CHITOSAN MODIFIED POLY(LACTIC-CO-GLYCOLIC) ACID NANOPARTICLES INTERACTION WITH
NORMAL, PRECANCEROUS KERATINOCYTES AND DENTAL PULP CELLS
18
Images were captured with Axi-oVision Rel 4.8 soft-
ware controlled by AxioCam MRm camera (Carl-
Zeiss, Germany). All images were representative of
at least two independent experiments.
The quantification of NPs uptake was obtained
with the help of the Icy software, using two plugins
(HK-MEANS) : one developed by Dufour A. 26 and
another one created by De Chaumont F. 27
2.4. Statistical analysis
NPs uptake and cytotoxicity data were compared
using Student’s t-test. A p value < 0.05 was con-
sidered statistically significant.
3. Results
3.1. PLGAChiNPs uptake by NOK, POE9 and
DPC cells
The PLGAChi NPs uptake by NOKs and POE9i cells
was determined by fluorescence microscopy (Fig.
1, Fig. 2), as well as by confocal imaging (Fig. 4,
Fig. 5). The quantification of NPs uptake revealed
a significant amount of NPs inside the cells, both
in normal human oral keratinocytes NOKs, as
well as in premalignant oral keratinocytes POE9i
(Fig. 6). Interestingly, the data obtained revealed
a penetration of PLGAChi NPs in almost all the
keratinocytes exposed to the NPs at the tested
concentrations and time points (Fig. 6). The uptake
of NPs inside NOKs varied from 91,83 % +/- 7, 37 to
100 % for NOKs, meaning that a significant amount
of keratinocytes incorporated the PLGAChi NPs.
In NOKs the percentage of cells which showed
incorporation of PLGAChi NPs was 91,83% +/- 7,37
after 12h exposure at a concentration of 20 μg/mL
PLGAChi NPs. 92,39+/- 1,34 of the exposed NOKs
showed NPs uptake after 24h exposure to 20 μg/
mL PLGAChi NPs. PLGAChi NPs entered 98,55%
+/-1,95 of the tested NOKs after incubation for
24h with 200 μg/mL PLGAChi NPs.
The total uptake inside NOKs was observed after
one day of incubation with 200 μg/mL PLGAChi
NPs. In POE9i cell line, all tested samples showed
a 100% uptake of PLGAChi NPs at all the tested
concentrations (20 μg/mL PLGAChi NPs and 200
μg/mL PLGAChi NPs) and exposure times (12h
and 24h) (Fig. 6) .
The fluorescence microscopic images showing
DPCs presented no relevant differences between
control images and images of DPCs exposed
to PLGAChi NPs (Fig. 3). No NPs were observed
inside the cells exposed to PLGAChi NPs, even at
the highest concentration 200 μg/mL and at the
longest exposure time, 24h.
A. NOKs control. B. NOKs 20 µg/mL PLGAChi NPs12h.
C. NOKs 20 µg/mL PLGAChi NPs 24h. D. NOKs 200 µg/mL PLGAChiNPs12h.
Stoma Edu J. 2017;4(1): 16-26. http://www.stomaeduj.com