Figure 1. a) Surface electric charge distribution on the GOx enzyme and BMV capsomer. The GOx surface mostly exhibits negative electric charges (red color), and the inner surface of BMV capsomers shows a more positive (blue color) electric charge distribution. The difference in electric charge distribution between GOx and the inner BMV surface results in electric charge complementarity. In silicoresults by Maestro Schrodinger®. b) Schematic representation of VLP-GOx nanoreactor self-assembly encapsulation and HSA functionalization.
To optimize the enzyme encapsulation, four different enzyme-coat protein molar ratios (GOx:CP) were assayed (Table 1). Nanoreactors containing enzymatic activity (VLP-GOx) were obtained in all mixtures tested. All nanoreactor preparations were purified by size exclusion chromatography (SEC) with an HPLC (Fig. 2). The purified preparation showing the highest enzymatic activity (1:3 molar ratio) was then functionalized with human serum albumin (HSA) (Fig. 1b). HSA shows binding affinity for specific receptors on the surface of different cells in diseased organs that permits the active targeting and the specific recognition of the albumin-based formulations. In vitro and in vivoexperiments HSA conjugated preparations showed higher cellular uptake efficiency, longer half-life, higher cytotoxicity, and accumulation in tumors to a much greater extent than the free drug preparations.[43, 44] The functionalization was carried out through previously reduced cysteine groups of HSA protein, producing di-thiol cross-linking with the cysteine (Cys108) of CP-BMV (SI 2).
Table 1. Hydrodynamic diameter determined by Dynamic Light Scattering (DLS) of proteins and catalytic contants of enzymatic nanoreactors assembled at different GOx:CP molar ratios.