International Journal of Pharmaceutics 473 (2014) 636–643

Pharmaceutical nanotechnology

Antibacterial efficacy of acridine derivatives conjugated with goldnanoparticles

Piyali Mitra a, Prabal Kumar Chakraborty b, Partha Saha b, Pulak Ray c, Samita Basu a,*aChemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, IndiabCrystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, IndiacBiophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India

A R T I C L E I N F O

Article history:Received 9 June 2014Received in revised form 25 July 2014Accepted 26 July 2014Available online 1 August 2014

Keywords:NanoparticlesCovalently modifiedAntibacterial efficacyTransmission electron microscope

A B S T R A C T

Adsorption of acridine derivatives viz. 9-aminoacridine hydrochloride hydrate (9AA-HCl), acridineyellow (AY), acridine orange (AO), and proflavine (Pro) on citrate stabilized gold nanoparticle surfacewere studied using different analytical techniques like UV–vis absorption spectroscopy, Fouriertransform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). The amine moietyof acridine derivative binds strongly to the gold nanoparticles as confirmed by spectroscopic studies. Theplasmon band observed for the wine red colloidal gold at 525 nm in the UV–vis spectrum is characteristicof gold nanoparticles. However, with the addition of acridine derivatives the intensity of the absorptionband at 525 nm decreases and a new peak emerges at red-end region – a signature of formation ofgold-drug complex. The TEM images show the average size of citrate stabilized gold nanoparticles as15–20 nm, which becomes larger in the presence of various drugs due to aggregation. From thethermogravimetric analyses (TGA) we have measured the number of drug molecules attached per goldnanoparticle (AuNP). These gold nanoparticles are very important as drug delivery vehicles and forclinical applications it is necessary to understand their activity in vivo. The antibacterial efficacy of drugscoated gold nanoparticles were studied against various strains of Gram positive and Gram negativebacteria. Among the four drugs, 9AA-HCl and AO showed antibacterial activity and for both of them theAuNP conjugated drug showed better antibacterial efficacy than the bare drug. Because of the highpenetrating power and large surface area of Au(0), a single gold nanoparticle can adsorb multiple drugmolecules, hence this total entity acts as a single group against the bacteria.

ã 2014 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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1. Introduction

In recent years, acridine and its derivatives have attractedmuch attention owing to their immense pharmacologicalimportance (Kragh-Hansen, 1981; Li et al., 2006; Salman et al.,2000). 9-Aminoacridine hydrochloride hydrate (9AA-HCl), acri-dine yellow (AY), acridine orange (AO), and proflavine (Pro) arethe important fluorescent dyes belonging to those families. Thesedyes and their derivatives are extensively used in biological fieldsowing to their strong interaction with biomolecules such as DNAand proteins (Bentin and Nielsen, 2003; Kononov, 2001; Lee andGalley, 1988; Long et al., 2007; Lyles and Cameron, 2002;Veldhuyzen et al., 2003; Wesley et al., 1990) and theirphotochemical reactions in different media (Junior et al.,

* Corresponding author. Tel.: +91 33 2337 5345; fax: +91 33 2337 4637.E-mail address: samita.basu@saha.ac.in (S. Basu).

http://dx.doi.org/10.1016/j.ijpharm.2014.07.0510378-5173/ã 2014 Elsevier B.V. All rights reserved.

2003). Additionally, these acridine-based drugs are well-knownfor their antifungal, antibacterial, and antimalarial properties(Loechler and King, 1986; Zhu et al., 1994). These dyes also act asmodel photosensitizers in photodynamic therapy (PDT) and thushave drawn the interest of researchers (Schmidt et al., 1981).Among them, 9AA-HCl has been proposed as a specific fluorescentprobe capable of binding the active center of guanidinebenzoatases (GB) (Murza et al., 2000). In fact, it has been usedto locate malignant cells in many tumor tissues (Steven et al.,1993) and used as fluorescent markers for detecting andinhibiting tumors. On the other hand AO is useful for studyingthe photophysics and molecular dynamics of DNA (Brauns et al.,1998). AO is also used as biological stains in fluorescencemicroscopy (Petit et al., 1993). The spectroscopic andphotophysical properties of these acridine derivatives inhomogeneous and micro heterogeneous media are very helpfulfor a better understanding of the nature of binding andbiodistribution of this kind of dye inside the living cells. In our

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earlier work we have already studied the photophysics of9AA-HCl in different micellar environments (Mitra et al., 2013).

Now a days, nanotechnology offers tremendous potential formedical diagnosis and therapy as it bears a successful impact onbiological and medicinal processes occurring at nanometer length(West and Halas, 2000; Zandonella, 2003). Recently, goldnanoparticles (AuNPs) based drug/gene delivery systems haveattracted attention due to their functional versatility, biocom-patibility, and low toxicity (Bhattacharya and Mukherjee, 2008;Connor et al., 2005; De et al., 2008; Templeton et al., 2000;Vigderman and Zubarev, 2013). AuNPs are multipurpose agentswith numerous applications in biomedicine like diagnostic assay(Goodman et al., 2004), thermal ablation, radiotherapy enhance-ment (Hainfeld et al., 2004; Hirsch et al., 2003) as well as used forthe detection of antigen in conjugation with an antibody (Namet al., 2003). The size of the AuNPs can be tuned from 2 to 100 nmwith correspondingly large surface-to-volume ratios (Daniel andAstruc, 2004). In addition, the shape and the orientation can alsobe manipulated by applying different techniques such aspolarization dependent two-photon luminescence (TPL), etc.(Senyuk et al., 2012). Due to their large surface area to volumeratio and biocompatibility, AuNPs are considered as idealcandidates for carrying large amounts of antibiotics withoutcompromising their activity and also significant progress wasobserved in the delivery of large biomacromolecules, includingDNA, siRNA, and proteins (Aryal et al., 2009; Ma et al., 2009;Pornpattananangkul et al., 2010; Wijaya et al., 2009; Yavuz et al.,2009). Many reports are available in the literature on theantimicrobial effect of antibiotics conjugated with differentnanoparticles (Grace and Pandian, 2007; Gu et al., 2003; Liet al., 2005; Nepal et al., 2008; Rosemary et al., 2006; Saha et al.,2007). The advantage of using both antibiotics and inorganicnanoparticles together is that if bacteria have resistance againstone of the components, a further component could kill them in adifferent manner. As an example, the antimicrobial activity ofvancomycin coated gold nanoparticles gets enhanced (Gu et al.,2003). Similarly the coatings of aminoglycosidic antibiotics ongold nanoparticles have an antibacterial effect on a range ofGram-positive and Gram-negative bacteria (Grace and Pandian,2007; Saha et al., 2007). On the other hand, GNPs have been usedin photothermal therapy for the destruction of cancer cells ortumors. When irradiated with a focused laser in the near-infraredregion (NIR) of suitable wavelength, targeted aggregates of GNPs,nanorods, or nanoshells can kill bacteria (Zharov et al., 2006) andcancer cells (Zharov et al., 2005).

In our present study, the conjugation of 9AA-HCl, AY, AO and Prowith citrate stabilized gold nanoparticles have been studied byvarious analytical techniques like UV–vis spectrum, Fouriertransform infrared spectroscopy (FT-IR) and transmission electronmicroscope (TEM). Further, the antibacterial efficacy of drugscoated gold nanoparticles were studied against strains of Grampositive and Gram negative bacteria. Thus, the study of conjugationof these biologically important drugs with gold nanoparticles(AuNPs) as exogenous and endogenous drug-delivery vehicles is ofutmost importance.

2. Materials and methods

2.1. Materials

HAuCl4�3H2O, trisodium citrate were purchased from Sigma.9AA-HCl, AY, AO and Pro were also purchased from Sigma–Aldrich.Potassium bromide (spectroscopic grade), used for infraredstudies, was purchased from Merck. All the solvents werepurchased from commercial sources and distilled prior to use.Double distilled deionized water was used for all the experiments.

2.2. Preparation of citrate capped gold nanoparticles (Turkevich et al.,1951)

To a boiling solution of HAuCl4�3H2O (1 mM, 0.5 cm3), trisodiumcitrate (10�2M, 0.5 cm3) and double distilled water (18.5 ml) wasadded as one portion and after the addition, the previously yellowsolution of gold chloride turned wine red in color.

2.3. Preparation of antibiotic coated gold nanoparticles

The drugs coated gold nanoparticles were prepared as follows:0.25 mM citrate stabilized gold nanoparticles was mixed withdrugs in water and stirred effectively for 2 h. This is marked as thestandard sample. Similarly, antibiotic protected Au(0) wereprepared at two different concentrations of gold particles viz.0.5 mM and 1.0 mM to study the function of nanoparticles on themicrobial activities.

2.4. Details of microbial assay

Antibacterial activity was studied by determining the zone ofinhibition following standard agar diffusion method. From overnightcultures of E. coli (Gram �ve) and B. subtilis (Gram +ve) strains, nextday secondary cultures were inoculated at a dilution of 1:100 in LBand allowed to grow at 37 �C in shaking condition to reach the OD600

to 0.6. Bacterial suspensions from these secondary cultures werethen used for 90 mm diameter petri dish filled with 25 ml LB-agar.After punching wells in the agar plates, 20 ml of each drug and goldnanoparticle conjugated drug were used to fill these wells and thezone of inhibitionwas measured in the next dayafter incubating theplates at 37 �C for overnight.

For TEM image, 400 ml of the above B. subtilis cell suspensionwas centrifuged and the cell pellet was washed twice with 1Xphosphate buffer saline (PBS). The cells were then fixed with 4%para-formaldehyde for 10 min at 37 �C followed by washing with1X PBS thrice. After re-suspension, the cells were allowed to attachon the grid, air dried and proceed for image acquisition.

2.5. Apparatus

TheabsorptionspectrawererecordedonaJascoV-650absorptionspectrophotometer at 298 K within a wavelength range of300–800 nm using a pair of 1 cm � 1 cm path length quartz cuvettes.For TEM imaging, the samples were observed in transmissionelectron microscope (FEI model Technie G2 20S). The infraredspectrawererecordedusingvacuumdriedsamplesintheformofKBrpellets in PerkinElmer Spectrum 100 FT-IR Spectrometer.Thermogravimetry (TG) measurements were carried out using athermogravimetryanalyzer (Netzsch, Germany, model: STA 449C) ata heating rate of 10 �C/min. Thermogravimetry analysis (TGA) is ahighly useful analytical technique which was performed with all thesamples to understand the nature and strength of binding of drugswith the gold nanoparticles (AuNPs). Through combining TGA withsize analyses of the nanoparticles by TEM, the number of ligandsattached to AuNPs can be determined.

3. Results and discussion

The colloidal solution containing citrate capped goldnanoparticles has very intense and characteristic wine red colororiginating from the coherent electron motion. The plasmon bandobserved for the wine red colloidal gold at 525 nm in the UV–visspectrum is characteristic of gold nanoparticles as depicted in Fig. 1.With the addition of acridine derivatives (9AA-HCl, AY, AO, and Pro)the intensity of the absorption band at 525 nm is decreased andaccompanied by a new peak emerging at red-end region due to the

Fig. 1. UV–vis spectrum of bare gold nanoparticle.

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formation of gold-drug complex. This was also reflected in thechange in color of bare citrate capped nanoparticles from red topurple to bluish purple and finally to blue as a consequence ofaggregation. Citrate ions are readily replaced by ��NH2 functionalgroup of ligand on gold nanoparticle surfaces. This ligand exchangereaction provides an important means for the chemical function-alization of the nanoparticles and greatly extends the versatility ofthese systems. Changes in the absorption spectroscopic character-istics of gold nanoparticles with wavelength upon adsorption ofacridine derivatives are depicted in Fig. 2A–D. The UV–vis spectra ofacridine derivative adsorbed gold nanoparticles were also recordedin different time (inset of Fig. 2A–D).

Fig. 2. UV–vis spectra of gold nanoparticles conjugated with (A) 9AA-HCl, (B) AY, (C) Aconjugated with drugs.

The aggregation of nanoparticles could be clearly seen from theTEM images. Fig. 3A shows the TEM image of citrate stabilized goldnanoparticles, which shows an average size of 15–20 nm. In thepresence of various drugs, aggregation could be clearly seen(Fig. 3B–E) which is the reason for color change. Aggregation ofthese metal nanoparticles yields both a shift in the plasmon bandenergy and substantial increase in longer wavelength absorptionas observed in the UV–vis spectrum.

The binding interaction of drug with Au(0) was furtherinvestigated using FT-IR studies. The IR spectra of free acridinederivatives and their conjugates (acridine derivatives coated goldnanoparticles) are shown in Fig. 4. Gold nanoparticles in generalhave strong affinity for amine groups (Leff et al., 1996). The NH2

stretching frequency of the amine group was observed at 3320,3310, 3440, and 3320 cm�1 for 9AA-HCl, AY, AO, and Pro,respectively. In the case of drug coated gold nanoparticles, nochanges are observed in the absorption frequencies of other groupsexcept the NH2 stretching peak, which is broadened and shifted tohigher wavelengths. From the above data, it can be concluded thatonly the nitrogen atoms of the free amino group that binds directlyon the gold surface.

The average number of gold atoms per AuNP can be calculatedfrom high resolution TEM analyses. Assuming a spherical shape ofthe individual nano particle the average number of gold atoms (N)for each type of nanosphere was calculated by Eq. (1),

N ¼ prD3

6M(1)

where r is the density for gold (19.3 g/cm3) and M stands foratomic weight of gold (197 g/mol) and D is the diameters of theparticles in nm, the number of gold atoms in a cluster comes out tobe 104,261 (Liu et al., 2007).

O, and (D) Pro, respectively, for 2 h. Inset shows the O.D. vs. time plot of the AuNP

Fig. 3. Transmission electron micrographs of (A) bare gold nanoparticles (Inset shows its size distribution diagram) and gold nanoparticles conjugated with (B) 9AA-HCl, (C)AY, (D) AO, and (E) Pro.

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The number of drug molecules attached to each AuNP particlewas determined qualitatively from the weight loss obtained fromTGA analyses as depicted in Fig. S1. Accordingly the averagenumbers of the 9AA-HCl, AY, AO, and Pro attached with singleAuNP are 41,896, 54,325, 38,121, and 24,538, respectively (Danieland Astruc, 2004; Gibson et al., 2007; Gopidas et al., 2003; Kumaret al., 2000, 2003; Mukherjee et al., 2007; Parab et al., 2011;Rai et al., 2010; Selvakannan et al., 2003; Selvakannan et al., 2004;Shi et al., 2004; Takae et al., 2005; Vigderman et al., 2012; Walkeret al., 2001). The drug molecules form dimer (Mitra et al., 2013) by

intertwining of a pair of monomeric species through the formationof a couple of hydrogen bonds between corresponding amino-Hand imino-N. In this case, it might be possible that the drugs whichare attached to gold surface by amine functional groups, the imine-N of that drug moiety can form dimer with amino-H of anotherdrug molecule. This might be the reason for the higher number ofdrug molecules attached to AuNPs. The three distinct weight lossesat three different temperature regions indicate that there are atleast three different modes of interactions between drug moietyand AuNPs which are electrostatic attraction of negatively charged

Fig. 5. Zone of inhibition for the drug D1 (9AA-HCl), D2 (AY), D3 (AO), and D4 (Pro) for B. subtilis (Gram +ve) and E. coli (Gram �ve) bacterial strains. (1) represents zone ofinhibition for bare drugs; (2)–(4) represent AuNP@drugs where [Au(0)] are 0.25 mM, 0.5 mM, and 1.0 mM, respectively.

Fig. 4. Infrared spectra of (a) 9AA-HCl, (b) AY, (c) AO, and (d) Pro free drug (black) and gold nanoparticles conjugated with drugs (red). (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

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Table 1Antibacterial activity of drug coated gold nanoparticles ([Au(0)] = 0.25 mM) and their level of zone of inhibition (Conc. of drug 1.0 mg/ml).

Micro organisms Nature of organisms Levels of zone of inhibition in mm

Pure 9AA-HCl AuNP@9AA-HCl Pure AO AuNP@AO

B. subtilis Gram-positive 15.06 � 0.296 15.83 � 0.247 10.65 � 0.021 11.64 � 0.311E.coli Gram-negative 20.31 � 0.084 20.60 � 0.311 10.98 � 0.169 12.04 � 0.091

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AuNPs toward positively charged drug molecules, covalentbonding, and hydrophobic interaction between respective drugsand AuNPs, respectively. These observations suggest that beingelectrostatically attracted to AuNPs, the drugs may bind covalentlyto the AuNPs through the amine groups. The small amount ofweight loss at relatively lower temperature region is suggestive ofphysical adsorption of drugs on the surface of AuNPs viarearrangement of bound drug moieties. A second weight loss athigher temperature indicates another distinctly different bondingmode that needs much higher energy to desorb/decompose thedrugs from the surfaces. The maximum weight loss at thistemperature is most likely due to release of covalent interactionbetween the AuNPs and respective drugs. A third weight lossat even higher temperature indicates the existence of electrostaticinteraction of the amine group with negatively chargedAuCl4� ions present on the surface of the AuNPs. Therefore, TGAdata indicate that after being electrostatically attached withAuNPs, the drug further binds to the nanoparticles throughcovalent and physical interactions.

Since amine functionalized chromophores have affinity towardgold by donating electron to the cluster, here we were interestedto check the ability of Au(0) as drug carrier for acridinebased antibiotics and also the antimicrobial efficacies of goldnanoparticles conjugated with acridine derivatives (AuNP@drug)complexes. We use two clinically significant micro-organism,B. subtilis and E. coli Gram positive and Gram negative bacteria,respectively, for our studies and the experiments were repeatedthree times for an accurate analysis. The anti-microbial efficacies ofthe drug coated gold colloids were compared against bare drugmolecules. Among the four drugs, D1 (9AA-HCl) and D3 (AO)showed antibacterial activity (Fig. 5) and for both of them the AuNPconjugated drug showed better antibacterial efficacy than the puredrug (Table 1). In case of the D2 (AY) and D4 (Pro), we could notdetect antibacterial activity for both AuNP conjugated drug andonly drug (Fig. 5). Due to the high penetrating power and largesurface area of Au(0) and affinity of the amine functionalized drugmolecules toward the gold nanoparticles, a single gold nanoparti-cle can adsorb multiple drug molecules (as mentioned in TGAanalyses) and hence this entity acts as a single group against thebacteria. D1 and D3 showed high efficacy toward Gram negativeE. coli than Gram positive B. subtilis as shown in Table 1. This can bedue to the fact that Gram positive bacteria possess thick cell wallwhich makes the AuNP conjugated drug difficult to penetratewhereas Gram negative possess thin cell wall and thus easy to

Table 2Antibacterial activity of drug coated gold nanoparticles at various concentrations of gold

Microorganisms AuNP@9AA-HCl

Concentration of gold nano particles

0.25 mM 0.50 mM 1.0

B. subtilis 15.83 16.15 16.�0.247 �0.289 �0

E. coli 20.60 20.94 22.�0.311 �0.325 �0

permeate. Moreover, the outermost layer for the Gram negativebacteria is the outer membrane containing lipopolysaccharide(LPS) which is negatively charged and the interaction of the drugwith LPS depends on cationic ionization of the drug molecule andsufficient molecular planarity. According to Albert’s hypothesis therequired parameters for antibacterial property for acridines are:(1) cationic ionization; (2) high level of ionization at neutral pH;and (3) planar molecular surface (Wainwright, 2001). In case of D1,there is a high degree of positive ionization at physiological pH aswell as sufficient planarity which makes the interaction easier andthis might be the reason of higher efficacy of D1 against Gramnegative bacteria. In case of D3, for the positive ionizationamino functional groups are present at 3 and 6 positions. Butdue to the di-methyl substitution [��N(CH3)2] steric hindrance willbe associated that lead to lack of co-planarity between the[��N(CH3)2] and the acridine system, making the interactiondifficult and finally to decreased antibacterial activities. This mightbe the reason for the lower and almost similar antibacterial activityagainst both Gram positive and Gram negative bacteria for D3. ForD2 and D4, due to reduced cationic ionization and loss of planarmolecular surface due to steric effect, the interaction through thebacterial wall is prohibited.

We also checked the effect of the gold nanoparticle on theantibacterial property of D1 and D3 by increasing the concentra-tion of gold nanoparticles from 0.25 to 0.5 mM and 1 mM. It wasfound that with increase in gold nanoparticle concentration, theantibacterial property is increased for both D1 and D3 (Table 2).This may be due to the fact that as Au(0) concentration is increasedmore amount of drug gets adsorbed on the nanoparticle which willincrease the antibacterial activity.

Gold nanoparticles do not have any antimicrobial activity.However, treatment of clinically important B. subtilis with goldnanoparticle conjugated D1 and D3 lead to severe alteration ofcellular boundaries as evident from TEM images. Conjugation ofgold nanoparticles with drug molecules lead to aggregation (Fig. 3)consisting of a number of gold particles surrounded by a number ofdrug molecules. Presence of these aggregates which act toward thebacterial cell can be found for both gold nanoconjugates of D1(Fig. 6a) and D3 (Fig. 6c). Cell wall and cell membrane both are verycrucial structures in maintaining cellular integrity throughregulation of osmotic balance and the cellular uptake andexclusion of small molecules and osmolites. Treatment withD1 and D3 lead to disruption of cell wall and membrane structureswith big pores or craters (Fig. 6b and d, respectively), through

nanoparticles and their level of zone of inhibition in mm (Conc. of drug 1.0 mg/ml).

AuNP@AO

0 mM 0.25 mM 0.50 mM 1.00 mM

97 11.64 11.98 12.52.268 �0.311 �0.296 �0.31814 12.04 12.59 13.31.346 �0.091 �0.042 �0.141

Fig. 6. TEM image of B. subtilis after being treated with AuNP@drug where (a) and (b) are for D1 (9AA-HCl) and, (c) and (d) are for D3 (AO) ([Au(0)] = 0.5 mM).

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which the cellular contents either leaked or burst out leading toosmotic imbalance, loss of cellular integrity and finally cell death.

4. Conclusion

Adsorption of acridine derivatives on gold nanoparticle surfacewas studied using different analytical techniques. The nitrogen atomof amine moiety binds strongly to the nanoparticles as confirmed byspectroscopic studies. Upon addition of acridine derivatives (9AA-HCl, AY, AO, and Pro) the intensity of the plasmon band at 525 nm,characteristic of gold nanoparticle, is decreased which is accompa-nied by emergence of a new peak at red-end region and color changefrom red to finally blue as a consequence of aggregation confirmingthe formation of gold-drug complex. This is also supported by TEMimages which showed aggregation of nanoparticles in presence ofvarious drugs and from TGA analyses we obtained the number ofdrug molecules attached per AuNP. Gold nanoparticle conjugated9AA-HCl and AO showed better antibacterial activity compared tothe bare drug and the activity increases with increase in the goldconcentration. TEM images confirmed that treatment with goldnanoparticle conjugated 9AA-HCl and AO on Gram positive andGram negative bacteria results severe alteration in the cell wallstructure which finally leads to leakage and/or burst out of thecellular content and finally cell death. Moreover, due to betterplanarity of the molecular structure, 9AA-HCl showed betterantibacterial activity compared to AO.

Acknowledgements

Financial assistance from the Chemical and BiophysicalApproaches for Understanding Natural Processes (CBAUNP) andBiomolecular Assembly, Recognition and Dynamics (BARD) projectof SINP under Department of Atomic Energy (DAE), Government of

India is greatly acknowledged. P. Mitra acknowledges the SeniorResearch Fellowship from Council of Scientific and IndustrialResearch (CSIR), India. We also acknowledge the kind support of Dr.Madhusudan Roy and Debaleen Biswas, Surface Physics andMaterial Science, SINP for the Thermogravimetric analyses.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2014.07.051.

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