Assessment of in vitro efficiency of multi-targeting liposomes containing zinc-phthalocyanine for photodynamic therapy

Background: Photodynamic therapy (PDT) yields suboptimal results in the treatment of various tumors due to the use of inferior photosensitizers, insufficient tumor photosensitization, and biological properties of the malignancy. To optimize PDT for these tumors, a novel photosensitization strategy is being developed in which zinc-phthalocyanine (ZnPC) is encapsulated in liposomes and targeted to the tumor endothelium, tumor cells, and interstitial spaces. We previously presented the development of these liposomes and provided proof-of-concept regarding photodynamic activity. The current work focuses on the in vitro uptake of these liposomes as well as cell viability and mechanisms of cell death following PDT. Study: Interstitially-targeted liposomes (ITLs) were composed of DPPC, cholesterol, and DSPE-PEG2000 (66:30:4 mol%). Vascular-targeting liposomes (VTLs) contained DPPC, DC-cholesterol, cholesterol, and DSPE-PEG2000 (66:25:5:4 mol%). Nanobody-conjugated liposomes were comprised of DPPC, cholesterol, and DSPE-PEG2000-maleimide (66:30:4 mol%), to which anti-EGFR nanobodies (10 nmol protein/5 μmol lipids) were coupled. All liposomes had a ZnPC:lipid ratio of 0.004. Sk-Cha1, HER14, and human vascular endothelial cells (HUVECs) were subjected to PDT (15 J/cm2) following incubation with ZnPC-liposomes. Cell viability was assayed using WST-1 and FLICA assays and propidium iodine staining. Liposomal uptake was examined using fluorescence spectrometry/microscopy of cells incubated with fluorescently-labeled liposomes. Results: ITLs were not taken up by Sk-Cha1 cells, whereas VTLs and nanobody-conjugated liposomes were effectively taken up by HUVECs and HER14 cells, respectively. PDT reduced cell viability to 62±16% (ITLs, 250 μM lipids, Sk-Cha1 cells), 19±2.0% (VTLs, 50 μM lipids, HUVECs) as measured 8 h post-PDT in comparison to light-only treated controls. Anti-EGFR coupled liposomes (250 μM lipids) reduced HER14 cell viability to 50±7.9%, 24 h post-PDT in comparison to light-only treated controls. Cell death assays 4 h post-PDT detected primarily necrosis for ITLs and VTLs, and mostly apoptosis for anti-EGFR liposomes. Conclusion: Liposomes were effectively taken up in vitro by the target cells and significantly reduced cell viability following PDT. These results verify the feasibility of these liposomes for PDT-application. Future research will focus on studying PDT-efficacy of multi-targeting ZnPC-liposomes in nude mice tumor xenografts.