pubs.acs.

org/molecularpharmaceutics Article

Assessment of Amphiphilic Poly‑N‑vinylpyrrolidone Nanoparticles’

Biocompatibility with Endothelial Cells in Vitro and Delivery of an
Anti-Inflammatory Drug
Aikaterini Berdiaki, Emmanouela Perisynaki, Antonios Stratidakis, Pavel P. Kulikov, Andrey N. Kuskov,
Polychronis Stivaktakis, Petra Henrich-Noack, Anna L. Luss, Mikhail M. Shtilman, George N. Tzanakakis,
Aristidis Tsatsakis, and Dragana Nikitovic*
Cite This: Mol. Pharmaceutics 2020, 17, 4212−4225 Read Online
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sı Supporting Information

ABSTRACT: Nanoparticles (NPs) produced from amphiphilic

derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of
various molecular weight polymeric hydrophilic fragments linked
into hydrophobic n-alkyl chains of varying lengths, were previously
shown to exert excellent biocompatibility. Although routes of
administration can be different, finally, most nanosystems enter the
blood circulation or lymphatic vessels, and by this, they establish
direct contact with endothelial cells. In this study, Amph-PVP NPs
and fluorescently labeled Amph-PVP-based NPs, namely “PVP”
NPs (Amph-PVP-NPs (6000 Da) unloaded) and “F”-NPs (Amph-
PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1
endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by
immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells.
The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as
shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range.
Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by
assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were
affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this
study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug
carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In
summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological
conditions. Amph-PVP NPs are a promising drug delivery system.
KEYWORDS: poly-N-vinylpyrrolidone, amphiphilic polymer, nanoparticle, endothelial cells, assessment, viability,
immunological activation, fluorescent probes

1. INTRODUCTION plethora of hydrophobic drugs is accomplished. The formation

Nanoparticles (NPs) are ultrafine units with dimensions of drug-carrier complexes enhances their biological compati-
measured in nanometers. Based on their morphology, shapes, bility, stability, and, importantly, bioavailability in aqueous
or sizes, they are classified into different categories. The biological solutions.7,8 The preliminary examination of Amph-
application of NPs systems in biomedicine is increasingly PVPNPs, in vitro and in vivo, demonstrated no adverse effects
popular due to their ability to deliver various drugs to specific on cell viability and satisfactory biocompatibility verifying,
biological targets, resolving, thus, unmet medical and thus, their application potential.8−10 Noteworthy, the utiliza-
pharmaceutical needs.1−4 Our previous studies have focused tion of some poly-N-PVP-based carriers has been approved for
on synthesizing a novel drug delivery system based on NPs
produced from amphiphilic derivatives of poly-N-vinylpyrroli- Received: June 25, 2020
done (Amph-PVP) composed of various molecular weight Revised: September 27, 2020
polymeric hydrophilic fragments which were linked into Accepted: September 28, 2020
hydrophobic n-alkyl chains of varying lengths.5,6 Importantly, Published: September 28, 2020
we have also shown that upon self-assembly of such core−shell
polymeric NPs in aqueous media, an efficient entrapment of a

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.molpharmaceut.0c00667

4212 Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
clinical studies.11 Importantly, nanocarrier-drug complexes are
delivered via various routes of administration, including
inhalation, transdermal application, oral, and injection into
blood vessels.12 In all cases, the NPs are eventually taken up by
blood or lymphatic vessels and enter the circulatory system.
Thus, the endothelial cells lining blood vessels are directly
exposed to NPs, which can result in possible deleterious
consequences regarding these cells’ homeostasis and immuno-
logical response.13−16 Endothelial cells create a semiselective
barrier that segregates the blood contents from the
parenchyma of peripheral organs and tissues, exhibiting a
pivotal role in propagating vascular system homeostasis
throughout the whole body.17 Primarily, the microvascular
endothelial cell barrier regulates crucial functions of tissues/
organs and enables homeostasis.18 The endothelial cell layer is
intimately associated with specific structures of the extrac-
ellular matrix (ECM) denominated basement membranes,
which contribute to the barrier function.19,20
The endothelium undergoes changes in function and
morphology, known as endothelial cell activation, to
participate in the inflammatory response.21,22 These changes
include the loss of vascular integrity, the expression of adhesion
molecules, cytokine production, and the upregulation of
human leukocyte antigen (HLA) molecules. The upregulation
of adhesion molecules, such as ICAM-1 (intercellular adhesion
molecule-1), VCAM-1 (vascular cell adhesion molecule-1),
and E-Selectin allows the adherence of monocytes on the
endothelium and their enhanced transmigration to the
subendothelium.23 Also, endothelial cells increase their
cytokine production, such as interleukins and tumor necrosis
factors, which further upregulate the immune response,
including leukocyte trafficking.24
Furthermore, platelets interact with an intact activated
endothelium, partially due to the attenuation of physiological
inhibitory mechanisms and partially due to de novo expression
of adhesion molecules on the surfaces of activated endothelial
cells and platelets. The above enhance the risk of thrombocytic
events.25 Importantly, patients with cardiovascular disease
whose endothelium function is dysregulated per se are
especially sensitive to added disturbances of endothelial
function.26
Therefore, it is of utmost importance to evaluate the
interaction of NPs with the healthy and activated endothelial
barrier. In this study, the effects of Amph-PVP-NPs (6000 Da)
(PVP) on viability and immune response of basal state
endothelial cells and an LPS-challenged model of activated
endothelium were characterized for the first time. For this
purpose, unloaded PVP-NPs (6000 Da) (PVP) and PVP-NPs
(6000 Da) loaded with indomethacin were produced. The
uptake of PVP-NPs by resting and activated endothelial cells
was assessed by separately synthesized fluorescently labeled
PVP (6000 DA) (F-NPs).
2. MATERIALS AND METHODS
2.1. Materials. N-Vinyl-2-pyrrolidone (VP), 2,2′-azobis-
(isobutyronitrile) (AIBN), 1,4-dioxane, and 6-hexane diamine
were obtained from Acros Organics (Thermo Fisher Scientific,
Geel, Belgium). Acrylic acid (AA), 5(6)-carboxyfluorescein
diacetate N-succinimidyl ester (CFSE), succinimide, N,N′-
dicyclohexylcarbodiimide (DCC, 1.0 M solution in methylene
chloride), octadecylmercaptan (ODM), 1,6-diphenyl-1,3,5-
hexatriene (DPHT), fluorescein 5-isothiocyanate (FITC),
and 3-chloroperoxybenzoic acid, Dulbecco’s Phosphate-Buf-
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fered Saline (DPBS), ethyl acetate, methanol, ethanol,
dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS),
(ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid) buffer (EGTA), indomethacin, and all other chemicals
used in this study were purchased from Sigma-Aldrich (Sigma-
Aldrich, St. Louis, MS, USA) unless otherwise specified and
used without further purification. Analytical grade preparations
were used for all solvents and buffer solution components. The
Millipore Milli-Q plus System (Merck KGaA, Darmstadt,
Germany) was used for the preparation of distilled−deionized
water.
2.2. Synthesis of Amphiphilic Poly-N-vinylpyrroli-
done-Based Polymeric Materials. Amphiphilic poly-N-
vinylpyrrolidone consisting of a PVP hydrophilic fragment
with a molecular weight of 6000 Da and an octadecyl
hydrophobic terminal fragment-namely, PVP (6000 Da), was
synthesized (Figure 1) and characterized, including TEM
analysis, as described in the previous studies.8,10
Figure 1. Synthesis scheme of amphiphilic poly-N-vinylpyrrolidone.
Briefly, 10.0 mL of N-vinyl-2-pyrrolidone was dissolved in 20
mL of 1,4-dioxane. Octadecyl mercaptan (0.286 g) and 0.107 g
of 2,2′-azobis(isobutyronitrile) (AIBN) were sequentially
added, and the reaction mixture was stirred at 70 °C for 3 h.
Afterward, 50 mL of distilled water was added, and the
resulting crude polymer mixture was purified by dialysis against
water (Slide-A-Lyzer Dialysis Flask, 2K MWCO, Thermo
Scientific, USA) and was lyophilized (Alpha 1-4 LD plus,
Martin Christ, Germany) before storing. 13C NMR (Figure 1),
IR-spectroscopy analysis (Figure 2), and 1H NMR (Supple-
Figure 2. 13C NMR spectra of amphiphilic poly(N-vinyl-2-
pyrrolidone) PVP-OD6000.
mentary Figure S1) determined the structure of the PVP-
OD6000 polymer (Figure 3). The average molecular weight of
the polymer is 6 kDa, whereas the hydrophobic fragment is
octadecyl (Figures 1 and 2).
The amphiphilic polymer’s critical aggregation concentra-
tion (CAC) was determined by fluorescence spectroscopy
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Molecular Pharmaceutics
Figure 3. IR spectra of amphiphilic poly(N-vinyl-2-pyrrolidone) PVP-OD6000. The average molecular weight of the polymer is 6 kDa, whereas the
hydrophobic fragment is octadecyl.
(Hitachi 650-10S, Japan) by using 1,6-diphenyl-1,3,5-hexa-
triene (DPHT) as the fluorescent probe.
The polymer NPs were prepared via the emulsification
method with solvent evaporation. Briefly, Amph-PVP was
dissolved in a minute amount of ethyl acetate to obtain a
solution which was then emulsified in an aqueous phase by
ultrasonic treatment (Sonoplus HD 2070, Bandelin, Ger-
many). The colloidal system resulting after removing the
organic solvent was concentrated by evaporation using a rotary
evaporator, Laborota 4010 (Heidolph, Germany). The
polymer NP solution was frozen and lyophilized using an
Alpha I-4LD freeze-dryer system (Martin Christ GmbH,
Germany) to obtain freeze-dried, solid NP. These NPs were
then resuspended in PBS (pH 7.4).
Indomethacin loaded PVP-NPs (6000 Da) were prepared
following the same method as for PVP-NPs (6000 Da), by
adding the appropriate amount of indomethacin to the initial
polymer solution in the organic solvent, in this case, methanol,
followed by an oil-in-water (O/W) emulsification, solvent
evaporation, freeze-drying, and resuspension in PBS. The
amount of IMC loaded in the NPs was determined by
measuring the UV absorbance at 318 nm using a Unico 2802
spectrophotometer (Unico, USA). The IMC content intro-
duced into the hydrophobic core of the NPs was evaluated
from the amount of drug incorporated in nanoparticles and the
total weight of drug-loaded NPs form using the following
equation:
weight of IMC in nanoparticles
IMC content (%) =
total weight of IMC loaded nanoparticles
The Drug Loading Efficiency (DLE) was evaluated using the
following equation:
weight of entrapped IMC in nanoparticles
DLE (%) =
initial weight of IMC used
× 100
Differential scanning calorimetry (DSC) and thermogravimet-
ric analysis (TGA), presented in Supplementary Figures S2
and S3, confirmed the absence of solvent residues in the final
pubs.acs.org/molecularpharmaceutics Article
preparations. For further investigations, all NPs samples were
resuspended in water or PBS (pH 7.4). The particle size
distribution and ζ-potential were determined by the dynamic
light scattering (DLS) method (Malvern Zetasizer Nano ZS,
UK).
2.3. Preparation and Characterization of PVP-NPs
(6000 Da) Carrying Fluorophores. F-NPs were prepared by
using an emulsification method. More specifically, appropriate
amounts of the PVP (6000 Da) polymer were dispersed in a
specific volume of H2O, and specific amounts of FITC were
dissolved in the organic solvent. In this case, chloroform,
followed by an oil-in-water (O/W) emulsification, solvent
evaporation, and freeze-drying.
The particle size distribution and ζ-potential were
determined by the dynamic light scattering (DLS) method
(Malvern Zetasizer Nano ZS, UK).
2.4. Cell Culture. In this study, the HMEC-1 human
dermal microvascular endothelial cell line was utilized. Cells
were grown in MCDB 131 (Biochrom AG; F0455)
supplemented with 10% fetal bovine serum (FBS; Invitrogen
10500-064; heat-inactivated), glutamine (10 mM; Biosera
XCT1715), hydrocortisone (1 μg/mL), gentamycin (Invitro-
gen; 15710-049), and penicillin/streptomycin (100 units/mL;
Biosera LMA4118). Before the addition of treatments, cells
were cultured in 2% medium for 24 h at 37 °C and 5% CO2.
2.5. Measurement of NP Uptake Utilizing Fluores-
cence. Growing cells were harvested and seeded in black 96-
well plates (Corning; 3603) at a density of 3,500 cells per well
× 100 in 200 μL of MCDB 131 (10% FBS) for 24 h, and then, the
medium was replaced with 200 μL of MCDB 131 (2% FBS)
for 24 h. Treatments were added for the next 48 h at 37 °C and
5% CO2 in 2% FBS medium. The cells were washed, and lysis
buffer was added before fluorescence measurements were
performed using the CyQUANT fluorometric assay (Thermo
Scientific; C7026) according to the manufacturer’s instruc-
tions. A standard curve of NP serial dilutions was calculated
and was used to convert fluorescence units to mg/mL of NPs
in the cell lysates. Fluorescence was measured in a fluorometer
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Molecular Pharmaceutics
Table 1. Characteristics of PVP (6000 Da), Indomethacin-Loaded PVP (6000 Da), and F NPs (Mean ± SD, n = 3) in PBS at
37 °C
particle size
NPs (nm)
hollow PVP (6000 Da) nanoparticles 178 ± 10
indomethacin loaded PVP (6000 Da) nanoparticles 194 ± 11
FITC loaded PVP (6000 Da) nanoparticles 180 ± 8
(F nanoparticles)
Figure 4. Structure of F-NPs. Structure of F-NPs - FITC dye molecules is encapsulated into Amph-PVP-NPs (6000 Da).
(Biotek, Winooski, Vermont, USA) using the proposed
excitation (494 nm) and emission filters (518 nm).
2.6. ΜΤΤ Assay. Growing cells were harvested and seeded
in 98-well plates (Corning; 3603) at a density of 15,000 cells
per well in 200 μL of MCDB 131 (10% FBS) for 24 h, and
then, the medium was replaced with 200 μL of MCDB 131
(2% FBS) for 24 h. Treatments were added for the next 48 h at
37 °C and 5% CO2 in 2% FBS medium. The labeling of the
cells and the measurements were performed according to the
manufacturer’s instructions (ThermoFisher, Vybrant ΜΤΤ
Assay).
2.7. Western Blot. Harvested cells were lysed with RIPA
solution (50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxy-
cholate, 150 mM NaCl, 1 mM EDTA with protease and
phosphatase inhibitors). Equal amounts of protein were
subjected to SDS-PAGE using 10% polyacrylamide gels
under reducing conditions. Separated protein bands were
transferred to nitrocellulose membranes in 10 mM (pH 11),
containing 10% methanol. Membranes were blocked overnight
at 4 °C with PBS containing 0.1% Tween-20 (PBS-Tween)
and 5% (w/v) low-fat milk powder. The membranes were
incubated for 1 h at room temperature (RT) with the primary
antibody in PBS containing 0.1% Tween-20 (PBS-Tween) and
1% (w/v) low-fat milk powder. The immune complexes were
detected after incubation with the appropriate peroxidase-
conjugated secondary antibody diluted (1:3,000) in PBS-
Tween, 2% low-fat milk, using the LumiSensorChemilumines-
cent HRP substrate kit (Genscript; L00221 V500), according
to the manufacturer’s instructions. Protein expression of Actin
was used to correct for the amount of each sample analyzed.
pubs.acs.org/molecularpharmaceutics Article
zeta potential indomethacin indomethacin loading
PDI (mV) content (%) efficiency (%)
0.162 ± 0.036 −7.14 ± 0.39
0.168 ± 0.044 −6.35 ± 0.32 20 94.8
0.149 ± 0.041 +8.08 ± 1.29
2.8. Fluorescence Microscopy. HMEC-1 cells were
seeded in 24-well plates, at 150,000 cells/well, and incubated
in complete medium for 24 h. The medium was replaced with
2% FBS medium for 24 h. Treatments were added, and the
cells were incubated for 48 h at 37 °C and 5% CO2. The
medium was removed; the cells were washed twice with 2%
FBS medium and replaced with a fresh one. Pictures were
obtained using a fluorescent microscope (Leica, DM IRE2).
2.9. Confocal Microscopy. HMEC-1 cells were seeded on
round coverslips placed in 24-well plates, at a concentration of
150,000 cells/well, and incubated in complete medium for 24
h. The media was replaced with 2% FBS medium for 24 h.
Treatments were added, and the cells were incubated for 48 h
at 37 °C and 5% CO2. The cells were fixed with 5%
formaldehyde and 2% sucrose in PBS for 10 min at RT. After
three washes with PBS, the permeabilizing agent Triton X100
was added for 10 min. TO-PRO-3 iodide (Molecular Probes;
T3605) diluted 1:500 in deionized H2O was applied for 40
min to stain nuclei. The coverslips were then mounted onto
slides using glycerol and visualized using confocal microscopy
(Leica, TCS SP2 SE).
2.10. Cell Growth Assay. The growth of human
microvascular endothelial HMEC-1 cells was assessed, as
previously reported.10 In short, HMEC-1 growing cells from
nonconfluent cultures were harvested and seeded in black 96-
well plates (Corning; 3603) at a density of 1.000 cells per well
in 200 μL of DMEM (10% FBS). The cell density number was
chosen from optimization experiments. The cells were allowed
to rest overnight. Treatments were added for the next 48 h at
37 °C and 5% CO2 in 0% FBS. The cells were then lysed, and
their number was calculated using the CyQUANT fluoro-
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Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
metric assay (Thermo Scientific; C7026) according to the
manufacturer’s instructions. Fluorescence was measured in a
fluorometer (Biotek, Winooski, Vermont, USA) using the
proposed excitation (485 nm) and emission (528 nm) filters. A
separate standard curve was used to convert fluorescence units
to cell numbers. All experiments were performed in triplicate.
2.11. Statistical Analysis. The statistical significance was
evaluated by the Student’s t test or one way-ANOVA analysis.
3. RESULTS
3.1. Synthesis and Characterization of Amph-PVP
NPs and Loaded with IMC Amph-PVP NPs. In this study,
IMC-loaded NPs were prepared using the amphiphilic polymer
PVP (6000 Da) and the solvent evaporation technique to
examine the influence of drug loading on NP properties
biocompatibility. The freeze-dried IMC-loaded or unloaded
PVP (6000 Da) was resuspended in PBS for further
characterization. The particle size distribution of the prepared
NPs formed from the PVP (6000 Da) polymeric materials was
determined by the DLS method and is presented in Table 1.
The drug loading percentage of the IMC-loaded NPs was 20%
w/w, whereas indomethacin loading efficiency was 94.8%.
3.2. Preparation and Characterization of PVP (6000
Da) Carrying Fluorophores. For better evaluation of the
interactions of PVP NPs with biological systems, fluorescently
labeled NPs were developed. Specifically, F-NPs were
developed by incorporating FITC molecules into the hydro-
phobic core of PVP NPs (6000 Da), as shown in Figure 4.
The concentration of FITC encapsulated into the NPs was
35 mg/g. The CAC of the synthesized PVP (6000 Da)
polymer, determined in the presence of DPHT, was 0.054
mmol/L (Figure 5). The mean particle size of F-NPs was 180
nm (Table 1), while the ζ-potential measured in PBS at +30
°C was +8.08 ± 1.29 mV.
Figure 5. Fluorescence intensity of the solution of PVP-ODM with
DPHT at 25 °C.
3.3. Uptake of F-NPs by Endothelial Cells. To evaluate
the uptake of PVP-NPs (6000 Da), we utilized F-NPs.
Fluorescently labeled NPs were diluted in PBS/cell culture
medium, and the fluorescence of samples was measured. The
sample fluorescence was converted to mg of NPs per ml of
diluted sample (mg/mL) utilizing a concentration curve. The
fluorescence of diluted NPs samples, denominated as arbitrary
units, was verified to be concentration-dependent, as shown in
Figure 6A. To determine the uptake of F-NPs by HMEC-1
endothelial cells, we exposed these cells to respective NPs
during 48 h, at which point the cells were harvested, and the
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fluorescence of cell lysate was measured. This approach
demonstrated that the uptake of F-NPs was concentration-
dependent (Figure 6B). In continuation, we wanted to
examine the uptake of F-NPs by activated endothelial
HMEC-1 cells. It is well established that LPS, when in contact
with endothelial cells, interacts with the TLR4 receptors and
induces their activation.27 A critical downstream mediator of
TLR4 is nuclear factor κβ (NF-κβ). Binding of LPS to TLR4
triggers the activation of phosphokinases, which phosphorylate
the cytoplasmic NF-κβ inducing its translocation to the
nucleus.28 HMEC-1 cells were challenged with LPS (100
μg/mL) during 2 h to generate a model of activated
endothelium. Western blot demonstrated a strong upregulation
of NF-κβ phosphorylation in LPS treated cells verifying their
activation (Figure 6C). Basal state and immunologically
activated HMEC-1 cells were exposed to F-NPs during 48 h,
at which point the cells were harvested and cell lysate was
collected. Measuring fluorescence of respective cell samples
demonstrated an increased uptake of F-NPs at 0.033 and 0.066
mg/mL compared to cells exposed to 0.01 mg/mL (p ≤ 0.01)
of both activated and nonactivated HMEC-1 cells (Figure 6D).
An enhanced uptake of F-NPs was observed in cells challenged
with LPS, compared to the nonactivated HMEC-1 cells (p ≤
0.05) at all concentrations tested (Figure 6D). These results
show not only that the uptake of PVP NPs is dependent on
their concentration but also that the activation status of
endothelial cells affects their uptake.
3.4. Effect of PVP-NPs (6000 Da) on HMEC-1 Cell
Viability at Basal and Activated States. We evaluated the
effect of PVP-NPs (6000 Da) and F-NPs on HMEC-1 cells’
viability utilizing the MTT assay. Cells were seeded at various
concentrations and absorbance, corresponding to the number
of viable cells, measured after 48 h following the
manufacturer’s instructions (Figure 7A). This approach
revealed that the ratio of absorbance/cell number when
utilizing 8000 cells was most suitable for further studies and
was duly used in all experiments. Exposing the HMEC-1 cells
with increasing concentrations (0.01 to 0.066 mg/mL) of both
PVPNPs and F-NPs during 48 h did not affect their viability
(Figure 7B). These experiments demonstrate the biocompat-
ibility of PVP NPs with the endothelial cells even at high
concentrations (66 μg/mL). FITC dye was found not to affect
endothelial cell viability at concentrations utilized (data not
shown).
Under pathological conditions or upon mechanical damage,
the endothelial cells acquire the activated phenotype. Thus, we
generated a model of activated endothelium by exposing
HMEC-1 cells to LPS (100 μg/mL) during 2 h. The LPS
challenge is well established to induce many endothelial cell
biological responses in a manner dependent on NF-kB
downstream signaling.29,30 Therefore, HMEC-1 cells chal-
lenged with LPS during 2 h were exposed to unloaded PVP
NPs and F-NPs for 48 h after which their viability was
evaluated. As shown in Figure 7C, neither unloaded PVP-NPs
(6000 Da) or F-NPs affected the viability of basal and activated
HMEC-1 cells (p = NS).
3.5. Effect of Amph-PVP NPs on HMEC-1 Cells’
Adhesion Molecule Expression. To evaluate the immunos-
timulatory effect of unloaded PVPNPs (6000 Da) and F-NPs
on basal state and activated endothelium, we treated HMEC-1
cells with NPs or with a combination of NPs and LPS at a
concentration verified to induce these cells’ activation (see
Figure 8A). Western blot of treated cells’ extracts showed that
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Figure 6. Uptake of PVP-NPs (6000 Da) by HMEC-1 endothelial cells. (A) Concentration-dependent fluorescence of F-NPs. (B) Uptake of F-
NPs by basal state endothelial cells, expressed as mg/mL of cells lysates. The cells were cultured in 96-well plates and treated with F-NPs for 48 h.
(C) Effect of LPS on HMEC-1 cells NF-κB activation. The position of the nearest respective protein marker band is depicted to the right. (D)
Uptake of F-NPs by activated endothelial cells expressed as mg/mL of NPs in the cells lysate; F+LPS treatments were compared to the respective F
treatments (same concentration). The cells were cultured in 96-well plates, challenged with LPS, and exposed to F-NPs for 48 h. The results
represent the average of three separate experiments. C = control; cells incubated with medium only. Statistical significance: * = p ≤ 0.05, ** = p ≤
0.01.

the exposure to PVP NPs (6000 Da) and F-NPs did not affect
the expression of ICAM-1 and E-selectin adhesion molecules.
As expected, increased expression of ICAM-1 and E-selectin
molecules upon LPS treatment was demonstrated, whereas
coadministration of PVPNPs (6000 Da) or F-NPs did not
further affect the LPS-dependent increase of the particular
adhesion molecules (Figures 8B and 8C). Endothelial cells
have a crucial role in regulating blood vessel tone, monocytes
recruitment, and thrombogenicity so that the dysfunction of
endothelial cells is directly correlated to the pathogenesis of
various diseases.26,31 These data demonstrate the PVP NPs
(6000 Da) do not exert synergistic effects on the dysregulation
of activated endothelium.
3.6. Uptake of PVP-NPs (6000 Da) by Living HMEC-1
Cells. Fluorescence microscopy and F-NPs were utilized to
visualize the uptake of PVP NPs (6000 Da) by living HMEC-1
cells. The FITC dye, which has been incorporated into F-NPs,
4217
fluoresces with green. As shown in Figure 9, a concentration-
dependent increase in the intracellular deposition of FITC
labeled PVP NPs (6000 Da) was determined. Furthermore, the
intensity of the green stain was more intense in activated
endothelial cells, suggesting that upon activation, the uptake of
PVP-NPs (6000 Da) is enhanced. These results confirm that
the ability of HMEC-1 endothelial cells to uptake PVP-NPs
(6000 Da) is dependent on their immunological status.
3.7. Intracellular Localization of PVP-NPs (6000 Da).
The cellular uptake not only is a key event for drug delivery but
also is immediately correlated to the biological consequences
of NPs utilization.3,13 HMEC-1 cells were treated with a high
dose (0.066 mg/mL) of F-NPs and were subsequently
investigated with confocal microscopy to observe the internal-
ization and intracellular localization of PVP-NPs (6000 Da).
Most FITC staining was located to the perinuclear cytoplasmic
space, but diffuse staining to the cytoplasm was likewise
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Figure 7. Effect of PVP-NPs (6000 Da) and F-NPs on the viability of HMEC-1 cells. (A) Optimization of the HMEC-1 cells’ seeding number. (B)
The effect of PVP-NPs (6000 Da) and F-NPs on HMEC-1 cells’ viability. Effect of F-NPs on the viability of LPS-activated HMEC-1 cells. The cells
were cultured in 96-well plates and treated with NPs for 48 h. C = control; cells incubated with medium only. The results represent the average of
three separate experiments. Statistical significance: NS = not significant.
evident (Figure 10A, B). Our data indicate that PVP-NPs
(6000 Da) are compatible with uptake/transport through the
endothelium barrier.
3.8. Effect of IMC-Loaded PVP-NPs (6000 Da) on
HMEC-1 Cell Growth. In continuation, we wanted to evaluate
the effect of a drug-carrier complex on endothelial cell growth
and viability. The effect of IMC-loaded PVP-NPs (6000 Da)
and unloaded PVP-NPs (6000 Da) on the proliferative
capacity of HMEC-1 cells was studied. In the experiments
performed, three different concentrations of 0.01, 0.033, and
0.066 mg/mL of indomethacin, unloaded PVP-NPs (6000
Da), and PVP-NPs (6000 Da) encapsulating Indomethacin
(PVP-IMC) were administered for 48 h, respectively. The
utilization of the CyQuant proliferative assay demonstrated
that the increased concentration of indomethacin inhibits
endothelial cells’ growth at higher concentrations of 0.033 and
0.066 mg/mL. The treatment of HMEC-1 cells with unloaded
PVP-NPs (6000 Da) and PVP-IMC did not lead to statistically
significant changes as compared to control (p = NS). As shown
in Figure 11, indomethacin exerts growth inhibitory and toxic
effects on endothelial cells at higher concentrations (p ≤
0.001). Previously, indomethacin was shown to downregulate
the migration of cancer cells due to dysregulation of the Ca
metabolism32 and to induce injury of endothelial cells by
disturbing mitochondria metabolism,33 well following the
results of the present study. Treating the cells with NPs
loaded with indomethacin at the low concentration (0.01 mg/
mL) likewise did not affect their growth. These results show
that the PVP (6000 Da) carrier, in combination with
indomethacin, did not affect HMEC-1 cell growth/viability
suggesting that the Amp-PVP carrier can in a biocompatible
manner transport the drug to tissue parenchyma.
The effect of indomethacin, indomethacin-loaded PVP-NPs
(6000 Da) (PVP-IMC), and unloaded PVP-NPs (6000 Da) on
HMEC-1 adhesion molecules was in continuation studied.
These cells were exposed to respective treatments at a
concentration of 0.01 mg/mL during 48 h, at which point
the cells were harvested. Cell extracts were probed with E-
pubs.acs.org/molecularpharmaceutics Article
selectin, ICAM-1, and VCAM-1 specific antibodies. As
presented in Figure 12, Western blot demonstrated that
none of the treatments induced significant changes in the
expression of E-selectin, ICAM-1, and VCAM-1 as compared
to the control.
4. DISCUSSION
Due to their capability to entrap hydrophobic drugs and
increase their bioavailability and pharmaceutical efficiency,
while decreasing side toxicity, polymeric NPs have been widely
investigated and applied in biomedical areas, especially for the
creation of novel drug delivery systems.9,10,33,34 Despite the
global use of different nanoscaled polymeric drug carriers for
research, more detailed information concerning their biological
effects, toxicity, and safety are still required.35−38 The vascular
endothelium consists of a monolayer of endothelial cells, lines
the cavities of arteries, veins, and capillaries, and is in direct
contact with the blood components. The endothelium is not
only just a barrier between blood and tissues but also the
largest endocrine gland.32 Indeed, the endothelial cells are
involved in the control of thrombosis and thrombolysis,
determine the dilation and contraction of blood vessels, and
participate in the interactions of platelets and leukocytes with
the vessel wall.39−41 Therefore, it is of utmost importance to
exclude possible deleterious effects of NPs on endothelial cells
homeostasis.
In our previous study, we determined that the NPs
comprising amphiphilic-PVP NPs (2000, 4000, 6000, 8000,
and 12000 Da) did not significantly affect the growth of
HMEC-1 endothelial cells.10 In this study, we evaluated the
effect of the amphiphilic-PVP (6000 Da) NPs on basal and
activated endothelial cells’ viability and immunological
activation. To examine the interaction of amphiphilic-PVP-
based polymeric materials with endothelial cells, we synthe-
sized PVP (6000 Da), F-NPs where the FITC molecules were
incorporated into the PVP (6000 Da) hydrophobic cores, and
PVP (6000 Da) loaded with the drug indomethacin.
4218 https://dx.doi.org/10.1021/acs.molpharmaceut.0c00667
Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article

Figure 8. Effect of PVP-NPs (6000 Da) and F-NPs on HMEC-1 cells’ adhesion molecule expression. (A) Activation of ICAM-1 expression by
increasing concentrations of LPS. (B) Effect of F- and PVP-NPs (6000 Da) on E-selectin expression of basal state and LPS-activated HMEC-1 cells.
(C) Effect of F- and PVP-NPs (6000 Da) on the expression of the ICAM-1 of basal state and LPS-activated HMEC-1 cells. C = control; cells
incubated with medium only. The position of the nearest respective protein marker band is depicted to the right. The results represent the average
of three separate experiments. Statistical significance: ** = p ≤ 0.01 compared to control.

Notably, some NPs exert cytotoxic effects on endothelial
cells. Thus, metal-based NPs, including ZnO or TiO2 NPs42−44
and silica-based NPs, were shown to cause oxidative stress and
endothelial dysfunction, in vitro, partly through the activation
of the MAPK/Nrf2 pathway and NF-κβ signaling.43,45
Intravenous administration of silver NPs, in in vivo models,
induced organ toxicity attributed to the disturbance of
interendothelial junctions due to the NP-dependent increase
of radical oxygen species production.46 In a previous study, we
showed that Amph-PVP NPs did not affect HMEC-1
endothelial cell growth.10 In the present study, we verified
their safety, demonstrating that PVP (OD6000) NPs do not
cause toxic effects up to 66 μg/mL. Previous studies likewise
showed that micelle-based NPs exhibit low toxicity concerning
endothelial cells. Indeed, polymeric micelles, such as methoxy
poly(ethylene glycol)-poly(D,L-lactide) (MPEG-PLA)-based
micelles, exerted no toxic effects on endothelial cells viability
up to concentrations of 200 μg/mL.47,48 Diglutamic acid-based
4219
micelles caused low or modest toxic effects on human
umbilical vein endothelial cells (HUVEC).48
One of the critical applications of nanomaterials is the
targeted transfer of drugs. Therefore, the possible effects of the
drug-carrier complex on endothelial cell viability need to be
taken into consideration. In previous studies, we showed that
drugs of high hydrophobicity, such as indomethacin, could be
efficiently encapsulated, during the self-assembly process, to
the hydrophobic core of amphiphilic polymeric NPs.5
Indomethacin is a nonsteroidal anti-inflammatory drug, a
derivative of indole, which also has analgesic and antipyretic
properties. Its anti-inflammatory activity is due to the
inhibition of prostaglandin synthesis and possibly other
actions, such as phosphodiesterase inhibition and leukocyte
migration.49,50 In this study to PVP-NPs (6000 Da), the model
drug, indomethacin, was incorporated, and the effects of the
complex were examined. No adverse effect of the complex on
endothelial cell viability was observed. Previously, Cu(II)
complex-loaded solid lipid NPs were shown to exert in vitro
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Figure 9. Detection of PVP-NPs (6000 Da) uptake by living HMEC-1 cells. (A) The uptake of F-NPs by living HMEC-1 cells was assessed using a
fluorescence microscope (green; FITC). (B) The uptake of F-NPs was evaluated after challenging HMEC-1 cells with LPS for 2 h (green; FITC).
C = control; cells incubated with medium only. Representative images are presented - magnification ×10.

cytotoxic effects on MCF-7 breast cancer cells and had toxic
effects on HUVEC control cells.51
Also, endothelium plays a prominent role in the inflamma-
tory response. The phenomenon through which the
endothelium undergoes modifications upon challenge is
denominated as endothelial activation.52 In anaphylaxis or
sepsis, there is a disintegration of intercellular contacts in
postcapillary venules under the conditions of an inflammatory
response, resulting in intercellular gap formation edema.53,54
Endothelial cells have a crucial role in the regulation of blood
vessel tone, monocytes recruitment, and thrombogenicity so
that dysfunction of endothelial cells is directly correlated to the
pathogenesis of cardiovascular diseases including atheroscle-
rosis.26 The expression of specific adhesion molecules, e.g.,
VCAM-1, ICAM-1, and selectins, is increased upon endothe-
lium activation facilitating, thus, the binding and the
penetration of endothelium barrier by immune cells as well
as their subsequent transmigration to tissue parenchyma.55
4220
Specifically, the overexpression of adhesion molecules,
including E-selectin, ICAM-1, and VCAM-1, initiates the
recruitment of monocytes, which are, under these conditions,
able to attach steadily onto the activated endothelial cells.26,31
A cascade is generated where monocytes differentiate into
macrophages and transform into macrophage foam cells upon
engulfing excessive lipids from the plasma.56 The resulting
agglomeration of macrophage foam cells into the arterial wall is
a hallmark of atherosclerosis.57 The exposure of endothelial
cells to different classes of NPs, e.g., metal-based,58,44
carbonaceous NPs,59,60 and silica NPs,43 enhanced the release
of inflammatory factors and increased the expression of
adhesion molecules (e.g., ICAM-1, VCAM-1, and selectins).
In the present study, PVP OD6000 NPs did not affect the
expression of adhesion molecules, including E-selectin, ICAM-
1, and VCAM-1, up to the high concentration of 66 μg/mL.
Likewise, no adverse effects on the expression of adhesion
receptors were evident when endothelial cells were treated
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Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics
Figure 10. Intracellular localization of PVP-NPs (6000 Da). (A, Β) HMEC-1 cell treated with 0.066 mg/mL of F-NPs (FITC; green stain). (C)
Cells stained with TOPRO, without NP treatments. Representative images are presented - magnification ×60 and zoom 4.
Figure 11. Effect of indomethacin, indomethacin-loaded PVP (6000
Da), and unloaded PVP-NPs (6000 Da) on the proliferative ability of
HMEC-1 cells. Cells were cultured in 96-well plates and treated with
respective agents 48 h. C = control; cells incubated with medium only.
The results represent the average of three separate experiments.
Statistical significance: *** = p ≤ 0.001 compared to control.
with PVP OD6000 NPs loaded with indomethacin, demon-
strating an absence of synergistic action.
Under various pathological conditions, the activation of the
endothelium is imminent; indeed, dysfunctional endothelium
is a hallmark of pathologies such as diabetes or cardiovascular
disease.26,31 Therefore, to anticipate the endothelial cell
response in vivo, it is vital to evaluate the biocompatibility of
various nanomaterials with endothelium under conditions that
4221
Article
mimic pathological states.61,62 It is well established that LPS,
when in contact with endothelial cells, interacts with the TLR4
receptors and induces their activation.27 A critical downstream
mediator of TLR4 is NF-κβ. Binding of LPS to TLR4 triggers
the activation of phosphokinases, which phosphorylate the
cytoplasmic NF-κβ. Upon activation, NF-κβ translocates to the
nucleus, where it exerts transcriptional regulation of inflam-
matory mediators.28 Therefore, the ratio p-NF-κβ/total-NF-κβ
can be used as a reliable indicator of cellular immune
activation.63−65
Generating a model of activated endothelium demonstrated
that PVP NPs (6000 Da), F-NPs, and loaded with
indomethacin PVP-NPs (6000 Da) did not exert adverse
effects on activated HMEC-1 cell viability. Furthermore,
neither PVP NPs nor the drug-carrier complex affected the
expression of adhesion receptors, including E-selectin, ICAM-
1, and VCAM-1. In previous studies, it was shown that ZnO-
based NPs facilitated the TNFα-dependent increase in ICAM-
1 expression in HUVECs66 but did not induce synergistic
effects in the presence of LPS of palmitate and NPs on
endothelial cell inflammatory responses.67 Moreover, both Ag
and silica NPs induced cytotoxicity and inflammatory response
of HUVEC cells under shear stress.68,69 Furthermore, repeated
exposure to rutile TiO2 NPs induced enhancement in plaque
progression in atherosclerotic mice, correlated with inflamma-
tion and oxidative stress.70 NPs based on chitosan derivatives
(Ch-der) or poly(lactic-co-glycolic acid) (PLGA), encapsulated
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Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article

Figure 12. Effect of indomethacin (IMC), indomethacin-loaded PVP-NPs (PVP-IMC), and unloaded PVP-NPs (6000 Da) (PVP) on protein
expression of HMEC-1 cells’ E-selectin, ICAM-1, and VCAM-1 adhesion molecules. The cells were treated with different agents for 48 h. (A) Effect
of indomethacin, PVP-IMC NPs, and hollow PVP-NPs (6000 Da) on E-selectin protein expression in HMEC-1 cells. (B) Effect of indomethacin,
PVP-IMC NPs, and hollow PVP-NPs (6000 Da) on the expression of ICM-1 protein in HMEC-1 cells. (C) Effect of indomethacin, PVP-IMC
NPs, and hollow PVP-NPs (6000 Da) on the expression of VCAM-1 protein in HMEC-1 cells. The results were normalized to Actin. The position
of the nearest respective protein marker band is depicted to the right. C = control; cells incubated with medium only. The results represent the
average of three separate experiments. Statistical significance: NS = not significant.

with natural cherry extract, exhibited a protective effect on
LPS-stressed HUVEC cells.71
The F-type (FITC included to the NP core) particles
demonstrated efficient uptake and no deleterious effects on
HMEC-1 cell viability and immunological activation. Fluo-
rescent NPs are especially suitable for living cells and may be
utilized for imaging of cellular uptake and intracellular
localization as well as tracking of diffusion processes in the
extracellular space. The synthesis of fluorescent NPs
demonstrated that FITC-labeled PVP NPs are nontoxic and
noninflammatory and that PVP NPs (6000 Da) are uptaken by
endothelial cells in a concentration-dependent manner. It is
4222
well established that the activated endothelium undergoes
structural and functional changes, which, among others, result
in the leakage of intravascular components.72,73 PVP NPs were
safe for use in an activated endothelium model, as no further
changes in the upregulation of adhesion molecules were
evident. However, activated HMEC-1 endothelial cells
exhibited an increased uptake of PVP NPs. Previous studies
have shown that conditions of shear stress can enhance the
HUVECs uptake of silica NPs.74 Moreover, the uptake of
poly[acrylonitrile-co-(N-vinylpyrrolidone)] [P(AN-co-NVP)]
NPs by HUVECs was facilitated by IL-1β treatment. In
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Mol. Pharmaceutics 2020, 17, 4212−4225
Molecular Pharmaceutics
contrast, a modest increase in Au NPs uptake was shown by
endothelial cells treated with TNFα.75
5. CONCLUSIONS
In this study, we demonstrated for the first time that PVP-NPs
do not affect the viability and the immune response of the
basal state and the challenged LPS endothelial cells. The
utilization of fluorescently labeled PVP-NPs showed that the
uptake of PVP-NPs is increased by activated HMEK-1
endothelial cells. In summary, PVP-NPs do not exert adverse
effects of either basal state or challenged endothelial cells. The
interactions of NP with the endothelium should be considered
when designing nanomaterials. In vitro assays are useful to
predict the better response of the endothelium to NPs in vivo.
Amph-PVP NPs are a promising drug delivery system and
could be utilized for specialized targeting of the desired tissues
while reducing the side effects in patients.
■ ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
h t t p s : / / p u b s . a c s . o r g / d o i / 10 . 1 02 1 / a c s .m o l p h a rm a -
ceut.0c00667.
Supplementary Figure S1, 1H NMR spectra; Supple-
mentary Figure S2, differential scanning calorimetry
(DSC); and Supplementary Figure S3, thermogravimet-
ric analysis (TGA) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Dragana Nikitovic − Laboratory of Histology-Embryology,
School of Medicine, University of Crete, 71003 Heraklion,
Greece; orcid.org/0000-0003-3882-7399;
Email: nikitovic@uoc.gr
Authors
Aikaterini Berdiaki − Laboratory of Histology-Embryology,
School of Medicine, University of Crete, 71003 Heraklion,
Greece
Emmanouela Perisynaki − Laboratory of Histology-
Embryology, School of Medicine, University of Crete, 71003
Heraklion, Greece
Antonios Stratidakis − Institute for Advanced Study (IUSS),
Environmental Health Engineering, 27100 Pavia, Italy;
Laboratory of Toxicology, School of Medicine, University of
Crete, 71003 Heraklion, Greece
Pavel P. Kulikov − Department of Biomaterials, D. Mendeleev
University of Chemical Technology of Russia, Moscow 125047,
Russian Federation; Centre for Strategic Planning of FMBA of
Russia, Moscow 119121, Russia
Andrey N. Kuskov − Department of Biomaterials, D. Mendeleev
University of Chemical Technology of Russia, Moscow 125047,
Russian Federation; orcid.org/0000-0001-8140-2754
Polychronis Stivaktakis − Laboratory of Toxicology, School of
Medicine, University of Crete, 71003 Heraklion, Greece
Petra Henrich-Noack − Clinic of Neurology with Institute of
Translational Neurology, University Clinic Muenster, Muenster,
Germany
Anna L. Luss − Department of Biomaterials, D. Mendeleev
University of Chemical Technology of Russia, Moscow 125047,
Russian Federation
pubs.acs.org/molecularpharmaceutics Article
Mikhail M. Shtilman − Department of Biomaterials, D.
Mendeleev University of Chemical Technology of Russia,
Moscow 125047, Russian Federation
George N. Tzanakakis − Laboratory of Histology-Embryology,
School of Medicine and Laboratory of Anatomy, School of
Medicine, University of Crete, 71003 Heraklion, Greece
Aristidis Tsatsakis − Laboratory of Toxicology, School of
Medicine, University of Crete, 71003 Heraklion, Greece;
Department of Biomaterials, D. Mendeleev University of
Chemical Technology of Russia, Moscow 125047, Russian
Federation
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00667
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was financially supported by the Ministry of Science
and Higher Education of the Russian Federation within the
framework of the state assignment under the project FSSM-
2020-0004 and by a grant from Era Net.RUS. Plus, initiative
(“NABUCO”, ID#169). The work of A.N.K. was supported by
D. Mendeleev University of Chemical Technology of Russia
(Project Number K-2020-018).
■
ABBREVIATIONS
NPs, nanoparticles; PVP, poly-N-vinylpyrrolidone; Amph-
PVP, amphiphilic poly-N-vinylpyrrolidone derivative consisting
of water-soluble polymer fragment with molecular weight 6000
Da and one terminal n-octadecyl hydrophobic group; F, PVP-
NPs (6000 Da) linked with fluorescent FITC; ICAM-1,
intercellular adhesion molecule-1; VCAM-1, vascular cell
adhesion molecule-1; ECM, extracellular matrix; LPS, lip-
opolysaccharides
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