Paul.xu at 14:48, 9 March 2012

2012-03-09T14:48:25Z

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== 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

 

{| width="600" cellspacing="1" cellpadding="1" border="1"
|-
|
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

   

[[Image:2012 1 5 day1 plate1.jpg]] 

[[Image:2012 1 5 day1.jpg]]

 

 

48 hours after transfection

[[Image:2012 1 6 day2 plate1.JPG]] 

[[Image:2012 1 6 day2.jpg]]

 

day3 

[[Image:2012 1 7 day3 plate1.jpg]]

 

 

Plate2 

{| width="600" cellspacing="1" cellpadding="1" border="1"
|-
|
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

   

[[Image:2012 1 5 day1 plate2.jpg]]

 

[[Image:2012 1 5 day1 plate2 2.jpg]]

 

48 hours after transfection

[[Image:2012 1 6 day2 plate2.JPG]]

[[Image:2012 1 6 day2 plate2 1.jpg]]

day3

[[Image:2012 1 7 day3 plate2.jpg]]

[[Category:Transfection]] [[Category:Paul]]

Synthesis PHP-sys-DSP-gPEI - Revision history

Paul.xu at 14:48, 9 March 2012