Multifunctional Cellulose Beads and Their Interaction with Gram

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Multifunctional cellulose beads and their interaction with Gram positive bacteria Leandro Schafranski Blachechen, Pedro Fardim, and Denise Freitas Siqueira Petri Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5009876 • Publication Date (Web): 06 Aug 2014 Downloaded from http://pubs.acs.org on August 11, 2014

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Multifunctional cellulose beads and their interaction with Gram positive bacteria Leandro S. Blachechen‡,§, Pedro Fardim*,§ and Denise F. S. Petri*,‡ ‡

Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-

000 São Paulo, SP, Brazil, [email protected] §

Laboratory of Fibre and Cellulose Technology, Åbo Akademi, Porthansgatan 3, 20500

Turku, Finland, [email protected]

*corresponding authors

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Abstract Cellulose beads with ~ 3 mm of diameter and high circularity were obtained by dripping cellulose

solutions (5

wt%,

6 wt% and

7

wt%) dissolved

in

NaOH7%/urea12%, into HCl 2M coagulation bath. Carboxylic groups were generated on beads surface through NaClO/NaClO2/TEMPO oxidation method, achieving total charge density of ~ 0.77 mmol/g. Pristine (CB) and oxidized (OCB) beads were characterized by means of optical images analyses, scanning electron microscopy (SEM) and compression tests. Both types of beads, CB and OCB, were used as adsorbent for poly(4-vinyl-N-pentyl pyridinium) bromide, QPVP-C5, a bactericidal agent. The adsorption of QPVP-C5 on CB and OCB was evaluated by means of FTIRATR, UV-Vis, CHN elemental analyses and X-ray photoelectron spectroscopy (XPS). The adsorbed amount of QPVP-C5 was remarkably higher on OCB than on CB, due to ionic interactions. Desorption was less than 5%. The interaction between neat OCB or OCB coated and two different amounts of QPVP-C5 and Gram-positive bacteria Micrococcus luteus was assessed by changes in turbidimetry, SEM and elemental analyses. Bacteria adsorbed on the surface of neat OCB and weakly QPVP-C5 coated OCB due to hydrogen bonding or ion-dipole interaction. Notorious bactericidal action was observed for OCB samples coated with large amount of QPVP-C5.

Keywords: Cellulose beads; oxidized cellulose beads; compressive properties; polycation adsorption; Gram-positive bacteria.


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Introduction Cellulose beads (CB) are versatile well-defined objects obtained by dissolution of cellulose fibers (or a derivative, which is then converted into cellulose) in a suitable solvent and subsequent regeneration of the same in a non-solvent bath, acquiring spherical forms with diameters ranging from 10 µm up to millimeter scale.1-6 The reactive hydroxyl groups of cellulose beads allow a fine-tuning of chemical properties through a wide range of chemical modifications by the insertion of specific functional groups or interactions with target compounds or ions.3 For instance, the oxidation of hydroxyl groups by optimized reactions with 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) yields cellulose beads rich in carboxyl groups,5,7,8 which serve as effective and selective adsorbents for heavy metal ions adsorption, e.g. silver, copper and lead.3,5 Similarly, cationic cellulose powders were used as adsorbents to selectively retain arsenic (V) and chromium (VI) from water.9,10 On the other hand, biocide agents can also be incorporated to cellulose beads either covalently or by adsorption. Silver ions, silver nanoparticles as well as quaternary ammonium salts (QAS) have been widely studied and exploited owing their bactericidal and fungicide properties.11-17 The biocide action of such materials relies on the interaction between their surface and microorganisms cell membrane. For polycations, while cationic charges of quaternary ammonium induce a loss of natural ions of membrane, the alkyl chain disturbs the lipoprotein interface, destabilizing and causing disruption of cell membrane.12,15,18-21 In the present study the creation and characterization of CB and oxidized CB (OCB) were carried out. The compressive behavior of wet beads and freeze-dried beads (conventional and with solvent exchange water to tert-butanol) was investigated. The main issue arisen here was the effect of oxidation on the adsorption of poly(4-vinyl-Npentyl) pyridinium bromide, QPVP-C5, a bactericidal agent. The interaction between 3 ACS Paragon Plus Environment

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M. luteus, a Gram-positive bacteria, and OCB or OCB coated with small amount of QPVP-C5 was strong enough to keep the bacteria adsorbed on the beads surfaces without disrupting them. Biocidal action was observed only for OCB coated with large amount of QPVP-C5.

Experimental Section Materials Eucalyptus pulp (ECF bleached) and Micrococcus luteus (ATCC 4698), a Grampositive bacteria, were purchased from UPM Kymmene (Uruguay) and Adolf Lutz Institute (Brazil), respectively. Poly(4-vinylpyridine) (PVP, Mw ∼ 60,000 g/mol, DP ∼ 600) and 1-bromopentane (purity 99%) were purchased from Aldrich (Brazil) and used without previous purification. Anhydrous ethanol (98%) was purchased from Labsynth (Brazil) and used as received. Bromide salt of PVP quaternized with linear pentyl chains was prepared in a flask with a reflux condenser by dissolving PVP (10 wt%) in anhydrous ethanol, adding the 1-bromopentane with an excess of 5 times the stoichiometric amount and keeping the mixture stirring under nitrogen atmosphere for 24h at 60 °C.22,23 The mixture was then precipitated in diethyl-ether to obtain a slightly yellow solid that was re-dissolved in ethanol and re-precipitated the same way. The product was washed with a cold HBr 0.1 mol/L solution in order to remove any eventual unreacted parts, washed with diethyl-ether once more, and dried under vacuum at room temperature. The effectiveness of the quaternization was evaluated by Fourier transform infrared (FTIR) vibrational spectroscopy observing the shift of the characteristic pyridine N−C


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stretching band from 1600 to 1640 cm−1, which is typical for pyridinium cations (Supporting Information, SI-1). The resulting polycation was coded as QPVP-C5. Methods Pulp pretreatment In order to ensure a good dissolution of cellulose fibers in a non-derivative solvent, NaOH-urea-water, the pulp was previously submitted to acid hydrolysis in ethanol media, as reported in the literature.24 The pulp amount of 150 g was added into a beaker containing 4 L of HCl (37%):ethanol (1:25, v/v) and the system was kept under moderate mechanical stirring during 3h, at 70 °C. Afterwards, the fibers were filtered, rinsed several times in distilled water and then freeze-dried.

Degree of polymerization The degree of polymerization (DP) of untreated and treated cellulose was determined by using capillary viscometry. Cellulose samples were dissolved in cupriethylenediamine, CUEN, (CUEN:water 1:1, v/v) at five different concentrations. The relative viscosity of each solution was measured in triplicate, at 25.0 ± 0.1 °C, according to an ASTM method.25 The linear coefficient of inherent viscosity versus cellulose concentration chart corresponds to the intrinsic viscosity, [η]. Therefore, DP values were calculated substituting the [η] values in the Mark–Houwink–Sakurada equation:26,27 DP0.905 = 0.75 x [η]

(1)


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Cellulose beads preparation Pretreated and freeze-dried Eucalyptus pulp was used to prepare cellulose beads (CB). Cellulose fibers were added to the dissolution media NaOH-urea-water (aqueous solution of 7 wt% NaOH and 12 wt% urea) so that the final concentrations were 5, 6 and 7 wt%; the resulting beads were coded as CB5%, CB6% and CB7%, respectively. The systems were stirred for 10 min at room temperature, becoming homogenous slurry, and then cooled down to -10 °C for at least 30 min, under moderate stirring. In order to avoid aging effects, all polymeric solutions were kept below zero until use. CB were prepared by dripping the cellulose solution from an Eppendorf 10 mL syringe into a beaker containing HCl 2M as coagulation bath. Parameters as temperature of bath (23 °C), size of beaker (1 L) and volume of acid solution (0.5 L) were kept constant, whereas the distance between the syringe tip and the bath surface was varied in the range of 2 cm to 5 cm aiming spherical beads. The coagulation lasted no longer than 5 min. Coagulated beads remained in the respective coagulation bath overnight so the complete regeneration of the cellulose chains was achieved. Afterwards CB were washed continuously with tap water for 20 min or until neutral pH and then stored in distilled water.

Beads oxidation The procedure for cellulose beads oxidation was based on the studies reported elsewhere.5,7,8,28 The mass of wet cellulose beads equivalent to 1 g of dry cellulose was placed in a screw cap Erlenmeyer flask containing 100 mL of NaH2PO4 50 mM and allowed to stand for 1h. NaClO2 (0.67 g, 7.4 mmol) and 2,2,6,6-tetramethylpiperidineN-oxyl (TEMPO; 0.03 g, 0.19 mmol) was added to 50 mL of NaH2PO4, dissolved and 6 ACS Paragon Plus Environment

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added to the flask. NaClO solution (10%, 0.06 g, 0.74 mmol) was then added to the system preheated in silicon bath at 60 °C, during 15 min. The flask was capped loosely and the reaction carried out for 5, 6 or 7h. Occasionally the system was manually shaken to ensure the homogenization of reaction medium. Afterwards, oxidized cellulose beads (OCB) were filtered, repeatedly rinsed and kept in distilled water.

Beads characterization Mass of wet beads (mwet) was averaged over at least 25 beads. Beads were taken from the storage container filled with distilled water, pre-dried with soft wipes in order to remove water meniscus, and then weighed. Beads were freeze-dried by two different ways: (i) swollen beads were frozen at -15 oC and then lyophilized and (ii) water of wet beads was exchanged by tert-BuOH, which has melting point of 25.5 oC, and then the beads swollen in tert-BuOH were freeze-dried. The latter is a method used to avoid drying artifacts.29 Size and shape of beads were obtained as follows. Images of at least 250 wet beads placed in a Petri dish were acquired with an image scanner. Furthermore, these images were binarized and processed using ImageJ software. Parameters as circularity and diameter (Ø) were obtained directly from these analyses.24 The negative charges contents in CB and OCB were determined by conductivity titration method.5,30 First, 1 g of designed beads was added to a beaker containing 100 mL of NaCl 1 mM and subsequently milled with a hand blender until slurry was obtained. Then, pH of mixture was set to 2.75 by adding known amount of standard HCl 0.1M solution with an automatic dosing system. Metrohm 718 STAT Titrino automatic titration system was used to titrate cellulose dispersions with NaOH 0.1M solution, at the rate of 0.1 mL/min 7 ACS Paragon Plus Environment

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up to pH 11. The remaining dispersion was put into an oven at 100 °C overnight and after that the dried content was weighed. Internal and external morphologies of freeze-dried CB and OCB were analyzed using JEOL 7401 field emission scanning electron microscope (FE-SEM). Beads were cut with a sharp blade at 24 °C and coated with a thin (~ 2 nm) gold layer.

Compressive behavior The compressive tests of wet and freeze-dried beads were performed using an Impac, Digital Dynamometer IP-90DI, with a 10 N load cell and at room temperature. The compression force required causing deformation of 5% the original size of the bead was measured with strain rate of 0.01 s-1, and then the interval of 30 s was taken prior to the next compression, until bead rupture was achieved. Calculations of pressure and ultimate compressive strength were done using the radius values obtained as described above.

Adsorption of polycation QPVP-C5 on beads The amount of wet beads, CB and OCB, equivalent to 30 mg of cellulose was placed in glass flasks containing 4 mL of QPVP-C5 solutions at 0.5, 2.5, 5.0, 12.5 or 17.5 g/L. Flasks were hermetically sealed and left under slightly magnetic stirring at 55 °C for 15h, at pH ~ 6. These conditions were set based on preliminary assays of optimization. Monitoring of the initial and final polycation concentration was done by measuring the absorption intensity at 256.5 nm of solutions before and after adsorption in a Shimadzu UV 2600 UV-Vis spectrophotometer. The amount of adsorbed QPVP-C5 8 ACS Paragon Plus Environment

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(ΓQPVP-C5) was determined through a calibration curve obtained from five different concentrations of specimen at the wavelength 256.5 nm, which corresponds to the maximum absorption of pyridinium groups due to the π → π* electronic transition. The amount of desorbed QPVP-C5 was also determined after beads had been washed and left in 4 mL of distilled water for 24h under orbital shaken. Cellulose spheres were then either immediately used or freeze-dried.

Internal Reflection Spectroscopy - FTIR-ATR Freeze-dried CB and OCB beads were analyzed before and after polycation adsorption by means of Attenuated Total Reflection Fourier Transform infrared (FTIRATR) spectroscopy. IR spectra were recorded in a Nicolet iS50 coupled to a crystal diamond with spectral resolution of 4 cm-1 and 32 scans. Background was taken from the air. At least three samples were measured in triplicate.

Elemental analyses The analysis of element composition of freeze-dried beads was performed using a Perkin Elmer – 2400 CHN Elemental Analyzer for C/H/N determination and a Spectro-Arcos ICP-OES Ciros CCD spectrometer for phosphorous content.

XPS surface analyses XPS measurements were performed for freeze-dried beads with a Physical Electronics PHI Quantum 2000 XPS instrument equipped with monochromatic Al Kα X-ray source. Three different spots were analyzed on each sample. Photoelectrons were 9 ACS Paragon Plus Environment

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collected using electrons detector with the pass energy 187 eV – step size 1.6 eV, in low-resolution survey mode, and 46.95 eV – step size 0.4 eV, in high-resolution mode C 1s. The O/C and N/C ratios were determined from the low-resolution XPS spectra. The high-resolution C1s spectra were deconvoluted to C1, C2, C3 and C4 partial curves using software provided by the instrument manufacturer. The following binding energies relative to C-C position were employed for the respective groups: C1 at 284.8 eV, +1.7 ± 0.2 eV for C-O (C2), +3.1 ± 0.3 eV for C=O or O-C-O (C3), and +4.6 ± 0.3 eV for O=C-O (C4) groups.

Biocide assays The antimicrobial action of beads samples was assessed by a standard protocol described in the literature.31 Three mL of Micrococcus luteus aqueous dispersion at 2.75 g/L (pH ~ 6) interacted with 35 mg of beads for 24 hours. The turbidity of dispersions was monitored before (τi) and after (τf) the contact with beads by spectrophotometry at 650 nm and 25 °C, using a Beckmann Coulter DU-600 equipment. The relative decrease of turbidity (∆τ) might be correlated to bacteria disruption, indicating antimicrobial activity. The larger is ∆τ, the more efficient is the antimicrobial agent: ∆τ = (τf - τi) / (τi) x 100 %

(2)


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Results and Discussion Cellulose beads formation and characterization Eucalyptus pulp was previously treated for 3h, at 70 °C in HCl-ethanol solution in order to partially hydrolyze cellulose chains leading to the decrease of cellulose degree of polymerization (DP) and removal of primary wall of cellulose fibers.24 This is necessary because the dissolution of cellulose in non-derivatizing solvents might require DP < 200.24,32,33 The DP values of untreated and treated pulp calculated from capillary viscometry data were 568 and 116, respectively. Thereafter, treated cellulose was dissolved in 7% NaOH–12% urea aqueous solutions at 5 wt%, 6 wt% and 7 wt%. Cellulose beads coded as CB5%, CB6% and CB7% were then prepared from the solutions by dropping method using HCl 2M solutions as coagulation bath. The final shape of the beads results fundamentally from factors as ejection speed, cellulose solution viscosity and the distance, d, between needle tip and bath surface.3 During the process solely d was manually varied and beads of distinct shapes were obtained as follows, tear-like (d ≤ 2.5 cm), spherical (d = 2.5-3.5 cm), ellipsoidal (d = 3.5-5 cm) and flattened beads (d > 5 cm). However, for this study only spherical beads were chosen (Supporting Information, SI-2). Spherical cellulose beads were oxidized (coded as OCB5%, OCB6% and OCB7%), using TEMPO-mediated method, in which the stoichiometry of reaction was kept constant (AGU:NaClO2:TEMPO:NaClO; 8:10:0.25:1), whereas the time was optimized. The yields of oxidized cellulose were 8%, 12% and 17%, for reaction periods of 5h, 6h and 7h, respectively.

The total charge content for pristine and

oxidized cellulose beads was determined by conductivity titration method (Supporting Information SI-3, Table 1). Pristine beads showed a very low content of negative charges, 6.0 10-3 mmol/g, which might stem from carboxylic acids of residual xylan. On 11 ACS Paragon Plus Environment

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the other hand, the reaction time of 6h with TEMPO increased the charge density to 0.77, 0.75 and 0.78 mmol/g for OCB5%, OCB6% and OCB7%, respectively. Coincidentally under similar oxidizing conditions Saito and Isogai7 reported 0.74 mmol/g of carboxylate content for cellulose cotton linter (Cellulose I), which corresponds to degree of substitution equal to DS = 0.12. No significant changes in the amount of carboxylic groups were observed for the beads obtained from different cellulose solutions concentrations, probably because the oxidation takes place predominantly on the beads surface. In average wet CB showed mean diameter (Ø) of 3.2 ± 0.1 mm and mean volume of 0.018 ± 0.002 cm3 (Supporting Information, SI-3 Table1). The circularity expresses how spherical is a given material by correlating both axis of an ellipse; a perfect sphere has circularity equal to 1. Mean circularity of 0.90 ± 0.01 was determined for CB and OCB. It is worth noting that even using a manual technique for beads production, the CB and OCB presented high circularity values with good reproducibility. On the other hand, the increase in the cellulose concentration from 5 wt% to 7 wt% for CB production led to mass increase from 1.21 ± 0.04 mg to 1.67 ± 0.06 mg per freeze-dried CB. The average mass per bead was not significantly influenced by the solvent used in the freeze-drying process. Moreover, the oxidation reaction time exerted remarkable influence on the conversion of cellulose into carboxylated cellulose. For instance, CB6% submitted to 5h or 7h (OCB6%-5h and OCB6%-7h) TEMPO/NaClO2/NaClO oxidation, presented 0.50 mmol/g and 1.02 mmol/g of charge content, respectively, corroborating with literature reports.5-7 Cross sections SEM images obtained for the beads freeze-dried in water (Figure 1), revealed variations in the morphology according to the concentration of cellulose. The beads interior was constituted of large cavities ranging from 200 µm to 300 µm for CB5% (Figure 1a), 300 µm to 400 µm for CB6% (Figure 1c) and 500 µm to 700 µm for 12 ACS Paragon Plus Environment

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CB7% (Figure 1e), separated by walls 30 µm (CB5%), 30 µm (CB6%) and 40 µm (CB7 %) thick. The oxidized beads (Figures 1b, 1d and 1f) also presented large pores. Such internal structures might have been formed during the ice crystallization process and do not necessarily represent the original pore structures. For this reason, water of wet beads was exchanged by tert-BuOH, and the beads were freeze-dried. As shown in Figure 2, the beads freeze-dried in tert-BuOH presented a more compact inner structure, with pores much smaller than those observed in Figure 1. The mean diameters of CB5%, CB6% and CB7% freeze-dried in water were comparable to those freeze-dried in tertBuOH, resulting in similar density values. On the other hand, oxidized beads freezedried in water and in tert-BuOH presented mean diameters of (3.0 ± 0.1) mm and (2.0 ± 0.1) mm, respectively. As a consequence, the densities of oxidized beads freeze-dried in tert-BuOH were larger than those determined for those freeze-dried in water (Supporting Information, SI-4 Table 2).


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(a)

(b)

(c)

(d)

(f) (e) Figure 1. SEM cross section micrographs of cellulose beads freeze-dried in water. (a) CB5%, (b) OCB5%, (c) CB6%, (d) OCB6%, (e) CB7% and (f) OCB7%.The scale bar corresponds to 1 mm. The oxidized beads resulted from 6h oxidation reaction. 14 ACS Paragon Plus Environment

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(a)

(b)

(c)

(d)

(e)

(f)

Figure 2. SEM cross section images of cellulose beads freeze-dried in tert-BuOH. (a) CB5%, (b) OCB5%, (c) CB6%, (d) OCB6%, (e) CB7% and (f) OCB7%. The white scale bar in the insets corresponds to 1 mm. The yellow scale bar corresponds to 1 µm. The oxidized beads resulted from 6h oxidation reaction.


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The compressive stress–strain curves determined for wet beads, beads freezedried in water and in tert-BuOH are presented in Figures 3a, 3b and 3c, respectively. In the case of wet CB5%, CB6% and CB7%, the deformation increased linearly with the compression up to 35% of deformation (Figure 3a). Under compression water molecules might evaporate from the surface, inducing water concentration gradient, and under high compression water can be released from the beads. The compressive behavior observed for wet OCB5%, OCB6% and OC7% was inferior in comparison to the wet pristine beads (inset in Figure 3a). These findings might be important for practical purposes; the choice between CB and OCB beads would be a compromise between compression resistance (CB are superior to OCB) and total amount of negative charges (larger in OCB than in CB). The compressive behaviors of CB5%, CB6% and CB7% freeze-dried in water (Figure 3b) and in tert-BuOH (Figure 3c) were similar up to 40% strain, probably because they presented comparable density values (Supporting Information, SI-4 Table 2). The linear increase of strain upon compression allowed estimating the corresponding Young’s moduli (Supporting Information, SI-4 Table 2), which ranged from ~ 2.5 MPa to ~ 5 MPa. For strains larger than 40% the CB beads freeze-dried in water starter to fail and fractures could be observed, while those CB beads freeze-dried in tert-BuOH became stiffer. The OCB5%, OCB6% and OCB7% freeze-dried in water presented Young’s moduli one order of magnitude smaller than the corresponding non-oxidized beads; this effect might be due to their lower density values and to microstructure complexity generated by ice crystallization. On the other hand, OCB5%, OCB6% and OCB7% freeze-dried in tert-BuOH presented the largest Young’s moduli (Supporting Information, SI-4 Table 2). As the compression load increased beyond the linear region, the beads became stiffer. Their hardening can be attributed to the collapsing and


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closuring of the porous, also called as densification regime, until the breaking point.34 In the case of CB and OCB freeze-dried in tert-BuOH the Young moduli presented a linear dependence on the density (Supporting Information, SI-5), as observed for polymeric foams.35

200 σ compressive (kPa)

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40

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Figure 3. Compression stress-strain curves determined for wet beads (a), beads freezedried in water (b) and beads freeze-dried in tert-BuOH (c). CB5% (solid square), CB6% (solid circle), CB7% (solid triangle), OCB5% (open square), OCB6% (open circle) and OCB7% (open triangle). The oxidized beads resulted from 6h oxidation reaction. The lines represent the linear fits for the determination of Young moduli values.

Adsorption of QPVP-C5 on beads Considering that CB7% presented the best compression properties among all beads prepared (Figure 3), they and their oxidized counterparts (OCB7%) were chosen as substrates for the adsorption of QPVP-C5. Figures 4a and 4b show that the adsorbed amount of QPVP-C5 (ΓQPVP-C5) onto CB7% and OCB7%, respectively, increased with the increase of QPVP-C5 concentration, favored by ion-dipole and electrostatic interactions. The adsorption of QPVP-C5 onto OCB7% was more pronounced than onto CB7% due to the higher charge density. Ion-pairs formation between quaternary ammonium of QPVP-C5 and carboxylate groups of OCB7% practically avoided


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desorption (less than 5%). It is particularly important, for example, in applications related to drinking water sterilization, where the release of water-soluble beads components might affect human health.36,37

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600

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Figure 4. The amount of adsorbed QPVP-C5 (ΓQPVP-C5) onto (a) CB7% and (b) OCB7% as a function of QPVP-C5 bulk concentration. Red and blue columns stand for amounts of adsorbed and desorbed polycation, respectively.


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The adsorption of QPVP-C5 onto CB7% and OCB7% was confirmed by FTIRATR spectroscopy. All spectra obtained for beads after adsorption-desorption processes presented the typical absorption band at 1640 cm-1, assigned to the C=N stretching vibrations (νC=N) of quaternary pyridine group belonging to QPVP-C5 (Supporting Information, SI-1). Two systems were chosen to be tested against bacteria, namely, QPVP-C5 adsorbed onto OCB7% from QPVP-C5 solutions at 5 g/L and 12.5 g/L; they were coded as OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5, respectively. They were chosen because in both cases the adsorbed amount of QPVP-C5 was high and desorption was as low as 1.0 ± 0.2 %. Considering that the biocide effect depends fundamentally on the presence of quaternary ammonium group on the surface, the chemical composition of OCB7%/QPVP-C5-5, OCB7%/QPVP-C5-12.5 and OCB7% (control) was determined by means of elemental analyses (bulk) and XPS analyses (surface). CHN analyses of OCB7% indicated contents of carbon and hydrogen slightly lower than those expected from empirical formula of ordinary cellulose (Supporting Information, SI-6 Table 3). After oxidation the O/C ratio naturally increases, leading to C and H contents lower than those for non-oxidized cellulose. Instead, H % showed to be slightly higher which indicated water adsorbed onto beads or entrapped inside the beads.38 The N content increased with the adsorbed amount of QPVP-C5; it increased from 0.7 wt%, in OCB7%/QPVP-C5-5, to 1.7 wt%, in OCB7%/QPVP-C5-12.5. Similarly, the increase in the C and H contents was assigned to the alkyl linear chains and backbone of QPVP-C5 adsorbed chains. XPS measurements (Supporting Information SI-6 Table 3 and SI-7) showed the contents of C, O and N on the beads surface (~7 nm of depth). The contents of N on the surface of OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5 amounted to 2.1 wt% and 20 ACS Paragon Plus Environment

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4.1 wt%, evidencing the presence of QPVP-C5 chains on the beads surfaces. Only traces (< 1.5 %) of Na, Ca and Br could be detected on the surface of OCB7%/QPVPC5-5 or OCB7%/QPVP-C5-12.5 beads, indicating that most Na+ ions were exchanged by pyridinium ion.

Bioactivity evaluation The bioactivity of beads containing QPVP-C5 was evaluated against the Grampositive bacteria Micrococcus luteus. Suspensions of microorganism are turbid when dispersed in distilled water due to the micrometric dimension of bacteria. However, upon contact with a biocidal material cell disruption takes place, causing size reduction and, consequently, a relative decrease of turbidity (∆τ). OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5 systems were chosen, considering the low desorption (Figure 4) and relative high polycation content on the surface (Supporting Information SI-6 Table 3). As control experiments the turbidity of bare bacteria dispersion was measured as a function of time (i) without any bead and (ii) in the presence of neat OCB7%. The turbidity of all systems decreased after 24h (Supporting Information SI-8 Table 4). The bare bacteria dispersion became less turbid, presenting ∆τ equal to 23%. This effect might be due to the osmotic lysis suffered by the bacteria dispersed in distilled water. As widely discussed in the literature, the bacteria envelope is porous and allows the transport of ions.15,39,40 An osmotic imbalance between the bacteria and a hypotonic medium, for example, may result in membrane disruption, if its capacity is exceeded by the overabundance of liquid.


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OCB7%/QPVP-C5-5

and

OCB7%/QPVP-C5-12.5 samples strongly affected the turbidity, which reached ∆τ values of 85% and 99%, respectively. These results corroborate with literature data.14,15,41 For instance, Silva and coworkers14 reported the use of QPVP-C5 thin films as effective bactericidal surface against diluted M. luteus suspensions reaching levels of turbidity close to zero (∆τ ~ 100%). The action of cationic biocidal materials can be described in two steps. First, the Gram-positive bacteria are attracted to the material surface through electrostatic interactions between bacteria envelope negatively charged phospholipids and material ammonium quaternary groups.11,42,43 Second, the compensation of the negative charges of the bacterial envelope is provided by the positive charges of the substrate, releasing the bacteria natural counter ions to the medium, causing an entropic gain to the system.19 Thus, solid substrate becomes bactericidal when the number of cationic sites is large enough to remove counter ions from bacteria, inducing disruption of the bacteria envelope.15,19 The biocide activity of OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5 is consistent with the N content on their surfaces (Supporting Information, SI-6 Table 3). Nevertheless, neat OCB7% unexpectedly also led to the reduction of M. luteus suspension turbidity, (∆τ = 65%), even in the absence of polycation on the surface (Supporting Information, SI-8 Table 4). This finding could indicate that either there is a nonconventional biocide mechanism or OCB7% work as sponges. Since there is no evidence that makes cellulose a candidate for antimicrobial material, the latter assumption becomes more likely. In order to test the hypothesis, the beads samples used in the antimicrobial assays were analyzed by scanning electron microscopy (Figure 5). M. luteus, which is spherical with diameter ~ 1 µm, was observed mainly on the surface of QPVP-C5 coated OCB7% beads (Figures 5b and 5c) and, in lesser amount, on neat OCB7% (Figure 5a). Bacteria was practically 22 ACS Paragon Plus Environment

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absent from the surface of OCB7%/QPVP-C5-12.5. However, particularly on OCB7%/QPVP-C5-12.5 some bacteria or bacteria debris were found close to the surface, but buried in the beads, as indicated in Figure 5c. These features suggest that the bacteria, which escaped from disruption, tried to diffuse to the bead interior. Considering that the images obtained from beads deep interior did not evidence their presence (Supporting Information, SI-9), the structures probably correspond to debris of bacterial cells. Such features were observed on the surface of hydrogels made of crosslinked cationic cellulose chains, which presented antibacterial effect against E. coli,44 and on the surface of quaternary ammonium polyethylenimine nanoparticles after interaction with Streptoccocus mutans.45 Thus, the surprising result of ∆τ = 65% determined for OCB7% seems to be due to adsorption of bacteria on beads surface. OCB7% beads carry negative charges on the surface, which would repel the bacteria phosphate groups, but there are many hydroxyl groups from cellulose unmodified chains that might interact with the peptidoglycans in the outermost layer of bacteria by H bonding or ion-dipole interactions.


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(a)

(b)

(c) Figure 5. SEM images of freeze-dried (a) OCB7%, (b) OCB7%/QPVP-C5-5 and (c) OCB7%/QPVP-C5-12.5 surfaces after 24h contact with M. luteus suspensions. The scale bars correspond to 1 µm and the circles highlight the presence of M. luteus or bacteria debris.


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Considering that the membrane of Gram-positive bacteria has teichoic acids in its composition, which are basically formed by polyglycerol phosphate, poly(glucosyl phosphate), or polyribitol phosphate, the quantification of P content in the beads was used to confirm the occurrence of bacteria incorporation in the beads,46,47 as shown in (Supporting Information, SI-9 Table 4). OCB7%/QPVP-C5-5 showed the highest P content (559 ± 11 ppm), followed by OCB7%/QPVP-C5-12.5 (353 ± 16 ppm) and OCB7% (252 ± 17 ppm). As a control, bare OCB7%, which had no contact with M. luteus presented only 4.6 ± 0.2 ppm of P. Thus, the presence of bacteria was more pronounced in OCB7%/QPVP-C5-5, probably due to electrostatic interaction and to the positive charge density on OCB7%/QPVP-C5-5, which was not high enough to cause bacteria disruption. Considering that the largest ∆τ value observed for the OCB7%/QPVP-C5-12.5 sample was due to bacteria envelope rupture, protoplast and phosphorous residues were released to the aqueous medium, explaining the lower P content and bactericidal action. In the case of OCB7%, the P content of 252 ± 17 ppm evidenced the bacteria incorporation. Based on these results one can conclude that after 24h: (i) the bacteria interacted with the beads, leading to reduction of relative turbidity of dispersions and (ii) considering that part of the positive charges of QPVP-C5 interact with the negatively charges on OCB7%, only when the adsorbed amount of QPVP-C5 onto beads is sufficiently high, the exceeding amount of positive charges might cause bacteria adhesion followed by bacteria disruption. Otherwise bacteria might adhere onto bare OCB7% or OCB7% coated with small amount of QPVP-C5 by ion-dipole interaction, without disruption. These trends are depicted in Figure 6. Figure 6a represents the partial adhesion of bacteria on OCB7%, Figure 6b depicts the interaction between bacteria and weakly charged OCB7%/QPVP-C5-5 without bacteria disruption and Figure 6c proposes the 25 ACS Paragon Plus Environment

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adhesion of bacteria onto highly charged OCB7%/QPVP-C5-12.5, followed by bacteria disruption.

(a)

(b)

(c)

Figure 6. Schematic representation of the interaction between bacteria (red solid circles) and beads after 24h. (a) partial adhesion of bacteria on OCB7%, (b) adsorption of bacteria and weakly charged OCB7%/QPVP-C5-5 without bacteria disruption and (c) adhesion of bacteria onto highly charged OCB7%/QPVP-C5-12.5, followed by bacteria disruption.


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Conclusions Cellulose beads were obtained using pretreated Eucalyptus pulp. The subsequent oxidation reaction increased the beads surface negative charge density, but reduced the compressive strength of beads in the wet state in comparison to pristine beads. The density and compressive behavior of pristine beads freeze-dried in water and in tertBuOH were comparable. On the other hand, oxidized beads freeze-dried in water presented poor compressive performance and low density in comparison to those freezedried in tert-BuOH, due to the complex internal pore structure resulting from ice crystallization. The adsorption of the biocidal polycation QPVP-C5 onto oxidized beads was driven by electrostatic interactions between the polycation and carboxylate groups on beads. The adsorbed amount of QPVP-C5 increased with polycation bulk concentration and bead surface charge density. Bacteria adsorbed onto oxidized beads and on beads carrying low adsorbed amount of polycation. On the other hand, beads with high adsorbed amount of QPVP-C5 exhibited biocidal properties. In this study we showcase multifunctional cellulosic materials of low cost and simple preparation for applications in aqueous media with the purpose either of binding or binding and killing Gram-positive bacteria just by tuning the beads oxidation and polycation adsorption conditions.

Supporting Information Available SI-1. FTIR-ATR spectra obtained for QPVP-C5, CB 7% OCB 7% before and after the adsorption QPVP-C5. SI-2. Photographs of typical CB7% and OCB7% beads. SI-3 Characteristics of cellulose beads (CB) and oxidized cellulose beads (OCB): mass, volume and density of wet beads and total negative charges per gram of bead. SI-4. 27 ACS Paragon Plus Environment

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Density (ρ) and Young’s modulus (ε) estimated for beads freeze-dried in water and in tert-BuOH. SI-5. Dependence of the Young moduli on the density determined for CB and OCB freeze-dried in tert-BuOH. SI-6. Elemental composition determined for OCB7%, OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5. SI-7 XPS spectra obtained for OCB7%, OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5. SI-8. Relative decrease of turbidity (∆τ, %) measured for dispersions of M. luteus after 24h contact with OCB7%, OCB7%/QPVP-C5-5 and OCB7%/QPVP-C5-12.5 and P elemental analysis of beads post biocide assay. SI-9. Typical SEM image obtained from the interior of OCB7%/QPVP-C5-12.5 after interacting 24h with bacteria. This material is available at free of charge via the Internet at http://pubs.acs.org

Acknowledgments L. S. Blachechen thanks Science without Boarders Program (Ciência sem Fronteiras) from Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil, for the Sandwich Doctorate scholarship. D. F. S. Petri acknowledges Brazilian Funding Agencies CNPq, FAPESP and CAPES Rede Nanobiotec for financial support. The authors thank Joanna Narewska for the XPS analyses and Jani Trygg for assistance in bead preparation and characterization.


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For Table of Contents Use Only Multifunctional cellulose beads and their interaction with Gram positive bacteria Leandro S. Blachechen, Pedro Fardim and Denise F. S. Petri


Multifunctional Cellulose Beads and Their Interaction with Gram

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