Addition of poly (propylene glycol) to multiblock copolymer to optimize siRNA delivery

Dai, Zhi; Arévalo, Maria T; Li, Junwei; Zeng, Mingtao

The following procedure to synthesize 3-benzyloxycarbonyll-lysine N-carboxyanhydrideLL(Z)-NCA was adapted from Zhou et al. 30 Ten grams of Nε-benzyloxycarbonyl-l-lysine was suspended in 100 ml of THF, the mixture was heated to 50 °C, and an equivalent amount of triphosgene was added. Three hours later, the mixture was poured into 300 ml of hexane and was stored for 16-20 h at -20 °C. Recrystallization from THF/hexane generated NCA-monomer crystals. PEG-PLL(Z) P1-Z, PLL(Z)-PEG-PLL(Z) P2-Z, and PLL(Z)-PPG-PEG-PPG-PLL(Z) P3-Z copolymers were prepared by ring opening polymerization of LL(Z)-NCA as initiated by PEG-NH 2 , NH 2 -PEG-NH 2 , and NH 2 -PPG-PEG-PPG-NH 2 . Briefly, PEG-NH 2 (200 mg) and LL(Z)-NCA (500 mg) were added to 10 ml dry DMF and were stirred at 30 °C for 72 h under N 2 atmosphere. Next, the reaction was poured into an excess of diethyl ether to precipitate the Figure 5 . Zeta potentials of complexes of copolymer P1, P2, and P3 with GFP siRNa at various N/P ratios. copolymer-siRNa complexes were prepared in aqueous solution using multiple N/P ratios while keeping siRNa (200 pmol) constant. Zeta potentials were determined at 25 °c using the Zetasizer Nano. PEG-PLL(Z) copolymer. The product was purified by repeated precipitation in diethyl ether, and then dried in a vacuum. We then proceeded with the synthesis of PEG-PLL P1, PLL-PEG-PLL P2, and PLL-PPG-PEG-PPG-PLL P3 copolymers using methods adapted from Zhou et al. with some modifications. 30 The PEG-PLL(Z) samples were first dissolved in 5ml of TFA. HBr (30% in acetic acid) was then added in excess and the mixtures were stirred for 5 h at room temperature. Deprotected polymers were isolated by precipitation using diethyl ether. The precipitated peptides were subsequently washed with excess diethyl ether and acetone, followed by dialysis against deionized water. The product was dried in a vacuum at -40 °C. Characterization by 1 H NMR spectrum was recorded by Chemtos LLC. DMSO-d-6 was used as the solvent for 1 H NMR measurements. Molecular weight (Mw) was determined from the 1 H NMR data.