Cellulose-Organic Montmorillonite Nanocomposites as

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Cellulose-organic montmorillonite nanocomposites as biomacromolecular quorum sensing inhibitor Deniz Demircan, Sedef Ilk, and Baozhong Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01116 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Cellulose-organic montmorillonite nanocomposites as biomacromolecular quorum sensing inhibitor Deniz Demircan1,2, Sedef Ilk3, Baozhong Zhang1* 1

Lund University, Centre for Analysis and Synthesis, P.O. Box 124, SE-22100 Lund, Sweden

2

Hacettepe University, Faculty of Science, Department of Chemistry, Beytepe, TR-06800

Ankara, Turkey 3

Ömer Halisdemir University, Central Research Laboratory, TR-51240, Niğde, Turkey

KEYWORDS: regenerated cellulose, anti-quorum sensing, cellulose nanocomposites, organic modified montmorillonite

ACS Paragon Plus Environment

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ABSTRACT. The aim of this study was to develop simple cellulose nanocomposites that can interfere with the quorum-sensing (QS) regulated physiological process of bacteria, which will provide a sustainable and inexpensive solution to the serious challenges caused by bacterial infections in various products like food packaging or biomedical materials. Three cellulose nanocomposites with 1-5 w% octadecylamine modified montmorillonite (ODA-MMT) were prepared by regeneration of cellulose from ionic liquid solutions in the presence of ODA-MMT suspension. Structural characterization of the nanocomposites showed that the ODA-MMT can be exfoliated or intercalated, depending on the load level of the nanofiller. Thermal gravimetric analysis showed that the incorporation of ODA-MMT nanofiller can improve the thermal stability of the nanocomposites compared to regenerated cellulose. Evaluation of the anti-QS effect against a pigment-producing bacteria C. violaceum CV026 by disc diffusion assay and flask incubation assay revealed that the QS-regulated violacein pigment production was significantly inhibited by the cellulose nanocomposites without interfering the bacterial vitality. Interestingly, the nanocomposite with the lowest load of ODA-MMT exhibited the most significant anti-QS effect, which may be correlated to the exfoliation of nanofillers. To our knowledge, this is the first report on the anti-QS effect of cellulose nanocomposites without the addition of any small molecular agents. Such inexpensive and nontoxic biomaterials will thus have great potential in the development of new cellulosic materials that can effectively prevent the formation of harmful biofilms. INTRODUCTION Cellulose, the most abundant class of natural biomacromolecules, has attracted growing attention in the last decades, due to its renewability, low cost, non-toxicity, environmental friendliness, biocompatibility, and various superior physical properties.1 Unfortunately, the high


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crystallinity and low solubility of cellulose make it difficult to be processed as melt or in solution.2 Regeneration of cellulose using a polar ionic solvent (e.g. LiCl/DMA, NMMO/H2O, LiOH/urea, or NaOH/thiourea) and a “non-solvent” (e.g. water, ethanol or acetone) is commonly applied to improve the solubility and processability,3 which has many practical applications such as membranes for dialysis or ultrafiltration,4 fibers for nonwoven textile,5 or hydrogels for biomedical applications.6 However, regenerated cellulose (RC) is highly porous and amorphous, which provides an ideal environment for the bacterial growth and thus creates challenges for biomedical applications. Common sterilization or disinfection (e.g. by UV light or oxidant) methods can provide temporary bacteria-free RC surface, but they cannot prevent the growth of bacteria during the use of the RC materials. To provide long-term bacterial resistance, antibacterial agents with bactericidal effect7 have been commonly used to kill the bacteria contacting the surface of RC materials. However, most antibacterial agents used or reported today are toxic, and their leaching into the environment can be a potential thread. Furthermore, the dead bacterial cells can be deposited on the RC surface to produce a biofilm,8 which can embed and protect the living bacteria from the influence of antibiotics.9 It was reported that over 60% of all bacterial infections are associated with biofilm formation.10 In this context, it is highly important and urgent to develop RC materials that can resist the bacteria by a mechanism that will not affect the vitality of bacteria. Therefore, the regulation of the quorum sensing (QS) process of bacteria has been considered as the next generation method for the development of effective bacteria-resistant materials. QS process is usually mediated by certain small molecules called autoinducers (AIs),11 which are produced inside the bacterial cells and diffuse out into the environment. Commonly used AIs include acylated homoserine lactones (AHLs) for Gram-negative bacteria, and oligopeptides for


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Gram-positive bacteria.12 When the AIs around the bacteria reached a critical concentration, they will re-enter the cells and affect various physiological processes of bacteria (e.g. bioluminescence, conjugation, expression of virulence factors, or biofilm formation).13 These physiological processes can thus be modulated by the interference in the production or recognition of AIs, without affecting the bacterial vitality.12 Such an external modulation is commonly referred to as anti-quorum sensing (anti-QS) process.14 In nature, anti-QS effect is usually achieved by certain naturally-occurring small molecules (e.g. essential oils, furanones, trans-cinnamaldehyde, farnesol, AHL-analogs etc.).15 These small molecules can be added to cellulose matrix to produce anti-QS materials, but such materials usually suffer from the leaching potential of the small molecules, which will not only exhaust the anti-QS effect but also cause various health or environmental issues. Therefore, macromolecular anti-QS agents have been of growing interest in various biomedical applications. For example, synthetic polymers with antiQS effect by the adsorption of AIs have been reported.16 Biomacromolecules like enzymes have also been used as anti-QS agents by degrading AIs.17 Unfortunately, neither pristine nor regenerated cellulose are able to interact with AIs, unless small molecular agents like resveratrol are incorporated.18 On the other hand, nanofillers have been widely used to prepare cellulose nanocomposites with improved thermal and mechanical properties, and some metallic nanofillers (e.g. silver or copper nanoparticles) are also known for their antibacterial effect.19 However, we are unaware of any report on RC nanocomposites without using small molecular agents that exhibit significant anti-QS properties. In this work, we are particularly interested in a class of RC nanocomposites containing octadecylamine (ODA) modified montmorillonite (MMT) as nanofillers. ODA-MMT is a green nontoxic nanofiller, which has been widely used for the production of nanocomposites of bio- or


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synthetic macromolecules.20 Since ODA is an amphiphilic molecule as AHLs, we expect the RC/ODA-MMT nanocomposites may interfere in the AHL-regulated physiological processes. To testify this hypothesis, a special Gram-negative bacterium, Chromobacterium violaceum CV026, was chosen for the anti-QS tests, because it can produce a purple pigment (violacein) in response to exogenous AHLs.15b It has been reported that the violacein production of CV026 can be induced or inhibited by AHLs with short (C4-C8) or long alkyl side chains (C10 to C14), respectively.21 Herein, we report on the preparation of green nanocomposites of RC and ODAMMT, characterization of their thermal stability, compatibility, and surface morphology, and evaluation of their anti-QS effect against CV026. Their bactericidal potential on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus was also evaluated. To our knowledge, this is the first reported cellulose nanocomposite without any added small molecular agents that exhibits significant anti-QS effect without bactericidal activities. EXPERIMENTAL SECTION Materials. Cellulose (microcrystalline and average particle size 90 m) and octadecylamine montmorillonite (ODA-MMT) (Nanomer I.30E, ODA content 25-30 %) were purchased from Sigma Aldrich, LiCl (AP, 97%). N,N-dimethylacetamide (anhydrous, 99.8%, CP), DMA, was purchased from Acros Organics. Luria-Bertani (LB) was supplied by LABM. Other chemicals and solvents were of analytical reagent grade. Preparation of RC and RC/ODA-MMT nanocomposites. RC was prepared according to a procedure reported in the literature (Figure 1).22 First, a solution of 4.3 w% cellulose in LiCl/DMA was prepared (Figure 1a), 23 which was poured into a mold and coagulated in ethanol bath (as non-solvent) at room temperature for 1 h to yield a gel. The gel was then collected by gravity filtration, washed with water (Figure 1b), and dried in a vacuum oven at 50 °C for 72 h.


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To prepare RC/ODA-MMT nanocomposites, dispersions of ODA-MMT in DMA were firstly prepared by stirring for 3 h at 60 °C and then sonicating for 1 h using Bransonic Ultrasonic Cleaner Branson 5510E-MT (Output 135 W, 42 kHz). The obtained dispersions were then mixed with the above-mentioned cellulose solution (4.3 w%) with ODA-MMT content as 1, 3 and 5 w%. The mixtures were stirred at 60 ◦C for 3 h and then sonicated for 1 h to produce a homogeneous suspension (Figure 1c), which was then spread and coagulated in ethanol bath to yield a transparent RC/ODA-MMT gel. The gel was collected by gravity filtration, washed with water (Figure 1d and Figure S1), and dried in a vacuum oven at 50 °C for 72 h. The dried nanocomposite samples were labeled as CN1, CN3 and CN5 (the subscribed numbers indicate the ODA-MMT content) and stored for further characterization.

Figure 1. Preparation of RC and RC/ODA-MMT nanocomposites. The gels in (b) and (d) were picked up by tweezers. Characterization of RC/ODA-MMT nanocomposites. Diffraction patterns of the nanocomposites were recorded with a Stoe Stadi MP X-ray powder diffractometer in transmission mode over 2 ranges 2–40° with Cu Kα radiation. The wave length of X-ray beam


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is 0.154184 nm. FTIR spectra were recorded on Bruker Alpha spectrometer using powder samples. Surface morphology of the nanocomposites was investigated by a scanning electron microscope (SEM) (Hitachi-4700, JEOL), after each sample was fixed on aluminum stub and coated with gold. Thermogravimetric analyses (TGA) were performed under nitrogen atmosphere with a Thermogravimetric Analyzer (TA Instrument Q500) at a heating rate 10 C/min. Evaluation of anti-QS properties Bacteria culture. The reporter strain C. violaceum CV026 was employed to evaluate the antiQS properties of nanocomposites, pristine cellulose and ODA-MMT. The bacterial suspension of CV026 was obtained by overnight aerobic growth (30 °C, 250 rpm) in Luria-Bertani (LB) broth. Disc diffusion test. Disc diffusion assay was carried out to detect the anti-QS activity. First, N-hexanoyl-l-homoserine lactone (HSL, a special AHL with C6 side chain, 0.25 μg/mL) was added into 100 mL of sterilized LB agar at an appropriate temperature, which was then gently mixed and poured into the petri plates. The overnight culture of CV026 was swabbed evenly onto a solidified agar surface. Sterile discs were loaded with solutions of the tested samples, placed on the agar plates, and then incubated at 30 C for 24 h. For comparison, only the solvent (DMA) or gentamicin (a commercial antibiotic) were also tested as controls. The anti-QS properties against CV026 were verified by the formation of a turbid halo (caused by the bacterial growth), without the production of purple pigment around the well on the plate. UV-vis quantification of violacein inhibition. Quantification of violacein inhibition was carried out by flask incubation assay. LB broth inoculated with bacteria mixed with HSL (5 μM) and the tested samples were incubated for 24 h at 30 C. Violacein extraction was carried out as described in literature24. First, the suspension from each flask (1 mL) was centrifuged at 11000


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rpm for 10 min to precipitate violacein. After the supernatant was discarded, the remaining pellet was vortexed in water for 1 min to extract the produced violacein. The supernatant was centrifuged again at 11000 rpm for 10 min to remove the insoluble particles, and measured by UV-vis spectrophotometer (PG Instruments, T-60). The absorbance (A) at λ = 585 nm was used to quantify the violacein inhibition (VI) according to the following formula, in which, Asample and Acontrol are UV-vis absorbance of the supernatants obtained from the discs containing the tested samples and no sample (only the solvent, DMA), respectively. VI % = (Acontrol–Asample)/Acontrol) × 100 % The anti-QS assay was performed 3 times and the standard deviation was presented as the error bars (Figure 6). RESULTS AND DISCUSSION Structural analyses of RC/ODA-MMT nanocomposites. As shown in Figure 1d (see also Figure S1), all the prepared RC/ODA-MMT gels were translucent, which indicates that ODAMMT nanofillers were well dispersed in the cellulose matrix. Further structural analyses based on FTIR, XRD, and SEM methods were carried out to investigate the morphology and the interactions between the nanofillers and the cellulose matrix.

First, FTIR analysis was performed to confirm the presence of both cellulose and ODA-MMT nanofillers in the nanocomposites, and study the interactions between the cellulose matrix and ODA-MMT (Figure 2). At the low frequency region, small bands at ~1155-1159 cm-1 due to C-O-C stretching of cellulose have been observed in pristine cellulose, RC and all the


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nanocomposites (Figure 2a-e), which confirms the presence of cellulose matrix in the nanocomposites.25 The characteristic bands of ODA-MMT at 449, 513 and 1005 cm-1 (Figure 2f) have also been observed in all the nanocomposites with some shifts toward higher frequency (457-461, 516-519, 1015-1016 cm-1) and increasing intensity as the content of ODA-MMT increased, which confirms the existence of the nanofiller in the nanocomposites. At the high frequency region, the N-H stretching of ODA-MMT at ~3628 cm-1 (Figure 2f)26 was not observed in the nanocomposites (Figure 2c-e), which indicates that the H-bonding in the original bulk ODA-MMT nanofiller has been largely replaced by the H-bonding between the OH and NH groups in the nanocomposites due to intercalation or exfoliation of ODA-MMT layers. This is further corroborated by the observed shift of the O-H vibrational band at 3300−3500 cm−1 toward higher frequency with the increased ODA-MMT content, which indicates that the Hbonding in pristine cellulose or RC is changed by the incorporation of ODA-MMT.


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Figure 2. FTIR spectra of (a) pristine cellulose, (b) RC, (c) CN1, (d) CN3, (e) CN5 and (f) ODAMMT. Note: The small band at 1600-1700 cm-1 region is attributed to the absorbed water. The negative bands at 2900-3100 cm-1 region are due to Teflon cap used to cover the pin to protect diamond crystal during the measurements. Demonstrations of the H-bonding effects (as blue dash lines) are shown on the right. Furthermore, the interaction between ODA-MMT and cellulose matrix has been evaluated by XRD analyses (Figure 3). Obviously, the characteristic basal reflection at 2 = 4.85 (d001 = 1.82 nm) of ODA-MMT was not observed for the nanocomposites, which can be attributed to the increased interlayer spacing between the MMT platelets due to the diffusion of cellulose matrix into the galleries of MMT.27 This is consistent with the results from FT-IR, which showed the


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formation of H-bonding between the OH groups of RC and the NH groups of ODA-MMT. Furthermore, a small peak at 2 = 3.59 (d001 = 2.46 nm) was observed for all the 3 nanocomposites with increasing intensity, which can be attributed to the intercalated MMT layers in the cellulose matrix. Similar effect has also been reported for MMT/cellulose acetate nanocomposites.28 It has been noted that the intensity of the peak at 2 = 3.59 is very low for CN1 (barely visible), which suggested that the ODA-MMT in CN1 contained partly exfoliated individual layers. For the nanocomposites containing higher ODA-MMT loads (i.e.CN3 and CN5), this peak become more visible, which suggested that these two nanocomposites contained increased amount of intercalated ODA-MMT tactoids. Such phenomenon is consistent with other reported MMT-containing nanocomposites.

Figure 3. XRD patterns of (a) ODA-MMT, (b) CN1, (c) CN3 and (d) CN5. Last, the surface morphology of the nanocomposites was studied by SEM. It is known that RC surface contains porous structures due to the exchange of solvent and non-solvent molecules during coagulation29. It is also known that the incorporation of MMT nanofillers into RC can


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significantly alter the surface morphology.30 According to our results (Figure 4), all the nanocomposites have smooth and uniform surface structures, which indicates good interaction and distribution of ODA-MMT layers in cellulose matrices. Furthermore, the three nanocomposites showed different surface morphologies depending on the ODA-MMT contents. For CN1, a dense structure with few voids was observed (Figure 4a), which can be attributed to the uniformly well dispersed clay platelets in the cellulose matrix. Such an observation suggests that the exfoliated ODA-MMT platelets are effectively wrapped by cellulose chains. When the content of ODA-MMT increased (CN3 and CN5), remarkable “macrovoids” were observed (Figure 4b and 4c), which is possibly due to the presence of more intercalated structures of the nanocomposites and aggregation of ODA-MMT platelets in the cellulose matrix.31 The increased extent of intercalation is consistent with the results from XRD discussed earlier.

Figure 4. SEM images of RC/ODA-MMT nanocomposites: (a) CN1, (b) CN3 and (c) CN5. Some “macrovoids” were circulated in the images as examples. Thermal stability of the nanocomposites. It is known that RC materials usually have lower thermal stability compared to pristine cellulose, which limits their processing conditions and applications.32 The incorporation of thermally stable nanoclays can greatly improve the thermal stability of matrix cellulose, because the silicate layers of nanoclay can act as thermal isolators and mass-transport barriers during the thermal degradation process. For example, Mahmoudian


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et al.22 has reported that the incorporation of 6 w% ODA-MMT in RC matrix could significantly improve the thermal stability and the char yield of the nanocomposites. It should be addressed that in their work, 1-butyl-3-methylimidazolium chloride (an ionic liquid) was used as the solvent for the preparation of nanocomposites. In our work, the RC/ODA-MMT nanocomposites were prepared by using a different solvent system that can result in different properties29, so TGA analyses were conducted.

Figure 5. (a) TGA and (b) DTG thermograms. Figure 5a and 5b show the TGA and DTG (derivative of weight loss) thermograms of pristine cellulose, RC, ODA-MMT and the nanocomposites. Table 1 lists the extrapolated onset temperature (To), the maximum degradation temperature (Tmax) and the char yields (CY) for all the samples. From our results, it is obvious that our RC regenerated from ethanol has considerably lower thermal stability compared to pristine cellulose. The lower thermal stability of RC can be attributed to three main reasons: First, the lower crystallinity and smaller crystallite size of RC compared to those of pristine cellulose can accelerate the degradation process 7b, 33 due to the different decomposition–gasification processes.32 Second, the thermal behavior can be affected by the presence of the anionic Cl groups (originated from LiCl in the solvent) in the nanocomposites34. Third, porous RC materials have larger surface area to be exposed to heat, which will further diminish their thermostability.35 Furthermore, the char yield (CY) of RC was


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found much higher than that of pristine cellulose, which was consistent with some other reports earlier.36 37 Table 1. Thermal parameters of cellulose, ODA-MMT, RC and RC/ODA-MMT nanocomposites

Sample

T0 (°C)

Tmax (°C)

CY (%)

Cellulose

306.0

326.3

3.6

ODA-

235.6

308.1

75.3

RC

213.4

229.7

24.7

CN1

254.7

267.8

30.4

CN3

303.1

344.5

9.8

CN5

268.7

311.0

19.8

MMT

The thermal stability of the 3 prepared RC/ODA-MMT nanocomposites was evaluated afterwards. All the 3 nanocomposites exhibited higher T0 and Tmax values compared to those of RC’s (Table 1), which suggests that the incorporation of ODA-MMT can significantly improve the thermal stability of the nanocomposites. This is due to not only the fact that ODA-MMT is a thermally more stable material compared to RC (Figure 5), but also the strong interfacial Hbonding interactions between cellulose and MMT, as revealed by the FT-IR results. Furthermore, the possible formation of a char layer network during the pyrolysis process may also retard the out-diffusion of gaseous decomposition products and lead to the observed higher degradation temperatures.27 Interestingly, the thermal stability of CN3 is the highest among all the three nanocomposites, which is similar or even higher than that of the pristine cellulose (~3 °C lower in T0 and ~18 °C higher in Tmax). Such an observed optimum thermal stability may be attributed


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to the dual effect of ODA-MMT organomodifiers. On one hand, ODA-MMT can improve the thermal stability by providing a barrier effect against mass-transport during the thermal degradation.

27, 38

. On the other hand however, higher amount of ODA-MMT may also increase

the thermal degradation at the initial stage of TGA measurements possibly because of the agglomeration of nanofillers.

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Such optimum thermal stability of ODA-MMT nanocomposites

has been reported for ODA-MMT/PET nanocomposites.40 Finally, we have also observed a second thermal degradation process of CN5 above 500 °C, which may be attributed to the dehydration/dehydroxylation process of MMT.41 Anti-QS activity tests. We assessed whether the prepared RC/ODA-MMT nanocomposites can disrupt the AHL-regulated QS response in biosensor strain C. violaceum 026 (CV026). Pristine cellulose, ODA-MMT and all the cellulose nanocomposites (CN1, CN3 and CN5) were screened, and the anti-QS effect was evaluated by two independent methods. First, the anti-QS effect was measured by the zone of pigment inhibition that results in an opaque, halo zone of clearance (Figure 6a).42 According to the results, the tested nanocomposites inhibited the production of purple violacein (no purple color production around the disc), while the bacterial growth was shown by the turbidity of the halo around the disc. The non-bactericidal effect RC/ODA-MMT nanocomposites was further verified by an antibacterial disc-diffusion assay against gram positive and negative bacteria (Figure S2). The samples containing only DMA, cellulose, or ODA-MMT did not show significant zone of pigment inhibition, and the disc containing gentamicin exhibited significantly larger zone of inhibition compared to the nanocomposites (~35 mm). All the 3 nanocomposites showed significantly larger zones of pigment inhibition (Figure 6a) compared to those of pure cellulose or ODA-MMT. Interestingly, the zone of pigment inhibition for the tested nanocomposites increased with the decreasing


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content of ODA-MMT (i.e. 12.27, 15.38 and 19.21 mm for CN5, CN3, CN1, respectively). This is somewhat counter-intuitive, because for many reported anti-QS agents (e.g. essential oils, metal nanowires), the zone of inhibition either increased with the increasing amount of anti-QS agents or showed no quantity-dependence.43 To understand the apparently peculiar “concentration” dependence for the anti-QS effect of our nanocomposites, we should address that in our cases neither the ODA-MMT nor the RC matrix should be regarded as the conventional “anti-QS agent”, since they did not show significant anti-QS effect according to our control tests. This means that the load level of ODA-MMT nanofillers in the nanocomposites should not be understood as the concentration of the anti-QS agent. For our materials without any additional anti-QS agents, the strong anti-QS effect is presumably related to the exfoliated ODA-MMT nanoparticles dispersed in the cellulose matrix, which is corroborated by the observation that CN1 with the highest level of exfoliation provided the strongest anti-QS effect. Further investigation will be needed in order to elucidate the mechanism of the observed anti-QS effect. Furthermore, the anti-QS effect was quantified by an independent UV-vis measurements of the pigment extracts from flask incubation. Similar as the disc diffusion assay, the samples containing only DMA, cellulose or ODA-MMT did not show significant anti-QS effect (VI

Cellulose-Organic Montmorillonite Nanocomposites as

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