Tumor gene targeting using microspheres: Cell culture and in vivo studies

In a previous study, Dass et al. (Pharm. Sci. 2:401-405, 1996), it was shown that 1 mg of two types of ion-exchange microspheres was capable of binding and releasing plasmids in a continuous-flow system to the-order of 10(11) copies in phosphate-buffered saline at 37 degrees C, However, the functionality of the plasmids was not evaluated. In this study, one of the plasmids, pCMV-CAT, was bound to both microspheres and the functionality of the biomolecule was examined in cell culture and in vivo transfection studies. Rat tumor cells incubated with the hydroxyapatite (HA) plasmids were transfected 5.4-fold better than when incubated with free plasmids and 56.0-fold better than polystyrene divinylbenzene (PDB) microspheres. Cells incubated with PDB microspheres were transfected 10.4-fold less than cells incubated with free plasmids. However, HA microspheres were highly cytotoxic to the cells whereas PDB spheres had no effect on cell numbers compared with control cells. Based on expression levels of delivered plasmids in the in vivo study, delivery on microspheres to kidneys was 2.7-fold better than plasmids delivered free. Microspherical delivery of plasmids to tumors was 1.6-fold better than free delivery. However, these results were not significantly different (p > .05). The tumor/normal tissue ratio of gene expression was 4.5:1 for free delivery and 2.6-fold when delivered on microspheres. Although the difference between deliveries in the two tissues was significant (p < 0.005) for free delivery, it was not so for microspherical delivery, Expression of the CAT gene was not noted in either liver or spleen of any of the animals. This present study has proved that ion-exchange microspheres have no detrimental effect on released plasmid DNA expression. In an in vivo setting, resistance to enzymatic degradation of plasmids that are bound to microspheres and subsequent release of plasmids once embolization has occurred in the tumor vascular bed effected better transfection than delivery of free plasmids.