European Polymer Journal 46 (2010) 958–967

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

Synthesis and characterization of thermo-responsive copolymeric nanoparticles 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, India Gude Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India

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b

a r t i c l e

i n f o

Article history: Received 9 April 2009 Received in revised form 22 December 2009 Accepted 9 January 2010 Available online 15 January 2010 Keywords: Nanoparticles Copolymer Microemulsion polymerization B16F10 melanoma cell lines Cytotoxicity

a b s t r a c t Copolymeric nanoparticles of methyl methacrylate (MMA) and N-vinylcaprolactam (VCL) were prepared through free radical polymerization using hydrogen peroxide and L-ascorbic acid as a redox initiator in o/w microemulsion containing sodium dodecyl sulphate (SDS). The copolymers were characterized by FTIR and gel permeation chromatography (GPC) and composition of copolymer was determined by 1H NMR spectroscopy. Reactivity ratio was determined by linear least square and non-linear least square methods. The morphology and particle size distribution of copolymer latexes was determined through transmission electron microscopy (TEM) and dynamic light scattering (DLS). Copolymers were of less than 50 nm size with spherical morphology and latexes were stable for more than 6 months. Phase transition temperature measured through UV–vis spectrometry, for the synthesized copolymer indicates their potential use in biosensors and targeted drug delivery system. Cytotoxicity of nanoparticles was determined by MTT assay on B16F10 melanoma cell lines. Cell viability data shows the IC50 values of copolymeric nanoparticles to be in the range of 0.01–0.1 mg/mL. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Recently, much attention has been focused on water soluble polymers, which respond to changes in pH, temperature, ionic strength and electric field [1,2]. Polymers responding to temperature change exhibit an abrupt change in volume at a phase transition temperature, which results in change, in specific volume of the macromolecules in solution, including a transition from a loose coiled structure to a more compact globule. This abrupt change in physical properties due to variation in temperature and pH can be monitored through balancing various attractive and repulsive forces between polymer backbone and functional

* Corresponding author. Tel./fax: +91 265 2795552. E-mail address: [email protected] (S. Devi). 1 Present address: Shah-Schulman Centre for Surface Science and Nanotechnology, Faculty of Technology, Dharmsinh Desai University, College Road, Nadiad 387001, Gujarat, India. 0014-3057/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2010.01.005

groups. When a repulsive force usually electrostatic in nature, overcomes an attractive force such as hydrogen bonding or hydrophobic interaction, sometimes the gel network swells, ‘‘discontinuously” leading to a volume transition. The variables that can trigger and shift the swelling and shrinking depend on the nature of intermolecular forces existing in the gel network [3]. Introduction of charged groups into a hydrophobic gel network either through hydrolysis or copolymerization with ionic comonomers can alter its swelling behavior in water or make it sensitive to the ionic strength and pH [4–7]. Among temperature sensitive polymers, poly(N-isopropylacrylamide) (PNIPAM) and its copolymers have been studied extensively [8]. Recently, poly(N-vinylcaprolactam) (PVCL), water soluble, biodegradable, temperature responsive polymer having lower critical solution temperature (LCST) near to body temperature (around 32 °C) has attracted much attention of researchers and technologist [9]. Even though PVCL has been commercially available

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

2. Experimental 2.1. Materials N-Vinylcaprolactam (98%, Sigma–Aldrich, Steinheim, Germany) and methyl methacrylate (Merck, Mumbai India) were distilled under vacuum and stored at 4 °C prior to 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 received. Double distilled deionised water (0.22 lm nylon filtered) was used throughout the experiments.

2.2. Cell cultures B16F10, a highly metastatic lung selected subline derived from C57/BL6 murine melanoma, was purchased from National Centre for Cell Science (NCCS), Pune, India. The cell line was maintained as a continuous culture in Iscove’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 streptomycin. Cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Media was replenished every third day. 2.3. Preparation of copolymeric nanoparticles Copolymeric nanoparticles of various compositions were prepared by o/w microemulsion polymerization technique. The ternary microemulsions comprising 6% w/ w MMA–VCL monomer mixture, with MMA:VCL weight ratios varying from 90:10 to 40:60, SDS (2% w/w) and water (92% w/w) were taken in three-neck reaction vessel equipped with a nitrogen inlet, thermometer, water condenser and magnetic stirrer. Typically, monomer mixture to surfactant ratio was kept constant at 3 for all recipes. The ternary microemulsion was deoxygenated by bubbling purified nitrogen for 15 min. Polymerization was initiated by redox initiator hydrogen peroxide and L-ascorbic acid at 40 ± 1 °C. MMA was added drop wise to maintain the monomer ratio in feed. The latex obtained after completion of the reaction was allowed to purify at Kraft temperature of surfactant to achieve surfactant free latex and then precipitated in 10-fold excess volume of diethyl ether. The copolymer was washed with cold water to remove unreacted monomer and homopolymer of VCL. The copolymer was further purified twice by dissolving the copolymer in small amount of tetrahydrofuran and reprecipitating in n-hexane. For the determination of reactivity ratio, copolymers were collected below 10% conversion and purified as described above. Monomer reactivities are being reported by using linear least square and non-linear least square methods. Attempts were made to find out the possible cytotoxicity effect of nanoparticles on B16F10 melanoma cell lines. 2.4. Characterization 2.4.1. Spectroscopic analysis FTIR spectrum of the copolymer was recorded on a Perkin-Elmer RX1 FTIR spectrophotometer (Massachusetts, USA) using 1-cm diameter KBr pellets. 1H NMR spectra were recorded using 400 MHz 1H NMR spectrometer (Bruker Specrospin Avance Ultra-shield, Germany) at room temperature (30 ± 2 °C). The spectra were obtained after accumulating 16 scans by using 1% sample in CDCl3. 2.4.2. Gel permeation chromatography Number average (M n ) and weight average (M w ) molecular weights of copolymers with different compositions were determined by using Perkin-Elmer Totalchrom Gel Permeation Chromatography instrument equipped with turbosec size exclusion software, PE-series 200 RI detector,

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for a long time from BASF (Baden Aniline and Soda Factory) the chemical company only a few studies related to its properties and applications has been reported so far. Much of the published work is concentrated on the PVCL based hydrogels as they have potential applications in controlled drug delivery system, separation science, and immobilization of enzymes and in oil-recovery technology [10–16]. For these applications colloidal systems can be equally attractive, which could be easily synthesized through free radical emulsion polymerizations. First time in 1986 Pelton and Chibante [17] reported synthesis of temperature-sensitive microgel from N-isopropylacrylamide (NIPAM) and N,N0 -methylene bisacrylamide (MBA). Thereafter, many research papers were published on copolymeric microgels based on NIPAM with other ionic monomers [18–21]. Recently, Laukkanen et al. have reported synthesis of thermosensitive PVCL microgel by emulsion polymerization using sodium dodecyl sulphate and potassium per sulphate as a surfactant and initiator, respectively [22]. Properties of the copolymer depend on the nature of the monomers and its composition, which depends upon the reactivity of the monomer in the given system. In the copolymerization of monomers with different reactivities, predominant incorporation of the more reactive monomer occurs within the copolymer chain even if its concentration in the feed is low. Okhapkin et al. [23] have reported 1:1 ratio of VCL to MMA in the copolymer synthesized at 9:1 feed ratio of VCL to MMA at less than 2% conversion. This is attributed to 20 times lower reactivity of VCL than MMA calculated with the help of linear least square and non-linear least square method for reactivity ratio determination. As a result more reactive monomer preferentially gets incorporated into the copolymer and the monomer ratio in the feed rapidly changes resulting into a significant drift in the copolymer composition with the conversion. To control the composition drift, Fedorov et al. [24] and Qiu and Sukhishvili [25] reported gradual continuous feeding concept for copolymerization of vinyl pyrrolidone and 4-vinylpyridine, and VCL and glycidyl methacrylate and they reported that the synthesized copolymers were not only with homogeneous composition but also retained their temperature sensitive character. Hence attempts have been made to synthesize thermoresponsive poly(MMA-co-VCL) nanoparticles through continuous feed o/w microemulsion polymerization technique.

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series 200 isocratic pump and rheodyne injector. The mixed column PLGel 5l, suitable for molecular weights up to 104–107, in polar solvent was used. Distilled, degassed THF (Merck, India, HPLC Grade) at flow rate of 1 mL/min was used as an eluent. Medium molecular weight polystyrene standards (POLYSCI, 1 mg/mL in THF, with molecular weight 1  103–3  105) were used for calibration of GPC.

were dissolved in 100 ll of DMSO; the optical density was measured in the enzyme-linked immunosorbent assay plate reader (Molecular Devices, Spectra Max 190 with Soft max Pro) at 540 nm with a reference wavelength of 690 nm. 3. Results and discussion

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3.1. FTIR analysis 2.4.3. Dynamic light scattering A Brookhaven’s 90 plus dynamic light scattering equipment with a solid state laser source operated at 688 nm was used to measure the particle size and size distribution of the polymerized latex in a dynamic mode. The particle size was obtained from the Stokes–Einstein relation D = KT /(3pgd), where d is the diameter of particles, D is the translational diffusion coefficient, K is the Boltzmann constant, T is the temperature and g is the viscosity of the medium. The scattering intensities from the samples were measured at 90° using photomultiplier tube. In order to minimize the inter-particle interactions, the analysis of the latex was done after 10 times dilution considering the refractive index and viscosity of water as that of latex. All the measurements were performed in triplicate. 2.4.4. Transmission electron microscopy TEM analysis of copolymeric nanoparticles was performed using CM 120 Philips transmission electron microscope (Tokyo, Japan) at accelerating voltage of 200 kV. One drop of latex was dispersed in 5 mL of water and was placed on the carbon coated copper grid. The grid was dried under IR lamp and the images of representative areas were captured at suitable magnifications. 2.4.5. Cloud point determination The cloud point of the copolymer solution in double distilled water (0.1 g L1) was obtained by spectrophotometric detection of the changes in turbidity at 500 nm using Perkin-Elmer lambda 35 UV–vis spectrophotometer (Massachusetts, USA). The water-jacketed sample and reference cell holders were coupled with a Julabo 5A circulating bath. Cloud point was considered as the temperature corresponding to a 10% reduction in the original transmittance of the solution [26]. 2.4.6. Cytotoxicity assay Cytotoxicity of the placebo copolymeric nanoparticles was evaluated using B16F10 melanoma cell lines. Cells were seeded in 96-well microplates at a density of 5  103 cells/well. Cells were allowed to grow and stabilize for 24 h. They were then treated with poly(MMA-co-VCL) nanoparticles for 24 h, in order to find their cytotoxic effect. The nanoparticles of different composition and concentrations of 0.00001 to 0.5 mg/mL were added directly to the medium in each well. On each plate, four wells were left untreated for their use as reference. Post treatment cell viability was determined colorimetrically by using 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reagent. MTT reagent (Sigma–Aldrich) was added to each well to make a final concentration of 1 mg/mL of media and incubated for 4 h at 37 °C. Formazan crystals

Representative FTIR spectra of purified (MMA-co-VCL) copolymers are presented in Fig. 1. The carbonyl stretching vibrations of the MMA and VCL units of the copolymer appear as strong absorption bands at 1732 and 1640 cm1, respectively. The copolymer being hydrogel in nature, the strong but broad band appearing at 3434 cm1 can be attributed to water of hydration. Yu et al. [27] have studied structural transformations and water association interactions in PVCL–water system by various methods such as IR-spectroscopy, quantum-chemical calculations, DSC and optical microscopy and have concluded that PVCL macromolecules in aqueous solution are the highly modified water associated structures, being affected by polarization action of highly polar amide groups due to specific configurational and conformational structures of the polymer. The bands appearing at 2993, 2949, 2857, 1443 and 1389 cm1 can be attributed to stretching and bending vibrations of –CH2 and –CH groups, respectively. Bands appearing at 1045, 1245 cm1 correspond to ester stretching vibration of acrylate polymer and bands observed around 2385 and 719 cm1 correspond to the –CN stretching vibrations and to the presence of more than three –CH2 groups in lactam ring structure providing evidence of copolymerization. 3.2. Determination of the reactivity ratio The high resolution 1H NMR spectra of poly(methyl methacrylate) (PMMA) homopolymer and poly(MMA-coVCL) copolymer are shown in Fig. 2. The composition of the copolymer was determined by the well separated signals that appeared at 4.798 ppm corresponding to the proton from a caprolactam ring of the VCL units and the signals that appeared near 0.842 ppm corresponding to the methyl proton of the MMA units [24]. The composition of the copolymer was evaluated from the relative intensities of –CH3 group of the MMA and >N–CH– group of the VCL units and the results obtained are summarized in Table 1. To determine reactivity ratios of MMA (rMMA) and VCL (rVCL), the widely used linear graphical Fineman–Ross method (FR) and Inverted Fineman–Ross (IFR) [28] and most accurate non-linear Tidwell–Mortimer (TM) least square method [29] were used and results are given in Fig. 3. The FR method is based on the following equation:

M ðP  1Þ P 2 M F¼ P

G¼

ð1Þ ð2Þ

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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for rVCL. The obtained values of rMMA and rVCL by this method were 2.63 and 0.19, respectively (Fig. 3(a)). Reactivity can also be calculated through Inverted Fineman–Ross method by plotting graph of G/F versus 1/F for all experiments. Slope (rMMA) and intercept (rVCL) of the best fitted line obtained by using Eq. (3) were 2.68 and 0.39, respectively (Fig. 3(b)). The necessary calculated parameters are given in Table 2. The non-linear least-square procedure as outlined by Tidwell and Mortimer is considered to be one of the most accurate procedures for determination of monomer reactivity ratios. The method is a modification of the curve fitting procedure so that sum of the squares of the differences between the observed and computed polymer composition is minimized. A brief description of the method consists, initial estimates of rMMA and rVCL. A set of computations is performed yielding the sum of the squares of the differences between the observed and computed polymer compositions. The summation is then minimized by iteration, yielding reactivity ratio. By this method, the reactivity ratio values obtained for rMMA and rVCL were 2.63 and 0.25 (Fig. 3(c)). The product of rMMA and rVCL decides the character of the obtained copolymer.

Table 1 Mole fraction of monomers in feed and copolymer. (MMA:VCL) mole ratio in feed

Mole fractions in feed MMMA

MVCL

PMMA

PVCL

90:10 80:20 70:30 60:40 50:50 40:60

0.9 0.8 0.7 0.6 0.5 0.4

0.1 0.2 0.3 0.4 0.5 0.6

0.96 0.92 0.9 0.8 0.7 0.69

0.04 0.08 0.1 0.2 0.3 0.31

% Conversion

Mole fraction in copolymer calculated from 1 H NMR

4.7 3.9 6.7 6.2 7.9 5.5

where M is the ratio of mole fraction of monomer in the feed and P is the ratio of mole fraction of monomer unit in copolymer. The linear relationship between G and F could be given as:

ð3Þ

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

(a)

(b) 3

10 8

2

G/F

G

6 4

1 2

R 2 = 0.987

R 2 = 0.8567

0 0

1

2

3

0

4

0

1

F

(c)

2

3

4

1/F

0.4

0.3

2.64, 0.25

r MMA

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G ¼ F r1 r2

0.2

0.1 1

2.5

4

5.5

r vcl Fig. 3. Reactivity ratio determinations by (a) Fineman–Ross method, (b) Inverted Fineman–Ross method and (c) Tidwell–Mortimer methods.

90:10 to 40:60 poly(MMA-co-VCL) copolymers are given in Fig. 4(A–E). The particle size was observed to be between 20 and 45 nm for all copolymer compositions and the data is compiled in Table 3. All the nanolatexes were stored at room temperature for a period of 6 months and its particle size was measured periodically in order to see the stability of the latex synthesized (Table 4). Particle size showed marginal increase with increasing incorporation of VCL unit in copolymer. Up to 1 month storage, no agglomeration of nanoparticles was observed. However percentage increment in particle size after 6 month was observed to be 30 ± 10%. Transmission electron microscopy was used to examine size and morphology of copolymeric nanoparticles. Fig. 5(a) and (b) show TEM images of 90:10 and 50:50 poly(MMA-co-VCL) copolymeric nanoparticles, respectively. The particle size observed from TEM analysis was approximately in the range of 30 ± 5 nm with spherical morphology and it was supported by the results obtained from dynamic light scattering technique. Table 3 summarizes the molecular weight and polydispersity of the copolymers synthesized. The molecular weight of the synthesized copolymers was in the range of 5–20  104 and polydispersity was in the range of 1.3–1.6. It was observed that as the VCL content increases in the copolymer,

Table 2 The calculated parameters needed for the determination of reactivity ratios at <8% conversion. (MMA:VCL) mole ratio in feed

M

P

G

F

G/F

1/F

90:10 80:20 70:30 60:40 50:50 40:60

9 4 2.33 1.5 1 0.66

24 11.5 9 4 2.3 2.2

8.62 3.65 2.07 1.12 0.57 0.36

3.37 1.39 0.60 0.56 0.43 0.29

2.55 2.62 3.43 2 1.3 1.2

0.29 0.72 1.65 1.78 2.33 4.19

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

In the present study product of rMMA and rVCL was observed to be less than 1 indicating the tendency of copolymer to be random in nature. 3.3. Particle size distribution and molecular weight determination Particle size and morphology of the poly(MMA-co-VCL) nanoparticles was determined through dynamic light scattering and transmission electron microscopy. A representative particle size histogram obtained through DLS for

(A)

100

90:10

20 nm

(B)

23 nm

Intensity

60 40

60 40 20

20

0

0 17

18

20

22

23

18

25

21

100

32 nm

70:30

(D) Intensity

Intensity

80 60 40

34 nm

100

33

60:40

60 40

0

29

30

32

33

28

34

31

50:50

39 nm

34

37

40

44

size (nm)

size (nm)

(F) 100

42 nm

40:60

80

Intensity

80

Intensity

29

80

0

100

26

20

20

(E)

23

size (nm)

size (nm)

(C)

80:20

80

80

Intensity

100

60 40

60 40 20

20 0

0 33

35

37

39

size (nm)

41

44

19

25

32

42

55

71

93

size (nm)

Fig. 4. A representative particle size histogram of poly(MMA-co-VCL) copolymer systems (A) (90:10) MMA-co-VCL, (B) (80:20) MMA-co-VCL, (C) (70:30) MMA-co-VCL, (D) (60:40) MMA-co-VCL, (E) (50:50) MMA-co-VCL, (F) (40:60) MMA-co-VCL.

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Table 3 Molecular weight, particle size and phase transition temperature of the copolymer latex. Copolymer Particle M w  104 Mn  104 PDI (MMA:VCL) size (nm)

Phase transition temperature (°C) By UV–vis spectrometry

PMMA 90:10 80:20 70:30 60:40 50:50 40:60

– 62 52 52 44 38 36

18 20 23 32 34 39 42

78.3 20.1 14.1 13.5 9.1 10.3 5.46

66.5 14.2 10.5 9.5 6.7 6.48 3.56

1.17 1.42 1.33 1.43 1.35 1.59 1.53

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

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3.4. Cloud point determination The temperature dependant phase separation of aqueous solutions of different compositions of poly(MMA-co-VCL) copolymer was investigated by visible spectrophotometry at 500 nm. Fig. 7 shows percentage transmittance versus temperature plots for three poly(MMA-co-VCL) compositions. Usually, a sample is transparent when its transmittance, is greater than 95% and becomes totally opaque when its transmittance, is less than 75%. In the present work, the temperature corresponding to a 10% reduction in transmittance was used as a measure of a cloud point [30]. The copolymer compo-

sitions and their corresponding phase separation temperature are given in Table 3. It was reported earlier that, a cloud point of poly(vinylcaprolactam) (PVCL) strongly depends on the molecular weight and composition of the polymer in case of copolymer [30]. Yin and Stover [26] have reported strong dependence of phase transition temperature on copolymer composition. While the dependence of phase transition temperature of the thermosensitive polymers on their molecular weight is a matter of dispute. Lessard et al. [31] found that the phase separation temperature of polyamide is independent of the molecular weight or the concentration. They argued that the coilto-globule transition takes place solely depending on the temperature of aqueous polymer solution at the initial stage of the phase separation, followed by the onset of aggregation of individual chain molecules mainly due to the intermolecular interaction between the hydrophobic groups distributed on the surface of the resulting globular particles of the polymer in aqueous solution. According to Schild and Tirrell [32,33], who studied the phase transition of poly(N-isopropylacrylamide) PNIPAAM samples, an increase in the phase separation temperature is to be expected with decreasing molecular weight and they argued that at higher concentration, where the coil-to-globule transition is followed by globular aggregation through intermolecular interactions. They have argued that, molecular weight should have an influence on cloud temperature, as the overlapping concentration is dependent on the chain length. The decrease in the phase separation temperature with increasing molecular weight has not been only established for polyNIPAAM but also for other thermosensitive polymers such as poly(N,N0 -diethylacryla-

Table 4 Time dependency of the hydrodynamic radius with respect to VCL content at 30 ± 2 °C. Copolymer composition poly(MMA-co-VCL)

Particle size (nm) 0 Month

1 Month

2 Months

3 Months

6 Months

% Increment after 6 months

90:10 80:20 70:30 60:40 50:50 40:60

20 23 21 31 32 42

22 20 22 31 32 42

21 21 25 33 33 45

24 24 27 35 38 43

28 28 28 37 39 54

40 20 38 20 20 30

Fig. 5. TEM images of (a) 90:10 poly(MMA-co-VCL) and (b) 50:50 poly(MMA-co-VCL) copolymer system.

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

mide) PDEAAM. Apart from the molecular weight influence, the differences between phase transition temperatures may also arise from the concentration of polymer samples and/or the conditions i.e. heating rate, experimental technique used in the measurements. Idziak et al. [34] have observed that an increase in heating rate results in a shift in phase separation temperature towards higher side. While Boutris et al. [30] pointed out that at low concentrations (below 0.5 wt%) the polymer particles fail or are slow in aggregating to a size that can be detected by the spectrophotometer. In the present case, it was observed that, aqueous solutions of copolymers with more than 40 mol % VCL show thermal phase transition near to body temperature

100

VCL-20 VCL-40 VCL-60

%T

75

50

25

0 30

40

50

60

70

o

Temperature ( C) Fig. 7. Percentage transmittance at 500 nm of aqueous solution containing 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) 40:60 poly(MMA-co-VCL).

(<44 °C) at heating rate 0.5 °C/min and polymer concentrations 0.1 g L1. Hence heating rate and polymer sample concentration are important factors in the cloud point determination. As reported earlier difficulty in determination of cloud point temperature at low concentration of PNIPAM based samples, in the present case, such difficulties were not observed for the synthesized copolymer nanoparticles. 3.5. In-vitro cytotoxicity As a part of our ongoing research on the development of novel controlled release (CR) systems attempts are made to synthesize various acrylate nanoparticles through emulsion and microemulsion polymerization technique for the entrapment of model drugs like acriflavin, carbamazepine and lamotrigine, for the investigation of their release pattern [35–38]. Such polymeric nanoparticles are being used for encapsulation of drug, but very few attempts are made for biological characterization of such systems. Polyacrylate nanoparticles are commonly used in biomedical applications, but toxicity associated with these nanoparticles has not been systematically examined. Similarly, evaluation of microcidal activity and cytotoxicity of surfactants, though thoroughly investigated, requires closer understanding in terms of their utilization in nanoparticle based drug delivery. Hoffmann et al. [39] synthesized methyl methacrylate based copolymeric nanoparticles by free radical polymerization and cytotoxicity of nanoparticles was determined in three different cell cultures including human foreskin fibroblast and two kidney cell lines MA-104 and Vero. They have reported IC50 values for nanoparticles in 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 formulations for life-threatening bacterial infections and cytotox-

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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 in B16F10 cell lines.

icity of nanoparticles emulsion was evaluated using human dermal fibroblast. They have reported the importance of various purification processes and have observed the formulation with more than 3 wt% of SDS remained cytotoxic to human keratinocytes even after purifications. Hence the concentration of surfactant used for preparing copolymeric nanoparticles plays an important role, not only in stabilizing the final latex but also for controlling the particle size. Indeed, it has been observed before that the toxicity of surfactants, including SDS in particular, depends on weather they are unassociated (in bulk) or bound to the surface of nanoparticles. This agrees with a prior report that SDS in aqueous medium is cytotoxic, but SDS associated with a matrix such as polymeric nanoparticles is not [42]. This led us to consider the possibility that any unassociated SDS present in the emulsion could perhaps be removed after the formation of nanoparticles, so as to decrease cytotoxic effect without changing the morphological features of the nanolatex. Thus our focus is to remove SDS and other contaminants from the microemulsion and evaluate the nanoemulsion for primary cell-viability studies of B16F10 melanoma cell lines. For purifications, nanolatex was stored at 16 °C (Kraft temperature of SDS) for a period of week in order to remove all the extra surfactant. The process was repeated till no further precipitation was observed. Polymeric nanolatex was further purified by ultra filtration unit (PALL, Mumbai, India) using omega membrane OAD65C12 minimate tangential flow filtration capsule (MWCO 650 Da) at room temperature (30 ± 2 °C). This step removes unassociated surfactant and other contaminants further. The possible effect of the polymeric

nanolatex on B16F10 melanoma cell lines, which ultimately is a decisive factor in the use of nanoparticles for drug delivery system, was examined. In-vitro cytotoxicity studies were conducted using a series of copolymeric nanoparticles against B16F10 melanoma cell lines. Fig. 8 shows the cytotoxicity profile of the different compositions of polymeric nanolatexes and the control SDS with a concentration range of 0.00001–0.5 mg/mL for 24 h. MTT-tests demonstrated that an increase in polymer concentration from 0.00001 to 0.01 mg/mL was not harmful for the survival of cell. Interestingly, it was observed from the preliminary data that, cytotoxicity of the nanoparticles decreases and cell-proliferation increase with respect to VCL content of the copolymer in the prescribed concentration range. This result provides important insight in terms of the design and intended use of SDS stabilized polymeric nanoparticles as a matrix for drug delivery system.

4. Conclusion Thermally responsive copolymeric nanolatex of VCL and MMA were synthesized through a continuous gradual feeding emulsion polymerization technique. The copolymers produced by the gradual feeding technique showed more homogeneous composition. Monomer reactivity ratios were determined by Fineman–Ross, Inverted Fineman–Ross and Tidwell–Mortimer methods shows good correlation between the linear and non-linear least square methods. It was observed that the studied monomer pair has a tendency to form random copolymer in the chosen

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