C O M M U N I C A T I O N S Figure 2. In Vitro and in ViVo function of NTA3-PTD. (A) H1299 cells were treated with NTA3-PTD + Ni plus 6xHis-rhodamine peptide or 6xHis- rhodamine peptide alone for 30 min, washed, and assayed by ﬂow cytometry. (B) H199 cells were treated with NTA3-PTD plus 6xHis- -Gal, 6xHis- -Gal alone, or positive control TAT- -Gal fusion protein for 2 h, washed, and incubated with C12FDG substrate for 45 min, followed by ﬂow cytometry. (C) CHO-ZEG cells were treated with NTA3-PTD plus 6xHis-Cre or 6xHis-Cre alone for 30 min, washed, trypsinized, and replated. After 24 h, cells were assayed for GFP expression via ﬂow cytometry. (D) ROSA-26R-LoxP-STOP-LoxP-luciferase mice were subcutaneously injected with 50 µ L of 20 µ M NTA3-PTD plus 6xHis-Cre or 6xHis-Cre alone and then assayed for luciferase expression at 4 days. found pH-sensitive release of 6xHis-rhodamine (Figure 1C) in the physiologically relevant range of pH 5-7. We tested the ability of NTA3-PTD to deliver rhodamine-labeled 6xHis-peptide to cells. NTA3-PTD-6xHis-rhodamine complexes were prepared and added to cells for 30 min, followed by three washes with PBS and heparin sulfate (0.5 mg/mL) and trypsiniza- tion.3 Flow cytometry (Figure 2A) demonstrates robust cell association of 6xHis-rhodamine via NTA3-PTD, but not with 6xHis- rhodamine either alone or in trans with TAT peptide. NTA3-PTD delivered the functional enzyme -galactosidase ( - Gal, 119 kDa) into H1299 cells. Recombinant 6xHis-tagged -Gal was prepared,18 and cells were treated for 2 h. After washing and trypsinization, -Gal activity was detected using the ﬂuorescent substrate C12FDG, which yields FITC ﬂuorescence after cleavage. Substantial -Gal activity was observed (Figure 2B) following coordination with NTA3-PTD but not with control -Gal in trans with TAT peptide. In Vitro delivery of 6xHis-Cre recombinase was facilitated by NTA3-PTD, resulting in genomic recombination of loxP sites to excise a stop sequence and initiate GFP expression (Figure 2C) in CHO reporter cells6 in a dose-dependent manner. 6xHis-Cre alone or in trans with TAT peptide induced negligible GFP expression. We did not detect any cytotoxicity with NTA-TAT delivery of peptides or proteins. Finally, we observed in ViVo delivery of Cre recombinase via NTA3-PTD in a luciferase-reporter mouse model.19 Mice (transgenic ROSA26 loxP-Stop-loxP luciferase) were injected with either 6xHis-Cre (left side) or NTA3-PTD 6xHis-Cre (right side) and assayed for luciferase expression after 4 days. NTA3-PTD coordina- tion resulted in genomic recombination and substantial luciferase activity (Figure 2D). We developed a pH-sensitive protein transduction domain, NTA3- PTD, capable of delivering functional 6xHis-tagged proteins into cells both in Vitro and in ViVo. This peptide immediately makes possible the intracellular delivery of thousands of existing 6xHis- tagged proteins.15 Future application of this technology may address both scientiﬁc and therapeutic challenges. J. AM. CHEM. SOC. 9 VOL. 132, NO. 31, 2010 10681