Spatially and temporally controlled release of nucleic acids has many potential applications, including disease treatment and discovery of molecular processes involved in gene delivery. For nonviral gene therapy applications, spatial targeting has been attempted though inclusion of receptor targeting ligands to facilitate vector uptake into specific cells populations (27
), and through the use of biotinylated cationic polymers for substrate-mediated delivery (14
We developed a novel compound, B-PC-PEI, that combines surface-mediated delivery with photolabile technology for capture and light-activated controlled release of nucleic acids from solid supports. B-PC-PEI consists of three functional domains designed to tether the compound to a surface, interact with nucleic acids, and release the nucleic acids from the surface through exposure to 365 nm light. This represents the first time this type of compound has been used for the controlled release of non-covalently modified nucleic acids from streptavidin-coated solid supports.
The use of biotinylated PEI for substrate-mediated delivery of pDNA can result in positive transfection (15
). The retention of nucleic acids within a PEI polyplex depends on the molecular weight of the polymer used (31
), the characteristics of the polymer (e.g. linear versus branched (32
)), and the N/P ratio (33
). Additionally, the extent of biotinylation will also affect the electrostatic interactions between PEI and nucleic acids because each biotin added should remove one free primary amine. The amount of release for a biotinylated PEI should be tunable by adjusting all of these properties, and therefore it should be easy to design the system to have negligible nucleic acid release from the polymer unless the whole polyplex is released from the substrate with light, as with B-PC-PEI. Further validating this point, the use of biotinylated PLL did not show enhanced transfection unless the majority of the polyplex was formed with a non-biotinylated PLL (15
For B-PC-PEI, photolysis of the PEI domain from the remaining molecule occurred within 5 min when in free solution () and 10 min when attached to streptavidin beads (). It is possible that increased light exposure was necessary to release the Cy5-labeled PEI domain from the streptavidin beads because the beads or the Cy5 attenuated some of the light. Also, PEI has been thought to form non-specific interactions with streptavidin (15
) so it is possible that for this experiment, those forces needed to be overcome as well. Comparing the fluorescence loss from the Cy5-labeled B-PC-PEI () and B-PEI () beads upon light exposure shows clearly that the loss is due to actual cleavage of the PEI domain from the bead and not photobleaching.
In addition to the two causes of fluorescence loss described previously, for the siRNA there could also be loss due to release of weakly associated siRNA from the bead. Similar to the results with the Cy5-labeled compounds, there was negligible fluorescence loss due to photobleaching or diffusion after 5 min of 365 nm light exposure (B-PEI polyplexes, ). For polyplexes made with B-PC-PEI there was a significant loss of bead associated fluorescence after only 5 min of light exposure (). As with the PEI domain, increasing the duration of light exposure further increased polyplex loss, however the majority of siRNA was released in the first 5 min. This differs from the PEI results possibly because PEI is less able to non-specifically interact with the streptavidin when it is electrostatically condensing the siRNA.
The loss of siRNA fluorescence from the bead was further validated by a gain in fluorescence in the supernatant (). At 0 min of 365 nm light exposure both B-PC-PEI and B-PEI polyplexes have a similar percent fluorescence in the supernatant (presumably due to diffusion), but by 5 min of light exposure the B-PC-PEI/siRNA polyplexes had released 71.03 ± 14.24% of the polyplexes into the supernatant while the B-PEI polyplexes remained unchanged from the 0 min time point. Additionally, increasing the duration of light to 10 min did not significantly enhance the release of the B-PC-PEI polyplexes. It is important to note that release of siRNA at 0 min for both B-PC-PEI and B-PEI polyplexes is most likely due to the low N/P ratio. Increasing the N/P ratio would cause greater retention of the siRNA within the polyplex; however N/P = 0.4 gave optimal fluorescence readings. Additionally, since it is the PEI and not the siRNA that is directly coupled to the beads and the light-activated mechanism, any released siRNA should be released as polyplexes with the PEI.
Finally, the integrity of the siRNA was tested with agarose gel electrophoresis, and under all conditions tested light exposure did not change the mobility or noticeably degrade the siRNA. This result is expected as experiments conducted by Quick and Anseth showed negligible change in pDNA integrity after 365 nm light exposure (34
). Additionally, Quick and Anseth showed retained pDNA activity after light exposure at 365 nm (34
While polymers have been previously engineered for the controlled release of nucleic acids (35
), the release has largely relied on cellular process. In this report, we have demonstrated the synthesis and functionality of a compound, B-PC-PEI, that is able to selectively release nucleic acids from a solid support after light application, and which has many potential applications ranging from biological studies to therapeutic applications.