Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells
Section snippets
Materials
The following compounds were obtained from the sources indicated: human serum albumin from the Central Blood Laboratory (Amsterdam, The Netherlands); dexamethasone and dexamethasone disodium phosphate from Bufa (Hilversum, The Netherlands); 3H-dexamethasone from Amersham International (Buckinghamshire, UK); LPS from Escherichia coli (serotype 026:B6) from Sigma Chemical Co. (St. Louis, MO, USA); Williams' medium E (WME) supplemented with Glutamax I and gentamicin from Gibco BRL (Paisley, UK);
Characterisation of Dexa10-HSA
Coupling of dexamethasone hemisuccinate to HSA resulted in a PBS-soluble conjugate with an average of 10 Dexa molecules covalently linked per HSA molecule as detected by HPLC analysis. Noncovalently attached Dexa was not detectable in the product. FPLC analysis showed a significant increase in negative charge as compared to HSA and an increase in molecular mass (Table 1). With hydrolysis under alkaline conditions, all Dexa was released within 1 h. HPLC analysis also showed that Dexa was only
Discussion
The present study has shown that cell-selective hepatic delivery of Dexa can be achieved by covalent linking of Dexa to HSA. The coupling of Dexa to lysine groups in HSA prevents subsequent protonation of the lysine amino group, leading to an increase in net negative charge (Table 1). In addition, the linking of Dexa molecules also increased the hydrophobicity of HSA. The endothelial and Kupffer cells contain scavenger receptors recognising macromolecules with negative charge 33., 34., 35., 36.
Acknowledgements
The following are gratefully acknowledged for their assistance: A. van Zanten for performing the radionation of Dexa10-HSA, A. van Loenen-Weemaes for all surgery on the rats, P. A. Klok for handling the rabbits to produce the anti-Dexa antibody, J. Visser for assistance with the HPLC technique, M.T. Merema and M.H. de Jager for making liver slices and Dr. A.P. Bruins for the mass spectral analysis.
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