“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

Abstract

Purpose Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. Methods Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. Results TAT/pDNA complexes were condensed into small, 50–100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these “soft” crosslinks could be competitively disrupted to facilitate DNA release. Conclusion Small and stable TAT-Ca/pDNA complexes were obtained via “soft” calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery.

Calcium Condensed LABL-TAT Complexes Effectively Target Gene Delivery to ICAM-1 Expressing Cells

“…The IC 50 value of CaCl 2 was ~210 mM for A549 cells. 15 Final concentrations of CaCl 2 used with cells in transfection studies ranged from 1–60 mM (corresponding to the reported starting concentrations of 5–300 mM) which were far below this IC 50 value.…”

“…15 Briefly, complexes were prepared by adding 10 µL (0.1 µg/µL) of DNA to 15 µL of PEI (polymer nitrogen to DNA phosphate (N/P) ratio of 10) or to 15 µL of TAT conjugates (at desired N/P ratios) followed by intensive pipetting. Fifteen microliters of DNase free water or known concentrations (e.g.…”

“…25–26 Recent work showed that TAT/DNA complexes have comparable transfection efficiency to polyethylenimine (PEI) when condensed using calcium. 15 Calcium was reported to bind both DNA phosphate groups and/or TAT amine groups resulting in compact complexes with optimal DNA release. 15 Translation of these complexes may require charge shielding to avoid clearance or targeting to promote accumulation at diseased tissue.…”

“…15 Calcium was reported to bind both DNA phosphate groups and/or TAT amine groups resulting in compact complexes with optimal DNA release. 15 Translation of these complexes may require charge shielding to avoid clearance or targeting to promote accumulation at diseased tissue.…”

Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

“…This may be attributed to the fact that although calcium was shown to condense copolymer/DNA complexes by interacting with both amines and phosphate, it also acted simultaneously as a competitive inhibitor of amine/phosphate interactions at high concentration. 41 Srinivasan et al showed that excessive Ca 2+ concentration (beyond 25 mM) led to particle aggregation and decreased transfection efficiency; therefore, the concentration of cations needs to be adequately controlled. 39 On the other hand, PA-I significantly outperformed PA in terms of transfection efficiency, in the presence of 15 mM Ca…”