Magnetically responsive biodegradable nanoparticles enhance adenoviral gene transfer in cultured smooth muscle and endothelial cells

Abstract

Replication-defective adenoviral (Ad) vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their clinical use is however limited by therapeutically suboptimal transduction levels in cell types expressing low levels of Coxsackie-Ad receptor (CAR), the primary receptor responsible for the cell entry of the virus, and by systemic adverse reactions. Targeted delivery achievable with Ad complexed with biodegradable magnetically responsive nanoparticles (MNP) may therefore be instrumental for improving both the safety and efficiency of these vectors. Our hypothesis was that magnetically driven delivery of Ad affinity-bound to biodegradable MNP can substantially increase transgene expression in CAR deficient vascular cells in culture. Fluorescently labeled MNP were formulated from polylactide with inclusion of iron oxide and surface-modified with the D1 domain of CAR as an affinity linker. MNP cellular uptake and GFP reporter transgene expression were assayed fluorimetrically in cultured endothelial and smooth muscle cells using lambda(ex)/lambda(em) of 540 nm/575 nm and 485 nm/535 nm, respectively. Stable vector-specific association of Ad with MNP resulted in formation of MNP-Ad complexes displaying rapid cell binding kinetics following a brief exposure to a high gradient magnetic field with resultant gene transfer levels significantly increased compared to free vector or nonmagnetic control treatment. Multiple regression analysis suggested a mechanism of MNP-Ad mediated transduction distinct from that of free Ad, and confirmed the major contribution of the complexes to the gene transfer under magnetic conditions. The magnetically enhanced transduction was achieved without compromising the cell viability or growth kinetics. The enhancement of adenoviral gene delivery by affinity complexation with biodegradable MNP represents a promising approach with a potential to extend the applicability of the viral gene therapeutic strategies.

Figure 1

Transmission electron micrograph of a magnetic particle (left) and a MNP–Ad affinity complex (right). The samples were observed without staining using FEI Tecnai G2 electron microscope, Netherlands (bar = 100 nm). Note the large number of magnetite nanocrystals incorporated in the polymeric matrix of the particle and the multivalent character of the MNP–Ad association in A and B, respectively.

Figure 2

Intracellular localization of fluorescent-labeled nanoparticles (first column) and GFP expression (second column) in confluent A10 cells treated under magnetic conditions either with formulations combining _GFPAd with D1-coated MNP (A, E), nIgG-coated MNP (B, F), analogous nonmagnetic NP (C, G), or with _GFPAd alone (D, H). The cells were incubated with the formulations containing 1.5 × 10⁷ viral particles/well for 15 min in the presence of a nonuniform magnetic field (see Experimental Section), and the GFP expression and NP-associated fluorescence were observed 3 days post-treatment (the original magnification was ×200). GFP expression was quantified fluorimetrically (λ_em/λ_ex = 485 nm/535 nm) over time (I).

Figure 3

GFP expression (first row) and cellular localization of complex-forming MNP (second row) in A10 and BAEC observed microscopically 3 days after treatment under magnetic vs nonmagnetic control conditions (columns titled: “+ field” and “− field”, respectively). Merged GFP and bright field images of A10 (A, B) and BAEC (C, D) correspond to the red fluorescent micrographs showing cell-associated MNP (E, F and G, H for A10 and BAEC, respectively).The cells were seeded at a density of 2 × 10³/well on day –1, and treated with MNP–Ad complexes formed using varying amounts of MNP and 7 × 10⁷ Ad/well on day 0. MNP uptake was determined by measuring the MNP-associated fluorescent signal (λ_em/λ_ex = 540 nm/575 nm) and expressed as a fraction of the initially applied MNP that remained stably associated with the cells (I). Transduction efficiency was determined fluorimetrically (λ_em/λ_ex = 485 nm/535 nm) 3 days post-treatment and presented as a fold increase in GFP expression vs free Ad for the sake of convenient comparison (J). The cell viability was determined by the Alamar Blue assay 3 days post-treatment using untreated cells as a control (K). The higher cell density of BAEC in comparison with A10 on the merged micrographs (3 days post-treatment) reflects the difference in the respective rates of cell growth under the employed experimental conditions. The original magnification was ×200.

Figure 4

GFP expression mediated by MNP–Ad in A10 (top row) and BAEC (bottom row) under magnetic vs nonmagnetic conditions (left and right columns, respectively) as a function of MNP and Ad formulation amounts. GFP expression was measured fluorimetrically (λ_em/λ_ex = 485 nm/535 nm) in live cells 3 days post-treatment. Each data point represents an average of three experimental replicates.

Figure 5

The kinetics of GFP expression by A10 cells (A) and BAEC (B) treated with magnetic affinity complexes formed at MNP and Ad formulation amounts of 1.9 μg PLA and 7.1 × 10⁷ per well, respectively. The kinetics of MNP elimination was expressed as the fraction of the initially applied MNP retained by the cells by a given time point (C).