Engineered human H-chain ferritin with reversed charge of the internal cavity exhibits RNA-mediated spongelike effect for loading RNA/DNA-binding molecules.

Ferritins are globular proteins with an internal cavity that enables the encapsulation of a plethora of low-mass compounds. Unfortunately, the overall negative surface charge of ferritin's internal cavity hampers efficient loading of negatively charged molecules. Therefore, we produced a genetically engineered human H-chain ferritin containing a cationic RKRK domain, reversing the natural net charge of the cavity to positive, thus allowing for efficient encapsulation of negatively charged siRNA. Due to the reversed, positive charge mediated by RKRK domains, the recombinant ferritin produced in E. coli inherently carries a load of bacterial RNA inside its cavity, turning the protein into an effective sponge possessing high affinity for DNA/RNA-binding substances that can be loaded with markedly higher efficiency compared to the wildtype protein. Using doxorubicin as payload, we show that due to its loading through the RNA sponge, doxorubicin is released in a sustained manner, with a cytotoxicity profile similar to the free drug. In summary, this is the first report demonstrating a ferritin/nucleic acid hybrid delivery vehicle with a broad spectrum of properties exploitable in various fields of biomedical applications.

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.