Characterization and real-time imaging of gene expression of adenovirus embedded silk-elastinlike protein polymer hydrogels

Abstract

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.

Figure 1

The amino acid sequence of SELP analogues: (A) SELP-47K (molecular weight: 69,814 Da) and (B) SELP-415K (molecular weight: 71,500 Da). Polymer analogs were composed of head and tail portions and a series of silk (GAGAGS) and elastin-like (GVGVP) repeats with the primary repetitive sequences in bold, and positively charged amino acids (at pH 7.4) highlighted in gray.

Figure 2

Typical time course of luciferase expression in HN12 tumor xenografts in athymic nude mice. Mouse A: PBS control. Mouse B: Ad-CMV-Luc control. Mouse C: 4 wt% SELP-47K + Ad-CMV-Luc. Mouse D: 10.9 wt% SELP-47K + Ad-CMV-Luc. Mouse E: 10.9 wt% SELP-415K + Ad-CMV-Luc.

Figure 3

Dynamic mechanical analysis of SELPs 47K 4 wt% (◆), 8 wt% (■), and 12 wt% (▲) as well as SELP-415K 12 wt% (×) cured for 4 hours. SELP-47K 12 wt% formed stiff gels that had significantly higher modulus than all other tested gels. SELP-415K 12 wt%, despite forming hydrogels that held their shape unaided, gave results similar to that of the weakest SELP-47K 4 wt% hydrogels.

Figure 4

Transmission electron microscope image of (A) 4 wt% SELP-47K, (inset) icosahedral Ad-CMV-Luc virion, (B) 12 wt% SELP-47K and (C) 12 wt% SELP-415K.