The Effect of Molecular Weight on the Antibacterial Activity of N,N,N-Trimethyl Chitosan (TMC)

N,N,N-trimethyl chitosan (TMC) with 93% degree of trimethylation was synthesized. TMC and the chitosan starting material were subjected to acidic hydrolysis to produce 49 different samples with a reduced average molecular weight (Mw) ranging from 2 to 144 kDa. This was done to allow the investigation of the relationship between antibacterial activity and Mw over a wide Mw range. NMR investigation showed that hydrolysis did not affect the degree of trimethylation (DSTRI) or the structure of the polymer backbone. The activity of TMC against Staphylococcus aureus (S. aureus) increased sharply with Mw until a certain Mw value (critical Mw for high activity, CMW) was reached. After the CMW, the activity was not affected by a further increase in the Mw. A similar pattern of activity was observed for chitosan. The CMW was determined to be 20 kDa for TMC and 50 kDa for chitosan.


Introduction
Chitosan is a biopolymer obtained from chitin. Chitin can be processed in different ways to produce chitosan having varying degrees of acetylation (DA) and chain length or weight average molecular weight (M w ). Several studies reported so far produced varying M w ranges for chitosan and chitooligosaccharides (COS, M w < 20 kDa) [1][2][3]. Low M w chitosan or COS possess a wide range of biological activities and potential applications in drug delivery [4], including antimicrobial, antioxidant, hemostatic, wound healing, hypoglycemic and enzyme inhibition activities, and various other applications [4].
A number of studies reported the effect of M w on the antimicrobial activity of chitosan and COS. Kulikov et al. [5] investigated COS within a M w range of 3.5-20 kDa for activity against methicillin-resistant Staphylococcus aureus (MRSA), within a pH range of 5.5-6.75. They have also explored the effect of M w on the growth and viability of a series of Candida species using COS in the M w range 5.9-600 kDa [6]. Chang et al. [7] studied chitosan and COS with M w values of 3-300 kDa for antibacterial efficacy against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), at pH 5 to 7, and at different temperatures (4-45 • C). The effect of size and pH on antibacterial activity of COS with was then utilized for investigating the relationship between antibacterial activity (MIC) toward S. aureus and M w .

Synthesis
Several studies reported the quaternization of chitosan at the 2-amino position and the use of various reaction conditions resulted in obtaining low to high degree of substitution for trimethylation (DS TRI ). Increasing the reaction time or repeated addition of the reagents to increase the DS TRI is generally accompanied by an increase in O-methylation in the products. In our previously published method [14,20], we have shown that the use of MeI in a DMF-H 2 O solvent system (Scheme 1) can be used to obtain high DS TRI , without any significant O-methylation. Hence, we utilized this method for the synthesis of the TMC used in this study. Chitosan was treated with the aforementioned reaction conditions before reprecipitation and repeated addition of the reagents. The spectra for the TMC synthesized can be seen in Figure 1A

Synthesis
Several studies reported the quaternization of chitosan at the 2-amino position and the use of various reaction conditions resulted in obtaining low to high degree of substitution for trimethylation (DSTRI). Increasing the reaction time or repeated addition of the reagents to increase the DSTRI is generally accompanied by an increase in O-methylation in the products. In our previously published method [14,20], we have shown that the use of MeI in a DMF-H2O solvent system (Scheme 1) can be used to obtain high DSTRI, without any significant O-methylation. Hence, we utilized this method for the synthesis of the TMC used in this study. Chitosan was treated with the aforementioned reaction conditions before reprecipitation and repeated addition of the reagents. The spectra for the TMC synthesized can be seen in Figure 1A and the protons appear as follows: GlcNAcCH3 (2.01 ppm), GlcN(CH3)3 (3.28 ppm), GlcN+GlcNAc H-2, H-5, H-6 (3.68-4.07 ppm), GlcN+GlcNAc H-3, H-4 (4.30-4.42 ppm) and GlcN H-1 (5.42 ppm). Small peaks, showing low levels of 3-O and 6-O methylation, are observed at 3.38 and 3.46 ppm respectively. The DSTRI was calculated from the integrals of the 1 H-NMR spectrum ( Figure 1) and was found to be 92.6%. The degree of substitution for dimethylation (DSDI) was 3% with traces of O-methylation (DSO-methyl = 2%).

Acidic Hydrolysis
Previous studies reported the hydrolysis of chitosan to produce low Mw chitosan and COS, mostly using chemical reagents, including treatment with acids and oxidative-reductive

Synthesis
Several studies reported the quaternization of chitosan at the 2-amino position and the use of various reaction conditions resulted in obtaining low to high degree of substitution for trimethylation (DSTRI). Increasing the reaction time or repeated addition of the reagents to increase the DSTRI is generally accompanied by an increase in O-methylation in the products. In our previously published method [14,20], we have shown that the use of MeI in a DMF-H2O solvent system (Scheme 1) can be used to obtain high DSTRI, without any significant O-methylation. Hence, we utilized this method for the synthesis of the TMC used in this study. Chitosan was treated with the aforementioned reaction conditions before reprecipitation and repeated addition of the reagents. The spectra for the TMC synthesized can be seen in Figure 1A and the protons appear as follows: GlcNAcCH3 (2.01 ppm), GlcN(CH3)3 (3.28 ppm), GlcN+GlcNAc H-2, H-5, H-6 (3.68-4.07 ppm), GlcN+GlcNAc H-3, H-4 (4.30-4.42 ppm) and GlcN H-1 (5.42 ppm). Small peaks, showing low levels of 3-O and 6-O methylation, are observed at 3.38 and 3.46 ppm respectively. The DSTRI was calculated from the integrals of the 1 H-NMR spectrum ( Figure 1) and was found to be 92.6%. The degree of substitution for dimethylation (DSDI) was 3% with traces of O-methylation (DSO-methyl = 2%).

Acidic Hydrolysis
Previous studies reported the hydrolysis of chitosan to produce low M w chitosan and COS, mostly using chemical reagents, including treatment with acids and oxidative-reductive depolymerization. The use of hydrochloric acid for hydrolysis of chitin and chitosan has been reported in a few studies [1, 3,6]. The hydrochloric acid catalyzed the transformation of chitin consists of two main reactions, hydrolysis of the glycosidic linkage and the N-acetyl linkage [4]. Hydrochloric acid has been used in different concentrations (0.5 M-12 M) and temperatures (40-100 • C) to produce chitosan and chitooligomers having different ranges of M w 7-2 [21], 37-15 [21], 40-20 [22], 64-7.5 [23], etc. Several other acids were also reported for chitosan hydrolysis, but they usually suffer from certain drawbacks. Sulfuric acid produces drastic hydrolysis but with sulfation and nitrous acid causes hydrolysis without deacetylation but involves the formation of 2,5-anhydro-D-mannose at the new reducing end [4,24,25]. On the other hand, fluorohydrolysis of chitosan results in the introduction of a fluoro group at the anomeric carbon of chitosan or oligomers [4,26]. Acidic hydrolysis of chitosan by 85% phosphoric acid was found to be dependent on the temperature and time of the reaction [2] while degradation of chitosan using an aqueous solution of acetic acid requires high temperature and high acid concentrations [27]. Reviewing the results from these studies, we concluded that hydrolysis of chitosan using hydrochloric acid would be the best choice for hydrolyzing the glycosylic bond without causing other changes in the chemical structure.
The acid concentration can be expected to have an effect on the hydrolysis of chitosan, as seen in previous studies [1]. Hence, we performed the acidic hydrolysis of chitosan and TMC using HCl at two different concentrations: 1 M (dilute) and 12 M (concentrated). The hydrolysis of TMC and chitosan using 1 M HCl resulted in polymers having a M w range of 144-60 kDa (TMC) and 138-35 kDa (chitosan) when the hydrolysis time was set between 0.25-206 h (Table 1). While the use of 12 M HCl resulted in faster hydrolysis within the same time interval, the lowest M w values obtained for TMC and chitosan and were 3 kDa and 2 kDa respectively. The percentage reduction in the M w value is plotted against time in Figure 2. Treatment with 1 M HCl at 60 • C resulted in a significant reduction in the M w of TMC, with M w decreasing by 22% of the original value (186 kDa) within the first 0.25 h. After 0.25 h, the M w decreased slowly and reached a maximum reduction of 67%. On the other hand, treatment with 12 M HCl resulted in a M w reduction of 47% and 98% after similar time intervals. In the case of chitosan, the hydrolysis was seen to follow a similar pattern  Initially, TMC and chitosan were treated with 1 M and 12 M HCl at 30°C. When the hydrolysis of chitosan was performed with 1 M HCl at a higher temperature of 60°C, the rate was found to increase by 2-7 fold. However, a further increase in temperature to 80°C did not result in a significant increase in the hydrolysis rate in the case of chitosan. Hence, for 1 M HCl, the optimized temperature for the hydrolysis of TMC and chitosan was 60°C. In the case of 12 M HCl, increasing temperatures above 30°C resulted in a color change of the materials; therefore, for 12 M HCl, the optimal temperature was 30°C.

Structural Characterization of Hydrolyzed Products
The depolymerized material was investigated by 1 H-NMR, COSY-NMR and FT-IR spectroscopy to determine if any structural changes in the polymer chain occurred during the hydrolysis process. The 1 H-NMR spectra for the anomeric region of the hydrolyzed TMCs are shown in Figure 3A, where the α-anomeric reducing end protons for the GlcN unit appear at 5.63 ppm. The intensity of this proton is seen to increase with the increase in the hydrolysis time. The 1 H-NMR spectra for the anomeric region of the chitooligomers obtained after time intervals 1 h, 4h, and 48 h are shown in Figure 3A. The α-anomeric reducing end protons appear at 5.47 ppm for the GlcN unit and at 5.23 for the GlcNAc unit. With a decrease in the length of the polymer chain, the intensity of the GlcN H-1α proton increases and becomes dominant, while the GlcNAc H-1α proton remains constant in all the spectra and could be detected in only traces, due to low DA in the polymer. The COSY-NMR spectra for the TMC and chitosan oligomer ( Figure 3C,D) further confirm that the structural units of the polymers remain unaltered during the hydrolysis process. Initially, TMC and chitosan were treated with 1 M and 12 M HCl at 30 • C. When the hydrolysis of chitosan was performed with 1 M HCl at a higher temperature of 60 • C, the rate was found to increase by 2-7 fold. However, a further increase in temperature to 80 • C did not result in a significant increase in the hydrolysis rate in the case of chitosan. Hence, for 1 M HCl, the optimized temperature for the hydrolysis of TMC and chitosan was 60 • C. In the case of 12 M HCl, increasing temperatures above 30 • C resulted in a color change of the materials; therefore, for 12 M HCl, the optimal temperature was 30 • C.

Structural Characterization of Hydrolyzed Products
The depolymerized material was investigated by 1 H-NMR, COSY-NMR and FT-IR spectroscopy to determine if any structural changes in the polymer chain occurred during the hydrolysis process. The 1 H-NMR spectra for the anomeric region of the hydrolyzed TMCs are shown in Figure 3A, where the α-anomeric reducing end protons for the GlcN unit appear at 5.63 ppm. The intensity of this proton is seen to increase with the increase in the hydrolysis time. The 1 H-NMR spectra for the anomeric region of the chitooligomers obtained after time intervals 1 h, 4h, and 48 h are shown in Figure 3A. The α-anomeric reducing end protons appear at 5.47 ppm for the GlcN unit and at 5.23 for the GlcNAc unit. With a decrease in the length of the polymer chain, the intensity of the GlcN H-1α proton increases and becomes dominant, while the GlcNAc H-1α proton remains constant in all the spectra and could be detected in only traces, due to low DA in the polymer. The COSY-NMR spectra for the TMC and chitosan oligomer ( Figure 3C,D) further confirm that the structural units of the polymers remain unaltered during the hydrolysis process. The structural characterization of the TMC and chitosan and their hydrolyzed products were also performed using FT-IR spectra. Figure 4A shows an overlay of TMC and chitosan spectra obtained for samples taken at different times of hydrolysis. The IR frequencies are as follows: 3239 (O-H), 2927 (C-H), 1636 (C=O amide I), 1473 (C-H), 1030 (C-O-C) and 880 (C-H for polysaccharide structure) cm -1 . When compared to the chitosan ( Figure 4B) spectrum, it is seen that the peak in the region 1508 cm -1 is absent. This is due to the absence of the N-H bending of the primary amine in TMC. However, no characteristic peak for the NMe3 group could be observed in the IR spectrum of TMC. No significant changes in the IR spectra for the TMC oligomers could be seen, confirming that TMC structure remains unchanged during the acidic hydrolysis process. In Figure 4B  The structural characterization of the TMC and chitosan and their hydrolyzed products were also performed using FT-IR spectra. Figure 4A shows an overlay of TMC and chitosan spectra obtained for samples taken at different times of hydrolysis. The IR frequencies are as follows: 3239 (O-H), 2927 (C-H), 1636 (C=O amide I), 1473 (C-H), 1030 (C-O-C) and 880 (C-H for polysaccharide structure) cm −1 . When compared to the chitosan ( Figure 4B) spectrum, it is seen that the peak in the region 1508 cm −1 is absent. This is due to the absence of the N-H bending of the primary amine in TMC. However, no characteristic peak for the NMe 3 group could be observed in the IR spectrum of TMC. No significant changes in the IR spectra for the TMC oligomers could be seen, confirming that TMC structure remains unchanged during the acidic hydrolysis process. In Figure 4B

Mw-Antibacterial Property Relationship
The hydrolyzed TMC and chitosan obtained within the Mw range of 144-3 kDa and 138-2 kDa, respectively, and their starting materials (TMC = 186 kDa and chitosan = 225 kDa) were investigated for antibacterial activity towards S. aureus. The aim was to investigate whether the Mw change within this range would have a significant impact on the antibacterial activity of TMC and chitosan. There was a 1000-fold variation in the measured activity in this study (Table 2). This data was therefore plotted as log(1/MIC) (MIC (g/mL) on a reverse logarithmic scale) versus Mw, as shown in Figure 5. The TMC and depolymerized TMC showed a clear correlation between Mw and activity against S. aureus ( Figure 5A). Within a Mw range of 144−20 kDa, the materials showed high activity, with log(1/MIC) value of 4.5 (MIC= 32 × 10 -6 g/ml = 32 µg/mL) in most cases. The antibacterial activity, however, decreased with decreasing Mw when the Mw value was less than 20 kDa and reached a minimum value of log(1/MIC) = 1.48 (MIC ≥ 32,768 µg/mL), at Mw = 3 kDa.

Mw-Antibacterial Property Relationship
The hydrolyzed TMC and chitosan obtained within the M w range of 144-3 kDa and 138-2 kDa, respectively, and their starting materials (TMC = 186 kDa and chitosan = 225 kDa) were investigated for antibacterial activity towards S. aureus. The aim was to investigate whether the M w change within this range would have a significant impact on the antibacterial activity of TMC and chitosan. There was a 1000-fold variation in the measured activity in this study (Table 2). This data was therefore plotted as log(1/MIC) (MIC (g/mL) on a reverse logarithmic scale) versus M w , as shown in Figure 5. The TMC and depolymerized TMC showed a clear correlation between Mw and activity against S. aureus ( Figure 5A). Within a M w range of 144-20 kDa, the materials showed high activity, with log(1/MIC) value of 4.5 (MIC= 32 × 10 −6 g/mL = 32 µg/mL) in most cases. The antibacterial activity, however, decreased with decreasing M w when the M w value was less than 20 kDa and reached a minimum value of log(1/MIC) = 1.48 (MIC ≥ 32,768 µg/mL), at M w = 3 kDa. Gentamicin (positive control) 0.5 × 10 −6 1 × 10 −6 As a control, a similar study was done with the depolymerized chitosan samples. Figure  To determine the antimicrobial efficacy of the lowest unit, glucosamine (M w = 0.179 kDa) was also tested against S. aureus, but this monomer did not show any activity within the measured range of dilutions (MIC ≥ 32,678 µg/mL).  Gentamicin (positive control) 0.5 × 10 −6 1 × 10 −6 As a control, a similar study was done with the depolymerized chitosan samples. Figure  To determine the antimicrobial efficacy of the lowest unit, glucosamine (Mw= 0.179 kDa) was also tested against S. aureus, but this monomer did not show any activity within the measured range of dilutions (MIC ≥ 32,678 µg/mL).
Thus, in both cases, the activity of the oligomer/polymer increases with Mw until a certain value is reached, which we have defined as critical mole weight for high activity (CMW). After this, the activity is virtually independent of Mw. According to Figure 3A the CMW for TMC is ~20 kDa and according to Figure 3B, the CMW for chitosan is ~50 kDa.  Thus, in both cases, the activity of the oligomer/polymer increases with M w until a certain value is reached, which we have defined as critical mole weight for high activity (CMW). After this, the activity is virtually independent of M w . According to Figure 3A the CMW for TMC is~20 kDa and according to Figure 3B, the CMW for chitosan is~50 kDa.

Synthesis and Acid Degradation
The procedure for the N,N,N-trimethylation of chitosan has been previously reported [20,28,29]. By repeated methylation reactions a product with a very high degree of trimethylation (quaternization) and a low degree of O-methylation was obtained.
The hydrolysis of TMC and chitosan was carried out in acidic medium. The temperature of the medium is often found to have a significant effect on the acidic hydrolysis of chitosan. In this study, it was found that the optimal temperature for the hydrolysis of chitosan and TMC was 60 • C. For 12 M HCl, the optimal temperature was 30 • C to avoid color change of the product. A previous study reported that the hydrolysis of chitosan oligomers using HCl showed a 6-fold increase from 3 M to 6 M acid concentration and another 6-fold increase from 6 M to 12 M acid concentration. Furthermore, when the reaction temperature was increased from 25 • C to 30 • C and from 30 • C to 35 • C, the rate of hydrolysis increased by 2-fold each time [1].

M w -Antibacterial Property Relationship
This is the first systematic study of the M W activity relationship for antimicrobial chitosan derivative where there is both a high degree of substitution (93% N,N,N-trimethylation) and a wide range of M w (186-3 kDa) in the investigated products. The current results are generally consistent with our previous studies of highly substituted N-acyl quaternary chitosan derivatives [16] where the activity was found to be independent of M w in the M w range 13-20 kDa, which is consistent with a slightly lower CMW for N-acyl derivatives than for TMC. The earlier study of similar N-(2-(N,N,N-trimethylammoniumyl)acetyl and N-(2-(N-pyridiniumyl)acetyl) derivatives found that glucose amine derivatives were inactive (MIC ≥ 8192 µg/mL) towards S. aureus (MRSA), E. faecalis and P. aeruginosa, the chitooligomer derivatives had low to moderate activity (MIC = 4006 to 512 µg/mL) and the chitosan derivatives had moderate to high activity (MIC = 523 to 32 µg/mL). These previous results other antimicrobial chitosan derivatives were therefore generally consistent with the current study of the M w activity relationship for TMC. The antimicrobial activity of chitosan derivatives is also influenced by the chemical structure, degree of acetylation, charge etc. The influence of these is discussed in detail in a recently published review paper [13].
In contrast to the few studies on chitosan derivatives, there are a number of studies that have reported the M w antimicrobial activity relationship for unmodified chitosan as discussed in the introduction. These studies have looked at different M w ranges and activity against different microorganisms. These authors reached somewhat different conclusions about the M w activity relationship and none of these studies have defined a specific M w that is comparable to CMW in the current study. However, a closer investigation of the previously reported data shows that the findings of the previous studies are not inconsistent with the current study. Similar relationships between M w and activity of chitosan have been observed but the conclusions vary partially because of differences in the focus of these studies and in the M w range that has been investigated.
Two of these studies have started from a relatively high M w . No et al. [9] studied activity properties of chitosan with Mw ranging from 28 to 1671 kDa and in most cases the materials had very similar activity with MIC values ranging from 0.05% to 0.1% and there was no clear trend with M w . However, the 28 kDa and 1106 kDa materials were found to be less active (MIC > 0.1%) against about half of the bacteria. Younes et al. [11] studied molecular weight 42 kDa to 135 kDa and DA ranging from 2% to 61%. The N-acetylation (DA) had a very significant negative effect on antimicrobial activity but the trend for molecular weight was much less marked. In most cases, there is only about a twofold difference (1 dilution) or no difference in activity between the highest and lowest M w chitosan with identical DA. This is a small difference relative to the current study where there was a 1000-fold difference in activity and between the lowest M w and the highest M w material in the case of TMC and an 8-fold difference in case of chitosan. Li et al. [8] on the other hand reported only on the activity of low molecular weight COS with M w 2 kDa and less. In their study, the M w had a very significant effect on activity. There was a very clear trend where the activity increased with M w . The difference in activity between the most active and highest M w material (>2 kDa) and the lowest molecular weight material (0.82 kDa), which showed activity, was 64 fold. Although Mellgård et al. [10] could not observe any trend in antibacterial activity with M w , they did report that 2KDa chitosan was inactive in all cases but one where it showed low activity against E. coli. Chitosan materials with a higher molecular weight (M w 28 to 224 M w ) were, on the other hand, active against the tested bacteria in most cases. Omura et al. [12] also found that COS with M w 0.18 to 0.99 kDa were inactive, whereas chitosan samples with M w 11 to 400 kDa were active and they could not report any clear trend in activity for the higher M w material. Kulikov et al. [6] reported that COS with M w 0.7 and 1.5 kDa are inactive against all Candida species except one and that chitosan with M w 8.4 to 70 kDa is more than 10 times more active than the low molecular weight COS, except for C. albicans where the difference was less but still more than fourfold. The activity was in most cases the same for 6, 8.4, 9.7, 20.0 and 70 kDa chitosan and in this M w range, there was no trend in the activity.
The general findings of these previous studies of chitosan are therefore very similar to the current study of TMC and chitosan. Thus, these previous studies also indicate that there is initially a very marked increase in activity with M w below a certain molecular weight and above this value the M w has limited or no effect on activity. This molecular weight value is therefore comparable to CMW as we have defined it in the current study. These previous studies also suggest that CMW for antimicrobial activity may, in general, be less than the value (50 kDa) found for the activity of chitosan against S. aureus in the current study and more similar to the CMW value we found for TMC.

Materials
Chitosan TM 4138 (M w = 225 kDa, and DA = 8%) was obtained from Primex ehf (Siglufjördur, Iceland). The degree of acetylation (DA) was obtained from the integrals of the 1 H NMR spectrum and the weight average molecular weight (M w ) was calculated using size exclusion chromatography. All chemicals were purchased from Sigma-Aldrich and used without any further purification. Dialysis membrane (RC, Spectra/Por, M w cutoff 3500 Da) was purchased from Spectrum ® Laboratories Inc. (Rancho Dominguez, CA, USA). Staphylococcus aureus (ATCC 29213) was obtained from the American Type Culture Collection. Mueller-Hinton Broth and blood agar [heart infusion agar with 5% (v/v) defibrinated horse blood] were purchased from Oxoid (Basingstoke, Hampshire, UK).

Synthesis N,N,N-Trimethyl Chitosan
Chitosan (4 g, 24.4 mmol) was stirred in DMF-H 2 O (100 mL, 1:1), followed by addition of NaOH (2.9 g, 73.4 mmol, 3 eq) under cooling (10 • C). Methyl iodide (9.15 mL, 146.9 mmol, 6 eq) was then added dropwise to the reaction mixture. After completion of addition, the reaction mixture was allowed to reach room temperature and then stirred for 48 h. The reaction mixture was then precipitated with acetone (150 mL) and the solid obtained was collected by filtration, followed by washing with fresh acetone (2 × 150 mL). The product was then subjected to the same reaction conditions three more times. The final product obtained was first purified by ion-exchange with 10% NaCl solution followed 1 mL/min. Each sample had an injection volume of 100 µL and a retention time of 25 min and all the measurements were done in triplicates.

Antibacterial Tests
The antibacterial activity was tested against the Gram-positive bacteria S. aureus (ATCC 29213). The broth microdilution method was used to determine the MIC values using Mueller-Hinton broth at pH 7.2 for TMC and 5.5 for chitosan. Samples for TMC were prepared in sterile water (pH = 7.2) while chitosan samples were prepared at pH = 5.5 at an initial concentration of 32,768 µg/mL. This was then serially diluted by twofold dilutions in a 96-well plate using Muller-Hinton broth, to get final concentrations varying from 16,384 to 4 µg/mL. Gentamicin was used as the performance control during the test. A standard 0.5 McFarland suspension (1-2 × 10 8 colony-forming units (CFU)/mL) was prepared by direct colony suspension in Mueller-Hinton broth. This suspension was further diluted to achieve a final test concentration of 5 × 10 5 CFU/mL in the wells of the microtiter plate. Mueller-Hinton broth without any addition was used as a sterility control and Mueller-Hinton broth with bacterial suspension as a growth control. The microtiter plates were then incubated at 35 • C for 18 h under moistened conditions. The MIC values were determined as the lowest concentration of the antibacterial agent that completely inhibited the visible growth of the microorganism in the microtiter plate.

Conclusions
N,N,N-trimethyl chitosan (TMC) with a very high degree of N,N,N-trimethylation and low O-methylation was synthesized from chitosan starting material with DA of 8%.
Acidic hydrolysis was performed on TMC and chitosan material and to produce samples of different M w for studying the relationship between antibacterial activity and M w . The polymers were treated at different temperatures with concentrated (12 M) or diluted (1 M) HCl. Rapid hydrolysis of the polymer was observed in the first 24 h with more than 50% reduction in the M w . Continued incubation up to 1388 h resulted in slow further degradation. NMR analysis confirmed the hydrolysis of the glycosidic bond. N,N,N-Trimethylation (DS TRI ) was not affected and no other change in the structure was observed with extensive acid treatment.
The hydrolysis of chitosan gave 26 samples with M w ranging from 133 to 2 kDa and 24 samples of TMC with M w ranging from 144 to 2 kDa.
The antimicrobial assay was performed to determine the MIC for activity against S. Aureus with all samples, as well as glucosamine.
The MIC values for chitosan and TMC against S. aureus decreased with M w until a certain M w value (CMW) was reached. After this, no change in activity with a further increase in M w was observed. The CMW was determined to be 20 kDa for TMC. The activity of chitosan also increased with M w up to a CMW value of 50 kDa after which the M w had no effect on activity This is the first study of molecular weight activity relationship for TMC. A number of studies have reported a similar pattern of activity for different M w chitosan against different microorganism and the relationship for other chitosan derivatives may also be similar.
These results on the Mw activity relationship may have implication for the possible mechanism of action for TMC and chitosan. This will be investigated in further studies.

Conflicts of Interest:
The authors declare no conflict of interest.