In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.
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 6h 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.
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy; Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Via De Sanctis 10, Caselle di Selvazzano Dentro, 35030 Padua, Italy. Electronic address: silvia.barbon@yahoo.it.
Silvia
S
Barbon
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy; Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Via De Sanctis 10, Caselle di Selvazzano Dentro, 35030 Padua, Italy. Electronic address: elena.stocco@gmail.com.
Elena
E
Stocco
Department of Biomedical Sciences, University of Padova, Via Colombo 3, 35121 Padua, Italy. Electronic address: alessandro.negro@unipd.it.
Alessandro
A
Negro
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy. Electronic address: daniele.dalzoppo@unipd.it.
Daniele
D
Dalzoppo
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy. Electronic address: borgio.luca@gmail.com.
Luca
L
Borgio
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy. Electronic address: senthilstem@gmail.com.
Senthilkumar
S
Rajendran
Department of Women's and Children's Health, Pediatric Surgery, University of Padua, Via Giustiniani 3, 35121 Padua, Italy. Electronic address: francesca.grandi7825@gmail.com.
Francesca
F
Grandi
Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua, Italy. Electronic address: andrea.porzionato@unipd.it.
Andrea
A
Porzionato
Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua, Italy. Electronic address: veronica.macchi@unipd.it.
Veronica
V
Macchi
Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Via Gabelli 65, 35121 Padua, Italy. Electronic address: raffaele.decaro@unipd.it.
Raffaele
R
De Caro
Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Via De Sanctis 10, Caselle di Selvazzano Dentro, 35030 Padua, Italy. Electronic address: pierpaolo.parnigotto@unipd.it.
Pier Paolo
PP
Parnigotto
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padua, Italy. Electronic address: claudio.grandi@unipd.it.
Claudio
C
Grandi
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020934:Ciliary Neurotrophic Factor / Q000737:chemistry / Q000627:therapeutic use*; D015696:Gene Products, tat / Q000737:chemistry*; D006801:Humans; D009416:Nerve Regeneration; D010525:Peripheral Nerves / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction
D020934:Ciliary Neurotrophic Factor; D015696:Gene Products, tat
Cell trafficking; Ciliary Neurotrophic Factor; Peripheral nerve regeneration; Polyvinyl alcohol; TAT sequence