Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 152

3. Formation of a complex known as lipoplex, in the size range of several nanometers in
diameter
4. Binding with the aid of excess positive charge to the negative charges on the cell surface,
specifically proteoglycans
5. Internalization through a vesicular pathway mainly endocytosis
6. Destabilization of endosomal membrane by lipoplexes, resulting in a flip-flop organization
of phospholipids
7. Endosomal release of DNA from the lipoplex into the cell cytoplasm, or diffusion of phos-
pholipids into the lipoplex and interaction with the cationic lipids, causing the DNA to dis-
sociate into the cytoplasm
8. Nuclear entry of part of the DNA, followed by transcription and translation to protein
Lipoplexes were found to be effective in binding to cells in vitro , and they also facil-
itate intracellular delivery of pDNA. Lipoplexes also lack specific surface proteins that
can complicate in vivo application [188] . However, the cationic lipids can be covalently
attached or comixed with proteins, glycoproteins, or glycosylated lipids to confer selec-
tive targeting of therapeutic DNA to a particular tissue or organ. It was observed that
functionally effective lipoplex concentrations are generally nontoxic in vitro .
The primary obstacle in development of lipoplex for in vivo gene delivery is low
transgene expression. Although the transfection occurs efficiently, the number of cells
modulated is insufficient; this inefficient cellular response was assumed to be caused
by intracellular degradation and/or endosomal escape [189,190] . New commercially
available cationic lipids were designed to overcome these drawbacks. Facilitation of
this endosomal release of pDNA can significantly improve transgene expression. It
was demonstrated that “helper” lipids that are usually neutral in charge, such as dio-
leoylphosphatidylethanolamine (DOPE), cholesterol and dioleoylphosphatidylcholine
(DOPC), [191,192] , and pH-sensitive liposomes assist endosomal release [193] .
Cationic lipids consist of three parts, as per conventional description: a polar
head and a hydrophobic anchor region connected by a spacer. However, based on
the reported literature, a cationic lipid could be considered as a molecule with five
components ( Fig. 4.8 ). Each of these components has been systematically altered to
gain higher efficiency. The polar head interacts with negatively charged DNA while
the hydrophobic region forms a lipid bilayer that self-assembles into a liposome. To
improve efficiency of cationic liposome-mediated gene delivery, a variety of active
structures were synthesized. On the basis of structure of polar heads, cationic lipids
were classified into four different categories:
1. Quaternary ammonium salt lipids
2. Lipoamines
3. Cationic lipids containing quaternary ammonium salt and lipoamines
4. Amidinium salt lipids and miscellaneous cationic entities
4.3.1 Quaternary Ammonium Salt Lipids
Cationic lipid DOTMA, the first cationic lipid used for gene delivery by Felgner
et al. in 1987 [185] , belongs to this class of quaternary ammonium salt lipids. These
positively charged groups were basically quaternary ammonium salts linked to a lipid