Stability of Alkyl Chain-Mediated Lipid Anchoring in Liposomal Membranes

Lipid exchange among biological membranes, lipoprotein particles, micelles, and liposomes is an important yet underrated phenomenon with repercussions throughout the life sciences. The premature loss of lipid molecules from liposomal formulations severely impacts therapeutic applications of the latter and thus limits the type of lipids and lipid conjugates available for fine-tuning liposomal properties. While cholesterol derivatives, with their irregular lipophilic surface shape, are known to readily undergo lipid exchange and interconvert, e.g., with serum, the situation is unclear for lipids with regular, linear-shaped alkyl chains. This study compares the propensity of fluorescence-labeled lipid conjugates of systematically varied lengths to migrate from liposomal particles consisting mainly of egg phosphatidyl choline 3 (EPC3) and cholesterol into biomembranes. We show that dialkyl glyceryl lipids with chains of 18–20 methylene units are inherently stable in liposomal membranes. In contrast, C16 lipids show some lipid exchange, albeit significantly less than comparable cholesterol conjugates. Remarkably, the C18 chain length, which confers noticeable anchor stability, corresponds to the typical chain length in biological membranes.


S1.3. Handling of Ethylene Oxide (EO)
The gaseous, flammable and highly toxic ethylene oxide (EO) must be handled carefully and has to be stored in pressure-proof gas bottles. It must be used only in an adequate fume hood under appropriate safety precautions. Polymerizations in which EO is involved are performed in flamedried glassware to enable the conversion of EO inside the sealed and evacuated glass apparatus and to guarantee secure handling via cryo-transfer techniques. To avoid abrupt detachment of the septum and hence release of EO the maximum batch-sizes of 8 g EO in a 500 mL flask must not be exceeded.

S2.3. Polymer Synthesis of BisOD-PEG
The synthesis is described for BisOD-PEG81 as a representative example. 1,2-Bis-n-octadecyl glyceryl ether (BisOD) (0.2 g, 0.36 mmol, 1 eq.) was placed in a dry Schlenk flask and dissolved in benzene (10 mL). The solution was stirred at 60 °C for 30 min and dried in vacuo for 16 h to remove moisture. Dry tetrahydrofuran (approx. 10 mL) was cryo-transferred to the Schlenk flask to dissolve the initiator. Afterwards, the initiator was deprotonated with a 0.5 M solution of potassium naphthalenide in THF (0.36 mL, 0.18 mmol, 0.5 eq.) while stirring. The solution was cooled down to -80 °C and ethylene oxide (EO) (1.70 mL, 37.57 mmol, 105 eq.) was cryotransferred using a graduated ampule. The polymerization was carried out at 60 °C for 24 h. In order to quench the polymerization, an excess of ethanol was added. The solvent was removed under reduced pressure, the crude product was dissolved in methanol and precipitated twice in cold diethyl ether to obtain the pure product. Yield: 99%.

S2.4. Functionalization of BisOD-PEG with Propargyl Bromide
The functionalization is described for BisOD-PEG81-alkyne as a representative example. BisOD-PEG81 (0.3 g, 0.074 mmol, 1 eq.) was placed in a dry Schlenk flask and dissolved in dry THF. The solution was cooled to 0 °C and sodium hydride (0.005 g, 0.223 mmol, 3 eq.) was added while stirring. Subsequently, propargyl bromide (0.020 mL, 0.223 mmol, 3 eq.) was added and the solution was stirred for 24 h at room temperature. The reaction mixture was filtered and the solvent was slightly reduced under reduced pressure. The remaining solution was precipitated twice in cold diethyl ether and the pure product was dried in vacuo. Yield: 66%.

S2.6. Synthesis of 1,2-Bis-N-Icosanyl Glyceryl Ether (BisID)
The synthesis was carried out according to literature. 3 Dry tetrahydrofuran (THF) was placed in three-necked round bottom flask equipped with Dimroth condenser and sealed precision glass (KPG) stirrer. Under argon atmosphere and stirring 3benzyloxy-1,2-propanediol (2.7 mL, 0.017 mol, 1 eq.), sodium hydride (1.62 g, 0.068 mol, 4 eq.) and 1bromoicosane (24.42 g, 0.068 mol, 4 eq.) was added. The reaction mixture was stirred at 80 °C for 9 days. Additional NaH was added (1.00 g, 0.042 mol, 2.5 eq) and the solution was stirred for another 23 days. The solvent was removed under reduced pressure to obtain a total volume of 250 mL. Water (250 mL) and diethyl ether (250 mL) was added and the mixture was stirred overnight at room temperature. To neutralize the reaction mixture, sulfuric acid (1 mol·L -1 , 15 mL, 0.015 mol) was added and again stirred overnight. The organic phase was extracted three times with diethyl ether (150 mL each) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified using flash column chromatography (petroleum ether/diethyl ether 40:1). The intermediate 1,2-bis-n-icosanyl-3-benzyl glyceryl ether (4.97 g, 0.007 mol) was obtained as colorless solid. Yield: 41%.

S2.7. Polymer Synthesis of BisID-PEG
The synthesis is described for BisID-PEG62 as a representative example. 1,2-Bis-n-icosanyl glyceryl ether (BisID) (0.2 g, 0.307 mmol, 1 eq.) was placed in a dry Schlenk flask and dissolved in benzene (10 mL). The solution was stirred at 60 °C for at least 30 min and dried in vacuo for 16 h to remove moisture. Dry tetrahydrofuran (approx. 10 mL) was cryo-transferred to the Schlenk flask to dissolve the initiator. Afterwards, the solution was stirred and the initiator was deprotonated with a 0.5 M solution of potassium naphthalenide in THF (0.31 mL, 0.153 mmol, 0.5 eq.). The solution was cooled down to -90 °C and ethylene oxide (EO) (0.95 mL, 20.919 mmol, 68 eq.) was cryo-transferred using a graduated ampule. The polymerization was carried out at 60 °C for 24 h. Subsequently, the reaction mixture was heated to 80 °C for 16 h. To quench the polymerization, an excess of ethanol was added and the solvent was removed under reduced pressure. The crude product was dissolved in methanol and precipitated three times in cold diethyl ether to obtain the pure product. Yield: 95%.

S2.8. Functionalization of BisID-PEG with Propargyl Bromide
The functionalization is described for BisID-PEG62-alkyne as a representative example. BisID-PEG62 (0.2 g, 0.059 mmol, 1 eq.) was placed in a Schlenk flask and dissolved in dry THF (10 mL). The solution was cooled to 0 °C and sodium hydride (0.004 g, 0.178 mmol, 3 eq.) was added. Afterwards, propargyl bromide (0.016 mL, 0.178 mmol, 3 eq.) was added, and the solution was stirred for 24 h at room temperature. The reaction mixture was filtered, and the solvent was reduced under reduced pressure. The remaining solution was precipitated twice in cold diethyl ether and the pure product was dried in vacuo. Yield: 61%. Scheme S1: Synthesis Route of the Dialkyl PEG-Lipids and Functionalization with propargyl bromide.

S2.9. Polymer Synthesis of Cholesterol-PEG-PEEGE
The synthesis protocol was described in previous reports.

S2.10. Polymer Synthesis of Cholesterol-PEG-linPG
The synthesis protocol was described in previous reports.

S2.11. Polymer Synthesis of Cholesterol-PEG-hbPG
The synthesis protocol was described in previous reports.

S2.12. Functionalization of Cholesterol-PEG-hbPG with Propargyl Bromide
The synthesis protocol was described in previous reports.