We recently reported on the physical characteristics of photo-triggerable liposomes containing dipalmitoylphosphatidylcholine (DPPC), and 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC) carrying a photo agent as their payload. When exposed to a low-intensity 514 nm wavelength (continuous-wave) laser light, these liposomes were observed to release entrapped calcein green (Cal-G; Ex/Em 490/517 nm) but not calcein blue (Cal-B; Ex/Em 360/460 nm). In this study, we have investigated the mechanism for the 514 nm laser-triggered release of the Cal-G payload using several scavengers that are known specifically to inhibit either type I or type II photoreaction pathways. Liposomes containing DPPC:DC(8,9)PC: distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-2000 (86:10:04 mole ratio) were loaded either with fluorescent (calcein) or nonfluorescent ((3)H-inulin) aqueous markers. In addition, a non-photo-triggerable formulation (1-palmitoyl-2-oleoyl phosphatidylcholine [POPC]:DC(8,9)PC:DSPE-PEG2000) was also studied with the same payloads. The 514 nm wavelength laser exposure on photo-triggerable liposomes resulted in the release of Cal-G but not that of Cal-B or (3)H-inulin, suggesting an involvement of a photoactivated state of Cal-G due to the 514 nm laser exposure. Upon 514 nm laser exposures, substantial hydrogen peroxide (H2O2, ≈100 μM) levels were detected from only the Cal-G loaded photo-triggerable liposomes but not from Cal-B-loaded liposomes (≤10 μM H2O2). The Cal-G release from photo-triggerable liposomes was found to be significantly inhibited by ascorbic acid (AA), resulting in a 70%-80% reduction in Cal-G release. The extent of AA-mediated inhibition of Cal-G release from the liposomes also correlated with the consumption of AA. No AA consumption was detected in the 514 nm laser-exposed Cal B-loaded liposomes, thus confirming a role of photoactivation of Cal-G in liposome destabilization. Inclusion of 100 mM K3Fe(CN)6 (a blocker of electron transfer) in the liposomes substantially inhibited Cal-G release, whereas inclusion of 10 mM sodium azide (a blocker of singlet oxygen of type II photoreaction) in the liposomes failed to block 514 nm laser-triggered Cal-G release. Taken together, we conclude that low-intensity 514 nm laser-triggered release of Cal-G from photo-triggerable liposomes involves the type I photoreaction pathway.
Keywords: photo-agents; photodynamic actions; photopolymerizable phospholipids; reactive oxygen species; visible laser-triggered payload release.