PLGAChi NPs, when all cells were penetrated by
the NPs (Fig. 6). What is mostly important is the fact
that the PLGAChi NPs entered in a higher amount
in the precancerous cells than in the normal oral
keratinocytes. Chitosan covered NPs demonstrated
a 100 % uptake at all tested concentrations and
time points in POE9i precancerous cells. Moreover,
the PLGAChi NPs were able to get internalized into
the epithelial cells in reasonable amounts and in
a time and concentration dependant manner. The
percentage of keratinocyte cells with internalized
NPs increased over incubation time, demonstrating
a growing and highly efficient process of
internalization of PLGAChi NPs by human NOKs
and POE9i keratinocytes. The results obtained in
3D studies confirm the fact that PLGAChi NPs can
enter the oral keratinocytes (Fig. 7).
Interestingly, what was observed was a higher and
more rapid uptakeof PLGAChi NPs in precancerous
keratinocytes compared to NOKs (Fig. 6). Based
on these data, we hypothesise a preference of
chitosan covered NPs for uptake byprecancerous
keratinocytes over normal keratinocytes. This
has been also hypothesised previously by 41 who
showed that epithelial cell cultures forming tight
junctions did not internalize NPs, while those lacking
tight junctions, i.e., the cancer cells, did internalize.
Although the NPs used in that study differ from
our study, this could provide a possible molecular
explanation. Interestingly, previous research
articles have also found a preference of chitosan
covered NPs for uptake by cancer stem cells. A
doxorubicin-encapsulated polymeric nanoparticle
surface-decorated with chitosan was able to target
and eliminate tumor reinitiating cancer stem-
like cells. 42 Moreover, hyaluronic acid-decorated
dual responsive nanoparticles of Pluronic F127,
PLGA, and chitosan were developed recently for
targeted co-delivery of doxorubicin and irinotecan
to eliminate cancer stem-like cells. 43 Also, chitosan-
coated hyaluronic acid, docetaxel containing NPs
were more effective against CD44+ cells than free
docetaxel. 44 We have not investigated this aspect
in our study, but this could be further investigated.
The findings of this study provide evidence for
the penetration of PLGAChi NPs not only in single
cells, but also in oral mucosal cells assembled in
3D tissus, as shown by the results on the human 3D
organotypic reconstructed human mucosa models
grown in vitro (Fig. 7). Altough the 3D organotypic
models replicate only to a certain extend the
structure of the human tissue, in this case the
human oral mucosa, they better resemble the oral
microenvironment of the oral keratinocytes than
the 2D models.
To our knowledge this is the first study that
assesed the penetration of PLGAChi NPs in the
reconstructed oral human mucosa. The NPs
crossed the superficial epithelial layers, reaching
the underlying conective tissue (Fig. 7).
Our results were in agreement with previous
studies which showed a high uptake of polymeric
NPs when fortified with chitosan, and that the
Stomatology Edu Journal
uptake of chitosan coated NPs was much higher
that that of uncoated NPs. 6, 30-31, 45-46 In a study from
2015, S.Alqahtani showed a significantly higher 3.5
fold cellular uptake of chitosan coated PLGAChi
NPs compared to PLGA NPs in Caco-2 cells. 31
In another study, positively charged chitosan
covered PLGA NPs exhibited enhanced
mucoadhesion, compared to negatively charged
PLGA NPs and enhanced intracellular uptake
in A549 cell line human lung carcinoma cells. 6
PLGAChi NPs managed to get internalized into
Caco-2 cells with reasonable amounts after just
1h. 31 Another research group stated that PLGAChi
NPs are internalized by hepatocytes 3A and
fibroblasts 3T6 in a few minutes. 30
Also, Chronopoulos reported that the uptake of
PLGAChi NPs appears faster than with PLGA NPs,
with major amounts of cytoplasmatic NPs found
after only 5 minutes. 30
Interestingly, uptake saturation is reached after
2-3h of incubation with PLGAChi NPs in human
3A hepatocytes and 3T6 fibroblasts although the
uptake of PLGA NPs still appears less extensive
than for PLAChi NPs. 30
The performance of a delivery system depends
on the polymeric composition, the size and
surface charge. 38,47 Therefore, smaller sizes of
NPs and a positive zeta potential lead to a better
internalisation inside cells due to the attractive
interaction with the negatively charged cell
membranes. 48,49
Hence, the size and zeta potential of the cureent
NPs fabricated in our study are in favour of particle
internalisation. Our data demonstrated that
PLGAChi NPs exhibited a significant internalisation
into the human oral keratinocytes .
However, the experiment presents a series of
limitations. As other studies showed the rapid
internalisation of chitosan covered NPs in minutes
or hours, 30,31 the exposure time used in our
study (12h and 24h) might have been too long.
Further investigat