Cell penetrating peptides (CPPs) efficiently enter cells while transporting proteins, drugs and other cargos. Tat (transactivating transcription factor) peptide is a non-structured,11-residue CPP with dense positive charge. Tat has been widely used to deliver covalently and noncovalently attached cargo to multiple cell lines and in vivo. It has been shown to enter cells via energy-dependent endocytosis, which is triggered by membrane interactions. Pep-1, a 21-residue amphiphilic CPP, is known for its superior drug delivery rates and low toxicity. Pep-1 is thought to directly penetrate cell membranes instead of using an endocytosis pathway, which could explain its higher internalization rates and the efficiency of its attached drugs. The interaction between these CPPs and lipid membranes is being studied using isothermal titration calorimetry (ITC). Results showed that Tat binds anionic lipid (POPG) vesicles but not zwitterionic (POPC) ones, suggesting an electrostatic mechanism. The contribution of electrostatics to the binding free energy was determined by altering the ionic strength of the buffer used in the ITC studies. The binding constant ranged from 2.2 x 106 M-1 at 15 mM NaCl to 2.2 x 105 M-1 at 250 mM NaCl, representing a decrease in affinity. 7/10 of lipids sites are electrostatically binding to Tat. Pep-1, however, was shown to bind both POPC and POPG vesicles, suggesting hydrophobic forces may play a role. With these results, the nature of binding between these CPPs and model membranes can be determined, which hopefully sheds light on their different cell uptake mechanisms.

Lipid membrane interaction thermodynamics of cell-penetrating peptides

253rd National Meeting (2017)

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