Artificial Membrane Fusion Triggered by Strain-Promoted Alkyne-Azide Cycloaddition.

Artificial systems for controlled membrane fusion applicable for drug delivery would ideally use triggers that are orthogonal to biology. To apply the strain-promoted alkyne-azide cycloaddition (SPAAC) to drive membrane fusion, oxo-dibenzocyclooctyne (ODIBO)-lipid 1 was designed, synthesized, and studied alongside azadibenzocyclooctyne (ADIBO)-lipids 2-4 to assess fusion with liposomes containing azido-lipid 5. Lipids 1-2 were first shown to be effective for liposome derivatization. Next, fusion was evaluated using liposomes containing 1 and varying ratios of PC and PE via a FRET dilution fusion assay, and a 1:1 PC-to-PE ratio yielded the greatest signal change attributed to fusion. Finally, lipids 1-4 were compared, and 1 yielded the greatest triggering of fusion, while 2-4 yielded varying efficacies depending on the structural features of each lipid. Fusion was further validated through STEM studies showing larger multilamellar assemblies after liposome mixing, and FRET assay results supporting the mixing of liposome aqueous contents. This work provides a platform for triggered fusion toward drug delivery applications and an understanding of the effects of lipid structure and membrane composition on fusion.

A clickable and photocleavable lipid analogue for cell membrane delivery and release.

For drug delivery purposes, the ability to conveniently attach a targeting moiety that will deliver drugs to cells and then enable controlled release of the active molecule after localization is desirable. Toward this end, we designed and synthesized clickable and photocleavable lipid analogue 1 to maximize the efficiency of bioconjugation and triggered release. This compound contains a dibenzocyclooctyne group for bioorthogonal derivatization linked via a photocleavable 2-nitrobenzyl moiety at the headgroup of a synthetic lipid backbone for targeting to cell membranes. To assess delivery and release using this system, we report fluorescence-based assays for liposomal modification and photocleavage in solution as well as through surface immobilization to demonstrate successful liposome functionalization and photoinduced release. In addition, fluorophore delivery to and release from live cells was confirmed and characterized using fluorescence microscopy and flow cytometry analysis in which 1 was delivered to cells, derivatized, and photocleaved. Finally, drug delivery studies were performed using an azide-tagged analogue of camptothecin, a potent anticancer drug that is challenging to deliver due to poor solubility. In this case, the ester attachment of the azide tag acted as a caging group for release by intracellular esterases rather than through photocleavage. This resulted in a dose-dependent response in the presence of liposomes containing delivery agent 1, confirming the ability of this compound to stimulate delivery to the cytoplasm of cells.

Triggered liposomal release through a synthetic phosphatidylcholine analogue bearing a photocleavable moiety embedded within the sn-2 acyl chain.

Liposomes represent promising carriers for drug delivery applications. To maximize this potential, there has been significant interest in developing liposomal systems encapsulating molecular cargo that are highly stable until their contents are released remotely in a controlled manner. Herein, we describe the design, synthesis, and analysis of a photocleavable analogue of the ubiquitous lipid phosphoatidylcholine (PC) for the development of highly stable and controllable photodisruptable membranes. Our strategy was to develop a lipid that closely mimics the structure of PC to optimize favorable properties including biocompatibility and stability of subsequent liposomes when mixed with lipids possessing a broad range of physicochemical properties. Thus, NB-PC was designed, which contains a photocleavable 2-nitrobenzyl group embedded within the acyl chain at the sn-2 position. Following the synthesis of NB-PC, liposome disruption efficacy was evaluated through photolysis studies involving the detection of nile red release. Studies performed using a range of liposomes with different percentages of NB-PC, PC, phosphatidylethanolamine (PE), cholesterol, and polyethylene glycol-PE (PEG-PE) demonstrated minimal background release in controls, release efficacies that correlate directly with the amount of NB-PC incorporation, and that release is only minimally impacted by the inclusion of the lipids PE and cholesterol that possess disparate properties. These results demonstrate that the NB-PC system is a highly stable, flexible, and tunable system for photoinitiated release from liposomal systems.