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Controlled Shape and Nucleation Switching of Interfacially Polymerizable Nanoassemblies by Methyl Substitution Mohammad-Ali Shahbazi,*,† Ermei Mak̈ ila,̈ ‡ Neha Shrestha,† Jarno Salonen,‡ Jouni Hirvonen,† and Hélder A. Santos*,† †

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland

‡

S Supporting Information *

ABSTRACT: Interfacial polymerization of uniform template-free nanostructures is very challenging since many factors play determinant roles in the final structure of the resulting nanoassemblies. Here, we present a single oxidative coupling method for the synthesis of different nanoshapes by addition or substitution of a methyl group on aniline monomers to freely alter the mechanism of monomer-to-polymer conversion. Well-defined nanotubes, nanohollows, and solid nanospheres are obtained from aniline, Nmethylaniline, and 2-methylaniline polymerizations, respectively. We found that the extent of hydrophobicity and protonation under mild acidic conditions determines the monomers’ arrangement in micelle or droplet form, reactivity, and nucleation mechanism. These can subsequently affect the final morphology through a fusion process to form tubular structures, external flux of monomers to form nanohollows, and intradroplet oxidation to form solid nanospheres. Altered biological responses, such as cytocompatibility, redox response, hemocompatibility, and cell proliferation, are also found to be dependent on the position of the methyl group in the nanostructures.

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monomer units attached in a head-to-tail manner7 can form onedimensional (e.g., nanorods, nanofibers, and nanotubes), twodimensional (e.g., nanoplates and nanobelts), or three-dimensional (e.g., nanohollows, nanospheres, and nanotubes) assemblies.23 While spherical and tubular nanohollows can be obtained by template-based methods and postsynthesis removal of the sacrificial core,24 we tried herein to develop a single facile self-assembly procedure in order to prepare both template-free hollow nanostructures and solid nanospheres with high particle monodispersity via altering the polymerization mechanism resulted from adding a methyl group to the structure of the aniline monomers during oxidation. In this case, we can avoid postsynthesis treatments to remove solid templates, minimize difficulties in retaining the original structure of the particles after template removal,25 reduce wide particle size distributions,26 and enhance the reproducibility of the nanoassemblies.

INTRODUCTION Controllable shape modification has proven to be a powerful tool for tailoring the properties of nanomaterials and optimizing their behavior. To this end, the synthesis of a variety of nanoshapes and understanding the growth mechanism have been established for some materials1,2 and are in progress for others, such as polymeric nanoconductives. Conductive polymers are of interest due to their low manufacturing costs, high thermal stability resulting from unique π-conjugated structures,3 stimuli-responsive properties,4 and shape dependency on the preparation parameters.5 These materials can exist in different oxidation states and respond to external stimuli by changing their conductivity, density, color, magnetic properties, morphology, hydrophilicity, and permeability, expanding the area of their practical applications from electronic devices (e.g., transistors, memory cells, and energy storage),6−8 nanolithography,9 membrane construction,10 antimicrobial activity,11 and catalysis12 to biomedical usage, such as tissue engineering (e.g., by tuning cell differentiation, cell growth, and cell adhesion),13,14 biosensors,15 neural probes,16 and drug delivery.17,18 The morphology of the conductive polymers is an important factor for controlling their practical properties in all of the abovementioned areas of research19 and can be affected by the experimental conditions of the polymerization process (e.g., pH, temperature, buffer solutions, and redox potential of the oxidant).20−22 These materials that comprise para-substituted © XXXX American Chemical Society

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RESULTS AND DISCUSSION The successful oxidative polymerization of the prepared nanostructures was evaluated by measuring the contact angles of the produced particles (Figure 1a,b). Typically, high contact angle values correspond to low hydrophilicity, and low contact Received: October 13, 2015 Revised: November 16, 2015

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DOI: 10.1021/acs.chemmater.5b03995 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials

Figure 1. Physicochemical characterization of the self-assembled nanostructures. Contact angle (a), schematically proposed mechanism of the wettability degree for the synthesized nanostructures (b), and FTIR band analysis of the tubular PA, PNMA nanohollows, and P2MA solid nanospheres (c). The presence of hydrogen and positive charge were responsible for the lower contact angle of PA. PNMA has less hydrogen and charge density on the surface that can reduce the hydrophilicity with a resulting increase in the contact angle. The contact angle of the P2MA was higher than PA because of the methyl groups available in the ortho-position of each monomer, and it was less than that of PNMA because of the charged nitrogen atoms and the presence of more hydrogen in the structure.

1040 cm−1 is associated with the aryl-S group, which may come from the hydrogen sulfate counterions or the partial sulfonation of the polymers during oxidation. In addition, the bands at 812 cm−1 are attributed to the out-of-plane C−H bending.29 FTIR band analysis of all of the nanostructures is shown in detail in Supporting Information Table S1. Figure 2a−c and Supporting Information Figure S1 show how chemical backbone alterations result in morphological changes of the aniline-derived polymers prepared with the same oxidation method. Well-defined regularly oriented nanotubes, nanohollows, and solid nanospheres were polymerized from aniline, N-methylaniline (NMA), and 2-methylaniline (2MA) monomers, respectively, via a similar method. The main aim was to overcome the problems associated with shape control in conductive polymeric structures because irregular structures are commonly observed after making small changes in the preparation parameters.32 The size distribution of aniline nanotubes was measured by scanning electron microscopy (SEM). Figure 1d shows that most of the polyaniline (PA) nanotubes have a length between 500 and 1300 nm. Poly(Nmethylaniline) (PNMA) nanohollows and poly(2-methylaniline) (P2MA) nanospheres showed average sizes of 571 ± 42 and 210 ± 17 nm, respectively (Figure 1e). Further analysis of the nanostructures revealed that, despite the positive zeta-potential of polymerized aniline (+10.9 ± 0.9 mV) and 2MA (+12.6 ± 1.1 mV), the P2MA nanohollows have negative zeta-potential (−18.8 ± 0.8 mV) due to the chemical structure of the polymers at different oxidation states (see Supporting Information Figures S2−S4). Thermogravimetric analysis (TGA) showed thermally stable structures up to 270 °C, and X-ray powder diffraction (XRPD) experiments revealed very low crystallinity as well as a change in the amorphous structure of the polymers after the

angle values are an indication of high hydrophilicity. Contact angles less than 90° for all of the polymers confirmed that the character of the monomers was switched from hydrophobic to hydrophilic polymers due to the protonation during the oxidation and the presence of free radicals in the polymer structure. While the contact angle of aniline-based nanotubes was the lowest, the PNMA nanohollows presented the highest value for the contact angle due to the methyl group attached to the amine groups in their constructive structure, reducing the hydrophilicity of the nanohollows after polymerization. The lower contact angle of P2MA compared to that of PNMA is attributed to the ortho-substituted methyl group in the aniline structure that allows the amine groups to be protonated more easily compared to that in PNMA. Since tubular morphology and surface roughness are other factors that may increase the contact angle values,27,28 we can conclude that the intrinsic hydrophilicity of PA and P2MA are even higher than that observed by the contact angle measurements, as tubular shape and surface roughness for aniline and surface roughness for P2MA were observed in the SEM imaging (Figure 2a−c). Attenuated total reflectance Fourier transform infrared (ATRFTIR) spectroscopy confirmed the oxidation and polymerization states of the polymers. In the spectrum of the polymers shown in Figure 2c, the bands around 1594 and 1499 cm−1 correspond to the CC stretching of quinoid and benzenoid rings,4 respectively, confirming the oxidation of the monomers. The overlapped wide band in the range of 1200−1330 cm−1 is ascribed to the C−N stretching of the benzene ring, the CN+ stretching in the polarized structure of the polymers, and the stretching of CN.29 The bands around 1140 cm−1 are assigned to electron delocalization due to oxidation and the in-plane bending of the aromatic C−H.30,31 The band located at around B


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Figure 2. Physicochemical characterization of self-assembled nanostructures. (a−c) SEM and transmission electron microscopy (TEM) images of PA (a), PNMA (b) and P2MA (c) synthesized in the presence of poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) (1.5%). Aniline and NMA monomers produced tubular and spherical nanohollows without using any template. Red arrow shows an empty space within PNMA. After oxidation, 2MA monomers produced solid spheres with a size significantly smaller than that of PNMA. Scale bars are 200 nm. SEM images with lower magnification and the chemical structures of monomers used for the synthesis of each particle are shown in Supporting Information Figure S1d,e. The respective size distribution of (d) PA (by measuring the size of 100 nanotubes imaged by TEM) and (e) PNMA and P2MA (measured by dynamic light scattering). (f, g) XRPD (f) and TGA (g) patterns of PA, PNMA, and P2MA prepared with a similar method. Details of the thermal and XRPD analysis are shown and explained in Supporting Information Figures S5−S7. (h) UV−vis spectra of the aniline, NMA, and 2MA (0.2 mol·L−1) after oxidation and polymerization initiated by ammonium persulfate (APS; 0.3 mol·L−1). Prior to the UV−vis study, the oxidized products were dried and then dissolved in dimethyl sulfoxide. Wavelengths of absorption peaks are given in nanometers.

Figure 3. Changes in the temperature and pH of the medium over the course of aniline (a), NMA (b), and 2MA (c) polymerization under the action of the oxidant, APS. The molar ratio of oxidant to monomers was 1.5 in all reaction media. It is evident that the methyl substitution induces a change in the monomer reactivity and modifies the exothermic profile, as well as the pH in the reaction media, synchronously. The sharper temperature increase corresponded to a more marked pH decline, suggesting that the thermal alterations were due to proton liberation and oxidation. For 2MA, the heat produced in the second exothermic phase is lower than that with aniline, indicating the presence of both neutral and ionized molecules in the reaction medium (less ionized molecules with 2MA compared to that with aniline). As for 2MA, the decrease of neutral molecules in the reaction medium and the protonated molecules being less prone to oxidation resulted in a considerably slower reaction between 20 and 70 min; see the slow decrease of the pH and constant temperature.

transition peak of the quinoid moieties around 520−750 nm.33 Such spectra indicate oxidation-based polymerization of the developed nanostructures. In order to understand the reason for the morphological differences of the nanostructures prepared with the same procedure, it is important to elucidate the correlation between methyl substitution and the monomer reactivity and formation

addition of the methyl group to the nitrogen atom or at the ortho position of aniline (explained in Supporting Information Figures S5 and S6). Other thermal characterization results are shown in the Supporting Information (Figure S7). To evaluate oxidation and subsequent protonation of the nanostructures, UV−vis spectroscopy was conducted (Figure 1h), showing a typical peak of a benzenoid ring at about 310−390 nm and the π−π* C


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Figure 4. Structure of monomers in aqueous solution, formation mechanism, successive shape evolution, and detailed schematic illustrating PA nanotube formation over the oxidation time. (a) Aniline monomers produced micelle-shaped assemblies with very small sizes due to their amphiphilic properties. (b) The numbers 1−6 show TEM images of the aniline monomers before adding APS and at 15, 30, 90, 180, and 960 min (16 h) after oxidation, respectively (scale bars are 500 nm). Additional TEM images are also shown in Supporting Information Figure S10. As depicted in the schematic (c), interfacial oligomerization and polymerization prevent most of the produced H+ from being released outside, increasing the density of protons residing within the micelles. Next, hydrogen bonding between the micelles helped them to fuse with each other and released the accumulated protons to the medium.

ionization potential. This explains the faster exothermic oxidation of neutral molecules compared to that for the protonated ones as a result of the presence of the electron pair on the nitrogen atoms. The formation mechanism of PA nanostructures has been previously explained by the homogeneous nucleation of ionized aniline monomers arranged in a semirod manner to produce nanotubes.31,34 We found that the monomers form micelles in under aqueous acidic conditions (Figure 4a,b 1) before ammonium persulfate (APS) addition due to the protonation and formation of amphiphilic anilinium structures consisting of hydrophobic −C6H5 and hydrophilic −NH3+ groups as the prevailing species. After the addition of APS to the solution, the chain propagation begins (Supporting Information Figure S9) and small spherical nanoparticles are produced in 15 min (Figure 4b2). At 30 min, most of the nanoparticles started to interconnect and form tubular structures as a result of the successive membrane fusion. After 90 min, no spherical micelles were observed and short length tubes were constructed (Figure 4b4). The tubes fused with each other further over time, becoming denser and forming longer structures of hundreds of nanometers to micrometers in length. The main driving force for the shape transition of PA from spherical to tubular is the interfacial polymerization and subsequent formation of efficient aggregated structures at the outermost layer that inhibits the release of liberated protons into the aqueous medium (Figure 4c). This results in enhanced charge density in the micelles and structural transitions with the assistance of hydrogen bonds between the heads of the nanodroplets.35,36 In general, the fusion of spherical vesicles during the process of polymerization has four successive steps: (1) contact of the micelles’ membranes (Figure 4b1,4b2), (2) center wall formation between micelles (Figures 4b1,4b2), (3) symmetric formation of fusion pores between the micelles (Figure 4b3), and (4) complete membrane fusion (Figure 4b4,b5). The one-step rapid exothermic trend observed and the high partition coefficient of NMA indicate that all of the molecules were neutral in the reaction medium and form relatively large

mechanism. Therefore, the temperature and pH of the reaction medium was measured over the course of polymerization. Figure 3 shows that monomer oxidation was exothermic in all of the samples, resulting in a decline in the pH of the reaction medium due to the protons released during the oxidation process of the monomers and the produced sulfuric acid during the reaction (Supporting Information Figure S8). For the oxidation of aniline and 2MA, the temperature profile showed two exothermic processes separated by an induction period, which is indicative of slow polymer chain growth. In both cases, the addition of the oxidizing agent resulted in a remarkable pH decrease from ∼4.5 to 3 due to the liberated protons and a corresponding increase in the temperature to ∼21 °C in the first step of polymerization. After that, the temperature stayed almost constant, and the pH decrease slowed. The next exothermic wave of the polymerization occurred after 30 and 70 min for aniline and 2MA, respectively, and was concomitant with an accelerated decrease in the pH from 1.7 to 1 in the reaction medium. A constant pH value at 1 indicates the consumption of all monomers during the reaction. Since neutral molecules polymerize faster than ionized monomers,31 the first and second separated steps of heat evolution correspond to the oxidation of neutral and protonated monomers, respectively, indicating a two-step nucleation in the reaction medium. The lower level of heat production in the second exothermic phase of 2MA compared to that of aniline confirms the lower degree of protonation of 2MA molecules compared to that of aniline due to the steric hindrance of the methyl group located in the ortho position. For NMA, a singlestage, rapidly occurring exothermic trend was observed, demonstrating a very high initial oxidation rate of neutral monomers with higher hydrophobicity (the measured partition coefficient values were 8.79 ± 0.24, 54.41 ± 1.33, and 24.89 ± 1.07 for aniline, NMA, and 2MA, respectively; the lower values for aniline and 2MA are due to the free amine group that can be protonated more easily, reducing the monomer’s hydrophobicity). NMA monomers are the most hydrophobic among the three due to the methyl substitution on the nitrogen atoms, making them neutral in the reaction medium with the lowest D


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Figure 5. Structure of the monomers in aqueous solution, formation mechanism, successive shape evolution, and detailed schematic representation of PNMA nanohollow formation over the oxidation time. (a) NMA monomers formed the largest droplets among the tested monomers in order to reduce the surface contact with the continuous aqueous phase due to their intrinsic high hydrophobicity. (b) The numbers 1−6 show TEM images of the NMA monomers before adding APS and at 15, 30, 90, 180, and 960 min (16 h) after oxidation, respectively (scale bars are 500 nm). Additional TEM images are shown in Supporting Information Figures S11 and S12. As depicted in the schematic (c), hole formation during oligomerization and water diffusion into the droplets push the hydrophobic monomers into the vicinity of the outer polymerized layer before they start to oxidize there due to the high amount of APS present. Over time and while the monomers are oxidized on the surface of the particles, they tend to integrate into the surface layer of the particles, where they will have less contact with the aqueous solution.

Figure 6. Structure of the monomers in aqueous solution, formation mechanism, successive shape evolution, and detailed schematic representation of P2MA solid nanosphere formation over the oxidation time. (a) 2MA monomers can have both amphiphilic and neutral molecules, resulting in their rearrangement to produce smaller nanodroplets compared to those with NMA, with ionized molecules assembled on the surface of the droplets. (b) The numbers 1−6 show TEM images of the 2MA monomers before adding APS and at 15, 30, 90, 180, and 960 min (16 h) after oxidation, respectively (scale bars are 200 nm). Additional TEM images are shown in Supporting Information Figure S13. (c) Interfacial oligomerization starts at the surface of the droplets and then continues inside the droplets by the penetration of APS within the droplets. Over time, the polymerization completes within the droplets, and the ionized molecules start to oxidize on the surface of the polymerized droplets.

eventually, nanohollow formation in the final step as the released monomers form a shell at the APS-rich outermost layer of the spheres (Figure 5b,c and Supporting Information Figures S11 and S12). In addition to the larger size of the initial droplet (Figure 5a and Supporting Information Figure S11), we hypothesize that the larger nanohollows compared to the solid P2MA nanospheres are formed due to three main factors: (i) the swelling occurring in the presence of high amounts of water penetrated into the initially formed hollows, (ii) the polymerization of released monomers on the surface of the nanohollows, and (iii) the heat released during oxidation that may facilitate the

nanodroplets to reduce the surface area in contact with the continuous aqueous phase due to the restricted miscibility in the aqueous solution (confirmed by TEM images and by the milky color of the nanoemulsion shown in Figure 5a). After the addition of APS to the monomers, the polymerization begins at the interface of the NMA/water nanodroplets, owing to the hydrophilicity of APS. As the monomer polymerization takes place in the outer surfaces of the NMA droplets and the water fluxes into the droplets, the hydrophobic monomers within the droplets tend to diffuse to the external polymerized surface, resulting in hole formation on the surface of the spheres25 and, E


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Figure 7. Redox-responsive behavior of PA, PNMA, and P2MA. SEM (a1, b1, c1) and TEM (a2, b2, c2) images of 100 μg·mL−1 of PA (a), PNMA (b), and P2MA (c), respectively, after 16 h of stirring in phosphate buffered saline (PBS) solution containing 10 mM GSH. Scale bars of SEM and TEM images are 1 μm and 500 nm, respectively. TEM images of particles at the same concentration in PBS at pH 7.4 without GSH are shown in Supporting Information Figure S15. Changes in the degree of oxidation of the polymers (d) were in agreement with the obtained imaging results as well as the observed changes in the color of the particles in suspension after dispersing them in PBS containing GSH for 16 h (e).

ogies were obtained by strictly obeying the experimental conditions during the preparation protocol. In all tested nanostructures, PMVEMA was used as a surfactant to make the droplets more stable and to facilitate the self-assembly of oligomers at the water/droplet interface during chain propagation. The interfacial polymerization of the nanostructures is demonstrated in Supporting Information Figure S14. Due to the recent increased interest in biomedical applications of aniline-based nanoparticles, we show herein how a simple change in the chemical structure of the monomers can affect the biological responses of the nanostructures. The redox bioresponsiveness of all of the prepared structures and the reversibility of the oxidation process were studied by visualizing the particles’ morphology and measuring the oxidation degree using ATRFTIR. SEM and TEM images of the particles (Figure 7a−c) showed no morphological changes for PNMA after reduction, whereas PA and P2MA exhibited collapsed structures due to the decreased rigidity and reorganization of the structure,37 indicating the redox responsiveness of PA and P2MA. The irreversible oxidation of PNMA is attributed to the involvement of all five electrons of the nitrogen atoms conjugated to the quinoid in the chemical conjugation in the oxidized state, whereas the nitrogen atoms of oxidized PA and P2MA have consumed four electrons for the chemical bonding to other surrounded atoms and they are more unstable than PNMA (Supporting Information Figures S3−S5). Before and after treating the nanoparticles with GSH (10 mM) for 16 h, the degree of oxidation was measured by dividing the absorbance peak of quinoid to the benzenoid4 obtained from the FTIR analysis. The highest value for the degree of oxidation is 100% for the fully oxidized pernigraniline structures (i.e., half of the polymerized monomers are alternatively quinoid), and the lowest value is 0 for the fully reduced leucoemeraldine state (there is no quinoid in the structure; Supporting Information Figures S2−S4). The degree of oxidation of the emeraldine state was 33.3%, which means that 1/4 of the polymerized monomers are quinoid.32 The results revealed that the degree of oxidation of

swelling of the droplets. The consecutive steps of PNMA formation (Figure 5c) include surface polymerization and hole formation on the surface shown with red arrows (Figure 5b2,b3), water penetration within droplets, monomer release to the surface of the particles shown with blue arrows (Figure 5b4,b5), and surface polymerization of monomers on the surface to cover the formed pores (Figure 5b6,c). As for 2MA (Figure 6a), the nanodroplets that formed are smaller than those of NMA in aqueous solution due to the lower hydrophobicity of the monomers. As can be observed from the TEM images (Figure 5b), it is not possible to follow the progress of the nanosphere synthesis due to the solid structure of the nanoparticles and their formation mechanism. The ionized and neutral molecules can coexist in the medium for 2MA due to the free amine groups. Hence, the neutral droplets can act as guiding templates for amphiphilic molecules to accumulate around them before they participate in polymer growth at the surface of the solid nanospheres. Since the electron pair on the nitrogen atom helps neutral molecules to polymerize earlier,31,34 by the addition of oxidant the heterogeneous nucleation initiates at the interface of the nanodroplets with water (Figure 6c). The surface polymerization of the droplet increases the hydrophilicity of the outer layer, enhancing the chance of both APS flux into the inner part of the droplet and polymerization of monomers inside the droplets since 2MA droplets are smaller than NMA due to their lower hydrophobicity. The pH decrease upon oxidation also facilitates facile protonation of the 2MA monomers inside the droplets, water attraction into the interior part, and subsequent polymer chain growth. Next, oxidation begins for the ionized monomers assembled around the droplets. In contrast to NMA monomers, 2MA monomers located within the droplet do not have a tendency to move outside the nanodroplets to form hollow structures due to their lower hydrophobicity. In general, as explained above, the oxidation of different monomers can produce different uniform structures over time. Moreover, despite the very high sensitivity of the nanostructures’ shapes to the polymerization conditions, reproducible morpholF


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Figure 8. Biological effects of different nanostructures. (a, b) Cytocompatibility results measured by an ATP-based luminescence assay showing that at a concentration of 160 μg·mL−1 none of the tested nanoparticles was cytocompatible with (a) MCF-7 and (b) MDA-MB-231 breast cancer cell lines (Supporting Information Figure S17). Values are expressed as mean ± SD of at least three independent experiments. Comparisons between the control and samples were analyzed by ANOVA, with levels of significance set at probabilities of *p < 0.05, **p < 0.01, or ***p < 0.001. (c−f) SEM images of RBCs after treatment with PBS at pH 7.4 used as control (c) and PBS containing 200 μg·mL−1 of PA (d), PNMA (e), and P2MA (f). PNMA showed the lowest interaction, and PA induced more changes in the RBCs’ morphology. P2MA had the highest rate of interaction with RBCs, but the change in morphology was less than that in the case of PA due to its spherical shape.

PA, PNMA, and P2MA decreased from 79.6 ± 0.8, 55.4 ± 0.4, and 82.8 ± 0.9 to 63.9 ± 1.7, 52.5 ± 0.5, and 49.1 ± 0.8, respectively (Figure 7d). These results represent the reduced degree of oxidation of PA and P2MA after they are treated with glutathione (GSH, 10 mM) for 16 h, whereas PNMA showed no remarkable redox responsiveness. Moreover, it can be concluded that, despite the fastest rate of oxidation being evident for NMA monomers due to their higher hydrophobicity and the presence of fewer ionized molecules in the reaction medium, the lower degree of oxidation of PNMA compared to that of the other two nanoassemblies is a result of the methyl group bound to the nitrogen atom. Photographs of changes in the color of the particles in suspension after dispersing them in PBS containing GSH are shown in Figure 7e. The cytocompatibility of polymerized aniline-based nanostructures was investigated in the MCF-7 and MDA-MB-231 breast cancer cell lines. Figure 8a,b shows high cytocompatibility values for the nanostructures at the concentrations tested up to 120 μg·mL−1 (except for P2MA, which shows ATP activity of ∼60% after 24 h at 120 μg·mL−1). At a concentration of 160 μg· mL−1, a considerable reduction in the cytocompatibility for all samples was observed. P2MA showed less cytocompatibility than PA and PNMA due to its positive charge, smaller particle size, and higher surface area compared to that of PNMA (16.5 ± 0.1 m2·g−1 vs 10.0 ± 0.1 m2·g−1 for PNMA and PA, respectively) and due to its lower hydrophilicity compared to that of PA. These results imply that the end-functional groups of the monomers play an important role in modulating the cytocompatibility of aniline-based nanoassemblies.

We also evaluated hemolysis and possible morphological changes of red blood cells (RBCs) induced by the aniline-based nanoderivatives. While the effect of the nanostructures on hemolysis was not concentration-dependent, a time-dependent reduction in hemocompatibility was observed (Supporting Information Figure S16), with the highest values being observed for P2MA. However, all of the concentrations tested presented low hemolysis values over 48 h. Because a low amount of hemolysis cannot guarantee the hemocompatibility of the tested materials,38 morphological changes were also investigated by SEM. The SEM images (Figures 8c−f) show that the cell membranes of RBCs were not ruptured after a 4 h incubation with the particles at 200 μg·mL−1. Nevertheless, the rate of nanoparticle localization onto the cell membrane and the change in the intact morphology of the RBCs were varied among the samples. PNMA had the lowest association with the cell membrane because of its negative surface charge, leading to excellent hemocompatibility. Compared to PA, P2MA showed more accumulation on the cell membrane due to its higher hydrophobicity; however, more changes in the normal structure of the RBCs were observed for the PA-treated samples due to its tubular structure. Cell proliferation results also showed that the number of cells in the medium decreased over time after exposure to 160 μg· mL−1 of the different particles (Figure 9a−c). By contrast, 5 μg· mL−1 of PA and PNMA stimulated cell proliferation, which was not the case for P2MA. These biological studies confirmed the importance of polymer chemistry as a crucial determinant for designing biocompatible nanostructures for biomedical applications. G



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METHODS

Methods and any associated references are available in the Supporting Information.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b03995. Detailed information on experimental measurements and procedures, including synthesis of aniline derived nanostructures, characterization of the nanostructures, TEM and SEM imaging, water contact angle measurements, thermal analyses, X-ray powder diffraction, oxidation speed and monomer reactivity, studies of the particle morphology over time, distribution coefficient of the monomers, bioreduction of the nanostructures, cell lines and culture conditions, ATP measurements, hemocompatibility studies, and cell proliferation studies, as well as Figures S1−S17 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(M.-A.S.) E-mail: m.a.shahbazi@helsinki.fi. *(H.A.S.) E-mail: helder.santos@helsinki.fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Santos thanks the financial support from the Academy of Finland (Grants 252215 and 281300), the University of Helsinki Research Funds, the Biocentrum Helsinki, and the European Research Council (FP/2007-2013, Grant 310892). The authors thank the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki, for providing all facilities for TEM and SEM imaging.

Figure 9. Effect of PA (a), PNMA (b), and P2MA (c) on MCF-7 cell proliferation over time at different concentrations. At a concentration of 5 μg·mL−1, PNMA had the highest stimulating effect on cell proliferation over time. The tested biological parameters showed more changes in the cell responses for P2MA due to its lower degree of hydrophilicity compared to that of PA and due to its positive zetapotential in contrast to the negatively charged PNMA nanostructures.

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REFERENCES

(1) Wang, Y.; Xie, S.; Liu, J.; Park, J.; Huang, C. Z.; Xia, Y. ShapeControlled Synthesis of Palladium Nanocrystals: A Mechanistic Understanding of the Evolution from Octahedrons to Tetrahedrons. Nano Lett. 2013, 13, 2276−2281. (2) Padmos, J. D.; Personick, M. L.; Tang, Q.; Duchesne, P. N.; Jiang, D. E.; Mirkin, C. A.; Zhang, P. The Surface Structure of Silver-Coated Gold Nanocrystals and Its Influence on Shape Control. Nat. Commun. 2015, 6, 7664. (3) MacDiarmid, A. G. ″Synthetic Metals″: A Novel Role for Organic Polymers (Nobel Lecture) Copyright((C)) the Nobel Foundation 2001. We Thank the Nobel Foundation, Stockholm, for Permission to Print This Lecture. Angew. Chem., Int. Ed. 2001, 40, 2581−2590. (4) Lv, L. P.; Zhao, Y.; Vilbrandt, N.; Gallei, M.; Vimalanandan, A.; Rohwerder, M.; Landfester, K.; Crespy, D. Redox Responsive Release of Hydrophobic Self-Healing Agents from Polyaniline Capsules. J. Am. Chem. Soc. 2013, 135, 14198−14205. (5) Zhang, X.; Manohar, S. K. Bulk Synthesis of Polypyrrole Nanofibers by a Seeding Approach. J. Am. Chem. Soc. 2004, 126, 12714−12715. (6) Zhao, H.; Wang, Z.; Lu, P.; Jiang, M.; Shi, F.; Song, X.; Zheng, Z.; Zhou, X.; Fu, Y.; Abdelbast, G.; et al. Toward Practical Application of Functional Conductive Polymer Binder for a High-Energy Lithium-Ion Battery Design. Nano Lett. 2014, 14, 6704−6710. (7) Li, D.; Huang, J.; Kaner, R. B. Polyaniline Nanofibers: A Unique Polymer Nanostructure for Versatile Applications. Acc. Chem. Res. 2009, 42, 135−145.

CONCLUSIONS

We discovered the feasibility of having structural control over polymerized assemblies by modifying the hydrophobicity and orientation of the monomers as a consequence of methyl substitution in aniline-based compounds. Our findings suggest that the addition of methyl groups to the amine group or the ortho position of the aniline can change the proportion of ionized and neutral molecules in the aqueous solution, subsequently leading to different nanostructures by different nucleation mechanisms. In addition, it is highlighted that switchable biological effects can appear as a consequence of modification in the end-functional group of the monomers and its subsequent effect on the morphology of the nanostructures. The present findings are a good starting point for understanding the correct growth mechanism of nanostructures prepared by an oxidationbased bottom-up approach and have implications that can be extended to cover shape control of other nanostructures prepared via monomer-to-polymer self-assembly and chain propagation. H


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Chemistry of Materials (8) Wu, H.; Yu, G.; Pan, L.; Liu, N.; McDowell, M. T.; Bao, Z.; Cui, Y. Stable Li-Ion Battery Anodes by in-Situ Polymerization of Conducting Hydrogel to Conformally Coat Silicon Nanoparticles. Nat. Commun. 2013, 4, 1943. (9) Luo, X.; Pedrosa, V. A.; Wang, J. Enzymatic Nanolithography of Polyaniline Nanopatterns by Using Peroxidase-Modified Atomic Force Microscopy Tips. Chem. - Eur. J. 2009, 15, 5191−5194. (10) McBee, T. W.; Wang, L.; Ge, C.; Beam, B. M.; Moore, A. L.; Gust, D.; Moore, T. A.; Armstrong, N. R.; Saavedra, S. S. Characterization of Proton Transport across a Waveguide-Supported Lipid Bilayer. J. Am. Chem. Soc. 2006, 128, 2184−2185. (11) Bogdanovic, U.; Vodnik, V.; Mitric, M.; Dimitrijevic, S.; Skapin, S. D.; Zunic, V.; Budimir, M.; Stoiljkovic, M. Nanomaterial with High Antimicrobial Efficacy–Copper/Polyaniline Nanocomposite. ACS Appl. Mater. Interfaces 2015, 7, 1955−1966. (12) Gallon, B. J.; Kojima, R. W.; Kaner, R. B.; Diaconescu, P. L. Palladium Nanoparticles Supported on Polyaniline Nanofibers as a Semi-Heterogeneous Catalyst in Water. Angew. Chem., Int. Ed. 2007, 46, 7251−7254. (13) Quigley, A. F.; Razal, J. M.; Kita, M.; Jalili, R.; Gelmi, A.; Penington, A.; Ovalle-Robles, R.; Baughman, R. H.; Clark, G. M.; Wallace, G. G.; et al. Electrical Stimulation of Myoblast Proliferation and Differentiation on Aligned Nanostructured Conductive Polymer Platforms. Adv. Healthcare Mater. 2012, 1, 801−808. (14) Stewart, E.; Kobayashi, N. R.; Higgins, M. J.; Quigley, A. F.; Jamali, S.; Moulton, S. E.; Kapsa, R. M.; Wallace, G. G.; Crook, J. M. Electrical Stimulation Using Conductive Polymer Polypyrrole Promotes Differentiation of Human Neural Stem Cells: A Biocompatible Platform for Translational Neural Tissue Engineering. Tissue Eng., Part C 2015, 21, 385−393. (15) Ruecha, N.; Rangkupan, R.; Rodthongkum, N.; Chailapakul, O. Novel Paper-Based Cholesterol Biosensor Using Graphene/Polyvinylpyrrolidone/Polyaniline Nanocomposite. Biosens. Bioelectron. 2014, 52, 13−19. (16) Abidian, M. R.; Corey, J. M.; Kipke, D. R.; Martin, D. C. Conducting-Polymer Nanotubes Improve Electrical Properties, Mechanical Adhesion, Neural Attachment, and Neurite Outgrowth of Neural Electrodes. Small 2010, 6, 421−429. (17) Abidian, M. R.; Kim, D. H.; Martin, D. C. Conducting-Polymer Nanotubes for Controlled Drug Release. Adv. Mater. 2006, 18, 405− 409. (18) Wang, C.; Xu, H.; Liang, C.; Liu, Y.; Li, Z.; Yang, G.; Cheng, L.; Li, Y.; Liu, Z. Iron Oxide @ Polypyrrole Nanoparticles as a Multifunctional Drug Carrier for Remotely Controlled Cancer Therapy with Synergistic Antitumor Effect. ACS Nano 2013, 7, 6782−6795. (19) Tran-Cong-Miyata, Q.; Nishigami, S.; Ito, T.; Komatsu, S.; Norisuye, T. Controlling the Morphology of Polymer Blends Using Periodic Irradiation. Nat. Mater. 2004, 3, 448−451. (20) Bláha, M.; Varga, M.; Prokeš, J.; Zhigunov, A.; Vohlídal, J. Effects of the Polymerization Temperature on the Structure, Morphology and Conductivity of Polyaniline Prepared with Ammonium Peroxodisulfate. Eur. Polym. J. 2013, 49, 3904−3911. (21) Ding, H.; Wan, M.; Wei, Y. Controlling the Diameter of Polyaniline Nanofibers by Adjusting the Oxidant Redox Potential. Adv. Mater. 2007, 19, 465−469. (22) Zhang, L.; Zujovic, Z. D.; Peng, H.; Bowmaker, G. A.; Kilmartin, P. A.; Travas-Sejdic, J. Structural Characteristics of Polyaniline Nanotubes Synthesized from Different Buffer Solutions. Macromolecules 2008, 41, 8877−8884. (23) Tran, H. D.; D’Arcy, J. M.; Wang, W.; Beltramo, P. J.; Strong, V. A.; Kaner, R. B. The Oxidation of Aniline to Produce “Polyaniline”: A Process Yielding Many Different Nanoscale Structures. J. Mater. Chem. 2011, 21, 3534−3550. (24) Wang, L.; Liang, H.; Wang, F.; Huang, L.; Liu, Z.; Dong, Z. OneStep Synthesis of Uniform Double-Shelled Polystyrene/Poly(OToluidine) Composite Hollow Spheres. Langmuir 2013, 29, 5863− 5868.

(25) Han, J.; Song, G.; Guo, R. A Facile Solution Route for Polymeric Hollow Spheres with Controllable Size. Adv. Mater. 2006, 18, 3140− 3144. (26) Luo, C.; Peng, H.; Zhang, L.; Lu, G. L.; Wang, Y.; Travas-Sejdic, J. Formation of Nano-/Microstructures of Polyaniline and Its Derivatives. Macromolecules 2011, 44, 6899−6907. (27) O’Kane, C.; Oliver, R. G.; Blunden, R. E. Surface Roughness and Droplet Contact Angle Measurement of Various Orthodontic Bonding Cements. Br J. Orthod 1993, 20, 297−305. (28) Bell, M. S.; Shahraz, A.; Fichthorn, K. A.; Borhan, A. Effects of Hierarchical Surface Roughness on Droplet Contact Angle. Langmuir 2015, 31, 6752−6762. (29) Wei, J.; Zhang, J.; Liu, Y.; Xu, G.; Chen, Z.; Xu, Q. Controlled Growth of Whisker-Like Polyaniline on Carbon Nanofibers and Their Long Cycle Life for Supercapacitors. RSC Adv. 2013, 3, 3957−3962. (30) Wang, Y.; Liu, Z.; Han, B.; Sun, Z.; Huang, Y.; Yang, G. Facile Synthesis of Polyaniline Nanofibers Using Chloroaurate Acid as the Oxidant. Langmuir 2005, 21, 833−836. (31) Shi, J.; Wu, Q.; Li, R.; Zhu, Y.; Qin, Y.; Qiao, C. The PhControlled Morphology Transition of Polyaniline from Nanofibers to Nanospheres. Nanotechnology 2013, 24, 175602. (32) Wang, Y.; Tran, H. D.; Liao, L.; Duan, X.; Kaner, R. B. Nanoscale Morphology, Dimensional Control, and Electrical Properties of Oligoanilines. J. Am. Chem. Soc. 2010, 132, 10365−10373. (33) Glavas, L.; Odelius, K.; Albertsson, A. C. Induced Redox Responsiveness and Electroactivity for Altering the Properties of Micelles without External Stimuli. Soft Matter 2014, 10, 4028−4036. (34) Stejskal, J.; Sapurina, I.; Trchová, M.; Konyushenko, E. N. Oxidation of Aniline: Polyaniline Granules, Nanotubes, and Oligoaniline Microspheres. Macromolecules 2008, 41, 3530−3536. (35) Wang, R.; Wang, C.; Liu, K.; Bei, F.; Lu, L.; Han, Q.; Wu, X. Nucleation of Polyaniline Nano-/Macrotubes from Anilinium Composed Micelles. J. Phys. Chem. B 2014, 118, 2544−2552. (36) Zhang, L.; Long, Y.; Chen, Z.; Wan, M. The Effect of Hydrogen Bonding on Self-Assembled Polyaniline Nanostructures. Adv. Funct. Mater. 2004, 14, 693−698. (37) Yu, Y.; Zhang, Y.; Jiang, Z.; Zhang, X.; Zhang, H.; Wang, X. Full View of Single-Molecule Force Spectroscopy of Polyaniline in Oxidized, Reduced, and Doped States. Langmuir 2009, 25, 10002−10006. (38) Shahbazi, M. A.; Hamidi, M.; Mäkilä, E. M.; Zhang, H.; Almeida, P. V.; Kaasalainen, M.; Salonen, J. J.; Hirvonen, J. T.; Santos, H. A. The Mechanisms of Surface Chemistry Effects of Mesoporous Silicon Nanoparticles on Immunotoxicity and Biocompatibility. Biomaterials 2013, 34, 7776−7789.

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