Shielding effect of ‘surface ion pairs’ on physicochemical and bactericidal properties of N,N,N-trimethyl chitosan salts

doi:10.1016/j.carres.2014.10.002

Carbohydrate Research

Volume 402, 30 January 2015, Pages 252-260

https://doi.org/10.1016/j.carres.2014.10.002 Get rights and content

Highlights

•
N,N,N-Trimethyl chitosan (TMC) free of O-methylation and with partial O-methylation were obtained.
•
The dialysis process allowed the removal of ‘surface ion pairs’ on the TMC salt structure, increasing the mobility of TMC chains.
•
After dialysis, TMC salts free of ‘surface ion pairs’ with amorphous character kill E. coli in only 6 h of incubation.

Abstract

Different methodologies were employed in this study to synthesize N,N,N-trimethyl chitosan salts (TMC). TMC free of O-methylation and with partial O-methylation were obtained and characterized through ¹H nuclear magnetic resonance, wide angle X-ray scattering, scanning electron microscopy coupled with X-ray energy dispersive spectroscopy, and thermogravimetric analysis. It was verified that the dialysis process allowed the removal of ‘surface ion pairs’ on TMC salt structure, increasing the mobility of TMC chains. The surface ion pairs considerably increased the material crystallinity, this property being independent of the used synthesis methodology. Biological tests showed that after dialysis, TMC salts free of ‘surface ion pairs’ kill Escherichia coli in only 6 h of incubation. So, the increase in the mobility of dialyzed TMC chains allowed a strong interaction with the cell envelope and the good bactericidal activity of TMC was enhanced.

Graphical abstract

N,N,N-trimethyl chitosan salt containing surface ion pairs (before dialysis) and N,N,N-trimethyl chitosan salt without surface ion pairs (after dialysis).

Introduction

Chitosan (CHT) is a polysaccharide consisting of β(1-4)-d-glucosamine and β(1-4)-N-acetyl d-glucosamine units and is obtained by partial deacetylation of the natural polymer chitin.¹ In recent years, CHT has been under investigation for various biomedical and pharmaceutical applications.2, 3, 4, 5 However, its poor aqueous solubility and loss of penetration-enhancing activity above pH 6.5 are major drawbacks for its use at physiological conditions.⁶ N,N,N-Trimethyl chitosan chloride (TMC) is the simplest form of quaternized CHT. TMC presents good solubility in water at neutral and basic pH values compared to raw CHT.7, 8, 9, 10

TMC is synthesized through the reaction of CHT with excess of methyl iodide in strong alkaline conditions, using N-methyl-2-pyrrolidone as solvent.8, 11, 12 The alkylation reaction of primary amines on CHT chains in strong alkaline conditions promotes partial and uncontrolled methylation of the hydroxyl groups present in the C3 and C6 carbons of the CHT structure.9, 11 Dimethylsulfate, is another methylating agent used for direct trimethylation of CHT. TMC with DQ = 52.5% and also containing O-methylation has been synthesized using dimethylsulfate.13, 14

O-Methyl free TMC can be synthesized from formic acid-formaldehyde methylation, followed by reaction with methyl iodide, without the presence of base.9, 15 In recent times, the new method proposed by Verheul et al.⁹ has been used to obtain TMC free of O-methyl groups. TMC without O-methylation has good properties relative to TMC with O-methylated groups, which include low cytotoxicity and good solubility at physiological pH.⁹ More homogeneously N-quaternized CHT-derivatives, in this case, TMC free of O-methylation could be synthesized by protecting the hydroxyl groups on the CHT backbone.14, 16 Protection groups have been used for the synthesis of CHT-derivatives, not only to allow more selective modifications but also to enhance the solubility of the polymer in organic solvents.¹⁴ Hydroxyl groups on the CHT structure can be protected by di-tert-butyl dimethylsilyl (di-TBDMS) protecting group, which presents good stability under acidic conditions and still can be easily removed under strongly basic or moderate acid conditions without affecting other functional groups.¹⁶ TBDMS protected CHT was used as a precursor in the synthesis of fully trimethylated TMC.¹⁶ By using the di-TBDMS CHT-derivative, TMC was 100% N-trimethylated, with no O-methylation.¹⁶

Compared to CHT, the O-methyl TMC and the TMC free of O-methylated groups display more effective bactericidal activity against Escherichia coli (E. coli).17, 18, 19, 20, 21 Although different mechanisms have been proposed to explain the antibacterial action of CHT and TMC, the exact mechanism is still unknown. Interactions between positively charged CHT or TMC molecules and negatively charged microbial cell membranes leads to the leakage of proteinaceous and other intracellular constituents which seem to be the basis of such mechanisms.²⁰

Studies have shown that at low pH conditions (pH <5.5) the protonated amino groups rather than N-trimethylated groups [–N(CH₃)₃⁺Cl⁻] in TMC, contributed to the antibacterial activity and the N-monomethylated [–NHCH₃] and N-dimethylated [–N(CH₃)₂] groups functioned in the same manner as free amino groups (–NH₂) at pH <5.5.¹⁹ According to Xu et al.¹⁹, Follmann et al.,²⁰ and Martins et al.²² the lower pH benefited the protonation of the –NH₂, –NHCH₃, and –N(CH₃)₂ groups, but inhibited the dissociation of –N(CH₃)₃⁺Cl⁻ sites, present on TMC salts. Therefore, TMC salts with more amounts of N-trimethylated groups in their chains presented greater flexibility in alkaline medium than that at low pH (<5.5). So, the TMC with elevated quaternization degree (DQ = 94.7%), exhibited strong antibacterial activity at pH 7.2.¹⁹ On the other hand, at acid medium the repulsive forces among –N(CH₃)₃⁺ groups and H⁺ ions favor the shielding process of N-quaternized sites and cause the TMC chains to become curled and more heavy than CHT; its interaction with the cell envelope is thus reduced.19, 20 So, –N(CH₃)₃⁺Cl⁻ groups could not interact with the negatively charged sites on bacteria cells below pH 5.5. Therefore, the ion chloride in TMC salts can shield the interaction of TMC with the bacteria cell envelope, decreasing the effectiveness of the bactericidal effect of TMC.¹⁹

Recently published studies established a relationship between biocide action of O-methylated TMC chloride and TMC chloride free of O-methyl groups with the pH, DQ, and molecular weight of CHT-derivative.19, 20, 23 However, the real effect of shielding process on –N(CH₃)₃⁺Cl⁻ promoted by the formation of ‘surface ion pairs’ among the sodium chloride and TMC salts has not been still demonstrated. Therefore, the goal of this study is to characterize TMC salts obtained through different purification processes and to evaluate the shielding effect of surface ion pairs on the physicochemical properties and bactericidal action of different TMC salts.

Section snippets

Characterization of N,N,N-trimethyl chitosan by ¹H NMR

The ¹H NMR spectra of CHT, DMC, N,N,N-trimethyl chitosan free of O-methylation (TMC-F), and O-methylated N,N,N-trimethyl chitosan (TMC-OM) are show in Figure 1. All the resonances were attributed and related with the polysaccharide structures presented in Scheme 1, Scheme 2. CHT spectrum shows resonances at 2.07 ppm, referring to the methyl hydrogen atoms of the acetamide groups; in 3.15 ppm (H2); 3.61–4.20 ppm (H3–H6); and H1 at 4.69 ppm, respectively) were referring to repeat units of CHT (Fig. 1

Conclusions

TMC was synthesized using different methods; however the DQ of the samples were approximately constant. From the ¹H NMR, TGA, SEM, EDS, and WAXS analyses it was found that the formation of ‘surface ion pairs’ among TMC and sodium chloride significantly influenced the physicochemical and biological properties of TMC salts. The surface ion pairs on TMC structure prevented the bactericidal action against E. coli. At low concentrations, TMC shielded by ionic pairs showed no significant microbial

Materials

CHT (CAS 9012-76-4) with deacetylation degree equal to 85% and average molecular weight 87 × 10³ g mol⁻¹ was purchased from Golden-Shell Biochemical (China). Methyl iodide (CAS 74-88-4), N-methyl-2-pyrrolidinone (NMP; CAS 872-50-4), formaldehyde stabilized with methanol (CAS 0050-00-0), and formic acid (CAS 77-92-9) were purchased from Sigma-Aldrich. Other reactants such as sodium hydroxide, sodium iodide, sodium chloride, hydrochloric acid, ethanol, and diethyl ether were also utilized in this

Acknowledgments

A.F.M and E.C.M. thank CNPq for financial support (Proc. 481424/2010-5 and 308337/2013-1).

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Shielding effect of ‘surface ion pairs’ on physicochemical and bactericidal properties of N,N,N-trimethyl chitosan salts

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Characterization of N,N,N-trimethyl chitosan by 1H NMR

Conclusions

Materials

Acknowledgments

Int. J. Biol. Macromol.

Biomaterials

Biomaterials

Carbohydr. Polym.

Carbohydr. Polym.

Biomaterials

Eur. J. Pharm. Biopharm.

Carbohydr. Polym.

J. Control. Release

Carbohydr. Res.

Carbohydr. Polym.

Carbohydr. Res.

Carbohydr. Polym.

Eur. Polym. J.

Characterization of N,N,N-trimethyl chitosan by ¹H NMR