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The public defense on 24th April 2020 at 12:00 will be organized via remote technology.

Link: https://aalto.zoom.us/j/6093297778

Zoom Quick Guide: https://www.aalto.fi/en/services/zoom- quick-guide

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Thesis advisor Dr. Vânia M. Moreira, University of Coimbra, Portugal and University of Helsinki, Finland

Preliminary examiners Professor Martti Toivakka, Åbo Akademi, Finland Professor Vincent Nierstatz, University of Borås, Sweden

Opponent Prodessor Aji Mathew, Stockholm University, Sweden

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sdrowyeK ecafrus ,noitacfiidom ,esolullec ,elitxet ,cibohpordyh ,elbahtaerb laiborcimitna )detnirp( NBSI )detnirp( 5-8283-06-259-879 NBSI )fdp( 2-9283-06-259-879 )detnirp( NSSI )detnirp( 4394-9971 NSSI )fdp( 2494-9971 rehsilbup fo noitacoL fo rehsilbup iknisleH noitacoL fo gnitnirp iknisleH raeY 0202 segaP 432 nru :NBSI:NRU/fi.nru//:ptth 2-9283-06-259-879

gardnammaS otlaA 67000 ,00011 BP ,tetetisrevinu-otlaA BP ,00011 67000 otlaA if.otlaa.www

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drolekcyN ,gnirefiidomty ,esolullec ,relitxet ,bofordyh ,ednadna lleiborkimitna )tkcyrt( NBSI )tkcyrt( 5-8283-06-259-879 NBSI )fdp( 2-9283-06-259-879 )tkcyrt( NSSI )tkcyrt( 4394-9971 NSSI )fdp( 2494-9971 trosgninvigtU srofgnisleH trokcyrT srofgnisleH rÅ 0202 latnadiS 432 nru :NBSI:NRU/fi.nru//:ptth 2-9283-06-259-879

Preface

This work was carried out at the Department of Bioproducts and Biosystems at Aalto Univeristy. Part of the work for Publications IV and V was also carried out at University of Helsinki. The work with the pigments was carried out at Na- tional Institute of Chemistry in Ljubljana, Slovenia. The research was funded by Aalto Univeristy and Business Finland, and I am grateful for their support mak- ing this research possible. Traveling and getting international experience was made possible thanks to Walter Ahlström Foundation and Climate-KIC: Pio- neers into Practice, and I would like to thank them for the useful experiences they enabled.

Monika, thank you so much! Thank you for accepting me as a student, helping (and pushing) me whenever I needed. Your door is always open and I have grown both as a scientist and as a person during this time. I enjoy having you as my supervisor and am proud to graduate in your group. Plus att få tala veten- skap på muminspråk ännu i det här skedet är ju lyx. Vânia, thank you for letting me be part of your reseach. You and Resicell have tought me plenty about sci- ence, and I am happy to have you as my advisor.

A warm thank you to the opponent Prof. Aji Mathew. I am grateful for you ac- cepting this laborious task. Similarly, thanks to the pre-examiners, Prof. Vincent Nierstratz and Prof. Martti Toivakka. Your careful examination pointed out the weaknesses and helped make a more solid thesis.

To all my co-authors, thank you so much! Alina, Ghada, Oona, thank you for the work we did together. Warm thanks also to Leena-Sisko, Matilda, Meggie, Leena and all others. It has been a great pleasue to get to know and work with you all.

Thanks to the whole Bioproduct Chemistry group! Science might not always be easy (well, it shouldn’t be easy) but nice colleagues help you get through the tougher parts and enjoy the better parts. Thank you all for that, I really enjoy our atmosphere and working in our group.

Teh Band, thank you for some of the best moments of my time at Puu. The or- chestra is my natural habitat, so thank you for reminding me that work not is everything, and that there are still wild horses. Meri, Kyösti, Paavo and all the rest, it is so much fun playing with you!

There are plenty of nice people at Puu, too many to mention, that creates our good atmosphere. Thank you for the coffee breaks, the smiles in the lab, help when needed. It is truly an amazing place to work, thank you for that. Thank i you, Rita, for being a great office mate! A warm thank you also to my temporary coworkers and hosts in Slovenia, I had a wounderful time and it was nice getting to know you!

My family has fueled my interest in science, thank you for that. Friends, outside of science, and orchestra mates are important as well, thank you all. And Joonas, thank you for standing by my side during the ups and especially downs of PhD.

Espoo, March 2020 Nina Forsman

ii

List of publications

I Forsman, N; Lozhechnikova A; Khakalo, A; Johansson, L-S; Vartiainen J; Österberg, M. Layer-by-layer assembled hydrophobic coatings for cellulose nanofibril films and textiles, made of polylysine and natural wax particles. Carbohydr. Polym. 173, 392–402 (2017).

II Forsman, N; Johansson, L-S; Koivula, Hanna; Tuure, M; Kääriäinen, P; Österberg, M. Open coating with natural wax particles enables scalable, non- toxic hydrophobation of cellulose-based textiles. Carbohydr. Polym. 227, 115363 (2020).

III Korhonen, O; Forsman, N; Österberg, M; Budtova, T. Eco-friendly surface hydrophobization of all-cellulose composites using layer-by-layer deposition. Accepted by Express Polymer Letters.

IV Hassan, G; Forsman, N; Wan, X; Keurulainen, L; Bimbo, L, M; Johansson, L-S; Sipari, N; Yli-Kauhaluoma, J; Zimmermann, R; Stehl, S; Werner, C; Saris, P; Österberg, M; Moreira, V, M. Dehydroabietylamine-based Cellulose Nanofibril Films: A new Class of Sustainable Biomaterials for Highly Efficient, Broad-Spectrum Antimicrobial Effects. ACS Sustain. Chem. Eng. 2019, 7: 5002- 5009, doi: 10.1021/acssuschemeng.8b05658.

V Hassan, G; Forsman, N; Wan, X; Keurulainen, L; Bimbo, L, M; Stehl, S; van Charante, F; Chrubasik, M; Prakash, A; Johansson, L-S; Mullen, D, C; Johnston, B; Zimmermann, R; Werner, C; Yli-Kauhaluoma, J; Coenye, T; Saris, P; Österberg, M; Moreira, V, M. Non-leaching, biocompatible abietane- nanocellulose surfaces that efficiently resist fouling by bacteria in the artificial dermis model. Submitted to ACS Applied Bio Materials.

iii

Authors’ contributions

Publication I: Layer-by-layer assembled hydrophobic coatings for cellulose nanofibril films and textiles, made of polylysine and natural wax particles

NF and AL contributed equally to the experimental design and shared experimental work. NF performed the QCM-' Dž-potential, FTIR, moisture buffering and part of the WCA measurements, and took active part in analysing the results together with AL. AL performed AFM and WCA measurements. NF and AL wrote the manuscript together under the supervision of MÖ. AK perfomed SEM imaging together with AL. JV was responsible for the OTR measurements and L-SJ for the XPS measurements.

Publication II: Open coating with natural wax particles enables scalable, non- toxic hydrophobation of cellulose-based textiles

NF was responsible for the experimental design, preparation of samples, WCA, FTIR and SEM measurements, took active part in analysing the results and drafted the manuscript under supervision of MÖ. L-SJ was responsible for the XPS measurements and HK for the WVTR measurements. MT designed and produced the garments under supervision of PK.

Publication III: Eco-friendly surface hydrophobization of all-cellulose com- posites using layer-by-layer deposition

NF and OK contributed equally to the experimental design and shared experimental work. NF performed the AFM and most of the WCA measurements, and took active part in analyzing the results together with OK. OK prepared the composites, performed the vapor sorption, tensile strength and some WCA measurements. NF and OK wrote the manuscript together under the supervision of MÖ and TB.

Publication IV: Dehydroabietylamine-based Cellulose Nanofibril Films: A new Class of Sustainable Biomaterials for Highly Efficient, Broad-Spectrum Antimicrobial Effects.

NF prepared the CNF films, performed the WCA, AFM and tensile strength measurements, analysed the results from aformentioned measurements as well as the XPS, OTR and WVTR results, and wrote part of the manuscript under supervision of MÖ and VM. NF, GH, LK, MÖ and VM were responsible for the experimental design. GH performed all synthetic work and wrote part of the manuscript, under the supervision of VM. LK was responsible for the design and synthesis of compounds and materials, and revised the manuscript. L-SJ was

iv responsible for the XPS measurements. XW performed the antimicrobial testing and SEM analysis under the supervision of PS. NS performed the mass analysis of the compunds. RZ, SS and CW were responsible for the streaming potential measurements. LB performed the biocompatibility testing.

Publication V: Non-leaching, biocompatible abietane-nanocellulose surfaces that efficiently resist fouling by bacteria in the artificial dermis model.

NF performed the WCA and AFM measurements, analysed the results from aformentioned analyses as well as the XPS results and wrote part of the manuscript under supervision of MÖ and VM. NF, GH, LK, MÖ and VM were responsible for the experimental design. GH performed all synthetic work and wrote part of the manuscript, under the supervision of VM. LK was responsible for the design and synthesis of compounds and materials, and revised the manuscript. L-SJ was responsible for the XPS measurements. XW performed the antimicrobial testing and SEM analysis under the supervision of PS. RZ, SS and CW were responsible for the streaming potential measurements. LB performed the biocompatibility testing. BJ, MC, AP, DM were responsible for the ToF-SIMS analysis. DM also helped to write and revise the manuscript. FvC and TC performed the biofilm and artificial dermis experiments and FvC also helped to write the manuscript. JY-K participated in writing and revision of the manuscript.

v

List of essential abbreviations

AFM Atomic force microscopy BL Bilayer BP Blue pigment CMC Carboxymethylated cellulose CNF Cellulose nanofibril CNF-CMC-1 Dehydroabietylamine coupled to CMC adsorbed onto CNF CNF-CMC-4 N-(3-Aminopropyl)dehydroabietylamine coupled to CMC adsorbed onto CNF CNF-CMC-7A Methyl 12-(3-aminopropoxy)abieta-8,11,13-trien- 18-oate coupled to CMC adsorbed onto CNF CNF-CMC-7B Methyl 12-(3-aminopropoxy)-N-(abiet-8,11,13- trien-18-oyl) cyclohexyl-L-alanine coupled to CMC adsorbed onto CNF CS Cationic starch FTIR Fourier transform infrared spectroscopy LbL Layer-by-layer NIR Near-infrared radiation QCM-D Quartz crystal microbalance with dissipation PLL Poly-L-lysine RH Relative humidity RT Room temperature SEM Scanning electron microscopy UV/VIS Ultraviolet/visual light spectra WCA Water contact angle XPS X-ray photoelectron spectroscopy Dž-potential Zeta-potential

vi

Table of Contents

Preface ...………………………………………………………………………………………..i

List of publications ..…….………………………………………………………………..iii

Authors’ contributions …………………………………………………………………...iv

List of essential abbreviations …………………………………………………………vi

1. Introduction ...... 1 1.1 General overview of the need for sustainable materials and coatings 1 1.2 Aims of the study ...... 2 1.3 Outline ...... 2 2. Background ...... 5 2.1 Cellulosic materials ...... 5 2.1.1 Plant-based textiles ...... 5 2.2 Carnauba wax ...... 6 2.3 Wetting ...... 6 2.3.1 Evaluating the water-repellance of textiles ...... 7 2.4 Hydrophobization methods for cellulosic materials ...... 8 2.4.1 Utilizing and increasing the intrinsic surface roughness ...... 8 2.4.2 Fluorine-based methods ...... 9 2.4.3 Silicon-based methods ...... 9 2.4.4 Polymer-based methods ...... 9 2.4.5 Bio-based methods ...... 9 2.5 Multifunctional cellulosic materials ...... 10 2.5.1 Hydrophobic and breathable materials ...... 10 2.5.2 Cooling pigments ...... 10 2.5.3 Antimicrobial and hydrophobic surfaces ...... 11 2.6 Surface modification methods ...... 12 2.6.1 Layer-by-layer deposition ...... 12 2.6.2 Sol-gel ...... 12 2.6.3 Gaseous application techniques ...... 12 2.6.4 Dye exhaustion ...... 13 2.6.5 Adsorption of polyelectrolytes ...... 13 3. Materials and methods ...... 14 3.1 Materials ...... 14 3.1.1 Polycations ...... 14 3.1.2 Carnauba wax dispersion ...... 14 vii

3.1.3 Ultrathin CNF films ...... 14 3.1.4 Free-standing CNF films ...... 15 3.1.5 All-cellulose composites ...... 15 3.1.6 Textiles ...... 15 3.2 Methods ...... 16 3.2.1 Layer-by-layer ...... 16 3.2.2 Dyeing of textiles ...... 16 3.2.3 Preparation of antimicrobial CNF films ...... 17 3.2.4 Contact angle ...... 17 3.2.5 Microscopy methods ...... 17 3.2.6 Chemical characterization methods ...... 18 3.2.7 Water vapor interaction ...... 19 3.2.8 Other methods ...... 19 4. Results and discussion ...... 22 4.1 Characteristics of the cellulosic surfaces studied ...... 22 4.2 Layer-by-layer deposition for surface modification ...... 23 4.2.1 The role of the polycation ...... 23 4.2.2 Effect of curing temperature on the wax coating ...... 24 4.2.3 Surface roughness ...... 26 4.2.4 Durability ...... 29 4.3 Breathability ...... 29 4.4 Hydrophobizing a surface using aqueous dispersions ...... 30 4.5 Scaling ...... 30 4.6 Functional pigments (unpublished results) ...... 31 4.7 Antimicrobial coatings ...... 33 4.7.1 Surface coverage and hydrophobicity ...... 33 4.7.2 Assessment of antimicrobial activity ...... 34 4.7.3 Biocompatibility ...... 37 4.7.4 Effect of the modification using dehydroabietane on other properties 38 5. Concluding remarks ...... 40 References ...... 42

viii

1. Introduction

1.1 General overview of the need for sustainable materials and coatings

A large part of the materials in everyday use are either composed of or contain plastics, i.e. synthetic polymers made from crude oil that are non-biodegrada- ble. The global textile industry is no exception as polyester composes a majority of the world fiber production (about 65%).1 Although, synthetic polymers have many attractive properties, such as durability, versatility and low price, more and more durability is also being considered as a major disadvantage as syn- thetic polymers tend to persist in the environment after use, resulting in long term pollution. The impact of microplastics released during the washing of tex- tiles, for example, has recently raised a lot of concern.2 Nevertheless, the versa- tility and low price of synthetic polymers results in their continued use in many applications, which - when combined with the current “throwaway society” cul- ture that discards still functional commodity products - sets the foundation for numerous environmental issues.

Bio-based and biodegradable materials offer a more sustainable alternative to synthetic polymers, especially in textiles. Cellulose is the most abundant biopo- lymer on Earth and is already widely used for fabrics e.g. hemp, linen and cotton (the most abundant textile material) are all derived from cellulosic plants. On the other hand, the large use of land, water and pesticides during cotton pro- duction has raised some concern regarding the sustainability of the cotton in- dustry. Consequently, technologies to convert other type of biomass sources into textiles have been developed and the methods related manufacturing are still evolving. Nevertheless, it is not just fiber production that can have negative en- vironmental impacts, as textiles are often chemically treated to in order to pro- vide different properties and finishes such as color, water-repellence, antimi- crobial resistance, fire-retardance, anti-wrinkling characteristics, softer feel and countless other attributes.3 Unfortunately, the methods used to achieve these types of desirable properties often involve harmful or even hazardous com- pounds.4 Conversly, within nature there are several well-functioning materials and surfaces with interesting features that can be mimicked or exploited for use with textiles and other materials in a more ecologically sustainable way. In this thesis, the main emphasis is on hydrophobic coatings that can retain fabric

1 breathability, although surface multifunctionality is also explored. Hydrophobic breathable textiles are commonly desired, although additional functionality im- prove the value and possible uses of the product. More functionality of bio- based materials also gives them an advantage over synthetic counterparts.

In addition to textiles, cellulose could replace plastics in many other applica- tions, e.g. packaging, construction materials and electronics. While the hydro- philicity of cellulose is behind many of the desirable properties of cellulosic ma- terials, it also is a drawback in many of these types of applications, though this could be overcome with a hydrophobic coating. Often - especially in textiles - it is desired that the coated material is still breathable, whilst extra functionality can also add value and potentially increase product usability. For example, dyes are an integral part of many products, but it can also possible have more func- tionality than just a nice color, e.g. thermal properties. In addition, properties like antimicrobial activity and resistance are not only especially useful in bio- medical applications, but also in a range of other products including indoor en- vironments.

1.2 Aims of the study

In this work, the aim was to understand, utlize and optimize the surface modi- fication of cellulosic materials with environmentally benign compounds, in or- der to develop more ecologically sustainable materials. Special emphasis was put on hydrophobic and breathable coatings, but cooling dyes and antimicrobial coatings were also studied. The most important research questions can be sum- marized as follows: How to achieve properties like hydrophobicity, cooling and antimicrobial activity on cellulosic surfaces while preserving the breathability and strength of the original material, by using only natural, non-toxic materials? How will different types of cellulosic materials respond to the treatments? How can the treatment be optimized? Hypotheses are the following: Wax particles will give high hydrophobicity yet not impair the breathability, although melting of the particles will reduce the breathability. The more wax on the surface the higher the hydrophobicity. The dehydroabietanes will provide both high hydro- phobicity and high antimicrobial activity, to different extent depending on the specific compound.

1.3 Outline

The main findings of this research work can be found in the included articles, which can be summarized as follows:

The aim of the first publication (I) was to understand the layer build-up process and the behavior of both polycationic substances and wax particles on cellulosic surfaces. The build-up of layers was systematically studied by quartz crystal mi- crobalance with dissipation (QCM-D) and water contact angle (WCA). It was found that the thickness of the poly-L-lysine (PLL) layer affected the amount of adsorbed wax particles and consequently, the resultant wetting properties. 2

Moreover, the higher the pH during PLL adsorption, the thicker the adsorbed layer and the optimal pH was determined to be 9.5 as this led to a looped and tailed PLL conformation that maximized wax particles adsorption. Two bilayers gave the highest WCA, and subsequent investigations with atomic force micros- copy (AFM) and X-ray photopelectron spectroscopy (XPS) confirmed that the surface was fully covered with wax particles. Further investigations demon- strated that the coating worked on different cellulosic substrates that included ultrathin model cellulose nanofibril (CNF) films, free-standing CNF films as well as two cotton and one linen textile. Moreover, results also showed that the higher the surface roughness of the substrate, the higher the WCA.

In Publication II the properties of the coating were studied in more detail with the goal of scaling up the method. WCA and XPS were utilized to systematically examine the effect of curing temperature and it was discovered that wax mole- cule reorientation and partial melting increased the hydrophobicity, whereas material breathability was high, regardless of the curing temperature. Cationic starch (CS) was also evaluated as a cheaper alternative to PLL and although the PLL anchoring layer provided a higher WCA, the properties of CS proved to be adequate. The surface roughness of the coated fabric was found to contribute to the surface hydrophobicity. Three jackets were made, and it was demonstrated that the coating could be applied using either spraying or brushing techniques. Additionally, pigments were also added in the wax dispersion in order to pro- duce finishes with combined color and hydrophobic functionalities.

The behavior of the polycation-wax particle coating on all-cellulose composites was the subject of Publication III. Unlike textiles, the mechanical properties of cellulose composites were seen to deteriorate on contact with water, therefore methods to minimize their contact with water were explored. Concentration of the dispersion with a rotavapor followed by application via spraying not only provided high hydrophobic characteristics but also retained the material’s me- chanical properties. Furthermore, the melting behavior of carnauba wax was in- vestigated in more detail with dynamic scanning calorimetry (DSC).

The aim of Publication IV was to make an antibacterial and hydrophobic cellu- losic material using dehydroabietylamines as an alternative to silver nanoparti- cle coatings. The dehydroabietylamines were covalently bound onto CNF films activated with carboxymethylated cellulose (CMC) as the goal was to create a more durable coating than used in the previous publications - covalent bonds are stronger than the electrostatic attractions utilized by LbL. Although the sur- face coverage was determined to be low, excellent antimicrobial properties were still achieved. In addition, the hydrophobicity of the treated materials was in- creased, whereas the mechanical and breathing properties remained almost un- changed.

Publication V was a follow up to Publication IV and the overall aim was similar, however dehydroabietic acids were utilized rather than dehydroabietylamines in this case. The modified films exhibited excellent antimicrobial properties and

3 were also found to be biocompatible as well as non-leaching. Experiments with the modified films, in conditions similar to those found in wounds, indicated that bacteria were unable to form a biofilm on these types of surface-modified cellulose films.

Figure 1. Outline of the study. To understand the surface interactions, model surfaces (thin films) are used and the modifications are then replicated on cel- lulosic textiles and composites. In A) a hydrophobic and breathable surface, in B) a hydrophobic material that remains cool in the sun and in C) a hydropho- bic and antimicrobial surface is desired.

4

2. Background

2.1 Cellulosic materials

Cellulose is produced by all photosynthesizing plants, some bacteria and tuni- cates, and is the most abundant biopolymer on Earth. Cellulose is a linear poly- saccharide that consists of two anhydroglucose rings, bound together with a ǃ 1o 4 glycosidic bond (Fig. 2), which as a natural material, is both renewable and biodegradable. Cellulose has both ordered crystallinic and disordered amorphous regions, and due to the accessible hydroxyl groups on the amor- phous regions cellulose is hydrophilic and swell in water.5 When exposed to hu- midity (water vapor), cellulose attracts the water molecules by sorption.6 Due to its strength, non-toxicity and availability, cellulose has already been used for a long time in a range of applications that include textiles, packaging materials, papers and tissues, biomedical applications and composites. The composites can either be all-cellulose composites7 or mixed with other materials.8 More re- cently, new types of applications for cellulose - and in particular cellulose nan- ofibrils (CNF) - have gained increasing interest, 9–11 and of the applications free- standing CNF films can be mentioned.12 Nevertheless, the hydrophilic nature of cellulose can be a drawback in some applications, and consequently methods that allow for the hydrophobization of cellulose has received plenty of atten- tion.13

Figure 2. The structure of cellulose.

2.1.1 Plant-based textiles

Textiles can be made from both synthetic (oil-based) and natural raw-materials like animal-based textiles that consist of proteins, e.g. wool and silk, and plant- based textiles composed of cellulose. Plant-based textiles are often divided into 5 two seperate catergories: (1) Natural textiles, i.e. cotton, linen, hemp, bamboo; (2) Man-made cellulosic or regenerated textiles, such as viscose/modal, lyocell (dissolved in NMMO, N-methylmorpholine-N-oxide)14 and Ioncell-F (dissolved in ionic liquids).15 Regenerated textile fibers where the cellulose has been dis- solved incuprammonium16 or LiCl/DMAc (lithiumchloride/dimethylacetam- ide),17,18 and textile fibers from oxidizing cellulose and cross-linking with chi- tosan19 have also been made. Chemically all plant-based textiles are cellulose and therefore no distinction between natural and man-made cellulosic textiles will be made in this work.

2.2 Carnauba wax

Carnauba wax comes from the leaves of the Copernicia prunifera palm tree, that grows in the northeast of Brazil. There have been attempts to plant the palm in other parts of the world, however, due to the specific weather conditions re- quired, the palm only produces wax in Brazil. Wax is obtained by first harvesting the leaves and then drying them to recover the wax. Carnauba wax is one of the hardest of all natural waxes, hypoallergenic and has a melting point of 83- 86°C.20,21 As a natural substance, carnauba wax is not chemically homogenous but consists of several different compounds, mainly wax esters (83-85%) and fatty acids (3%), fatty alcohols (3%), lactides (3%), hydrocarbons (2%) and res- ins (5%).20,22

2.3 Wetting

Classical wetting behavior of a surface depends on the interfacial tensions be- tween the liquid-VROLG DŽSL), liquid-YDSRU DŽLV) and solid-YDSRU DŽSV SKDVHVDŽSV

LVDOVRNQRZQDVVXUIDFHIUHHHQHUJ\DQGDŽLV as the surface tension of the liquid (Fig. 3). The thermodynamic equilibrium of a static water droplet on a homog- enous flat surface is described by the Young’s equation:23

ఊ ିఊ ܿ݋ݏߠ = ೄೇ ೄಽ ఊಽೇ

ZKHUHLJLVWKHVWDWLFFRQWDFWDQJOHEHWZHHQWKHOLTXLGGURSOHWDQGWKHVXUIDFH,W is generally accepted that a surface is hydrophobic if the water contact angle 24,25 (WCA or LJw) is > 90° and hydrophilic if LJw < 90°. In contrast, a surface is considered to be superhydrophobic if it has a LJw > 150° and a roll-off angle (RA) < 10°.26–28 Nonetheless, some researchers have suggested that the 90° threshold for hydrophobicity is not truly representative and that 65° would be a better limit.29,30 Additionally, a surface where the droplet easily rolls off can also be regarded as self-cleaning. Instead of measuring RA, the contact angle hysteresis – the difference between advancing and receding contact angle – can be used as an alternative (Fig. 3). For a smooth surface with low surface energy, the highest 31,32 LJw attained is around 120°. In order to achieve higher LJw surface roughness, preferably on several scale lengths, is needed in addition to the low surface en- ergy.33–37

6

There are two types of wetting on rough, structured surfaces: the Cassie state where the droplet lies on top of the surface with air pockets between the surface structures,38 and the Wenzel state when the droplet completely wets the surface 39 with are no air pockets. Generally, the Wenzel state has a higher RA – or LJw hysteresis – since the droplet is more constricted when compared to the Cassie state.40 A water-repellent (or water-resistant) material refers to a material that keeps water out from the inner surface under normal conditions (e.g. rain), whereas a waterproof material keeps the water out even in more challenging conditions (e.g. fully submerged in water under pressure). Overall, hydropho- bicity is a surface phenomenon, whilst water-repellency and -proofness refer to the whole material. Water-repellent materials can be attained with a high LJw and water-proof materials need sufficiently high denseness in addition.

Figure 3. A) Contact angle and interfacial tensions. BC) Superhydrophobic surface in the B) Cassie state and C) Wenzel state. D) Advancing and receding contact angle. E) Roll-off angle (RA).

2.3.1 Evaluating the water-repellance of textiles

LJw is the most common and representative method to evaluate the hydrophobi- city of a surface.24 A droplet of water is placed on the surface (textile), and the angle between the sides of the droplets and surface is measured. This method is however associated with some limitations. Experimental errors arise due to ir- regularities in the baseline of a textile surface, effects of gravity due to the size of the liquid and camera aspects such as lightning contrast and lens focus. Zim- merman et al found that such experimental parameters caused the LJto vary by more than 10° of the same surface.41 The substrate can also absorb the water while the droplet retain its shape, falsely giving results that the surface would be hydrophobic. This can be eliminated by including measurents of water ab- VRUSWLRQRYHUWLPH5HJDUGLQJ5$DQGLJK\VWHUHVLVHUURUVPD\EHFDXVHGby the angle for slanting the plate, difficulties to get clear images of the rolling droplet as well as setting an accurate baseline.

7

LJw is not extensively used in the textile industry. Instead, a set of other methods that evaluate the water-resistance are used, such as rain imitation tests (spray test, rain test and impact penetration test), hydrostatic pressure test and sorp- tion tests.42,43 In the rain imitation tests, a specified amount of water is sprayed onto the textile, that is then visually evaluated for water stains or a blotting pa- per under the textile is weighted.44 These methods evaluate both hydrophobicity and absorption. In the hydrostatic head test, a water column of increasing height is put onto the textile and the height of the column when three places of the fabric underside are wet is reported.45 In other words, the absorption is measured. In sorption test the textile is placed in water and the absorbed mass is taken,43 and as the name suggests this test measures absorption.

To evaluate the washability and durability of the water-repellant textiles stand- ard tests for washing and abrasion are used. The textile is first washed a speci- fied number of cycles or brushed a certain time whit a specific load, and after- wards the water-repellance is evaluated.46–48

2.4 Hydrophobization methods for cellulosic materials

There are two main ways to make a material water-resistant, one is to make the structure so dense that the water cannot penetrate through the material. The second, is to make the surface hydrophobic so that the water does not spread but rather is repelled from the material. Since the mechanical properties of cel- lulosic films and composites are negatively affected by water and highly dense textiles are undesirable due to a lack of breathability, this work focuses on the hydrophobization of cellulosic surfaces.

2.4.1 Utilizing and increasing the intrinsic surface roughness

The highest hydrophobicities are obtained via a combination of low surface en- ergy and high surface roughness, and thus it is beneficial to increase the surface roughness in addition to chemical modifications. Roughness can be increased by either etching, lithography, electrospinning or by increasing roughness while simultaneously modifying the surface chemistry via techniques like layer-by- layer deposition, sol-gel, electrochemical reaction and deposition, chemical va- por deposition, chemical grafting or any type of nanoparticle deposition.49,50 However, the added roughness (i.e. nanoparticles) can also a be disadvanta- geous, as the surface may be easily damaged by erosion or scratching.35

Due to their structure, textiles already have some inherent surface roughness and as a consequence hydrophobic treatments can further enhanced this effect compared to flat surfaces.39 When a textile is made the yarns are either woven or knitted together and the thickness of the yarn, the thread count and type of weave/knit will all affect the microscale roughness of the textile material. There- fore by tuning the parameters of the textile structure the surface roughness, and in addition water-resistance, can be enhanced.51

8

2.4.2 Fluorine-based methods

Fluorocarbons, i.e. alkyl chains of 8-12 carbon atoms where most of the hydro- gen atoms are replaced by fluorine, are used extensively in textiles52 and other applications.53 Fluorocarbons have a very low surface energy and can function very well as surface coatings. Consequently, different types of fluorocarbons and deposition techniques are used in the textile industry today, including fluori- nated acrylate polymers,54,55 fluorinated silicon-based coatings56–58 and ex- panded polytetrafluoroethylene, commonly known as GoreTex. To si- moultaneuosly achieve breathability, porous membranes of the fluorocarbons are used.59 The main disadvantage related to C8 fluorocarbons is that they de- grade into perfluorooctanic acid (PFOA) and perfluorooctanic sulfonate (PFOS), which are both toxic, bioaccumulative and persistent in nature.52 Cur- rently C8 fluorocarbons are being phased out, and being replaced by alternative C6 or shorter fluorocarbons.60 Shorter fluorocarbons degrade into perfluoro- hexanoic acid (PFHxA), which is more easily removed from the environment, however the toxicity of PFHxA has not been extensively studied to be deemed totally safe.52 Notwithstanding, as similar chemical compounds tend to have similar properties, it is not unlikely that PFHxA also would be toxic.

2.4.3 Silicon-based methods

In addition to fluorinated substances, silicon-based treatments, which include alkylsilanes,61 siloxanes,62 silsesquioxanes,41,63 or a combination of several sili- con-based substances,64,65 are also popular. Often nanoparticles (silica or other materials) are used in addition to silicon-based treatments,66–68 to provide sur- face roughness.69 Silicon-based coatings are also available as water-borne sys- tems,46,70 which offers additional benefits from an industrial and environmental point-of-view. Nonetheless, some silicon-based methods have poor mechanical durability67 and the safety of nanoparticles for humans and the environment is still under debate.71

2.4.4 Polymer-based methods

Polymers with plain alkane chains or chains with hydrophobic functional groups, have low surface energy and can therefore be used to hydrophobize cel- lulose. Polymers can be covalently bound by grafting,72–74 adsorbed as charged layers (see Chapter 2.6.1),75,76 adsorbed as polymeric surfactants,77 or otherwise applied.78,79 Modification of a material’s chemistry by covalent bonds, however, may reduce the biodegradability of the material as many synthetic polymers themselves are not biodegradable. This is unfavourable in terms of ecological sustainability.

2.4.5 Bio-based methods

In addition to fluorinated and silicon-based materials and some polymers, also natural alkenes, e.g. waxes and oils, possess low surface energy. Historically wax

9 has been applied onto textiles and other cellulosic surfaces to make them hydro- phobic, but a solid wax or oil film lacks the surface roughness that is needed for high hydrophobicity and can reduce the breathability. In nature there are many surfaces that are hydrophobic – even superhydrophobic – without the use of fluorocarbons, e.g. the lotus and other plant leaves, the wings of butterflies and the feet of some insects.80 Even though waxy substances cover these surfaces, the key to their superhydrophobicity is their multilayered, hierarchical surface roughness.50,81 As mentioned in Chapter 2.4.1, the structural surface roughness can easily be damaged, however in nature (bio)surfaces can be regenerated. Moreover, it can be argued that all methods, which utilize surface roughness (by deposition of nanoparticles or otherwise) and low surface energy, are biomi- metic.

Currently, although the literature on bio-based hydrophobization methods of cellulose is limited, it includes organic compounds like alkyl ketene dimers found in waxes,82 other wax, fatty acid or oil-based components like calcium carbonate and wax,83 nano- and microstructured cellulose stearoyl fatty acid es- ters84 and epoxidized soy-bean oil.85,86 Other methods used involve carbon- based compounds like graphene oxide87 and diamond-like-carbon88 to create structure. Other hydrophobicizing agents include betulin combined with a bet- ulin-copolymer,89 and PLA-functionalized nanoclay.90

2.5 Multifunctional cellulosic materials

2.5.1 Hydrophobic and breathable materials

In some applications, it is desirable that the material breaths (allows for passive water vapor and gas flow), while still being hydrophobic. For example, wearable textiles needs to be breathable for comfort and health,59 whereas in buildings moisture buffering hygroscopic materials provide comfort and reduce the need and cost of ventilation.91 In order to fulfill the criteria of porosity to let water vapor through but not liquid water, the coating must possess holes rather than being a continuous, sealing film. In this case, hydrophobic coatings made from particles are ideal as air and water vapor can move through the spaces between the particles. Furthermore, due to the naturally holey structure of textiles, a coating that only covers the fiber portion of the material allows at least some of a textile’s breathability to be preserved.

2.5.2 Cooling pigments

Although native cellulose is naturally white, everyday aesthetics require that a far wider range of colors is available. Consequently, numerous dyes, pigments and coloration techniques have been developed, however, a pigment can do more than simply look appealing as the color can also impart some additional functionality. For example, cooling pigments that scatter light in the near-infra- red region but absorb visible light have gained plenty of research interest92–96 – though primarily on surfaces other than cellulose. Cooling pigments can reduce

10 the need for air conditioning in hot climates and provide comfort when in direct sunlight. Moreover, in some applications a surface that combines cooling and hydrophobic properties is also desired.97

2.5.3 Antimicrobial and hydrophobic surfaces

In many applications, an antimicrobial surface is beneficial, both for health rea- sons and general cleanliness. Additionally, for some uses it is beneficial if the surface also is hydrophobic as well as antimicrobial (e.g. textiles), although this is not always the case (e.g. wound healing). In this work, CNF films were modi- fied to be have antimicrobial and hydrophobic properties, therefore the litera- ture related to CNF films with similar properties is presented. Silver nanoparti- cles, for example, are well-known for their antimicrobial properties,98–101 and have been used extensively to modify CNF surfaces.102,103 Nonetheless, issues related to the toxicity due to the widespread Ag nanoparticles use is of increas- ing concern.104

In general, cationic surfaces have antimicrobial activity and methods to make CNF cationic have been previously reported, including quarternary ammonuim compound surface treatments either by grafting105,106 or etherification.106 Pre- treatment with 2,3-epoxypropyl trimethylammonium chloride (EPTMAC) has also been used to produce a cationic cellulose surface, which showed clear anti- microbial activity and negligible leaching, although no hydrophobic properties were reported.107 The mode of action for the cationic surfaces is that the cationic moieties electrostatically interact with the anionic proteins and phospholipids, impairing the permeability of the cytoplasmic membrane leading to the death of the cell.108 Utilization of silyl compounds to couple quaternary ammonium compounds to cellulose surfaces could potentially introduce hydrophobicity, as could aminosilanes grafted onto CNF.108,109 The benefit of all these methods is that they can produce contact-active and non-leaching materials.

In nature, plants have little defense against predation other than chemically de- rived methods and as a result, there is an abundance of natural antimicrobial agents, comprised mostly of resin-type materials. Resins and resin-derived compounds can be used on cellulose, where they also provide some hydropho- bicity.110,111 Ganewatta grafted quaternary ammonium-decorated abietic acids onto silylated CNF and achieved strong acitivty against both Gram-positive and -negative bacteria,111 whereas de Castro used esterification with resin acids and obtained strong activity against Gram-negative and only modesta acitivty against Gram-postivie bacteria.110 Neither achieved WCAs higher than 90°. CNF modified with resins can also be used in composites, in which the whole bulk, not just the surface, is modified.112 Another approach utilizing bio-based mate- rials is the adsorption of chitosan to produce antimicrobial and hydrophobic CNF materials.113,114

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2.6 Surface modification methods

There are many benefits to the use of surface modification when compared to that of changes to the bulk. When only the surface is modified, less material is required and through the tuning of the surface specific properties alone, all the beneficial properties of the bulk can remain intact.

2.6.1 Layer-by-layer deposition

Layer-by-layer deposition, introduced for polyelectrolytes by Decher in the early 90’s,115 is a gentle and straightforward surface modification method that does not require any special equipment or conditions like high temperature, pH, pressure, activation steps, catalysts or harsh solvents. With the correct combi- nation of substances, LbL can even be performed in the domestic environment. LbL utilizes electrostatic interaction to hold the layers together on the surface, and no covalent bonds are formed.116 Cellulose, and most surfaces and particles are anionic in nature such that the immersion of a cellulosic material in an (aqueous) solution of a cationic polymer or dispersion of cationic particles re- sults in a cationic layer being formed on the surface. Not all of these cationic components become firmly adhered to the cellulose and the loosely attached molecules can be readily removed by rinsing, to leave a monolayer of the cation on the surface. If the same procedure is repeated with an anionic substance, a double layer – or bilayer – composed of cation and anion layers can be pro- duced. With repeated alternation of cationic and anionic substances more layers can be added and there is no theoretical limit of to the number of layers that can be produced.117 Although dipping is the most common method used to produce such layers, spraying can also be used.117 In recent years, LbL has been used to make cellulose flame reatardant,118 antifogging,119 and thermally conductive,120 to mention a few areas of use.

2.6.2 Sol-gel

In the sol-gel technique, a precursor solution with the modifying agent is hydro- lyzed into a colloidal sol, and then the (textile) sample is dip-coated or spin- coated with the sol. Evaporation of the solvent and gelation of the reimaning sol takes place, after which the sample is heat-treated to cure the coating. This method is widely used for hydrophobizing textiles mostly with silica com- pounds,47,121 even though other substances can be used to achieve different prop- erties.122 This method could be used in large scale.

2.6.3 Gaseous application techniques

There are several thin film application techniques utilizing the gaseous phase. In chemical vapor deposition (CVD), a volatile precursor is caused by heating to react and form a solid coating on the (textile) substrates.123 E.g. fluorocarbons124 and carbon nanoparticles125 have been deposited onto cellulosic surfaces with CVD. Sometimes the CVD subtechnique atomic layer deposition (ALD), where alternating gaseous precursors are exposed to the surface to form a thin layer, 12 is used, e.g. with aluminium oxide and silicon compounds.126,127 To deposit na- nosized metal and metal oxide particles liquid flame spraying (LFS) can be used.128 In the LFS process the precursors are in liquid form and fed together with the combustion gases into a special spray gun. Plasma-based techniques has been widely studied and hold the advantage of not producing any liquid waste and being highly efficient, but are not yet suitable for large scale.129

2.6.4 Dye exhaustion

In the dye exhaustion method, the (textile) material is immersed in dye liquor and the fibers gradually absorb the colorant. Chemical agents or mordants can be used to enhance the absorption, but are not always essential for the success- ful completion of the process.130,131 Depending on the dye and fibers used, there may not be any attractive forces between the dye and fibers – hence the need for mordants. It can be argued that this is not a surface specific method.

2.6.5 Adsorption of polyelectrolytes

Although cellulose has many beneficial properties, its use is hampered not only by its hydrophilicity, but also its poor reactivity.132 To increase the reactivity or enhance the affinity of cellulosic materials towards other subsances used for functionalization, the surface of cellulose can be modified by adsorption of dif- ferent polyelectrolytes133,134 or polysaccharides that possess a natural affinity to- wards cellulose.135 Carboxymethylated cellulose (CMC) has high affinity to cel- lulose and due to its structure it has more reactive groups than unmodified cel- lulose.136 The adsorption process between CMC and cellulose is almost irre- versible and once modified, the CMC can further be functionalized via addi- tional chemical compounds.137 Adsorption of CMC was selected for investigation in this work, due to its high number of carboxyl groups, which are needed to covalently bind the dehydroabietanes required for the desired antimicrobial properties.

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3. Materials and methods

3.1 Materials

3.1.1 Polycations

Two different polycations were used to bind the anionic wax particles to the cel- lulosic surface: poly-L-lysine (PLL) purchased from Sigma-Aldrich, Germany and Classic 145 cationic starch (CS) obtained from Chemigate Oy, Lapua, Fin- land. The 0.1 % (w/v) PLL was used without further purification in solutions with pH 9.5, unless stated otherwise. According to the manufacturer, the potato- based cationic starch had a degree of substitution of quaternary ammonium groups of 0.42 and a CS solution was prepared by adding 5 g of the material to 1 L of boiling water, before subsequently being simmered – with stirring – for 15 min.

3.1.2 Carnauba wax dispersion

Refined carnauba wax was purchased from Sigma-Aldrich and used without any further purification. As the wax is insoluble in water, a wax particle dispersion was prepared via the following process steps: Water was first heated to 100 °C and wax was added prior to sonication with an Ultrasonic Probe Sonifier S-450 with a 1/2” extension (Branson Ultrasonics) for 5 min. Immediately following sonication, the system was cooled to allow the melted wax to form solid parti- cles. This dispersion was then filtered through a funnel with a nominal pore size of 100 - 160 μm to remove large particles. Following filtration, the average par- ticle size within the dispersion was determined to be approximately 0.5 μm and more detailed information about the dispersion can be found elsewhere.138 The dispersion was found to be stable at room temperature for several years.

3.1.3 Ultrathin CNF films

Never-dried bleached birch pulp from a Finnish pulp mill was used to prepare cellulose nanofibrils (CNF). No chemical or enzymatic pre-treatment was ap- plied, but the pulp was washed to create the sodium form prior to the disinte- gration, to better control the counterion type and the ionic strength.139 The pulp was disintegrated and circulated for 12 passes through a high-pressure fluidizer (Microfluidics, M- 110Y, Microfluidics Int. Co., Newton, MA).

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For the preparation of the ultrathin films, only the finest fraction of the CNF was used. This was obtained by diluting the CNF dispersion after 12 passes to 1.67 g/L and thoroughly mixing the solution before centrifugation at 10400 rpm for 45 min. Once this treatment was complete, the upper phase containing the su- pernatant fine fraction CNF was immediately collected. Polyethyleneimine (PEI), purchased from Polysciences, was used to anchor the CNF onto gold QCM-D crystals for adsorption studies. 0.34 g of 30 wt-% aqueous PEI was di- luted with 100 ml MilliQ-water to produce a solution for surface preparation. Clean QCM-D Au crystals were spin-coated at 3000 rpm for 1 min firstly with PEI and then 1 min with supernatant CNF, before the films were dried at 80 °C for 10 min.

3.1.4 Free-standing CNF films

To prepare the free-standing CNF films, 100 ml of 0.85 % CNF – that had been subjected to 6 passes rather than the 12 described in Chapter 3.1.3 – was filtered through a 10 μm pore size Sefar Nitex polyamine monofilament open-mesh fab- ric at a pressure of 2.5 bar for a filtration time of 30 min. After filtration, the films were dried for 2h at 100 °C under 1800 kg/cm2 pressure in a hot press (Carver Laboratory Press).

3.1.5 All-cellulose composites

All-cellulose composites were prepared via the short-fiber dispersion approach using a method previously reported by Korhonen et al.140 In brief, 5 wt-% of cellulose was dissolved into 8 wt-% aqueous NaOH at – 7 °C for 2 h using a mixing rate of 300 rpm. After dissolution, 2.4 wt-% of softwood kraft fibers were added into the solution, whilst the stirring speed was 125 rpm. The resultant suspension was placed in an oven at 50 °C for 1 h to allow the sample to gel prior to coagulating and washing in water for 2 days. Finally, a two-step drying method was applied to produce the desired composites: Firstly, the sample was pressed at room temperature for 2 min at 0.37 MPa, in a pneumatic sheet press (L&W SE 040, Ab Lorentzen & Wettre) to remove a majority of the aqueous solution. This was followed by the second stage that involved the material being hot-pressed at 100°C for 2 h (Carver Laboratory Press) to remove the remaining water.

3.1.6 Textiles

Several different textiles from both natural and man-made cellulosic fibres were used, however, all were plant-based and consisted of cellulose. The textiles and their related properties are presented in Table 1. All textiles were white and washed with ethanol prior to use.

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Table 1. The textiles used in this work and their properties.

Name Composition Gram- Source mage, g/m2 Cotton 100 % cotton 156 Eurokangas, Espoo, Finland Microviscose 100 % viscose 122 Aalto University, Finland Tencel 100 % tencel 118 Lenzing, Austria Hemp 55 % hemp, 283 Aalto University, Finland 45 % cotton Knitted cotton 100 % cotton 629 Huafu cotton yarn supplier

3.2 Methods

3.2.1 Layer-by-layer

The layer-by-layer surfaces used in this work were prepared by sequential im- mersion steps as follows: Firstly, samples were immersed in the cationic solu- tion for 5 min, before being rinsed three times by immersion for a few minutes in three different beakers of deionized or MilliQ-water. Once fully rinsed, sam- ples were then immersed in the wax dispersion for a similar duration and then rinsed in triplicate with deionized water. This procedure was repeated for as many times as desired, although typically the whole process was repeated twice to form two bilayers. In some specific cases, spray coating provided an alterna- tive method, for example, during coating optimization for the composites (Pub- lication III) in order to provide high hydrophobicity and mechanical properties and when coating textiles larger than an A4 (i.e. garments, Publication II). When spraying was used, samples were not rinsed in between layer adsorption; garments were dried in between layer adsorption, whereas in the case of com- posite materials the next layer was added immediately.

After the samples were coated, they were either left to dry either at room tem- perature or were heat treated in an oven for at least 10 min. The temperature used was 70 °C or 105 °C, although temperatures in the range between 30 - 105 °C were used to find the optimum temperature. The exact temperature is speci- fied for each experiment.

3.2.2 Dyeing of textiles

A dye dispersion was made by stirring 8 g of blue pigment (YIn1-xMnxO3, YInO3 doped with Mn, EX1456-Lot:L2037, Shepherd Color Company, USA) in 200 g deionized water for 15 min at 900 rpm with 0.4 g of non-ionic surfactant (Addi- tol VXW 6208/60, Cytec Austria GmbH, Austria). After stirring, the dispersion was milled for 10 min with 0.4 mm diameter Zr beads in a Dyno-mill Research Laboratory agitator bead mill (WAB, Willy A. Bachofen AG Maschinenfabrik, Switzerland).

16

The dye exhaustion method was used for the dyeing process and this involved heating the stirred dye dispersion initially to 50 °C, before adding a piece of cot- ton (1:50 weight ratio cotton:dispersion). The solution was then further heated to 90 °C for 20 min and the material was left in the dispersion for a further 1 h with continuous stirring. After the dyeing process was complete, the material was removed and dried at room temperature.

Further functionalization was carried out by spraying the dry, dyed textiles with the wax dispersion or waterborne fluoroalkyl siloxane (Dynasylan F 8815, Evo- nik Resource Efficiency GmbH) with a concentration of 100 g/L.

3.2.3 Preparation of antimicrobial CNF films

CNF films were activated with CMC by stirring in a solution of 0.05 M/0.01 M CaCl2/NaHCO3 in water (75 mL) to which 270 mg of Na-CMC had been added. The mixture was stirred for 4 h at 80 °C, after which the activated film was rinsed with deionized water, CH3COOH and NaHCO3. The activated films were then amidated by agitation with a mixture of 0.02 M N-(3-dimethylaminopro- pyl)-NĻ-ethylcarbodiimide hydrochloride (EDC) and 0.02 M 1-hydroxyben- zotriazole hydrate (HOBt) in 94% w/v ethanol (60 mL) to which 0.02 M dehy- droabietylamine (compound 1, 4, 7A or 7B) and 0.04 M N,N-diisopropylethyl- amine (DIPEA) were added. The mixture was left at room temperature for 24 h, and afterwards rinsed with ethanol and water. The films were left to dry overnight at 103 °C. More detailed information can be found in Publications IV and V.

3.2.4 Contact angle

WCAs were measured with a CAM 2000 (KSV Instruments Ltd, Finland), using a droplet size of 6.5 μL and the results were determined with the Young-Laplace equation via the proprietary software. The WCA was measured for a total of 60 s and the value at 5 s from all samples were compared with a minimum of four different measurements taken for each sample. Advancing and receding WCAs were measured on a plane with a 5° slope.

For determination of long-term hydrophobicity, a droplet of water was placed onto the surface of interest and photographed at preselected intervals for at least 2 h.

3.2.5 Microscopy methods

Atomic Force Microscopy (AFM): A Nanoscope V MultiMode scanning probe microscope (Bruker Corporation, Massachusetts, USA) was used to image ultrathin cellulose films on QCM-D crystals and silicon wafers spin coated with ultrathin CNF. The images were recorded with tapping mode in air using silicon cantilevers (NSC15/AIBS, MicroMasch, Tallinn, Estonia) that had a tip radius of < 10 nm according to the manufacturer.

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Scanning Electron Microscopy (SEM): The surface morphology of compo- sites and textiles were studied with a Zeiss Sigma VP (Carl Zeiss NTS Ltd, Ger- many) field emission-scanning electron microscope using an acceleration volt- age of 1.5 kV. Neither cellulose nor wax possess conducting elements, therefore samples were sputter-coated (Emitech K100X) with a thin ׽10–15 nm layer of Pt/Pd. All samples were attached to Al SEM stubs with conductive carbon tape prior to sputtering and imaging.

3.2.6 Chemical characterization methods

X-ray Photoelectron Spectroscopy (XPS): The chemical composition of the cellulosic surfaces was determined with a Kratos Analytical AXIS Ultra elec- tron spectrometer with monochromatic A1 Kɲ irradiation at 100 W. The nomi- nal analysis area was 300 x 700 μm and 2 to 3 separate locations were examined for each sample. Low resolution wide survey scans were used for elemental sur- face analysis, while high resolution scans of C 1s, O 1s and N 1s were carried out for more detailed investigations. CasaXPS software was utilized for data analy- sis. A specific four component fitting routine tailored for cellulosic specimen was used for the four carbon regions.141

Quartz Crystal Microbalance with Dissipation (QCM-D): Adsorption studies were carried out with a QCM-D (E4, Q-Sense AB, Västra Frölunda, Swe- den). Gold QCM-D crystals were spin-coated with cellulose nanofibrils using PEI as an anchoring agent. Prior to the QCM measurements, all samples were filtered through a 0.45 μm filter – except for the wax dispersion, which was fil- tered with a 1 μm filter – and then sonicated for five minutes before use, to make them as uniform as possible. The concentration of PLL was 10 mg/L, and of the wax dispersion 100 mg/L. Adsorption was monitored until a stable plateau in frequency was reached and then the surface was rinsed with MilliQ water. The next layer was adsorbed only after a stable plateau was acquired during the rins- ing procedure. A constant pumping rate of 0.1 mL/min was utilized for all stages and mass changes were calculated according to the Johannsmann equation,142 later simplified by Naderi:143

௣௙మௗమ (݂)଴1+ଔƸ݉ = כ ෝ݉ ଷ

is the equivalent mass, ݉଴ is the true sensed mass, ଔƸ(݂) is the complex כ where ݉ෝ shear assumed independent of frequency and ݀ is the thickness of the film. The true sensed mass was obtained by plotting the equivalent mass as a function of the square of the resonance frequency.

Differential Scanning Calorimetry (DSC): The melting of carnauba wax was studied with DSC. The wax was placed in hermetically sealed Al pans, with an empty pan used as a reference, and the thermal behavior was measured from 23 °C to 120 °C. The rate of temperature change was 5 °C/min and the system was constantly purged during the measurements with a 100 mL/min N2 gas flow.

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Dž-potential: Electrophoretic mobility of PLL was measured using a Zeta-sizer Nano-ZS90 (Malvern Instrument Ltd., Worcestershire, U.K.). This electropho- retic mobility data ZDVVXEVHTXHQWO\XVHGWRFDOFXODWHWKHDž-potential by the in- strument software using the Smoluchowski model.

3.2.7 Water vapor interaction

Dynamic Vapor Sorption (DVS): Sorption isotherms were measured using a dynamic vapor sorption device (DVS Intrinsic). The relative humidity (RH) was decreased to 0 % in order to determine the dry weight of the specimen. Sorption behavior was then monitored by the stepwise increase of RH to 95 % – a first step of 5 % was subsequently followed by 10 % increments. Desorption behavior was measured by decreasing the RH from 95 % to 0 % in the reverse order. Each relative humidity step was maintained until the weight change was detected to be less than 0.002 %/min for a duration of 10 minutes and all mea- surements were performed at 25 °C.

Water Vapor Transmission Rate (WVTR): Textile breathability was evalu- ated by WVTR and the experiments were performed in custom built chambers, according to ASTM standard E 96/E 96M – 05 (Desiccant method). Two paral- lel samples were analysed in each experiment at a chamber RH of 71 % and 23 °C with the treated side of the sample placed toward the higher vapor pressure. Sample thicknesses required for calculations were determined using a Schraeder micrometer (Lorenzen & Wettre, Sweden) that had a NjPDFFXUDFy.

3.2.8 Other methods

White Light Interferometry: Roughness of the coated and uncoated textiles was determined using a scanning white light interference microscope (Con- tourGT-K, Bruker Corporation, USA). In-built software was used to calculate the arithmetical mean height Sa. The scanned DUHDZDVNjP × NjPWKH focus was 20 × 0.55x and 640 × 480 pixels were measured. Three replicates of each sample were analyzed.

Ultraviolet–visible Spectroscopy (UV-Vis): Spectral properties of the col- ored textiles were obtained by a UV-Vis equipped with an integrating sphere (PerkinElmer Lambda 950 UV–Vis spectrometer).

Washing fastness: The washing fastness of samples was determined accord- ing to the ISO 105-C10:2006 standard. Samples were laundered for 30 min at 40 °C using a soap solution of 5 g/L with pH 7 and liquor to fabric ratio of 50:1. The samples were then air dried after washing. Color properties were deter- mined with a Datacolor Spectraflash® SF 600 PLUS-CT spectrophotometer with illuminant D65 using the 10° standard observer, d/8° measurement geom- etry and a measurement area diameter of 20 mm (on 4 layers of a fabric). Ten measurements were performed on each sample. CIE L*a*b*were determined using Datacolor Datamaster software.

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Assessment of antimicrobial activity: Unmodified and modified CNF films were cut into pieces (1x1 cm2) and incubated in 1.35 mL of bacterial sus- pensions with ~105 CFU/mL (Colony forming unit/mL) at 37 °C for 24 h. After the treatment with the films, serial dilutions of the bacterial suspensions were spread onto a plate and incubated at 37 °C overnight to determine the number of viable cells by manually counting. The bacteria used were S. aureus ATCC12598, S. aureus ATCC25923, S. aureus MRSA 14TK301 and E. coli DH5- Į.

Assessment of biofilms formation: The strain S.aureus UAMS-1 was used for the experiments with biofilms. Circles with a 0.5 cm diameter of both CNF and CNF-CMC-7B were placed in a flat-bottmed well microtiter plate (Greiner Bio-One, Kremsmünster, Austria). In the plate well, the samples were sub- merged in 400 μL Mueller-Hinton broth and inoculated with 104 CFU S.aureus UAMS-1. Incubation at 37 °C took place for 4 h, and afterwards the medium was removed, and the samples were washed with 400 μL buffer solution (0.9% w/v NaCl). New medium was added, and removed after 20 h when the samples were moved to a new well and washed in a similar fashion as described above. The biofilms were collected by 3 cycles of vortexing and sonication for 30 s. After the treatment, serial dilutions of the bacterial suspensions were spread onto a plate and incubated at 37 °C overnight to determine the number of viable cells by manually counting.

Artificial dermis experiment: The artificial dermis consisted of two layers and details of its preparation can be found elsewhere.144 The dermis was put into a well microtiter plate (Grenier Bio-One, Kremsmünster, Austria). The medium used was composed of Bolton Broth (Oxoid, Basingstoke, UK) containing 50% plasma (Sigma-Aldrich), 5% freeze-thawed horse blood and 0.5 U/mL heparin (Calbiochem, San Diego, USA). 500 μL of the medium and subsequently 10 μL of an overnight culture of S.aureus UAMS-1 (ca 104 CFU) was pipetted onto the artificial dermis. To avoid dehydration, the medium was added to the wells to a final volume of 1 mL. Pieces of CNF and CNF-CMC-7B were cut to fully cover the artificial dermis, placed on top of it and pushed until they stuck to the dermis surface. Cultivation took place for 24 h at 37 °C and after that the material was taken off and washed with 1 mL PS and the artificial dermis was collected in a tube containing 10 mL buffer solution. The biofilms were collected by changing between vortexing and sonication 3 times for 30 s. After the treatment, serial dilutions of the bacterial suspensions were spread onto a plate and incubated at 37 °C overnight to determine the number of viable cells by manually counting.

Blood hemolysis (biocompatibility): Erythrocytes were derived from the whole blood of healthy humans (Red Cross, Finland) via centrifugation. The erythrocytes were diluted with phosphate buffered saline (PBS) solution (ratio 1:50) in 50 mL flasks (Corning Inc. Life Sciences). 12 mm circles of unmodified and modified CNF films were placed individually at the bottom of a 24-well plate. The films were washed with sterile PBS, and 2 mL of the erythrocyte dilu- tion was placed into each predetermined well and 200 μL of 20% Triton X-100

20 and 1.8 mL of PBS in any remaining empty wells as a control. The plates were incubated for 1 h at 37 °C under a 5% CO2 atmosphere in a Heracell 150i incu- bator (Thermo Fischer Scientific). To pellet the intact erythrocytes, the system was centrifuged at 500 × g for 5 min, after which 200 μL of supernatant from each well was transferred to a 96-well plate (PerkinElmer Inc,) and the absorb- ance measured at 415 nm using a Varioska Lux (Thermo Fischer Scientific). More detailed information can be found in Publications IV and V.

Cell viability (biocompatibility): Fibroblasts obtained from human skin were cultured in a 75 cm2 flask in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) to which 10% foetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acids, 1% L-glutamine, penicillin (100 IU/mL), and strep- tomycin (100 mg/mL) (all from Hyclone) had been added. All cultures were kept in a Heracell 150i incubator (ThermoFischer Scientific) under an atmosphere comprised of 5% CO2 with 95% relative humidity and the growth medium was changed every other day. 12 mm circles of unmodified and modified CNF films were placed at the bottom of a 24-well plate (Corning Inc. Life Sciences), washed with sterile phosphate buffered saline (PBS), after which the fibroblasts were detached, and 4 × 104 human fibroblasts were placed on top of the materials in the well plate. Cells were allowed to attach and incubate with the films for 72 hours, after which the medium was aspirated, and the cells and materials were washed with fresh PBS. A 1 mL CellTiter-Glo® reagent assay (Promega Corpo- ration) was added to each well, and the plate was examined for luminescence using a Varioskan Lux (Thermo Fischer Scientific). The luminescence of the samples wells was compared with PBS and Triton X-100 controls. More detailed information can be found in Publications IV and V.

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4. Results and discussion

4.1 Characteristics of the cellulosic surfaces studied

In this work, different types of cellulosic surfaces were studied: ultrathin CNF films, free-standing CNF films, all-cellulose composites and different types of cellulose-based textiles. Ultrathin CNF films spin-coated on Au crystals were used as model surfaces for the surface chemistry studies due to their low surface roughness and thicknesses. Free-standing CNF films were significantly thicker (70 μm cf. < 10 nm) and had surface roughness at different length scale, both nm and μm, which originated from the Nitex films used during the CNF film preparation process. Although the CNF films were very dense and were used as model films, they could also be directly used in real-world applications. All-cel- lulose composites – which can be used applications like packaging and furniture – were also dense, had an even higher surface roughness than the free-standing films and thicknesses ranged from mm to cm. The surface roughness and po- rosity varied from textile to textile, but generally textiles were porous with high surface roughness. Thickness also varied but was in the range of mm and the most notable of the many applications of textiles is in wearable garments. An overview of the studied surfaces and their properties can be found in Fig. 4.

Figure 4. The different cellulosic surfaces studied, and a qualitative guidance of their properties. 22

4.2 Layer-by-layer deposition for surface modification

The layer-by-layer coating method was used to modify the cellulosic surfaces in order to render them hydrophobic yet breathable in an environmentally benign fashion. Both PLL and CS were used as cations and the anionic component was always the wax dispersion. Experiments showed that the modified surface was already hydrophobic after one bilayer, but the presence of two bilayers gave a higher WCA (Publication I). Although additional bilayers could have been added, it was found that with more than two bilayers the hydrophobicity did not increase. From a practical point of view, the less layers the better as the addition of extra layers requires time and rinsing water. Moreover, different cations or anions can be utilized for the different layers and by variation of the (poly)elec- trolytes, a greater level of surface functionality can be achieved.

4.2.1 The role of the polycation

Poly-L-lysine (PLL) and cationic starch (CS) were the polycations used in this work. Lysine is an essential amino-acid, whereas PLL is industrially produced by a fermentation process using Streptomyces albulus İ-PLL)145,146 or chemi- cally synthesized (Į-PLL).147 Starch is found in potatoes, rice, corn and other plants and is naturally anionic, but can be cationized by substitution of quarter- nary ammonium groups using glycidyltrimethylammonium chloride or 3- chloro- 2-hydroxypropyltrimethylammonium chloride.148,149 PLL is difficult to produce in large enough quantities150 and is rather expensive,151 in contrast, CS is considerably cheaper and already used commercially in the textile and paper industries.152 A third option – not explored in this study – is chitosan, a deacety- lated form of the polysaccharide chitin that originates from the shells of crusta- ceans.153 Furthermore, there is a wide range of synthetic polycations that could be used for such applications, however, the focus of this thesis is on naturally sourced compounds in order to make a fully bio-based coating and enhance the use of natural materials.

The polycations were used as anchoring agents for the wax particles on cellulose and the objective was to achieve an as hydrophobic coating as possible. Conse- quently, water contact angles (WCAs) were measured and it was determined that higher WCAs were achieved with PLL rather than CS when the same num- ber of bilayers were used (Publication II, Fig. 5). This results from the fact that PLL itself is slightly hydrophobic, which adds to the hydrophobicity of the whole surface, whereas CS is hydrophilic. The WCA of PLL is high, however not stable. The droplet absorbed after a few minutes, neither did it move on the surface. The conformation of the cationic polyelectrolyte layer also affects how much wax is adsorbed. In the case of PLL, the thickness of the layer could be adjusted E\PRGLILFDWLRQRIWKHS+DVWKHDž-potential of PLL is pH dependent. A similar effect was not observed for CS and this can be explained by their slightly differ- ent chemistries: CS has only tertiary amino groups and overall fewer amino groups than PLL, which has both primary and secondary amino groups. This means that PLL has more groups that can be ionized depending on pH, hence

23 the stronger effect on PLL of different pH. The shape of PLL is also changed with pH; there are more ‘loops and tails’ with high pH, whereas the structure of the starch backbone is more rigid. It could be possible however, to tune the CS layer thickness by the addition of salt, which would be interesting for future studies.

Figure 5. A) WCA of one layer of PLL (gray), one bilayer (BL) of PLL/wax (blue), two bilayers of PLL/wax (purple) one bilayer of CS/wax (brown) and two bilayers of CS/wax (green) on cotton. Lighter shades WCA at 5 s, and darker shades at 60 s. One layer of CS is missing due to the low (<10°) WCA. B) Sensed mass during QCM-D of one bilayer PLL/wax with PLL at pH 4.5, 6.5, 7.5 and 9.5. Vertical lines indicate rinsing with MilliQ-water. & Dž-potential of PLL at different pH.

4.2.2 Effect of curing temperature on the wax coating

As majority of the hydrophobic properties of the modified surface were provided by the wax particles, the ability to get enough wax particles on the surface was thus necessary and this was achieved by optimization of the polycation coating. Nevertheless, there are also other treatments that can be undertaken to further enhance hydrophobicity following the initial LbL coating application. The cur- ing temperature was found to have an impact on the hydrophobicity and water absorption of the treated samples. The WCA increased when the curing temper- ature was raised to 70 °C, which resulted in the highest WCA achieved (150°, Fig. 6A). Above 70 °C, the WCA was found to be slightly reduced, although over- all, long-term hydrophobicity was improved with a higher curing temperature. The reason for this is two-fold: molecular rearrangement and partial melting of the wax.

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Figure 6. Cotton textiles modified with two bilayers of PLL/wax and cured at different temperatures. A) Water contact angle, B) surface composition, deter- mined by XPS, and C) surface morphology (SEM) as a function of curing tem- perature. D) Melting point of carnauba wax measured with differential scan- ning calorimetry. Figure modified from Publications II and III.

Wax consists of several different functional groups that are mostly comprised of wax esters, fatty alcohols and fatty acids.20,154,155 A surface tries to minimize its energy by matching its chemical composition to the surrounding. From the XPS analysis (Fig. 6B) it is seen that the amount of hydrophilic C-O decreases and hydrophobic C-C increases on the surface with increasing temperature. When the wax particles are dispersed in water, the hydrophilic parts of carnauba wax graduate towards the surface of the particle. Once the water is removed, hydro- phobic air surrounds the particles (except the small area where they touch the substrate) which causes the hydrophilic groups to move away from the surface and the hydrophobic aliphatic carbon-chains take their place. This process hap- pens naturally over time, but it is also aided by the kinetic energy provided by curing at elevated temperatures.

The second explanation for the observed behavior is the partial melting of the wax. Several different molecules make up the wax, and some melt at lower tem- peratures than others (Fig. 6D). Nonetheless, the overall melting temperature of wax is 83-86 °C and most of the wax particles remain in the solid state until melting initiates in this range and the wax only completely melts above 90 °C (Fig. 6C). Melting improves the coverage of the fibers, which gives better long- term hydrophobicity (Fig. 7A). The effect of melting is also seen from the de- crease in the N signal (coming from PLL) at higher temperatures (Fig. 6B). Par-

25 tial melting increases the coverage as well and may be more ideal as the na- noscale surface roughness provided by the particles is retained when only part of the wax molecules melt.

Figure 7. A) Colored water droplets photographed over time on cotton coated with two bilayers of PLL/wax and cured at different temperatures. B) Advanc- ing and receding WCA, and WCA hysteresis of cotton coated with two bilayers of PLL/wax and cured at 70 °C and 105 °C. Five measurements were taken for both samples, although for the sample cured at 105 °C one droplet rolled away before it was measured. Figure modified from Publication II.

In order to better understand the hydrophobicity, WCA hysteresis was meas- ured (Fig. 7B). The hysteresis was low for the cotton sample cured at 70 °C, and very low when cured at 105 °C. On a structurally rough surface, the water droplet can either be on top of the air pockets between the structural cavities (Cassie state)34,38 or fill the cavities (Wenzel state).34,39 When the wax particles melted there were no cavities to fill and this in, combination with the increased wax coverage of the surface, could explain the observed enhancement in water mo- bility when the particles were melted – nevertheless, water moved easily on both surfaces. If the water easily moves away from the surface, long-term hydropho- bicity might not even be needed, as the water never will stay long on the surface. For flat-lying, non-moving surfaces (e.g. tables) no hysteresis will provide high enough mobility that the water rolls off, but for surfaces that move (e.g. gar- ments beign worn) even low hysteresis is enough for the water to roll off.

4.2.3 Surface roughness

Even though low surface energy leads to more hydrophobic surfaces, the key to very high hydrophobicity is high surface roughness. Therefore, although wax has a low surface energy, its application in the form of particles rather than as an even film provided additional roughness. The effect of the wax particles and their melting was studied using ultrathin CNF films to avoid the effect of the substrate on roughness. It was found that the presence of two bilayers gave a

26 higher surface roughness than only one (Fig. 8E). When the particles were melted, the surface roughness that originated from the particles was signifi- cantly reduced, but not fully lost as the particles formed crater-like structures (Fig. 8A-E) and the roughness of the underlying substrate did not significantly change due to the heat treatment.

It is known that multiscale, hierarchical surface roughness is beneficial for high hydrophobicity.156 In this case, the wax particles provided nanoscale roughness, whereas the substrates themselves also provided additional surface roughness, especially textiles which had microscale roughness. As stated previously, melt- ing was beneficial for the long-term water-repellence, however, it needs to be remembered that hydrophobicity and absorption are two different phenomena. A surface can have a high WCA, but still absorb water under pressure if the hy- drophobic coating is discontinuous, like in the case of a particle coating. This is what occurs when a water droplet changes from the Cassie state to the Wenzel state due to pressure and the substrate absorbs water. In contrast, a surface can also have a low WCA, yet still be completely impermeable (e.g. metal, glass etc.) It depends on the application if high WCA or strong impermeability is needed, however cellulosic surfaces naturally absorb water and thus in this case, high WCA and decreased absorbance are targeted. Therefore, melting of the particles has discernable benefits, even if some of the surface roughness is lost.

The roughness of the substrate also contributes to the overall surface roughness and consequently, to the wetting properties. An ultrathin and smooth model CNF film – coated during QCM-D – did not reach WCAs much higher than 90° regardless of the number of bilayers added (Publication I). In contrast, free- standing CNF films achieved WCAs of approximately 140° (Publication I) and for textiles the WCAs ranged from below 130° to above 155°, depending on the initial surface roughness of the textiles (Publication II, Fig 8F). The highest WCA measured on the coated composites was 133° (Publication III), but since the more hydrophilic polycation CS was used instead of PLL these results are not directly comparable. When using fluorocarbons on textiles WCAs in the range of 110° to 172° have been reported and for silicon-based treatments the range is 117° to 167°, even though both treatments typically render surfaces with a WCA > 150°.52

Measurement of surfaces with multiscale roughness is complicated and many methods fail to provide an accurate evaluation,157 hence the roughness results should be treated with caution. Different measurement techniques give infor- mation about the scale of roughness. The roughness data in Fig. 8 A-D is ob- tained by AFM, and as the image size used was 10 × 10 μm the measured surface roughness for this type of surface is well below 1 μm. AFM is a technique for thin films and can give accurate roughness results in the nm range. The data from Fig. 8E-F comes from white light interferometer measurements and the meas- ured size was NjP × NjP, which – as opposed to AFM – better can include the roughness of the fabric itself. The roughness scale is in μm, although the accuracy of the highest values obtained is questionable. It could be observed by

27 naked eye that the textiles had roughness at mm length scale that was not pos- sible to accurately detect with any of the methods used.

Figure 8. AFM height images of ultrathin CNF films spin-coated with two bi- layers of CS/wax and A) dried at room temperature, or cured at B) 70 °C and C) 100 °C, respectively. D) Surface roughness of an ultrathin CNF film and aforementioned samples. E) Surface roughness (white light interferometer) and WCA of freestanding CNF films, untreated and treated with one and two bilayers of PLL/wax, dried in room temperature and at 105 °C. F) Surface roughness (white light interferometer) of untreated textile samples and the WCA of same textiles coated with two bilayers PLL/wax.

Nonetheless, the findings outlined here indicate that a substrate’s surface roughness does contribute to the final hydrophobicity. When utilizing these findings in a practical sense, this means that the surface can be specifically de- signed to have high surface roughness. This especially true in the case of textiles, for which different weaving and knitting structures exist. If the surface of the textile – or other substrate – has a sufficiently high level of roughness it may reduce the number of bilayers required to hydrophobize the material, which could allow for simpler processing. As previously stated, the wax particles pro- vide roughness on the nanoscale and the (textile) surface provides roughness at the microscale, but it is difficult to determine how a coated textile with a slightly 28 higher surface roughness would feel when handled. Surface roughness affects the hand feel of textiles158,159 and while it has been suggested that particles smaller than 100 μm would increase the feeling of softness,160 tactile perception of textiles is still affected by a number of different factors.160,161

4.2.4 Durability

Durability of modifications is of high practical relevance. The durability of the LbL coated textiles was not evaluated numerically, however some assessment can still be made. The coating was not removed by extensive rinsing with water, but it did not whitstand washing with detergents, as it is attached to the sub- strate by electrostatic interactions. Producing a wax particle coating that can endure washing with detergents would likely require covalently binding the wax particles to the surface, which might affect the biodegradability and thus eco- logical sustainability of the method. Abraison was not tested, but the coating was not removed by rubbing or other normal touching. Carnauba wax is one of the hardest natural waxes, which indicates that the abrasion resistance would be high. The hydrophobicity over time was stable, the WCA had not noticeably decreased even after a couple of years of storage of the sample. Carnauba wax is only partly soluble in ethanol and acetone, and the coating could thus whitstand different solvents. In future research, the durability would need to be systemat- ically studied.

4.3 Breathability

An important factor in clothing is breathability, the comfort of the wearer suf- fers if the garments do not breathe. Even in applications other than apparel, breathability has benefits, i.e. moisture buffering can increase both comfort and reduce any required ventilation or heating.91,162 It was found that even the fabric with a fully melted wax layer had the same level of breathability as the uncoated reference (Publication II, Fig. 9). This is due to that the wax coating only covers the fibers and is very thin. Consequently, air can still pass through the pores in the fabric that result from the knitted/weaved structure. Overall, the defining factor for materials hydrophobized with this method is the initial breathability of the material itself as a very dense material with the cellulose fibers very close together will not breathe as well as a more loosely fibrous network. In the case of composites, these do not breathe as the surface is absorbing and releasing water vapor, rather than that water vapor flows through them. Coated compo- sites had higher sorption than the reference (Publication III, Fig. 9B), which can be attributed to the hydrophilicity of CS, which attracts additional water va- por.

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Figure 9. A) Water vapor permeability and WCA of untreated cotton, and cot- ton treated with two bilayers of PLL/wax, cured at 70 °C and 105 °C. B) Water vapor sorption of untreated reference all-cellulose composite, and composites coated with two bilayers CS/wax and cured at 70 °C and 90 °C.

4.4 Hydrophobizing a surface using aqueous dispersions

In this work, water-borne substances were used to hydrophobize cellulosic sur- faces and it may seem contradictory that a water-based method is used to hy- drophobize a surface that needs protection from water. Nevertheless, some cel- lulosic materials, like textiles, can withstand water and aquesous dispersions, and their mechanical properties are not significantly affected, whereas other materials, like all-cellulose composites, are not as resistant to water exposure. In general, the most important properties of textiles are not their mechanical characteristics, whilst in other applications – e.g. packaging and building mate- rials – mechanical behavior is crucial. Cellulose is hydrophilic and when af- fected by water it is the mechanical properties that most often suffer. Therefore, there is a clear need for a reduction in the amount of water used during the coat- ing procedure. Consequently, use of spraying instead of immersion as the dep- osition technique was investigated to preserve mechanical strength of all-cellu- lose composites through reduced contact with water (Publication III). Tuning of the surface roughness prior to coating also might help to reduce the number of bilayers needed, as discussed in Chapter 4.2.3. In addition, foam applications, where air bubbles replace some of the water, might be interesting for further studies. In order to completely remove water from the coating process an alter- native, non-aqueous solvent could be used. However, many solvents wet cellu- lose and use of other solvents rather than water could also compromise the over- all sustainability of this methodology. Another possibility might be application during pulping when the cellulose is still wet and has never been dried yet, and this would require more research but could be interesting to explore.

4.5 Scaling

The ability to obtain a well-defined structure on a small-scale sample is often more easily facilitated in a laboratory environment, therefore it can be a chal- lenge to replicate the same method on a larger scale. Dipping as a deposition method worked well at the small scale, i.e. specimens with a size of less than 10 30

× 10 cm, but for larger samples like garments, spraying and brushing were found to be more convenient methods for coating (Publication II). It needs to be kept in mind that the coating was added afterwards to ready-made garments and not as part of the textile production process. Nonetheless, dye baths are quite com- monly used in the textile industry and therefore dipping could also be suitable for large scale for textile modification.

Rinsing, to obtain a monolayer of the polyelectrolyte, is an important step in the process and is challenging to perform while spraying or brushing. In an indus- trial process with dye baths, rinsing may not be an issue. If rinsing was found to be too complex to achieve for some reason, the amount of the polyelectrolytes required would need to be optimized such that they were present in the correct ratio. Consequently, the coating would be easier to apply industrially, however as the coating may always be compatible with some washing detergents, an al- ternative method that allows the perfect polyelectrolyte ratio to be obtained in a domestic setting would also be needed for home use. For larger scale applica- tions, it would also be beneficial if the dispersion had higher concentrations as less water would be required, nonetheless water could be recycled through in- telligent production process plant design and the concentration of the disper- sion can be increased by controlled evaporation in a rotavapor.

The drying/curing of the applied coating is critical. With industrial methods curing can be performed in a precise way, whereas for home use, the controlled curing is a bit more difficult to achieve, although hair-dryers and saunas can possibly be used. Another possibility is ironing, however direct contact of a heated iron with the coated textile may result in coating loss as the wax could attach to the surface of the iron.

4.6 Functional pigments (unpublished results)

Water-resistance and breathability are not the only properties desired and, in some applications, additional functionality is required. For example, color is an important feature for almost all materials and an inorganic blue pigment with cooling properties has recently been developed.163 In this case, the pigment scat- ters and does not absorb light in the near-infrared spectrum.164 As a result, the dark blue pigment does not heat up when exposed to direct sunlight, unlike tra- ditional dark surfaces, but remains cool like a light material would. Use of these types of pigments in construction would lead to a reduction in air condition- ing/cooling requirements and lower CO2 emissions that result from energy con- sumption. On the otherhand, the pigment could be added to textiles in order to cool the user instead of the whole building and this would also have the added advantage of cooling users when outside in the sun. To achieve cooling fabrics, textiles were dyed with the inorganic blue pigment (Table 2). It was found that dyed textiles had the same temperature as the equivalent white textiles in the sun and were considerably cooler than surfaces coated with other dark pig- ments, even though the solar reflectance was in between that of white and dark surfaces. These types of garments could be very promising in personal thermal 31 control systems and would be easier to fabricate than some other proposed tex- tiles.165

Table 2. Thermal properties of white cotton, blue pigment on cotton, carbon black and cobalt-blue.

Sample Heat build-up, °C Total solar reflectance, % White cotton 27.4 58.29 Blue pigment on cotton 27.2 38.75 Carbon black 42.0164 4.6164 164 164 CoAl2O4 37.3 27.8

In order to add further functionality and protect the dyed textiles, hydrophobi- city was introduced by spraying wax particles or fluoroalkylsilane (FAS) onto the textiles, respectively. Both methods showed clear increase in hydrophobi- city, with FAS found to perform slightly better than the wax particles (Fig. 10). This treatment with FAS also improved the washing fastness, whereas the wax particles did not when compared to unhydrophobized textiles. This observation might be due to differences in the surface energies as fluorinated compounds have lower surface energies than waxes, which may result in less interactions with the washing water, leading to better protection and washing fastness of the pigment.

Figure 10. A) WCA prior to washing and B) washing fastness of textiles dyed with blue pigment and coated with wax particles and FAS, respectively. The washing fastness is determined as the change in color (E*) according to the CIEL*a*b* system, and the smaller the change the better washing fastness.

Another way to incorporate color with the wax particles is to add pigments to the dispersion (Publication II). This method also has the added benefit of more clearly showing where the coating has already been applied – the almost white dispersion is nearly invisible on white cellulose-textiles, whereas with additional pigment it could be seen.

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4.7 Antimicrobial coatings

Four different dehydroabietanes with varying degrees of antimicrobial activity were used to modify the surface of CNF-films (Fig. 11). The surface of the films was first activated with CMC, and then the dehydroabietane compounds were coupled, via amine bonds, to the COOH-groups of CMC. The reaction took place at room temperature and in ethanol, which is beneficial from an environmental point of view compared to N,N-dimethylformamide (DMF), the most common solvent for coupling reactions. Compounds 1 is dehydroabietylamine and com- pound 4 (Fig. 11) is a synthetic derivative, while compounds 7A and 7B, are de- rivatives of dehydroabietic acids, which is a naturally occurring compound from the resin of pine trees. The differences between 1 and 4 are protonable amino groups and the 3 carbon atom spacer chain between the CMC and the diterpe- noid skeleton. Compounds 7A and 7B share the same diterpenoide skeleton as 1 and 4, but the spatial orientation towards the surface of the CNF-CMC film is changed. Compound 7A and 7B have a methyl ester and a cyclohexyl-L-alanine moiety, respectively. The cyclohexyl-L-alanine group was previously reported to be relevant for the antimicrobial activity. 166–169

Figure 11. Molecular structure and nomenclature of the modified CNF films. Full names of the compounds can be found in the list of abbreviations.

4.7.1 Surface coverage and hydrophobicity

XPS results of the modified films showed an increase in the C-C and N content as well as a decrease in the C-O content (Fig. 12, Publication IV and V). Neither cellulose nor CMC have any N or C-C in their structure (even though trace amounts of C-C residue always remain after pulping) and changes in the che- mical composition of the surface only originate from the presence of the dehy- droabietanes. As a result, N and C-C can be used as markers that allow the mo- lecular surface coverage of the dehydroabietanes molecules to be calculated. The calculations showed that the surface coverage was relatively low, with a highest value of 30% measured (Fig. 12). This is not unexpected as dehydroabietanes are large molecules and due to steric hindrance, these molecules are unlikely to 33 be able to functionalize every free COOH group at the surface to cover it com- pletely. Nevertheless, this mild modification method still improved the hydro- phobicity of all the samples, especially for CNF-CMC-1 (Fig. 12). Since the WCAs of the samples other than CNF-CMC-1 were < 90°, CNF-CMC-1 is technically the only hydrophobic sample, although the hydrophobicity of the other samples was increased significantly.

Figure 12. A) WCA at 5 s (lighter shades) and 60 s (darker shades) and B) wide atomic scans (XPS), high resolution C 1s carbon fits (XPS) and surface cover- age of pure and modified CNF films.

4.7.2 Assessment of antimicrobial activity

The antimicrobial activity of the modified films was assessed against different strains of bacteria including Gram-positive S. aureus and Gram-negative E. coli by immersion in a bacterial suspension for 24 h (Fig. 13). All modified films showed clear antimicrobial activity against the tested bacteria, compared to un- modified CNF films, but to different extents (Fig. 13, Publication IV and V).

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CNF-CMC-1 showed a 99.9% reduction of S. aureus ATCC12598, a 95.3% re- duction of methicillin-resistant S. aureus MRSA 14TK301 and a 99.2% reduc- tion of E. coli. In orther words, almost all S. aureus ATCC12598 bacteria were dead or inactivated after 24 h of incubation with CNF-CMC-1, and the other bacteria strain counts were significantly reduced. CNF-CMC-4 was even more potent and reduced virtually all bacteria strain counts of ATCC25923 and ATCC12598, and additionally significantly reduced both MRSA 14TK30 (99.1%) and E. coli. (98.9%) counts. Similarly, CNF-CMC-7B showed robust activity, but targeting different strains of bacteria. CNF-CMC-7B caused a 98% reduction of ATCC25923 and E.coli, and reduced virtually all counts of strains ATCC12598 and MRSA 14TK301. CNF-CMC-7A was less potent, but still showed some ac- tivity with a reduction of 99.1% of MRSA 14TK301. This test shows that all sam- ples were able to reduce bacterial counts at least to some extent, with CNF-CMC- 4 and CNF-CMC-7B being the most potent.

Figure 13. Antimicrobial assessment of pure and modified CNF films on differ- ent strains of bacteria, after contact with the films for 24 h. CFU/mL (Colony Forming Units/milliliter) refer to the number of viable bacterial cells. As dif- ferent batches of CNF was used as reference, two CNF references are showed with CNF being reference for CNF-CMC-1 and CNF-CMC-4 and CNF2 being reference for CNF-CMC-7A and CNF-CMC-7B. * = Bacterial count to small to be shown, ** = not tested. Modified from Publications IV and V.

Additional SEM images of the films after contact with bacterial solution for 24 h revealed that the surface of the bacterial cells on CNF-CMC-4 and CNF-CMC- 7B had significant morphological changes. The bacteria on CNF-CMC-1 showed

35 no such changes and looked similar to the bacteria on unmodified CNF. This further suggests that contact with CNF-CMC-4 and CNF-CMC-7B kills the bac- teria.

However, a surface might not necessarily have to kill the bacteria to function as an antimicrobial surface. In many applications, inhibition of biofilm growth on the surface is enough, or even more important. Killing the bacteria upon contact limits the ability to form biofilms and other factors include interfering with ad- hesion factors of the microbes. Fouling experiments with the UAMS-1 strain showed that biofilms did not form very efficiently at the surface of CNF-CMC- 7B, when compared to CNF (Publication V). Additionally, when placed on top of an infected artificial dermis (chronic wound model), CNF-CMC-7B caused a 1.4-log reduction in the CFU which were retrieved from its surface after incuba- tion, when compared to CNF. No samples other than CNF-CMC-7B were tested in biofilms models.

Some of the main reported factors affecting the bacteriocidal activity of a surface are topography, charge, hydrophobicity and chemical surface composition. AFM images of the samples revealed that the surface topography remained largely unchanged after the modification with the dehydroabietanes (Fig. 14). Streaming current experiments showed that all samples had net anionic poten- tial. Local differences might still occur and CNF-CMC-4 and CNF-CMC-7B, the more potent surfaces, had both amine-group(s) that most likely contributed to the antimicrobial activity. The hydrophobicity was increased for all samples, to varying extents. The surface coverage of the films was however low, and the more hydrophobic regions were unevenly distributed, leading to the modified films showing a mosaic distribution of polarity. The chemical environment seems to be the most important factor towards understanding the mode of ac- tion, and the introduction of protonizable groups in CNF-CMC-4 and CNF- CMC-7B was not the only change that sets the molecules apart. The difference between weakly active CNF-CMC-7A and strongly active CNF-CMC-7B is the methyl ester of 7A versus the cyclohexyl-L-alanine moiety of 7B, that seems to be of clear importance. At least part of the differences in antimicrobial efficiency stem from the spatial orientation of the dehydroabietane molecule and the bind- ing distance to the CMC-backbone. These factors affect the distribution of po- larity and how specific features of the surface of the bacteria are likely targeted as part of the antimicrobial action.

36

Figure 14. AFM images height images with corresponding surface roughness. Modified from Publications IV and V.

As mentioned, (positive) surface charge is a factor that gives a surface antimi- crobial activity. The cationic moieties electrostatically interact with the anionic proteins and phospholipids, impairing the permeability of the cytoplasmic membrane leading to the death of the cell.108 Cationic substances like PLL,170chi- tosan171 and cationic starch172 show antimicrobial activity at least to some extent and LbL modified surfaces might also have some antimicrobial activity. Their antimicrobial activity was however not investigated and in the LbL coating, the outermost layer is comprised of the anionic wax particles, not the cationic com- pound. Therefore, it would be reasonable to assume that any potential antimi- crobial effects are significantly reduced as the cationic groups are unable to in- teract with the bacterial cell membranes. On the otherhand, the hydrophobicity of the wax particles may provide some protection against microbes as they keep the surface dry, however, a dry environment is not always conducive for antimi- crobial activity as some bacteria thrive in dry conditions.173 Therefore, the po- tential antimicrobial effect of the LbL-coated surfaces is likely to be negligible.

4.7.3 Biocompatibility

As antimicrobial compounds and materials may also be harmful and toxic to humans, the biocompatibility needs to be assessed for materials that are in- tended for human exposure. Hemolysis testing shows the rate and extent to which red blood cells die upon contact with a surface. During testing no cells were found to have died on the unmodified CNF surface and cellulose is gener- ally considered a biocompatible material (Table 3, Publications IV and V). In contrast, with CNF-CMC-1 and CNF-CMC-7B some cells died, whereas a major- ity of cells were found to perish upon contact with CNF-CMC-4. However, lack of cell proliferation does not automatically correlate with cell death, as cells need to establish contact points with the surface (i.e. via their surface proteins) for them to live on the surface. For example, only about 60% of the cells prolif- erated at the surface of CNF, even though cellulose (or CNF) is not expected to kill the cells. Compared to unmodified CNF, the cells proliferated reasonably well on CNF-CMC-1 and even better on CNF-CMC-7B, whereas the cell prolif- eration on CNF-CMC-4 was very poor at only about 3%. Overall, the samples tested – except CNF-CMC-4 – showed high biocompatibility, particularly CNF-

37

CMC-7B. Furthermore, when the high antimicrobial activity of CNF-CMC-7B is considered, its biocompatibility is remarkable.

Table 3. Biocompatibility analysis. Hemolysis testing (% of hemolyzed eryth- rocytes relative to a 2% Triton X-100 solution) and human fibroblast viability (% of viable fibroblasts after 72 h on top of each material compared to % of viable fibroblast on a sterile tissue). CNF-CMC-7A was not tested. Modified from Publications IV and V.

Assay CNF CNF-CMC- CNF-CMC- CNF-CMC- Triton X- 1 4 7B 100 2% Hemolysis 0 0.9 ± 0.6 6.5 ± 1.9 1.8 ± 1.7 100 ± 3.0 Fibroblast pro- 59.8 ± 7.3 47.2 ± 17.7 2.7 ± 0.4 84.9 ± 19.8 1.2 ± 0.4 liferation 72h

4.7.4 Effect of the modification using dehydroabietane on other proper- ties

When modifying some property, other properties are often easily affected and thus all the important characteristics need to be evaluated after modification. The CNF films modified with the dehydroabietanes showed increased hydro- phobicity and antimicrobial activity. However, other properties barely changed at all. No changes in surface morphology were observed in AFM images (Figure 14, Publication IV and V). The tensile strength of the films was also unchanged, as well as the water vapor permeability and (WVP) and oxygen permeability (OP), although the OP was slighty higher at low humidity (Publication IV). The change in OP can be explained by the hydrophobicity of the surface. Air is hy- drophobic as well, and thus the modified surface attracts more air, which may be detected as a slight increase in OP.

Apart from slightly increased OP, no other changes in the morphology, tensile or barrier porierties were seen. This shows how surface specific the modification is – only the outermost surface of the films is modified. It is not possible to as- sess the thickness of the modification but considering that no change in surface morphology is observed in the AFM images, we can assume that it is in the range of a few nanometers. The surface coverage is also low (30% at highest) and there is no film formation, so it is reasonable to believe that the modification does not affect bulk properties like tensile and barrier properties. However, since the modification was done in wet state (ethanol), some weakening of the film could occur and hence the tensile strength of the modified films was measured. The tensile strength remained unchanged, in contrast to the all-cellulose composites described in Chapter 4.4, that were sensitive to soaking in water. This difference in behavior may both be due to the fact than CNF films are homogeneous, made out of only CNF and are hence more robust than the composites or from the fact that ethanol leads to less swelling than water.

The chemical durability of the samples was tested for CNF-CMC-1. The modified films retained their antimicrobial activity after exposure to different solvents, 38 including a strong base, for 24 h (Publication IV). The film did, however, not endure treatment with strong acid. The abrasion durability was not measured and is difficult to assess due to the low coverage of the dehydroabietanes. Dura- bility over time was neither tested, but would be interesting to study in further experiments.

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5. Concluding remarks

In this work, the functionalization of different cellulosic surfaces using natural, non-toxic materials were studied. The utilized substrates all had different struc- tures, surface roughness and thickness, but were all based on pure cellulose.

Wax particles were deposited onto cellulosic surfaces using the layer-by-layer method and PLL or CS as anchoring agents. Through a combination of high sur- face roughness and low surface energy, the modified surfaces achieved high hy- drophobicity already after applying two bilayers. By optimizing the thickness of the cationic layer, the wax particle adsorption and hydrophobicity could be fur- ther increased. Heat treatment below the melting point of wax caused molecular reorientation and partial melting of the wax particles, which was beneficial for the hydrophobicity and long-term water-resistance. The surface roughness of the substrate was also found to contribute to the overall hydrophobicity. Re- gardless of whether the wax particles remained unmelted or melted, the coated materials retained their breathabilty. While the durability of the modified sur- faces was not tested, it can be speculated that the long-term surface resiliance is probably poor as no covalent bonds were formed. The coating can be reapplied, but even though it is bio-based and non-toxic it is neither practical nor ecologi- cal to reapply the coating constantly. Moreover, as the modification uses aque- ous solutions and dispersions, it is not a straightforward method for the modi- fication of water-sensitive material.

Colors could be added to the wax dispersion and could also provide further func- tionality. A blue pigment that does not absorb light in the NIR-region was used to dye the textiles, which were then further functionalized by hydrophobization. The dark blue textiles displayed similar behavior to that of white textiles when exposed to sunlight and they also demonstrated high levels of hydrophobicity when using wax particle or FAS coating. The washing fastness was however poor and the effect of the pigment when released into the environment is unknown as currently there is no ecotoxicological assessment available. Nevertheless, if the indoor temperature can be kept closer to the outside (high) temperature by using materials dyed with the pigment, major energy savings can be made.

Dehydroabietanes were found to provide both hydrophobicity and antimicro- bial properties and the amount of the molecules required to achieve these de- sired properties was very small, which makes them economic. In addition to the beneficial antimicrobial activity, the modified surfaces were also found to be bi- ocompatibile. The antimicrobial activity of the modified surfaces was contact- 40 active, i.e. no potentially toxic compounds were released, which further en- hances the material’s biocompatibility and rage of possible applications. More research is required on how these modified materials behave in a long-term per- spective and whether such materials can be produced on a larger scale. The con- tact-active mode for killing the microbes is a beneficial feature from a safety and environmental point of view, since the material is unlikely to induce microbial- resistance – although more research is needed in this area – and does not re- lease harmful substances into the environment.

All the modified materials investigated in this work show promise for use in the larger scale and to reduce the amount of toxic substances that are currently used in cellulosic surface functionalization. Furthermore, these modification meth- ods utilized only small amounts of different chemical treatments to achieve the desired functionalites demonstrating that materials with excellent surface prop- erties can be made without adversely affecting the inherent features of the un- derlying bulk material. The key conclusion of this work is that the properties of cellulose can be immensely changed by using only a small amount of bio-based, non-toxic material on the surface.

The research work related to this topic is not by far finished. Ideas for further research include systematic studies about the durability, both mechanical, chemical and over time, for the surface modification methods studied. How the structure of the textiles or other substrates affect both the hydrophobicity and the breathability is not yet well understood. Perhaps the wax particles could be covalently bond to the surface, for increasing durability, and perhaphs the de- hydroabietanes could be used on textiles. A different wax than carnauba would also be interesting, or a combination of several waxes. A study where wax parti- cles and dehydroabietanes would be combined would also be very interesting.

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