Preparation, stability and antimicrobial activity of cationic cross-linked starch–iodine complexes

International Journal of Biological Macromolecules 51 (2012) 800–807

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Preparation, stability and antimicrobial activity of cationic cross-linked starch–iodine complexes Rima Klimaviciute a,∗ , Joana Bendoraitiene a , Ramune Rutkaite a , Jurate Siugzdaite b , Algirdas Zemaitaitis a a b

Laboratory of Biopolymer Research, Faculty of Chemical Technology, Kaunas University of Technology, Radvilenu pl. 19, LT-50254 Kaunas, Lithuania Veterinary Academy, Lithuanian University of Health Sciences, Tilzes Str. 18, LT-47181 Kaunas, Lithuania

a r t i c l e

i n f o

Article history: Received 13 June 2012 Received in revised form 16 July 2012 Accepted 27 July 2012 Available online 3 August 2012 Keywords: Cationic cross-linked starch Iodine Adsorption Antibacterial properties Biodegradability

a b s t r a c t Cationic cross-linked starch (CCS)–iodine complexes containing different amounts of quaternary ammonium groups (different degrees of substitution (DS)) and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solution. Equilibrium adsorption studies showed that with an increase of DS the amount of iodine adsorbed on CCS and the affinity of iodine to CCS increased linearly. The influences of the DS of CCS and the amount of adsorbed iodine on the stability of CCS–iodine complexes in a solution of 0.02 M sodium acetate and reactivity toward l-tyrosine have been investigated. At the same DS, the stability of CCS–iodine complexes decreased with an increase of the amount of adsorbed iodine. With increasing the DS, the stability of CCS–iodine complexes increased. The iodine consumption in the reaction with l-tyrosine increased significantly with an increase of the amount of adsorbed iodine. The influence of DS on iodine consumption was lower and depended on the amount of adsorbed iodine. The antibacterial activity of CCS–iodine complexes against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was determined by the broth-dilution and spread-plate methods. The obtained results have demonstrated that an appropriate selection of the CCS–iodine complex composition (the DS of CCS and the amount of adsorbed iodine) could ensure good antimicrobial properties by keeping a low concentration of free iodine in the system. The main advantage of using CCS–iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Iodine is one of the most efficacious antiseptics and has traditionally been available in solutions or tinctures but shows a high degree of instability [1]. This problem was overcome by the development of iodophores. Iodophores are complexes of iodine and a solubilizing agent or carrier. In an aqueous iodophore solution, iodine is present in the form of different thermodynamically stable anionic iodine species and diatomic iodine [2]. The two most important iodophores used nowadays are polyvinylpyrrolidone-iodine (or povidone-iodine) (PVP-I) and cadexomer-iodine (CI). PVP with iodine forms a stable charge-transfer complex. In PVPI solutions, free species of iodine are formally controlled by the mass action law including a coupled reversible interaction between iodine–iodide, triiodide–polymer and iodine–triiodide–polymer complexes [3]. The antibacterial action of PVP-I increases with an

∗ Corresponding author. Tel.: +370 37 456081; fax: +370 37 456081. E-mail addresses: [email protected], [email protected] (R. Klimaviciute). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.07.025

increase of dilution degree [4]. One of the hypotheses is that the dilution of PVP-I results in weakening the iodine linkage to the carrier [4,5]. The amount of free iodine and the antibacterial action of PVP-I depends also on the formulation of PVP-I [3]. Cadexomer is a derivative of dextrines (containing some number of carboxyl groups) cross-linked with epichlorohydrin and exits in the form of water-insoluble microbeads; 0.9% of molecular iodine is physically (not chemically) trapped in the core of these microbeads [6]. As a cadexomer microbead absorbs moisture, the dextrin matrix swells, the iodine molecules are released in a controlled manner and react with microbes and proteins in the wound milieu. Although PVP-I and CI have shown the same clinical results, PVP-I and CI ointments exhibit different iodine-releasing properties [7]. The amount of molecular iodine released from CI ointment is 9-fold higher than that released from PVP-I ointment when they are placed in a phosphate-buffered saline. At the same time, the amount of iodine reacted with wound exudates in PVP-I ointment was twofold higher than the amount of iodine that had reacted with wound exudates in CI ointment, suggesting that for PVP-I sugar ointment iodine is rapidly consumed by its protein component, and the antiseptic effect is promptly attenuated. For the CI

R. Klimaviciute et al. / International Journal of Biological Macromolecules 51 (2012) 800–807

Nomenclature amount of adsorbate adsorbed per one dm3 of solution at equilibrium (mmol/l) Ce equilibrium concentration of the solute in the bulk solution (mmol/l) initial concentration of the solute in the bulk soluCo tion (mmol/l) CCS cationic cross-linked starch degree of substitution calculated according to NDS (2-hydroxypropyl)-3-N,N,N-trimethylammonium groups EF effectiveness of cationic groups in the binding of iodine (mol I2 /mol cationic groups) EPTMAC 2,3-epoxypropyltrimethylammonium chloride G◦ Gibbs free energy of adsorption (kJ/mol) KC thermodynamic distribution constant Langmuir equilibrium constant (l/mmol) KL qe amount of the adsorbate adsorbed by adsorbent at the equilibrium (mmol/g) Langmuir maximum adsorption capacity (mmol/g) QL R universal gas constant (J/(mol K)) R2 linear correlation coefficient temperature (K) T CAe

ointment, iodine could be gradually consumed by protein components, and its antiseptic effect could be sustained longer. The difference in PVP-I and CI properties is related to different water adsorption rates and water diffusion into the polymer matrix [8]. In stable iodine solutions, i.e. in the presence of iodide ions and at pH < 7, the formation of triiodide (I3 − ) and/or pentaiodide (I5 − ) ions can take place. Anionic species of iodine could interact with cationic groups of various polymers and form polymeric iodophores as ionic complexes [9–11]. Anion exchangers with strong basic properties adsorbed polyiodides (containing up to 4.75 molecules of iodine) directly from water solutions, and the rate of interaction between such substances was high [9]. In aqueous solution, in the presence of KI, quaternary ammonium groups of polydialyldimethylammonium salts rapidly attached iodine during the formation of polymer–iodine complexes [10]. Insoluble iodinesaturated materials with primary, secondary, tertiary amino or quaternary ammonium groups are also suggested as bactericides for water treatment [11]. Starch is a low-cost natural renewable polymer that can be cross-linked with epichlorohydrin and cationized with 2,3epoxypropyltrimethylammonium chloride with a high reaction efficiency [12]. The obtained cationic cross-linked starches (CCS) with the degree of substitution (DS) varying from about 0.2 to 0.6 can adsorb from aqueous solutions various anions, such as anionic dyes [13], anionic complexes of disperse dyes and dispersing agents [14], hexavalent chromium [15] as well as anionic species of iodine [16]. The aim of this work was to prepare CCS–iodine complexes with different contents of cationic quaternary ammonium groups and iodine, to determine the stability of such complexes in buffer solution, their reactivity toward l-tyrosine, and to evaluate their antimicrobial properties. 2. Experimental 2.1. Materials The native potato starch (Antanavas Starch Plant, Lithuania), 2,3-epoxypropyltrimethylammonium chloride (70%, Fluka), and

801

epichlorohydrin (99%, Aldrich) were used as received. I2 -KI fixanal was purchased from Fluka. l-tyrosine (98%) was obtained from Sigma–Aldrich. Sodium tiosulphate (Na2 S2 O3 ), sodium hydroxide (NaOH) and sodium acetate (CH3 COONa) were of analytical grade. The bacterium ATCC and NCTC strains were used for investigations: Gram-positive spore-forming rods – Bacillus cereus (ATCC 11778), Gram-positive Staphylococcus aureus (ATCC 9144), Gramnegative rod-like Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (NCTC 6750). Tryptic soy agar (TSA) and tryptic soy broth (TSB) were used for bacterium cultivation and for antibacterial activity tests.

2.2. Preparation of CCS CCS was obtained by a two-stage process. The molecular mass of the anhydroglucoside unit (AGU) was assumed as a mole of starch. The native potato starch was suspended in water in order to obtain a 50% (w/w) slurry. The macromolecules of starch were cross-linked with 0.1 mol/AGU of epichlorohydrin (EPCH) in the presence of NaOH added until the pH value of the slurry reached 11. The cross-linking at 45 ◦ C was completed after 24 h. The cross-linked starch was washed with cold water, dried and then cationized with 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) in the presence of NaOH as a catalyst (the molar ratio AGU:EPTMAC:NaOH:H2 O was 1:0.2–1:0.04:16) at 45 ◦ C for 24 h. After the reaction, CCS was washed 5 times with water and 2 times with a water–isopropanol mixture, and dried. The number of cationic groups in CCS was expressed as the degree of substitution (DS), which was calculated from the nitrogen content estimated by the Kjeldahl method [17] after purification by Soxhlet extraction with methanol for 16 h. The cross-linked starch obtained in the reaction of starch with EPCH gathers additionally only hydroxyalkyl groups, so it is very difficult to determine the number of formed cross-links. The degree of CCS cross-linking was expressed as the amount of EPCH used in the cross-linking reaction (0.1 mol/AGU).

2.3. Equilibrium adsorption studies Iodine potassium iodide (I2 -KI) solutions were prepared from I2 KI fixanal (molar ratio of I2 to KI was 1–3) by dilution with distilled water to obtain the required concentration. The pH of I2 -KI solution was in the range 5.4–5.6 in all experiments. 0.1 g of dry CCS was placed into an Erlenmeyer flask, and 100 cm3 of I2 -KI solution of a desired concentration was added. The flask was stoppered and shaken for 30 min at a temperature of 30 ◦ C at a fixed shaking intensity in thermostated water bath with a temperature control of ±1 ◦ C (Memmert GmbH, Germany). Then the mixture was filtered through a glass filter, and the residual iodine concentration in solution was estimated by titration with Na2 S2 O3 solution. The hydrolysis of iodine was taken into account as described in [16].

2.4. Preparation of CCS–iodine complexes The precisely weighed dry CCS microgranules were poured over I2 -KI solution of a desired concentration and mixed with a magnetic stirrer for 30 min at a temperature of 20 ◦ C. To obtain CCS–iodine complexes with a maximum possible content of iodine, an excess of I2 -KI solution was used. The obtained suspension of the CCS–iodine complex was filtered through a glass filter and used without drying. The iodine content in prepared CCS–iodine complexes was determined by titration with Na2 S2 O3 solution and expressed as mole of I2 per mole of CCS cationic groups.

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2.5. Reactivity of iodine toward l-tyrosine

0.008

Damp microgranules of CCS–iodine complexes containing 0.0127 g of iodine were dispersed in 20 ml of 0.02 M sodium acetate solution, and 5 ml of 10 mM l-tyrosine solution was added. The initial concentrations of both iodine and l-tyrosine in the reaction mixture were equal to 2 mM each. The mixture was stirred with a magnetic stirrer at room temperature. The start of mixing was set as the beginning of the reaction. After desired time intervals, the excess of 0.005 M Na2 S2 O3 solution was added to each sample and after 10 min titrated with 0.005 M I2 -KI solution to determine the residual amount of non-reacted iodine.

q e (mmol/g)

0.006 0.004

0.002 0 0

Damp microgranules of CCS–iodine complexes containing a desired amount of iodine were dispersed in 20 ml of 0.02 M sodium acetate solution and stirred with a magnetic stirrer at a temperature of 20 ◦ C for 10 min. Then the mixture was filtered through a glass filter, and the concentration of free iodine in the filtrate was estimated. The residual amount of iodine was calculated as a difference between added and free amounts of iodine. 2.7. Antibacterial testing The antibacterial activity of CCS–iodine complexes was determined by testing different concentrations of samples against B. cereus, S. aureus, P. aeruginosa and E. coli bacteria by the brothdilution and spread-plate methods. CCS–iodine complexes were prepared just before the antibacterial testing and dispersed in distilled water. A range of concentrations (from 1024 to 2 ␮g/ml according to the concentration of added iodine) for each sample were prepared. The test bacteria (B. cereus, S. aureus, E. coli and P. aeruginosa) were streaked out on TSA plates and incubated at 37 ◦ C for 24 h. A representative colony was placed in 5 ml of TSB and incubated at 37 ◦ C for 24 h. S. aureus, B. cereus, E. coli and P. aeruginosa cultures containing 108 CFU/ml (colony-forming units, corresponding to Mc Farland’s 0.5) were prepared by dilution with TSB and used for antibacterial tests. The test organisms (100 ␮l) were added to each tube and incubated at 37 ◦ C for 24 h. At the end of this period, a small amount of the diluted mixture from each tube was pulled out and spread on TSA. The plates were incubated at 37 ◦ C for 48 h. The growth of bacterial cells was observed on agar plates. The lowest concentration of the bactericidal material at which no growth was observed was determined as the minimum bactericidal concentration (MBC) value. 3. Results and discussion 3.1. Equilibrium adsorption of iodine on CCS with different DS The microgranules of cross-linked N-(2-hydroxypropyl)-3N,N,N-trimethylammonium starch chloride (CCS) were prepared by reacting cross-linked starch with EPTMAC. Depending on the amount of EPTMAC used in the reaction, the CCS with DS of 0.14, 0.35, 0.54 and 0.85 was obtained. In stable iodine aqueous solutions, i.e. in the presence of iodide ions and at pH < 7, anionic species of iodine could be adsorbed by quaternary ammonium groups of CCS according to the ionexchange mechanism. The influence of DS on the equilibrium adsorption of iodine on CCS from aqueous I2 -KI solutions at a temperature of 30 ◦ C was investigated and expressed in mmol of iodine per one gram of CCS. The adsorption isotherms of iodine on CCS with different DS are presented in Fig. 1. According to the Langmuir adsorption model [18], the driving forces of adsorption were

0.004

0.008

0.012

C e (mmol/l)

2.6. Assessment of free iodine concentration in sodium acetate solution

Fig. 1. Adsorption isotherms of iodine onto CCS with different DS: 0.14 (♦), 0.35 (), 0.54 () and 0.85 (䊉). Symbols represent experimental data, and lines represent fitted curves of the Langmuir model. Adsorption temperature 30 ◦ C.

electrostatic interactions between quaternary ammonium groups of CCS and the anionic species of iodine. The considerable increase in adsorption capacity by upon increasing the DS of CCS (Fig. 1) confirms this presumption. The Langmuir equation may be presented as qe =

QL KL Ce , 1 + KL Ce

(3.1)

where qe (mmol/g) is the amount of the adsorbate adsorbed by the adsorbent at the equilibrium, Ce (mmol/l) is the equilibrium concentration of the adsorbate, QL (mmol/g) is the maximum adsorption capacity, and KL (l/mmol) is the Langmuir equilibrium constant. Eq. (3.1) can be linearized into five different linear forms [19]. For iodine adsorption on CCS, the highest values of the correlation coefficient (R2 > 0.99) were obtained when the first linear Langmuir equation was used: 1 1 Ce = + · Ce . qe QL KL QL

(3.2)

The values Ce /qe versus Ce were plotted, and the values of QL were calculated from the slope of the obtained straight line. The obtained values of QL were used to calculate the effectiveness of CCS cationic groups in iodine binding (EF) expressed as the number of moles of iodine per one mole of CCS cationic groups. A linear relationship was observed between the values of EF and the DS of CCS (see Fig. 2A). With an increase of DS, more than one molecule of iodine was adsorbed by one mole of cationic groups, i.e. some molecules of adsorbed iodine were present in the form of I3 − and others as I5 − or I7 − . The higher was DS, the higher portion of iodine was introduced into the complex with CCS as I5 − or I7 − . When the DS was 0.85, a marked part of adsorbed iodine was in the form of I7 − . The increase in EF with an increase of DS could be related to changes in the hydrophobicity of modified starch microgranules during iodine adsorption. The higher is DS, the higher number of cationic groups is located on the surface of microgranules and, thus, a higher number of I3 − ions covers the surface with iodine molecules exposed outside. The hydrophobicity of such microgranules is higher as compared to that of CCS microgranules with a lower DS. More hydrophobic microgranules could more easily adsorb iodine molecules from the solution. The fact that iodine adsorption onto CCS with a higher DS is favorable was confirmed by the values of changing the Gibbs free energy (G◦ ) (see Fig. 2B) determined by using the following equations: KC =

CAe , Ce

(3.3)

R. Klimaviciute et al. / International Journal of Biological Macromolecules 51 (2012) 800–807

2.5

DS of CCS

A 0

2

0

1.5

ΔG ( kJ / mol )

EF (mol/mol )

803

1 0.5 0

0.3

0.6

0.9

B

-5 -10 -15

0

0.3

0.6

0.9

DS of CCS

-20

Fig. 2. Dependence of EF (A) and G◦ (B) on DS of CCS.

(3.4)

where KC is the thermodynamic distribution constant, CAe is the amount of iodine adsorbed on CCS in one dm3 of the solution at equilibrium (mmol/l), Ce is the equilibrium concentration of iodine in solution (mmol/l), T is the solution temperature in K, and R is the universal gas constant (J/(mol K)). The thermodynamic distribution constant KC was determined by a linear regression analysis of the dependence ln qe /Ce versus qe [20]. The value of G◦ gives some information about the adsorbate affinity to the adsorbent and the driving forces of adsorption. The more negative the value of G◦ , the larger is the affinity, and the adsorption proceeds more spontaneously. One can see from data presented in Fig. 2B, with increasing DS the values of G◦ become more negative, i.e. the affinity of iodine to CCS increases with an increase of DS, and this dependence is linear. Moreover, according to Eq. (3.4), the more negative is the value of G◦ , the higher is the value of the distribution constant KC , i.e. the major part of the adsorbate on the adsorbent is at equilibrium. 3.2. Stability of CCS–iodine complexes in buffer solution The amount of iodine available for bactericidal action in iodophore (called “available” or “free” iodine) is the amount of iodine in equilibrium in the solution at the time of use. The total amount of iodine in a iodophore consists of free and reservoir iodine. It could be expected that the increase of iodine affinity to CCS with increasing, DS alongside the higher hydrophobicity of CCS–iodine complexes, could have a hold on the stability of CCS–iodine complexes. For this reason, the amount of free iodine released from CCS–iodine complexes of different compositions into the aqueous solution of 0.02 M sodium acetate has been determined. Fig. 3 reflects the stability of complexes prepared by adsorption of different amounts of iodine (different values of EF) onto CCS with the DS of 0.35. The stability of CCS–iodine complexes decreased and the amount of free iodine released from CCS–iodine complex increased with an increase of the EF value. Although the amount of total iodine in complexes increased from 0.28 to 1 mol/mol, the amount of free iodine increased only from 0.125 to 0.2125 mmol/l. Furthermore, the concentration of free iodine in buffer solution was independent of the amount of total iodine added to the solution when the value of EF < 1 mol/mol and slightly increased when EF = 1 mol/mol. The concentration of free iodine in the solution rose significantly for the CCS–iodine complex with a maximum possible amount of bound iodine (EF = 1.98 mol/mol) and depended on the amount of added iodine (see Fig. 3). It could be suggested that iodine incorporated into CCS–iodine complexes as triiodide

0.5

Free iodine (mmol/l )

G◦ = −RT ln KC ,

0.4 0.3 0.2 0.1 0 0

2

4

6

8

Added iodine (mmol/l ) Fig. 3. Concentration of free iodine as a function of added concentration of iodine in 0.02 M sodium acetate solution. EF of CCS–iodine complexes was 0.28 (), 0.425 (), 0.60 (), 1 () and 1.98 () mol/mol. DS of CCS was 0.35.

(EF ≤ 1 mol/mol) was more stable than in the case of pentaiodide (EF > 1 mol/mol). In order to evaluate the influence of DS on the stability, three groups of CCS–iodine complexes with a different amount of bound iodine were assessed. In the first group the DS of CCS was different (0.14, 0.35, 0.54 and 0.85), but the amount of bound iodine was the same (0.127 g per 1 g of CCS) (see Fig. 4A). Depending on the DS, 0.127 g/g of bound iodine gave different values of EF (0.615, 0.28, 0.21 and 0.16 mol/mol, respectively), i.e. EF < 1 mol/mol. The second group of complexes consisted of CCS with different DS and the amount of bound iodine so as to have the value of EF = 1 mol/mol (see Fig. 4B). The third group of complexes contained the maximum possible amount of bound iodine (according to data in Fig. 2A), i.e. maximum possible value of EF was 1.55, 1.98, 2.04 and 2.11 mol/mol at the DS value of 0.14, 0.35, 0.54 and 0.85, respectively (see Fig. 4C). By analyzing data presented in Fig. 4, some summing-up could be done. Primarily, the stability of CCS–iodine complexes increased with increasing the DS of CCS used for the complex formation. As an exception, the complex of CCS with DS of 0.14 and containing 0.127 g/g iodine could be mentioned (see Fig. 4A). When the content of iodine in complexes was less than 1 mol/mol (the first group of complexes), the concentration of free iodine in buffer solution was independent of the amount of iodine added with complexes at all DS values (see Fig. 4A). With an increase of iodine content in CCS–iodine complexes, the manner of iodine release changed (see Fig. 4B and C). When the amount of iodine in a complex was 1 mol/mol, an increase of free iodine concentration with increasing the amount of iodine added was observed when the DS of CCS

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0.4

A

0.12

Free iodine (mmol/l )

Free iodine (mmol/l )

0.15

0.09 0.06 0.03 0.00

B

0.3

0.2

0.1

0.0 0

2

4

6

8

2

0

Added iodine (mmol/l )

Free iodine (mmol/l )

1.0

4

6

8

Added iodine (mmol/l )

C

0.8 0.6 0.4 0.2 0.0 0

2

4

6

8

10

Added iodine (mmol/l ) Fig. 4. Concentration of free iodine as a function of added concentration of iodine in 0.02 M sodium acetate solution when DS of CCS was 0.14 (), 0.35 (), 0.54 () and 0.85 (). (A) Amount of bound iodine was 0.127 g/g; (B) EF = 1 mol/mol; (C) maximum possible EF.

3.3. Reaction of l-tyrosine with CCS–iodine complexes It is known [21] that free iodine reacts with the proteins of bacteria by iodization of tyrosine residuals and thus kills the bacterial organisms. For this reason, the reactivity of CCS–iodine complexes toward l-tyrosine has been investigated to evaluate their possible antimicrobial activity. The initial concentrations of total iodine and l-tyrosine were the same in all experiments and equaled 2 mmol/l. However, the amount of CCS–iodine complexes added to the reaction mixture, like the amount of free iodine released from the complexes (see Section 3.2) was the subject of DS and EF value. For comparison, the reaction of l-tyrosine with I2 -KI was examined. In this case, the concentration of total and free iodine in the reaction mixture was the same. As one can see from data presented in Fig. 5, the consumption of iodine in the reaction with l-tyrosine increased with an increase of the EF value of the CCS–iodine complex formed

2 Total iodine consumed ( mmol/l )

was 0.14 and 0.35 (especially in the case of DS = 0.14), whereas the concentration of free iodine remained constant when the DS of CCS was 0.54 or 0.85 (Fig. 4B). The concentration of free iodine in buffer solution was increasing with the increasing amount of added iodine when CCS–iodine complexes with maximum possible iodine content were used independently of the DS of CCS (Fig. 4C). These results clearly indicate that iodine stability in CCS–iodine complexes is related to the form of iodine species attached to CCS. CCS complexes with triiodide ions (EF ≤ 1 mol/mol) are more stable as compared to those with pentaiodide ions (EF > 1 mol/mol). By varying the DS of CCS and the amount of introduced iodine, CCS–iodine complexes of different stabilities in buffer solution can be prepared. Consequently, a different concentration of free iodine in the solution and a different ratio of total and free iodine content in the solution could be obtained.

1.5

1

0.5

0 0

25

50

75

100

Time (min ) Fig. 5. Iodine consumption versus time during l-tyrosine reaction of with I2 -KI (×) and CCS–iodine complexes with EF: 0.28 (), 1 () and 1.98 () mol/mol. DS of CCS was 0.35. Initial concentrations of total iodine and l-tyrosine were 2 mmol/l each.

from CCS with DS = 0.35. The consumption of iodine correlated with the initial concentration of free iodine in the reaction mixture (see Fig. 3). It could be pointed out that when bound iodine was in the form of pentaiodide (EF = 1.98 mol/mol), iodine consumption in reaction with l-tyrosine was very similar to that of I2 -KI despite the much lower concentration of free iodine in the solution (see Fig. 3). The antimicrobial activity of I2 -KI solution is limited in its clinical use due to a high concentration of free iodine, leading to an intensively irritating effect on the abraded tissue and mucous membranes. The obtained results demonstrate that by appropriately selecting the CCS–iodine complex composition could ensure the good antimicrobial properties at a low concentration of free iodine.

R. Klimaviciute et al. / International Journal of Biological Macromolecules 51 (2012) 800–807

80

A

50

Total iodine consumed (%)

Total iodine consumed ( % )

60

40 30 20 10

805

B

70 60 50 40 30 20 10 0

0 0

25

50

75

30

0

100

Time (min ) 90 Total iodine consumed ( % )

60

90

Time (min )

C

80 70 60 50 40 30 20 10 0 0

25

50

75

100

Time (min ) Fig. 6. Iodine consumption versus time during l-tyrosine reaction with CCS–iodine complexes when DS of CCS was 0.14 (), 0.35 (), 0.54 () and 0.85 (). (A) Amount of bound iodine was 0.127 g/g; (B) EF = 1 mol/mol; (C) maximum possible EF. Initial concentrations of total iodine and l-tyrosine were 2 mmol/l each.

The reactivity of three groups of CCS–iodine complexes, discussed in Section 3.2, toward l-tyrosine is presented in Fig. 6. When the amount of iodine in complexes was the same, i.e. in the first group of complexes, the reaction rate was unaffected by the DS of CCS (see Fig. 6A). Within 90 min of reaction, about 50% of total iodine was consumed in the iodization of l-tyrosine. It could be assumed that the concentration of free iodine (see Fig. 4A) was too low to influence the reaction rate. When iodine was released from CCS–iodine complexes with EF = 1 mol/mol (see Fig. 6B), approximately 70% of iodine was consumed within 90 min of reaction. A noticeable decrease in iodine consumption was observed only in the case of a complex of CCS with DS = 0.14, although the concentration of free iodine released from this complex was high (see Fig. 4B). Interesting results were obtained for CCS–iodine complexes with the maximum possible EF values (see Fig. 6C). Iodine consumption in the reaction was decreasing in the following order of DS: 0.35 ≈ 0.54 > 0.14 > 0.85, and the maximum iodine consumption was about 90% after 90 min. At the same time, the concentration of free iodine in the system decreased differently from the order of DS: 0.14 > 0.35 > 0.54 > 0.85 (see Fig. 4C). It could be suggested that both DS of CCS and the amount of incorporated iodine (composition of a complex) play a significant role in the reactivity of CCS–iodine complex toward l-tyrosine. In some cases the reactivity of CCS–iodine complexes could not be related directly to the concentration free iodine in the system. 3.4. Antimicrobial properties of CCS–iodine complexes The consumption of iodine in the reaction with l-tyrosine was most effective in the case of CCS–iodine complexes with the maximum value of EF (see Fig. 6C). The antibacterial activities of such

CCS–iodine complexes (the DS of CCS was 0.14, 0.35, 0.54, 0.85 and the maximum values of EF were 1.60, 1.82, 2.01 and 2.48 mol/mol, respectively) were determined by testing different concentrations of the complexes against Gram-positive spore-forming rods – B. cereus (ATCC 11778), Gram-positive S. aureus (ATCC 9144), Gramnegative rod-like E. coli (ATCC 8739), and P. aeruginosa (NCTC 6750) by the broth-dilution and spread-plate methods. The MBC values of the samples obtained in spread-plate tests, taking into account the concentration of added iodine and the concentration of CCS–iodine complexes, are summarized in Table 1, which shows that the antibacterial activity of the complexes depended on the type of bacteria. All the complexes studied show a high antibacterial activity against S. aureus (MBC value 256 ␮g/ml). Against E. coli, the MBC value of 256 ␮g/ml was obtained for CCS–iodine complexes from CCS with the DS of 0.14 and 0.54, and 512 ␮g/ml – for CCS–iodine complexes obtained from CCS with the DS of 0.35 and 0.85. The MBC value of 512 ␮g/ml was determined for P. aeruginosa for all complexes. Slightly higher MBC values obtained in the case of B. cereus bacteria could be related to the ability of such bacteria to produce protective endospores. The obtained data on the antibacterial activities of CCS–iodine complexes are similar to the results obtained for iodine–lithium–␣-dextrin complexes [22]. In order to reach the same concentration of total iodine in the dispersion, a lower concentration of CCS–iodine complex with higher DS and bounded iodine levels is sufficient (see Table 1). For example, to achieve the same antibacterial activity against S. aureus, i.e. when the MBC value established according to the added iodine concentration was equal to 256 ␮g/ml, the amount of added CCS–iodine complex decreased from 1073 to 398 ␮g/ml with increasing the DS of CCS. As a result, a lower residual level of CCS will remain in the dispersion when all iodine will be consumed

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Table 1 MBC values of KI-I2 and CCS–iodine complexes of different compositions against investigated bacteria species. Composition of CCS–iodine complex

MBC (according to the concentration of added iodine) (␮g/ml)

DS

EF (mol/mol)

B. cereus

0.14 0.35 0.54 0.85

1.60 1.82 2.01 2.43

512 256 1024 512

MBC (according to the concentration of CCS–iodine complex) (␮g/ml)

S. aureus

P. aeruginosa

E. coli

B. cereus

S. aureus

P. aeruginosa

E. coli

256 256 256 256

512 512 512 512

256 512 256 512

2146 599 1930 796

1073 599 482 398

2146 1198 964 796

1073 1198 482 796

Fig. 7. SEM microphotographs of CCS (DS = 0.35) microgranules before (A) and after (B) treatment with ␣-amylaze.

to kill the bacteria. Another advantage of CCS–iodine complexes as antimicrobial agents is the biodegradability of their polymeric matrix. As one could see in the SEM photographs presented in Fig. 7, the CCS microgranules were easily destroyed during the enzymatic treatment. Thus, after iodine consumption for antibacterial effects, the remaining matrix of CCS will be biodegradable.

4. Conclusions CCS–iodine complexes containing different amounts of quaternary ammonium groups and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solutions. The equilibrium adsorption studies showed that with an increase of DS from 0.14 to 0.85 the amount of iodine adsorbed on CCS increased linearly from 1.55 to 2.39 mole of iodine per one mole of cationic groups (EF value). The increase in EF value was related to the increase in the affinity of iodine to CCS, i.e. the values of changing the Gibbs free energy (G◦ ) became more negative with an increase of the DS. The influence of the DS of CCS and EF on the stability of CCS–iodine complexes in the solution of 0.02 M sodium acetate has been investigated. At the same DS, the stability of CCS–iodine complexes decreased with an increase of the EF. With an increase of the DS of CCS, the stability of CCS–iodine complexes increased. When the content of iodine in the complexes was below 1 mol/mol, the concentration of free iodine in buffer solution was independent of the amount of total iodine added with the complexes. The effect of the DS of CCS on iodine consumption in l-tyrosine iodination depended on the EF value of CCS–iodine complexes. The reaction with l-tyrosine was unaffected by changes in DS when the EF value was below 1 mol/mol. However, iodine consumption increased significantly with an increase of the EF value. In most cases, the reactivity of CCS–iodine complexes toward l-tyrosine was independent of the concentration of free iodine in solution.

The antibacterial activities of CCS–iodine complexes against B. cereus, S. aureus, P. aeruginosa and E. coli were determined by the broth-dilution and spread-plate methods. All compositions showed a high antibacterial activity in the range 256–512 ␮g/ml depending on the concentration of added iodine. The obtained results demonstrate that an appropriate selection of the CCS–iodine complex composition (the DS of CCS and EF) could ensure good antimicrobial properties while keeping a low concentration of free iodine in the system. The main advantage of using CCS–iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix.

Acknowledgement The authors are grateful to the Research Council of Lithuania for the financial support of the project MIP 53/2010.

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