DNA Delivery to Cells in Culture Using Peptides
互联网
680
There are now several cationic peptide carriers that efficiently import plasmids and oligonucleotides into cells. As a result, we anticipate that cationic peptides will play an increasingly important role with in vitro and in vivo gene delivery systems. Cationic peptides usually bind through ionic interactions to the negatively charged phosphate backbone of DNA, although additional noncovalent bonds may stabilize the interaction between the polymer and DNA (Fig. 1 ). Alternatively, cationic peptides may be covalently conjugated to DNA to promote entry into the cell. Regardless of the type of linkage between the peptide and DNA, peptide-mediated delivery can be characterized by the pathway of entry into cells: endosomolytic (1 -5 ) or membrane-penetrating (6 -9 ). Endosomolytic peptides enter cells through endocytosis, whereas membrane-penetrating peptides bypass the endocytotic pathway and may fuse directly with the cellular membranes. Although this chapter will discuss several methods for preparing peptide/DNA complexes, we will focus on the solid phase synthesis of peptides and the complexes that these peptides form with DNA.
Fig. 1. Comparison of poly-L -lysine with HK polymer. In contrast to polylysine (K), serum has minimal effect on gene expression with the H-K triplex. Poly-L -lysine (K) or H-K polymers were first incubated with the PCI-Luc for 30 min followed by incubating with DOTAP liposomes. After incubating the triplex with MDA-MB-435 cells for 4 h, luciferase expression was measured 48 h later. Although K has nearly twice as many positive charges per molecule to interact with DNA compared to HK, the transfection complex containing the K-polymer is very sensitive to the presence of serum. In contrast, the transfection complex containing the HK polymer is resistant to serum and consequently transfection remains high. These results are consistent with the occurrence of noncovalent bonds other than ionic interactions.
