Nanostructured Composites Obtained by ATRP Sleeving of Bacterial

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Nanostructured Composites Obtained by ATRP Sleeving of Bacterial Cellulose Nanofibers with Acrylate Polymers Paula S. S. Lacerda, Ana M. M. V. Barros-Timmons,* Carmen S. R. Freire,* Armando J. D. Silvestre, and Carlos P. Neto CICECO and Chemistry Department, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal

ABSTRACT: Novel nanostructured composite materials based on bacterial cellulose membranes (BC) and acrylate polymers were prepared by in situ atom transfer radical polymerization (ATRP). BC membranes were functionalized with initiating sites, by reaction with 2-bromoisobutyryl bromide (BiBBr), followed by atom transfer radical polymerization of methyl methacrylate (MMA) and n-butyl acrylate (BA), catalyzed by copper(I) bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), using two distinct initiator amounts and monomer feeds. The living characteristic of the system was proven by the growth of PBA block from the BC-g-PMMA membrane. The BC nanofiber sleeving was clearly demonstrated by SEM imaging, and its extent can be tuned by controlling the amount of initiating sites and the monomer feed. The ensuing nanocomposites showed high hydrophobicity (contact angles with water up to 134°), good thermal stability (initial degradation temperature in the range 241−275 °C), and were more flexible that the unmodified BC membranes.

1. INTRODUCTION Bacterial cellulose (BC) is an extracellular polysaccharide produced by several bacteria, mainly belonging to the Gluconacetobacter genus. It can be produced in both stationary and agitated cultures, resulting in the accumulation of a gelatinous cellulose membrane at the air/liquid interface or fibrous suspensions, respectively. The high purity of this biopolymer, along with its unique physical properties that arise from its 3D structure of highly crystalline nano- and microfibrils, and biocompatibility have triggered considerable interest mainly in the biomedical field,1−4 electronics,5 and in nanocomposite materials.6−10 The most common strategy to prepare bacterial cellulose nanocomposites is the simple blending with natural and synthetic polymers or fillers.6−10 Alternatively, BC production in the presence of polymers previously added to the culture medium has also been reported.11 The in situ polymerization of monomers inside of BC network is another attractive alternative to produce BC-based nanocomposites.12,13 Bacterial cellulose nanocomposites of poly(methyl methacrylate) have also been prepared by the mechanical fracturing of the cellulose chains producing chain-end-type radicals that initiated the polymerization of methyl methacrylate.14 Reversible-deactivation radical polymerization (RDRP) mechanisms allow the synthesis of polymers with controlled © XXXX American Chemical Society

molecular weights, relatively low polydispersities, and controlled molecular architecture in terms of chain topology, composition, and functionality. Among these mechanisms, ATRP is currently one of the most widely used radical polymerization mechanisms due to its compatibility with a variety of functional monomers and reaction conditions as well as its ability to produce polymers with high chain-end functionality.15 ATRP has been used to prepare new materials based on natural and synthetic polymers, as well as synthetic polymers linked to biomolecules such as peptides, proteins, nucleic acids, and polysaccharides, yielding promising new functional nanocomposite materials.16,17 Specifically, for polysaccharides, Bernard et al.18 have reviewed the use of ATRP to modify pullulan, dextran, chitosan, and acetylated starch using acrylate derived monomers under homogeneous conditions. The fibrous nature of cellulose has impaired the use of ATRP in homogeneous conditions, however, the use of alkyl or acyl cellulose derivatives19 and ionic liquids20 or dimethylacetamide/lithium chloride21 as solvents, allowed the grafting of, for example, polystyrene and poly(methyl methacrylate) in homogeneous media. In addition, ATRP has been also explored for Received: March 27, 2013

A

dx.doi.org/10.1021/bm400432b | Biomacromolecules XXXX, XXX, XXX−XXX

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The same procedure was followed to prepare BC-BiB(I0.5) using half of the number of moles of BiBBr used for the conditions referred above. 2.3. Grafting of Methyl Methacrylate or n-Butyl Acrylate from Initiator-Functionalized BC Membranes. Monomers MMA or BA were grafted from BC-BiB using Cu(I)Br and the complexing ligand PMDETA as catalytic system. A mixture of DMF/H2O 80:20 (9.82 mL/2.45 mL) was used as solvent.31 Typically, in a glass vial equipped with a rubber septum CuBr was vigorously dispersed in degassed water and next PMDETA was added. The drained BC-BiB membrane (10 mg, estimated dry weight) was introduced into an Erlenmeyer flask equipped with magnetic stirring bar and caped with a rubber septum. Afterward, degassed DMF and the freshly prepared catalytic solution were introduced into the flask and the mixture was purged with N2 for 30 min. Finally, an amount of previously degassed monomer (MMA or BA) was injected into the reaction mixture which was then immersed in an oil bath at 50 °C under constant stirring. After 3 h, the mixture was removed from the oil bath and exposed to air to stop the reaction. Thereafter, the membrane was thoroughly washed with DMF (first overnight and then twice more for an hour with fresh solvent). Next the membrane was further washed with water for 24 h (renewing the solvent three times) to remove residual reactants and byproducts, rendering a white membrane. Finally, the ensuing BC-g-PMMA or BC-g-PBA membranes were lyophilized. Table 1 summarizes the different molar proportions of the reagents used.

heterogeneous modification of different cellulosic substrates, such as paper,22 cellulose nanocrystals,23,24 and microfibrillated cellulose.25 Despite the promising advantages, the limitations of the use of ATRP in heterogeneous systems have also been addressed by researchers working in this field, namely, in connection with the occurrence of side reactions that may hinder the whole polymerization or at least compromise its kinetics.22,26 Nevertheless, while ATRP has been successfully used in the preparation of plant cellulose-based grafted copolymers,22,27 its versatility has not yet been explored for bacterial nanocellulose. This strategy could be very interesting and promising for the development of novel nanostructured nanocomposite materials because of the conjugation of the intrinsic morphology of BC with the functionalities of the grafted polymers. In fact, other nanocellulose forms, as microfibrillated cellulose and cellulose nanocrystatals, could not be explored in the same way because they do not possess nanostructured morphologies (are obtained in the form of individualized nanofibrils or crystals) and therefore the features of the final materials are completely different, as well as their potential applications. In the present study, novel nanocomposites from BC membranes and grafted acrylic polymers were prepared. The BC membranes were first functionalized with initiating sites for ATRP and then polymerization of methyl methacrylate and nbutyl acrylate was carried out. The effects of the degree of substitution of the macroinitiator and of the polymerization components ratios on the properties of the ensuing nanocomposites were studied. The living characteristic of the system was also proven using a second monomer to highlight the potential of this synthetic path to tune different properties and applications.

Table 1. Molar Proportions Used for ATRP Grafting of MMA or BA from Initiator-Functionalized Bacterial Cellulose Membrane copolymer BC-g-PMMA(I1M1) BC-g-PMMA(I1M0.5) BC-g-PMMA(I0.5M1) BC-g-PBA(I1M1) BC-g-PMMA-co-PBA (I1M1)

2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals were of analytical grade and were used as received unless otherwise stated, except for monomers (methyl methacrylate and n-butyl acrylate), which were passed through a column of neutral aluminum oxide (Carlo Erba, particle size 63−200 μm) prior to use to remove the inhibitor. Bacterial cellulose (a tridimensional network of nano- and microfibrils with 10−200 nm width) in the form of wet membranes was produced in our laboratory using the bacteria Gluconacetobacter sacchari28 and conventional culture conditions.29 2.2. Immobilization of Initiator on BC Membranes. As an example, the synthesis of the BC macroinitiator (BC-BiB(I1)) with the highest number of initiator groups is presented. Wet BC membranes (99% water) were cut into small pieces of approximately 1.5 × 1.5 cm2, lyophilized after liquid nitrogen immersion and kept in a desiccator until use. The synthesis of the BC macroinitiator was adapted from a reported procedure.30 Typically a lyophilized BC sample (15 mg) was introduced into an Erlenmeyer flask equipped with a rubber septum, containing 4-(dimethylamino)pyridine (DMAP; 536.2 mg, 4.4 mmol) in anhydrous DMF (20 mL) at room temperature. The mixture was purged with N2 for 30 min and kept under stirring for a total of 2 h in order to ensure full intumescence of the BC membrane. Next, 148 μL of 2bromoisobutyryl bromide (BiBBr) was added (1.2 mmol, 4.32 BC OH equiv) dropwise to the ice-cold reaction mixture. The mixture was left to thaw to room temperature and the reaction was allowed to proceed under stirring for more 2 h. Thereafter, the initiator-modified BC (BC-BiB) was thoroughly washed in DMF (first overnight and then twice more for an hour with fresh solvent) to remove residual reactants and byproducts. The obtained sample was cut into two pieces: a small one that was washed in water, lyophilized, and characterized; the other was drained and used as such in the subsequent polymerization step.

monomer (mmol)

CuBr (mmol)

PMDETA (mmol)

18.4 9.2 18.4 18.4 18.4

0.369 0.369 0.369 0.369 0.369

0.369 0.369 0.369 0.369 0.369

2.4. Grafting of Methyl Methacrylate-co-Butyl Acrylate from Initiator-Functionalized BC Membrane. First, MMA was grafted from the drained BC-BiB(I1) membrane following the procedure described above. The ensuing lyophilized BC-g-PMMA(I1M1) membrane was then used as macroinitiator to grow the PBA block using exactly the same conditions as those used for the PBA grafting (Table 1). 2.5. Isolation of Grafted Polymer Chains by Hydrolysis from the BC Backbone. Grafted PMMA and PBA chains were cleaved from the BC membrane under basic hydrolysis conditions. Specifically, the lyophilized grafted BC samples were shredded, transferred to a round-bottom flask, and dispersed into THF (3 mL) with 8 mg of KOH, and the suspension was stirred for 24 h at 50 °C under a nitrogen atmosphere.32 The resulting suspension was filtered through a filter crucible, washed with dichloromethane, and the grafted polymer containing solution was concentrated using a rotary evaporator. The polymer was then further precipitated from cold methanol and dried at 40 °C in a vacuum oven. The dried polymer was then dissolved (10 mg/mL) in a 0.1 mol/L lithium chloride in DMF solution for GPC analysis. 2.6. Weight-Gain Calculation. To estimate the weight-gain, the specimens were weighed before and after polymerization. The weightgain (G, wt%) was calculated as follows:

G=

W2 − W1 × 100 W1

where W1 was the estimated dry weight of BC-BiB macroinitiator and W2 was the dry weight of the BC nanocomposite samples. The value of W1 was estimated since, as mentioned above, BC-BiB wet membranes B


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Figure 1. Bacterial cellulose functionalization with the ATRP initiator (step 1) and ATRP grafting of MMA or BA from modified bacterial cellulose (step 2).

Figure 2. ATR-FTIR spectra of pristine BC and macroinitiators BC-BiB(I1) and BC-BiB(I0.5). In the inset, the correspondent amplification of the spectra in the 1800−1700 nm range is presented. were cut in two pieces, one of which was dried and characterized and the other was used to anchor the polymer chains after draining. Hence, the estimation of W1 is based on the difference between the dry weights of the original BC and the BC-BiB portion not used in the polymerization and considering that the weight-gain resulting from the introduction of the initiator groups is negligible. 2.7. Characterization Methods. 13C solid-state cross-polarized magic-angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectra were recorded at 9.4 T on a Bruker Avance 400 spectrometer using a 4 mm double-bearing probe, 9 kHz spinning rate, and MAS with proton 90° pulses of 40 μs. 1H NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer operating at 300 MHz, using CDCl3 as solvent. FTIR spectra were acquired using a Perkin-Elmer FTIR System Spectrum BX Spectrometer equipped with a single horizontal Golden Gate ATR cell. Thirty-two scans were acquired in the 4000−500 cm−1 range with a resolution of 1 cm−1. Thermogravimetric analyses (TGA) were carried out using a Setsys Evolution 1750 (Setaram) from room temperature up to 800 °C, at a heating rate of 10 °C/min, under a nitrogen flow (200 mL/min). The thermal degradation temperatures were taken at the onset of 5 wt % weight loss from the heated sample. Dynamic mechanical analysis (DMA) measurements were carried out with a Tritec 2000 DMA Triton equipment operating in the tension mode. Tests were performed at 1 Hz, and the temperature was varied from −100 to 200 °C at 2 °C/min. BC and BC-modified

membranes were dried over a day under gentle pressing in a ventilated oven at 40 °C to yield smooth films. Prior to analyses the ensuing films were previously equilibrated at 50% humidity and at 25 °C inside a permeation apparatus. Size exclusion chromatography (SEC) was carried out using a Varian PL-GPC 110 instrument equipped with an IR-PD 2020 light scattering detector, using 0.1 mol/L lithium chloride in DMF as the mobile phase, a run time of 30 min and a column temperature of 70 °C. Polystyrene standards were used for calibration. Water contact angle (CA) measurements were conducted at room temperature on a Contact Angle System OCA from Dataphysics, equipped with the SCA20 software. The sample’s water contact angle was evaluated by static contact angle measurements using the sessile drop (2 μL) method. Scanning electron microscopy (SEM) micrographs were obtained using a Hitachi SU-70 SEM and energy dispersive X-ray spectroscopy (EDX) analysis was performed using a EDX Bruker Quantax 400. Samples of lyophilized membranes were fractured by immersion in liquid nitrogen, deposited on an aluminum plate and coated with a carbon layer, approximately 15−50 nm thick, by evaporation of carbon rods to afford a conductive film. X-ray diffraction (XRD) patterns of pristine and modified BC were measured using a Phillips X’pert MPD diffractometer using Cu Kα radiation. The scattered radiation was detected in the angular range from 4 to 40° (2θ). C


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3. RESULTS AND DISCUSSION Bacterial cellulose nanocomposites were obtained using a “grafting from” approach in two steps (Figure 1): first the

Figure 3. SEM and EDX spectroscopy bromine mapping (a and b, respectively) of the surface of macroinitiator BC-BiB(I1).

Table 2. Initial Reagents Proportions and Key Properties of Bacterial Cellulose Nanocomposites Obtained from ATRP Grafting of MMA or BA copolymer BC-g-PMMA(I1M1) BC-g-PMMA(I1M0.5) BC-g-PMMA(I0.5M1) BC-g-PBA(I1M1) BC-g-PMMA-coPBA(I1M1)

[M]0/[CuBr]0/[PMDETA]0

[M]0 (mol/L)

G (%)

50:1:1 25:1:1 50:1:1 50:1:1 50:1:1

1.55 0.78 1.55 1.55 1.55

887 341 59 110 616a

Figure 5. 13C CP-MAS solid state NMR of pristine BC, BC-gPMMA(I1M1), BC-g-PMMA(I1M0.5), BC-g-PMMA(I0.5M1), and BC-gPBA(I1M1).

a After first block grafting, the weight-gain (G) of nanocomposite BCg-PMMA was 576%.

macroinitiator. These procedures were performed in heterogeneous media, with BC used as a swollen membrane, because the nanofibers did not dissolve under the reaction conditions used.

immobilization of the ATRP initiator on BC, followed by the grafting of MMA, BA, or both from the bacterial cellulose

Figure 4. ATR-FTIR spectra of pristine BC, BC-g-PBA(I1M1), BC-g-PMMA(I1M1), and of homopolymers PMMA and PBA (produced by ATRP). D


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throughout all membranes (including the bulk nanofibril) and further confirming the effectiveness of initiator groups grafting. 3.2. Grafting of MMA or n-BA from Bacterial Cellulose Macroinitiator. ATRP reactions were carried out using two different proportions of starting reagents (Tables 1 and 2). MMA concentration values of 1.55 mol/L and half (referred to as M1 and M0.5, respectively) were used. Keeping constant the time and reaction temperature as well as the catalytic system, nanocomposites BC-g-PMMA(I1M1) and BC-g-PMMA(I1M0.5) were obtained from BC-BiB(I1) in order to assess the effect of the chain length of the grafts. Additionally, to evaluate the effect of the number of initiation sites BC-g-PMMA(I0.5M1) was obtained from macroinitiator BC-BiB(I0.5). To further explore the versatility of this procedure a more flexible polymer was grafted from bacterial cellulose, using nbutyl acrylate, the macroinitiator BC-BiB(I1), and the higher monomer concentration, leading to the formation of BC-gPBA(I1M1). Finally, the living characteristic of the system was also explored using BC-g-PMMA(I1M1) as macroinitiator to grow a BA block and obtain BC-g-PMMA-co-PBA(I1M1). Grafting of polymer chains from BC macroinitiators, BC-BiB, led in all cases to a substantial weight-gain (G), as indicated in Table 2. The PMMA grafting nanocomposite BC-g-PMMA(I1M1) afforded the highest weight-gain (887%). This was expected, as this nanocomposite was produced from the most substituted macroinitiator and using the higher monomer concentration. When the monomer to catalytic system ratio was reduced to half, the G value of the ensuing nanocomposite BCg-PMMA(I1M0.5) was roughly proportionally reduced to 341%. For the polymerization using BC-BiB(I0.5), containing half of polymerization docking sites, the G was considerably lower (59%). This may be due to the reduced number of initiator groups, which may require a longer reaction time to achieve a similar degree of monomer conversion to that of BC-gPMMA(I1M1).33 In the case of BC-g-PBA(I1M1), the weight-gain (110%) was considerably lower than that reported for the PMMA counterpart prepared under similar reaction conditions. This could be due to the difference in solubility and reactivity between the two monomers. Finally, when the diblock-type graft was performed, a total G of 616% was obtained for BC-g-PMMA-co-PBA corresponding to a 576% weight-gain registered after grafting the first PMMA block followed by a more moderate weight-gain (40%) upon grafting of the second PBA block. The differences registered for the G values during the preparation of this nanocomposite when compared to those obtained for the corresponding counterparts (BC-g-PMMA(I1M1) and BC-g-PBA(I1M1)) may be attributed to the heterogeneity of the system. In the case of PBA grafting this is further aggravated by the solubility and reactivity issues mentioned above, together with the low blocking efficiency associated with these meth(acrylate) systems.34,35 Nevertheless, the result obtained confirms the living characteristic of the system. The FTIR spectra of unmodified BC and representative BCcopolymers, namely BC-g-PMMA(I1M1) and BC-g-PBA(I1M1) are shown in Figure 4. For comparison purposes, PMMA and PBA homopolymers were produced by ATRP (under the same reaction conditions used for the production of BC hybrid copolymers) and their FTIR spectra are also shown in Figure 4. In the spectra of BC composites, the presence of the strong and narrow carbonyl peak at 1726 cm−1 confirms the presence of the acrylate functionality as well as the band at 1146 cm−1

Figure 6. Digital photograph of (a) pristine bacterial cellulose and (b) BC-g-PMMA(I1M1) wet membranes.

Table 3. Thermal Properties of Pristine Bacterial Cellulose, BC Macroinitiators, BC Hybrid Copolymers, and Homopolymers material

Td,5a (°C)

BC PMMAc PBAc BC-BiB(I0.5) BC-BiB(I1) C-g-PMMA(I0.5M1) BC-g-PMMA(I1M0.5) BC-g-PMMA(I1M1) BC-g-PMMA-co-PBA(I1M1) BC-g-PBA(I1M1)

290 277 294 258 168 282 241 262 275 269

Tdb (°C) 365 280, 408 339 314 363 272, 271, 285, 268,

391

400 382 380 320, 371, 397

a

Temperature at 5% mass loss detected using TGA. bTemperature at maximum mass loss rate detected using DTG. cHomo-PMMA or homo-PBA polymer obtained by ATRP.

3.1. Immobilization of Initiator on BC. The ATRP BCbased macroinitiator was obtained by partial esterification of BC hydroxyl groups with bromoisobutyryl bromide. An excess of BiBBr was necessary to achieve a moderate substitution degree, as most of the reactive is decomposed by the intrinsic residual water present in BC membranes (∼10%). With this in mind, 4.32 and 2.16 equiv per OH group (referred to as I1 and I0.5, respectively) were used, aiming at obtain two samples with distinct degrees of substitution in order to investigate the influence of the density of macroinitiator grafting sites on the properties of BC grafted materials. The functionalization of BC with ATRP initiators was confirmed by FTIR, based on the appearance of the carbonyl stretching band of the 2-bromoisobutyrate group at 1737 cm−1 (Figure 2). As expected, the intensity of this band is stronger in the spectrum of BC-BiB(I1) than in that of BC-BiB(I0.5), confirming that the degree of substitution can be modulated by varying the amount of BiBBr. Regarding the broad stretching band of hydroxyl groups at 3342 cm−1, no significant changes in their intensity are observed by comparison with the BC FTIR spectrum. These results confirm the partial surface acylation of the BC fibers. The SEM analysis of BC-BiB samples confirms that the pristine BC nanofibrilar structure is largely preserved, as illustrated in Figure 3a for BC-BiB(I1), which is clearly in tune with the low degree of substitution evidenced by the FTIR analysis. Furthermore, SEM coupled with EDX spectroscopy (Figure 3b) confirmed the presence of bromine on the nanofibrills surface (bromine presence is evidenced by the blue dots), showing that these are uniformly distributed E


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Figure 7. Thermograms of bacterial cellulose BC, BC-based macroinitiators (BC-BiB(I1), BC-BiB(I0.5)), BC nanocomposites (BC-g-PMMA(I1M1), BC-g-PMMA(I1M0.5), BC-g-PMMA(I0.5M1), BC-g-PBA(I1M1), and BC-g-PMMA-co-PBA(I1M1), and ATRP produced PMMA and PBA homopolymers.

107.1 ppm (C1);36 and of PMMA at 15.7−22.7 ppm (α-CH3), 44.8 ppm (quaternary C), 51.7 ppm (OCH3), together with a shoulder assigned to CH2 and finally at 177.7 ppm assigned to CO37 or of PBA, at δ14.0 ppm (CH3), 19.5 (CH2-CH2CH3), 31.1 (CH2-CH2-CH3), and 174.5 ppm (CO).37 The spectrum of BC-g-PMMA(I0.5M1) clearly shows the presence of BC and PMMA resonances, whereas for BC-gPMMA(I1M0.5), the BC resonances are nearly vanished, and for BC-g-PMMA(I1M1), they can no longer be detected. This behavior is in agreement with the above-discussed weight increments and FTIR profiles. In the case of BC-g-PBA(I1M1), apart from BC carbon resonances, typical PBA resonances are

attributed to the methylene C−H twisting vibration. Furthermore, the intensity of characteristic cellulose bands (at 3300 and 1100 cm−1, associated to the vibrations of the OH and C−O−C groups of cellulose)7,28 are substantially reduced, especially in the case of BC-g-PMMA(I1M1), which is in line with the considerable weight-gain resulting from PMMA grafting. The grafting of polymers from BC was further confirmed by 13 C CP-MAS solid state NMR of the resulting nanocomposite materials (Figure 5). The NMR spectra of the nanocomposites show the presence of the typical 13C resonances of bacterial cellulose at δ 65.2 (C6), 71.6−74.5 (C2,3,5), 90.0 (C4) and F


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Figure 8. SEM micrograph of the cross sections (magnifications 6.00K and 20.0K) and surfaces (magnification 20.0K) of pristine BC and grafted nanocomposites BC-g-PMMA(I1M0.5), BC-g-PMMA(I1M1), and BC-g-PBA(I1M1).

copolymerized to confer different functionalities, such as pH or temperature response, flexibility, and so on. 3.3. Characterization of the BC Nanocomposites. The grafting of PMMA and PBA chains onto the BC membranes led to significant changes of their visual aspect, as shown in Figure 6 for BC-g-PMMA(I1M1). The translucid gelatinous BC membrane (a) turned into a white opaque nanocomposite material (b). These novel nanocomposites were then characterized in terms of thermal stability, morphology, crystalline structure, hydrophobicity, and thermomechanical performance. Thermogravimetric analysis (TGA) was used to investigate the thermal decomposition of the BC nanocomposites, as summarized in Table 3 and Figure 7. BC macroinitiators, as well as PMMA and PBA homopolymers (produced under the same ATRP reaction conditions used for the production of BC hybrid copolymers) were also analyzed for comparison purposes. Unmodified BC displayed a typical single mass-loss step degradation profile, initiating its thermal decomposition (Td,5) at 290 °C and reaching a maximum decomposition rate at 365 °C.40,41 The introduction of BiBBr initiator groups resulted in a decrease of the thermal stability of the BC membrane, with initial degradation temperatures at 258 and 168 °C, and maximum degradation temperatures at 339 and 314 °C, for BCBiB(I0.5) and BC-BiB(I1) macroinitiators, respectively (Table 3). The decrease of thermal stability for this type of macroinitiators has been reported for other cellulose substrates42 and lignocellulosic materials43 based macroinitiators, and it was attributed to the degradation of the

also detected with low intensity, which once more is related with the low weight-gain of this material, comparable with, for example, BC-g-PMMA(I0.5M1). Finally, the nanocomposites were submitted to hydrolysis of the ester linkage between the grafted polymer chains and BC backbone32 aiming at analyzing by GPC the released PMMA, PBA, and PMMA-co-PBA polymers. Some of the samples were not suitable for GPC analysis as they were not soluble in the GPC eluent. Nevertheless, notice should be made that the hydrolysates of BC-g-PMMA(I1M1) and BC-g-PMMA-co-PBA(I1M1) samples, which were adequately soluble, were analyzed by 1H NMR spectrometry. The spectra obtained only revealed the presence of typical resonances of PMMA and PBA.38 The molar mass (Mw) and corresponding polydispersity index (PDI) values obtained were 2.7 × 105 Da (PDI = 1.5) and 3.7 × 105 Da (PDI = 1.6) for BC-g-PMMA(I1M1) and BCg-PMMA-co-PBA, respectively. The Mw values are in agreement with the G values registered for the corresponding nanocomposites. The polydispersity PDI values are high indicating that the polymerization is poorly controlled. The lack of control over chain growth can be attributed to limitations intrinsic to the use of ATRP based on Cu catalysts and aqueous media.22,26,39 Nevertheless, the higher Mw of the BC-gPMMA-co-PBA hydrolysate relative to that of BC-g-PMMA confirms the living characteristic of the system as the ATRP initiation of BA has effectively taken place. With this result the versatility of ATRP to tune the properties of the ensuing materials has been proven because as long as the catalytic/ initiator system is available to participate in radical polymerization, other monomers with distinct characteristics can be G


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temperature of the second degradation step of the PMMA homopolymer. Finally, for BC-g-PBA(I1M1), apart from a substantial increment in thermal stability (initial degradation temperature of 269 °C), relative to the corresponding macroinitiator, the mass loss profile is distinct from that of the PMMA nanocomposites showing four consecutive degradation steps between 268 and 397 °C. In general, the thermostability of the nanocomposites prepared depends on the weight-gain. For materials with a higher incorporation of synthetic polymer, the TGA profile is closer to that of the corresponding synthetic homopolymer while for those with lower G values it resembles more that of BC. To evaluate the effect of the grafting of polymer chains on the microstructure of BC, SEM micrographs of the surface as well as of the cross section of the membranes were collected, as illustrated in Figure 8 for BC-g-PMMA(I1M1) and BC-gPBA(I1M1). The characteristic tridimensional network of nano and microfibrils of BC is clearly visible on the surface and crosssection of the BC and grafted BC-membranes. However, after grafting an increment of the diameter of the cellulose fibrils is observed which is obviously associated with the chemical sleeving by the PMMA or PBA polymeric chains. This effect is more pronounced for PMMA nanocomposites which is in agreement with the higher weight-gains previously discussed. This increase of diameter of the cellulose fibrils with weightgain is clearly evidenced by the SEM images of BC-gPMMA(I1M0.5) and BC-g-PMMA(I1M1), as well as BC-gPBA(I1M1) (Figure 8). The effect of PMMA and PBA grafting on the crystallinity of BC membranes was assessed by XRD (Figure 9). BC exhibited a diffractogram typical of Cellulose I (native cellulose), with the main peaks at 2θ = 14.4, 16.7, 22.6, and 34.3°. PMMA and PBA display a diffraction profile typical of amorphous polymers with broad bands centered at around 2θ = 14 and 31° for PMMA and at 2θ = 8 and 20° for PBA. As regards the diffraction pattern of the PMMA- and PBA-grafted nanocomposites, it is clear that it is strongly dependent on the amount of grafted polymers. As expected, the diffraction peaks of cellulose are more pronounced in the diffractograms of the nanocomposites with lower weight-gains, namely, BC-g-PMMA(I0.5M1), BC-gPMMA(I1M0.5), and BC-g-PBA(I1M1). The surface phobic behavior of the grafted BC membranes was investigated by water static contact angle (CA) measurements (Figure 10). For pristine BC, a water CA of 32° was obtained. Grafting PMMA or PBA yielded highly hydrophobic membranes, as indicated by water contact angles of 134° for BC-g-PMMA(I1M0.5) and BC-g-PMMA(I1M1) and 116° BC-gPBA(I1M1) nanocomposites. Indeed, these values did not vary appreciably with time over 15 min. These data prove that the hydrophobicity of BC is considerably increased via ATRP grafting of PMMA or PBA. Furthermore, it seems that it is not directly affected by the length of the grafts, as indicated by the similar CA values for BC-g-PMMA nanocomposites produced from macroinitiator with similar number of initiating sites. This observation is in agreement with previous studies where we have demonstrated that only a small extent of cellulose fibers surface coverage was required to promote a drastic change in the contact angles, and above that limit, the contact angle is governed mainly by the properties of the graft.44 As for BC-gPMMA(I0.5M1) prepared using a macroinitiator with less anchoring sites, a different phobic behavior was observed. In

Figure 9. X-ray diffractograms of pristine BC, grafted nanocomposites (BC-g-PMMA(I1M1), BC-g-PMMA(I1M0.5), BC-g-PMMA(I0.5M1), and BC-g-PBA(I1M1)) and ATRP produced PMMA and PBA homopolymers.

Figure 10. Contact angle pictures of water droplet over (a) pristine BC, (b) BC-g-PMMA(I1M1), and (c) BC-g-PBA(I1M1).

bromoalkyl groups with HBr release upon heating, which induces cellulose degradation at lower temperatures. The lower stability of BC-BiB(I1) is in agreement with the higher number of initiator groups introduced, as demonstrated by FTIR analysis. The thermograms of the BC-g-PMMA nanocomposites show a substantial increment in stability, when compared to the corresponding macroinitiators. While for BC-BiB(I1) degradation starts at 168 °C, for BC-g-PMMA(I1M0.5) and BC-gPMMA(I1M1) it starts at 241 and 262 °C, respectively. Furthermore, the maximum degradation temperatures depend on the amount of PMMA incorporated (weight-gain). For BCg-PMMA(I0.5M1), the maximum degradation temperature takes place in one step at 363 °C (near to BC maximum degradation temperature) and for BC-g-PMMA(I1M0.5) and BC-g-PMMA(I1M1) (and even for BC-g-PMMA-co-PBA(I1M1)), the degradation occurs in two steps. Notice should be made that the temperatures of the second degradation step of these composites are higher than that of BC and closer to the H


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Figure 11. Temperature dependence of the logarithm of the storage modulus E′ and of tan δ (at 1 Hz) of pristine BC, BC-g-PMMA(I1M1), and BCg-PBA(I1M1).

polymer (i.e., lower mass gain, Table 2). As regards the tan δ curves of the nanocomposites as a function of temperature, besides the transitions attributed to the presence of water between 0 and 20 °C, in the case of BC-g-PMMA, a second phase transition at 145.2 °C is also observed, which is associated with the glass transition of PMMA moiety. As confirmed by the analysis of the PMMA homopolymer (data not shown). This transition is also revealed as a drastic reduction of the elastic module as the synthetic part of the nanocomposite becomes softer. In the case of PBA grafted BC nanocomposite, the tan δ curve shows only a plateau in the range of −42 to 34 °C. This could be attributed to a sequential effect of both the PBA moiety and water present is the nanocomposite. At the down part of this range, a transition was expected due to the PBA moiety, as indicated by the DMA measurements of the PBA homopolymer (Tg at −49.3 °C, data not shown). However, as a result of the low content of PBA grafted polymers in the nanocomposite, only a shoulder can be detected at low temperatures. This is in line with what was already discussed for the low grafting of PBA onto BC. Nevertheless, in the log (E′/ Pa) curve this transition is cleared detected by the drop of E′ registered at this temperature. As the production of BC nanocomposites involving ATRP was performed using copper as catalyst, despite of the extensive washing after polymerization the effect of a second washing step to further reduce it was assessed by ICP. A BC-g-PMMA

this case, the water drops were readily absorbed by the film. This observation suggests a lower surface coverage with PMMA grafts, which is in line with the FTIR, 13C CP/MAS NMR, TGA, and XRD data. Dynamic mechanical measurements were performed for ungrafted BC and BC-g-PMMA(I1M1) and BC-g-PBA(I1M1) nanocomposites. Figure 11 shows the curves of (E′/Pa) (storage tensile modulus) and tan δ (tan δ vs temperature at 1 Hz) of these samples. In view of the hygroscopic nature of BC and the plasticizing effect of water, all the samples were equilibrated at 50% relative humidity prior to testing. For ungrafted BC, the log (E′/Pa) curve only shows a small kink, possibly due to the presence of water. Furthermore, the corresponding tan δ reveals a broad phase transition between 0 and 20 °C confirming this hypothesis. This was further confirmed by the significant reduction of this transition when this sample was dried in situ (in the DMA chamber) prior to analyses (Data not shown). For the grafted nanocomposites BC-g-PMMA and BC-g-PBA the log (E′/Pa) curves show a different trend. The values registered for elastic moduli across the whole temperature range are lower than that of the ungrafted BC membrane. This is associated with the fact that the acrylate polymers are more flexible than the BC nanofibrillar network. Indeed, despite of the fact that PBA is more flexible than PMMA, the BC-g-PBA nanocomposite is more rigid than the BC-g-PMMA nanocomposite due to the lower content of grafted synthetic I


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(2) Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Angew. Chem., Int. Ed. 2011, 50, 5438−5466. (3) Trovatti, E.; Freire, C. S. R.; Pinto, P. C.; Almeida, I. F.; Costa, P.; Silvestre, A. J. D.; Neto, C. P.; Rosado, C. Int. J. Pharm. 2012, 435, 83− 87. (4) Trovatti, E.; Silva, N. H. C. S.; Duarte, I. F.; Rosado, C. F.; Almeida, I. F.; Costa, P.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P. Biomacromolecules 2011, 12, 4162−4168. (5) Shah, J.; Brown, R. M. Appl. Microbiol. Biotechnol. 2005, 66, 352− 355. (6) Nogi, M.; Yano, H. Adv. Mater. 2008, 20, 1849−1852. (7) Trovatti, E.; Fernandes, S. C. M.; Rubatat, L.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P. Cellulose 2012, 19, 729−737. (8) Fernandes, S. C. M.; Oliveira, L.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P.; Gandini, A.; Desbriéres, J. Green Chem. 2009, 11, 2023−2029. (9) Martins, I. M. G.; Magina, S. P.; Oliveira, L.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P.; Gandini, A. Compos. Sci. Technol. 2009, 69, 2163−2168. (10) Pinto, R. J. B.; Neves, M. C.; Neto, C. P.; Trindade, T. Eur. J. Inorg. Chem. 2012, 5043−5049. (11) Choi, J.; Park, S.; Cheng, J.; Park, M.; Hyun, J. Colloids Surf., B 2012, 89, 161−166. (12) Müller, D.; Rambo, C. R.; Recouvreux, D. O. S.; Porto, L. M.; Barra, G. M. O. Synth. Met. 2011, 161, 106−111. (13) Shi, Z.; Zang, S.; Jiang, F.; Huang, L.; Lu, D.; Ma, Y.; Yang, G. RSC Adv. 2012, 2, 1040−1046. (14) Sakaguchi, M.; Ohura, T.; Iwata, T.; Takahashi, S.; Akai, S.; Kan, T.; Murai, H.; Fujiwara, M.; Watanabe, O.; Narita, M. Biomacromolecules 2010, 11, 3059−3066. (15) Matyjaszewski, K. Macromolecules 2012, 45, 4015−4039. (16) Le Droumaguet, B.; Nicolas, J. Polym. Chem. 2010, 1, 563−598. (17) Siegwart, D. J.; Oh, J. K.; Matyjaszewski, K. Prog. Polym. Sci. 2012, 37, 18−37. (18) Tizzotti, M.; Charlot, A.; Fleury, E.; Stenzel, M.; Bernard, J. Macromol. Rapid Commun. 2010, 31, 1751−1772. (19) Porsch, C.; Hansson, S.; Nordgren, N.; Malmström, E. Polym. Chem. 2011, 2, 1114−1123. (20) Meng, T.; Gao, X.; Zhang, J.; Yuan, J.; Zhang, Y.; He, J. Polymer 2009, 50, 447−454. (21) Raus, V.; Štěpánek, M.; Uchman, M.; Šlouf, M.; Látalová, P.; Č adová, E.; Netopilík, M.; Kříž, J.; Dybal, J.; Vlček, P. J. Polym. Sci., Polym. Chem. 2011, 49, 4353−4367. (22) Malmström, E.; Carlmark, A. Polym. Chem. 2012, 3, 1702−1713. (23) Morandi, G.; Heath, L.; Thielemans, W. Langmuir 2009, 25, 8280−8286. (24) Yi, J.; Xu, Q.; Zhang, X.; Zhang, H. Polymer 2008, 49, 4406− 4412. (25) Xiao, M.; Li, S.; Chanklin, W.; Zheng, A.; Xiao, H. Carbohydr. Polym. 2011, 83, 512−519. (26) Sankhe, A. Y.; Husson, S. M.; Kilbey, S. M. Macromolecules 2006, 39, 1376−1383. (27) Roy, D.; Semsarilar, M.; Guthrie, J. T.; Perrier, S. Chem. Soc. Rev. 2009, 38, 2046−2064. (28) Trovatti, E.; Serafim, L. S.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P. Carbohydr. Polym. 2011, 86, 1417−1420. (29) Hestrin, S.; Schramm, M. Biochem. J. 1954, 58, 345−352. (30) Bontempo, D.; Masci, G.; De Leonardis, P.; Mannina, L.; Capitani, D.; Crescenzi, V. Biomacromolecules 2006, 7, 2154−2161. (31) De Leonardis, P.; Mannina, L.; Diociaiuti, M.; Masci, G. Polym. Int. 2010, 59, 759−765. (32) Gonçalves, G.; Marques, P. A. A. P.; Barros-Timmons, A.; Bdkin, I.; Singh, M. K.; Emami, N.; Grácio, J. J. Mater. Chem. 2010, 20, 9927−9934. (33) Pyun, J.; Kowalewski, T.; Matyjaszewski, K. Macromol. Rapid Commun. 2003, 24, 1043−1059. (34) Shipp, D. A.; Wang, J.-L.; Matyjaszewski, K. Macromolecules 1998, 31, 8005−8008.

membrane was chosen and part of the sample was further washed in water for another 24 h in order to verify the efficiency of this washing process. The copper content upon the standard washing procedure was 5.85 × 102 μg/g of composite membrane sample. The second washing step reduced it to 1.97 × 102 μg/g of composite membrane sample. In fact, the deposition of copper on cellulose substrates when using ATRP makes it difficult to remove, as has already been reported.21 Besides the negative aspects associated with the presence of residual copper, the need to significantly reduce the quantity used has to be addressed as it is known that when catalytic systems involving this metal are used in aqueous media parallel reactions can take place, thus, jeopardizing polymerization control.39 Nevertheless, new approaches in conducting ATRP based on the significant reduction of copper, namely, activators regenerated by electron transfer (ARGET) ATRP have already been used for surface grafting of cellulosic materials.43,45 Hence, efforts to adjust the reaction conditions to this system are now under current investigation.

4. CONCLUSIONS It has been demonstrated, for the first time, that ATRP is a suitable method for the modification of bacterial cellulose (nanostructured membrane) in heterogeneous medium. Indeed, BC-g-PMMA and BC-g-PBA nanocomposites have been successfully prepared using ATRP, mediated by Cu(I)Br and PMDETA. The versatility of this procedure has been proven using different initiator/monomer ratios offering the possibility to tune the hydrophobicity of the ensuing nanocomposites as well as the thermal and mechanical properties. Noteworthy, is also the covalent link between BC and the synthetic grafts which prevents leaching during use, a common limitation of many composites. Moreover, the living nature of the system was also proven by the growth of the second PBA block, which opens a variety of possibilities to tune the phobic behavior of these materials, their mechanical properties, pH, and temperature responsive behavior, and even bioapplications upon adequate modification of the graft end groups with molecules with biological activity. Hence, it is envisaged that these novel nanostructured porous materials could find potential applications in several fields, namely, in controlled release of drugs or other bioactive compounds.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Paula Lacerda would like to thank CICECO for the awarding of a research grant. CICECO acknowledges FCT for Pest-C/ CTM/LA0011/2011 project. Thermal Analysis Laboratory was funded by FEDER Funds through Programa Operacional Factores de Competitividade − COMPETE and by National Funds through FCT under Project REEQ/515/CTM/2005. Dr Dmitry Evtyugin is acknowledged for the GCP Analysis.

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REFERENCES

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Nanostructured Composites Obtained by ATRP Sleeving of Bacterial

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