Synthesis of PAA
Synthesis of Poly(amido amine)s (PAAs) homopolymers by Michael addition
p(HMBA-AEP) and p(CBA-AEP)
1. Add HMBA(0.55 g, 2.46 mmol) or CBA (0.64 g, 2.46 mmol) and AEP (0.32 g, 2.46 mmol) into a brown reaction flask with methanol as a solvent (2.5 mL).
2. Let polymerization in the dark at 40°C under nitrogen atmosphere.
3. The reaction mixture will become homogeneous in less than 1 hour, allow the reaction for 6 days. You will see a viscous solution.
4. Add 10 mol% excess AEP (0.03 g, 0.2 mmol) to consume any unreacted acrylamide groups, let stir for 2 days at 40°C.
5, Dilute the resulting solution with water to about 30 ml, acidify with 6 M HCl to pH~4, and then dialyze through a dialysis membrane (MWCO 1000g/mol) for 3 days.
6. Freeze-drying. p(MBA-AEP) and p(BAP-AEP)
Polymerization in water as the solvent, terminate the reactions in 6 hours and 3 hours, respectively.
co-polymer
mono-polymer
p(HMBA80/CBA20-AEP) Copolymers: PAA copolymers with varying amounts of disulfide groups
AEP + HMBA and CBA at various ratios (i.e. 80/20, 60/40 and 40/60).
1. Add CBA (0.16 g, 0.60 mmol), HMBA (0.54 g, 2.41 mmol) and AEP (0.39 g, 3.01 mmol) into a brown reaction flask with methanol as a solvent (3.0 mL).
2. Stir the reaction mixture at 40°C in the dark under nitrogen atmosphere for 6 days.
3. Add 10 mol% excess of AEP (0.04 g, 0.03 mmol) to consume any unreacted acrylamide groups, stir for 2 days at 40°C.
4, Dilute the resulting solution with water to about 30 ml.
5, Acidify with 6 M HCl to pH~4.
6. Dialyze through dialysis membrane (MWCO 1000g/mol) for 3 days.
7. Freeze-drying the copolymer to get HCl-salt.
p(CBA-AEP) has best transfection efficiency, 1:3 to 1:6. CBA: 100% has the best transfection efficiency.
Bioreducible poly(amido amine)s (SS-PAAs).
1. Add N, Nâ-cystaminebisacrylamide CBA (0.250 g, 1.1 mM) and 5-amino-1-pentanol (APOL) (1.1 mM) to a brown reaction flask.
2. Add methanol/water mixture (1.1 mL, 4/1, v/v).
3. Polymerize in the dark at 45 °C under nitrogen atmosphere for 10 days yielding a viscous solution.
4. Add 10 mol% excess 5-amino-1-pentanol to consume any unreacted acrylamide, stir for 2 days at 45 °C.
5. Dilute with 30 ml water, acidify with 6 M HCl aqueous solution to pH~3.
6. Purify by ultrafiltration with acidic deionised water (pH~4).
7, freeze-dry to yield solid powder.
