Nanoformulation of the Broad-Spectrum Hydrophobic Antiviral Vacuolar ATPase Inhibitor Diphyllin in Human Recombinant H-ferritin.

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.

BODIPY-labelled acetylcholinesterase reactivators can be encapsulated into ferritin nanovehicles for enhanced bioavailability in the CNS.

The BODIPY-labelled oxime reactivator was prepared and used to study its biodistribution into central nervous system. The newly synthesized oxime was found to be weak inhibitor of acetylcholinesterase and strong inhibitor of butyrylcholinesterase. Its reactivation ability for organophosphate inhibited acetylcholinesterase was found similar to a parent oxime. The BODIPY-labelled oxime was further encapsulated into recombinant human H-ferritin and evaluated in vitro and in vivo. The oxime or encapsulated oxime were found to be bioaccumulated primarily in liver and kidneys of mice, but some amount was distributed also to the brain, where it was detectable even after 24 h. The BODIPY-labelled oxime encapsulated to human H-ferritin showed better CNS bioaccumulation and tissue retention at 8 and 24 h time points compared to free oxime, although the fluorescence results might be biased due to BODIPY metabolites identified in tissue homogenates. Taken together, the study demonstrates the first utilization of recombinant ferritins for changing the unfavourable pharmacokinetics of oxime reactivators and brings promising results for follow-up studies.

Passive Diffusion vs Active pH-Dependent Encapsulation of Tyrosine Kinase Inhibitors Vandetanib and Lenvatinib into Folate-Targeted Ferritin Delivery System.

INTRODUCTION: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system. METHODS: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities. RESULTS: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs' structural and functional properties. CONCLUSION: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.