Latest Developments in Antimicrobial Functional Materials for

Latest Developments in Antimicrobial Functional Materials for Footwear

M. M. Sánchez-Navarro, M. A. Pérez-Limiñana, N. Cuesta-Garrote, M. I. Maestre-López, M. Bertazzo, M. A. Martínez-Sánchez, C. Orgilés-Barceló and F. Arán-Aís,

Footwear Technological Institute INESCOP, Polígono Industrial Campo Alto s/n, 03600 Elda, Alicante, Spain

This review is an outline of the research carried out by INESCOP, the Footwear Research Institute in Spain, on antimicrobials for footwear. First of all, the microbiology associated with shoes worn by different users was investigated using modern molecular methods, including Polymerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE). Secondly, natural compounds with demonstrated antimicrobial properties were microencapsulated by different procedures in order to obtain innovative antimicrobial coating materials with different control release properties. Microencapsulation is a powerful way to control the release of antimicrobial agents to achieve better efficiency and a longer-lasting effect. Finally, the improvement of shoe comfort through the development of innovative materials based on nanotechnologies was investigated. Silver@silica nanocomposites were synthesised and incorporated into different tanned leathers including metal-tanned and oxazolidine tanned leather, as a suggestive alternative to create leather with enhanced antifungal and antimicrobial properties.

Keywords antibacterial; microencapsulation; silver nanoparticles; essential oils.

1. Introduction

Footwear is one of the only garments that is not usually washed and is taken only care of for aesthetic and/or preservation reasons. However, it is subjected to extreme conditions of use and since footwear encompasses a closed and poorly ventilated space it creates the basis for microorganism growth which are mainly responsible for shoe bad odour. Odour is a key factor in limiting footwear lifetime as the causative compounds do not disappear immediately. It arises when sweat from feet is trapped inside the footwear materials and their components are degraded by feet microbiota [1]. Feet have more sweat glands than any other part of the body and in combination with the feet being trapped inside footwear the conditions for microbial growth are ideal. The growth of microorganisms may also compromise the health of the feet, e.g. for people with foot ulcers due to chronic diseases such as diabetes. Other factors involved in the development of odour in shoes may be the nature of the shoe lining material (mainly textile or leather, synthetic or natural) and also the duration, extent of wear that the shoe is exposed to, and breathability. A way to avoid or reduce bad shoe odours apart from via foot care and hygiene measures is through the use of antimicrobial and/or deodorising agents. A wide range of antimicrobial compounds are commercially available. In the footwear industry, suitable antimicrobial technologies are mainly focused on metals and metal compounds (e.g. silver, copper, zinc, metal oxides, etc.). A few other compounds such as quaternary ammonium salts, borates, 2,4,4-trichloro-2-hydroxydiphenil ether (Triclosan) or 3-iodo-2-propynyl-butylcarbamate (IPBC), etc., are also used. However some of them may in some degree be extremely irritant, harmful and toxic for the Environment and human health. Therefore, there is much interest in finding ways to formulate new types of safe and ecological materials. In this sense, a number of natural antimicrobial agents have been identified over the last decades such as citral, nerol, citronellol, linalool, geraniol, limonene, Origanum, Thymus vulgaris, etc [2-5]. Several of these compounds have a proven effect against bacteria and fungi found on feet. In addition, in recent years silver has been used extensively, as silver ions exhibit good antimicrobial effects on a range of bacteria and fungi [6-7]. Silver ions have been employed in a wide range of applications such as refrigerators, washing machines, clothes and duvets, in all cases to reduce microbiological growth [8-9]. Silver ions are known to reduce microbiological growth with a low inhibition concentration and the historical use of silver for various applications shows no evidenced adverse effects [10-11]. Several technologies can be used to control the amount of released silver ions from a product, thereby ensuring a long-lasting effect. Nowadays, these properties have been further enhanced through nanotechnology that has allowed the size of metal nanoparticles to be modularly and accurately reproduced. Previous studies revealed high antimicrobial activity of silver nanoparticles (AgNPs) against a broad spectrum of microorganisms. The advantage of the silver antimicrobial mechanism is the ability to produce an antibacterial effect at very low concentrations (oligodynamic effect). Innovative solutions for footwear. The footwear sector is currently working on improving all factors that influence comfort and lifetime of the product. Through research and market analysis, all of these are known to be important decision factors in consumer purchase decisions. However, all of these features require the use of materials with new performance and functionalities as well as thorough understanding of the way in which the components react to these changes. In this sense, the new footwear materials should be able to withstand temperature and humidity fluctuations as well as heavy wear and abrasion.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

102

This chapter is a review of some previous studies carried out by INESCOP, the Footwear Research Institute in Spain, on antimicrobials for footwear. First of all, the microbiology associated with shoes worn by different users was investigated using modern molecular methods, including Polymerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE). Secondly, natural compounds with proven antimicrobial properties were microencapsulated by different procedures in order to obtain innovative antimicrobial coating materials with different control release properties. Microencapsulation is a powerful way to control the release of antimicrobial agents to achieve better efficiency and a longer-lasting effect. Finally, the improvement of shoe comfort through the development of innovative materials based on nanotechnologies was investigated. Silver@silica nanocomposites were synthesised and incorporated into different tanned leathers including metal-tanned and oxazolidine tanned leather, as a suggestive alternative to create leather with enhanced antifungal and antimicrobial properties.

2. Determination of the microorganisms present in footwear components in contact with the foot

In order to carry out the selection of antimicrobials able to eliminate in a specific and precise way the microorganisms responsible for bad odour and other problems related to the foot, it is necessary to identify the microorganisms responsible for this smelly behaviour. For this reason, the analysis of the microbiota associated with leather insoles worn by healthy users was undertaken [12]. In this study, leather insoles that had been continuously worn by several users for 15 days were analysed. DNA samples from unused leather insoles were used as a negative control. The total nucleic acid extraction of the leather insoles was carried out throughout semi wet scrapping. The surface of the leather insole was initially scraped by using sterile swabs soaked in 2 mL of saline 0.9 % NaCl solution. This step was repeated three times so as to ensure that as much material as possible was extracted. Subsequently, the extraction of intact total nucleic acid (TNA) was carried out using the Masterpure DNA Purification Kit MCD8520 (Epicentre Biotechnologies, USA). The gentle salt – precipitation protocol permits rapid purification of high – molecular – weight nucleic acids from the leather insole samples. The purified nucleic acid samples were visualised in a 1% agarose gel using the Quantity One 1-D Analysis Software V4.6.8 from the Documentation System Gel Doc XR (BIORAD, Spain). Once the DNA purification was finalised, the DNA concentration (ng/μL, as measured absorbance at 260 nm (A260) and purity (A260/A280 ratio) were checked using the Nanodrop ND–1000 Spectrophotometer (NanoDrop Technologies, Inc., USA) by means of the ND-1000 V3.5.2. nucleic acid application module. The resulting nucleic acids had a concentration in the range of 150 and 200ng/μL and an A260/A280 ratio of 1.8–2.0, indicating that they are substantially free of proteins, phenol or other contaminants that absorb strongly at or near 280nm. In order to obtain a high number of copies of the phylogenetically conserved 16S and 18S fragments, PCRs (polymerase chain reactions) were carried out using specific primers for Bacteria, Archaea and Eukarya. The original DNA template amounts were 250ng, 150ng, 100ng, 50ng and 25ng. Dilutions from the original DNA template were done, as well as the addition of BSA (bovine serum albumin) in order to improve PCR specificity. The rRNA 16S and 18S primers for the three phylogenetic domains are shown in Table 1. Table 1 rRNA 16S and 18S primers for the three phylogenetic domains.

Genes Domain Primer name Sequence (5’-3’)

rRNA 16S

Archaea

907r 5’ CCG TCA ATT CMT TTG AGT 3’

344(GC)f 5’ CGC CCG CCG CGC CCC GCG CCG GTC CCG

CCG CCC CCG CCC GAC GGG GYG CAG CAG GCG CGA 3’

Bacteria 907r 5’ CCG TCA ATT CMT TTG AGT 3’

341 (GC)f 5’ CGC CCG CCG CGC CCC GCG CCG GTC CCG

CCG CCC CCG CCC GCC 3’

rRNA 18S Eukarya

EUK1Ar 5’ CTG GTT GAT CCT GCC AG 3’

EUK 156GCf 5’ CGC CCG CCG CGC CCC GCG CCG GTC CCG

CCG CCC CCG CCC GAC GGG ACC AGA CTT GGC CTC C 3’

Once the amplification products were obtained, microbial composition was analysed using denaturing gradient gel electrophoresis (DGGE), which is a molecular fingerprinting method that separates polymerase chain reaction (PCR)-generated DNA products. Each of the bands obtained was further amplified again on a second DGGE in order to check the purity and get enough DNA products for advanced sequencing. Furthermore, the bands pattern was analysed by means of FPQuestTM Bioinformatic Software (BIORAD, Spain) to determine the degree of similarity between them. After having obtained the amplification products of Bacteria and having analysed the microbial composition in a DGGE gel, the banding pattern obtained for the bacteria community associated with the templates of two different users is shown in Fig.1.


© FORMATEX 2013

____________________________________________________________________________________________

103

Fig. 1 DGGE Gel. Degree of similarity between the microbiota bands pattern obtained on the DGGE gel and the software FPQuest. A) Staphylococcus epidermidis: Credit: INESCOP; B) Bacillus sp.: Credit: INESCOP; C) Pseudomonas sp: Credit: Deligianni et al. [13]; D) Brevibacterium sp: Credit: M.E. Mcride et al. [13]. For amplification of 16S and 18S rRNA gene with primers respectively for Archaea and Eukarya no bands were obtained in test conditions. Amplification products with Bacteria specific primers were obtained, corresponding with the partial sequences of the rRNA 16S gen of: Bacillus sp. (HM021765.1) and Brevibacterium sp. (GU726866.1) for user 1 and Staphylococcus epidermidis (FJ957856.1) and Pseudomonas sp. (EU239155.1) for user 2. Throughout this assay several bacterial species were identified, which are associated with leather insoles worn by healthy users. Some species are common to both users (such as Staphylococcus sp. and Brevibacterium sp.) and some specific to each user (such as Pseudomonas sp. and Bacillus sp.). This data give us an idea of the microbial variability associated with these samples. This variability is due to the high number of factors that contribute to generating the biotope where the microorganisms coexist. The microbiota is determined not only by the user genotype, but also by the phenotype, i.e. the environmental conditions around each user. Therefore, it would be useful to get further information by repeating the assay with a larger number of users to obtain statistically comparable data, as well as by repeating the amplifications for Achaea and Eukarya, whilst modifying working conditions tested.

3. Microencapsulation of natural antimicrobial compounds

In recent years, the interest in natural extracts has increased as an alternative for the control of pathogen microorganisms [14]. Essential oils such as Melaleuca alternifolia (tea tree oil, TTO), lemon oil, clove oil, eucalyptus oil or lavender oil, among others, have been successfully employed for their germicidal activity to control wound and feet infections caused by dermatophytes, as well as in traditional medicine for severe atopic and bacterial dermatitis caused by human skin bacteria such as Staphylococcus or Streptococcus [15-16]. Nevertheless, due to their high volatility and in order to be incorporated in the shoe components (lining, insole, etc.) they should be protected by a physical barrier which slows down the evaporation rate and ensures its controlled release exactly when needed in contact with feet during shoe wearing. In this sense, microencapsulation is a powerful way to control the release of antimicrobial agents to achieve better efficiency and a longer-lasting effect. Microencapsulation technology could be defined as a coating process of active substances with materials of different natures, generally polymeric shells, to obtain micrometric particles known as microcapsules or submicrometric particles known as nanocapsules [17-18]. The mechanism and release rate of microencapsulated active substances depend on the properties of the coating shell: chemical nature, morphology, glass transition temperature, crosslinking degree, etc. Therefore, its selection is an important factor for the microcapsule performance. Additionally, the selection of the encapsulation method also depends on the active substance as well as the microcapsule requirements for an intended application. The advantages offered by microencapsulation when compared with a conventional process can be summarised as follows: the protection and masking of the encapsulated substance whilst being confronted with unstable or harsh mediums, allowing its subsequent progressive release. These factors have prompted certain industrial sectors, such as the medical, pharmaceutical, cosmetics, food and nutrition, agriculture industries, etc. to base some of their most innovative products on this technology. When it comes to the footwear industry, microencapsulation is still an emerging technique, which can contribute a high level of innovation in terms of the materials used, in as much as its application could thus transform everyday traditional footwear into authentically smart products, capable of genuinely improving the quality of our lives. This could be attained, for example, via the incorporation of different products with therapeutic and/or antimicrobial


© FORMATEX 2013

____________________________________________________________________________________________

104

properties, such as essential oils, which would allow us to talk of a completely new concept of active footwear that ensures the continuous care of our feet. Recent researches carried out by INESCOP aiming to perform the microencapsulation of different natural compounds with proven antimicrobial properties in order to obtain innovative antimicrobial coating materials with different control release properties. For such purpose, the microencapsulation was carried out by two different methods, in situ polymerisation and complex coacervation processes, allowing the use of different shell polymeric materials, synthetic and semi-synthetic polymers such as melamine-formaldehyde and gelatine, respectively.

3.1. Microencapsulation of natural antimicrobial oils by in situ polymerisation

In situ polymerisation allows the formation of microcapsules containing water-immiscible dispersed phase, with improved mechanical properties [19] and thermal stability [20]. The properties of the membrane depend not only on its chemical structure but also on all the synthesis conditions. The polycondensation of the amino resin occurs in the continuous phase, and the phase separation is linked to the pH and the formaldehyde/melamine molar ratio. Among various shell materials presumably impermeable amino resins, especially melamine–formaldehyde resins play a main role in the patent literature [21]. Melamine–formaldehyde microcapsules (MF) were prepared by selecting different antimicrobial oils as core materials using an in situ polymerisation process. The microcapsules obtained were characterised to evaluate their properties as well as the effect of their incorporation into different footwear materials. The microencapsulation process by in situ polymerisation consists of two main steps. In the first one the melamine-formaldehyde prepolymer is obtained from the individual monomers. In a second step, the oil core is emulsified; the emulsion properties such as drop size determine the properties of the final microcapsules [20-23]. After that, the melamine formaldehyde prepolymer is added to the emulsion and the amino resin is precipitated on the oil emulsion interface under the correct pH and temperature conditions. As a result, spherical and smooth shell microcapsules containing the antimicrobial oil cores were obtained. The mean particle size of the microcapsules obtained is around 1-5 μm. The microencapsulation of the antimicrobial oils was determined by the resin/oil ratio [24-25]. For small resin/oil ratios, 1:1 and 2:1, the precipitation of the resin did not occur during the microcapsule formation process, so the oil was not entrapped into the shell (Fig. 2A). As the resin/oil ratio increased, resin/oil ratios from 3:1 to 7:1, microcapsules with a perfect spherical morphology and smooth surface were obtained (Figs.2B and 2C).

A B C

Fig. 2 Images of MF microcapsules containing TTO as the core material with resin/oil ratios 1:1, 4:1 and 6:1 respectively. Therefore, the encapsulation efficiency improved as the resin/oil ratio increased, as well as the stability and mechanical properties of the shell (Fig.3) [26].

Fig. 3 Microencapsulation efficiency as a function of resin/oil ratio.

16.1622.35

45.32

58.74

68.73

14.97

0

1020

30

40

5060

70

80

EE (%

)

2:1 3:1 5:1 4:1 6:1 7:1


© FORMATEX 2013

____________________________________________________________________________________________

105

This fact indicated that the release of oil from the microcapsules was easier for the lower resin ratio, according to a lower degree of degradation observed. Shoe wearing implies several conditions related to the foot environment, such as the pressure distribution, temperature, humidity, pH, etc, which might affect the release profile of the encapsulated substance from the microcapsules in the active shoe. For a long lasting and optimum effect of the encapsulated substance, the study of these parameters and its influence on the controlled release as well as the durability of the microcapsules were analysed. Therefore, the influence of foot sweat on the microcapsules shell properties was evaluated [27], since it is constituted by a mixture of different salts which could modify the amino resin shell permeability. In this study, microcapsules synthesised by in situ polymerisation were treated with an artificial sweat solution prepared according to a standardised procedure [28]. After the treatment, the physicochemical properties of the nanocapsules were characterised by different techniques such as size distribution measurements, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and scanning electronic microscopy (SEM). The physicochemical properties of the amino resin in the presence of artificial sweat were modified, and therefore their morphology changed (Fig.4) and the aggregation increased.

A B

Fig. 4 Electronic microscopy images of the MF microcapsules: A) before and B) after the artificial sweat treatment. The artificial sweat affected the morphology of the microcapsules with lower resin/oil ratio. They appeared to deflate due to the oil release from the microcapsules core after sweat treatment. Microcapsules with higher resin/oil ratios were not apparently affected by the artificial sweat. According to the thermogravimetric analysis results, the changes in physicochemical properties might be attributed to ionic interactions between the emulsifier (sodium dodecyl sulphate, SDS) and the components of artificial sweat (sodium chloride, urea, tris(hydroxymethyl)amino methane and nitrilotriacetic acid) and mainly salts. The melamine-formaldehyde microcapsules containing an antimicrobial oil core showed a sweat responsive behaviour depending on the resin/oil mass ratio content of the shell. As the resin/oil mass ratio increased, the microcapsules were more resistant to the sweat effect as well as more resistant to breaking by pressure, avoiding the oil release by this mechanism which should be the most suitable in wearing conditions. The antimicrobial properties of the melamine formaldehyde microcaspules were determined by in vitro and in vivo assays. Firstly, in vitro assays in solid media were done to analyse the antimicrobial properties of the melamine-formaldehyde microcapsules containing TTO against four bacteria typically present in feet and used footwear (Escherichia coli SG13009 (QIAGEN), Bacillus subtilis 168 ATCC 23857, Klebsiella pneumoniae CECT 141 and Staphylococcus aureus CECT 239).The evaluation of the inhibition halos assay was carried out by seeding medium plates for each type of bacteria. A cloth disc impregnated with the melamine formaldehyde microcapsules containing TTO was placed on them, as well as a control one with no antimicrobial substance. After 24h of growth, the inhibitory halos were measured. The results showed that the cloth discs impregnated with melamine-formaldehyde microcapsules with TTO core showed an inhibitory effect for K. pneumoniae and S. aureus. In both cases there was an inhibition halo around the impregnated disc that was not on the control one. In addition, further in vivo studies were conducted, which were aimed to evaluate the antimicrobial activity of the melamine-formaldehyde microcapsules containing different antimicrobial essential oils (tea tree oil, lemon oil, clove oil, chamomile oil and neem oil) incorporated in footwear components [29]. Healthy subjects recruited from within INESCOP staff carried out controlled wear trials using two different kinds of shoes, men’s and women’s styles, both models were sandals (Fig.5A). For such shoes, insoles made of split leather were coated with the microcapsules containing the different essential oil mixture emulsions and left to dry at room temperature. The antimicrobial activity wear trial was carried out as follows: Firstly, the selected shoes were sterilised in an ozone chamber. Then, the different microencapsulated antimicrobial formulations were applied as a coating on the insole material, only in the left shoe, with a brush, and left to dry at room temperature. The shoes were worn by the recruits for an average total of 50h over 7 days. After that, antimicrobial assays were performed with two different mediums, Tryptone Soy Agar (TSA) and Mannitol Salt Agar (MSA). Sampling was performed by pressing and rubbing the surface of the Rodac® contact plate


© FORMATEX 2013

____________________________________________________________________________________________

106

filled with the different culture media against the treated and untreated insole, with the samples being taken as close as possible to the metatarsal area (forefoot area). The plates were immediately incubated at 32 °C overnight. Finally, the microbial growth was evaluated by colony counting means (Figs. 5B and 5C).

A B C

Fig. 5 Antimicrobial assays by means of in vivo wear trials with footwear incorporating antimicrobial microcapsules. A) Sampling by pressure; B) Untreated sample; C) Oil treated sample.

As a conclusion, a different inhibition effect of the microorganisms was observed depending on the kind of essential oil and the microencapsulation process used. For the majority of the samples studied, on the plates corresponding to the untreated shoe, the growth of the microorganisms was higher than that of the shoe treated with antimicrobial oil microcapsules. Lemon and TTO oil based microencapsulated formulations seemed to be the most effective, as they provided higher inhibition results.

3.2. Microencapsulation of natural antimicrobial oils by complex coacervation

Instead of the outstanding properties of the melamine-formaldehyde microcapules, the current tendency to reduce or avoid the use of synthetic materials led to the authors using biodegradable polymers such as gelatine and carboxymethylcellullose (CMC) to produce green and sustainable microcapsules by a complex coacervation process as an alternative to synthetic microcapsules such as melamine-formaldehyde ones. Microencapsulation by the complex coacervation process is based on the phase separation which takes place spontaneously in an aqueous medium when two or more colloids bearing opposite charges (polycation and polyanion) are mixed in the presence of an active principle dispersion [30-31]. Usually, a protein and a polysaccharide are used, such as gelatine and CMC. Gelatine was the first shell-forming material used in microencapsulation and, nowadays, it remains a significant potential material. As mentioned, the use of biopolymers, such as gelatine, for the creation of the capsule wall makes it possible to obtain natural and biodegradable microcapsules as an alternative to other artificially produced substances deriving from synthetic polymers, like melamine-formaldehyde, polyurethane, or polystyrene. Gelatine is an amphoteric protein (presents positive charge at pH values below its isoelectric point (pI), and negative charge at higher pH values) which is derived from collagen and turns out to be very adequate for the coacervation owing to its special configuration that facilitates the occlusion of a considerable amount of water [32]. The CMC presents negative charge over the entire pH range. As a consequence, at pH below its pI, gelatine is positively charged and interacts with the CMC molecules, with which it produces a neutralisation of charges and a desolvation of the polymeric mixture, which separates into a liquid phase referred to as a complex coacervate. The microencapsulation of natural antimicrobial oils by the complex coacervation method consisted of several fundamental stages. Firstly, the emulsification of the essential oil was completed in an aqueous gelatine solution at 50ºC, where a 5 wt% CMC solution was added. Then the formation of the coacervate was induced through the reduction of the pH with 10 wt% acetic acid. Following this, the system was cooled to 5-10ºC and the coacervated gelated capsule was hardened by adding a crosslinker like formaldehyde, glutaraldehyde, glyoxal, etc. The temperature was maintained at 5-10ºC for 2h after the addition of the hardener. After that, the pH had to be increased to alkaline conditions for 1h to favour the crosslinking process. Previous studies carried out by the authors aimed to microencapsulate several natural antimicrobial oils, such as neem, tea tree, calendula, limonene oil, etc., among others, by complex coarcervation. Fig. 6 shows as an example the typical morphology of the microcapsules obtained by a complex coacervation process. It is an oil core surrounded by a gelatineous shell forming a spindle-shaped microcapsule.

During the microencapsulation by a complex coacervation process, pH plays an important role because it affects the formation of protein-carbohydrate complexes (coacervate) by influencing the degree of ionisation of the functional group of protein (amino group) and carbohydrate (carbonyl group). In a mixture containing an anionic polysaccharide and a protein, adjustment of pH below the pI would result in maximum electrostatic attraction as the two biopolymers are carrying opposite charges and therefore facilitate the microcapsule production. The relative amount of colloids and shell-forming polymers to core ratio, among others parameters, also determines the microencapsulated oil yield. The


© FORMATEX 2013

____________________________________________________________________________________________

107

shell-forming polymer ratio (gelatine-CMC ratio, as an example) has a large effect on the coacervate formation and also on the encapsulation efficiency [33], as it is shown in the Table 2.

A B

Fig. 6 A) Optical and B) SEM micrographs of typical microcapsules obtained by complex coacervation. Table 2 Effect of gelatine-CMC ratio on the encapsulation efficiency of Tea Tree Oil by complex coacervation, as an example.

Gelatine-CMC ratio Encapsulation Efficiency (%)

6,5 18,7 7,9 61,4 10,0 63,3 13,6 48,9 21,4 12,8

The hardening process of the gelatine-based microcapsules obtained by complex coacervation has been also studied. Due to the fact that the complex coacervation method is determined by temperature (gelatine melting) and pH conditions (at which coacervation occurs), the hardening process must be optimum for such pH values [34]. The crosslinking process of the gelatine-CMC microcapsule shell using more sustainable hardeners such as glutaraldehyde, glyoxal, metallic salts or vegetable tannins to crosslink gelatine-based shell as an alternative to formaldehyde was evaluated [35]. Metallic hardeners and vegetable tannins did not harden enough the shell at the pH at which the coacervation process takes place (pHcoacervation), so when pH>pHcoacervation the coacervate was destroyed and microcapsules were not obtained. However, aldehyde-based hardeners crosslinked the coacervate enough to maintain its structure under alkaline conditions, improving the crosslinking degree. The use of glutaraldehyde enhanced the thermal resistance of the microcapsules in a similar way to formaldehyde (Fig. 7A) and it seemed to be the best alternative to formaldehyde for effectively hardening gelatine-CMC microcapsules.

Fig. 7 DTA thermograms corresponding to gelatine-CMC microcapsules crosslinked with:

A) Different hardeners after annealing treatment; B) Glutaraldehyde at different pH values after annealing treatment. Due to the fact that the pH is an important parameter to obtain an optimally crosslinked shell, the influence of pH on the crosslinking efficiency using glutaraldehyde was undertaken. For such a purpose, the crosslinking efficiency at different pH values was evaluated by thermal gravimetric analysis (TGA) after an annealing treatment at 40ºC for 30 min Fig. 7B shows the corresponding thermograms. In addition, the solubility in water of the crosslinked coacervate at 50ºC was determined, resulting in a decrease in the solubility of the coacervate when it is crosslinked at higher pH values.

1000x

0 100 200 300 400 500 600Temperature (ºC)

227ºC268ºC

376ºC

pH=4.4

pH=6

pH=7

pH=10

microencapsulated oilshell

206ºC 261ºC

193ºC

Firs

t der

ivat

e (d

m/d

t)

0 100 200 300 400 500 600Temperature (ºC)

shell-forming

microencapsulated Tea Tree

239ºC

275ºC

water retention and free-oil

formaldehyde

glutaraldehyde

glioxal

Firs

t der

ivat

e (d

m/d

t)


© FORMATEX 2013

____________________________________________________________________________________________

108

Glutaraldehyde is an effective hardener for gelatine microcapsules alternative to formaldehyde and its efficiency as a crosslinker greatly depends on pH values, the optimum results being obtained for neutral or alkaline conditions. This is due to the fact that glutaraldehyde polymerises at neutral or slightly alkaline pH values to form α,β-unsaturated aldehyde polymers which increase in length as the pH is raised. In this study it was confirmed that those reactive species are responsible for the crosslinking of protein-based shells, improving the thermal resistance of the microcapsules. Finally, the antimicrobial effect of gelatine-CMC containing an antimicrobial oil such as TTO was evaluated against the strain of Klebsiella pneumoniae [36]. The microcapsules were subjected to different oil release conditions, such as mechanical rupture and artificial sweat treatment using glass pearls under vigorous agitation. As a result, regardless of the releasing mechanism used, the microcapsules containing TTO oil showed a remarkable antimicrobial effect. This effect could be due to the presence in the medium of a residual amount of formaldehyde (a strong biocide) which was used as a crosslinking agent during the hardening of microcapsules; and therefore, it could mask the antimicrobial effect of the TTO. After evaluating the effect of 300 ppm of formaldehyde, the amount of residual formaldehyde in the microcapsule slurry, a huge inhibition of Klebsiella pneumoniae growth was observed. After that, in order to evaluate the only antimicrobial effect of the microencapsulated TTO, microcapsules without a crosslinker and the shell-forming polymer, gelatine-CMC complex (coacervate), were prepared and their antimicrobial effect was evaluated. The coacervate did not inhibit the Klebsiella pneumoniae growth at all; meanwhile, the TTO microcapsules without a crosslinker produced a 100% inhibition rate (UFC/mL). This data allowed the confirmation of the antimicrobial effect of the microencapsulated oil and its potential use to obtain active footwear materials with an added value.

3.3. Application of microencapsulated biocides in footwear materials

The incorporation of the synthesised microcapsules containing natural antimicrobial oils into two reference materials, commonly used as lining or insole materials in shoemaking (split leather and polyester textile) was evaluated in order to produce functional materials. Fig. 8 show SEM images of the microencapsulated biocides, obtained by in situ polymerisation and complex coacervation respectively, attached to the leather-fabric fibres.

A B C D

MF microcapsule - leather fibers MF microcapsules - fabric fibers GC microcapsules - leather fibers GC microcapsules - fabric fibers

Fig. 8 SEM images of the materials treated with MF (A, B) and GC microcapsules (C, D) containing a natural antimicrobial oil. In order to predict the mechanical behaviour of the microcapsules anchored to those footwear materials under shoemaking and wearing conditions, different standardised physical tests for footwear materials such as rubbing and ironing at different temperatures were undertaken. A rubbing fastness test was selected to simulate friction stresses produced during shoe wearing which can favour the rupture of the microcapsules and, therefore release the oil. Moreover, ironing fastness and also rubbing fastness tests can also evaluate the durability of the microcapsules during shoemaking process. Some of the steps of the manufacturing process are carried out under high pressure, high temperature and humidity conditions, such as heat setting, toe and heel lasting, etc. Therefore functional materials including microcapsules should be resistant under the strictest footwear manufacturing conditions. After the completion of these tests, the samples were qualitatively assessed by scanning electron microscopy (SEM). Fig.9 shows some examples of SEM images obtained from split leather and polyester that had been modified with microcapsules containing a natural essential oil after being subjected to different physical tests. According to the results, melamine-formaldehyde microcapsules showed higher mechanical resistance than gelatine-CMC microcapsules. This might be due to a lower mean particle size as well as a different chemical nature of the polymeric shell and different crosslinking degree. In addition, these results were corroborated by shoe manufacturing trials using different leathers and textiles, used as lining and insole materials, including melamine-formadehyde and gelatine-CMC microcapsules containing natural antimicrobial oils. After manufacture, samples of such materials from different parts of the shoe (toe area, mid-foot and rear area) were analysed by scanning electron microscopy. The results showed that the durability of the microcapsules applied to the lining material depends on the type of shell material of the microcapsule, the average particle size and area of the shoe where they were incorporated. Eg. gelatine microcapsules applied to the lining material only withstood the manufacturing process conditions in the mid-foot area. The GC microcapsules applied to the toe and heel areas were


© FORMATEX 2013

____________________________________________________________________________________________

109

completely destroyed during the process. However, the melamine-formaldehyde microcapsules, that had smaller particle sizes, exhibited greater durability and resistance, being resistant to temperature and pressure conditions of the whole manufacturing process. On the contrary, the microcapsules applied to the insole materials were in any case resistant to the process conditions, regardless of the microcapsule type.

I A II A III A

I B II B III B

Fig. 9 SEM images of the footwear materials treated with GC (A) and MF (B) microcapsules containing a natural antimicrobial oil after different physical tests: I) Rubbing fastness test; II) Ironing fastness test at 120ºC; III) laundering fastness test.

In conclusion, the antimicrobial oil release of gelatine microcapsules will be proposed by shell rupture by rubbing during footwear wearing in contact with the foot. As controlled release systems for long-lasting natural antimicrobial oils will be employed, the use of melamine-formaldehyde microcapsules is proposed. In this case, the antimicrobial oil release occurs through a mechanism of diffusion through the polymeric shell favoured by the temperature, humidity and sweat conditions in the shoe which affect the shell microcapsule permeability.

4. Silver@silica nanocomposites as antimicrobial agents

Current studies carried out by the authors aim to develop coated leathers with enhanced antimicrobial properties, thus providing a solution to avoid the problems mentioned above. To achieve this objective, nanosilver colloidal solutions were synthesised using different reducing agents such as borohydride [37], gelatine/glucose [38] and an aloe vera phenolic extract [39]. The first one acts as a control of the others, which are considered greener methods. In all cases, the formation of AgNPs was further determined by using the UV–visible spectroscopy as a function of time. The UV-visible spectrum of the AgNPs showed a well-defined absorption band at 396 nm attributed to the phenomenon of plasmon resonance characteristic of the AgNPs, which may be related to an average size of 10-20 nm, which is in agreement with TEM measurements (Fig. 10A). Fig. 10B shows the AgNP lattice fringes as an evidence of their high crystallinity [40], the interplanar spacing being of about 0.25 nm, that corresponds to a cubic crystal arrangement.

A B C D

Fig. 10 TEM micrographs corresponding to: A) AgNPs colloidal solution; B) AgNPs lattice fringes; C) AgNPs@silica nanocomposites; D) SEM micrograph corresponding to leather fibers treated with the AgNPs@silica nanocomposites.


© FORMATEX 2013

____________________________________________________________________________________________

110

Furthermore, in order to improve nanoparticle stability and avoid their aggregation, AgNPs@silica nanocomposites were synthesised by a modified Stöber method [41]. The antimicrobial properties of a novel silver-silica nanocomposite were evaluated as an antimicrobial additive for leathers and compared to AgNPs antimicrobial properties. A transmission electron micrograph of the AgNPs@silica nanocomposites is shown in Fig.10C. The nanocomposite consists of aggregate silica matrix particles where AgNPs nanoparticles are dispersed and embedded within the matrix. Synthesised AgNPs and Ag@silica nanocomposites were applied to different leathers to obtain antimicrobial coatings. Treated leathers studied include conventional (chrome, aluminium), and novel metal-tanned leather (titanium, oxazolidine), in order to verify the effectiveness of free and encapsulated silver nanoparticles in both types of leather with ecological potential [42-43]. For that purpose, leather samples were treated with the corresponding silver solutions by applying 50 μL of the solution on the grain, and 50 μL on the flesh sides of the leather, allowing them to dry at least for 24h at room temperature. In order to verify the presence of the AgNPs and AgNPs@silica nanocomposites on the leather matrix, SEM analysis was performed on both the grain and flesh sides of the leather and also, with the leather matrix, on the side of cryofractured leather samples, to test nanoparticles and nanospheres penetration (Fig.10D). The antimicrobial activity of the treated leathers was tested by means of liquid and solid (agar diffusion) antibacterial tests against both Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae). All the different treated leather samples showed a strong antibacterial activity in liquid media. In the agar diffusion test, titanium and aluminium tanned leathers showed also a synergistic effect along with silver on B. subtilis survival (Fig.11). As a conclusion, AgNPs, and specially AgNPs@silica nanocomposites represent a suggestive alternative to create leather with enhanced antifungal and antimicrobial properties [44]. Some advantages of the AgNP@silica nanocomposites are the dispersion of the discrete Ag nanoparticles throughout the silica matrix (which prevents agglomeration of the silver particles), the small diameter of the silver particles (which results in a large surface area and release of a large amount of Ag+, which results in high antimicrobial efficiency), and the small size of the AgNPs@silica nanocomposite (which allows the material to be uniformly dispersed and readily incorporated into leathers but also into a variety of substrates, including synthetic fibres, coatings, plastics, etc.). A further advantage is that the immobilisation of silver nanoparticles within the silica structure limits the potential for release and disposal of the nanoparticles themselves. This property may be highly desirable because of the possible abilities of nanoparticles to go through biological membranes and other barriers.

Fig. 11 Petri dish showing the antimicrobial effect of tanned leather samples treated with silica-coated AgNPs against Bacillus Subtilis.

5. Conclusions

The footwear sector is currently working on improving all factors that influence comfort, lifetime or malodour of the product. However, all of these features require the use of materials with new performance and functionalities as well as a thorough understanding of the way in which the components react to these changes. The new footwear materials should be able to withstand temperature and humidity fluctuations as well as heavy wear and abrasion. In this sense, INESCOP is working on the development of innovative antimicrobial materials based on advanced technologies such as microencapsulation and nanotechnology. A different inhibition effect of the microorganisms was observed depending on the kind of biocide and the microencapsulation process used. For the majority of the samples studied, Gram positive bacteria seem to be the most sensitive ones towards antimicrobial essential oils and biocides based on Ag, as they show higher inhibition results (Table 3).


© FORMATEX 2013

____________________________________________________________________________________________

111

Table 3 Inhibition effect of the different biocides studied.

Acknowledgements The research described in this chapter has been undertaken within several projects partially supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the Institute for Small and Medium Industry of the Generalitat Valenciana (IMPIVA) through its R&D Program for Technological Institutes and the European Regional Development Fund (ERDF).

References

[1] K. Ara, M. Hama, S. Akiba, K. Koike, K. Okisaka, T. Hagura, T. Kamiya, F. Tomita. Foot odour due to microbial metabolism and its control. Canadian Journal of Microbiology. 2006;52 (4):357–364.

[2] Dwijendra Singh, T.R.S. Kumar, Vivek K. Gupta, Pushplata Chaturvedi. Antimicrobial activity of some promising plantoils, moleecules and formulations. Indian Journal of Experimental Biology. 2012;50:714-717.

[3] Gallucci, N; Oliva, M , Carezzano, E, Zygadlo, J; Demo, M. Terpenes antimicrobial activity against slime producing and non–producing staphylococci. Molecular Medicinal Chemistry. 2010; 21:132-136.

[4] R. R. Thanighai arassu Balwin Nambikkairaj P. Sivamani. Chemical Composition of four Essential Oils by GC-MS and their Antifungal Activity Against Human Pathogenic Fungi. Indian Journal of Applied Research. 2013;3(3):375-379.

[5] Leopold Jirovetz, Gernot Eller, Gerhard Buchbauer, Erich Schmidt, Zapriana Denkova, Albena S. Stoyanova, Radosveta Nikolova, Margit Geissler. Chemical composition, antimicrobial activities and odor descriptions of some essential oils with characteristic floral-rosy scent and of their principal aroma compounds. Recent Research Developments in Agronomy & Horticulture. 2006; 2: 1-12.

[6] Lansdown, A.B. Silver. 1. Its antibacterial properties and mechanism of action. Journal of Wound Care. 2002;11:125–130. [7] Zhao G, Stevens SE. Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver

ion. Biometals. 1998;11:27–32 [8] Jones SA, Bowler PG, Walker M, Parsons D. Controlling wound bioburden with a novel silver-containing Hydrofiber dressing.

Wound Repair and Regeneration Journal. 2004;12(3):288-94. [9] Catauro M, Raucci MG, De Gaetano FD, Marotta A. Antibacterial and bioactive silver-containing Na2O_CaO_2SiO2 glass

prepared by sol-gel method. Journal of Materials Science: Materials in Medicine. 2004;15(7):831–837. [10] Zhang L, Yu JC, Yip HY, Li Q, Kwong KW, Xu A, Wong PK. Ambient light reduction strategy to synthesize silver

nanoparticles and silver-coated TiO2 with enhanced photocatalytic and bactericidal activities. Langmuir. 2003;19:10372–10380. [11] Pal S, Tak YK, Song JM. Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A

study of the Gram-negative bacterium Escherichia coli. Applied and Environmental Microbiology. 2007;73:1712–1720. [12] N. Cuesta Garrote, M.M. Sánchez Navarro, F. Arán Aís and C. Orgilés Barceló. Natural antimicrobial agents against the

microbiota associated with insoles in Science and Technology against Microbial Pathogens. Research, Development and Evaluation. Antonio Mendez-Vilas, eds. WorldScientific;2011.

[13] E. Deligianni, S. Pattison, D. Berrar, N.G. Ternan, R.W. Haylock, J.E. Moore, S.J. Elborn, J.S.G. Dooley. Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro. BMC Microbiology. 2010;10:38.

[14] Reichling J., Schnitzel P., Suschke U, Saller R. Essential oils from aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties-an overview. Forch Komplementärmed. 2009;16:79-90.

[15] Syed TA, Qureshi ZA, Ali SM, Ahmad S, Ahmad SA. Treatment of toenail onychomycosis with 2% butenafine and 5% Melaleuca alternifolia (tea tree) oil in cream. Tropical Medicine and International Health. 1999;4:284-287.

[16] Sadlon A. E., Lamson D. W. Inmune modifying and antimicrobial effects of eucalyptus oil and simple inhalation devices. Altenative Medicine Review. 2010;15(1): 33-47.

[17] Ghosh SK. 2006. Functional Coatings by Polymer Microencapsulation”. WILEY-VCH Verlag GmbH & KGaA, Weinheim. [18] Benita, S, ed. Microencapsulation: Methods and Industrial Applications. New York: Marcel Dekker, Inc.;1996. [19] Sun G, Zhang Z. Mechanical properties of melamine-formaldehyde microcapsules. Journal of Microencapsulation.

2001;18(5):593-602. [20] K. Dietrich, E. Bonatz, H. Geistlinger, H. Herma, R. Nastke, HJ.Purz. Amino resin microcapsules II. Preparation and

morphology. Acta Polym. 1989;40(5):325-31. [21] Dietrich K, Herma H, Nastke R, Bonatz E, Teige W, Amino resins microcapsules. I. Literature and patent review. Acta Polym.

1989;40(4):243-51. [22] K. Dietrich, E. Bonatz, H. Herma, R. Nastke, W. Teige. "Amino resin microcapsules IV. Surface tension of the resins and

mechanisms of capsule formation. Acta Polym. 1990;41(2):91-95.

Essential oils microencapsulated biocides

++

MF (Lemon)

++

MF (TTO + Neem)

Use test (in vivo)

-

-

+

++

Lemon oil

-

-

+

-

Neem oil

+

+

++

++

Tree Tea oil

Inhibitory halos assay (in vitro)

Essential oils biocides

++K. pneumoniae

++E. coliGram -

++S. aureus -

++++B. subtilisGram +

Gelatine (TTO)AgNPsSiO2AgNPs


Ag based biocides Essential oils microencapsulated biocides

++

MF (Lemon)

++

MF (TTO + Neem)

Use test (in vivo)

-

-

+

++

Lemon oil

-

-

+

-

Neem oil

+

+

++

++

Tree Tea oil


Essential oils biocides

++K. pneumoniae

++E. coliGram -

++S. aureus -

++++B. subtilisGram +

Gelatine (TTO)AgNPsSiO2AgNPs


Ag based biocides

++: high inhibitory effect; +: inhibitory effect; -: no significant inhibitory effect


© FORMATEX 2013

____________________________________________________________________________________________

112

[23] HY Lee, SJ Lee, W Cheong, JH Kim. Microencapsulation of fragrant oil via in situ polymerisation: Effects of pH and melamine-formaldehyde molar ratio. Journal of Microencapsulation. 2002;16(5):559-69.

[24] M. M. Sánchez Navarro, F. Payá Nohales, F. Arán-Ais, C. Orgilés Barceló. Polymer Shell Nanocapsules Containing a Natural Antimicrobial Oil for Footwear Applications. Progress in Colloid and Polymer Science. 2012; 139:73-77.

[25] M. Magdalena Sánchez Navarro, Natalia Cuesta-Garrote, Francisca Arán-Ais, César Orgilés-Barceló. Microencapsulation of Melaleuca Alternifolia (Tea Tree) oil as Biocide for Footwear Applications. Journal of Dispersion Science and Technology. 2012;32:1-6.

[26] M. Sánchez Navarro, F. Arán Aís, C. Orgilés Barceló. Microencapsulación de aceite esencial de eucalipto para su aplicación en calzado. V Congreso Jóvenes Investigadores en Polímeros. Calella Palafrugell (Gerona, Spain), 26 May 2010.

[27] M Sanchez, F Payá, F. Arán, C. Orgilés. Sweat responsive nanocapsules for footwear applications. Congress Particles 2011. Berlin (Germany), 11-12 July 2011.

[28] Standard ISO 11641 - Leather -- Tests for colour fastness -- Colour fastness to perspiration [29] M. Sánchez Navarro, M. Ángeles Pérez Limiñana, M. Bertazzo, Francisca. Arán-Ais, César Orgilés Barceló. Antimicrobial

effect of microencapsulated essential oil mixtures in footwear. II International Conference on Antimicrobial Research. Lisboa (Portugal), 20-23 November 2012.

[30] Zhijian D; Yong M; Khizar H. Morphology and release profile of microcapsules encapsulating peppermint oil by complex coacervation. Journal of Food Engineering. 2011;104(3):455-460.

[31] Nakagawa k, Nagao H. Microencapsulation of oil droplets using freezing-induced gelatine-acacia complex coacervation. Colloid Surface A. 2012;411:129-139.

[32] Fakirov S, Bhattacharyya D. Munich, eds. Handbook of engineering biopolymers: homopolymers, blends and composites. Germany: Hanser; 2010.

[33] Perez-Limiñana MA, Arán-Aís F, Payá-Nohales FJ, Orgilés-Barceló C. Effect of the shell-forming polymer ratio on the encapsulation of Tea Tree Oil by complex coacervation as natural biocide. Journal of Microencapsulation. TMNC-2013-0018.R1. In press

[34] M. A. Pérez Limiñana, F. Arán Ais, C. Orgilés Barceló. Influence of pH on the cross-linking efficiency of glutaraldehyde in the microcapsules obtained by complex coacervation. Third International Symposium Frontiers in Polymers Science. Sitges (Barcelona), 21-23 May 2013.

[35] M. A. Pérez Limiñana, F. Arán Ais, C. Orgilés Barceló. Use of more sustainable cross-linkers for gelatine-CMC microcapsules as an alternative to formaldehyde. Third International Symposium Frontiers in Polymers Science. Sitges (Barcelona), 21-23 May 2013.

[36] M.A. Pérez-Limiñana, N. Cuesta-Garrote, M. Sánchez-Navarro, F. Arán-Aís, C. Orgiles-Barceló. A natural biocide microencapsulated by complex coacervation. II International Conference on Antimicrobial Research (ICAR2012). Lisboa, 21-23 November 2012.

[37] J.A. Creighton, C.G. Blatchford and M.G. Albrecht. Journal of the Chemical Society, Faraday Transactions 2: Molecular and Chemical Physics. 1979;75:790-798.

[38] M.B. Ahmad, J.J. Lim, K. Shameli, N.A. Ibrahim and M.Y. Tay. Synthesis of Silver Nanoparticles in Chitosan, Gelatin and Chitosan/Gelatin Bionanocomposites by a Chemical Reducing Agent and Their Characterization. Molecules, 2011;16(9):7237-7248.

[39] Y. Zhang, D. Yag, Y. Kong, X. Wang, O. Pandoli and G. Gao. Synergetic Antibacterial Effects of Silver Nanoparticles@Aloe Vera Prepared via a Green Method. Nano Biomedicine and Engineering. 2010;2(4):252-257.

[40] D- Philip. Biosynthesis of Au, Ag and Au–Ag nanoparticles using edible mushroom extract. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2009;73:374-381.

[41] W. Stöber, A. Fink. Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science. 1968;26:62-69.

[42] M. Roig, V. Segarra, M.A. Martinez, M. Bertazzo, J. Ferrer. Oxazolidine metal – free leather. Leather International. 2012:16-20.

[43] M. Bertazzo, D. Poveda, A. Albert, N. García–Gras, V. Segarra–Orero, M. Roig, M.A. Martínez–Sánchez. System for biodegradability evaluation on leather used in the footwear industry. Journal of AQEIC. 2012;63(3):61-69.

[44] M. Isabel Maestre-López, Federico J. Payá-Nohales, Francisca Arán-Ais, Miguel A. Martínez- Sánchez, César Orgilés-Barceló, Marcelo Bertazzo. Antimicrobial effect of coated leather based on silver@silica nanocomposites. Imagenano–NanoBio&Med 2013. Bilbao, 23-26 April 2013.


© FORMATEX 2013

____________________________________________________________________________________________

113

Documents

Latest Developments in Antimicrobial Functional Materials for