Screening for Improved Cationic Lipids

COS-7 or CHO cells (for initial transfection screening) or cells of therapeutic interest (e.g., dendritic cells and various cancer cells) at a confluence of 50%, grown in 96-well culture plates, were placed into the robot by the robotic conveyor. In a fully automated process, the robot removes the lid from the cell culture microtiter plate, dispenses lipoplexes into the wells (triplicates), replaces the lid and returns the plate to the incubator. After four hours, the cells are automatically retrieved, the cell monolayers are carefully washed using a special drop mode of the integrated plate washer, fresh medium is added, and the cells are incubated for further 42 hours before harvesting. 

COMBINATION OF THE SCREENING APPROACH WITH COMBINATORIAL SOLID PHASE SYNTHESIS OF CATIONIC LIPIDS 

In this section, an example will be given in which a (small) library of a new type of cationic lipids was synthesized and screened for TE (63). For synthesis, combinatorial solid phase chemistry was used. All cationic lipids of the example library are structurally based on 3-methylamino-1,2-dihydroxy-propane as the polar, cationic lipid part. As nonpolar lipid part, different hydrocarbon chains are boimd to the amino group of the scaffold and the amino group was further methylated to get constantly cationic-charged lipids. Lipids were synthesized in both configurations and as racemats, and the counterions were varied as well. Table 1 summarizes the structural features of these lipids. 

All lipids were synthesized in R- or S-configuration or as racemat. Chloride, sulfate, methylsulfate, and acetate were used as counterions. (R1 and R2 Fig. 5). 

3-methylamino-1,2-dihydroxy-propane as the polar, cationic lipid part. As nonpolar lipid part, different hydrocarbon chains are bound to the amino group of this scaffold. The amino group is further methylated to get a constantly cationic-charged lipid. This synthesis strategy allows to synthesize the new lipids in different configuration, and with different counterions. 

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