Overall Charge Of Complexes And Entry Into The Cell

In addition, our delivery system is efficient because the complexes deliver DNA into cells by fusion with the cell membrane and avoid the endocytic pathway (Fig. 6). Cells are negatively charged on the surface, and specific cell types vary in their density of negative charge. These differences in charge density can influence the ability of cells to be transfected. Cationic complexes have nonspecific ionic charge interactions with cell surfaces. Efficient transfection of cells by cationic complexes is, in part, contributed by adequate charge interactions. In addition, recent publications report that certain viruses have a partial positive charge around key subunits of viral proteins on the virus surface responsible for binding to and internalization through target cell surface receptors. Therefore, this partial positive charge is required for virus entry into the cell. Thus, maintenance of adequate positive charge on the surface of targeted liposome complexes is essential for optimal delivery into the cell. Different formulations of liposomes interact with cell surfaces via a variety of mechanisms. Two major pathways for interaction are by endocytosis or by direct fusion with the cell membrane (27,29-34). Preliminary data suggest that nucleic acids delivered in vitro and

Figure 6 Mechanisms for cell entry of nucleic acid-liposome complexes. Two major pathways for interaction are by endocytosis or by direct fusion with the cell membrane. Complexes that enter the cell by direct fusion allow delivery of more nucleic acids to the nucleus because the bulk of the nucleic acids do not enter endosomes.

Figure 6 Mechanisms for cell entry of nucleic acid-liposome complexes. Two major pathways for interaction are by endocytosis or by direct fusion with the cell membrane. Complexes that enter the cell by direct fusion allow delivery of more nucleic acids to the nucleus because the bulk of the nucleic acids do not enter endosomes.

in vivo using complexes developed in our lab enter the cell by direct fusion (Fig. 6). Apparently, the bulk of the nucleic acids do not enter endosomes, and therefore, far more nucleic acid enters the nucleus. Cell transfection by direct fusion produced orders of magnitude, increased levels of gene expression, and numbers of cells transfected vs. cells transfected through the endocytic pathway.

We believe that maintenance of adequate positive charge on the surface of complexes is essential to drive cell entry by direct fusion. Therefore, we create targeted delivery of our complexes in vivo without the use of PEG. These ligand-coated complexes also reexpose the overall positive charge of the complexes as they approach the target cells. Through ionic interactions or covalent attachments, we have added monoclonal antibodies, Fab fragments, proteins, partial proteins, peptides, peptide mimetics, small molecules, and drugs to the surface of our complexes after mixing. These ligands efficiently bind to the target cell surface receptor, and maintain entry into the cell by direct fusion. Using novel methods for addition of ligands to the complexes for targeted delivery results in further increased gene expression in the target cells after transfection. Therefore, we design targeted liposomal delivery systems that retain predominant entry into cells by direct fusion vs. the endocytic pathway. Figure 7 shows our optimized strategy to achieve targeted delivery, deshielding, fusion with the cell membrane, entry of nucleic acids into the cell and to the nucleus, and production of gene expression of a cDNA cloned in a plasmid.

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