Dimethyldioctadecylammonium
bromide (DDA) (Sigma, U.K.), 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP) (Avanti Polar Lipids, Alabaster, AL, USA), and cephalin (soy
phosphatidylethanolamine) (Avanti Polar Lipids, Alabaster, AL, USA)
were used as received. C12–200 was synthesized by reacting
1 mol equiv of N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine
(Enamine Ltd., Kyiv, Ukraine) with 7 mol equiv of 1,2-epoxydodecane
(Sigma, U.K.) at 80 °C for 2.5 days, according to previous protocols.21 (link) LNPs were prepared on a μEncapsulator
1 System (Dolomite Bio, Royston, U.K.). The lipid solution was prepared
by dissolving lipids in 90% EtOH at a total concentration of 1.5 mg/mL,
consisting of 35 mol % complexing lipid (C12–200, cephalin,
DDA, or DOTAP), 49 mol % cholesterol (Sigma, U.K.) and 16 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE) (Avanti Polar Lipids, Alabaster, AL, USA). For high lipid concentration
particles, the total lipid concentration was increased to 7.5 mg/mL.
One hundred microliters (100 μL) of the lipid was loaded into
one side of the μEncapsulator reservoir, while the other side
was loaded with 100 μL of citrate buffer (pH 3), and the solutions
were then loaded into the corresponding pumps. A 50 μm fluorophilic
chip with a T-junction and subsequent PBS dilution channel was used.
LNPs were prepared using the following conditions: chip temperature,
70 °C; lipid solution pump pressure, 2000 Pa; citrate buffer
pump pressure, 666 Pa; and PBS pump pressure, 2000 Pa. LNPs were purified
by dialyzing against PBS in a 3500 MWCO dialysis cartridge (Thermo
Fisher, U.K.) overnight. In these studies, high, medium, and low particle
concentrations correspond to 109, 108, and 107 particles/mL, respectively, diluted in PBS. For combinations
of cationic and zwitterionic lipids, the lipid solutions were prepared
such that the total complexing lipid mole percentage was maintained
at 35 mol % by varying the ratio of DDA/DOTAP to cephalin (10:1,
1:1, or 0.1:1).
bromide (DDA) (Sigma, U.K.), 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP) (Avanti Polar Lipids, Alabaster, AL, USA), and cephalin (soy
phosphatidylethanolamine) (Avanti Polar Lipids, Alabaster, AL, USA)
were used as received. C12–200 was synthesized by reacting
1 mol equiv of N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine
(Enamine Ltd., Kyiv, Ukraine) with 7 mol equiv of 1,2-epoxydodecane
(Sigma, U.K.) at 80 °C for 2.5 days, according to previous protocols.21 (link) LNPs were prepared on a μEncapsulator
1 System (Dolomite Bio, Royston, U.K.). The lipid solution was prepared
by dissolving lipids in 90% EtOH at a total concentration of 1.5 mg/mL,
consisting of 35 mol % complexing lipid (C12–200, cephalin,
DDA, or DOTAP), 49 mol % cholesterol (Sigma, U.K.) and 16 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE) (Avanti Polar Lipids, Alabaster, AL, USA). For high lipid concentration
particles, the total lipid concentration was increased to 7.5 mg/mL.
One hundred microliters (100 μL) of the lipid was loaded into
one side of the μEncapsulator reservoir, while the other side
was loaded with 100 μL of citrate buffer (pH 3), and the solutions
were then loaded into the corresponding pumps. A 50 μm fluorophilic
chip with a T-junction and subsequent PBS dilution channel was used.
LNPs were prepared using the following conditions: chip temperature,
70 °C; lipid solution pump pressure, 2000 Pa; citrate buffer
pump pressure, 666 Pa; and PBS pump pressure, 2000 Pa. LNPs were purified
by dialyzing against PBS in a 3500 MWCO dialysis cartridge (Thermo
Fisher, U.K.) overnight. In these studies, high, medium, and low particle
concentrations correspond to 109, 108, and 107 particles/mL, respectively, diluted in PBS. For combinations
of cationic and zwitterionic lipids, the lipid solutions were prepared
such that the total complexing lipid mole percentage was maintained
at 35 mol % by varying the ratio of DDA/DOTAP to cephalin (10:1,
1:1, or 0.1:1).