Abstract
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.
Highlights
In the past two decades, gene therapy has been a focus of various therapeutic approaches for cancer treatment [1,2,3]
The folic acid (FA)-poly(ethylene glycol) (PEG)-NH2 polymer was used as a macroinitiator to afford the diblock copolymer, FA-PEG-PBLG, through ring-opening polymerization of BLG-NCA. 1 H NMR spectrum in Figure 1D shows the presence of FA, PEG, and PBLG characteristics peaks, indicating successful polymer synthesis
KB cells (p < 0.001), whereas these two targeted nanocomplexes showed much lower level of internalization than those of Ad-fluorescein isothiocyanate (FITC)/P5 N5 LG in folate receptor (FR)-negative A549 cells. These results suggest that cellular uptake efficiency of nanocomplexes composed with cationic and FR-targeted nanomaterials (P5 N2 LG-FA and P5 N5 LG-FA) on the surface of Ad is greatly affected by cellular FR expression level, and these results are in line with our findings from Figures 3 and 5A
Summary
In the past two decades, gene therapy has been a focus of various therapeutic approaches for cancer treatment [1,2,3]. Engineered oncolytic adenovirus (Ad) is one of the most promising vectors in cancer gene therapy because of its advantageous characteristics including cancer-specific replication, potent cancer cell killing effect, and high expression level of therapeutic transgenes. Oncolytic Ad can elicit potent antitumor effects when administered locally to the tumor in a similar manner to other clinically approved oncolytic viruses. A system that is capable of efficiently delivering oncolytic viruses via the systemic route to tumor tissues is necessary to further improve the clinical efficacy of oncolytic vectors. Systemic delivery of oncolytic Ad is a challenging task due to the immunogenicity and hepatic tropism of Ad. The immunogenic viral capsid can be recognized by the host immune system and lead to rapid clearance from the blood as well as adverse inflammatory response, leading to poor safety and therapeutic efficacy of systemically administered oncolytic viruses. Nonspecific hepatic sequestration and accumulation of oncolytic Ad can lead to hepatotoxicity [4]
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