European Polymer Journal 46 (2010) 958–967

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Contents lists available at ScienceDirect

European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Macromolecular Nanotechnology

Synthesis and characterization of thermo-responsive copolymericnanoparticles of poly(methyl methacrylate-co-N-vinylcaprolactam)

Sunil Shah a,1, Angshuman Pal a, Rajiv Gude b, Surekha Devi a,*

a Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Indiab Gude Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar,Navi Mumbai 410210, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 April 2009Received in revised form 22 December 2009Accepted 9 January 2010Available online 15 January 2010

Keywords:NanoparticlesCopolymerMicroemulsion polymerizationB16F10 melanoma cell linesCytotoxicity

0014-3057/$ - see front matter � 2010 Elsevier Ltddoi:10.1016/j.eurpolymj.2010.01.005

* Corresponding author. Tel./fax: +91 265 279555E-mail address: surekha_devi@yahoo.com (S. De

1 Present address: Shah-Schulman Centre forNanotechnology, Faculty of Technology, DharmsinCollege Road, Nadiad 387001, Gujarat, India.

Copolymeric nanoparticles of methyl methacrylate (MMA) and N-vinylcaprolactam (VCL)were prepared through free radical polymerization using hydrogen peroxide and L-ascorbicacid as a redox initiator in o/w microemulsion containing sodium dodecyl sulphate (SDS).The copolymers were characterized by FTIR and gel permeation chromatography (GPC) andcomposition of copolymer was determined by 1H NMR spectroscopy. Reactivity ratio wasdetermined by linear least square and non-linear least square methods. The morphologyand particle size distribution of copolymer latexes was determined through transmissionelectron microscopy (TEM) and dynamic light scattering (DLS). Copolymers were of lessthan 50 nm size with spherical morphology and latexes were stable for more than6 months. Phase transition temperature measured through UV–vis spectrometry, for thesynthesized copolymer indicates their potential use in biosensors and targeted drug deliv-ery system. Cytotoxicity of nanoparticles was determined by MTT assay on B16F10 mela-noma cell lines. Cell viability data shows the IC50 values of copolymeric nanoparticles to bein the range of 0.01–0.1 mg/mL.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Recently, much attention has been focused on watersoluble polymers, which respond to changes in pH, tem-perature, ionic strength and electric field [1,2]. Polymersresponding to temperature change exhibit an abruptchange in volume at a phase transition temperature, whichresults in change, in specific volume of the macromole-cules in solution, including a transition from a loose coiledstructure to a more compact globule. This abrupt change inphysical properties due to variation in temperature and pHcan be monitored through balancing various attractive andrepulsive forces between polymer backbone and functional

. All rights reserved.

2.vi).Surface Science andh Desai University,

groups. When a repulsive force usually electrostatic in nat-ure, overcomes an attractive force such as hydrogen bond-ing or hydrophobic interaction, sometimes the gel networkswells, ‘‘discontinuously” leading to a volume transition.The variables that can trigger and shift the swelling andshrinking depend on the nature of intermolecular forcesexisting in the gel network [3]. Introduction of chargedgroups into a hydrophobic gel network either throughhydrolysis or copolymerization with ionic comonomerscan alter its swelling behavior in water or make it sensitiveto the ionic strength and pH [4–7].

Among temperature sensitive polymers, poly(N-isopro-pylacrylamide) (PNIPAM) and its copolymers have beenstudied extensively [8]. Recently, poly(N-vinylcaprolac-tam) (PVCL), water soluble, biodegradable, temperatureresponsive polymer having lower critical solution temper-ature (LCST) near to body temperature (around 32 �C) hasattracted much attention of researchers and technologist[9]. Even though PVCL has been commercially available

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for a long time from BASF (Baden Aniline and Soda Factory)the chemical company only a few studies related to itsproperties and applications has been reported so far. Muchof the published work is concentrated on the PVCL basedhydrogels as they have potential applications in con-trolled drug delivery system, separation science, andimmobilization of enzymes and in oil-recovery technol-ogy [10–16]. For these applications colloidal systems canbe equally attractive, which could be easily synthesizedthrough free radical emulsion polymerizations. First timein 1986 Pelton and Chibante [17] reported synthesis oftemperature-sensitive microgel from N-isopropylacrylam-ide (NIPAM) and N,N0-methylene bisacrylamide (MBA).Thereafter, many research papers were published oncopolymeric microgels based on NIPAM with other ionicmonomers [18–21]. Recently, Laukkanen et al. have re-ported synthesis of thermosensitive PVCL microgel byemulsion polymerization using sodium dodecyl sulphateand potassium per sulphate as a surfactant and initiator,respectively [22].

Properties of the copolymer depend on the nature of themonomers and its composition, which depends upon thereactivity of the monomer in the given system. In the copo-lymerization of monomers with different reactivities, pre-dominant incorporation of the more reactive monomeroccurs within the copolymer chain even if its concentra-tion in the feed is low. Okhapkin et al. [23] have reported1:1 ratio of VCL to MMA in the copolymer synthesized at9:1 feed ratio of VCL to MMA at less than 2% conversion.This is attributed to 20 times lower reactivity of VCL thanMMA calculated with the help of linear least square andnon-linear least square method for reactivity ratio deter-mination. As a result more reactive monomer preferen-tially gets incorporated into the copolymer and themonomer ratio in the feed rapidly changes resulting intoa significant drift in the copolymer composition with theconversion. To control the composition drift, Fedorov etal. [24] and Qiu and Sukhishvili [25] reported gradualcontinuous feeding concept for copolymerization of vinylpyrrolidone and 4-vinylpyridine, and VCL and glycidylmethacrylate and they reported that the synthesizedcopolymers were not only with homogeneous compositionbut also retained their temperature sensitive character.

Hence attempts have been made to synthesize thermo-responsive poly(MMA-co-VCL) nanoparticles throughcontinuous feed o/w microemulsion polymerization tech-nique.

2. Experimental

2.1. Materials

N-Vinylcaprolactam (98%, Sigma–Aldrich, Steinheim,Germany) and methyl methacrylate (Merck, Mumbai In-dia) were distilled under vacuum and stored at 4 �C priorto use. L-Ascorbic acid, hydrogen peroxides (30% w/v,Merck, Mumbai, India) and sodium dodecyl sulphate(SDS) from SD Fine Chem., Baroda, India were used as re-ceived. Double distilled deionised water (0.22 lm nylonfiltered) was used throughout the experiments.

2.2. Cell cultures

B16F10, a highly metastatic lung selected subline de-rived from C57/BL6 murine melanoma, was purchasedfrom National Centre for Cell Science (NCCS), Pune, India.The cell line was maintained as a continuous culture inIscove’s minimum Dulbecco’s medium (IMDM; GIBCO,BRL, MD, USA) supplemented with 10% fetal bovine serum(GIBCO–BRL), 100 U/mL penicillin and 100 lg/mL strepto-mycin. Cells were grown in a humidified atmosphere of5% CO2 and 95% air at 37 �C. Media was replenished everythird day.

2.3. Preparation of copolymeric nanoparticles

Copolymeric nanoparticles of various compositionswere prepared by o/w microemulsion polymerizationtechnique. The ternary microemulsions comprising 6% w/w MMA–VCL monomer mixture, with MMA:VCL weightratios varying from 90:10 to 40:60, SDS (2% w/w) andwater (92% w/w) were taken in three-neck reaction vesselequipped with a nitrogen inlet, thermometer, water con-denser and magnetic stirrer. Typically, monomer mixtureto surfactant ratio was kept constant at 3 for all recipes.The ternary microemulsion was deoxygenated by bubblingpurified nitrogen for 15 min. Polymerization was initiatedby redox initiator hydrogen peroxide and L-ascorbic acidat 40 ± 1 �C. MMA was added drop wise to maintain themonomer ratio in feed. The latex obtained after completionof the reaction was allowed to purify at Kraft temperatureof surfactant to achieve surfactant free latex and then pre-cipitated in 10-fold excess volume of diethyl ether. Thecopolymer was washed with cold water to remove unre-acted monomer and homopolymer of VCL. The copolymerwas further purified twice by dissolving the copolymer insmall amount of tetrahydrofuran and reprecipitating inn-hexane. For the determination of reactivity ratio, copoly-mers were collected below 10% conversion and purified asdescribed above. Monomer reactivities are being reportedby using linear least square and non-linear least squaremethods. Attempts were made to find out the possiblecytotoxicity effect of nanoparticles on B16F10 melanomacell lines.

2.4. Characterization

2.4.1. Spectroscopic analysisFTIR spectrum of the copolymer was recorded on a Per-

kin-Elmer RX1 FTIR spectrophotometer (Massachusetts,USA) using 1-cm diameter KBr pellets. 1H NMR spectrawere recorded using 400 MHz 1H NMR spectrometer (Bru-ker Specrospin Avance Ultra-shield, Germany) at roomtemperature (�30 ± 2 �C). The spectra were obtained afteraccumulating 16 scans by using 1% sample in CDCl3.

2.4.2. Gel permeation chromatographyNumber average (Mn) and weight average (Mw) molec-

ular weights of copolymers with different compositionswere determined by using Perkin-Elmer Totalchrom GelPermeation Chromatography instrument equipped withturbosec size exclusion software, PE-series 200 RI detector,

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series 200 isocratic pump and rheodyne injector. Themixed column PLGel 5l, suitable for molecular weightsup to 104–107, in polar solvent was used. Distilled, de-gassed THF (Merck, India, HPLC Grade) at flow rate of1 mL/min was used as an eluent. Medium molecularweight polystyrene standards (POLYSCI, 1 mg/mL in THF,with molecular weight 1 � 103–3 � 105) were used for cal-ibration of GPC.

2.4.3. Dynamic light scatteringA Brookhaven’s 90 plus dynamic light scattering equip-

ment with a solid state laser source operated at 688 nmwas used to measure the particle size and size distributionof the polymerized latex in a dynamic mode. The particlesize was obtained from the Stokes–Einstein relationD = KT /(3pgd), where d is the diameter of particles, D isthe translational diffusion coefficient, K is the Boltzmannconstant, T is the temperature and g is the viscosity ofthe medium. The scattering intensities from the sampleswere measured at 90� using photomultiplier tube. In orderto minimize the inter-particle interactions, the analysis ofthe latex was done after 10 times dilution consideringthe refractive index and viscosity of water as that of latex.All the measurements were performed in triplicate.

2.4.4. Transmission electron microscopyTEM analysis of copolymeric nanoparticles was per-

formed using CM 120 Philips transmission electron micro-scope (Tokyo, Japan) at accelerating voltage of 200 kV. Onedrop of latex was dispersed in 5 mL of water and wasplaced on the carbon coated copper grid. The grid wasdried under IR lamp and the images of representative areaswere captured at suitable magnifications.

2.4.5. Cloud point determinationThe cloud point of the copolymer solution in double dis-

tilled water (0.1 g L�1) was obtained by spectrophotomet-ric detection of the changes in turbidity at 500 nm usingPerkin-Elmer lambda 35 UV–vis spectrophotometer (Mas-sachusetts, USA). The water-jacketed sample and referencecell holders were coupled with a Julabo 5A circulating bath.Cloud point was considered as the temperature corre-sponding to a 10% reduction in the original transmittanceof the solution [26].

2.4.6. Cytotoxicity assayCytotoxicity of the placebo copolymeric nanoparticles

was evaluated using B16F10 melanoma cell lines. Cellswere seeded in 96-well microplates at a density of5 � 103 cells/well. Cells were allowed to grow and stabilizefor 24 h. They were then treated with poly(MMA-co-VCL)nanoparticles for 24 h, in order to find their cytotoxic ef-fect. The nanoparticles of different composition and con-centrations of 0.00001 to 0.5 mg/mL were added directlyto the medium in each well. On each plate, four wells wereleft untreated for their use as reference. Post treatment cellviability was determined colorimetrically by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide(MTT) reagent. MTT reagent (Sigma–Aldrich) was addedto each well to make a final concentration of 1 mg/mL ofmedia and incubated for 4 h at 37 �C. Formazan crystals

were dissolved in 100 ll of DMSO; the optical densitywas measured in the enzyme-linked immunosorbent assayplate reader (Molecular Devices, Spectra Max 190 with Softmax Pro) at 540 nm with a reference wavelength of690 nm.

3. Results and discussion

3.1. FTIR analysis

Representative FTIR spectra of purified (MMA-co-VCL)copolymers are presented in Fig. 1. The carbonyl stretchingvibrations of the MMA and VCL units of the copolymer ap-pear as strong absorption bands at 1732 and 1640 cm�1,respectively. The copolymer being hydrogel in nature, thestrong but broad band appearing at 3434 cm�1 can beattributed to water of hydration. Yu et al. [27] have studiedstructural transformations and water association interac-tions in PVCL–water system by various methods such asIR-spectroscopy, quantum-chemical calculations, DSC andoptical microscopy and have concluded that PVCL macro-molecules in aqueous solution are the highly modifiedwater associated structures, being affected by polarizationaction of highly polar amide groups due to specific config-urational and conformational structures of the polymer.The bands appearing at 2993, 2949, 2857, 1443 and1389 cm�1 can be attributed to stretching and bendingvibrations of –CH2 and –CH groups, respectively. Bandsappearing at 1045, 1245 cm�1 correspond to ester stretch-ing vibration of acrylate polymer and bands observedaround 2385 and 719 cm�1 correspond to the –CN stretch-ing vibrations and to the presence of more than three –CH2

groups in lactam ring structure providing evidence ofcopolymerization.

3.2. Determination of the reactivity ratio

The high resolution 1H NMR spectra of poly(methylmethacrylate) (PMMA) homopolymer and poly(MMA-co-VCL) copolymer are shown in Fig. 2. The composition ofthe copolymer was determined by the well separated sig-nals that appeared at 4.798 ppm corresponding to the pro-ton from a caprolactam ring of the VCL units and thesignals that appeared near 0.842 ppm corresponding tothe methyl proton of the MMA units [24]. The compositionof the copolymer was evaluated from the relative intensi-ties of –CH3 group of the MMA and >N–CH– group of theVCL units and the results obtained are summarized inTable 1.

To determine reactivity ratios of MMA (rMMA) and VCL(rVCL), the widely used linear graphical Fineman–Rossmethod (FR) and Inverted Fineman–Ross (IFR) [28] andmost accurate non-linear Tidwell–Mortimer (TM) leastsquare method [29] were used and results are given inFig. 3.

The FR method is based on the following equation:

G ¼ MPðP � 1Þ ð1Þ

F ¼ M2

Pð2Þ

Fig. 1. FTIR spectra of poly(MMA-co-VCL) copolymer system. (A) 90:10 poly(MMA-co-VCL) and (B) 50:50 poly(MMA-co-VCL).

Fig. 2. 1H NMR spectra of PMMA and poly(MMA-co-VCL) copolymer.

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Table 1Mole fraction of monomers in feed and copolymer.

(MMA:VCL)mole ratioin feed

Molefractionsin feed

Mole fractionin copolymercalculated from1H NMR

% Conversion

MMMA MVCL PMMA PVCL

90:10 0.9 0.1 0.96 0.04 4.780:20 0.8 0.2 0.92 0.08 3.970:30 0.7 0.3 0.9 0.1 6.760:40 0.6 0.4 0.8 0.2 6.250:50 0.5 0.5 0.7 0.3 7.940:60 0.4 0.6 0.69 0.31 5.5

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where M is the ratio of mole fraction of monomer in thefeed and P is the ratio of mole fraction of monomer unitin copolymer. The linear relationship between G and Fcould be given as:

G ¼ Fr1�r2 ð3Þ

By plotting G versus F for all experiments, one can ob-tain a straight line where the slope of the straight line isthe value for rMMA and the intercept of the line is the value

R 2 = 0.9870

2

4

6

8

10

0 1 2 3 4

F

G

(a) (

2.64, 0.25

0.1

0.2

0.3

0.4

2.51

r

r MM

A

(c)

Fig. 3. Reactivity ratio determinations by (a) Fineman–Ross method, (b) I

for rVCL. The obtained values of rMMA and rVCL by this meth-od were 2.63 and 0.19, respectively (Fig. 3(a)). Reactivitycan also be calculated through Inverted Fineman–Rossmethod by plotting graph of G/F versus 1/F for all experi-ments. Slope (rMMA) and intercept (rVCL) of the best fittedline obtained by using Eq. (3) were 2.68 and 0.39, respec-tively (Fig. 3(b)). The necessary calculated parameters aregiven in Table 2. The non-linear least-square procedureas outlined by Tidwell and Mortimer is considered to beone of the most accurate procedures for determination ofmonomer reactivity ratios. The method is a modificationof the curve fitting procedure so that sum of the squaresof the differences between the observed and computedpolymer composition is minimized. A brief description ofthe method consists, initial estimates of rMMA and rVCL. Aset of computations is performed yielding the sum of thesquares of the differences between the observed and com-puted polymer compositions. The summation is then min-imized by iteration, yielding reactivity ratio. By thismethod, the reactivity ratio values obtained for rMMA andrVCL were 2.63 and 0.25 (Fig. 3(c)). The product of rMMA

and rVCL decides the character of the obtained copolymer.

R2 = 0.85670

1

2

3

0 1 2 3 4

1/F

G/F

b)

5.54

vcl

nverted Fineman–Ross method and (c) Tidwell–Mortimer methods.

Table 2The calculated parameters needed for the determination of reactivity ratiosat <8% conversion.

(MMA:VCL) moleratio in feed

M P G F G/F 1/F

90:10 9 24 8.62 3.37 2.55 0.2980:20 4 11.5 3.65 1.39 2.62 0.7270:30 2.33 9 2.07 0.60 3.43 1.6560:40 1.5 4 1.12 0.56 2 1.7850:50 1 2.3 0.57 0.43 1.3 2.3340:60 0.66 2.2 0.36 0.29 1.2 4.19

M = MMMA/MVCL; P = PMMA/PVCL; G = M(P � 1)/P; F = M2/P.

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In the present study product of rMMA and rVCL was observedto be less than 1 indicating the tendency of copolymer tobe random in nature.

3.3. Particle size distribution and molecular weightdetermination

Particle size and morphology of the poly(MMA-co-VCL)nanoparticles was determined through dynamic light scat-tering and transmission electron microscopy. A represen-tative particle size histogram obtained through DLS for

0

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17 18 20 22 23 25

29 30 32 33 34

0

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size (nm)

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33 35 37 39 41 44

20 nm (A)

32 nm (C)

39 nm (E)

90:10

70:30

50:50

Fig. 4. A representative particle size histogram of poly(MMA-co-VCL) copolymeMMA-co-VCL, (D) (60:40) MMA-co-VCL, (E) (50:50) MMA-co-VCL, (F) (40:60) MM

90:10 to 40:60 poly(MMA-co-VCL) copolymers are givenin Fig. 4(A–E). The particle size was observed to be be-tween 20 and 45 nm for all copolymer compositions andthe data is compiled in Table 3. All the nanolatexes werestored at room temperature for a period of 6 months andits particle size was measured periodically in order to seethe stability of the latex synthesized (Table 4). Particle sizeshowed marginal increase with increasing incorporation ofVCL unit in copolymer. Up to 1 month storage, noagglomeration of nanoparticles was observed. Howeverpercentage increment in particle size after 6 month wasobserved to be 30 ± 10%. Transmission electron microscopywas used to examine size and morphology of copolymericnanoparticles. Fig. 5(a) and (b) show TEM images of 90:10and 50:50 poly(MMA-co-VCL) copolymeric nanoparticles,respectively. The particle size observed from TEM analysiswas approximately in the range of 30 ± 5 nm with spheri-cal morphology and it was supported by the results ob-tained from dynamic light scattering technique. Table 3summarizes the molecular weight and polydispersity ofthe copolymers synthesized. The molecular weight of thesynthesized copolymers was in the range of 5–20 � 104

and polydispersity was in the range of 1.3–1.6. It was ob-served that as the VCL content increases in the copolymer,

0

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size (nm)

Inte

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0

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18 21 23 26 29 33

28 31 34 37 40 44

size (nm)

Inte

nsit

y

0

20

40

60

80

100

19 25 32 42 55 71 93

size (nm)

Inte

nsit

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23 nm (B)

34 nm (D)

42 nm (F)

80:20

60:40

40:60

r systems (A) (90:10) MMA-co-VCL, (B) (80:20) MMA-co-VCL, (C) (70:30)A-co-VCL.

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Table 3Molecular weight, particle size and phase transition temperature of thecopolymer latex.

Copolymer(MMA:VCL)

Particlesize(nm)

Mw � 10�4 Mn � 10�4 PDI Phasetransitiontemperature(�C)By UV–visspectrometry

PMMA 18 78.3 66.5 1.17 –90:10 20 20.1 14.2 1.42 6280:20 23 14.1 10.5 1.33 5270:30 32 13.5 9.5 1.43 5260:40 34 9.1 6.7 1.35 4450:50 39 10.3 6.48 1.59 3840:60 42 5.46 3.56 1.53 36

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the peak molecular weight of the copolymer decreases andpolydispersity increases (Fig. 6).

3.4. Cloud point determination

The temperature dependant phase separation ofaqueous solutions of different compositions of poly-(MMA-co-VCL) copolymer was investigated by visiblespectrophotometry at 500 nm. Fig. 7 shows percentagetransmittance versus temperature plots for three poly-(MMA-co-VCL) compositions. Usually, a sample is trans-parent when its transmittance, is greater than 95% andbecomes totally opaque when its transmittance, is lessthan 75%. In the present work, the temperature corre-sponding to a 10% reduction in transmittance was usedas a measure of a cloud point [30]. The copolymer compo-

Table 4Time dependency of the hydrodynamic radius with respect to VCL content at 30 ±

Copolymer compositionpoly(MMA-co-VCL)

Particle size (nm)

0 Month 1 Month 2 Months

90:10 20 22 2180:20 23 20 2170:30 21 22 2560:40 31 31 3350:50 32 32 3340:60 42 42 45

Fig. 5. TEM images of (a) 90:10 poly(MMA-co-VCL) and

sitions and their corresponding phase separation tempera-ture are given in Table 3. It was reported earlier that, acloud point of poly(vinylcaprolactam) (PVCL) stronglydepends on the molecular weight and composition of thepolymer in case of copolymer [30]. Yin and Stover [26]have reported strong dependence of phase transitiontemperature on copolymer composition. While the depen-dence of phase transition temperature of the thermosensi-tive polymers on their molecular weight is a matter ofdispute. Lessard et al. [31] found that the phase separationtemperature of polyamide is independent of the molecularweight or the concentration. They argued that the coil-to-globule transition takes place solely depending on thetemperature of aqueous polymer solution at the initialstage of the phase separation, followed by the onset ofaggregation of individual chain molecules mainly due tothe intermolecular interaction between the hydrophobicgroups distributed on the surface of the resulting globularparticles of the polymer in aqueous solution. According toSchild and Tirrell [32,33], who studied the phase transitionof poly(N-isopropylacrylamide) PNIPAAM samples, anincrease in the phase separation temperature is to be ex-pected with decreasing molecular weight and they arguedthat at higher concentration, where the coil-to-globuletransition is followed by globular aggregation throughintermolecular interactions. They have argued that, molec-ular weight should have an influence on cloud tempera-ture, as the overlapping concentration is dependent onthe chain length. The decrease in the phase separationtemperature with increasing molecular weight has notbeen only established for polyNIPAAM but also for otherthermosensitive polymers such as poly(N,N0-diethylacryla-

2 �C.

3 Months 6 Months % Increment after 6 months

24 28 4024 28 2027 28 3835 37 2038 39 2043 54 30

(b) 50:50 poly(MMA-co-VCL) copolymer system.

Fig. 6. Effect of copolymer composition on molecular weight distribution by GPC.

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mide) PDEAAM. Apart from the molecular weightinfluence, the differences between phase transition tem-peratures may also arise from the concentration of poly-mer samples and/or the conditions i.e. heating rate,experimental technique used in the measurements. Idziaket al. [34] have observed that an increase in heating rate re-sults in a shift in phase separation temperature towardshigher side. While Boutris et al. [30] pointed out that atlow concentrations (below 0.5 wt%) the polymer particlesfail or are slow in aggregating to a size that can be detectedby the spectrophotometer.

In the present case, it was observed that, aqueous solu-tions of copolymers with more than 40 mol % VCL showthermal phase transition near to body temperature

0

25

50

75

100

30 40 50 60 70

Temperature ( oC)

% T

VCL-20VCL-40VCL-60

Fig. 7. Percentage transmittance at 500 nm of aqueous solution contain-ing a 0.1 mg/mL poly(MMA-co-VCL) solution at different temperature. (s)80:20 poly(MMA-co-VCL), (h) 60:40 poly(MMA-co-VCL), (4) 40:60poly(MMA-co-VCL).

MA

CRO

(<44 �C) at heating rate 0.5 �C/min and polymer concentra-tions 0.1 g L�1. Hence heating rate and polymer sampleconcentration are important factors in the cloud pointdetermination. As reported earlier difficulty in determina-tion of cloud point temperature at low concentration ofPNIPAM based samples, in the present case, such difficul-ties were not observed for the synthesized copolymernanoparticles.

3.5. In-vitro cytotoxicity

As a part of our ongoing research on the development ofnovel controlled release (CR) systems attempts are made tosynthesize various acrylate nanoparticles through emul-sion and microemulsion polymerization technique for theentrapment of model drugs like acriflavin, carbamazepineand lamotrigine, for the investigation of their release pat-tern [35–38]. Such polymeric nanoparticles are being usedfor encapsulation of drug, but very few attempts are madefor biological characterization of such systems. Polyacry-late nanoparticles are commonly used in biomedical appli-cations, but toxicity associated with these nanoparticleshas not been systematically examined. Similarly, evalua-tion of microcidal activity and cytotoxicity of surfactants,though thoroughly investigated, requires closer under-standing in terms of their utilization in nanoparticle baseddrug delivery. Hoffmann et al. [39] synthesized methylmethacrylate based copolymeric nanoparticles by free rad-ical polymerization and cytotoxicity of nanoparticles wasdetermined in three different cell cultures including hu-man foreskin fibroblast and two kidney cell lines MA-104and Vero. They have reported IC50 values for nanoparticlesin the range of 27.2–500 lg/mL for above studied cell lines.Similarly Garay-Jimenez et al. and Abeylath et al. [40,41]synthesized various polyacrylate based antibiotic formula-tions for life-threatening bacterial infections and cytotox-

Fig. 8. Cytotoxicity profiles of PMMA and poly(MMA-co-VCL) nanoparticles with different composition (75:25, 50:50, 25:75) and SDS as a control, tested inB16F10 cell lines.

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icity of nanoparticles emulsion was evaluated using hu-man dermal fibroblast. They have reported the importanceof various purification processes and have observed theformulation with more than 3 wt% of SDS remained cyto-toxic to human keratinocytes even after purifications.Hence the concentration of surfactant used for preparingcopolymeric nanoparticles plays an important role, notonly in stabilizing the final latex but also for controllingthe particle size. Indeed, it has been observed before thatthe toxicity of surfactants, including SDS in particular, de-pends on weather they are unassociated (in bulk) or boundto the surface of nanoparticles. This agrees with a prior re-port that SDS in aqueous medium is cytotoxic, but SDSassociated with a matrix such as polymeric nanoparticlesis not [42]. This led us to consider the possibility that anyunassociated SDS present in the emulsion could perhapsbe removed after the formation of nanoparticles, so as todecrease cytotoxic effect without changing the morpholog-ical features of the nanolatex. Thus our focus is to removeSDS and other contaminants from the microemulsion andevaluate the nanoemulsion for primary cell-viability stud-ies of B16F10 melanoma cell lines. For purifications, nano-latex was stored at 16 �C (Kraft temperature of SDS) for aperiod of week in order to remove all the extra surfactant.The process was repeated till no further precipitation wasobserved. Polymeric nanolatex was further purified by ul-tra filtration unit (PALL, Mumbai, India) using omega mem-brane OAD65C12 minimate tangential flow filtrationcapsule (MWCO 650 Da) at room temperature (30 ± 2 �C).This step removes unassociated surfactant and other con-taminants further. The possible effect of the polymeric

nanolatex on B16F10 melanoma cell lines, which ulti-mately is a decisive factor in the use of nanoparticles fordrug delivery system, was examined. In-vitro cytotoxicitystudies were conducted using a series of copolymericnanoparticles against B16F10 melanoma cell lines. Fig. 8shows the cytotoxicity profile of the different compositionsof polymeric nanolatexes and the control SDS with a con-centration range of 0.00001–0.5 mg/mL for 24 h. MTT-testsdemonstrated that an increase in polymer concentrationfrom 0.00001 to 0.01 mg/mL was not harmful for the sur-vival of cell. Interestingly, it was observed from the preli-minary data that, cytotoxicity of the nanoparticlesdecreases and cell-proliferation increase with respect toVCL content of the copolymer in the prescribed concentra-tion range. This result provides important insight in termsof the design and intended use of SDS stabilized polymericnanoparticles as a matrix for drug delivery system.

4. Conclusion

Thermally responsive copolymeric nanolatex of VCL andMMA were synthesized through a continuous gradualfeeding emulsion polymerization technique. The copoly-mers produced by the gradual feeding technique showedmore homogeneous composition. Monomer reactivity ra-tios were determined by Fineman–Ross, Inverted Fin-eman–Ross and Tidwell–Mortimer methods shows goodcorrelation between the linear and non-linear least squaremethods. It was observed that the studied monomer pairhas a tendency to form random copolymer in the chosen

S. Shah et al. / European Polymer Journal 46 (2010) 958–967 967

monomer feed ratios as the product of r1 and r2 is less than1. TEM and DLS analysis of nanolatex confirmed the forma-tion of well defined reasonably well mono-dispersed be-low 30 ± 10 nm particles with spherical morphology andlatex remains stable for a period of more than 6 month atroom temperature. Lower critical solution temperature(LCST) values of the copolymers are observed to vary withcopolymer composition and more than 40% VCL contentshows LCST near to body temperature. MTT-cytotoxicitytests demonstrated that an increase in polymer concentra-tion from 0.00001 to 0.01 mg/mL was not harmful for thecell survival in each composition.

Acknowledgements

The authors are thankful to GUJCOST (Gandhinagar,Gujarat) for the financial support. We appreciate the helpprovided by Dr. V.A. Kalamkar, Department of Statistics,The M.S. University of Baroda, Baroda and Dr. Paresh Sang-hvi, Product technologist, Deltech Europe Ltd., UK, in thecomputational work.

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