Enhanced antimicrobial activity and structural transitions of a

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Enhanced antimicrobial activity and structural transitions of a nanofibrillated cellulose-nisin bio-composite suspension Ramon Weishaupt, Lukas Heuberger, Gilberto Siqueira, Beatrice Gutt, Tanja Zimmermann, Katharina Maniura-Weber, Stefan Salentinig, and Greta Faccio ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04470 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Applied Materials & Interfaces

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Enhanced antimicrobial activity and structural

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transitions of a nanofibrillated cellulose-nisin bio-

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composite suspension

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Ramon Weishaupt†, Lukas Heuberger†, Gilberto Siqueira§, Beatrice Gutt†, Tanja Zimmer-

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mann§, Katharina Maniura-Weber†, Stefan Salentinig†,*, Greta Faccio†,*

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†

Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Bi-

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ointerfaces, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland §

Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for

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Applied Wood Materials, Überlandstrasse 129, CH-8600, Dübendorf, Switzerland

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ETH,

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ABSTRACT

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The occurrence of resistance to antibiotics has posed a high demand for novel strategies to

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fight bacterial infections. Antimicrobial peptides (AMPs) are a promising alternative to con-

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ventional antibiotics. However, their poor solubility in water and sensitivity to degradation

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has limited their application. Here we report the design of a smart, pH-responsive antimicro-

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bial nanobiocomposite material based on the AMP nisin and oxidized nanofibrillated cellulose

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(TONFC). Morphological transformations of the nano-scale structure of nisin functionalized 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TONFC fibrils were discovered at pH values between pH 5.8 and 8.0 using small angle X-ray

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scattering (SAXS). Complementary zeta potential measurements indicate that electrostatic-

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attractions between the negatively charged TONFC surface and the positively charged nisin

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molecules are responsible for the integration of nisin. Contrary, shifting the pH level or in-

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creasing the ionic strength reduce the nisin binding capacity of TONFC. Biological evaluation

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studies using a bioluminescence-based reporter strain of Bacillus subtilis and a clinically rele-

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vant strain of Staphylococcus aureus indicated a significantly higher antimicrobial activity of

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the TONFC-nisin biocomposite compared to the pure nisin against both strains under physio-

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logical pH and ionic strength conditions. The in-depth characterization of this new class of an-

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timicrobial bio-composite material based on nanocellulose and nisin, may guide the rational

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design of sustainable antimicrobial materials.

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KEYWORDS

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Nanocellulose, TEMPO-oxidation, antimicrobial peptide, biocomposite, peptide immobiliza-

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tion, SAXS, bioactive material.

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Introduction

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ized patients. With the rise of antibiotic resistance,1 treatments of infection are increasing in

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complexity and alternative approaches to fight bacteria are urgently sought.2-3 Antimicrobial

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peptides (AMPs) represent a valuable alternative to conventional antibiotics and are currently

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gaining interest in biomedical research.4-6 In contrast to most conventional antibiotics, AMPs

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affect pathogenic bacteria via multiple routes of action, such as membran disintegration,

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intracellular effects and growth inhibition, thus rendering the development of resistances

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greatly more difficult.7-9 Nisin, a 34-amino-acid-long antimicrobial model AMP10 produced

Nosocomial infections are today one of the most common complication affecting hospital-

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by lactic acid bacteria to compete antagonistic strains in their habitat; is one of the few AMPs

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approved as food preservative11 and has a long history in clinical research.12-13 The molecular

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structure of nisin is characterized by intramolecular thioether-based (lanthionine)-rings that

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confer rigidity to the 3D structure of the molecule14 and promote both, specific binding to the

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peptidoglycan cell wall biosynthesis key-precursor lipid II and general charge-interactions

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with the often negatively charged microbial cell wall envelope.7 The unique combination of

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these high-affinity and moderate-affinity targets enables nisin to antagonize a broad spectrum

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of Gram positive bacterial strains at nanomolar concentrations.15 The application of AMPs

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like nisin has however been limited by their sensitivity to chemical degradation by bacterial-

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and/or patient-derived proteases,16 limited solubility in aqueous solutions17 and therefore,

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poor penetration efficiency to bacterial infection sites.18

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Immobilization of biomolecules is known to alter their functionality by enhancing their activi-

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ty19 or improving their stability to degradation in some cases.20-21 Encapsulation of AMPs in

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an appropriate carrier system such as polyelectrolyte carrier matrices or liposomes can protect

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them from degradation and increases their solubility, augmenting delivery to the site of infec-

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tion effectively.22 Incorporation into a delivery system would additionally allow reducing the

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required dosage23 and possible systemic toxicity.5 Understanding the structure of polyelectro-

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lyte carrier matrices like TONFC, under variable conditions could have a tremendous impact

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in various biomedical applications.24 Nanocelluloses, are natural polymers and produced by a

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wide variety of organisms including animals, plants and microbial organisms.25-26 These fibers

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combine the characteristic cellulose features like hydrophilicity, broad chemical-modification

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capacity with the specific benefits of nanoscale materials like high specific surface area and

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superior mechanical properties.25

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Oxidation with TEMPO (2,2,6,6-Tetramethyl-1-piperidinyloxyl)- regio-selectively converts

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hydroxyl groups at the C6 position of surface-exposed glucose units of cellulose fibers into

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carboxylic groups and, followed by mechanical disintegration, produces a nanofibrillated sus3 ACS Paragon Plus Environment


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pension, here termed TONFC (TEMPO-Oxidized Nanofibrillated Cellulose).27 The high den-

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sity of negative surface charges in combination with the large specific surface area improves

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the dispersion capability of TONFC in water28 and allows the controlled immobilization and

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stabilization of active biomolecules at high densities.19, 29 The use of TONFC fibrils in bio-

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medical applications may require their processing as a never-dried, hydrated material, and

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hence, there is great interest to further study their three-dimensional nano- and microstructure

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in the wet state.30 However, the influence of an interacting AMP such as nisin on the TONFC

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network structure is not yet understood.

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This manuscript describes the design of an antimicrobial bio-nanocomposite material based

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on the self-assembly of nisin with TONFC fibrils. At pH levels higher than the pKa of carbox-

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ylic groups and low ionic strength, the TONFC surface provides an amount of negative sur-

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face charges sufficient for the adsorption of the positively charged cationic nisin molecules.31-

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32

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(Scheme 1, A) while the effects of nisin and pH on the nanocellulose surface properties and

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network morphology were characterized using zeta potential measurements and SAXS re-

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spectively (Scheme 1, B).

The adsorption of nisin onto the TONFC surface is studied by using biochemical assays


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Scheme 1. (A) Biofunctionalization of TONFC with nisin via physical assembly. The nisin molecule presents hydrophobic (grey), hydrophilic (green), and positively charged (blue) amino acids; few residues are post-translationally modified to dehydroalanine (Dha) and dehydrobutyrine (Dhb), aminobutyric acid (Abu), (PDB: 1wco33). (B) Analysis of surface and structural properties of the TONFC-nisin biocomposite using SAXS and zeta potential measurements. (C) Assessment of the antimicrobial activity of TONFC-nisin with both its interaction with a specific cell surface receptor (1) and the growth inhibitory effects (2).

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These investigations, together with in vitro evaluation studies using a nanomolar sensitive

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bioluminescence-based bioreporter strain of B. subtilis and a clinically relevant S. aureus

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strain (Scheme 1, C) established the TONFC-nisin biocomposite as a more active antimicro-

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bial material with dynamic response features. The detailed insights into nanostructure and bi-

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oactivity gained following this approach, will ultimately contribute to the development of ad-

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vanced antimicrobial biomaterials relevant for consideration in various fields from food

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science to biomedicine.

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Results and Discussion The physical interaction of nisin with TONFC suspensions.



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Physical interactions between biomolecules and a carrier substrate are often dependent on

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the environmental conditions. Hence, first the loading capacity of TONFC fibrils for nisin

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molecules is quantified over a range of relevant pH- and ionic strength conditions (Scheme

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1A). The highest loading of 202 ± 10 µg/mg TONFC (yield: 49 % w/w of added nisin) was

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obtained at pH 3.8 while slowly decreasing to 135 ± 6 µg/mg towards pH 8.0 (yield: 31 %),

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i.e. close to the isoelectric point (pI) of nisin at 8.534 as shown in Figure 1A. A more pro-

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nounced drop in nisin loading was observed at pH values above the pI with 79 ± 12 µg/mg

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TONFC at pH 9.5 (yield: 19 %). At a salt concentration of ≥ 200 mM, the binding capacity

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drastically dropped to ~14 µg/mg (yield: 3.4 %) under all tested conditions. Further it could

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be shown that a significant amount of nisin remained bound to the TONFC substrate between

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pH 3.8 and 8.0 and at an ionic strength of 100 or 200 mM thus covering physiological cultur-

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ing conditions.35 No binding was observed at pH 2.0 unless tested at the highest ionic strength

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of 500 mM when a loading capacity of ~30 µg nisin/ mg TONFC was achieved. SDS PAGE

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analysis at relevant buffering conditions of the nisin solution and TONFC-nisin suspension

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prepared at pH 5.8 and at an ionic strength of 20 mM further showed the non-covalent interac-

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tion between nisin and the material and its permanence in a monomeric state (Figure S1, C).


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Figure 1. (A) Nisin adsorption to TONFC evaluated under different pH and ionic strength

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conditions in McIlvaine buffer. Capacity values are reported in µg peptide/mg TONFC. (B)

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pH-dependent zeta potential measurements for TONFC (grey) and TONFC-nisin biocompo-

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site suspensions (blue) measured in McIlvaine buffer at an ionic strength of 20 mM. The isoe-

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lectric point of nisin of 8.534 (vertical red line) is marked. All graphs show mean values and

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standard deviations (n ≥ 6).

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Both pH and ionic strength of the peptide solutions had a critical influence on the spontaneous

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interaction of nisin molecules with TONFC leading to a different degree of loading. Under

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conditions compromising the electrostatic interactions such as pH 2.0 or 9.5 or elevated salt

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concentrations (≥ 200 mM), the binding of nisin on TONFC appeared limited. In particular,

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cosmotropic or chaotropic effects of solvent-derived counter ions can either stabilize or desta-

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bilize hydrophobic interactions as well as hydrogen bonds between nisin and the TONFC sur7 ACS Paragon Plus Environment


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face residues.36-37 Hence, the screening of the charge interactions between the positively

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charged nisin and the negatively charged TONFC surface caused by the high ionic strength of

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the buffer at salt concentrations ≥ 200 mM may cause the observed change in trend of

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TONFC-nisin interactions. The exact contributions of such interactions is fairly difficult to

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predict and require more detailed analyses, for instance through molecular dynamics simula-

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tions.38

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Fibril surface properties of TONFC-nisin

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The biochemical analysis revealed a clearly condition dependent loading capacity of TONFC

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for the AMP nisin (Figure 1A). To analyze the effect of peptide loading on the fibril surface

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properties more deeply, zeta potential measurements were carried out under conditions of pH

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between 2.0 and 10.0 (Figure 1B & Table S1).

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TEMPO-mediated selective oxidation of surface-exposed primary hydroxyl groups was re-

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ported to introduce carboxylic groups.39 As shown in Figure 1B, the zeta potential of TONFC

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in 20 mM buffer at pH > 5.8 remained negative under all tested conditions reaching -50 mV at

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pH above 8.0. These observations are in good agreement with previous findings on this sys-

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tem.32 In contrast to bare TONFC, TONFC-nisin showed significantly more positive zeta po-

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tential values at acidic conditions between -17.3 ± 3.13 mV at pH 3.8 and 0.63 ± 1.48 mV at

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pH 2.0. The more positive zeta-potentials observed for TONFC-nisin compared to TONFC

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between pH 2.0 and 3.8 indicate the electrostatic interactions of the surfaces with the positive-

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ly charged nisin molecules. At pH 5.8 and 9.5, above the pKa of the carboxylic groups on the

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TONFC surface, the excess of negative charges from deprotonated carboxylic groups on the

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surface may exceed the impact of the positive charges from interacting nisin molecules.32

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Comparing the surface charge- and loading capacity profiles suggests that the interaction

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between nisin and TONFC depended on the level of available surface charges on the TONFC

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fibrils as long as the pH was significantly below the pI of nisin (Figure 1). Gradual protona8 ACS Paragon Plus Environment

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tion of the carboxylic groups on TONFC fibrils below their apparent pKa value that was re-

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ported between 4.8 and 8.0 and/or the association of counter-ion reduced the number of avail-

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able negative surface charges could explain the decreased binding capacity for cationic nisin

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molecules via electrostatic interactions.31-32

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Structural transitions of the TONFC-nisin fibril network

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The influence of pH change and nisin adsorption on the surface charge of TONFC could be

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elucidated (Figure 1). These findings prompted the further investigation how changing condi-

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tions and the interaction with the peptides influences the dimensional- and structural proper-

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ties of the TONFC fibrils. To analyze the condition dependent nanoscale structure of TONFC

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small angle X-ray scattering (SAXS) measurements were carried out at pH 5.8 and 8.0 at an

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ionic strength of 20 mM (Figure 2).

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In Figure 2, the experimental SAXS data of bare TONFC and TONFC-nisin suspensions

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investigated at pH 5.8 and 8.0 are presented. The I(q) curves are characteristic for worm-like

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fibrils with local stiffness.40 As a result of the large fibril dimensions, above the resolution

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limit of the used SAXS set-up, the Guinier region that is associated with the total size of the

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fibrils and the following power-law regime with q-2 for theta solvent and q-5/3 for good solvent

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conditions was not accessible. The scattering patterns showed the subsequent q-1 behavior of

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locally stiff fibrils, followed by the Guinier scattering from the fibril cross-section and a q-4

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decrease of the I(q) at higher q values, characteristic for the scattering from the smooth fibril

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surface. Hence it is possible to decouple the data into the individual contributions from the

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worm-like chain at lower q-values and the contributions from the local cylinder cross section

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at higher q-values.41



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Figure 2. Experimental SAXS curves of bare TONFC (grey symbols) and TONFC-nisin bio-

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composite suspensions (blue symbols) at mass fractions of 0.4% (w/v) TONFC at pH 5.8 (A)

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and pH 8.0 (B). The form factor of flexible cylinders with ellipsoidal core was selected to ob-

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tain the best possible fit to the corresponding experimental data (full lines in black for

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TONFC and blue for TONFC-nisin).

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As shown in Figure 2 the q-1 regime extended to lower q-values upon increasing pH from

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5.8 to 8.0. This indicated a decrease in the nanofibril cross-section, potentially combined with

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an increase in the local stiffness (Kuhn-length) of the fibril upon increase of the pH. To fur-

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ther evaluate these transformations in fibril morphology of nanocellulose coils, a model-

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dependent fitting of the scattering data was performed according to equations 1-3 with a form

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factor model for flexible cylinders with an ellipsoidal cross-section.40

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This model provided characteristic parameters including the minor radius and the ratio be-

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tween the minor and major axis of the elliptical core and the Kuhn length (Table 1). One

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needs to consider that inter-fibril interactions may influence the data at intermediate q-range

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and thus the determination of the Kuhn length. On the contrary, the data at higher q-values,

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revealing the cross section dimensions of the fibrils, were reported to be rather independent of

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concentration.42 10 ACS Paragon Plus Environment

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Table 1. Parameters for model-dependent fitting of SAXS data using a model for flexible cyl-

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inders with ellipsoidal core.

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suspension

bare TONFC

TONFC-nisin bare TONFC

TONFC-nisin

pH/mMa

5.8/20

5.8/20

8.0/20

8.0/20

Radius “R” [Å]

12.2 ± 0.28

13.2 ± 0.33

5.2 ± 0.31

10.9 ± 0.35

Axis ratio “a”

5.8 ± 0.17

5.4 ± 0.17

8.3 ± 0.42

4.1 ± 0.16

Kuhn length “b” [Å]

233 ± 5.1

344 ± 9.5

10’000b

10’000b

a

Ionic strength of the suspension, bThe Kuhn length at pH 8 is an estimate, this value does not influence the fit and parameters above as the corresponding Guinier regime is below the lowest accessible q value of 0.008 Å-1

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The cross-section at pH 8.0 is well comparable with previous findings stating a cross sec-

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tion of ~20-30 Å43 for fibrils of TONFC suspensions prepared similarly while at pH 5.8, c is

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clearly higher. Further information on the calculation of the specific surface area (SSA) is

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presented in the SI. The increase in fibril radius upon nisin loading from ~12 to 13 Å at pH

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5.8 and from ~5 to 11 Å at pH 8.0 together with the decrease in axis ratio further confirms

13

that TONFC fibrils interacted with the cationic nisin molecules. However, the dimensional

14

change was less obvious at pH 5.8 as compared to pH 8.0. The peptide-fibril interaction at pH

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5.8 is indicated indirectly by an increase in Kuhn length as represented in Scheme 2, A. How-

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ever the increase in this local stiffness could also be related to inter-fibril interactions.



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Scheme 2. Schematic representation of the dimensional changes of bare TONFC (grey) and TONFC-nisin (blue) and ellipsoid fibril chain organization at pH 5.8 (A) and pH 8.0 (B) obtained by the model dependent fitting of SAXS data (Table 1). The parameters: b is the Kuhn length, r is the radius and a the axis ratio. Ellipsoids, fibrils and peptide structure are represented true to scale.

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Given the relationship between the Kuhn length b and the persistence length l, which is:

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2, the measured persistence lengths for TONFC at pH 5.8 of l = 116-172 Å were compa-

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rable to previous theoretical simulations predictions of similar TONFC materials, reporting l-

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values of ~100-300 Å.44 The increase in b-values upon adsorption of the nisin may result from

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the reduced degree of conformational freedom of the polymer chains within the nanocellulose

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fibril caused by integration of nisin molecules in a linear conformation into the polymer net-

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work. Interestingly, peptide-induced structural transitions persisted, even if pH was shifted to

15

pH = 8.0. This was surprising because a significantly different loading was obtained under

16

these two conditions (Figure 1A). The dimensional increase at pH = 8.0 of approx. 5 Ǻ after

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modification could be directly related to interacting nisin molecules. The average fibril vol-

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ume of bare TONFC at pH 8.0 decreased approx. 3.8-fold as compared to pH 5.8 (Scheme 2). 12 ACS Paragon Plus Environment

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The decrease in fibril radius and the distinct increase in stiffness upon increasing pH from 5.8

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to 8.0 may result from the higher negative charge density on the polymer chains of the fibrils.

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The charge density between pH 5.8 and 8.0 can differ between 10 and 40%, depending on

4

which model is considered for modelling the specific interactions of ions (including peptides)

5

and the carboxylic groups.32 Charge repulsion between these subunits potentially leads to de-

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fibrillation of some fibril aggregates, which reduces the effective radius and stretching of the

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fibril coils resulting in an increase in stiffness. However, a clear distinction between stiffness

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of the fibrils and potential inter-fibril interactions affecting the scattering in this intermediate

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q-region is difficult to achieve.45

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A further microscopic analysis of the fibril network organization of TONFC suspensions

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prepared inside the SAXS capillaries using cross-polarized light revealed epitopes of oriented

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fibril aggregates in all analyzed conditions. This was experimentally confirmed by the bire-

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fringent regions (iridescence)46 observed in the samples as shown in Figure S2. Most likely,

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the orientation was caused by shear-induced alignment of fibrils or fibril bundles during injec-

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tion into the SAXS capillaries as proposed recently.47 Nevertheless, the orientation of TONFC

16

fibrils at pH 8.0 (Figure S2, C) was more homogeneous and the epitopes of orientation longer

17

than at pH 5.8 (Figure S2, A & B). This effect was reproducible and observed even after ul-

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trasonic treatment of the TONFC suspension after filling the X-ray capillary. The phenome-

19

non is consistent with the proposed dimensional reconstruction and higher stiffness at pH 8.0

20

shown in Scheme 2 and possibly due to both a higher aspect ratio together with a more pro-

21

nounced electrostatic repulsion of the elongated fibrils at pH 8.0, as compared to pH 5.8 (Ta-

22

ble 1). This could lead to an increase of the dynamic shear modulus and mesh size of the pol-

23

ymer network48 and therefore an increase in the propensity for side-by-side association of

24

single fibrils along the direction of injection.

25



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Antimicrobial activity of the biocomposite suspensions on a B. subtilis bioreporter strain

3

The nanostructure of TONFC fibrils was clearly affected by the pH of the buffer and the ad-

4

sorption of nisin molecules (Scheme 2, Figure 2). However, effects related to physical immo-

5

bilization on the bioactivity of nisin on the other hand, remained elusive. To gain insight into

6

how the formation of the TONFC-nisin biocomposite affects the antimicrobial potential of

7

nisin, an engineered bioluminescent strain of B. subtilis was used. The nisin-responsive biore-

8

porter strain B. subtilis WT168 sacA::pSDlux102 (pAH328-PpsdA) was obtained from the

9

“Mascher Strain Collection” which is based on the work of Radeck et al.49 Briefly, cellular in-

10

teraction with lantibiotics such as nisin activate promoter PpsdA that was deployed in front of

11

the luxABCDE-operon from Photorabdus luminescence. Promoter activation subsequently

12

leads to detectable and quantifiable luminescence signals that correspond to the antimicrobial

13

activity of nisin. The use of this B. subtilis bioreporter strain in which the biosensor and the

14

native detoxification module PpsdA are interconnected might clarify how the association of

15

nisin with TONFC alters its delivery and availability to the specific cell surface target while

16

signaling how the antibacterial activity is modified.

17

The B. subtilis bioreporter strain was grown in LBM9 growth medium at pH 7.0 and an ion-

18

ic strength of 120 mM. The bioreporter strain B. subtilis responded to the presence of soluble

19

nisin over a narrow interval of 0.5-1 µg/mL (Figure 3A, B & S3). The determined range of

20

sensitivity was highly consistent with previous reports.50 In presence of the TONFC-nisin bio-

21

composite suspension that was washed in in LBM9 growth medium in advance, no linear in-

22

crease in the bioluminescence signal pattern could be measured within the tested range of

23

concentrations. Biocomposite suspensions containing the smallest tested quantity of immobi-

24

lized nisin of 0.06 µg/mL induced a nearly maximal activation of the bioreporter strain in con-

25

trast to a concentration of 0.5 µg/mL of soluble nisin that was necessary to achieve a similar

26

output (Figure 3A). In both soluble and immobilized nisin, an up to ten-fold higher cell sur14 ACS Paragon Plus Environment

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face receptor activation for the biocomposite suspension was detected. In terms of the mini-

2

mal nisin concentration necessary to induce significant growth inhibition, a similar trend

3

could be observed with a two-fold improvement for the biocomposite suspensions (Figure

4

3B).

5

In fact, lipid II residues, one of the main cellular targets of nisin33 are involved in trans-

6

peptidation and trans-glycosylation as critical steps in successful cell wall biosynthesis.51 Se-

7

questration and blocking of such residues through affinity binding could be achieved by nisin

8

molecules physically immobilized to a substrate.52-53 As previously reported,54 the superficial

9

binding of nisin to the target receptor lipid II might already be sufficient to deploy potent an-

10

timicrobial activity of nisin against vegetative cells of Gram positive strains. If this holds true

11

in the investigated system, complete detachment from the carrier substrate TONFC would not

12

be necessary to maintain the antibacterial efficacy of nisin. Moreover, the delivery of nisin as

13

part of a nanocellulose-based carrier system described in this study, potentially allows a more

14

complex mode-of-action as proposed by Vukomanović et al.55 for gold nanoparticles carrying

15

high densities of cationic AMPs. In this case, effects such as accumulation of high densities of

16

ligands on the substrate surface56 could prolong the exposure to target residues and simultane-

17

ously hinder nisin molecules from entering the cellular detoxification machinery57 through

18

sustained binding to the TONFC-nanofibril surface. Limitations in structural flexibility related

19

to physical immobilization of nisin to TONFC, may explain the observed discrepancy in in-

20

creasing efficiency rates between surface receptor activation and growth inhibition, see Figure

21

3A and 3B. It can be speculated that physical binding retarded at least partially the capability

22

of nisin to efficiently insert into the cell membrane.33



Page 16 of 40

1 2

Figure 3. Measurement of the luciferase activities of the B. subtilis bioreporter strain grown

3

in LBM9 medium in presence of increasing concentrations of soluble nisin (grey) or TONFC-

4

nisin suspensions (blue) (A) and corresponding growth yields (B). Concentration-dependent

5

enhancement of PpsdA induction is measured as relative fractional area (FA)58 under the curves

6

represented in Figure S2. The differences in concentration between onset of maximal receptor

7

activity responses and inhibition of growth are indicated (red arrow). Values in A and B rep-

8

resent mean ± standard deviation (n ≥ 6) from two independent measurements. RLU; relative

9

luminescence units. (C) Observed growth inhibition profiles and fitted four parametric logistic

10

regression (4PLS) dose-response curves for soluble nisin (grey) and TONFC-nisin biocompo-

11

site suspensions (blue) in Tris-HCl buffer (triangles) and LBM9 growth medium (circles)

12

against susceptibility test strain S. aureus ATCC 6538. Data are normalized to control; IC50

13

(half maximal inhibitory concentration), are shown as red dashed lines. (D) Calculated mini-

14

mal inhibitory concentrations (MIC) for soluble nisin (grey bars) or biocomposite suspension

15

(blue bars) based on parameters collected in Table S2. Values in graphs represent mean ± 16 ACS Paragon Plus Environment

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1

standard deviation for experiments in LBM9 medium (n ≥ 8) or Tris-HCl buffer (n ≥ 4) from

2

2 independent experiments. (***P < 0.001, *P < 0.01, ns = not significant).

3 4 5

Antimicrobial activity of the biocomposite suspensions on a clinical S. aureus test strain

6

To assess the validity of the several fold increased antimicrobial activity of the TONFC-

7

nisin biocomposite previously observed using the B. subtilis bioreporter strain (Figure 3 A &

8

B), this was tested against a clinically relevant Gram positive strain S. aureus (ATCC 6538) in

9

growth medium (LBM9) and sole buffer (TrisHCl) (Figure 3C, 3D & Table S2).35 All tested

10

systems pinpointed a characteristic concentration-dependent decrease in growth yield over 24

11

h of measurement. Co-incubation of S. aureus with soluble nisin or the biocomposite

12

TONFC-nisin in a complex growth medium with a defined salt composition such as LBM9

13

shown in Figure 3C led to a similar trend as previously described for the bioreporter strain

14

(Figure 3A & 3B). MICLBM9 values of 7.0 ± 0.6 µg/mL for soluble nisin and 3.4 ± 0.4 µg/mL

15

for the TONFC-nisin biocomposite indicated an approximately 2-fold higher quantity of solu-

16

ble nisin necessary to completely inhibit growth (Figure 3D).

17

Susceptibility testing in sole buffer (Tris-HCl) on the other hand, revealed no significant

18

difference with MICTrisHCl = 0.3 ± 0.2 µg/mL for soluble nisin and MICTrisHCl = 0.5 ± 0.2

19

µg/mL for the TONFC-nisin biocomposite. Considering the low MIC values obtained in sole

20

buffer, bacteria respond to different cultivation conditions also by modifying the amount and

21

quality of proteins that are presented on their surfaces.59 Cultivation in sole buffer, even for a

22

short period of time might have thus affected the expression of lipid II, the molecule recog-

23

nized by nisin.33 In addition, it cannot be excluded that the incorporation of nisin into a

24

TONFC matrix prevented unspecific interactions with growth medium components that are

25

known to have an adverse influence on the functionality of AMP-antibiotics.60 As statistical 17 ACS Paragon Plus Environment


Page 18 of 40

1

analysis revealed, soluble nisin and TONFC-nisin were both equally effective to induce sig-

2

nificant growth reduction (non-inhibitory concentration, NIC) and completely prevented

3

growth (MIC) in sole buffer medium verifying that no adverse effect on the bioactivity of

4

nisin was accompanied by the immobilization process (Figure 3D & S4).

5

Contrary to the physisorption-based approach presented here, nisin has been previously

6

immobilized to other nanosized material, for example multi-walled carbon nanotubes

7

(MWCNTs) using a chemical approach but no enhancement effect was detected. This immo-

8

bilization approach might have directly modified the peptide functional group thus giving a

9

reduction in bioactivity.61-62 However, this is not a general outcome as an 18-fold enhance-

10

ment of the bioactivity against Pseudomonas aeruginosa was reported for a derivative of the

11

antimicrobial peptide LL37 integrated into liposomes.63 Similarly, an intermediate enhance-

12

ment of bactericidal activity of indolicidin was detected against Gram positive and negative

13

bacterial strains when immobilized to quantum dots while reducing the toxic side-effects of

14

the material.64

15 16

Interaction of bacterial cells with TONFC fibril networks

17

The morphology of TONFC-fibril suspensions containing 12 µg nisin/mL and the interac-

18

tion with S. aureus cells were additionally investigated by scanning electron microscopy

19

(SEM). Figures 4 and S5 show homogeneously packed networks of predominantly microme-

20

ter sized, interconnected fibers composed from aggregates of single TONC-fibrils.


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1 2

Figure 4. Representative SEM images of the (A) bare TONFC suspensions, (B) TONFC sus-

3

pensions incubated with S. aureus (C) or TONFC-nisin biocomposite suspensions incubated

4

with S. aureus at different magnifications. The scale bars represent 10 µm (top row) and 1 µm

5

(middle row); Single bacteria are recolored and indicated (arrows) as a visual aid. (D/E) Bare

6

TONFC- and TONFC-nisin biocomposite suspensions incubated with S. aureus deposited and

7

prepared as dry films. The scale bars represent 0.5 µm (bottom row). Engulfing TONFC fi19 ACS Paragon Plus Environment


Page 20 of 40

1

brils (arrows) and groves/porous structures related to nisin activity on the bacterial cell sur-

2

face (stars) are indicated.

3

Microcolonies and single bacterial cells with an intact, viable morphology (round cell enve-

4

lope) were visible when co-incubated with bare TONFC suspensions shown in Figure 4B &

5

D. This result is similar to S. aureus cells incubated in sole buffer in absence of nisin (Figure

6

S6). If co-incubated with TONFC-nisin suspensions as shown in Figure 4C & E single bacte-

7

rial cells developed ill-shaped morphologies such as the roughly waved and disrupted cell en-

8

velope. However, in both cases smaller structural units of single TONFC-fibril bundles in the

9

nanometer range could be detected next to, and even engulfing, the bacterial cells (Figure 4D

10

& E). The clearly altered morphological features like grooves and hole-like structures on the

11

envelope of bacterial cells indicate the typical antimicrobial activity of nisin.9, 15, 56 These ob-

12

servations further underline previous findings demonstrating the highly antimicrobial proper-

13

ties of the elaborated TONFC-nisin biocomposite material. Moreover, the findings indicated

14

that the incorporation into the TONFC suspension significantly enhanced the antimicrobial

15

activity of nisin and demonstrated the great potential of TONFC as a carrier system, also for

16

cationic AMP drugs.

17

18 19

Conclusions

20

which combine the strength and stimuli-responsive functionalities of oxidized wood-derived

21

nanofibrillated cellulose, TONFC with the specific bioactive features of antimicrobial pep-

22

tides. Our study demonstrates the integration of the antimicrobial peptide nisin into the na-

23

noscale structure of the TONFC fibril network depending on buffer pH and peptide loading.

24

The non-covalent interactions between nisin and TONFC fibrils induced an increase in the fi-

25

bril radius and reduction of the fiber flexibility. To the best of our knowledge, this is the first

TONFC-based materials provide abundant opportunities to design effective biocomposites,


Page 21 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

report on the self-assembly of TONFC suspensions with nisin into a biocomposite material

2

with high antimicrobial activity. This activity was significantly higher as compared to the free

3

nisin in solutions on both a nisin-specific B. subtilis bioreporter strain and a clinical S. aureus

4

strain. We believe that, if structural effects and related bioactivity patterns are taken into ac-

5

count as shown, this work can pave the way for the development of a wide range of advanced,

6

nanocellulose-based composite materials for application in various fields such as biomedicine

7

and food science ranging from stimuli-responsive drug delivery systems to antimicrobial sur-

8

faces.



Page 22 of 40

1 2

Materials and Methods

3

softwood pulp (Picea abies and Pinus spp.) were obtained from Stendal GmbH (Berlin, Ger-

4

many) and used for the NFC production. 2,2,6,6-Tetramethyl-1-piperidinyloxyl (TEMPO) and

5

sodium hypochlorite (NaClO) solution (12-14% chlorine) were purchased from VWR interna-

6

tional. Sodium bromide (NaBr ≥ 99%) and sodium hydroxide (NaOH ≥ 99%) were supplied

7

by Carl Roth GmbH & Co. Nisin (N5764) from Lactococcus lactis was purchased from Sig-

8

ma-Aldrich (Buchs, Switzerland) as crude lyophilized powder containing ˷2.5 % (w/w) pure

9

nisin and further purified by dialysis as described below. Bovine serum albumin (BSA) stand-

10

ard (prod. no. P5619), BHI (Brain heat infusion broth, prod. no. 53286), casein-based tryptone

11

(prod. no. T7293), yeast extract (prod. no. Y1625) 5 × M9-Minimal Salts (prod. no. M6030),

12

and all other chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland) in analyti-

13

cal grade, and used without further purification unless otherwise stated. Staphylococcus aure-

14

us ATCC 6538 was obtained from American Type Culture Collection (Manassas, USA). Ba-

15

cillus subtilis WT168 sacA::pSDlux102 (pAH328-PpsdA) was received from the “Mascher

16

Lab Strain Collection” (Prof Dr. Thorsten Mascher, Technische Universität Dresden, Institute

17

of Microbiology, Zellescher Weg 20b, 01217 Dresden, Germany) through personal communi-

18

cation.

Materials. Never-dried Elemental Chlorine Free (ECF) cellulose fibers from bleached

19

TEMPO mediated oxidation of nanofibrillated cellulose (TONFC). The decoration of

20

pristine nanocellulose fibril surface with carboxylic groups and analysis of the resulting mate-

21

rial was performed as previously described elsewhere65 and had an average carboxylic group

22

content of 1.91 mmol/g TONFC.19

23

Preparation of nisin solutions. Approx 30 mL solution of 20 mM McIlvaine66 buffer pH

24

5.8 containing 13 mg/mL was prepared, mixed for 45 min at RT on overhead shaker, trans-

25

ferred to 10 mL Float-A-Lyzer G2 dialysis tubes (500-1000 Da, SpectrumLabs, Rancho

26

Dominguez, CA-USA) pre-conditioned according to the manufacturer’s instructions. Dialysis 22 ACS Paragon Plus Environment

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1

was performed for 18 h at RT in a volume of 2 L of identical buffer under constant stirring

2

with complete buffer exchange steps after 1 h, 3 h and 6 h and concentrated according to the

3

manufacturer’s instructions for approx. 3 hours at RT. The ionic strength of 6 mL aliquots

4

was adjusted by adding 4 M NaCl stock solution and pH was set finally, by dropwise addition

5

of either 1 M NaOH or 1 M HCl. The prepared nisin solutions were mixed for 3 h on an over-

6

head shaker and potential insoluble fractions removed by centrifugation at 4’000 × g for 10

7

min at RT followed by filtration using standard 0.22 µm PES-filters (Titan2, ThermoFisch-

8

erScientific, Waltham, MA-USA).

9

Characterization and quantification of TONFC-nisin interactions. The spontaneous

10

physical interaction of TONFC suspensions and nisin at different conditions was analyzed as

11

previously described.19 Briefly, 1.6 mL suspensions containing 0.75 mg TONFC and 0.318

12

mg nisin per mL were prepared per condition in 2 mL protein low binding plastic tubes

13

(022431102, Vaudaux-Eppendorf AG, Schönenbuch-CH), incubated for 3 h at RT under con-

14

stant agitation of 120 rpm, washed twice by centrifugation at 14’000 × g for 3 min and by re-

15

placing the supernatant with an equal volume of corresponding fresh buffer or growth medi-

16

um to remove loosely bound nisin. The bicinchoninic acid assay (BCA protein assay,

17

ThermoFisherScientific, Waltham, MA-USA) and BSA as a standard was used to quantify all

18

sample preparations according to the manufacturer’s instructions after validation as described

19

in the SI (Figure S1 and related description of the BCA peptide quantification assay in

20

the SI).

21

Analysis of the TONFC-nisin fibril surface- and network properties

22

Zeta potential measurements. The zeta potentials of the bare TONFC- and biocomposite

23

suspension samples were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Mal-

24

vern UK) in plastic capillary cells (DTS1070, Malvern Instruments, Malvern UK) using a He–

25

Ne Laser beam at a wavelength of 633 nm. The measurements were performed at a scattering

26

angle of 90° and with a laser power of 4 mW. The temperature was kept at 25 °C in all meas23 ACS Paragon Plus Environment


Page 24 of 40

1

urements. The samples were prepared in 20 mM McIlvaine buffer at different pH as described

2

above, homogenized by pipetting up and down, followed by 5 min sonication in a water bath

3

(Bransonic 52, Branson Ultrasonics SA, Carouge-CH). The final suspensions had a TONFC

4

concentration of 0.5 mg/mL.

5

Small angle X-ray scattering (SAXS). Bare TONFC- and TONFC-nisin buffered suspen-

6

sions were injected into SAXS glass capillaries, followed by 5 min sonication in water bath

7

(Bransonic 52, Branson Ultrasonics SA, Carouge-CH) in advance of SAXS measurements.

8

The final suspensions had a TONFC concentration of 0.4 % w/v. SAXS measurements were

9

performed on a Bruker Nanostar (Bruker AXS, Karlsruhe, Germany) connected to an X-ray

10

source (Incoatec IµSCu, Geest-hacht, Germany) operating at 50 kV and 600 µA with a sealed-

11

tube Cu anode. A Göbel mirror was used to convert the divergent polychromatic X-ray beam

12

into a focused beam of monochromatic Cu Kα radiation (λ = 1.54 Å). The beam cross section

13

diameter was 0.3 mm. A sample to detector distance of 107.5 cm gave the q-range of around

14

0.008 < q < 0.30 Å-1, where q is the length of the scattering vector, defined by q = (4π/λ)

15

sin(θ/2), λ is the wavelength and θ the scattering angle. The 2D SAXS patterns were acquired

16

within 1 h using a VÅNTEC-2000 detector (Bruker AXS, Karlsruhe, Germany) with active

17

area 14 x 14 cm2 and with a pixel size of 68µm x 68µm. All experiments were performed at T

18

= 25°C. Two dimensional scattering patterns were integrated into the one-dimensional scatter-

19

ing function I(q). Glassy carbon was measured with all samples for transmittance corrections,

20

relative to the transmittance of the buffer (background). Normalized scattering curves are

21

plotted as a function of relative intensity, I, versus q. The scattering of the specific buffer was

22

subtracted as background from all samples.

23

Analysis of SAXS data. Model dependent analysis of the SAXS data was undertaken to

24

gain insight into the TONFC-nisin self-assembly. The form factor of flexible cylinders with

25

an ellipsoidal core was used to describe the scattering data in these systems. The length of the 24 ACS Paragon Plus Environment

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1

TONFC fibrils is up to several micrometers with a cross-section in the range of several na-

2

nometers.43 Hence, the scattering data can be described using equation 1:40

3

∝ , , , ,

(1)

4

Pfiber(q,L,b) is the scattering function of a flexible cylinder with excluded volume effects

5

calculated with Method 1 described by Pedersen and Schurtenberger40 with the variables q, L

6

and b being the magnitude of the scattering vector, the contour length and the Kuhn length re-

7

spectively. Pcross(q, Rmin, a) describes the ellipsoidal cross section of the fibrils using:

8 9

&/ ! ,",#$ ,",#$ % )* !

, , (

(2)

in which J1(x) is the first order Bessel function and

10

+ , ,, ∝ -./0 * + ,2. *

11

Rmin being the cross sectional radius, a the major/minor axis ratio of the elliptical cross section

12

and α the angle between the axis of the ellipse and the q-vector.

(3)

13

Antibacterial susceptibility testing of biocomposite suspensions

14

Media preparation. 2 × LB (lysogeny broth) was mixed by resolving 20 g tryptone, 10 g

15

NaCl and 10 g yeast extract in 1 L nanopure water and adjusted to pH 7.0 by dropwise addi-

16

tion of 1M NaOH/HCl. 2 × LB and 2 × M9-minimal salts solution were autoclaved and prior

17

to use, mixed 1:1 and supplemented with 2 mL/L of a sterile 1 M solution of MgSO4*7H2O

18

and 0.1 mL/L of 1 M solution of CaCl2. The prepared LBM9 medium had an ionic strength of

19

200 mM and was diluted to 120 mM by adding sterile nanopure water. 1 × BHI medium was

20

prepared according to manufacturer’s instructions. Both test media were sterilized by auto-

21

claving after preparation. All nisin solutions used were sterile filtered by using disposable,

22

sterile 33 mm filter units with a 0.22 µm pore size (Millex GP, Sigma-Aldrich (Buchs, Swit-

23

zerland) before further use. All TONFC-nisin suspensions used were prepared from bare

24

TONFC suspensions pasteurized at 70 °C for 3 h and sterile filtered nisin solutions. The pre-

25

pared TONFC-nisin suspensions were washed twice in LBM9 medium or sole buffer before 25 ACS Paragon Plus Environment


Page 26 of 40

1

quantification of the nisin loading density to avoid detachment of nisin from TONFC during

2

antibacterial susceptibility tests. All antibacterial susceptibility tests were performed in sterile

3

2 mL protein low binding plastic tubes (022431102, Vaudaux-Eppendorf AG, Schönenbuch-

4

CH) and reference samples were either prepared by adding nanopure water or bare TONFC at

5

a concentration equal to the highest concentration of biocomposite suspension experimentally

6

analyzed.

7

Luminescence based bioreporter assay. B. subtilis WT168 sacA::pSDlux102 (pAH328-

8

PpsdA) was used to test the delivery of nisin to cell surface receptor and correlating growth

9

reducing effects. Bacteria from glycerol stocks were streaked on LB (lysogeny broth) agar

10

plates supplemented with 10 µg/mL Chloramphenicol (Cm), a single colony transferred to 15

11

mL of fresh LBM9 + Cm medium, incubated overnight at 160 rpm and 37 °C, diluted 1:50 in

12

fresh medium to a final volume of 100 mL and grown to approx. OD600 0.45 (log-phase). Bac-

13

terial suspensions were diluted to OD600 0.1 in fresh 120 mM LBM9 + Cm medium pH 7.0

14

and 180 µL distributed per well of a 96-well plate with a white walled and transparent flat

15

bottom (3610, Corning Incorporated, Corning, NY-USA). Stock solutions/suspensions, by

16

mixing 10 × stocks containing between 40-0.625 µg nisin/mL in 120 mM McIlvaine buffer

17

pH 7.0. 10 µL of the 10 × stock solutions/suspensions were then added to the bacterial sus-

18

pensions to a final bacterial cell density of OD600 = 0.09 and 1 × final nisin concentrations.

19

The mixed samples were incubated in a multi well-plate spectrophotometer (Synergy H1, Bi-

20

oTek Instruments, Luzern-CH) for 24 h at 160 rpm with temperature fixed at 37 °C while

21

OD600 and luminescence intensity (exposure: 100 ms, gain: 135) were recorded every 3 min.

22

Susceptibility testing against S. aureus test strain. S. aureus (ATCC 6538) was used to

23

test growth reducing effects, following a standardized test procedure.67 Bacteria from glycerol

24

stocks were streaked on brain-heart infusion (BHI) agar plates, a single colony transferred to

25

15 mL of fresh BHI medium, incubated overnight at 160 rpm and 37 °C, diluted 1:50 in fresh

26

medium and grown to approx. OD600 = 0.35 (log-phase). Bacterial suspensions were diluted to 26 ACS Paragon Plus Environment

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1

OD600 0.2 in either fresh 120 mM LBM9 medium pH 7.0 (complex growth medium) or 120

2

mM TrisHCl at pH 7.0 (sole buffer), washed twice in identical medium by 3 min centrifuga-

3

tion 3’000 × g at RT. Stock solutions/suspensions were prepared in LBM9 or TrisHCl medi-

4

um as 1.05 × stocks containing between 25.2-0.02 µg nisin/mL. 10 µL of the washed cultures

5

(OD600 = 0.2) were mixed per well of a transparent 96-well plate (781662, Brand plates pure-

6

GradeTM S, Brand GmbH, Wertheim, Germany) with 190 µL of the 1.05 × nisin stock solu-

7

tions/suspensions (final OD600 = 0.01). The mixed samples were incubated in a multi-well

8

plate spectrophotometer (Synergy H1, BioTek Instruments, Luzern-CH) for 24 h at 160 rpm

9

with temperature fixed at 37 °C while OD600 was recorded every 30 min.

10

Growth inhibiting- and bacterial cell surface receptor activating effects were manifested by

11

a reduction in the fractional area (FA). The FA derives from the area under OD600/time

12

(growth inhibition) and RLU/OD600/time (receptor activation) curves relative to the corre-

13

sponding reference sample, see supplementary Figure S3. Briefly, FA was calculated as de-

14

scribed elsewhere58 by integration of the background corrected curve between t0 and tend ac-

15

cording to the trapezoidal rule using the integration tool of the PrismTM software (Version

16

6.07, June 12, 2015, Graphpad Software Inc. San Diego CA, USA) and normalized to refer-

17

ence (FA: OD600) or maximal luminescence signal (FA: RLU/OD600). The obtained (FA) data

18

points were either directly analyzed (bioreporter assay) or fitted to a four parametric dose re-

19

sponse model (susceptibility testing)68 described by Equation 4 using the PrismTM software.

20

With a = upper asymptote ( ≅ 1), d = lower asymptote () ≅ 0), c = log concentration at the

21

half maximal dose response level (c = logIC50), b = Hill slope and x = log of the antimicrobial

22

concentration (log[µg/mL]).

23 24 25

6789:;; ) +

"? B A

(4)

The minimal inhibitory concentration “MIC” [µg/mL] is defined, as the line tangential to the curve inflexion point58 c respectively calculated according to Equation 5: 27 ACS Paragon Plus Environment


G

DEF 10 HB

1 2 3

"

Enhanced antimicrobial activity and structural transitions of a

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