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

Abstract

Artificial systems for controlled membrane fusion applicable for drug delivery would ideally use triggers that are orthogonal to biology. To apply the strain-promoted alkyneazide cycloaddition (SPAAC) to drive membrane fusion, ODIBO lipid 1 was designed, synthesized and studied alongside 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/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. This work provides a platform for triggered fusion towards drug delivery applications and an understanding of the effects of lipid structure and membrane composition on fusion.

A General Approach to Enzyme‐Responsive Liposomes

“…We have previously found that heating of liposomesc an be necessary to achievee ffects such as liposomef usion. [45] Different control sets were also tested using the exact same liposomef ormulation by treating with only buffer or with heat-denatured enzyme, leadingt od iminished fluorescencec hanges (background releaser anging from % 8-12 %, Figure 1A). Similar results were also observed for liposomes lacking ERL in which this lipid was replaced by PC.…”

“…After screening a variety of different lipid mixtures, liposomes composed of 30 % ERL , 50 % DOPE, 10 % PC and 10 % DOTAP treated with 0.45 U of porcine liver esterase and incubated in a 30 °C water bath in between measurements showed an ≈30 % decrease in fluorescent intensity upon esterase treatment within 40 minutes (Figure A and Table S1, Entry 1). We have previously found that heating of liposomes can be necessary to achieve effects such as liposome fusion . Different control sets were also tested using the exact same liposome formulation by treating with only buffer or with heat‐denatured enzyme, leading to diminished fluorescence changes (background release ranging from ≈8–12 %, Figure A).…”

Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy

“…Numerous model systems for membrane fusion have been developed in recent years, all of which have employed a diverse range of molecules as fusogens. Examples of such fusogens include: hydrogen bonding motifs 1 , DNA [2][3][4][5][6][7][8][9][10][11] , PNA [12][13][14] , coiled-coil peptides [15][16][17][18] , and small molecule recognition motifs [19][20][21][22][23] . These systems exhibit varying efficiencies of fusion: some only facilitate hemifusion, or lipid-mixing; whereas others promote full fusion, resulting in content-mixing.…”

Budding and Division of Giant Vesicles Linked to Phospholipid Production

“…The addition of alkyne- or azide-functional moieties in the molecular structure of amphiphiles coupled with the formation of triazoles provide various opportunities for functionalization and modification of vesicular surfaces 24–26 . These synthetic amphiphiles can be incorporated in lipid bilayers and further reacted with complementary molecules without damaging the vesicles 27,28 . Moreover, in the synthesis of 1,2,3-triazoles, the CuAAC reaction is an effective chemoselective and water-tolerant ligation tool that can be used for synthesizing stable cycloaddition products, such as novel phospholipids, to induce growth and splitting of vesicles 19,23 .…”