Phase behavior, microstructure and cytotoxicity in mixtures of a charged triblock copolymer and an ionic surfactant

European Polymer Journal 75 (2016) 461–473

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

Macromolecular Nanotechnology

Phase behavior, microstructure and cytotoxicity in mixtures of a charged triblock copolymer and an ionic surfactant Bárbara Claro a,c, Kaizheng Zhu a, Shahla Bagherifam a,b, Sandra G. Silva c, Gareth Griffiths b, Kenneth D. Knudsen d, Eduardo F. Marques c, Bo Nyström a,⇑ a

Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway Department of Biosciences, University of Oslo, 0371 Oslo, Norway c Centro de Investigação em Química (CIQ-UP), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, P-4169-007 Porto, Portugal d Department of Physics, Institute for Energy Technology, P.O. Box 40, N-2027 Kjeller, Norway b

a r t i c l e

i n f o

Article history: Received 6 December 2015 Accepted 8 January 2016 Available online 9 January 2016 Keywords: Surfactant Amphiphilic block copolymer Micelles Phase behavior Drug delivery

a b s t r a c t In the present study, aqueous solutions of a thermo-responsive negatively charged triblock copolymer methoxy-poly(ethylene glycol)-block-poly(N-isopropylacrylamide)-block-poly (2-succinic acid-propyloxyl methacrylate) (MPEG45-b-PNIPAAM48-b-PSAPMA10), have been characterized in the presence of sodium dodecyl sulfate (SDS) or dodecyltrimethylammonium bromide (DTAB) surfactant, at a constant concentration of polymer and various levels of surfactant addition. For this purpose, dynamic light scattering (DLS) was used to probe the effect of the ionic surfactants on the size of the block copolymer species, and smallangle neutron scattering (SANS) was applied as a complementary technique to probe the structure on a mesoscopic length scale. The results obtained revealed that the addition of a surfactant to the copolymer solution leads to a decrease of the particle size, due to electrostatic repulsions and solubilization of the hydrophobic microdomains. By zeta potential analysis it was shown that the charge density of the surfactant-coated polymer moieties increases with increasing surfactant concentration. The turbidities of the polymer– surfactant mixtures were measured using a cloud point analyzer. Our data revealed that the behavior not only depends on the surfactant concentration, but it is also affected in some cases by temperature. In addition, cytotoxicity studies were carried out on mouse fibroblasts cells NIH–3T3 to evaluate the potential of the systems as drug delivery carriers. Results showed that the cytotoxicity of the polymer changes with surfactant addition, rising as the concentration of SDS increases but falling off with increasing DTAB concentration. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Block copolymers have been widely studied in recent years due to their ability to self-assemble into nanostructures in aqueous solution, and for their potential in applications such as enhanced oil recovery, targeted drug delivery and sensor design [1–4]. Given the functional versatility of block polymers, the main focus in the present work has been to develop

⇑ Corresponding author. E-mail address: [email protected] (B. Nyström). http://dx.doi.org/10.1016/j.eurpolymj.2016.01.018 0014-3057/Ó 2016 Elsevier Ltd. All rights reserved.

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systems based on this type of polymers with potential for drug delivery applications. For this purpose, it is necessary to find block copolymers capable of forming tunable and responsive aggregates that also possess high levels of biocompatibility. Poly(N-isopropylacrylamide) (PNIPAAM) is one of the most intensively studied temperature-responsive polymers [5]. In aqueous solution, it exhibits a low critical solution temperature (LCST), around 32 °C [6–8]. When the solution temperature is increased above the transition temperature, a coil to globule transition occurs, which is then followed by the creation of aggregates, and if the solution is not too dilute, macroscopic phase separation takes place [7]. To create nano-structures and load them with drugs, one usually takes advantage of their ability to form polymer micelles at elevated temperatures. By designing copolymers consisting of PNIPAAM and other blocks of different hydrophobicity, it is possible to tune the physical properties of the copolymer and the self-assembling features. There are several publications [9–13] dealing with the self-assembly of responsive amphiphilic copolymers and the formation of core–shell structures. However, there is a lack of studies that address the interplay between charged amphiphilic copolymers and ionic surfactants, especially a comparison of polymer–surfactant effects induced by anionic and cationic surfactants, and the subtle balance between electrostatic and hydrophobic interactions. The formed polymer–surfactant complexes are of great interest as potential drug and gene delivery vehicles [14,15]. They may be used as carriers of nucleic acids and other biological components into living cells for therapeutic purposes [16–20]. In this work, the interactions between methoxy-poly(ethylene glycol)-block-poly(N-isopropylacrylamide)-block-poly(2succinic acid-propyloxyl methacrylate) (MPEG45-b-PNIPAAM48-b-PSAPMA10), a temperature- and pH-responsive negatively charged triblock copolymer, and the ionic surfactants sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) were analyzed in aqueous solution by various experimental methods. Methoxy-poly(ethylene glycol) (MPEG) is a hydrophilic block and in combination with more hydrophobic blocks in the copolymer, the species may form core–shell structures, and in aqueous solutions this block frequently constitutes the corona that stabilizes the supramolecular structure [21–23]. Poly(2-succinic acid-propyloxyl methacrylate) (PSAPMA) is a hydrophobic block, completely insoluble in water, that deprotonates in aqueous solutions hence becoming negatively charged; this block provides the pH-sensitivity of the copolymer. The combination of the three blocks in this amphiphilic triblock copolymer (MPEG45-b-PNIPAAM48-bPSAPMA10) gives rise then to a dual responsive negatively charged polymer, which exhibits special interactions in the presence of an anionic surfactant (SDS) as well as with the cationic surfactant (DTAB). In this investigation, the polymer– surfactant interactions were probed by different experimental techniques at a constant concentration of polymer and various levels of surfactant addition. The principal objective of this work has been to study the interactions between ionic surfactants and a dual-responsive triblock copolymer to gain insight into how polymer, surfactant concentration, and temperature affect the size and structure of these complexes. Investigation of the properties of the copolymer–surfactant systems that can be modulated is of utmost importance as this type of systems can be used in drug delivery applications.

2. Experimental 2.1. Materials All the chemicals used for the synthesis of the triblock copolymer were purchased from Sigma–Aldrich and Fluka, and they were employed as received, unless mentioned otherwise. Sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) were purchased from Sigma–Aldrich and used as received. N-isopropylacrylamide (NIPAAM, Acros) was recrystallized from a toluene/n-hexane mixture and dried under vacuum before use. 2-hydroxypropyl methacrylate (HPMA) from Fluka comprises an isomeric mixture of 75% HPMA and 25% 2-hydroxyisopropyl methacrylate, but for clarity only the chemical structure of the major HPMA isomer is considered in this study. HPMA was purified to remove the trace inhibitor present in the sample by allowing the component to pass through a short basic Al2O3 column, prior to use. Triethylamine (TEA, Aldrich) was dried over anhydrous magnesium sulfate (Aldrich), filtered, distilled under N2 atmosphere and stored over 4 Å molecular sieves. Copper (I) chloride from Aldrich was washed with glacial acetic acid (Aldrich), followed 0000

by washing with methanol, diethyl ether, and then dried under vacuum and kept under N2 atmosphere. N,N,N0 ,N00 ,N000 ,N (hexamethyl triethylene tetramine) (Me6TREN) was synthesized according to a previous description [24]. The synthesis of the MPEG macroinitiator was performed in accordance with a reported procedure by the reaction of monomethoxycapped poly(ethylene glycol) (MPEG45-OH with an average number molecular weight of Mn = 2000 was provided by the manufacturer) with 2-bromoisobutyryl bromide in the presence of trimethylamine [25,26]. All water used in this study was purified with a Millipore Mill-Q system with the resistivity of 18 MX cm.

2.2. Synthesis and characterization of polymer The responsive copolymer used in this study was synthesized via a ‘one-pot’ two-step aqueous atom transfer radical polymerization procedure, followed by esterification with succinic anhydride of the hydroxylated precursor copolymer (Scheme 1) according to a modified reported procedure [27,28]. The chemical structure of this polymer and the approximate length of the three blocks were ascertained by the 1H NMR spectra (Fig. 1). The 1H chemical shifts in CD3OD are referred to

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Scheme 1. Synthetic route for the preparation of the MPEG-b-PNIPAAM-b-PSPMA triblock copolymer via an aqueous ATRP procedure.

the tetramethylsilane (TMS) signal. The values of the m, n and o indexes in MPEGm-b-PNIPAAMn-b-PHPMAo were evaluated by comparing the typical peak of the integral area of the end-capped methoxylproron peak (1) of MPEG (d = 3.30 ppm, –OCH3, Ia), the characteristic peak of NIPAAM (5) (d = 2.1 ppm, –CH2CH–, Ib), and of the methyl group of HPMA monomer (8, 80 ) (d = 1.5–1.75 ppm, –CH2–C3, Ic) based on a simple equation: n(NIPAAM) = 3(Ia/Ib); o(HPMA) = (2Ia/3Ic). The composition of the triblock copolymer is estimated to be: m/n/o = 45/48/10, i.e., MPEG45-b-PNIPAAM48-b-PHPMA10. It should be mentioned that the appearance of the new peaks (13, 130 ) in d = 2.5 ppm, was assigned to the succinic acid group and confirmed the successful synthesis of the esterified product.

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Fig. 1. 1H NMR spectra recorded for MPEG-b-PNIPAAM-b-PHPMA (bottom, hydroxylated precursor) and MPEG-b-PNIPAAM-b-PSPMA (top) in CD3OD-d4 (600 MHz, 25 °C), ⁄ and ⁄⁄ indicate residual NMR solvent (CH3OD and HDO).

2.3. GPC measurement GPC was performed on a Tosoh EcoSEC dual detection (RI and UV) GPC system coupled to an external Wyatt Technologies miniDAWN TREOS multi-angle static light scatting (MALS) detector. Samples were run in 0.02 M LiBr in DMF at a flow rate of 0.3 mL/min at 35 °C. The column set was one Tosoh TSKgel SuperH4000 column (6⁄150 mm). Refractive index increment values (dn/dc) were calculated online assuming 100% mass recovery (RI as the concentration detector) using the Astra 6 software package (Wyatt Technologies) by selecting the entire trace from the analyze peak to the onset of the solvent peak or flow marker. Absolute molecular weights and molecular weight distribution were calculated using the Astra software package. The polymer concentration was 5.0 mg/mL. The final esterified triblock copolymer (MPEG-b-PNIPAAM-b-PSPMA) has a fairly narrow molecular weight distribution (see Fig. 2) with a polydispersity index of Mw/Mn = 1.1.

Fig. 2. GPC measurement of MPEG45-b-PNIPAAM48-b-PSPMA10 in 0.02 M LiBr in DMF at 35 °C at a flow rate of 0.3 mL/min.

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2.4. Preparation of polymer–surfactant mixtures A stock polymer solution was prepared in Ultra-pure water from Milli-Q system and the solution was allowed to stir for more than 24 h in an ice bath to ensure a homogeneous solution and it was kept at 5 °C to avoid formation of aggregates. Stock solutions for both surfactants were prepared and stored at 25 °C. Aqueous solutions of 0.25 wt% of polymer in the presence of different levels of surfactant addition were prepared and stored at 25 °C. 2.5. Dynamic light scattering (DLS) DLS measurements were performed to determine the size of the polymer/surfactant species. The experiments were conducted using an ALV/CGS-8F multi-detector compact goniometer system with eight off fiber-optical detection units, made by ALV-GmbH, Langen, Germany. The laser light (He–Ne, k = 632.5 nm) was focused on the sample cell (10 mm NMR tube) and the intensity of scattered light was measured simultaneously at eight scattering angles in a range of 22–141°. The temperature in the measuring cell is controlled to within ±0.01 °C with a heating/cooling circulator circulating water around a cylindrical quartz container filled with a refractive index-matching liquid (cis-decalin). The copolymer solutions were filtered in an atmosphere of filtered air through a 5 lm filter (Millipore) directly into precleaned NMR tubes. In this study, the experimentally recorded intensity autocorrelation function g(2)(t), measured for different solutions, is directly related to the theoretically amenable first-order electric field correlation function g(1)(t), through the Siegert relation [29] g(2)(t) = 1 + B|g(1) (t)|2, where B (61) is usually treated as an empirical factor and the wave vector q is defined by q = (4pn/ k) sin(h/2), where k is the wavelength of the incident light in a vacuum, h is the scattering angle, and n is the refractive index of the medium [30]. In the analysis of correlation functions of copolymer/surfactant systems, it was found that the decays were described by a single exponential followed at longer times by a stretched exponential [31]:

"
 # b t gð1Þ ðtÞ ¼ exp 

sse

ss ¼

sse b

C


 1 b

ð1Þ

ð2Þ

where C is the gamma function of b1. The stretched exponent b (0 < b61) is a measure of the width of the distribution of relaxation times and sse is some effective relaxation time. The Stokes–Einstein relationship can be used to calculate the hydrodynamic radius from the relaxation modes: Rh = (kBT/6pgD) where kB is Boltzmann’s constant, T is the absolute temperature, and g is the viscosity of the water-surfactant medium [22]. The mutual diffusion coefficient D can be expressed as s1 = Dq2 [31–33]. 2.6. Small angle neutron scattering (SANS) The SANS-instrument at the JEEP-II reactor at Kjeller, Norway was employed for the measurements. The neutron detector was a 128  128 pixel, 3He-filled RISØ type, mounted on rails inside the detector chamber. The detector distance was 1.0 and 3.4 m, and the investigated scattering vector q-range was 8  103 6 q 6 0.3 Å1. In all the SANS measurements, deuterium oxide (D2O) was used as a solvent instead of H2O in order to obtain good contrast and low background for the neutronscattering experiments. Due to the low concentration (0.25 wt%), 5 mm cuvettes were used for all samples. All the measurements were performed at 25 °C. To ensure good thermal contact, the measuring cells were placed onto a copper-base and mounted onto the temperaturecontrolled sample stage. The detector chamber was evacuated to reduce the scattering caused from air. Standard reductions of the scattering data, including transmission corrections, were done by including data collected from empty cell, beam without cell, and blocked-beam background. The data were converted to an absolute scale (coherent differential cross section dR/ dX) via normalization based on direct beam measurements. 2.7. Zeta-potential The polymer–surfactant solutions prepared with different levels of surfactant addition were analyzed by Laser Doppler Micro-electrophoresis with a Zeta-sizer Nano ZS instrument, MAL1049741 (Malvern instruments Ltd., United Kingdom), at the temperatures of 25, 32, and 45 °C. A ‘‘dip” cell was used as sample cell, including palladium electrodes with 2 mm spacing, and disposable cuvettes. One milliliter of the sample was transferred to a disposable cuvette and depending on the temperature of each measurement the time of thermal equilibrium was 2 min at 25 °C, 5 min at 32 °C and 10 min at 45 °C. Five measurements were done for each sample and the values of the electrophoretic mobility were converted into zeta-potential values. To check the instrument calibration, a PS-latex standard suspension with 68 mV zeta-potential was first measured.

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2.8. Turbidimetry A NK60-CPA cloud point analyzer from Phase Technology, Richmond, B.C., Canada, was used to analyze the turbidity and determining the cloud point (CP) of the solutions. This instrument uses a scanning diffuse light scattering technique in order to characterize the phase changes of the sample with high sensitivity and accuracy. A light beam is applied with the peak wavelength of the employed AlGaAs light source at 654 nm and with an 18 nm spectral half-width in the measuring sample [9]. Directly above the sample, there is an optical system with light-scattering detectors that monitors the scattered intensity signal (S) of the sample while it is subjected to prescribed temperature alterations [9,34]. The measured signal S from the cloud point analyzer can empirically be related to the turbidity s which was determined from measurements of the transmittance on a standard spectrophotometer in a 1 cm cuvette, using the empirical expression s (1/L)ln(It/I0), where L is the path length through which the light passes, It is the transmitted light intensity, and I0 is the incident light intensity [9,34]. A direct relationship between the calculated turbidity from the spectrophotometer experiments and the signal from the cloud point analyzer is given by the equation [22] s (cm1) = 9.0  109S3.751 The heating rate was set to 0.2 °C/min between 20 °C and 50 °C and all the data from the cloud point analyzer is reported in terms of turbidity in the present work. 2.9. Cytotoxicity To explore the biocompatibility of the synthesized copolymer and copolymer–surfactant solutions, they were subjected to in vitro cytotoxicity testing on NIH/3T3 as a standard fibroblast cell line. A colorimetric assay was used to evaluate cell viability after treating the cells with copolymer, surfactant and polymer–surfactant solution at different concentrations. NIH/3T3 cells were purchased from ATCC (American type culture collection) and cultured in T75 cell culture flasks in Dulbecco’s modified Eagle’s cell culture medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Sigma Aldrich). The monolayer was then trypsinized and the cells were seeded in 96-well plates at a density of 5  103 cells/well (100 lL/well) in the culture medium. Following 24-h incubation and attachment, the cells were treated with blank surfactants (surfactant solutions without triblock copolymer), blank triblock copolymer (triblock copolymer solution without surfactant) and triblock-copolymer solutions with surfactants. Treated cells were incubated for more than 24 h and cell viability was evaluated using WST-8 (water-soluble tetrazolium salt) based cell counting kit (Sigma Aldrich) (CCK8). Water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. 10 lL of CCK-8 was added to each well and incubated for 2 h. The absorbance of soluble formazan was measured by a plate reader at 460 nm (Synergy Mx Monochromator-based Multi-mode Microplate Reader, BioTek Instruments, Vermont, USA). The experiment was done four times for each group and relative cell viability was calculated as below:

 Relative cell viability ð%Þ ¼

 Asample  Aref  100 Acontrol  Aref

ð3Þ

where Asample is the absorbance obtained from the wells containing the treated cells, Aref the absorbance of blank medium (100 lL medium + 100 lL MilliQ + 20 lL MTS) and Acontrol the absorbance of control cells (untreated cells). 3. Results and discussion 3.1. Turbidimetry Turbidity measurements were carried out on aqueous solutions of the temperature-sensitive MPEG45-b-PNIPAAM48-bPSAPMA10 block copolymer to monitor major temperature-induced changes of the thermodynamic conditions at different polymer concentrations. This was done both in presence and absence of ionic surfactants, to reveal formation of aggregates and possible macroscopic phase separation. Fig. 3 depicts the temperature-induced turbidity change of the copolymer at different polymer concentrations in the absence of surfactant. The general trend is that the turbidity increases with increasing temperature, with this trend becoming stronger as the polymer concentration rises. This finding indicates that elevated temperature and high polymer concentration favor the growth of large aggregates. As the temperature increases, the PNIPAAM block becomes stickier and the sticking probability increases, thereby promoting aggregation. Higher polymer concentrations will lead to shorter average distance between the moieties and higher collision frequency and this facilitates the aggregation process. The minimum concentration measured was 0.1 wt%, since for lower concentrations no turbidity alteration is observed in the considered temperature interval. The cloud point (CP) is taken at the temperature at which the first deviation from the baseline of the scattered intensity takes place. Upon increase of the copolymer concentration, the cloud point is shifted toward lower temperatures (see inset in Fig. 3). The trend can be ascribed to more intensive intermolecular associations at higher concentrations, as reported previously [22].

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Fig. 3. (a) Effects of concentration and temperature on the turbidity of aqueous solutions of MPEG45-b-PNIPAAM48-b-PSAPMA10 in the absence of surfactant. The inset shows effect of polymer concentration on the cloud point in the absence of surfactant. (b) Effect of surfactant concentration on the cloud point at a fixed polymer concentration of 0.25 wt%. The solid and dashed curves are guides for the eye.

Effects of DTAB and SDS addition on the cloud point temperature for a 0.25 wt% copolymer solution are displayed in Fig. 3b. The general trend is that addition of surfactant shifts the cloud point to higher temperatures. This is expected as a result of the gradual solubilization of the hydrophobic microdomains with increasing level of surfactant addition. At higher surfactant concentrations, the cloud point curve levels off, and this is ascribed to the saturation of the polymer with surfactant. It is interesting to note that in spite of the opposite sign of the charges for DTAB and the copolymer, the anionic SDS surfactant has the largest impact on the change of the value of CP. This suggests that the solubilization power of the hydrophobic patches is more efficient for SDS than for DTAB. Similar behavior has also been reported previously in the literature [35]. We further note that though SDS and polymer are both negatively charged, this surfactant can still bind to the more hydrophobic microdomains of the copolymer. Hence, the higher values of the cloud point are attributed to repulsive electrostatic interactions and solubilization of hydrophobic microdomains of the polymer; these effects are expected to suppress the aggregation of the hydrophobic chains. For the polymer–DTAB system, a different feature appears: in this case there is an abrupt rise of the cloud point at very low surfactant concentration, followed by a plateau-like region at higher surfactant concentrations. The rise of the cloud point is much smaller than for the polymer–SDS system, and the plateau domain suggests that the polymer is saturated with surfactant at fairly low DTAB concentration. These features indicate that the interaction of DTAB with the polymer is less efficient than for SDS, in spite of the fact that DTAB is oppositely charged compared to the polymer. However, we also note that the zeta-potential (cf. Fig. 5) of the species is low even at a high DTAB concentration (f  10 mV), whereas for the polymer–SDS system the maximum zeta-potential amounts to 55 mV. In the presence of DTAB, it seems that both the solubilization ability of the hydrophobic patches of the copolymer and the repulsive electrostatic interactions are weaker than in the presence of SDS. 3.2. Zeta-potential experiments The zeta-potential results for aqueous solutions of MPEG45-b-PNIPAAM48-b-PSAPMA10 of various polymer concentrations in the absence of surfactant at different temperatures are shown in Fig. 4. At a given low polymer concentration, the negative value of the zeta-potential decreases with increasing temperature; this feature is ascribed to the augmented aggregation taking place at elevated temperatures, because of the enhanced hydrophobicity of the PNIPAAM and PSAPMA blocks. At a fixed temperature, the zeta-potential assumes lower negative values with increasing polymer concentration, but the change of the zeta-potential is more accentuated at the highest temperature due to the combined effect of temperature and concentration on the aggregation state. Effects of DTAB and SDS addition on the zeta-potential in a 0.25 wt% aqueous solution of MPEG45-b-PNIPAAM48-bPSAPMA10 are illustrated in Fig. 5. At low DTAB concentrations, the negative sign of the zeta-potential is due to the charge of the polymer (the zeta-potential decreases from 30 mV to 4 mV in the presence of 0.5 mM DTAB), but at higher levels of DTAB addition the curve passes through the charge neutralization point (0 mV) and the sign of the zeta-potential becomes positive (Fig. 5a). It subsequently increases up to approximately 10 mV at 18 mM DTAB, and then the curve seems to flatten out. This behavior of the zeta-potential suggests that more surfactant is gradually bound to the polymer with a corresponding rise in surface charge of the particles, and at sufficiently high surfactant concentration the polymer is saturated with surfactant (plateau-like region of the zeta-potential curve) and surfactant micelles are formed in the bulk. Studies on the temperature dependence of MPEG45-b-PNIPAAM48-b-PSAPMA10/DTAB system were conducted; however no effect on zetapotential values was observed. Zeta-potential was also measured for 0.25 wt% MPEG45-b-PNIPAAM48-b-PSAPMA10 in the presence of SDS at 25 °C (Fig. 5b). Since both the copolymer and the SDS are negatively charged, we expected that the negative zeta-potential would

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Fig. 4. Zeta-potential of MPEG45-b-PNIPAAM48-b-PSAPMA10 for different temperatures as a function of polymer concentration. The line is a visual guide for the eye.

Fig. 5. Zeta-potential of 0.25 wt% polymer: (a) MPEG45-b-PNIPAAM48-b-PSAPMA10/DTAB mixtures and (b) MPEG45-b-PNIPAAM48-b-PSAPMA10/SDS mixtures as a function of surfactant concentration at 25 °C. The inset plot shows the effect of temperature on the zeta potential.

increase upon binding of the negatively charged surfactant to the copolymer; this is evident from Fig. 5b. The charge density is seen to increase with increasing surfactant concentration up to approximately 15 mM, while at higher SDS concentrations a plateau-like region is reached as the polymer species are saturated with surfactant. Similar to DTAB, the increase of zeta-potential is a consequence of the adsorption of the negatively charged SDS onto the negatively charged copolymer chain, increasing the number of negative charges of the species present in the solution. In the plateau-like region at higher surfactant concentrations, the solution is saturated by surfactant micelles. These micelles will enter the copolymer chain and solubilize its hydrophobic microdomains. This plateau is a consequence of increased number of surfactant micelles presented in the copolymer solution, without interaction with the copolymer. A comparison of the zeta-potential results for copolymer solutions with SDS or DTAB discloses that at higher surfactant concentrations the surface charge of the particles assume significantly higher values in the presence of SDS. These trends seem to indicate that in the presence of DTAB, the binding of DTAB to the polymer is generated mainly through electrostatic generations, whereas with SDS the hydrophobic interactions should play an essential role for the high values of the zetapotential. The results reveal that the effect of surfactant concentration on the zeta-potential is significantly stronger at the highest temperature (45 °C). This can probably be ascribed to a higher degree of binding of SDS to the polymer, because an elevated temperature favors a higher degree of surfactant binding to the polymer since the PNIPAAM and PSAPMA blocks become more hydrophobic.

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To gain insight into the structure, dynamics, and size of the polymer species on a mesoscopic length scale, SANS experiments and DLS measurements have been carried out for these systems. 3.3. Small angle neutron scattering The SANS intensity profiles for 0.25 wt% of MPEG45-b-PNIPAAM48-b-PSAPMA10, in absence and presence of different dSDS concentrations in D2O, at a temperature of 25 °C, can be observed in Fig. 6 in a semilogarithmic representation. To obtain more detailed information about the polymer structure of the complexes formed in the presence of SDS, the scattering from MPEG45-b-PNIPAAM48-b-PSAPMA10 in the mixtures has been obtained using contrast-matching conditions, where deuterated sodium dodecyl sulfate (d-SDS) is added to the hydrogenated copolymer instead of SDS. By this procedure, the surfactant will not be visible. An upturn in the scattered intensity can be observed in the low q range. In the absence of d-SDS, this upturn is quite strong, signaling the presence of large species in the solution. The addition of 1 mM d-SDS gives a slightly stronger upturn than the sample without d-SDS. This effect can probably be ascribed to the formation of larger aggregates at this low surfactant concentration as indicated from the DLS experiments (see the inset in Fig. 7). With further increase of the d-SDS concentration, a clear and highly systematic reduction is observable in the scattered intensity at low q values. This indicates a gradual reduction in aggregate size, as confirmed by the DLS results shown below. The trend is very pronounced from 1 to 4 mM, but less above 8 mM (cmc of SDS). This may suggest that at higher levels of SDS addition, most of the hydrophobic microdomains of the polymer have already been solubilized and higher surfactant concentration will only lead to more micelles in the bulk. At 8 mM d-SDS and above, there are some weak signs of a correlation peak around q = 0.04 Å1. Although the alkyl chain of SDS is invisible (since it is deuterated), this peak occurs because the charged head group (OSO 3 ) in the micellar shell still gives a weak contribution. We therefore effectively see the contribution to the scattering signal from interacting shells of SDS micelles, with a specific interaction distance: 2p/q  p/0.04 Å1, or ca. 150 Å. SANS measurements for DTAB in polymer solution were not performed because d-DTAB was not commercially available. The inset of Fig. 6 shows the data for the 8 mM dSDS sample (expanded in the vertical direction) fitted to a core–shell sphere model [36], where the core is nearly invisible (since the SDS chain is deuterated). A Coulombic interaction term (Hayter–Penfold formalism) [37], was included to account for the charges of the SDS head groups. With this model, we obtain a core radius of 16 Å and a shell thickness of 5 Å, which is close to the expected values for pure SDS micelles. Furthermore, an effective charge of 9 (e) per micelle was found. The weak scattering pattern thus represents mainly the contribution from SDS-micelles (possibly with a small amount of polymer incorporated), but we also see clearly some excess scattering at the lowest q, which is due to SDS–polymer complexes larger than the SDS micelles. A similar fitting was also intended for the sample with 4 mM d-SDS (not shown), although the signal was very weak. In this case one does not expect individual SDS micelles (4 mM is below cmc for SDS), but rather mixed polymer/SDS-complexes, probably with the SDS decorating the surface. Here, larger sizes were obtained (approx. 20 Å radius), as expected if the polymer and SDS are mixed, but also there was additional low-q scattering due to the presence of some larger complexes.

Fig. 6. SANS intensity profile plotted versus the scattering vector q at 0.25 wt% of MPEG45-b-PNIPAAM48-b-PSAPMA10 with and without d-SDS, at 25 °C. The inset shows the sample with 8 mM d-SDS (expanded) fitted to a core–shell model.

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Fig. 7. Effect of DTAB on the hydrodynamic radius of the particles for 0.25 wt% MPEG45-b-PNIPAAM48-b-PSAPMA10 at different temperatures. The inset plot shows the concentration dependence of the hydrodynamic radius at 25 °C in the absence of surfactant. The lines are visual guides for the eye.

3.4. Dynamic light scattering In order to obtain more detailed information about the sizes (hydrodynamic radii, Rh) of the polymer–surfactant complexes formed in solution, DLS measurements were carried out at different polymer concentrations and various polymer– surfactant compositions, at 25 °C. To obtain the results of these measurements, a correlation function was fitted by means of Eq. (1) [9,38]. The hydrodynamic radius increases with the increment of polymer concentration as shown in the inset of Fig. 7. This feature indicates that the concentration-induced increase in size of the polymer moieties can be associated with an enhanced tendency of formation of larger aggregates as the polymer concentration increases. The DLS results of the copolymer support the findings from the previous characterization techniques. It is possible to relate the decrease of surface charge observed in the zeta-potential and the increase in turbidity with the growth of the particles size observed in DLS. Let us first consider the general trend of Rh when DTAB is added to solutions of MPEG45-b-PNIPAAM48-b-PSAPMA10 (see Fig. 7). A salient feature is the pronounced drop of Rh at quite low levels of DTAB addition, and at higher surfactant concentrations the hydrodynamic radius falls off slowly. This trend is compatible with the cloud point features observed for this system (Fig. 3). This suggests that the polymer–surfactant interaction is strong initially, but as the level of surfactant addition increases the binding effect of surfactant to the polymer is modest and saturation appears. The effect of temperature on Rh is also depicted in Fig. 7. It is obvious that even in the presence of a low amount of DTAB, the temperature effect of Rh has practically disappeared; this is probably due to the solubilization of hydrophobic patches in the copolymer and thereby the packing effect of hydrophobic moieties is reduced. The effect of SDS addition on the hydrodynamic radius in 0.25 wt% solutions of MPEG45-b-PNIPAAM48-b-PSAPMA10 at 25 °C is displayed in Fig. 8a. In this case, a maximum of Rh is observed at low surfactant concentration; this may indicate the formation of mixed polymer–surfactant micelles. This interpretation is consistent with the SANS observation that the strongest upturn of the scattered intensity is found in the presence of a low surfactant concentration and not for the solution without surfactant. At higher SDS concentration, a monotonous decrease of Rh is registered and this can probably be ascribed to solubilization of hydrophobic microdomains and break-up of hydrophobic associations. The influence of temperature on Rh in 0.25 wt% solution of MPEG45-b-PNIPAAM48-b-PSAPMA10 at different concentrations of SDS is shown in Fig. 8b. It is obvious that the decrease of Rh with increasing SDS concentration becomes much stronger as the temperature rises. A possible scenario to explain this finding is to realize that elevated temperatures promote an augmented density of hydrophobic segments in the PNIPAAM-based copolymer species, and this leads to a higher sensitivity to solubilization upon addition of SDS and disruption of association complexes. 3.5. Cytotoxicity Fig. 9a depicts the cell viability of fibroblast cells NIH–3T3 in the presence and absence of SDS in a 0.25 wt% solution of MPEG45-b-PNIPAAM48-b-PSAPMA10. The general tendency is a decrease of the cell viability with increasing SDS concentration.

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Fig. 8. (a) SDS concentration dependence of the hydrodynamic radius of the species formed in 0.25 wt% solution of MPEG45-b-PNIPAAM48-b-PSAPMA10 at 25 °C. (b) Influence of temperature on the hydrodynamic radius in copolymer solutions in the presence of different levels of SDS. The line is a visual guide for the eye.

Fig. 9. (a) Cell viability of NIH–3T3 in the presence of copolymer/SDS solution, after 24 h. The standard deviation in the graphs is less than 0.1%. (b) Cell viability of NIH–3T3 in the presence of copolymer/DTAB solution, after 24 h. The standard deviation in the graphs is less than 0.1%.

The 0.25 wt% blank copolymer solution showed a negligible cytotoxic effect (>83%), making this polymer a good candidate to be used as a nanocarrier for drug delivery applications. The results obtained show clearly that concentrations of SDS in the range between 0.25 and 8 mM induce a cytotoxic effect, thus an increased SDS concentration decreases the cell viability of the fibroblast cell line. However, in most cases the polymer–SDS mixtures resulted in higher viability than the corresponding pure SDS solutions. There is no doubt that by keeping the polymer concentration constant and increasing the level of SDS addition, presence of free SDS in the solution enhances the cytotoxicity. Fig. 9b shows the cell viability for the same cell line in the presence and in the absence of DTAB in 0.25 wt% solution of MPEG45-b-PNIPAAM48-b-PSAPMA10. The trend for this system is that – in contrast to what occurred with the addition of SDS – for a range between 2 mM and 12.5 mM of DTAB, the cell viability increases by increasing the DTAB concentration. This finding is rather unexpected, because it has previously been reported that positively charged nanoparticles are more inclined than uncharged or negatively charged ones to cause disruption of the negatively charged cell membrane [39–41]. The difference between the DTAB solution and DTAB/copolymer solution is not so large, except for 12.5 mM of DTAB. These differences may reflect variations in the cell membranes, differences in growth conditions or even the charge density, since it is positive in all systems. The increase of the cell viability may also be related to the size of the particles in the solution. In the case of the copolymer/DTAB system, the size of the polymer was found to decrease with increasing DTAB concentration. Similar results have been observed in previous studies [42]. Surfactant toxicity is dependent upon the ability of the surfactant to partition between the aqueous phase and the cell membrane and may also be dependent on its ability to subsequently cross the membrane and enter the cytoplasm, as seen in previous studies [43,44]. In this work, our findings show that the surfactants may interact with the cell membrane, depending on the chemical nature of their polar head, and the sizes of the aggregates.

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4. Conclusions In this study, we have shown how the self-assembly properties of a negatively charged thermo-sensitive copolymer (MPEG45-b-PNIPAAM48-b-PSAPMA10) can be tuned by adding sodium dodecyl sulfate (SDS) or dodecyltrimethylammonium bromide (DTAB) to the solution. Binding of an ionic surfactant to the polymer significantly increases the cloud point of the sample, and this effect is stronger in the presence of the anionic SDS than for DTAB. It is clear that SDS is more efficient to solubilize the hydrophobic microdomains than the oppositely charged DTAB. The zeta-potential results disclose that the surface charge of the particles is gradually increased as the level of surfactant addition rises. The binding of SDS to the polymer is more pronounced at elevated temperatures, probably because more hydrophobic microdomains are developed at higher temperatures. At moderate levels of d-SDS addition to the solution of the polymer, the SANS results reveal that the scattering profile can be portrayed by a core–shell model (we obtain a core radius of 16 Å and a shell thickness of 5 Å) and at d-SDS concentrations of 8 mM and above, there are some weak signs of a correlation peak around q = 0.04 Å1; this peak is ascribed to the charged head group (OSO 3 ) in the micellar shell that gives a weak contribution. As the d-SDS concentration rises, a clear and highly systematic reduction is observable in the scattered intensity at low q values. This indicates a gradual reduction in aggregate size, as confirmed by the DLS results. The DLS results demonstrate that the size of the polymer–surfactant complexes decrease upon addition of moderate amounts of surfactant. In the case of DTAB, an abrupt decrease of the hydrodynamic radius is found already at low surfactant concentration, whereas in the presence of SDS a more gradual drop of the hydrodynamic radius is observed over an extended surfactant concentration domain. This suggests that the copolymer species are saturated with DTAB at a lower concentration than with SDS. At elevated temperatures, the drop of the hydrodynamic radius upon addition of SDS is significantly stronger than at 25 °C. This finding is attributed to an enhanced solubilization power of the hydrophobic microdomains at higher temperature in the presence of SDS. The results on cytotoxicity on mouse fibroblasts cells NIH–3T3 disclosed poor cell viability in the presence of SDS, but in general the cell viability was high for the polymer/SDS complexes. In the case of DTAB, the cell viability was high both for the bare DTAB and the polymer/DTAB complexes. 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