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Bioengineering and Drug design Lab, De

Chennai-600036, India. E-mail: mukeshd@

† Electronic supplementary informa10.1039/c3ra47073c

Cite this: RSC Adv., 2014, 4, 11393

Received 27th November 2013Accepted 7th January 2014

DOI: 10.1039/c3ra47073c

www.rsc.org/advances

This journal is © The Royal Society of C

Characterization and applications of cyclic b-(1,2)-glucan produced from R. meliloti†

Geetha Venkatachalam, Venkatesan Nandakumar, Ganesan Sureshand Mukesh Doble*

Cyclic b-(1,2)-glucan, with a degree of polymerization ranging from 17–28, without any substitution and a

molar mass of 3101.5 Da, was produced from Rhizobium Meliloti MTCC-3402 in glutamic acid and a

mannitol medium. The size of this glucan was less than those reported from oats and yeast. The main

fraction was with 19 glucose residues (cavity size of 0.92 nm) and a melting temperature of 134.1 �C.Glucan encapsulates drugs (85–99%) such as curcumin, dexamethasone, reserpine, 6-methylcoumarin,

4-hydroxycoumarin and 4 methyl umbelliferone very efficiently. The encapsulation efficiency was better

for hydrophobic than hydrophilic drugs (correlation coefficient of 0.92). Glucan was not cytotoxic

towards L6 myoblast and 3T3 fibroblast cells and could be produced on the nanometer scale (average

particle size 50–200 nm). Glucan exhibited dose dependent radical scavenging antioxidant activity. It

was able to bind to dyes including methyl violet, trypan blue and bromocresol green indicating that the

latter could be used in vivo at very low concentrations. Glucan decolorized coomassie brilliant blue R,

bromophenol blue and bromocresol purple opening up applications in effluent treatment industries.

Introduction

The family of Rhizobiaceae (Mesorhizobium, Rhizobium, Sino-rhizobium and Bradyrhizobium japonicum) form symbiotic asso-ciations with host plants including alfalfa, clover, and soy bean,and produce cyclic b-(1,2)-glucans as extracellular poly-saccharides which help the organism during osmoregulationand plant infection.1–3 In general, these oligosaccharides areD-glucose residues connected by b-(1,2) linkages with a degree ofpolymerization (DP) ranging from 17–25 and in some species itreaches up to 40 glucose units. Cyclic b-(1,2)-glucans producedfrom Rhizobium and Agrobacterium species have exible back-bone structures and narrow cavity sizes with varying molecularweights.4,5 These glucans are biocompatible and more watersoluble (250 g L�1) than cyclodextrin (solubility 18 g L�1) andhave negligible cytotoxicity.6

Linear glucans are biopolymers (b-1,3) produced by species ofAgrobacterium, Rhizobium and Alcaligenes faecalis. They are wellcharacterized and hence used extensively as gelling and bio-thickening agents in food for the improvement of texture, forexample in tofu (bean curd), bean jelly and sh pastes.7 In Japan,curdlan is widely used in the food industry for its water-retentioncapacity. Curdlan possesses properties like anti-tumorigenicity,anti-inammatory, immunomodulating, anti-infective activities

partment of Biotechnology, IIT-Madras,

iitm.ac.in; Tel: +91-44-2257-4107

tion (ESI) available. See DOI:

hemistry 2014

against fungus, bacteria, protozoa and viruses, wound repair,protection against radiation and anti-coagulant activity. Incontrast, reports on the applications of cyclic b-(1,2)-glucan arevery limited since its physico-chemical and biological propertieshave not been fully elucidated. A detailed characterization ofcyclic b-(1,2)-glucan produced from R. meliloti is reported here.This may help the biopolymer nd applications in medical,pharmaceutical, food and avouring industries.

Drugs for anticancer, antipsychotic, blood pressure, inam-matory and autoimmune conditions are hydrophobic and havepoor water solubility. Polymeric nanoparticles including lacticacid and glycolic acid (PLGA), cyclodextrin, dextrin, chitosan, zeinnanoparticles, albumin, micelles, pullulan and peptides havebeenmodied by ionic crosslinking, covalent crosslinking, and alayer-by-layer method for encapsulation of such drugs. Deoxy-cholic acid, cholesterol, carboxylic acids, and hydrophobic poly-mers are used as hydrophobic segments in polysaccharidenanoparticles for hydrophobic drug release.8,9 Use of syntheticpolymers for drug encapsulation is limited due to their poorwater solubility and may require a prolonged stay in the bodybefore they undergo degradation. In contrast, the cyclic glucanreported here is isolated from a natural source, not modied orcross linked and it is biocompatible. In this paper the encapsu-lation efficiency of glucan towards several drugs is reported.

The interaction and binding of glucan with several dyesincluding methyl violet (MV), tryphan blue (TB) and bromoc-resol green (BCG) are investigated here. These dyes are used inmedical imaging and are reported to be cytotoxic. Paper mills,color photography and textile industries produce synthetic dyes

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as a major pollutant in their waste water. These dyes are carci-nogenic, hazardous and affect the gas solubility in water,aesthetics and aquatic life.10 The decolorization of dyes usingbiological methods is an environmentally acceptable technique.Here the decolorizing ability of glucan is investigated againstdyes such as coomassie brilliant blue R (CBBR), bromophenolblue (BPB) and bromocresol purple (BCP).

The present study explores three possible applications ofcyclic b-(1,2)-glucan namely as a drug carrier, as a dye binderand as a dye decolorizer. The cytotoxicity of this polymer is alsoinvestigated, since this knowledge is necessary if it is to betested for pharmaceutical applications.

Fig. 1 MALDI-TOF MS positive-ion mass spectrum of cyclic b-(1,2)-glucan. Values indicated on the peaks are the masses of sodium-cationized glucan.

Results and discussionPurication of the cyclic b-(1,2)-glucan

A chromatogram of the ethanolic extract (aer fermentation)puried by size exclusion chromatography is shown in the ESI(Fig. S1†) and it is visualized by TLC as a single black spot(Fig. S2†).

Physicochemical characteristics

The 1H NMR spectrum indicates the presence of methylene (d3.79), hydroxyl (d 3.72, 3.70, and 3.40) and methine protons (d3.57, 3.68, 3.60, and 3.44) (Fig. S3†). The chemical shi at 4.78ppm (H-1) is the characteristic peak of the b-conguration of allthe glucose residues. The shi at d 3.44 ppm (H-2) correspondsto the H-2 protons involved in the b-(1,2) glycosidic linkage inglucan (Table S1†). The methylene protons from the succinylsubstitution which appear as signals at 2.44 and 2.66 ppm areabsent here. From the 1H NMR spectrum it could be concludedthat the cyclic b-(1,2)-glucan isolated from R. meliloti is similarto the one produced by other Rhizobial strains and is neutralwithout any substitution.5 The 2D NMR spectra also conrm theproton–proton and proton–carbon coupling of glucan at3.44 ppm which includes 1H-1H COSY (H6 4 H5, H4 4 H5,H44H3), HSQC (H54 63.19, H64 59.31, H4 & H34 70.79,69.23) and HMBC (H4 4 70.79, H2 4 59.31, H5 4 68.23,H3 4 70.79, H6 4 63.19) (S4–S6†).

Table S2† lists the bands present in the FT-IR spectrum of thecyclic b-(1,2)-glucan (hydrogen bonded hydroxyl groups andglycosidic bonds) which match well with data reported in theliterature for similar glucans from Rhizobiummeliloti.11Molecularions at a m/z of 2777.17, 2939.85, 3101.46, 3263.82, 3425.0,3587.92, 3749.22, 3911.03, 4073.23, 4234.61, 4397.82, 4560.53 Dain the MALDI-TOF MS spectra indicate the presence of glucancorresponding to a degree of polymerization (DP) rangingbetween 17 and 28 (Fig. 1). These molecular ions are identical tothe values reported for glucan produced by Mesorhizobium lotiwhich are reported to have DPs ranging from 17–28.5 There is nosubstitution in the glucan structure which is conrmed fromTLCand MALDI-TOF MS analysis. The present product is differentfrom the ones produced by other bacterial species of Rhizobiaceaewhich have substitutions including acetyl, succinyl, phospho-glycerol, methylmalonyl or phosphocholine.12 The ion at a m/z of3101.46 is the most intense peak, which represents the main

11394 | RSC Adv., 2014, 4, 11393–11399

component with a ring size of 19 glucose residues. It is inter-esting to note that the major glucan species containing 19glucose residues reported in this study, is close to the rangetypical for the cyclic b-(1,2)-glucan predominantly produced byRhizobium (DP of 17–28 glucose residues per ring).1,13 Our dataalso suggest that the b-glucan produced by Rhizobium melilotishares a similar structure with those isolated fromMesorhizobiumloti,5 Mesorhizobium huakuii13 and Brucella abortus.14

DSC (differential scanning calorimetry) shows an endo-thermic transition peak at 134.08 �C (Fig. S7†). This is an indi-cation of its thermal stability, which depends on the degree ofpolymerization. Transitions may be due to the changes in itssize.15 Two conformational transitions in the triple helix andtriple helix–single coil of scleroglucan are observed as twoendothermic peaks, which are due to the changes in the struc-ture.16 This could be attributed to the differences in the DPbetween the two.17 To date there is no report on the thermalstability of cyclic b-glucan. Thermogravimetric analysis shows thedegradation pattern of the glucan, which occurs in fourtemperature ranges (Fig. S8†). An initial weight loss (5.36%) isobserved between 42 and 200 �C. A sharp and major weightreduction is observed between 200 and 400 �C with a maximumoccurring at 300 �C. The percentage weight loss from 200 to400 �C is 50%, and from 400 to 600 �C it is 15%. The nal weightreduction from 600 to 850 �C is 30.5%. No residue is observedfrom the TGA, which indicates complete degradation of thepolymer.

The percentage of carbon and hydrogen (from CHN analysis)is 33.2 and 4.6 respectively, which indicates an average degree ofpolymerization of 19. The ratios of C and H in b-glucan fromoats (100–200 kDa) and yeast (105 kDa) are reported to be 37.9and 5.8%, and 43.8 and 6.2% respectively. These glucans arelarger in size than the bacterial glucan reported here (DP ¼17–28). The percentage of carbon and hydrogen identiedthrough elemental analysis reported here is lower than thelevels reported for (1,3)-b-glucan from brewer's yeast andoats.18,19 This may be due to the major differences in their

This journal is © The Royal Society of Chemistry 2014

Fig. 3 Transmission electron microscopy image of (A) cyclic b-(1,2)-glucan and (B) after curcumin encapsulation.

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molecular weight and shape. Glucan from oats is reported toform aggregates with sizes ranging from 5–100 mm (20 mm onaverage).20 The morphology of cyclic b-(1,2)-glucan in pure formappears as rod like structures, but upon simultaneous sonica-tion and acetone precipitation it appears (in TEM) as sphericalparticles of 50 to 200 nm in diameter.

Encapsulation of hydrophobic drugs by cyclic b-(1,2)-glucan

The FTIR spectra of the cyclic b-(1,2)-glucan (Fig. 2a), and drugencapsulated (Fig. 2b curcumin, c. dexamethasone, d. reser-pine, e. 6-methylcoumarin, f. 4-hydroxycoumarin, g. 4-methyl-umbelliferone) show changes in the 1000–1200 cm�1 region aswell as the appearance of new peaks corresponding to theencapsulated drugs (Table S2†). The 1H NMR spectra of theencapsulated product show resonances corresponding toglucan along with the drugs. In some regions the drug peaksmerge with the glucan peaks (Fig. S9†).

Glucan encapsulated curcumin appears as spheres (Fig. 3)with an average particle size of 58 nm (Fig. S10†). The encapsu-lation efficiency is 98.7% which matches well with the literaturereports for chitosan–poly(butyl cyanoacrylate) encapsulated cur-cumin nanoparticles.21 In the case of dexamethasone, theencapsulation efficiency here is 92.0%, whereas the literaturereports only a 44.5–76.0% encapsulation efficiency with chito-san.22 97.8 and 87.5% of reserpine and 6-methylcoumarinrespectively are encapsulated by cyclic b-(1,2)-glucan. Polylactide-co-glycolide acid (PLGA) nanoparticles are reported to encapsu-late 88% of 4-methyl-7-hydroxy coumarin.23 85.2% and 92.2% of4-hydroxycoumarin and 4-methylumbelliferone respectively areencapsulated by cyclic b-(1,2)-glucan. In our previous study wereported a 92.6% umbelliferone encapsulation by this polymer.2

The current study shows that cyclic b-(1,2)-glucan has a higherloading efficiency than other polymers reported in the literature.An excellent positive correlation (r ¼ 0.92) exists between log P

Fig. 2 FTIR spectra of drug encapsulated cyclic b-(1,2)-glucan. (a)glucan alone, (b) curcumin, (c) dexamethasone, (d) reserpine, (e) 6-methylcoumarin, (f) 4-hydroxycoumarin, (g) 4-methylumbelliferone.

This journal is © The Royal Society of Chemistry 2014

(hydrophilic–hydrophobic balance) of the drugs and the encap-sulation efficiency (Fig. 4), indicating that this process is betterwith hydrophobic than with hydrophilic drugs. This is expectedsince the pocket of the cyclic glucan is hydrophobic in nature.

Cyclic b-(1,2)-glucan as a dye binder

Glucan does not absorb in the UV-visible range, but when it isused with the dyes, methyl violet (MV), trypan blue (TB) andbromocresol green (BCG), it increases the absorption maximain a concentration dependant manner as well as shis the lmax

(Fig. 5A–C). The rst two dyes are used to stain lens capsules forcapsulorhexis creation (removal of the lens capsule duringcataract surgery). When compared to MV, TB is reported toexhibit cytotoxicity at a concentration of 2.5 mg ml�1.24 So thesedyes with glucan can be used in biomedical applications at alower concentration than using the dye alone as a binder forstaining the capsule. BCG is used as an inhibitor of the pros-taglandin E2 transport protein.25 At a concentration of 30 mM inKrebs bicarbonate solution it is used for staining the trachealsegments in mice. Along with glucan its toxicity could bereduced and possibly be used in humans.

Fig. 4 log P versus % of drug encapsulation (curcumin, dexametha-sone, reserpine, 6-methylcoumarin, 4-hydroxycoumarin, 4-methylumbelliferone).

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Fig. 5 Cyclic b-(1,2)-glucan absorption of different dyes. (A) Methyl violet, (B) tryphan blue, (C) bromocresol green, (D) brilliant blue R, (E)bromophenol blue, and (F) bromocresol purple.

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Cyclic b-(1,2)-glucan as a dye decolorizer

200 mgml�1 of glucan when incubated with brilliant blue R (BBR),bromophenol blue (BPB) and bromocresol purple dyes, decreasesthe absorbance of the dyes (by 36.5, 22 and 10.9% respectively)which indicates that the polymer decolorizes them (Fig. 5D–F).Some natural polysaccharides such as galactomannans, locustbean gum, guar gum and cassia gum are reported to decolorizedyes from effluents and it is reported that the interaction isthrough van derWaals forces, hydrogen bonding and electrostaticattraction.26 These observations indicate that glucan can be usedas a decolorizing agent in effluent treatment.

Fig. 6 Fluorescence microscopy images of L6 cells stained withNucBlue®. (A) Untreated and (B) treated with cyclic b-(1,2)-glucan(magnification 10�, scale bar 85 mm).

Cytotoxicity of glucan

L6 and 3T3 cells are viable even up to a concentration of 200mg ml�1 of glucan, which proves that it is biocompatible(Fig. S11†). NucBlue® stained uorescence images of untreatedand glucan treated L6 cells have intact and round nuclei (Fig. 6Aand B respectively) which further conrms that the polymer isnot toxic. It is reported that the viability of Human EmbryonicKidney 293 cell lines slightly decreased in the presence of cyclicb-(1,2)-glucan.27 However, no such inhibition is observed herewith L6 cells. It is reported that glucan in general acts as animmune enhancer through the activation of macrophages and

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it accelerates the production of cytokines including TNF-a, IL-6and IL-12.28

Antioxidant properties

Cyclic glucan shows dose dependant antioxidant activity atincreasing concentrations (from 100 to 500 mg ml�1) in DPPH(16.0 � 1.8 to 40.8 � 0.8%) and hydroxyl radical (24.0 � 0.9 to60.8 � 1.9%) scavenging assays (Fig. S12a and b†). Thepercentage of antioxidant activity of ascorbic acid (standard) at

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100 mg is 82% � 2.3 and 76% � 3.1 in DPPH and hydroxylradical scavenging assays respectively.

Experimental proceduresChemicals

Curcumin, reserpine, dexamethasone, 6-methylcoumarin, 4-hydroxycoumarin and 4-methylumbelliferone were purchasedfrom TCI chemicals, India, and dyes are from Himedia, Merckand Sigma Aldrich. Solvents used for encapsulation are ofanalytical grade (Merck, India). NMR solvents namely D2O,acetone D, ethanol D, methanol D and chloroform D were fromMerck, Germany. All other chemicals used here were purchasedfrom Merck, Himedia and Sigma, India and L6 cells were fromthe National Centre for Cell Sciences (NCCS), Pune, India. R.meliloti from the Microbial Type culture collection (MTCC 3402)was obtained from the Institute of Microbial Technology(IMTECH), Chandigarh, India.

Microorganism and cultivation

R. meliloti MTCC 3402 was used for the production of cyclic b-(1,2)-glucan. The media contained (in grams); glutamic acid –

2.0, mannitol – 10, K2HPO4 – 0.1, MgSO4$7H2O – 0.4, CaCO3 –

0.5, FeCl3$6H2O – 0.0025, MnCl2$4H2O – 0.001, Na2MoO4$2H2O– 0.00001, ZnSO4$7H2O – 0.00001, CuSO4$5H2O – 0.00001,H3BO3 – 0.00001, CoCl2$6H2O – 0.00001, biotin – 2 mg andthiamine – 5 mg in one litre (at a pH of 7.0).2 The cultivation wascarried out on a 1 L scale for 8 days in a 3 L Erlenmeyer ask at33 �C on a rotary shaker at 180 rpm. Cells and culture super-natants were separated by centrifugation at 35 000 g for 30 minand the amount of cyclic b-(1,2)-glucan was quantied by theanthrone–sulfuric acid method.1

Characterization of the glucan

The aqueous ethanol phase was concentrated using a rotaryevaporator and puried by size exclusion chromatography withBiogel P6 (Bio rad, USA) (24–96 cm) as the stationary phase and5% acetic acid owing at a rate of 1 ml min�1 as the mobilephase. The purity of the samples was assessed by thin layerchromatography (TLC) using a silica gel coated glass plate as thestationary phase and a butanol–ethanol–water (5 : 5 : 4 vol/vol)solvent mixture as the mobile phase. The active compoundswere visualized by rst spraying 5% H2SO4 in methanol andthen heating it to 110 �C for 15 min.29

1H and 2D NMR (nuclear magnetic resonance) spectroscopy(Bruker 500 MHz spectrometer) was carried out with 10 mg ofsample dissolved in D2O (2 ml). 5 mg of the sample was mixedwith potassium bromide (KBr) at a ratio of 1 : 20 and pulverized,dried under vacuum for 1 h and compressed into a thin pelletand analyzed with a Fourier transform infrared spectrophotom-eter (FT-IR, Perkin Elmer ATR/FTIR) in the range of 400–4000cm�1. The mass spectrum of the glucan was recorded with amatrix assisted laser desorption ionization time-of-ight massspectrometer (MALDI-TOF MS Voyager-DE™ PRO Bio-spectrometry™ workstation, Applied Biosystems) in the positiveion mode using 2,5-dihydroxybenzoic acid as the matrix.5

This journal is © The Royal Society of Chemistry 2014

The thermal stability of the degassed glucan was determinedin the temperature range of 30 to 300 �C with a differentialscanning calorimeter (DSC7 from Perkin Elmer, Q200 MDSCfrom TA instruments). The scans were performed at a rate of10 �C min�1, and thermogravimetric analysis of the glucan wascarried out with the TGA7, Perkin Elmer, Q500 Hi-Res TGA7from TA instruments. Here, 1.06 mg of the sample was placed inan aluminum oxide crucible and was heated from 50 to 850 �Cat a rate of 20 �Cmin�1.17 Measurements were carried out undera nitrogen atmosphere at a ow rate of 80 ml min�1. Theamount of carbon and hydrogen in the lyophilized glucan wasestimated with the help of a Perkin Elmer 2400 Series II CHNanalyzer.30 The particle size and the zeta potential of the glucanwere determined using a Zetatrac-Zeta potential analyzer(Microtrac Inc, USA) at a wavelength of 780 nm and a backscattering angle of 180�.

Encapsulation of poorly water soluble drugs by cyclic b-(1,2)-glucan

10 mg of curcumin, reserpine, dexamethasone, 6-methyl-coumarin, 4-hydroxycoumarin and 4-methylumbelliferone (ESIFig. S9†) was solubilized in a suitable solvent (acetone ormethanol) and added slowly to the glucan (100 mg) and kept inthe dark for 24 h to attain equilibrium. Later the mixture wascentrifuged at 14 000 rpm for 30 min and the pellet waslyophilized. The loading efficiency of the encapsulated polymerwas quantied by a method reported earlier.2,31 FTIR and NMRspectroscopy were used to characterize the polymer encapsu-lated drug. The particle size and changes in the morphology ofthe glucan were determined with a particle size analyzer andtransmission electron microscope (Philips/Fei CM-20),respectively.

Cyclic b-(1,2)-glucan as a dye binder

The interaction and binding efficiency of glucan towards dyessuch as methyl violet, trypan blue, Bromocresol green, brilliantblue R, bromophenol blue and bromocresol purple (ESIFig. S13†) were tested by a method reported earlier.2

Cytotoxicity of glucan

The biocompatibility of the glucan was tested against L6myoblast and 3T3 cell lines. The cells were grown to conuence,trypsinized with 1X trypsin and the number of cells was countedwith the help of a haemocytometer (Marienfeld, Germany). Theywere then diluted (104 cells per well) with Dulbecco's modiedeagle's medium (DMEM) and seeded in 96 well plates andcultured for 24 h. The glucan was solubilized in DMEM, dilutedto different concentrations (200, 150, 100, 75 and 50 mg ml�1)and added to each culture well and incubated for 24 hours.32 20ml of 5 mg ml�1 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) was added to each well and incubatedfor 4 hours. The precipitates were solubilized in DMSO(dimethyl sulfoxide) and the extent of the reduction of the MTTwas quantied by measuring the absorbance at 570 nm using amultimode plate reader (Enspire, Perkin Elmer, Singapore) andthe percentage of viable cells was calculated.

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Simultaneously L6 cells were also cultured for 24 h in DMEMsupplemented with 10% fetal bovine serum and 1% antibioticat 37 �C in a 5% CO2 atmosphere on polystryrene petriplateswith cover-slips. The cells were incubated with (200 mg ml�1)and without glucan for 24 h. Then the cells were stained withNucblue® (NucBlue® Live ReadyProbes) and xed in a glassslide and visualized under a uorescence microscope (LeicaMicrosystems, Germany) to determine the changes in theirmorphology. DPPH (2,2-diphenyl-1-picrylhydrazyl)33 andhydroxyl radical scavenging assays34 was performed to deter-mine the antioxidant potential of glucan, with ascorbic acid asthe standard.

Structural features

The log P (hydrophilic to lipophilic ratio) of these drugs anddyes was calculated using ALOGPS 2.1 soware (On-line Lip-ophilicity/Aqueous Solubility Calculation Soware).

Conclusions

Cyclic b-(1,2)-glucan from Rhizobium Meliloti MTCC-3402 has aDP of 17–28 (cavity size of �0.83 to 1.36 nm) and it could beused as a drug carrier for poorly water soluble drugs. Theencapsulation efficiency is 85 to 99% for hydrophobic drugssuch as curcumin, dexamethasone, reserpine, 6-methyl-coumarin, 4-hydroxycoumarin and 4-methylumbelliferone. Thebinding ability of glucan with dyes such as MV, TB and BCGdemonstrates its possible application in medical imaging todecrease the cytotoxicity of the latter. The decolorizing ability ofglucan with BBR, BPB and BCP indicates its possible applica-tion in effluent treatment industries. Glucan could be an idealdelivery system for hydrophobic drugs but not for hydrophilicones. This property is very useful in cancer treatment wheremost of the drugs used are hydrophobic. High thermal stabilityand good biocompatibility of this polymer facilitates its use inin vivo applications including drug delivery and tissue engi-neering. Moreover, there is no substitution in the cyclic struc-ture which could be exploited for preparing polymer blends tofurther improve its physico-chemical properties.

Acknowledgements

Geetha Venkatachalam gratefully acknowledges the nancialsupport from the Department of Science and Technology, India,under the women scientist scheme (DST-SR/WOS-A/LS-15/2010). A special thanks to Mr Rakesh Nankar and Mrs Anju VNair for helpful discussions. The authors also thank theSophisticated Analytical Instrument Facility, IIT-Madras foranalytical help.

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RSC Adv., 2014, 4, 11393–11399 | 11399