Paul.xu at 18:25, 12 July 2011

2011-07-12T18:25:01Z

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4-amino-1-butanol (ABOL, Aldrich)

histamine (HIS, Fluka) Mono-Boc-protected diaminobutane (MBDAB, Fluka)

N,Nâ-cystaminebisacrylamide (CBA,Polysciences)

1,4-bis(acryloyl)piperazine (BAP, Sigma-Aldrich)

1. Add CBA (2.63 g, 10 mmol) and ABOL (0.91 g, 10 mmol) to a brown reaction flask 

2. Add MeOH/DI water mixture (5 ml, 4/1, v/v) to dissolve monomers. 

3. React in the dark at 45°C under nitrogen atmosphere. The reaction mixture becomes homogeneous in less than two hours. Let the reaction  to proceed for 10 days yielding to a viscous solution. During the polymerization a gradual increase in viscosity can be seen. In all cases gelation should not occur. 

4. In a final stage, add 10 mol% excess ABOL (0.091 g, 1 mmol)  to the reaction mixture in order to consume any unreacted acrylamide groups after polymerization and ensure that all potentially toxic acrylamide end groups are consumed, and that polymers contained only amino end groups. Stir for two days at 45°C. 

5. Dilute the resulting solution with 30 ml DI water, acidified with 4M HCl aqueous solution to pH ~5, and then purify and isolate by ultrafiltration operation (3000/mol cut-off) with filtrated acidic DI water (pH ~5), followed by freeze-drying. 

 
 
Average molecular weight (Mw) of p(CBA-ABOL), p(CBA-HIS) and p(BAP-ABOL) was 4.1, 9.0 and 11.0 kg/mol, respectively. 

p(CBA-ABOL): White solid. Yield: 1.49 g p(CBA-HIS): White solid. Yield: 1.92 g p(BAP-ABOL): White solid. Yield: 1.10 g 

Protein Transfection 

1. Prepare PAA/Î²-galactosidase nanocomplexes at different polymer/Î²-galactosidase weight ratios  by adding a HEPES buffer solution (10 mM, pH 7.4) of PAA (800 ÎŒl) to a HEPES buffer solution (10 mM, pH 7.4) of Î²-galactosidase (200 ÎŒl, 75 ÎŒg/ml).

2. Vortex for 5 seconds and incubate at room temperature for 30 minutes. 

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Synthesis CBA ABOL - Revision history

Paul.xu at 18:25, 12 July 2011