Design and Validation of Nanofibers Made of Self-Assembled Peptides to Become Multifunctional Stimuli-Sensitive Nanovectors of Anticancer Drug Doxorubicin

Abstract

Self-assembled peptides possess remarkable potential as targeted drug delivery systems and key applications dwell anti-cancer therapy. Peptides can self-assemble into nanostructures of diverse sizes and shapes in response to changing environmental conditions (pH, temperature, ionic strength). Herein, we investigated the development of self-assembled peptide-based nanofibers (NFs) with the inclusion of a cell-penetrating peptide (namely gH625) and a matrix metalloproteinase-9 (MMP-9) responsive sequence, which proved to enhance respectively the penetration and tumor-triggered cleavage to release Doxorubicin in Triple Negative Breast Cancer cells where MMP-9 levels are elevated. The NFs formulation has been optimized via critical micelle concentration measurements, fluorescence, and circular dichroism. The final nanovectors were characterized for morphology (TEM), size (hydrodynamic diameter), and surface charge (zeta potential). The Doxo loading and release kinetics were studied in situ, by optical microspectroscopy (fluorescence and surface-enhanced Raman scattering–SERS). Confocal spectral imaging of the Doxo fluorescence was used to study the TNBC models in vitro, in cells with various MMP-9 levels, the drug delivery to cells as well as the resulting cytotoxicity profiles. The results confirm that these NFs are a promising platform to develop novel nanovectors of Doxo, namely in the framework of TNBC treatment.

Unveiling the mechanism of action of acylated temporin L analogues against multidrug-resistant Candida albicans

“…We calculated the critical aggregation concentration (CAC) of peptide Dec-1B in aqueous solution using Nile Red (NR) as fluorescent probe ( Figure 3 , panel A). NR is not highly soluble in water, and it has a large preference to partition into hydrophobic environments producing a blue shift and hyperchromic effect 36 . Despite the presence of the longer lipid tail (C-10), the peptide Dec-1B has the same CAC of 39.9 µM of the peptide Pent-1B , thus showing similarly a low ability to aggregate in aqueous solution at the concentration used for the antimicrobial assays.…”

“…The critical aggregation concentration (CAC) of Dec-1B was calculated using Nile Red (NR) as the fluorescent probe. This probe has poor solubility in water, while in presence of hydrophobic environments such as aggregated produces a hyper-hypsochromic effect consisting of a blue-shift and an intensity increase 36 . The peptide stock concentration of 400 µM was prepared dissolving Dec-1B in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), and then different peptide aliquots (0.8, 1, 5, 10, 15, 20, 30, 50, 100, 150, 200, 250, 300 µM) were prepared.…”

“…The peptide stock concentration of 400 µM was prepared dissolving Dec-1B in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), and then different peptide aliquots (0.8, 1, 5, 10, 15, 20, 30, 50, 100, 150, 200, 250, 300 µM) were prepared. The organic solvent was removed under nitrogen stream, 0.5 ml of water was added to each peptide aliquots, and then were sonicated for 15 min and freeze-dried 36 . Then, each peptide solution was hydrated with NR solution of 500 nM for 1 h. The NR spectra were recorded at a fluorescence emission between 570 and 700 nm (slit width, 5 nm), and an excitation wavelength of 550 nm (slit width, 10 nm).…”

Synthetic Amphipathic β-Sheet Temporin-Derived Peptide with Dual Antibacterial and Anti-Inflammatory Activities

“…NR is not very soluble in water and has a large preference to move into hydrophobic environment as aggregates, producing a blue shift and hyperchromic effect. The peptide stock (400 μM) was prepared in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as solvent and different aliquots were taken to prepare each peptide solution at different concentrations (1, 5, 10, 15, 20, 30, 50, 100, and 200 μM) [ 60 ]. Then, HFIP was removed under nitrogen stream, 1 mL of water was added, and the solution was sonicated for 15 min and freeze-dried.…”

“…The measurements were performed in triplicate. The data were analyzed by plotting the maximum emission fluorescence corresponding wavelength (y) as a function of peptide concentration (x) and fitting with the sigmoidal Boltzmann equation as described previously [ 27 , 60 ].…”