Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Delivery

Background:

• Many therapeutics fail due to poor cell membrane penetration.
• Cell-penetrating peptides (CPPs) can deliver cargo across membranes but often lack metabolic stability.
• The human calcitonin fragment hCT(9-32) is a CPP with limited stability.

Purpose of the Study:

• To enhance the proteolytic resistance of the CPP hCT(9-32).
• To create metabolically stable CPP derivatives for improved drug delivery.
• To evaluate the cell internalization, metabolic stability, and toxicity of modified CPPs.

Main Methods:

• Synthesis of six N-terminally labelled hCT(9-32) derivatives with modified amino acid residues.
• Confocal laser scanning microscopy to assess peptide internalization in HeLa and HEK 293T cells.
• RP-HPLC and MALDI-TOF mass spectrometry to analyze metabolic stability in human blood plasma and cell culture supernatant.
• Circular dichroism (CD) spectroscopy to study peptide conformation and cell toxicity assays.

Main Results:

• All synthesized derivatives maintained efficient cell internalization in tested cell lines.
• Five of six modified peptides exhibited significantly increased half-lives in human blood plasma compared to the unmodified peptide.
• Degradation analysis revealed enhanced stability in the N-terminal region of modified peptides, with minor C-terminal cleavage.
• Modified peptides showed no significant cell toxicity and retained their membrane-permeating capabilities.

Conclusions:

• N-terminal modifications of hCT(9-32) with N-methylphenylalanine or d-phenylalanine significantly improve metabolic stability.
• These stabilized CPPs retain their ability to cross cell membranes and are non-toxic.
• The developed CPP derivatives represent promising tools for enhancing therapeutic agent delivery.