Heparin activity-assay of liposomal formulations

Abstract

It is very important for a liposomal formulation that the encapsulated substance will be active upon release from the vesicle. Chemical reactions between the drug and its liposome carrier or the active site of the drug may result in an inactive form of the drug that will have no desirable effects whatsoever. The heparin activity assay was carried out in order to determine: a) the activity of the liposome-encapsulated heparin and b) the concentration of heparin encapsulated an alternative strategy of using liposome lysis with triton. The assay is based on the inhibitory effect of antithrombin III (ATIII) on the reaction between factor Xa and factor Xa substrate. This reaction yields a yellowish solution in which the intensity of the color is irreversible to the inhibition of ATIII. The activity of heparin, of which the presence enhances the inhibitory effect of ATIII, was able to be determined by the intensity of the resulted color of the solution. Blank wells (absent heparin) were used in order to determine the background absorbance (noise). A standard curve was produced relating the absorbance (reaction yield) with heparin activity by using free heparin. Two different kinds of liposomes were also used for this assay. One containing phosphatidic acid (used with cholesterol and lecithin, denoted PA) and the other containing stearylamine (used with phospatidylcholine and cholesterol, denoted ST). Lysed and non-lysed liposomes were used to determine the activity of encapsulated heparin after release. The results showed clearly that encapsulated heparin detained its activity after liposomal lysis. However there was also an activity of non-lysed liposomal heparin which could be attributed to the heparin attached to the outside of the liposomal structure. The supematants extracted from liposomal centrifugations were also measured for their absorbance in order to confirm their heparin-activity. Results also confirmed the theory of coated liposomal heparin. ©PHARMAKON-Press.