In order for genes to be expressed they must be in the cell nucleus. Plasmids are very large molecules. Typically, they contain between 3 and 15 kb which equals, at approximately 660 Da and two negative charges per base pair, a molecular weight of 2 to 10 million Da and 6000 to 30,000 negative charges per molecule. Knowing that cell membranes are effective permeability barriers to even small molecules, one can appreciate the problem of intracellular delivery of plasmids. The problem is even more pronounced in in vivo transfection because free DNA is quickly degraded by extracellular DNA nucleases, which protect the body against exogenous genes. This is especially true for systemic administration. The only exception seems to be intramuscular injection, where upon local injection of naked DNA some activity was observed. However, the entry of DNA molecules into intact cells still presents an enigma, and we shall discuss some possibilities of DNA internalization in Chapter 10. It could be a consequence of mechanical rupture, osmotic imbalance, or hydrodynamic or local pressure (upon bolus injection) effects.