Characterization Methods for Nanoparticle-Skin Interactions: An Overview.

Research progresses have led to the development of different kinds of nanoplatforms to deliver drugs through different biological membranes. Particularly, nanocarriers represent a precious means to treat skin pathologies, due to their capability to solubilize lipophilic and hydrophilic drugs, to control their release, and to promote their permeation through the stratum corneum barrier. A crucial point in the development of nano-delivery systems relies on their characterization, as well as in the assessment of their interaction with tissues, in order to predict their fate under in vivo administration. The size of nanoparticles, their shape, and the type of matrix can influence their biodistribution inside the skin strata and their cellular uptake. In this respect, an overview of some characterization methods employed to investigate nanoparticles intended for topical administration is presented here, namely dynamic light scattering, zeta potential, scanning and transmission electron microscopy, X-ray diffraction, atomic force microscopy, Fourier transform infrared and Raman spectroscopy. In addition, the main fluorescence methods employed to detect the in vitro nanoparticles interaction with skin cell lines, such as fluorescence-activated cell sorting or confocal imaging, are described, considering different examples of applications. Finally, recent studies on the techniques employed to determine the nanoparticle presence in the skin by ex vivo and in vivo models are reported.

CH vs. HC-Promiscuous Metal Sponges in Antimicrobial Peptides and Metallophores.

Histidine and cysteine residues, with their imidazole and thiol moieties that deprotonate at approximately physiological pH values, are primary binding sites for Zn(II), Ni(II) and Fe(II) ions and are thus ubiquitous both in peptidic metallophores and in antimicrobial peptides that may use nutritional immunity as a way to limit pathogenicity during infection. We focus on metal complex solution equilibria of model sequences encompassing Cys-His and His-Cys motifs, showing that the position of histidine and cysteine residues in the sequence has a crucial impact on its coordination properties. CH and HC motifs occur as many as 411 times in the antimicrobial peptide database, while similar CC and HH regions are found 348 and 94 times, respectively. Complex stabilities increase in the series Fe(II) < Ni(II) < Zn(II), with Zn(II) complexes dominating at physiological pH, and Ni(II) ones-above pH 9. The stabilities of Zn(II) complexes with Ac-ACHA-NH2 and Ac-AHCA-NH2 are comparable, and a similar tendency is observed for Fe(II), while in the case of Ni(II), the order of Cys and His does matter-complexes in which the metal is anchored on the third Cys (Ac-AHCA-NH2) are thermodynamically stronger than those where Cys is in position two (Ac-ACHA-NH2) at basic pH, at which point amides start to take part in the binding. Cysteine residues are much better Zn(II)-anchoring sites than histidines; Zn(II) clearly prefers the Cys-Cys type of ligands to Cys-His and His-Cys ones. In the case of His- and Cys-containing peptides, non-binding residues may have an impact on the stability of Ni(II) complexes, most likely protecting the central Ni(II) atom from interacting with solvent molecules.

Divalent metal ion binding to Staphylococcus aureus FeoB transporter regions.

Transition metal ions such as iron, copper, zinc, manganese or, nickel are essential in many biological processes. Bacteria have developed a number of mechanisms for their acquisition and transport, in which numerous of proteins and smaller molecules are involved. One of the representatives of these proteins is FeoB, which belongs to the Feo (ferrous ion transporter) family. Although ferrous iron transport system is widespread among microorganisms, it is still poorly described in Gram-positive pathogens, such as Staphylococcus aureus. In this work, combined potentiometric and spectroscopic studies (UV-Vis, CD and EPR) were carried out to determine Cu(II), Fe(II) and Zn(II) binding modes to FeoB fragments (Ac-IDYHKLMK-NH2, Ac-ETSHDKY-NH2, and Ac-SFLHMVGS-NH2). For the first time iron(II) complexes with peptides were characterized by potentiometry. All studied ligands are able to form a variety of thermodynamically stable complexes with transition metal ions. It was concluded that among the studied systems, the most effective metal ion binding is observed for the Ac-ETSHDKY-NH2 peptide. Moreover, comparing preferences of all ligands towards different metal ions, copper(II) complexes are the most stable ones at physiological pH.

Effect of Copper(II) Ion Binding by Porin P1 Precursor Fragments from Fusobacterium nucleatum on DNA Degradation.

Fusobacterium nucleatum is one of the most notorious species involved in colorectal cancer. It was reported that numerous outer membrane proteins (OMP) are actively involved in carcinogenesis. In this paper, the structure and stability of certain complexes, as well as DNA cleavage and ROS generation by fragments of OMP, were investigated using experimental and theoretical methods. Mass spectrometry, potentiometry, UV-Vis, CD, EPR, gel electrophoresis and calculations at the density functional theory (DFT) level were applied. Two consecutive model peptides, Ac-AKGHEHQLE-NH2 and Ac-FGEHEHGRD-NH2, were studied. Both of these were rendered to form a variety of thermodynamically stable complexes with copper(II) ions. All of the complexes were stabilized, mainly due to interactions of metal with nitrogen and oxygen donor atoms, as well as rich hydrogen bond networks. It was also concluded that these complexes in the presence of hydrogen peroxide or ascorbic acid can effectively produce hydroxyl radicals and have an ability to cleave the DNA strands. Surprisingly, the second studied ligand at the micromolar concentration range causes overall DNA degradation.

Coordination pattern and reactivity of two model peptides from porin protein P1.

It has been reported that numerous of Fusobacterium nucleatum outer membrane proteins take part in cancerogenesis. Therefore, it is very interesting to study their interactions with metal ions and the ability to produce reactive oxygen species, which may be involved in cancer progression. Since investigations of metal binding to proteins are often based on fragments that contain the metal-binding domains, designing model peptides should be very mindful. As was shown in this paper, very similar protein fragments may behave differentially. Herein, combined potentiometric, spectroscopic, and computational studies were performed to determine metal ion binding by ligands constituting fragments of porin protein P1. Two studied tetrapeptides (Ac-KEHK-NH2 and Ac-EHKA-NH2) that have common EHK motif have different coordination properties and reactivity. Therefore, we should be cautious when transferring the behavior of small peptide fragments to whole protein.

Phleomycin complex - Coordination mode and in vitro cleavage of DNA.

Phleomycin is one of the anticancer glycopeptide antibiotics which cause DNA cleavage. It is commonly used as a copper(II) complex. Therefore, it is important to study the metal ion binding process and to define the coordination mode. In this paper, we describe the acid-base properties of phleomycin and the coordination sphere of the Cu(II) cation. In the metal binding process up to five nitrogen donor atoms can be involved. Four of them in the same plane deriving from: the pyrimidine ring, secondary amine of ß-aminoalanine, imidazole and amide of the nearest peptide bond (from ß-hydroxyhistidine) and in the apical position from the α-amino functional group of ß-aminoalanine, resulting complex has a square-pyramidal geometry. Phleomycin complexes are able to induce single- and double-stranded DNA damage when they are accompanied by one-electron reductants, such as dithiothreitol, glutathione, 2-mercaptoethanol or ascorbic acid. In such conditions they produce reactive oxygen species which are responsible for DNA cleavage. The metal ion binding site is relatively close to the nucleic acid interacting moiety. This supports the hypothesis that copper ion is important in the anticancer activity which involves DNA degradation.