,
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H., Veiga, F., Pina, M. E., & Sousa, 2005; Sinha, Mittal, Bhutani, & Kumaria, 2004) whereas
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in liquid formulations, it is used as thickening agent, suspending agent and emulsion
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stabilizer (Desplanques, Renou, Grisel, & Malhiac, 2012).
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Recently, XG has also been used in the preparation and stabilization of inorganic iron
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and palladium nanoparticles (Fan et al., 2013; Xue & Sethi, 2012; Comba , Dalmazzo,
110
Santagata, & Sethi, 2011; Vecchia, Luna, & Sethi, 2009). Nanoparticles are solid structures
111
with a size below 200 nm and have found their applications in sensing, imaging and in drug
112
and gene delivery. Gold nanoparticles (GNP) are one of the most commonly explored and
113
used nanoparticles in drug delivery because of controlled size, improved efficacy and
114
targeted delivery (Almeida, Figueroa, & Drezek; 2013; Pissuwan, Niidome, & Cortie, 2011).
115
As drug delivery carrier, GNP has been used for the delivery of both hydrophilic and
116
hydrophobic drugs (Oliveira et al., 2013; Chen et al., 2007; Gibson, Khanal, & Zubarev,
117
2007; Aryal, Grailer, Pilla, Steeber, & Gong, 2009; Prabaharan M, Grailer JJ, Pilla S, Steeber
118
DA, & Gong, 2009). But, the conventional synthesis of GNP involves the use of chemicals
119
like sodium borohydrate, tri-sodium citrate etc as reducing agent (Burygin et al., 2009;
120
Duncan, Kim, & Rotello, 2010; Khan, Jung et al., 2013; Vishakante, & Siddaramaiah, 2013;
121
Saha et al., 2007). These GNP have been found to be very unstable and form aggregates with
122
the slight change in pH and electrolyte concentration (Mirza and Shamshad, 2011; Rouhana,
123
Jaber, & Schlenoff, 2007). The GNP prepared with gellan gum showed the stability between
124
pH 4 to 8 and addition of NaCl up to 0.1M (Dhar, Reddy, Shiras, Pokharkar, & Prasad,
125
2008). Natural gums stabilize the inorganic nanoparticles by two mechanisms: first, by
126
adsorbing to the surface of nanoparticles which creates steric repulsion among the particles.
127
In second mechanism, they increase the viscosity of nanoparticle suspension, and therefore
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5 Page 5 of 36
slow down the aggregation processes (Tiraferri, Chen, Sethi, & Elimelech, 2008; Xue &
129
Sethi, 2012; Comba & Sethi, 2009). The aim of this investigation was to synthesize the
130
environment friendly gold nanoparticles using XG as reducing agent. The nanoparticle
131
preparation was optimized for gum concentration and volume, gold concentration,
132
temperature and reaction time. Gum stabilized GNP were studied for its utility as drug
133
delivery carrier using Doxorubicin hydrochloride as model drug. Doxorubicin hydrochloride
134
is hydrophilic in nature and has been clinically used for the treatment of various cancers,
135
haematological malignancies, soft tissue sarcomas and solid tumors (Carvalho et al., 2009;
136
Laginha, Verwoert, Charrois, & Allen, 2005). Two measure limitations of DOX are non-
137
specific cytotoxicity and multi drug resistance. Multi drug resistance to DOX is due to drug
138
efflux by P-glycoproteins. Doxorubicin is substrate for P-glycoproteins which efflux DOX
139
and decrease intracellular drug level. DOX loaded GNP have shown to overcome both the
140
problems of non-specific toxicity and drug resistance (Gu, Cheng, Man, Wong, & Cheng,
141
2012). The prepared nanoparticles were also investigated for biocompatibility, stability and
142
cytotoxicity study in lung cancer cells.
143
2.
144
2.1.
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Materials and methods Materials
Xanthan gum and Tetrachloroauric acid (HAuCl4) were purchased from Sigma-
146
Alderich (St Louis, MO, USA). Doxorubicin hydrochloride was received as gift sample from
147
TherDose pharma pvt ltd (Hyderabad, India). The chemical used for buffer preparations were
148
of analytical grade and were purchased from sd Fine-Chem Ltd. (Hyderabad, India). MTT (3-
149
(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromide, Dulbecco’s modified eagle
150
medium (DMEM), trypsin–EDTA, fetal bovine serum (FBS) and antibiotic solution (10,000
151
U/ml penicillin, 10 mg/ml streptomycin) were purchased from Sigma-Aldrich (St Louis, MO, 6 Page 6 of 36
152
USA). Cell culture plastic wares were obtained from Tarsons Products Pvt. Ltd. (Kolkata,
153
India). All the formulations were prepared in MilliQ water.
154
2.2.
Preparation of gum solution The stock solution of gum was prepared by dissolving 500 mg of the gum in 100 ml
156
water and was stirred overnight at room temperature. The solution was centrifuged to remove
157
the insoluble materials and supernatant was lyophilized. The lyophilized dry powder was
158
dissolved in water to get desired concentration of xanthan gum.
159
2.3.
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Synthesis of gold nanoparticles
Gold nanoparticles were prepared by reducing the aqueous solution of HAuCl4 by
161
heating at 80 ºC in the presence of XG solution (1.5 mg/ml). The change in colour was
162
obtained from colourless to purple to rubey-red after 2 h. The colloidal solution was cooled at
163
room temperature and stored in amber colour vials at 4 ºC. The nanoparticle formulation
164
showed the absorption maxima at 525 nm.
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2.4.
Optimization of formulation and process variables
The effect of formulation and process variables such as gum concentration, gold
168
concentration, gold to gum volume ratio, reaction time and temperature were studied by
169
changing one parameter at a time and keeping other constant.
170
2.5.
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2.5.1. Hydrodynamic diameter and polydispersity
Characterizations of Xanthan gum stabilized gold nanoparticles(XGNP)
172
The hydrodynamic diameter and polydispersity index of GNP were determined by
173
dynamic light scattering using Zetasizer Nano-ZS (Malvern instrument Ltd., Malvern, UK). 7 Page 7 of 36
Before measurement, the samples were diluted appropriately to get particle count rate
175
between 100-300 kcps and instrument was set up at 25 ºC with a backscattering angle of
176
173°.
177
2.5.2. Transmission electron microscopy (TEM)
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A drop of sample was placed on carbon coated copper grid, air dried at room
179
temperature and stained with 2% uranyl acetate. The nanoparticle size measurement was
180
done using transmission electron microscope (Hitachi, H-7500) and average of 10
181
nanoparticle size was considered as the size of the sample.
182
2.5.3. Surface charge
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The electrokinetic properties of the GNP were determined by measuring the zeta
184
potential using Zetasizer Nano-ZS. The samples were diluted 10 times with MilliQ water
185
before the measurement.
186
2.5.4. Fourier transform infrared analysis
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An amount of 2 mg of sample was mixed with 100 mg of potassium bromide and
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compressed to form a pellet. The pellet was placed in pellet holder and scanned for the
189
measurement of % transmittance in the wave number range of 4000 to 450 cm-1 using FTIR
190
spectrophotometer (Perkin Elmer, USA). The spectra requisition was carried out using the
191
software Spectrum One (Perkin Elmer, USA).
192
2.6.
Doxorubicin loading to nanoparticles
193
Blank gold nanoparticles were dispersed in phosphate buffer saline and were incubated
194
with 1 mg of DOX solution (1 mg/ml) at room temperature overnight. The nanoparticles
195
dispersion was centrifuged at 15000 rpm for 30 min. DOX loading was determined by
8 Page 8 of 36
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measuring the free drug content in supernatant. Percent drug loading was calculated as
197
follows: % DOX loading = (1-DS/DT) × 100
199
Whereas, DS is amount of DOX present in supernatant, DT = Total amount of DOX
200
loaded initially.
201
2.7.
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In vitro drug release studies
The in vitro drug release studies were performed using dialysis method. In dialysis
203
tubing, the nanoparticles equivalent to 1 mg of DOX were dispersed in 1 ml of distilled water
204
and placed in 100 ml of release medium (phosphate buffer saline pH 7.4 and sodium acetate
205
buffer pH 4.5) at 37 ºC in dark. Three millilitres of sample was withdrawn at different time
206
intervals up to 12 hours and was replaced with same volume of fresh medium. The samples
207
were analyzed for drug content using UV/VIS spectrophotometer (Perkin Elmer, Lambda
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25, USA) at 480 nm.
209
2.8.
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The biocompatibility of prepared gold nanoparticles was determined by estimating the
211
% hemolysis, caused by nanoparticles. The assay procedure reported by Kumar et al. 2011,
212
was used with slight modification (Kumar, Paul, Sharma, 2011). Blank gold nanoparticles
213
were dispersed in normal physiological saline (0.9 %w/v NaCl). Varying concentrations (50-
214
200 µg/ml) of the dispersion were added into red blood cells suspension (2 %v/v), mixed well
215
and incubated at 37 °C. After 1 h, the samples were centrifuged at 3000 rpm for 5 min and
216
absorbance of supernatant was measured at 540 nm spectrophotometrically. The supernatant
217
absorbance of RBC suspension treated with distilled water and normal saline were taken as
218
standard and control; respectively. The % hemolysis was calculated as: % Hemolysis =
219
(Asample – Acontrol) / (Astandard - Acontrol) × 100
220
2.9.
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Cytotoxicity studies 9 Page 9 of 36
Cellular toxicity of pure DOX and DXNP was evaluated by MTT assay in A549 human
222
lung cancer cells. The assay is based on the mitochondrial reduction of yellow MTT, a
223
tetrazolium dye, to a highly coloured blue formazan product. About 10000 cells per well were
224
cultured in 96 wells plate and allowed to attach overnight. Next day, cells were incubated
225
with different concentration (0.1-10 µg/ml) of pure doxorubicin, blank gold nanoparticles
226
(positive control) and doxorubicin loaded gold nanoparticles. Cells were incubated in CO2
227
incubator at 37 °C for 48 h. The solution of each well was replaced with fresh serum free
228
media and 10 µl of MTT reagent (5 mg/ml) was added. After 4 h, the media was removed,
229
200 µl DMSO was added and incubated for 20 min. The absorbance was measured at 570 nm
230
using Spectra Max plus 384 UV-Visible plate reader. The cell viability was determined as a
231
percent based on the absorbance measured relative to the absorbance of untreated or control
232
cells. The half maximum inhibitory concentration (IC50) values were determined by non-
233
regression analysis using GraphPad Prism v. 5.03 (GraphPad Software Inc. CA).
234
2.10. Stability studies
235
2.10.1. Storage stability
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Doxorubicin loaded nanoparticles were stored at 4 °C and were studied for particle
237
size, zeta potential and drug content up to 1 month. Particle size and zeta potential were
238
determined by particle size analyzer and DOX content was determined using UV/VIS
239
spectrophotometer.
240
2.10.2. Effect of pH and ionic concentration of medium
241
For the determination of effect of pH on the stability, DXGP were incubated in
242
different pH medium (pH 3 to pH 11). Nanoparticles incubated in phosphate buffer saline pH
243
7.4 were taken as control. Absorbance was measured after 24 h and compared with
244
absorbance of control. 10 Page 10 of 36
The effect of ionic strength on the stability of DXNP was determined by incubating the
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nanoparticles in deionised water containing varying concentrations (0.1-2M) of sodium
247
chloride (NaCl).
248
2.10.3. Serum Stability of DXGP
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Serum stability of DXGP was evaluated at two different serum concentration
250
levels (10%v/v and 100% serum). Nanoparticles were dispersed in serum and particles
251
size, zeta potential and absorbance were measured at different time intervals up to 72 h.
252
2.11. Statistical analysis
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All the experiments were performed in triplicates. Data are expressed as Mean ± SD
254
(standard deviation) (n=3). The values of in vitro drug release, cytotoxicity and stability
255
studies were statistically analyzed using student t-test. Statistical significance level was set at
256
p < 0.05.
257
3.
Result and discussion
258
3.1.
Preparation and optimization of gold nanoparticles
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Gold nanoparticles were prepared by chemical reduction method using xanthan gum as
260
reducing agent. The role of gum concentration was studied by using gum solution in the
261
range of 0.5-2 mg/ml containing 10 mM of HAuCl4 concentration (Fig. 1a). The absorbance
262
in the UV spectrum which describes the reduction of GNP, was found to be directly
263
related to gum concentration. The intensity of the absorption band gradually increased and
264
the maximum intensity of absorption was attained at 1.5 mg/ml which indicated the
265
maximum gold nanoparticle concentration.
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The effect of gold to gum volume ratio was evaluated by varying the gum volume in
267
the range of 2.5-10 ml (Fig. 1b). At lower gum volume (2.5 ml) a peak shift was observed 11 Page 11 of 36
and the absorption band shifted to 550 nm which suggested that lower gum volume was not
269
sufficient to reduce all Au ions. When the gum volume was changed from 5 to 7.5 ml
270
followed by 10 ml, there was no significant shift in the characteristic absorption band but the
271
absorbance was decreased. The maximum intensity of absorption was attained at 5 ml and
272
was considered optimum gum volume for the synthesis of XGNP. The effect of gold
273
concentration (Fig. 1c) and volume (Fig. 1d) was also studied and was found as per earlier
274
reports. A volume of 100 µl of 10 mM gold solution showed better absorbance at 525 nm.
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Reaction temperature and time are two important process parameters in the synthesis
276
on gold nanoparticles. In this study, the temperature was changed in the range of 60-90 °C
277
with 10 mM HAuCl4 concentration and 1.5 mg/ml gum solution (Fig. 2a). The maximum
278
intensity of the characteristic absorption band was attained at 80 ˚C which indicates the
279
maximum gold nanoparticle concentration. For the optimization of reaction time,
280
nanoparticle synthesis was studied up to 4 h. The absorbance was found to be increased up to
281
2 h and after that there was no change in the absorbance (Fig. 2b).
282
3.2.
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Characterization of Xanthan gum stabilized gold nanoparticles The hydrodynamic diameter and polydispersity index of optimized XGNP were
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determined using dynamic light scattering method. The observed mean hydrodynamic
285
diameter was 41.2 ± 3.78 nm. The prepared nanoparticles showed high polydispersity (0.35-
286
0.6) which can be attributed to presence of wide range of particles. The high negative surface
287
potential (-47.2±2.59) indicated the high stability of nanoparticles and presence of gum
288
molecules on nanoparticle surface. The presence of acetyl group on O-6 position and pyruvic
289
acid linked with mannose sugar are responsible for the negative charge. After DOX loading,
290
the surface charge was decreased to -29.1±2.78 mV indicating the interaction between
291
positively charged amine group of DOX and negative acidic group of gum. The nanoparticle
292
surface morphology and actual particle size were determined by transmission electron 12 Page 12 of 36
293
microscopy. Figure 3 showed that nanoparticles were spherical in shape and 15-20 nm in
294
size. The chemical interactions between DOX and nanoparticles were studied by FTIR
296
analysis (Fig. 4). In XG spectra, the vibration peak of hydrogen bonded −OH and C–H
297
stretching were observed at 3416 and 2902 cm−1, respectively. The vibration peak of the
298
acetal was found at 1051.8 cm−1 whereas C=O stretching peak was found at 1728.4 cm−1. The
299
FITR spectra of blank nanoparticles exhibited all the characteristic peaks of XG at 3398,
300
1728 and 1042 cm−1. The peak at 1254.2 cm−1 presented the C-O-C group. The DXGP
301
showed principal IR peaks of both XG and DOX showing the non-covalent binding of DOX
302
to nanoparticles.
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DSC is a useful technique to explain the thermal stability and phase transitions. Figure
304
5 illustrates the DSC thermograms of pure DOX, native XG and DXGP. DSC scan of XG
305
exhibited endothermic transition at 125 °C. The endothermic peak of DOX appeared at its
306
melting point 195 °C. This peak was disappeared in DXGP spectra indicating that DOX was
307
not precipitated on nanoparticle surface but was present as molecular or amorphous
308
dispersion.
309
3.3.
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DOX loading
The observed doxorubicin loading was found to be 71.4 ± 2.53%. The high drug
311
loading can be attributed to presence of XG on the nanoparticle surface. Xanthan gum
312
contains negatively charged moieties such as pyruvic acid which interacts with positively
313
charged amine group of doxorubicin.
314
3.4.
In vitro drug release studies
13 Page 13 of 36
In vitro drug release from the nanoparticles was studied in PBS and SAB buffer (Fig.
316
6). The nanoparticles showed complete drug release within 10 h. Up to 4 h, there was no
317
significant difference (p > 0.05) in doxorubicin release in both the medium but at the end
318
DOX release was higher in SAB (98.1%) as compared to PBS (83.6%). The faster release of
319
DOX in SAB can be attributed to protonation of amine groups of DOX molecules which
320
increases hydrophilicity and solubility at lower pH. The protonation of amine group also
321
leads to decreased interaction between DOX and GNP (Minati et al., 2012). The PBS pH 7.4
322
is a simulated condition for blood plasma whereas SAB pH 4.5 is for the internal
323
environment of cancer cell. Thus the slower release of DOX in PBS and faster release in SAB
324
would help to release the maximum amount of drug in tumour and hence to enhance the
325
therapeutic efficacy of the formulation.
326
3.5.
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Hemolytic toxicity
Nanoparticles are reported to cause oxidative-stress induced hemolysis (Teodora,
328
2013). Therefore, the suitability for intravenous administration of nanoparticles is generally
329
determined by hemolytic study. In this study, dose-dependent hemolytic toxicity of xanthan
330
gum stabilized gold nanoparticle was carried out. The prepared nanoparticles did not show
331
hemolysis up to 200 µg/ml.
332
3.6.
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Cytotoxicity studies
Anti-proliferative activity of doxorubicin loaded xanthan gum stabilized gold
334
nanoparticles (DXGP) and free DOX was studied in A549 human lung cancer cells. The cell
335
viability was determined by MTT assay after 48 h of incubation with different concentrations
336
(Fig. 7). Blank xanthan stabilized gold nanoparticles (BXNP) were taken as positive control
337
and did not decrease the cell viability at any test concentration. It suggested that the prepared
338
nanoparticles were non-toxic and biocompatible. Drug loaded nanoparticles showed 14 Page 14 of 36
concentration-dependent cytotoxicity. The cell viability was decreased with the increase in
340
DOX concentration. The IC50 value for pure DOX and DXGP was 2.46 and 0.79 µg/ml,
341
respectively. The lower IC50 value of DXGP indicated the more cytotoxicity which can be
342
attributed to enhanced cellular uptake and hence increased intracellular drug concentration.
343
However, the exact mechanism and role of carbohydrate moieties present on nanoparticle in
344
cellular uptake require detailed investigation. One possible reason may be due to the
345
presence of mannose sugars in the composition of xanthan gum which was used for the
346
stabilization of GNP. It is documented that mannose receptors are up regulated in
347
human lung cancer cells A549 (Basuki et al., 2014; El-Boubbou et al., 2010). Basuki et al
348
reported the improvement in cell uptake of mannose functionalized iron nanoparticles
349
in human lung cancer cells A549. Hence, the presence of mannose in xanthan gum
350
might have contributed to increase in uptake of drug through receptor-mediated
351
endocytosis pathway.
d Stability studies
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3.7.
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The colloidal stability of DXGP was studied for both physical and chemical
355
stabilities. The change in particle size and zeta potential are shown in Fig. 8. No significant
356
(p>0.05) changes in particle size and zeta potential were observed up to 15 days. But,
357
chemical degradation of DXNP was faster than physical instability. The DOX content was
358
less than 90% within 24 hours. However, the DOX degradation was slower with DXGP in
359
comparison to pure DOX. The observed drug degradation rate constant (K) was 8.28 × 10-3
360
and 5.07 × 10-3 day-1 for pure DOX and DXGP (Fig. 8c). Therefore, decease in drug content
361
was due to light sensitivity of DOX itself not because of formulation.
15 Page 15 of 36
DXGP showed a wide range of pH stability (pH 5-pH 9). The observed absorbance of
363
DXGP at 480 nm was 1.109, 0.899, 0.908, 0.927 and 0.776 for pH 3, 5, 7, 9 and pH 11
364
respectively (Fig. 9a). The absorbance of control sample was 0.931. The results suggested
365
that XG or carbohydrate capping of nanoparticles helped to increase the colloidal stability of
366
gold nanoparticles. The decrease in absorbance at pH 11 may due to gel formation properties
367
of XG at higher pH levels (pH > 10) (Sharma et al., 2006).
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In other study, DXGP were treated with different NaCl concentration to determine the
369
stability of nanoparticles in varying ionic concentrations. Figure 9b showed the absorbance
370
pattern of DXGP after 24 h of incubation with different NaCl concentrations. The control
371
group was incubated in deionised water without NaCl. The DXGP were found to be stable up
372
to 0.5M NaCl concentration.
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The stability of DXGP was further evaluated at two serum conditions-diluted
374
(10% v/v serum) and undiluted or 100% serum. Figure 10a showed the particle
375
diameter of DXGP exposed to serum, after different time period. In both the conditions,
376
particle size was increased continuously up to 72 h. But, the increase in particle
377
diameter was more in undiluted serum than diluted serum. After 2 h, particle size was
378
increased by 7.1 nm in 10% serum wile it increased by 31.5 nm in 100% serum. From
379
the figure 10a it is clear that DXGP were stable up to 24 h in both the conditions but
380
after that size of nanoparticle was increased tremendously. The increase in particle size
381
can be explained by the adsorption of serum proteins or other components on
382
nanoparticle surface. (Yang, Zhang, Wang, White, & Jiang, 2014; Zang & Zang, 2010).
383
The nanoparticle dispersion in distilled water had a high negative zeta potential of -30.2
384
mV and thus the electrostatic interactions between electronegative nanoparticles and
385
positive serum components can also not be ruled out. This possibility was well
386
supported by the zeta potential values observed after addition in to serum (Fig. 10b).
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16 Page 16 of 36
Zeta potential of nanoparticle was increased (less negative values) dramatically due to
388
charge neutralization. After 24 h, surface charge of DXGP was -18.2 and -14.5 mV in
389
10% and 100% serum, respectively. Higher zeta potential in 100% serum indicated the
390
more interactions between nanoparticles and serum proteins leading to higher particle
391
size.
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The change in absorption pattern of DXGP with time was also observed over the
393
range of 350-750 nm. Interestingly, the absorption pattern in diluted serum (Fig. 10c)
394
was similar to that of spectra observed throughout the study but new absorption
395
spectrum was observed in 100% serum (Fig. 10d). It may be due to, as earlier
396
mentioned, adsorbed or electro-statically interacted serum components. However, no
397
change in absorption spectra was observed up to 24 h in both serum conditions. Overall,
398
DXGP were stable physically and chemically up to 24 h even under the extreme
399
conditions of serum.
400
402
4.
Conclusions
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The applications of gold nanoparticles as multifunctional vehicle (drug delivery carrier,
403
imaging) are increasing because of ease of synthesis, biocompatibility and opportunity for
404
surface conjugation. In this investigation, gold nanoparticles were synthesized using natural
405
and biocompatible Xanthan gum as reducing and capping agent. Xanthan gum at very low
406
concentration of 1.5 mg/ml converted the ionic gold to neutral gold particles with a particle
407
size of 15-20 nm. Nanoparticles were found to be non-toxic and biocompatible in hemolysis
408
study. Doxorubicin was loaded to the nanoparticles through non-covalent interaction. The
409
prepared carbohydrate-rich gold nanoparticles showed high drug loading, good colloidal
410
stability and enhanced cytotoxicity in A549 lung cancer cells. High physical stability of gum 17 Page 17 of 36
stabilized nanoparticles can be attributed to steric repulsion among the particles provided by
412
same charge of gum moieties present on the nanoparticle surface. Receptor-mediated
413
endocytosis of nanoparticles could be a reason for better anticancer activity of DXGP than
414
free drug. Presence of mannose in xanthan gum might have contributed this effect.
415
Hence, natural gum based approach could be a better method for the synthesis of gold
416
nanoparticles.
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Acknowledgements
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The research work was financially supported by Council of Scientific and Industrial
420
Research (CSIR) grant under project Advanced Drug Delivery (ADD-CSC 0302).
421
Doxorubicin hydrochloride was kindly provided by Therdose Pharma. HK thanks to the Head
422
of IICT-RMIT research centre for providing the Junior Research Fellowship. DP is awarded
423
Senior Research Fellowship by CSIR, New Delhi. Authors are also thankful to the Director,
424
CSIR-Indian Institute of Chemical Technology, Hyderabad for providing the necessary
425
facilities.
426
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547 548 549
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552
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553 554
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Figure legends
557 558 559
Fig. 1. Optimization of different formulation variables for the synthesis of Xanthan gum stabilized gold nanoparticles. Effect of a) Xanthan gum concentration b) gum volume c) gold volume and d) gold concentration
560 561
Fig. 2. Effect of temperature a) and reaction time b) in the synthesis of XG stabilized gold nanoparticles
562 563
Fig. 3. Transmission electron microscopy (TEM) image of DOX loaded Xanthan gum stabilized gold nanoparticles (DXGP)
564
Fig. 4. FTIR Spectra of a) pure Dox b) Native XG c) Blank GNP and d) Dox loaded GNP
565 566
Fig. 5. Differential scanning thermogram of a) pure DOX b) Dox loaded xanthan gum stabilized nanoparticles and c) Native xanthan gum
567 568
Fig. 6. In vitro drug release from DXNP in sodium acetate buffer pH 4.5 and phosphate buffer saline pH 7.4
569 570
Fig. 7. Percent cell viability of A549 human lung cancer cells after 48 h of treatment with free
571
Fig. 8. Storage stability of DXGP: a) particles diameter b) zeta potential and c) DOX content
572
Fig. 9. Effect of pH and ionic strength on the stability of DXGP
573 574
Fig. 10. Serum stability of DXGP a) particle size b) zeta potential c) absorption spectra in diluted (10%) serum and d) absorption spectra in undiluted (100%) serum
Ac ce p
te
d
M
556
DOX, BGNP and DXNP (Mean ± SD, n=3)
23 Page 23 of 36
575
Figures
577 579
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581 583
cr
585 587
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589 591
595 596
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a)
597
606 608 610 612 614
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Ac ce p
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d
598 600
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593
616 617 618
b)
619 620 24 Page 24 of 36
622 624 626
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628 630
cr
632 634
us
636 638
642 643
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c)
645
655 657 659 661 663
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653
Ac ce p
651
d
647 649
an
640
664 665
d)
666 667 668
Fig. 1. Optimization of different formulation variables for the synthesis of Xanthan gum stabilized gold nanoparticles. Effect of a) Xanthan gum concentration b) gum volume c) gold volume and d) gold concentration
669 25 Page 25 of 36
671 673 675 677
ip t
679 681
cr
683
us
685 687
690
a)
M
691 692
702 704 706 708 710
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Ac ce p
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d
694 696
an
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711 712
b)
713 714
Fig. 2. Effect of temperature a) and reaction time b) in the synthesis of XG stabilized gold nanoparticles
715 716 26 Page 26 of 36
ip t cr us
718 719
an
717
Fig. 3. Transmission electron microscopy (TEM) image of DOX loaded Xanthan gum stabilized gold nanoparticles (DXGP)
M
720 721
725 726
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Ac ce p
723
d
722
27 Page 27 of 36
a) 911.53 870.17 1523.92
3525.46
1729.37
1212.41 1235.55
2931.91 3336.70 1617.36 1582.51
1413.68 1384.31
b)
466.13 583.32
969.48 1114.22 1072.51 1005.97
1285.36
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2255.89 1984.06 2165.66 2079.45
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728 729
3600
3200
1413.94
2800
2400
2000
734 735 736 737 738
1200
1000
800
600
450.0
Fig. 4. FTIR Spectra of a) pure Dox b) Native XG c) Blank GNP and d) Dox loaded GNP
d te
733
1400
Ac ce p
732
1600
cm-1
730 731
1800
M
727
4000.0
2934.15
731.16 465.91 969.69 804.16 688.06 536.88 991.36 870.16 721.63 600.45 1005.37 761.60 583.10 1072.42 911.75 709.22 950.20 846.07 1114.33
1211.94 1235.28
739 740
28 Page 28 of 36
ip t cr us
741
743 744
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Fig. 5. Differential scanning thermogram of a) pure DOX b) Dox loaded xanthan gum stabilized nanoparticles and c) Native xanthan gum
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750 751 752 753
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29 Page 29 of 36
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Fig. 6. In vitro drug release from DXNP in sodium acetate buffer pH 4.5 and phosphate buffer saline pH 7.4
an
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30 Page 30 of 36
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Fig. 7. Percent cell viability of A549 human lung cancer cells after 48 h of treatment with free DOX, BGNP and DXNP (Mean ± SD, n=3)
M
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31 Page 31 of 36
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b)
32 Page 32 of 36
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c)
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Fig. 8. Storage stability of DXGP: a) particles diameter b) zeta potential and c) DOX content
M
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33 Page 33 of 36
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Fig. 9. a) Effect of pH and b) ionic strength on the stability of DXGP
814 815 816 34 Page 34 of 36
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Fig. 10. Serum stability of DXGP a) particle size b) zeta potential c) absorption spectra in diluted (10%) serum and d) absorption spectra in undiluted (100%) serum
835 836 837 838
36 Page 36 of 36