Protecting redesigned supercharged ferritin containers against protease by integration into acid-cleavable polyelectrolyte microgels

Abstract

HypothesisThe application of ferritin containers as a promising drug delivery vehicle is limited by their low bioavailability in blood circulation due to unfavorable environments, such as degradation by protease. The integration of ferritin containers into the polymeric network of microgels through electrostatic interactions is expected to be able to protect ferritin against degradation by protease. Furthermore, a stimuli-responsive microgel system can be designed by employing an acid-degradable crosslinker during the microgel synthesis. This should enable ferritin release in an acidic environment, which will be useful for future drug delivery applications. ExperimentsNanoparticle/fluorophores-loaded ferritin was integrated into microgels during precipitation polymerization. The integration was monitored by transmission electron microscopy (TEM)22Transmission electron microscopy (TEM). N-isopropylacrylamide (NIPAM). Drug delivery systems (DDS). Human heavy chain ferritin (HF). Transferrin receptor 1 (TfR-1). Polyethylene glycol (PEG). Doxorubicin (DOX). Ferritin(neg) (Ftn(neg)). Ferritin(pos) (Ftn(pos)). 1-vinylimidazole (VIM). 3,9-Divinyl-2,4,8,10-tetra-oxaspiro[5.5]undecane (VOU). Size exclusion chromatography (SEC). Fluorescein (Fscn). Rhodamine B (Rho B). Rhodamine 6G (Rho 6G). Polymerase chain reaction (PCR). Atto Rhodamine 6G (AR 6G). Alexa Fluor 488 (AF 488). Alexa Fluor 647 (AF 647). Dimethylsulfoxide (DMSO). Potassium persulfate (KPS). Tetramethylethylendiamine (TEMED). Sodium dodecyl sulfate (SDS). Dynamic Light Scattering (DLS). Volume phase transition temperature (VPTT). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). CeO2 nanoparticle loaded Ferritin(neg) (Ce-Ftn(neg)). Ethylene glycol dimethacrylate (EGDMA). N,N’ Methylenebisacrylamide (BIS). and fluorescence microscopy, respectively. After studying ferritin release in acidic solutions, we investigated the stability of ferritin inside microgels against degradation by chymotrypsin. FindingsAbout 80% of the applied ferritin containers were integrated into microgels and around 85% and 50% of them could be released in buffer pH 2.5 and 4.0, respectively. Total degradation of the microgels was not achieved due to the self-crosslinking of N-isopropylacrylamide (NIPAM). Finally, we prove that microgels could protect ferritin against degradation by chymotrypsin at 37 °C.