125. Formulation and In Vitro Properties of Adenovirus-Polylactide Nanoparticle Affinity-Based and Magnetic Composites
Authors: Michael Chorny, Ilia Fishbein, Ivan S. Alferiev, Origene Nyanguile, Robert J. Levy
Journal: Molecular Therapy (2004)

Top of pageAbstract
Background

Adenoviral vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their use is however limited by the reduced efficacy and systemic adverse reactions resulting from the inability to effectively localize and provide the sustained presence of the vector in the target tissue, while minimizing its escape from the delivery site. To address these problems we investigated the affinity immobilization of adenovirus (AdV) on a biodegradable nanoparticulate platform. Additionally we explored the possibility of enhancing the gene expression by rendering the composite magnetically responsive.

Methods

Two polylactide-based nanoparticle (NP) formulations were formed by the nanoprecipitation and emulsification-solvent evaporation methods. The NP surface modification with an anionic thiol-reactive derivative of polyallylamine was accomplished photochemically by a brief exposure to long-wave UV light. After separation from unreacted polymer NP were coated with a thiolated form of D1 domain of the Coxsackie-AdV receptor and finally incubated with GFP-encoding AdV suspension in presence of 5% bovine serum albumin. Magnetic NP were formed by inclusion of iron oxide nanocrystals in the polymeric matrix. The particle core and the virus were stained with green BODIPY and red Cy3 dyes, respectively, for fluorimetric studies. AdV-NP binding efficacy was evaluated by measuring the residual fluorescence of unbound virus following magnetic separation of AdV-NP composite. The formulation uptake and efficacy were studied on rat arterial smooth muscle cell culture. The intracellular localization was determined on live cells as a function of incubation time and particle size. The effect of magnetic force on gene expression was assessed by measuring GFP fluorescence in cell lysates using free AdV as a control.

Results

NP sized 160 and 360 nm were surface-modified with a thiol-reactive polymer having a particle stabilizing effect due to its negative charge. The colloidal stability of the NP was not adversely affected by their subsequent modification with D1 protein and AdV. Five and 20 min photoactivation resulted in an equally high NP capacity (~92%) for AdV binding, whereas unspecifically bound AdV fraction was consistently below 25% in the studied concentration range. Smaller NP exhibited a 1.2-fold higher in vitro cellular uptake and a more rapid elimination than that of larger NP; about 14% of the 360 nm NP initially taken up were found in the cells after 48 hr, while 160 nm NP were undetectable at this timepoint.

GFP expression mediated by AdV-particle composite was 2.4 and 10.2-fold higher than that of free AdV when incubated with or without magnet, respectively. The magnetic field exposure resulted in a 4.7-fold increase in gene expression for the composite formulation, having no effect on the free AdV treated cells.

Conclusions

Immobilization of AdV on a magnetic polymer-based NP platform resulted in a substantial increase in the efficacy of gene transfer in vitro. Rendering this formulation magnetically-responsive provides a potential means for optimizing its biodistribution and efficacy in vivo, and makes this novel gene delivery system an interesting candidate for therapeutic applications.