FT Raman investigation of novel chitosan sulfates exhibiting osteogenic capacity

Abstract

Chitosan sulfates (CHS) exhibiting various total degrees of substitution ascribed to sulfate groups (DS_S) were synthesized. The sulfation could be under homogeneous or non-homogeneous conditions. The obtained CHS were characterized and total DS_S of up to 1.73 were determined. Using chlorosulfonic acid as sulfating agent, CS with total DS_S between 0.86 and 1.67 were obtained and the total DS_S can be regulated by varying the sulfation parameters. Using other sulfating agents, CS with distinct total DS_S of up to 1.73 were prepared. By means of FT Raman spectroscopy, marker bands at 1070 cm⁻¹ or 1014 cm⁻¹ attributed to vibrations of sulfation groups can be applied for quantifying the total DS_S of CHS. Calibration curves with correlation coefficients of more than 0.95 were established, suggesting the feasibility of Raman spectroscopy for quantifying the total DS_S of CHS. Finally, the capacity of CHS to improve the osteogenic activity of bone morphogenetic protein-2 (BMP-2) was presented.

Introduction

Chitosan is the deacetylated form of naturally occurred chitin with a degree of acetylation below 0.4. Chitosan consists of 2-amino- and 2-acetamino-2-deoxy-β-d-glucopyranose (Muzzarelli and Muzzarelli, 2005, Rinaudo, 2006). In order to prepare products with desired properties based on this biopolymer, chemical modifications of chitosan including carboxymethylation and sulfation have been frequently carried out and investigated (Kumar et al., 2004, Muzzarelli and Muzzarelli, 2005, Muzzarelli et al., 1984, Rinaudo, 2006, Zhou et al., 2009).

Chitosan sulfate (CHS), a half-ester of chitosan, is proved to be anticoagulant, antiviral, antimicrobial, and antioxidant (Huang et al., 2004, Nishimura et al., 1998, Vikhoreva et al., 2005, Xing et al., 2004, Xing et al., 2005). CHS could be synthesised heterogeneously or quasi-homogeneously in aprotic organic solvents, such as N,N-dimethylformamide (DMF) (Huang et al., 2004, Vikhoreva et al., 2005). CHS could also be obtained after homogeneous sulfation of chitosan. For this purpose, chitosan was dissolved in dichloroacetic acid or formic acid. Then, the solution was diluted with an aprotic organic solvent before adding sulfating agents. Usually applied sulfating agents are SO₃-DMF complex and chlorosulfonic acid (Gamzazade et al., 1997, Xing et al., 2005).

The determination of the amounts of sulfate groups in CHS was normally realised via elemental analysis. Other analysis methods including IR or NMR spectroscopy can also be applied to analyse CHS (Huang et al., 2004, Xing et al., 2005, Zhou et al., 2009). Raman spectroscopy is a rapid and non-destructive analysis method with beneficial properties, such as ultra-sensitive characterization and no requirement of sample preparation. It has been applied to characterise biological systems and polymer derivatives (Li et al., 2010, Schenzel and Fischer, 2001, Yuen et al., 2009, Zhang et al., 2010). Raman spectroscopy can not only qualify but also quantify the polymer derivatives, such as carboxymethyl cellulose and cellulose sulfate. Characteristic vibrations derived from substituents could be used to determine the total DS attributed to these substituents (Yuen et al., 2009, Zhang et al., 2010).

In this report, diverse novel CHS were prepared with various sulfating agents and their total DS_S were determined. Then, FT Raman analysis of CHS was carried out and strong linear correlations between Raman analysis parameters and the total DS_S were observed, suggesting that FT Raman can be another alternative for determining the total DS_S of CHS. Finally, the feasibility of CHS for stimulating the biological activity of BMP-2 was examined with selected CHS.