Editing Synthesis of PEI Cho (section)

== Synthesis of LPC-L, LPC-LT and LPC-T  ==

=== [[Image:LPC-L synthesis.png]]  ===

=== Synthesis of LPC-L  ===

Dissolve LPEI (MW 25,000; 1.0 g=0.04 mmol; Polysciences, Warrington, Pa.) in 150 mL of anhydrous methylene chloride (Sigma Chemical Co., St. Louis, Mo.) with heating in a hot water bath for about 20 minutes. 

Cool the solution and add additional methylene chloride adjust the final volume of 150 mL. 

Add dropwise of 18 mg (0.04 mmol) of cholesteryl chloroformate (Aldrich Chemical Co., Inc., Milwaukee, Wis.) dissolved in 3 mL of anhydrous methylene chloride to the LPEI solution 

 over about 10 minutes. 

Stir the mixture over night 

Remove the solvent by rotary evaporation, 

Purify the product by solvent precipitation in acetone (Sigma Chemical), ethyl acetate (Sigma Chemical), and excess methylene chloride. 

Dry the conjugate 

Lyophilize 

Store at −20° C. 

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=== Synthesis of LPC-LT  ===

Follow the procedure of Synthesis of LPC-L 

Add an additional 0.036 g (0.08 mmol) of cholesteryl chloroformate to the solution after the first addition of cholesteryl chloroformate. 

<br> 

[[Image:LPC-T synthesis.png]] 

[[Image:LPC-T synthesis 2.png]] 

<br> 

=== Synthesis of LPC-T  ===

Dissolve LPEI (MW 25,000; 1.0 g=0.04 mmol) in 150 mL of anhydrous methylene chloride (Sigma Chemical Co., St. Louis, Mo.) with heating in a hot water bath for about 20 minutes. 

Remove the clear solution from the hot water bath, cool, and then add additional methylene chloride to adjust the volume to 150 mL. 

Stir the solution for 15 min. at room temperature. 

Add 200 μL of anhydrous triethylamine (TEA; Sigma Chemical) 

Stir another 5 min. 

Add 60 μL (0.06 mmol) of 1 M '''chlorotrimethyl silane''' (Aldrich Chemical) to protect the terminal hydroxyl group 

You will see the forming of an HCl(g) cloud over the solution. 

Stir the mixture overnight. 

Add dropwisely of 36 mg (0.08 mmol) of cholesteryl chloroformate dissolved in 3 mL of anhydrous methylene chloride to the trimethylsilane (TMS)-protected LPEI over 10 min. 

The cholesterol is conjugated to the secondary amine on the LPEI by a carbamate bond. 

Stir the solution overnight. 

Add 3 mL of trifluoroacetic acid (TFA) to d eprotect LPC-T-TMS. This will result in precipitation of LPC-T. 

Dry the sample on a rotary evaporator. 

Dissolve the residue in 20 mL of ultrapure water 

Purify by dialysis (MWCO 6,000 to 8,000) for three days. 

Lyophilize t he conjugate 

<br> Store at −20° C. 

 [[Image:LPC-Transfection.png]] 

[[Image:LPC-viability.png]] 

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