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Related Experiment Video

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pH-responsive PepFect cell-penetrating peptides.

Jakob Regberg1, Luis Vasconcelos1, Fatemeh Madani2

  • 1Department of Neurochemistry, the Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrhenius väg 16B, 106 91 Stockholm, Sweden.

International Journal of Pharmaceutics
|January 29, 2016
PubMed
Summary

New cell-penetrating peptides (CPPs) were developed with pH-responsive histidine modifications. The PepFect 3 analogue, PepFect 132, showed enhanced cellular bioactivity and membrane interactions, demonstrating improved peptide drug delivery potential.

Keywords:
Calcein leakageCell-penetrating peptideCellular uptakeOligonucleotide deliveryPepFectpH-responsive

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Delivery Systems

Background:

  • Cell-penetrating peptides (CPPs) facilitate the delivery of therapeutic molecules into cells.
  • Modifying CPPs can enhance their efficiency and introduce functionalities like pH-responsiveness.
  • Galanin-derived peptides offer a scaffold for developing novel CPPs.

Purpose of the Study:

  • To design and synthesize novel cell-penetrating PepFect peptide analogues with pH-responsive properties.
  • To investigate the impact of specific sequence modifications on peptide-oligonucleotide complex formation and stability.
  • To evaluate the cellular bioactivity, membrane interaction, and pH-responsiveness of the developed peptide analogues.

Main Methods:

  • Synthesis of PepFect peptide analogues with N-terminal modifications, including histidine substitutions.
  • Formation of non-covalent peptide-oligonucleotide complexes via co-incubation.
  • Characterization of complexes using dynamic light scattering (DLS) and circular dichroism (CD).
  • Assessment of peptide-cargo affinity using a developed assay.
  • Evaluation of cellular bioactivity in HeLa cells via a luciferase-based splice correction assay.
  • Study of membrane interactions using a calcein leakage assay in large unilamellar vesicles (LUVs).

Main Results:

  • Histidine-modified analogues showed pH-responsive behavior, with complexes size changing with pH.
  • The effect of modifications depended on the C-terminal sequence; PepFect 3 analogues showed increased activity, while PepFect 14 analogues showed decreased activity.
  • PepFect 132, a novel PepFect 3 analogue, exhibited significantly higher bioactivity and membrane activity compared to the parent peptide.
  • Leucine/histidine modifications conferred pH-responsiveness, with smaller complexes at neutral pH and larger complexes at acidic pH.

Conclusions:

  • Sequence modifications, particularly histidine incorporation, can yield pH-responsive cell-penetrating peptides.
  • The C-terminal sequence significantly influences the efficacy of N-terminal modifications.
  • PepFect 132 represents a promising candidate for enhanced drug delivery applications due to its improved bioactivity and membrane interaction.
  • The developed PepFect analogues offer potential for targeted and stimuli-responsive delivery of oligonucleotide cargo.