Matej Bračič a,b *, Lourdes Pérez c , Rosa Infante Martinez-Pardo c , Ksenija Kogej d , Silvo
Hribernik b , Olivera Šauperl b , Lidija Fras Zemljič b * a Savatech d.o.o., Industrial Rubber Products and Tyres, Škofjeloška cesta 6, 4000 Kranj,
Slovenia. b Institute for the Engineering and Design of Materials, University of Maribor, Smetanova
17, 2000 Maribor, Slovenia. c Departamento de Tecnología de Tensioactivos, Instituto de Química Avanzada de
Cataluña, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain d Chair of Physical Chemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
Corresponding authors:
*mattej.bracic@gmail.com
Tel.: 00386 2207887
* lidija.fras@um.si
Tel.: 00386 2207909
1

Keywords: Viscose, hyaluronic acid, natural cationic surfactant, pH-potentiometric titration, antimicrobial activity.
Abstract
In this investigation, a novel coating for viscose fabric surface modification was developed using a synergistic formulation between a natural antimicrobial cationic surfactant from lysine (MKM) and a biopolymer hyaluronic acid (HA). The interaction between MKM and HA in aqueous solutions, as well as the interactions between their synergistic formulation (HA-MKM) and viscose fabric (CV) were studied using pH-potentiometric titrations’, turbidity measurements, the
Kjeldahl method for the determination of nitrogen amounts, attenuated total reflection infrared spectroscopy (ATR-IR), and scanning electron microscopy (FESEM). The hydrophilic and antimicrobial properties of the functionalised CV were examined in order to evaluate its usages for medical applications. The results of the interaction studies showed that MKM and HA interact with each other by forming a precipitate when the binding sites of HA are saturated. The precipitate has a slightly positive charge at neutral pH due to excess binding of the MKM to HA.
The excess positive charge was also detected on CV coated with HA-MKM. This was proven to be very beneficial for functionalised CV antimicrobial properties. The antimicrobial tests showed exceptional antimicrobial activity of the coated CV against Escherichia Coli, Staphylococcus
Aureus, Streptococcus Agalactiae, Candida Albicans, and Candida Glabrata which gives to fabrics high impact of potential use.
2

Medical textiles have attracted a lot of interest over the past decade and are amongst the fastest
growing textile fields today (Yuan Gao and Cranston 2008). They are predominantly used as
implants, for wound healing and disease curing, etc.(Czajka 2005). One of the more desirable
properties of medical textiles, especially within the wound healing field, is their antimicrobial activity. Special surface modifications of textile fibres and fabrics are generally applied to introduce such properties to their surfaces, as conventional natural and man-made fibres do not
exhibit antimicrobial activity (Ristić et al. 2011). These surface modifications usually include the
incorporation of an antimicrobial agent in the fibre matrix prior to extrusion or during the fibreforming process when working with man-made fibres. Functional coating of the fibre’s outer
surface can also be applied and is more common for natural fibres (Yuan Gao and Cranston
2008). A wide variety of chemical agents have been used to impart antimicrobial activities to textile materials, including metals and metal salts (Shateri Khalil-Abad et al. 2009; Yadav et al.
2006), iodophors (Singhal and Ray 2002), phenols and thiophenols (Fillat et al. 2012), antibiotics
(Qian et al. 2009), and many others. The vast majority of these agents is unfortunately toxic to
humans and is difficult to degrade within the environment. Therefore, natural, biodegradable, and environmentally-friendly textile materials, as well as polymer surface modifying agents, are of great interest because of greater awareness of consumers regarding their health and their
environment (Ristić et al. 2011). Natural polymers such as polysaccharides (cellulose) fulfil the
above-mentioned requirements for the production of medical textiles. Natural antimicrobial and wound-healing agents such as polysaccharides chitosan and HA, and natural proteins like sericin, represent amongst others more promising natural agents in the field of functional coatings for
medical textiles (Fras Zemljič et al. 2009; Rajendran et al. 2011). Each of these agents has its
unique qualities but also ‘drawbacks’, therefore combining them in order to create multi- functional coatings for targeted applications has become a very interesting task in the medical textiles field.
In our work, a novel synergistic formulation between polysaccharide HA and surfactant MKM was developed as a functional textile material coating. The idea behind the synergistic formulation is
3

to combine the wound healing properties of HA ( )with the antimicrobial properties of the MKM within one product. HA is a polysaccharide consisting of repeating β 1-4 D-glucoronic acid (GlcA) and β 1-3 N-acetyl-D-glucoseamine (GlcNAc) units (Figure 1). It can be found in tissue types such as skin and joints. It has viscoelastic and unique moisturizing properties and is biocompatible and biodegradable. It is therefore also being used for its excellent wound healing abilities within
various medical applications, one of the more notable ones being wound dressings (Brown and
Jones 2005; Dumitriu 1998; Lee et al. 2009).
MKM is a natural cationic surfactant derived from lysine. It is an Nε – acyl lysine methyl ester and has been synthesised at the Institute of Advanced Chemistry of Catalonia (Figure 1). Its
antimicrobial activity and low toxicity were studied in a previous paper (Pérez et al. 2009).
Natural surfactants from amino-acids can be orally administrated, are non-irritating, biodegradable and have, unlike synthetic surfactants, a minimal aquatic impact. This is important when synthesising products in accordance with the current human health and environment regulations. All this guarantees their ultimate commercial development within the food and cosmetic sectors thus highlighting their potential for biochemical applications including drug
delivery, DNA transfection, and antimicrobial activity (Holmberg 2001). Other benefits, beside the
healing properties of HA and the antimicrobial properties of MKM, are also gained from the HA-
MKM synergistic formulation. One of them is the stability of the functional coating agent dispersion when conducting adsorption to the textile material. Polysaccharide dispersions often have low stability. This can be improved by the addition of a surfactant that can also ensure equal accumulation of the dispersed particles on the textile material’s surface. Although some work has been done on the use of polysaccharide-surfactant synergistic formulations within the medical field, no studies have been conducted so far on textile material functionalisation using such formulations. Even more, to our knowledge no studies have been done regarding the development of the synergistic formulation between MKM and HA as a fibre coating. Lu and co-
authors (2000) have reported an increased healing effect of meconium-induced acute lung
injuries regarding non-ionic polymers when combined with a phospholipid-based cationic
surfactant. Panyam and Chavanpatil (2011) used a commercially-available anionic surfactant in
combination with alginate to form nano-capsules for the controlled release of compounds.
4

Khdair and co-authors (2008) prepared nanoparticles from alginate and the anionic surfactant
dioctyl sodium sulphosuccinate in which a photosensitiser methylene blue was encapsulated. The synergistic effect of the polysaccharide and the surfactant nanoparticles enhanced the anticancer photodynamic efficacy in vitro.
Interactions between HA and MKM as well as interactions between the HA-MKM formulation and
CV were studied in this paper using pH-potentiometric titrations, turbidity measurements, the
Kjeldahl method for the determination of the nitrogen amount, and tensiometry. Antimicrobial tests were conducted in accordance with the international standard E2149-01.
Fig.1 Chemical structures of the surfactant MKM (Pérez et al. 2009) (left) and HA (right).
1.1 Interaction between surfactant and polymers
The interactions between ionic polymers and ionic surfactants of opposite charge are described
in detail elsewhere (Holmberg et al. 2003). Therefore, only a brief description is given in the
following text, which will help the reader to understand why these complex interactions are important during an adsorption process on textile materials. The interactions can be described by a simple phase diagram (Figure 2) where the concentration of the polymer is plotted on the ordinate axis and the concentration of the surfactant on the abscise axis. The diagram is divided into four regions. In the first region (I) at low surfactant concentrations no interactions occur between both species of interest until the so-called critical association concentration (CAC) of the surfactant is reached. At that point the second region (II) begins and the surfactant starts to bind cooperatively to the oppositely-charged polymer. The binding process involves micelle formation at the polymer, and therefore the CAC often lies far below the critical micelle concentration
(CMC) of the surfactant (Merta and Stenius 1995). The amount of bound surfactant within this
5

region linearly increases with polymer concentration. The binding process stops at the second critical association concentration of the surfactant, denoted as CAC
2
. At CAC
2
, all binding sites on the polymer are occupied with surfactant molecules and any further addition of surfactant in the system results in an increased concentration of free surfactant molecules, which is characteristic of region (III) beginning from this point on. The concentration of free surfactant molecules increases until the critical micelle concentration designated as CMC’ is reached. The CMC’ strongly depends on the interaction between the polymer and the surfactant and therefore shouldn’t be confused with the CMC of the surfactant in the absence of a polymer. It is normally higher than CMC. At surfactant concentrations above CMC’ the fourth region begins (IV) and single surfactant molecules start to self-associate to form micelles. In region IV, free micelles and polymer-bound micelles coexist in the system.
Fig.2 Phase diagram of interactions between oppositely-charged polymer and surfactant. The
diagram is adapted from Holmberg et al. (Holmberg et al. 2003).
Two major driving forces contribute to the interactions between oppositely-charged polymers and surfactants. In the first phase, the electrostatic attraction forces dominate and attract surfactant ions to the polyion. In the second phase, the cooperative interactions between bound surfactant ions are the dominant ones, which lead to micellisation of the surfactant on the polymer’s backbone or in its vicinity and a polymer-surfactant complex, PSC, is formed. Thalberg
and Lindman (1989) studied the interactions between HA and cationic surfactants
(alkyltrimethylammonium bromides of various chain lengths) and found out that a precipitate is
6

formed in the region of cooperative surfactant binding to the polymer. At low surfactant concentrations (region II in Figure 2) the precipitate appears as a pale opalescence growing to a milky opaque dispersion when the surfactant concentration increases (region III in Figure 2). At very high surfactant concentrations (region IV in Figure 2) the two-phase dispersion turns more transparent again as dispersed phase sediments and physical separation into two macroscopic phases, a dilute upper phase and a dense precipitate, is achieved. In some cases re-dissolution of the precipitate is possible. This is achieved by additional hydrophobic binding of surfactant to the precipitated complex. The binding of the cationic surfactant to hyaluronic acid has proven to be of a strong cooperative nature, indicating that binding is in the form of micelle-like clusters
(Thalberg and Lindman 1989).
Knowing the interaction behaviour between hyaluronic acid and cationic surfactants allows us to choose appropriate conditions for textile material functionalisation. In this paper the CV was subjected to the HA-MKM system in the first and third binding regions as explained above. In the first region both HA and MKM will predominantly react with the fabric surface as individual components, whilst in the third region both compounds will bind to the surface in the form of an insoluble complex. In this third region the synergistic formulation is generally uncharged and thus precipitates from the solution to form a milky-like dispersion. It is in this case energetically favourable for the precipitate, which is rather hydrophobic, to adsorb to the fabrics surface. This increases the amount of bound material compared to adsorption of individual components from a solution, where it is energetically favourable for the dissolved molecules to stay in solution rather than to bind to the fabric surface. The free MKM molecules in region (III) can further bind to the uncharged complex with their hydrophobic tails. This can be an advantage for overcoming the repulsion forces between the HA backbone and the small negative charge of the CV. The cationic MKM can interact by electrostatic forces (which are pH dependant) with the CV acidic groups and thus neutralise the surface-charge of the fabric. A simple scheme of HA-MKM complex bound to CV is proposed in Figure 3.
7

Fig.3 Scheme of HA-MKM complex bound to a slightly negatively-charged CV surface. The positive charge of MKM micelles in the complex is emphasised.
2.1 Materials
A canvas woven type CV with a mass per unit area of 153 g/m 2 and with warp and weft densities of 37 yarns/cm and 29 yarns/cm respectively was purchased from Lenzing, Austria. Sodium salt of
HA, NaHA, with a molecular weight of the monomer unit of 401.3 g/mol was purchased from Lex,
Slovenia. MKM was kindly provided by the Institute of Advanced Chemistry of Catalonia.
Hydrochloric acid and potassium chloride for pH-potentiometric titrations, sodium carbonate for alkaline washing of CV, concentrated sulphuric acid, and the catalyst for the Kjeldahl analysis were purchased from Sigma-Aldrich, USA. Potassium hydroxide for pH potentiometric titrations was purchased from J.T. Baker, USA. The non-ionic wetting agent Sandoclean PC was purchased from Clariant, Switzerland.
2.2 Viscose fabric pre-treatment
The CV was alkaline washed and demineralised prior to use. The alkaline washing was carried out in a sodium carbonate and non-ionic wetting agent bath at 60 °C for 30 minutes in order to remove impurities and other chemicals used during the process of viscose fabrication. The demineralisation step was carried out in 0.01 M hydrochloric acid in order to exchange the sodium counter ions of the carboxylic groups for hydrogen atoms. The demineralisation step is necessary prior to fabric analysis using pH-potentiometric titrations. The fabric was thoroughly rinsed with demineralised water after alkaline washing and demineralisation until a constant conductivity of demineralised water was reached (σ < 1 μS/cm). The pre-treated fabric was
8

stored in an air-conditioned chamber under standard atmospheric conditions (temperature = 20
± 2 °C and relative humidity = 65 ± 2 %), until further use.
2.3 Viscose fabric functionalisation
The pre-treated CV was immersed in the aqueous HA-MKM in the first (CV-HA-MKM I) and the third region (CV-HA-MKM III) (c.f. Fig. 2) at pH ≈ 6 for 2 hours at ambient temperature. The immersion step was followed by pressing the impregnated fabric through a laboratory padder
(Werner Mathis AG, Switzerland) until a wet pick-up of 90 % was reached. The fabric was dried for 60 minutes at 40°C and stored under standard atmospheric conditions (section 2.2) for 24 hours. The unbound material was removed by subsequent demineralisation and rinsing of the fabric, as described in section 2.2. The CV was also functionalised with HA and MKM separately to be used as blank samples. The functionalisation process was conducted in the same manner as described above. Sample notifications and their descriptions are presented in Table 1.
Table1: Sample notations of the synergistic formulation and viscose fabric samples.
Sample notation
HA-MKM (a)
Description
Synergistic formulation of hyaluronic acid (c
1
= 2.50 x 10 -4
HA-MKM (b)
CV mol/L) and the lysine based cationic surfactant
Synergistic formulation of hyaluronic acid (c
2
= 1.25 x 10 -3 mol/L) and the lysine based cationic surfactant
Pre-treated viscose fabric
CV-HA
CV-MKM
Pre-treated viscose fabric functionalised with hyaluronic acid
(c
1
= 2.50 x 10 -4 mol/L)
Pre-treated viscose fabric functionalised with the surfactant
MKM (c = 1.3 x 10
-3 mol/L)
CV-HA-MKM I Pre-treated viscose fabric functionalised with hyaluronic acid
(c
1
= 2.50 x 10 -4 mol/L) and the lysine based cationic surfactant
(2.00 x 10 -4 mol/L) - the first region of the binding process
(Figure 2)
9

CV-HA-MKM III (a)
CV-HA-MKM III (b)
Pre-treated viscose fabric functionalised with hyaluronic acid
(c
1
= 2.50 x 10 -4 mol/L) and the lysine based cationic surfactant
(5.00 x 10 -4 mol/L) - the third region of the binding process
(Figure 2)
Pre-treated viscose fabric functionalised with hyaluronic acid
(c
2
= 1.25 x 10 -3 mol/L) and the lysine based cationic surfactant
(1.20 x 10 -3 mol/L) - the third region of the binding process
(Figure 2)
* In all CV-HA-MKM samples the HA-MKM was prepared and adsorbed to CV at pH ≈ 6.
2.4 Methods
Turbidity measurements
Turbidimetric titrations were performed in order to observe turbidity changes during the binding process between HA and MKM. The information about the CAC and CAC2 is collected from the obtained data. Knowing these values allowed adsorbing of the synergistic complex on the fabric either from region I or from region III. The titrations were performed by filling a glass titration cell with 30 mL of aqueous HA solution at two concentrations (c
1
= 2.5 x 10
-4 mol/L and c
2
= 1.25 x 10
-3 mol/L) and at pH ≈ 6, adjusted by small additions of 0.1 M HCl. Incremental additions of 0.1 mL
(MKM solution, c = 5 x 10
-3 mol/L) were added stepwise every 5 minutes using a Mettler Toledo
DL 53 automatic titration unit (Mettler Toledo, USA). The turbidity was measured spectroscopically using a DP660 phototrode (Mettler Toledo, USA) and the results were presented as Figure where the electrode potential, E, is function of MKM concentration, c
MKM
(E
= f(c
MKM
)). pH-potentiometric titration pH-potentiometric titrations were performed for i) HA and MKM solutions, ii) for the HA-MKM complex, iii) for CV and iv) for all functionalised viscose fabric samples as described in section 2.3.
(see table 1). i) A glass titration cell was filled with HA (c = 1.25 x 10
-3 mol/L) or MKM (c = 1.3 x 10
-
3 mol/L) solutions and titrated in a forward (from acidic to alkaline) and backward (from alkaline
10

to acidic) manner in the pH region between 2.8 and 11 using 0.1 M hydrochloric acid and 0.1 M potassium hydroxide. The ionic strength of the solutions was set to 0.1 M using potassium chloride. The titrants were added to the system in a dynamic mode using a double burette
Mettler Toledo T70 automatic titration unit. The pH value was measured using a Mettler Toledo
DG-117 combined glass electrode. The equilibrium criteria for a stable pH reading was set to dE/dt = 0.1 mV /20 s. The minimum time for the reading was thus set to 20 seconds, while the maximum time was set to 180 seconds. ii) The pH of the initial HA solution was adjusted to pH = 2
(degree of ionization = 0). This disabled any electrostatic interactions between HA and MKM when the MKM solution was added to the system. The mixture was then titrated in the same manner as described above. iii and iv) all fabric samples where immersed in 200 mL of potassium chloride solution at an ionic strength of 0.1 M. The titration was performed in the same manner as the titrations for the solutions, only the equilibrium criteria for a stable pH reading was set to dE/dt = 0.1 mV/150 s in this case. The minimum time for the reading was set to 150 seconds, while the maximum time was set to 7200 seconds. Determination of the amount of charged
functional groups is described elsewhere (Cakara et al. 2009; Zemljič et al. 2011). Only a brief
description is presented in this paper. In the titration system, as described above, the ionic species present are H + , OH , their counter ions K + and Cl as well as the species of interest, denoted as k n
A , where n is the charge number and k is the enumerator. The total charge Q, due to the presence of n
A is calculated using the electro-neutrality condition according to Eq. 1: k
Q (pH)

F V t
 k n
 

          k
F V t
, (1) where square brackets denote the ion concentrations in mol/dm 3 , V t
the total volume and F the
Faraday's constant. The potassium and chloride ion concentrations, [K + ] and [Cl ], respectively, are known from the titrant additions, while the hydrogen and hydroxyl ion concentrations, [H + ] and [OH ], respectively, are measured with a pH metre. In a blank titration without the species of interest only H + , OH , K + , and Cl - ions are present, thus Q = 0 for any given pH. This allows replacing the [OH ] – [H + ] term in Eq. 1 by the difference [K + ] blank
– [Cl ] blank
and results in (Eq.2):
Q (pH)

F V t
 k n
  k

F V t
       blank

  blank

. (2)
11

The latter approach is recommended because it permits eliminating the error due to the presence of dissolved carbon dioxide in the titration system. The titrant volume was normalised to the mass of the titrated samples and expressed as charges per mass (in mmol/g) versus pH curve. The Q(pH) curves are referred to as charging isotherms. From the charging isotherm of the forward titration, the dissociation constant (as a pK value) was calculated by the half neutralisation value method.
Attenuated infrared spectroscopy (ATR-IR)
ATR-IR spectra were recorded using a PerkinElmer Spectrum GX Series-73565 spectrometer at a scan range of 4000 to 650 cm -1 . A total of 32 scans were performed at all measurements with a resolution of 4 cm -1 .
Scanning electron microscopy (FESEM)
FESEM analysis was performed on a small piece of each fabric sample placed on a microscope sample holder with a double-sided adhesive carbon tape. A Carl Zeiss FE-SEM SUPRA 35 VP electron microscope was used for observation of the fabric morphology. The images were recorded with an acceleration voltage of 1 keV at a working distance of 4,5 mm.
Kjeldahl Analysis
About 1.5 g of the sample was digested in concentrated sulphuric acid and a catalyst containing
2.8 % TiO
2
, 3.0 % CuS0
4
5H
2
O, and 94.2 % K
2
SO
4
. The residue was treated with sodium hydroxide to liberate ammonia, which was subsequently absorbed in boric acid and titrated with hydrochloric acid. All samples were analyzed at least in triplicate to ensure reproducibility.
Tensiometry
The CV was cut into 2 × 3.5 cm 2 rectangular pieces with a mass of 0.1 g and fixed in a special sample holder of a K12 Mk5 processor tensiometer (Krüss, Germany). The sample holder acts as a balance enabling continuous measurement of the sample weight throughout the sorption process. The sample edge was brought into contact with the surface of the measuring liquid (nheptane or water) by automatic linear movement of the glass container in which the liquid was stored. The sample weight-gain during the sorption process (m 2 ) was monitored as a function of time (t). The initial slope of the plot m 2 = f(t) is known as the capillary velocity, from which the
12

contact angle can be calculated according to the modified Washburn equation (Eq.3)(Peršin et al.
2004):
cos
  m
2 t


 2    c
, (3) where Ѳ is the contact angle between the solid and liquid phases, m 2 /t is the capillary velocity, η is the liquid viscosity, ρ is the liquid density, γ is the surface tension of the liquid, and c is the material constant. The constant c was determined for each sample from contact angle measurements using n-heptane, for which the contact angle on the fabric was zero and η = 0.4 mPas, ρ = 0.6836 g/cm 3 , and γ = 20.4 mN/m for n-heptane. The results were statistically processed (a set of parallel measurements until the standard deviation was less than 2°) and represent average values of 10 measurements of the water contact angle.
Mechanical properties
The uncoated and all coated CV fabric samples were subjected to mechanical testing according to the ISO 13934-1 test method: Determination of maximum force and elongation at maximum force using the strip method. The maximum breaking strength was determined on 50 mm x 200 mm large samples previously stored in standard atmospheric conditions (T = 25 ± 2 °C and RH =
65 ± 2 °C) for 24 hours. The test was performed in the weft and warp directions of the fabric.
Antimicrobial activity
The antimicrobial testing of all samples was carried out in accordance with the international standard E2149-01. This is a standard test method for determining the antimicrobial activities of
textiles (Fras et al. 2012; Fras Zemljič et al. 2009; Genco et al. 2012; Zemljic et al. 2013; Zemljič and Šauperl 2012). The textile material is suspended in a bacterial inoculum and continuously
shaken for 65 seconds and for 65 minutes. Serial dilutions and standard plate count techniques are applied to determine the average colony forming units per millilitre (CFU/mL) for samples exposed to the inoculums for the above mentioned times. The reduction rate is calculated using
Equation 4. The bacterial inoculum is prepared by growing a fresh 18 hour shake culture of the
13

desired bacteria in a sterile nutrient broth. The culture is later diluted with a sterile phosphate buffer solution (pH = 6.8) until a working concentration of 1.5 – 3 x 10 5 CFU/mL is reached. reduction[ %]

B

A

100
B , (4) where A is CFU/mL for the sample exposed to the bacterial inoculums for 65 minutes and B is
CFU/mL for the sample exposed to the bacterial inoculums for 65 seconds. Details regarding this technique may be found in the mentioned standard.
3.1 Interaction between MKM and HA
Turbidity measurements
In Figure 4 the turbidimetric titration isotherms E = f(c
MKM
) for the samples HA-MKM (a) and HA-
MKM (b) is shown (for sample details see Table 1). At lower concentrations of MKM, the potential of the electrode is stable, thus indicating a clear solution. At higher MKM concentrations the potential drops steeply, indicating a rapid change in the visual appearance of the system. The rapid change in turbidity can be attributed to the strong and rather fast cooperative binding of MKM to the charged HA backbone, which is in accordance with the
findings of Thalberg and Lindman (1989) for the binding of cationic surfactants to HA. The CAC
value, indicating the beginning of the binding process, is located at the point of the first change in slope, while the CAC2 value indicates the end of the binding process and is located at the end of the slope change, where the potential of the electrode stabilizes again (Figure 4). The determined
CAC and CAC2 for HA-MKM (a) and HA-MKM (b) are reported in Table 2.
14

Fig.4 Turbidity isotherms (E = f(c
MKM
) curves) for MKM binding to HA at different HA concentrations.
In the case of the lower HA concentration (c
1
= 2.50 x 10 -4 mol/L), CAC was 3.8 x 10 -4 mol/L and
CAC2 was 4.8 x 10 -4 mol/L. Both values were very close to each other due to the very steep part of the isotherm between CAC and CAC2 (Figure 4). At higher HA concentration (c
2
= 1.25 x 10 -3 ), the CAC and CAC2 values were correspondingly higher (Table 2). The number of binding sites on the HA backbone increases with its concentration; therefore, more MKM can bind until an uncharged complex is formed.
Table 2: CAC and CAC2 values for the HA-MKM system in aqueous medium determined by turbidity measurements at two different HA concentrations (for details on samples HA-MKM (a) and HA-MKM (b) see Table 1).
Sample
HA-MKM (a)
HA-MKM (b)
CAC (mol/L)
3.89 x 10 -4
1.07 x 10 -3
CAC
2
(mol/L)
4.80 x 10 -4
1.15 x 10 -3
Note that the MKM concentration in fabric functionalisation was two times lower than the experimental CAC in order to ensure that the functionalisation really took place in region (I) of the binding process (see Figure 2). A concentration slightly higher than the CAC2 was chosen to ensure the functionalisation of the fabric in region (III) of the binding process (See Table 1 for the concentration values). pH-potentiometric titration pH-potentiometric titrations were performed for HA and MKM solutions as well as for HA-MKM complex in order to identify their unique pH dependent-charging behaviour. This is essential when identifying both species on functionalised viscose fibres using the same technique. The data in Figure 5 demonstrates that the carboxyl functional groups of HA protonate/deprotonate in the pH region from 3 to 5 and that their net charge Q is negative within the whole pH region.
The pK a
of HA was determined at the protonation degree α = 0.5 and is pK a
= 3.6. The HA net charge per mass is Q/m = 0.94 mmol/g. The pK a
of the HA value determined in this work is in good agreement with the pK a
values determined by other authors (Malay et al. 2007; Tømmeraas
15

and Wahlund 2009). The literature data on pK
a
vary from 2.5 to approximately 3.6, which mainly depends on the type and concentration of the added electrolyte, the nature of the solvent and
the HA origin (Brown and Jones 2005; Dumitriu 1998). The amine functional groups of MKM
protonate/deprotonate in the pH region from 5 to 7 and their net charge Q is positive within the whole pH region. The pK a
of the amine groups at α = 0.5 is pK a
= 5.8 and their net charge per mass is Q/m = 2.03 mmol/g.
Fig.5 pH potentiometric neutralisation titration curves (Q/m as a function of pH) for HA, MKM, and HA-MKM (b).
The Q/m = f(pH) isotherm of the HA-MKM complex exhibits positive charge up to pH ≈ 7 and a slightly negative charge towards the end of the titration, at pH > 8. The positive charge can be explained by initial hydrophobic binding of MKM to the un-ionised HA (at low pH), with surfactant’s alkyl chains oriented towards the un-ionised macromolecules. In this case, the positively-charged surfactant head groups are oriented towards the polar solvent and assign the complex a positive net charge. During the course of titration with KOH the HA macromolecule is gradually ionised and the positively-charged MKM molecules start to reorient, i.e. they bind to the negatively-charged carboxyl groups of HA with their positively-charged head groups. In this way positive and negative charges are neutralised and the overall charge gradually decreased to zero. The HA-MKM isotherm exhibits only one pK a
value (pK a
= 6.0). This coincides with the value obtained for pure MKM. The net positive charge per mass for the HA-MKM complex is Q/m = 1.0 mmol/g, while the net negative charge amounts to 0.2 mmol/g. Results for the HA-MKM complex support the proposed model of formulation adsorption to the slightly negatively-charged viscose
16

fabric. The complex is slightly positively-charged at pH 6-7, confirming that accessible positive charges are present on its surface. Their presence was also confirmed later on viscose fabric functionalised with HA-MKM.
Attenuated infrared spectroscopy
ATR-IR spectra of MKM, HA and their complex are shown in Figure 6. The peaks most significantly describing the chemical structure of both substances are depicted in the spectra. The MKM surfactant exhibits peaks at wavenumbers of 3318 cm -1 (a), 2915 cm -1 and 2847 cm -1 (b and c),
1747 cm -1 (d), 1644 cm -1 (e), and 1227 cm -1 (f) corresponding to the N-H bond of the amide, the
C-H stretching vibrations and the NH 3+ , the C=O stretching vibrations of the amide, the C=O stretching vibrations of the ester, and the C-O stretching vibrations of the ester respectively
(Pretsch et al. 2009; Rozenberg and Shoham 2007). The HA exhibits typical peaks at
wavenumbers of 3300 cm -1 (g), 1605 cm -1 (h), 1407cm -1 (i), and 1037 cm -1 (j) corresponding to the
O-H and N-H stretching vibrations, the asymmetric C=O stretching vibrations, the symmetric C-O
stretching vibrations, and the C-OH group respectively (Pretsch et al. 2009; Wu 2012). The ATR-IR
spectrum of the insoluble synergistic formulation HA-MKM exhibits a broad peak at 3300 cm -1
(ag) corresponding to the O-H and N-H stretching vibrations of HA. The peak at 3318 cm -1 (a) arising from the N-H amide bond of MKM is overlapping with this one and cannot be separated.
The two sharp peaks at 2915 cm-1 and 2847 cm-1 (b and c) correspond to the C-H stretching vibrations and the NH 3+ of MKM. One can also clearly observe the peaks at 1747 cm -1 (d) from the
C=O stretching vibrations of the amide of MKM, the overlapping peaks at 1644 cm -1 (e) from the
C=O stretching vibrations of the ester of MKM and at 1605 cm -1 (h) from the C=O stretching vibrations of HA, the C-O stretching vibrations of HA at 1407cm -1 (i), the C-O stretching vibrations of the ester of MKM at 1227 cm -1 (f), and the C-OH group of HA at 1037 cm -1 (j).
17

Fig.6 ATR-IR spectra of MKM (bottom), HA (middle) and their complex HA-MKM (top).
3.2 Functionalised viscose fabric pH-potentiometric titration pH -potentiometric charge isotherms for the fabric samples CV, CV-MKM-HA I, and CV-MKM-HA
III (a) are shown in Figure 7. Total amounts of accessible charged species on the fabric surfaces are presented in Table 3. The isotherm for the CV sample exhibits very similar pH-dependent behaviour as did the HA sample (Figure 5). The CV deprotonates/protonates within the pH region
3-6, with a pKa of 4.30 at

=0.5. It is well known that viscose cellulose possesses carboxylic end
groups with pKa values usually ranging from 3.4 to 4.5 (Fras et al. 2004). The results obtained in
this paper agree with the ones from other authors. The total amount of free carboxylic groups per mass of fabric, determined from the plateau values of the isotherm, was 2 mmol/kg. The isotherm shape, as well as the pKa of the CV, greatly resembled the HA ones. This made a reliable determination of HA on the surface nearly impossible for all the CV-MKM-HA samples. It could be
18

clearly ascertained from the isotherms that positive charges are present on the surfaces of both
CV-MKM-HA samples. Positive charges arise from the free amino groups of the MKM. The total positive charges amount to 6 mmol/kg for the CV-MKM-HA I sample and to 27mmol/kg for the
CV-MKM-HA III sample. The amount of negative-charges, which in both cases can arise from carboxylic groups of the HA as well as the groups of the CV fabric, slightly deviates from the amount of the CV sample (Table 3). The difference is somehow more pronounced in the case of
CV-MKM-HA III (a). In the case of CV-MKM-HA I (no cooperative interaction between MKM and
HA) both species bind to the CV surface individually. Regarding the neglectable difference in positive and negative charges between CV-MKM-HA I and CV it could be concluded that the adhesion is poor and mostly mechanical, and that most of the material is removed during the rinsing process. A small positive charge suggests that a small amount of MKM molecules are still bound to the carboxylic groups on the viscose by electrostatic interactions. Differently, in region
(III) of the MKM-HA binding process, the uncharged complex precipitates and eventually some excess of MKM binds to its surface. In this case, the MKM-HA complex might have covered some of the carboxylic groups of CV during fabric functionalisation, making them inaccessible and thus reducing the total amount of negative charges present in the original CV surface. The increase in positive-charges, on the other hand, clearly suggests that the complex with excess of MKM is strongly bound/attached to the fabric surface after functionalisation and rinsing.
Table 3: Values of Q/m for CV, CV-MKM-HA III (a), and CV-MKM-HA I samples determined by pHpotentiometric titrations.
Sample
CV
CV-MKM-HA III (a)
CV-MKM-HA I
Q/m positive (mmol/kg)
-
27
6
Q/m negative (mmol/kg)
23
18
21
The presence of the MKM-HA complex attached on the fibre surface could also be evaluated by observing the changes in the pKa values of the samples. The CV-MKM-HA I protonates/deprotonates within the pH region 3-6 and its pKa is 4.10, which was similar to that of CV (pKa = 4.30). The difference is negligible and it is therefore not possible to confirm the presence of other species on CV. The slight shift of the pKa towards the acidic pH region may be because of the presence of a minor amount of HA. The CV-MKM-HA III on the other hand
19

protonates/deprotonates within the pH region 3-8 with a pKa of 5.6. This is significantly shifted towards the alkaline region in comparison with the CV sample. The protonation/deprotonation behaviour, as well as the pKa value resemble those of pure MKM and of the HA-MKM complex.
This leads to a conclusion that the surfactant was indeed present on the surface of the functionalised fabric. The CV-MKM-HA III (a) isotherm exhibits only one pKa value, even though two types of functional species are present in the CV-MKM-HA III (a) sample with two different pKa values. This can be explained by proposing that the MKM-HA complex behaves as an independent species; individual properties of MKM and HA arising from their charged groups are lost as a result of complexation .
Fig.7 pH-potentiometric titration isotherms of the pre-treated CV, and for CV functionalised with the HA-MKM synergistic formulation
Attenuated infrared spectroscopy
ATR-IR spectra of CV, CV-HA, CV-MKM, CV-HA-MKM I and CV-HA-MKM III are presented in Figure
8. The CV fabric exhibits characteristic peaks at wavelengths of 3308 cm -1 (k), 2890 cm -1 (l), 1638 cm -1 (m), 1155 cm -1 (n) 897 cm -1 (o) corresponding to O-H stretching vibration, C-H stretching vibration, O-H bending vibration of the adsorbed water, C-O-C asymmetric stretching vibration,
and C-O-C stretching vibration at the β-(1→4)-glycosidic linkage respectively (Pretsch et al. 2009;
Rojo et al. 2013). The CV-HA, CV-MKM as well as CV-HA-MKM I samples exhibited the same peaks
as the CV sample because most of the coating was washed out during the rinsing process. The
CV-HA-MKM III samples a and b on the other hand both exhibit characteristic peaks at 2915 cm-1 and 2847 cm-1 (b and c) correspond to the C-H stretching vibrations and the NH 3+ of MKM and
20

the overlapping peaks at 1644 cm -1 (e) from the C=O stretching vibrations of the ester of MKM and at 1605 cm -1 (h) from the C=O stretching vibrations of HA confirming the presence of the synergistic formulation between HA and MKM on the surface of the CV fabric. Eve more, the same as by potentiometric titration, the accessible protonated amino groups on the fabrics were detected.
Fig.8 ATR-IR spectra of CV (bottom), CV-HA, CV-MKM as well as CV-HA-MKM I (middle), and CV-
HA-MKM III (top).
Scanning electron microscopy
FESEM images in Figure 9 show the morphology of uncoated and coated CV samples. One can observe a rather smooth fibre surface in the CV and CV-MKM fabric sample, indicating that no
MKM is bound to the fabric after the rinsing step. In the CV-HA and CV-HA-MKM I samples one can observe little deposited material on the fibre surface. In the CV-HA case the HA forms thin fibre-like structures in certain places of the CV fibres while in the CV-HA-MKM I a negligible
21

amount of agglomerated material can be observed which indicates that little of the HA and MKM do react in region I (Figure 2) of the binding process. Bigger agglomerates of the synergistic formulation can be observed on the CV-HA-MKM III (a) and CV-HA-MKM III (b) samples. The fibre coverage is only partial in the CV-HA-MKM III (a) case, where one can observe randomly distributed agglomerates, while the fibres are almost fully covered in the case of CV-HA-MKM III
(b) which confirms the AT-IR and pH-potentiometric measurements. The FESEM images supported our explanation of interactions between surfactant and polymers in 1.1. In the first region where components bound to the fabrics individually and mostly in the extended conformation thin coated layers were observed, whilst in the third region complexes like precipitates are bound to fabrics in coiled structure, providing agglomerates onto fabric surfaces.
Fig.9 FESEM images of CV (a), CV-HA (b), CV-MKM (c), CV-HA-MKM I (d), CV-HA-MKM III a (e), and
CV-HA-MKM III b (f) at a 5000 fold magnification.
Kjeldahl method
Nitrogen content was confirmed in both CV-MKM-HA I and CV-MKM-HA III (a) samples. The amount in CV-MKM-HA III (a) is 20 % higher, which confirms the results obtained from pH-
22

potentiometric titrations. The values for both samples were compared with the nitrogen content for CV and CV-MKM samples. Both contained negligible amounts of nitrogen, below 1 %, which is also in agreement with the titration data. The presence of trace amounts of nitrogen on the CV and CV-MKM samples can be attributed to the presence of some minor amount of MKM left on the surface after rinsing in the case of CV-MKM and to the presence of impurities in the case of
CV.
Tensiometry
The contact angle results of the pre-treated CV and of the functionalised CV are reported in Table
4 and show that all CV samples have angles lower than 90 °. This indicates their hydrophilic characteristics. The values did not change significantly between individual samples. Slightly higher values were recorded for samples in the presence of MKM. This might be because of the introduction of a hydrophobic domain (surfactants tail) to the fabric. Namely, the hydrophobic tails of the surfactant repel water molecules. However, differences in contact angles are minor, suggesting that the impact of MKM is insignificant and that the initial viscose fabric maintained its wetting abilities after functionalisation.
Table 4: Contact angles and the total mass of adsorbed water for the pre-treated CV and the functionalized CV determined by tensiometry measurements.
Sample Contact angle (°) Stdev Mass (g)
CV
CV-MKM
CV-HA
CV-MKM-HA I
83
86
85
88
1
1
1
0.3
0.45
0.46
0.53
0.44
CV-MKM-HA III (a) 86 2 0.46
23

Fig. 10 Sorption isotherms of CV and CV functionalised with MKM, HA and HA-MKM synergistic formulation.
From the sorption isotherms (Figure 10) and the equilibrium adsorbed mass of liquid (data in
Table 4), more information can be obtained about the effect of HA and MKM on the water sorption properties of CV fabric. Most of the water is absorbed by the fabric in the first 10 minutes and the equilibrium sorption time occurs after one hour. The CV-MKM sample exhibits very similar sorption curve and also similar values of the total mass of adsorbed water as does the CV sample. This is a consequence of the poor binding of MKM by the fabric. The surfactant is easily removed during the rinsing process and does not alter the fabrics properties. This was quite different in the case of CV-HA, where the mass of adsorbed water is the largest; it amounts to 0.53 g. This significant difference is attributed to the excellent sorption properties of hyaluronic acid bound to the fabric surface. It is also reasonable to expect that sorption is more pronounced at higher HA concentration. On the other hand, both CV-MKM-HA III (a) and CV-
MKM-HA I failed to improve the sorption properties of the fabric. In the case of CV-MKM-HA I the sorption isotherm stabilizes at the lowest value for the adsorbed mass of water (0.44 g). Because both HA and MKM under these conditions (region I) bind to the fabric individually, it might be expected that MKM is rinsed away, whereas HA stays on the surface and improves the sorption properties, in the same way as does the CV-HA case (see above). However, as the pHpotentiometric titrations show, some of the MKM still remains on the surface of the fabric after rinsing. This confirms that interaction between MKM and HA is weak (uncooperative). MKM occupies binding sites on the fabric surface and thus reduces the possibilities for HA to bind to it.
24

Consequently, without bound HA, properties of the fabric surface are not beneficial for water sorption. Similarly, HA-MKM III did not alter the sorption properties of the CV fabric; the adsorbed amount of water in this case is only 0.46 g, similar to CV, CV-MKM and also CV-MKM-
HA I. In the CV-MKM-HA III (a) case, HA and MKM are bound to the fabric in the form of an insoluble and uncharged complex, followed by extensive additional binding of MKM. It is possible that the complex swells during its formation, which can result in a reduced sorption capacity.
Additional contribution to the reduced sorption capacity may stem from the low net charge of the complex.
Mechanical properties
The breaking strength of uncoated and coated CV fabric samples is presented in Table 5. The CV exhibits a breaking strength of 614 N in the warp and 439 N in the weft direction. This is in accordance with the data regarding the warp and weft densities of the fabric being 37 yarns/cm and 29 yarns/cm respectively. The lower weft density reflects in the lower breking strength in the weft direction compared to the warp direction. The breaking strength of the coated CV samples in the weft direction differs very slightly from the uncoated CV ranging from 429 N for CV-HA-
MKM III (a) to 435 for the CV-MKM and the CV-HA-MKM III (b) samples. The difference can be neglected as the standard deviation values range up to ± 4.1 N. The breaking strength of the coated CV samples in the warp direction on the other hand differ from the uncoated CV for around 5 % ranging from 574 N for the CV-HA-MKM III (a) sample to 587 N for the CV-HA sample.
The drop in breaking strength in the warp direction after functionalisation is not significant when considering the use of the fabric as a textile material based wound dressing but one should consider the influence of wetting and subsequent drying at elevated temperatures on the mechanical properties of CV fabrics when preparing such materials.
Table 5: Breaking strength of uncoated and coated CV fabric determined in accordance with the
ISO 13934-1 testing method.
Force (N)
Sample warp weft
CV 614 ± 2.6 439 ± 3.2
25

CV-HA
CV- MKM
CV-HA-MKM I
CV-HA-MKM III (a)
CV-HA-MKM III (b)
587 ± 4.2
575 ± 4.9
580 ± 2.6
574 ± 1.5
582 ± 1.7
433 ± 1.5
435 ± 2.9
430 ± 4.1
429 ± 1.2
435 ± 3.7
Antimicrobial activity
Results of antimicrobial testing, given as the reduction of the number of pathogenic microorganisms calculated according to Equation 4 in %, are shown in Table 6. The abbreviations used in the table that indicate the names of the micro-organisms, are as follows: S.Aureus for
Staphylococcus Aureus, E.Coli for Escherichia Coli, S.Agalactiae for Streptococcus Agalactiae,
C.Albicans for Candida Albicans and C.Glabrata for Candida Glabrata. As stated for standardized tests, the substances are antimicrobially active if the reduction of microorganisms is greater than
80 %.
Table 6: Reduction of pathogenic micro-organisms (in %) for solutions of HA and MKM and for the pre-treated and functionalised CV. These values were determined in accordance with the international standard E2149-01.
Sample
Reduction [%]
S.Aureus E.Coli S.Agalactiae C.Albicans C.Glabrata
HA
MKM
CV
-66
62
20
2
43
18
-13
59
72
15
83
20
20
66
58
CV-MKM-HA I
CV-MKM-HA III (a)
CV-MKM-HA III (b)
12
84
98
0
52
100
78
84
100
1
61
100
42
95
100
* The samples CV-HA and CV-MKM are not presented in the table because the coating was washed out during the rinsing step and the reduction rates did not differ from the CV ones.
The results in Table 6 show negative reduction rates of the HA solution in the case of grampositive bacteria S.Aureus and S.agalactiae, -66 % and -13 % respectively. The reduction rates are
26

slightly positive in the case of the gram-negative E.Coli and both fungi types C.Albicans and
C.Glabrata, 2%, 15 % and 20 % respectively. The negative values of the reduction rates in the case of S.Aureus and S.agalactiae indicate that HA stimulates the growth of gram-positive bacteria. The MKM solution on the other hand exhibits higher reduction rate values against gram-positive bacteria S.Aureus and S.agalactiae, 62 % and 59 % respectively. The reduction rates are also higher against the gram-negative E.Coli and both fungi types C.Albicans and
C.Glabrata, 43 %, 83 % and 66 % respectively. It has been shown in the text below that the synergistic formulation between HA and MKM boosts the antimicrobial activity compared to pure
MKM, even though the reduction rates of HA and MKM differ from each other. The reduction rates of the CV fabric treated with the synergistic formulation were compared to the untreated
CV sample which exhibits reduction rates of 20 % and 72 % for the gram-positive bacteria
S.Aureus and S.agalactiae respectively. The reduction rates against the gram-negative E.Coli and both fungi types C.Albicans and C.Glabrata, are 18 %, 20 %, and 58 % respectively. The CV-HA-
MKM I sample exhibits similar reduction rates as the CV sample. The reduction rates for the gram-positive bacteria S.Aureus and S.agalactiae are 12 % and 78 % respectively. The reduction rates for the gram-negative E.Coli and both fungi types C.Albicans and C.Glabrata, are 0 %, 1 %, and 42 % respectively. This confirms the pH-potentiometric, AT-IR and FESEM results suggesting that the HA and MKM bind separately to the surface of the CV fabric when the binding process is conducted in region I (Figure 2) and are later mostly washed out during the rinsing step. The same phenomenon was observed fot the CV-HA and CV-MKM samples (not shown in Table 6).
The CV-HA-MKM III (a) and CV-HA-MKM III (b) samples on the other hand exhibit higher antimicrobial activity compared to CV. As indicated by the pH-potentiometric titration, AT-IR, and
FESEM results the synergistic formulation and excess of protonated amino groups are present on the fabric surface in both cases. The reduction rates of CV-HA-MKM III (a) against the grampositive bacteria S.Aureus and S.agalactiae are much higher compared to CV, both 84 %. The reduction rates against the gram-negative E.Coli and both fungi types C.Albicans and C.Glabrata, are 52 %, 61 %, and 95% respectively. The antimicrobial activity of both samples can be attributed to the free amine groups of MKM on the surface of the synergistic formulation. The reduction rate of the synergistic complex is lower against gram-negative bacteria compared to
27

gram-positive ones which could also be observed for the MKM solution. The antimicrobial activity against both fungi types behaves in an opposite fashion. The formulation shows lower activity against C.Albicans and a higher one against C.Glabrata when compared to the MKM solution. The antimicrobial activity was further enhanced by increasing the HA concentration in the formulation (sample CV-HA-MKM III (b)) resulting in more free MKM molecules bound to the fabric surface. Higher HA concentration leads to a higher number of binding sites for the surfactant, which results in a larger amount of the complex which finally precipitates to the fabrics surface as can be seen in Figure 9. In this case the reduction rates against gram-positive bacteria S.Aureus and S.agalactiae reach values of 98 % and 100 % respectively. The reduction rates reach the value of 100% for the gram-negative E.Coli and both fungi types C.Albicans and
C.Glabrata as well. This points to the importance of the relation between the concentration of free amino groups on the material surface and its antimicrobial activity and supports the findings of pH-potentiometric titrations.
A synergistic formulation between a cationic surfactant MKM and a polysaccharide HA was prepared and used as a novel coating for CV, which is intended for use in textile medicine. The combination of MKM and HA was adsorbed to CV below the CAC and above the CAC2. In this way, the effect of individually bound MKM and HA (below CAC) was compared to the binding of a synergistic complex of MKM and HA (as precipitate above CAC2). the pH-potentiometric titration,
AFT-IR and the Kjeldahl method, confirmed that binding of the HA-MKM complex to CV above
CAC2, in comparison to the binding below CAC, is more successful in terms of the deposited amount of amino groups compared to binding below the CAC value. The precipitated complex adsorbs to the fabric to a greater extent than do individual HA and MKM, and remains on the fabric surface even after rinsing. The amount of amino groups deposited on the CV surface is of primary importance when aiming for an antimicrobial functionalisation of textiles. The tensiometry measurements show no significant changes in the initial hydrophilic character of CV after the adsorption of MKM and HA. The same was observed for fabric mechanical properties.
28

Pure HA adsorbed to viscose fabric improves its water uptake but fails to do so when bound to the fabric in the form of a HA-MKM complex. The antimicrobial activity results confirm these findings by showing that the viscose fabric with the bound HA-MKM complex above CAC2 exhibits excellent antimicrobial activities towards gram-positive (S. Aureus and S. Agalactiae) and gram-negative (E. Coli) pathogens as well as towards two types of pathogen fungi (C. Albicans and C. Glabrata). These kinds of functionalised fibres could find applications as medical textiles within several branches.
Acknowledgements
The financial support of Savatech d.o.o., Industrial rubber products and tyres is gratefully acknowledged. The authors also acknowledge the financial support from the Ministry of
Education, Science and Sport of the Republic of Slovenia through the program P2 0118 as well l as ARRS project L2-4060 .
and the financial support from Spanish Plan National I+D+I MAT2012-
38047-C02-02.
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Fig.1 Chemical structures of the surfactant MKM (Pérez et al. 2009) (left) and HA (right).
Fig.2 Phase diagram of interactions between oppositely-charged polymer and surfactant. The
diagram is adapted from Holmberg et al. (Holmberg et al. 2003).
Fig.3 Scheme of HA-MKM complex bound to a slightly negatively-charged CV surface. The positive charge of MKM micelles in the complex is emphasised.
31

Fig.4 Turbidity isotherms (E = f(c
MKM
) curves) for MKM binding to HA at different HA concentrations.
Fig.5 pH potentiometric neutralisation titration curves (Q/m as a function of pH) for HA, MKM, and HA-MKM (b).
Fig.6 ATR-IR spectra of MKM (bottom), HA (middle) and their complex HA-MKM (top).
Fig.7 pH-potentiometric titration isotherms of the pre-treated CV, and for CV functionalised with the HA-MKM synergistic formulation
Fig.8 ATR-IR spectra of CV (bottom), CV-HA, CV-MKM as well as CV-HA-MKM I (middle), and CV-
HA-MKM III (top).
Fig.9 FESEM images of CV (a), CV-HA (b), CV-MKM (c), CV-HA-MKM I (d), CV-HA-MKM III a (e), and
CV-HA-MKM III b (f) at a 5000 fold magnification.
Fig. 10 Sorption isotherms of CV and CV functionalised with MKM, HA and HA-MKM synergistic formulation.
Table1: Sample notations of the synergistic formulation and viscose fabric samples.
Table 2: CAC and CAC2 values for the HA-MKM system in aqueous medium determined by turbidity measurements at two different HA concentrations (for details on samples HA-MKM (a) and HA-MKM (b) see Table 1).
Table 3: Values of Q/m for CV, CV-MKM-HA III (a), and CV-MKM-HA I samples determined by pHpotentiometric titrations.
Table 4: Contact angles and the total mass of adsorbed water for the pre-treated CV and the functionalized CV determined by tensiometry measurements.
Table 5: Breaking strength of uncoated and coated CV fabric determined in accordance with the
ISO 13934-1 testing method.
Table 6: Reduction of pathogenic micro-organisms (in %) for solutions of HA and MKM and for the pre-treated and functionalised CV. These values were determined in accordance with the international standard E2149-01:
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