Editing Synthesis of PEI Cho

[[Image:Cholesterol.png]]--&gt;[[Image:Cholesteryl_chloroformate.png]]



1, Stir 3 g PEI (mw 1800) on ice in a mixture of 10 mL of anhydrous methylene chloride and 100 uL of triethylamine for 30 min. &nbsp;<br> 

2. Dissolve 1 g cholesteryl chloroformate in 5 mL of ice-cold anhydrous methylene chloride. 

3. Slowly add cholesteryl chloroformate to the PEI solution for 30 min. 

4. Stir the mixture for 12 h on ice. 

5. Dry mixture on a rotary evaporator. 

6, Dissolve the powder in 50 mL of 0.1 N HCl, then filter through the glass microfiber filter. 

7, Remove nonreacted cholesteryl chloroformate by extracting three times with 100 mL of methylene chloride 

8, Filter resulting aqueous solution through a glass microfiber filter. 

9. Concentrate by solvent evaporation 

10. Precipitate with large excess acetone. 

11, Dry under vacuum. 

12, Dissolve powder in water 

13. Precipitate three times with acetone. 

14. Wash three times with methanol then three times with diethyl ether. 

<br> 

Small scale: 

300 mg PEI 18 + 1 ml&nbsp;Dichloromethane + 10 ul&nbsp;triethylamine 

100 mg&nbsp;cholesteryl chloroformate in 0.5 ml&nbsp;1 ml Dichloromethane 

<br> 

'''#2: Synthesis of PEI-Chol (P10C, P17C and P30C).''' 

'''[[Image:PEI-Chol synthesis.png]]''' 

[[Image:PEI-Chol efficiency.png]] 

Ratio of PEI/CHOL: 10.5/1 17/1, 29.6/1<br> 

1 P17C as a sample,. Dissolve 90 mg of cholesteryl chloroformate (Mw: 449.12) in mixed solution of 15 mL of chloroform and 15 mL of methanol.<br> 

2. Dissolive PEI 2 kDa 200 mg in the same solution. <br> 

3. Add cholesteryl chloroformate dropwise to PEI for 2 hours. 

4. Allow the reaction proceed at 37 oC for 48 hrs. 

5. Dissolve final product in water and remove unreacted cholesterol particles (aggregates) by filtration. 

6. Dialyze vs water at 4 oC for 1 day to exclude insoluble cholesteryl chloroformate. 

7. lyophilize the aqueous solution.<br> 

Best transfection: P17C + liposome made DOPE<br> 

<br> 

'''Liposome preparation method:''' 

Dissolve polycationic lipid and dioleoylphosphatidyl ethanolamine (DOPE) in chloroform in a weight ratio of 1&nbsp;: 2. 

Dry with N2 gas and reduced under pressure to thin film. 

Then rehydrate in distilled water, suspend by vortexing and stored overnight at 4 oC.<br>Sonicate the solution for 10-15 min with a bath-type sonicator.<br> 

<br> 

'''PEI-Myr (P10M, P16M and P26M). ''' 

Ratio of PEI'''/'''ethyl myristate: 10.5/1, 16/1, 26.2/1<br> 

1. P26M as a sample, add 30 mL of ethyl myristate (Fw: 256.43) to PEI 2 kDa in 4 mL of methanol. 

2. Allow the reaction to proceed at 57 oC for 3 days. 

3. Dissolve the final product in water and centrifuge at 13000 rpm. 

4. Discard the supernatant dialyse the precipitate against water at 4 oC for 1 day to exclude remaining ethyl myristate. 

5. lyophilize aqueous solution. 

<br> '''Modify PEI with cholesteryl chloroformate''' 

1. Add 50 mg (0.006 mmol) of 25-kDa PEI (Aldrich) to 450 ul anisole while heating and intensive mixing to facilitate solubility. 

2. Add Cholesteryl chloroformate in 450 ul anisole dropwise to PEI solution while stirring 

Amount Modification 8.1 mg (0.018 mmol) 0.5% of amines 16.2 mg (0.036 mmol) 1% 81 mg (0.18 mmol) 5% 244 mg (0.72 mmol) 20% 

3, Stir for 2 h at room temperature 

4, Add 2-pyrrolidone to final vol of 5 ml (final conc is 1.2 mM PEI-cholesterol conjugate). 

5. For 24 well plate: add 1 ul PEI-Chl in PBS to 24 ul of OptiMEM, add 0.5 ug of plasmid DNA to 25 ul of OptiMEM. 

6, Mix two solutions were mixed and incubate for 20 min at 25 oC. 

7. Add mixture to cells. 

8, Remove medium 4 h after transfection, add complete medium (DMEM containing 10% FBS). 

1% modification give best transfection efficiency 

== Protocol 2<br>  ==

Synthesis of Branched PEI-Chol. 

<br> 

[[Image:Branch PEI Chol synthesis.png]] 

Stage I. <br>1. Dissolve Benzylchloroformate (1.76 g, 1.5 mL, 10.36 mmol) in methylene chloride (5 mL) and place in a three-neck flask under nitrogen atmosphere. 

2. Dissolve imidazole (1.4 g, 20.6 mmol) in methylene chloride (20 mL) and place in an addition funnel. 

3. Cool three-neck flask to 0 °C using ice and add imidazole solution gradually over 30 min. 

4. Stir the mixture at room temperature for 2 h. 

5. Add methylene chloride (25 mL) and citric acid (10%; 25 mL). 

6. Separate layers and wash the organic fraction with citric acid (10%; 25 mL). 

7. Dry organic component over magnesium sulfate and concentrate in vacuo. 

8. Dry the residue under high vacuum for 2 h at ambient temperature. 

Stage II. 

9. Add DMAP (35 mg) and methylene chloride (25 mL) to the product of Stage I 

10. Cool the mixture to 0 °C under nitrogen atmosphere. 

11. Add PEI (MW 1800; 1.7 g, âŒ10 mmol PEI repeat unit) in methylene chloride (25<br>mL) gradually over 30 min. 

12. Stir for 18 h at ambient temperature. 

13. Concentrate in vacuo and thoroughly wash three times with ethyl ether. 

Stage III. 

14. Dissolve cholesteryl chloroformate (600 mg, 1.34 mmol) in methylene chloride (10 mL) 

15. Add dropwise quickly with the product of Stage II (PEI-CBz). 

16. Add triethylamine (1 mL) in methylene chloride (10 mL) cooled to under -10 °C by ice-salt bath, under nitrogen atmosphere, under stirring 

17. Stir the reaction mixture for 6 h at 0 °C and for another 6 h at ambient temperature. 

18. Concentrate the product in vacuo at ambient temperature. 

19. Wash three times with ethyl ether. 

20. Remove residual cholesteryl chloroformate with ethyl ether. 

<br>Stage IV. 

21. Dissolve the product of stage III (Chol-PEI-CBz) in 25 mL of acetic acid 

22. Add 50 mg of 10% palladium on activated carbon (Pd/C). 

23. Purge the solution with nitrogen. 

24. Stir under hydrogen at atmospheric pressure for âŒ8 h. 

25. Filter the reaction mixture 

26. Wash with 200 mL of ethyl acetate/acetic acid (9/1 v/v). 

27. Concentrate in vacuo to give a residue. 

28. Dissolve the crude product in 20 mL of 1 N NaOH. 

29. Precipitate with excessive acetone below 0 °C. 

30. Wash three times with ethyl ether.<br> 

<br> 

== Synthesis of PEI-Bu<br>  ==

1, 4-butanediol bis (chloroformate) (0.43 g, 2 mmol) in chloroform (40 mL) was added dropwise to a PEI 800 Da (1.8 g, 3 mmol) chloroform solution (6 mL) at 0°C with stirring in anhydrous environment. The reaction was vigorously stirred for 24 hours at room temperature. The solvent was then removed under reduced pressure to obtain the crude product. After dialysis by 3500 Da dialysisbag for 24 h to remove small fragments, the dialysate was freeze-dried to obtain the final product. The PEI-Bu polymer was kept at −80°C in a dry environment before use<br> 

<br> 

== Synthesis of Water Soluble PEI-Chol ==

[[File:Water soluble PEI Chol synthesis.png|RTENOTITLE]]



Dissolve 3 g PEI (1800 Da) in a ice-cold mixture of 10 ml anhydrous methylene chloride (Dichloromethane, DCM) and 10 ul triethylamine on ice for 30 min.

Dissolve 1 g Cholesteryl chloroformate in 5 ml methylene chloride on ice.

Add Cholesteryl chloroformate solution slowly to the PEI solution over 30 min

Stire the mixture for 12 h on ice.

Dry the resulting product on a rotary evaporator.

Dissolve the powder in 50 ml 0.1 N HCl.

Extract the aqueous solution with 100 ml methylene chloride to remove unreacted cholesteryl chloroformate

Filter through a glass microfiber filter.

Evaporate the solvent.

Dissolve the powder in water<br/>Precipitate three times with large excess acetone

Wash with methanol then three times with diethyl ether.

Dry under vacuum.







'''Method 2:'''

Add 30 mL of dehydrated chloroform to 100 μL dehydrated triethylamine<br/>Add 3 g of PEI<br/>place on ice

Stir for 30 minutes

Add 1 g cholesterol chloroformate dissolved in 10 mL chloroform at 0°C

stir overnight.

dissolve resulting yellowish viscous gel in 50 mL hydrochloric acid (0.1 mol/L).

Add 200 mL dehydrated chloroform to remove other polymers and cholesterol

filter through filter paper.

Repeat washing/extraction 2X<br/>freeze-dried --> a yellow powder.



'''Method 3.'''

Add 3&nbsp;grams of PEI (mw 1800) &nbsp;to a mixture&nbsp;of 10 mL of anhydrous methylene chloride and 100 µL of&nbsp;triethylamine, cool&nbsp;<span style="line-height: 20.8px;">on ice</span>&nbsp;for 30 min. 

Dissolve 1 gram of cholesteryl chloroformate&nbsp;in 5 mL of anhydrous ice-cold&nbsp;methylene chloride

Add <span style="line-height: 20.8px;">cholesteryl chloroformate solution</span>&nbsp;slowly (dropwise) &nbsp;to the PEI&nbsp;solution over&nbsp;30 min.

Stire the mixture for 12 h on&nbsp;ice.

Dry the reaction product&nbsp;on a rotary&nbsp;evaporator. 

Dissolve the powder in 50 mL of 0.1 N&nbsp;HCl and then filtered through the glass microfiber filter.

<br/>Cholesteryl chloroformate is well soluble in methylene&nbsp;chloride, diethyl ether, and acetone. Therefore, nonreacted&nbsp;cholesteryl chloroformate can be&nbsp;removed from the<br/>product by washing three times in these different organic&nbsp;solvents as follows: the aqueous solution is extracted&nbsp;three times with 100 mL of methylene chloride to remove<br/>nonreacted cholesterol chloroformate, and then extracted&nbsp;aqueous solution is filtered through a glass microfiber&nbsp;filter. The product is&nbsp;concentrated by solvent evaporation,<br/>precipitated with large excess acetone, and dried&nbsp;under vacuum. The powder is dissolved in water and&nbsp;precipitated three times with acetone. After washing with<br/>methanol, the product is washed three times with&nbsp;diethyl ether to yield&nbsp;white powder.

[[File:Water soluble PEI CHol.png|RTENOTITLE]]



[[File:Water soluble PEI Chol transfection1.png|RTENOTITLE]]

== 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. 

<br> 

=== 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]]