The feasibility of cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) as oral gene delivery carriers was investigated in nude mice with eight 40 μg 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 × 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 × 10 −10 cm/s of naked DNA to 395.6 ± 142.2 × 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, β-galatosidase (β-Gal) activity in tissues was quantitatively assessed using chlorophenol red-β-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 β-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.

The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential

Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport.

Elevation of corticosteroids and excessive glutamate release are the two major stress responses that occur sequentially during traumatic CNS injury. We have previously reported that sequential application of corticosterone and kainic acid (CORT + KA) mimicking the nerve injury condition results in synergistic enhancement of neurite outgrowth and expression of growth-associated protein 43 (GAP-43) in cultured dorsal root ganglion (DRG). GAP-43 is known to promote neurite extension when phosphorylated by protein kinase C (PKC). In addition, PKC can phosphorylate the signal transducer and activator of transcription 3 (STAT3) at Ser727, which is phosphorylated primarily by Janus kinase (JAK) at Tyr705. In this study, we further examine the role of PKC in this stress-induced growth-promoting effect. In the cultured DRG neurons, the JAK inhibitor AG-490 and the PKC inhibitor Ro-318220 reduced the CORT + KA-enhanced neurite growth effect when applied prior to CORT and KA treatment, respectively. Both AG-490 and Ro-318220 diminished the CORT + KA-enhanced GAP-43 expression, phosphorylation, and axonal localization. Furthermore, CORT + KA treatment synergistically phosphorylated STAT3 at Ser727 but not at Tyr705. Similar phenomena were observed in an animal model of acute spinal cord injury (SCI), in which phosphorylation of GAP-43 and phospho-Ser727-STAT3 was elevated in the injured DRG 4 hr after the impact injury. Further treatment with the therapeutic glucocorticoid methylprednisolone enhanced the phosphorylation of GAP-43 in both the DRG and the spinal cord of SCI rats. These results suggest that elevated glucocorticoids and overexcitation following CNS injury contribute to nerve regeneration via induction of JAK/PKC-mediated GAP-43 and STAT3 activities.

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