Delivery of c-myb antisense oligodeoxynucleotides to human neuroblastoma cells via disialoganglioside GD(2)-targeted immunoliposomes: antitumor effects.
BACKGROUND
Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro.


METHODS
Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis.


RESULTS
Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells.


CONCLUSIONS
Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.
Pagnan
G
G
Laboratory of Oncology, G. Gaslini Children's Hospital, Genoa, Italy.
Stuart
D D
DD
Pastorino
F
F
Raffaghello
L
L
Montaldo
P G
PG
Allen
T M
TM
Calabretta
B
B
Ponzoni
M
M
D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D015153:Blotting, Western; D016503:Drug Delivery Systems; D005732:Gangliosides; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008081:Liposomes; D009447:Neuroblastoma / Q000188:drug therapy*; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage* / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb / Q000037:antagonists & inhibitors* / Q000235:genetics; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D005732:Gangliosides; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense; D020598:Proto-Oncogene Proteins c-myb; C025447:sialogangliosides; C112746:dinutuximab