Synthesis of PAM-Chol

Polyamidoamine Cholesterol

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Add cholesteryl chloroformate to excess ethylenediamine to prepare EDA-Chol (ethylenediamine-cholesterol):

PAM-Chol (polyamidoamine-cholesterol, generation 0.5) is synthesized by Michael addition of the primary amine of ethylenediamine to double bonds of two methyl acrylate molecules,   use methyl acrylate/MeOH

Add ethylenediamine to PAM-Chol:  the resulting two ester groups of PAM-Chol (generation 0.5) is attacked by ethylenediamine to produce PAM-Chol (generation 1), use ethylenediamine/MeOH

To avoid side reaction and to obtain a perfect dendritic lipid, excess methyl acrylate and ethylenediamine should be added in each step

Methyl acrylate:

Synthesis

Synthesis of EDA-Chol (A)

Dissolve 80 g (1.3 mol) of ethylenediamine and 4 g (8.9 mmol) of cholesteryl chloroformate  in 10 mL of chloroform .

Warm the mixture to 35 oC and stirred for 48 hours.

Wash the reaction mixture with brine (5M NaCl in water) three times.

Collect organic layer

Dry over anhydrous sodium sulfate, and evaporate.

Yield is 2.8g of colorless powder.

Synthesis of PAM-Chol g =0.5 (B)

Dissolve 2.1g (4.5 mmol) of EDA-Chol and 300 mL (3.3 mol) methyl acrylate in 300 mL MeOH.

Warm the mixture to 35 oC and stirred for 48 hours.

Evaporate the organic solvent and excess methyl acrylate.

PAM-Chol g = 0.5 (B) is obtained as 3.0 g of yellow viscous oil.

Synthesis of PAM-Chol g = 1 (C)

Dissolve 3.0 g PAM-Chol g =0.5 (B) in 300 mL MeOH.
Add 300 mL (4.5 mol) of ethylenediamine to the solution dropwise at 0^oC.

Warm the mixture to 35 ^oC
Stir for 48 hours.

Evaporate the organic solvent and excess ethylenediamine.

Purify PAM-Chol g = 1.0 (C) on silica gel with an eluent of n-propanol: ammonia water: water (70:10:20 to 70:20:10 v/v/v).

 The transfection efficiency followed the order of PAM-Chol/DOPE g = 1.0 > DC-Chol/DOPE > PAMAM g = 6.0 (with chloroquine) > PAM-Chol/DOPE g = 2.0 > PAMAM g = 6.0 (without chloroquine).  The transfection efficiency of PAM-Chol/DOPE g = 1.0 at the optimal condition was about 3- and 100-fold more efficient than those of DC-Chol/DOPE and PAMAM g = 6.0 (without chloroquine), respectively. PAM-Chol g = 1.0, which requires only one Michael addition/amidation step, showed significantly higher efficiency than PAMAM g = 6.0, which
requires seven iterative Michael addition/amidation steps.

Liposome Preparation: 0.5 mg of DOPE (Sigma, St. Louis, MO) and 0.5 mg of synthetic lipids (PAM-Chol g = 1.0 and g = 2.0) in chloroform were dried under a stream of N2 gas and vacuum. After overnight hydration with 1 mL of distilled water at 4 oC, the mixture was sonicated using a bath-type sonicator

Conclusion:  PAM-Chol g = 1 is good enough for transfection, and easy to make