1111. TAT-Streptavidin: A Novel Drug Delivery Vector for the Intracellular Uptake of Macromolecular Cargo

Abstract

Top of pageAbstract Summary |[ndash]| The TAT protein transduction domain has been used extensively for directing the intracellular delivery of proteins to which it is conjugated, complexed, or fused 1,2,3,4. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-Streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. We demonstrate that TAT-SA can be used to direct intracellular delivery of various bioactive cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired. Results |[ndash]| FACS analysis of TAT-SA-treated Jurkat cells confirmed nearly 100% uptake as compared with WT-SA-treated cells after 3 h. Trypsinization and treatment with an impermeable fluorescence quenching antibody seperately confirmed that the majority of cell-associated fluorescence was from internalized protein and not due to an extracellular membrane-bound fraction. Confocal microscopy of live WT-SA-treated Jurkat cells 3 h after treatment showed little to no fluorescence, consistent with low-level, non-specific uptake, while cells treated with TAT-SA displayed punctate patches of intracellular fluorescence, indicative of an endosomal entry pathway. The ability of a pH-responsive polymer poly(propylacrylic acid) (PPAA) to enhance cytoplasmic delivery of TAT-SA was investigated 5,6. Images of live Jurkat cells 4 h after incubation with TAT-SA:PPAA complexes were characterized by a diffuse cytoplasmic distribution, in contrast to the compartmentalization seen in the absence of an endosomal disrupting polymer. Subcellular fractionation was performed to quantify the uptake of TAT-SA and the effect of PPAA on the intracellular distribution within various subcellular compartments. The ability of TAT-SA:PPAA complexes to deliver a bioactive cargo protein to the cytoplasm was investigated with the nonphosphorylatable, nondegradable superrepressor I|[kappa]|B|[alpha]| (srI|[kappa]|B|[alpha]|) mutant which has been demonstrated as a potent inhibitor of NF-|[kappa]|B activity when expressed in cells 7. Here, purifed, biotinylated srI|[kappa]|B|[alpha]| was delivered to a 57A HeLa cell line which had been genetically engineered with a Luc reporter gene downstream of NF-|[kappa]|B promoter elements 8. TAT-SA:PPAA:srI|[kappa]|B|[alpha]| complexes inhibited TNF-|[alpha]|-induced NF-|[kappa]|B activation by nearly 50% (p<0.001) in treated cells after 4 h, demonstrating the potential of the TAT-SA:PPAA complex as an effective intracellular delivery vector. This work thus demonstrates that TAT-SA and PPAA can be used jointly as a biomolecular vector for delivering heterogeneous cargo proteins of large size in an apparent step-wise uptake and endosomal release mechanism.