Purification and Quantitative Determination of Carboxymethylchitin Incorporation Into Submicron Bilayer-Lipid Membrane Artificial Cells (Liposomes) Encapsulating Hemoglobin

Abstract

The separation of carboxymethylchitin-coated hemoglobin-loaded liposomes (CMC-LEHb)s from the non-adsorbed CMC has been achieved by gel chromatography. This purification takes place at the physiological pH of 7.4 favoring HbO₂ preservation. A comparative study between experimental techniques for the quantitative determination of the adsorption of carboxymethylchitin (CMC) onto liposomes encapsulating hemoglobin (LEHb)s has been conducted. Results suggest that FT-IR spectroscopy gives a more accurate quantitative adsorption index while the chitinase-based enzymatic assay should be used as a qualitative detection tool. Quantitative bilayer and surface characterization show that the RBC membrane composition has been closely simulated by that of CMC-LEHbs in terms of total lipids and carbohydrates at 87.8% (phospholipids and cholesterol) and 12.2% CMC respectively.