Elsevier

Carbohydrate Polymers

Volume 72, Issue 4, 10 June 2008, Pages 616-624
Carbohydrate Polymers

Precise derivatization of structurally distinct chitosans with rhodamine B isothiocyanate

https://doi.org/10.1016/j.carbpol.2007.10.004Get rights and content

Abstract

Work to date shows that structurally distinct chitosans have reacted inefficiently and unpredictably with fluorescein isothiocyanate (FITC) in an acid–methanol solvent that maintains both chitosan and fluorophore solubility. Since isothiocyanate preferentially reacts with neutral amine groups, and chitosan solubility typically depends upon a minimal degree of protonation, we tested the hypothesis that precise derivatization of chitosan with rhodamine isothiocyanate (RITC) can be achieved by controlling the reaction time and the degree of protonation. Addition of 50% v/v methanol reduced the chitosan degree of protonation in acetic acid but not HCl solutions. At various degrees of protonation, chitosan reacted inefficiently with RITC as previously observed with FITC. Nevertheless, precise derivatization was achieved by allowing the reaction to proceed overnight at a given degree of protonation (p < 0.0001, n = 18) and fixed initial fluorophore concentration. A reproducible 2% to 4% fraction of neutral amines reacted with RITC in proportion to the initial fluorophore concentration (p < 0.005). Using our optimized protocol, chitosans with different degree of deacetylation and molecular weight were derivatized to either 1% or 0.5% mol/mol RITC/chitosan-monomer with a precision of 0.1% mol/mol. The average molecular weight of fluorescent RITC-chitosan was similar to the unlabeled parent chitosan. Precise molar derivatization of structurally distinct chitosans with RITC can be achieved by controlling chitosan degree of protonation, initial fluorophore concentration, and reaction time.

Introduction

Chitosan is a biocompatible polysaccharide employed in medical applications where the rate of cell uptake and clearance is of paramount importance. Chitosan structure can vary in terms of molecular weight and molar percent glucosamine [glucosamine/(glucosamine + N-acetyl glucosamine)] which is also known as the degree of deacetylation (DDA). Fluorescent chitosans derivatized with fluorescein isothiocyanate (FITC) have been previously used to show that chitosan DDA and molecular weight can influence cell binding, in vivo absorption and in vivo clearance (Chae et al., 2005, de Campos et al., 2004, Huang et al., 2004, Onishi and Machida, 1999). To pin-point structural features that affect chitosan-cell interactions, specifically in regenerative medicine applications involving transient in situ chitosan residency (Buschmann et al., 2006, Hoemann et al., 2005, Shive et al., 2006), the derivatization level using any given fluorophore should be held constant when comparing structurally distinct chitosans. Using this principle, and a library of equally derivatized FITC-chitosans, Chae et al. (2005) determined that increasing chitosan molecular weight suppresses in vivo intestinal absorption, although the authors of this study did not provide a detailed description of the labeling procedure in DMSO.

A procedure for coupling chitosan with FITC in acid–methanol has been described (Huang et al., 2002, Qaqish and Amiji, 1999). However we determined that this method has previously yielded widely varying and uncontrolled 24–91% coupling efficiencies between FITC and chitosan (Table 1). We therefore aimed to optimize the acid–methanol labeling procedure to produce structurally distinct chitosans precisely derivatized with 1.0% mol/mol rhodamine B isothiocyanate (RITC). Our strategy intended on the one hand to minimally alter chitosan DDA and on the other hand to have acid-stable fluorescent derivatives for comparative intracellular tracking in live cells. We hypothesized that the reaction could be best controlled by holding constant the time, temperature, and solution pH. Given that isothiocyanate optimally reacts with neutral amine groups at pH 9.0, we considered that solution pH was a most critical variable because homogenous labeling requires chitosan to be fully soluble, while most chitosans are insoluble when a substantial number of amine groups are neutral at pH  6.5 (Nordtveit et al., 1994, Rinaudo et al., 1999a). Using a labeling protocol optimized for reaction time and chitosan degree of protonation, we determined the effect of performing the chitosan labeling reaction in dilute acetic acid versus dilute HCl with chitosans of variable deacetylation levels and different molecular weights generated either by nitrous acid degradation or hydrochloric acid hydrolysis (Fig. 1).

Section snippets

Materials

Medical-grade free-base chitosans with defined DDA and molecular weight (Table 2) were provided by BioSyntech (Laval, QC, Canada) and certified to contain <0.2% w/w protein, <500 EU/g endotoxin, and <10 ppm heavy metals. All chitosans were either lyophilized or weighed taking into account water content (loss on drying). Rhodamine B isothiocyanate (C29H30ClN3O3S···Cl, 536 g/mol, mixed isomers, Product No. R1755), rhodamine B (C28H31ClN2O3, 479 g/mol, Product No. 25-242-5), and 1 N HCl (tissue

Chitosan degree of protonation in acid–methanol

To optimize the reaction pH, we determined the chitosan degree of protonation in acid–methanol at specific [acid]/[glucosamine] ratios. Chitosans dissolved in acetic acid had a higher solution pH and a therefore a lower degree of protonation compared to chitosans dissolved in HCl at the same concentration (Fig. 2, Fig. 3), consistent with previous literature (Rinaudo et al., 1999a). Methanol had a striking effect on chitosan dissolved in acetic acid, namely an increase in pH and a remarkable

Conclusion

Reaction conditions were established that permit precise coupling of rhodamine B isothiocyanate with structurally distinct chitosans. Precise labeling (0.5% mol/mol or 1.0% mol/mol) was achieved by generating chitosan solutions at specific degree of protonation in the reaction media (α = 0.8 or α = 0.5, respectively), by adding 2% mol/mol (6.6% w/w) fluorophore, and by allowing the reaction to proceed for 18 h. Using this protocol, other isothiocyanate-reactive compounds could also be conjugated to

Acknowledgements

Funding was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). O.M. was supported by a fellowship from the Canadian Arthritis Network. M.L. was supported by a fellowship from the Canadian Institutes for Health Research. C.H. received salary support from the Fonds de la recherche en santé Quebec (FRSQ). We gratefully acknowledge Alessio Serreqi and Mariana Anca for production and characterization of autoclave sterile chitosan solutions, Stephane Methot for

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