CHITOSAN MODIFIED POLY(LACTIC-CO-GLYCOLIC) ACID NANOPARTICLES INTERACTION WITH
NORMAL, PRECANCEROUS KERATINOCYTES AND DENTAL PULP CELLS
22
that hinders their employment for biomedical
applications. 5-6
In order to improve PLGA NP proprieties, several
research groups have tried to cover the polymers
with a chitosan coating. Chitosan is obtained from
chitin which is a positively charged polysaccahride
found in crustaceans. 28 Chitosan contains many
amino and hydroxyl groups, thus it can bind
effectively to negatively charged substances
(such as the cells membranes) via electrostatic
interactions or hydrogen bonding, thus improving
the intracellular uptake. 9 Chitosan by itself is
known to strongly adhere to negatively charged
surfaces due to its high charge density at pH<
6.5. 29 The advantages of modifying the surface of
PLGA NPs with a mucoadhesive polymer, such as
chitosan, may potentially include the inversion
of zeta potential, the ability to promote cellular
adhesion and retention of the delivery system at
the target site. 30
In order to fabricate polymeric NPs for future dental
applications, we have tested the positive charged
chitosan coated PLGA NPs (PLGAChi NPs) on oral
cavity cells. NPs interaction with the oral epithelial
cells was assessed by cytotoxicity measurements.
After 24h of incubation with PLGAChi NPs (20 μg/
mL NPs and 200 μg/mL NPs) no significant statistic
differences were observed in the viability of
samples and controls (Fig. 8). PLGA Chi NPs were
biocompatible to all the tested cell lines: NOKs and
POE9i cells. We found no association between the
significant increase in the cell uptake of chitosan
containing NPs and cell toxicity, at the tested
concentrations. Due to PLGA and chitosan’s well
known biodegradability and biocompatibility, it
was expected that NPs made of chitosan and PLGA
would be well tolerated by the cells. Our results
are in agreement with other comparable studies.
Following a 2015 experiment, S. Alqahtani stated
that chitosan covered PLGANPs did not affect the
viability of Caco-2 cells. 31 Caco-2 cells displayed
a viability above 95%, even after incubation for
one day with a higher concentration of NPs than
it was used in our study: 500 μg/mL PLGAChi
NPs. 31 Moreover, chitosan covered NPs did not
contribute additional toxicity to colorectal cancer
cells after 3 days of exposure at a 75 μM solution
of NPs. 32 The viability of the cells incubated with
chitosan NPs remained at about 90 % relative to
the untreated cells on day 1 and at about 89 % on
day 3. 32 Another recent study also found that the
surface modification of PLGA NPs with chitosan did
not show any significant diffference in cytotoxicity
of PLGA NPs. 6 The results indicated that A549
cell lung carcinoma cells exhibited around 80
% cell survival as compared to positive controls
(10 % cytotoxic) for the following concentrations
of PLGAChi NPs: 0.25, 0.5, 0.75, 1,1.25, 1.5, 1.75
and 2 mg/mL. 6 Other investigators have reported
PLGAChi NPs to be safe even at much higher
concentrations of 20 mg/mL. 33-35 In addition, other
research groups revealed interesting cytotoxicity
results of chitosan NPs effect on human skin
keratinocytes HaCaT cells. Tretinoin containing
chitosan solid lipid NPs were not cytotoxic to
HaCaT cells even at the highest concentration 500
μg/mL used, which led to around 5 % less viability
compared with the control. 36 Also lecithin/chitosan
NPs can be applied to skin cells at concentrations
up to 200
µg/mL without inducing plasma
membrane damage or cell viability decrease. 37
Similarly, in a recent study, HaCaT cells exposed
to melatonin containing lecithin/chitosan NPs in a
concentration of chitosan of 1.25-20 μg/mL for 2
hours showed no relevant cytotoxicity. 38 However,
a significant reduction in the cell viability of HaCaT
cells was observed in the case of cells treated with
NPs at a chitosan concentration of 20 μg/mL. 38
Chitosan-alginate NPs did not have a toxic effect
on human monocytes but there was mild toxicity
to skin keratinocytes at higher concentration of
NPs. 29 Moreover, chitosan and PLGA NPs loaded
with chlorexidine dihydrochloride in vitro toxicity
evaluation on human gingival fibroblasts was
between 20 % and 60 % in all experimental
conditions. 9 Poly-γ-glutamic acid/glycol chitosan
NPs incorporating p-phenylenediamine (PDA)
showed lower cytotoxicity against HaCaT
human skin keratinocyte cells than PDA alone. 39
Interestingly, PDA-incorporated NPs showed
reduced apoptosis and necrosis reaction in HaCaT
cells. 39
A possible explanation for the chitosan NPs high
biocompatibilty could be that chitosan is much
more cytotoxic in a free soluble form than when
it is incorporated into NPs, due to the fact that
in the case of NPs, a significant portion of the
positive amino groups of chitosan are engaged in
electrostatic interractions. 38, 40
To confirm PLGAChi NPs efficiency in intracellular
penetration, the cellular internalization of PLGAChi
NPs conjugated with fluorescein was investigated
by fluo