Different membrane behaviour and cellular uptake of three basic arginine-rich peptides

Area of Science:

• Biochemistry and Biophysics
• Molecular Biology
• Cell Biology

Background:

• Cell penetrating peptides (CPPs) facilitate cellular uptake of cargo.
• Understanding CPP-membrane interactions is crucial for their entry mechanisms.
• CPPs and antimicrobial peptides share common characteristics.

Purpose of the Study:

• To investigate the differential uptake mechanisms of three basic peptides: RL9, RW9, and R9.
• To elucidate the role of lipid membrane properties in peptide-cell entry.
• To explore potential antimicrobial activities of these CPPs.

Main Methods:

• Differential scanning calorimetry (DSC) to study peptide effects on lipid phase transitions.
• Isothermal titration calorimetry (ITC) to quantify peptide-lipid binding affinity.
• Circular dichroism (CD) and NMR spectroscopy to determine peptide secondary structures.
• Cellular uptake studies in Chinese Hamster Ovary (CHO) cells.
• Antimicrobial activity assays against *Escherichia coli* and *Staphylococcus aureus*.

Main Results:

• RW9 and R9 were internalized into CHO cells, while RL9 was not.
• Peptide interaction with cell membranes required negative charges on lipid headgroups.
• RW9 and R9 significantly disturbed the phase transition of anionic lipids (DMPG), unlike RL9.
• RW9 and R9 exhibited approximately tenfold higher affinity for DSPG LUVs compared to RL9.
• RW9 showed mild bacteriostatic activity against *E. coli*.

Conclusions:

• The presence of negative charges on lipid membranes is essential for the internalization of R9 and RW9.
• Differences in peptide-lipid interactions, specifically affinity and membrane perturbation, explain the differential cellular uptake.
• RL9's inability to interact effectively with anionic lipid headgroups prevents its cellular entry.
• RW9 possesses dual functionality as a CPP and a mild antimicrobial agent.