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Maria Justina Roxana Virlan1a, Bogdan Calenic1b, Cimpan Mihaela Roxana2c, Daniela Elena Costea3d, Maria Greabu1e*
¹Department of Biochemistry, Faculty of Dentistry, University of Medicine and Pharmacy Carol Davila, Bucharest, Romania
²Department of Clinical Dentistry - Biomaterials, Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway
The Gade Laboratory of Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway

DDS, MSc, PhD student
DDS, PhD, Lecturer
DDS, PhD, Associate Professor
DDS, PhD, Professor
PhD, Professor, Head of Department
Received: February 28, 2017
Revised: March 22, 2017
Accepted: April 02, 2017
Published: April 03, 2017

Academic Editor: David Wray, MD (Honours), BDS, MB ChB, FDS, RCPS (Glasgow), FDS RCS (Edinburgh) F Med Sci Professor Em.,
Professor, University of Glasgow, Glasgow, UK

Cite this article:

Virlan MJR, Calenic B, Cimpan MR, Costea DE, Greabu M. Chitosan modified poly(lactic-co-glycolic) acid nanoparticles interaction with normal,
precancerous keratinocytes and dental pulp cells. Stoma Edu J. 2017;4(1)


Introduction: Nanoparticles (NPs) can carry molecules to different body tissues. Due to their
controlled delivery properties, chitosan covered poly-lacto-co-glycolic NPs (PLGAChi NPs) could be
used to deliver drugs to oral tissues for the treatment of dental diseases or in anticancer therapy. The
aim of this study was to determine the uptake and cytotoxicity of PLGAChi NPs on different types of
cells found in the oral cavity.
Methodology: Normal oral keratinocytes (NOKs), precancerous keratinocytes (POE9i) and dental
pulp cells (DPCs) were exposed for 12h and 24h to 20 g/mL and 200 g/mL PLGAChi NPs covalently
tagged with fluorescein. 3D organotypic tissues of oral mucosa were grown in vitro and exposed to
200g/mL PLGAChi NPs for 24h.
Results: Both normal and premalignant oral mucosa cells (NOK and POE9i) displayed uptake
of PLGAChiNPs in a time and concentration-dependent manner, both in 2D and 3D models. A
higher and more rapid uptake of PLGAChi NPs by precancerous cell line POE9i was observed when
compared to NOKs.Interestingly, DPCs did not display internalized PLGAChi NPs, even at the highest
concentration of 200 g/mL.
Conclusion: Chitosan-coated PLGAChi NPs proved to be able to cross the cellular membrane of oral
keratinocytes, in 2D as well as in 3D cultures. The polymeric NPs used in the present study seem not
to be suitable for applications that require NPs uptake by DPCs, as no evidence of uptake in these
cells was found in this study. The finding that PLGAChi NPs showed significant internalization by
human keratinocytes indicate that they could be used for drug delivery purposes to oral mucosa.
Keywords: chitosan, PLGAChi, nanoparticles, oral keratinocytes, dental pulp.

1. Introduction with the negatively charged cell membrane(6-7).

Polymeric nanoparticles (NPs) have been consi- PLGA NPs can be surface modified to carry a
dered as the most efficient vehicles for drug positive charge by the addition of a chitosan
delivery due to their excellent pharmacokinetic shell. PLGA-chitosan NPs combine the positive
properties such as particle size, surface charge, charge of chitosan and PLGA’s ability to efficiently
surface chemistry, hydrophobicity, degree of entrap hydrophobic and hydrophilic drugs (8-9).
rigidity and degradation speed (1-3). Specifically, Chitosan, the deacetylated derivative of chitin, is
poly-lacto-co-glycolic NPs (PLGA NPs) can mostly preferred as the coating polymer, because
transport molecules to different tissues in the body, it is a cationic, nontoxic, biocompatible and bio-
facilitating intracellular uptake of various drugs (4-5). degradable polymer and has mucoadhesive,
However, the overall negative charge of PLGA NPs antibacterial, antifungal, antitumor and stimulating
has been reported to diminish their interaction immunoenhancing properties (10-13).

*Corresponding author:
Prof. Dr. Maria Greabu, PhD, Professor, Department of Biochemistry, Faculty of Dentistry, „Carol Davila” University of Medicine and Pharmacy of Bucharest, Bucharest, Romania
8 Blvd. Eroii Sanitari, Sector 5, RO-050474 Bucharest, Romania
Tel/Fax: +40.721.274.932 / +, e-mail:

1 STOMA.EDUJ (2017) 4 (1)


Chitosan-modified NPs were develloped for the mL epithelial growth factor, 25 μg/mL bovine
transport of active molecules through nasal, ocular, pituitary extract, 20 μg/mL l-glutamine, 100 U/mL
vaginal or intestinal mucosa(7,14-16). penicillin, 100 μg/mL streptomycin and 0.25 μg/
Chitosan nanocarriers could be used in mL amphotericin B (all supplements were aquired
future dental applications(17) such as in dentin from InVitrogen, Massachusetts, USA ).
pulp regeneration procedures(18, 19), in bone DPCs and NOFs were grown in DMEM medium
regeneration techniques(20), in endodontics(21-23) (Sigma St Louis, Missouri) containing 10% fetal
or in periodontal therapy(24, 20). Moreover, chitosan bovine serum, 20 μg/mL  l-glutamine, 100 U/mL
containing NPs were able to transport antitumour penicillin, 100 μg/mL streptomycin, and 0.25 μg/
substances to different cancer cell lines(25-31), mL amphotericin B (all supplements were aquired
including oral cancer cells(29-30). Nanocarriers of from InVitrogen,   Massachusetts, USA). The pro-
chitosan incorporating anticancer substances tocol for growing normal human organotypics
demonstrated higher toxicity than the free drugs (OTs) was previously described by Costea
and significant inhibition of tumor growth in The multilayered epithelium was elaborated
animals(27). using NOKs grown on top of collagen matrices
Despite numerous scientific reports regarding populated with NOFs.
organic nanomaterials in medicine, more 2.2. Viability Test
experiments are needed in order to asses the NOKs and POE9i cells were cultured in 6 well
effects of organic NPs on the oral mucosa. It has plates (150.000 cells/well) with 3.5mL of culture
been shown that the interactions between NPs medium. Cells were allowed to set for 24h into
and cells depends on the cell type, as well as the incubator at 37°C and supplemented with
on the size and surface charge of NPs(31-42). NPs 5 % CO2. Afterwards, the media was removed
behave completely differently depending on their and the cells were washed with PBS. Then, 3.5mL
surface coverings and size, while the concentration media containing PLGAChi NPs at the tested
and the exposure time to such NPs makes them concentrations was added in each well: 5μg/mL,
cytotoxic or biocompatible. Moreover, the oral 20 μg/mL and 200 μg/mL.The viability was counted
mucosa is composed of a variety of cells with with trypan blue and an automatic cell counter
different properties which may react differently to (Sigma-Aldrich, St. Louis, MO). The counting was
the same NPs. The pathologic conditions can also done in triplicates for every cell culture well.
modify the response of human oral cells to NPs, 2.3. Exposure of cells to PLGAChi NPs
due to changes in cell physiological status. The cells were seeded in two-well glass chambers
The aim of our study is to determine the uptake (Thermo Fisher Scientific; Nunc™ Lab-Tek™)
and effect of chitosan covered poly-lacto-co- at a density of 75.000 cells/well. Every cell type
glycolic NPs (PLGAChi NPs) on the cells found was incubated with 1.5mL of their own culture
in the oral cavity, in normal and patologic medium. The cells were kept for 48 h at 37°C till
conditions. NPs were tested on normal human oral they became 70 % – 80 % confluent. Afterwards,
keratinocytes (NOKs) and human dental pulp cells the media were removed and washed twice with
(DPCs), harvested from healthy human donors, PBS. NOKs, DPCs and POE9i cells were exposed
as well as on POE9i cell line used as a model for for 12h and 24h at the following concentrations of
precancerous oral keratinocytes. In the attempt to fluorescein marked PLGAChi NPs: 20 μg/mL and
create a stronger resemblance to the natural 3D 200 μg/mL. 1.5mL of media containing PLGAChi
structure of the oral mucosal tissue, the PLGAChi NPs at the mentioned concentrations was placed
NPs were also exposed to 3D organotypic (OT) in every chamber slide: 20 μg/mL and 200 μg/
oral mucosa tissues grown in vitro. The PLGAChi mL. The solutions thus prepared were rotated for
NPs tested in our study were previously fabricated 30 minutes before exposure. The glass chambers
and characterized by Navarro et al. (43- 44). were placed in the incubator in a humidified
atmosphere at 37°C and supplemented with 5%
2. Materials and methods CO2 for 12 h or 24 h. At the end of the exposure
2.1. Cell culture time, the cells were washed three times with PBS
NOKs, DPCs and normal oral fibroblasts (NOFs) in order to remove unattached particles, followed
were primary cells isolated from clinically healthy by fixation and staining. The controls were run in
adult volunteers (n=5). Samples of gingival mu- duplicate in each experiment and were placed into
cosa showing no sign of clinical inflammation at the incubator for one day.
collection time were used to generate NOKs and The organotypic cultures were exposed to NPs
NOFs. The protocol for the isolation of NOKs has after a total period of 10 days of coculture. The
previously been described by Costea et al. (45). OTs were exposed to 200 μg/mL PLGAChi NP
DPCs were isolated following a protocol adapted and let into the incubator for 24h.At the end of the
from from Ishkitiev. et al. (46) and Lee et al. (47). exposure time, the OTs were washed three times
POE9i cells are dysplastic, premalignant human with PBS in order to remove unattached particles,
immortalized oral keratinocytes. NOKs and POE9i followed by fixation and staining.
keratinocytes were grown in Keratinocyte Serum- Imaging and image analysis was performed using
Free Growth Medium ( KSFM) (from Sigma-Aldrich, an optical microscope. The fluorescent microscopy
St. Louis, MO)  medium supplemented with 1ng/


( AxioImager.M2 with ApoTome.2, company lo- (Fig.6). Interestingly, the data obtained revealed
cation). The cells were mounted in Vectashield a penetration of PLGAChi NPs in almost all the
mounting medium with DAPI for nuclear staining keratinocytes exposed to the NPs at the tested
and were visualized at a Zeiss up-right Axio Imager concentrations and time points (Fig.6). The uptake
microscope with ApoTome slider module, using of NPs inside NOKs varied from 91,83 % +/- 7, 37 to
the 403 or 603 oil immersion objective lens. Images 100 % for NOKs, meaning that a significant amount
were captured with Axi-oVision Rel4.8 software of keratinocytes incorporated the PLGAChi NPs.
controlled by AxioCam MRm camera (Carl-Zeiss, In NOKs the percentage of cells which showed
Germany). All images were representative of at incorporation of PLGAChi NPs was 91,83 % +/-
least two independent experiments. 7,37 after 12h exposure at a concentration of 20
The quantification of NPs uptake was obtained μg/mL PLGAChi NPs. 92,39+/- 1,34 of the exposed
with the help of the Icy software, using two plugins NOKs showed NPs uptake after 24h exposure to 20
(HK-MEANS) : one developed by Dufour A (48) μg/mL PLGAChi NPs. PLGAChi NPs entered 98,55
and another one created by De Chaumont F (49). % +/-1,95 of the tested NOKs after incubation
2.4. Statistical analysis for 24h with 200 μg/mL PLGAChi NPs. The total
NPs uptake and cytotoxicity data were compared uptake inside NOKs was observed after one day of
using Student’s t-test. A p value < 0.05 was con- incubation with 200 μg/mL PLGAChi NPs.
sidered statistically significant. In POE9i cell line, all tested samples showed a
100 % uptake of PLGAChi NPs at all the tested
3. Results concentrations (20 μg/mL PLGAChi NPs and 200
3.1. PLGAChiNPs uptake by NOK, POE9 and μg/mL PLGAChi NPs) and exposure times (12h
DPC cells. and 24h) (Fig.6) .
The PLGAChi NPs uptake by NOKs and POE9i cells The fluorescence microscopic images showing
was determined by fluorescence microscopy(Fig. DPCs presented no relevant differences between
1, Fig. 2), as well as by confocal imaging (Fig. 4 and control images and images of DPCs exposed
Fig.5). The quantification of NPs uptake revealed to PLGAChi NPs (Fig. 3) . No NPs were observed
a significant amount of NPs inside the cells, both inside the cells exposed to PLGAChi NPs, even at
in normal human oral keratinocytes NOKs, as the highest concentration 200 μg/mL and at the
well as in premalignant oral keratinocytes POE9i 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

3 STOMA.EDUJ (2017) 4 (1)


E. NOKs 200 µg/mL PLGAChi NPs 24h F. PLGAChi NPs detection ( NOKs 200 µg/mL
PLGAChi NPs 24h )

Figure 1. Fluorescence images showing uptake of PLGAChi NPs byNOKs

A. POE9i Control B. POE9i 20 µg/mL PLGAChi NPs12h

C. POE9i 20 µg/mL PLGAChi NPs 24h D. POE9i 200 µg/mL PLGAChi NPs 12h


E. POE9i 200 µg/mL PLGAChiNPs 24h F. Detection of PLGAChi NPs internalization

inside POE9i (image of POE9i 200 µg/mL
PLGAChi NPs 12h )

Figure 2. Fluorescence images showing uptake of PLGAChi NPs inside POE9i. The fluorescent
green dots are the internalized PLGAChi NPs

A. DPCs Control B. DPCs 200 µg/mL PLGAChi NPs 24h

Figure 3. Fluorescence microscopy images showing no signs of PLGAChi NPs uptake by DPCs dots are the internalized PLGAChi NPs

Figure 4. Confocal microscopy image Figure 5. Confoccal Images of POE9i exposed

demonstrating PLGAChi NPs uptake inside to 200 µg / mL PLGAChi NPs for 12H. The
NOKs.The green fluorescent PLGAChi NPs are fluorescent PLGAChi NPs green NPs are
observed inside cells observed inside the cells

5 STOMA.EDUJ (2017) 4 (1)


Figure 6. Percentage of cells which have internalized PLGAChi NPs from the total amount of
exposed cells. NOKs and POE9i cells ( average values )

3.2. PLGAChi NPs uptake in 3D organotypic cell human oral mucosa grown in vitro. The images
cultures of normal human mucosa -in 3D cell obtained by fluorescence microscopy revealed
cultures that PLGAChi NPs were able to penetrate the
Intracellular uptake of the PLGAChi NPs was visible superficial layers of the reconstituted epithelial
in the epithelial compartment of the reconstituted mucosa after 24h exposure at a concentration of
200 µg/mL.
3.3. Cytotoxicity evaluation of PLGAChi NPs in
NOKs and POE9i cells
After 24h exposure to PLGAChi NPs, NOKs
demonstrated no significant difference in the
viability values at all tested concentrations: 5μg/
mL, 20 μg/mL and 200 μg/mL PLGAChi NPs. A
slight decrease in viability was observed inthe
POE9i cell line exposed to 20 μg/mL PLGAChi
NPs. However, the POE9i samples exposed to 20
Figure 7. Penetration of PLGAChi NPs in the μg/mL PLGAChi NPs showed 81% percentage of
3D organotypic models of human mucosa. The viable cells, as compared to 88 % live cells, in the
green fluorescent dots represent the PLGAChi control sample.
NPs which were able to enter the reconstituted In the NOKs and POE9i cell there was no statistical
OT of normal human mucosa. The sample was difference between the control and treated cells
exposed to 200µg/mL PLGAChi NPs for 24h (Fig. 8).

Figure 8. Viability of NOKs and POE9i after exposure to PLGAChi NPs


4. Discussion to colorectal cancer cells after 3 days of exposure

Polymeric NPs are still viewed as the first option at a 75 μM solution of NP (57). The viability of
for drug delivery and also widely used in the the cells incubated with chitosan NPs remained at
research of other diseases (1,2,3). Recently, a about 90% relative to the untreated cells on day 1
wide variety of studies has been undertaken and at about 89 % on day 3 (57). Another recent
leading the way for possible future applications study also found that the surface modification
of PLGA NPs in a high number of dental fields, of PLGA NPs with chitosan did not show any
from periodontology and endodontics to tissue significant diffference in cytotoxicity of PLGA
regeneration of skin, bone, or cartilage (50). NPs (7). The results indicated that A549 cell lung
Biocompatibility, biodegradability, flexibility, and carcinoma cells exhibited around 80% cell survival
minimal side effects are the main advantages as compared to positive controls (10% cytotoxic)
when using PLGA for biomedical applications (4). for the following concentrations of PLGAChi NPs:
However, the overall negative charge of these NPs 0.25, 0.5, 0.75, 1,1.25, 1.5, 1.75 and 2 mg/mL (7).
has been reported to diminish their interaction Other investigators have reported PLGAChi NPs
with the negatively charged cell mebrane, while to be safe even at much higher concentrations
the rapid opsonization of hydrophobic PLGA NPs of 20 mg/mL (58-60). In addition, other research
is a major limitation that hinders their employment groups revealed interesting cytotoxicity results of
for biomedical applications (6-7). chitosan NPs effect on human skin keratinocytes
In order to improve PLGA NP proprieties, several HaCaT cells. Tretinoin containing chitosan solid
research groups have tried to cover the polymers lipid NPs were not cytotoxic to HaCaT cells even at
with a chitosan coating. Chitosan is obtained from the highest concentration 500 μg/mL used, which
chitin which is a positively charged polysaccahride led to around 5 % less viability compared with
found in crustaceans (50-53). Chitosan contains the control (61). Also lecithin/chitosan  NPs  can
many amino and hydroxyl groups, thus it can be applied to skin cells at concentrations up to
bind effectively to negatively charged substances 200 µg/mL without inducing plasma membrane
(such as the cells membranes) via electrostatic damage or cell viability decrease (62). Similarly, in
interactions or hydrogen bonding, thus improving a recent study, HaCaT cells exposed to melatonin
the intracellular uptake (17). Chitosan by itself is containing lecithin/chitosan NPs in a concentration
known to strongly adhere to negatively charged of chitosan of 1.25-20 μg/mL for 2 hours showed
surfaces due to its high charge density at pH < 6.5 no relevant cytotoxicity (63). However, a significant
(54). The advantages of modifying the surface of reduction in the cell viability of HaCaT cells was
PLGA NPs with a mucoadhesive polymer, such as observed in the case of cells treated with NPs
chitosan, may potentially include the inversion at a chitosan concentration of 20 μg/mL (63).
of zeta potential,the ability to promote cellular Chitosan-alginate NPs did not have a toxic effect
adhesion and retention of the delivery system at on human monocytes but there was mild toxicity
the target site (55) . to skin keratinocytes at higher concentration
In order to fabricate polymeric NPs for future of NPs (54).Moreover, chitosan and PLGA NPs
dental applications, we have tested the positive loaded with chlorexidine dihydrochloride in vitro
charged chitosan coated PLGA NPs (PLGAChi toxicity evaluation on human gingival fibroblasts
NPs) on oral cavity cells. NPs interraction with the was between 20 and 60% in all experimental
oral epithelial cells was assessed by cytotoxicity conditions (17). Poly-γ-glutamic acid/glycol
measurements. After 24h of incubation with chitosan NPs incorporating p-phenylenediamine
PLGAChi NPs (20 μg/mL NPs and 200 μg/mL NPs) (PDA) showed lower cytotoxicity against HaCaT
no significant statistic differences were observed human skin keratinocyte cells than PDA alone
in the viability of samples and controls (Fig. 8). (64). Interestingly, PDA-incorporated NPs showed
PLGA Chi NPs were biocompatible to all the tested reduced apoptosis and necrosis reaction in HaCaT
cell lines: NOKs and POE9i cells. We found no cells (64). A possible explanation for the chitosan
association between the significant increase in NPs high biocompatibilty could be that chitosan is
the cell uptake of chitosan containing NPs and cell much more cytotoxic in a free soluble form than
toxicity, at the tested concentrations. Due to PLGA when it is incorporated into NPs, due to the fact
and chitosan’s well known biodegradability and that in the case of NPs, a significant portion of the
biocompatibility, it was expected that NPs made positive amino groups of chitosan are engaged in
of chitosan and PLGA would be well tolerated by electrostatic interractions (63, 65).
the cells. Our results are in agreement with other To confirm PLGAChi NPs efficiency in intracellular
comparable studies. Following a 2015 experiment, penetration, the cellular internalization of PLGAChi
S. Alqahtani stated that chitosan covered PLGANPs NPs conjugated with fluorescein was investigated
did not affect the viability of Caco-2 cells (56). by fluorescence microscopy. The results indicated
Caco-2 cells displayed a viability above 95%, significant differences in NPs uptake between the
even after incubation for one day with a higher different cell lines used in this study. Fluorescence
concentration of NPs than it was used in our study: microscopy experiments conducted after 12h and
500 μg/mL PLGAChi NPs (56). Moreover, chitosan 24 h of incubation revealed a rate of inglobation
covered NPs did not contribute additional toxicity influenced by the cell type.

7 STOMA.EDUJ (2017) 4 (1)


DPCs did not internalize PLGAChi NPs, even at The findings of this study provide evidence for
the highest concentration (200 μg/mL PLGAChi the penetration of PLGAChi NPs not only in single
NPs) and the longest incubation time (24h) (Fig.3). cells, but also in oral mucosal cells assembled in
But the microscopic results showed supporting 3D tissus, as shown by the results on the human 3D
evidence of incorporation of the tested NPs in oral organotypic reconstructed human mucosa models
keratinocytes, both in normal and in pathologic grown in vitro (Fig. 7). Altough the 3D organotypic
precancerous conditions (Fig. 1, Fig. 2, Fig. 4, Fig. models replicate only to a certain extend the
5). structure of the human tissue, in this case the
The data obtained revealed a significant uptake human oral mucosa, they better resemble the oral
of PLGAChi NPs into oral keratinocyte cells microenvironment of the oral keratinocytes than
after 12h exposure with 20 μg/mL NPs : 91,83 the 2D models. To our knowledge this is the first
% in NOKs and 100% in POE9i cells (Fig. 6). The study that assesed the penetration of PLGAChi NPs
percentage of NOKs which have internalized the in the reconstructed oral human mucosa. The NPs
chitosan covered PLGA NPs increased gradually crossed the superficial epithelial layers, reaching
with the incubation time and concentration of the the underlying conective tissue (Fig. 7).
solution of polymeric NPs. The highest uptake Our results were in agreement with previous
of PLGAChi NPs inside both keratinocyte types studies which showed a high uptake of polymeric
was observed after 24 h exposure to 200 μg/mL NPs when fortified with chitosan, and that the
PLGAChi NPs, when all cells were penetrated by uptake of chitosan coated NPs was much higher
the NPs (Fig. 6). What is mostly important is the fact that that of uncoated NPs (70,71,56,55,7). In a study
that the PLGAChi NPs entered in a higher amount from 2015, S.Alqahtani showed a significantly
in the precancerous cells than in the normal oral higher 3.5 fold cellular uptake of chitosan coated
keratinocytes. Chitosan covered NPs demonstrated PLGAChi NPs compared to PLGA NPs in Caco-
a 100% uptake at all tested concentrations and time 2 cells (56). In another study, positively charged
points in POE9i precancerous cells. Moreover, the chitosan covered PLGA NPs exhibited enhanced
PLGAChi NPs were able to get internalized into mucoadhesion, compared to negatively charged
the epithelial cells in reasonable amounts and in PLGA NPs and enhanced intracellular uptake
a time and concentration dependant manner. The in A549 cell line human lung carcinoma  cells
percentage of keratinocyte cells with internalized (7). PLGAChi NPs managed to get internalized
NPs increased over incubation time, demonstrating into Caco-2 cells with reasonable amounts after
a growing and highly efficient process of just 1h (56). Another research group stated that
internalization of PLGAChi NPs by human NOKs PLGAChi NPs are internalized by hepatocytes 3A
and POE9i keratinocytes. The results obtained in and fibroblasts 3T6 in a few minutes (55). Also,
3D studies confirm the fact that PLGAChi NPs can Chronopoulos reported that the uptake of
enter the oral keratinocytes ( Fig. 7). PLGAChi NPs appears faster than with PLGA NPs,
Interestingly, what was observed was a higher and with major amounts of cytoplasmatic NPs found
more rapid uptakeof PLGAChi NPs in precancerous after only 5 minutes (55). Interestingly, uptake
keratinocytes compared to NOKs (Fig. 6). Based saturation is reached after 2-3h of incubation with
on these data, we hypothesise a preference of PLGAChi NPs in human 3A hepatocytes and 3T6
chitosan covered NPs for uptake byprecancerous fibroblasts although the uptake of PLGA NPs still
keratinocytes over normal keratinocytes. This has appears less extensive than for PLAChi NPs (55).
been also hypothesised previously by (66) who The performance of a delivery system depends
showed that epithelial cell cultures forming tight on the polymeric composition, the size and surface
junctions did not internalize NPs, while those lacking charge (72, 63).
tight junctions, i.e., the cancer cells, did internalize. Therefore, smaller sizes of NPs and a positive
Although the NPs used in that study differ from zeta potential lead to a better internalisation
our study, this could provide a possible molecular inside cells due to the attractive interaction with
explanation.Interestingly, previous research the negatively charged cell membranes (73, 74).
articles have also found a preference of chitosan Hence, the size and zeta potential of the cureent
covered NPs for uptake by cancer stem cells. A NPs fabricated in our study are in favour of particle
doxorubicin-encapsulated polymeric nanoparticle internalisation. Our data demonstrated that
surface-decorated with chitosan was able to target PLGAChi NPs exhibited a significant internalisation
and eliminate tumor reinitiating  cancer  stem- into the human oral keratinocytes .
like cells (67). Moreover, hyaluronic acid-decorated However, the experiment presents a series of
dual responsive  nanoparticles  of Pluronic F127, limitations. As other studies showed the rapid
PLGA, and  chitosan  were developed recently internalisation of chitosan covered NPs in minutes
for targeted co-delivery of doxorubicin and or hours (56, 55), the exposure time used in our
irinotecan to eliminate cancer stem-like cells (68). study (12h and 24h) might have been too long.
Also,chitosan-coated hyaluronic acid, docetaxel Further investigations using a wider variety of
containing  NPs   were more effective against concentrations and time points are needed in
CD44+ cells than free docetaxel (69). We have not order to asses the differences in uptake of PLGAChi
investigated this aspect in our study, but this could NPs between normal and patologic conditions.
be further investigated.


The use of organotypic models of reconstructed We acknwoledge Dr. G Negroiu, Institute of

human mucosa in vitro resemble much more the Biochemistry, Bucharest, Romania for assisting us
natural conditions in vivo than the usual tests in the acquisition of microscopy images. Many
on monolayer cell cultures. But the organotypic thanks to Macedon SE from Institute of Electrical
models cannot substitute the in vivo experiments and Electronics Engineers for the help in the
as they are composed only of a collagen biomatrix quantification of the uptake of nanoparticles inside
and epithelial cells, without other components of cells.
the natural mucosa, such as the imune cells and
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Maria Justina Roxana VIRLAN

DDS, MSc, PhD Student
Department of Biochemistry
Faculty of Dental Medicine, U.M.F.”Carol Davila “Bucharest, Romania

Dr Justina Virlan graduated from the Faculty of Dental Medicine in 2012 and completed an Endodontics
residency. In 2015 she finished a master programme at the Faculty of Medical Engineering. A laureate of the
National Chemistry Competition and a PhD student at the Biochemistry Department of the Dental Medicine
Faculty, she has a high interest in nanomaterials and their applications in dentistry.


Chitosan modified poly(lactic-co-glycolic) acid nanoparticles (PLGAChi NPs) could be used to
deliver drugs:
qa. to the dental pulp cells;
qb. to the oral mucosa;
qc. to the dental pulp cells and oral mucosa;
qd. none of the above.

The PLGAChi NPs (used in this study) can enter :

qa. normal oral keratinocytes (NOKs);
qb. precancerous oral keratinocytes (POE9i);
qc. dental pulp cells (DPC);
qd. normal oral keratinocytes (NOKs) and precancerous oral keratinoctes (POE9i).

The multilayered epihelia of oral mucosa was grown in vitro using:

qa. collagen matrix;
qb. collagen matrix; normal oral fibroblasts (NOFs);
qc. collagen matrix; normal oral fibroblasts (NOFs); normal oral keratinocytes (NOKs);
qd. collagen and matrigel matrix; normal oral fibroblasts (NOFs); normal oral keratinocytes

In the cell lines that have internalized PLGAChi NPs, the maximum uptake of NPs was observed
after exposure to:
qa. 200 g/mL PLGAChi NPs for 24 h;
qb. 200 g/mL PLGAChi NPs for 12 h;
qc. 20 g/mL PLGAChi NPs for 12 h;
qd. 20 g/mL PLGAChi NPs for 24 h.

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