4. Discussion
Thanks to the complementarity of charges between the negative charge of the glucose oxidase protein and the positively charged internal side of the coat protein of BMV, it was possible to encapsulate the GOx inside the BMV capsid. An average of 0.85 GOx molecules per capsid was obtained. Considering the hydrodynamic diameter of the dimeric enzyme (≈12.2 nm) and the internal diameter of the BMV capsid of 17.8 nm,[45] it seems not possible the encapsulation of more than one GOx dimeric molecule per capsid. GOx was previously encapsidated in cowpea chlorotic mottle virus (CCMV) for cascade reaction study with the so-called DNAzyme, a peroxidase mimic formed in situ by a specific sequence of ssDNA in the
presence of hemin, and gluconokinase.[46] However, no catalytic information of nanoreactors containing only GOx was reported.
As expected, the nanoreactors showed a higher KM than the free enzyme, especially in the HSA-functionalized nanoreactors. This could be due to the substrate mass transfer limitations when the enzyme is inside a protein cage. This mass transfer limitation seems to be also the origin of the decrease of transformation rate (kcat) of nanoreactors VLP-GOx and VLP-GOx@HSA to 14% and 5.5% of the value of free enzyme, respectively. Nevertheless, both preparations showed high GOx activity.
Unexpectedly, we found a slight cytotoxic effect of unloaded VLPs, especially on 4T1 cell line (Fig. 4). These results should be taken cautiously due to the high protein concentration. It is known thatin vivo virus and virus-like nanoparticles are able to enhance specific anti-cancer immune reactions when delivered directly to the tumor.[47-49] VLPs from Cowpea mosaic virus (CPMV) have shown potential as safe anti-cancer immunotherapy (Beatty and Lewis, 2019).[50] Systemically administered CPMV can stimulate an additive therapeutic immune response following treatment of the tumor with a conventional agent such as radiation.[51] However, scarce information is available on in vitro cytotoxicity of empty and unfunctionalized VPLs. Unfunctionalized P22 VLPs showed a cell survival rate of 90% of at concentrations as high as 200 μg/mL, indicating that VLP had almost no toxicity to bone marrow-derived dendritic cells.[52] On the other hand, unloaded Human hepatitis B virus-derived VLPs also showed no cytotoxicity on 293 kidney cell line.[53]
The LD50 in both cell lines is similar for VLP-GOx and VLP-GOx@HSA when evaluated in GOx content or GOx activity (Fig. 4 and 5). Significant damage in cancer cells was found in the triple-negative breast cancer cell line MDA-MB-231/eGFP (Fig. 6 and videos SI). MDA-MB-231/eGFP stable cells express a green fluorescent protein (GFP) as cytoplasmatic proteins. This mammalian cell is transfected with a cDNA chimera composed of a fusion of the genes encoding the fluorescent protein and β-actin. After 1-hour incubation in the presence of GOx enzymatic nanoreactors, a drastic change in the cell morphology is noted. A plasmatic membrane protrusion after 2 hours of incubation was clearly observed in phase contrast images (videos SI). This seems to be due to the formation of gas bubbles inside the cells produced by the metabolic oxygen production by the antioxidant system (e.g. catalase) induced by te excess of hydrogen peroxide produced by the GOx activity. As shown in the videos (SI), when the cell “explodes” the fluorescence immediately disappears due to the cell membrane destruction and the subsequent dilution of the fluorophore in the medium. Glucose oxidase has been reported as a cytotoxic agent for tumor cells and its capacity to reduce the tumor tissue in vivoexperiments.[54] The cytotoxic effect of GOx is originated from the production of hydrogen peroxide, causing oxidative damage in the cells, hypoxia as oxygen is consumed, and glucose consumption that starves the cells.[55-57] The advantages of VLPs and nanocarriers of enzymatic activity are the protection against the proteases,[38] the reduction of immunogenic response, especially when the nanoreactors are covered with compatible proteins or non-immunogenic polymers such as polyethylene glycol.[58]
It has been reported that HSA functionalization increases tumor cell affinity.[43] The HSA provides an interaction between the nanocage surface and secreted protein acidic and rich in cysteine (SPARC). HSA-SPARC interaction allowed breast tumor cell internalization.[44] The role of SPARC in cancer development is still controversial. Higher levels of SPARC expression have been reported in glioblastomas,[59]pancreatic cancer,[60] gastrointestinal cancer,[61] cervical cancer,[62] bone cancer,[63]and breast cancer.[64-66] In contrast, lower levels of SPARC expression have been found in pancreatic cancers,[67] ovarian,[68]and colorectal.[69,70] HSA-based drugs can bind to SPARC.[71,72] With this in mind, VLP-GOx nanoreactors were functionalized with HSA. The HSA covered nanoparticles (Fig. 3B) significantly increased the protein content of nanoparticles. However, no advantage in cytotoxicity in cell cultures was found when the cytotoxicity results were based on GOx content or GOx activity (Fig. 5 and 6). The effect of HSA functionalization in vivo experiments is currently being explored.