1) In order to achieve the visibility of the chitosan macromolecule for the UV optical system of the analytical ultracentrifuge on investigation of the molecular characteristics and polymers interactions, the labeling of chitosan by a new fluorophore of fluorescein-5-isothiocyanat was carried out. 2) Samples of fluorescent chitosan with two different degrees of fluorophore substitution and various degrees of acetylation were obtained. 3) The labeled chitosans with the fluorescein-5-isothiocyanat allowed estimating the sedimentation coefficient and the molecular characteristic in the analytical ultracentrifuge. 4) The sensitivity of the UV-optical system of the analytical ultracentrifuge for the obtained fluorescent samples of chitosan relatively to the fixation of the meniscus and the influence of the wavelength and rotation speed were estimated.
Due to the features of the chemical structure and organization of the macromo lecules, chitosan possesses many properties, that enable to use in many spheres [
As is known, one possible way of overcoming this invisibility is to label the polysaccharide with a chromophore containing compounds. At present time a labeling procedure effectively used in the field of polymers and on the strength of practical needs this method which develops with searching of the new systems of fluoroagents and polymers.
For example, a non-invasive analytical tool was developed to assess the use of in situ biomaterials for surgical implants or scaffolds in tissue engineering and on the basis of polymeric methods of treatment. In this study, a method for fluorescence monitoring of the chitosan membrane framework degradation was established for in vitro use in bioreactors and, ultimately, in vivo. The basis of this tracking system is the fluorescence-radiating biomaterial obtained by covalent binding of tetramethylboramine isothiocyanate (TRITC) fluorophore based on chitosan [
At present work the samples with high extinction of UV-adsorption fluorescent chitosan modified with fluorescein-5-isothiocyanat (FITC) were obtained and possibilities of using this fluorescence labeled chitosans for characterization in the analytical ultracentrifuge were investigated. Labeled with FITC chitosans intended to use especially for identification of polymers interactions by synthetic boundary methods in AUS [
Fluorescein-5-isothiocyanat. As fluoroagent is used fluorescein-5-isothiocyanat (FITC, the product of ALDRICH, F22502-1G), M = 389.4 g/mol.
Chitosan samples with different viscosities (η) and fractions of N-acetylated units (FA), were provided by SIGMA. The chitosan-1, η = 400 mPa, FA = 0.15; The chitosan-2, η = 200 mPa, FA = 0.18.
Chitosans were labelled with the fluorescein-5-isothiocyanat by method described in work (Coelfen [
So, chitosan is hygroscopic polymer therefore for correlation density and solutions concentration the thermogravimetric analysis has performed the dates which are given in
The chitosans dissolved in acetate buffer which made up with the following composition (Dawson et al., 1986) [
An Optima XL-A analytical ultracentrifuge (Beckman, Palo Alto, CA, USA) was used for all the experiments. It included two integrated detection systems, scanning UV:vis and Rayleigh interference optics systems. For the sedimentation analysis the rotor speed of 50,000 rpm, temperature of 20˚C and scanning wavelengths of 210 nm - 270 nm were employed. Sedimentation coefficient distributions were calculated using of SEDFITBETA2 data evaluation program.
UV/VIS Spectrometer Lambda 2 (Perkin Elmer) was used for determining of efficiency of the fluorescence labelling reaction of chitosans and to calculate the extinction coefficient of chitosan solutions.
Chitosan samples | LOD, % | DA, % | ρ (g/ml) | υ (ml/g) | MW, g/mol |
---|---|---|---|---|---|
Chitosan-1 | 9.90 | 85 | 1.817 | 0.550 | 100,000 |
Chitosan-2 | 7.53 | 82 | 2.000 | 0.465 | 50,000 |
All dialyzing equilibrium of polymer solutions was performed with Spectra Por Dialysis Membrane, MWCO 3500. Vacuum drying of samples was performed with equipment CHRIST LOC-1M, The freezing conditions was performed in vacuum 0.431 mbar and temperature 20˚C.
The modification of chitosan with FITC realized by method described in works Coelfen [
Preliminary dissolved the FITC in DMSO is added to solution of chitosan in acetate buffer 0.4 MCH 3 COOH / 0.4 MCH 3 COONa / 0.2 MNaCl . The amount of the reacting compounds correlated as for the first case to 100 structured units of chitosan corresponded one molecule of FITC (DS 1.0%) and in the second case to 200 units corresponded one molecule of FITC (DS 0.5%). The reactionary mixture was stirred 24 h. Excesses amount of FITC removed with dialyzing in system Acetic buffer/DMSO taken in 12:1 ratio. With evaporation of the solution under vacuum and low temperature, the sample of fluorescent chitosan is received.
The fluorescein 5-isotiocyanatewas introduced to the different chitosans (
Scheme 1. Reductive amination by chitosan (I) of fluorescein 5-isotiocyanate (II) to obtain the fluorescent chitosan (III).
The molar ratios of compounds in the coupling reaction as mentioned before were 100:1, 200:1 in order to obtain the chitosans with a chromophore which can conveniently be visualized and quantified by UV or fluorescence spectroscopy, without altering the conformation of the chitosan.
The qualitative estimation of productivity to reactions was performed with UV spectroscopy measurements of modified chitosans solutions with different concentrations in acetate buffer. The maximum emission wavelength of 240 nm with excitation ε = 9.934 was found in specters. Fluorescent chitosans with DS 0.5% show the same maximum emission wavelength of 240 nm but with decreased excitation. No significant difference was observed between the maximum emission wavelengths (εmax) of different MW chitosans.
The quantity estimation of degree substitution of the fluoroagent is definite with measuring of compactness charge in macromolecule with the help of devise ParticleCarge Detector PCD 3. According to this measurement for Chitosan-1 with DS 1% it was 322.556 k/g and for Chitosan-1 with DS 0.5% was 162.52 k/g.
The fluorescent chitosans were characterized and some compared analysis of the sedimentation coefficients as a function of the degree of substitution of the chromophore and UV extinction for the two chitosans by treatment of UV absorption scans of the XL-A optic system of the analytical ultracentrifuge was carried out. The data of measurements show that for the chitosan-1 with DA 85%, MW = 100,000 and substituted with fluoroagent 0.5% in macrochain has average sedimentation coefficient value 2.3 Svb and extinction coefficient at 240 nm wavelength of UV corresponded to 5.582. For this chitosan sample with contents of fluoreagent 1% the sedimentation coefficient is negligibly increased, at extinction coefficient increased on 9.934 as it was expected. For another chitosan sample with DA 82%, MW = 50,000 also observed the same sequence of dependence of the sedimentation coefficient (S = 1.49 Svb) with DS and extinction coefficient.
Sedimentation behavior of fluorescein modified chitosans determines that the chromophor group does not render the essential influence upon molecular and hydrodynamic parameters of chitosan. In the following discussion, we have chosen the modified chitosan-1 (see
As mentioned before, the fluorescein modified chitosan intended to use to obtain the membrane by synthetic boundary method in the analytical ultracentrifuge through interpolymer interactions [
The fluorescent chitosan without significant depolymerisation of the polysaccharide with high extinction was prepared. Like chitosan, fluorophore 5-isotioceanat chitosan was water soluble at acidic pH values.
The increased incorporation of the fluorophore 5-isotioceanat at least up to a degree of substitution of 1% has no deleterious effects on the molar mass of two chitosans of differing degrees of acetylation. The inclusion of the fluorophore allows the use of the absorption optical system of the analytical ultracentrifuge to evaluate the sedimentation velocity and membrane formation process in synthetic boundary methods, for which these samples of chitosan will be used.
I express my gratitude to Professor of University of Konstanz, Germany Helmut Coelfen who provided laboratories and help on carrying out this work, also I thank the professor of Institute of chemistry and physics of polymers of the Academy sciences of Uzbekistan Sayora Rashidova for her assistance.
The author declares no conflicts of interest regarding the publication of this paper.
Kodirkhonov, M.R. (2019) Obtaining the Fluorescent Chitosan for Investigations in the Analytical Ultracentrifuge. Advances in Biological Chemistry, 9, 23-30. https://doi.org/10.4236/abc.2019.91002