Pirisinu, Marco (2015) Rational design and application of a new cell-penetrating peptide targeting mitochondria. Doctoral Thesis.
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Aim: Cell-penetrating peptides (CPPs) are short, no toxic peptides with cationic or/and amphipathic properties able to get inside cellular organelle. For this reason, CPPs are used in delivery of a wide variety of cargoes, such as peptides, drugs and nucleic acids. The aim of the present study was to synthesize a small novel cell-penetrating peptide based on CPPs Szeto-Schiller peptide sequence with low toxicity and able to targeting mitochondria.
Methods: All peptides used were synthesized manually using Fmoc chemistry. In the first step of project, Hela 705 cells were seeded at 1x104cells/100µL and 24h later they were treated with different concentrations of our peptide. Cell proliferation assay was performed to evaluate cell viability and further biological effects, such as mitochondrial potential membrane and antioxidant activity, were valued. During last step of the project, uptake studies were performed using a carboxyfluorescein-conjugated peptide. Fluorescent microscopy was used to determine presence of peptide inside cells and isolated mitochondria to confirm as one of its target.
Results: Microscopy studies confirmed ability of this peptide to get inside cell and isolation of mitochondria showed its high concentration in mitochondrial compartment. Biological studies showed absence of toxicity and no effect on mitochondrial potential membrane making MIP1 an important tool applicable in delivery molecules of therapeutic strategies. Moreover, antioxidant properties confirmed protective actions of MIP1 against H2O2 insult.
Conclusion: We obtained a promising peptide MIP1. CPPs provide a promising delivery strategy for gene regulation or gene therapy. MIP1 conjugated with different cargoes could be used as important delivery machine, making easier mitochondrial treatments. Moreover, due to its own antioxidant activities, MIP1 could be a really useful tool against oxidative stress.
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