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.