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Transient expression levels, vector dissemination and toxicities associated with adenoviral vectors have prompted the usage of matrices for localized and controlled gene delivery. Two recombinant silk-elastinlike protein polymer analogues, SELP-47K and SELP-415K, consisting of different lengths and ratios of silk and elastin units, were previously shown to be injectable hydrogels capable of matrix-mediated controlled adenoviral gene delivery. Reported here is a study of spatiotemporal control over adenoviral gene expression with these SELP analogues in a human tumor xenograft model of head and neck cancer using whole animal imaging. Real-time images of viral expression levels indicate that polymer concentration and polymer structure are predominant factors that affect viral release and, thus, viral transfection. Decrease in polymer concentration and increase in polymer elastin content results in greater release, probably due to changes in the network structure of the hydrogel. To better understand this relationship, macro- and microstructural properties of the hydrogels were analyzed using dynamic mechanical analysis (DMA) and transmission electron microscopy (TEM). The results confirm that the concentration and the elastin content of the protein polymer affect the pore size of the hydrogel by changing the physical constraints of the SELP fibril network and the degree of hydration of the SELP fibrils. The potential to modulate viral release using SELP hydrogel delivery vehicles that can be injected intratumorally by minimally invasive techniques holds significant promise for the delivery of therapeutic viruses.

Previous studies from our group demonstrated visual evidence that endocytosis mechanism(s) contribute to the internalization and intracellular trafficking of cationic and anionic poly(amidoamine) (PAMAM) dendrimers across Caco-2 cells. These dendrimers colocalized with established endocytosis markers, which suggested PAMAM dendrimers may be internalized by a clathrin-dependent endocytosis mechanism and are rapidly trafficked to endosomal and lysosomal compartments. In the present study, generation 4 PAMAM-NH 2(G4NH 2) dendrimer was labeled with tritium to measure the rate of uptake and permeability in Caco-2 cells. The effect of endocytosis inhibitors brefeldin A, colchicine, filipin, and sucrose on G4NH 2absorption and transport was examined to give further insight into the endocytosis mechanisms that transport PAMAM dendrimers across Caco-2 cell monolayers. G4NH 2showed linear uptake at lower concentrations, and rapidly increased as a function of concentration. The rate of G4NH 2uptake significantly declined at high concentrations in the presence of the endocytosis inhibitors, and the apparent permeability similarly reduced in the presence of these inhibitors. A significant reduction in G4NH 2permeability was observed in the presence of brefeldin A and colchicine, which generally disrupt vesicular trafficking and formation during the endocytosis process. Coincubation with filipin and sucrose reduced G4NH 2permeability to a lesser extent, which suggests G4NH 2could be nonspecifically internalized in coated vesicles at the plasma membrane. The observations from this study further confirm that G4NH 2internalization and transport involves an endocytosis pathway.