Antimicrobial Chitosan and Chitosan Derivatives: A Review of the

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Antimicrobial Chitosan and Chitosan Derivatives: A Review of the Structure-Activity Relationship Priyanka Sahariah, and Már Másson Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01058 • Publication Date (Web): 21 Sep 2017 Downloaded from http://pubs.acs.org on September 22, 2017

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Antimicrobial Chitosan and Chitosan Derivatives: A Review of the Structure-Activity Relationship Priyanka Sahariaha, Mar Massona* a

Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland

KEYWORDS. Chitosan, structure-activity relationship, antimicrobial chitosan derivatives, applications of chitosan, mode of antibacterial action of chitosan.

ABSTRACT. This review gives an updated overview of the current state-of-the-art for antimicrobial chitosan and chitosan derivatives and the effects of structural modifications on activity and toxicity. The various synthetic routes introduced for chemical modification of chitosan are discussed and the most common functional groups are highlighted. Different analytical techniques used for structural characterization of the synthesized chitosan derivatives are discussed and critically evaluated. For the purpose of this review, the antimicrobial chitosan derivatives have been classified on the basis of the type of functional group conjugated to the polymer backbone. In each case, the influence of the degree of substitution on the biological properties has been examined. Finally, we have summarized the collective information and suggested future directions for further research in order to improve our understanding of the bioactivity and to develop more useful chitosan conjugates.

ACS Paragon Plus Environment

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1. Introduction Antibiotics have played a major role in preventing and controlling the spread of pathogenic microorganisms. However, the emergence of antibiotic resistance and new pathogenic strains, as well as a lack of appropriate therapeutics, has led to infections which still remain a significant cause of disease and mortality in modern societies. There has been a growing interest in the use of new agents, such as antimicrobial polymers, as alternatives for therapy and disinfection. Chitosan, a biopolymer of marine origin, has recently attracted attention due to its significant antimicrobial properties and the advantages of being non-toxic, biodegradable and biocompatible1, 2. However, the antimicrobial properties are mostly limited to pH values below 6. This can restrict applications and bioactivity studies under neutral and physiological conditions. In order to overcome this limitation, chitosan has been synthetically modified to give derivatives having increased activity and improved aqueous solubility. The most common functional groups present in antimicrobial chitosan derivatives are quaternary ammoniumyl, guanidinyl, carboxyalkyl, hydroxyalkyl, thiol-containing groups and hydrophobic groups, such as long alkyl chains, and substituted phenyl and benzyl rings3-10. However, many other substituents on chitosan have also been studied for other applications such as adsorption enhancement11,

12

.

These antimicrobial chitosan derivatives have been widely applied in various fields, as is evident from the large number of publications in recent years.

Although there is a growing interest in the chemical derivatization of chitosan and a number of promising chitosan derivatives have been reported13-17, there has been no agreement on the structural features that might contribute to optimal antimicrobial activity and low toxicity. This lack of consensus can partially be attributed to differences in the methods used for


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characterization of synthesized derivatives, different activity assays and the conditions used in these assays. These differences can be significant, leading to conflicting conclusions. Therefore, a careful and critical examination of existing literature on antimicrobial chitosan and chitosan derivatives is needed to provide a good overview of the structure-activity relationship for these materials. In this review, we aim at classifying the antimicrobial chitosan derivatives on the basis of the type of functional group conjugated to the polymer backbone and the degree of substitution. The various synthetic methods used for obtaining such modifications are also discussed. Special attention is given to the more common functional groups that are known to improve bioactivity. Studies of the correlation between structure and activity are discussed in detail, and we have summarized what we consider to be the collective findings of these studies. Furthermore, this review addresses by what means the methods used for synthetic modification of chitosan and their structural characterization can affect the antimicrobial activity, the study of the modes of action, toxicity as well as the investigation of applications in various fields. 2. General properties of chitosan Chitosan is a linear polymer occurring naturally only in certain fungi (Mucoraceae)18 and is chemically composed of glucosamine and N-acetylglucosamine monomers linked through β(1−4)glycosidic linkages (Figure 1). The fully N-acetylated form of this polymer, called chitin, can be derived from the exoskeleton of insects and crustaceans, such as shrimps, lobsters and crabs. Chitin can be chemically converted to its partially deacetylated form called chitosan19 and is the main commercial source of chitosan. Chitin is the second most abundant biopolymer after cellulose19 and its structure differs from cellulose in that a hydroxyl group has been replaced by


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an amino group at the C-2 position. Chitosan contains three types of nucleophilic functional groups: a C-2 NH2 group, a C-3 secondary OH group and a C-6 primary OH group. Chitosan has been chemically modified either at the amino group or at the hydroxyl groups to produce derivatives containing cationic or other hydrophilic and hydrophobic moieties. The importance of chitosan as an antimicrobial agent has been observed in various studies20-22. Its ability to inhibit microbial growth is observed only in acidic medium, where the polymer is soluble and carries a net positive charge20, 23. This property limits the use of chitosan in several bioactivity measurements which are water based. Therefore, derivatization of chitosan is particularly aimed at improving the solubility of chitosan in aqueous medium and at the same time enhancing its antimicrobial properties. For example, quaternizing the 2-amino group or introduction of quaternary ammonium groups and cationic groups have improved the solubility and antimicrobial activity of chitosan24-26, whereas introduction of hydrophobic groups such as Nacetyl (up to DS = 0.5) increases the solubility in the case of low Mw chitosan without significantly improving its antimicrobial properties27.


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Primary C-6 OH group

O

OH *

O

O

OH

NH HO O

O O O

HO

NH2

NH2

OH

*

O

n

SecondaryC-2 NH2 group

Secondary C-3 OH group

Chemical structure of Chitosan O

OH **

O

O

O

NH O

OH

* Deacetylation

O

O O

HO

OH

OH

NH HO O

*

O

OH

NH2 HO O

O O O

HO

n

NH O

O NH

NH2

OH

O

Chitin

O

*

n

Chitosan pH = 5.5 OH *

O

O

OH

NH3 HO O

O O O

HO

NH O

OH

NH3

O

*

n

Protonated form of chitosan

Figure 1. Figure showing the structure of chitin, chitosan and the protonated form of chitosan.

3. Antimicrobial chitosan derivatives Owing to the poor solubility of chitosan in aqueous medium, synthetic modifications have mostly been carried out in acidic-aqueous medium or under heterogeneous conditions where the polymer is only partially dissolved in the reaction medium. Such conventional methods usually result in products that can be substituted at all three reactive centers of chitosan, i.e., the amino group and the two hydroxyl groups, and this ultimately results in a heterogeneous product or a product having a low degree of substitution. To overcome these issues, different types of protecting groups were introduced in chitosan chemistry to mask either the 2-amino group or the 3 and 6-hydroxyl groups. The commonly used protecting groups for the amino group are


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phthaloyl28, 29 and acetyl, while triphenylmethyl30, trimethylsilyl31, tertiarybutyldimethylsilyl32, 33 and acetyl are used for the hydroxyl groups (Scheme 1). The advantages of using protecting groups are that it allows selective modifications at the reactive centers, allows reactions in homogeneous medium and gives a high degree of substitution in the products4, 26, 34. Examples of antibacterial chitosan derivatives synthesized using protecting groups are N,N,N-trimethyl, Nalkyl-N,N-dimethyl, N,N-dialkyl-N-methyl, N-guanidinyl, N-2-acetyltrimethylammoniumyl, N-2pyridiniumyl and N-2-acetyl piperidine4, 25, 26, 34, 35. The introduction of several functional groups, such as trimethyl, 2-hydroxy-3-trimethylammoniumyl, guanidinyl, trimethyl ammmoniumyl, pyridiniumyl, and quaternary alkyl groups, provides a permanent positive charge to the polymer, which improves its solubility in aqueous medium, thereby enabling bioactivity measurements at pH 7.

Scheme 1. Scheme showing the common protecting groups used in chitosan chemistry.


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3.1. Chitosan containing quaternary ammonium groups Chitosan derivatives carrying a permanent positive charge have been obtained by direct quaternization of the amino N-atom or by introducing quaternary ammonium groups at any of the three reactive centers (the two OH groups and the NH2 group). The common method for quaternizing the N-atom in chitosan is by treatment with methyl iodide in the presence of NaOH to form the N,N,N-trimethyl chitosan (TMC) (2a, 2b; Scheme 2). Several studies have been reported which have used this approach to obtain different degrees of quaternization in TMC24, 36

. However, when N-methylation is performed using this condition, it often leads to partial O-

methylation37. Later studies have reported procedures to obtain TMC without O-methylation using acidic medium38 or heterogeneous medium39. However, fully quaternized TMC (DS = 1) can only be obtained by treating the O-protected chitosan precursor (TBDMS-chitosan) with MeI and Cs2CO3 in NMP as the reagents (2c)34. Quaternary ammonium groups have been introduced into unmodified chitosan by reaction with epoxides, such as glycidyl-trimethylammonium chloride (2d, 2e)40, by reacting chitosan with alkyl halides, such as 3-Chloro-2hydroxypropyltrimethyl ammonium chloride (2f)41, or by reductive alkylation followed by methylation (2g)42. The use of several protecting groups has also been observed in the synthesis of quaternary chitosan derivatives. The 6-O-triphenylmethyl protecting group was used for preparing quaternary piperazine (2h–k) pyridine (2l) and N-2-acetyl trimethylammonium (2m) chitosan derivatives35, 43. The 3,6-O-di-tertiarybutyldimethyl silyl protecting group was used for obtaining N-selective modifications, such as quaternary N-(2-(N,N,N-trialkyl ammoniumyl (2n) and 2-pyridiniumyl)acetyl) chitosan (2o)4,

44

and N-2-acetyl quaternary ammonium chitosan

derivatives containing longer alkyl chains (2p, 2q)44. This protected chitosan precursor was also used for preparing longer alkyl chain quaternary derivatives by introducing the alkyl groups first by reductive alkylation to form the monoalkyl or dialkyl derivative, followed by treatment with


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methyl iodide to produce its quaternary form (2r, 2s)25, 34. This method gave a higher degree of substitution of alkyl groups in the products in comparison to the ones obtained using unmodified chitosan45-47.

*

OH O

O HO

*

*

OH O

O HO

N

OH *

O

O HO

N

3

Ph

O

OH O

O HO

Cl

*

O

*

O TBDMSO

N

O

R1

n = 1,4

i) RCHO; NaBH4 ii) MeI;NMP N

OH

Br n

Br

*

Cl HO

O

OH O

* Cl

O HO

O

HN

*

O 3

N

O HO

N

N

O HN

3

*

OH O

O HO

* O

HN

*

OH O

O HO

* O

HN

Cl *

Cl

O HO

H N

N

2i

N N

2k

*

NH

N

N

2q

O HO

* O

N

HO

2d 2f

n n = 1,4

*

HN

HO

O R

O

N

2j

2o

OH O

O

OH O

*

OH NH

11

N

*

pH = 7 *

N

*

O

O HO

NH2

2p

O HO

2b 2g

n

O

N

*

N R 3 R2

R

H N O

OH O

MeI;NMP

*

NH2

NH2

n = 1,3

Cl

2n

O HO

O

Cl

O N

HN

OTBDMS O *

*

N H

N N

N R 1 R2

*

OH O

N

n

2h

O HO

*

Me3N

O

HN

n

OH O

i) RCHO; NaBH 4 ii) MeI;NMP R1=alkyl, 2r R1,R2=alkyl, 2s

N

O O HO

Cl

*

Ph

Ph *

*

MeI/NMP R1,R2,R3=H, 2c

O Cl

2l

*

2m

N

O

HN

O HO R3

N

MeI/NaOH NMP

*

OH O

O

HN

2a

*

*

N

pH = 10

O O

* O HO

*

NH2

2e 11

Scheme 2. Synthetic scheme for quaternary chitosan derivatives. 3.2. Chitosan containing alkyl and aromatic groups Introduction of alkyl groups at the O or N positions of chitosan is commonly carried out by heating chitosan in the presence of alkyl halides and a strong base, such as NaOH48, 49. Using an ionic liquid as a medium under basic conditions, different alkyl halides, namely ethyl, butyl, dodecyl and cetyl, have been reacted to give a series of mixed N,O-alkylated product (3a, Scheme 3) with a degree of substitution (DS) ranging from 0.35–0.778. Selective alkylation at the 2-amino position of chitosan is usually carried out by reductive amination whereby the


8

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initially formed Schiff base intermediate is reduced with the help of a reducing agent to yield the final product (Scheme 3). This method has been used to react various substituted benzaldehydes with chitosan in acetic acid resulting in derivatives with DS varying from 0.12 to 0.307 (3b). Another study has reported the formation of Schiff bases of chitosan in methanol using citral (DS=0.82) and cinnamaldehyde (DS=0.84)50 (3c). Alkyl and aromatic groups have also been introduced into the amino group of chitosan by acylation reactions50. Such reactions are usually carried out by reacting chitosan with acyl chlorides in the presence of bases, or by heating with anhydrides. Acetyl chloride and dipropionyl chloride were used for the synthesis of the acylated chitosans O,O-diacetylchitosan (DACT) (3d) and O,O-dipropionyl chitosan (DPPCT) (3e) having DS values of 0.55 and 0.77 for the hydroxyl groups, and 0.05 and 0.13 for the amino groups, respectively51. Another study reported the synthesis of N-(4-carboxybutyroyl) chitosan (3f) using varying equivalents (0.1, 0.3, 0.6 and 1.0 mol) of glutaric anhydride per glucosamine unit to give derivatives having DS values of 0.10, 0.25, 0.48 and 0.5352. Low Mw chitosan (5.5 kDa) was acylated with the N-hydroxysuccinimide ether of 3-hydroxytetradecanoic acid in a water-DMF mixture to produce a water soluble derivative (3g)53. The synthesis of a chitosanthioglycolic acid derivative (3h) and a N-acetyl-L-cysteine-chitosan conjugate (3h) was carried out by carbodiimide activation of thiogycolic acid and N-acetyl-L-cysteine, respectively, with 1ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride followed by reaction with the amino group of chitosan54,

55

. A similar carbodiimide-activated procedure was also used for

preparing a range of 4-carboxybenzenesulfonamide chitosan derivatives with DS varying from 0.04–0.13 (3h)56.


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2 (C

H

2) 2N

H

2

60° C, 3h ClC HC 2 O OH /N aO H

N H

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Scheme 3. Synthetic scheme for chitosan derivatives containing alkyl, aminoalkyl, aromatic, carboxyalkyl and guanidinyl groups. 3.3. Chitosan with substituents having free amino or hydroxyl groups Alkyl halides containing free amino groups (aminoethyl) and substituted amino groups (dimethylaminoethyl and diethylaminoethyl) have been reacted with chitosan in the presence of NaOH to obtain N- or O-modified chitosan derivatives (3i). These derivatives were synthesized from chitosan having an average Mw of 310 kDa and two different degrees of acetylation (DA),


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50% and 90%57,

58

. A series of N- and O-modified aminoethyl chitosan derivatives having

different molecular weights (1.5, 34, 99 and 358 kDa) were also synthesized by a similar method. In this study, it was concluded that due to the high reactivity of the terminal amines, in comparison to the reactive centers of chitosan (including the 2-amino group), the architecture of the final product could be a polyethylimine derivative or even a branched polyethylimine derivative57. An aminoalkyl group was selectively attached to the O-positions of chitosans by initially activating the OH groups with p-benzoquinone, followed by reaction with ethylenediamine (3j). The O-modified chitosan derivative was then deacetylated using NaOH and hydrolyzed using acetic acid to produce a range of derivatives having different DA and Mw59. The amine containing functional group guanidine was attached to chitosan by treating with aminoiminomethanesulfinic acid (AIMSOA) (3k) in aqueous medium. The conjugates were synthesized from starting chitosan having different Mw, which resulted in the final products having Mw values between 13-164 kDa. The use of AIMSOA in the molar ratios 1:1, 1:2 and 1:3 gave a DS of 0.14-0.50 in the products3. Other studies have reported chitosan treatment with cyanamide at elevated temperatures in acidic medium to give guanidinylated chitosan derivatives (3k) (DS of 0.08 and 0.01)60. Chitosan biguanidine (containing three free amino groups) was synthesized by reacting chitosan with dicyandiamide (1:2 ratio) under acidic conditions to get partly biguanidinylated chitosan (3l)61 . 3,6-O-di-TBDMS chitosan was also used for the synthesis of different guanidine containing derivatives. The protected chitosan was reacted with N,N'-di-boc-N''-triflylguanidine in the presence of triethylamine/DCM to produce N-(N,N-di-bocguanidiniumyl)-3,6-O-di-TBDMS chitosans having different degrees of substitution 0.1, 0.3, 0.5 and 1.0). The removal of the protecting groups TBDMS and Boc yielded the final product Nguanidiniumyl chitosan (3m). A similar synthetic strategy was utilized to attach guanidine


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groups having spacers, for example N-(2-guanidiniumylacetyl) chitosans and N-(6guanidiniumylhexanoyl) chitosans (3n)26. Hydroxyalkyl derivatives of chitosan are commonly prepared by treating chitosan with epoxides under heating. In some studies, however, the epoxide is generated in situ under alkaline conditions which then undergoes a nucleophilic substitution reaction with the 2-amino or 6hydroxyl group of chitosan62,63. In such reactions, the pH is a critical factor for obtaining high DS, since use of high pH would result in hydrolysis of the reagent. The reaction was therefore carried out under slightly alkaline conditions (pH 8 was maintained) and a low NaOH concentration was used in the synthesis. N-halamine modified chitosan derivatives (chitosan modified with 3-glycidyl-5,5-dimethylhydantoin) (3o) were synthesized by reacting varying molar equivalents (0.8 to 1.5) of 3-glycidyl-5,5-dimethylhydantoin with chitosan in 1% acetic acid at 65 °C to obtain a DS range of 0.35–0.4564. Selective 3,6-O-di-hydroxyethyl chitosan derivatives (3p) were synthesized by reacting chlorohydrin with N-phthaloyl chitosan65, while 3,6-O-di-hydroxypropyl chitosan derivatives (3q) having different DS and Mw were obtained by reacting chitosan with propylene oxide66 using NaOH and isopropanol in both the reactions. 3.4. Chitosan containing carboxyalkyl groups Carboxymethyl chitosan (CMC) derivatives are commonly synthesized by addition of NaOH solution to a suspension of chitosan in isopropanol, followed by addition of monochloroacetic acid5, 6, 67-70. A study reported that the introduction of the carboxymethyl functional group at the reactive centers (NH2 and OH) of chitosan can be directed by controlling the reaction conditions. N,O-CMC (3r) was prepared by stirring chitosan in isopropanol and NaOH for 1 h, followed by heating the mixture at 60 °C for 3 h in the presence of monochloroacetic acid, while O-CMC (3s) was synthesized by suspending chitosan in NaOH solution and reacting with monochloroacetic


12

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acid at 0–30 °C for 5–24 h71. The synthesis of O-CMC has been observed in many studies, where selective O-methylcarboxylation has been obtained by using similar reagents but maintaining the temperature between 18–30°C6,

72, 73

. Thus, the reaction temperature seems to be the decisive

factor for producing exclusively O-CMC. The synthesis of N,O-carboxyethyl chitosan (CEC) (3t) has been reported by treating chitosan with NaOH/isopropanol and chloropropionic acid at 60 °C74. The formation of mono- and di-N-CEC has been reported by treating chitosan with NaHCO3 and chloropropionic acid in water. The use of varying equivalents of chloropropionic acid in the reaction resulted in products having DS varying from 0.2-1.675. Regioselectivity of carboxyethylation at the amino position of chitosan has been studied using varying equivalents of chloro-, bromo- and iodopropionic acids. The results led to the conclusion that N-CEC (3u) can be formed exclusively using chitosan/NaHCO3/H2O and that the DS in the product increases in the order iodo˂bromo˂chloro76. 3.5. Chitosan containing multiple functional groups Chitosan derivatives containing multiple functional groups at either the N or O positions or both positions have been synthesized and studied for antimicrobial properties. Quaternary chitosan, namely N,N,N-trimethyl-O-[(2-hydroxy-3-trimethylammonium)propyl] chitosan (DS at O = 0.22-0.42 and DS at O = 0.05, 0.10 and 0.20)77 has been prepared using TMC as a precursor. TMCs with varying degrees of trimethylation were O-carboxymethylated (DS = 0.44-0.61) to different extents using various reaction times77,

78

. In another study, water soluble chitosan

derivatives carrying dual functional groups: O-(2-hydroxy-3-trimethylammonium)propyl and Nbenzylimine, N-benzyl or N-benzyl-N,N-diethyl were synthesized by epoxidation, reductive amination and alkylation40 (Figure 2). Chitosan derivatives having N-alkyl or aryl groups, such as octyl, phenyl containing electron donating and electron withdrawing groups, pyridine and


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thiophene, were synthesized by reductive amination and then quaternized by introduction of an O-(2-hydroxy-3-trimethylammonium)propyl group at the 6-O positon79. Introduction of multiple functional groups at the 2-amino position of chitosan has been carried out by first protecting the hydroxyl groups with TBDMS. Addition of different equivalent quantities of acetyl chloride, stearoyl chloride and an excess of methyl iodide, followed by deprotection, resulted in the product N-acetyl-N-stearoyl-N',N'',N'''-trimethyl chitosan, where DSacetyl = 0.05−0.97, DSstearoyl = 0−0.28 and DStriemthyl = 0.03−0.9527. The O-CMC derivative has been used for the introduction of several functional groups at the amino position, such as the introduction of dendrimers80, derivatives82, quaternized derivatives73,

74, 83

81

, Schiff's bases72, acylthiourea

, terephthaloylthiourea cross-linked derivatives84,

thiosemicarbazone85, and poly(N-vinylimidazole)86, as well as the N,N,N-trimethyl group78. Quaternization of the amino group is a commonly performed reaction on carboxylmethyl chitosan derivatives. Examples are quaternization of various O-carboxymethyl-N-aryl chitosans by the (2-hydroxy-3-trimethylammonium)propyl group87 and quaternization of N,O-(2carboxymethyl)chitosan or N,O-(2-carboxyethyl) chitosan using glycidyldimethylbenzyl ammonium chloride under basic conditions88, 89. Such a quaternary chitosan derivative, namely O-carboxymethyl-N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan, was utilized for introducing polyamidoamine (PAMAM) dendrimers. The reaction was performed by EDC/NHS coupling between the carboxylic group of O-CMC and the free amino group of the PAMAM dendrimer. The grafting ratio of PAMAM on O-CMC was calculated to be 0.17 and 0.2081. N,Ocarboxyethyl chitosan was quaternized by introducing a (2-hydroxy-3-trialkylammonium)propyl group (where alkyl = trimethyl, triethyl, tripropyl, tributyl and dimethylbenzyl) at the amino group74.


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Figure 2. Figure showing chitosan derivatives containing multiple functional groups; groups: (a ) N,N,N-trimethyl chitosan containing various substituents at 6-OH position; (b) Ocarboxymethyl chitosan containing various substituents at 2-NH2 position; (c) O-carboxyethyl chitosan containing various substituents at 2-NH2 position; (d) N-hydroxypropyl-N'.N'.N'trimethyl chitosan containing various substituents at 6-OH position and (e) chitosan containing three different substituents at the 2-NH2 position. 3.6. Chitosan containing amino acids and peptides Chitosan has been conjugated to several cationic amino acids as well as peptides (Figure 3). The synthesis of chitosan-arginine and chitosan-guanidine conjugates has been observed in many studies90, 91. The carboxyl group of arginine was first activated by NHS/EDC at a first molar ratio and then coupled to the amino group of chitosan during different reaction times. This resulted in chitosan-arginine derivatives having DS in the range 0.08-0.2892. Chitosan-peptide conjugates have also been studied for their antibacterial properties. Acid functionalized chitosan was grafted


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onto a protected polypeptide by a coupling reaction and thereafter the protecting group was removed, followed by formation of a nanoparticle93. Another study reported two series of cationic peptidopolysaccharides, namely chitosan-polylysine and chitosan-poly(lysine-ranphenylalanine) having different grafting ratios (DS = 0.01–0.23). In this study, 6-Otriphenylmethyl chitosan was treated with NCA monomers (containing lysine and phenylalanine) in DMF to form the copolymers by NCA ring-opening polymerization initiated from the amine groups of chitosan. The final conjugates were obtained by deprotection using HBr. These conjugates were then investigated for antimicrobial efficacy towards a series of Gram positive bacteria, Gram negative bacteria and fungi94. The short antimicrobial peptide anoplin has been conjugated to azido-functionalized chitosan using Cu-mediated 'click chemistry'. Alkynefunctionalized anoplin monomers were grafted on the biopolymer using both the C- and the Nterminal to produce conjugates having three different DS (0.06−0.23). These conjugates were then tested for antibacterial activity against S. aureus and E. coli and hemolytic activity towards human red blood cells95.

Figure 3. Figure showing the amino acids and peptides commonly attached to chitosan.


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4. Characterization of chitosan derivatives Characterization of chitosan derivatives is an essential step following synthesis which helps in determining the attachment of the functional group and any other structural changes within the molecule. This includes determination of degree of substitution and molecular weight or chain length of the resulting polymer derivative. 4.1. Structural determination and calculation of degree of substitution. Structure−activity relationship studies require precise characterization of the synthesized materials so that the contribution of the structural modification or the covalently attached functional groups on the activity or toxicity can be correctly evaluated. A number of different spectroscopic techniques have been used for identification and characterization of the modified chitosan derivatives, such as 1H-NMR, 13C NMR, FT-IR, and UV, as well as elemental analysis. The chemical modification of chitosan by a particular functional group is usually expressed as the degree of substitution (DS) of chitosan. The most commonly used spectroscopic technique for the characterization of the chitosan derivatives is FT-IR. This method can provide useful information about the presence of certain functional groups on chitosan. Chitosan derivatives containing groups such as azide, guanidine, carboxyl, carbonyl, amides, alkyl, aromatic, etc., have been analyzed using IR spectroscopy25,

26, 92, 95-101

. Similarly, UV spectrophotometry has

also served as a useful tool for detecting the presence of functional groups such as salicyloyl, guanidine (Sakaguchi color indication test), aromatic rings, 3-glycidyl-5,5-dimethylhydantoin3, 26, 64, 98, 102

in chitosan. FT-IR and UV-vis spectroscopy can be used for structural confirmation of

new materials and identification of contaminants in the products. These two techniques are also useful for determining product mixtures and the quality of starting materials. However, the peak intensities in FT-IR and UV-vis spectroscopy are dependent on the quantity or concentration in


17

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Page 18 of 65

the sample and contaminants or other substituents with overlapping absorption can interfere in the measurement. Therefore, such methods are not very reliable for determining the exact DS in the products. Again, since elemental analysis provides only the % of the different atoms present in the polymeric chain, with this method it is difficult to identify whether the desired modification in the product has been obtained without any intermediate or side product formation. This method can also be misleading when the modification occurs at the different reactive centers of chitosan. The presence of contaminants can also have a significant effect. The most useful and accurate technique for characterizing the products and calculating the DS in the chitosan derivatives is the use of 1H-NMR spectroscopy5,

8, 56, 77, 81, 93, 94

. While the other

techniques can give us only an indication of what kind of product has been formed, 1H-NMR spectroscopy is useful for detecting the attachment of functional groups on the chitosan backbone and also the position of attachment (-OH or –NH2). It is often possible to distinguish the peaks belonging to contaminants, reagents or solvent residues from those of the product peaks. Absolute purity is therefore not a requirement. A comparison of the integrals of the signals in the monomeric unit with that of the newly attached functional group can provide a very accurate determination of the DS in the products. The degree of acetylation for chitosans can also be calculated in a similar manner using 1H-NMR103. Other NMR techniques used for determining the structure of the chitosan derivatives are 13C-NMR49, 97, 104-106, 1H-1H COSY44, 79, 95

and 1H-13C HSQC44, 52, 95. The distribution of the substituents along the polymer chain (random or clustered) can also be

an important parameter affecting bioactivity and physicochemical characteristics. Distribution of the N-acetyl groups in chitosan can affect the antimicrobial activity107,

108

but similar detailed

studies of the effect of the distribution of other substituents is still lacking.


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Biomacromolecules

4.2. Molecular weight analysis Determination of the size of the chitosan and the modified chitosan derivative is important, as the size of the polymer chain can be an influential factor in determining its physicochemical properties as well as its antimicrobial properties. An approximate method for separating the chitosans or their derivatives above a certain Mw range is by dialyzing the samples using membranes having different Mw cut-offs, where the low Mw polymers or the oligomers can be easily removed109. Other methods that have been used for determining the average Mw for chitosan and its derivatives are viscometry32, 44 or the end-reducing assay24, 32, 39, 44, which is only suitable low molecular weight substances. The viscometric average molecular weight Mv is determined by measuring the intrinsic viscosity of the polymer and then calculating the Mw using the Mark-Houwink equation110. In this method, the determination of Mw for chitosan is calculated using viscosity constants,. The viscosity constant depends strongly on the chemical structure, solubility, molecular weight range, temperature and solvent. Some error is therefore likely to be present in Mv obtained with this method as the correct viscosity constant values are not available for most derivatives. In the end-reducing assay, the reducing end of the sugar is being oxidized by the dinitrosalicylic acid reagent to produce color111. Since this method is highly dependent on the production of color, the results can sometimes be misleading111. A more accurate method for determining the weight average Mw or the number average Mw as reported in most studies is size exclusion chromatography (SEC), which is based on the size of a molecule in solution and is measured using either a light scattering detector79, 112, 113 or a viscometer4, 25-27, 35, 95

in combination with a concentration detector, such as refractive index (RI) or UV detector.

The light scattering method is an absolute method for determining the molar mass directly and is ideal for high molar mass samples (homopolymers) if the dn/dc value is known. It can also be used for co-polymers but in such case the dn/dc value can vary within the elugram, which


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complicates the analysis. SEC combined with viscometry works on a different principle. The hydrodynamic volume is determined based on the elution time and the molecular weight can then be calculated as the product of the hydrodynamic volume divided by the intrinsic viscosity114. The response of a light scattering detector is a function of polymer concentration and the weight average molecular weight, therefore the size of the light scattering response increases dramatically with increase in molecular weight. GPC Light scattering detectors in combination with refractive index (RI) and viscosity detectors are therefore required for accurate and complete characterization of high molecular weight polymers. This technique is known as Triple Detection GPC115. To apply this technique, it is important that the polymer does not have any interaction with the stationary phase or the mobile phase. The selection of column material and mobile phase is therefore important and should be based on the type or nature of the polymer being tested114. Furthermore, in SEC, it is important to know the increment of refractive index (dn/dc) for the material tested which depends mainly on the chemical structure and the solvent used.

5. Characteristics affecting antimicrobial activity 5.1.

Molecular weight

Chitosan has been found to be an effective antimicrobial agent and several studies have reported that this antimicrobial efficacy is related to the molecular weight (Mw) of the polymer. An average molecular weight chitosan (Mw > 60 kDa) is seen to inhibit the growth of several groups of Gram positive and Gram negative bacteria, as reported in many studies20-22 (Table 1). In Table 1 we can see the studies reporting the activity of chitosan with different Mw towards 5 Gram positive species and 6 Gram negative species. There is no clear difference in activity towards these two groups. The activity is rather seen to vary with the bacterial species that is


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Biomacromolecules

tested. It is also observed that the variation in activity of chitosan between different strains of the same species is up to 30 fold and therefore the selection of bacterial strain is also an important factor in such investigations. Many studies have reported the antifungal activity of chitosan towards different fungal species, and this activity is found to vary between low to high (MIC = 10–2500 µg/mL). The relation between the antibacterial activity and Mw is seen to be affected by the type of bacterium used. In one study, it was seen that the growth of Gram positive and Gram negative bacteria was effectively controlled using chitosan of 470 kDa, while 1106 kDa chitosan was capable of inhibiting only the Gram negative strains20. Another study showed that chitosan showed increasing inhibitory effect towards Staphylococcus aureus (S. aureus) when the Mw was varied from 5 to 305 kDa, whereas towards Escherichia coli (E. coli), a reverse trend in activity was observed116. Chitosan (300 kDa) and its (dodec-2-enyl) succinoyl derivatives were hydrolyzed to obtain low Mw chitooligosaccharide (COS) (4.6 kDa) and analogous derivatives, which were found to be active against different kinds of bacteria, yeast and filamentous fungi117. Table 1. MIC/MLC values for chitosan against various micro-organisms. Microorganism StrainRef

MIC/MLCvalue (µg/mL or ppm)

Molecularweight (kDa)

pH

Degreeof Acetylation (%)

Staphylococcus aureus NR71

20

NR

NR

NR

Staphylococcus aureus ATCC 2973720

800–>1000

28–1106

5.9

NR

Bacillus cereus LMG6924118

60

43

5.5

6

Bacillus cereus ATCC 2136620

500–>1000

28–1106

5.9

NR

Bacillus cereus ATCC 14579119

80–>2000

2.3–224

6

16–48

Listeria monocytogenes NR120

150

49–1100

6

2–53

Listeria monocytogenes LMG13305118

200

43

5.5

6

Listeria monocytogenes Scott A20

800–≥1000

470–1670

5.9

NR

Bacillus megaterium KCTC 300720

500–800

28–1670

5.9

NR

Gram positive bacteria


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Lactobacillus brevis IFO 1310920

500–≥1000

224–1106

5.9

NR

Lactobacillus bulgaricus IFO 353320

≥1000

28–1670

5.9

NR

Escherichia coli CCRC 10674120

100–500

49.1–1100

6

NR

Escherichia coli ATCC1177520

800–≥1000

28–1670

5.9

2–53

Escherichia coli NVH 3793119

30–>2000

2.3–224

6

16–48

Pseudomonas aeruginosa CCRC 10944120

150–200

49.1–1100

6

2–53

Pseudomonas fluorescens LMG 1794118

80

43

5.5

6

Pseudomonas fluorescens ATCC 2154120

800

224–746

5.9

NR

Salmonella typhimurium CCRC 10746120

1500–>2000

49.1–1670

6

2–53

Salmonella typhimurium ATCC 1402820

≥1000

746–1671

5.9

NR

Enterobacter aerogenes LMG 2094118

60

43

5.5

6

Vibrio cholera CCRC 13860120

≥200

49.1–1100

6

2–53

Botrytis cinerea71

10

NR

NR

NR

Drechstera sorokiana71

10

NR

NR

NR

Fusarium oxysporum CCRC 32121120

500–>2000

49–1100

6

2–57

Microsporum canis71

1000

NR

NR

NR

Micronectriella nivalis71

10

NR

NR

NR

Trichophyton equinum71

2500

NR

NR

NR

Candida lambica 194118

400

43

5.5

6

Gram negative bacteria

Fungi

*NR = Not Reported Some studies have shown that the variation of Mw of the COS within a narrow range greatly influences the microorganism’s growth. A study of growth inhibition in methicillin-resistant Staphylococcus aureus (MRSA) was performed and the results showed that COS of 9.6–20 kDa remained highly active, 2–4.2 kDa COS were moderately active, while 0.73–1.52 kDa COS materials remained completely inactive towards this highly resistant strain121. Similar studies have shown that COS having a Mw range 5–10 kDa were capable of inhibiting the growth of several strains of bifidobacteria (B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum), while COS with Mw < 5 kDa did not display any activity towards


22

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these strains122. In general, studies have also shown that chitosan, low Mw chitosan and COS are very effective towards several fungi and plant pathogens123, 124. A study of the antibacterial activity of a series of aminoethyl modified chitosan derivatives showed that derivatives having Mw values of 99, 51 and 27 kDa exhibited equivalent antibacterial activity against E. coli, while the derivative having the low Mw value of 1.4 kDa did not display any activity towards the investigated strain of this bacteria57. The effect of Mw on the antibacterial activity of two series of quaternized carboxymethylated chitosans was studied and they reported that with an increase in the degree of quaternization and a decrease in the Mw values from 11-48 kDa, the antimicrobial activity either remained identical or varied only by a two-fold dilution73. A study of the structure-activity relationship of quaternary N,N-dimethyl-Ndodecyl, N-(2-(N,N,N-trialkylammoniumyl)acetyl) and N-(2-(N-pyridiniumyl)acetyl) derivatives of chitosan (Mw = 8.1-8.5 kDa), chitooligosaccharide (Mw = 0.95 kDa) and glucosamine was carried out in order to investigate the effect of these substituents when attached to different Mw chitosan or its monomer. The study concluded that in the case of chitooligomer and glucosamine, the dodecyl quaternary derivative was more active than the remaining quaternary derivatives, while in the case of chitosan, this trend in activity was reversed and the shorter alkyl quaternary chitosans showed higher activity than the quaternary derivative carrying the dodecyl chain44. A series of N-2-hydroxypropyl trimethylammonium chitosans (QTS) having similar DS (0.86– 0.88) and a range of Mw (1.7, 35.7, 90.2, 415.5 kDa) was studied for antibacterial activity towards Gram positive and Gram negative strains. The results showed that the QTS were not effective against Gram negative strains of E. coli and P. aeruginosa, whereas the whole Mw range of this derivative were almost equally effective towards Candida albicans. The activity towards the Gram positive strains of S. aureus, S. epidermidis, and B. subtilis decreased with


23

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Page 24 of 65

decreasing Mw values125. A comparison of the antibacterial properties of a series of trimethylammoniumyl chitosan (DQ = 0.3 and 0.65, Mw = 281-35 kDa) and methylated N-3pyridylmethyl chitosans (DQ = 0.8, Mw = 82-8 kDa) towards E. coli and S. aureus showed that irrespective of the Mw variation, the activity towards these two strains remained identical in most cases within each series of derivatives, with a maximum variation of one-dilution126. Similar studies where five series of chitosan derivatives, namely TMC, trimethylammoniumyl chitosan and pyridiniumyl chitosan having C-2 and C-6 spacers, were found to have no significant effect on S. aureus and E. coli within a small Mw range of 7-23 kDa4. From these studies we can conclude that there seems to be some consistency in the reported results and that a certain number of monomeric units are required for displaying high antimicrobial activity, but this value is found to be dependent on other factors, such as the nature of the substituent that is attached and the type of microorganism tested. 5.2. pKa and cationic charge density Chitosan (pKa = 6.3–6.5) in its unprotonated form is insoluble in aqueous medium. When the pH is lowered below 6.5, the amino groups will protonate and get converted to the quaternary form (−NH3+), thereby giving a positive charge to the polymer backbone and making it water soluble. The presence and the density of this cationic charge is believed to be responsible for efficient binding of chitosan to the anionic components present in the bacterial membrane127. One study has shown that water soluble chitosans with low viscosity exhibit lower inhibitory effect towards several strains of Gram positive and Gram negative bacteria when compared to acid soluble chitosan (cationic chitosan) with high viscosity23. In another study, chitosan was investigated for inhibitory effect towards six varieties of bacterial strains under a pH variation of 4.5–5.9. The results showed that chitosan was most effective towards these strains at the lowest


24

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Biomacromolecules

measured pH (4.5)20. Similar observations were reported in another study where the water soluble chitosan (DA= 50% and Mw = 53–1.4 kDa) inhibited the growth of Candida albicans, while remaining inactive against S. aureus and E. coli at pH values 4–6. On the other hand, the chitosan (water insoluble) (DA= 14% and Mw = 400–17 kDa) significantly inhibited the growth of all the types of microorganisms when tested at low pH (4–6)128. Chitosan exhibited a MIC ≥8192 µg/mL for S. aureus when measured at a pH of 7.2, while the same chitosan showed significantly enhanced activity (MIC = 64 µg/mL) towards this strain when the measurements were performed at a pH value of 5.524. The above studies lead us to the conclusion that when the pH of the medium containing chitosan goes below its pKa, the polymer is seen to have enhanced antimicrobial effect. The presence of the positive charge on the polymer backbone rather than the solubility is thus the critical factor for antimicrobial activity. This has been further confirmed in studies where chitosan derivatives carrying a cationic charge show increased antimicrobial effect relative to chitosan itself. A comparison of the activity of TMC with mono- and di-methylated chitosan showed that only the quaternary derivative TMC was active against S. aureus, while the other derivatives as well as chitosan were inactive at pH 7.2

24

. This study concluded that

quaternization is essential for antimicrobial activity at neutral pH. A water soluble quaternary ammonium salt of chitosan, chitosan-N-hydroxy-2,3-propyl-N-methyl-N,N-diallylammonium methyl sulfate, was found to have lower MIC for S. aureus (135 µg/mL) and Klebsiella pneumoniae (330 µg/mL) than chitosan (MIC = 640 and 680 µg/mL, respectively)129. While chitosan is almost inactive (MIC = ≥8192)24 at pH 7.2, several quaternary chitosan derivatives (Figure 4) have been found to exhibit high activity against S. aureus and E. coli under identical conditions (Table 2). Table 2 shows that cationic chitosan derivatives are the most widely


25

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Page 26 of 65

investigated derivatives for antibacterial activity. Many derivatives have been reported in a single study and thus have only one MIC value for each type of bacteria, whereas, for the more common derivatives, such as TMC, where more studies have been carried out, there are a very wide range of reported MIC values. This may be due to differences in the degrees of quaternization for products and also the assay conditions. The reported MIC values in different studies should therefore be interpreted with care, as they may not be comparable. The MIC values of some highly active cationic chitosan derivatives for S. aureus and E. coli are as follows:

TMC

(4

µg/mL

and

64

µg/mL,

respectively)4,

N-(2-(N,N,N-

trimethylammoniumyl)acetyl) chitosan (32 µg/mL and 16,384 µg/mL)4, N-ethyl-N,N-dimethyl chitosan (4 µg/mL and 64 µg/mL)25, N-butyl-N,N-dimethyl chitosan (32 µg/mL and 32 µg/mL)25, N-hexyl-N,N-dimethyl chitosan (256 µg/mL and 16 µg/mL)25, N-guanidinyl chitosan (32 µg/mL and 512 µg/mL)26, N,N-dibutyl-N-methyl chitosan (512 µg/mL and 32 µg/mL)25, N,O-(2-hydroxy propyl trimethylammonium)-N-benzyl chitosan (8 µg/mL and 32 µg/mL)79, N,O-(2-hydroxy propyl trimethylammonium)-N-octyl chitosan (16 µg/mL and 32 µg/mL)79, and N,O-(2-hydroxy propyl trimethylammonium)-N-4-pyridinyl methyl chitosan (64 µg/mL and 64 µg/mL)79. The presence of temporary or permanent positive charges on the chitosan backbone is accompanied by the presence of a counter ion. A study was performed to investigate the effect of the type of counter ion present in quaternized chitosan (TMC) on its bactericidal activity130. The study concluded that the bactericidal activity of TMC towards S. aureus and E. coli is correlated to the availability of the quaternized groups in the TMC backbone. The highest activity of TMC was found in the presence of acetate and sulfate ions, possibly due to their delocalized system. The lower activity of TMC in the presence of halide ions was in the order Cl– > I– ~ Br–130.


26

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Biomacromolecules

To conclude, it can be stated that while the solubility of chitosan and its derivatives is important for performing any bioactivity measurements, the main factor responsible for their activity is the presence of cationic charges in the backbone.

Cationic chitosan derivatives OH O

O HO

*

OH

NH2

n

n

Chitosan derivative

where N X X

X=

N X

* R

Chitosan

N

O

O HO

*

*

R=

X=

H N

5N

N

3

2

2

OH

H N

N

O

4

4

H N

X

X=

N

N

N

N

H N

N

N

O

3X

X=

N

N

H N

N N

O

H N

N

N

5X

X=

NH2 NH

N

N

H N

O

NH2 NH

Figure 4. Figure showing the quaternary ammonium (cationic) moieties commonly present in chitosan derivatives. Table 2. Antimicrobial activity of various chitosan derivatives against S. aureus, E. coli and P. aeruginosa. Bacteria DA

DS

Mw

Chitosan derivative

pH (%)

Cyanoethylchitosan131

21

(%)

NR

(kDa)

NR

NR

Staphyloco ccus aureus

Escherichia coli

(µg/mL)

(µg/mL)

19

312

Pseudomon as aeruginosa (µg/mL) NR


27

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Aminoethylchitosan132

10

NR

300c

5.5

125

62.5

7.8

Dimethylaminoethyl chitosan132

50/10

NR

140–310c

5.5

125

62.5

15.6–31.25

Diethylaminoethyl chitosan132

50/10

NR

140–310c

5.5

250

250–500

62.5–125

N-ethyl chitosan8

9.8

51.8

50c

7

32

64

128

N-butyl chitosan8

9.8

50.7

50c

7

64

128

128

N,N,N-trimethyl chitosan4, 44, 126, 133-135

4–20

28–100

7.8–9420

7.2/7

4–>2000

1–>2000

NR

N-(Propyl/Furfuryl)-N,N-dimethylchitosan135

4

81.6– 91.8

7.8–214

7

NR

0.5–1

NR

N-(2-(N,N,N-Trimethylammoniumyl)acetyl) chitosan4, 35, 44

0–18

0–100

8–48

7.2/5.5

8–2048

128–16384

64

N-(2-(1-Pyridiniumyl)acetyl) chitosan4, 44

0–34.2

81–100

8–18.9

7.2

8–1024

512–1024

512

N-(3-Pyridylmethyl) chitosan79, 126, 134

6–10

35–94

58–276

7.2

8–125

32–250

NR

N-(Oct/Benz/CH3-benz/OH-benz)-N-(2hydroxypropyl trimethylammoniumyl)chitosan79

6

90.4– 93.3

276

7

8–16

32

NR

N-(3,4-CH3O/F/Cl/CF3/4-Br/NO2/COOH)benzyl-N(2-hydroxypropyltrimethylammoniumyl) chitosan79

6

91–96.5

276

7

16–32

32

N-dodecyl chitosan8

9.8

50.7

50c

7

64

128

>256

Chitosan8

9.8

35.3– 77.2

50c

7

32–>256

128–>256

256–>256

(Acetyl/Chloroacetyl/Benzoyl)thiourea chitosan136

4

81–91.5

50c

NR

62

15

15–62

o-biguanidinylbenzoyl chitosan HCl137, 138

7.7

14.9– 39.5

346–410

7

8–32

16–64

NR

p-biguanidinylbenzoyl chitosan HCl138

7.7

16.2– 44.7

349–424

7

8–32

16–64

NR

(Acetyl/Chloroacetyl/Benzoyl)Phenylthiosemicarba zone139-141

4

35.6

7–200

NR

56–>250

7

225–900

Acetyl(o-Me/p-Me/p-Chloral/pnitro)phenylthiosemicarbazone139

4

33.7– 35.3

7–200

NR

250

7

450

(O-carboxymethyl/N,O-carboxylethyl) chitosan6, 74

6.7–25

NR /72

NR /347

NR /7

320/31.3

320/62.5

NR

Quaternary(Me/Et/Pr/But/Benz)carboxylethylchitos an74

6.7

51–59

409–475

7

6.3–31.3

6.3–31.3

NR

(Acetyl/Chloroacetyl/Benzoyl(thioureacarboxymeth yl chitosan99

12

88.8– 89.6

200

NR

7.8–62.5

62.5–500

NR

*NR = Not Reported; DA = Degree of acetylation; DS = Degree of substitution; Mw = Average molecular weight; Mwc = Average molecular weight for starting chitosan. Details on DA, DS, Mw, pH and MIC are provided in the supporting information (Table S1). 5.3.

Degree of acetylation

Antimicrobial activity of chitosan can only be assessed when the polymer has good solubility and this in turn requires the presence of a greater number of free amino groups in the chain.


28

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Chitosan with a greater number of N-acetyl groups increases the hydrophobicity of the polymer. Chitosan with N-acetyl DS of up to 0.5 has aqueous or even organic solubility in the case of low Mw142, while a DS value above 0.5 or high Mw reduces its solubility in aqueous or acidicaqueous mediums143. The optimum solubility obtained in the case of half N-acetylated chitosan is possibly due to the disordered H-bonding between the adjacent polymer chains which results in a loose packing arrangement and facilitates easy water penetration and hydration143. Highly Nacetylated chitosan can be partially deacetylated to produce water soluble chitosan144. Chitosan having a lesser degree of acetylation (DA) or a higher number of free amino groups has led to enhancement of antimicrobial activity towards various strains of fungi, Gram positive and Gram negative bacteria23,145, especially in the case of S. aureus and E. coli71, 146. A series of N-acetyl chitosans having varying DA values (8, 12, 35, 44, 55 and 61%) and a narrow Mw range (5.4-9.4 kDa) were tested towards Gram positive S. aureus and B. subtilis and Gram negative E. coli and P. aeruginosa. The study showed that the relative inhibition time was highest (>85 h) for chitosans having 8% and 12% DA, while the remaining acylated chitosans (35−61 %) showed considerably lower inhibition time (5−30 h)147. Different findings have been reported in other studies. Chitosans having three different DA values (50%, 25% and 10%) were tested for activity towards Gram positive and Gram negative strains and the results showed that chitosan with DS = 25% was found to have optimum activity. However, no significant difference in the minimum inhibitory concentrations (MIC) could be observed, since the values were either identical or differed at most by only 1-2 dilutions (MIC = 1.25–5 mg/mL)148. Similarly, minimal changes in the activity of chitosans having DA values from 2–53% were obtained when tested against various bacteria and fungi120. In another study, the antimicrobial activity of aminoethyl chitosan, dimethylaminoethyl chitosan and diethylaminoethyl chitosan prepared from chitosan having DA


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values of 50 and 90% was measured towards E. coli, S. typhimurium and P. aeruginosa. The results showed that the MIC values for each derivative were either identical or varied at most by one dilution, irrespective of the difference in the DA values58. The effect of DA on the antimicrobial activity of a series of quaternary ammonium chitosan derivatives was also evaluated. In this study, no particular trend in activity of the derivatives towards S. aureus and E. coli was observed when the DA was varied in the range 6–34%4. Thus, the DA appears to have very limited effect on antimicrobial activity when the DA value is less than 50%. 5.4.

Degree of substitution

The degree of substitution (DS) or the ratio of the functional groups attached to the chitosan backbone is reported either specifically for the amino or the hydroxyl substitution or collectively for all the three functional groups. The DS of a substituent in the polymer chain has been shown to have a pronounced effect on the antimicrobial properties of the polymer towards various strains of bacteria and fungi. The antimicrobial activity of water soluble N-(4-carboxybutyroyl) chitosans (5a, Figure 5) towards plant pathogenic bacteria (Agrobacterium tumefaciens and Erwinia carotovora) and fungi (Botrytis cinerea, Pythium debaryanum and Rhizoctonia solani) was found to be greater than that of unmodified chitosan and the activity increased with increasing DS from 0.10−0.5352. A study of a series of o-biguanidinyl benzoyl chitosans (5b) having DS values of 0.14, 0.24, 0.32 and 0.39 showed that the MIC values (at pH = 7) varied from 32−8 µg/mL towards S. aureus and from 64−16 µg/mL towards E. coli as the DS increased137. Similar increasing activity with DS towards S. aureus and E. coli was observed in several other studies involving O-fumaryl chitosan (5c)104, p-biguanidinyl benzoyl chitosan (5d)138,

salicyloyl

chitosan

(5e)98,

2-hydroxypropyldimethylbenzylammonium

N,O-(2-

carboxyethyl)chitosan chloride (5f)149, N,N,N-trimethyl-O-[(2-hydroxy-3-trimethylammonium)


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propyl]chitosan (5g)77 (Figure 6). Two different arginine-functionalized chitosans (5h) having DS values of 0.06 and 0.3 were tested for antibacterial activity and the results showed that although both derivatives were capable of strongly inhibiting the growth of P. fluorescens and E. coli, the derivative having higher DS was more effective in permeabilizing the cell membrane of both Gram negative strains90. Similar activity towards Gram negative bacteria E. coli was seen in another study involving chitosan-arginine derivatives (5i) having different DS. It was observed that in the concentration range of 50-150 ppm, the inhibitory effect towards E. coli increased with increasing DS values from 0.08 to 0.18 to 0.28, but this trend in activity was reversed in the case of Gram positive S. aureus92. A comparison of the antibacterial efficacy of chitosan and hydroxypropyltrimethylammonium chitosans (5j) (DS = 0.06, 0.18 and 0.44) was performed against S. aureus, Methicillin-resistant S. aureus and Staphylococcus epidermis. The results showed that at a concentration of 2.5 mg/mL, the derivatives having DS values 0.18 and 0.44 were significantly more active (P ˂0.01) in comparison to the derivative having DS = 0.06 and chitosan150. The antibacterial activity of a series of N-guanidinyl chitosans (5k) (DS = 0.11, 0.35, 0.55 and 1), N-(2-guanidiniumylacetyl) chitosans (5l) (DS = 0.14, 0.55 and 1) and N-(6guanidiniumyl-hexanoyl) chitosans (5m) (DS = 0.12, 0.53 and 1) against S. aureus and E. coli increased with increasing DS in the polymer chain; however, an optimum activity was observed at a DS value of 0.5 and above in most cases26 (Figure 6). This increasing activity with increasing DS was also observed in a series of N,N,N-trimethylammoniumyl chitosan derivatives (5n, 5o) having similar DS and spacer lengths26. A study of N-dodecyl chitosan reported that when the DS value of the derivatives increased from 0.35−0.77, the derivatives showed decreasing activity towards a number of Gram positive and Gram negative bacterial strains. However, the difference in the MIC values was only one or two dilutions8.


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Structure-activity relationship R1

Effect of degree of substitution * O

R2 =

R2 n

OH

O

O DS = 0.10-0.53 (5a)

Chitosan derivative

NH

R2 =

3

NH

HN

NH

N H

R1 =

NH2.HCl

R2 =

O

where

O N H

R2 =

NH

O 5 X

NH spacer = 6

O NH

X

spacer = 2

X spacer = 0

where

Increasing degree of substitution

Decreasing antibacterial activity

N

DS = 0.16-0.44 H (5d)

DS = 0.07-0.48 (5c)

R2 =

N

H N

R2 =

DS = 0.22-0.42

(5g)

H N

R2 =

NH2 NH

R2 =

NH2

H N

O

NH

(5f)

N OH

DS = 0.57-0.76

R2 =

NH

NH2

DS = 0.13-1.0 (5n)

NH2

N H

NH2

R2 =

H N

R2 =

N HO

DS = 0.06-0.44 (5j)

NH

H N O

N

3N H

DS = 0.06-0.30 (5h, 5i) NH

H N

5N

O

DS = 0.14-1.0 (5l)

O

(5r)

and

N

DS = 0.11-1.0 (5k)

NH

(5q)

O N H

N HO

DS = 0.69

R2 =

OH

DS = 0-0.85

DS = 0.10-0.71 (5e)

O

and

N

O

NH2.HCl

X=

N

(5p, 5s)

N H

R1 =

R2 =

R1 =

OH

O

R2 =

NH

N H

OH

N H DS = 0.14-0.39 (5b)

Effect of spacer length

O

O

H

NH2

DS = 0.12-1.0 (5m)

Increasing antibacterial activity

O

* O HO

Increasing spacer length

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5N

O

DS = 0.13-1.0 (5o)

Figure 5. Figure showing the effect of the positioning of the cationic charge (spacer length) and the degree of substitution (DS) on the antimicrobial activity of chitosan derivatives.

Figure 6. Figure showing the variation of antimicrobial activity with increasing degree of substitution (DS) for chitosan derivatives. In A, guanidiniumyl acetyl) chitosan and trimethyl chitosan,

= N-guanidiniumyl chitosan,

= N-(2-

= N-(6-guanidiniumylhexanoyl) chitosan. In B, = N,N,N-

= N-(2-(N,N,N-trimethylammoniumyl)acetyl) chitosan and

= N-(6-


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(N,N,N-trimethylammoniumyl)hexanoyl) chitosan. Reproduced in part from ref 26, Copyright 2015, with permission from Elsevier. Thus, studies have shown that when the polymer carries positively charged moieties, the antibacterial efficacy is usually enhanced with increasing ratio of the functional groups attached to the chain, while with more hydrophobic moieties, an increasing DS value tends to reduce the activity. 5.5.

Positioning of the cationic charge

The importance of the cationic charge has been discussed in the previous section, but studies have shown that the location of the permanent positive charge in the polymer backbone can also be a deciding factor with regard to its antimicrobial activity. This has been observed in studies where the activity of the TMC derivative is found to be higher than O-[(2-hydroxy-3trimethylammonium)propyl] chitin and N-[(2-hydroxy-3-trimethylammonium)propyl] chitin towards S. aureus and E .coli133. TMC was also seen to exhibit higher activity than a series of acetyl quaternary chitosan derivatives towards S. aureus, E. faecalis and P. aeruginosa44. A study of a series of N-acetyl-piperidine chitosan derivatives showed that the derivatives were active towards Gram positive and Gram negative strains at pH 5.5 only when the DS in the products were low, or in other words, when the derivatives carried a greater number of protonated amino functional groups151. In another study, a series of highly substituted chitosan N-betaines having different DS remained low in activity at pH 7.2. The derivatives showed activity when the pH was lowered to 5.5 and the activity was found to increase with decrease in the DS of the polymer. This study concluded that the presence of the cationic charge on the polymer backbone is essential for exhibiting high antimicrobial properties35. A study of the antimicrobial activity of a series of trimethylammonium (5p) (Figure 5) and pyridinium moieties


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(5q) having C-0, C-2 and C-6 spacer lengths were studied. The results showed that TMC was the most active compound towards S. aureus and E. coli and that the inhibitory effect of the compounds decreased with increasing distance of the cationic charge from the polymer backbone4. A series of chitosan derivatives were prepared by introducing the guanidinium group (5r) and the trimethylammonium group (5s) at the 2-amino position of chitosan either directly or using C-2 and C-6 spacers. The authors concluded that in the case of both series of cationic chitosan derivatives, the compounds having direct substitution at the amino group showed the highest antimicrobial activity towards the strains of S. aureus and E. coli26. From these studies we can conclude that the antimicrobial effect seems to be the highest when the cationic moieties or the positive charge is closest to the polymer backbone and tends to decrease when the functional groups are present at an increasing distance from the polymer chain. This effect is clearly observed when the same functional group is linked to the polymer through different spacers of different lengths. 5.6.

Hydrophobicity

Hydrophobicity is generally found to be related to the antimicrobial activity of chitosan. However, the relationship between the hydrophobic groups, such as alkyl or aromatic, and activity has not been clearly established. Introduction of long alkyl chains, particularly at the 2amino group, has been reported in many studies (Figure 7). Chitosan and N-(docec-2-enyl) or Nsuccinoyl derivatives (DS = 0.03, 0.09 and 0.16) were investigated for antimicrobial activity against different types of bacteria and yeast. In this study, the modified derivatives showed similar inhibition of growth (% cell viability) towards P. aureofaciens, E. agglomerans and C. kruisei as chitosan and the reduction in the cell viability in E. coli (16%) was also the same, but an increase in the cell viability of B. subtilis (34%) was observed in comparison to that of


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unmodified chitosan117. The antimicrobial activity of N-(4-carboxybutyroyl) chitosans was found to be enhanced, with the lowest MICs of 800 and 725 µg/mL for A. tumefaciens and E. carotovora in comparison to chitosan (>3000 µg/mL). The mycelial growth inhibition was also strengthened in the case of the modified derivatives52. Another study showed that the introduction of hydrophobic moieties in chitosan enhanced its ability to permeabilize the cell wall of Gram positive and Gram negative bacteria152. The Schiff base of chitosan with citral and cinnamaldehyde, sorbyl chitosan and p-amino benzoyl chitosan were found to have better antimicrobial activity than chitosan towards S. aureus, E. coli and A. niger. The activity of the derivatives was found to increase with increasing concentration from 0.005−2% (w/v)50. Similarly, when chitosan was functionalized with the salicyloyl group at different ratios, the derivatives were found to be capable of exhibiting a greater inhibitory zone towards both S. aureus and E. coli98. Substituted N-benzyl chitosans were found to have an improved inhibitory effect compared to chitosan (>2500 µg/mL) towards plant pathogenic bacteria A. tumefaciens (MIC = 1050−1300 µg/mL), E. carotovora (MIC = 500−1100 µg/mL) and fungi F. oxysporum (EC50 = 400−2086 µg/mL) and P. debaryanum (EC50 = 468−1625 µg/mL)7. Similarly, the inhibitory effect of a series of substituted N-cinnamyl chitosans determined using a radial growth test also showed improved activity towards several fungi as compared to unmodified chitosan153.


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OH *

O HO

R1 O

*

* NH2

O

O HO

* R2

n

Chitosan

n

Chitosan derivative

O

O R1 =

NH2

N

N N H

O

O

R2 =

O

NH

O

COOH 3

SH

H N

SH O S O NH2

HS

O

X = p-CH3, p-N(CH3)2, o-C2H5, p-C2H5, o-OCH3, m,p-di-OCH3, o,p-di-OC2H5

X R1

R2

N

N

n R1 and

NH2

R2 =

R3

R1, R2, R3 = H or CH2CH2NH2

2

10

R2=

14

Figure 7. Figure showing the alkyl, aromatic and aminoalkyl groups commonly present in chitosan derivatives. A few other studies have reported different findings. The activity of a series of N-acetyl, Npropionyl and N-hexanoyl chitosans were found to have shorter relative inhibition times as compared to unmodified chitosan when tested at a concentration of 1 mg/mL against S. aureus, B. subtilis, E. coli and P. aeruginosa147. The effect of an increase in hydrophobicity due to the differences in the length of the alkyl chains was used to study the antimicrobial activity. In this study, the N- or O-alkyl chitosan derivatives having ethyl, butyl, dodecyl and cetyl groups with combined DS = 0.50–0.51 were found to have decreasing activity with increasing chain length towards a number of Gram positive and Gram negative bacteria8. The antibacterial activity of quaternized N-alkyl and N,N-dialkyl chitosan derivatives (alkyl = methyl, ethyl, butyl and hexyl)


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was investigated against two Gram positive and two Gram negative bacterial strains. The results showed that the short alkyl chain derivatives showed high activity towards S. aureus, the more hydrophobic N-hexyl derivatives were more active against E. coli and E. faecalis, while the activity towards P. aeruginosa remained independent of any variations in chain length25. These studies show that the presence of hydrophobic groups may increase the antimicrobial activity relative to the parent chitosan, but in some cases, increased hydrophobicity caused by longer alkyl chains can lead to a decrease in activity. The effect of hydrophobicity can also depend on the type of bacteria tested. However, some caution is advised when interpreting results from this type of study, as derivatives with different DS are not directly comparable and therefore may not give accurate results. 5.7.

Multiple functional groups

Sometimes more than one type of functional group is attached to chitosan, which is usually aimed at improving the antimicrobial efficacy of the polymer. The presence of additional substituents seems to have an impact on the overall activity of the polymer in most cases. The antibacterial activity of TMC has been found to be superior to most of the quaternary chitosan derivatives against Gram positive and Gram negative bacteria126,

133

. This activity is found to

decrease when the C-6 OH group of TMC was modified with carboxymethyl groups78. When the C-6 OH group of TMC (DS = 0.72) was substituted by a quaternary ammonium moiety, such as 2-hydroxy-3-trialkylammonium propyl (DS = 0.05, 0.10 and 0.20), the activity of TMC was enhanced up to a maximum of 3 dilutions towards both S. aureus and E. coli154. O-CMC has been utilized as a scaffold for introducing several different substituents at either the O- or the Nposition. Studies have shown that the antimicrobial efficacy of this derivative was enhanced towards different strains of bacteria and fungi when groups such as acetyl, chloroacetyl,


37

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benzoylthiourea99, crosslinked terephthaloyl thiourea moieties84, thiosemicarbazone moieties100, polyvinyl86 and N-alkyl-N-2-hydroxy-3-trialkylammonium propyl87 were added to the derivative. N,O-CMC (MIC = 62.5 µg/mL) showed higher activity in the presence of the N-2-hydroxypropyl dimethylbenzylammoniumyl group towards both S. aureus (MIC = 6.3 µg/mL) and E. coli (MIC = 12.5 µg/mL)83,

155

. Quaternary (N-2-hydroxy-3-trialkylammoniumyl propyl and N-2-

hydroxypropyl dimethylbenzylammoniumyl) carboxyethyl chitosan derivatives showed higher antimicrobial activity as compared to N,O-carboxyethyl chitosan when its activity was measured against S. aureus and E. coli. The high activity was thought to be due to the synergistic effect between the carboxyethyl and the quaternary ammonium groups, which increased with increasing alkyl chain length74, 149. When the 6-O position of N-2-hydroxy-3-trialkylammonium propyl chitosan was substituted with a CMC-PAMAM dendrimer, its antibacterial activity towards E. coli was increased from 62.5 µg/mL to 15.6 µg/mL (MIC), while towards S. aureus its activity dropped from 500 µg/mL to 1000 µg/mL (MIC). The antibacterial efficacy of chitosan containing varying ratios of three distinct functional groups, (N-acetyl, N-stearoyl and N,N,N-trimethyl) was determined against S. aureus and E. coli. The results were interpreted with the aid of Design of Experiments software (MODDE) which showed the effect of the individual groups (single or quadratic terms) and also the interaction between the groups on the antibacterial activity. It was observed that the N,N,N-trimethyl group had the highest positive effect on S. aureus and E. coli and the N-acetyl group did not have any significant influence. On the other hand, the N-stearoyl functionality by itself had a negative impact on the antibacterial activity but its quadratic term (stearoyl*stearoyl) significantly improved the activity27. Thus, these studies indicate that the addition of a second functionality can enhance the antimicrobial effect. However, in some cases they tend to further reduce the activity of the parent


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derivative. Although the added functionality may not have significant antimicrobial effect by itself, it can show improved activity when it is in combination with other substituents. It is therefore also important to take into account the interaction between the different substituents. 5.8. Effects of anionic groups, polar groups and peptides The effects of other substituents, such as anionic, polar groups and peptides, on antimicrobial activity have been less studied. One of the most widely studied anionic groups is CMC (N- or OCMC). However, in most studies, CMC has been utilized in combination with other functional groups for measuring the antimicrobial efficacy of the products, as discussed in section 5.7. The presence of polar functional groups, such as hydroxyalkyl, has been less studied. One study had investigated the antibacterial activity of 3,6-O-hydroxyethyl chitosan towards Enterococcus and E. coli. In this study, 3,6-O-hydroxyethyl chitosan showed higher reduction in the colonization of Enterococcus than chitosan, while this derivative was less active than chitosan towards E. coli, as observed through SEM65. In another study, a series of N,O-hydroxypropyl chitosans (DS = 1.5–3.1) showed no growth inhibition in S. aureus and E. coli, but the derivatives remained moderately active towards several species of fungi (MIC = 5,000−31,000 µg/mL), the lower DS derivatives being more active66. The antimicrobial activity of chitosan-thioglycolic acid derivatives (low and medium Mw, DD>85%) were evaluated against Gram positive and Gram negative bacteria as well as fungi. This study concluded that the low Mw chitosan-thioglycolic acid derivative showed superior antimicrobial activity as compared to TMC and CMC, and was capable of reducing the colony counts of Streptococcus sobrinus by 100%, Neisseria subflava by 99.99% and Candida albicans by 99.97%5. In another study, two thiolated chitosan derivatives containing 2-iminothiolane HCl (TC-IMI) and N-acetyl-L-cysteine (TC-NAC) were studied for antibacterial activity towards S. aureus and E. coli. The results showed that TC-IMI had higher


39

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activity (MIC = 2,900µg/mL and 3,000 µg/mL) than chitosan (MIC = 4,100 µg/mL and 4,400 µg/mL) and TC-NAC (MIC = 6,200µg/mL and 4,900 µg/mL) towards S. aureus and E. coli, respectively55. The introduction of peptides into chitosan has been shown to enhance the antibacterial activity of chitosan as well as the peptide. Two series of cationic peptidopolysaccharides, i.e., chitosanpolylysine and chitosan-poly(lysine-ran-phenylalanine), having different grafting ratios (0.01– 0.23) were investigated for antimicrobial activity against P. aeruginosa, E. coli, S. aureus, C. albicans and F. solani. The study showed that the chitosan-polylysine series showed very high activity MICs (5–20 µg/mL) while the peptides themselves were not active (MIC >1,000 µg/mL) within the measured range of dilutions towards all the microbes. It was also observed that the antimicrobial activity of the conjugates was optimized only in the presence of the cationic residues (lysine) and in the absence of the hydrophobic residues (phenylalanine)94. Similar results were obtained when a short antimicrobial peptide, namely anoplin, was grafted onto chitosan using different reactive end terminals and different grafting ratios of the peptide to chitosan. The conjugates were investigated for activity towards S. aureus, E. faecalis, E. coli and P. aeruginosa. It was observed that the conjugates were capable of enhancing the activity of the peptide (MICE .coli = 64 µg/mL) in most cases, especially towards E. coli with an MIC as low as 4 µg/mL95.

6. Mechanism of antibacterial action Chitosan and its derivatives exhibit a difference in activity towards the Gram positive and Gram negative bacteria, as is evident in most studies. This difference in activity can be correlated to the difference in the composition of the cell wall. In Gram positive bacteria, the cell wall is


40

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made up of a thick peptidoglycan layer where negatively charged teichoic acids are covalently linked to N-acetylmuramic acid, while lipopolyteichoic acids form covalent bonds with the cytoplasmic membrane. These teichoic acids perform functions such as providing strength to the cell wall and arranging uniform high density charges in the cell wall, thereby affecting the passage of ions across the outer surface layers156. In case of Gram negative bacteria, a thin peptidoglycan layer above the cytoplasmic membrane is further covered by an additional outer envelope called the outer membrane (OM). Lipoprotein and lipopolysaccharide (LPS) are the principal components of the OM and therefore the hydrophilic O-specific side chains present in the LPS help in identifying bacteria157. Hydrophobic compounds and macromolecules are usually not active towards Gram negative bacteria, and in order to interact with the Gram negative bacteria it is therefore essential to overcome the outer membrane barrier158. The mode of antibacterial action of chitosan is presumably due to interactions with the bacterial surface (either cell wall or outer membrane), and to explain this mechanism, four models have been proposed so far.

Figure 8. Figure showing one of the proposed159-162 modes of action of chitosan on Gram positive and Gram negative bacteria. A: Structural composition of the outer envelope of Gram positive and Gram negative bacteria where a = outer membrane, b = peptidoglycan layer and c = cytoplasmic membrane; B: Effect of chitosan binding to the outer envelope of Gram positive and Gram negative bacteria.


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The first and most widely accepted model of interaction involves the electrostatic attraction of the cationic groups on chitosan towards the negatively charged components present on the bacterial surface. At pH ˂ 6, the quaternary ammonium groups (R-NH3+) compete with the divalent metal ions, such as Ca2+ and Mg2+, present in the bacterial cell wall for binding with the polyanions163. Therefore, the presence of cationic charges in the chitosan backbone is a key requirement for displaying activity towards bacteria164. It has been proposed that the electrostatic interaction operates differently according to the type of bacterial species. In Gram positive bacteria, the peptidoglycans in the cell wall are said to be hydrolyzed leading to the leakage of intracellular components (Figure 8). One study confirmed the leakage of electrolytes and proteinaceous materials from the bacterial cells by an increased absorption at 260 nm159. Other studies have shown that the hydrolysis of the peptidoglycans also results in an increased electrical interaction which is detected by the increased conductivity in the cell suspension160 and cytoplasmic β-galactosidase release132, 161. A second proposed mechanism is that, in the case of the Gram negative bacterial species, chitosan can bring about changes in the permeability of the outer envelope by forming an ionic type of bonding. These changes lead to prevention of transport of nutrients into the cell and builds up an internal osmotic pressure. Ultimately, the cell dies due to lack of nutrients165. However, other studies have shown that the electrostatic binding between the cationic groups in chitosan and the negatively charged components of the bacterial surface helps in opening up the OM in Gram negative bacteria166.


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Figure 9. Figure showing the effect of oleoyl-chitosan nanoparticles on S. aureus and E. coli. TEM of S. aureus, 1A): untreated cells, B), C) and D) cells treated with 300 mg/l oleoyl-chitosan nanoparticles for 5, 15 and 30 min respectively. TEM of E. coli, 2A): untreated cells, B), C) and D) cells treated with 300 mg/l oleoyl-chitosan nanoparticles for 5, 15 and 30 min respectively. Reprinted from ref 167, Copyright 2008, with permission from Elsevier. A few studies have been performed to determine the mode of action of chitosan derivatives towards bacteria. Fluorescence and SEM studies performed on Gram negative bacteria treated with chitosan-arginine derivatives have revealed that the initial site of action is the OM in E. coli and P. fluorescens. By affecting the membrane permeability, it eventually leads to the leakage of the intracellular components and cell lysis90. The effect of oleoyl-chitosan nanoparticles on E. coli and S. aureus at a concentration of 300 mg/l was investigated by transmission electron microscope (TEM). The TEM results after exposure for 30 min (Figure 9), shows that serious damage was observed on the bacterial surface of E. coli and S. aureus with hole formation and loss of cytoplasmic materials167. Field Emission Scanning Electron Microscopic pictures (FESEM) (Figure 10) of pathogenic cells treated with cationic peptidopolysaccharide (peptide-


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chitosan conjugate) showed wrinkled surfaces on E. coli and F. solani, leakage of intracellular contents in the case of P. aeruginosa and cells collapsing for S. aureus and C. albicans94.

Figure 10. Morphology of various pathogens before (i) and after (ii) treatment with cationic peptidopolysaccharide. Reproduced with permission from ref 94. Copyright 2012 WILEY-VCH Verlag Gmbh & Co. KGaA, Weinheim. In the third proposed mode of action, chitosan is assumed to target the microorganism intracellularly. According to this theory, chitosan is capable of perforating the microbial cell in the case of bacteria and fungi and interact with the DNA. Studies have concluded that chitosan can penetrate the multilayered (murein cross-linked) bacterial cell wall as well as the cytoplasmic membrane168. Chitosan then destroys the cell by binding to the DNA which prevents DNA transcription and interrupts protein and mRNA synthesis169, 170. The antibacterial activity of chitooligomers on E. coli cells studied using a confocal laser scanning microscope showed that chitooligomers were present inside the cell and the probable cause of inhibition was reported to


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be prevention of DNA transcription71. The validity of this model of interaction between chitosan and the intracellular target is highly dependent on the ability of chitosan to penetrate the multilayered cell wall and the cytoplasmic membrane, and therefore this mode of action is less likely to occur in high Mw chitosan and chitosan derivatives171. Chitosan has therefore generally been regarded as a membrane disruptor rather than a penetrating material168. '' A fourth mechanism of action is proposed where metal ions present in the bacterial surface are chelated by the amino groups of chitosan. This chelation effect is found to overpower the electrostatic effect when the pH of the medium is higher than the pKa of chitosan172. Chitosan is well known to have excellent chelating properties towards metal ions173,

174

and this property

arises due to the ability of the unprotonated amino groups (when pH > 6) in chitosan to donate its lone pair of electrons to the metal to form complexes. The chelation process occurs either by the bridge model, where the amino groups of the same or different chitosan chains are bound to the metal ions, or by the pendant model, in which the metal ions are attached to the amino groups forming a pendant175. This bonding between the free amino groups and the divalent metal ions, such as Ca2+and Mg2+, that are present in the microorganism cell wall prevents the production of toxins and thereby inhibits the overall growth of the microorganism176. This type of chelating property can only be shown by chitosan carrying unprotonated amino groups and thus this mode of action can be operative only after compromising the polycationic moiety of chitosan. Therefore, in the case of chitosan derivatives where the amino group has been modified with cationic moieties or electron withdrawing groups, the chelating effect is less likely to be responsible for bacterial killing or growth inhibition. There have been only a few studies reported on the mode of action of chitosan towards fungi, and most of them have proposed that chitosan is capable of penetrating the fungal cell surface


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and interacting with the plasma membrane177. When fungal cells were treated with FITC labelled low Mw chitosans (1-10 kDa), the fluorescent polymers were found to penetrate the cell surface of the fungal cells and accumulate on the plasma membrane. Furthermore, the morphological changes observed under SEM showed that the surface of the treated cells was disrupted178. Another detailed study on the antifungal action of low MW chitosan (96.5 kDa) towards Candida albicans proposed that the strong binding of chitosan to the fungal cells (through electrostatic interactions) results in hyperpolarization of the plasma membrane and this leads to the efflux of negatively charged molecules in the cells, such as phosphate, nucleotides and substrates of enzyme reactions177. Similar studies have apparently not been carried out for studying the effect of chitosan derivatives on fungi. Thus, the most commonly cited and suggested mechanism of action of chitosan is the interaction with the outer surface of bacteria or fungi due to electrostatic attractions, followed by rupture of the surface to release the intracellular components. On the other hand, the ability of chitosan to reach the intracellular targets or chelate the divalent metal ions is dependent on the Mw of the polymer or on the presence of free amino groups, and therefore these modes of action can only be encountered in certain types of chitosan. Additionally, the mechanism of antibacterial action has mostly been studied for chitosan and only a limited number of studies have been reported for the chitosan derivatives. Therefore, more insights into the mode of action of chitosan derivatives carrying different kinds of substituents are needed.

7. Cytoxicity Several antibacterial chitosan derivatives have been measured for toxicity against human red blood cells (RBCs). This includes derivatives such as quaternary N-monoalkyl and N,N-dialkyl chitosan derivatives25, chitosan carrying trimethylammoniumyl (TMA) and pyridiniumyl (Py)


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functional groups with different spacer lengths4, chitosan carrying multiple functional groups27 and chitosan-peptide conjugates95 (Table 3). In the case of quaternary monoalkyl and dialkyl chitosan derivatives (DS = 0.61 to 0.75) , it has been observed that an increase in length of the alkyl chain from methyl to hexyl in the quaternary group led to an increase in the toxicity towards RBCs. These derivatives were also found to display a similar trend of increasing toxicity with chain length towards the Caco-2 human epithelial cell line25. In another study, a comparison of a series of derivatives carrying TMA (DS = 1.0) and Py groups (DS = 1.0) showed that TMC, which exhibited the highest antibacterial activity (MIC = 8−32 µg/mL) in the whole series, showed moderate hemolytic activity with HC50 between 640−6114 µg/mL and the toxicity decreased with decrease in the DA values in the derivatives. All the other derivatives carrying TMA and Py groups, which were lower in antibacterial activity (MIC = 500−4092 µg/mL), remained non-hemolytic (HC50 > 32,768 µg/mL), even at the highest measured concentration. However, the derivatives were found to be cytotoxic towards the human colorectal adenocarcinoma-derived cell line Caco-2, with EC50 values in the range 10−108 µg/mL in most cases4. A short antimicrobial peptide, anoplin, was grafted onto chitosan to produce conjugates having high antibacterial properties (MIC = 4−1024 µg/mL) against a series of Gram positive and Gram negative strains (DS= 0.06−0.23). These conjugates were found to display significantly lower hemolytic activity (HC50 = 16,384 or >32,768 µg/mL) towards human red blood cells than the unmodified peptide and exhibited high selectivity towards the bacterial strains, particularly towards E. coli (Selectivity = 8)95. A [(lysine)11–(phenylalanine)10] peptidegrafted chitosan nanocapsule having high antibacterial properties against E. coli (MIC = 16 µg/mL) and S. aureus (MIC = 16 µg/mL) showed lower HC50 values of 700 µg/mL against RBCs in comparison to the peptide (HC50 = 110 µg/mL). The cytotoxicity results of these


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nanocapsules towards Lm3 cells showed that the cell viability was not affected up to a concentration of 100 µg/mL93. In another study, a poly-lysine-grafted chitosan having high antimicrobial activity against bacteria and fungi (MIC = 5–10 µg/mL) showed a lack of hemolytic activity (HC50 >50,000–>100,000 µg/mL) and low toxicity towards human aorta Smooth Muscle Cells in vitro and female BALB/c mice in vivo in preliminary experiments94.

Cytotoxicity experiments using different chitosan derivatives have also been reported in various studies. Hydroxypropyltrimethylammoniumchloride (HACC) chitosans having three different DS (0.06, 0.18 and 0.44) were investigated for their biocompatibility towards mouse fibroblasts (L929 cells) according to the ISO standards in an in vitro cytotoxicity evaluation. The results showed that chitosan, HACC (DS = 0.06), and HACC (DS = 0.18) remained non-toxic to the cells, while the HACC (DS = 0.44) was rated as slightly toxic150. In another study, HACCchitosan fiber extracts were also used to treat mouse fibroblasts and the results showed that at concentrations of 55 and 100%, the cells showed 88% viability. At the lower concentration of 25%, no change in the cell viability was noticed in comparison to the negative control. In other words, the HACC-chitosan fibers did not show any toxicity at lower concentrations179. A water soluble

quaternary

salt

of

chitosan,

chitosan-N-hydroxy-2,3-propyl-N-methyl-N,N-

diallylammonium methyl sulfate (MDAACS), was also evaluated for cytotoxicity against the L929 fibroblasts using the MTT colorimetric assay. After 24, 48 and 72 h incubation, the cell viability with the MDAACS was higher than with chitosan, especially after the 72 h period129. In another study, an in vitro cytotoxicity analysis of N-2-hydroxypropyl trimethylammonium chloride chitosan against chicken embryo kidney cells was performed and the results showed that the cells had 90% viability and no significant changes in the cell morphology were observed


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during this treatment68. Chitosan itself have not been investigated much for cytotoxicity. Some studies have evaluated the cytotoxicity of chitosan in the form of water soluble chitosan nanoparticles against the HepG2 human liver cancer cell line180 and mouse hematopoietic stem cells181. The cytotoxic effect of chitosans with different degrees of acetylation (DA = 13%, 30% and 60%) were studied against mouse 3T3 and two pulp fibroblast cell-lines. The results showed that, in general, 13% DA chitosan showed a higher cell survival rate than others. However, the authors also concluded that the different cell lines reacted differently to the different DA chitosans182. A few other studies have used chitosan as a control to compare its cytotoxicity to the synthesized chitosan derivatives129, 150.

The results from the above studies lead us to the conclusion that, in general, the presence of a greater number of cationic groups (or charges) and the closeness of this charge to the polymer backbone increases the toxicity as well as the antibacterial activity of chitosan derivatives. Also, in the case of quaternary ammonium chitosan derivatives, increasing hydrophobicity due to increasing alkyl chain length will lead to an increase in hemolysis and cytotoxicity. The toxic effect of antimicrobial peptides towards red blood cells can be significantly reduced when the peptides are grafted onto chitosan. However, more in vitro studies on various cell lines and indepth in vivo studies are required to determine the suitability of antimicrobial chitosan derivatives for clinical applications. Table 3. Table showing the hemolytic activity and cytotoxicity for the chitosan derivatives. Hemolysis

Cytotoxicity

HC50 (µg/mL)

EC50 in µg/mL, (Cell line)

N,N,N-trimethyl chitosan

6114

86.20(Caco-2), 45 (VA10)

N-ethyl-N,N-dimethyl chitosan

>1024

73.03(Caco-2),

Polymer


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N-butyl-N,N-dimethyl chitosan

36

12.27(Caco-2), 28 (VA10)

N-hexyl-N,N-dimethyl chitosan

16

13.50(Caco-2), 7 (VA10)

N,N-diethyl-N-methyl chitosan

384

39.24(Caco-2)

N,N-dibutyl-N-methyl chitosan

20

8.61(Caco-2)

N,N-dihexyl-N-methyl chitosan

40

14.37(Caco-2)

N-(2-(N,N,N-trimethyl ammoniumyl)acetyl) chitosan

16,384

40(Caco-2)

N-(6-(N,N,N-trimethyl ammoniumyl)hexanoyl) chitosan

>32,768

26(Caco-2)

N-(2-(1-pyridiniumyl)acetyl) chitosan

>32,768

38(Caco-2)

N-(6-(N,N,N-trimethyl ammoniumyl)hexanoyl) chitosan

>32,768

644 (Caco-2)

N-acetyl-N'-stearoyl-N″,N″,N″-trimethyl chitosan

597−6144

−

Chitosan-poly(ethylene glycol)

>25,000

N-acetyl-N″,N″,N″-trimethyl chitosan

1288−8598

−

Anoplin-chitosan

16,384 − >32,768

−

[Poly(Z-Lys11-Phe10)-chitosan]x

700

>200 (HCCLM3)

>50,000

−

Chitosan-(Lys)8-(Phe)8

12,500

−

Chitosan-(Lys)12.5-(Phe)12.5

7500

−

Chitosan-Lys, Chitosan-(Lys)3, Chitosan-(Lys)9, Chitosan-(Lys)16, Chitosan-(Lys)25

8. Applications Chitosan and its derivatives have been widely used in different fields, such as medicine, the pharmaceutical industry, the food industry, cosmetics, water treatment, tissue engineering and agriculture. These applications have utilized chitosan either in its pure form, as a salt or in a formulation where chitosan has been combined with other polymers, chemicals, scaffolds, textiles or other materials. Chitosan is used as a natural pesticide in controlling diseases in crops such as rice, wheat, barley, oilseed rape, tobacco, etc. Chitosan in combinations with various essential oils have been investigated and found to be effective against a wide range of microbes present in fruits and cooked food183. Studies have shown that chitosan solutions prepared in


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acetic acid and the use of chitosan with dental materials such as hydroxyapatite increases the biocompatibility and prevents the adherence of oral bacteria to the tooth184. Chitosans used in different formulations, such as mouth rinses, toothpaste and chewing gums, have also been effective as antibacterial agents in controlling the Streptococcus group of bacteria185,

186

. The

order of inhibitory effect of chitosan and its derivatives on the adherence of oral bacteria onto human anterior teeth surfaces are low molecular chitosan > phosphorylated chitosan > amorphous chitosan > carboxymethyl chitosan177. Some notable investigated applications of chitosan derivatives include chitosan acetate in combination with polyphosphate/silver and quaternized trimethylammonium modified chitosan as bandages for wound treatment187, 188 and chitosan-lauric acid and CMC in combination with NaCl to provide preservation for freshly cut fruits189. Some of the investigated applications involving the antimicrobial properties of chitosan and its derivatives against different bacteria are listed in Table 4. Table 4. Proposed applications of antimicrobial chitosan, chitosan formulations and chitosan derivatives. Chitosan/chitosan derivative

Microbial strain

Application

Chitosan

Streptococcus

Dental materials185, 190

Chitosan

Listeria monocytogenes, Pseudomonas aeruginosa and S. aureus

Dairy food packaging191

Chitosan

F. acuminatum, Cylindrocladium floridanum, Aspergillus flavus, Magnaporthe grisea, Bipolaris sorokiniana, F. graminearum, Phytophthora parasitica, Sclerotinia sclerotiorum

Plant protection192

Chitosan-polyphosphate-silver

P. aeruginosa and S. aureus

Wound dressing193

Chitosan acetate

P. aeruginosa, Proteus mirabilis and S. aureus

Wound dressing194

Carboxymethyl chitosan

E. coli

Fruit preservation189

Chitosan-sulfonamide derivatives

Staphylococcus aureus, Sarcina lutea, Bacillus cereus, Bacillus subtilis, Escherichia coli, Candida albicans, Candida glabrata and Candida sake

Wound dressing and wound healing17

S. aureus

Wound dressing195

N,N,N-trimethyl polylactide/polypropylene fibers

chitosan-


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N-(carboxymethyl) chitosan

F. solani and C. lindemuthianum

Plant protection192

N,N,N-dimethylalkyl chitosans

A. tumefaciens and E. carotovora and fungi B. cinerea, F. oxysporum, and P. debaryanum

Crop protection45

N-(o,p-diethoxybenzyl)chitosan

F. oxysporum and P. debaryanum

N-(o,o-dichlorobenzyl) chitosan, N-(o,odichlorobenzyl) chitosan, N,O-(p-chlorobutyryl) chitosan, N,O-decanoyl chitosan, N,O-cinnamoyl chitosan and N,O-(p-methoxybenzoyl) chitosan

B. cinerea

Crop protection45

N-phenylalanine-O-carboxymethyl chitosan

S. aureus and E. coli

Food preservative coating196

Chitosan/Quaternary chitosan-polylactide

S. aureus and E. coli

Wound healing197

Chitosan, chitosan-hydroxyapatite, N-[1Hydroxy-3-(trimethylammonium) propyl]chitosan chloride, carboxymethyl chitosan

Streptococcus

Dental care185, 198-200

N-[1-Hydroxy-3-(trimethylammonium) propyl]chitosan

Bacillus subtilis

Paper packaging201

carboxymethyl chitosan

E. coli and S. aureus

Cotton fabric202

poly(n-butyl acrylate)-chitosan

S. aureus

Cotton fabric203

Chitosan-cellulose

E. coli and S. aureus

Membranes204

O-hydroxyethyl chitosan-cellulose

E. coli

Textile205

Chitosan-lauric acid-starch

B. subtilis and E. coli

Antimicrobial film206

Dodecenyl succinylated phthaloyl chitosan

E.coli, S. aureus and B. subtilis

Antimicrobial film207

9. Conclusion and future perspectives Chemical modification of chitosan with distinct single or multiple functional groups and varying degrees of substitution will increase the structural diversity relative to the natural polymer. It can simultaneously enhance the aqueous solubility and antimicrobial activity. Research into the structure activity relationship has shown that relatively small variations in the structure can have a significant influence on the bioactivity. Therefore, in addition to choosing an efficient synthetic route for derivatization, selecting a proper method for characterization is equally important. This review has touched on the different synthetic approaches that have been used for preparing antimicrobial chitosan derivatives utilizing either unmodified chitosan or


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protected chitosan and has discussed the advantages and limitations of the methods used for the characterization of these derivatives. Our evaluation of a number of relevant publications suggests that the structure, chemical properties and the DS for substituents attached to the polymer backbone are the most important factors affecting the antimicrobial activity and the toxicological profile. Other factors, such as molecular weight and degree of acetylation, can also be a significant factor. Cationic groups, such as quaternary ammoniumyl and guanidinyl, will, in general, have a positive effect on the antimicrobial activity, but this effect is dependent on factors such as positioning of the cationic charge and the degree of substitution. The effect of other substituents is less clear and the structure activity relationship has not been studied as thoroughly in these cases. The antimicrobial effect may also vary depending on the type of microorganism being tested. The activity of antimicrobial peptides can be enhanced by grafting them to chitosan and the toxicity towards human cells can simultaneously be reduced. Only few systematic studies have taken into account more than one structural parameter for certain types of derivatives, like for example the DS for certain substituents and the Mw. Results from such studies are not always consistent with other studies where only one parameter is considered. Thus there is a need for more studies that can simultaneously consider more than one variant in the structure and any synergistic effects. The antimicrobial activity of chitosan derivatives will also depend on the assay conditions, especially the pH and the strain of the microorganism used in the test. Therefore, the results from different studies cannot be directly compared and it can be difficult to know which material is the most active. Establishing some kind of standard conditions for evaluation of new materials would be beneficial for further development of the field.


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There is no generally accepted mechanism of action for antimicrobial chitosan derivatives, although the electrostatic interaction between the chitosan and the microbial cell wall/membrane is thought to play a significant role and most studies suggest that chitosan and chitosan derivatives act as membrane disruptors. Most studies related to the mechanism of action have only been carried out for unmodified chitosan, so it is unclear if the proposed mechanism of action is valid for chitosan derivatives as well. Further studies of the mechanism of action, should therefore focus not only on chitosan but also on chitosan derivatives with different structures and differentiate the mechanisms for various micro-organisms. Structure activity investigation can be a part of such studies to show whether certain structural property is important for activity or not. This review has focused on soluble chitosan, chitosan derivatives and conjugates which are soluble in aqueous solution and where it is assumed that individual polymer chains will interact with the microbial cell. It is not well understood to what extent these polymer chains could interact to form nanostructures in solution and whether this property could influence the observed activity. This should be considered in future studies. Chitosan and chitosan derivatives have also been used for preparation of antimicrobial films, coatings and nanoparticles. It is not known if structures that show optimal activity in solution will also have good activity is such form and this needs to be studied further. It is very common to state that chitosan is a non-toxic, biodegradable and biocompatible biopolymer146,

208, 209 206, 210

. Many reports of novel antimicrobial chitosan derivatives do not

include studies of toxicological properties and little is known about the biological fate of these materials. There are a number of possible applications for antimicrobial chitosan derivatives which could be used as therapeutic agents, disinfectants and preservatives. A number of studies


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have been carried out with promising results, as reported in this review. Further studies into the clinical potential of these materials would be very welcome. However, before an application is filed for regulatory approval or clinical investigation, it is desirable to carry out studies to fully optimize the structure of the chitosan derivative for the intended application. Rigorous structure activity studies will therefore be an essential part of the progress towards realizing the full potential of antimicrobial chitosan derivatives. These studies should not only consider antimicrobial activity but also toxicology, mechanism of action, biodegradability and pharmacokinetic aspects.

ASSOCIATED CONTENT Supporting Information. Table showing details on DA, DS, Mw, pH and MIC (Table S1). The supporting information is available free of charge on the ACS publication website at DOI: XX

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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We thank financial support from the Icelandic Technical Development Fund (Rannis grant no. 179012-0611) as a part of the jointly funded European Marine Biotech-ERA Net project Blueteeth.

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