# Cationic Lipid Derived from a Basic Amino Acid: Design and Synthesis
## 3 Results
### Lipid Mixtures Containing DOPE as a Helper Lipid Increase Transfection Efficiency  
The highest transfection efficiencies were achieved when using lipoplexes with \(w/w\) total lipid/pDNA ratios of 1:0.4. In general, transfections were carried out with lipoplexes containing the CholCadLys and CholCad lipids, in combination with cholesterol or DOPE in different molar proportions. Very few transfected cells were observed with the CholCadLys lipid containing cholesterol, resulting in a transfection efficiency of 2% (Figures 9 and 10C,D). However, when transfection was performed with lipoplexes formed with CholCad and DOPE (Figures 9 and 10E-G), and CholCadLys lipid and DOPE (Figures 9 and 10 IJ), a transfection efficiency of up to 10% was achieved.  
Given that the best transfections were obtained with the CholCadLys and the CholCad lipids, and these lipids mixed with DOPE, a mixture of these lipids was prepared to observe the effect on transfection efficiency. Stable liposomes were formed with the lipid mixture as follows: CholCadLys-CholCad in a molar ratio of 1:2; CholCadLys-CholCad in a molar ratio of 2:1; CholCadLys-CholCad-DOPE in a molar ratio of 1:1:2; CholCadLys-CholCad-DOPE in a molar ratio of 2:1:1. Lipoplexes were then formed, and the He  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{2}{c}{**Molar Ratios**} & \multicolumn{2}{c}{**Average Size (nm)**} & \multicolumn{2}{c}{
\begin{tabular}{c} **Concentration** \\ **(Particles/mL)** \\ \end{tabular} } \\ \cline{2-6} \multicolumn{2}{c}{**(CholCadLys–Helper Lipid)**} & \multicolumn{2}{c}{**Liposomes**} & \multicolumn{2}{c}{**Lipoplexes**} & \multicolumn{2}{c}{**Liposomes**} & \multicolumn{2}{c}{**Lipoplexes**} \\ \hline \multirow{2}{*}{Cholesterol} & 1:1 & 136.6 & 132 & \(1.72\,\times\,10^{8}\) & \(2.53\,\times\,10^{8}\) \\ \cline{2-6}  & 2:1 & 114.8 & 92.3 & \(1.35\,\times\,10^{8}\) & \(1.80\,\times\,10^{8}\) \\ \hline \multirow{2}{*}{DOPE} & 1:1 & 102.4 & 142 & \(3.39\,\times\,10^{7}\) & \(2.33\,\times\,10^{8}\) \\ \cline{2-6}  & 1:2 & 116.7 & 107.2 & \(2.16\,\times\,10^{7}\) & \(3.04\,\times\,10^{7}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Size and count of liposomes and lipoplexes.  
Figure 9: Transfection efficiency in Hek-293 FT, defined as the percentage of GFP-positive cells detected by fluorescence microscopy.  
Figure 10: Transfection in Hek-293 FT Cell Line. Different lipoplex mixtures were prepared using the synthesized lipid and a helper lipid in various ratios. (**A**) Untransfected control cells; (**B**) CholCadLys; (**C**) CholCadLys–cholesterol 1:1; (**D**) CholCadLys–cholesterol 2:1; (**E**) CholCad-DOPE 1:1; (**F**) CholCad–DOPE 1:2; (**G**) CholCad-DOPE 2:1; (**H**) CholCad; (**I**) CholCadLys–DOPE 1:1; (**J**) CholCadLys–DOPE 1:2; (**K**) CholCadLys–CholCad 1:2; (**L**) CholCadLys–CholCad 2:1; (**M**) CholCadLys–CholCad–DOPE 1:1;2; (**N**) CholCadLys–CholCad–DOPE 2:1:1; (**O**) Lipofectamine 0.5 \(\upmu\)g–0.400 \(\upmu\)g DNA.  
An increase in transfection efficiency was observed with the mixture of CLs in different proportions. Using CholCadLys-CholCad in a 1:2 ratio and CholCadLys-CholCad in a 2:1 ratio, a transfection efficiency of up to 30% was achieved (Figures 9 and 10K,L). Meanwhile, mixing CholCadLys-CholCad-DOPE in a 1:1:2 ratio and CholCadLys-CholCad-DOPE in a 2:1:1 ratio resulted in a transfection efficiency of up to 40% (Figure 10M,N), which was superior to the LipofectamineTM 2000 (Thermo Fisher Scientific) used as a comparison method for this transfection system in this cell line.