Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems

Two series of new hybrid gamma/gamma-peptides, gamma-CC and gamma-CT, formed by (1S,2R)-3-amino-2,2,dimethylcyclobutane-1-carboxylic acid joined in alternation to a N-alpha-functionalized cis- or trans-gamma-amino-l-proline derivative, respectively, have been synthesized and evaluated as cell penetrating peptides (CPP) and as selective vectors for anti-Leishmania drug delivery systems (DDS). They lacked cytotoxicity on the tumoral human cell line HeLa with a moderate cell-uptake on these cells. In contrast, both gamma-CC and gamma-CT tetradecamers were microbicidal on the protozoan parasite Leishmania beyond 25 mu M, with significant intracellular accumulation. They were conjugated to fluorescent doxorubicin (Dox) as a standard drug showing toxicity beyond 1 mu M, while free Dox was not toxic. Intracellular accumulation was 2.5 higher than with Dox-TAT conjugate (TAT = transactivator of transcription, taken as a standard CPP). The conformational structure of the conjugates was approached both by circular dichroism spectroscopy and molecular dynamics simulations. Altogether, computational calculations predict that the drug-gamma-peptide conjugates adopt conformations that bury the Dox moiety into a cavity of the folded peptide, while the positively charged guanidinium groups face the solvent. The favorable charge/hydrophobicity balance in these CPP improves the solubility of Dox in aqueous media, as well as translocation across cell membranes, making them promising candidates for DDS.