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Elsevier Editorial System(tm) for Ciência & Tecnologia dos Materiais Manuscript Draft Manuscript Number: CTMAT-D-14-00005R1 Title: Bioactive microsphere-based coating for biomedical-textiles with encapsulated antimicrobial peptides (AMPs) Article Type: Biomaterials Special Issue Keywords: Antimicrobial peptides; antimicrobial textiles; medical textiles; Microspheres Corresponding Author: Dr. Isabel Gouveia, PhD Corresponding Author's Institution: Univ Beira Interior First Author: Laura Antunes, MSc Order of Authors: Laura Antunes, MSc; Graça Faustino; Claudia Mouro, MSc; Joana Vaz; Isabel Gouveia, PhD Abstract: In this study a sonochemical method was used to prepare chitosan-based-micro/nanoparticle with incorporated antimicrobial-peptides as a novel antimicrobial coating for cotton-gauzes. Characterization in terms of size, morphology and stability was evaluated and microspheres were approximately 2 µm in-size and were further coated by the layer-by-layer deposition of alginate (Alg) and chitosan (CS). In addition, an antimicrobial-peptide Dermicidin-1-L was incorporated into the microspheres (CS/AMP microspheres) to give more effective antimicrobial activity against a wider range of microorganisms. Results showed a much higher antimicrobial activity of the CS/AMP-microsphere-coated-cotton in comparison with LbL-Alg/CS-microsphere-coating, without cytotoxicity. Suggested Reviewers: Opposed Reviewers: Response to Reviewers: Subject: Revision of manuscript Ref. No.: CTMAT-D-14-00005 Title: Bioactive microsphere-based coating for biomedical-textiles with encapsulated antimicrobial peptides (AMPs) Dear Editor, We would like to thank you for giving us the chance to revise the paper. As a consequence, the manuscript was revised and detailed responses were given to the questions raised. We greatly acknowledge the comments, suggestions and questions and the careful review that the Editor and Reviewers have provided. The paper was significantly improved thanks to your contributions. We really hope that the changes made will able this manuscript to be published In Ciência e Tecnologia dos Materiais, giving the originality and the importance in open new avenues for cellulose-based materials application in health-care. Looking forward to hearing your final decision. Sincerely yours, Isabel C. Gouveia

Response to reviewers: Reviewers' comments: Reviewer #1: 1.This work is about the development of bioactive microsphere-based coating for biomedical textiles. In this context the authors should define the concept "Bioactive". The reviewer may be right about the confusion of the word and we have remove it from the title and when appropriate. Bioactive in this study case means antimicrobial. 2. Giving the importance of antibacterial and cytotoxicity analysis, and to support the discussion, additional information, such as microscope images and pH evolution/behaviour (if results are available), should be presented. There is a wide range of methods available to examine the interaction of microorganisms with textiles. Several testing methods are published on the qualitative and quantitative evaluation of antimicrobial activity of textiles. Qualitative test methods are used widely for evaluation of bacteriostatic activity and include procedures such as measurement of the zone of inhibition for evaluation of samples treated with antimicrobials. Quantitative test methods are used to evaluate the bactericidal activity of textile materials by measuring the reduction in bacterial numbers when contacted by test samples under defined conditions. The methods described in AATCC-100, AATCC-147 and JIS L 1902 appears to be the most commonly employed. In the effort to move toward global standardization, the Japanese Standard JIS L 1902 has been revised and harmonized with the European Standard and International Standard EN ISO 20743. This standardization reflects the opinions of multiple countries and will help to promote the development of textile products with antibacterial functions that can be used around the world. In the review of the literature it was found that for the qualitative tests antibacterial AATCC 147 and JIS L 1902-Halo method, no differences were observed between them. Concerning the two quantitative methods, AATCC 100 and JIS L 1902-Absorption method, the results showed that the JIS L 1902 method is more sensitive to the amount of antimicrobial agent than the AATCC 100 test (Askew, 2009; Pinho, Magalhaes, Henriques & Oliveira, 2011). Therefore in this work the antibacterial activity of cotton samples was evaluated using the antibacterial test JIS L 1902:2002, and cytotoxicity using E DIN EN ISO 10993-5 which are recommended by the renowned German Hohenstein institute. 3.The adhesion force of the microspheres onto the fibers should be discussed more deeply. Tables 1, 2 and 3 are established from microscope images. They should be Figures not Tables! and should be bigger. When it is reported: "To author´s knowledge…" should the meaning be "To authors' knowledge..."? All exponent numbers are wrongly represented. Some References are not correctly indicated (see 2.3.3) Avoid expressions like: "The adhesion of the microspheres onto the fibbers was evaluate by the optical microscope"! … was analysed by optical microscopy? Explanations like "The agglomeration is in agreement with … because the small particles tendency to form agglomerate" are weak/not acceptable. There are many word mistakes along the text (Misrospheres, trough, …). Finally, the English writing of the manuscript should be improved.

We have provided all the changes as requested by the reviewer. Reviewer #2: Comments to the author: The authors present a method to prepare a novel antimicrobial coating for cooton-gauzes. Although the results are very intersting and, besides few small writing mistakes that should be avoided (and that the authors will easily identify), in order to improve the manuscript I would suggest the following revisions: 1. Experimental details Some more details are needed in the experimental part, mainly related with the techniques that have been used: A - The authors claim ( in 2.3.5) that "the size and morphology of microspheres were characterized by SEM and an inverted microscopy" Was it a fluorescence? The size and the morphology of the microspheres were observed under a HITACHI S2700 scanning electron microscopy (SEM) and an inverted microscopy OLYMPUS CKX 41 which are not fluorescence ones. 2.How was evaluated the size distribution? Only by SEM? Laser particle size analyser? The size distribution was analysed by SEM only since we don´t have a zeta-sizer available, but the software of the SEM equipment give very accurate results. 3. A - it is not clear and must be explicited how the authors evaluated the microsphere stability - for example in paragraph 2.3.6 the authors claim "the stability of microspheres was assessed by following the changes in microspheres..." Changes were provide to better elucidate, as you can see in the revised manuscript. 4. B - how was monitored the process of layers deposition? How was done the characterization of alternate deposit layers? By flow cytometry analysis? We haven’t analyse this by flow cytometry since it was not the aim of this study. The purpose of LbL assembly on microspheres aimed to provide stronger shells and this was accomplished with success. 5. 2 - Results a. My main objection is in the way how the authors presented the micrographs - all the micrographs shown in Tables 1, 2 and 3 must as Figures and not included as Tables. Besides the legends of the micrographs should be very clear and the magnification used in each micrograph has to be clear in the picture. It is a pity that we will be not able to notice the details that the authors want to point out. We have provided all the changes as requested by the reviewer. b. In paragraph 3.2.1. the authors say: " In all conditions studied for the production of CS microspheres, they have shown very low stability. ... In contrast, the microspheres produced under the optimum conditions are very stable, in solution, at all pH..." I don,t understand quite well what the authors mean. They must rewrite the paragraph in order to make it more clear. We have provided all the changes as requested by the reviewer.

Dear Editor

Please find enclosed the REVISED manuscript entitled “Bioactive microsphere-based

coating for biomedical-textiles with encapsulated antimicrobial peptides (AMPs)” for

consideration for publication in journal of Ciência e Tecnologia dos Materiais.

The study is totally new and innovation lies on the use of antimicrobial peptides

incorporated in microrpheres as a novel bioactive process to give antimicrobial

properties using very low concentrations of the antimicrobial agents. It is, of course,

the Editor and the referee’s choice to decide the originality and importance of the

manuscript. We greatly acknowledge the comments and recommendations.

Kind regards

Isabel Gouveia

PhD – Textile Engineering/Biotechnology

FibEnTech research unit, University of Beira Interior

6201-001Covilhã - Portugal

Cover Letter

Subject: Revision of manuscript Ref. No.: CTMAT-D-14-00005

Title: Bioactive microsphere-based coating for biomedical-textiles with encapsulated

antimicrobial peptides (AMPs)

Dear Editor,

We would like to thank you for giving us the chance to revise the paper. As a consequence, the

manuscript was revised and detailed responses were given to the questions raised. We greatly

acknowledge the comments, suggestions and questions and the careful review that the Editor

and Reviewers have provided. The paper was significantly improved thanks to your

contributions.

We really hope that the changes made will able this manuscript to be published In Ciência e

Tecnologia dos Materiais, giving the originality and the importance in open new avenues for

cellulose-based materials application in health-care.

Looking forward to hearing your final decision.

Sincerely yours,

Isabel C. Gouveia

*Detailed Response to Reviewers

Response to reviewers:

Reviewers' comments:

Reviewer #1:

1.This work is about the development of bioactive microsphere-based coating for biomedical

textiles. In this context the authors should define the concept "Bioactive".

The reviewer may be right about the confusion of the word and we have remove it from the

title and when appropriate. Bioactive in this study case means antimicrobial.

2. Giving the importance of antibacterial and cytotoxicity analysis, and to support the

discussion, additional information, such as microscope images and pH evolution/behaviour

(if results are available), should be presented.

There is a wide range of methods available to examine the interaction of microorganisms with

textiles. Several testing methods are published on the qualitative and quantitative evaluation

of antimicrobial activity of textiles. Qualitative test methods are used widely for evaluation of

bacteriostatic activity and include procedures such as measurement of the zone of inhibition

for evaluation of samples treated with antimicrobials. Quantitative test methods are used to

evaluate the bactericidal activity of textile materials by measuring the reduction in bacterial

numbers when contacted by test samples under defined conditions.

The methods described in AATCC-100, AATCC-147 and JIS L 1902 appears to be the most

commonly employed. In the effort to move toward global standardization, the Japanese

Standard JIS L 1902 has been revised and harmonized with the European Standard and

International Standard EN ISO 20743. This standardization reflects the opinions of multiple

countries and will help to promote the development of textile products with antibacterial

functions that can be used around the world. In the review of the literature it was found that

for the qualitative tests antibacterial AATCC 147 and JIS L 1902-Halo method, no differences

were observed between them. Concerning the two quantitative methods, AATCC 100 and JIS L

1902-Absorption method, the results showed that the JIS L 1902 method is more sensitive to

the amount of antimicrobial agent than the AATCC 100 test (Askew, 2009; Pinho, Magalhaes,

Henriques & Oliveira, 2011).

Therefore in this work the antibacterial activity of cotton samples was evaluated using the

antibacterial test JIS L 1902:2002, and cytotoxicity using E DIN EN ISO 10993-5 which are

recommended by the renowned German Hohenstein institute.

3.The adhesion force of the microspheres onto the fibers should be discussed more deeply.

Tables 1, 2 and 3 are established from microscope images. They should be Figures not

Tables! and should be bigger.

When it is reported: "To author´s knowledge…" should the meaning be "To authors'

knowledge..."?

All exponent numbers are wrongly represented.

Some References are not correctly indicated (see 2.3.3)

Avoid expressions like: "The adhesion of the microspheres onto the fibbers was evaluate by

the optical microscope"! … was analysed by optical microscopy?

Explanations like "The agglomeration is in agreement with … because the small particles

tendency to form agglomerate" are weak/not acceptable.

There are many word mistakes along the text (Misrospheres, trough, …).

Finally, the English writing of the manuscript should be improved.

We have provided all the changes as requested by the reviewer.

Reviewer #2: Comments to the author:

The authors present a method to prepare a novel antimicrobial coating for cooton-gauzes.

Although the results are very intersting and, besides few small writing mistakes that should

be avoided (and that the authors will easily identify), in order to improve the manuscript I

would suggest the following revisions:

1. Experimental details

Some more details are needed in the experimental part, mainly related with the techniques

that have been used:

A - The authors claim ( in 2.3.5) that "the size and morphology of microspheres were

characterized by SEM and an inverted microscopy" Was it a fluorescence?

The size and the morphology of the microspheres were observed under a HITACHI S2700

scanning electron microscopy (SEM) and an inverted microscopy OLYMPUS CKX 41 which are

not fluorescence ones.

2.How was evaluated the size distribution? Only by SEM? Laser particle size analyser?

The size distribution was analysed by SEM only since we don´t have a zeta-sizer available, but

the software of the SEM equipment give very accurate results.

3. A - it is not clear and must be explicited how the authors evaluated the microsphere

stability - for example in paragraph 2.3.6 the authors claim "the stability of microspheres

was assessed by following the changes in microspheres..."

Changes were provide to better elucidate, as you can see in the revised manuscript.

4. B - how was monitored the process of layers deposition? How was done the

characterization of alternate deposit layers? By flow cytometry analysis?

We haven’t analyse this by flow cytometry since it was not the aim of this study. The purpose

of LbL assembly on microspheres aimed to provide stronger shells and this was accomplished

with success.

5. 2 - Results

a. My main objection is in the way how the authors presented the micrographs - all the

micrographs shown in Tables 1, 2 and 3 must as Figures and not included as Tables.

Besides the legends of the micrographs should be very clear and the magnification

used in each micrograph has to be clear in the picture. It is a pity that we will be not

able to notice the details that the authors want to point out.


b. In paragraph 3.2.1. the authors say: " In all conditions studied for the production of CS

microspheres, they have shown very low stability. ... In contrast, the microspheres produced

under the optimum conditions are very stable, in solution, at all pH..."

I don,t understand quite well what the authors mean. They must rewrite the paragraph in

order to make it more clear.


Available online at

Ciência & Tecnologia dos Materiais

MBioactive microsphere-encapsulated antimicrobial peptides (AMPs)

Laura Antunesa, Graça Faustinoa FibEnTech – Fiber R&D Unit of Textile and Paper Materials

Abstract

In this study a sonochemical method was used to prepare chitosanpeptides as a novel antimicrobial coating for cottonevaluated and microspheres were approximately 2 µm in(Alg) and chitosan (CS). In addition, an antimicrobialmicrospheres) to give more effective antimicrobial activity against a wider range of microorganisms. Results showed a much higher antimicrobial activity of the CS/AMP-microspherewithout cytotoxicity. © 2013 Sociedade Portuguesa de Materiais (SPM). Published by Elsevier España, S.L. All rights reserved

Keywords: Antimicrobial peptides; antimicrobial textiles; medical textiles; Microspheres

1. Introduction*

Recently, several methods depending on the active agent and type of fiber have been developed or are being developed to confer antimicrobial activity to textiles. The antimicrobial agents can be applied to the textile substrates by exhaustion techniqueexhaustion, impregnationng, coating, spraying, microencapsulation and foams. However, desired bioactive antimicrobial properties related to the effectiveness of these application methods are not always achieved with a high success rate. Thus, new developments and processes are expected around the world [1,2]. The area of functional fibers and technical textiles has encouraged industry to use microencapsulation processes as a mean of transmitting properties and finishes for textiles, which would not be profitable

* Corresponding author.

E-mail address: [email protected]

Available online at www.sciencedirect.com

Ciência & Tecnologia dos Materiais 25 (2013) 01–80

http://ees.elsevier.com/ctmat

Special Issue Biomaterials

-based coating for biomedical-textiles with encapsulated antimicrobial peptides (AMPs)

Graça Faustinoa, Claúdia Mouroa, Joana Vaza, Isabel C. Gouveia

Materials and Environmental Technologies Research Center,,University of Beira Interior, Adress, 6201-001Covilhã, Portugal

method was used to prepare chitosan-based-micro/nanoparticle with incorporated antimicrobialpeptides as a novel antimicrobial coating for cotton-gauzes. Characterization in terms of size, morphology and stability was

ximately 2 µm in-size and were further coated by the layer-by-layer deposition of alginate (Alg) and chitosan (CS). In addition, an antimicrobial-peptide Dermicidin-1-L was incorporated into the microspheres (CS/AMP

imicrobial activity against a wider range of microorganisms. Results showed a much microsphere-coated-cotton in comparison with LbL-Alg/CS-microsphere

Published by Elsevier España, S.L. All rights reserved

Antimicrobial peptides; antimicrobial textiles; medical textiles; Microspheres

Recently, several methods depending on the active agent and type of fiber have been developed or are being developed to confer antimicrobial activity to textiles. The antimicrobial agents can be applied to the

techniquess of , coating, spraying,

microencapsulation and foams. However, desired properties related to the

effectiveness of these application methods are not always achieved with a high success rate. Thus, new developments and processes are expected around the

The area of functional fibers and technical textiles has aged industry to use microencapsulation

processes as a mean of transmitting properties and finishes for textiles, which would not be profitable

using other techniques. Microcapsules have been used in several areas such as adhesives, cosmetics, pesticides, pharmaceuticals, medicine, food, etc. it is only for a few years that they have beenrecently introduced in textile area [controlled release of bioactive substances encapsulated in polymeric microspheres and microcapsules deposited on textiles may be a new strategy that opens new perspectives for applications of textiles. The sonochemical method developed by Suslick and co-workers [6] can be used to produce micrometersized gas-or liquid-filled proteinaceous microspheres from various kinds of proteins. This oneprocedure yields microspheres with a long shelf life and high stability. Ultrasound offers the prospect of an escalation of reaction rates, improved yields, or a better quality product due to the betterhomogenation of the constituent chemicals In addition, the ability to readily tailor the properties (e.g., size, composition, porosity, stability, surface

http://ees.elsevier.com/ctmat

textiles with

, Isabel C. Gouveiaa*

,University of Beira Interior,

micro/nanoparticle with incorporated antimicrobial-gauzes. Characterization in terms of size, morphology and stability was

layer deposition of alginate L was incorporated into the microspheres (CS/AMP

imicrobial activity against a wider range of microorganisms. Results showed a much microsphere-coating,

using other techniques. Microcapsules have been used in several areas such as adhesives, cosmetics,

pharmaceuticals, medicine, food, etc. bBut they have been more

[3-5]. Therefore, controlled release of bioactive substances encapsulated in polymeric microspheres and

ed on textiles may be a new strategy that opens new perspectives for applications

The sonochemical method developed by Suslick and can be used to produce micrometer-

filled proteinaceous microspheres ious kinds of proteins. This one-step

procedure yields microspheres with a long shelf life and high stability. Ultrasound offers the prospect of an escalation of reaction rates, improved yields, or a

the better improved nation of the constituent chemicals [7,8].

In addition, the ability to readily tailor the properties (e.g., size, composition, porosity, stability, surface

Formatted: English (U.K.)

*Manuscript

Author name / Ciência & Tecnologia dos Materiais 25 (2013) 01–80

functionality, colloidal stability), capsules prepared by the layer-by-layer (LbL) technique have also attracted particular interest. These capsules allow the introduction of multiple functionalities, thus providing opportunities to engineer a new class of materials with unprecedented structure and function and can be assembled from suite materials, including synthetic and natural polyelectrolytes, nanoparticles and bio-macromolecules. Moreover, various bioactive compounds can be sequestered into the capsule for drug delivery, sensing, or catalysis applications, and capsule surface can be modified to alter the functionality and/or improve the colloidal stability of the capsules [9,10]. The polyelectrolyte multilayer coatings may serve multiple purposes such as stabilizing alginate hydrogels against dissolution in biological environments and providing barrier membranes for alginate hydrogels to slow release of encapsulated drug or biomolecules [11]. On the other hand, many of the antimicrobials used as finishing agents for textile materials are toxic which means that there is a growing public concern about the possible effects of antimicrobial finishes in environmental and biological systems. Thus, the antimicrobial finishing of textiles should be able to destroy undesirable microorganisms, as well as be safe and environmentally friendly world [1,2,12]. A wide range of extracts and natural products with antimicrobial properties has been reported during the last years, due to the increased multidrug resistance of many human pathogenic microorganisms as well as the appearance of undesirable side-effects of certain antibiotics [12]. In accordance to this, antimicrobial peptides (AMPs) [13-15] and natural bioactive compounds [2,12] have been recently reported as new candidates to overcome the disadvantages of the currently used in use antibiotics and synthetic biocides. Consequently, considerable efforts have been expended to exploit the therapeutic potential of AMPs, especially regarding the pharmaceutical industry [14]. Moreover, because of the membrane-disturbing mode of action of most AMPs, there is a reduced likelihood of the acquisition of resistance by bacteria [13-15]. In this, way, the association between chitosan and AMPs may be suitable for further enhancing the antimicrobial properties of chitosan and/or other antimicrobial polymers and increase the action against a wide broader spectrum of microorganisms. Consequently, a new approach that is developed in this study aimed the to investigateion of the more suitable conditions for surface functionalization of

textile materials with potential use as wound-dressings, with embedded bioactive microspheres embedded, capable of providing a controlled antimicrobial action increased by the AMPs in order to prevent microbial infections. Therefore, this study aims to investigate the development of chitosan-based microspheres using the sonochemical and LbL techniques in order to produce more stable and resistant microspheres to be used in the functionalization of textile materials. More important, the embedment of an antimicrobial peptide (AMP) was analyzed to assess the increase and effectiveness of the microsphere-based coating as an antimicrobial method for textiles. To author’ s knowledge, this is the first report associating the latest innovation in terms of new biocides, i.e. AMPs and textiles, in particular a new finishing method based on chitosan-AMPs microsphere-coating for textiles as a potential for the design of new and non-toxic bioactive textiles with a high effectiveness of antibacterial activity, showing no cytotoxicity. 2. Experimental

2.1. Materials and reagents

Terephthalic Acid (98%), Sodium Hydroxide (> 98%), Phosphate Buffer pH 7.4, Chitosan (low molecular weight), Alginic Acid sodium salt, Acetic Acid glacial (95%), Dodecane (≥ 90%), Sodium Chloride (≥ 99.5%) was purchased from Sigma-Aldrich. AMP Dermicidin was chosen due to the anionic charge and the present application in this study, with potential as a wound-dressing. Therefore, Dermcidin DCD – 1L (from AnaSpec) was purchase to Genentec (Brussels). The textile materials used were 100% cotton gauzes and were selected due to their common use in medical textiles.

2.2. Microorganisms

The microorganisms used in all assays were clinical multiresistant species of Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) as described in the international standard JIS L 1902-2002.

2.3. Methods

Formatted: Font: Italic



2.3.1. Minimal Inhibition Concentration of DMD-1L

The minimal inhibitory concentration (MIC) against multiresistant (clinical) Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) was determined using the broth macrodilution method, as described by CLSI M7-A6 standard method (International standard CLSI M7-A6). The choice of using clinical species aimed to assess the effectiveness against multiresistant bacteria. According to the guidelines, the minimal inhibitory concentration was determined by serial dilution in Mueller-Hinton Broth (MHB) (Sigma-Aldrich, St. Louis, MO) with concentrations of DCD-1L ranging from 0.2 µg/mL to 8 µg/mL. The inoculums were prepared from fresh overnight liquid cultures and the turbidity was adjusted to 0.5 McFarland (approximately 1x108 CFU/mL) with 0.85% (w/v) NaCℓ, and then diluted to give a final concentration of 1x105 CFU/mL. 1mL of inoculum was added to each tube containing 1 mL of antimicrobial agent in the dilution range prepared from a stock solution of 10 µg/mL. The inoculated macrodilution tubes were incubated for 24 hours (h) at 37 ⁰C to assess antibacterial activity. Control tubes of the medium, broth and broth with DCD-1L were also incubated.

2.3.2. Preparation of the Chitosan MisrospheresMicrospheres

Chitosan microspheres were synthesized sonochemically from aqueous solutions of chitosan. Concentration ranged from 0.20 to 1.0 mg/mL. The n-dodecane was used as the co-solvent to make the emulsion which is necessary for microsphere formation. Temperature ranged from 20, 25, 30, 35 and 40 ⁰C, and sonication time from 5, 10, 15, 20, 25 and 30 min. sonicated with 32 kHz. It was also tested an AMP (DCD-1L, 5.0 µg/mL. highest MIC value) dissolved in the CS solution at the optimum conditions described below, in order to evaluate the possibility of incorporating bioactive molecules into the microsphere which, in this case, may provide a higher antimicrobial effect against a larger spectrum of microorganisms. After the synthesis, the mixture solution was stored at 4ºC during 12 hours to able the separation of all phases (dodecane, microspheres and chitosan or chitosan/AMP solution).

2.3.3. LBL Self-Assembly of Alg/CS layers on Chitosan Microspheres

LbL deposition of polyelectrolytes over the microsphere shell was assessed to evaluate the influence on microsphere stability overtime and in storage conditions. The polyelectrolyte solutions used for alternating deposition of micro layers of Alg/CS on the CS microspheres were prepared at concentrations of 1 mg/mL For the LbL deposition alginate was dissolved in an aqueous solution of 0.5M sodium chloride as described elsewhere 16 and Chitosan was dissolved in aqueous solution of 0.1 M acetic acid. The pH values were adjusted to 3 using HCℓ 0.1 M and NaOH 0.1 M solutions. For the deposition of each layer, 900 µL of polyelectrolyte solution was added to a test-tube containing 100 µL of microsphere suspension and stirred for 10 min on a magnetic stirrer. Afterwards they were washed with 400 µL of desioniated water, and stirred on a magnetic stirrer for 5 min. This process was repeated (2-6 layers) using the oppositely charged polyelectrolyte until expected multilayer pattern was obtained, where chitosan was used as polycation and alginate as polyanion. The layer sequence was (Alg/CS)n. as described by LiuShao and co-workers [10] and illustrated in figure 1[11].

Fig. 1. Chitosan-template polyelectrolyte multilayer microcapsules loaded with Alginate (Adapted from Liu and co-works [11]).

2.3.4. Functionalization of the cotton gauzes

Optimum conditions, namely: 3.9 mL of CS solution (0.8 mg/L) and 2.6 mL of n-dodecane or 5 µg/mL AMP dissolved in the CS solution, in the case of CS-AMP microspheres, were used to produce and bind simultaneously the microspheres onto textile materials (50 mg), which were immersed in the solution, before sonication, and sonicated for 10 min at 37 ⁰C. For the case of the LbL CS/Alg coated CS microspheres, the cotton gauzes (50 mg) were immersed into a test-tube with microsphere suspension prepared in the above described optimum conditions, in a proportion to obtain a solution volume of 6.5 mL

LbL Assembly

chitosan alginate

Chitosan microsphere

Core-Shell Microparticle




(5.85 mL polyelectrolyte and 0.65 mL microspheres) and was sonicated with 32 kHz for 10 min at 37 ⁰C.

2.3.5. Characterization of microspheres

The size and the morphology of the microspheres were observed under a HITACHI S2700 scanning electron microscopy (SEM) and an inverted microscopy OLYMPUS CKX 41. SEM samples were prepared by the application of a drop of microspheres suspension onto the glass and then dried at room temperature (20 ± 2 ⁰C); which were further coated with a thin layer of sputtered gold in a gold EMITECH-K550 evaporator. The samples for the inverted microscopy were prepared by the application of a drop of microspheres suspension in a plate and observed under a microscope.

2.3.6. Measurement of the Stability of Chitosan Microspheres and LBL Chitosan Microspheres

The stability of the chitosan microspheres, CS/AMP microspheres and LbL Alg/CS coated chitosan microspheres, was assessed by following the changes in the microspheres which were monitored under different temperatures (-5, 5, 20, 35, 37 and 40 ⁰C), taking into account the possible conditions of storage (-5, 5 and 20 ⁰C) and the possible contact with human body (37 and 40 ⁰C), in case of wound-dressings. Stability to pH (4.5, 7 and 9.5) was also evaluated due to the different pH of the skin. Stability in all conditions was monitored for 30 days.

2.3.7. Assessment of antibacterial activity by the JIS L 1902-2002 Halo-method

Antibacterial activity of LbL2-Alg/CS and CS/AMP microspheres, the more stable microspheres produced, as well as controls (unfunctionalized cotton gauzes), was tested against two bacterial multiresistant clinical strains - a Gram positive strain Staphylococcus aureus (ATCC 6538) and a Gram negative strain Klebsiella pneumoniae (ATCC 4352), adapted from the Japanese Industrial Standard JIS L Standard 1902:2002 (Testing for antibacterial activity and efficacy on textile products). Bacterial inoculums were prepared from an overnight liquid culture in Nutrient Broth and incubated at 110 rpm, 37 ⁰C for 24 h. Bacterial concentrations were then adjusted to 1-2x108 cel/mL (equivalent to 0.5 McFarland) and working standards were prepared to a final concentration of 1±0.3 x105 CFU/mL. The

cotton samples coated with the different microspheres were placed in a 50 mL Falcon tube and 200 µL of the working standards previously prepared was added. The T24h tubes were incubated for 24 h at 37 ⁰C and the T0h samples were analyzed. To release the bacterial cells from the cotton samples, before and after the 24 hours incubation period, 20 mL of 0.85 % NaCℓ with surfactant Tween80 (0.2 % (v/v)) was added to the samples in 50 mL The resulting suspensions were diluted in sterile 0.85 % sodium chloride solution (1:10; 1:100; 1:1000) and plated to determine the viable counts. The plates were incubated at 37 ⁰C for 24 h, and the number of colonies was determined. This procedure was performed in triplicate. The growth reduction rate of the bacteria was calculated using the equation (Eq. 1)

��

�� 100% ��%� (1)

where, T0h is the CFU/mL of bacterial colonies at the initial stage (0h) and T24h is the CFU/mL of bacterial colonies after 24 h incubation.

2.3.8. Evaluation of cytotoxicity

This procedure was performed according to E DIN EN ISO 10993-5. Initially, it was prepared a perspiration extract of the CS/AMPs bioactive microsphere-coated cotton gauzes, which presented the major inhibition of bacterial growth. The bioactive material was incubated with an acid perspiration solution, for 24 h at 37 ⁰C under slight shaking. The resulting perspiration extract was set up to a pH value of 7.3–7.4 with sodium hydroxide and filtrated in sterile conditions. A cell culture of Connective tissue cells L 929 [ATCC No. CCL1, NCTC clone 929 L (DSMZ), was treated for 68–72 h with the perspiration extract diluted about 33.3% – 4.4%. To confirm the validity of the test system, controls were carried out along the experiment. Control tests made included: solvent control (phosphate buffered solution diluted in culture medium corresponding to the test material), positive control (5% DMSO in culture medium) and negative control (culture medium). Then, the test group of the material presented concentrations of the test material in culture medium of 4.4%, 6.6%, 9.9%, 14.8%, 22.2%, and 33.3%. After the incubation period, the protein content of the test groups cultures (Culture medium: DMEM with 10% FBS) was compared with the protein content of the controls and from that the cell growth was determined in the presence of the test material. In the presence of cell-toxic substances there is a modified proliferation and partition rates of the cells (growth inhibition test).

Author name / Ciência & Tecnologia dos Materiais 25 (2013) 01

3. Results and discussion

3.1. Determination of the minimal inhibitory concentration of DCD-1L

The results showed a MIC for Klebsiella pneumoniaeof 2.92 µg/mL and for Staphylococcus aureusµg/mL. Therefore, the 5.00 µg/mL (5x10-5concentration was used in the preparation of the CS/AMPs microspheres. The different MIC for the two strains can be due to the fact that most anionic and amphipathic antimicrobials act easier against against Gram negative bacteria (pneumoniae) because of the composition of Gram negative outer membrane, most possible through hydrophobic interaction with the phospholipids of the membrane. The results are as expected very important since these MIC values (5x10-5 % (w/v) in the case K.pneumoniae) are significantly lower in comparison with others used as antimicrobial agents for textiles, as for example for chitosan (0.05% (w/v) against aureus and K. pneumoniae) and for triclosan (6 % (w/v) against S. aureus) [16-17].

3.1.1. Production and characterization of the Chitosan Microspheres

Chitosan microspheres were prepared by sonication (Ultrasons-H, Selecta) in optimal conditions at frequency of the 32 kHz, for 10 min. The optimum conditions were found to be, 3.9 mL of chitosan 0.8 mg/mL dissolved in aqueous solution of 1 M acetic acid placed in a test-tube with 2.6 mL dodecane, to give a total volume of 6.5 mL, which was also found to be the best amount in the equipment used (data not shown). The results showed that at temperature of 37⁰C sonicated for 10 min, occurred the higher formation of CS microspheres, i.e., ± 1 mL. With regards with the sonication time, results showed that the best time was 10 min, where microspheres resulted stable for many hours. In the above described conditions, the CS microspheres have spherical shape and a size about 2 µm, as it can be seen in the inverted microscopyimages and SEM images presented in figureThus they have the shape and size that are in accordance with the previous results obtained by Shao and co-workers [10] with the advantage of being obtained in a much faster and simple method than the reported by Agnihotri and co-workers[18] who studied the preparation of chitosan nanoparticles by emulsion


Determination of the minimal inhibitory

Klebsiella pneumoniae Staphylococcus aureus of 5.00

5 % (w/v) concentration was used in the preparation of the

The different MIC for the two strains can be due to the fact that most anionic and amphipathic antimicrobials

inst Gram negative bacteria (K. ) because of the composition of Gram

negative outer membrane, most possible through hydrophobic interaction with the phospholipids of the

The results are as expected very important since these 5 % (w/v) in the case

) are significantly lower in comparison with others used as antimicrobial agents for textiles, as for example for chitosan (0.05% (w/v) against S.

) and for triclosan (6 %

Production and characterization of the

Chitosan microspheres were prepared by sonication H, Selecta) in optimal conditions at

frequency of the 32 kHz, for 10 min. The optimum conditions were found to be, 3.9 mL of chitosan 0.8 mg/mL dissolved in aqueous solution of 1 M acetic

tube with 2.6 mL dodecane, to lume of 6.5 mL, which was also found

to be the best amount in the equipment used (data not shown). The results showed that at temperature of 37 C sonicated for 10 min, occurred the higher

formation of CS microspheres, i.e., ± 1 mL. With onication time, results showed that

the best time was 10 min, where microspheres resulted

In the above described conditions, the CS microspheres have spherical shape and a size about 2 µm, as it can be seen in the inverted microscopy

figuretable 1. Thus they have the shape and size that are in accordance with the previous results obtained by Shao

with the advantage of being obtained in a much faster and simple method than the

who studied the preparation of chitosan nanoparticles by emulsion

cross-linking or by Shao and co-workers [10]ultrasonication required 30 min to generate monodisperse CS microspheres with 1 µm size and spherical shape, still a little more time in comparison of the 3 minutes reposted by Shimanovihworkers [4], probably due to the difference in using BSA instead of CS, that can provide protein crosslinking through SS bonds. Therefore, in comparison with other methods which used CS as the primer polymer for the microsphere shell formation, the results obtained are promising since they show microspheres with size ± 2 µm through a simple and fast process (only 10 minutes sonication). Table 1. Micrographs of the different produced microspheres. CS/AMP LbL4 Alg/CS

IMnv

erte

d m

agni

ficat

ion

100

0x

mic

rosc

ope

SE

M

Mag

nific

atio

n 40

00x

Figure 1. Micrographs of the different produced

microspheres (IM- inverted microscopy and SEM)

3.1.2. LbL deposition of Alg/CS nanolayers on Chitosan Microspheres

The morphology of microspheres with deposition of Alg/CS layers was characterized by inverted and SEM. Inverted microscope images show that it appears that the microspheres of chitosan with LBLwell, spherical and the layers are deposited on the shell of the CS microspheres as shown in the figure Observing the SEM images (figuretablethe microspheres with LbL layers have spherical shape and monodispersitivity of about 1 µm in size, although it was also visible some microspheres with nonshape most probably because the microsphere cores were totally removed when completely dried before testing in SEM analysis, resulting in microcapsules with shrunken surfaces as reported by Xie and co-workers

workers [10] whereas ultrasonication required 30 min to generate monodisperse CS microspheres with 1 µm size and

l shape, still a little more time in comparison Shimanovih and co-

, probably due to the difference in using BSA instead of CS, that can provide protein

er methods which used CS as the primer polymer for the microsphere shell formation, the results obtained are promising since they show microspheres with size ± 2 µm through a simple and fast process (only 10 minutes

the different produced

CS

Figure 1. Micrographs of the different produced

inverted microscopy and SEM).

Alg/CS nanolayers on

The morphology of microspheres with deposition of Alg/CS layers was characterized by inverted and SEM. Inverted microscope images show that it appears that the

LBL4 (Alg/CS) are, as ell, spherical and the layers are deposited on the shell of

figuretable 2. table 2) it appears that

layers have spherical shape of about 1 µm in size, although it

was also visible some microspheres with non-spherical shape most probably because the microsphere cores were totally removed when completely dried before testing in SEM analysis, resulting in microcapsules with shrunken

workers [19].

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Table 2. Images of the cross-linking and agglomeration effect of the LbL6 Alg/CS microspheres (inverted microscopy and SEM).

CS/AMP LbL4 Alg/CS

IM

Mag

nific

atio

n 10

00x

nve

rted

SE

M

Mag

nific

atio

n 10

00x

FigureTable 2. Images of the cross-linking and agglomeration effect

of the LbL6 Alg/CS microspheres (IM- inverted microscopy and

SEM).

Chitosan microspheres with LbL (Alg/CS)6 presented some problems such as cross-linking, as visible in figuretable 3 due to the agglomeration promoted by the solutions of CS and Alg for the LbL deposition. However, cross-linking of chitosan microspheres was expected, in this case, because it’s in agreement with the results obtained by Mei and co-workers [20]. CS microspheres took the shape of irregular long strips interconnecting with each other, which is typical for macromolecular structure because of the straight chain. The agglomeration observed is in agreement with results of Genç and co-workers [21] because the small particles tendency to form agglomerate. The results show the success of the LbL assembly of the polyelectrolytes on the CS microsphere shell as expected, increasing the thickness of the membrane and the stability of the microspheres, as shown in the stability assays discussed below.

3.1.3. Chitosan Microspheres with AMPs

Chitosan microspheres with AMP DCD-1L were sonicated using the same optimal conditions studied for CS microspheres. The images acquired of CS/AMP microspheres with AMPs in the inverted microscope and SEM are shown in figuretable 1. The microspheres present a spherical shape and monodispersivity, being in agreement with the previous results where CS solutions were used instead of CS/AMP solution, because they present the same size (2 µm). Pedro and co-workers [22] studied the entrapping of citronella oil in chitosan microspheres produced by the emulsions technique, with different particles sizes, ranging from 11 ± 3 µm to 225 ± 24 µm. In addition, it was also reported that the smallest microparticles showed

the biggest release rate, most probably because they have a larger specific surface area, causing the oil release rate to be faster. Therefore, for certain applications in which a smaller size might be required, this sonication process reveals to be very effective.

3.2. Stability of the microspheres

3.2.1. CS microspheres

In all the preliminary assays conditions studied for the production of CS microspheres, they have shown very low stability. For the different concentrations, temperatures and sonication time, microspheres weare stable for only several hours, in the solution (data not shown). In contrast, the microspheres produced under the optimum conditions (at frequency of the 32 kHz, for 10 min, the optimum conditions were 3.9 mL of chitosan 0.8 mg/mL dissolved in aqueous solution of 1 M acetic acid placed in a test-tube with 2.6 mL dodecane, to give a total volume of 6.5 mL) are very stable, in solution, at all pH and all temperature studied for at least 1 month. However, at pH 7 and temperature of 40 ⁰C they present lower stability (only some hours). This is in agreementeing with previous studies performed by Avivi and Gedanken [23]. The microspheres have to stay in solution in order to last several weeks, otherwise, as for example in dry air, they break out very easily (in few hours).

3.2.2. Microspheres with multilayers of Alg/CS and AMP/CS microspheres

The microspheres with 4 and 6 layers of Alg/CS showed high stability at all pH and temperatures for at least 1 month, in solution being in accordance with the stability studies reported by Shao and co-workers [10]. In the case of 6 layers of Alg/CS deposition, it was however observed some interconnection and agglomeration of the microspheres, as is visible in figuretable 2. Therefore, LbL Alg/CS4, with 4 layers of LbL Alg/CS were further investigated concerning the antibacterial activity. Moreover, the microspheres with LbL Alg/CS layers and the CS/AMP microspheres do not break out for one week, when exposed to dry air, which is in agreement with the study of Shao and co-workers [10] being much more stable than the microspheres of chitosan that only last in moisture conditions. In the case of the CS/AMPs this might be due to the ionic binding through the amino and carboxylic groups of the CS and of the peptide, since no LbL deposition

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Author name / Ciência & Tecnologia dos Materiais 25 (2013) 01

was made in this case. In addition, disulfide bondsmay occur between the AMP molecules, as described for L-cysteine-BSA microspheres Gouveia a higher stability to the structure, comparable to the LbL coated CS microspheres. All these results are in agreement with the expected and the LbL deposition has shown to be fundamental to provide microsphere´s stability.

3.2.3. Microspheres-based Coating for Textile Materials

The adhesion of the microspheres onto the fibers was evaluated by optical microscopythe optical microscope. FigureTable 3 shows the AMP/CS microspheres adhered to the fibers tested. Adhesion is visible most probably due to the surface structure of the fiber and the negative charge exhibit by these cellulosic fibers, especially because the outer layer is composed by CS which has an opposite charge to the fiber and may enhance electrostatic interaction.

Table 3. Images of microsphere-based coating on the cotton fibers.

Fibers Microspheres

CS/AMP Microspheres

Alg/CS

Cotton Magnification

1000x

FigureTable 3. Images of microsphere-based coating on

the cotton fibers.

3.2.4. Antibacterial activity

In this work, the antibacterial efficacy of the Alg/CS multilayer’s and CS/AMPs coated cotton samples was assessed against multiresistantby the evaluation of bacterial activity according with qualitative method JIS L 1902-2002. The Control samples (cotton gauze without microspheres) did not present any inhibition, as expected (see Table 1). Quite different were the results presented by the samples coated with Alg/CS multilayers in chitosan microspheres, since inhibition


was made in this case. In addition, disulfide bonds may occur between the AMP molecules, as described

[5] giving a higher stability to the structure, comparable to the

All these results are in agreement with the expected deposition has shown to be fundamental

based Coating for Textile

The adhesion of the microspheres onto the fibers was the optical e AMP/CS

microspheres adhered to the fibers tested. Adhesion is visible most probably due to the surface structure of the fiber and the negative charge exhibit by these cellulosic fibers, especially because the outer layer is

posite charge to the fiber and may enhance electrostatic interaction.

based coating on the cotton fibers.

Microspheres LbL4 Alg/CS

based coating on

In this work, the antibacterial efficacy of the LbL Alg/CS multilayer’s and CS/AMPs coated cotton samples was assessed against multiresistant bacteria by the evaluation of bacterial activity according with

The Control samples (cotton gauze without microspheres) did not present any inhibition, as

. Quite different were the results ted by the samples coated with Alg/CS

multilayers in chitosan microspheres, since inhibition

rates of 28.33% and 29.00% were obtained against pneumoniae and S. aureus, respectively, an antibacterial effect comparable with the qualitative results given by Shimanovih and co-workersno quantitative average was provided.On the other hand, samples treated with chitosan microspheres produced with AMPs showed significant higher inhibition rates against the test specimens revealing that the sonication process and the embedding into CS microspheres doesn’t alter the peptide activity and that a major antibacterial activity is achieved in comparison with microspheres coating of the cotton gauzes. Rates were 75.33% and 99.86% against K. pneumoniaeaureus multiresistant species, respectively, being in agreement with the fact that there is a greater inhibition growth in the case of K. pneumoniaeshown by the MIC values. This is in accordance with the expected due to the release of the AMP, in this case DCD-1L, that is entrapped in the CS layer of the microspheres being released over time, increasing the antibacterial effect of the bioactive textile, in comparison with LbL Alg/CS microspherecoating. These inhibition rates are very high and target multirisistance bacteria with success avoiding their natural to growth when in close contact with this novel CS/AMP microsphere coated gauzes, as it is expected in the case of new biocides as AMPs these samples tested against K. pneumoniaeaureus species can be observed in table

Table 14. Antimicrobial activity of the LbL Alg/CS and CS/AMP coated cotton samples.

control LbL4 Alg/CS

S. aureus

reduction rate (%) 0 28.33

K. pneumoniae

reduction rate (%) 0 29.00

3.2.5. Cytotoxicity analysis

The perspiration extract of the test material (Cotton gauze coated with CS/AMPs microspheres) showed a growth inhibition of 20 % in the cytotoxicity test under the mentioned conditions (see Table growth inhibition of more than 30% in comparison with the solvent control is assessed as a clear celltoxic effect. Therefore, the cytotoxicity test for these novel bioactive cotton gauzes revealed less than 30%

rates of 28.33% and 29.00% were obtained against K. respectively, an

antibacterial effect comparable with the qualitative workers [4] since

ntitative average was provided. On the other hand, samples treated with chitosan microspheres produced with AMPs showed significant higher inhibition rates against the test specimens revealing that the sonication process and the embedding into CS microspheres doesn’t alter the

tivity and that a major antibacterial activity is achieved in comparison with LbL-Alg/CS microspheres coating of the cotton gauzes. Rates were

K. pneumoniae and S. multiresistant species, respectively, being in

th the fact that there is a greater K. pneumoniae, as

shown by the MIC values. This is in accordance with the expected due to the release of the AMP, in this

1L, that is entrapped in the CS layer of the s being released over time, increasing the

antibacterial effect of the bioactive textile, in Alg/CS microsphere-based

coating. These inhibition rates are very high and target multirisistance bacteria with success avoiding their

to growth when in close contact with this novel CS/AMP microsphere coated gauzes, as it is expected

as AMPs [14]. Results of K. pneumoniae and S.

species can be observed in table 14.

Alg/CS and CS/AMP

4 Alg/CS CS/AMP

75.33

99.86

The perspiration extract of the test material (Cotton gauze coated with CS/AMPs microspheres) showed a growth inhibition of 20 % in the cytotoxicity test under the mentioned conditions (see Table 25). A growth inhibition of more than 30% in comparison

the solvent control is assessed as a clear cell-toxic effect. Therefore, the cytotoxicity test for these novel bioactive cotton gauzes revealed less than 30%

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of cellular viability reduction, making CS/AMP-microspheres a safe antimicrobial agent.

Table 25. Cytotoxic effect of the CS/AMP coated cotton samples.

Average of perspiration extracts

Standard deviation

Growth Inhibition (%)

Blanc

Positive control

Negative control

Solvent control

Test material AMP/CS cotton

0.1902

0.1935

1.3504

1.3604

1.2707

± 0.0082

± 0.0310

± 0.0253

± 0.0405

± 0.0502

99

0

0

20

In accordance to this, it can be concluded that no cytotoxic substances are released from the functionalised novel CS/AMP microsphere-based coating process avoiding the risk of irritations with skin contact. Moreover, despite some emerging reports in this area [2-3], none of them clearly indicated a safe and non-toxic effect.

Conclusions

This work describes a novel method to give a CS/AMPs antimicrobial microsphere-based coating for cotton gauzes using non-toxic and biodegradable agents. To the best of author’ s knowledge, this is the first report on the simultaneous formation and coating of textiles through a single-step sonochemical method, incorporating AMPs and giving antibacterial effect against clinical multiresistant bacteria without cytotoxicity. The major advantages of this method in comparison with other techniques that are commonly used to incorporate microspheres/microcapsules onto textile materials, are the non-toxicity both to the potential users and to the environment, and the possibility of being carried out in a simple step process with short reaction time and without using cross-linking agents such glutaraldehyde or epoxy resins that are normally required to produce microspheres or to bind them onto the textile materials. The other advantage is the incorporation of antimicrobial agents such as AMPs, effective against a wide range of microorganisms, inclusive multiresistant bacteria that where other agents failed, and which is a major issue for medical textiles.

Consequently, this can be a very promising strategy that may open new avenues for the design of in situ textile-based antimicrobial delivery systems for skin-contact interaction.

Acknowledgements

The authors would like to thank Fundação para a Ciência e Tecnologia (FCT) for the funding granted concerning the project - PTDC/EBB-BIO/113671/2009 (FCOMP-01-0124-FEDER-014752) Skin2Tex. Also we would like to thank Fundo Europeu de Desenvolvimento Regional (FEDER) through COMPETE – Programa Operacional Factores de Competitividade (POFC) for the co-funding.

References

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