The feasibility of cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) as oral gene delivery carriers was investigated in nude mice with eight 40 mu g doses of pCMV-lacZ in 2 days at 3 h intervals. The association between DNA and PNTs, the DNase I stability of PNTs-associated DNA, and in vitro permeability of DNA were estimated. The results showed that the cyclo-(D-Trp-Tyr) PNTs self-associated at concentrations above 0.01 mg/mL. Plasmid DNA associated with PNTs with a binding constant of 3.2 X 10(8)M(-1) calculated by a fluorescence quenching assay. PNTs were able to protect DNA from DNase I, acid, and bile digestion for 50 min, 60 min, and 180 min, respectively. The in vitro duodenal apparent permeability coefficient of pCMV-lacZ calculated from a steady state flux was increased from 49.2 +/- 21.6 X 10(-10) cm/s of naked DNA to 395.6 +/- 142.2 X 10(-10) cm/s of pCMV-lacZ/PNT formulation. The permeation of pCMV-lacZ formulated with PNTs was found in an energy-dependent process. Furthermore, beta-galatosidase (beta-Gal) activity in tissues was quantitatively assessed using chlorophenol red-beta-D-galactopyranoside (CPRG) and was significantly increased by 41% in the kidneys at 48 h and by 49, 63, and 46% in the stomach, duodenum, and liver, respectively, at 72 h after the first dose of oral delivery of pCMV-lacZ/PNT formulation. The organs with beta-Gal activity were confirmed for the presence of pCMV-lacZ DNA with Southern blotting analysis and intracellular tracing the TM-rhodamine-labeled DNA and the presence of mRNA by reverse transcription-real time quantitative PCR (RT-qPCR). Another plasmid (pCMV-hRluc) encoding Renilla reniformis luciferase was used to confirm the results. An increased hRluc mRNA and luciferase in stomach, duodenum, liver, and kidney were detected by RT-qPCR, ex vivo bioluminescence imaging, luciferase activity quantification, and immunostaining, respectively.