Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

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Colloids and Surfaces A: Physicochemical andEngineering Aspects

j ourna l h om epa ge: www.elsev ier .com/ locate /co lsur fa

Poly(methyl methacrylate) (core)biosurfactant (shell) nanoparticles:Size controlled sub-100 nm synthesis, characterization, antibacterialactivity, cytotoxicity and sustained drug release behavior

ChinmayAmbalal a School of Life b University Ins

h i g h l

A novel hticles by omicroemu

Non-toxic,antibacterilate) (core)

pH-dependibuprofen,cumin.

a r t i c l

Article history:Received 23 DReceived in reAccepted 23 FAvailable onlin

Keywords:Modied atomBiosurfactantPoly(methylm

CorresponE-mail add

1 These auth

http://dx.doi.o0927-7757/ Hazraa,1, Debasree Kundua,1, Aniruddha Chatterjeeb,,Chaudharia, Satyendra Mishrab

Sciences, North Maharashtra University, Jalgaon, Maharashtra, Indiatitute of Chemical Technology, North Maharashtra University, Jalgaon, Maharashtra, India

i g h t s

ybrid core/shell nanopar-il/water green atomizedlsion.

biocompatible andal poly(methyl methacry-biosurfactant (shell).ent sustained release of

anthraquinone and cur-

g r a p h i c a l a b s t r a c t

e i n f o

ecember 2013vised form 21 February 2014ebruary 2014e 1 March 2014

ized microemulsion

ethacrylate) nanoparticles

a b s t r a c t

A facile oil/water (O/W) modied atomized microemulsion process for the synthesis of novel poly(methylmethacrylate) (nPMMA) (core)biosurfactant (shell) particles designed for drug delivery applications isreported. The amount of biosurfactant required was 1/35 of the monomer amount by weight and thesurfactant/water ratio could be as low as 1/210. These surfactant levels are much lower in comparisonwith those used in a conventional microemulsion polymerization system. The particles were sphericaland 2050 nm in diameter with an average molecular weight of (0.91.5) 105 g mol1 (polydispersityindex 1.642.65). These nanoparticles were non-toxic, biocompatible and exhibited strong antibacterialactivity against Bacillus subtilis and Pseudomonas aeruginosa. Glutathione depletion with a concomitantincrease in malondialdehyde levels (increasing reactive oxygen species) and lactate dehydrogenase activ-ity demonstrates that nPMMAs induce oxidative stress leading to genotoxicity and cytotoxicity in B.subtilis. The release of ibuprofen, anthraquinone and curcumin from the particles was sustained andstrongly dependent on the initial drug loading content and medium pH. The system showed sustaineddrug release through three stages; although the release in stage I followed non-Fickian diffusion, Fickiandiffusion was proven to be the release mechanism of stages II and III. The unique biosurfactant coatednPMMA enables it not only to be a pH-responsive nanocarrier, but also to possess a tailored release pro-le. Thus, this work manifests the potential of biosurfactantpolymer hybrids as next generation deliveryvehicles for biomedical applications.

2014 Elsevier B.V. All rights reserved.

ding author. Tel.: +91 257 2258420; fax: +91 257 2258403.resses: aniruddha chatterjee2006@yahoo.co.in, aniruddhachatterjee2006@gmail.com (A. Chatterjee).ors contributed equally as rst authors in this manuscript.

rg/10.1016/j.colsurfa.2014.02.0512014 Elsevier B.V. All rights reserved.

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 97

1. Introduction

Coreshell nanostructured polymers, which are composed ofat least two distinguished domains in the core and shell phase,respectivelyfabrication,manufacturcolloidal m(PMMA), alone of the of its bioc[12,14,15]. vehicle forgenes, enzlenge to deproducing swith a compolymerizapolymer-bapreparing ctoxicologicasurfactantsnever be osion polym(i) generallto reach suof ionic mnucleation

To explotant amounwhich the microemulscore/shell crecent progin preparinthe synthesization andand nPMMA[20,21]. Unfods were diis not desiralabile bioacto develop hydrochloriNeverthelestoxic natureno surprisepenetrate c

In our polystyrenesion procesnovel atom(core)biosnanocompodelivery vesion of ourprepare corsurfactantstrehalose lippatible and such nPMMpotentially polymerizalarger than solid contenlarge amou

impact of excess surfactant on the properties and post-treatment ofsynthesized polymeric lattices; and (v) need of designing and devel-oping new surfactant systems with improved emulsifying proper-ties. Therefore, we report here a modied atomized microemulsion

s fornd iitiatoy of d naties aluaal anidan

teria

ateri

thyl nol w

Ammtd. (Moid cd an

and

oduc

mno isolin ws peris wehodod as

chemolipi), w.8%), and tructeptiddroxose lse-sd 2al ch1.

nthenano

nspa nm wmod) [26etersh bioed i

withitiatto thaledeforecatin, have attracted signicant research interest in the lm drug delivery, conducting materials, paper and textileing, and impact modiers [113]. Of these polymericicro- and nanoparticles, poly(methyl methacrylate)so designated as poly(methyl 2-methylpropenoate), ismost widely explored biomedical materials becauseompatibility, non-toxicity and non-immunogenicityAlthough PMMA has a long heritage as a carrier

bioactive substances (therapeutic drugs, proteins,ymes, vaccines, etc.), there is a tremendous chal-velop robust and economical techniques capable ofize-tunable and morphology-controlled nanoparticlesplex architecture [79,12]. Conventional emulsion

tion (seeded tandem polymerization) and pre-formedsed techniques are still the methods of choice fororeshell PMMA nanoparticles (nPMMA). However,l issues arising from the use of organic solvents,

and free radicals during these polymerizations canbviated [6,7]. On the contrary, surfactant-free emul-erization suffers from several drawbacks, includingy >200 nm particle size and it seems to be difcultb-100 nm and (ii) polydispersity due to the presenceonomers, thereby bring the possibility of secondary[1,5].re a practical technical route in which the surfac-t required could be signicantly decreased and bynanoparticle size could be controlled, differentialion polymerization [6,7] and biofunctionalization ofolloidal nPMMA was proposed [2,4,8,16]. Signicantress in bioinspired approaches, in particular progressg biomolecules based coreshell nPMMA, has allowedis of chitosan-modied nPMMA by emulsion polymer-

emulsier-free emulsion copolymerization [4,1719],-BSA using by Cu2+-mediated graft copolymerization

ortunately, these biomolecules used in the above meth-ssolved in an excessively acidic aqueous milieu, whichble for medical application and are detrimental to acid-tive substances. Since then, there were many attemptschitosan derivatives from glutamate, aspartate, andde salts to reduce the excessive acids and its fatality.s, their biodegradability, biocompatibility and non-

are not yet established conclusively [12]. It comes as that such PMMA particulate carrier systems are yet toommercial market.previous work, we have synthesized amorphous

(nPS) and particles (spherical) by modied microemul-s as well as crystalline nPS particle (hexagonal) byized microemulsion process [2226]. Similarly, PSurfactant (shell) biocompatible and biodegradable bio-sites were synthesized for their feasibility as drughicle [27]. The present work is therefore an exten-

preceding efforts to develop a new facile route toeshell nPMMAbiosurfactant nanoparticles. The bio-

we have chosen here are rhamnolipids, surfactin andids of microbial origin and are biodegradable, biocom-

non-toxic with very low CMCs. Hence, we believe that ifA (core)biosurfactant (shell) can be made then it couldnullify ve-fold disadvantages of classical emulsiontion: (i) high surfactant/monomer weight ratios, usually1, are required to produce small particles; (ii) only a lowt, usually less than 10 wt.%, could be made; (iii) use of

nts of expensive surfactant; (iv) considerable negative

procesticles aand instabilitthesizeproperalso evsteroid(antiox

2. Ma

2.1. M

MemethaIndia).India Lcumindistilleization

2.2. Pr

Rhaduced,SurfactBS02 aanalysfrom Rpurie

Therhamn(75.5%C10 (0(15%), their sof a pa -hyTrehaltrehaloC11 anchemicTable S

2.3. Sy(shell)

Tra2050(O/W) (Fig. 1paramtor, eacdissolvstirredmal inadded was sesure brecipro synthesizing nPMMA (core)biosurfactant (shell) par-n particular, we have studied the effect of emulsierr concentration on the particle size, number and thethe colloidal particles as well as cytotoxicity of the syn-noparticles. Finally, we investigated their antibacterialand their potential as possible drug delivery vehicle isted with three hydrophobic drugs: ibuprofen (IB; non-ti-inammatory), anthraquinone (AQ) and curcumint and anticancer agents).

ls and methods

als

methacrylate (MMA) monomer, n-hexanol (n-Hx) andere procured from S.D. Fine Chemicals Ltd. (Mumbai,onium persulfate (APS) was sourced from Qualigensumbai, India). IB, AQ (97%) and curcumin (94% cur-

ontent) was purchased from SigmaAldrich. Doubled deionized water (DDIW) was used during polymer-

in other experiments.

tion and purication of the biosurfactants

lipids from Pseudomonas aeruginosa BS01 were pro-ated and puried as previously described [2729].as obtained from the cell free broth of Bacillus clausii

Liu et al. [30]. The isolation, purication and structuralre done according to Namir et al. [31]. Trehalose lipidscoccus pyridinivorans NT2 were produced, isolated anddetailed by Kundu et al. [32].

ical structure of the most abundant component in thed biosurfactant product was identied as Rha-C10-C10hile the others were characterized as Rha-Rha-C8-Rha-C8-C10 (1.5%), Rha-C10-C12:1 (10%), Rha-C10-C12Rha-Rha-C10-C14:1 (2%), with small contributions ofural congeners (Fig. S1a). Puried surfactin consistede loop containing seven amino residues bonded toyl fatty acid chain with 15 carbon atoms (Fig. S1b).ipids consisted of trehalose-succinic acid-C7-C11-C11,uccinic acid-C9-C10-C10, trehalose-succinic acid-C9-C9-,3,4,2-trehalose tetraester (Fig. S1c). The physico-aracteristics of these three biosurfactants are listed in

sis and purication of PMMA (core)biosurfactantparticles

rent or translucent dispersions of nPMMA in the range ofith spherical shape were synthesized by an oil/water

ied atomized microemulsion process patented by us]. Typical recipes for nPMMA with different process

are shown in Table 1. In a at 1 l stainless steel reac-surfactant (rhamnolipid/surfactin/trehalose lipid) was

n a rst part of DDIW water and the mixture was an agitator at 200 rpm and 55 2 C. APS, as a ther-

or, dissolved in a second part of DDIW water wase reactor for formation of free radicals and the reactor. Monomer (puried and distilled under reduced pres-

use) was sprayed through the nozzle of atomizer byg compressor at controlled pressure and at a constant

98 C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

Table

1Rec

ipes

use

d

in

the

pre

sent

wor

k.

Run

MM

A

(ml)

Bio

surfac

tant

(g)

APS

(g)

Wat

er

(ml)

(1st

+

2nd

par

t)Con

vers

ion

(%)

Solid

conte

nt

(%)

Appea

rance

Part

icle

size

(nm

)M

w(

105

)

Mn(

105

)

Poly

disper

sity

index

Np(

1018

) N

DLS

TEM

1

14

0.4

(RL)

0.08

80

+

569

212

.17

0.1

Tran

sluce

nt

39.3

24.5

1.43

0.54

2.65

7.41

282

14

0.4

(RL)

0.08

55

+

5

72

3

15.5

8

0.2

Tran

sluce

nt

38.6

26.5

1.26

0.65

1.94

7.22

293

28

0.8

(RL)

0.16

55

+

584

622

.69

0.1

Visco

us

tran

sluce

nt

39.5

30.5

0.89

0.47

1.89

2.87

614

28

0.8

(RL)

0.16

40

+

5

89

1

28.8

8

0.3

Visco

us

tran

sluce

nt

44.5

31.5

0.91

0.39

2.33

2.55

565

14

0.4

(SR)

0.08

80

+

575

411

.15

0.1

Tran

sluce

nt

33.0

23.6

1.13

0.69

1.64

7.44

256

14

0.4

(SR)

0.08

55

+

581

313

.68

0.2

Tran

sluce

nt

38.0

29.6

1.47

0.76

1.93

7.56

297

28

0.8

(SR)

0.16

55

+

5

88

1

20.0

7

0.1

Visco

us

tran

sluce

nt

32.0

31.7

0.89

0.47

1.89

2.93

668

28

0.8

(SR)

0.16

40

+

594

127

.53

0.2

Visco

us

tran

sluce

nt

40.5

36.5

0.91

0.53

1.72

2.55

549

14

0.4

(TL)

0.08

80

+

5

65

1

13.1

5

0.3

Tran

sluce

nt

36.0

30.5

1.57

0.67

2.34

7.33

2710

14

0.4

(TL)

0.08

55

+

561

716

.18

0.2

Tran

sluce

nt

43.5

38.2

1.46

0.61

2.39

7.09

2411

28

0.8

(TL)

0.16

55

+

577

122

.69

0.3

Visco

us

tran

sluce

nt

50.5

41.5

1.22

0.49

2.48

2.04

6112

28

0.8

(TL)

0.16

40

+

5

82

3

29.8

4

0.1

Visco

us

tran

sluce

nt

34.5

31.0

1.19

0.66

1.80

2.11

6213

a14

0.08

80

+

5

61

1

11.1

3

0.4

Tran

sluce

nt

24.5

18.0

1.51

0.63

2.29

6.36

27

RL,

rham

nol

ipid

;

SR, s

urfac

tin;

TL, t

rehal

ose

lipid

;

Mw, w

eigh

t-av

erag

e

and

Mn, n

um

ber-

aver

age

mol

ecula

r

wei

ghts

of

the

nPM

MA

par

ticl

es, r

espec

tive

ly. n

PMM

A

par

ticl

es

pre

par

ed

from

run

1,

5

and

9

wer

e

furt

her

take

n

for

anal

ytic

al

char

acte

riza

tion

s,

in

vitr

o

cyto

toxi

city

assa

y,

and

antiba

cter

ial a

ctiv

ity

exper

imen

ts. n

PMM

A

par

ticl

es

pre

par

ed

from

run

5

wer

e

use

d

for

IB

load

ing

and

rele

ase

studie

s.a

nPM

MA

SFpre

par

ed

using

a

surfac

tant-

free

emulsio

n

pol

ymer

izat

ion

pro

cess

.

Fig. 1. Atomizparticles via th

rate over thenvironmenmaintainedstream fromdistillation was maintaplete additithe polymeto room temformed, whparticles. Ator discharby controllspeed, reaction zone, e

For comwas perfor(nPMMASF)methacrylataining ammmixture. Potemperatura typical SFatures resupolymerizato room tem

2.4. Isolatio

PMMA nlatex into mkept overnitated partitimes with iccator for 4ed reaction vessel for synthesis of nPMMAbiosurfacatnt core/shelle modied atomized microemulsion technique.

e entire surface of the clear solution, where a uniquet of the microemulsion proceeds. The bafes were

at the top of the reactor to bounce back the monomer outgoing air. The exhaust was then led through the

column for the recovery of monomer. The temperatureined at 55 2 C throughout the reaction. After the com-on of monomer, the reaction was continued for 1 h andrization reaction was stopped by cooling the mixtureperature. A transparent or translucent dispersion was

ich indicates the formation of microemulsion of nPMMA

buttery valve located at the dish bottom of the reac-ges the reaction mixture. The process was monitoreding the orice size, reciprocating compressor pressure,tion temperature, distance between atomizer and reac-tc.parison, a soap-free emulsion polymerization (SFEP)med synthesizing surfactant-free nPMMA particles, according to Camli et al. [1]. The monomer methyl-te (MMA) was added into a sealed glass reactor con-onium persulfate (APS) in a pre-mixed acetonewaterlymerizations were carried out for 3 h at 75 C in ae-controlled water bath under magnetic mixing. UnlikeEP system, the presence of acetone at elevated temper-lts in a clear monomer solution resembling a dispersiontion procedure. PMMA dispersions were cooled downperature, isolated and then characterized.

n of nanoparticles

anoparticles were isolated by drop wise addition of theethanol with constant stirring, and the mixture was

ight for uniform dispersion of precipitate. The precip-cles were ltered under vacuum and washed severalmethanol/water (1:1) and then dried in a vacuum des-8 h at 50 C.

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 99

2.5. Characterization

2.5.1. Determination of monomer conversion and solid contentThe total number of latex particles in the system (NP) and the

number of p(Xm) are cal

NP =60VXD

N = 43

(D

Xm (%) = WWwhere 0 itotal volumdensity of Pcle, NA is 6.weight, andrespectively

2.5.2. ParticThe Z-av

size were mUK). The diwere measEach data p

2.5.3. MoleThe nu

average mo(PDI) were an Agilent PL-Gel Agilconstructedrange of 44solved in teltered wittion.

2.5.4. MorpAtomic F

plates wereing in semi-of 10 N m1

microscopycover glass imens werecarbon conuum with gThe sputter(S-4800, Hi

A transmoperating aof the sampsamples dis

2.5.5. ElectrThe -po

zetasizer (zsolution at at room temwere adjustdetermined

2.5.6. FTIR spectroscopyFourier transforms infrared (FTIR) spectra of the biosurfactants

and nPMMA particles were recorded on Nicolet 5700 FT-IR spec-trometer at resolution of 0.5 cm1 with an average of 32 scans.

Therm wasimadratur

in al0 toheat

min

rmaleter, Toknnee of 1

FunctountATL wratioH gla

isoptrantsion pano

easd bynosse lipetho

amoined

assancenbuffeodiuS aqt 37s wa

erkin was

alani

totox

itro of Bs weAs wll Pamono

the C wiabilityltetfromcuba1 ml)empngthed olymer chains per particle (N) as well as the conversionculated according to the following equations:

m3

(1)

/2)3NAMn

(2)

1

2 100 (3)

s the density of MMA (0.94 g cm3 at 25 C), V is thee of MMA, Xm is polymerization conversion, is theMMA (g cm3 at 25 C), D is the diameter of the parti-02 1023 mol1, Mn is the number-average molecular

W1 and W2 are the weights of the polymer and MMA,.

le size and particle size distributionerage particle size and the distribution of the particleeasured using a Zetasizer (Malvern Zetasizer Nano-ZS,ameters of highly diluted dispersions (in 103 M HCl)ured, at several temperatures between 20 and 50 C.oint was the average of at least three measurements.

cular weights and their polydispersity index (PDI)mber-average-molecular weight (Mn) and weight-lecular weight (Mw) as well as the polydispersity indexdetermined by gel permeation chromatography withGPC-Addon Rev A02.02 series HPLC system using aent column and THF solvent. A calibration curve was

using standard polystyrene having a molecular weight90 to 1,112,000 g mol1. The dried nPMMA was dis-trahydrofuran at a concentration of 0.3% (w/v) and thenh a nylon membrane (pore size 0.45 m) before injec-

hology of the coreshell nanoparticlesorce Microscopy (AFM) images of dried samples in glass

captured using an AFM (Solver P20M, NT-MDT) work-contact mode. A microcantilever with a spring constantwas used to scan the samples. For scanning electron

(SEM), a colloidal emulsion (1%, v/v) was dropped on aand dried in a dust-free environment. The dried spec-

stuck on the sample holders with a double-coatedductive tab and then were sputter-coated under vac-old by using a sputter coater (E-102, Hitachi, Japan).-coated samples were then observed by the microscopetachi, Japan) at 15 kV.ission electron microscope (TEM, FEI Tecnai 2083)

t 120 kV was used to image and study the morphologyles. The TEM samples were prepared by drop castingpersion in the carbon coated copper (200 mesh) grid.

okineticstentials of nanoparticle latexes were measured using aetasizer 3000, Malvern Instruments, UK) in 1 mM NaClroom temperature. The measurements were also doneperature. The pH values of nanoparticle dispersions

ed to be in a range of 112. The isoelectric points were at a pH where the -potential is zero.

2.5.7. DSC

60, Shtempesealedfrom 3at the (50 ml

Thewere dmadzuand scaing rat

2.5.8. Am

nPMMThe titwith pKOH inas a tidisperisoprothree massessel-Rhamtrehaloacid m

Thedeterm(TNBS)nal cophate of 4% s1% TNBbated asample900, Psurfacethe -

2.6. Cy

In vceduresamplenPMMRosweblood cate toat 37

cell vidiphen20 l (and inide (0.room twaveleperformal analysis performed on Differential Scanning Calorimeter (DSC-zu, Tokyo, Japan) to investigate the glass transitione (Tg) of nPMMA and bulk PMMA. The samples (5 mg)uminum pans were scanned in the temperature range

300 C along with a reference sample as standard,ing rate of 10 C min1 under a nitrogen atmosphere1).

degradation properties of nPMMA and bulk PMMAmined on Thermo Gravimetric Analyzer (TGA-50, Shi-yo, Japan). Sample (10 mg) was put on a platinum pand in the temperature range from 30 to 500 C at the heat-0 C min1 under a nitrogen atmosphere (50 ml min1).

ional group analysis on the coreshell nanoparticles of carboxylic group on the surface of nPMMARL andas determined by using potentiometric titration [33].

n was carried out by Autotitrator (T50, Mettler Telodo)ss sensor (DGi115-SC, Mettler Relodo) by using 0.01 Mropanol standardized by potassium hydrogen phthalate. The sample preparation was performed by mixing aof the puried nanoparticles (20 mg ml1, 0.5 ml) withl (50 ml). Each value reported was an average of at leasturements. In addition, rhamnolipid in nPMMARL was

quantication of l-rhamnose by the orcinol assay [34].e was used for making the standard curve. Presence ofids in nPMMATL was determined by the phenol-sulfuric

d [35]. The stand curve was prepared with d-glucose.unt of amine groups on the surface of nPMMASR was

by the conventional 2,4,6-trinitrobenzene sulfonic acidy [8]. The nanoparticle dispersion was diluted to give atration of 10 mg ml1 and suspended in 100 l of phos-red saline solution (10 mM PBS, pH 7.6). Then, 200 lm hydrogen carbonate solution (pH 8.5) and 200 l ofueous solution were added. These mixtures were incu-C for 2 h. After incubation, the absorbance of 200 l ofs measured at 415 nm in a spectrophotometer (Lambda-Elmer). The amount of amine groups on the particle

calculated based on the calibration curve prepared byne standard solution.

icity assay in peripheral blood mononuclear cells

cytotoxicity assay was carried out following the pro-orase et al. [36]. Different working stocks of nPMMAre prepared, and 0.1 ml of twofold dilution series ofas added in a 96-well microtiter plate by using 10%rk Memorial Institute medium. Stimulated peripheralnuclear cells at 2 105 per well were added in dupli-

dilution suspension and the plates incubated for 5 daysth humidied 5% CO2 atmosphere. After incubation,y was determined by (4,5-dimethylthiazol-2-yl)-2,5-razolium bromide assay (Sigma, St. Louis, MO). Then,

a stock of 5 mg ml1) reagent was added in each wellted at 37 C for 4 h in a CO2 incubator. Dimethyl sulfox-

was added to each well and kept in the dark for 1 h aterature. Optical density was taken at 550 and 630 nm, the latter as a reference wavelength. The assays werein triplicate on 2 different days (n = 6).

100 C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

2.7. Antihemolytic activity

The nPMMA samples were exposed to anticoagulated (sodiumcitrate) human blood for 1 h under agitation (70 5 rpm) usingan orbital sat 37 C 6 for 15 min ared blood cwater-dispemal saline-the incubatThe absorbameasured bwas calcula

% hemolys

where As, Apositive cotests were c

2.8. nPMMA

The antpositive Badomonas aea nutrient bobtained wtrations of plate, followmicrotiter bacteria ancontrol. TheP. aeruginosculture medtained onlywithout bainhibited thinhibitory cmedium is

For mornPMMA trprocedure o

2.9. Oxidati

To elucidticles in Bacby three mrelease, inttathione lev

2.9.1. LactaMembra

assay kit (Swas measucentage rele

2.9.2. DeterOne mill

10% (w/v) tfor 10 min. Tcentrifugatiticles, cells,Three milli

acid (TBA; Sigma) solution was then added to the supernatant andwas incubated at 95 C for 60 min. It was cooled to room temper-ature and was centrifuged at 3500 rpm for 15 min. The absorptionspectrum of the supernatant was recorded at max = 532 nm to esti-

he fte. Fdiald

betwt difysis o

Glutauced

desia wen alie wain. Awas by m

of sution ing the Bormmol m

rug

nPMcurcucuba), theQ, anphouentted bloadtion capsing e(

wt(ini

rug l the ith mmer

C wie relectedeasemetinat

inet

nalylowin

ord

= k0haker (Orbitek, Scigenics Biotech, India) thermostated 1 C. The samples were then centrifuged at 4000 rpmnd plasma was aspirated immediately. The blood had

ell (RBC) concentration of 1 108 cells ml1. Deionizedrsed RBC was used as the positive control, and the nor-dispersed RBC was used as the negative control. Afterion, RBC was centrifuged under 2200 rpm for 5 min.nce of the released hemoglobin in the suspensions wasy spectrophotometry at 546 nm. The hemolysis ratioted with the following formula:

is =(

As AnAp An

) 100 (4)

p, and An are the absorbances of the test suspensions,ntrol, and negative control, respectively. Two parallelarried out to obtain a reliable value.

bacteria interaction

ibacterial activity was investigated against gram-cillus subtilis NCIM 2718 and gram-negative Pseu-ruginosa NCIM 5029. Both the strains were inoculated inroth (NB) at 37 C for 24 h. These bacterial suspensionsere then diluted to 108 CFU ml1. The different concen-nPMMA samples were poured in a 96-well microtitered by adding 10 l of the bacterial suspension. The

plate was incubated at 37 C for 2 h. The untreatedd nanoparticle suspensions were referred as positive

antibacterial activities of nPMMA against B. subtilis anda were obtained by measuring the absorbance of theium at 600 nm compared with the control, which con-

different concentrations of nPMMA suspensions andcteria. The lowest concentration of nanoparticles thate growth of bacteria was considered as the minimumoncentration (MIC), where the absorbance of the culturethe lowest and thereafter becomes constant.phological observation of the bacterial cells post-eatment, SEM images were taken following thef Inphonlek et al. [8].

ve stress markers

ate the mechanism of toxicity induced by nPMMA par-illus subtilis NCIM 2718, oxidative stress was assessed

ethods, viz., extracellular lactate dehydrogenase (LDH)racellular ROS in the bacterial cells and reduced glu-els.

te dehydrogenase (LDH) releasene integrity of the treated culture was assessed an LDHigma) as per the manufacturers protocol. Absorbancered at 490 and 690 nm and data were reported as per-ase of LDH compared to control.

mination of lipid peroxidation (LPO)iliter of treated bacterial culture was mixed with 2 ml ofrichloroacetic acid (TCA) and left at room temperaturehe pellet was removed and supernatant was taken afteron at 10,000 rpm for 30 min to ensure that the nanopar-

and precipitated proteins were completely removed.liters of a freshly prepared 0.67% (w/v) thiobarbituric

mate ttriplicamalonplottedagainshydrol

2.9.3. Red

viouslyBacterMAs. Amixturfor 5 mbuffer tied 500 lincubaremainusing after nGSH

2.10. D

TheIB/AQ/and in30 minof IB, AspectroSubseqcalculain the centraand enfollow

LC% =

EE% =

A dtainingbags wwas imat 37

tion thAt selethe relspectrodeterm

2.11. K

To athe fol

1. Zero

MtM0ormation of TBARS. All analyses were carried out inrom the TBARS intensities the corresponding levels ofehyde (MDA) were deduced from a calibration curveeen the intensity of TBARS measured at max = 532 nm

ferent concentrations of MDA synthesized by acidicf 1,1,3,3-tetramethoxypropane.

thione levels glutathione levels were estimated by method pre-cribed by Hazra et al. [28], with slight modication.re cultured to 1 109 cfu ml1 and treated with nPM-

quot of 0.6 ml was extracted with 30 l of 100% TCA. Thes kept in ice for 10 min and centrifuged at 10,000 rpmfter precipitation, 200 l of 30 mM TrisHCl (pH 8.9)used to neutralize the sample. Further, GSH was quan-easuring absorbance at 412 nm by reaction betweenpernatant and 2.5 ml of 0.01% dithionitrobenzoate afterfor 1520 min; simultaneously, a 0.4 ml portion of thetreated culture was used for determination of proteinradford method. Glutathione levels were estimated

alization to cellular protein levels and expressed asg1 protein.

loading and in vitro release studies

MA particles (50 mg) were suspended in 10 ml of DDIW.min (0.15 mg ml1) was added to this suspension

ted for 48 h. Following centrifugation (10,000 rpm for particles were washed thrice with DDIW. The amountd curcumin in the supernatant was assayed using a UVtometer at 222 nm, 274 nm, and 430 nm, respectively.ly, the amount of drugs loaded onto the particles wasy subtracting the sum of the nal drug concentrationing solution and the wash from the initial drug con-in the loading solution. The drug loading content (LC)ulation efciency (EE) were then calculated using thequations:

wt of drug in nanoparticles of nanoparticles + wt of drug

) 100 (5)

wt of drug in nanoparticlestial wt of drug in the loading solution

) 100 (6)

oaded nanoparticle suspension (5 ml) or solution con-free drug, as a control, was placed in dialysis membraneolecular weight cut-off 25,000 g mol1. The sealed bagsed in 200 ml of 0.15 M Tris buffer solutions (pH 7.4)th continuous magnetic stirring. To ensure sink condi-ase medium was replaced with fresh buffer every 24 h.

time intervals, 200 l of sample was withdrawn from medium. The drug content in the sample was assayedrically at 222 nm. Each data point was a mean of threeions standard deviation.

ics studies

ze release kinetics and mechanism, data were tted tog four mathematical models:

er

t (7)

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 101

MA/bi

2. First orde

MtM0

= 1

3. Higuchi m

MtM0

= kH

4. Korsmey

MtM0

= ktn

where, trelease dthe drugand k repconstantrespectivmine thelinear relation co

2.12. Statis

Data weexperimentMiniTab Veby Tukeys controls. Fudetermine p-value of nPMMARL > nPMMATL. There are

on sub-100 nm nPMMA [1,5,39] and some degree of and aggregation is often reported [8]. Compared to pre-ts, our method does not require use of toxic organicd co-surfactants and thus is advantageous over reversesion or dispersion polymerization method to producelled and monodisperse sub-100 nm PMMA.terature reports, it was found that conventionalarticles had a surface charge of 75.4 mV [40]. The sur-s of nPMMASR particles were positive at pH 28 due totion of amine groups of surfactin (Fig. S4, Supplemental), which strongly indicated that the surface component

ticles was made of surfactin. From the changes of size charges, it was evident that the positively charged sur-

be coated on the negatively charged PMMA colloidal electrostatic interaction. Upon increasing solution pH,tials began to decrease conrming that the isoelec-

for the latexes coincides with the pKa of the aminee groups [41,42]. It even reached to negative valuesasic pH, resulting from the transformation of proton-s to neutral ones and then combined with hydroxylition, highly positive charge of +49 mV can keep thees stable in acid and neutral solutions due to the chargerce. However, for nPMMARL and nPMMATL coreshellative surface charges were recorded which increasessing medium pH from 2 to 7 due to the ionization ofnits. When the basicity increased, the particle surfaceore and more hydroxyl ions from the solution (possibly

rhamnose and trehalose sugar moiety) resulting in arface and a negative zeta-potential. This is the evidencevely charged rhamnolipids or trehalose lipids could bed on the nPMMA particles, leading to the lower surfaces known that nanoparticles dispersed in aqueous solu-

stabilized either by electrostatic stabilization (surfaceby steric stabilization (surfactants or other moleculesicle surface), or by a combination of both [4,8,18].-potential values beyond 20 mV are considered char-f a stable colloidal dispersion. According to the DLVOregation occurs when attractive van der Waals forcese particles prevail over the electrostatic repulsive forces

did not occur in our case. Thus, these size-controlled,rse, stable nano latex could serve as good drug delivery

iation of chemical functional groups present in thees was identied by FTIR spectroscopy (Fig. 7). Ford, the broad peak at 35003200 cm1 correspondedetching (for O H bonds) and the signals at 2850 andwas attributed to CH2 CH3 stretching, ( CH3) sym-rmation vibration, (C H) bending vibrations of CH3roups, which are characteristics of polysaccharides.retching band at 1732 cm1 is characteristic of estercarboxylic acid groups while characteristic frequen-8 and 1029 cm1 represent C O C ethereal vibration. 7a). The FTIR spectrum of surfactin showed a broad03200 cm1, which was attributed to an O H stretch-H stretching. The signal at 3092 cm1 corresponded

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 105

to C H strwas assigngroups on [43,44]. Simand at 1654conrming [32] (Fig. 7astretching oand O H de

The FTIRFor nPMMappeared awave numbfrequency onPMMA restion of COby ellipse inof PMMA. F(C O adsor3440 cm1,stretching owere obser1730 cm1

in PMMA anrespectivelylipid or trestrength ofshift its streof rhamnolFig. 6. TEM photographs of (a) nPMMARL, (b) nPMMASR, (c) nPMMATL and (d) s

etching vibrations. The peak observed at 1634 cm1

ed to NH3+ groups due to the protonation of aminesurfactin and amide linkage I appeared at 1008 cm1

ilarly, the broad negative bands at about 3400 cm1

cm1 are attributed to O H stretching (for O H bonds)the presence of glycolipid moieties of trehalose lipids). Other notable frequencies were at 1732 cm1 (C Of ester bonds and carboxylic acids) and 1454 cm1 (C Hformation vibrations, typical for carbohydrates).

spectrum of nPMMA nanoparticles is shown in Fig. 7(b).ARL, the stretching vibration frequency of ( CH3)t wave numbers of 2925 cm1 and 2850 cm1 and theer 1730 cm1 corresponds to the stretching vibrationf ( C O). The broad peak at 34403450 cm1 for all theults from OH stretching vibrations due to incorpora-OH [810]. The peaks from 2800 to 3100 cm1 (marked

Fig. 7b) exhibit various characteristic vibration bandsor nPMMASR, both the characteristic peaks of PMMAption at 1730 cm1) and surfactin (N H stretching at

asymmetric stretching of CH2 at 2950 cm1, symmetricf CH2 at 2845 cm1, and NH3+ vibration at 1634 cm1)ved. For nPMMATL, broad peak at 34403450 cm1 andwere assigned to OH stretching vibrations of COOHd C O stretching of ester bonds of trehalose tetraester,. The formation of hydrogen bond between rhamno-halose lipids and nPMMA is expected to weaken the

carbonyl bond in rhamnolipid or trehalose lipids andtching vibration to lower frequencies. The interaction

ipid/trehalose lipids with nPMMA particles has shifted

its carbonylnolipid or trrespectivelydence of thto the nPMMilar to surfaobserved, wcomponentand surfactstretching icompared tthe thicker incident beor the corestretching i

To ensuand the PMcles were redecantationnatant was(DDIW) usesupernatanmethod andthe biosurfaand nPMMAsis. The FTIRhydrolysis wther conrmparticles.urfactant-free nPMMA (nPMMASF) particles.

stretching vibration from 1732 cm1 in the pure rham-ehalose lipids to 1704 cm1 in nPMMARL and nPMMATL,. This chemical interaction provided a substantial evi-

e presence of rhamnolipid/trehalose lipids coating onA. For the nPMMASR particles, their spectra were sim-

ctin, however, the additional signal at 1730 cm1 washich corresponded to the C O stretching of PMMA core. This could conrm the complexation between PMMAin. In addition, it can be observed that the signal of C On nPMMARL/nPMMASR/nPMMATL particles is lower aso the previous reports. This may be due to the fact thatshell of biosurfactants could limit the penetration of theam on the reection mode of the FTIR to the inner layer

of particles. This would result in the decrease of C Ontensity of PMMA.re covalent linkage between the biosurfactant shellMA core, the nPMMAbiosurfactant core/shell parti-peatedly washed with water through a centrifugation,, and redispersion cycle until conductivity of the super-

equal to that of double distilled and deionized waterd, and rhamnolipid, surfactin and trehalose lipid in thet was not detectable with the orcinol method, Bradford

phenol-sulfuric acid method, respectively. To separatectants from the PMMA core, the nPMMARL, nPMMASRTL core/shell particles were subjected to acid hydroly-

spectrum of the dried products obtained from after acidas identical to that reported for the PMMA, which fur-ed the formation of nPMMAbiosurfactant core/shell

106 C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

DSC curvhalose lipidbulk PMMAInformationfactin and tat 60, 130 aS5a). It waof nPMMAR128 C for nnPMMATL thtive peaks oof correspothin shell lasecond scanTg of nPMMand 114 C Tg of polymnano-scale surface eneof surface ehigher tempparticles (sjoined to fogained enenPMMARL,S(107 C) (FiFig. 7. FTIR spectra (a) pure biosurfactants only and (b) nPMMA/b

es of rst scan of pure rhamnolipid, surfactin and tre-s; and rst as well as second scan of nPMMARL,SR,TL and

are shown in Fig. S5ab and Table S2 (Supplemental). Melting temperature (Tm) of pure rhamnolipid, sur-rehalose lipids showed only broad endothermic peaksnd 90 C, respectively (Supplemental Information, Fig.s observed from Fig. S5(b i, iii and v) that rst scanL,SR,TL showed two step exothermic peaks at 62 andPMMARL; 130 and 136 C nPMMASR; and 93 and 126 Cat are attributed to Tg1 [22,24,45,46] along with respec-f Tm arising due to the presence of very little amount

nding biosurfactants. This nding corroborates with theyer of biosurfactants observed in TEM. On immediatening after constant cooling to ambient temperature, theAs shifted at 115 C for nPMMARL, 117 C for nPMMASRfor nPMMATL (Fig. S5b ii, iv and iv). The reason for higher nanoparticles might be a decrease in particle size tothat results in an increase in surface area and higherrgy [47]. So during rst scanning of nPMMA, the lossnergy results in the energy release which shifted Tg1 toerature. But during second scan, there were no polymer

intering phenomenon occurred) [2224,46], and theyrm a single lm layer with lesser surface area, whichrgy and showed single Tg2 during second scanning ofR,TL respectively. The lower value of Tg1 for bulk PMMAg. S5b; vii) was due to its large size and lower surface

area, whichdata obtainmodied ch

Fig. S6 thermal staMAs showefor nPMMAdon = 358 C(WL) = 100%with 100% WnPMMARL,Sin the therarise from tand radicalther investibehind the

To furthlipids on thevaluated tsurface by aqueous sowhile in nPobserved th7.29 mM g

primary amby the TNBiosurfactant coreshell particles.

was much lower than nPMMA particles [2224]. Theed here is in line with the Tg reported in PMMA-aciditosan nanoparticles (114122 C) [4,46].and Table S2 (Supplemental Information) show thebility (dt) of nPMMARL,SR,TL and bulk PMMA. nPM-d higher thermal stability (don = 364 C and doff = 411 CRL; don = 370 C and doff = 417 C for nPMMASR and

and doff = 398 C for nPMMATL) with % weight loss [19] than bulk PMMA (don = 283 C and doff = 360 C

L), which further proved a close molecular packing inR,TL than bulk PMMA. The mechanism for this increasemal degradation temperature of nPMMAs appears towo mechanisms, i.e., steric restriction of chain motion

deactivation by nanoparticles [46,48]. However, fur-gations are necessary to reach a meaningful conclusionthermal degradation pattern.er conrm the presence of rhamnolipid and trehalosee surface of nPMMARL and nPMMATL, respectively, wehe amount of carboxylic groups on the nanoparticleusing acidbase titration of free fatty acid in non-lution. The acid content of nPMMARL was 0.08 mM g1,MMATL it was found to be 2.04 mM g1. Besides, it wasat nPMMARL and nPMMATL contain 11.13 mM g1 and1 of rhamnose and trehalose, respectively. Moreover,ine groups on the surface of nPMMASR were determinedS assay. This method is based on the reaction of TNBS

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 107

Fig. 8. (a) Cytoperipheral blocoreshell pamean S.D. (stcate. *Statisticeach).

and primarwere 28.05

3.3. In vitromononuclea

Unwantphagocytic clear leukoPMMA partnPMMA comononuclematerials (eas the concvival rate siin the rangecentrationsto the cells(ANOVA) fotal Informatshows pairs

on grouping information using Tukeys multiple comparisons at95% condence. This study would be, therefore, helpful for devel-opment of carriers for drug delivery and llers for cosmetic surgery.

r studies with in vivo models are however necessary to delin-e un

ood c

emolASR win hu

highconcytic asignolysiMMfor aASR p

tiba

evabiosous Furtheeate th

3.4. Bl

A hnPMM1 g l1

icantlyin the hemoltically a hemSince Pjugate nPMM

3.5. An

To (core)at varitoxicity of nPMMA/biosurfactant coreshell particles against humanod mononuclear cells. (b) Hemolysis assay of nPMMA/biosurfactantrticles using human blood erythrocytes. Data represented areandard deviation) of three identical experiments made in three repli-ally signicant difference as compared to the controls (p < 0.05 for

y amine. Free amine groups on the nanoparticle surface M ml1.

cytotoxicity test on human peripheral bloodr cells

ed biological effects induced by the suppression of theand antibacterial activity of human polymorphonu-cytes is claimed to be a serious health concern due toiculate carrier toxicity. Hence, the cytotoxicity of theated with biosurfactants on human peripheral bloodar cells was investigated. Irrespective of the type ofither biosurfactant alone or the nanoparticles) used,entration of the nanoparticles increased, the cell sur-gnicantly decreased (Fig. 8a). This is particularly true

of 0.10.4 g l1 (p < 0.05 for each). However, higher con- (0.50.7 g l1) did not produce signicant cytotoxicity

(p > 0.05 for each). The results of analysis of variancer % cell viability are given in Table S3 (see Supplemen-ion). Moreover, Table S4 (in Supplemental Information)

of signicance of particle type and concentration based

The antibasame voluma 2 h exposbulk PMMAkilled both where the were then dthe nanopaB. subtilis anthat the MIwhen intronanoparticl(p < 0.001 foThe same observed asurfactantshigher (0.2Apparentlyactivities aefcacy wanumber of nanoparticldivided by As the consignicantleach) (Fig. fastest antiGram-positactivity betsignicant. factant mosurface arethe surfacesize of biocprovides mimproved k

Fig. S7 graphs of subtilis aftedisintegratideath. It seederlying mechanisms of reduced cytotoxicity.

ompatibility

ysis assay was conducted (Fig. 8b), which showed thatas less toxic than nPMMARL and nPMMATL, even at

man blood. It was observed that % hemolysis was signif-er in cells exposed to nPMMARL and nPMMATL particlesentration range of 0.61 g l1 (p < 0.05 for each). Thectivity in cells exposed to nPMMASR was not statis-

icant. According to ASTM standards, a material withs value less than 2% is considered as hemocompatible.A is one of the most commonly studied polymer con-pplication in nanomedicine, a superior performance byresents blood compatibility for this system.

cterial properties

luate the antibacterial properties of nPMMAurfactant (shell) nanoparticles, the growth of bacteriaconcentrations of nanoparticles was assessed at A600.cterial photographs were obtained by culturing thee of each sample solution in nutrient agar plates after

ure to the bacteria. Compared with the control and the plates, the coreshell nanoparticles (30 nm) perfectlyB. subtilis (Fig. 9 a) and P. aeruginosa (Fig. 9b). The MICsconcentration at which the O.D. becomes minimum,etermined (Fig. 9c). It was found that when increasing

rticles concentration, the MICs of nanoparticles againstd P. aeruginosa decreased. For B. subtilis, it could be seenC of pure biosurfactants were 0.40.55 g l1. However,ducing them into the polymer (PMMA/biosurfactantes), the MICs were lowered to a range of 0.10.32 g l1

r each) when compared to the control or bulk PMMA.trend of the nanoparticles antibacterial activity wasgainst P. aeruginosa. However, the MICs of the bio-

only and PMMA/biosurfactant nanoparticles were50.7 g l1 and 0.30.6, respectively; p < 0.05 for each)., all nanoparticles exhibited stronger antibacterialgainst B. subtilis than P. aeruginosa. The antibacterials also further investigated by the kinetic test. Thesurviving bacterial colonies for each type of coreshelles was counted as a function of contact time (min) andthat of the control to acquire the fractional survival.tact time increases, the fractional survival of bacteriay decreases in a time-dependent manner (p < 0.05 for9d and e). In addition, the nPMMASR particles exhibitmicrobial properties against both Gram-negative andive bacteria. However, the difference in bactericidalween nPMMARL and nPMMATL was not statisticallyThis effective antimicrobial performance of the biosur-died nPMMA particles can be explained by the largea of the nano-size particles. Based on the same weight,

area of biocides increases drastically with decreasedides and the expanded surface area of nanoparticlesore active sites to contact the bacteria, leading to theilling efciency [8,4850].(Supplemental Information) displays the SEM micro-the interaction between nPMMASR particles with B.r incubation. The arrow marked area shows cellularon and fragmentation which may be the onset of cellms plausible that the nPMMASR particles can bind with

108 C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

Fig. 9. Photographs showing the bacterial culture plates of (a) B. subtilis and (b) P. aeruginosa upon a 2 h exposure to the control, bulk PMMA, and the 30 nmnPMMA/biosurfactant coreshell particles. (c) The MICs of nPMMA/biosurfactant coreshell particles. Data presented are mean S.D. of three identical experiments madein three replicate. Statistically signicant differences were assessed as compared to the controls. *p < 0.05, **p < 0.001. Antimicrobial kinetic test graphs for the 30 nmnPMMA/biosurfactant coreshell particles as a function of contact time against (d) P. aeruginosa and (e) B. subtilis. The fractional survival was calculated as fractional sur-vival = (B/A) (where A is the number of surviving bacteria colonies in the control and B is that in the coreshell nanoparticle sample). Data represented are mean S.D. ofthree identical experiments made in three replicate. *Statistically signicant difference as compared to the controls (p < 0.05 for each).

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 109

the bacterial membranes via electrostatic interaction, and eventu-ally kill the cells. The addition of surfactin particularly can increasethe amine groups, and more positive charges, which led to moreantibacterial activity compared to other nanoparticles. The differ-ential behavto the diffeGram-negasubtilis cell could rendetion by the nthe cell waloglycan andlipoproteinattack of thactivity of t

Since higorganism wthe bacteriers like LDHlevels wereticles show(p < 0.05) intion) and LPincrease in saturated fanPMMATL anicantly dcompared tand it followS8c). The cGSH depletioxidative stnPMMASR prial cells. Thbacterial cea compromROS. Basedcellular toxH2O2 in bacphospholipDNA damagphology and(Scheme 3)the model that could ities responsenvironmenassessment

3.6. Drug lo

The nPMciency for I91.45, 75.87and nPMMA33.57 and 2amount of dionic interagroups of suacid group ipH and saltmaximum lto the loss omer as a reswas selecte

3. Po

10 pes usysis tarticlslownopag eff

lowe < 15; Trist 10 h is observed for the particles with 2.5% and 15.5% LC,tively. In contrast, the release prole for particles with 33.3%lted in only 40% of the drug being released even after 50 h.noparticles with high drug loading started to aggregate inlysis bag beyond 50 h and thus the release experiment wasated thereafter.

dependence of release kinetics on IB loading content maylained by the change in nanoparticles properties and phys-ate of the drug in the nanoparticles. It is believed thatg-loaded nanoparticles are much more hydrophobic than

ank particles due to the charge neutralization and thehobic nature of IB. With increasing IB loading, the hydropho-of the particles increases resulting in lower swelling and

rates. Similar results have been observed in doxorubicinhloride/PMAA-PS-80-g-St system [10], dox loaded sulfo-

dextran microsphere [54] and liposomal doxorubicin [55].llustrated in Fig. 10(b), at the same drug loading (i.e., 15.5%release from the nanoparticles is much faster at pH 5 (simu-endosome pH) than that at pH 7.4 (simulating normal tissuee higher release rate at acidic pH may be due to the pH-ence of IB-nPMMASR interaction, which is weakened atH as a result of PMAA protonation and existence of surfactincation leading to its faster dissolution. The above resultssistent with recent literature using similar carboxylic aciding polymers [10,52,53]. Recently many researchers have

efcient pH responsive delivery systems, especially usingior of these nanoparticles toward these bacteria is duerence in cell wall architecture of Gram-positive andtive bacteria [4951]. The peptidoglycan layer of the B.wall is composed of networks with plenty of pores thatr them more susceptible to the intracellular transduc-anoparticles leading to cell disruption. On the contrary,

l of P. aeruginosa is made up of thin membrane of peptid- an outer membrane composed of lipopolysaccharide,

, and phospholipids, which would be less prone to thee nanoparticles. This explains the higher antibacterialhe nanoparticles against B. subtilis than P. aeruginosa.her antibacterial activity was observed in B. subtilis, thisas further probed for underlying the mechanism behindcidal effects. For this purpose, oxidative stress mark-

release, lipid peroxidation and reduced glutathione estimated. B. subtilis cells exposed to nPMMASR par-ed a concentration-dependent, statistically signicantcrease in LDH release (Fig. S8a; Supplemental Informa-O (Fig. S8b; Supplemental Information) as evident by anthe formation of MDA, an oxidized product of polyun-tty acids compared to that observed with nPMMARL andt similar concentrations. Cellular GSH levels were sig-epleted (p < 0.05) after 1 h exposure to nPMMA particles,o control (i.e., without nanoparticles in culture media)ed the order as nPMMASR > nPMMARL > nPMMATL (Fig.

orrelation between LDH release, LPO induction, andon in a concentration-dependent manner suggests thatress could act as an important pathway by which thearticles induce DNA damage in Gram-positive bacte-ese ndings conrm that nPMMAs are internalized bylls and induce signicant oxidative stress leading toised cellular antioxidant defense and accumulation of

on these observations a hypothetical mechanism foricity could be through the generation of OH, O2, andterial cells resulting in the oxidation of polyunsaturatedids. The lipid peroxidation reaction subsequently causese, GSH depletion, and disruption of membrane mor-

the electron transport chain, which leads to cell death. Our observations on the toxic response to nPMMAs inbacterium B. subtilis reect the possible perturbationsnict damage to other members of microbial communi-ible for biogeochemical cycles in aquatic and terrestrialt. Our observations substantiate the need for impact

of such novel materials in environmental settings.

ading onto and in vitro release from the nanoparticles

MASR particles exhibited high loading capacity and ef-B. The loading efciency of IB, AQ and curcumin was

and 77.22%, respectively, whereas in case of nPMMARLTL the encapsulation efciency was rather low (23.79,

5.66%, respectively). It can be explained that the higherrug loading in nPMMASR particles was due to the strongction between the degree of protonation of the aminorfactin in the nPMMASR shell, as well as the carboxylicn IB. This interaction is greatly affected by the medium

contents [52,53]. In this work, we observed that theoading capacity decreased to 3.7% at pH 4, attributablef the ionic interaction between the drug and the poly-ult of PMAA protonation. Based on these data, nPMMASRd for in vitro IB release studies.

Scheme

Fig.particlof dialnanopmuch the naa stronwith a30% LCpH 7.4the rsrespecLC resuThe nathe diatermin

Thebe expical stthe druthe blhydropbicity releasehydrocpropyl

As iIB), IB lating pH). Thdependacidic pas polyare concontainstudiedssible mechanism of nPMMA-induced genotoxicity and cytotoxicity.

resents the release proles of IB from the nPMMASRing free IB as a reference considering the barrier effectubing membrane. It was seen that IB release from thees to the release medium outside the dialysis tubing waser than free IB, indicating prolonged release of IB fromrticles. Fig. 10 (a) shows that the initial IB loading hasect on the release kinetics of IB from the nanoparticles,r release rate at higher initial drug loading content, i.e.,.5% LC < 2.5% LC. In the same release medium (0.15 M;

buffer), an initial burst release of 17% and 11% within

110 C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113

80

100

10 20 30 40 50

80

100(a) Free IB LC = 2.5 %

LC = 15.5 % LC = 30.0 %

Time (h)

(b) Free IB LC = 15.5 %; pH 5.0

LC = 15.5 %; pH 7.4

Fig. 10. (a) Eff articles at 37 C in 0.15 M pH 7.4 Tris/NaCl buffer. (b) Effect of pH on kineticsof IB release fr ee IB from the dialysis bag was used as control. For each buffer system theionic strength dependent trials.

charged spebiosurfactaleaching (achieving sindings. Thutilized as and extracethan that inare expectetemic circutherapeutic

Fig. 11(aticles with in the relearelease of Aof the entraphase was to the AQ rreleased frodition, the the very fasrate of theues. A similfor chitosansulated polMoreover, anicantly pcondition [5

The in visimulating illustrates of the drugobserved dfor approxito (a) the acof the polymthe drug intmatrix erodthe drug mosurface defprised of a sof the drug decrease in0 10 20 30 40 50

0

20

40

60

0

0

20

40

60

Cu

mu

lative

re

lea

se (

%)

Time (h)

Cu

mu

lative

re

lea

se (

%)

ect of initial loading content on the kinetic of IB release from nPMMASR coreshell pom the nanoparticles with drug loading content of 15.5% at 37 C. The release of fr

was kept constant at 0.15 M by adding NaCl. Data points are mean S.D. of three in

cies as gate keepers [56]. Our system utilizes strongernt coating to protect the release of IB with minimum27%) at pH 7.4. This makes our system more reliable inte specic drug delivery compared to some of the recente above results prove that surfactin can be effectivelya pH responsive gate keeper to target various tissuesllular tumors. Since extracellular pH in tumor is lower

normal tissue and the plasma [10], the nanoparticlesd to release more drug in tumor tissue than in the sys-lation. As a result, lower systemic toxicity while higher

efcacy could be obtained.) shows the release proles of AQ from nPMMASR par-a loading capacity of 15.5% for various time intervalsse media at various pH values at 37 C. Initially theQ at the pH 5.0 and 7.4 provided a continuous releasepped AQ for up to 50 h and the initial burst releasenot as obvious as IB release data. This is in contrastelease rate at pH 3.0 where 90% of the loaded AQ wasm the nanoparticles within 50 h. At strong acidic con-nanoparticles would dissolve quickly, which leads tot release thereby demonstrating that the drug releasese nanoparticles is possible by changing the pH val-ar effect of sustained release was previously observed/poly (lactic acid) [57] and anti-cancer drugs encap-

y (glycolic acid) stabilized by polyvinyl alcohol [58].s an acidic drug, solubility of AQ can be improved sig-ossibly due to ionization into anions under the alkaline7].tro release of curcumin from nPMMASR particles underphysiological conditions (37 C, PBS buffer; pH 7.4)three distinguishable stages (Fig. 11b). The release

undergoes a burst release in the stage I, which wasuring the rst 10 h of the release, and it is responsiblemately 35% of the release. This burst is most likely duecumulation of the drug molecules at or near the surfaceer matrix which subsequently facilitates the release ofo the surrounding solution and (b) if the surface of thees as a result of the incubation in the aqueous solution,lecules could escape much more simply through these

ects [3]. The stage II for all the drug contents is com-teady state drug release which accounts for almost 50%was released in between 10 and 40 h. Finally, a gradual

the release rate (stage III) until they reached their

Fig. 11. Release prole of (a) AQ and (b) curcumin from nPMMASR coreshell parti-cles at 37 C in Tris/NaCl buffer (0.01 M) at 37 C. Values reported as the mean S.D.(n = 3).

C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113 111

Table 2Drug release kinetics and mechanisms for different stages of the release of three types of drugs following the KorsmeyerPeppas (Power law model).

Druga Release stage Model parameters Release mechanism

n R2

IB

AQ

Curcumin

a Drug-carri

maximum rare requiredmatrix [3].

3.7. Kinetic

The valucient R2 aimportant Informationlaw (Korsmthe three tylowed the noccurred mpolymeric mutilized to orelease procformation oticle is an eAs opposedand III undedrug molec

4. Conclus

A one-psion procesPMMA nanosulfate (SDScompatiblelipid). The monomer abe as low in comparision polymnanosized cshells of thand 2050 0.91.5 10content of ntant was incwas ascertFTIR and t(shell) partstrong antiGlutathionealdehyde ledehydrogentive stress

d ched oquines wwithtem ith aed ontentgatioorpoatio

whie relfusiog coa. Thiss as n.

wled

ruddn (Uh BSRdate

(D.Sch (C

Seni, resp

the Tl of M, Ind

dix AI 0.8157 II 0.4804 III 0.2677

I 0.7811 II 0.2558 III 0.1928

I 0.6638 II 0.2166 III 0.2419

er formulation: nPMMA (5 mg ml1) + IB/AQ/curcumin (5 mg ml1).

elease occurred. It is believed that the drug molecules to follow longer routes in order to escape the polymeric

s of the drug release

es of the release exponent n and the correlation coef-fter tting with the power law led to a number ofobservations (Table 2 and Table S5 in Supplemental). Firstly, tabular values of R2 showed that the powereyerPeppas) was best tted with release kinetic data ofpes of drug used here. Secondly, stage I of the release fol-on-Fickian diffusion. Therefore, the release of the drugainly through the combination of the diffusion from theatrix and the surface erosion. The FESEM analysis was

bserve the morphology of the polymer surface once theess was over (Fig. S9 in Supplemental Information). Thef the cracks on the surface of the polymeric nanopar-vidence of the proposed release mechanism for stage I.

to the proposed release mechanism for stage I, stages IIrwent the Fickian diffusion in which the release of theules was solely by diffusion [3].

ions

ot oil/water (O/W) modied atomized microemul-s was used to synthesize biosurfactant-functionalizedparticles, by which conventional toxic sodium dodecyl) was replaced by non-toxic, biodegradable and bio-

biosurfactants (rhamnolipid, surfactant and trehaloseamount of biosurfactant required was 1/35 of themount by weight and the surfactant/water ratio couldas 1/210. These surfactant levels are much lowerson with those used in a conventional microemul-erization system. These nanoparticles are composed ofores of high molecular weight PMMA and nano-thin

Detaileanchoranthraparticldrugs ery sysdays woccurring coninvestithe inccombinmatrixafter thian difvaryinagentshybridcations

Ackno

Animissiothroug(BSR), nologyResearvidingKunduviding(SchooJalgaon

Appene biosurfacatnts. The particles were spherical in shapenm in diameter with an average molecular weight of5 g mol1 (polydispersity index 1.642.65). The solidPMMA increased when the amount of added biosurfac-reased. Surface coating by biosurfactants onto nPMMAained by AFM, TEM, zeta potential measurements,hermal analysis. These nPMMA (core)biosurfactanticles were non-toxic, biocompatible and exhibitedbacterial activity against B. subtilis and P. aeruginosa.

depletion with a concomitant increase in malondi-vels (increasing reactive oxygen species) and lactatease activity demonstrates that nPMMAs induce oxida-leading to genotoxicity and cytotoxicity in B. subtilis.

Supplemfound, in th2014.02.05

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Poly(methyl methacrylate) (core)biosurfactant (shell) nanoparticles: Size controlled sub-100nm synthesis, characterizatio...1 Introduction2 Materials and methods2.1 Materials2.2 Production and purification of the biosurfactants2.3 Synthesis and purification of PMMA (core)biosurfactant (shell) nanoparticles2.4 Isolation of nanoparticles2.5 Characterization2.5.1 Determination of monomer conversion and solid content2.5.2 Particle size and particle size distribution2.5.3 Molecular weights and their polydispersity index (PDI)2.5.4 Morphology of the coreshell nanoparticles2.5.5 Electrokinetics2.5.6 FTIR spectroscopy2.5.7 Thermal analysis2.5.8 Functional group analysis on the coreshell nanoparticles

2.6 Cytotoxicity assay in peripheral blood mononuclear cells2.7 Antihemolytic activity2.8 nPMMAbacteria interaction2.9 Oxidative stress markers2.9.1 Lactate dehydrogenase (LDH) release2.9.2 Determination of lipid peroxidation (LPO)2.9.3 Glutathione levels

2.10 Drug loading and in vitro release studies2.11 Kinetics studies2.12 Statistical analysis

3 Results and discussion3.1 Preparation of the nanoparticles3.1.1 Effect of monomer amount on nPMMA particle size3.1.2 Effect of initiator amount on nPMMA particle size3.1.3 Effects of surfactant type and concentration on particle size

3.2 Characterization of the PMMA (core)biosurfactant (shell) nanoparticles3.3 In vitro cytotoxicity test on human peripheral blood mononuclear cells3.4 Blood compatibility3.5 Antibacterial properties3.6 Drug loading onto and in vitro release from the nanoparticles3.7 Kinetics of the drug release

4 ConclusionsAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data