We recently synthesized a lipophilic prodrug of 9-(2-phosphonyl-methoxyethyl)adenine (PMEA), designated PMEA-LO, and incorporated it into reconstituted lactosylated high-density lipoprotein (LacNeoHDL). In a rat model, LacNeoHDL-associated PMEA-LO was internalized by the asialoglycoprotein receptor on parenchymal liver cells and converted into its active diphosphorylated metabolite. To further evaluate the therapeutic potential of the carrier-associated prodrug, we examined in this study the processing of (125)I-labeled PMEA-LO--loaded LacNeoHDL by HepG2 cells. Upon incubation with HepG2 cells, PMEA-LO--loaded LacNeoHDL became rapidly cell-associated. The association was saturable and of high-affinity (k(d) = 3.8 +/- 0.4 nM). Asialofetuin, an established ligand for the asialoglycoprotein receptor, inhibited the association by >75%, which confirms the role of the asialoglycoprotein receptor. Association of the prodrug-loaded particles to HepG2 cells was coupled to degradation. Radiolabeled degradation products appeared in the culture medium with a lag phase of 2 h. Asialofetuin and chloroquine inhibited secretion of degradation products by 75 to 80%, indicating that PMEA-LO--loaded LacNeoHDL is internalized via the asialoglycoprotein receptor and lysosomally processed. The therapeutic potential of LacNeoHDL-associated PMEA-LO was studied by measuring its effects on hepatitis B virus (HBV) replication in Hep AD38 cells (HBV-transfected HepG2 cells). LacNeoHDL-associated PMEA-LO effectively inhibited HBV DNA synthesis. The EC(50) value of carrier-associated PMEA-LO was 35 times lower than that of free PMEA (3.4 +/- 0.4 and 120 +/- 18 ng of PMEA/ml, respectively). We conclude that the present results, combined with our earlier in vivo disposition data, underscore the therapeutic potential and utility of PMEA-LO--loaded LacNeoHDL for treatment of chronic hepatitis B.