Optimizing Active Tumor Targeting Biocompatible Polymers for Efficient Systemic Delivery of Adenovirus

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P₅N₂LG-FA and P₅N₅LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N₂ < N₅ in terms of charge), along with a P₅N₅LG control polymer without FA. Our findings demonstrate that P₅N₅LG, P₅N₂LG-FA, and P₅N₅LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P₅N₂LG-FA and P₅N₅LG-FA (Ad/P₅N₂LG-FA and Ad/P₅N₅LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P₅N₅LG (Ad/P₅N₅LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P₅N₅LG, Ad/P₅N₂LG-FA, and Ad/P₅N₅LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P₅N₂LG-FA and Ad/P₅N₅LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P₅N₅LG, Ad/P₅N₂LG-FA, and Ad/P₅N₅LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P₅N₂LG-FA and Ad/P₅N₅LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P₅N₅LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P₅N₂LG-FA was significantly lower than Ad/P₅N₅LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.