International Journal of Pharmaceutics 589 (2020) 119776

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Chitosan-coated PLGA nanoparticles for the nasal delivery of ropinirole T

hydrochloride: In vitro and ex vivo evaluation of efficacy and safety
Aikaterini-Theodora Chatzitakia, Sandra Jesusb, Christina Karavasilia, Dimitrios Andreadisc,
Dimitrios G. Fatourosa, Olga Borgesb,d,
⁎

a
Department of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece
b
Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
c
Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece
d
Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

A RT ICLE INFO ABSTRACT

Keywords: Nose-to-brain delivery is an attractive route for direct drug delivery to the central nervous system (CNS),
Nasal delivery avoiding hepatic first-pass metabolism and solving blood–brain barrier passage issues. Therefore, the aim of the
PLGA/chitosan nanoparticles present study was the development of PLGA and PLGA/chitosan (chit) nanoparticles (NPs) with mucoadhesive
Ropinirole properties, able to encapsulate ropinirole hydrochloride (RH), an anti-Parkinsonian dopaminergic agonist, and
Mucoadhesion
suitable to promote RH delivery across the nasal mucosa.
Cytotoxicity
NPs produced by nanoprecipitation showed spherical shape and a mean average size of 98.8 nm and 468.0 nm
Ex vivo permeability
(PLGA and PLGA/chit, respectively). RH loaded PLGA/chit NPs showed a complete release of the drug in si­
mulated nasal electrolyte solution (SNES) over the period of 24 h and increased the permeation of RH through
sheep nasal mucosa by 3.22-fold in comparison to PLGA NPs. None of RH loaded NPs induced hemolysis in
whole blood or the production of reactive oxygen species (ROS) in Raw 264.7 cells. On their turn, PLGA/chit NPs
decreased cell viability of Raw 264.7 cells and Peripheral Blood Mononuclear Cells (PBMCs) in a concentration-
dependent manner.
These results revealed that, particularly PLGA/chit NPs, could be a valuable carrier for the delivery of RH to
the CNS, opening a new path for Parkinson’s disease therapy.

1. Introduction
Neurologic disorders such as Parkinson’s disease require direct
transport of therapeutic substances to brain in order to maximize drug
concentration in their action site. Parkinson’s disease is characterized
by neurodegeneration and loss of dopaminergic neurons in the Central
Nervous System (CNS) and the treatment approaches include the ad­
ministration of dopamine agonists. Among them, ropinirole hydro­
chloride (RH) is a non-ergoline agonist that acts on D2- and D3-re­
ceptors in the brain (Barcia et al., 2017). RH is indicated for the
treatment of Parkinson’s disease, as a monotherapy or in combination
with levodopa to decrease its on–off variations, and for the manage­
ment of restless leg syndrome (Khan et al., 2010). Its precise mechanism
of action is still unknown, though it is believed to stimulate the post-
synaptic D2-receptors within the caudate-putamen in the brain. RH is
chemically a 4-[2-(dipropylamino)-ethyl]-1,3-dihydro-2H-indol-2-one
and is characterized by high aqueous solubility with logP 2.7 (Pardeshi
and Belgamwar, 2017). Currently, the most common delivery of neu­
rotherapeutics, including for RH, is via the oral route which is asso­
ciated with an elimination half-life of approximately 6 h and an ex­
tensive hepatic first-pass metabolism resulting in low bioavailability in
the range of 36–57 % and insufficient drug levels in the CNS (Pardeshi
and Belgamwar, 2017). Also, a great challenge in CNS delivery is the
blood brain barrier (BBB) limitation of drug penetration (Gänger and
Schindowski, 2018), which is controlled by the drug’s physicochemical
properties and cerebral defensive mechanisms (Pardeshi and
Belgamwar, 2013). Therefore, the nose-to-brain route for drug admin­
istration has attracted much attention considering the plethora of
benefits that nasal cavity offers. The relatively large surface area of the
nasal epithelium, high vascularization and direct brain targeting render
nasal cavity as a promising route of administration for molecules tar­
geting the brain (Bourganis et al., 2018). Following this route, the ad­
ministered drugs avoid the first hepatic metabolism and have direct
access to the brain through the olfactory and trigeminal pathways

⁎
Corresponding author at: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
E-mail address: olga@ci.uc.pt (O. Borges).

https://doi.org/10.1016/j.ijpharm.2020.119776
Received 16 April 2020; Received in revised form 11 August 2020; Accepted 13 August 2020
Available online 17 August 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.
A.-T. Chatzitaki, et al.
(Bourganis et al., 2018) which are localized at the roof of nasal cavity,
offering the opportunity for direct drug transport to CNS (Pardeshi and
Belgamwar, 2013).
The suitability of nose-to-brain delivery of RH has been previously
investigated by Pardeshi et al. (2017) who formulated a nanoplex of RH
utilizing dextran sulfate sodium that provided increased ex vivo drug
permeability across sheep nasal mucosa (Pardeshi and Belgamwar,
2017). Rao et al. (2017) encapsulated ropinirole in a thermo-reversible
in situ nasal gel showing an approximately 5-fold bioavailability en­
hancement in the brain after in vivo intranasal administration, com­
pared to the intravenously administered drug solution (Rao et al.,
2017). Polymeric NPs have been also evaluated as suitable vectors for
the intranasal delivery of RH, as they prevent drug degradation, allow
sustained drug release and consequently decrease the administration
frequency (Pardeshi et al., 2013).
However, the presence of mucociliary clearance, an innate clear­
ance mechanism that hinders the efficacy of intranasal drug delivery,
creates the necessity for novel pharmaceutical formulation approaches.
Exploitation of mucoadhesive agents and adsorption enhancers may
prolong the time of interaction between drug formulation and mucus
epithelial layer and open tight junctions, respectively (Gänger and
Schindowski, 2018). Chitosan and its derivatives have been widely in­
vestigated as mucoadhesive agents to enhance the adsorption of nasally
administered hydrophilic drugs and macromolecules, hampering the
mucociliary clearance and increasing membrane permeability (Gänger
and Schindowski, 2018). Previous work from our group on mu­
coadhesive PLGA-lipid microparticles containing different concentra­
tions of trimethyl chitosan (TMC) have been previously evaluated for
the nasal administration of RH. Ex vivo results demonstrated a 2.35-fold
enhancement of RH permeability across sheep nasal mucosa, compared
to the drug solution (Karavasili et al., 2016).
In that context, the main objective of the present study was the
development of mucoadhesive polymeric NPs combining PLGA and
chitosan (PLGA/chit), able to encapsulate RH and enhance RH ex vivo
permeability across sheep nasal mucosa, demonstrating suitability for
RH nose-to-brain delivery. In parallel, to guarantee that the developed
formulation was not only effective but also safe, several biocompat­
ibility studies were performed with human derived cells.
2. Materials and methods
2.1. Materials
Poly(D,L-lactide-co-glycolide) acid [ester terminated lactide:glyco­
lide 75:25, molecular weight (Mw): 76 kDa to 115 kDa], polyvinyl al­
cohol [Mowiol 4–88, Mw ̴ 31 kDa], 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT), bovine serum albumin [BSA,
96 % fraction V], myoglobin [95 %-100 %] and lysozyme [≥ 80 %],
BSA-FITC (A9771) and lipopolysaccharide (LPS) derived from
Salmonella enterica serotype minnesota and Rhodamine-B were ob­
tained from Sigma-Aldrich Corporation (St.Louis, MO, USA). RH was
kindly donated from Pharmathen SA (Marousi Attica, Greece) and
chitosan (ChitoClear™ − 95 % DD and 8 cP viscosity measured in 1 %
solution in 1 % acetic acid) was purchased from Primex Bio-Chemicals
AS (Avaldsnes, Norway). Micro-Bicinchoninic acid (micro-BCA) protein
assay kit was purchased from Pierce Chemical Company (Rockford, IL,
USA). Mouse RAW 264.7 macrophage cells (ECACC, Salisbury, UK)
were maintained in Dulbecco’s modified Eagle medium (DMEM) sup­
plemented with HEPES 10 mM, 3.7 g/L sodium bicarbonate, 1 %
Penicillin/Streptomycin and 10 % of inactivated fetal bovine serum
(FBS). Roswell Park Memorial Institute (RPMI 1640) supplemented
with 20 mM HEPES, 2 mM L-Glutamin, 1 % Penicillin/Streptomycin
and 10 % of FBS was used to culture PBMCs. The nuclear stain Hoechst
33342, Alexa Fluor-594 WGA were purchased from Invitrogen
(Carlsbad, CA, USA). Pyrogen-free water was kindly given by Labesfal
Farma (Viseu, Portugal). All other chemicals and reagents were of
2
International Journal of Pharmaceutics 589 (2020) 119776
Table 1
Concentrations of the raw materials used for the production of the optimized
NPs.
Formulations Concentrations
PLGA (mg/ PVA (% Chitosan (% w/ RH (%
mL) w/v) v) w/v)
PLGA NPs 5 0.8 – –
RH – PLGA NPs 5 0.8 – 0.02
PLGA/chit NPs 5 0.8 0.1 –
RH – PLGA/chit NPs 5 0.8 0.1 0.02
analytical grade.
2.2. Methods
2.2.1. Nanoparticle preparation
NPs were produced via nanoprecipitation method (Table 1). Briefly,
PLGA NPs were prepared by adding 2 mL of the organic phase, con­
sisting of PLGA (5 mg/mL) dissolved in acetone, dropwise in 6 mL of
0.8 % w/v PVA aqueous solution. For PLGA/chit NPs the aqueous phase
contained the same concentration of PVA and 0.1 % w/v chitosan dis­
solved in acetic acid 2 % v/v. The ratio between the organic and the
aqueous phase was set at 1:3. The NPs remained under magnetic stir­
ring for 60 min at room temperature to achieve maturation and were
then centrifuged at 3000 g using Vivaspin 20 centrifugal concentrator
tubes (Mw 300 kDa Sigma-Aldrich Corporation, St. Louis, MO, USA).
The NPs were washed with pyrogen free water, centrifuged and finally
resuspended with an appropriate volume of water to the final con­
centration required in each of the following assays (calculated from
weighted freeze-dried NP pellets or according to drug content, when
applicable). RH loaded NPs were obtained under the same procedure as
blank NPs with the addition of RH in the aqueous phase (0.02 % w/v)
(Karavasili et al., 2016). BSA-FITC adsorbed NPs were prepared by
incubating the protein (100 μg/mL) with NPs suspension (960 μg/mL)
for 2 h, protected from light. Rhodamine-B loaded NPs were prepared
by adding 10 µg/mL of Rhodamine-B to the aqueous phase during NP
preparation. The procedure was performed in the dark.
2.3. Characterization of nanoparticles
2.3.1. Size, ζ-potential and morphological analysis
Particle size and ζ-potential analysis were performed by dynamic
light scattering (DLS) and electrophoretic light scattering (ELS), re­
spectively, using Zetasizer Nano-ZS (Malvern Instruments Ltd, Malver,
United Kingdom) at 25 °C. Size and ζ-potential were determined by
diluting nanoparticle suspensions in milli-Q water.
Morphological characteristics of unloaded nanoparticles suspended
in the original medium were examined using a high-resolution
Transmission Electron Microscope (TEM) (FEI-Tecnai G2 Spirit
Biotwin). A drop of each sample was placed in copper grids (300 mesh)
and was dried out before visualization.
2.3.2. Determination of drug loading efficiency and production yield
estimation
Accurate volumes of the blank and RH loaded NP formulations,
corresponding to 0.5 mg/mL of NPs, were dissolved with 50 μL
chloroform and 3.5 mL water and stirred for 15 min. Samples were
centrifuged at 20800 g for 20 min, followed by drug quantification in
the supernatants at 250 nm using an High Performance Liquid
Chromatography (HPLC) method (Karavasili et al., 2016). The cali­
bration curve of RH in water was linear in the concentration range of
0.098 μg/mL to 0.78 μg/mL (R2 = 0.995). The experiments were re­
peated five times and results were calculated according to the following
equation.
A.-T. Chatzitaki, et al.
Drug Loading Efficiency (LE%) =
weight of RH in NPs (mg)
× 100
total weight of RH added (mg)
The production yield was determined as the percentage weight of
lyophilized NPs over the total amount of RH and raw materials initially
used to produce NPs via nanoprecipitation method. Eq. (2) was used to
calculate the production yield.
weight of freeze dried NPs
Yield (%) = × 100
weight of raw materials and drug
2.4. In vitro release studies
In vitro release studies of RH encapsulated in PLGA and PLGA/chit
NPs were carried out in SNES pH 5.5 at 35 °C ± 0.2 °C, using the
dialysis bag diffusion method (cellulose membrane, MWCO
12.000–14.000, avg. flat width 10 mm, Orange Scientific, Belgium).
Dialysis bags were soaked in distilled water for 30 min under agitation
prior to being filled with 1 mL aqueous NP suspension of each for­
mulation, diluted to obtain 49 μg RH, and were then sealed at both
ends. Samples were immersed in glass vials filled with 20 mL pre­
warmed SNES 35 °C, sealed with parafilm and placed in a shaking water
bath. At predetermined time intervals 500 μL of sample were with­
drawn and replaced with an equal volume of fresh and preheated re­
lease medium. Samples were analyzed for RH content in accordance to
the following chromatographic conditions. RH was detected at 250 nm
using a Discovery HS C18 (15 cm × 4.6 mm, 5 μm) with the mobile
phase consisting of 0.05 M methanol:ammonium acetate buffer (pH 7)
80:20 (v/v) at a flow rate of 0.9 mL min−1 and injection volume of
90 μL. Calibration curve of RH in SNES showed good linearity in the
concentration range of 0.01 μg/mL to 2.5 μg/mL (R2 = 0.998).
2.5. Differential scanning calorimetry (DSC) studies
DSC analysis were performed on the raw materials and the lyophi­
lized NP formulations using 204 F1 Phoenix DSC apparatus (Netsch
GmBH, Germany). Six mg of raw materials and each formulation were
placed in aluminum pans with perforated lids and their thermal beha­
vior was monitored at a heating rate of 10 °C/min from 40 °C to 260 °C
under a nitrogen purge of 70 mL/min. Proteus ver. 5.2.1 software
(Netsch GmBH, Selb, Germany) was used for data analysis.
2.6. Stability studies
Accelerated stability studies were conducted by monitoring changes
in the particle size and ζ-potential of the NPs over a 3-month period
during storage at 25 °C ± 2 °C and relative humidity (RH) of 60 ± 5 %
as recommended by International Conference on Harmonisation (ICH)
Q1A (R2) guidelines, 2003 (Patil and Surana, 2013). Measurements
were performed periodically in a monthly-based period after dilution of
the aqueous NP suspensions in milli-Q water on a Zetasizer (Nano-ZS,
Malvern Panalytical, Marlvern Ltd, Malvern, United Kingdom) at 25 °C
(Patil and Surana, 2013).
2.7. Mucoadhesion studies
The mucoadhesive properties of the NPs were investigated by
monitoring alterations on the surface charge properties of the NPs upon
incubation with mucin (Takeuchi et al., 2005). The ζ-potential of the
NP suspension and mucin (0.5 mg/mL) in SNES was determined in­
itially. Mucin solution in SNES was incubated in a 1:1 ratio (w:w) with
NP suspensions at room temperature under middle agitation. After 1 h,
samples were centrifuged at 20800 g for 30 min and supernatants were
discarded in order to remove free soluble protein. Pellets were re­
suspended in SNES and the ζ-potential of the dispersion was recorded.
International Journal of Pharmaceutics 589 (2020) 119776
2.8. Hemolysis assay
(1) Hemolysis assay was performed as previously described (Jesus
et al., 2020). Blood collected from healthy volunteers after informed
written and signed consent, was stabilized with Lithium heparin and
diluted to a final total hemoglobin concentration of 10 mg/mL. Dif­
ferent concentrations of the formulations with and without RH were
added to 100 μL of the diluted blood and 700 μL of PBS in a final vo­
lume of 900 μL. The same procedure was repeated also without blood to
(2) determine if nanoparticles interfere with the hemolysis assay. PBS and
Triton-X-100 were used as a negative (NC) and positive (PC) controls of
the assay. The samples were mixed and incubated for 3 h at 37 °C with a
mild shake every 30 min and after that period, they were centrifuged at
800 g for 15 min. Hemolysis was evaluated by mixing 100 μL of each
supernatant with 100 of cyanmethemoglobin (CMH) reagent in to a 96-
well plate, shaking for 2 min to 3 min and reading the absorbance at
540 nm using a microplate reader. The percentage of hemolysis was
calculated using the following Eq. (3):
Abs sample Abs negative control
Hemolysis (%) = × 100
Abs positive control Abs negative control
(3)
2.9. Studies on peripheral blood mononuclear cells (PBMCs)
2.9.1. PBMCs isolation
Peripheral blood was kindly given by IPST,IP (Coimbra, PT) and
was obtained from normal donors in heparinized syringes followed by
serum depletion. According to the provider’s guidance protocol and as
published by Jesus et al. (2017). PBMCs were isolated on a density
gradient using Lymphoprep (Axis-Shield, Dundee, Scotland). Isolated
cells were maintained in RPMI. For cytotoxicity and uptake studies,
PBMCs were plated in well plates at a density of 5 × 106 monocytes/
mL.
2.9.2. Cytotoxicity studies
The cytotoxicity of NPs was estimated by MTT cytotoxicity assay. In
a 96-well plate plated with 100 µL of 5 × 106 monocytes/mL, serial
dilutions of unloaded and RH loaded NPs as well as free RH were added
to cells. After 24 h of incubation, 20 µL of MTT (5 mg/mL in PBS,
pH = 7.4) was added to each well and plates were incubated for ad­
ditional 4 h. After that time, plates were centrifuged at 800 g for 25 min.
Supernatants were aspirated from all plates and prewarmed DMSO was
added to each well to solubilize formazan crystals. Optical density (OD)
was measured at 540 nm and 630 nm and cell viability was calculated
by Eq. (4):
OD sample (540 nm) OD sample (630 nm)
Cell Viability(%) = × 100
OD control (540 nm) OD control (630 nm)
(4)
The concentration of NPs that inhibits cell viability by 50 % (IC50 -
half maximal inhibitory concentration) was estimated by plotting the
log concentration of the NP vs inhibition percentage of cell viability and
extrapolating the value from a non-linear regression using Prism 6.0
(GraphPad Software, San Diego, CA, USA).
2.9.3. Uptake studies by peripheral blood mononuclear cells (PBMCs)
2.9.3.1. Flow cytometry analysis. To investigate NPs cellular uptake,
two different conditions (BSA-FITC adsorbed NPs and Rhodamine-B
loaded NPs) were evaluated. For the uptake studies, PBMCs were plated
in a 48 well-plate at a concentration of 5 × 106 monocytes/mL.
In the next day, 40 μL of BSA-FITC adsorbed NPs or Rhodamine-B
loaded NPs (prepared as previously described) as well as free protein
(BSA-FITC) and rhodamine-B solutions (controls) were added to the
wells and incubated at 37 °C for 4 h. After incubation, the plate was
centrifuged at 800 g for 25 min and after the removal of supernatants,
3
A.-T. Chatzitaki, et al.
300 μL cold PBS was added to each well and kept at 4 °C until the
analysis in a BD FACSCalibur Flow Cytometer (BD Biosciences, Bedford,
MA, USA). The BSA-FITC labeled NP samples were also analyzed after
adding 20 μL of 0.4 % trypan blue to quench the extracellular fluores­
cence of BSA-FITC (resultant from cellular adsorption) and therefore
estimate the cellular internalization of the protein.
2.9.3.2. Confocal microscopy. PBMCs were plated on glass coverslips
treated with Poly-D-lysine in 24 well plates at a density of
5 × 106 monocytes/mL and incubated at 37 °C in 5 % CO2 overnight.
In the next day, different formulations (either BSA-FITC adsorbed NPs
or Rhodamine-B loaded NPs) were incubated with cells for 4 h.
Following that period, cells were washed 3 times with phosphate
buffer saline (PBS) pH 7.4 and fixed with 4 % paraformaldehyde in
PBS for 20 min at 37 °C. After fixation, cells were washed again 3 times
and then, cells were stained with Hoechst 33342 (1 µM) for nucleus
stain and Alexa Fluor 594 wheat germ agglutinin (1 μg/mL) for
membrane stain. In case of Rhodamine-B loaded NPs only Hoechst
33342 was used to stain the nucleus. After labeling for 10 min, cells
were washed twice with PBS and coverslips mounted on microscope
slides with DAKO mounting medium and examined under an inverted
laser scanning confocal microscope (Zeiss LSM 710, Carl Zeiss,
Oberkochen, DE) equipped with imaging software (Zen Black 2012
software, Carl Zeiss).
2.10. Studies on RAW 264.7 macrophage cell line
2.10.1. Cytotoxicity studies
Raw 264.7 (ATCCR TIB-71TM) were cultured in Dulbecco’s mod­
ified Eagle medium (DMEM) supplemented with 10 mM HEPES, 3.7 g/L
Sodium Bicarbonate, 1 % Penicillin/Streptomycin and 10 % of in­
activated fetal bovine serum (FBS) and incubated overnight at 37 °C and
5 % CO2. MTT cytotoxicity assay was also performed in macrophages,
as described previously, for PBMCs with some modifications. Cells were
seeded in a 96-well plate at a density of 2 × 105 cells/well and in­
cubated with MTT solution for 1 h 30 min.
2.10.2. Nanoparticle effect on production of the reactive oxygen species
(ROS Assay)
The ROS production was evaluated using the dichlorofluorescein
diacetate probe (DCFH-DA) (Thermo Fisher Scientific Inc., Waltham,
MA, USA) as previously described (da Silva et al., 2019). The RAW
264.7 cells were seeded in a black 96-well plate at density of 5 × 105
cells/mL and incubated for 24 h at 37 °C and 5 % CO2. After 24 h, dif­
ferent concentrations (serial dilutions) of unloaded and RH loaded
PLGA and PLGA/chit NPs as well as LPS as positive control and cell
culture medium (negative control) were incubated with cells. After 24 h
incubation, cell culture medium was replaced by DCFHDA (50 μM) in
serum free DMEM and the plates incubated for another 2 h. The re­
sulting fluorescence was measured at 485/20 nm and 528/20 nm (ex­
citation/emission wavelengths).
The following equation (Eq. (5)) was used to calculate ROS pro­
duction:
ROS production (mean fluorescence increase)
Fluoroscence (SAMPLE)
= × 100
Fluoroscence (NEGATIVE CONTROL) (5)
2.11. Ex vivo permeability studies
Ex vivo permeability studies were carried out across sheep nasal
mucosa obtained from the local slaughterhouse according to a pre­
viously described protocol (Karavasili et al., 2016). Immediately after
isolation, tissues were maintained in phosphate buffer (pH 7.4) until
use. The tissues were mounted between the donor and the receptor
4
International Journal of Pharmaceutics 589 (2020) 119776
compartment of a Franz diffusion cell which was filled with 20 mL SNES
with the mucosal surface facing the donor compartment. Five hundred
μL of each formulation (corresponding to 52 μg RH) were placed on the
donor compartment under stirring at 100 rpm and 35 °C ± 0.2 °C
keeping the donor compartment sealed with Parafilm®. Nasal mucosa
treated only with SNES under the same conditions was used as control.
At regular time intervals 500 μL were withdrawn from the receptor and
replaced with an equal volume of fresh prewarmed SNES. Samples were
centrifuged at 20800 g for 20 min and drug quantification in the su­
pernatants was performed with HPLC.
Steady state flux (Jss) was calculated based on the plot of drug
permeation across nasal mucosa per unit area (μg/cm2) against time
(min) and corresponds to the slope of the linear section of the obtained
line. Eq. (6) was used to calculate apparent permeability coefficient
(cm/min) (Paap), while Eq. (7) was used to calculate the steady state
diffusion coefficient (D).
Jss
Paap =
initial concentration of drug in the donor compartment
(6)
D= Papp·L/K (7)
where K is the partition coefficient and L is the diffusion path length.
(Pardeshi and Belgamwar, 2018).
2.12. Histopathological examination
At the end of the ex vivo permeability studies, control tissues and
tissues treated with the NP formulations were collected, washed prop­
erly with distilled water and maintained in formalin 10 %. Tissue sec­
tions were stained with hematoxylin and eosin and observed under a
light microscope (Olympus CX31 optical microscope). Images were
acquired using the OLYMPUS analySIS getIT software.
2.13. Statistical analysis
Results were expressed as mean ± standard error or mean ± SEM.
Statistical analysis was performed using one-way ANOVA and
GraphPad Prism v 5.03 (GraphPad Software Inc., La Jolla, CA, USA).
Data obtained by flow cytometry were analyzed with CellQuest Modfit
LT analysis software (BD Biosciences, Bedford, MA, USA). P
value ≤ 0.05 was considered as statistically significant.
3. Results
3.1. PLGA NPs are smaller and have lower polydispersity when compared to
PLGA/chit NPs
It has been previously established that a hydrodynamic diameter
smaller than 200 nm leads to an easier transportation to the brain by
olfactory receptor neuron (ORN) via olfactory region (Kumar et al.,
2008). However, the presence of a cationic ligand, as chitosan in our
study, promotes nose-to-brain delivery via absorption-mediated trans­
cytosis through BBB for NPs with size less than 500 nm (Kumar et al.,
2013). In this work, PLGA NPs yielded a mean average size of
98.8 nm ± 10.3 nm with low PDI (0.14) and a negative surface charge
in water (−9.28 mV ± 4.45 mV). The negative charge of PLGA nano­
particles is attributed to the carboxyl groups of PLGA and PVA (Guo and
Gemeinhart, 2008). On the other hand, the blend of chitosan and PLGA
resulted in a significant increase in particle size (468.0 nm ± 40.0 nm)
and also in the PDI (0.29). The presence of chitosan in PLGA NPs is
responsible for the high positive charge of the NPs, due to the amino
groups of chitosan. The highly positive surface charge (+54.4 mV ±
2.6 mV), suggested that nanoparticle suspensions were stable and
protected from aggregate formation (Sharma et al., 2015). The en­
capsulation of RH did not affect significantly the hydrodynamic
A.-T. Chatzitaki, et al. International Journal of Pharmaceutics 589 (2020) 119776

Fig. 1. TEM images of NPs dispersed in pyrogen-free water. A) and B) PLGA NPs; C) and D) PLGA/chit NPs.

diameter, the polydispersion and the surface charge of NPs, which

followed the same tendency as the unloaded NPs (Supplementary ma­
terial, Table S1). Particle size measurements were further corroborated
with TEM imaging of the unloaded NPs revealing their spherical shape
(Fig. 1).

3.2. Chitosan blend enhanced water permeation resulting in higher in vitro

RH release

As previously showed, PLGA and PLGA/chit NPs were successfully

produced by nanoprecipitation. The production yield achieved for the
PLGA and PLGA/chit NPs was 60.8 ± 4.0 % and 33.6 ± 4.9 %, re­
spectively. These values were slightly increased when RH was loaded
into NPs (66.0 ± 9.3 % and 45.7 ± 7.9 %, respectively). Considering
RH loading efficiency (LE), the methodology employed allow to have
5.4 ± 0.7 % LE on PLGA NPs and 5.7 ± 2.5 % on PLGA/chit NPs. Fig. 2. In vitro RH release profiles from RH loaded NPs in SNES. Data are
These low values might be attributed to the hydrophilic nature of the presented as mean ± SD and , n ≥ 3 (more than 3 independent experiments).
RH resulting in low LE during the production process (Govender et al.,
1999).
3.3. Thermal analysis
The in vitro release pattern of RH loaded NPs was evaluated in SNES
(pH 5.5) at 35 ± 0.2 °C for 24 h and results are shown in Fig. 2. RH -
DSC studies were performed for the raw materials and lyophilized
PLGA/chit NPs exhibited a biphasic release profile with a burst RH
NPs and the illustrative spectra are presented in supplementary mate­
release of 34 % in the first 5 min, probably due to the surface adsorbed
rial, Figure S1. PLGA 75:25 polymer exhibited a glass transition at
RH on NPs, followed by a sustained release during 24 h. RH loaded
54.7 °C. PVA had a glass transition temperature at 60 °C and melting
PLGA/chit NPs achieved a release of 89 % of the total drug content,
point at 193 °C. For chitosan an endothermic peak at 100 °C was ob­
whereas only 20 % of RH was released from RH - PLGA NPs. The slow
served due to polymer’s dehydration, as it has been previously de­
release of RH from PLGA NPs might be attributed to the hydrogel ob­
scribed elsewhere (Neto et al., 2005). A sharp endothermic peak at
stacle that PVA residuals form (Sahoo et al., 2002) and to the crystalline
248.1 °C was observed for pure RH. The DSC thermograms of the lyo­
nature of PVA and PLGA polymers. On the other hand, the addition of
philized formulations with and without drug presented an endothermic
the hydrophilic chitosan favors the hydration of the polymeric blend
peak in the temperature range of 50 °C to 100 °C due to the dehydration
matrix and promotes the diffusion of the drug, therefore enhancing RH
of the polymer’s (PVA) hydrophilic moieties (Azevedo et al., 2011; El-
release (Mohammed et al., 2017).
Houssiny et al., 2016) and one endothermic peak at 193 °C corre­
sponding to the melting point of PVA. On the other hand, the absence of

5
A.-T. Chatzitaki, et al.
Fig. 3. Stability of unloaded and RH loaded PLGA and PLGA/chit NPs. A) Average size (left y-axis) and PDI (right y-axis) and B) ζ-potential of NP suspensions
(pyrogen-free water) after production and 3 months after storage at environmental conditions (25 ± 2 °C, 60 ± 5 % RH). Data are presented as mean ± standard
deviation (SD), n ≥ 3 (minimum of 3 independent batches).
the drug’s characteristic peak, suggests either the presence of RH in an
amorphous state in the NPs or that the drug concentration was lower
than the limits of equipment detection (Patil and Surana, 2013).
3.4. Stability studies revealed that RH loaded NPs were stable over a period
of 3 months
Stability studies were performed at 25 °C ± 2 °C and 60 % ± 5 %
RH over a period of 3 months. As demonstrated in Fig. 3 and in sup­
plementary Figure S2 (size distribution), no significant changes were
observed in the physicochemical characteristics of the NPs (size, PDI
and ζ-potential) showing good stability over a period of three months.
Despite both formulations presented comparable stability, different
mechanisms might be present. For PLGA NPs stability is likely attrib­
uted to PVA stabilization due to steric repulsion of the adsorbed sta­
bilizer at their surface (Gossmann et al., 2015). On their turn, for PLGA/
chit NPs the repulsive forces originated from the high surface charge
might be the major stabilizer (Sharma et al., 2015).
3.5. Chitosan presence in PLGA/chit NPs promotes mucoadhesion
The mucoadhesion ability of PLGA and PLGA/chit NPs was in­
directly evaluated by assessing the ability of the NPs to adsorb mucin, a
protein typically secreted by mucosal epithelial tissues (Takeuchi et al.,
2005). The surface charge of NPs and mucin was determined in SNES
prior to incubation of their mixture. Following the incubation and the
removal of non-adsorbed mucin, ζ-potential of NPs in SNES were re­
corded. The presence of mucin had little or no effect on the surface
negative charge of PLGA NPs (p > 0.05), as indicated in Table 2,
possibly due to their anionic charge and the repulsive forces with mucin
(Takeuchi et al., 2003). On the contrary, the change of the ζ-potential of
PLGA/chit NPs towards negative values indicated the development of
electrostatic interactions between the negatively charged sialic acid
residues of the mucin glycoprotein and the positively charged amino
Table 2
Assessment of the mucoadhesive properties of PLGA and PLGA/chit NPs
through ζ-potential measurements. NPs were suspended in SNES and incubated
for 1 h with mucin solution. ζ-potential measurements were performed before
and after incubation. Data (expressed in mV) are presented as mean ±
standard deviation (SD), n ≥ 3 (minimum of 3 independent batches).
Formulation ζ-potential (mV)
NPs suspended in SNES After 1 h incubation with mucin
in SNES
PLGA NPs −3.68 ± 1.90 −4.80 ± 1.26
PLGA/chit NPs +10.45 ± 2.04 −2.03 ± 0.78
6
International Journal of Pharmaceutics 589 (2020) 119776
groups of chitosan, which may favor the interpenetration of the chit­
osan NPs and mucin. Apart from the electrostatic interactions, being the
major underlying mechanism of chitosan mucoadhesion, hydrophobic
and hydrogen bonding interactions have been also found to contribute
to the mucoadhesive properties of the cationic polymer (M Ways et al.,
2018). Similar results were previously reported, where chitosan coated
PLGA NPs have higher contact with intestinal mucosa (de Lima et al.,
2018) and chitosan coated liposomes developed ionic interactions with
the negatively charged mucus layer (Takeuchi et al., 2003) suggesting
the potential mucoadhesive properties of the chitosan coated NPs.
3.6. Only PLGA/chit NPs at high concentration induced a hemolytic effect
Hemolysis is the result of decomposition of erythrocytes membrane
and release of hemoglobin, which is oxidized to methemoglobin and at
the end is converted to cyanmethemoglobin (Dobrovolskaia et al.,
2008). Hemolytic values above 5 % were considered as hemolysis, as
suggested by the American Society for Testing and Materials Interna­
tional (ASTM, 2013) (da Silva et al., 2019).
From the formulations tested, only blank PLGA/chit NPs, at the
higher (1700 μg/mL) concentration tested, induced hemolysis
(∼18 %). All other formulations had an hemolytic value below 5 % in
the concentration rage tested (417–1700 µg/mL). These results are il­
lustrated in supplementary Figure S3. Regarding PLGA NPs, we can
hypothesize that the negative surface charge contributed to the lack of
hemolytic effect. Instead, the high positive charge of PLGA/chit NPs
from the presence of amino groups of chitosan could be responsible for
the hemolytic action (Dobrovolskaia et al., 2008). Similar results to the
ones observed by us can be found in scientific literature. The absence of
hemolytic action of PLGA NPs after different incubation’s time with red
blood cells in the concentration of 3000 μg/mL (Fornaguera et al.,
2015) and in another study, NPs modified with chitosan derivatives
incubated (range of 250 μg/mL to 2000 μg/mL), caused hemolysis (Zhu
et al., 2007). On the other hand, the encapsulation of RH diminished
the hemolytic action of PLGA/chit NPs. This result is in agreement with
Zhu et al. (2007), who referred the diminishing of hemolytic action of
cationic NPs after insulin encapsulation, due to the development of
electrostatic interactions between NPs and insulin instead NPs and er­
ythrocytes (Zhu et al., 2007). In the present study, the absence of he­
molytic effect of the RH loaded PLGA/chit NPs may also be explained
by the possible presence of the RH on the surface of the particles which
hides the effect of chitosan.
3.7. Only PLGA/chit NPs induce a decrease in the cellular viability of
PBMCs and RAW 264.7
Peripheral blood mononuclear cells (PBMCs) contain the precursors
A.-T. Chatzitaki, et al.
Fig. 4. Cytotoxicity assays (MTT) performed in A) PBMCs and B) RAW 264.7 cell line, after 24 h of incubation with NPs without and with drug. Data are presented as
mean ± SEM, n ≥ 4 (four or more independent experiments, each in triplicate).
of important immune cells present on mucosal surfaces. The macro­
phages in particular, can be found in the nasal cavity, presenting a
major role in mucosal immunity (Kiyono and Fukuyama, 2004).
Therefore, PBMCs isolated from human blood and Raw 264.7 murine
macrophages were treated with different doses of unloaded and RH
loaded PLGA and PLGA/chit NPs as well as RH solution in order to
investigate the influence of these materials on cellular viability of cells.
Results in Fig. 4A illustrate that PLGA NPs and RH loaded PLGA NPs did
not cause a decrease in cellular viability in PBMCs in all tested con­
centrations, since cell viability was above 70 % of the control (Paul
et al., 2013) and the same happened with RH solution. On the other
hand, a decrease in cell viability was induced by PLGA/chit NPs (un­
loaded and loaded), probably due to NPs positive surface charge (Deka
et al., 2016) and apoptotic and membrane rupture properties of chit­
osan (de Lima et al., 2018). This decrease allowed to calculate the half
maximal inhibitory concentration (IC50) induced by unloaded and RH
loaded PLGA/chit NPs, at 1414 μg/mL and 1086 μg/mL, respectively.
Similar results were found in Raw 264.7 cells (Fig. 4B). Again, PLGA
NPs and RH solution, in concentration range of 1.8 μg/mL to 1875 μg/
mL did not induce cell death. On its turn, unloaded and RH loaded
PLGA/chit NPs induced a decrease in cell viability at concentrations
higher than 14.8 μg/mL, with the IC50 calculated at 19.3 μg/mL and
15.6 μg/mL, respectively. The difference in the IC50 of PLGA/chit NPs,
observed between PBMCs and Raw 264.7, may be explained by the
sensitivity of macrophage cell line, which is related to their higher
degree of cellular differentiation (Pilling et al., 2017; Way et al., 2009).
7
International Journal of Pharmaceutics 589 (2020) 119776
3.8. The presence of chitosan in the NPs increases the NPs cellular
interaction, but does not increase the cellular uptake
Protein adsorption studies to NPs surface were initially performed
with different proteins including BSA. From the range of protein con­
centrations tested (10 μg/mL to 100 μg/mL), the results revealed that
the highest protein adsorption efficacy was observed at the ratio
100:960 (µg protein:µg NPs) (Supplementary material, Table S2, Figure
S4). Therefore, this ratio was used to prepare the protein loaded NPs for
the cellular (PBMCs) uptake studies. For that, we have used BSA-FITC
adsorbed PLGA and PLGA/chit NPs, as well as rhodamine B loaded
PLGA and PLGA/chit NPs. Results from BSA-FITC adsorbed NPs are
illustrated in Fig. 5. Both PLGA and PLGA/chit NPs enhanced the up­
take of BSA-FITC. Nonetheless, PLGA/chit NPs induced a higher uptake,
which may be due to the electrostatic interactions between the positive
charged NPs surface and the negatively charged cell membrane. The
positive effect of chitosan on cellular uptake has already been described
by Sheng et al. (2015), who used trimethyl chitosan coated PLGA NPs
and reported the enhancement of cellular uptake in comparison to
uncoated PLGA NPs (Sheng et al., 2015). To distinguish between the
amount of BSA-FITC that was effectively internalized by the cells and
the BSA-FITC that was adsorbed at the cell surface, the cells were
treated with trypan blue to quench the extracellular fluorescence
(Fig. 5A). With this approach, the decrease in the fluorescence intensity
revealed that BSA-FITC and hypothetically the NPs were mainly ad­
sorbed at cell surface as previously reported elsewhere for chitosan NPs
(Huang et al., 2004). These results were confirmed by a second
A.-T. Chatzitaki, et al. International Journal of Pharmaceutics 589 (2020) 119776

Fig. 5. Evaluation of cellular uptake of BSA-FITC adsorbed NPs by PBMCs. A) Total uptake and cellular uptake by lymphocytes and monocytes after incubation with
protein adsorbed NPs1 for 2 h. Bars represent mean ± SEM, n = 3. B) Confocal images of PBMCs after incubation with BSA-FITC adsorbed NPs (green). Cells were
labeled with Hoechst 33342 (blue) to stain the nucleus and Alexa 594 to identify cell membrane. Scale bar: 5 μm.

technique, the confocal laser scanning microscopy (CLSM) (Fig. 5B).
The MTT assay was carried out after 2 h incubation of cells with BSA-
FITC NPs, to elucidate the effect of these NPs under the conditions of
the uptake studies. Results revealed that cell viability during the assay
was superior to 70 % of control for all the formulations tested.
Rhodamine-B loaded PLGA and PLGA/chit NPs were prepared to
study the delivery of RH and also to follow the NPs location after in­
cubation with cells. Both NPs were able to be internalized by lympho­
cytes and monocytes (Fig. 6A). Despite we did not exclude the extra­
cellular signal of Rhodamine-B as performed with trypan blue for FITC
signal, confocal images revealed the cell cytoplasm full of Rhodamine-B
loaded NPs (Fig. 6B). These images support the conclusion that drug

8
A.-T. Chatzitaki, et al. International Journal of Pharmaceutics 589 (2020) 119776

Fig. 6. Assessment of cellular uptake of different Rhodamine-B loaded NPs by PBMCs. A) Cellular uptake by lymphocytes and monocytes after incubation with
Rhodamine B loaded PLGA and PLGA/chit NPs for 2 h. Bars represent mean ± SEM, n = 3 (3 independent blood donors). B) Confocal images of PBMCs after
incubation with Rhodamine-B loaded NPs2 (red). Cells were labeled with Hoechst 33342 (blue) to stain the nucleus. Scale bar: 10 μm.

loaded PLGA and PLGA/chit NPs were able to enter into cells, where
they have the opportunity to efficiently delivering the loaded cargo. As
described for BSA-FITC loaded NPs, Rhodamine-B loaded PLGA and
PLGA/chit NPs did not induce cellular death, as determined with the
MTT assay at the conditions of experiment.
In conclusion, both delivery systems had a similar ability to promote
the intracellular delivery of proteins and hydrophilic drugs, but PLGA/
chit NPs showed a higher ability to interact with the cellular mem­
branes, due to electrostatic forces between positive charge of chitosan
and anionic cell surface characteristic.
3.9. None of the developed formulations induces the production of reactive
oxygen species
It has been reported that NPs can induce cells to produce ROS as a
result of metabolic activity (Fu et al., 2014; Kim et al., 2015; Magder,
2006). On a regular basis, there is a defence mechanism in order to
counterbalance ROS production. However, upon stimulus, the over­
production of ROS by activated macrophages can promote oxidative
stress and consistently cell injury (da Silva et al., 2019).
The RAW 264.7 cell line was incubated for 24 h with RH loaded and
unloaded PLGA and PLGA/chit NPs to assess ROS production. Both
formulations, loaded and unloaded did not induce cells to produce ROS
as presented in Fig. 7A. Importantly, by using the same concentrations
for both formulations, we could observe that PLGA NPs (loaded and
unloaded) at the higher tested concentrations did not induce cellular
death, while PLGA/chit NPs (loaded and unloaded), decreased cellular
viability near to 70 % at 100 μg/mL (Fig. 7B). The absence of ROS
production in this concentration, might suggest that PLGA/chit NPs
induced cellular death is not dependent on ROS signaling and may be
attributed to apoptosis or cell membrane rupture, induced by chitosan
(de Lima et al., 2018).

9
A.-T. Chatzitaki, et al.
3.10. Ex vivo permeability studies
Ex vivo permeation studies with RH loaded NPs were performed
across sheep nasal mucosa and results are depicted in Table 3 and
Fig. 8. The steady state diffusion coefficient was calculated to be
0.16·10−6 cm2 min−1 for the PLGA NPs and 0.52·10−6 cm2 min−1 for
the PLGA/chit NPs. A slow drug permeation profile across nasal mucosa
was observed for the RH loaded PLGA NPs (5.5 µg/cm2 in 4 h), which is
in accordance with the slow in vitro release of RH from the same NPs,
indicating that drug diffusion from the NPs might be the rate limiting
factor for drug permeation across the tissue (Seju et al., 2011). On the
contrary, PLGA/chit NPs exhibited a higher permeation profile (32 µg/
cm2 in 4 h) resulting in a 3.22-fold increase of apparent permeability
coefficient from 2.16 ± 0.4·10−6 cm min−1 for PLGA NPs to
6.96 ± 2.7·10−6 cm min−1. In comparison to previous work from our
group on RH loaded PLGA microparticles containing TMC (Karavasili
et al., 2016), the RH – PLGA/chit NPs exhibited a better permeability
profile. In detail, these NPs induced a 1.68-fold increase in RH per­
meability when compared to the microparticles and a 3.96-fold increase
when compared to n RH solution (Karavasili et al., 2016).
In reports from Pardeshi et al. (2013), solid lipid NPs and lipid
combined with polymer NPs loaded with RH were evaluated regarding
their permeation properties across sheep nasal mucosa. Their results
Table 3
Steady state flux (Jss) and apparent permeability values (Papp) of RH-PLGA NPs
and RH-PLGA/chit NPs across sheep nasal mucosa. Results are the mean
value ± SD, n ≥ 3.
Jss (μg cm−2 min−1) Papp (10−6 cm min−1)
RH – PLGA NPs 0.056 ± 0.01 2.16 ± 0.4
RH – PLGA/chit NPs 0.18 ± 0.07 6.96 ± 2.7
10
International Journal of Pharmaceutics 589 (2020) 119776
Fig. 7. ROS assay. A) ROS production
after 24 h incubation of RAW 264.7
macrophage cell line with different
concentrations of unloaded and RΗ
loaded NPs. LPS (1 µg/mL) was used as
a positive control and unstimulated
cells as a negative control. Results are
presented in fluorescence increase fold
compared to the negative control. B)
Cell viability assay (MTT) after the
performance of ROS production assay.
****p < 0.0001.
confirmed lipid-based NPs and lipid-polymer NPs as efficient and safety
carriers, releasing more than 61 % RH across sheep nasal mucosa
(Pardeshi et al., 2013a,b). On their turn, Patil and Surana (2013) de­
veloped PLGA NPs for the nasal administration of RH and showed
significantly higher ex vivo drug permeation across sheep nasal mucosa
(97.9 ± 2.9 %) compared to RH solution (74.43 ± 4.6 %) (Patil and
Surana, 2013). Unfortunately, no direct comparisons can be performed
with our results since both authors present the percentage of permeated
RH (from the total added) instead of μg of drug permeated per mucosal
surface area (cm2).
It has been previously reported that the mechanism of RH per­
meation following intranasal administration occurs through the para­
cellular pathway, passing through the hydrophilic passages between
adjacent epithelial cells (Pardeshi and Belgamwar, 2016). Nevertheless,
the higher RH permeability observed for the PLGA/chit NPs could be
attributed not only to the presence of chitosan, acting as both a per­
meation enhancer and an opener of the tight junctions of the nasal
epithelium (Khan et al., 2010; Mistry et al., 2009), but also to the higher
drug release from the chitosan coated NPs. In addition to that, it has
been previously shown that NPs with size less than 500 nm, as in the
present study, interact efficiently with the mucus layer of the nasal
cavity, therefore increasing NP retention time and hence drug release
and absorption (Sonvico et al., 2018). Similar observations were made
for chitosan-grafted PLGA NPs with a particle size of 463.9 ± 12 nm
formulated for the intranasal delivery of chloropromazine (Chalikwar
et al., 2013).
The effect of the NP-based formulations on tissue integrity was as­
sessed with histopathological analysis after the end of the ex vivo per­
meability studies. Untreated epithelium was used as the control. No
significant differences on mucosal morphology were observed after
tissue treatment with RH loaded PLGA and PLGA/chit NPs compared to
the control, whereas only PLGA NPs were found to induce a minor
A.-T. Chatzitaki, et al.
Fig. 8. Ex vivo permeability studies of RH loaded from PLGA and PLGA/chit NPs across sheep nasal mucosa. A) Permeability studies across nasal mucosa at several
time points (PLGA NPs in red color, PLGA/chit NPs in blue color). Results are presented as mean value ± SEM, n ≥ 3. Histopathological studies of (B) untreated and
(C) treated nasal mucosa with RΗ loaded PLGA NPs and (D) RΗ loaded PLGA/chit NPs after 4 h incubation. Scale bar: 100 μm.
epithelial superficial desquamation. The integrity of the epithelial layer
and the presence of goblet cells and mucus layer indicated the absence
of harmful properties of both formulations.
4. Conclusions
In the present work, mucoadhesive polymeric PLGA/chit NPs were
efficiently developed through a simple nanoprecipitation technique for
the nasal administration of RH. The drug loaded PLGA/chit NPs had
acceptable particle size and a strong positive charge, rendering them
highly mucoadhesive, compared to PLGA NPs, as indicated by the in
vitro mucin adsorption studies. In vitro drug release and ex vivo per­
meability studies through nasal mucosa revealed a significantly faster
RH release and a 3.22-fold enhancement in the Papp of RH from the
PLGA/chit NPs, while the presence of chitosan also favored PBMC
cellular interaction. Although PLGA/chit NPs demonstrated in vitro
lower biocompatibility, toxic effects induced were dose dependent, and
observed only at very high concentrations, not relatable to in vivo do­
sages. Overall, these results show that RH loaded PLGA/chit NPs is a
valuable delivery system that needs to be tested in a suitable animal
model through nasal administration. These results are the basis of hope
needed to find new therapeutic approaches for Parkinson’s disease.
CRediT authorship contribution statement
Aikaterini-Theodora Chatzitaki: Investigation, Writing - original
draft. Sandra Jesus: Methodology, Validation. Christina Karavasili:
Methodology. Dimitrios Andreadis: Methodology. Dimitrios G.
Fatouros: Conceptualization, Validation, Resources, Supervision. Olga
Borges: Conceptualization, Validation, Resources, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
11
International Journal of Pharmaceutics 589 (2020) 119776
influence the work reported in this paper.
Acknowledgements
This work was financed by the European Regional Development
Fund (ERDF), through the Centro 2020 Regional Operational
Programme and through the COMPETE 2020 – Operational Programme
for Competitiveness and Internationalization and Portuguese national
funds via FCT – Fundação para a Ciência e Tecnologia, under project
PROSAFE/0001/2016, POCI-01-0145-FEDER-030331 and the strategic
project UIDB/04539/2020. The authors thank Dr. Ana Donato for as­
sistance in hemolysis studies in the Clinical Analysis Laboratory of the
Faculty of Pharmacy of Coimbra University (Portugal), Dr. Mónica
Zuzarte for the TEM microscopy observations performed at
iLAB—Bioimaging Laboratory of the Faculty of Medicine of the
University of Coimbra and Dr. Luísa Cortes for the Confocal microscopy
analysis performed at the Microscopy Imaging Center of Coimbra, at the
Center for Neuroscience and Cell Biology.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2020.119776.
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