Tailoring the Antimicrobial Response of Cationic Nanocellulose

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Tailoring the antimicrobial response of cationic nanocellulose-based foams through cryo-templating Caio Gomide Otoni, Juliana Figueiredo, Larissa Capeletti, Mateus Borba Cardoso, Juliana da Silva Bernardes, and Watson Loh ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00034 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Applied Bio Materials

Tailoring the antimicrobial response of cationic nanocellulose-based foams through cryo-templating

Authorship Caio G. Otoni*,†, Juliana S.L. Figueiredo†, Larissa B. Capeletti‡, Mateus B. Cardoso‡, Juliana S. Bernardes‡, and Watson Loh*,†

Author affiliations †

Institute of Chemistry, University of Campinas (UNICAMP), P.O. Box 6154, Zip Code

13083-970, Campinas, São Paulo, Brazil (Otoni: [email protected]; Figueiredo: [email protected]; Loh: [email protected]);

‡

Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for

Research in Energy and Materials (CNPEM), Zip Code 13083-970, Campinas, Sao Paulo, Brazil (Capeletti: [email protected]; Bernardes: [email protected]; Cardoso: [email protected]).

* Corresponding authors Watson Loh, Ph. D. and Caio G. Otoni, Ph.D. Institute of Chemistry, University of Campinas (UNICAMP) Caixa Postal 6154, Campinas, SP, 13083-970, Brazil Telephone: +55 19 35213148; Fax: +55 19 35213023 E-mail addresses: [email protected] and [email protected]

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Abstract graphic


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Abstract To shed light on novel sustainable materials with antimicrobial functionality, in this contribution we describe the use of cationic nanocellulose to produce foams featuring antibacterial activity against the powerful human pathogen Escherichia coli. Dialdehyde cellulose was cationized with Girard’s reagent T (GRT), mechanically disintegrated into nanofibrillated cellulose (NFC), and shaped into foams through different protocols. All steps were carried out in aqueous media and in the absence of hazardous chemicals. While evaporative drying led to compact films (density of 1.3 g cm-3), freeze-casting (i.e., freezing and freeze-drying) produced monolithic cryogels with low densities (< 50 mg cm-3) and porosities of ca. 98%. Although highly porous, the cryogels obtained through rapid freezing remarkably presented smaller pores than those that were previously frozen in a slow fashion. The quaternary ammonium groups of GRTcationized NFC removed E. coli to different extents depending upon sample morphology. We innovatively demonstrated that not only porosity, which is directly associated to surface area, but also pore size plays an essential role on the antimicrobial performance. This outcome arises from the inaccessibility of bacterial cells to cationic surfaces inside monoliths comprising small pores. We herein present an uncomplicated, environmentally friendly protocol for fine-tuning the porosity and pore size of all-cellulose materials through cryo-templating. Controlling these morphometric parameters allowed us to achieve a ca. 85% higher anti-E. coli activity when comparing samples made up of the very same material (i.e., same NFC concentration and degree of substitution), but presented as dense films. These findings bear clear implications for the pursuit of sustainable materials presenting multifunctionality. 3 ACS Paragon Plus Environment


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Keywords: cellulose nanofiber; nanofibrillated cellulose; foam; cryogel; aerogel; antibacterial.


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Introduction The increasing requirements for sustainability and multifunctionality in nowadays’ society encourage research on and development of active materials derived from renewable supplies. In this sense, polysaccharides denote promising resources for the production of multifunctional materials due to the high occurrence of hydroxyl groups in their backbones, providing them a high potential for functionalization. In addition to the environmentally friendly aspects that typify most polysaccharides, cellulose stands out due to its multiscale hierarchical structure arising from the self-assembly of nanocelluloses – i.e., cellulose nanocrystals (CNC) or nanocrystalline cellulose (NCC) and cellulose nanofibers (CNF) or nanofibrillated cellulose (NFC) – into higher-order structures featuring low density, high mechanical strength, and good flexibility and thermal stability.1-2 Since the pioneer studies on woody materials by Anselme Payen, reported back in 1839,3 cellulose, cellulose derivatives, and cellulose-based composites have been widely exploited for the production of multifunctional materials suitable to a range of applications, as comprehensively reviewed elsewhere.4-10 NFC denotes a specific class of nanocelluloses that is built up by the alternated association of highly ordered and amorphous domains, being typically obtained through the mechanical disintegration of cellulose fibrils. It combines the aforementioned characteristics of cellulose with high flexibility as well as the inherent features of nanosized materials, particularly high specific surface area. This further potentiates the abundance of hydroxyl groups and broadens the possibilities for surface modifications, including cationization for introducing antimicrobial functionality.11 Indeed, few are the examples of naturally occurring cationic species, which do not include cellulose. 5 ACS Paragon Plus Environment


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However, it is possible to produce cationic cellulose with potential antimicrobial properties through the physical immobilization or chemical anchoring of nisin,12-14 the grafting of L-lysine moieties,15 the adsorption of chitosan or polyvinylamine layers,16-17 and the covalent attachment of quaternary ammonium compounds (QAC), such as (2,3epoxypropyl)trimethylammonium chloride (EPTMAC)18-20 and (2-hydrazinyl-2-oxoethyl)trimethylazanium chloride (Girard’s reagent T, hereafter referred to as GRT).21-22 QAC have been extensively demonstrated to inhibit the growth of bacteria, fungi, and viruses.23 In the case of bacteria, specifically, positively charged species are believed to disrupt the ionic integrity of cell membrane by electrostatically interacting with the negatively charged phospholipid bilayer and leading to the cationic displacement of divalent cations.24 Quaternization involving the permanent grafting of QAC onto cellulose is of particular interest for preventing the active compound to leach out to the environment. This, in turn, fades concerns on their potential toxicity and resistance as well as prolonging the lifetime of the antimicrobial cellulosic material.17 Regarding cytotoxicity, it is worth mentioning that mammalian cell membranes are electrically neutral, making these cells not as susceptible to cationic species as bacterial cells.20, 25 Even if cellulose backbone comprises plenty of hydroxyl groups (three per anhydroglucose unit), thus being expected to be highly reactive, most of them are tightly connected through hydrogen bonds. In this sense, the sterically hindered hydroxyls have limited accessibility to reactants and, as a result, depleted reactivity.26 Periodate oxidation has been used as a means of introducing highly reactive aldehyde groups into cellulose macromolecules by regioselectively oxidizing the vicinal secondary hydroxyl groups at carbon atoms 2 and 3, which were originally bonded.27 Dialdehyde cellulose 6 ACS Paragon Plus Environment

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(DAC) is more prompt to further derivatization, including Schiff base reactions for imine bond formation upon reaction with GRT.21 Other oxidation and quaternization strategies have been proposed, but this route is particularly relevant because both oxidation with sodium periodate and cationization with GRT are carried out in aqueous medium and involve mild conditions as well as inexpensive chemicals (except for sodium periodate that can be easily regenerated and recycled).28 The pioneer cellulose cationization by GRT dates back to 2003 and it was intended to improve the binding power and wet strength of paper and tissue products.29 However, to the best of our knowledge, limited information is available on the antimicrobial action of GRT-cationized nanocellulose. The elongated aspect of NFC (aspect ratio may achieve several hundreds)30 grants their aqueous dispersions high entanglement and gel-like behavior even at low solid contents (overlap concentrations as low as 0.01-0.05 wt.% have been reported).31-32 This opens up the possibility of producing, upon the controlled removal of the dispersant medium, lightweight foams and foam-like materials (namely aerogels, cryogels, and xerogels). Porous nanocellulose-based materials have been extensively investigated recently for a wide range of applications, including thermal and acoustic insulations,33-34 (bio)medicine and tissue engineering,35 supercapacitor for energy storage,36 superabsorbency,37 selective removal of contaminants from wastewater,38 and antimicrobial materials.17, 39 In this context, this study was devoted to the cationization of nanocellulose using GRT as well as the pioneer production of contact-active antimicrobial foams envisaged to be relevant for air and liquid filtration, absorbency, functional coatings, and products for healthcare, hygiene, and packaging. Because freeze-casting has been demonstrated to 7 ACS Paragon Plus Environment


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play a role on the morphology and mechanical performance of nanocellulose-based foams,40-41 it also set out to investigate the effects of the foam-forming protocol on sample morphology and to elucidate whether or not morphological aspects play a role on the antibacterial performance of the herein produced multifunctional materials.


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Experimental Section Materials Commercial bleached Eucalyptus kraft pulp (initial moisture content of ca. 55 wt.%) was kindly provided by Suzano Papel e Celulose (Limeira, Brazil). The following chemicals were bought from Sigma-Aldrich Co. (St. Louis, MO) and used as received: 99% pure (2-hydrazinyl-2-oxoethyl)-trimethylazanium chloride (GRT; CAS No. 123-46-6), 99.8+% pure sodium periodate (NaIO4; CAS No. 7790-28-5), 98% pure hydroxylamine hydrochloride (NH2OH.HCl; CAS No. 5470-11-1), and grade II glutaraldehyde solution (CAS No. 111-30-8). Water (18.2 MΩ cm) was deionized with a Milli-Q system (Barnstead Nanopure Diamond, Van Nuys, CA).

Surface modification of cellulose pulp Cellulose pulp was firstly oxidized using sodium periodate and thereafter cationized with GRT according with the procedure described by Sirviö et al.,21 schematically illustrated in Scheme 1, with modifications based on preliminary experiments for optimization purposes. Never-dried cellulose pulp was dispersed in ultrapure water at 2.5 wt.% and heated up in a water bath at 50 °C. Sodium periodate was then added at 1 mmol g-1 (dry basis) under mechanical stirring in the absence of light to prevent photochemical degradation of sodium periodate. After 3 h of reaction at 50 °C under mechanical stirring, the suspension was filtered and thoroughly washed with ultrapure water.



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Scheme 1. Summary of the synthetic pathway for oxidation and cationization of cellulose with sodium periodate and Girard’s reagent T, respectively. Reproduced with permission from reference 21. Copyright 2011 Elsevier.

Periodate-oxidized cellulose pulp was then cationized with GRT. The aqueous suspension of oxidized pulp was again adjusted to 2.5 wt.% and its pH was adjusted to 4.5 using 0.1 M HCl solution. Then, the oxidized pulp was allowed to react with 1 mmol g-1 (dry basis) of GRT for 1 h at 55 °C under mechanical stirring. The cationized product was thoroughly washed and dialyzed against water to eliminate unreacted chemicals.

Mechanical disintegration of cationic cellulose pulp Never-dried pristine (i.e., surface unmodified), sodium periodate-oxidized, and GRTcationized cellulose pulps at 2.5 wt.% were disaggregated using a flat-based blender for 10 min at 10,000 revolutions. The solid contents of the cellulose dispersions were adjusted to 3 wt.% and then these were passed 30 times through a MKCA6-5 SuperMasscoloider grinder (Masuko Sangyo Co., Ltd., Kawaguchi, Japan) at ca. 1500


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rpm and using a decreasing gap width between disks to produce their nanofibrillated cellulose analogues named NFC, oNFC, and cNFC, respectively.

Apparent zeta (ζ) potential measurements Cellulose nanofibers were dispersed in ultrapure water (0.1 wt.%, pH 6.5), added by 10 mM NaCl as recommended by Foster et al.,42 sonicated for 10 min in an ultrasound bath, and submitted to electrophoretic mobility measurements on a ZetaSizer Nano ZS (Malvern Panalytical Ltd., Malvern, UK) at 25 °C. Three measurements were performed for each sample.

Determination of degree of substitution Because GRT was the unique component comprising a nitrogen atom in its structure, NFC and cNFC was frozen and freeze-dried into powder and submitted to elemental analysis to quantify the nitrogen content on a 2400 CHNS/O Series II analyzer (Perkin Elmer Inc., Waltham, MA). The aldehyde content was determined through elemental analysis after the oxime reaction among the aldehyde groups in oNFC and hydroxylamine hydrochloride (Scheme 2) at pH 4.5 and room temperature for 48 h, and under magnetic stirring, as described in details by Sirviö et al.27 The content of quaternary ammonium groups in cNFC was directly determined by measuring the total nitrogen content.



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Scheme 2. Oxime reaction between aldehyde groups of periodate-oxidized cellulose and hydroxylamine hydrochloride (NH2OH·HCl). Reproduced with permission from reference 27. Copyright 2011 Elsevier.

Surface elemental composition X-ray photoelectron spectroscopy (XPS) was used to provide further insights on the surface chemical composition of pristine and surface modifies NFC samples, which were frozen, freeze-dried, and analyzed on a K-Alpha spectrometer (Thermo Fisher Scientific, UK) operating with monochromatic Al Kα radiation. Survey spectra were acquired using a pass energy of 200 eV. At least two specimens were analyzed per treatment by focusing the 300-μm-diameter X-ray beam at two random spots within each specimen. Acquired data were processed using the Thermo Avantage software, version 5.921 (Thermo Fisher Scientific) and the contents of carbon, oxygen, and nitrogen are reported as average values.

Fourier-transform infrared spectroscopy (FTIR)


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Frozen and freeze-dried NFC, oNFC, and cNFC samples were also characterized on a Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, CA) operating with an attenuated total reflectance (ATR) module. Spectra were acquired at wavenumbers ranging from 4000 to 400 cm-1 at a resolution of 2 cm-1, and 128 spectra were accumulated for each sample.

Atomic force microscopy (AFM) Dispersions of pristine and surface modified NFC in water were deposited onto mica and allowed to dry overnight before being imaged on an AFM, model MultiMode VIII (Bruker AXS GmbH, Germany) equipped with a NanoScope V controller and a Si/Au probe, model ScanAsyst-Fluid (Bruker AXS GmbH). Images were generated using PeakForce tapping mode, a nominal resonance frequency of 150 kHz and a nominal force constant of 0.7 N m-1 and was processed in Gwyddion software, version 2.52.

Foam-forming protocol Never-dried NFC samples were mixed with proper amounts of ultrapure water to produce 1.5 wt.% dispersions, which were mechanically stirred (model 713D, Fisatom Equipamentos Científicos Ltda., São Paulo, Brazil) at 1500 rpm for 15 min at room temperature. Then, 2.5 g of such dispersions were poured into 14-mm-diameter, 18mm-high cylindrical molds and allowed to freeze and/or dry for 48 h at the conditions depicted in Table 1.



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Table 1. Conditions used to produce foams from pristine nanofibrillated cellulose (NFC) and cationic NFC (cNFC) aqueous dispersions using polypropylene (PP) or copper molds. Freezing Drying temperature Sample*

Mold material

Temperature

Time /°C

/°C

/h

NFC_SF1

PP

-10 ± 1

24

-58 ± 1

cNFC_ED

PP

-

-

40 ± 1

cNFC_RF

Cu

-196

0.25

-58 ± 1

cNFC_SF1

PP

-10 ± 1

24

-58 ± 1

cNFC_SF7

PP

-10 ± 1

168

-58 ± 1

* Pristine nanofibrillated cellulose (NFC) and Girard’s reagent T-cationized NFC (cNFC) submitted to evaporative drying (ED) or freeze-drying preceded by rapid freezing (RF) or slow freezing for 1 day (SF1) or 7 days (SF7).

Physical properties For volume calculations, the diameters of the circular bases of the dried cylindrical samples were measured to the nearest 0.01 mm with a digital caliper (Mitutoyo Sul Americana Ltda., Suzano, Brazil). The heights of monolithic samples were measured with the same caliper, while the thickness of flat samples was measured to the nearest 0.001 mm with a digital micrometer (Mitutoyo Sul Americana Ltda.). All measurements 14 ACS Paragon Plus Environment

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were performed at least in triplicates, and the average values were used to calculate the volume of the cylindrical samples. Volume shrinkage upon drying was calculated as follows (Eq. 1, where 𝑉𝑓𝑜𝑎𝑚 is the volume of the dried foam and 𝑉𝑚𝑜𝑙𝑑 is the volume of the mold that was occupied by the dispersion prior to drying):

𝑆ℎ𝑟𝑖𝑛𝑘𝑎𝑔𝑒 (%) = (1 −

𝑉𝑓𝑜𝑎𝑚 𝑉𝑚𝑜𝑙𝑑

) ∙ 100

(1)

Bulk density (𝜌𝑓𝑜𝑎𝑚 ) was calculated by dividing the mass of a dried sample, determined to the nearest 0.00001 g on a scale (model AP250D, Ohaus Corp., Parsippany, NJ), by its 𝑉𝑓𝑜𝑎𝑚 . Sample porosity was calculated as follows (Eq. 2, where 𝜌𝑠𝑘𝑒𝑙𝑒𝑡𝑎𝑙 is the skeletal density of cellulose: 1500 mg cm-3):

𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 (%) = (1 −

𝜌𝑓𝑜𝑎𝑚 𝜌𝑠𝑘𝑒𝑙𝑒𝑡𝑎𝑙

) ∙ 100

(2)

The foam presenting the best antimicrobial response was used for water absorption and wet resilience tests. Briefly, dried foams of a known mass (wd) were allowed to swell in water without stirring for 24 h and weighed again (ws). Then, these were dipped again in water and magnetically stirred at ca. 200 rpm for another 24 h, when the remaining foam was dried and weighed (wf). Water-holding capacity and wet resilience were



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estimated by swelling degree (Eq. 3) and mass retention after redispersion (Eq. 4), respectively.

𝑤𝑠

𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑑𝑒𝑔𝑟𝑒𝑒 (𝑔 𝑔−1 ) = (

− 1)

(3)

) ∙ 100

(4)

𝑤𝑑

𝑀𝑎𝑠𝑠 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 (%) = (1 −

𝑤𝑓 𝑤𝑑

X-ray micro-computed tomography (XRµCT) High-resolution XRµCT projections were acquired on a SKYSCAN 1272 (Bruker microCT, Kontich, Belgium) scanner operating at source voltage and current of 20 kV and 175 µA, respectively. The NRecon software, version 1.6.10.4 (Bruker microCT), was used to reconstruct projections into three-dimensional images, which were processed using the CTVox software, version 3.3 (Bruker microCT), for 3D viewing and cutting, and CTAn software, version 1.13 (Bruker microCT), for quantifying morphometric parameters (e.g., porosity). Two-dimensional images were processed using the DataViewer software, version 1.5.6.2 (Bruker microCT). ImageJ software, version 1.52a,43 was used to measure the equatorial diameter of at least 250 circular pores randomly selected in at least four cross-sectional layers within specimen height. For quasi-circular pores, both the maximum and minimum Feret diameters were measured and averaged, being the mean value considered for calculations.


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Antimicrobial activity A single colony of Escherichia coli (DH5α) bacteria was collected from an agar plate grown at 37 °C during 16-20 h and transferred to 5 mL of Luria Bertani broth (LB, containing 10 g L-1 of peptone and NaCl, and 5 g L-1 of yeast extract). After incubation of this mixture overnight at 37 °C and 250 rpm in an orbital shaker, the bacteria were separated from culture medium by centrifugation at 4000 rpm during 5 min. The pellet was washed twice by redispersion in 10 mL of saline solution (NaCl 0.9%) followed by another centrifuging cycle. The final pellet was then resuspended in 2 mL phosphatebuffered saline (PBS) and the optical density of the suspension was determined at 600 nm (OD600). For bacterial susceptibility tests, samples were first embedded in a certain amount of sterile water in a test tube, which was then added by the same volume of LB broth suspension with bacteria. All test suspensions had an initial concentration of 106 colony-forming units per mL (CFU mL-1) as well as 10 mg mL-1 of sample. The weight of sample and the volume of bacterial suspension used in all experiments were precisely the same, i.e., a given mass of sample was always exposed to the same number of CFU, allowing proper comparison. After incubating the initial suspension for 18 h at 37 °C and 250 rpm in an orbital shaker, the supernatant was sampled and the antimicrobial response was evaluated by counting the number of E. coli CFU on LB agar plates. The same procedure was carried out in parallel for pure sterile water, which was used as control. A dispersion of never-dried cNFC (i.e., GRT-cationized NFC that did not undergo any foam-forming procedure) was tested likewise for comparison purposes. The microbiological assays were performed in three repetitions, each of these being carried out at different days and using fresh E. coli suspensions. In each repetition, all 17 ACS Paragon Plus Environment


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treatments (including blank and control) were simultaneously incubated, plated, and counted in triplicates. The reported figure refers to the average and standard deviation values among the three repetitions.

Scanning electron microscopy (SEM) Dried foams were cryo-sectioned in liquid nitrogen using a razor blade and fixed onto stubs using a carbon conductive adhesive tape and then gold-sputtered on a BAL-TEC SCD 050 Sputter Coater equipped with a planetary drive and a rotary stage. A ca. 16nm-thick gold layer was deposited for 60 s at 40 mA. Samples were then imaged on an Inspect F50 scanning electron microscope (FEI Company, Hillsboro, OR) operating at an acceleration voltage of 5 kV and high vacuum as well as using secondary electrons (SE) mode. After incubation, foams were washed with PBS, fixed overnight in a 4% solution of glutaraldehyde, washed twice again with PBS, and dehydrated by ascending ethanol solution grades at 25, 50, 75, and 100 vol.% (twice), 1 h each. Ethanol was allowed to evaporate for 48 h in a desiccator before samples were cryo-sectioned, goldcoated, and imaged (surfaces and cross-sections) as described above, except for the acceleration voltage that was 2 kV.

Statistical treatment of data


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MINITAB® Release 14.12.0 (Minitab Inc., U.S.), was used to submit quantitative data to one-way analysis of variance (ANOVA) at 5% of significance. If p < 0.05, Tukey’s multiple comparison test was carried out at 5% of significance as well.



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Results and Discussion Overview of nanofibrillated celluloses Apparent ζ potential values are presented in Table 2. The negative charge value on pristine cellulose is expected due to the presence of carboxyl groups mostly associated with hemicelluloses and lignin, which remain in residual amounts even after pulping treatments.44 Similar ζ potential values (-19.1 ± 0.4 mV) have been reported for sonicated aqueous dispersions of Eucalyptus micro/nanofibrils.45 Apparent ζ potential values of periodate-oxidized samples remained negative, because aldehyde groups do not show liquid charges, whereas their GRT-cationized counterparts showed positive apparent ζ potential, confirming the grafting of positively charged groups onto NFC surface.26 Cationization of NFC surface was also confirmed by XPS (Table 2), provided that nitrogen was only detected in cNFC. This was accompanied by an increase in the atomic percentage of carbon and a decrease in the content of oxygen (Table 2), as each grafted GRT molecule provides oNFC with extra five atoms of carbon while not altering oxygen content (Scheme 1), the latter being reduced only in relative terms. Typical XPS spectra are presented in Figure S1 (Supporting Information). Finally, NFC cationization was corroborated by FTIR. As expected, representative spectra of all samples (Figure S2, Supporting Information), regardless of surface modification, were characteristic of cellulose, as indicated by broad peaks at 3660-3000 (O–H stretching) and 3000-2800 cm-1 (C–H stretching) as well as sharp peaks at 1427 and 1367 (C–H deformation), 1336 (O–H in-plane deformation), 1315 (CH2 wagging), 1202 (O–H deformation), 1158 (C–O–C asymmetric stretching), 1054 and 1030 (C–O stretching), 1106 and 897 cm-1 (glucose ring stretching).46 NFC and oNFC showed remarkably 20 ACS Paragon Plus Environment

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similar spectra, except for the peaks at 1720 and 868 cm-1 in the latter due to nonconjugated C=O stretching assigned to aldehyde carbonyl group, hemiacetals, and hydrated forms of aldehyde groups, all introduced upon periodate oxidation.21, 47-48 cNFC spectrum differs from the previous ones due to the shoulders at 1480 and 1411 cm-1, assigned to the methyl group and C–N bonds in the quaternary ammonium group from GRT molecule, respectively;49 the shoulder at 924 cm-1, characteristic of N–N bonds from GRT;21, 26, 50 and the peak at 1686 cm-1, attributed to the C=N double bond vibration from GRT with possible overlapping with the residual carbonyls from oNFC.26, 51

The broad peak at 1644 cm-1 could arise from adsorbed water46 or N–H bond

bending,51 while the peak at 1318 cm-1 (more intense, in relative terms, when compared to that at 1315 cm-1 in NFC and oNFC) could be an overlapping of the CH2 wagging and the C–N bond vibration.26 Together, ζ potential, XPS and CHNS/O elemental analyses, and FTIR demonstrate the successful oxidation of NFC into oNFC using sodium periodate and the cationization of DAC into cNFC using GRT.

Table 2. Apparent ζ potential, degree of substitution, and contents of carbon, oxygen, and nitrogen in pristine nanofibrillated cellulose (NFC), sodium periodate-oxidized NFC (oNFC), and Girard’s reagent T-cationized NFC (cNFC). Sample

NFC

Apparent ζ

Degree of

potential

substitution

Carbon

Oxygen

Nitrogen

/mV

/mmol g-1

/at.%

/at.%

/at.%

-16 ± 2 a

-

65 ± 2 ab

35 ± 2 ab

-

Surface elemental composition



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oNFC

-9 ± 1 b

0.60 ± 0.01 a

62 ± 1 a

38 ± 1 a

-

cNFC

+12 ± 1 c

0.25 ± 0.02 b

67 ± 1 b

31 ± 1 b

1.3 ± 0.3

a-c

Average ± standard deviation values followed by different letters within a column

indicate different values (p < 0.05).

Table 2 also shows the content of substituent groups on NFC surface. By using an anhydroglucose unit (AGU)/sodium periodate molar ratio of 0.41, an aldehyde content of 0.6 mmol g-1 was achieved after 3 h at 55 °C. As for the cationization, we used GRT at a concentration of 1 mmol g-1 (i.e., 1-fold excess relative to aldehyde groups) and achieved an effective cationicity of 0.25 mmol g-1. This suggests that about 42% of the aldehyde groups were grafted with quaternary ammonium groups, and a substantial amount of aldehyde groups remained unreacted. A considerably smaller concentration of sodium periodate was used here in comparison with other periodate oxidations reported in the literature.21,

27

In similar reaction

conditions (3 h at 50 °C), Sirviö, Hyvakko, Liimatainen, Niinimaki and Hormi

27

used an

AGU/periodate molar ratio of 1.6 to oxidize birch cellulose and obtained an aldehyde content of 1.7 mmol g-1. Similarly, a cationicity of 1.93 mmol g-1 was achieved by Sirviö, Honka, Liimatainen, Niinimäki and Hormi

21

when using a highly oxidized birch cellulose

(aldehyde content of 13.67 mmol g-1) as well as a 3.9-fold excess GRT. In this sense, even though we prioritized low consumption of chemicals and mild surface modification conditions in order to be in line with industrial operations, oxidation/cationization degree and charge density may be easily tuned. Surface modifications were carried out prior to


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mechanical disintegration because the provided electrical charges are known to improve the nanofibrillation efficiency due to the electrostatic repulsion among fibrils.19, 22

One might perform surface modifications as both pretreatment and post-treatment for

mechanical disintegration in order to increase surface charge density. Although technically feasible, this would require multiple steps and potentially compromise the economic feasibility of this product. Also, it is to be noted that too harsh oxidation and cationization conditions might lead to cellulose dissolution in water and loss of its hierarchical, fibrous characteristic.21 In fact, the morphological integrity and fibrous aspect of pristine NFC were maintained after surface modifications through the mild oxidation and cationization conditions used in the present study, as demonstrated by AFM (Figure 1) and SEM (Figure S3, Supporting Information) images.



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Figure 1. Atomic force microscopy images of pristine nanofibrillated cellulose (NFC, left) and Girard’s reagent T-cationized NFC (cNFC, right). Imaged areas: 3 x 3 µm2 (top; white scale bars: 500 nm) and 10 x 10 µm2 (bottom; black scale bars: 1000 nm).

Foam preparation and physical properties


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Aqueous dispersions of pristine and GRT-cationized NFC were used to produce foamlike materials. As expected, the different foam-forming protocols led to two-dimensional or monolithic samples featuring remarkably different morphologies, which are shown in Figure 2 and whose physical properties are presented in Table 3.

a

b

c

d

e

Figure 2. Monoliths made up of pristine (a, NFC_SF1) and Girard’s reagent Tcationized (b-e) nanofibrillated cellulose and produced through evaporative drying (b, cNFC_ED) or freeze-drying preceded by rapid freezing (c, cNFC_RF) or slow freezing for 1 d (a and d, cNFC_SF1) or 7 d (e). Scale bar: 10 mm.

Table 3. Physical properties of monoliths made up of pristine nanofibrillated cellulose (NFC) and Girard’s reagent T-cationized NFC (cNFC) and produced through evaporative drying (ED) or freeze-drying preceded by rapid freezing (RF) or slow freezing for 1 d (SF1) or 7 d (SF7). Sample

Volume

Shrinkage

Bulk density

/cm3

/%

/mg cm-3

Porosity /%A

Pore size /%B

/µm



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NFC_SF1

1.7 ± 0.4 b

40 ± 14 b

27 ± 7 a

98.2 ± 0.5 c

83.5

146 ± 85

cNFC_ED

0.022 ± 0.001 a

99.19 ± 0.02 c

1270 ± 28 c

15 ± 2 a

0.03

NDC

cNFC_RF

2.4 ± 0.1 c

13 ± 3 a

25 ± 1 a

98.3 ± 0.1 c

87.4

30 ± 17

cNFC_SF1

1.8 ± 0.2 b

36 ± 6 b

33 ± 3 a

97.8 ± 0.2 c

89.1

170 ± 65

cNFC_SF7

1.4 ± 0.1 b

48 ± 4 b

44 ± 3 b

97.0 ± 0.2 b

76.9

264 ± 99

a-c

Average ± standard deviation values followed by different lowercase letters within a

column indicate different values (p < 0.05); A

Calculated by Eq. 2;

B

Calculated by CTAn software, based on reconstructed XRµCT images;

C

Pores were not detected by XRµCT even at its highest resolution (pixel size: 4 µm).

Periodate oxidation has been demonstrated to weaken cellulose chains by opening the anhydroglucose ring, possibly leading to chain scission once mechanically disintegrated.52 Then, while materials made up of lower aspect ratio NFC might present weakened mechanical properties, aldehyde groups of DAC are known to crosslink with hydroxyl groups of neighboring fibrils into hemiacetals,34 potentially counterbalancing the deleterious effect of oxidation on mechanical properties upon compression. Indeed, the Young’s modulus (Figure S4, Supporting Information) of NFC_SF1 and cNFC_SF1 (164 ± 34 and 188 ± 53 kPa, respectively) were equal (p > 0.05), indicating no effect of the combination between oxidation and cationization on monoliths’ stiffness. These compression moduli are comparable with values reported for similar systems, e.g., 180 and 249 kPa for foams fabricated with NFC suspensions at 1.2 and 1.9 wt.%, respectively,53 as well as 69 and 207 kPa for regenerated cellulose-based aerogels 26 ACS Paragon Plus Environment

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originated from 2.0 and 2.5 wt.% solutions, respectively.54 Therefore, from the rigidity standpoint, the herein produced materials should be suitable for a range of industrial applications.55 Concerning morphology, unlike the white, opaque three-dimensional foams obtained through freeze-casting, the NFC dispersion submitted to evaporative drying presented an extremely high volume shrinkage, resulting in a yellowish film featuring low porosity and high density, similarly to previous reports on cellulose-based xerogels.35 Apropos, within the class of solid foam-like materials, such films are closer to xerogels, which classically have porosities ranging from 15 to 50% as well as a combination among small mesopores and micro-sized pores.35 Indeed, volume shrinkages of ca. 89-93% (depending on the dispersant medium) have been reported for NFC-based xerogels.56 The strong pore collapse arises from the high capillary stresses developed during the evaporation of water molecules and leads to high shrinkage and density as well as low porosity and specific surface area.35, 56 The compact structure of the evaporative dried sample is corroborated by the strong X-ray attenuation in XRµCT (Figure 3). Conversely, freeze-casting led to quasi-cylindrical monolithic samples (Figure 2) that experienced remarkably low volume shrinkages upon drying, therefore featuring low densities and high porosities (Table 3). Differently from evaporative drying, freezedrying preserves the open porosity created by ice sublimation under high vacuum. In this sense, as consequence of the growth of ice crystals, neighboring NFCs are pushed together into compact walls, creating pores that are as large as the ice crystals themselves.35 Cryo-templating was then used to tailor the porous structure of the NFCbased monoliths. Dispersions submitted to slow freezing created cryogels that shrunk 27 ACS Paragon Plus Environment


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just as well (p > 0.05), therefore presented the same volume (p > 0.05). Rapidly frozen cryogels, in turn, showed a much less pronounced volume shrinkage (p < 0.05), resulting in the bulkiest and most porous (hence, the least dense) monoliths.


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Figure 3. Three-dimensional (left, side view, and center, interior view) and crosssectional (right) X-ray micro-computed tomography images of monoliths made up of pristine nanofibrillated cellulose (NFC) and Girard’s reagent T-cationized NFC (cNFC) and produced through evaporative drying (ED) or freeze-drying preceded by rapid freezing (RF) or slow freezing for 1 d (SF1) or 7 d (SF7). For sample positions, please refer to Figure S5 (Supporting Information). Scale bars: 1 mm.

Antimicrobial activity The antimicrobial activities of the herein produced samples were tested against E. coli, a Gram-negative bacterium that has been extensively studied and characterized and with some strains that are known as human pathogens.57 The percentages of E. coli CFU that remained in suspension after incubation with the monoliths are presented in Figure 4, wherein the isolated effects of cationization, porosity, and pore size can be discerned by comparing samples highlighted in red, green, and blue, respectively.


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Figure 4. Escherichia coli colony-forming units that remained in suspension after incubation alongside monoliths made up of pristine nanofibrillated cellulose (NFC) and Girard’s reagent T-cationized NFC (cNFC) and produced through evaporative drying (ED) or freeze-drying preceded by rapid freezing (RF) or slow freezing for 1 d (SF1) or 7 d (SF7), as compared to control (sample-free suspension of E. coli cells) and aqueous dispersion of never-dried (ND) cNFC. The roles played by cationization, porosity, and pore size are depicted in red, green, and blue, respectively.

The samples labeled as cNFC_ED (evaporative dried into a cNFC film) and cNFC_ND (suspension of never-dried cNFC) were taken as boundary porosity condition, i.e., as 31 ACS Paragon Plus Environment


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analogues of ideal monoliths featuring quasi-zero and quasi-total porosities, respectively. Indeed, the latter presented a higher anti-E. coli efficiency than the former, indicating that sample morphology plays a role on the antibacterial performance. Consequently, the effect of cationization itself must be investigated by comparing samples produced through the same protocol, but using dispersion of pristine and cationic NFC, i.e., NFC_SF1 and cNFC_SF1, respectively. As expected, the quaternary ammonium groups provided cNFC with antimicrobial activity against E. coli (Figure 4). Differently from sessile cations, when grafted onto surfaces (e.g., NFC), these cationic compounds have limited diffusion and are believed to play their antibacterial roles by the “phospholipid sponge effect”, in which they subtract anionic phospholipids from bacterial cell membranes, creating holes and impairing their structural integrity.58-59 The interaction among cNFC and E. coli cells is evidenced in Figure 5 – typical E. coli cells are presented in Figure S6 (Supporting Information) for comparison purposes.


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Figure 5. Scanning electron microscopy image of the surfaces of foams made up of pristine (left) and Girard’s reagent T-cationized nanofibrillated cellulose (right), with arrows indicating representative Escherichia coli cells. Magnification: 10,000 x. Scale bars: 5 µm.

After removing the cNFC_SF1 monolith from the LB broth, the medium was replaced 9 times during fixation/dehydration process, and there was still a crowded population of E. coli cells that were not leached out and remained adhered to the cationic surface, which further corroborates the strong cNFC/bacterial cell interaction. No E. coli cells were detected on the surface of NFC_SF1 monolith after incubation, fixation, washing, and drying. It is hypothesized that, due to the weak interaction between unmodified NFC and bacterial cells, the latter were washed out during the fixation/dehydration steps. Actually, pristine NFC in expected to neither interact nor inactivate bacteria, and this was corroborated here and elsewhere.20 Even though it was not significant, the minor trend of decreasing E. coli counts observed here might be related to biomolecular corona from LB medium onto the residual, negatively charged hemicellulose.60 When comparing the level of bacterial removal by the cNFC-based samples (all made up of the same formulation, i.e., NFC morphology, cationicity, and concentration in foam-forming dispersion), it is clear that the morphology of the material intended for antimicrobial purposes is a key aspect. The evaporative dried sample showed a slight antimicrobial effect when compared to control, but not significantly different from the monolith made up of pristine NFC (NFC_SF1). The rapidly frozen cryogel, in turn,



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presented significant E. coli removal (counts were lower than both control and NFC_SF1). This difference is attributed to the different porosities of such samples. In the dense film featuring extremely low porosity (Table 3), the area available for cNFC/bacterial cell interaction is basically its two faces, whereas in the highly porous cNFC_RF cryogel the surface area is remarkably increased. When cNFC was frozen in a slow fashion for 24 h (cNFC_SF1), the antibacterial response was further increased (Figure 4). If one compares the antimicrobial outcome of such sample with that of cNFC_ED, a straightforward conclusion may be drawn regarding the effect of porosity. Nevertheless, the cNFC_RF and cNFC_SF1 monoliths feature the same porosity (p > 0.05), suggesting that this is not the unique morphometric parameter affecting antimicrobial activity. As a matter of fact, should porosity be high (and so would specific surface area) but the inner surface not be accessible to bacterial cells, and given that GRT immobilization onto NFC is permanent, the cationic surface of cNFC would be underexploited. A point that should be taken into account is pore interconnectivity, which was high in all samples, except in cNFC_ED, according to three-dimensional XRµCT reconstructions (percentages of closed porosity lower than 1% in cNFC_RF, cNFC_SF1, and cNFC_SF7). Another point that may prevent bacterial cells to reach inner cationic surface is pore size, in the case that the pores themselves (or the openings resulting from the superposition among them) are smaller than E. coli cells, which are of a few micrometers in size (Figure S6). As seen in Table 3, pores in cNFC_RF are remarkably smaller than those in cNFC_SF1, which can be also observed in XRµCT profiles (Figure 3, in which dark regions indicate empty


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spaces, i.e., pores, where X-ray attenuation is low) and, in more details, SEM images (Figure 6). Pore size distributions are presented in Figure S7 (Supporting Information).



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Figure 6. Cross-sectional scanning electron microscopy images of monoliths made up of pristine nanofibrillated cellulose (NFC) and Girard’s reagent T-cationized NFC (cNFC) and produced through evaporative drying (ED) or freeze-drying preceded by rapid freezing (RF) or slow freezing for 1 d (SF1) or 7 d (SF7). Magnifications: 1000 x (left; white scale bars: 100 µm) and 1000-10,000 x (right; yellow scale bars: 40 µm).

The different antimicrobial activities of rapidly and slowly frozen cryogels are then attributed to the smaller pores of the former, which arise from the smaller ice crystals allowed to form upon freezing within a copper mold (featuring much higher heat conductivity than PP) and exchanging heat with a medium at much lower temperature (liquid nitrogen). The smaller pores and sterically hindered openings resulting from their overlapping therefore obstruct the movement of bacterial cells within the monoliths. This information can be corroborated in Figure 7, which shows E. coli cells on the surfaces of both cryogels, but only inside the sample comprising larger pores.



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Figure 7. Surface and cross-sectional scanning electron microscopy images of cryogels made up of Girard’s reagent T-cationized nanofibrillated cellulose and rapidly (cNFC_RF) or slowly (cNFC_SF1) frozen for 24 h. Scale bars: 5 µm. The arrows indicate representative Escherichia coli cells.

Cryo-templating NFC dispersion for a longer period (cNFC_SF7) did not provide further advantage. As expected, this sample presented larger pores because ice crystals were


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allowed to grow for a higher extent, but this did not reflect in improved antimicrobial activity. Instead, there was a minor trend of decreasing the antibacterial effect, observation that is likely due to the higher density and lower porosity (p < 0.05) of cNFC_SF7 monoliths than cNFC_SF1 ones. The anti-E. coli response of the latter did not differ from that of the never-dried NFC dispersion (cNFC_ND), sample that is considered to have the highest antimicrobial efficiency among this set because of its maximized specific surface area and absence of pores or channels that, as demonstrated here, may prevent bacterial cells from reaching cationic surfaces. It thus makes sense to highlight cNFC_SF1 as the optimum condition. It combines the best antimicrobial outcome (which remains unclear if it can be attributed to cell inactivation by cationic species, cell scavenging from suspension, or both – new experiments in the direction of trying to elucidate this mechanism are planned as a sequence to this study) with an intermediate cryo-templating time and the possibility (because of the three-dimensional structuring and wet stability) of being applied in several systems for which loose NFC would not be suitable, including filtration operations, adsorption processes (e.g., for water purification), functional cushioning, as well as hygiene, healthcare, and packaging items, to mention a few. These materials could play their functional roles not only in air, but also in liquid media as they were suitably water-swellable and water-durable. More specifically, cNFC_SF1 displayed good water-holding capacity (indicated by a swelling degree of 24 ± 2 g g-1) and wet resilience (mass retention of 88 ± 3% after being dipped in water for 48 h) – the latter is attributed not only to entanglements and physical interactions (e.g., hydrogen bonding and electrostatic interactions), which could be depleted upon swelling in water, but also 39 ACS Paragon Plus Environment


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to the likely formation of hemiacetals by the combination of hydroxyl and aldehyde groups of adjacent NFCs.34 Regarding shape memory, compression curves (Figure S4) demonstrate that dried cNFC_SF1 foams could theoretically retain their original dimensions up to ca. 10% compression, as the transition from the linear viscoelastic behavior to the region involving permanent deformation occurred at 10 ± 3%. However, regardless of the compression extent, such foams presented an impressive water-activated shape recovery because their original dimensions were instantaneously achieved once swollen in water – this behavior can be observed in the video available as Supporting Information. This characteristic opens up the possibility of reusing the foams even if they have been compressed, which would involve additional swelling and drying steps. Nonetheless, further investigations are needed to confirm whether or not their antimicrobial performance is maintained after recycling.

Conclusions We herein report the pioneer production of cationic all-cellulose foam-like materials featuring antimicrobial activity against E. coli as a result of grafting the Girard’s reagent T onto nanofibrillated cellulose. We also demonstrated a straightforward means of controlling the morphology of foam-like materials by playing with the parameters involved in the foam-forming protocol, which in turn were also demonstrated to play a major role on the antibacterial response of such multifunctional materials. From our experiments, it becomes evident that it is desirable to have surface areas as large as 40 ACS Paragon Plus Environment

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possible, which was achieved here by producing highly porous monoliths. Still, high porosity may not be enough to ensure suitable antimicrobial activity in case the inner surface is not reachable by microbial cells. Therefore, pore size and interconnectivity denote variables that are as important as porosity when it comes to such applications. This finding is relevant because one may boost the efficiency of a material intended for antimicrobial purposes without the need for modifying synthetic routes and NFC concentrations. These morphometric parameters as well as anti-E. coli efficiency were herein demonstrated to be fine-tuned by cryo-templating, but other means of regulating the morphology of solid foam-like materials (particularly density, porosity, surface area, and pore size) include adjusting solid content in the precursor dispersion/solution,35, 61 emulsion templating,56 and addition of porogens (e.g., PMMA spheres and paraffin wax).62 Freeze-casting involving slow freezing was shown to be suitable for producing ultralight, highly porous monoliths from physical-mechanical and antimicrobial standpoints. Although freeze-drying on a laboratory scale may be too time-consuming, more industrially relevant apparatuses are available for the scaled-up accomplishment of such an environmentally friendly process.61 Finally, the antimicrobial efficiency of the foams produced here was confirmed using a Gram-negative bacterium as a model microorganism, but new investigations are encouraged to validate this system for its Gram-positive counterparts, even though cationic nanocellulose has been shown to inactivate Gram-positive and Gram-variable bacteria.12-14, 16, 19-20, 23

Acknowledgments



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This study was supported by São Paulo Research Foundation (FAPESP, grants #2017/07013-1, #2017/01167-7, and #2015/25406-5) and LNNano - Brazilian Nanotechnology National Laboratory, CNPEM/MCTIC. The authors are thankful to Dr. E.P. Maia at Suzano Papel e Celulose (Brazil) for kindly donating cellulose samples, Dr. M. Mariano for providing copper molds, D.B. Silva for FTIR runs, Dr. R.A.R. Giorjao for support with XPS, Dr. C.A.R. Costa and C.A. Biffe for help with AFM, Dr. R.F. Gouveia and B. Massucato for support in XRµCT runs.

Supporting Information. XPS spectra of pristine and chemically modified NFC; FTIR spectra of pristine and chemically modified NFC; SEM images of pristine and chemically modified NFC; Force versus deformation curves obtained in compression assays; Sample positions presented in XRµCT images; E. coli cells; Histograms of pore size distributions in cryogels. This material is available free of charge at http://pubs.acs.org.

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