Summary The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.Antisense oligodeoxynucleotides (ODNs) have proven useful for selective inhibition of gene expression (Holt et al., 1988; Szczylik et al, 1991). However, their rate of cellular uptake appears to be quite slow, and consequently attempts have been made to enhance their stability and their delivery into cells. For instance, receptor-mediated endocytosis has been used to increase the uptake of synthetic ODNs and other foreign molecules such as proteins complexed to specific ligands (Wu & Wu, 1987, 1988Cotten et al., 1990;Leamon & Low, 1991;Citro et al., 1992;Manfredini et al., 1993). Since the receptors for some growth factors, vitamins and hormones are overexpressed in rapidly dividing tumour cells (Rothemberg & Da Costa, 1971;Asok et al., 1981;Sclhub & Franklin, 1984; Lacey et al., 1989), the ligands of these receptors can be exploited to selectively introduce therapeutic compounds into the cells. The use of modified ligands for specific cell-surface receptors as carriers of oncogene-targeted antisense ODNs represents a potentially useful therapy to be used alone or in combination with antineoplastic drugs.We have previously reported that a c-myb antisensetransferrin-polylysine complex produces an enhanced uptake into HL-60 cells, resulting in an increased biological effect. Recently, we have also observed that a polylysine chain covalently linked to compounds such as insulin, folic acid, retinoic acid, oestrone and testosterone can be used for specific interactions with nucleic acids in physiological ionic conditions (G. Citro, unpublished observation). The presented study describes the efficacy of folic acid receptor-targeted c-myb antisense in the HL-60 cell line. The effect of the complexed phosphodiester (PO) ODNs was compared with that of phosphorothioate (PS) ODN antisense given alone.With doses of 20 and 30 fig mlVl, we found that PS c-myb antisense actively inhibited the rate of the cell proliferation while free PO c-myb antisense had no effect. However, when free PO c-myb antisense ODNs were complexed to FApolylysine, their inhibitory effect on the cell proliferation was even greater than that obtained using the free PS oligos. Furthermore, whereas recent research has indicated there...