"Soft" calcium crosslinks enable highly efficient gene transfection using TAT peptide.
OBJECTIVE
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.


CONCLUSIONS
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.
Department of Pharmaceutical Chemistry, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA.
Abdulgader
A
Baoum
Sheng-Xue
SX
Xie
Amir
A
Fakhari
Cory
C
Berkland
D002118:Calcium / Q000737:chemistry*; D002460:Cell Line; D003432:Cross-Linking Reagents; D004587:Electrophoresis, Agar Gel; D004847:Epithelial Cells / Q000502:physiology; D018014:Gene Transfer Techniques; D015756:Genes, tat / Q000235:genetics; D006801:Humans; D008168:Lung / Q000166:cytology; D010316:Particle Size; D010455:Peptides; D014162:Transfection
D003432:Cross-Linking Reagents; D010455:Peptides; D002118:Calcium