Synthesis PHP-sys-DSP-gPEI

Guanidinylation of PEI using 1H-pyrazole-carboxamidine hydrochloride

Protocol 1.

1. Dissolve PEI (0.010 mol amine residues/ L) in 50 mM potassium carbonate aqueous solution. 

2. Add pyrazole-1-carboxamidine (5 eq. per amine residue) to the PEI solution.

3. Stir for 20 h.

4. Dialyse (M.W.C.O = 1,000 Da; Han- kook Spectrum Co.) against distilled water to obtain their corresponding PEGus. T

5. Analyse PEI and PEGu by 1H-NMR spectrums of PEI and PEGu.

6. The conversion yield is 12% and 19% for PEI 25 kDa and PEI 2 kDa, respectively.

Protocol 2.

1. Add  equimolar amount of 1H-pyrazole-carboxamidine hydrochloride to 15 wt % aqueous solutions 

2. React at neutral pH for 24 h at room temperature under stirring

3. Dialyse the reaction products against DI water (MWCO,1 kDa) and lyophilized.

4. Titrate guanidine groups in the gPEI and gPEI-Jeffamine copolymers spectrophotometrically using the Sakaguchi reaction of guanidine with 1-naphthol-2-sulfonic acid and hypobromite (1% Br2 v/v in 1.25 M NaOH). Calibration curves were developed using L-arginine as a standard (20).

Approximately 5 meq/g of guanidine groups were detected in either gPEI1 or gPEI2.

Protocol 3.

1. Dissolve PEI-Jeffamine conjugate (4 g) in 50 mL of water.

2. Add 1H-pyrazole-carboxamidine hydrochloride (2 g, 13.6 mmol).

3. Stir the solution at room temperature for 24 h.

4. Dialyse the product against DI water (MWCO 1 kDa).

5. Lyophilize.

Yield, 75%. Calculated, on the basis of a 1:1 mol ratio of gPEI1/Jeffamine

My Protocol

12/31/2011

0.75 g 1,3-dap (~0.79 g w some water)

1.84 g 1,3,dbp

1.5 ml MeOH

react at 50 oC for 1 hour, add 10 mg cystamine, react at 50 oC for 2 days

dissolve polymer in 5 ml water then, purify polymer using 40 ml ethanol, spin down polymer. Wash two times.

dissolve polymer in 5 ml water.--> PHP-ss

DSP conjugation is absolutely required for long term stability of the transfection reagent.

Dissolve 1 g PEI18 in 10 ml water, dissolve 0.5 g 1H-pyrazole-carboxamidine hydrochloride, mix A & B, react at RT o/n-->GuPEI18.

Guanidinylation

PHP-ss-DSP-guPEI18: 20 mg DSP + 0.2 ml GuPEI18 + 5 ml PHP-ss, react at 37 oC o/n

PHP-ss-guPEI18: 0.2 ml GuPEI18 + 5 ml PHP-ss, react at 37 oC o/n

Transfection

dilution: 1:10 to 1:80 in PBS

transfection 1 ug DNA + 3 ul diluted transfection reagent

PHP-ss-DSP-guPEI18, 1:10	PHP-ss-DSP-guPEI18, 1:20	PHP-ss-DSP-guPEI18, 1:30	PHP-ss-DSP-guPEI18, 1:40
PHP-ss-guPEI18, 1:10	PHP-ss-guPEI18, 1:20	PHP-ss-guPEI18, 1:30	PHP-ss-guPEI18, 1:40
guPEI18, 1:10	guPEI18, 1:20	guPEI18, 1:30	guPEI18, 1:40

24 hours after transfection

   

48 hours after transfection

day3

Plate2

PHP-ss-DSP-guPEI18, 1:50	PHP-ss-DSP-guPEI18, 1:60	PHP-ss-DSP-guPEI18, 1:70	PHP-ss-DSP-guPEI18, 1:80
PHP-ss-guPEI18, 1:50	PHP-ss-guPEI18, 1:60	PHP-ss-guPEI18, 1:70	PHP-ss-guPEI18, 1:80
guPEI18, 1:50	guPEI18, 1:60	guPEI18, 1:70	guPEI18, 1:80

24 hours after transfection

   

48 hours after transfection

day3