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EU ITN-PEOPLE-2013 # 608407

"Nanocarriers for the delivery of antimicrobial agents to fight resistance mechanisms"  

Activities and Main Results 2014

  Cyclodextrin derivatives for applications in biotechnology and nanobiotechnology: focus on photoactive molecules and oligomers

Recent research has showed the versatility of cyclodextrins in smart applications: grafting of δ-aminolevulinic acid moieties on the narrow periphery of a β-cyclodextrin through hydrolysable bonds was implemented. The aim was to generate a water-soluble, molecular/drug carrier with the capacity to undergo intracellular transformation into protoporphyrin IX (PpIX), an endogenous powerful photosensitizer for photodynamic therapy (PDT). It was demonstrated that the prepared water-soluble colorless cyclodextrin derivative upon incubation with cells resulted in intracellular intense red fluorescence registered by confocal microscopy, evidently due to the engagement of the intracellular machinery towards formation of PpIX. Moreover, the compound effectively transported intracellularly a fluorescein-labeled model guest molecule thereby demonstrating the bimodal action of the derivative (Beilstein J. Org. Chem. (2014) 10, 2414–2420.)

A recently prepared cyclodextrin-porphyrin (CD-mTHPP) conjugate was studied using fluorescence steady-state and lifetime measurements as well as confocal and fluorescence lifetime imaging microscopy on human squamous carcinoma cells (A431). A cytoplasmic uptake of CD-mTHPP predominantly in monomeric form was shown. When delivered to human skin ex vivo, CD-mTHPP exhibited improved skin distribution compared to mTHPP alone using aqueous vehicles and the skin penetration was deeper, as assessed using two-photon fluorescence microscopy (Photochem. Photobiol. Sci. (2014) 13, 1185-1191).

Aiming to develop cyclodextrin oligomers as potential multi-drug carriers using aqueous solutions and one step procedures, the biocompatible Staudinger ligation strategy was applied for the first time in cyclodextrin substrates. The dynamic conformational equilibria of the products and their ability to capture guest molecules was studied by 1D and 2D and DOSY NMR spectroscopy, as well as quantum chemical computations. The products adopted open and closed conformations and were able to host model guest molecules in aqueous solution (Beilstein J. Org. Chem. (2014) 10, 774–783).

Structure of proteins and nucleic acids by X-ray crystallography

Macromolecular structure elucidation is actively pursued in the laboratory. Biological macromolecules are the main drug targets and determination of their structures at high resolution is a prerequisite for rational (structure-guided) drug design and for the understanding of the biology and disease mechanisms of organisms. The structures of two variants of the C-terminal and one of the reduced form of the N-terminal soluble domain of bacterial electron transfer transmembrane protein DsbD have been solved and analysed (J. Biol. Chem. (2014) 289, 8681-8696 and previous publications). Structures of the antigen-processing aminopeptidases ERAP2 and IRAP in complex with phosphinic pseudopeptides and other rationally designed inhibitors which are drug candidates are being solved (first ERAP2-peptide structure published in PNAS in 2013, several more structures of complexes are in preparation).

Crystallisation Methodology for Biological Macromolecules We are developing novel methodology for macromolecular crystallization, using heterogeneous nucleation-inducing agents (Nature Protoc. (2014) 9, 1621-1633) as well as theoretical and biophysical approaches, in collaboration with Imperial College London (Prof. N. Chayen) and with the G. Nounesis lab at INRASTES.