A self-assembled nanostructured material with photosensitising properties

Abstract

Using a combination of molecular modelling and spectroscopic experiments, the naturally-occurring pharmacologically active hypericin compound is shown to form a stable 1 : 1 complex with apomyoglobin that preserves its fluorescence and singlet oxygen photosensitising properties. The binding equilibrium constant has been determined as K a = (2.4 ¡ 0.5) 6 10 5 M 21 , equivalent to a dissociation constant K d = 4.2 ¡ 0.8 mM. The kinetic details of singlet oxygen production have been characterised and indicate that the protein scaffold protects hypericin from oxygen. The complex is active against Staphylococcus aureus bacteria and shows lower dark toxicity than free hypericin. In view of its superb biocompatibility, apomyoglobin should be considered as a nanovehicle for hypericin in theranostic applications.

The complex of hypericin with β-lactoglobulin has antimicrobial activity with potential applications in dairy industry

“…When Hyp is dissolved in the presence of 2β-LG, its absorption spectrum shows sharper bands instead and, most importantly, the fluorescence emission increases substantially and becomes structured ( Figure 3B). These facts indicate that Hyp is located in a substantially less polar environment than water, as previously observed with apomyoglobin and human serum albumin (Miskovsky et al, 1998;Das et al, 1999;Comas-Barceló et al, 2013), as a result of binding to the protein. These conditions are a prerequisite for preservation of the singlet oxygen photosensitization via energy transfer from the Hyp triplet state.…”

“…Accordingly, the affinity of Hyp for 2β-LG can be evaluated by monitoring the fluorescence emission as a function of Hyp concentration ( Figure 3C). A model for the binding equilibrium Hyp + 2β-LG ⇆ 2β-LG-Hyp (Comas- Barceló et al, 2013) affords an association constant, K a = 1.40 ± 0.07 µM −1 , corresponding to a dissociation constant, K d = 0.71 ± 0.03 µM.…”

“…We have recently shown that Hyp binds to the hydrophobic pocket of apomyoglobin, preserving its fluorescence and 1 O 2 photosensitizing properties (Comas- Barceló et al, 2013). Furthermore, we have shown that the complex is active against Staphylococcus aureus bacteria.…”

“…Assuming a dimerization constant, K D = 8.6 µM −1 , this corresponds to 2β-LG ≈ 40 µM. The solid line is the result of the fit with a model for a single binding site for hypericin on 2β-LG (Comas-Barceló et al, 2013) and afford an association constant, K a = 1.40 ± 0.07 µM −1 , corresponding to a dissociation constant of K d = 0.71 ± 0.03 µM.…”

Tuning the local solvent composition at a drug carrier surface: the effect of dimethyl sulfoxide/water mixture on the photofunctional properties of hypericin–β-lactoglobulin complexes

“…19 Among other carriers, proteins are highly biocompatible systems that warrant bioavailability of the PS. We recently demonstrated that the complex between Hyp and apomyoglobin (apoMb) is an effective photosensitizing agent for Gram positive bacteria, 20 with fluorescence properties that allow us to localize the PS with subdiffraction resolution on bacterial cells, using STED (Stimulated Emission Depletion) microscopy. 21 We also demonstrated the use of b-lactoglobulin (bLG) as a carrier for Hyp in aqueous solutions such as phosphate-buffered saline (PBS), with the advantage, relative to apoMb, that it could carry more Hyp molecules per protein unit.…”