Riboflavin-mediated delivery of a macromolecule into cultured human cells

Cell surface receptors for the vitamins folic acid and biotin have been pre viously reported to mediate the endocytosis of vitamin-conjugated macromole cules into cultured cells. To evaluate whether a similar uptake pathway for riboflavin-conjugated macromolecules might exist, riboflavin was covalentl y linked to bovine serum albumin (BSA) via the vitamin's ribityl side chain , and uptake of the protein by cultured human cells was examined. Whereas u nconjugated BSA was not internalized by KB, A549, SK-LU-1 or SK-OV cells, r iboflavin-conjugated BSA was readily internalized (> 10(6) molecules/cell). Analysis of the uptake pathway revealed that the riboflavin-BSA conjugate likely docks on cells at a carrier/transport protein that is distinct from the uptake pathway for free riboflavin and then enters via normal membrane cycling. Evidence for this contention is: (i) the internalized conjugate ac cumulates in endosomal compartments, (ii) uptake into cells is halted at te mperatures near 0 degrees C where membrane trafficking is abrogated, (iii) cell association is inhibited by unlabeled riboflavin-BSA, but not by free riboflavin, and (iv) cellular uptake of [H-3]riboflavin is only partially i nhibited by riboflavin-BSA. Regardless of the pathway of internalization, t hese data demonstrate that riboflavin conjugation can facilitate protein en try into human cells in culture. (C) 1999 Elsevier Science B.V. All rights reserved.