Fusion of Bipolar Tetraether Lipid Membranes Without Enhanced Leakage of Small Molecules

A major challenge in liposomal research is to minimize the leakage of encapsulated cargo from either uncontrolled passive permeability across the liposomal membrane or upon fusion with other membranes. We previously showed that liposomes made from pure Archaea-inspired bipolar tetraether lipids exhibit exceptionally low permeability of encapsulated small molecules due to their capability to form more tightly packed membranes compared to typical monopolar lipids. Here, we demonstrate that liposomes made of synthetic bipolar tetraether lipids can also undergo membrane fusion, which is commonly accompanied by content leakage of liposomes when using typical bilayer-forming lipids. Importantly, we demonstrate calcium-mediated fusion events between liposome made of glycerolmonoalkyl glycerol tetraether lipids with phosphatidic acid headgroups (GMGTPA) occur without liposome content release, which contrasts with liposomes made of bilayer-forming EggPA lipids that displayed ~80% of content release under the same fusogenic conditions. NMR spectroscopy studies of a deuterated analog of GMGTPA lipids reveal the presence of multiple rigid and dynamic conformations, which provide evidence for the possibility of these lipids to form intermediate states typically associated with membrane fusion events. The results support that biomimetic GMGT lipids possess several attractive properties (e.g., low permeability and non-leaky fusion capability) for further development in liposome-based technologies.


Supplemental Figures
Hydrodynamic radius of liposomes made of EggPA (A) or GMGTPA (B) measured by dynamic light scattering after content mixing experiments. Experiments were carried at room temperature in TES buffer (10 mM, 2 mM Histidine, 0.1 mM EDTA, NaCl 100 mM, pH 7.4) supplemented with 2 or 5 mM CaCl 2 . Liposomes in TES buffer without Ca 2+ were used as controls (black traces). Each sample had been quenched with EDTA (100 mM) prior to measurement in order to stop the aggregation process. 31 P spectra of looping model (lipid 14), spanning model (lipid 13), and bipolar tetraether D-GMGTPA at 30°C; 10240 scans, 1024 points, and a recycle delay of 2.5 s. A typical experiment used a 90° pulse width of 3.6μs, and a 412 ppm spectral width. All data were processed with 100 Hz of line broadening.

General Information
All reagents were purchased from commercial sources and used without further purification. EggPA (#840101P), PE-NDB (#810144P) and PE-Lissamine (#810158P) lipids were purchased from Avanti Polar Lipids. EggPA lipid was stored under Argon at -20°C and used within 3 months of purchase. Glassware was dried at 115°C overnight. Air and moisture-sensitive reagents were transferred using a syringe or stainless steel cannula. Intermediates were purified over silica (60Å, particle size 40-63 µm) purchased from Dynamic Adsorbents, Inc. Reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm silica gel plates (60F-254) from Dynamic Adsorbents, Inc. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. 1 H, 2 H, 13 C, 31 P NMR spectra were obtained on either JEOL ECA 500 spectrometer or Varian 500MHz spectrometer. Chemical shifts are reported in ppm relative to residual solvent. The FID file was analyzed using NMRnotebook version 2.70 build 0.10 by NMRTEC.
Dynamic Light Scattering (DLS) measurements were performed on a Wyatt DynaPro NanoStar (Wyatt Technology, Santa Barbara, CA) instrument using a disposable cuvette (Eppendorf UVette 220 nm -1,600 nm) and data processed using Wyatt DYNAMICS V7 software. Each analysis involved an average of 10 measurements. The data was exported for final plotting using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA).
Low resolution MS analysis was performed on a Micromass Quattro Ultima triple quadrupole mass spectrometer with an electrospray ionization (ESI) source. High resolution MS analysis was performed using Agilent 6230 Accurate-Mass TOFMS with an electrospray ionization (ESI) source by Molecular Mass Spectrometry Facility (MMSF) in the department of chemistry and biochemistry at University of California, San Diego.
Fluorescence measurements were taken on a Perkin Elmer Enspire multimode plate reader (Corning 96-well, half area, non-treated black polystyrene plates were used). The data were exported for final plotting using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA).

2
Then, a solution of 2-cyanoethyl N,N,N′,N′tetraisopropylphosphorodiamidite (1.22 g, 4.06 mmol) in 10 mL of degassed DCM was added dropwise and the reaction mixture was stirred at room temperature (rt) for 2 h. The reaction was diluted with DCM and then washed with saturated aqueous NaHCO 3 solution, brine and then dried over MgSO 4 . The solvent was evaporated under vacuum and purified over silica using hexane/ethyl acetate/triethylamine (8:2:0.5 then 0:10:0.5) as the eluent. Phospharamidite 2 was obtained (0.64 g, 73%) as a yellow oil.

NBD-N 3
Trifluoracetic acid (0.2 mL) was added to a solution of t-Boc-N-Amido-PEG1-azide (99 mg, 0.43 mmol) in DCM (1.8 mL), and the reaction mixture was stirred at rt for 4 h. Upon completion of the reaction, excess acid and solvent was removed under vacuum, and the residue was dried in vacuo overnight. The resulting product was used without further purification and dissolved in dry tetrahydrofuran (THF) (5 mL), and then NBD-Cl (90 mg, 0.45 mmol) and triethylamine (1.29 mmol, 0.2 mL) were added successively. The reaction was allowed to proceed with stirring at rt for 1 h and removal of the solvent in vacuo gave a brown liquid which was further purified over silica using hexane/EtOAc (75:25 to 25:75) as eluent. NBD-N 3 (51 mg, 40%) was obtained as a brown oil.

Rho-N 3
Trifluoracetic acid (0.2 mL) was added to a solution of t-Boc-N-Amido-PEG1-azide (97 mg, 0.42 mmol) in DCM (1.8 mL), and the reaction mixture was stirred at rt for 4 h. Upon completion of the reaction, excess acid and solvent was removed under vacuum, and the residue was dried in vacuo overnight. The resulting product was used without further purification and dissolved in a mixture of dry DCM and dimethylformamide (DMF) (4 mL, 1:5). Then, lissamine rhodamine B sulfonyl chloride (219 mg, 0.38 mmol), 4dimethylaminopyridine (5 mg, 0.04 mmol) and N,N-diisopropylethylamine (2.28 mmol, 0.4 mL) were added successively. The reaction was allowed to proceed with stirring at rt for 16 h and removal of the solvent in vacuo gave a dark liquid which was further purified over silica using DCM/MeOH (9:1) as eluent. An isomeric mixture of Rho-N 3 (62 mg, 22%) was obtained as a purple solid.

3-(benzyloxy)-2-(tetradecyloxy)propan-1-ol (S4)
A solution of S3 (1.73 g, 4.25 mmol) in dry DCM (11 mL) was cooled to -78°C. Then, DIBAL-H (10.6 mmol) in DCM (1M, 11 mL) was added dropwise to the cold solution and reacted for 16 h at rt. After the reaction was complete, a small amount of MeOH was used to carefully quench the remaining DIBAL-H followed by the addition of aqueous NaOH (5 M, 15 mL). The solution was then extracted using diethylether (Et 2 O) (3x50 mL) and then washed with water (1x100 mL). The extracted organic layer was dried using Na 2 SO 4 and the solvent was removed under vacuum. The crude was purified over silica using hexane/EtOAc (8:2) as the eluent and alcohol S4 (1.51 g, 94%) was obtained as a clear oil.

3-(benzyloxy)propane-1,2-diol (S9)
A solution of 1,3-O-benzylideneglycerol (1.50 g, 8.3 mmol) in dry DCM (34 mL) was cooled to -78°C. Then, DIBAL-H (33.0 mmol) in DCM (1M) was added dropwise to the cold solution and reacted for 16 h at rt. After the reaction was complete, a small amount of MeOH was used to carefully quench the remaining DIBAL-H followed by the addition of aqueous NaOH (5 M, 40 mL). The solution was then extracted using diethyl ether (Et 2 O) (3x100 mL) and then washed with water (1x100 mL). The extracted organic layer was dried using Na 2 SO 4 and the solvent was removed under vacuum. The crude was purified over silica using EtOAc as the eluent and alcohol S9 (1.18 g, 78%) was obtained as a clear oil.