Dotma

DOTMA, DOPE, DOPG, DPPE PEG 2000, and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The structures of the lipids, peptides, and siRNAs are shown in Table 1. Peptides Y (K₁₆GACYGLPHKFCG), ME27 (K₁₆RVRRGACRGDCLG), ME72 (K₁₆RVRRGACRGECLG), KG31 (K₁₆RXSXGACYGLPHKFCG, Hydrophobic Y, X = epsilon-aminohexanoic acid), and KG32 (K₁₆RVRRGACYGLPHKFCG, Cleavable Y) were synthesized by ChinaPeptides (Shanghai, China) and AMS Biotechnology (Abingdon, UK). The siRNAs used in this study were custom synthesized and purchased from Dharmacon (Cambridge, UK). The siRNAs were dissolved in sterile RNase-free water at a concentration of 2.5 mg/mL.Table 1

Structures of the Different Lipids and Sequences of Peptides and siRNAs

Lipoplexes were manufactured by mixing cationic liposomes with the RNA using dedicated protocols as described previously [3 (link), 7 (link)]. Briefly, the RNA-lipoplexes were generated by dilution of RNA in H₂O and 1.5 M NaCl prior to addition of liposomes to obtain the ratios given below and a final NaCl concentration of 150 mM. To formulate spleen-targeting RNA-lipoplexes, liposomes consist of a 2:1 (mol:mol) mixture of the synthetic cationic lipid 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) (Merck & Cie, Schaffhausen, CH) and the phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL, USA, or Corden Pharma, Liestal, CH). To make the lung-targeting RNA-lipoplexes, liposomes consisting of DOTMA and cholesterol (Avanti Polar Lipids, Alabaster, AL, USA) at a 1:1 molar ratio were used.
The charge ratio for particle formation was calculated using the molar mass of 670 Da for the positively charged DOTMA and a mean molar mass of 330 Da for each negatively charged nucleotide of the RNA. For particles targeting the lung the charge ratio was 4:1, resulting in particles with an average size of ~ 260 nm and a polydispersity index of ~ 0,23. For particles to target the spleen the charge ratio was 1.3:2, resulting in an average size of ~ 250 nm and a polydispersity index of ~ 0,25.