In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

Journal Article · Sat Oct 15 04:00:00 EDT 2016 · Toxicology and Applied Pharmacology

DOI:https://doi.org/10.1016/J.TAAP.2016.09.001· OSTI ID:22690819

Barbon, Silvia ^[1]; Stocco, Elena ^[1]; Negro, Alessandro ^[2]; Dalzoppo, Daniele ^[1]; Borgio, Luca ^[1]; Rajendran, Senthilkumar ^[1]; Grandi, Francesca ^[3]; Porzionato, Andrea ^[4]; Macchi, Veronica ^[4]; others, and

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1. Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua (Italy)
2. Department of Biomedical Sciences, University of Padova, Via Colombo 3, 35121 Padua (Italy)
3. Department of Women's and Children's Health, Pediatric Surgery, University of Padua, Via Giustiniani 3, 35121 Padua (Italy)
4. Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua (Italy)

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was accomplished after cell treatment. • TAT-CNTF can be efficiently loaded on oxidized PVA cylinders for local delivery. • TAT-CNTF features make it ideal for peripheral nerve regeneration therapies.

• OSTI ID: 22690819
• Journal Information: Toxicology and Applied Pharmacology, Journal Name: Toxicology and Applied Pharmacology Vol. 309; ISSN TXAPA9; ISSN 0041-008X
• Country of Publication: United States
• Language: English

Related Subjects

54 ENVIRONMENTAL SCIENCES
60 APPLIED LIFE SCIENCES
ABUNDANCE
AMINO ACID SEQUENCE
CELL PROLIFERATION
CONCENTRATION RATIO
GROWTH FACTORS
IN VITRO
INJURIES
MOLECULAR WEIGHT
NERVES
OXIDATION
PVA
REGENERATION
THERAPY
UPTAKE