Dendrimers are unique and well-defined nanostructured (5-15nm) and highly branched polymers that have gained wide attention as promising intracellular drug delivery vehicles . The surface functionality can be °¥tailored' to attach a variety of drugs, genes, targeting agents and imaging agents either by complexation or conjugation. Cellular interactions and cytotoxicity of dendrimers are mainly governed by the physicochemical properties including the size, generation, surface functionality and the net charge on the dendrimer. In addition, the surface functionality plays a strong role in the intracellular drug release. We are investigating the effect of dendrimer surface functionality on the intracellular uptake and drug release.

Different PAMAM dendrimers including G4 amine terminated, G4 hydroxyl terminated and G3.5 carboxyl terminated were labeled with fluoroisothiocyanate (FITC). The cell uptake studies were carried out using A549 lung eiphtelial cells and the intracellular fluorescence was quantified using flow cytometry. Intracellular localization of dendrimers was imaged using fluorescence and confocal microscopy. Inhibition studies were carried out to identify the endocytotic pathway for the cellular uptake of different dendrimers. Various endocytotic blockers including sucrose, filipin, nocodazole, ammonium chloride and cytochalasin were used to identify the endocytoic pathway for the transport of different dendrimers. Our results suggest that these dendrimers are taken up by fluid phase endocytosis, but the rate of uptake and the mechanism of uptake were dependant on the surface functionality. Cationic dendrimers are taken up faster than anionic dendrimers. The cationic dendrimers are mainly endocytosed by fluid phase endocytosis through clathrin coated pits, while anionic dendrimers are mainly transported through caveolae mediated endocytosis. Neutral dendrimer appear is transported by clathrin coated endocytosis through nonspecific interactions. By tailoring the dendrimer surface, it is possible to target the dendrimer to a specific group of cells by exploiting the constitutive endocytotic pathway of the specific group of cells.

The dendrimer surface charge also appears to play a significant role on the intracellular drug release. Our preliminary results suggest that cationic dendrimers may adversely affect the lysosomes, perhaps preventing drug release from the dendrimer-drug conjugate. On the other hand, anionic dendrimers appear to have longer lysosomal residence times, enabling drug release and enhancing therapeutic activity. To quantify the differences in the drug release, two sets of release studies will be performed: (1) To test whether the drug is released differently from the conjugates, we will assay for the presence of free MTX-FITC in the cytosol as a function of time, using an established protocol; (2) To confirm the role of lysosomes in accumulation and release of free MTX, we will add lysomotropic agents (NH4Cl [50mM] or chloroquine [100 ƒİM]), which destabilize the lysosomes by increasing the pH, leading to lysosomal membrane disruption. If the intracellular release is different because of lysosomal residence times, we expect to see a difference in the release of free MTX-FITC levels as a function of treatment with these agents.