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.