- Email: [email protected]
Developing an antibacterial biomaterial
Accepted Manuscript DEVELOPING AN ANTIBACTERIAL BIOMATERIAL Selda Guler, Emine Erdogan Ozseker, Alper Akkaya PII: DOI: Reference:
S0014-3057(16)30579-1 http://dx.doi.org/10.1016/j.eurpolymj.2016.09.031 EPJ 7502
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
13 June 2016 6 September 2016 18 September 2016
Please cite this article as: Guler, S., Erdogan Ozseker, E., Akkaya, A., DEVELOPING AN ANTIBACTERIAL BIOMATERIAL, European Polymer Journal (2016), doi: http://dx.doi.org/10.1016/j.eurpolymj.2016.09.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DEVELOPING AN ANTIBACTERIAL BIOMATERIAL Selda GULER, Emine Erdogan OZSEKER, Alper AKKAYA Ege University, Faculty of Science, Biochemistry Department, Bornova-Izmir 35100, Turkey
*Corresponding author: Assoc. Prof. Dr. Alper AKKAYA, Ege University, Faculty of Science, Biochemistry Department, Bornova-Izmir 35100, Turkey Tel: +90 232 311 23 67 Fax: +90 232 311 54 85 e-mail: [email protected]
1
ABSTRACT Development and production of antibacterial materials were gained momentum because of the increasing infectional diseases in recent years. Antibacterial products are used in many industries, such as, textile, dye and package industries and medical technology. Short duration antibacterial affect is the main problem for these products. Therefore, the development of materials that exhibit long duration antimicrobial activity, is needed for industry. In this study, the
free
carboxylic
acid
group
of
alginate
were
activated
using
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and amoxicillin (AMX) was covalently immobilized to alginate. AMX, D-α-amino-p-hydroxybenzyl penicillin trihydrate, is one of the most frequently used β-lactam antibiotics that has been employed to treat and reduce the spread of bacterial infections in human, with the benefits of maintaining animal welfare. At the end of the study, an antibacterial biomaterial was developed. The optimum reaction medium, that immobilization of AMX to Ca-alginate beads, was determined as; 1 mg of EDC, pH 5, 50 mM of phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature was 4oC, mixing speed was 150 rpm, initial amount of AMX (IAA) was 4 mg and incubation time 1.5 h. Ca-alginate beads and AMX immobilized Ca-alginate beads were tested for antibacterial affects against Escherichia coli (ATCC 8739, gram negative) and Staphylococcus aureus (ATCC 6538, gram positive)
bacterial strain. According to the SEM images and ATR-FTIR spectrums of AMX
immobilized Ca-alginate and Ca-alginate beads were analyzed, the immobilization process was proved successfully. Developed biomaterial was antibacterial according to antibacterial tests.
Keywords: Alginate, biomaterial, amoxicillin, antibacterial materials 2
1. Introduction Polymers and polymer composites have been widely used in tremendous engineering fields because of their advantages including light weight, good processability, chemical stability in any atmospheric conditions, etc. [1]. For biomedical composites, even though excellent mechanical performance is desirable and often targeted for improvement, the biocompatibility of the material is of paramount concern. The biological compatibility is more important than the mechanical compatibility. Being composed of two or more types of materials, composites carry an enhanced probability of causing adverse tissue reactions [2]. In this study, alginate was used as a support for AMX immobilization. Sodium alginate contains pendant carboxylate and is known as polyanionic copolymer of 1,4-linked-α-Lguluronic acid and β-D-mannuronic acid residues found in brown seaweeds [3]. Alginate is soluble in aqueous solutions and forms stable gels in the presence of certain divalent cations, such as calcium, barium and strontium [4]. The disintegration of the alginate beads happens due to cation exchange, exchange of Ca2+ ions (participating in crosslinking points) with monovalent ions such as Na+ [5]. The gelation and crosslinking of the polymers are mainly achieved by the exchange of sodium ions from the guluronic acids with the divalent cations, and the stacking of these guluronic groups to form the characteristic egg-box structure [6]. The divalent cations bind to the guluronic acid blocks in a highly cooperative manner and the size of the cooperative unit is more than 20 monomers. Each alginate chain can dimerize to form junctions with many other chains and as a result, gel networks are formed rather than insoluble precipitates [7]. Ca-alginate bead is commonly used in the encapsulation process [8]. However, free carboxylic acids, which are situated at Ca-alginate bead, were activated with EDC and AMX was immobilized to Ca-alginate covalently instead of the encapsulation at this process.
3
AMX is a bactericidal antibiotic that belongs to group of penicillin (Fig. 1). It binds to penicillin-binding proteins, and thus interferes with bacterial cell wall synthesis, resulting in the lyses of replicating bacteria [9]. Figure 1 In general, AMX has antibacterial effects on Streptococcus sp., Bacillus subtilis, Enterococcus sp., Haemophilus sp., Helicobacter sp., Moraxella sp., Clostiridium sp. etc. [10]. AMX was immobilized Ca-alginate beads by covalent bounds. Therefore, biocomposite material was developed for against resistant external influences. The unique properties of alginate, combined with its biocompatibility, hydrophilicity and relatively low cost, have made it an important polymer in pharmaceutical applications [11]. Alginate hydrogels can be prepared by various cross-linking methods, and their structural similarity to extracellular matrices of living tissues allows wide applications in wound healing, delivery of bioactive agents such as small chemical drugs and proteins, and cell transplantation. Alginate wound dressings maintain a physiologically moist microenvironment, minimize bacterial infection at the wound site, and facilitate wound healing. Drug molecules, from small chemical drugs to macromolecular proteins, can be released from alginate gels in a controlled manner, depending on the cross-linker types and cross-linking methods. In addition, alginate gels can be orally administrated or injected into the body in a minimally invasive manner, which allows extensive applications in the pharmaceutical arena. Alginate gels are also promising for cell transplantation in tissue engineering. Tissue engineering aims to provide man-made tissue and organ replacements to patients who suffer the loss or failure of an organ or tissue [4]. In this study, alginate molecules were functionalized with AMX and antibacterial product was
4
developed. This Ca-alginate beads can be transformed back to the solution form and application of different materials is possible to achieve a permanent antibacterial product. This antibacterial product can be applied to textile materials and new generation wound dressing, surgical clothes etc. could be developed. It can also be applied to cosmetic materials such as cream, soap etc.
5
2. Materials and methods 2.1. Materials EDC was purchased from Merck; KH2PO4, methanol, AMX, sodium alginate, CaCl2 were purchased from Sigma; nutrient broth, nutrient agar were purchased from Fluka. 2.2. Microorganisms S. aureus (ATCC 6538) and E. coli (ATCC 8739) were used in antibacterial tests. The microorganisms (S. aerous (ATCC 6538), E. coli (ATCC 8739)), were obtained commercially from American Type Culture Collection (ATCC). S. aerous (ATCC 6538) was isolated from human lesion and E. coli (ATCC 8739) was isolated from the feces. 2.3. Preparation of Ca-alginate Beads Sodium alginate was dissolved in distilled water at the final concentration of 2% (w/w) overnight. Ca-alginate beads were prepared by dropping aqueous alginate into a 3% 100 ml calcium chloride solution into ice bath and the form of Ca-alginate beads were allowed to cure for overnight. Ca-alginate beads were washed with distilled water for once and buffer for twice to remove excess Ca2+ and finally stored at +4oC until immobilization process [12]. 2.4. Immobilization of AMX to Ca-alginate Beads The initial reaction medium (IRM), where immobilization of AMX to Ca-alginate beads was performed, was determined as; pH 5, 25 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature was 25oC, mixing speed was 100 rpm, initial amount of AMX (IAA) was 6 mg, incubation time was 1 hour.
6
The reaction medium was prepared as including 5 pieces of Ca-alginate beads and dissolved EDC in 2 ml phosphate buffer solution then incubated for 20 minutes. At the end of the incubation time, 1 ml of dissolved AMX in 4:1 methanol-distilled water solvent system was added to reaction medium and incubated at 25oC and 100 rpm for 1 hour. Ca-alginate beads were separated from reaction medium and washed with 1 ml of 4:1 methanol-distilled water solvent system for 4 times. All washing waters (WW) were collected and put together with IRM. Samples were taken from the medium and absorbance values were measured by spectrophometer (Perkin Elmer UV/Visible Lambda 25, USA) at 272 nm and amount of AMX was determined which was in the reaction medium after immobilization process. The difference between IAA and last was determined the amount of AMX gave the amount of immobilized AMX (AIA) Eq. (1). –
(1)
In this study, Na-alginate was prepared in bead form and immobilization was performed using bead formed Ca-alginate. Ca-alginate beads were predisposed to reaction with EDC. However, the modification method of Ca-alginate beads was varied depending of biomolecule, which was used for immobilization [13, 14]. 2.5. Optimization of AMX Immobilization Amount of EDC (AE), concentration of buffer solution, pieces of Ca-alginate beads, immobilization temperature, mixing speed and IAA parameters were optimized for AMX immobilization as shown in Table 1. Table 1
7
2.6. Antimicrobial Tests In the experiment, antimicrobial effects of Ca-alginate beads, AMX immobilized Caalginate beads and composite materials were tested by Gram positive (S. aerous (ATCC 6538)) bacteria and Gram negative (E. coli (ATCC 8739)) bacteria. Similar to the Disk Diffusion Method (Spread Plate Technique) were used for antimicrobial tests [15]. In Disk Diffusion Method, the sterile disk was impregnated with the antibacterial sample but the AMX was bound to Ca-alginate beads covalently. S. aureus (ATCC 6538) and E. coli (ATCC 8739) were obtained from bacterial stock cultures and incubated slant agar at 37oC overnight. Bacterial cells were obtained from slant agar by sterile loop and incubated in nutrient broth at 37oC, 200 rpm for 24 hours. After incubation, 50 µl culture of antibacterial test microorganisms were inoculated onto nutrient agar and spread with drigalski spatula. Ca-alginate and AMX immobilized Ca-alginate beads were placed in the middle of the petri dish and incubated at 37oC for 24 hours. Antimicrobial effects of the beads were observed after incubation. Ca-alginate and AMX immobilized Ca-alginate beads were stored at 4oC and 25oC to determine the time-dependent antibacterial effects. Antimicrobial tests were done regularly for 4 months. 2.7. Scanning Electron Microscopy Surfaces of Ca-alginate and AMX immobilized Ca-alginate beads were visualized with scanning electron microscope (FEI Quanta 250 FEG) (SEM). 2.8. Structural Analysis by Fourier Transform Infrared Spectroscopy (FT-IR)
8
ATR-FTIR spectrums were obtained with Perkin Elmer Spectrum 100 FT-IR with attenuated total reflection (ATR) equipment (USA). FT-IR analysis was performed on Caalginate and AMX immobilized Ca-alginate beads. Spectrums were obtained the spectral range of 400–4000 cm−1.
9
3. Results and discussion 3.1. Effect of Amount of EDC on Immobilization Carbodiimides have been utilized in a number of different research areas including, for example, modify carboxyl groups in enzymes, peptide synthesis [16, 17], conjugation of proteins in structural studies [18, 19] quantification of carboxyl moieties in proteins [20], and coupling reactions in an anticalcification treatment for porcine bioprosthetic valves. EDC was chosen as cross-linking reagent. This chemical compound is a zero-length cross-linker that forms amide bonds between closely neighboring carboxyl and amino groups [21]. In this experiment, EDC was used to activate carboxylic acid groups of Ca-alginate beads for immobilization AMX from amine group [22]. The optimum AE was observed 1 mg (Fig. 2). As shown in Figure 2, the immobilization yield Eq. (2) of AMX was got decreasing after 1 mg EDC, because of EDC was started activating of phosphate group in buffer solution [23].
(2)
All the free carboxylic acid groups of Ca-alginate beads were activated until amount of 1 mg of EDC and then, more over AE was attempted the phosphate molecules. Therefore, it was affected the yield of immobilization of AMX. Figure 2 3.2. Effects of Types of Buffer and Buffer Concentration A buffer is made by mixing a volume of a weak acid or weak base together with its conjugate. Buffer solutions prevent pH changes because of proton or hydroxyl ions that are 10
observed end of the reaction. In this experiment, phosphate buffer was chosen because of EDC. EDC has reaction affinity with other kinds of buffer solution. EDC is reacted with phosphate buffer but it is more specific carboxylic acid groups than phosphate groups. The reaction of EDC with carboxyl groups is most efficient at pH 3.5-4.5. The optimum pH for EDC- mediated amide bond formation is often a little higher, usually between pH 4-6. This optimum reflects a balance of a number of factors, including the rate of formation and hydrolysis of the o-acylisourea and the pKa of the amine nucleophile, which may be substantially above the pH at which the oacylisourea intermediate is preferentially formed. Phosphate and carboxylic acid are first reactant groups for carbodiimides. If phosphate buffer must be used for reaction acceleration, polycarboxylic acid substance is added into reaction medium for contrast with phosphate molecule [23]. pH value of the phosphate buffer was chosen as pH 5, because EDC was affected carboxylic acid groups at acidic pH. However, other pH values were affected solubility of molecules because of dielectric constant. The solvent system was included 4:1 methanol-distilled water because of solubility of AMX. Dielectric constant of solvent system was lower than dielectric constant of water. Solubility of Ca and phosphate were effected and calcium diphosphate was precipitated. Therefore, pH 5 was chosen as optimum pH value [24, 25]. Caalginate beads were precipitated other pH values. High phosphate buffer concentration, egg-box structure of Ca-alginate bead was broken and precipitated. Because calcium was interacted with phosphate, which was came from buffer solution, and precipitated as calcium phosphate. Because phosphate groups in the buffer was competed with carboxylate groups of alginate in the reaction with calcium ions [26] and calcium ions were bound phosphate ions more specifically than alginate.
11
The optimum buffer concentration was determined 50 mM (Fig. 3). Higher concentration values than 50 mM, phosphate was precipitated because of interferences. Therefore, the values are not shown on curve. Figure 3 3.3. Effect of Number of Ca-alginate Beads Effect on Immobilization The most important feature of alginate’s physical properties is almost temperature independent and biocompatible hydrogel formation in the presence of multivalent cation (such as Ca2+) which is the basis for gel formation [27]. Ca-alginate beads were prepared with syringe owing to the feature of alginate. The yield of immobilization was calculated according to Eq. (3) Optimum number of Ca-alginate beads was determined as 5 pieces.
(3)
AMX efficiency was stabilized after 5 beads (Fig. 4). The Ca-alginate beads have egg-box structure that was leaved free carboxylic groups after formation of the alginate beads [28]. As a result, while number of Ca-alginate bead was increased, the number of carboxylic acid groups was increased. Therefore, limited EDC concentration could not activate all carboxylic acid groups in reaction medium. Figure 4 3.4. Effect of Temperature Collision number of particles increases with increasing temperature in many chemical reactions. However, biologic molecules structure such as antibiotics were denaturized because of 12
high temperature. Commercial AMX is stored at +2-8oC [29]. Also AMX, which is determined in milk sample, is retained stability throughout the chosen storage period when kept at -20oC [30]. In this study, commercial AMX was used and optimum incubation temperature was determined as +4ºC at the end of experiment (Fig. 5). This value was situated within the storage temperature. When the temperature was increased, the AIA was decreased and morphology of Ca-alginate beads was deteriorated. Figure 5 3.5. Effect of Mixing Speed One of the most important factors of reaction rate is increasing surface contact between the reactant particles. This is achieved by increasing mixing speed and contact surface. The effective number of collision between reactants increases with mixing speed [31]. Effect of mixing speed on immobilization was given in Figure 6. The AIA increased regularly until 150 rpm and then it was stabilized. Therefore, 150 rpm was chosen as optimum mixing speed. Figure 6 3.6. Effect of Amount of AMX Optimum amount of AMX was determined as 4 mg and the yield of AIA was calculated with Eq. (4). (Fig. 7). The maximum yield of AMX immobilization to Ca-alginate beads was observed at amount of 4 mg AMX. AMX molecules have prone groups (COOH, NH2, ect.) (Fig. 1) and they could be activated with EDC [21]. So, the AMX molecules were connected with each other and created the polymer. Also the polymer structures of AMX were known dimer and
13
trimeric structures [30]. Lower values than 4 mg AMX samples could not cover surface of the Ca-alginate beads, so 4 mg AMX was given the best immobilization yield.
(4)
Figure 7 3.7. Effect of Immobilization Time Effect of incubation time on AMX immobilization was determined and optimum incubation time was chosen as 1.5 hours (Fig. 8). AIA was increased until 1.5 hours and then AIA was decreased. Because, molecular structure of AMX was deformed over time. Figure 8 3.8. Antimicrobial Effects of AMX Immobilized Ca-alginate Antimicrobial effects of Ca-alginate beads and AMX immobilized Ca-alginate beads were tested by Gram positive (S. aerous (ATCC 6538)) bacteria and Gram negative (E. coli (ATCC 8739)) bacteria. Ca-alginate and AMX immobilized Ca-alginate beads were stored at 4 and 25 oC to determine the time-dependent antibacterial effects.1st month antimicrobial test results were shown in Fig. 9. Figure 9 As a result of tests, Ca-alginate beads has no antimicrobial effects on S. aerous (ATCC 6538) and E. coli. (ATCC 8739). At the same time, AMX immobilized Ca-alginate beads had antimicrobial effects and the diameter of zones were measured. The diameter zone of AMX
14
immobilized Ca-alginate beads were measured 1 cm (stored at 4oC) against for E. coli (ATCC 8739). For all that, microbial growth was observed in petrie dish which was contained AMX immobilized Ca-alginate beads (stored at 25oC). The diameter of zones of beads were measured as 2.8 cm (stored at 4oC) and 2.1 cm (stored at 25oC) against for S. aerous (ATCC 6538). After 4 months later, AMX immobilized Ca-alginate beads were lost antimicrobial effects on E. coli (ATCC 8739) strain. And an antimicrobial effect on S. aerous (ATCC 6538) was decreased (data not shown). 3.9. ATR-FTIR Analysis Results of Ca-alginate and AMX Immobilized Ca-alginate Beads Ca-alginate beads have characteristic band at 1603 and 1428cm-1. These bounds are refers to asymmetric stretching vibration and symmetric vibration of carbonyl groups. Also there were observed 1113 cm-1 peak; stretching of C-C, 1079 cm-1 peak; stretching of C-O and 991 cm-1 ; OH bounds in carboxylic acid [32, 33]. When ATR-FTIR spectrum of Ca-alginate beads was analyzed, 1428-1113-1079-991 cm-1 peaks were not existed on AMX immobilized of Ca-alginate beads spectrum. 1603 cm-1 peak was gotten smaller on AMX immobilized Ca-alginate beads. Therefore, the activating of carboxylic acid groups was done successfully. Instead of 1197-983-869 cm-1 peaks were observed on the spectrum of AMX immobilized Ca-alginate bead. These peaks were match with ATR-FTIR spectrum of AMX (Fig. 10). 869 cm-1 peak refer benzene ring (C18-C20-C21-C22-C23-C24) of AMX, and 983 cm-1 (N19H2) bending and out of plane bending CH to benzene ring-1197 cm-1 was shown (N10H, N19H2) and CH bending [33]. Figure 10
15
3.10. SEM Images of Ca-alginate and AMX Immobilized Ca-alginate Beads AMX immobilization process was occurred successfully as shown on Figure 11. SEM image of Ca-alginate bead was given in Fig. 11 (A). The surface structure of Ca-alginate bead had reveals cracks caused by partial collapsing of the polymer network during dehydration [34]. In Fig. 11 (B) AMX immobilized of Ca-alginate bead appearance was looked different from Caalginate beads. The surface of Ca-alginate bead was changed topological. AMX was bounded on surface of Ca-alginate bead like plate structures and Ca-alginate bead had rough appearance. It was improved with reference SEM image of AMX as shown in Fig. 11 (C). The reference SEM image was belonged to amoxicillin and obtained using Jeol Jem-1200 EX II Electron Microscope at an acceleration voltage of 25 kV [35]. Figure 11
16
4. Conclusion This study presents a different idea that can be used for the development of the antibacterial products. Generally, Ca-alginate beads are used for encapsulation. At encapsulation method, biomolecules are mixed with alginate to trap inside of the material. In this study, AMX was linked Ca-alginate beads with covalent bounds so the beads have resistance to external influences. The major disadvantage of the antibacterial materials, which are in the market, is effected short-term antibacterial activity. AMX immobilized Ca-alginate beads were washed long term to eliminate the non-specific bounding of AMX. However, antibacterial effect of AMX immobilized Ca-alginate beads was protected and storage stability of AMX immobilized Caalginate beads were taken about 4 months. Alginate is a biocompatible material and it is shown no penetration through the skin. Immobilized AMX molecules does not penetrate to skin because AMX was immobilized to Ca-alginate beads covalently. The improved biocomposite material is incorporated with different material such as fabric and used in medical textile as stupe, surgical gown etc. Also, alginate can be functionalization by this method with other biomolecules and could be used for column stationary phase.
17
5. References [1] G. Li, H. Meng, Recent Advances in Smart Self-Healing Polymers and Composites, Elsevier, 2015. [2] H. Liu, Nanocomposites for Musculoskeletal Tissue Regeneration, first ed., Woodhead Publishing, 2016. [3] G.R. Mahdavinia, Z. Rahmani, S. Karami A. Pourjavadi, Magnetic/pH-sensitive κcarrageenan/sodium alginate hydrogel nanocomposite beads: preparation, swelling behavior, and drug delivery, J. Biomater. Sci. Polym Ed. 25:17 (2014) 1891-1906, [4] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications Prog Polym Sci. 37:1 (2012) 106-126. [5] G.R. Mahdavinia, S. Mousanezhad, H. Hosseinzadeh, F. Darvishi, M. Sabzi, Magnetic hydrogel beads based on PVA/sodium alginate/laponite RD and studying their BSA adsorption, Carbohyd. Polym. 147 (2016) 379-391. [6] F. Khan, S.R. Ahmad, Polysaccharides and their derivatives for versatile tissue engineering application, Macromol. Biosci. 13 (2013) 395-421. [7] W.R. Gombotz, S. F. Wee, Protein release from alginate matrices, Advanced Drug Delivery Reviews 64 (2012) 194-205. [8] S.N.A. Rahima, A. Sulaimana, F. Hamzah, K.H.K. Hamid, M.N.M. Rodhi, M. Musa, N.A. Edama, Enzymes encapsulation within calcium alginate-clay beads: Characterization and application for cassava slurry saccharification, Procedia Engineering 68 (2013) 411-417.
18
[9] K.Y. Liu, F.C. Du, Q. Fu, W.J. Zhang, H.W. Sun, Y. Zhang, G. Guo, Efficacy and pharmacological mechanism of pronase-enhanced low-dose antibiotics for Helicobacter pylori eradication, Antimicrob. Agents. Ch. 58:6 (2016) 3348-3353. [10] Amoxicillin sodium salt susceptibility and minimum inhibitory concentration (MIC) data. http://www.toku-e.com/Assets/MIC/Amoxicillin%20sodium%20salt.pdf (accessed 15.03.03). [11] H. Daemi, M. Barikani, M. Barmar, A simple approach for morphology tailoring of alginate particles by manipulation ionic nature of polyurethanes, Int. J. Biol. Macromol. 66 (2014) 212220. [12] E.E. Ozseker, A. Akkaya, Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads, Carbohyd. Polym. 151 (2016) 441-451. [13] K. Sarkar, Z. Ansari, K. Sen, Detoxification of Hg (II) from aqueous and enzyme media: Pristine vs. tailored calcium alginate hydrogels, Int. J. Biol. Macromol. 91 (2016) 165-173. [14] K. Sarkar, K. Sen, On the design of Ag–morin nanocomposite to modify calcium alginate gel: framing out a novel sodium ion trap, RSC Adv. 5 (2015) 57223-57230. [15] Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013. [16] I.W. Hamley, PEG−Peptide Conjugates, Biomacromolecules 15 (2014) 1543-1559. [17] Y.T. Zhua, X.Y. Rena, Y.M. Liub, Y. Weic, L.S. Qinga, X. Liao, Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays, Mater. Sci. Eng. C 38 (2014) 278-285. 19
[18] E. Lepvrier, C. Doigneaux, L. Moullintraffort, A. Nazabal, C. Garnier, Optimized protocol for protein macrocomplexes stabilization using the EDC, 1‑ Ethyl-3-(3-(dimethylamino) propyl) carbodiimide, zero-length cross-linker, Anal. Chem. 86 (2014) 10524-10530. [19] A. Leitner, L.A. Joachimiak, P. Unverdorben, T. Walzthoeni, J. Frydman, F. Förster, R. Aebersolda, Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes, P. Natl. Acad. Sci. USA 111:26 (2014) 9455-9460. [20] S. Tanco, K. Gevaert, P.V. Damme, C-terminomics: Targeted analysis of natural and posttranslationally modified protein and peptide C-termini, Proteomics 15 (2015) 903–914. [21] G.T. Hermanson, Bioconjugate Techniques, second ed., Academic Press, London, 2008. [22] F. Kazenwadel, H. Wagner, B.E. Rapp, M. Franzre, Optimization of enzyme immobilization on magnetic microparticles using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent, Anal. Methods. 7 (2015) 10291-10298. [23] N. Gee, A. Draghi, Conjugation reactions, EP2566845 A1 patent apply, Innova Biosciences Limited, 2013. [24] U. Demirci, A. Khademhosseini, Gels Handbook: Fundamentals, Properties and Applications World Scientific, New Jersey, 2016. [25] A. Wang, S.E. Duncan, K.F. Knowlton, W.K. Ray, A.M. Dietrich,
Milk protein
composition and stability changes affected by iron in water sources, J. Dairy Sci. 99:6 (2016) 4206-4219. [26] K.Y. Lee, D.J. Mooney, Alginate properties and biomedical applications, Prog. Polym. Sci. 37:1 (2012) 106-126. 20
[27] J. Sun, H. Tan, Alginate-based biomaterials for regenerative medicine applications, Materials 6 (2013) 1285-1309. [28] F.A. Loewus, W. Tanner, Plant carbohydrates I: Intracellular carbohydrates, Vol. 13, Springer Science & Business Media, Berlin, 2012. [29] Amoxicillin. http://www.sigmaaldrich.com/catalog/product/sigma/a8523?lang=en®ion =TR (accessed 15.03.08). [30] J. Wang, J.D. MacNeil, J.F. Kay, Chemical analysis of antibiotic residues in food, John Wiley & Sons, New Jersey, 2011. [31] D.F. Anderson, T.G. Kurtz, Stochastic analysis of biochemical systems, first ed.. Springer, Berlin, 2015. [32] Q. Qian, W. Bonani, D. Maniglio, J. Chen, C. Migliaresi, Modulating the release of drugs from alginate matrices with the addition of gelatin microbeads, J. Bioact. Compat. T. Pol. 29:3 (2014) 193-207. [33] A. Bebu, L. Szabo, N. Leopold, C. Berindean, L. David, IR, Raman, SERS and DFT study of amoxicillin, J. Mol. Struct. 933 (2011) 52-56. [34] L. Segale, L. Giovannelli, P. Mannina, F. Pattarino, Calcium alginate and calcium alginatechitosan beads containing celecoxib solubilized in a self-emulsifying phase, Scientifica (2016). [35] M.S. Refat, H.M.A. Al-Maydama, F.M. Al-Azab, R.R. Amin, Y.M.S. Jamil, Synthesis, thermal and spectroscopic behaviors of metal–drug complexes: La(III), Ce(III), Sm(III) and
21
Y(III) amoxicillin trihydrate antibiotic drug complexes, Spectrochim. Acta, Part A 128 (2014) 427-446.
Figure Captions Figure 1. Molecular structure of AMX Figure 2. Amount of EDC effects on AMX immobilization (conditions: pH 5, 25 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 3. Effect of pH and buffer concentration on AMX immobilization (conditions: amount of EDC 1 mg, 5 pieces of Ca-alginate beads, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 4. Pieces of Ca-alginate beads effect on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 5. Effect of incubation temperature on AMX immobilization (conditions: amount of EDC 1 mg pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 6. Effect of mixing speed on AMX immobilization (conditions: amount of EDC 1mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, amount of AMX 6 mg, incubation time 1 hour).
22
Figure 7. Amount of AMX concentration on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, mixing speed 150 rpm, incubation time 1 hour,) Figure 8. Effect of incubation time on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, mixing speed 150 rpm, amount of AMX 4 mg). Figure 9. Results of antimicrobial tests (A) Ca-alginate beads (B) AMX immobilized Ca-alginate bead (E. coli, stored at 4 and 25oC) (C) AMX immobilized Ca-alginate bead (S. aerous, stored at 4 and 25oC). Figure 10. (A) ATR-FTIR Spectrum of Ca-alginate bead (B) ATR-FTIR Spectrum of AMX (C) ATR-FTIR Spectrum of AMX immobilized Ca-alginate bead. Figure 11. (A) SEM Image of Ca-alginate bead (B) SEM Image of AMX immobilized Caalginate bead (C) Reference SEM image of AMX
23
Fig. 1
24
Fig. 2
25
Fig. 3
26
Fig. 4
27
Fig. 5
28
Fig. 6
29
Fig. 7
30
Fig. 8
31
Fig 9 (A)
32
Fig 9 (B)
33
Fig. 9 (C)
34
Fig 10 (A)
35
Fig 10 (B)
36
Fig. 10 (C)
37
Fig 11 (A)
38
Fig 11 (B)
39
Fig 11 (C)
40
Table 1: Optimization of immobilization parameters Parameters Amount of EDC (mg) Concentration of Buffer Solution (mM) Pieces of Ca-alginate Beads (N) Temperature of Incubation (oC) Mixing speed (rpm) Amount of AMX (mg) Incubation Time (hour)
Quantity of Parameters 0.25-0.50-0.75-1.00-2.50-5.00-7.50-10.0025.00-50.00 2.5–5.0–10.0–25.0-50.0 2-5-10-15-25 4-15-25-37-45-55-80 0-50-100-150-200-250 2-3-4-5-6-7 0.25-0.50-0.75-1.00-1.50-2.00-3.00-4.00
41
Antibacterial tests of Ca-alginate
SEM image of Ca-alginate
SEM image of AMX imm. Ca-alginate
Antibacterial tests of AMX imm. Ca-alginate
AMX O
O OH Ca-alginate
EDC
Ca-alginate
N H
Statement of Significance 1. Amoxicillin (AMX) was bonded Ca-alginate bead covalently. 2. Biomaterial has long-term antibacterial effect. 3. The improved biomaterial is incorporated with different materials to impart antibacterial properties
42
S0014-3057(16)30579-1 http://dx.doi.org/10.1016/j.eurpolymj.2016.09.031 EPJ 7502
To appear in:
European Polymer Journal
Received Date: Revised Date: Accepted Date:
13 June 2016 6 September 2016 18 September 2016
Please cite this article as: Guler, S., Erdogan Ozseker, E., Akkaya, A., DEVELOPING AN ANTIBACTERIAL BIOMATERIAL, European Polymer Journal (2016), doi: http://dx.doi.org/10.1016/j.eurpolymj.2016.09.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DEVELOPING AN ANTIBACTERIAL BIOMATERIAL Selda GULER, Emine Erdogan OZSEKER, Alper AKKAYA Ege University, Faculty of Science, Biochemistry Department, Bornova-Izmir 35100, Turkey
*Corresponding author: Assoc. Prof. Dr. Alper AKKAYA, Ege University, Faculty of Science, Biochemistry Department, Bornova-Izmir 35100, Turkey Tel: +90 232 311 23 67 Fax: +90 232 311 54 85 e-mail: [email protected]
1
ABSTRACT Development and production of antibacterial materials were gained momentum because of the increasing infectional diseases in recent years. Antibacterial products are used in many industries, such as, textile, dye and package industries and medical technology. Short duration antibacterial affect is the main problem for these products. Therefore, the development of materials that exhibit long duration antimicrobial activity, is needed for industry. In this study, the
free
carboxylic
acid
group
of
alginate
were
activated
using
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and amoxicillin (AMX) was covalently immobilized to alginate. AMX, D-α-amino-p-hydroxybenzyl penicillin trihydrate, is one of the most frequently used β-lactam antibiotics that has been employed to treat and reduce the spread of bacterial infections in human, with the benefits of maintaining animal welfare. At the end of the study, an antibacterial biomaterial was developed. The optimum reaction medium, that immobilization of AMX to Ca-alginate beads, was determined as; 1 mg of EDC, pH 5, 50 mM of phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature was 4oC, mixing speed was 150 rpm, initial amount of AMX (IAA) was 4 mg and incubation time 1.5 h. Ca-alginate beads and AMX immobilized Ca-alginate beads were tested for antibacterial affects against Escherichia coli (ATCC 8739, gram negative) and Staphylococcus aureus (ATCC 6538, gram positive)
bacterial strain. According to the SEM images and ATR-FTIR spectrums of AMX
immobilized Ca-alginate and Ca-alginate beads were analyzed, the immobilization process was proved successfully. Developed biomaterial was antibacterial according to antibacterial tests.
Keywords: Alginate, biomaterial, amoxicillin, antibacterial materials 2
1. Introduction Polymers and polymer composites have been widely used in tremendous engineering fields because of their advantages including light weight, good processability, chemical stability in any atmospheric conditions, etc. [1]. For biomedical composites, even though excellent mechanical performance is desirable and often targeted for improvement, the biocompatibility of the material is of paramount concern. The biological compatibility is more important than the mechanical compatibility. Being composed of two or more types of materials, composites carry an enhanced probability of causing adverse tissue reactions [2]. In this study, alginate was used as a support for AMX immobilization. Sodium alginate contains pendant carboxylate and is known as polyanionic copolymer of 1,4-linked-α-Lguluronic acid and β-D-mannuronic acid residues found in brown seaweeds [3]. Alginate is soluble in aqueous solutions and forms stable gels in the presence of certain divalent cations, such as calcium, barium and strontium [4]. The disintegration of the alginate beads happens due to cation exchange, exchange of Ca2+ ions (participating in crosslinking points) with monovalent ions such as Na+ [5]. The gelation and crosslinking of the polymers are mainly achieved by the exchange of sodium ions from the guluronic acids with the divalent cations, and the stacking of these guluronic groups to form the characteristic egg-box structure [6]. The divalent cations bind to the guluronic acid blocks in a highly cooperative manner and the size of the cooperative unit is more than 20 monomers. Each alginate chain can dimerize to form junctions with many other chains and as a result, gel networks are formed rather than insoluble precipitates [7]. Ca-alginate bead is commonly used in the encapsulation process [8]. However, free carboxylic acids, which are situated at Ca-alginate bead, were activated with EDC and AMX was immobilized to Ca-alginate covalently instead of the encapsulation at this process.
3
AMX is a bactericidal antibiotic that belongs to group of penicillin (Fig. 1). It binds to penicillin-binding proteins, and thus interferes with bacterial cell wall synthesis, resulting in the lyses of replicating bacteria [9]. Figure 1 In general, AMX has antibacterial effects on Streptococcus sp., Bacillus subtilis, Enterococcus sp., Haemophilus sp., Helicobacter sp., Moraxella sp., Clostiridium sp. etc. [10]. AMX was immobilized Ca-alginate beads by covalent bounds. Therefore, biocomposite material was developed for against resistant external influences. The unique properties of alginate, combined with its biocompatibility, hydrophilicity and relatively low cost, have made it an important polymer in pharmaceutical applications [11]. Alginate hydrogels can be prepared by various cross-linking methods, and their structural similarity to extracellular matrices of living tissues allows wide applications in wound healing, delivery of bioactive agents such as small chemical drugs and proteins, and cell transplantation. Alginate wound dressings maintain a physiologically moist microenvironment, minimize bacterial infection at the wound site, and facilitate wound healing. Drug molecules, from small chemical drugs to macromolecular proteins, can be released from alginate gels in a controlled manner, depending on the cross-linker types and cross-linking methods. In addition, alginate gels can be orally administrated or injected into the body in a minimally invasive manner, which allows extensive applications in the pharmaceutical arena. Alginate gels are also promising for cell transplantation in tissue engineering. Tissue engineering aims to provide man-made tissue and organ replacements to patients who suffer the loss or failure of an organ or tissue [4]. In this study, alginate molecules were functionalized with AMX and antibacterial product was
4
developed. This Ca-alginate beads can be transformed back to the solution form and application of different materials is possible to achieve a permanent antibacterial product. This antibacterial product can be applied to textile materials and new generation wound dressing, surgical clothes etc. could be developed. It can also be applied to cosmetic materials such as cream, soap etc.
5
2. Materials and methods 2.1. Materials EDC was purchased from Merck; KH2PO4, methanol, AMX, sodium alginate, CaCl2 were purchased from Sigma; nutrient broth, nutrient agar were purchased from Fluka. 2.2. Microorganisms S. aureus (ATCC 6538) and E. coli (ATCC 8739) were used in antibacterial tests. The microorganisms (S. aerous (ATCC 6538), E. coli (ATCC 8739)), were obtained commercially from American Type Culture Collection (ATCC). S. aerous (ATCC 6538) was isolated from human lesion and E. coli (ATCC 8739) was isolated from the feces. 2.3. Preparation of Ca-alginate Beads Sodium alginate was dissolved in distilled water at the final concentration of 2% (w/w) overnight. Ca-alginate beads were prepared by dropping aqueous alginate into a 3% 100 ml calcium chloride solution into ice bath and the form of Ca-alginate beads were allowed to cure for overnight. Ca-alginate beads were washed with distilled water for once and buffer for twice to remove excess Ca2+ and finally stored at +4oC until immobilization process [12]. 2.4. Immobilization of AMX to Ca-alginate Beads The initial reaction medium (IRM), where immobilization of AMX to Ca-alginate beads was performed, was determined as; pH 5, 25 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature was 25oC, mixing speed was 100 rpm, initial amount of AMX (IAA) was 6 mg, incubation time was 1 hour.
6
The reaction medium was prepared as including 5 pieces of Ca-alginate beads and dissolved EDC in 2 ml phosphate buffer solution then incubated for 20 minutes. At the end of the incubation time, 1 ml of dissolved AMX in 4:1 methanol-distilled water solvent system was added to reaction medium and incubated at 25oC and 100 rpm for 1 hour. Ca-alginate beads were separated from reaction medium and washed with 1 ml of 4:1 methanol-distilled water solvent system for 4 times. All washing waters (WW) were collected and put together with IRM. Samples were taken from the medium and absorbance values were measured by spectrophometer (Perkin Elmer UV/Visible Lambda 25, USA) at 272 nm and amount of AMX was determined which was in the reaction medium after immobilization process. The difference between IAA and last was determined the amount of AMX gave the amount of immobilized AMX (AIA) Eq. (1). –
(1)
In this study, Na-alginate was prepared in bead form and immobilization was performed using bead formed Ca-alginate. Ca-alginate beads were predisposed to reaction with EDC. However, the modification method of Ca-alginate beads was varied depending of biomolecule, which was used for immobilization [13, 14]. 2.5. Optimization of AMX Immobilization Amount of EDC (AE), concentration of buffer solution, pieces of Ca-alginate beads, immobilization temperature, mixing speed and IAA parameters were optimized for AMX immobilization as shown in Table 1. Table 1
7
2.6. Antimicrobial Tests In the experiment, antimicrobial effects of Ca-alginate beads, AMX immobilized Caalginate beads and composite materials were tested by Gram positive (S. aerous (ATCC 6538)) bacteria and Gram negative (E. coli (ATCC 8739)) bacteria. Similar to the Disk Diffusion Method (Spread Plate Technique) were used for antimicrobial tests [15]. In Disk Diffusion Method, the sterile disk was impregnated with the antibacterial sample but the AMX was bound to Ca-alginate beads covalently. S. aureus (ATCC 6538) and E. coli (ATCC 8739) were obtained from bacterial stock cultures and incubated slant agar at 37oC overnight. Bacterial cells were obtained from slant agar by sterile loop and incubated in nutrient broth at 37oC, 200 rpm for 24 hours. After incubation, 50 µl culture of antibacterial test microorganisms were inoculated onto nutrient agar and spread with drigalski spatula. Ca-alginate and AMX immobilized Ca-alginate beads were placed in the middle of the petri dish and incubated at 37oC for 24 hours. Antimicrobial effects of the beads were observed after incubation. Ca-alginate and AMX immobilized Ca-alginate beads were stored at 4oC and 25oC to determine the time-dependent antibacterial effects. Antimicrobial tests were done regularly for 4 months. 2.7. Scanning Electron Microscopy Surfaces of Ca-alginate and AMX immobilized Ca-alginate beads were visualized with scanning electron microscope (FEI Quanta 250 FEG) (SEM). 2.8. Structural Analysis by Fourier Transform Infrared Spectroscopy (FT-IR)
8
ATR-FTIR spectrums were obtained with Perkin Elmer Spectrum 100 FT-IR with attenuated total reflection (ATR) equipment (USA). FT-IR analysis was performed on Caalginate and AMX immobilized Ca-alginate beads. Spectrums were obtained the spectral range of 400–4000 cm−1.
9
3. Results and discussion 3.1. Effect of Amount of EDC on Immobilization Carbodiimides have been utilized in a number of different research areas including, for example, modify carboxyl groups in enzymes, peptide synthesis [16, 17], conjugation of proteins in structural studies [18, 19] quantification of carboxyl moieties in proteins [20], and coupling reactions in an anticalcification treatment for porcine bioprosthetic valves. EDC was chosen as cross-linking reagent. This chemical compound is a zero-length cross-linker that forms amide bonds between closely neighboring carboxyl and amino groups [21]. In this experiment, EDC was used to activate carboxylic acid groups of Ca-alginate beads for immobilization AMX from amine group [22]. The optimum AE was observed 1 mg (Fig. 2). As shown in Figure 2, the immobilization yield Eq. (2) of AMX was got decreasing after 1 mg EDC, because of EDC was started activating of phosphate group in buffer solution [23].
(2)
All the free carboxylic acid groups of Ca-alginate beads were activated until amount of 1 mg of EDC and then, more over AE was attempted the phosphate molecules. Therefore, it was affected the yield of immobilization of AMX. Figure 2 3.2. Effects of Types of Buffer and Buffer Concentration A buffer is made by mixing a volume of a weak acid or weak base together with its conjugate. Buffer solutions prevent pH changes because of proton or hydroxyl ions that are 10
observed end of the reaction. In this experiment, phosphate buffer was chosen because of EDC. EDC has reaction affinity with other kinds of buffer solution. EDC is reacted with phosphate buffer but it is more specific carboxylic acid groups than phosphate groups. The reaction of EDC with carboxyl groups is most efficient at pH 3.5-4.5. The optimum pH for EDC- mediated amide bond formation is often a little higher, usually between pH 4-6. This optimum reflects a balance of a number of factors, including the rate of formation and hydrolysis of the o-acylisourea and the pKa of the amine nucleophile, which may be substantially above the pH at which the oacylisourea intermediate is preferentially formed. Phosphate and carboxylic acid are first reactant groups for carbodiimides. If phosphate buffer must be used for reaction acceleration, polycarboxylic acid substance is added into reaction medium for contrast with phosphate molecule [23]. pH value of the phosphate buffer was chosen as pH 5, because EDC was affected carboxylic acid groups at acidic pH. However, other pH values were affected solubility of molecules because of dielectric constant. The solvent system was included 4:1 methanol-distilled water because of solubility of AMX. Dielectric constant of solvent system was lower than dielectric constant of water. Solubility of Ca and phosphate were effected and calcium diphosphate was precipitated. Therefore, pH 5 was chosen as optimum pH value [24, 25]. Caalginate beads were precipitated other pH values. High phosphate buffer concentration, egg-box structure of Ca-alginate bead was broken and precipitated. Because calcium was interacted with phosphate, which was came from buffer solution, and precipitated as calcium phosphate. Because phosphate groups in the buffer was competed with carboxylate groups of alginate in the reaction with calcium ions [26] and calcium ions were bound phosphate ions more specifically than alginate.
11
The optimum buffer concentration was determined 50 mM (Fig. 3). Higher concentration values than 50 mM, phosphate was precipitated because of interferences. Therefore, the values are not shown on curve. Figure 3 3.3. Effect of Number of Ca-alginate Beads Effect on Immobilization The most important feature of alginate’s physical properties is almost temperature independent and biocompatible hydrogel formation in the presence of multivalent cation (such as Ca2+) which is the basis for gel formation [27]. Ca-alginate beads were prepared with syringe owing to the feature of alginate. The yield of immobilization was calculated according to Eq. (3) Optimum number of Ca-alginate beads was determined as 5 pieces.
(3)
AMX efficiency was stabilized after 5 beads (Fig. 4). The Ca-alginate beads have egg-box structure that was leaved free carboxylic groups after formation of the alginate beads [28]. As a result, while number of Ca-alginate bead was increased, the number of carboxylic acid groups was increased. Therefore, limited EDC concentration could not activate all carboxylic acid groups in reaction medium. Figure 4 3.4. Effect of Temperature Collision number of particles increases with increasing temperature in many chemical reactions. However, biologic molecules structure such as antibiotics were denaturized because of 12
high temperature. Commercial AMX is stored at +2-8oC [29]. Also AMX, which is determined in milk sample, is retained stability throughout the chosen storage period when kept at -20oC [30]. In this study, commercial AMX was used and optimum incubation temperature was determined as +4ºC at the end of experiment (Fig. 5). This value was situated within the storage temperature. When the temperature was increased, the AIA was decreased and morphology of Ca-alginate beads was deteriorated. Figure 5 3.5. Effect of Mixing Speed One of the most important factors of reaction rate is increasing surface contact between the reactant particles. This is achieved by increasing mixing speed and contact surface. The effective number of collision between reactants increases with mixing speed [31]. Effect of mixing speed on immobilization was given in Figure 6. The AIA increased regularly until 150 rpm and then it was stabilized. Therefore, 150 rpm was chosen as optimum mixing speed. Figure 6 3.6. Effect of Amount of AMX Optimum amount of AMX was determined as 4 mg and the yield of AIA was calculated with Eq. (4). (Fig. 7). The maximum yield of AMX immobilization to Ca-alginate beads was observed at amount of 4 mg AMX. AMX molecules have prone groups (COOH, NH2, ect.) (Fig. 1) and they could be activated with EDC [21]. So, the AMX molecules were connected with each other and created the polymer. Also the polymer structures of AMX were known dimer and
13
trimeric structures [30]. Lower values than 4 mg AMX samples could not cover surface of the Ca-alginate beads, so 4 mg AMX was given the best immobilization yield.
(4)
Figure 7 3.7. Effect of Immobilization Time Effect of incubation time on AMX immobilization was determined and optimum incubation time was chosen as 1.5 hours (Fig. 8). AIA was increased until 1.5 hours and then AIA was decreased. Because, molecular structure of AMX was deformed over time. Figure 8 3.8. Antimicrobial Effects of AMX Immobilized Ca-alginate Antimicrobial effects of Ca-alginate beads and AMX immobilized Ca-alginate beads were tested by Gram positive (S. aerous (ATCC 6538)) bacteria and Gram negative (E. coli (ATCC 8739)) bacteria. Ca-alginate and AMX immobilized Ca-alginate beads were stored at 4 and 25 oC to determine the time-dependent antibacterial effects.1st month antimicrobial test results were shown in Fig. 9. Figure 9 As a result of tests, Ca-alginate beads has no antimicrobial effects on S. aerous (ATCC 6538) and E. coli. (ATCC 8739). At the same time, AMX immobilized Ca-alginate beads had antimicrobial effects and the diameter of zones were measured. The diameter zone of AMX
14
immobilized Ca-alginate beads were measured 1 cm (stored at 4oC) against for E. coli (ATCC 8739). For all that, microbial growth was observed in petrie dish which was contained AMX immobilized Ca-alginate beads (stored at 25oC). The diameter of zones of beads were measured as 2.8 cm (stored at 4oC) and 2.1 cm (stored at 25oC) against for S. aerous (ATCC 6538). After 4 months later, AMX immobilized Ca-alginate beads were lost antimicrobial effects on E. coli (ATCC 8739) strain. And an antimicrobial effect on S. aerous (ATCC 6538) was decreased (data not shown). 3.9. ATR-FTIR Analysis Results of Ca-alginate and AMX Immobilized Ca-alginate Beads Ca-alginate beads have characteristic band at 1603 and 1428cm-1. These bounds are refers to asymmetric stretching vibration and symmetric vibration of carbonyl groups. Also there were observed 1113 cm-1 peak; stretching of C-C, 1079 cm-1 peak; stretching of C-O and 991 cm-1 ; OH bounds in carboxylic acid [32, 33]. When ATR-FTIR spectrum of Ca-alginate beads was analyzed, 1428-1113-1079-991 cm-1 peaks were not existed on AMX immobilized of Ca-alginate beads spectrum. 1603 cm-1 peak was gotten smaller on AMX immobilized Ca-alginate beads. Therefore, the activating of carboxylic acid groups was done successfully. Instead of 1197-983-869 cm-1 peaks were observed on the spectrum of AMX immobilized Ca-alginate bead. These peaks were match with ATR-FTIR spectrum of AMX (Fig. 10). 869 cm-1 peak refer benzene ring (C18-C20-C21-C22-C23-C24) of AMX, and 983 cm-1 (N19H2) bending and out of plane bending CH to benzene ring-1197 cm-1 was shown (N10H, N19H2) and CH bending [33]. Figure 10
15
3.10. SEM Images of Ca-alginate and AMX Immobilized Ca-alginate Beads AMX immobilization process was occurred successfully as shown on Figure 11. SEM image of Ca-alginate bead was given in Fig. 11 (A). The surface structure of Ca-alginate bead had reveals cracks caused by partial collapsing of the polymer network during dehydration [34]. In Fig. 11 (B) AMX immobilized of Ca-alginate bead appearance was looked different from Caalginate beads. The surface of Ca-alginate bead was changed topological. AMX was bounded on surface of Ca-alginate bead like plate structures and Ca-alginate bead had rough appearance. It was improved with reference SEM image of AMX as shown in Fig. 11 (C). The reference SEM image was belonged to amoxicillin and obtained using Jeol Jem-1200 EX II Electron Microscope at an acceleration voltage of 25 kV [35]. Figure 11
16
4. Conclusion This study presents a different idea that can be used for the development of the antibacterial products. Generally, Ca-alginate beads are used for encapsulation. At encapsulation method, biomolecules are mixed with alginate to trap inside of the material. In this study, AMX was linked Ca-alginate beads with covalent bounds so the beads have resistance to external influences. The major disadvantage of the antibacterial materials, which are in the market, is effected short-term antibacterial activity. AMX immobilized Ca-alginate beads were washed long term to eliminate the non-specific bounding of AMX. However, antibacterial effect of AMX immobilized Ca-alginate beads was protected and storage stability of AMX immobilized Caalginate beads were taken about 4 months. Alginate is a biocompatible material and it is shown no penetration through the skin. Immobilized AMX molecules does not penetrate to skin because AMX was immobilized to Ca-alginate beads covalently. The improved biocomposite material is incorporated with different material such as fabric and used in medical textile as stupe, surgical gown etc. Also, alginate can be functionalization by this method with other biomolecules and could be used for column stationary phase.
17
5. References [1] G. Li, H. Meng, Recent Advances in Smart Self-Healing Polymers and Composites, Elsevier, 2015. [2] H. Liu, Nanocomposites for Musculoskeletal Tissue Regeneration, first ed., Woodhead Publishing, 2016. [3] G.R. Mahdavinia, Z. Rahmani, S. Karami A. Pourjavadi, Magnetic/pH-sensitive κcarrageenan/sodium alginate hydrogel nanocomposite beads: preparation, swelling behavior, and drug delivery, J. Biomater. Sci. Polym Ed. 25:17 (2014) 1891-1906, [4] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications Prog Polym Sci. 37:1 (2012) 106-126. [5] G.R. Mahdavinia, S. Mousanezhad, H. Hosseinzadeh, F. Darvishi, M. Sabzi, Magnetic hydrogel beads based on PVA/sodium alginate/laponite RD and studying their BSA adsorption, Carbohyd. Polym. 147 (2016) 379-391. [6] F. Khan, S.R. Ahmad, Polysaccharides and their derivatives for versatile tissue engineering application, Macromol. Biosci. 13 (2013) 395-421. [7] W.R. Gombotz, S. F. Wee, Protein release from alginate matrices, Advanced Drug Delivery Reviews 64 (2012) 194-205. [8] S.N.A. Rahima, A. Sulaimana, F. Hamzah, K.H.K. Hamid, M.N.M. Rodhi, M. Musa, N.A. Edama, Enzymes encapsulation within calcium alginate-clay beads: Characterization and application for cassava slurry saccharification, Procedia Engineering 68 (2013) 411-417.
18
[9] K.Y. Liu, F.C. Du, Q. Fu, W.J. Zhang, H.W. Sun, Y. Zhang, G. Guo, Efficacy and pharmacological mechanism of pronase-enhanced low-dose antibiotics for Helicobacter pylori eradication, Antimicrob. Agents. Ch. 58:6 (2016) 3348-3353. [10] Amoxicillin sodium salt susceptibility and minimum inhibitory concentration (MIC) data. http://www.toku-e.com/Assets/MIC/Amoxicillin%20sodium%20salt.pdf (accessed 15.03.03). [11] H. Daemi, M. Barikani, M. Barmar, A simple approach for morphology tailoring of alginate particles by manipulation ionic nature of polyurethanes, Int. J. Biol. Macromol. 66 (2014) 212220. [12] E.E. Ozseker, A. Akkaya, Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads, Carbohyd. Polym. 151 (2016) 441-451. [13] K. Sarkar, Z. Ansari, K. Sen, Detoxification of Hg (II) from aqueous and enzyme media: Pristine vs. tailored calcium alginate hydrogels, Int. J. Biol. Macromol. 91 (2016) 165-173. [14] K. Sarkar, K. Sen, On the design of Ag–morin nanocomposite to modify calcium alginate gel: framing out a novel sodium ion trap, RSC Adv. 5 (2015) 57223-57230. [15] Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013. [16] I.W. Hamley, PEG−Peptide Conjugates, Biomacromolecules 15 (2014) 1543-1559. [17] Y.T. Zhua, X.Y. Rena, Y.M. Liub, Y. Weic, L.S. Qinga, X. Liao, Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays, Mater. Sci. Eng. C 38 (2014) 278-285. 19
[18] E. Lepvrier, C. Doigneaux, L. Moullintraffort, A. Nazabal, C. Garnier, Optimized protocol for protein macrocomplexes stabilization using the EDC, 1‑ Ethyl-3-(3-(dimethylamino) propyl) carbodiimide, zero-length cross-linker, Anal. Chem. 86 (2014) 10524-10530. [19] A. Leitner, L.A. Joachimiak, P. Unverdorben, T. Walzthoeni, J. Frydman, F. Förster, R. Aebersolda, Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes, P. Natl. Acad. Sci. USA 111:26 (2014) 9455-9460. [20] S. Tanco, K. Gevaert, P.V. Damme, C-terminomics: Targeted analysis of natural and posttranslationally modified protein and peptide C-termini, Proteomics 15 (2015) 903–914. [21] G.T. Hermanson, Bioconjugate Techniques, second ed., Academic Press, London, 2008. [22] F. Kazenwadel, H. Wagner, B.E. Rapp, M. Franzre, Optimization of enzyme immobilization on magnetic microparticles using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent, Anal. Methods. 7 (2015) 10291-10298. [23] N. Gee, A. Draghi, Conjugation reactions, EP2566845 A1 patent apply, Innova Biosciences Limited, 2013. [24] U. Demirci, A. Khademhosseini, Gels Handbook: Fundamentals, Properties and Applications World Scientific, New Jersey, 2016. [25] A. Wang, S.E. Duncan, K.F. Knowlton, W.K. Ray, A.M. Dietrich,
Milk protein
composition and stability changes affected by iron in water sources, J. Dairy Sci. 99:6 (2016) 4206-4219. [26] K.Y. Lee, D.J. Mooney, Alginate properties and biomedical applications, Prog. Polym. Sci. 37:1 (2012) 106-126. 20
[27] J. Sun, H. Tan, Alginate-based biomaterials for regenerative medicine applications, Materials 6 (2013) 1285-1309. [28] F.A. Loewus, W. Tanner, Plant carbohydrates I: Intracellular carbohydrates, Vol. 13, Springer Science & Business Media, Berlin, 2012. [29] Amoxicillin. http://www.sigmaaldrich.com/catalog/product/sigma/a8523?lang=en®ion =TR (accessed 15.03.08). [30] J. Wang, J.D. MacNeil, J.F. Kay, Chemical analysis of antibiotic residues in food, John Wiley & Sons, New Jersey, 2011. [31] D.F. Anderson, T.G. Kurtz, Stochastic analysis of biochemical systems, first ed.. Springer, Berlin, 2015. [32] Q. Qian, W. Bonani, D. Maniglio, J. Chen, C. Migliaresi, Modulating the release of drugs from alginate matrices with the addition of gelatin microbeads, J. Bioact. Compat. T. Pol. 29:3 (2014) 193-207. [33] A. Bebu, L. Szabo, N. Leopold, C. Berindean, L. David, IR, Raman, SERS and DFT study of amoxicillin, J. Mol. Struct. 933 (2011) 52-56. [34] L. Segale, L. Giovannelli, P. Mannina, F. Pattarino, Calcium alginate and calcium alginatechitosan beads containing celecoxib solubilized in a self-emulsifying phase, Scientifica (2016). [35] M.S. Refat, H.M.A. Al-Maydama, F.M. Al-Azab, R.R. Amin, Y.M.S. Jamil, Synthesis, thermal and spectroscopic behaviors of metal–drug complexes: La(III), Ce(III), Sm(III) and
21
Y(III) amoxicillin trihydrate antibiotic drug complexes, Spectrochim. Acta, Part A 128 (2014) 427-446.
Figure Captions Figure 1. Molecular structure of AMX Figure 2. Amount of EDC effects on AMX immobilization (conditions: pH 5, 25 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 3. Effect of pH and buffer concentration on AMX immobilization (conditions: amount of EDC 1 mg, 5 pieces of Ca-alginate beads, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 4. Pieces of Ca-alginate beads effect on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, incubation temperature 25oC, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 5. Effect of incubation temperature on AMX immobilization (conditions: amount of EDC 1 mg pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, mixing speed 100 rpm, amount of AMX 6 mg, incubation time 1 hour). Figure 6. Effect of mixing speed on AMX immobilization (conditions: amount of EDC 1mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, amount of AMX 6 mg, incubation time 1 hour).
22
Figure 7. Amount of AMX concentration on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, mixing speed 150 rpm, incubation time 1 hour,) Figure 8. Effect of incubation time on AMX immobilization (conditions: amount of EDC 1 mg, pH 5, 50 mM phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature 4oC, mixing speed 150 rpm, amount of AMX 4 mg). Figure 9. Results of antimicrobial tests (A) Ca-alginate beads (B) AMX immobilized Ca-alginate bead (E. coli, stored at 4 and 25oC) (C) AMX immobilized Ca-alginate bead (S. aerous, stored at 4 and 25oC). Figure 10. (A) ATR-FTIR Spectrum of Ca-alginate bead (B) ATR-FTIR Spectrum of AMX (C) ATR-FTIR Spectrum of AMX immobilized Ca-alginate bead. Figure 11. (A) SEM Image of Ca-alginate bead (B) SEM Image of AMX immobilized Caalginate bead (C) Reference SEM image of AMX
23
Fig. 1
24
Fig. 2
25
Fig. 3
26
Fig. 4
27
Fig. 5
28
Fig. 6
29
Fig. 7
30
Fig. 8
31
Fig 9 (A)
32
Fig 9 (B)
33
Fig. 9 (C)
34
Fig 10 (A)
35
Fig 10 (B)
36
Fig. 10 (C)
37
Fig 11 (A)
38
Fig 11 (B)
39
Fig 11 (C)
40
Table 1: Optimization of immobilization parameters Parameters Amount of EDC (mg) Concentration of Buffer Solution (mM) Pieces of Ca-alginate Beads (N) Temperature of Incubation (oC) Mixing speed (rpm) Amount of AMX (mg) Incubation Time (hour)
Quantity of Parameters 0.25-0.50-0.75-1.00-2.50-5.00-7.50-10.0025.00-50.00 2.5–5.0–10.0–25.0-50.0 2-5-10-15-25 4-15-25-37-45-55-80 0-50-100-150-200-250 2-3-4-5-6-7 0.25-0.50-0.75-1.00-1.50-2.00-3.00-4.00
41
Antibacterial tests of Ca-alginate
SEM image of Ca-alginate
SEM image of AMX imm. Ca-alginate
Antibacterial tests of AMX imm. Ca-alginate
AMX O
O OH Ca-alginate
EDC
Ca-alginate
N H
Statement of Significance 1. Amoxicillin (AMX) was bonded Ca-alginate bead covalently. 2. Biomaterial has long-term antibacterial effect. 3. The improved biomaterial is incorporated with different materials to impart antibacterial properties
42