No abstract

The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems.

The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking Escherichia coli (E. coli). To characterize the effects of Pep-1 and combi-2 on E. coli membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of E. coli membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue-shift and decreased in a nonconcentration-dependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with E. coli membrane mimicking vesicles having shown a significant blue-shift in fluorescence intensity were then prepared and studied in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 3-helix structure in the presence of E. coli membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on E. coli membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on E. coli at any concentration while Pep-1 stabilizes the E. coli membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in E. coli membrane. SEM images reveal that Pep-1 and combi-2 form superstructures including fibrils in the presence of E. coli membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide-lipid interactions.

While the majority of known antimicrobial peptides are cationic, a small number consist of short Asp-rich sequences that are anionic. These require metal ions to become biologically active. Here, we report the study of the zinc complexes of the peptide GADDDDD (GAD5), an antimicrobial peptide. Using a combination of dynamic light scattering (DLS), ζ-potential, infrared, Raman, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM), we find that adding zinc ions to GAD5 forces it into a compact structure. Higher amounts of zinc ions favor a larger structure, possibly a dimer. SEM images show that zinc ions reduce the size of the fibrillar structures of GAD5. TGA curves show that the addition of zinc ions increases the thermal stability of the structure of the peptide. TGA and DSC indicate that the association of GAD5 with a zwitterionic phospholipid in the presence of zinc ions is the most stable. The stability of that complex is due to the presence of a sharp endothermic peak in the 200–300 °C range, suggesting the presence of interlamellar water that is essential to the stabilization of the structure. These results indicate that the Zn–GAD5 complex prefers the bacteria-mimicking neutral (zwitterionic) membranes. In the presence of negatively charged phospholipids, the complex remains unordered and unstable. In terms of mechanism of action, the Zn–GAD5 complex promotes a possible endocytic uptake with respect to neutral (zwitterionic) membranes while promoting membrane disruption by forming pores with respect to negatively charged phospholipids.