(182u) Characterization of Microfluidic Calcium Phosphate-Mediated DNA Delivery

Calcium phosphate-based delivery of nucleic acids has existed for decades and the technique is known for its ease and simplicity, but high variability in transfection efficiency between cell lines. Despite the variability, calcium phosphate precipitates remain a promising vector for nucleic acid delivery in part because they form hydroxyapatite, the naturally occurring biomaterial in bone and teeth, which is safer than the immunogenic viruses that initially dominated gene delivery.

Gene delivery vectors, including polymer-based DNA complexes and calcium phosphate-DNA co-precipitates, have most often been prepared by simple mixing of the individual components. Our group, however, has recently has shown the controlled mixing and complex formation of polymer/DNA polyplexes under laminar flow in a microfluidic device gives rise to a more uniform particle size distribution and higher transfection efficiency than bulk-mixed particles. The present study applies such microfluidic devices to characterize their ability to fabricate calcium phosphate-DNA co-precipitates.

Higher transfection efficiency and lower cytotoxicity were achieved for select concentrations and device styles in our microfluidic devices compared to bulk mixing. DLS sizing results indicate calcium phosphate-DNA co-precipitates formed by bulk mixing exhibit a bi-modal size distribution, with particles between 200 and 1000 nm and aggregates of several microns. However, the size distribution of calcium phosphate-DNA co-precipitates formed in the microfluidic device exhibit a single peak with diameters on the order of 100 nm.

In addition to improved size distribution and transfection efficiencies, this method may also fabricate multifunctional calcium phosphate-DNA nanoparticles. Additional shell layers can be added to the calcium phosphate-DNA co-precipitate base in a diffusion-controlled manner to carefully design the architecture of the nanoparticle with a characteristically tight distribution of physiochemical properties that is difficult to impossible to achieve by bulk mixing.