1 s2.0 S092777571400199X Main

Colloids and Surfaces A: Physicochem. Eng.
Aspects 449 (2014) 96113
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa
Poly(methyl methacrylate) (core)biosurfactant (shell) nanoparticles:

Size controlled sub-100 nm synthesis, characterization, antibacterial
activity, cytotoxicity and sustained drug release behavior
Chinmay Hazra a,1 , Debasree Kundu a,1 , Aniruddha Chatterjee b, ,
Ambalal Chaudhari a , Satyendra Mishra b
a
b
School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra, India

University Institute of Chemical Technology, North Maharashtra University, Jalgaon, Maharashtra, India
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
A novel hybrid core/shell nanoparticles by oil/water green atomized

microemulsion.
Non-toxic,
biocompatible
and
antibacterial poly(methyl methacrylate) (core)biosurfactant (shell).
pH-dependent sustained release of
ibuprofen, anthraquinone and curcumin.
a r t i c l e
i n f o
Article history:
Received 23 December 2013
Received in revised form 21 February 2014
Accepted 23 February 2014
Available online 1 March 2014
Keywords:
Modied atomized microemulsion
Biosurfactant
Poly(methylmethacrylate) 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(methyl
methacrylate) (nPMMA) (core)biosurfactant (shell) particles designed for drug delivery applications is
reported. The amount of biosurfactant required was 1/35 of the monomer amount by weight and the
surfactant/water ratio could be as low as 1/210. These surfactant levels are much lower in comparison
with those used in a conventional microemulsion polymerization system. The particles were spherical
and 2050 nm in diameter with an average molecular weight of (0.91.5) 105 g mol1 (polydispersity
index 1.642.65). These nanoparticles were non-toxic, biocompatible and exhibited strong antibacterial
activity against Bacillus subtilis and Pseudomonas aeruginosa. Glutathione depletion with a concomitant
increase in malondialdehyde levels (increasing reactive oxygen species) and lactate dehydrogenase activity 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 and
strongly dependent on the initial drug loading content and medium pH. The system showed sustained
drug release through three stages; although the release in stage I followed non-Fickian diffusion, Fickian
diffusion was proven to be the release mechanism of stages II and III. The unique biosurfactant coated
nPMMA enables it not only to be a pH-responsive nanocarrier, but also to possess a tailored release prole. Thus, this work manifests the potential of biosurfactantpolymer hybrids as next generation delivery
vehicles for biomedical applications.
2014 Elsevier B.V. All rights reserved.
Corresponding author. Tel.: +91 257 2258420; fax: +91 257 2258403.
E-mail addresses: aniruddha chatterjee2006@yahoo.co.in, aniruddhachatterjee2006@gmail.com (A. Chatterjee).
1
These authors contributed equally as rst authors in this manuscript.
http://dx.doi.org/10.1016/j.colsurfa.2014.02.051
0927-7757/ 2014 Elsevier B.V. All rights reserved.
C. Hazra et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 96113
1. Introduction
Coreshell nanostructured polymers, which are composed of
at least two distinguished domains in the core and shell phase,
respectively, have attracted signicant research interest in the lm
fabrication, drug delivery, conducting materials, paper and textile
manufacturing, and impact modiers [113]. Of these polymeric
colloidal micro- and nanoparticles, poly(methyl methacrylate)
(PMMA), also designated as poly(methyl 2-methylpropenoate), is
one of the most widely explored biomedical materials because
of its biocompatibility, non-toxicity and non-immunogenicity
[12,14,15]. Although PMMA has a long heritage as a carrier
vehicle for bioactive substances (therapeutic drugs, proteins,
genes, enzymes, vaccines, etc.), there is a tremendous challenge to develop robust and economical techniques capable of
producing size-tunable and morphology-controlled nanoparticles
with a complex architecture [79,12]. Conventional emulsion
polymerization (seeded tandem polymerization) and pre-formed
polymer-based techniques are still the methods of choice for
preparing coreshell PMMA nanoparticles (nPMMA). However,
toxicological issues arising from the use of organic solvents,
surfactants and free radicals during these polymerizations can
never be obviated [6,7]. On the contrary, surfactant-free emulsion polymerization suffers from several drawbacks, including
(i) generally >200 nm particle size and it seems to be difcult
to reach sub-100 nm and (ii) polydispersity due to the presence
of ionic monomers, thereby bring the possibility of secondary
nucleation [1,5].
To explore a practical technical route in which the surfactant amount required could be signicantly decreased and by
which the nanoparticle size could be controlled, differential
microemulsion polymerization [6,7] and biofunctionalization of
core/shell colloidal nPMMA was proposed [2,4,8,16]. Signicant
recent progress in bioinspired approaches, in particular progress
in preparing biomolecules based coreshell nPMMA, has allowed
the synthesis of chitosan-modied nPMMA by emulsion polymerization and emulsier-free emulsion copolymerization [4,1719],
and nPMMA-BSA using by Cu2+ -mediated graft copolymerization
[20,21]. Unfortunately, these biomolecules used in the above methods were dissolved in an excessively acidic aqueous milieu, which
is not desirable for medical application and are detrimental to acidlabile bioactive substances. Since then, there were many attempts
to develop chitosan derivatives from glutamate, aspartate, and
hydrochloride salts to reduce the excessive acids and its fatality.
Nevertheless, their biodegradability, biocompatibility and nontoxic nature are not yet established conclusively [12]. It comes as
no surprise that such PMMA particulate carrier systems are yet to
penetrate commercial market.
In our previous work, we have synthesized amorphous
polystyrene (nPS) and particles (spherical) by modied microemulsion process as well as crystalline nPS particle (hexagonal) by
novel atomized microemulsion process [2226]. Similarly, PS
(core)biosurfactant (shell) biocompatible and biodegradable bionanocomposites were synthesized for their feasibility as drug
delivery vehicle [27]. The present work is therefore an extension of our preceding efforts to develop a new facile route to
prepare coreshell nPMMAbiosurfactant nanoparticles. The biosurfactants we have chosen here are rhamnolipids, surfactin and
trehalose lipids of microbial origin and are biodegradable, biocompatible and non-toxic with very low CMCs. Hence, we believe that if
such nPMMA (core)biosurfactant (shell) can be made then it could
potentially nullify ve-fold disadvantages of classical emulsion
polymerization: (i) high surfactant/monomer weight ratios, usually
larger than 1, are required to produce small particles; (ii) only a low
solid content, usually less than 10 wt.%, could be made; (iii) use of
large amounts of expensive surfactant; (iv) considerable negative
97
impact of excess surfactant on the properties and post-treatment of

synthesized polymeric lattices; and (v) need of designing and developing new surfactant systems with improved emulsifying properties. Therefore, we report here a modied atomized microemulsion
process for synthesizing nPMMA (core)biosurfactant (shell) particles and in particular, we have studied the effect of emulsier
and initiator concentration on the particle size, number and the
stability of the colloidal particles as well as cytotoxicity of the synthesized nanoparticles. Finally, we investigated their antibacterial
properties and their potential as possible drug delivery vehicle is
also evaluated with three hydrophobic drugs: ibuprofen (IB; nonsteroidal anti-inammatory), anthraquinone (AQ) and curcumin
(antioxidant and anticancer agents).
2. Materials and methods
2.1. Materials
Methyl methacrylate (MMA) monomer, n-hexanol (n-Hx) and
methanol were procured from S.D. Fine Chemicals Ltd. (Mumbai,
India). Ammonium persulfate (APS) was sourced from Qualigens
India Ltd. (Mumbai, India). IB, AQ (97%) and curcumin (94% curcuminoid content) was purchased from SigmaAldrich. Double
distilled and deionized water (DDIW) was used during polymerization and in other experiments.
2.2. Production and purication of the biosurfactants
Rhamnolipids from Pseudomonas aeruginosa BS01 were produced, isolated and puried as previously described [2729].
Surfactin was obtained from the cell free broth of Bacillus clausii
BS02 as per Liu et al. [30]. The isolation, purication and structural
analysis were done according to Namir et al. [31]. Trehalose lipids
from Rhodococcus pyridinivorans NT2 were produced, isolated and
puried as detailed by Kundu et al. [32].
The chemical structure of the most abundant component in the
rhamnolipid biosurfactant product was identied as Rha-C10 -C10
(75.5%), while the others were characterized as Rha-Rha-C8 C10 (0.8%), Rha-C8 -C10 (1.5%), Rha-C10 -C12 :1 (10%), Rha-C10 -C12
(15%), and Rha-Rha-C10 -C14:1 (2%), with small contributions of
their structural congeners (Fig. S1a). Puried surfactin consisted
of a peptide loop containing seven amino residues bonded to
a -hydroxyl fatty acid chain with 15 carbon atoms (Fig. S1b).
Trehalose lipids consisted of trehalose-succinic acid-C7 -C11 -C11 ,
trehalose-succinic acid-C9 -C10 -C10 , trehalose-succinic acid-C9 -C9 C11 and 2,3,4,2 -trehalose tetraester (Fig. S1c). The physicochemical characteristics of these three biosurfactants are listed in
Table S1.
2.3. Synthesis and purication of PMMA (core)biosurfactant
(shell) nanoparticles
Transparent or translucent dispersions of nPMMA in the range of
2050 nm with spherical shape were synthesized by an oil/water
(O/W) modied atomized microemulsion process patented by us
(Fig. 1) [26]. Typical recipes for nPMMA with different process
parameters are shown in Table 1. In a at 1 l stainless steel reactor, each biosurfactant (rhamnolipid/surfactin/trehalose lipid) was
dissolved in a rst part of DDIW water and the mixture was
stirred with an agitator at 200 rpm and 55 2 C. APS, as a thermal initiator, dissolved in a second part of DDIW water was
added to the reactor for formation of free radicals and the reactor
was sealed. Monomer (puried and distilled under reduced pressure before use) was sprayed through the nozzle of atomizer by
reciprocating compressor at controlled pressure and at a constant
7.41
7.22
2.87
2.55
7.44
7.56
2.93
2.55
7.33
7.09
2.04
2.11
6.36
Np (1018 )
2.65
1.94
1.89
2.33
1.64
1.93
1.89
1.72
2.34
2.39
2.48
1.80
2.29
Polydispersity
index
0.54
0.65
0.47
0.39
0.69
0.76
0.47
0.53
0.67
0.61
0.49
0.66
0.63
Mn (105 )
TEM
24.5
26.5
30.5
31.5
23.6
29.6
31.7
36.5
30.5
38.2
41.5
31.0
18.0
39.3
38.6
39.5
44.5
33.0
38.0
32.0
40.5
36.0
43.5
50.5
34.5
24.5
0.08
0.08
0.16
0.16
0.08
0.08
0.16
0.16
0.08
0.08
0.16
0.16
0.08
14
14
28
28
14
14
28
28
14
14
28
28
14
1
2
3
4
5
6
7
8
9
10
11
12
13a
0.4 (RL)
0.4 (RL)
0.8 (RL)
0.8 (RL)
0.4 (SR)
0.4 (SR)
0.8 (SR)
0.8 (SR)
0.4 (TL)
0.4 (TL)
0.8 (TL)
0.8 (TL)
80 + 5
55 + 5
55 + 5
40 + 5
80 + 5
55 + 5
55 + 5
40 + 5
80 + 5
55 + 5
55 + 5
40 + 5
80 + 5
69
72
84
89
75
81
88
94
65
61
77
82
61
2
3
6
1
4
3
1
1
1
7
1
3
1
12.17
15.58
22.69
28.88
11.15
13.68
20.07
27.53
13.15
16.18
22.69
29.84
11.13
0.1
0.2
0.1
0.3
0.1
0.2
0.1
0.2
0.3
0.2
0.3
0.1
0.4
Translucent
Translucent
Viscous translucent
Viscous translucent
Translucent
Translucent
Viscous translucent
Viscous translucent
Translucent
Translucent
Viscous translucent
Viscous translucent
Translucent
DLS
1.43
1.26
0.89
0.91
1.13
1.47
0.89
0.91
1.57
1.46
1.22
1.19
1.51
Mw (105 )
Particle size
(nm)
Appearance
Solid content (%)
Conversion (%)
Water (ml)
(1st + 2nd part)
APS (g)
Biosurfactant (g)
MMA (ml)
Run
Table 1
Recipes used in the present work.
RL, rhamnolipid; SR, surfactin; TL, trehalose lipid; Mw , weight-average and Mn , number-average molecular weights of the nPMMA particles, respectively. nPMMA particles prepared from run 1, 5 and 9 were further taken for
analytical characterizations, in vitro cytotoxicity assay, and antibacterial activity experiments. nPMMA particles prepared from run 5 were used for IB loading and release studies.
a
nPMMASF prepared using a surfactant-free emulsion polymerization process.
28
29
61
56
25
29
66
54
27
24
61
62
27
98
Fig. 1. Atomized reaction vessel for synthesis of nPMMAbiosurfacatnt core/shell

particles via the modied atomized microemulsion technique.
rate over the entire surface of the clear solution, where a unique
environment of the microemulsion proceeds. The bafes were
maintained at the top of the reactor to bounce back the monomer
stream from outgoing air. The exhaust was then led through the
distillation column for the recovery of monomer. The temperature
was maintained at 55 2 C throughout the reaction. After the complete addition of monomer, the reaction was continued for 1 h and
the polymerization reaction was stopped by cooling the mixture
to room temperature. A transparent or translucent dispersion was
formed, which indicates the formation of microemulsion of nPMMA
particles. A buttery valve located at the dish bottom of the reactor discharges the reaction mixture. The process was monitored
by controlling the orice size, reciprocating compressor pressure,
speed, reaction temperature, distance between atomizer and reaction zone, etc.
For comparison, a soap-free emulsion polymerization (SFEP)
was performed synthesizing surfactant-free nPMMA particles
(nPMMASF ), according to Camli et al. [1]. The monomer methylmethacrylate (MMA) was added into a sealed glass reactor containing ammonium persulfate (APS) in a pre-mixed acetonewater
mixture. Polymerizations were carried out for 3 h at 75 C in a
temperature-controlled water bath under magnetic mixing. Unlike
a typical SFEP system, the presence of acetone at elevated temperatures results in a clear monomer solution resembling a dispersion
polymerization procedure. PMMA dispersions were cooled down
to room temperature, isolated and then characterized.
2.4. Isolation of nanoparticles

PMMA nanoparticles were isolated by drop wise addition of the
latex into methanol with constant stirring, and the mixture was
kept overnight for uniform dispersion of precipitate. The precipitated particles were ltered under vacuum and washed several
times with methanol/water (1:1) and then dried in a vacuum desiccator for 48 h at 50 C.
2.5. Characterization
2.5.1. Determination of monomer conversion and solid content
The total number of latex particles in the system (NP ) and the
number of polymer chains per particle (N) as well as the conversion
(Xm ) are calculated according to the following equations:
60 VXm
NP =
D3
(1)
3
N=
4 (D/2) NA
n
3
M
Xm (%) =
W1
100
W2
(2)
(3)
where 0 is the density of MMA (0.94 g cm3 at 25 C), V is the

total volume of MMA, Xm is polymerization conversion, is the
density of PMMA (g cm3 at 25 C), D is the diameter of the particle, NA is 6.02 1023 mol1 , Mn is the number-average molecular
weight, and W1 and W2 are the weights of the polymer and MMA,
respectively.
2.5.2. Particle size and particle size distribution
The Z-average particle size and the distribution of the particle
size were measured using a Zetasizer (Malvern Zetasizer Nano-ZS,
UK). The diameters of highly diluted dispersions (in 103 M HCl)
were measured, at several temperatures between 20 and 50 C.
Each data point was the average of at least three measurements.
2.5.3. Molecular weights and their polydispersity index (PDI)
The number-average-molecular weight (Mn ) and weightaverage molecular weight (Mw ) as well as the polydispersity index
(PDI) were determined by gel permeation chromatography with
an Agilent GPC-Addon Rev A02.02 series HPLC system using a
PL-Gel Agilent column and THF solvent. A calibration curve was
constructed using standard polystyrene having a molecular weight
range of 4490 to 1,112,000 g mol1 . The dried nPMMA was dissolved in tetrahydrofuran at a concentration of 0.3% (w/v) and then
ltered with a nylon membrane (pore size 0.45 m) before injection.
2.5.4. Morphology of the coreshell nanoparticles
Atomic Force Microscopy (AFM) images of dried samples in glass
plates were captured using an AFM (Solver P20M, NT-MDT) working in semi-contact mode. A microcantilever with a spring constant
of 10 N m1 was used to scan the samples. For scanning electron
microscopy (SEM), a colloidal emulsion (1%, v/v) was dropped on a
cover glass and dried in a dust-free environment. The dried specimens were stuck on the sample holders with a double-coated
carbon conductive tab and then were sputter-coated under vacuum with gold by using a sputter coater (E-102, Hitachi, Japan).
The sputter-coated samples were then observed by the microscope
(S-4800, Hitachi, Japan) at 15 kV.
A transmission electron microscope (TEM, FEI Tecnai 2083)
operating at 120 kV was used to image and study the morphology
of the samples. The TEM samples were prepared by drop casting
samples dispersion in the carbon coated copper (200 mesh) grid.
2.5.5. Electrokinetics
The -potentials of nanoparticle latexes were measured using a
zetasizer (zetasizer 3000, Malvern Instruments, UK) in 1 mM NaCl
solution at room temperature. The measurements were also done
at room temperature. The pH values of nanoparticle dispersions
were adjusted to be in a range of 112. The isoelectric points were
determined at a pH where the -potential is zero.
99
2.5.6. FTIR spectroscopy

Fourier transforms infrared (FTIR) spectra of the biosurfactants
and nPMMA particles were recorded on Nicolet 5700 FT-IR spectrometer at resolution of 0.5 cm1 with an average of 32 scans.
2.5.7. Thermal analysis
DSC was performed on Differential Scanning Calorimeter (DSC60, Shimadzu, Tokyo, Japan) to investigate the glass transition
temperature (Tg ) of nPMMA and bulk PMMA. The samples (5 mg)
sealed in aluminum pans were scanned in the temperature range
from 30 to 300 C along with a reference sample as standard,
at the heating rate of 10 C min1 under a nitrogen atmosphere
(50 ml min1 ).
Thermal degradation properties of nPMMA and bulk PMMA
were determined on Thermo Gravimetric Analyzer (TGA-50, Shimadzu, Tokyo, Japan). Sample (10 mg) was put on a platinum pan
and scanned in the temperature range from 30 to 500 C at the heating rate of 10 C min1 under a nitrogen atmosphere (50 ml min1 ).
2.5.8. Functional group analysis on the coreshell nanoparticles
Amount of carboxylic group on the surface of nPMMARL and
nPMMATL was determined by using potentiometric titration [33].
The titration was carried out by Autotitrator (T50, Mettler Telodo)
with pH glass sensor (DGi115-SC, Mettler Relodo) by using 0.01 M
KOH in isopropanol standardized by potassium hydrogen phthalate
as a titrant. The sample preparation was performed by mixing a
dispersion of the puried nanoparticles (20 mg ml1 , 0.5 ml) with
isopropanol (50 ml). Each value reported was an average of at least
three measurements. In addition, rhamnolipid in nPMMARL was
assessed by quantication of l-rhamnose by the orcinol assay [34].
l-Rhamnose was used for making the standard curve. Presence of
trehalose lipids in nPMMATL was determined by the phenol-sulfuric
acid method [35]. The stand curve was prepared with d-glucose.
The amount of amine groups on the surface of nPMMASR was
determined by the conventional 2,4,6-trinitrobenzene sulfonic acid
(TNBS) assay [8]. The nanoparticle dispersion was diluted to give a
nal concentration of 10 mg ml1 and suspended in 100 l of phosphate buffered saline solution (10 mM PBS, pH 7.6). Then, 200 l
of 4% sodium hydrogen carbonate solution (pH 8.5) and 200 l of
1% TNBS aqueous solution were added. These mixtures were incubated at 37 C for 2 h. After incubation, the absorbance of 200 l of
samples was measured at 415 nm in a spectrophotometer (Lambda
900, Perkin-Elmer). The amount of amine groups on the particle
surface was calculated based on the calibration curve prepared by
the -alanine standard solution.
2.6. Cytotoxicity assay in peripheral blood mononuclear cells
In vitro cytotoxicity assay was carried out following the procedure of Borase et al. [36]. Different working stocks of nPMMA
samples were prepared, and 0.1 ml of twofold dilution series of
nPMMAs was added in a 96-well microtiter plate by using 10%
Roswell Park Memorial Institute medium. Stimulated peripheral
blood mononuclear cells at 2 105 per well were added in duplicate to the dilution suspension and the plates incubated for 5 days
at 37 C with humidied 5% CO2 atmosphere. After incubation,
cell viability was determined by (4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, MO). Then,
20 l (from a stock of 5 mg ml1 ) reagent was added in each well
and incubated at 37 C for 4 h in a CO2 incubator. Dimethyl sulfoxide (0.1 ml) was added to each well and kept in the dark for 1 h at
room temperature. Optical density was taken at 550 and 630 nm
wavelength, the latter as a reference wavelength. The assays were
performed in triplicate on 2 different days (n = 6).
100
2.7. Antihemolytic activity

The nPMMA samples were exposed to anticoagulated (sodium
citrate) human blood for 1 h under agitation (70 5 rpm) using
an orbital shaker (Orbitek, Scigenics Biotech, India) thermostated
at 37 C 6 1 C. The samples were then centrifuged at 4000 rpm
for 15 min and plasma was aspirated immediately. The blood had
red blood cell (RBC) concentration of 1 108 cells ml1 . Deionized
water-dispersed RBC was used as the positive control, and the normal saline-dispersed RBC was used as the negative control. After
the incubation, RBC was centrifuged under 2200 rpm for 5 min.
The absorbance of the released hemoglobin in the suspensions was
measured by spectrophotometry at 546 nm. The hemolysis ratio
was calculated with the following formula:
% hemolysis =
As An
Ap An
100
(4)
where As , Ap , and An are the absorbances of the test suspensions,

positive control, and negative control, respectively. Two parallel
tests were carried out to obtain a reliable value.
2.8. nPMMAbacteria interaction
The antibacterial activity was investigated against grampositive Bacillus subtilis NCIM 2718 and gram-negative Pseudomonas aeruginosa NCIM 5029. Both the strains were inoculated in
a nutrient broth (NB) at 37 C for 24 h. These bacterial suspensions
obtained were then diluted to 108 CFU ml1 . The different concentrations of nPMMA samples were poured in a 96-well microtiter
plate, followed by adding 10 l of the bacterial suspension. The
microtiter plate was incubated at 37 C for 2 h. The untreated
bacteria and nanoparticle suspensions were referred as positive
control. The antibacterial activities of nPMMA against B. subtilis and
P. aeruginosa were obtained by measuring the absorbance of the
culture medium at 600 nm compared with the control, which contained only different concentrations of nPMMA suspensions and
without bacteria. The lowest concentration of nanoparticles that
inhibited the growth of bacteria was considered as the minimum
inhibitory concentration (MIC), where the absorbance of the culture
medium is the lowest and thereafter becomes constant.
For morphological observation of the bacterial cells postnPMMA treatment, SEM images were taken following the
procedure of Inphonlek et al. [8].
acid (TBA; Sigma) solution was then added to the supernatant and
was incubated at 95 C for 60 min. It was cooled to room temperature and was centrifuged at 3500 rpm for 15 min. The absorption
spectrum of the supernatant was recorded at max = 532 nm to estimate the formation of TBARS. All analyses were carried out in
triplicate. From the TBARS intensities the corresponding levels of
malondialdehyde (MDA) were deduced from a calibration curve
plotted between the intensity of TBARS measured at max = 532 nm
against different concentrations of MDA synthesized by acidic
hydrolysis of 1,1,3,3-tetramethoxypropane.
2.9.3. Glutathione levels
Reduced glutathione levels were estimated by method previously described by Hazra et al. [28], with slight modication.
Bacteria were cultured to 1 109 cfu ml1 and treated with nPMMAs. An aliquot of 0.6 ml was extracted with 30 l of 100% TCA. The
mixture was kept in ice for 10 min and centrifuged at 10,000 rpm
for 5 min. After precipitation, 200 l of 30 mM TrisHCl (pH 8.9)
buffer was used to neutralize the sample. Further, GSH was quantied by measuring absorbance at 412 nm by reaction between
500 l of supernatant and 2.5 ml of 0.01% dithionitrobenzoate after
incubation for 1520 min; simultaneously, a 0.4 ml portion of the
remaining treated culture was used for determination of protein
using the Bradford method. Glutathione levels were estimated
after normalization to cellular protein levels and expressed as
GSH mol mg1 protein.
2.10. Drug loading and in vitro release studies
The nPMMA particles (50 mg) were suspended in 10 ml of DDIW.
IB/AQ/curcumin (0.15 mg ml1 ) was added to this suspension
and incubated for 48 h. Following centrifugation (10,000 rpm for
30 min), the particles were washed thrice with DDIW. The amount
of IB, AQ, and curcumin in the supernatant was assayed using a UV
spectrophotometer at 222 nm, 274 nm, and 430 nm, respectively.
Subsequently, the amount of drugs loaded onto the particles was
calculated by subtracting the sum of the nal drug concentration
in the loading solution and the wash from the initial drug concentration in the loading solution. The drug loading content (LC)
and encapsulation efciency (EE) were then calculated using the
following equations:
LC% =
wt of drug in nanoparticles
wt of nanoparticles + wt of drug
100
wt of drug in nanoparticles
initial wt of drug in the loading solution
(5)
2.9. Oxidative stress markers
EE% =
To elucidate the mechanism of toxicity induced by nPMMA particles in Bacillus subtilis NCIM 2718, oxidative stress was assessed
by three methods, viz., extracellular lactate dehydrogenase (LDH)
release, intracellular ROS in the bacterial cells and reduced glutathione levels.
A drug loaded nanoparticle suspension (5 ml) or solution containing the free drug, as a control, was placed in dialysis membrane
bags with molecular weight cut-off 25,000 g mol1 . The sealed bag
was immersed in 200 ml of 0.15 M Tris buffer solutions (pH 7.4)
at 37 C with continuous magnetic stirring. To ensure sink condition the release medium was replaced with fresh buffer every 24 h.
At selected time intervals, 200 l of sample was withdrawn from
the release medium. The drug content in the sample was assayed
spectrometrically at 222 nm. Each data point was a mean of three
determinations standard deviation.
2.9.1. Lactate dehydrogenase (LDH) release

Membrane integrity of the treated culture was assessed an LDH
assay kit (Sigma) as per the manufacturers protocol. Absorbance
was measured at 490 and 690 nm and data were reported as percentage release of LDH compared to control.
2.9.2. Determination of lipid peroxidation (LPO)
One milliliter of treated bacterial culture was mixed with 2 ml of
10% (w/v) trichloroacetic acid (TCA) and left at room temperature
for 10 min. The pellet was removed and supernatant was taken after
centrifugation at 10,000 rpm for 30 min to ensure that the nanoparticles, cells, and precipitated proteins were completely removed.
Three milliliters of a freshly prepared 0.67% (w/v) thiobarbituric
100
(6)
2.11. Kinetics studies

To analyze release kinetics and mechanism, data were tted to
the following four mathematical models:
1. Zero order
Mt
= k0 t
M0
(7)
101
Scheme 1. Pictorial representation of the formation of nPMMA/biosurfactant coreshell particles.
2. First order
Mt
= 1 exp(k1 t)
M0
(8)
3. Higuchi model
Mt
= kH t 1/2
M0
(9)
4. KorsmeyerPeppas (Power law model)

Mt
= kt n
M0
(10)
where, t is time and Mt and M0 are the concentration of the

release drug at desired times and the total concentration of
the drug encapsulated in the matrices, respectively. k0 , k1 , kH ,
and k represent zero-order release constant, rst-order release
constant, Higuchi constant, and KorsmeyerPeppas constant,
respectively. The release exponent n could be used to determine the release mechanisms. Values of n were obtained by
linear regression, and accompanied with the respective correlation coefcient R2 values.
2.12. Statistical analysis
Data were expressed as the mean S.D. from three independent
experiments. Two-way ANOVA statistical analysis was run using
MiniTab Ver 16.0 (MiniTab, Inc., State College, PA, USA) followed
by Tukeys multiple comparison post hoc analysis to compare to
controls. Furthermore, Tukeys post hoc analysis was performed to
determine differences between the different treatment groups. A
p-value of <0.05 was considered to indicate signicance.
3. Results and discussion
3.1. Preparation of the nanoparticles
The modied atomized microemulsion polymerization was
accomplished by the aid of three types of biosurfactant/APS initiating system. The nucleation and growth of nanoparticles occurred
as follows. Since rhamnolipid and trehalose lipid consists of a
relatively hydrophilic carboxyl moiety and hydrophobic moiety

bearing -hydroxy fatty acids which are expected to present
at the interface between the micelles and water medium to
strengthen the hydrophobicity. The hydrophilic moiety is located
outside of the micelles by existing in the aqueous milieu, but
the hydrophobic moiety tends to exist inside the hydrophobic
monomer-swollen micelles. This concept is similar to that of the
microemulsion polymerization in which the mid-chain alcohols
(usually C4 C8 ) functioning as costabilizers are aligned with the
interface between the micelles and water phase due to the same
hydrophilichydrophobic nature of the molecules. The third biosurfactant, i.e., surfactin possesses plenty of amine groups on their
backbones capable of forming active radicals with APS. The resulted
radicals on the nitrogen atoms of amines then propagated MMA
monomer. By using this polymerization method, we report here
stability-enhanced functionalized coreshell nanoparticle latex
with either rhamnolipid/trehalose lipids/surfactin as a shell and
PMMA as a core (Schemes 1 and 2). Table 1 summarizes how the
recipes inuence the monomer conversions, solid contents and
nanoparticles size after polymerization for 1 h. It can be observed
that the conversion of MMA does not inuence much and are comparable by the change of biosurfactant concentration for all the
system. The likely explanation behind this observation is that the
concentration of three biosurfactants used here is much more than
their corresponding critical micelle concentration (CMC). Above
CMC, the polymerization occurs inside the micellar core and hence
polymerization kinetics remains slow as compared to below CMC
[6]. The solid contents of the nanoparticles latexes were in a range
of 11.1529.84%, which directly depends on the amounts of loaded
polymer, and the monomer conversion percentages. The increase
in monomer conversion percentages upon the amount of loaded
surfactin was more pronounced than rhamnolipid and trehalose
lipids. This could be due to the increasing amount of amine groups
from surfactin. The amine groups are capable of forming redox pairs
with initiator. Hence, more amine groups led to the more redox
pair formation, and thus more free radicals [4,8]. The more amine
groups could subsequently enhanced polymerization of the MMA
monomer and resulted in higher monomer conversion percentages
[4,8,18]. At lower solid content, for example, run 5 (11.15%), the
102
Scheme 2. Mechanism of atomized microemulsion polymerization adopted in this study.
average particle size is 23.6 nm in diameter. As the water/monomer

ratio was relatively high, the probability that one particle collides
with another particle was lower, and hence the size of the particles was smaller. This is not the case with other samples obtained
at higher solid content even though the biosurfactant/monomer
ratio was the same. Besides the changes in the sizes of the nPMMA
particles, the appearance changed from translucent dilute liquid to
a translucent viscous one with increasing solid content (Supplemental Information, Fig. S2). These observations are in agreement
with the principle of emulsion polymerization and hence these
biosurfactants can be utilized as emulsiers in the emulsion polymerization very satisfactorily since they do not violate the bare
minimum criterion of emulsion polymerization.
3.1.1. Effect of monomer amount on nPMMA particle size
The MMA amount in the reaction system affects the particle size
of the resultant polymer. The experimental results are shown in
Fig. 2(a), which indicate that an increase in the MMA consumption
results in an increase in the particle size. When the monomer
amount is 10 ml, the particle size is in the range of 1421 nm.
With increasing monomer amount, the particle size also increases.
0.40
(a)
nPMMA RL
nPMMA SR
nPMMA TL
0.30
Particle size*10 (nm)
0.35
0.25
0.20
0.15
10
0.16
(b)
20
25
Monomer amount (ml)
30
nPMMA RL
nPMMA SR
nPMMA TL
0.15
Particle size distribution
15
0.14
0.13
0.12
0.11
0.10
10
15
20
25
Monomer amount (ml)
30
Fig. 2. Effect of monomer amount on the particle size of nPMMA (experimental

condition: biosurfactant, 0.4 g; APS, 0.08 g; water, 85 ml and 55 2 C). Small (nonvisible) standard deviations are within the symbols.
Moreover, the growth rate of particle size becomes faster as

monomer loading further increases. For example, when 30 ml of
MMA is added, the particle size increases to 3039 nm, and the
particle size is about twice of the size when 10 ml of MMA is used.
The particle size distribution also becomes large with increasing
the monomer amount (Fig. 2b). While from Fig. 2(a), it can be
inferred that surfactin is better emulsier than rhamnolipid and
trehalose lipids in terms of the particle size, Fig. 2(b) shows that
surfactin and rhamnolipid have quite similar effect on the particle
size distribution.
3.1.2. Effect of initiator amount on nPMMA particle size
The effects of the amount of APS on particle size were investigated and the results are shown in Fig. 3, which shows that as the
initiator concentration increases, the particle size in the resulting
latex decreases at rst and then increases gradually. This phenomenon could be explained by the co-existence of two processes:
(i) oligomeric radicals could transfer between the aqueous phase
and the polymer particles; and (ii) oligomeric radicals could continue to undergo the nucleation process and form new particles, and
these two processes could dominate the reaction in the different
stages. At a low initiator concentration, the active sites which could
grow into particles would be less. Then with the same monomer
amount, the particle size would be larger; with the increase of
initiator content, more active sites could be formed. Therefore,
the monomer amount that each active site could share becomes
less and resulting particles would be small; when initiator concentration is high enough, the number of free radicals shared by
one particle would increase and then some dead polymer particles
could be initiated again resulting in a larger particle size [9]. Fig. 3
also indicates that with an increase in the amount of initiator, the
particle size gradually tends to a narrower distribution. The effect
of the APS amount on the characteristics of nPMMASR emulsions
also could be observed as shown in Fig. S3 (Supplemental Information), which indicates that with a decrease in the particle size the
emulsion becomes more transparent, as observed earlier [9,37].
3.1.3. Effects of surfactant type and concentration on particle size
The experimental data indicated that using the three types of
surfactant, the particle size of nal product is different: for rhamnolipid it is 24.5 nm, for surfactin it is 23.6 nm and for trehalose lipid
it is 30.5 nm (run 1, 5 and 9, respectively), which indicate surfactin
is the best in terms of reducing the particle size of nPMMA. This
is reasoned from these facts: (i) the CMC of surfactin (7.59.5 M)
is much smaller than rhamnolipid (0.1 mM) and trehalose lipids
(300 M), and when adding the same biosurfactant amount, surfactin possibly generate more micelles than the other two; and (ii)
being anionic, surfactin can bring electrostatic charge onto the latex
particle surface, thereby preventing ion aggregation and result in
good mechanical stability of the emulsion.
With the increase in the amount of the biosurfactants, the particle sizes become smaller (Fig. 4a). However, beyond a certain level,
the amount of biosurfactant had little effect on particle size. The
particle size of run 5 was <25 nm (23.6 nm) at a surfactin/water
0.40
(b) 0.22
nPMMA RL
nPMMA SR
nPMMA TL
0.35
(a)
0.30
0.25
0.20
0.15
0.10
0.00
0.04
0.08
0.12
APS amount (g)
103
nPMMA RL
nPMMA SR
nPMMA TL
0.20
0.18
0.16
0.14
0.12
0.00
0.16
0.04
0.08
0.12
0.16
APS amount (g)
Fig. 3. Effect of initiator amount on the particle size of nPMMA (experimental condition: MMA, 14 ml; biosurfactant, 0.4 g; water, 85 ml and 55 2 C). Small (non-visible)
standard deviations are within the symbols.
weight ratio of 1:210 (ca. 0.85 wt.% of water phase) and a surfactin/MMA weight ratio of 1:35. These ratios are much lower than
those reported in the literature. We could not compare our data
with those of previous works as there are no reports on the use
of biosurfactants in atomized microemulsion system. Additional
co-surfactant was used in earlier works [6,37,38] whereas no cosurfactant was used in this work. Moreover, oil-soluble initiators
(AIBN and BPO) were used by other researchers [6,37], while a
water-soluble initiator (APS) was used in the present work. The data
demonstrated by Fig. 4 shows that particle size is quite in the same
range (2050 nm) like those reported in the literature. Hence, the
use of the co-surfactant does not show any benet, and we anticipate that the co-surfactant can be eliminated in this reaction system
to simplify the recipe.
In our process, particle size of the microemulsion did not change
even after six months, which means that it is highly stable. The
amount of surfactant used here is much lower than the usual levels
reported in the literature described above.
The dependence of the particle size and its distribution on
the mixed biosurfactant (rhamnolipid + surfactin) is illustrated in
Fig. 4(b). The experimental data indicate that the particle size
decreases from 25 nm to 21 nm as the molar ratio of rhamnolipid/surfactin increases from 0.125 to 0.25, and then the particle
size increases from 26 nm to 37 nm as the molar ratio of rhamnolipid/surfactin increases from 0.3 to 3. Similar data existed in
the literature although with TritonX-100/SDS system [9]. This
phenomenon can be explained by the properties of the two biosurfactants. It seems that both rhamnolipid and surfactin produced
a synergistic effect that results in the decrease of the particle size
at the molar ratio of 0.25. Since the CMC and emulsifying effect
of rhamnolipid surfactant is less than surfactin, the particle size
increases with increasing the amount of rhamnolipid [in the range
of the molar ratio of rhamnolipid/surfactin (0.33)]. Fig. 4(b) also
indicates that with increasing rhamnolipid, the particle size distribution gradually narrows.
3.2. Characterization of the PMMA (core)biosurfactant (shell)
nanoparticles
Particle sizes and size distributions of the nanoparticles
were determined using DLS analysis and TEM micrographs, as
shown in Table 1. Compared to the pristine PMMA particles,
i.e., nPMMASF particles (18 nm; see Fig. 6d), the complexation
between PMMA particles and biosurfactants resulted in the larger
size particles, which were in the range of 23.641.5 nm. The
nPMMAbiosurfactant core/shell particles exhibited larger average hydrodynamic diameters (3250.5 nm) than those observed by
TEM (23.641.5 nm). The result of AFM images (Fig. 5) corroborated
with the average particle diameters from TEM. The particle size
determined by DLS was based on the intensity of scattered light and
measurement of the movement of particles undergoing Brownian
motion. In the solution, the biosurfactant shell highly swelled due
50
0.40
(a)
nPMMA RL
nPMMA SR
nPMMA TL
35
Particle size (nm)
40
30
25
20
Particle size
0.35
45
(b)
0.30
0.25
0.20
0.15
15
0.10
10
0.2
0.4
0.6
0.8
Biosurfactant (g)
1.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Molar ratio of rhamnolipid/surfactin
Fig. 4. Effect of (a) biosurfactant amount and (b) mixed biosurfactant (rhamnolipid/surfactin) ratio on the particle size of nPMMA (Experimental condition: MMA, 14 ml;
APS, 0.08 g; water, 85 ml and 55 C). Small (non-visible) standard deviations are within the symbols.
104
Fig. 5. AFM images of nPMMA synthesized using (a) rhamnolipid, (b) surfactin and
(c) trehalose lipid.
to the expansion of charged groups, both ammonium groups on the

backbone and the intact counter groups (amino acids in surfactin
and carbohydrates in rhamnolipid and trehalose lipid), on the particle surface. In addition, there would be some possible aggregation of
the particles, especially in the case of amino acids, because of their
amphoteric property. For these reasons, particle sizes measured by
DLS tended to be larger. In AFM and TEM, a thin lm of polymer

latex was scanned at dry state, the expansion of the biosurfactant
shell was limited. Therefore, the sizes determined by these methods
were smaller. However, the particle sizes measured from the two
methods were in similar order. Apparently, all the nanoparticles
possessed spherical shape with uniform size distribution (Fig. 6)
along with the well-dened coreshell nanostructures (Fig. 6;
inset of b), wherein the hydrophobic PMMA cores (darker part)
were 20 nm in diameter. This was coated with biosurfactant shells
(light black ring) of 3.6 nm. The particle sizes were increasing in
the following order: nPMMASR > nPMMARL > nPMMATL . There are
fewer data on sub-100 nm nPMMA [1,5,39] and some degree of
irregularity and aggregation is often reported [8]. Compared to previous reports, our method does not require use of toxic organic
solvents and co-surfactants and thus is advantageous over reverse
microemulsion or dispersion polymerization method to produce
well-controlled and monodisperse sub-100 nm PMMA.
From literature reports, it was found that conventional
nPMMASF particles had a surface charge of 75.4 mV [40]. The surface charges of nPMMASR particles were positive at pH 28 due to
the protonation of amine groups of surfactin (Fig. S4, Supplemental
Information), which strongly indicated that the surface component
of nanoparticles was made of surfactin. From the changes of size
and surface charges, it was evident that the positively charged surfactin could be coated on the negatively charged PMMA colloidal
particles via electrostatic interaction. Upon increasing solution pH,
the -potentials began to decrease conrming that the isoelectrical point for the latexes coincides with the pKa of the amine
and amidine groups [41,42]. It even reached to negative values
at strong basic pH, resulting from the transformation of protonated amines to neutral ones and then combined with hydroxyl
ions. In addition, highly positive charge of +49 mV can keep the
nanoparticles stable in acid and neutral solutions due to the charge
repulsion force. However, for nPMMARL and nPMMATL coreshell
latexes, negative surface charges were recorded which increases
with increasing medium pH from 2 to 7 due to the ionization of
the MAA units. When the basicity increased, the particle surface
adsorbed more and more hydroxyl ions from the solution (possibly
coming from rhamnose and trehalose sugar moiety) resulting in a
negative surface and a negative zeta-potential. This is the evidence
that negatively charged rhamnolipids or trehalose lipids could be
immobilized on the nPMMA particles, leading to the lower surface
charges. It is known that nanoparticles dispersed in aqueous solutions can be stabilized either by electrostatic stabilization (surface
charge) or by steric stabilization (surfactants or other molecules
at the particle surface), or by a combination of both [4,8,18].
Generally, -potential values beyond 20 mV are considered characteristics of a stable colloidal dispersion. According to the DLVO
theory, aggregation occurs when attractive van der Waals forces
between the particles prevail over the electrostatic repulsive forces
[10] which did not occur in our case. Thus, these size-controlled,
monodisperse, stable nano latex could serve as good drug delivery
system.
The variation of chemical functional groups present in the
nanoparticles was identied by FTIR spectroscopy (Fig. 7). For
rhamnolipid, the broad peak at 35003200 cm1 corresponded
to O H stretching (for O H bonds) and the signals at 2850 and
2925 cm1 was attributed to CH2 CH3 stretching, ( CH3 ) symmetric deformation vibration, (C H) bending vibrations of CH3
and CH2 groups, which are characteristics of polysaccharides.
The C O stretching band at 1732 cm1 is characteristic of ester
bonds and carboxylic acid groups while characteristic frequencies at 1028 and 1029 cm1 represent C O C ethereal vibration
[27,29] (Fig. 7a). The FTIR spectrum of surfactin showed a broad
peak at 35003200 cm1 , which was attributed to an O H stretching and N-H stretching. The signal at 3092 cm1 corresponded
105
Fig. 6. TEM photographs of (a) nPMMARL , (b) nPMMASR , (c) nPMMATL and (d) surfactant-free nPMMA (nPMMASF ) particles.
to C H stretching vibrations. The peak observed at 1634 cm1

was assigned to NH3 + groups due to the protonation of amine
groups on surfactin and amide linkage I appeared at 1008 cm1
[43,44]. Similarly, the broad negative bands at about 3400 cm1
and at 1654 cm1 are attributed to O H stretching (for O H bonds)
conrming the presence of glycolipid moieties of trehalose lipids
[32] (Fig. 7a). Other notable frequencies were at 1732 cm1 (C O
stretching of ester bonds and carboxylic acids) and 1454 cm1 (C H
and O H deformation vibrations, typical for carbohydrates).
The FTIR spectrum of nPMMA nanoparticles is shown in Fig. 7(b).
For nPMMARL , the stretching vibration frequency of ( CH3 )
appeared at wave numbers of 2925 cm1 and 2850 cm1 and the
wave number 1730 cm1 corresponds to the stretching vibration
frequency of ( C O). The broad peak at 34403450 cm1 for all the
nPMMA results from OH stretching vibrations due to incorporation of COOH [810]. The peaks from 2800 to 3100 cm1 (marked
by ellipse in Fig. 7b) exhibit various characteristic vibration bands
of PMMA. For nPMMASR , both the characteristic peaks of PMMA
(C O adsorption at 1730 cm1 ) and surfactin (N H stretching at
3440 cm1 , asymmetric stretching of CH2 at 2950 cm1 , symmetric
stretching of CH2 at 2845 cm1 , and NH3 + vibration at 1634 cm1 )
were observed. For nPMMATL , broad peak at 34403450 cm1 and
1730 cm1 were assigned to OH stretching vibrations of COOH
in PMMA and C O stretching of ester bonds of trehalose tetraester,
respectively. The formation of hydrogen bond between rhamnolipid or trehalose lipids and nPMMA is expected to weaken the
strength of carbonyl bond in rhamnolipid or trehalose lipids and
shift its stretching vibration to lower frequencies. The interaction
of rhamnolipid/trehalose lipids with nPMMA particles has shifted
its carbonyl stretching vibration from 1732 cm1 in the pure rhamnolipid or trehalose lipids to 1704 cm1 in nPMMARL and nPMMATL ,
respectively. This chemical interaction provided a substantial evidence of the presence of rhamnolipid/trehalose lipids coating on
to the nPMMA. For the nPMMASR particles, their spectra were similar to surfactin, however, the additional signal at 1730 cm1 was
observed, which corresponded to the C O stretching of PMMA core
component. This could conrm the complexation between PMMA
and surfactin. In addition, it can be observed that the signal of C O
stretching in nPMMARL /nPMMASR/ nPMMATL particles is lower as
compared to the previous reports. This may be due to the fact that
the thicker shell of biosurfactants could limit the penetration of the
incident beam on the reection mode of the FTIR to the inner layer
or the core of particles. This would result in the decrease of C O
stretching intensity of PMMA.
To ensure covalent linkage between the biosurfactant shell
and the PMMA core, the nPMMAbiosurfactant core/shell particles were repeatedly washed with water through a centrifugation,
decantation, and redispersion cycle until conductivity of the supernatant was equal to that of double distilled and deionized water
(DDIW) used, and rhamnolipid, surfactin and trehalose lipid in the
supernatant was not detectable with the orcinol method, Bradford
method and phenol-sulfuric acid method, respectively. To separate
the biosurfactants from the PMMA core, the nPMMARL , nPMMASR
and nPMMATL core/shell particles were subjected to acid hydrolysis. The FTIR spectrum of the dried products obtained from after acid
hydrolysis was identical to that reported for the PMMA, which further conrmed the formation of nPMMAbiosurfactant core/shell
particles.
106
Fig. 7. FTIR spectra (a) pure biosurfactants only and (b) nPMMA/biosurfactant coreshell particles.
DSC curves of rst scan of pure rhamnolipid, surfactin and trehalose lipids; and rst as well as second scan of nPMMARL,SR,TL and
bulk PMMA are shown in Fig. S5ab and Table S2 (Supplemental
Information). Melting temperature (Tm ) of pure rhamnolipid, surfactin and trehalose lipids showed only broad endothermic peaks
at 60, 130 and 90 C, respectively (Supplemental Information, Fig.
S5a). It was observed from Fig. S5(b i, iii and v) that rst scan
of nPMMARL,SR,TL showed two step exothermic peaks at 62 and
128 C for nPMMARL ; 130 and 136 C nPMMASR ; and 93 and 126 C
nPMMATL that are attributed to Tg1 [22,24,45,46] along with respective peaks of Tm arising due to the presence of very little amount
of corresponding biosurfactants. This nding corroborates with the
thin shell layer of biosurfactants observed in TEM. On immediate
second scanning after constant cooling to ambient temperature, the
Tg of nPMMAs shifted at 115 C for nPMMARL , 117 C for nPMMASR
and 114 C for nPMMATL (Fig. S5b ii, iv and iv). The reason for high
Tg of polymer nanoparticles might be a decrease in particle size to
nano-scale that results in an increase in surface area and higher
surface energy [47]. So during rst scanning of nPMMA, the loss
of surface energy results in the energy release which shifted Tg1 to
higher temperature. But during second scan, there were no polymer
particles (sintering phenomenon occurred) [2224,46], and they
joined to form a single lm layer with lesser surface area, which
gained energy and showed single Tg2 during second scanning of
nPMMARL,SR,TL respectively. The lower value of Tg1 for bulk PMMA
(107 C) (Fig. S5b; vii) was due to its large size and lower surface
area, which was much lower than nPMMA particles [2224]. The
data obtained here is in line with the Tg reported in PMMA-acid
modied chitosan nanoparticles (114122 C) [4,46].
Fig. S6 and Table S2 (Supplemental Information) show the
thermal stability (dt ) of nPMMARL,SR,TL and bulk PMMA. nPMMAs showed higher thermal stability (don = 364 C and doff = 411 C
for nPMMARL ; don = 370 C and doff = 417 C for nPMMASR and
don = 358 C and doff = 398 C for nPMMATL ) with % weight loss
(WL ) = 100% [19] than bulk PMMA (don = 283 C and doff = 360 C
with 100% WL ), which further proved a close molecular packing in
nPMMARL,SR,TL than bulk PMMA. The mechanism for this increase
in the thermal degradation temperature of nPMMAs appears to
arise from two mechanisms, i.e., steric restriction of chain motion
and radical deactivation by nanoparticles [46,48]. However, further investigations are necessary to reach a meaningful conclusion
behind the thermal degradation pattern.
To further conrm the presence of rhamnolipid and trehalose
lipids on the surface of nPMMARL and nPMMATL , respectively, we
evaluated the amount of carboxylic groups on the nanoparticle
surface by using acidbase titration of free fatty acid in nonaqueous solution. The acid content of nPMMARL was 0.08 mM g1 ,
while in nPMMATL it was found to be 2.04 mM g1 . Besides, it was
observed that nPMMARL and nPMMATL contain 11.13 mM g1 and
7.29 mM g1 of rhamnose and trehalose, respectively. Moreover,
primary amine groups on the surface of nPMMASR were determined
by the TNBS assay. This method is based on the reaction of TNBS
107
on grouping information using Tukeys multiple comparisons at

95% condence. This study would be, therefore, helpful for development of carriers for drug delivery and llers for cosmetic surgery.
Further studies with in vivo models are however necessary to delineate the underlying mechanisms of reduced cytotoxicity.
3.4. Blood compatibility
A hemolysis assay was conducted (Fig. 8b), which showed that
nPMMASR was less toxic than nPMMARL and nPMMATL , even at
1 g l1 in human blood. It was observed that % hemolysis was significantly higher in cells exposed to nPMMARL and nPMMATL particles
in the concentration range of 0.61 g l1 (p < 0.05 for each). The
hemolytic activity in cells exposed to nPMMASR was not statistically signicant. According to ASTM standards, a material with
a hemolysis value less than 2% is considered as hemocompatible.
Since PMMA is one of the most commonly studied polymer conjugate for application in nanomedicine, a superior performance by
nPMMASR presents blood compatibility for this system.
3.5. Antibacterial properties
Fig. 8. (a) Cytotoxicity of nPMMA/biosurfactant coreshell particles against human

peripheral blood mononuclear cells. (b) Hemolysis assay of nPMMA/biosurfactant
coreshell particles using human blood erythrocytes. Data represented are
mean S.D. (standard deviation) of three identical experiments made in three replicate. *Statistically signicant difference as compared to the controls (p < 0.05 for
each).
and primary amine. Free amine groups on the nanoparticle surface

were 28.05 M ml1 .
3.3. In vitro cytotoxicity test on human peripheral blood
mononuclear cells
Unwanted biological effects induced by the suppression of the
phagocytic and antibacterial activity of human polymorphonuclear leukocytes is claimed to be a serious health concern due to
PMMA particulate carrier toxicity. Hence, the cytotoxicity of the
nPMMA coated with biosurfactants on human peripheral blood
mononuclear cells was investigated. Irrespective of the type of
materials (either biosurfactant alone or the nanoparticles) used,
as the concentration of the nanoparticles increased, the cell survival rate signicantly decreased (Fig. 8a). This is particularly true
in the range of 0.10.4 g l1 (p < 0.05 for each). However, higher concentrations (0.50.7 g l1 ) did not produce signicant cytotoxicity
to the cells (p > 0.05 for each). The results of analysis of variance
(ANOVA) for % cell viability are given in Table S3 (see Supplemental Information). Moreover, Table S4 (in Supplemental Information)
shows pairs of signicance of particle type and concentration based
To evaluate the antibacterial properties of nPMMA

(core)biosurfactant (shell) nanoparticles, the growth of bacteria
at various concentrations of nanoparticles was assessed at A600 .
The antibacterial photographs were obtained by culturing the
same volume of each sample solution in nutrient agar plates after
a 2 h exposure to the bacteria. Compared with the control and the
bulk PMMA plates, the coreshell nanoparticles (30 nm) perfectly
killed both B. subtilis (Fig. 9 a) and P. aeruginosa (Fig. 9b). The MICs
where the concentration at which the O.D. becomes minimum,
were then determined (Fig. 9c). It was found that when increasing
the nanoparticles concentration, the MICs of nanoparticles against
B. subtilis and P. aeruginosa decreased. For B. subtilis, it could be seen
that the MIC of pure biosurfactants were 0.40.55 g l1 . However,
when introducing them into the polymer (PMMA/biosurfactant
nanoparticles), the MICs were lowered to a range of 0.10.32 g l1
(p < 0.001 for each) when compared to the control or bulk PMMA.
The same trend of the nanoparticles antibacterial activity was
observed against P. aeruginosa. However, the MICs of the biosurfactants only and PMMA/biosurfactant nanoparticles were
higher (0.250.7 g l1 and 0.30.6, respectively; p < 0.05 for each).
Apparently, all nanoparticles exhibited stronger antibacterial
activities against B. subtilis than P. aeruginosa. The antibacterial
efcacy was also further investigated by the kinetic test. The
number of surviving bacterial colonies for each type of coreshell
nanoparticles was counted as a function of contact time (min) and
divided by that of the control to acquire the fractional survival.
As the contact time increases, the fractional survival of bacteria
signicantly decreases in a time-dependent manner (p < 0.05 for
each) (Fig. 9d and e). In addition, the nPMMASR particles exhibit
fastest antimicrobial properties against both Gram-negative and
Gram-positive bacteria. However, the difference in bactericidal
activity between nPMMARL and nPMMATL was not statistically
signicant. This effective antimicrobial performance of the biosurfactant modied nPMMA particles can be explained by the large
surface area of the nano-size particles. Based on the same weight,
the surface area of biocides increases drastically with decreased
size of biocides and the expanded surface area of nanoparticles
provides more active sites to contact the bacteria, leading to the
improved killing efciency [8,4850].
Fig. S7 (Supplemental Information) displays the SEM micrographs of the interaction between nPMMASR particles with B.
subtilis after incubation. The arrow marked area shows cellular
disintegration and fragmentation which may be the onset of cell
death. It seems plausible that the nPMMASR particles can bind with
108
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 nm
nPMMA/biosurfactant coreshell particles. (c) The MICs of nPMMA/biosurfactant coreshell particles. Data presented are mean S.D. of three identical experiments made
in 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 nm
nPMMA/biosurfactant coreshell particles as a function of contact time against (d) P. aeruginosa and (e) B. subtilis. The fractional survival was calculated as fractional survival = (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. of
three identical experiments made in three replicate. *Statistically signicant difference as compared to the controls (p < 0.05 for each).
the bacterial membranes via electrostatic interaction, and eventually kill the cells. The addition of surfactin particularly can increase
the amine groups, and more positive charges, which led to more
antibacterial activity compared to other nanoparticles. The differential behavior of these nanoparticles toward these bacteria is due
to the difference in cell wall architecture of Gram-positive and
Gram-negative bacteria [4951]. The peptidoglycan layer of the B.
subtilis cell wall is composed of networks with plenty of pores that
could render them more susceptible to the intracellular transduction by the nanoparticles leading to cell disruption. On the contrary,
the cell wall of P. aeruginosa is made up of thin membrane of peptidoglycan and an outer membrane composed of lipopolysaccharide,
lipoprotein, and phospholipids, which would be less prone to the
attack of the nanoparticles. This explains the higher antibacterial
activity of the nanoparticles against B. subtilis than P. aeruginosa.
Since higher antibacterial activity was observed in B. subtilis, this
organism was further probed for underlying the mechanism behind
the bactericidal effects. For this purpose, oxidative stress markers like LDH release, lipid peroxidation and reduced glutathione
levels were estimated. B. subtilis cells exposed to nPMMASR particles showed a concentration-dependent, statistically signicant
(p < 0.05) increase in LDH release (Fig. S8a; Supplemental Information) and LPO (Fig. S8b; Supplemental Information) as evident by an
increase in the formation of MDA, an oxidized product of polyunsaturated fatty acids compared to that observed with nPMMARL and
nPMMATL at similar concentrations. Cellular GSH levels were signicantly depleted (p < 0.05) after 1 h exposure to nPMMA particles,
compared to control (i.e., without nanoparticles in culture media)
and it followed the order as nPMMASR > nPMMARL > nPMMATL (Fig.
S8c). The correlation between LDH release, LPO induction, and
GSH depletion in a concentration-dependent manner suggests that
oxidative stress could act as an important pathway by which the
nPMMASR particles induce DNA damage in Gram-positive bacterial cells. These ndings conrm that nPMMAs are internalized by
bacterial cells and induce signicant oxidative stress leading to
a compromised cellular antioxidant defense and accumulation of
ROS. Based on these observations a hypothetical mechanism for
cellular toxicity could be through the generation of OH , O2 , and
H2 O2 in bacterial cells resulting in the oxidation of polyunsaturated
phospholipids. The lipid peroxidation reaction subsequently causes
DNA damage, GSH depletion, and disruption of membrane morphology and the electron transport chain, which leads to cell death
(Scheme 3). Our observations on the toxic response to nPMMAs in
the model bacterium B. subtilis reect the possible perturbations
that could inict damage to other members of microbial communities responsible for biogeochemical cycles in aquatic and terrestrial
environment. Our observations substantiate the need for impact
assessment of such novel materials in environmental settings.
3.6. Drug loading onto and in vitro release from the nanoparticles
The nPMMASR particles exhibited high loading capacity and efciency for IB. The loading efciency of IB, AQ and curcumin was
91.45, 75.87 and 77.22%, respectively, whereas in case of nPMMARL
and nPMMATL the encapsulation efciency was rather low (23.79,
33.57 and 25.66%, respectively). It can be explained that the higher
amount of drug loading in nPMMASR particles was due to the strong
ionic interaction between the degree of protonation of the amino
groups of surfactin in the nPMMASR shell, as well as the carboxylic
acid group in IB. This interaction is greatly affected by the medium
pH and salt contents [52,53]. In this work, we observed that the
maximum loading capacity decreased to 3.7% at pH 4, attributable
to the loss of the ionic interaction between the drug and the polymer as a result of PMAA protonation. Based on these data, nPMMASR
was selected for in vitro IB release studies.
109
Scheme 3. Possible mechanism of nPMMA-induced genotoxicity and cytotoxicity.
Fig. 10 presents the release proles of IB from the nPMMASR

particles using free IB as a reference considering the barrier effect
of dialysis tubing membrane. It was seen that IB release from the
nanoparticles to the release medium outside the dialysis tubing was
much slower than free IB, indicating prolonged release of IB from
the nanoparticles. Fig. 10 (a) shows that the initial IB loading has
a strong effect on the release kinetics of IB from the nanoparticles,
with a lower release rate at higher initial drug loading content, i.e.,
30% LC < 15.5% LC < 2.5% LC. In the same release medium (0.15 M;
pH 7.4; Tris buffer), an initial burst release of 17% and 11% within
the rst 10 h is observed for the particles with 2.5% and 15.5% LC,
respectively. In contrast, the release prole for particles with 33.3%
LC resulted in only 40% of the drug being released even after 50 h.
The nanoparticles with high drug loading started to aggregate in
the dialysis bag beyond 50 h and thus the release experiment was
terminated thereafter.
The dependence of release kinetics on IB loading content may
be explained by the change in nanoparticles properties and physical state of the drug in the nanoparticles. It is believed that
the drug-loaded nanoparticles are much more hydrophobic than
the blank particles due to the charge neutralization and the
hydrophobic nature of IB. With increasing IB loading, the hydrophobicity of the particles increases resulting in lower swelling and
release rates. Similar results have been observed in doxorubicin
hydrochloride/PMAA-PS-80-g-St system [10], dox loaded sulfopropyl dextran microsphere [54] and liposomal doxorubicin [55].
As illustrated in Fig. 10(b), at the same drug loading (i.e., 15.5%
IB), IB release from the nanoparticles is much faster at pH 5 (simulating endosome pH) than that at pH 7.4 (simulating normal tissue
pH). The higher release rate at acidic pH may be due to the pHdependence of IB-nPMMASR interaction, which is weakened at
acidic pH as a result of PMAA protonation and existence of surfactin
as polycation leading to its faster dissolution. The above results
are consistent with recent literature using similar carboxylic acid
containing polymers [10,52,53]. Recently many researchers have
studied efcient pH responsive delivery systems, especially using
110
100
100
(a)
Free IB
LC = 15.5 %
Free IB
LC = 15.5 %; pH 5.0
LC = 15.5 %; pH 7.4
80
Cumulative release (%)
80
Cumulative release (%)
(b)
LC = 2.5 %
LC = 30.0 %
60
40
20
60
40
20
0
0
10
20
30
40
50
Time (h)
10
20
30
40
50
Time (h)
Fig. 10. (a) Effect of initial loading content on the kinetic of IB release from nPMMASR coreshell particles at 37 C in 0.15 M pH 7.4 Tris/NaCl buffer. (b) Effect of pH on kinetics
of IB release from the nanoparticles with drug loading content of 15.5% at 37 C. The release of free IB from the dialysis bag was used as control. For each buffer system the
ionic strength was kept constant at 0.15 M by adding NaCl. Data points are mean S.D. of three independent trials.
charged species as gate keepers [56]. Our system utilizes stronger

biosurfactant coating to protect the release of IB with minimum
leaching (27%) at pH 7.4. This makes our system more reliable in
achieving site specic drug delivery compared to some of the recent
ndings. The above results prove that surfactin can be effectively
utilized as a pH responsive gate keeper to target various tissues
and extracellular tumors. Since extracellular pH in tumor is lower
than that in normal tissue and the plasma [10], the nanoparticles
are expected to release more drug in tumor tissue than in the systemic circulation. As a result, lower systemic toxicity while higher
therapeutic efcacy could be obtained.
Fig. 11(a) shows the release proles of AQ from nPMMASR particles with a loading capacity of 15.5% for various time intervals
in the release media at various pH values at 37 C. Initially the
release of AQ at the pH 5.0 and 7.4 provided a continuous release
of the entrapped AQ for up to 50 h and the initial burst release
phase was not as obvious as IB release data. This is in contrast
to the AQ release rate at pH 3.0 where 90% of the loaded AQ was
released from the nanoparticles within 50 h. At strong acidic condition, the nanoparticles would dissolve quickly, which leads to
the very fast release thereby demonstrating that the drug release
rate of these nanoparticles is possible by changing the pH values. A similar effect of sustained release was previously observed
for chitosan/poly (lactic acid) [57] and anti-cancer drugs encapsulated poly (glycolic acid) stabilized by polyvinyl alcohol [58].
Moreover, as an acidic drug, solubility of AQ can be improved signicantly possibly due to ionization into anions under the alkaline
condition [57].
The in vitro release of curcumin from nPMMASR particles under
simulating physiological conditions (37 C, PBS buffer; pH 7.4)
illustrates three distinguishable stages (Fig. 11b). The release
of the drug undergoes a burst release in the stage I, which was
observed during the rst 10 h of the release, and it is responsible
for approximately 35% of the release. This burst is most likely due
to (a) the accumulation of the drug molecules at or near the surface
of the polymer matrix which subsequently facilitates the release of
the drug into the surrounding solution and (b) if the surface of the
matrix erodes as a result of the incubation in the aqueous solution,
the drug molecules could escape much more simply through these
surface defects [3]. The stage II for all the drug contents is comprised of a steady state drug release which accounts for almost 50%
of the drug was released in between 10 and 40 h. Finally, a gradual
decrease in the release rate (stage III) until they reached their
Fig. 11. Release prole of (a) AQ and (b) curcumin from nPMMASR coreshell particles at 37 C in Tris/NaCl buffer (0.01 M) at 37 C. Values reported as the mean S.D.
(n = 3).
111
Table 2
Drug 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
R2
IB
I
II
III
0.8157
0.4804
0.2677
0.9581
0.9617
0.9825
Non-Fickian diffusion
Fickian diffusion
Fickian diffusion
AQ
I
II
III
0.7811
0.2558
0.1928
0.9916
0.9863
0.9978
Fickian diffusion
Fickian diffusion
Curcumin
I
II
III
0.6638
0.2166
0.2419
0.9947
0.9519
0.9855
Fickian diffusion
Fickian diffusion
Drug-carrier formulation: nPMMA (5 mg ml1 ) + IB/AQ/curcumin (5 mg ml1 ).
maximum release occurred. It is believed that the drug molecules

are required to follow longer routes in order to escape the polymeric
matrix [3].
3.7. Kinetics of the drug release
The values of the release exponent n and the correlation coefcient R2 after tting with the power law led to a number of
important observations (Table 2 and Table S5 in Supplemental
Information). Firstly, tabular values of R2 showed that the power
law (KorsmeyerPeppas) was best tted with release kinetic data of
the three types of drug used here. Secondly, stage I of the release followed the non-Fickian diffusion. Therefore, the release of the drug
occurred mainly through the combination of the diffusion from the
polymeric matrix and the surface erosion. The FESEM analysis was
utilized to observe the morphology of the polymer surface once the
release process was over (Fig. S9 in Supplemental Information). The
formation of the cracks on the surface of the polymeric nanoparticle is an evidence of the proposed release mechanism for stage I.
As opposed to the proposed release mechanism for stage I, stages II
and III underwent the Fickian diffusion in which the release of the
drug molecules was solely by diffusion [3].
4. Conclusions
A one-pot oil/water (O/W) modied atomized microemulsion process was used to synthesize biosurfactant-functionalized
PMMA nanoparticles, by which conventional toxic sodium dodecyl
sulfate (SDS) was replaced by non-toxic, biodegradable and biocompatible biosurfactants (rhamnolipid, surfactant and trehalose
lipid). The amount of biosurfactant required was 1/35 of the
monomer amount by weight and the surfactant/water ratio could
be as low as 1/210. These surfactant levels are much lower
in comparison with those used in a conventional microemulsion polymerization system. These nanoparticles are composed of
nanosized cores of high molecular weight PMMA and nano-thin
shells of the biosurfacatnts. The particles were spherical in shape
and 2050 nm in diameter with an average molecular weight of
0.91.5 105 g mol1 (polydispersity index 1.642.65). The solid
content of nPMMA increased when the amount of added biosurfactant was increased. Surface coating by biosurfactants onto nPMMA
was ascertained by AFM, TEM, zeta potential measurements,
FTIR and thermal analysis. These nPMMA (core)biosurfactant
(shell) particles were non-toxic, biocompatible and exhibited
strong antibacterial activity against B. subtilis and P. aeruginosa.
Glutathione depletion with a concomitant increase in malondialdehyde levels (increasing reactive oxygen species) and lactate
dehydrogenase activity demonstrates that nPMMAs induce oxidative stress leading to genotoxicity and cytotoxicity in B. subtilis.
Detailed characterizations showed that surfactin was effectively

anchored on the surface of the nPMMA, preventing release of IB,
anthraquinone and curcumin under undesirable conditions. The
particles were able to efciently load high contents of these three
drugs with no loss of colloidal stability. The designed drug delivery system indicates a three stages drug release over a period of 50
days with a sustained manner and suppressed burst release, which
occurred only for 10 h and strongly dependent on the initial loading content and medium pH. The release kinetics and mechanistic
investigations implied that the rst stage for all the contents of
the incorporated drugs followed the non-Fickian diffusion, i.e. the
combination of the diffusion and the surface erosion of the polymer
matrix which was conrmed by FESEM analysis of the polymers
after the release process; however, stages II and III obeyed the Fickian diffusion. Future work will involve investigating the effect of
varying coating thickness on the release prole of chemopreventing
agents. This work manifests the potential of biosurfactantpolymer
hybrids as next generation delivery vehicles for biomedical applications.
Acknowledgements
Aniruddha Chatterjee is thankful to the University Grants Commission (UGC), New Delhi for providing the nancial support
through BSR Start-up Grant [F. No. 20-1/2012 (BSR)/20-4 (10)/2012
(BSR), dated October 30, 2012]. Department of Science and Technology (D.S.T), New Delhi and Council for Scientic and Industrial
Research (C.S.I.R), New Delhi are gratefully acknowledged for providing Senior Research Fellowship to Chinmay Hazra and Debasree
Kundu, respectively. We are thankful to SAIF, IIT, Bombay for providing the TEM facility. Thanks are also due to Dr. Kirtee Kamalja
(School of Mathematical Sciences, North Maharashtra University,
Jalgaon, India) for her help in statistical analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.
2014.02.051.
References
[1] S.T. Camli, F. Buyukserin, O. Balci, G.G. Budak, Size controlled synthesis
of sub-100 nm monodisperse poly(methylmethacrylate) nanoparticles using
surfactant-free emulsion polymerization, J. Colloid Interface Sci. 344 (2010)
528532.
[2] S.M. Desai, S.S. Solanky, A.B. Mandale, K. Rathore, R.P. Singh, Controlled grafting
of N-isopropyl acrylamide brushes onto self-standing isotactic polypropylene
thin lms: surface initiated atom transfer radical polymerization, Polymer 44
(2003) 76457649.
112
[3] A. Sohrabi, P.M. Shaibani, H. Etayash, K. Kaur, T. Thundat, Sustained drug

release and antibacterial activity of ampicillin incorporated poly(methyl
methacrylate)-nylon6 core/shell nanobers, Polymer 54 (2013) 26992705.
[4] N. Pimpha, U. Rattanonchai, S. Surassmo, P. Opanasopit, C. Rattanarungchai,
P. Sunintaboon, Preparation of PMMA/acid-modied chitosan coreshell
nanoparticles and their potential as gene carriers, Colloid Polym. Sci. 286 (2008)
907916.
[5] S.T. Camli, F. Buyukserin, M.S. Yavuz, G.G. Budak, Fine-tuning of functional
poly(methylmethacrylate) nanoparticle size at the sub-100 nm scale using
surfactant-free emulsion polymerization, Colloids Surf. A: Physicochem. Eng.
Aspects 366 (2010) 141146.
[6] C. Norakankorn, Q. Pan, G.L. Rempel, S. Kiatkamjornwong, Synthesis of
poly(methyl methacrylate) nanoparticles initiated by 2,2 -azoisobutyronitrile
via differential microemulsion polymerization, Macromol. Rapid Commun. 28
(2007) 10291033.
[7] G. He, Q. Pan, G.L. Rempel, Synthesis of poly(methyl methacrylate) nanosize
particles by differential microemulsion polymerization, Macromol. Rapid Commun. 24 (2003) 585588.
[8] S. Inphonlek, N. Pimpha, P. Sunintaboon, Synthesis of poly(methyl methacrylate) core/chitosan-mixed-polyethyleneimine shell nanoparticles and their
antibacterial property, Colloids Surf. B: Biointerfaces 77 (2010) 219226.
[9] L. Yuan, Y. Wang, M. Pan, G.L. Rempel, Q. Pan, Synthesis of poly(methyl
methacrylate) nanoparticles via differential microemulsion polymerization,
Eur. Polym. J. 49 (2013) 4148.
[10] A. Shalviri, H.K. Chan, G. Raval, M.J. Abdekhodaie, Q. Liuc, H. Heerklotz, X.Y.
Wu, Design of pH-responsive nanoparticles of terpolymer of poly(methacrylic
acid), polysorbate 80 and starch for delivery of doxorubicin, Colloids Surf. B:
Biointerfaces 101 (2013) 405413.
[11] G. Kim, S. Lim, B.H. Lee, S.E. Shim, S. Choe, Effect of homogeneity of
methanol/water/monomer mixture on the mode of polymerization of MMA:
soap-free emulsion polymerization versus dispersion polymerization, Polymer
51 (2010) 11971205.
[12] N. Sahiner, S. Butun, P. Ilgin, Hydrogel particles with core shell morphology for
versatile applications: Environmental, biomedical and catalysis, Colloids Surf.
A: Physicochem. Eng. Aspects 386 (2011) 1624.
[13] M. Hazarika, D. Arunbabu, T. Jana, Formation of core (polystyrene)shell (polybenzimidazole) nanoparticles using sulfonated polystyrene as template, J.
Colloid Interface Sci. 351 (2010) 374383.
[14] V.L. Schade, T.S. Roukis, The role of polymethylmethacrylate antibiotic-loaded
cement in addition to debridement for the treatment of soft tissue and osseous
infections of the foot and ankle, J. Foot Ankle Surg. 49 (2010) 5562.
[15] P. Sunintaboon, S. Duangphet, P. Tangboriboonrat, Polyethyleneiminefunctionalized poly(methyl methacrylate) colloidal nanoparticles for directly
coating natural rubber sheet, Colloids Surf. A: Physicochem. Eng. Aspects 40
(350) (2009) 114120.
[16] Y.S. Siu, L. Li, M.F. Leung, K.L.D. Lee, P. Li, Polyethylenimine-based amphiphilic
coreshell nanoparticles: study of gene delivery and intracellular trafcking,
Biointerphases 7 (2012), http://dx.doi.org/10.1007/s13758-011-0016-24.
[17] N. Saengkrit, P. Sanitrum, N. Woramongkolchai, S. Saesoo, N. Pimpha,
S. Chaleawlert-umpon, T. Tencomnao, S. Puttipipatkhachorn, The PEIintroduced CS shell/PMMA core nanoparticle for silencing the expression
of E6/E7 oncogenes in human cervical cells, Carbohydr. Polym. 90 (2012)
13231329.
[18] C. Petchthanasombat, T. Tiensing, P. Sunintaboon, Synthesis of zinc oxideencapsulated poly(methyl methacrylate)-chitosan coreshell hybrid particles
and their electrochemical property, J. Colloid Interface Sci. 369 (2012) 5257.
[19] N. Kanjanathaworn, D. Polpanich, K. Jangpatarapongsa, P. Tangboriboonrat,
Reduction of cytotoxicity of natural rubber latex lm by coating with PMMAchitosan nanoparticles, Carbohydr. Polym. 97 (2013) 5258.
[20] C. He, J. Liu, X. Ye, L. Xie, Q. Zhang, X. Ren, G. Zhang, C. Wu, Preparation
of well-dened coreshell particles by Cu2+ -mediated graft copolymerization of methyl methacrylate from bovine serum albumin, Langmuir 24 (2008)
1071710722.
[21] C. He, J. Liu, Q. Zhang, C. Wu, A novel stable amperometric glucose biosensor
based on the adsorption of glucose oxidase on poly (methyl methacrylate)bovine serum albumin coreshell nanoparticles, Sens. Actuators B 166167
(2012) 802808.
[22] S. Mishra, A. Chatterjee, Particle size, morphology and thermal properties of
polystyrene nanoparticles in microemulsion process, Polym. Plast. Technol.
Eng. 49 (2010) 791795.
[23] S. Mishra, A. Chatterjee, Effect of nano-polystyrene (nPS) on thermal, rheological, and mechanical properties of polypropylene (PP), Polym. Adv. Technol. 22
(2011) 15471554.
[24] S. Mishra, A. Chatterjee, V.K. Rana, Polymer nanoparticles: their effect on rheological, thermal, and mechanical properties of linear low-density polyethylene
(LLDPE), Polym. Adv. Technol. 22 (2011) 18021811.
[25] S. Mishra, A. Chatterjee, Novel synthesis of polymer and copolymer nanoparticles by atomized microemulsion technique and its characterization, Polym.
Adv. Technol. 22 (2011) 15931601.
[26] S. Mishra, A. Chatterjee, A process for synthesis of polymer latex nanoparticles by monomer atomization in microemulsion, Indian Patent Application No.
254969 (10/01/2013).
[27] C. Hazra, D. Arunbabu, D. Kundu, A. Chaudhari, T. Jana, Biomimetic fabrication of biocompatible and biodegradable coreshell polystyrene/biosurfactant
bionanocomposites for protein drug release, J. Chem. Technol. Biotechnol. 88
(2013) 15511560.
[28] C. Hazra, D. Kundu, A. Chaudhari, T. Jana, Biogenic synthesis, characterization,

toxicity and photocatalysis of zinc sulde nanoparticles using rhamnolipids
from Pseudomonas aeruginosa BS01 as capping and stabilizing agent, J. Chem.
Technol. Biotechnol. 88 (2013) 10391048.
[29] C. Hazra, D. Kundu, P. Ghosh, S. Joshi, N. Dandi, A. Chaudhari, Screening
and identication of Pseudomonas aeruginosa AB4 for improved production, characterization and application of a glycolipid biosurfactant using
low-cost agro-based raw materials, J. Chem. Technol. Biotechnol. 86 (2011)
185198.
[30] X.Y. Liu, H. Namir, S.Z. Yang, B.Z. Mu, Structural characterization of eight cyclic
lipopeptides produced by Bacillus subtilis HSO121, Prot. Pept. Lett. 14 (2007)
766773.
[31] H. Namir, X.Y. Liu, S.Z. Yang, B.Z. Mu, Surfactin isoforms from Bacillus subtilus
HSO121: separation and characterization, Prot. Pept. Lett. 15 (2008) 265269.
[32] D. Kundu, C. Hazra, N. Dandi, A. Chaudhari, Biodegradation of 4-nitrotoluene
with biosurfactant production by Rhodococcus pyridinivorans NT2: metabolic
pathway, cell surface properties and toxicological characterization, Biodegradation 24 (2013) 775793.
[33] N. Pimpha, S. Chaleawlert-umpon, P. Sunintaboon, Core/shell polymethyl
methacrylate/polyethyleneimine particles incorporating large amounts of iron
oxide nanoparticles prepared by emulsier-free emulsion polymerization,
Polymer 53 (2012) 20152022.
[34] E.V. Chandrasekaran, J.N. Bemiller, Constituent analysis of glycosaminoglycans,
Methods Carbohydr. Chem. 8 (1980) 8996.
[35] M. Dubois, K. Gilles, J. Hamilton, P. Rebers, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956)
350356.
[36] H.P. Borase, C.D. Patil, I.P. Sauter, M.B. Rott, S.V. Patil, Amoebicidal activity of
phytosynthesized silver nanoparticles and their in vitro cytotoxicity to human
cells, FEMS Microbiol. Lett. 345 (2013) 127131.
[37] H. Wang, Q.M. Pan, G.L. Rempel, Micellar nucleation differential microemulsion
polymerization, Eur. Polym. J. 47 (2011) 973980.
[38] G.W. He, Q.M. Pan, Synthesis of polystyrene and polystyrene/poly(methyl
methacrylate) nanoparticles, Macromol. Rapid Commun. 25 (2004) 1545
1548.
[39] I. Freris, P. Cristofori, A. Riello, J. Benedetti, Encapsulation of submicrometersized silica particles by a thin shell of poly(methyl methacrylate), J. Colloid
Interface Sci. 331 (2009) 351355.
[40] S. Jenjob, P. Sunintaboon, P. Inprakhon, N. Anantachoke, V. Reutrakul, Chitosanfunctionalized poly(methyl methacrylate) particles by spinning disk processing
for lipase immobilization, Carbohydr. Polym. 89 (2012) 842848.
[41] A.M. Santos, A. Elassari, J.M.G. Martinho, C. Pichot, Synthesis of cationic
poly(methyl methacrylate)-poly(N-isopropyl acrylamide) coreshell latexes
via two-stage emulsion copolymerization, Polymer 46 (2005) 1181
1188.
[42] L. Nabzar, D. Duracher, A. Elassari, G. Chauveteau, C. Pichot, Electrokinetic properties and colloidal stability of cationic amino-containing
N-isopropylacrylamide-styrene copolymer particles bearing different shell
structures, Langmuir 14 (1998) 50625069.
[43] B.R. Singh, S. Dwivedi, A.A. Al-Khedhairy, J. Musarrat, Synthesis of stable
cadmium sulde nanoparticles using surfactin produced by Bacillus amyloliquifaciens strain KSU-109, Colloids Surf. B: Biointerfaces 85 (2011) 207
213.
[44] C. Sivapathasekaran, P. Das, S. Mukherjee, J. Saravanakumar, M. Mandal, R. Sen,
Marine bacterium derived lipopeptides: characterization and cytotoxic activity
against cancer cell lines, Int. J. Pept. Res. Ther. 16 (2010) 215222.
[45] S.C. Pilcher, W.T. Ford, Structures and properties of poly(methyl methacrylate) latexes formed in microemulsions, Macromolecules 31 (1998) 3454
3460.
[46] W. Viratyaporn, R.L. Lehman, Effect of nanoparticles on the thermal stability
of PMMA nanocomposites prepared by in situ bulk polymerization, J. Therm.
Anal. Calorim. 103 (2011) 267273.
[47] X. Qu, Y. Shi, Y. Tang, L. Chen, X.J. Jin, Novel sintering behavior of polystyrene
nanolatex particles in the lming process, J. Colloid Interface Sci. 250 (2002)
484491.
[48] A. Laachachi, M. Cochez, M. Ferriol, J.M. Lopez-Cuesta, E. Leroy, Inuence of
TiO2 and Fe2 O3 llers on the thermal properties of poly(methyl methacrylate)
(PMMA), Mater. Lett. 59 (2005) 3639.
[49] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, A mechanistic study of the
antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus,
J. Biomed. Mater. Res. A 52 (2000) 662668.
[50] Y. Li, X.G. Chen, N. Liu, C.S. Liu, C.G. Liu, X.H. Meng, L.J. Yu, J.F. Kenendy, Physicochemical characterization and antibacterial property of chitosan acetates,
Carbohydr. Polym. 67 (2007) 227232.
[51] M. Kong, X.G. Chen, C.S. Liu, C.G. Liu, X.H. Meng, L.J. Yu, Antibacterial mechanism
of chitosan microspheres in a solid dispersing system against E. coli, Colloids
Surf. B 65 (2008) 197202.
[52] Y. Tian, L. Bromberg, S.N. Lin, T. Alan Hatton, K.C. Tam, Complexation and release
of doxorubicin from its complexes with Pluronic P85-b-poly(acrylic acid) block
copolymers, J. Control. Release 121 (2007) 137145.
[53] Y. Tian, P. Ravi, L. Bromberg, T.A. Hatton, K.C. Tam, Synthesis and aggregation
behavior of Pluronic F87/poly (acrylic acid) block copolymer in the presence of
doxorubicin, Langmuir 23 (2007) 26382646.
[54] Z. Liu, R. Cheung, X.Y. Wu, J.R. Ballinger, R. Bendayan, A.M. Rauth, A study of
doxorubicin loading onto and release from sulfopropyl dextran ion-exchange
microspheres, J. Control. Release 77 (2001) 213224.
[55] X. Li, D.J. Hirsh, D. Cabral-Lilly, A. Zirkel, S.M. Gruner, A.S. Janoff, W.R. Perkins,
Doxorubicin physical state in solution and inside liposomes loaded via a pH
gradient, Biochim. Biophys. Acta 1415 (1998) 2340.
[56] J. Song, H. Kong, J. Jang, Enhanced antibacterial performance of cationic
polymer modied silica nanoparticles, Chem. Commun. 36 (2009) 5418
5420.
113
[57] D. Jeevitha, K. Amarnath, Chitosan/PLA nanoparticles as a novel carrier for the

delivery of anthraquinone: synthesis, characterization and in vitro cytotoxicity
evaluation, Colloids Surf. B 101 (2013) 126134.
[58] M.Y. Murali, K.G. Brij, J. Meena, C.C. Subhash, Fabrication of curcumin encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer
cells, J. Colloid Interface Sci. 351 (2010) 1929.

1 s2.0 S092777571400199X Main

Загружено:

Сведения о документе

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

1 s2.0 S092777571400199X Main

Загружено:

Авторское право:

Доступные форматы

Colloids and Surfaces A: Physicochem. Eng.

Aspects 449 (2014) 96113

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Poly(methyl methacrylate) (core)biosurfactant (shell) nanoparticles:

School of Life Sciences, North Maharashtra University, Jalgaon, Maharashtra, India

A novel hybrid core/shell nanoparticles by oil/water green atomized

impact of excess surfactant on the properties and post-treatment of

Fig. 1. Atomized reaction vessel for synthesis of nPMMAbiosurfacatnt core/shell

2.4. Isolation of nanoparticles

where 0 is the density of MMA (0.94 g cm3 at 25 C), V is the

2.5.6. FTIR spectroscopy

2.7. Antihemolytic activity

where As , Ap , and An are the absorbances of the test suspensions,

2.9. Oxidative stress markers

2.9.1. Lactate dehydrogenase (LDH) release

2.11. Kinetics studies

Scheme 1. Pictorial representation of the formation of nPMMA/biosurfactant coreshell particles.

4. KorsmeyerPeppas (Power law model)

where, t is time and Mt and M0 are the concentration of the

relatively hydrophilic carboxyl moiety and hydrophobic moiety

Scheme 2. Mechanism of atomized microemulsion polymerization adopted in this study.

average particle size is 23.6 nm in diameter. As the water/monomer

Particle size*10 (nm)

Fig. 2. Effect of monomer amount on the particle size of nPMMA (experimental

Moreover, the growth rate of particle size becomes faster as

Particle size distribution

Particle size*10 (nm)

APS amount (g)

Particle size (nm)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

to the expansion of charged groups, both ammonium groups on the

DLS tended to be larger. In AFM and TEM, a thin lm of polymer

to C H stretching vibrations. The peak observed at 1634 cm1

on grouping information using Tukeys multiple comparisons at

Fig. 8. (a) Cytotoxicity of nPMMA/biosurfactant coreshell particles against human

and primary amine. Free amine groups on the nanoparticle surface

To evaluate the antibacterial properties of nPMMA

Scheme 3. Possible mechanism of nPMMA-induced genotoxicity and cytotoxicity.

Fig. 10 presents the release proles of IB from the nPMMASR

Cumulative release (%)

Cumulative release (%)

charged species as gate keepers [56]. Our system utilizes stronger

Drug-carrier formulation: nPMMA (5 mg ml1 ) + IB/AQ/curcumin (5 mg ml1 ).

maximum release occurred. It is believed that the drug molecules

Detailed characterizations showed that surfactin was effectively

[3] A. Sohrabi, P.M. Shaibani, H. Etayash, K. Kaur, T. Thundat, Sustained drug

[28] C. Hazra, D. Kundu, A. Chaudhari, T. Jana, Biogenic synthesis, characterization,

[57] D. Jeevitha, K. Amarnath, Chitosan/PLA nanoparticles as a novel carrier for the

Вам также может понравиться