Co-Delivery of CPT-11 and Panobinostat with Anti-GD2 Antibody Conjugated Immunoliposomes for Targeted Combination Chemotherapy

SIMPLE SUMMARY: In targeted cancer therapies, liposomes conjugated with antibody (Ab) can selectively deliver drugs to antigen-expressing cancer cells through active targeting and improve anti-cancer efficacy. Many glioblastoma cell lines and primary biopsies express high levels of disialoganglioside GD2 antigen, making it an excellent candidate for targeted cancer therapy. In this study, we prepared anti-GD2 Ab conjugated immunoliposomes (ImmuLipCP) for co-delivery of CPT-11 and panobinostat, which is intended for combination targeted chemotherapy. To compare the GD2 targeting mechanism, we used glioma cells with low GD2 expression (U87MG) and its drug-resistant variant with high GD2 expression (U87 DR). We demonstrate that ImmuLipCP show enhanced cytotoxicity against U87DR through Ab-mediated intracellular trafficking and drug delivery, for synergistic cancer cell killing effects. Using a xenograft tumor model by subcutaneous implantation of U87DR in nude mice, we also validate the targeting and anti-cancer efficacy of ImmuLipCP in vivo. ABSTRACT: The consistent expression of disialoganglioside GD2 in neuroblastoma tumor cells and its restricted expression in normal tissues open the possibility to use it for molecularly targeted neuroblastoma therapy. On the other hand, immunoliposomes combining antibody-mediated tumor recognition with liposomal delivery of chemotherapeutics have been proved to enhance therapeutic efficacy in brain tumors. Therefore, we develop immunoliposomes (ImmuLipCP) conjugated with anti-GD2 antibody, for targeted co-delivery of CPT-11 and panobinostat in this study. U87MG human glioma cell line and its drug resistant variant (U87DR), which were confirmed to be associated with low and high expression of cell surface GD2, were employed to compare the targeting efficacy. From in vitro cytotoxicity assay, CPT-11 showed synergism drug interaction with panobinostat to support co-delivery of both drugs with ImmuLipCP for targeted synergistic combination chemotherapy. The molecular targeting mechanism was elucidated from intracellular uptake efficacy by confocal microscopy and flow cytometry analysis, where 6-fold increase in liposome and 1.8-fold increase in drug uptake efficiency was found using targeted liposomes. This enhanced intracellular trafficking for drug delivery endows ImmuLipCP with pronounced cytotoxicity toward U87DR cells in vitro, with 1.6-fold increase of apoptosis rate. Using xenograft nude mice model with subcutaneously implanted U87DR cells, we observe similar biodistribution profile but 5.1 times higher accumulation rate of ImmuLip from in vivo imaging system (IVIS) observation of Cy5.5-labelled liposomes. Taking advantage of this highly efficient GD-2 targeting, ImmuLipCP was demonstrated to be an effective cancer treatment modality to significantly enhance the anti-cancer therapeutic efficacy in U87DR tumors, shown from the significant reduced tumor size in and prolonged survival time of experiment animals as well as diminished expression of cell proliferation and enhanced expression of apoptosis marker proteins in tumor section.