Intracellular Delivery Of Cytotoxic Diselenides And Fluorescent Imaging Agents Using An Engineered Nonviral Protein Capsid

Protein capsids are attractive as drug delivery vehicles due to their uniform and nanoscale sizes,straightforward genetic or chemical modification,high biocompatibility,and biodegradability.The lumazine synthase from Aquifex aeolicus（Aa LS）forms a dodecahedral protein capsid consisting of 60 identical subunits with an outer diameter of 16 nm and an inner diameter of 9 nm.The Aa LS capsid has 12 pores at its five-fold symmetry axes that are 8.9（?）wide at the narrowest point,which allow small molecules（<1000 Da）to diffuse freely across the capsid shell.As a consequence of making two point mutations in Aa LS（C37A/E122C）,the variant Aa LS-IC possesses a single cysteine per subunit that projects into the capsid lumen,which enables the efficient covalent capture of thiol reactive guests.Selenocysteine（Sec）,also known as the 21st amino acid,is an analogue of cysteine in which selenium takes the place of sulfur.Sec was found in various selenoproteins and selenoenzymes such as glutathione peroxidases and thioredoxin reductases that have essential biological functions in oxidoreductions,redox signaling,and antioxidant defense.Therefore,selenoproteins are essential for life;however,diselenide dimer of free Sec（Sec₂）,has been shown to be broadly cytotoxic.Its cytotoxic properties make Sec a promising anti-cancer agent,but the wide spectrum of its toxic activity limits its potential usefulness.In previous study,the engineered protein capsid Aa LS-IC reacted with Sec₂ to form a selenenylsulfide conjugate,Aa LS-IC-Sec,with a yield of 81%.Thus,one Aa LS-IC capsid can carry about 48 Sec molecules in its interior.Also,Sec can be released from the capsid by reducing agents such as glutathione and DTT,which could allow for the triggered release in the cell.Aa LS-IC-Sec also exhibited cytotoxicity to various human cancer cell lines,but with a higher degree of selectivity compared to free Sec₂.In addition,confocal microscopy studies showed that a dye labeled version of the capsid,in which the engineered cysteine of Aa LS-IC was conjugated to a fluorescein derivative via a thioether bond,had different levels of cell uptake and different intracellular distributions in different cell lines.Further,the intracellular distribution of dye-labeled Aa LS-IC intriguingly correlated with the cytotoxic sensitivity to encapsulated Sec.Therefore,cell uptake and intracellular trafficking should be important for drug delivery by the Aa LS-IC capsid.However,in order to better understand the potency and cell-type specificity of Aa LS-IC-Sec,the cytotoxic mechanism of Sec and the intracellular trafficking pathway of Aa LS-IC capsid still need to be further investigated.Here,to investigate the contribution of selenium redox reactivity and overall molecular structure of Sec on its cytotoxicity,we measured the cytotoxicity of five different diselenide compounds and their Aa LS-IC encapsulated selenenylsulfide counterparts towards He La cells.All of the free diselenides tested displayed some cytotoxicity,however,most of them were less toxic than Sec₂,which revealed that the inherent reactivity of the diselenide bond might cause a general,but moderate,cytotoxic effect.The relatively high cytotoxicity of Sec₂ might reflect a specific interaction between Sec and a cellular target that leads to a more potent response than the non-specific diselenide reactivity alone.Among the compunds tested,the selenenylsulfide conjugate between Aa LS-IC and methyl selenol had the highest cytotoxicity,which suggests that the cytotoxic mechanism of Sec could involve interaction with Sec-β-lyase and/or methylase enzymes.To further elucidate the intracellular distribution of Aa LS-IC,confocal microscopy was used to visualize the fluorescently labeled capsids in cells,and immunofluorescence was used to visualize capsids bearing an immunological tag.Both approaches indicated that,after being taken up from the growth medium,Aa LS-IC was located on the outside of the nucleus in both He La and HCT116 cells.Endosome co-staining experiments further revealed that Aa LS-IC was mostly trapped in endosomes after cell internalization for both He La and HCT116 cell lines.In addition,I also investigated the relative capabilities of protein capsids with different sizes to deliver small-molecule cargoes by comparing the cytotoxicity towards He La and MCF7 cells of Aa LS-IC-Sec with Aa LS-13-IC-Sec,which is the selenenylsulfide conjugate of Sec with a variant of Aa LS-IC（Aa LS-13-IC）that has an expanded capsid structure.The cell uptake efficiency of these two capsids was measured by FACS as well.Aa LS-13-IC encapsulated Sec showed less cytotoxicity towards both He La and MCF-7 cell lines compared to Aa LS-IC-Sec,however,Aa LS-13-IC capsid delivered more fluorescent dye into He La cells.In conclusion,this study extended the protein capsid based endosomal delivery of diselenides and further demonstrated the intracellular distribution of the capsid,which provides a promising foundation for the development of this protein capsid based cell delivery system.