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Accepted Manuscript
Mechanisms of interaction of biodegradable
nanocapsules with non-phagocytic cells
polyester
Izabel Cristina Trindade, Gwenaelle Pound-Lana, Douglas
Gualberto Sales Pereira, Laser Antônio Machado de Oliveira,
Margareth Spangler Andrade, José Mário Carneiro Vilela, Bruna
Bueno Postacchini, Vanessa Carla Furtado Mosqueira
PII:
DOI:
Reference:
S0928-0987(18)30389-0
doi:10.1016/j.ejps.2018.08.024
PHASCI 4661
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
17 May 2018
10 August 2018
18 August 2018
Please cite this article as: Izabel Cristina Trindade, Gwenaelle Pound-Lana, Douglas
Gualberto Sales Pereira, Laser Antônio Machado de Oliveira, Margareth Spangler
Andrade, José Mário Carneiro Vilela, Bruna Bueno Postacchini, Vanessa Carla Furtado
Mosqueira , Mechanisms of interaction of biodegradable polyester nanocapsules with nonphagocytic cells. Phasci (2018), doi:10.1016/j.ejps.2018.08.024
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ACCEPTED MANUSCRIPT
Mechanisms of interaction of biodegradable polyester nanocapsules with nonphagocytic cells
Izabel Cristina Trindade1, Gwenaelle Pound-Lana1, Douglas Gualberto Sales Pereira1, Laser
Antônio Machado de Oliveira,2 Margareth Spangler Andrade3, José Mário Carneiro Vilela3,
Laboratory of Pharmaceutics and Nanobiotechnology (LDGNano) – School of Pharmacy,
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Bruna Bueno Postacchini4 and Vanessa Carla Furtado Mosqueira1*
Laboratory of Biomaterials and Experimental Pathology – NUPEB, Universidade Federal de
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Universidade Federal de Ouro Preto, Minas Gerais, 35400-000 Brazil.
Ouro Preto, Minas Gerais, Brazil.
Technological Center - CETEC SENAI - Minas Gerais Regional Department – Belo
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Horizonte, Minas Gerais, Brazil.
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Photophysics Laboratory – Department of Physics- Institute of Exact and Biological Sciences,
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Universidade Federal de Ouro Preto, Minas Gerais, Brazil.
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*Corresponding author: Vanessa Carla Furtado Mosqueira
Laboratory of Pharmaceutics and Nanobiotechnology, School of Pharmacy/Universidade
Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro, Ouro Preto, Minas
Gerais/Brazil, CEP 35400-000. Tel: +55 31 35591032; Fax: +55 31 35591367
E-mail address: vamosqueira@gmail.com; mosqueira@ufop.edu.br (V.C.F. Mosqueira)
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ABSTRACT
The interaction of polymer nanocapsules (NC) prepared from four biodegradable polyesters
with variable polymer hydrophobicity (PCL, PLA, PLGA and PLA-PEG) was investigated in
the non-phagocytic Vero, Caco-2 and HepG2 cell lines. The NC, labeled with the highly
lipophilic fluorescent indocarbocyanine dye DIL, had very similar sizes (approx. 140 nm) and
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negative zeta-potentials. Asymmetric flow field-flow fractionation evidenced NC colloidal
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stability and negligible transfer of the dye to serum proteins in the incubation medium. The
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cytotoxicity of the NC was evaluated via MTT assay over a large polymer concentration
range (0-1000 µg/mL) and time of exposure (2, 24 and 48 h). The NC were safe in vitro up to
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a concentration of approx. 100 µg/mL or higher, depending on the cell line and nature of the
polymer. Vero cells were more sensitive to the NC, in particular NC of the more hydrophobic
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polymer. The cells were exposed to endocytosis inhibitors, incubated with NC, and the cellassociated fluorescence was quantified by spectrofluorometry. HepG2 cells presented a 1.5-2-
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fold higher endocytic capacity than Caco-2 and Vero cells. The main mechanism of NC
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uptake was caveolin-mediated endocytosis in HepG2 and Vero cells, and macropinocytosis in
Caco-2 cells. Polymer hydrophobicity had an effect on the level of NC associated to HepG2
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cells and to a lesser extent on the endocytosis mechanisms in Vero and Caco-2 cells. The NC
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uptake levels and endocytosis mechanisms differed significantly between cell lines tested.
Keywords: nanocarrier, endocytic pathways, confocal microscopy, cell uptake, field flow
fractionation, biodegradable polyester, carbocyanine dye.
Chemicals: chlorpromazine, cytochalasin D, 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DIL), methyl-β-cyclodextrin, poly(ε-caprolactone),
polylactide, poly(lactide-co-glycolide), poly(D,L-lactide)-block-polyethylene glycol
copolymer.
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1. INTRODUCTION
Polymeric nanoparticles (NP) offer excellent drug nanocarriers, in particular for lipophilic
cargos, and are widely used in pharmaceutical research to increase the therapeutic efficacy of
drugs and decrease their undesirable side-effects. However, a better understanding of the
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mechanisms of interaction of NP with cells/organs and intracellular fate is still needed to enable
translation of these nanocarriers from bench to bedside (Yameen et al., 2014). The study of the
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pathways of NP internalization by cells can help in understanding the pharmacological and
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toxic effects of these NP, being of great importance for the design of more efficient and safer
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nanocarriers (Behzadi et al., 2017).
Most NP interacts with cells via surface binding and receptor-mediated endocytosis
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mechanisms, as elegantly discussed in several recent articles (Behzadi et al., 2017; Nel et al.,
2009). However, many pitfalls are frequently found in the characterization (Nel et al., 2009)
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and imaging of fluorescent-NP, particularly in cell culture media containing serum proteins
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(Meindl et al., 2017, Feliu et al., 2017). Besides, detailed studies concerning the stability of
non-covalently bound dyes used to label particles and study their interactions with cells in the
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presence of serum proteins in non-static conditions are much less frequent (Bastiat et al., 2013;
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Miller et al., 2012).
Extensive literature is available regarding polymeric matrix-based nanospheres and their
interactions with different cells in vitro (Meindl et al., 2017; Zhang et al., 2013). In contrast,
polymeric nanocapsules (NC) comprising an oil or lipid filled core (Mosqueira et al., 2001a)
with different structural organization, polymer and lipid compositions, are more complex
structures compared to matrix systems (Figure 1) and have been poorly investigated concerning
their interactions with cells and respective endocytic pathways. Biodegradable polyester-based
NC have been proven to be useful to deliver high contents of lipophilic cargos by parenteral
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and recently oral route (Branquinho et al., 2014; de Mello et al., 2016), enhancing the intestinal
tract absorption of drugs (Attili-Qadri et al. 2013), the body exposure (Branquinho et al., 2017;
Garcia et al., 2015; Oliveira et al., 2017), the delivery across the blood-brain barrier (Rodrigues
et al., 2016), the efficacy (Benvegnú et al., 2011; Branquinho et al., 2014; de Mello et al., 2016;
Mosqueira et al., 2004) and also to reduce the cardiotoxicity of drugs (Branquinho et al., 2017;
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Leite et al., 2007; Souza et al., 2017), even with the most common polymer surfaces (non
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ligand-decorated nor surface-modified). These aspects are a great incentive to translate such
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nanocarriers into pharmaceutical products. However, the origin of NC outstanding
performance as nanocarriers has not been fully elucidated and requires detailed investigation
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at the cellular level.
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NP endocytosis depends dramatically on the NP physico-chemical properties, such as size,
shape, surface charge, chemical composition (Cheng et al., 2015; Hühn et al., 2013; Meindl et
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al., 2017; Oh and Park, 2014) and also on cell type (Hillaireau and Couvreur, 2009; Kroll et
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al., 2011). The polymers poly-ε-caprolactone (PCL), poly-D,L-lactide (PLA), poly-D,L-lactideco-glycolide (PLGA) and poly(D,L-lactide)-block-polyethylene glycol (PLA-PEG) are widely
used in pharmaceutical products already approved by the US food and drug administration
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(FDA) for use in humans and are currently under investigation for their use as polymer-based
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drug nanocarriers (FDA; Marin et al., 2013). These polymers can all form a hydrophobic wall
that stabilizes the interface of drug-loaded NC. Although they belong to the same family of
biodegradable polyesters, careful selection of the copolymer is necessary because its chemical
composition and hydrophobicity may affect the rate of drug release and the nanocarrier
behavior in vitro and in vivo (Branquinho et al., 2014; de Paula et al., 2013; Mosqueira et al.,
2006). In particular, these polymers vary in hydrophobicity, and this parameter influences the
interaction of the derived NC with the biological medium, complement system and with each
specific type of cell (Mosqueira et al., 2001a).
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This work evaluates in different non-phagocytic cell lines wide concentration range (1 to 1000
μg/mL) of NC in vitro to guarantee low toxicity and investigates the internalization pathways
of NC stabilized by biodegradable polyesters of variable polymeric composition (Figure 1). To
quantify NC association to cells and also to visualize the NC within cells, the NC were labeled
with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DIL), a highly
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lipophilic fluorescent dye. The stability of this dye loading in polyester NC was evaluated under
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flow conditions by AF4 with multi-angle light scattering and fluorescence detection (AF4-
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MALS-FLD) and in the presence of serum proteins. In order to strictly evidence effects of the
polyester hydrophobicity, the NC were prepared with very similar sizes and zeta potential (ZP)
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values and were fully characterized in media reproducing conditions used in in vitro studies.
Thus, the first part of this work presents a detailed characterization of the NC in terms of size,
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fluorescence stability and ability to retain the dye in the oily core so as to validate the use of
DIL as an NC marker in cell interaction and imaging. The Rose Bengal assay was carried out
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to quantitatively study the NC surface hydrophobicity (Müller et al., 1997). In a second part,
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toxicity in a wide concentration range (1 to 1000 μg/mL µg/mL) against non-phagocytic Caco2 and HepG2 (tumor-derived) and Vero (normal) cells, representative of cellular parenchyma
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of organs highly exposed to nanoparticulate systems following oral and intravenous
administration. The endocytic capacity of cells was monitored by quantitative steady-state
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fluorescence spectroscopy. The contribution of energy-dependent endocytosis was evaluated
using thermal inhibition (incubation at 4°C), and the contribution of caveolin (CvME), clathrin
(CME) and macropinocytosis mediated endocytosis determined using the chemical inhibitors
methyl-β-cyclodextrin (MβCD), chloropromazine and cytochalasin D, respectively. Finally,
the intracellular distribution of the NC was studied qualitatively by confocal laser scanning
microscopy.
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Figure 1: Simplified scheme representing nanocapsules (NC) produced from different
homopolyesters (PCL and PLA), copolyesters (PLGA) and block polymer (PLA-PEG) as the
polymer wall (black) and an oily core in a reservoir type nanostructure. To stabilize the NC in
aqueous medium poloxamer, a PEG containing surfactant (PEG-PPO-PEG, red and green) is
adsorbed to the NC surface for PCL, PLA and PLGA NC. In PLA-PEG NC, the PEG block
(red) is covalently linked to the PLA block and no additional surfactant is used. The order of
increasing polyester hydrophobicity indicated by the black arrow was estimated according to
calculated LogP (supplementary material).
2. MATERIALS AND METHODS
2.1. Materials
PLGA (Mw 45,000 g/mol), PLA (Mw 75,000 g/mol), PCL (Mw 65,000 g/mol), Pluronic® F68 (poloxamer 188, PEG-polypropylene oxide-PEG triblock copolymer surfactant),
fluorescent dyes DIL and DAPI, Triton X-100 solution, methyl-β-cyclodextrin (MβCD),
cytochalasin D and acetonitrile (ACN, HPLC grade) were acquired from Sigma-Aldrich
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(USA), acetone (PA grade) from Vetec (Brasil), Miglyol® 812N (triglyceride of caprylic
(C8)/capric (C10) acids) from Sasol (Gmbh, Germany), Lipoid® S75 from Lipoid (Gmbh,
Germany), phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM,
4.5 g/L glucose and L-Glutamine), trypsin-EDTA (0.25%) and antibiotic stock solution
(10.000 I.U of penicillin +10 mg/mL of streptomycin) from Lonza (USA), inactivated fetal
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bovine serum (FBS) from Cultlab (Brazil), trypan Blue solution from LGC Biotecnologia
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(Brazil) and chlorpromazine was kindly donated by Cristália Chemical and Pharmaceutical
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Products (Brazil). All above mentioned products were used without further purification. PLAPEG (Mn 26,000 g/mol, Ð=1.26) was synthesized and characterized as previously described
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(Pound-Lana et al., 2017). Ultrapure water was obtained on a Simplicity-MilliQ system
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(Millipore, USA).
2.2. NC preparation
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The NC were prepared by polymer nanoprecipitation (Fessi et al., 1989). Briefly, the polymer
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(6 mg), Lipoid S75 (7.5 mg), Miglyol (25 µL) and 30 µL of DIL (2.0 mg/mL in methanol)
were solubilized in acetone (2 mL). This mixture was poured through a syringe into 4 mL of
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ultrapure water containing Pluronic (7.5 mg) under magnetic stirring, except in PLA-PEG NC,
where ultrapure water was used without the addition of any surfactant. The resulting suspension
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was kept under magnetic stirring for 10 min at 25°C, the solvents were removed and the
aqueous colloidal suspension concentrated under reduced pressure to a final volume of 750 µL
(80 µg/mL of DIL and 8 mg/mL of polymer).
2.3. DIL quantification by spectrofluorimetry
Spectra of the dye in solution were recorded on a RF-5301 spectrofluorimeter (Shimadzu
Scientific Instruments, Japan) in quartz cuvettes (10 mm optical path). In PBS:ACN (23:77
v/v) and under an excitation wavelength of 525 nm DIL shows emission maximum at 561 nm.
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The emission intensity increased linearly with DIL concentration in the 0.001 to 0.20 µg/mL
range (Supplementary Figure S1) and thus DIL was quantified in samples using the
corresponding linear regression y = 1.2 + 2289 x; (R2 = 0.990; supplementary Figure S1).
Fluorescence spectra of DIL in medium containing Triton X-100 (Triton X-100:PBS:ACN
0.1:23.4:76.5 v/v), the medium used to extract the dye in experiments of NC association with
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cells, present a new emission band with maximum intensity at 597 nm related the presence of
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Triton (Supplementary Figure S1). Since no changes were observed in the spectral shape and
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fluorescence intensity of DIL, the fluorescence spectra recorded in Triton X-100:PBS:ACN
medium between 530 and 640 nm with an excitation wavelength of 525 nm were deconvoluted
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to exclude the contribution of the surfactant to the sample fluorescence, then determining the
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intensity at 561 nm corresponding to DIL fluorescence only.
2.4. NC characterization
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2.4.1. DIL loading
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To determine the % of DIL loading, the NC suspension (400 µL) was filtered in a 50 kDa
centrifugal filter (Amicon® Ultra-0.5 mL device, Millipore, USA) at 900 × g for 5 min. DIL
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was quantified in the ultrafiltrate as described in section 2.3 and the DIL encapsulation
efficiency was determined as the fraction of DIL in the ultrafiltrate with respect to the total in
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the NC suspension, in percentage.
2.4.2. NC size and surface charge characterization
The NC hydrodynamic diameter (Z-average) and polydispersity index (PdI) were determined
at 25°C by dynamic light scattering (DLS) on a Zetasizer Nano ZS equipment (Malvern
Instruments, UK) equipped with a 633 nm laser and detector at backscattered 173º angle. The
zeta potential (ZP) was determined by microelectrophoresis coupled to laser Doppler
anemometry on the same equipment. The samples were diluted at 1:1000 in water or NaCl
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solution (1 mM, to guarantee a low and constant ionic strength in all measurements) for size
and ZP readings, respectively. Each mean value represents the set of at least 10 sequential
measurements conducted in three different batches of the formulation.
2.4.3. Morphological analysis by atomic force microscopy (AFM)
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A NC suspension sample (2 µL) was diluted in ultrapure water (1:100 v/v) and spread on a
freshly cleaved mica support fixed on a glass blade and dried under a flow of argon gas. The
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images were obtained in air at room temperature in tapping mode on a Dimension V
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microscope (Digital Instruments, USA) monitored by a Nanoscope IIIa controler (Santa
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Barbara, CA, USA), using commercial Si probes from Nanosensors with cantilevers having a
length of 228 µm, resonance frequencies of 75-98 kHz, spring constants of 3.0-7.1 N/m,
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nominal tip curvature radius of 5 nm to 10 nm and at a scan rate of 1 Hz. Dimensional analyses
were performed with the Nanoscope 5.31r1 “section analyses” software. Geometrical
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diameters were measured on the height images considering the width of the spheres at half
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height and are reported as the average of values obtained from 40 particles.
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2.4.4. Nanoparticle tracking analysis (NTA)
The NC mean diameter were determined by NTA at 25°C on a NanoSight LM10 equipment
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(Malwern Instrument, UK), equipped with a sample chamber with a 532 nm laser, lens of
magnification of 20 × coupled to a sensitive camera. The samples were diluted at a ratio of
1:4000 or 1:8000 in ultrapure water.
2.4.5. Rose Bengal adsorption study
The original method of Rose Bengal (RB) adsorption to NP in aqueous medium (Müller et al.,
1997) was adapted to quantify the hydrophobicity of the NC surface. Blank NC suspensions
were incubated at 0, 40, 80, 120, 160, 200 and 240 µg of polymer/mL with Rose Bengal at a
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fixed concentration of 20 µg/mL in ultrapure water at 25°C under mechanical shaking in a final
volume of 1 mL for 3h. A 400 µL aliquot was placed in a 50 kDa centrifugal device (Amicon®,
Millipore), centrifuged at 2,000 × g for 5 min. RB was quantified in the ultrafiltrate by steadystate fluorescence spectroscopy by placing 50 to 150 µL of ultrafiltrate in a total volume of 2.0
mL of ACN:water (90:10) at 525 and 560 nm excitation and emission wavelengths ,
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respectively. A calibration curve in the same solvent system was linear (R2>0.999) in the 4 –
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500 ng /mL range with 3 nm slit widths. To avoid interference with RB quantification by
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spectrofluorimetry blank NC were used (no DIL). The partition coefficient (PQ) was calculated
as the ratio of bound divided by free RB, where the bound RB mass was calculated as the
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difference between the concentration of RB in the ultrafiltrate without NC and RB in the sample
ultrafiltrate. To express PQ as a function of the total NC surface area in the incubation medium,
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the surface area of a single NC (ANC) was calculated using the equation ANC = π × Dh2, where
Dh is the NC hydrodynamic diameter by DLS, and the number of NC per mL of suspension
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was calculated using the mass of dry ingredients (55.0 mg/mL) divided by the mass of a single
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NC (mNC) calculated using the equation mNC = d × VNC, where d is the value of the average
density of the NC (1.03 g/L, as determined in Mosqueira et al., 2001a and VNC is the volume
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of one NC calculated using the equation VNC = 4/3 × π × (Dh/2)3.
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2.4.6. Colloidal stability upon incubation in culture media
NC suspension samples in ultrapure water, pure FBS, or DMEM medium supplemented with
10% of FBS (1000 µg polymer/mL) were maintained at 37°C for 2 h, 24 h and 48 h. Samples
were collected, diluted at a ratio of 1:100 in ultrapure water or 1 mM NaCl (aq) to determine
the size and ZP, respectively.
2.4.7. Size characterization and DIL loading stability by AF4-MALS-FLD
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Fractionation of the NC was performed on a Postnova analytics AF2000 MT system (Landberg,
Germany) comprising two PN1130 HPLC pumps (tip and focus pumps), an AF2000 module
(crossflow pump), PN5300 autosampler, PN4020 channel oven, a separation channel lined with
a Postnova AF2000 MT Series NovaRC AQU 5 kDa cut-off regenerated cellulose membrane
and a 350 µm spacer, with UV absorption (254 nm, PN3211 UV-Vis detector), fluorescence
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intensity (PN 3412 fluorescence detector, 561 nm with excitation at 525 nm), MALS (PN3621
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detector with a 532 nm laser) and DLS as described above, in series. The eluent was 10 mM
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NaCl in ultrapure water filtered on a 0.1 µm PTFE membrane filter (Millipore®). The
autosampler, separation channel and detectors were kept at 25 °C or 37 °C and the detector
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flowrate was maintained at 0.5 mL/min. The injection flowrate was 0.2 mL/min with injection
time of 3 min, injection volume of 20 µL and a transition time of 1 min. The initial crossflow
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(1.5 mL/min) was maintained for 3 min, followed an exponential decrease to 0.05 mL/min over
a period of 15 min and was maintained at 0.05 mL/min for 15 min followed by 7 to 15 min
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without applied crossflow to confirm complete elution of the sample. The diluent (10 mM NaCl
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or DMEM/FBS, 1450 µL) was placed in an amber vial and NC suspension (50 µL) was added;
the vial was vortex-mixed, kept in the AF4 system autosampler at 25 or 37°C and submitted to
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200 shaking cycles prior to injection. The sample was injected (20 µL) and eluted with 10 mM
NaCl (aq). Controls included pure DMEM/FBS (no NC added) and DIL solution (DIL in
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methanol (2 mg/mL, 2 µL, to provide the same DIL to DMEM/FBS as in NC samples) diluted
with 1498 µL of DMEM/FBS). To evaluate the stability of the suspensions over time,
successive injections were carried out from the same vial at 1.5 - 2 h intervals for up to 66 h.
Dg was determined at 3.8 s time intervals using the angular variation of the scattered light
intensity at angles 12°-164° (20 angles, MALS detector) and the Postnova AF2000 software
calculation for spherical shape model, and Dh by DLS at 5.0 s intervals using the Malvern
instrument Zetasizer software. The differential and cumulative Dg-distributions and
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distribution parameters D5, D50 and D95 were determined using the intensity of the UV or FLD
signal as the value of concentration for each fraction relative to the total sample, whereas D hdistribution parameters were based on the 173° backscattered light intensity of the DLS
measurements.
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2.4.8. DIL release
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DIL release profile from the NC in PBS was determined using the direct dialysis method (Shen
and Burgess, 2013), whereby NC suspensions (250 µL) and PBS (pH=7.2, 750 µL) were placed
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in a previously hydrated 12-14,000 MWCO dialysis membrane (Spectrapor®) bag and
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submitted to dialysis in PBS with 10% FBS (pH=7.2, 19 mL) during 3 h at 37°C. Samples were
withdrawn from the external phase at predetermined time intervals, evaporated to dryness,
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dissolved in ACN, filtered (0.45 µm) and quantified by spectrofluorimetry.
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2.5.1. Cell culture
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2.5. In vitro experiments
Human colorectal adenocarcinoma (Caco-2, ATCC® HTB-37™), human hepatocellular
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carcinoma (HepG2, ATCC® HB-8065™) and African green monkey normal epithelial kidney
cell (Vero, ATCC® CCL-81™) lines were purchased from the Cell Bank of Rio de Janeiro,
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originally provided by The American Type Culture Collection (ATCC, USA). The cells were
maintained as described in supplementary material. The cells were cultured in DMEM medium
supplemented with 10% FBS and 1% of penicillin/streptomycin stock solution. The cells were
maintained in an incubator humidified and saturated with 5% CO2 at 37°C. Cells were routinely
subcultured until reaching 70-80% confluency and detached with trypsin-EDTA. Cells were
counted on a Neubauer counting plate to a desired cell concentration after labelling with trypan
blue.
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2.5.2. Cell viability
The viability of each cell line was assayed in complete DMEM culture medium after exposure
to NC in a concentration range of 1 to 1000 μg/mL for 2h, 24 h and 48 h by the MTT assay
(Mosmann, 1983). Briefly, Caco-2, HepG2 and Vero cells were seeded with complete DMEM
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culture medium (200 μL) in 96-well plates at densities of 3.0 x 103, 2.4 x 104 and 3.0 x 103/well,
respectively, to guarantee log phase growth upon incubation for 24 h for adhesion and
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adaptation. The culture medium was removed and replaced with 200 μL of fresh medium
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containing NC at 0, 1, 10, 50, 100, 500, 1000 µg of polymer/mL. The plates were incubated
for 2h, 24 h and 48 h, the medium containing NC removed and the wells washed three times
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with PBS containing Ca2+ and Mg2+ to prevent cell detachment and remove NC. Then, 200 μL
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of medium containing MTT (0.5 mg/mL) were added to each well and the plates were incubated
for 4 h. The plate was centrifuged at 400×g for 5 min at 4ºC, the medium carefully aspirated
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from the wells and formazan crystals solubilized with dimethyl sulfoxide (200 µL per well).
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Complete dissolution of the formazan crystals was verified by optical microscopy. The
absorbance was measured at 570 nm with reference at 650 nm on a Microplate Reader Emax
(Molecular Devices, USA). Cell viability was expressed in percentage compared to cells
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incubated without NP. For all experiments three controls were carried out in parallel: cells in
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complete medium (100% viability), cells in complete medium containing ultrapure water and
NP in complete medium supplemented with MTT without cells. The experiments were
performed in triplicate and three repetitions. Quality control wells were included in each plate
and the number of viable cells counted in a Neubauer's chamber after staining with trypan blue.
Maximal inhibitory concentrations for 50% of cells (IC50) were calculated to compare the
toxicity of the NP using GraphPad Prism® version 6.01.
2.5.3. NC uptake
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Caco-2, HepG2 and Vero cell lines were seeded at a density of 6.0×104, 1.2×105 and 8.0×104
cells/well, respectively, in 12-well plates, which corresponds to suitable cell confluency (7080%), incubated for adhesion and adaptation on complete medium. Then the medium was
replaced by new medium containing DIL-labeled NC (at a polymer concentration of 100
μg/mL) and the cells were incubated for 2 h. After, the medium was rapidly removed and the
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wells were washed three times with cold PBS containing Ca2+ and Mg2+ to abort endocytosis
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and remove unbound particles or weakly bound particles at the cell surface. To determine the
cellular DIL content, the dye was extracted from the cell pool, as follows: Triton X-100 in PBS
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(0.5 wt%, 400 μL) was added for cell membrane disruption and release of the intracellular
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content, and total cell removal from the well was aided with a scraper to form a cell pool. The
contents of each well were collected and pooled in microtubes and three aliquots of ACN were
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subsequently added to extract the fluorescent dye from the cellular pellets, mixed, separated
and centrifuged (600 x g for 10 min at 4°C) and pooled. The supernatant was collected, pooled
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(up to 1.3 mL) and fluorescence measured at 525 nm excitation wavelength and emission
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collection between 530-640 nm at 25°C. All measurements were performed in quintuplicate.
Fluorescence values were determined and converted to mass of DIL using the equation
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obtained from a calibration curve (Supplementary Figure S1), considering that under our
types.
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experimental conditions the dye loading was 1 wt% with respect to polymer mass for all NC
To verify the number of cells in the wells and normalize the number of viable cells for
comparisons, additional wells (quality control) were used in each plate and the number of cells
determined using trypan bleu exclusion followed by Neubauer chamber counting. To allow
comparison between cell types and account for variations in the number of cells per well
required for cell confluency the results were expressed as fluorescence of DIL associated per
60,000 cells.
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2.5.4.
Endocytic pathways
The mechanisms of NC endocytosis were probed incubating the cells with endocytosis
inhibitors, followed by incubation with NC at 37°C. The types of inhibitors, their
concentrations and incubation time were selected for each cell type based on methods
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previously described in the literature for Caco-2 and HepG2 cells with MβCD (Roger et al.,
2009), chlorpromazine (Roger et al., 2009) or cytochalasin D (Shurety et al., 1996) and Vero
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cells with MβCD (Vercauteren et al., 2010) or cytochalasin D (Nawa et al., 2003). Briefly, the
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culture medium was removed 24 h post cell adhesion and the cells were incubated for 1 h with
new medium containing endocytosis inhibitors: Caco-2 cells were incubated with MβCD (10
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mM) or chlorpromazine (10 μg/mL) or cytochalasin D (10 µM) for 1 h, HepG2 cells were
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incubated with MβCD (5 mM), chlorpromazine (7 μg/mL) or cytochalasin D (4 µM) and Vero
cells were incubated with MβCD (5 mM) or cytochalasin D (5 µg/mL). NC suspension was
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added at a polymer concentration of 100 μg/mL and the cells incubated for a further 2 h.
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Experiments were also carried out at 4°C to evaluate the participation of energy dependent
pathways and estimate NC binding. For all experiments, controls were carried out in parallel:
i) cells incubated only with culture medium, ii) cells incubated with culture medium containing
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NC and no inhibitors, iii) cells incubated with culture medium containing inhibitors and no
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NC, to verify potential cytotoxicity of the inhibitors. The medium was removed and the wells
were washed three times with cold PBS containing Ca2+ and Mg2+ to remove non-associated
NC. Cell lysis followed by DIL extraction and quantification by spectrofluorimetry was
performed as described in sections 2.5.3 and 2.3, respectively. Three separate and distinct
experiments were performed. All measurements were performed in triplicate in each plate,
converted to a single average for the total statistical analysis in the experimental series (n=9).
2.5.5. Confocal laser scanning microscopy analysis
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Confocal microscopy images were recorded to visualize DIL and identify its location within
the cells. Caco-2, HepG2 and Vero cells were grown in 75 cm2 flasks under the conditions
described above. Sterile round glass coverslips were placed in 24-well plates and the cells were
seeded (24 h) with complete culture medium at optimized cell densities of 5 × 104, 10 × 104
and 6 × 104 cells/well suitable for confocal analysis, respectively, followed by 24 h incubation
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for adhesion and adaptation. Then the cells were incubated for 2 h with new medium containing
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NC (100 μg of polymer and 1 μg of DIL/mL). Control cells were incubated with free DIL at
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the same concentration as used in the NC. The medium was removed and the wells washed
three times with cold PBS to remove non-associated NC and abort endocytosis. The coverslips
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were fixed with 10% formaldehyde in PBS (pH 7.2, 300 μL) for 20 min at 25°C, the wells
washed three times with PBS and stained with DAPI solution (0.1 μg/mL) for 20 min at 25°C
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for nuclear DNA labeling. The DAPI solution was removed and the wells washed three times
with PBS. The glass coverslips were mounted on glass slides spotted with glycerol: PBS
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(90:10, pH 9) for preservation and adherence of the material. The coverslips were analyzed on
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a LSM 780 confocal scanning microscope (Zeiss, Germany) at excitation wavelength of 358
nm (for DAPI fluorescence) and 549 nm (for DIL fluorescence). The images were processed
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in Zen Lite 2.3 software. Controls included cells incubated with blank NC and cells incubated
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with free DIL showed no fluorescence (Supplementary Figure S2)
2.6. Statistical analysis
Results were expressed as mean ± standard deviation (SD). Unpaired student´s t-test and
one-way ANOVA test followed by Tukey’s test were used and other specific statistical tests
are stated in the figure legends. The sizes and zeta potential values of NC were compared
using unpaired student´s t-test. Values of p <0.05 were considered statistically significant.
All statistical analysis was performed using GraphPad Prism® version 6.01.
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3. RESULTS
3.1. NC characterization and stability in cell culture medium
The % of DIL loading was approximately 100% for all types of NC (Table 1), indicating high
solubility of the dye in the oily core with no influence of the type of polymer. The NC prepared
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with the different polymers were monodisperse in size with similar hydrodynamic diameters
(115-138 nm) determined in batch by DLS in water (Figure 2). The sizes obtained by NTA
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were similar to the values obtained by DLS, but NTA values presented larger standard
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deviations. The AFM images of the NC show little differences in morphological aspects
(Figure 2). Well-separated, spherical (although flattened) structures, are visible in the AFM
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images of NC, together with small aggregates.
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ZP lower than -38 mV (Table 1 and Figure 3) were obtained for all types of NC in low ionic
strength medium. However, upon incubation of the NC in higher ionic strength medium with
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serum proteins (DMEM/FBS) the ZP values were reduced by 13-22 mV in modulus (Figure 3)
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and their diameter remained similar, except for PLGA NC, which diameter increased
significantly according to DLS (batch) measurement, but not in flow measurements. The ZP of
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PLA-PEG NC similar to that of the other NC indicates that the amount of PEG at the surface
was efficient to maintain colloidal stability, but was unable to shield the negative charge. The
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ZP of all DIL-loaded NC were similar (p > 0.05) to blank NC (data not shown), suggesting that
the fluorescent dye was located in the oily core of these nanostructures and have no influence
on the NC surface charge.
Table 1: Physicochemical characterization of the nanocapsules (NC) in batch and flow mode.
NC
% DIL
Batch mode (DLS)
Flow mode ‡
loading
AF4-MALS-DLS
(80
Diluent
Zeta
Dh (nm)
Dh Dg
D0.05 D0.5 D0.95
†
type
µg/mL)
potential
(PdI)
(nm) (nm)
(mV)*
PCL
99.9
NaCl (aq)
- 44 ± 1 138 ± 1 (0.14) 133 106
50 110 168
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PLGA
PLA-PEG
99.5
98.5
99.8
- 22 ± 2
124 ± 2 (0.21)
142
110
50
112 170
NaCl (aq)
- 39 ± 2
132 ± 1 (0.14)
151
122
70
118 184
DMEM/FBS
- 22 ± 1
146 ± 1 (0.21)
141
110
52
110 168
NaCl (aq)
- 38 ± 3
128 ± 1 (0.13)
144
116
74
112 164
DMEM/FBS
- 25 ± 2
183 ± 2 (0.25)
139
116
66
114 164
NaCl (aq)
- 39 ± 1
115 ± 1 (0.14)
44
74 118
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PLA
DMEM/FBS
110
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DMEM/FBS - 19 ± 1 112 ± 1 (0.11) 143 104
44 106 162
†
at 25°C in 1 mM NaCl (aq) or DMEM/FBS diluted 1:10 in ultrapure water; at 25°C in ultrapure water or
DMEM/FBS diluted 1:10 in ultrapure water; ‡samples incubated at 37°C in 10 mM NaCl (aq) or pure
DMEM/FBS and fractionated by asymmetric flow field flow fractionation in 10 mM NaCl (aq). PdI
polydispersity index; Distribution values in flow mode, including Dh: average hydrodynamic diameter by
dynamic light scattering; Dg: average gyration diameter by multi-angle laser light scattering; D0.05, D0.5 and
D0.95 were determined as described in the supplementary material.
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Figure 2: Atomic force microscopy images of the polyester nanocapsules in height (left) and
in amplitude (right) mode. All images were scanned at 2 × 2 µm and show lateral grey scale in
z-axis.
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3.2. Figure 3: Mean zeta potentials and
hydrodynamic mean diameters of the
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nanocapsules in water, pure fetal bovine serum (FBS) and DMEM cell culture medium
with 10% FBS (DMEM/FBS) over time determined by dynamic light scattering (DLS,
top) and comparison between DLS and nanoparticle tracking analysis in water
(bottom). Results were expressed as mean ± standard deviation (SD).Rose Bengal
adsorption study
RB adsorbed to the NC in a concentration-dependent manner (the higher the NC
concentration in the incubation medium, the lower the amount of free RB found in the
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ultrafiltrate) and in similar proportions regardless of the polymer (Figure 4A). Plotting the
partition coefficient against the NC total surface area (Figure 4B) resulted in a straight line at
lower NC content (at higher NC content free RB was scarce or depleted), which slope is often
used to compare nanoparticle hydrophobicity. The values obtained (148-203 m-2) were in a
narrow range and increased in the order PLA-PEG<PLGA~PCL<PLA, thus did not strictly
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follow the order of hydrophobicity of the bulk polyesters based on logP calculations
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(supplementary material).
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Figure 4: Rose Bengal (RB) adsorption study. The slope of the linear regression of the linear portion
of the plot partition coefficient (mass of bound vs free RB) as a function of surface area (insert in B)
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was 167, 203, 177 and 148 m-2 for PLC, PLA, PLGA and PEG-PLA nanocapsules, respectively.
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3.3. Size characterization and DIL loading stability by AF4-MALS-FLD
Additional size characterization of the NC suspensions by AF4-MALS-FLD revealed that the
NC did not suffer significant changes in size upon incubation in media of higher ionic strength
(10 mM NaCl) also used as the fractionation eluent (Figure 5). Fractionation in flow mode by
AF4 shows that less than 5 % of the NC population had gyration diameters (by MALS) below
50 nm or above 170 nm even upon incubation in DMEM/FBS, confirming that more than 90%
of the particles (by weight) were comprised in a narrow gyration diameter distribution centred
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around 110 nm, corresponding to a hydrodynamic diameter of 140 nm (Table 1). PLA-PEG
NC were significantly smaller than PCL, PLA and PLGA in water at 25°C or in 10 mM aqueous
NaCl and increased in size (p<0.05) when incubated with DMEM/FBS at 37°C for 2-3 h
(Figure 5B and Table 1), reaching size-distribution parameters similar to all other NC (Figure
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5D and Table 1).
Figure 5: Nanocapsule characterization by asymmetric flow field flow fractionation.
Size distributions calculated using UV detection as the concentration detector and gyration
radius (Rg) by multi-angle light scattering (A). Fractograms of PLGA NC (B) incubated at
37°C for 3h in 10 mM aqueous NaCl (dotted line) or DMEM with 10% FBS (full line).
Fractograms of PLA-PEG NC incubated in DMEM/FBS at 37°C recorded with fluorescence
detection at time intervals between 2 and 66 h (C). Fractograms recorded with fluorescence
detection of PCL, PLA, PLGA, PLA-PEG NC and DIL solution incubated in DMEM/FBS at
37 °C for 3h (D).
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Additionally, a kinetic study of DIL fluorescence in the NC, its release and association to
proteins present in DMEM/FBS was carried out by AF4-MALS-FLD (Figure 5C). Only large
macromolecules and nanoparticles are detected upon AF4 fractionation and DMEM/FBS
shows a strong UV absorption signal at 7-13 min retention time, corresponding to elution of
the proteins present in FBS, with no fluorescence signal at the wavelength selected for DIL
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detection (selectivity). Upon DMEM/FBS incubation with a solution of free-DIL (no NC), a
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strong fluorescence signal appeared, associated to FBS proteins (red line in Figure 5D),
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demonstrating that under our experimental conditions DIL in its free form was able not only
to associate to FBS proteins, but also to remain associated during fractionation. In another
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experiment, the level of fluorescence associated to the NC (retention times in the range of 12
to 35 min) was monitored as a function of time upon incubation of the NC in 10 mM aqueous
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NaCl or in DMEM/FBS at 37°C (Figure 5C). The proportion of fluorescence intensity
associated to the NC in this kinetic study was similar in all NC and remained higher than
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approximately 90% of the initial fluorescence during the experimental time. Only limited
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transfer of the fluorescent dye from the NC population to FBS proteins, identified by the
appearance of a shoulder at retention time 7-13 min, was detected within 2 to 12 h of
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incubation (Figure 5C and D). These results indicate that DIL did not leak from the NC and
that the NC fluorescence was maintained associated to NC upon prolonged incubation in cell
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culture medium containing serum proteins even under dynamic flow conditions.
3.4. Dye release in vitro
No fluorescence was detected in the release media (data not shown) indicating that for up to 3
h at 37°C the DIL was retained within the NC in the presence of proteins in the acceptor
medium.
3.5. Cell viability
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The viability of the three cell lines exposed to NC from 1 to 1000 µg/mL of polymer is shown
in Figure 6 and is dose-dependent in polymer. All NC exhibited very low cytotoxicity up to
100 µg/mL of polymer. On the other hand, the viability remained close to 100% for a 2 h
exposure (data not shown), higher than 70% for a 24 h exposure and was slightly lower at 48 h,
showing an incubation time-, cell type-, polymer type- and concentration-dependency (Figure
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6). Vero cells (normal) were more sensitive than HepG2 and Caco-2 tumor-derived cell lines.
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In the Vero cell line an effect of polymer hydrophobicity on cell viability at 24 and 48 h was
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observed, where NC prepared from the more hydrophobic PCL and PLA NC had IC50 values
of 248 and 128, respectively, whereas the NC from the less hydrophobic PLGA and PLA-PEG
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NC had IC50 values of 900 and >1000 µg/mL, respectively (Table imbedded in Figure 6).
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3.6. Total NC association to cells
The total fluorescence associated to Caco-2, HepG2 and Vero cells was very low (< 2% of the
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total dye in the incubation medium at 1000 ng DIL per mL) (Figure 7). Higher levels of NC
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were associated to HepG2 cells than to Caco-2 and Vero cells. Caco-2 cells showed similar
interaction with all NC types (4.9–7.7 ng per 6×104 cells). In contrast, HepG2 cells associated
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significantly higher amounts of PCL NC (13 ng per 6×104 cells) followed by PLA and PLAPEG NC (11 ng per 6×104 cells) compared to Vero and Caco-2 cells and strikingly lower
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amounts of PLGA NC (4.5 ng per 6×104 cells). No cell lines were able to distinguish between
PLA and PLA-PEG NC.
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Figure 6: Viability of cells incubated with nanocapsules at concentrations from 0 to 1000
µg/mL of polymer for 24 h (A, left) or 48 h (A, right) and corresponding IC 50, the polymer
concentration that reduces cell viability by 50% (B). Results were expressed as mean ± standard
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deviation (SD). Statistical differences were determined by comparing data between two groups
with normal distribution using an unpaired student´s t-test and also by one-way ANOVA with
Tukey´s post-test.
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Figure 7: Percentage of DIL associated to different cell lines. Horizontal white bars denote
significant differences (p < 0.05) between polymeric NC types for the same cell line. Letters c,
h, v refers to significant differences (p < 0.05) for the same NC type between Caco-2, HepG2
and Vero cell lines, respectively. Results were expressed as mean ± standard deviation (SD) of
three independent experiments performed in triplicate. Statistical differences were determined
by comparing data between two groups with normal distribution using an unpaired student´s ttest and also by one-way ANOVA with Tukey´s post-test.
3.7. Endocytosis mechanisms
Inhibition of uptake by Caco-2 cells via energy-dependent internalization pathways, by
maintaining the cells at low temperature (4°C), resulted in a 34-50% decrease in uptake
compared to the values obtained at 37°C (Figure 8A). Significant differences (p<0.05) were
observed between NC types, particularly in HepG2 cells, where the higher the hydrophobicity
of the polymer, the higher the binding at 4°C (PCL>PLA>PLGA NC). In all non-phagocytic
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cell lines PLA-PEG NC showed cell association levels similar to PLA NC. Non-energy
mediated NC binding was much lower in HepG2 cells than in Vero and Caco-2 cells (p<0.05).
In Vero cells, the more hydrophilic polymers (PLGA and PLA-PEG) resulted in higher binding
at 4°C (50-75%) than the more hydrophobic (PCL and PLA, 30-40%), oppositely to HepG2
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cells.
Among the energy-dependent mechanisms, inhibition of CME by chlorpromazine in Caco-2
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cells did not reduce the uptake of the NC containing the more hydrophobic PCL and PLA
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polymers (p>0.05) and had only a small effect on the uptake of NC prepared from more
hydrophilic PLGA (p<0.05). In general the CME mechanism of NC internalization in Caco-2
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cells was less evident than in other cells under our experimental conditions. Interestingly, cells
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treated with MβCD showed higher uptake of PCL NC than the control cells (p<0.001) at 37°C
and no inhibition for the other NC types, indicating poor involvement of CvME in Caco-2 cell
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uptake. Inhibition of macropinocytosis by cytochalasin D in Caco-2 cells induced a significant
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reduction of the uptake only for the NC composed of the more hydrophilic polymers (PLGA
and PLA-PEG, 60% and 50%, respectively) and no significant effect on PCL and PLA NC
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uptake.
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Figure 8: DIL fluorescence associated to cells following incubation of nanocapsules with
Caco-2 (A), HepG2 (B) and Vero (C) cells and inhibition of endocytosis mechanisms with
chlorpromazine, MβCD, cytochalasin D or incubation at 4°C. Data was normalized for 60,000
cells to allow comparison between cell lines. AU: arbitrary units of fluorescence intensity.
Results were expressed as mean ± standard deviation (SD) of three independent experiments
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performed in triplicate. Statistical differences were determined by comparing data between two
groups with normal distribution using an unpaired student´s t-test and also by one-way
ANOVA with Tukey´s post-test. White bars indicate p<0.05 relative to total association
(control at 37°C without inhibitors).
Maintenance of HepG2 cells at low temperature (4°C) was also able to reduce the uptake of
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NC by approximately 70-80% for all types of NC (Figure 8B), indicating that this cell type
internalized particles mainly by energy-dependent mechanisms. Inhibitors of endocytic
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pathways were able to reduce the uptake of NC by HepG2 cells indicating that all tested
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pathways were used for NC uptake. Inhibition of the CME reduced the uptake of PCL, PLA,
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PLGA and PLA-PEG NC by approximately 30%. CvME inhibition reduced the uptake of all
NC by up to 60 % being the main mechanism of uptake in this cell line. In addition,
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macropinocytosis inhibition increased with polymer hydrophilicity as the uptake of PCL, PLA,
PLGA and PLA-PEG NC was reduced by 20%, 15%, 66% and 37%, respectively. Strikingly,
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PLGA were the NC least internalized by HepG2 cells and interacted via energy-dependent
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pathways, particularly macropinocytosis to a higher extent than the other NC types.
Incubation of Vero cells at 4°C reduced the uptake of PCL, PLA, PLGA and PLA-PEG NC by
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70%, 63%, 49%, and 30%, respectively (Figure 8C), showing that the involvement of physical
binding in this cell type followed a direct correlation with polymer hydrophilicity. The CME
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was not evaluated in Vero cells since this cell line does not respond to chlorpromazine
treatment (Vercauteren et al., 2010). Inhibition of CvME and macropinocytosis reduced the
uptake by approximately 34-50% and 15-34%, respectively. CvME (p<0.05) and
macropinocytosis (p <0.001) uptake were significantly higher for PLGA NC in Vero cells
compared to the other NC.
3.8. Intracellular localization of the NC
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The NC intracellular localization was evaluated qualitatively by confocal microscopy (Figure
9). In Caco-2 cells (Figure 9A), PCL and PLA NC were distributed in the cytoplasm, with
higher diffuse fluorescence intensity in the cytoplasm and punctate pattern in the region near
the cytoplasmic membrane. PLGA NC related fluorescence was found near the cell nucleus,
oppositely to PCL NC. PLGA and PLA-PEG NC were more uniformly distributed in Caco-2
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cells with a diverse fluorescence pattern without significant association with the cell
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membrane. In 3D images of single cells, the fluorescence was located very close to the nucleus
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(perinuclear region), except for PCL NC.
In HepG2 cells (Figure 9B) no fluorescence accumulation was found near the plasma
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membrane (absence of a cell ghost pattern). Upon incubation with PLGA NC the fluorescence
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was weaker and concentrated in vesicular structures (punctuate pattern) rather than distributed
homogeneously through the cell cytoplasm, and presented many cells without DIL (red)
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fluorescence. PCL, PLA and PLA-PEG NC related fluorescence was much more diffuse and
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dispersed in the cytoplasm compared to the pattern with PLGA NC. The three-dimensional
(3D) Z series images showed fluorescence in the same axis as that of the nucleus, confirming
that in HepG2 cells all NC were located near the perinuclear region, inside the cellular
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compartment (Figures 9B). PLGA NC particularly highly accumulated in the perinuclear
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region and possibly inside cell nuclei.
Much lower fluorescence intensities were observed in Vero cells, with diffuse pattern of
distribution throughout the cytoplasm and no association to the plasma membrane of the cell
(Figure 9 C). Only traces of fluorescence were observed in the perinuclear region of Vero cells
after exposure to any of the NC with no clear differences between intracellular distribution
profiles. The three-dimensional (3D) Z series image show more concentrated fluorescence
related to PLGA NC near the nucleus of Vero cells.
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Figure 9: Confocal laser scanning images of Caco-2, HepG2 and Vero cells (A) and
tridimensional image analysis of single cells in Z series (B) exposed to 100 μg/mL of
nanocapsules for 2 h. Bars correspond to 20 µm. DAPI stained nuclei (blue) and DIL-labeled
nanocapsules (red). White arrows indicate DIL near the plasma membrane.
4. DISCUSSION
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It is vastly documented in the literature that NP uptake and endocytic mechanisms depend on
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the nanoparticle size (Oh and Park, 2014) and surface charge (Hühn et al., 2013). Therefore,
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fixing these parameters is of great importance for this type of evaluations. This was achieved
in our study, where DIL-labeled NC of similar size distributions and surface charge were
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obtained with four different polyesters. NC physical stability, size and surface charge were also
maintained after incubation in DMEM medium with 10% FBS under flow conditions, as
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confirmed by AF4-MALS-FLD and DLS analyses. Thus, without interference of these
parameters, the cellular pathways of NC uptake evaluated in our experiments are related
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exclusively to the differences in NC surface characteristics provided by the hydrophobicity of
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the polyester walls and NC structural organization. The core-shell organization of the NC with
a lipid core capable of bursting under the pressure of the AFM tip was demonstrated in previous
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studies (Leite et al., 2005, Assis et al., 2008), also confirmed by small angle neutron scattering
2001).
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(Rübe et al., 2005) and transition electron microscopy with cryo-fracture (Mosqueira et al.,
The Rose Bengal dye adsorption assay is currently the method of choice to evaluate the surface
hydrophobicity of nanoparticles and in particular pharmaceutical nanocarriers (Xiao and
Wiesner 2012, Gao and Lowry 2018). Doktorovova et al. (2012) proposed two modified
procedures for the Rose Bengal assay to characterize other lipid-based nanocarriers, which
circumvent the difficult nanoparticle removal from the continuous phase. However, in our case
another modification of the method had to be included with a filtration step to quantify the dye
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in the ultrafiltrate. The NC efficiently adsorbed Rose Bengal, removing the dye from the
aqueous phase even at low NC concentrations (low surface area), and resulting in higher
partition coefficients compared to other literature reports with different polymeric nanocarriers
(Gaumet, Gurny et al. 2009, Doktorovova, Shegokar et al. 2012, Xiao and Wiesner 2012).
However, the differences in partition coefficient between our polyester NC were small. The
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same was observed by other authors with nanospheres composed of polymers of drastically
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different degrees of hydrophobicity such as PLGA and polystyrene (Gaumet, Gurny et al.
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2009). This could be due the method’s many pitfalls (Xiao and Wiesner 2012), or to
interference of the surfactant stabilizer (Pluronic). However, in the presence of serum proteins
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an exchange can occur between the surfactant and the proteins modifying the surface of the
NC. Thus, the values obtained in the Rose Bengal assay may not be representative of the NC
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hydrophobicity as it is experienced by the cells in vitro tests and in vivo (Coty and Vauthier
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2018).
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Dye-loaded polymeric nanoparticles have gained prominence in the improvement of
fluorescence bioimaging techniques in vitro and in vivo (Reisch and Klymchenko, 2016). In
such applications, an important source of experimental bias is the potential release of the
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fluorescence marker from the NP in medium containing proteins and other complex
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components. We selected DIL due to its highly lipophilic character (log P = 23), which favors
its strong association to the oil core of NC. Furthermore, the stability of lipophilic
indocarbocyanine dyes for labeling similar oily core lipid NP was already confirmed by
different methods (Bastiat et al., 2013). Compared to lipid NC, polyester NC have an additional
barrier consisting of the polymer wall expected to further prevent dye release and transfer.
Nonetheless, serum proteins are known to accelerate the release of lipophilic cargos from
nanocarriers, as was reported in the case of DIL encapsulated in PLA-PEG micelles (Chen et
al., 2008).
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Therefore, we studied the encapsulation and release of DIL from the NC by an alternative and
original method in DMEM supplemented with FBS via AF4-MALS-FLD. AF4 is particularly
valuable in nanoparticle characterization because a mixture of nanosized objects can be
separated with respect to their hydrodynamic volume while low molar mass compounds are
removed during the fractionation process. In particular, AF4 coupled to light scattering
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detectors has been used to examine changes in NP size and colloidal stability before and after
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incubation in biological media (Ashby et al., 2013; Koshkina et al., 2016; Miller et al., 2012).
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Drug release from nanocarriers (liposomes) can be studied by AF4 (Hinna et al., 2016a; Hinna
et al., 2016b), suggesting that it would be a valuable tool to investigate the transfer of a
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fluorescent label physically entrapped in NP to proteins of the incubation medium used in cell
culture. Better than static release experiments the method presented herein can simulate dye
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release kinetics in flow conditions with the quantification of the dye transfer to serum proteins.
We confirmed that free DIL was able to associate with serum proteins when incubated with
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medium containing proteins, in agreement with literature data (Chen et al., 2008). In contrast,
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encapsulated DIL remained associated with the polyester NC with very little transfer to the
serum proteins, even under flow conditions. Thus, DIL “jumping” from NC to serum proteins
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can be considered negligible under our in vitro experimental conditions. Therefore, the amount
of cell-associated DIL quantified in our in vitro cell uptake studies could only result from NC
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binding and uptake by the cells, although a contribution of direct transfer of DIL from NC to
the cell membrane by physical contact could not be excluded, as schematically represented in
Figure 9. In this work, DIL was confirmed as a suitable dye to quantify the association of
polymeric NC to cells. The method used to quantify the dye involves extraction from the cells
using a strong non-ionic surfactant (Triton X-100) to disrupt the cell membrane and organic
solvent to fully solubilize the dye followed by steady-state fluorescence spectroscopy.
Although this method is time-consuming, it has the advantage of quantifying the dye in its fully
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soluble state. This is particularly important, considering that confined dyes within nanocarriers
may suffer extensive quenching effects, whereby fluorescence emission significantly decreases
(Reisch and Klymchenko, 2016). Fluorescence quenching in NP is mostly due to two factors:
high local dye concentration within the NP, leading to reabsorption of emitted fluorescence by
nearby molecules (“inner filter effect”) or aggregation of the dye within the NP, leading to
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alterations in the electronic molecular orbital and loss of the fluorophoric properties. In this
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extraction from cells in the concentration range studied.
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work, no concentration quenching was observed in the medium used to quantify DIL following
Finally, cytotoxicity typically is a concentration-dependent phenomenon (Oliveira et al., 2017).
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Therefore, safe concentrations in vitro were determined in this work for each cell and NC type.
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The nature of the polymer in the NC and cell type both influenced the IC50. IC50 values higher
than 1000 μg/mL were obtained for all NC in Caco-2 and HepG2 cells in 24 h, whereas Vero
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cells were more sensitive, showing the lowest IC50 values with PLA and PCL NC. Our study
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shows that normal cells may be more sensitive to NC toxicity than tumor-derived cells,
although the NC were quantitatively less internalized by the normal Vero cells under our
experimental condition. To allow comparisons between cell types and polymer composition,
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the cells were exposed to the same relatively high concentration of NC (100 μg of
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polymer/mL), which was considered the limiting concentration that does not cause significant
toxicity for up to 24 h (viability > 70%, Figure 5) and maximal ability to quantify and visualize
particle interaction with cells using confocal microscopy. The shorter (2 h) incubation time,
typically used in NC uptake studies, guarantees even lower cytotoxicity, to compensate for the
presence of endocytosis inhibitors in these experiments that are generally toxic to cells. No
morphological changes were detected in the studied cell lines under our experimental
conditions (Figures 8).
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Even though the cells were exposed to a high NP concentration, the total fluorescent dye
associated to cells was very little (0.45-1.3 %), in agreement with literature data on other
negatively charged polymeric NP in non-phagocytic cells (Gaumet et al., 2009; Platel et al.,
2016). HepG2 was the cell type that presented the highest capacity to internalize NC under the
conditions evaluated. This is expected since HepG2 are tumor-derived cells and perform
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detoxification/metabolization functions in vivo, interacting with particles in the blood
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circulation administered orally or by parenteral administration, a role for which a higher
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endocytic capacity is required than in other cell types (Loh et al., 2010). In the present work,
polymer hydrophobicity was also found to have a significant impact on the level of NC uptake
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in HepG2 cells, which presented a three-fold higher uptake of PCL NC compared to PLGA
NC. It can be speculated that in hepatic cells the higher complement activation by alternative
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pathways for more hydrophobic surfaces such as PCL may favor NC uptake as reported in the
case of macrophages (Mosqueira et al., 2001a).
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By incubating the cells at 4ºC, non-specific physical binding at the cell surface or
internalization by unknown non-energy mediated mechanisms are estimated. Physical binding
is an obligatory step to further internalization. Our results clearly show that the NC wall
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forming polymer hydrophobicity has a strong effect on physical binding in all the three cell
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lines, but this effect depends on the cell type and their contribution to the physiology of the
body. HepG2 cells involved specific endocytic energy-dependent mechanisms to a large extent
and little physical binding. Caco-2 and Vero cells interaction with NC had a significant
involvement of physical binding (50-65% and 30-70%, respectively). In Vero cells of epithelial
origin (kidneys), the higher the polymer hydrophobicity the lower the involvement of physical
binding.
Switching from conventional to PEG-stabilized nanoparticles is a well-known strategy to delay
blood clearance and enhance the pharmacokinetic profile of encapsulated drugs. PLA-PEG NC
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were demonstrated to reduce the uptake by J774A.1 macrophages (Mosqueira et al., 2001a)
and alter the NC blood clearance in vivo (Mosqueira et al., 2001b), affecting the blood/liverdistribution in a dose-dependent effect (Oliveira et al., 2017), and the efficacy of encapsulated
compounds (Branquinho et al., 2014). One of the aims of this study was to compare cell uptake
of a PEG-stabilized versus PLA NC, considering that some molecular drug targets are
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intracellular in non-phagocytic cells. Previous data with bidimensional electrophoresis
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indicated that complement activation in PLA-PEG NC may occur, although at a lower extent
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than in PLA NC (Mosqueira et al., 2001a). Our characterization data in serum medium suggest
that proteins may have adsorbed at the PLA-PEG NC surface. Accordingly, little differences
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were found between PLA and PLA-PEG in terms of level of cell-associated NC, endocytosis
mechanisms or intracellular distribution in non-phagocytic cells, which contrasts with previous
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reports in phagocytic cells (J774.A1 macrophages) (Mosqueira et al., 2001a). This suggests
that the differences in drug efficacy observed in vivo due to the PEG corona are not related to
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differences in direct interactions between the NC and non-phagocytic cells, but may be due to
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the rate of drug release, the type of protein corona adsorbed at the NC surface or to the altered
in vivo biodistribution (Bertrand et al., 2017). Furthermore, differences in levels of physical
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binding and macropinocytosis between PLA and PLA-PEG NC appear in Caco-2 and HepG2
cells, possibly related to differences in hydrophilicity of the NC surface. This confirms the
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importance of polymer hydrophobicity in the interaction with cells in the presence of different
incubation media simulating conditions as close as possible to in vivo conditions.
Caco-2 cells were expected to interact to a larger extent with NC in comparison with Vero
cells, due to their ability to actively transport matter across monolayers related to their
physiological function of absorption following the oral administration route. However, in these
two cell types no differences (p>0.05) between the levels of NC interaction according to
polymer hydrophobicity were observed under our experimental conditions. Several authors
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observed the involvement of CME and CvME in the uptake of different NP by Caco-2 cells,
such as lipid NC (50 nm, composed of phosphatidylcholine and tricaprylin) (Roger et al.,
2009), but not in negatively charged polystyrene NP (100 nm) (Bannunah et al., 2014). These
differences in uptake mechanisms could be due to the differences in size, surface charge and
uptake kinetics of the particles studied (Gaumet et al., 2008). However, in our study using
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140 nm negatively charged NC, macropinocytosis was identified as a major internalization
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route for most NC in Caco-2 cells.
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Multiple endocytic pathways were found to contribute to the uptake of NC by HepG2 cells and
CvME was the main entry route for the NC evaluated in our study (Figure 7). Other studies
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also reported the use of multiple pathways for NP uptake by this cell type. For example, CME
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and CvME inhibition in the same magnitude order as in the present work was observed in the
uptake by HepG2 cells of NP stabilized by a D-α-tocopheryl-polyethylene-glycol1000-poly(β-
D
amino ester) block copolymer (100 nm), where additionally no participation of
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macropinocytosis was observed (Chen et al., 2015). Although we did not observe a direct
correlation between polymer hydrophobicity and energy-dependent endocytic pathways in
HepG2 cells, the variability in literature data suggests that NC composition and polymer
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hydrophobicity may contribute to the prevalence of a given uptake mechanism. The higher
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uptake and greater ability of HepG2 cells to use all energy-dependent endocytic pathways
suggests a major role of hepatocytes in the metabolism of ~100 nm biodegradable polymeric
nanocarriers after intravenous administration.
Few studies are available regarding the internalization pathways of polymeric NP in Vero cells.
These are generally directed at viral infection models (Hernaez et al., 2016) and a few studies
report endocytosis of metallic or inorganic NP (Li et al., 2017; Sun et al., 2017). Vero cells,
although originated from the kidney, were selected to represent a normal cell line for
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comparison with the other tumor cell lines. Vero cells are not considered to be a good model
to study filtration ability and nephrotoxicity (Huang et al., 2014). In our study, we observed a
significant contribution of CvME in this normal cell line (34-50%) and lower contribution of
macropinocytosis (20%). Passive NC binding to Vero cell seems to play an important role with
the NC prepared from the more hydrophilic polymers. Similarly to this study the internalization
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of mono- and dihydrated calcium oxalate nanocrystals of similar sizes also showed a great
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reduction in uptake (44%) when Vero cells were incubated at 4°C (Sun et al., 2017). Our results
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together with the literature show that CvME and passive interaction pathways participate
significantly in NP association with Vero cells, even though the overall NP uptake is very
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limited.
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Thus, our findings evidence dramatic differences in the NP uptake behavior of different cell
lines, as summarized in Figure 9. Consequently, comparative studies using different cell
models are always necessary to predict the in vivo safety or in vitro interaction of NP with cells.
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Similar conclusions were reported in the work of Kroll and collaborators (2011), who tested
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the cytotoxicity of inorganic NP in a set of ten cell lines (Kroll et al., 2011).
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Figure 9: Schematic summary of main mechanisms of nanoparticle interaction with Caco-2,
HepG2 and Vero cells. ND: not determined; X: no effect; percentages were quantitatively
determined by steady-state spectrofluorimetry.
The pattern of NC-related fluorescence intracellular distribution depends on the NC type.
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Qualitative analysis shows a diffuse pattern of fluorescence, in agreement with the multiple
mechanisms of endocytosis employed by the three cell lines. The images show PLGA NC in
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all the three cells located close to nucleus. This work suggests that the biodegradable polyesterbased NC, in particular PLGA NC, can be selected when the subcellular therapeutic target is
the nuclear region, as in cancer chemotherapies, even though the level of cell uptake in the nonphagocytic cells tested here was low. PCL NC were present near the cell membrane in Caco-2
cells only, which is in agreement with the significant involvement of physical binding.
Interestingly, in HepG2 cells only PLGA NC showed a punctuate pattern without fluorescence
near the plasma membrane and this NC type was the one most internalized by
macropinocytosis. We observed a higher involvement of macropinocytosis when hydrophilic
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polymers (PLGA and PLA-PEG) were used in the NC wall and this generated a punctuate
pattern of fluorescence in Caco-2 and HepG2 cells. When CvME mechanisms were
predominant, a more diffuse fluorescence pattern was observed, particularly significant and
clear in Vero cells. Accordingly, macropinosomes are expected to internalize larger objects
and may appear as larger dots in the cell cytoplasm than clathrin and caveolae pits, which are
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expected to uptake smaller particles. In this context, identifying the endocytic pathways of
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nanocarriers in each target cell could assist in optimizing the treatments of numerous diseases
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where the use of these nanostructures may be employed (Hillaireau and Couvreur, 2009).
5. CONCLUSIONS
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The NC prepared with four biodegradable polyesters of very similar sizes and zeta potentials
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were stable in cell culture medium. The AF4 technique was valuable to study the colloidal
stability and simultaneously evidence the kinetics of dye transfer to serum proteins,
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demonstrating that DIL was retained within the NC, even in the presence of serum proteins.
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Our cell viability study over a large concentration range reinforces the safety of these
nanocarriers, and indicates an effect of the polymer used on the IC50 of the NC, identified in
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the most sensitive cell line tested (Vero). The cells associated low levels of NC, in agreement
with their non-phagocytic nature. HepG2 cells showed the highest levels of NC
AC
internalization among the three tested cell lines and the level of NC uptake followed the order
of polymer hydrophobicity: PLGA NC < PLA NC < PCL NC, but no differences in uptake
levels were found between PLA and PEG-PLA NC. Polyester hydrophobicity seemed to
induce differences in NC physical binding at the cell surface, depending on cell type.
Altogether our data indicates that the nature of the polymer forming wall in biodegradable
polyester NC affects the level and the mechanisms of NC uptake by cells.
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In recent years, more emphasis has been placed on studying the endocytic pathways of nonphagocytic cells in comparison with phagocytic cells, because the former are common
therapeutic targets. Considering that the level of NC association and the uptake mechanisms
varied largely according to NC and cell type, this study can assist in the selection of polymers
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used in drug nanocarrier design based on the characteristics of the target organ cell.
Acknowledgements: we thank Dr. NC Nogueira-Paiva for assistance with confocal
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microscopy. This work was financially supported by the Brazilian agencies Conselho Nacional
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de Desenvolvimento Científico e Tecnológico (CNPq, Grant #310463/2015-7 and
#481872/2013-2), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG,
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#APQ-02864-16), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
NANOBIOMG Network (RED-00007-14) Minas Gerais and INCT-NANOFARMA (Grant
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E
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#2014/50928-2 and #465687/2014-8).
REFERENCES
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Declarations of interest: none
AC
Ashby, J., Schachermeyer, S., Pan, S., Zhong, W., 2013. Dissociation-Based Screening of Nanoparticle–
Protein Interaction via Flow Field-Flow Fractionation. Analytical Chemistry 85, 7494-7501.
Attili-Qadri, S., Karra, N., Nemirovski, A., Schwob, O., Talmon, Y., Nassar, T., Benita, S., 2013. Oral delivery
system prolongs blood circulation of docetaxel nanocapsules via lymphatic absorption. PNAS 110,
17498-17503.
Bannunah, A.M., Vllasaliu, D., Lord, J., Stolnik, S., 2014. Mechanisms of Nanoparticle Internalization and
Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge. Molecular
Pharmaceutics 11, 4363-4373.
Bastiat, G., Pritz, C.O., Roider, C., Fouchet, F., Lignières, E., Jesacher, A., Glueckert, R., Ritsch-Marte, M.,
Schrott-Fischer, A., Saulnier, P., Benoit, J.-P., 2013. A new tool to ensure the fluorescent dye labeling
stability of nanocarriers: A real challenge for fluorescence imaging. Journal of Controlled Release
170, 334-342.
Behzadi, S., Serpooshan, V., Tao, W., Hamaly, M.A., Alkawareek, M.Y., Dreaden, E.C., Brown, D., Alkilany,
A.M., Farokhzad, O.C., Mahmoudi, M., 2017. Cellular uptake of nanoparticles: journey inside the cell.
Chemical Society Reviews 46, 4218-4244.
42
ACCEPTED MANUSCRIPT
AC
CE
PT
E
D
MA
NU
SC
RI
PT
Benvegnú, D.M., Barcelos, R.C.S., Boufleur, N., Reckziegel, P., Pase, C.S., Ourique, A.F., Beck, R.C.R.,
Bürger, M.E., 2011. Haloperidol-loaded polysorbate-coated polymeric nanocapsules increase its
efficacy in the antipsychotic treatment in rats. European Journal of Pharmaceutics and
Biopharmaceutics 77, 332-336.
Bertrand, N., Grenier, P., Mahmoudi, M., Lima, E.M., Appel, E.A., Dormont, F., Lim, J.-M., Karnik, R.,
Langer, R., Farokhzad, O.C., 2017. Mechanistic understanding of in vivo protein corona formation on
polymeric nanoparticles and impact on pharmacokinetics. Nature Communications 8, 777.
Branquinho, R.T., Furtado Mosqueira, V.C., Valamiel de Oliveira-Silva, J.C., Simoes-Silva, M.R., SaudeGuimaraes, D.A., de Lana, M., 2014. Sesquiterpene Lactone in Nanostructured Parenteral Dosage
Form Is Efficacious in Experimental Chagas Disease. Antimicrobial Agents and Chemotherapy 58,
2067-2075.
Branquinho, R.T., Roy, J., Farah, C., Garcia, G.M., Aimond, F., Le Guennec, J.-Y., Saude-Guimarães, D.A.,
Grabe-Guimaraes, A., Mosqueira, V.C.F., de Lana, M., Richard, S., 2017. Biodegradable Polymeric
Nanocapsules Prevent Cardiotoxicity of Anti-Trypanosomal Lychnopholide. Scientific Reports 7,
44998.
Chen, F.Q., Zhang, J.M., Wang, L., Wang, Y.T., Chen, M.W., 2015. Tumor pH(e)-triggered charge-reversal
and redox-responsive nanoparticles for docetaxel delivery in hepatocellular carcinoma treatment.
Nanoscale 7, 15763-15779.
Chen, H., Kim, S., He, W., Wang, H., Low, P.S., Park, K., Cheng, J.X., 2008. Fast release of lipophilic agents
from circulating PEG-PDLLA micelles revealed by in vivo Forster resonance energy transfer imaging.
Langmuir 24, 5213-5217.
Cheng, X.J., Tian, X., Wu, A.Q., Li, J.X., Tian, J., Chong, Y., Chai, Z.F., Zhao, Y.L., Chen, C.Y., Ge, C.C., 2015.
Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and Nonphagocytic
Cells in a Size-Dependent Manner. Acs Applied Materials & Interfaces 7, 20568-20575.
D'Addio, S. M.; Saad, W.; Ansell, S. M.; Squiers, J. J.; Adamson, D. H.; Herrera-Alonso, M.; Wohl, A. R.;
Hoye, T. R.; Macosko, C. W.; Mayer, L. D.; Vauthier, C.; Prud'homme, R. K., 2012. Effects of block
copolymer properties on nanocarrier protection from in vivo clearance. Journal of Controlled
Release, 162 (1), 208-217.
de Mello, C.G., Branquinho, R.T., Oliveira, M.T., Milagre, M.M., Saude-Guimaraes, D.A., Mosqueira, V.C.,
Lana, M., 2016. Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous
Administration in Murine Experimental Chagas Disease. Antimicrob Agents Chemother 60, 52155222.
de Paula, C.S., Tedesco, A.C., Primo, F.L., Vilela, J.M.C., Andrade, M.S., Mosqueira, V.C.F., 2013.
Chloroaluminium phthalocyanine polymeric nanoparticles as photosensitisers: Photophysical and
physicochemical characterisation, release and phototoxicity in vitro. European Journal of
Pharmaceutical Sciences 49, 371-381.
FDA, Food and Drug Administration (U.S.) Code of Federal Regulations Title 21
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=177.
Feliu, N., Sun, X., Alvarez Puebla, R.A., Parak, W.J., 2017. Quantitative Particle–Cell Interaction: Some
Basic Physicochemical Pitfalls. Langmuir 33, 6639-6646.
Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S., 1989. Nanocapsule formation by
interfacial polymer deposition following solvent displacement. International Journal of
Pharmaceutics 55, R1-R4.
Garcia, G.M., Oliveira, L.T., Pitta, I.d.R., de Lima, M.d.C.A., Vilela, J.M.C., Andrade, M.S., Abdalla, D.S.P.,
Mosqueira, V.C.F., 2015. Improved nonclinical pharmacokinetics and biodistribution of a new PPAR
pan-agonist and COX inhibitor in nanocapsule formulation. Journal of Controlled Release 209, 207218.
Gaumet, M., Gurny, R., Delie, F., 2009. Localization and quantification of biodegradable particles in an
intestinal cell model: The influence of particle size. European Journal of Pharmaceutical Sciences 36,
465-473.
43
ACCEPTED MANUSCRIPT
AC
CE
PT
E
D
MA
NU
SC
RI
PT
Gaumet, M., Vargas, A., Gurny, R., Delie, F., 2008. Nanoparticles for drug delivery: The need for precision
in reporting particle size parameters. European Journal of Pharmaceutics and Biopharmaceutics 69,
1-9.
He, B., Lin, P., Jia, Z.R., Du, W.W., Qu, W., Yuan, L., Dai, W.B., Zhang, H., Wang, X.Q., Wang, J.C., Zhang, X.,
Zhang, Q., 2013. The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells.
Biomaterials 34, 6082-6098.
Hernaez, B., Guerra, M., Salas, M.L., Andres, G., 2016. African Swine Fever Virus Undergoes Outer
Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from
Multivesicular Endosomes. Plos Pathogens 12.
Hillaireau, H., Couvreur, P., 2009. Nanocarriers' entry into the cell: relevance to drug delivery. Cellular
and Molecular Life Sciences 66, 2873-2896.
Hinna, A.H., Hupfeld, S., Kuntsche, J., Bauer-Brandl, A., Brandl, M., 2016a. Mechanism and kinetics of the
loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionationbased drug release-/transfer-assay. Journal of Controlled Release 232, 228-237.
Hinna, A.H., Hupfeld, S., Kuntsche, J., Brandl, M., 2016b. The use of asymmetrical flow field-flow
fractionation with on-line detection in the study of drug retention within liposomal nanocarriers and
drug transfer kinetics. Journal of Pharmaceutical and Biomedical Analysis 124, 157-163.
Huang, J.X., Blaskovich, M.A., Cooper, M.A., 2014. Cell- and biomarker-based assays for predicting
nephrotoxicity. Expert Opin. Drug Metab. Toxicol, 14, 1621-1635.
Hühn, D., Kantner, K., Geidel, C., Brandholt, S., De Cock, I., Soenen, S.J.H., Rivera_Gil, P., Montenegro, J.M., Braeckmans, K., Müllen, K., Nienhaus, G.U., Klapper, M., Parak, W.J., 2013. Polymer-Coated
Nanoparticles Interacting with Proteins and Cells: Focusing on the Sign of the Net Charge. ACS Nano
7, 3253-3263.
Koshkina, O., Westmeier, D., Lang, T., Bantz, C., Hahlbrock, A., Wurth, C., Resch-Genger, U., Braun, U.,
Thiermann, R., Weise, C., Eravci, M., Mohr, B., Schlaad, H., Stauber, R.H., Docter, D., Bertin, A.,
Maskos, M., 2016. Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on
Protein Adsorption in Serum and Cellular Uptake. Macromolecular Bioscience 16, 1287-1300.
Kroll, A., Dierker, C., Rommel, C., Hahn, D., Wohlleben, W., Schulze-Isfort, C., Göbbert, C., Voetz, M.,
Hardinghaus, F., Schnekenburger, J., 2011. Cytotoxicity screening of 23 engineered nanomaterials
using a test matrix of ten cell lines and three different assays. Particle and Fibre Toxicology 8, 9.
Leite, E.A., Grabe-Guimaraes, A., Guimaraes, H.N., Lins Machado-Coelho, G.L., Barratt, G., Mosqueira,
V.C.F., 2007. Cardiotoxicity reduction induced by halofantrine entrapped in nanocapsule devices. Life
Sciences 80, 1327-1334.
Li, Y.H., Lin, Z.F., Xu, T.T., Wang, C.B., Zhao, M.Q., Xiao, M.S., Wang, H.Z., Deng, N., Zhu, B., 2017. Delivery
of VP1 siRNA to inhibit the EV71 virus using functionalized silver nanoparticles through ROSmediated signaling pathways. Rsc Advances 7, 1453-1463.
Loh, J.W., Yeoh, G., Saunders, M., Lim, L.Y., 2010. Uptake and cytotoxicity of chitosan nanoparticles in
human liver cells. Toxicology and Applied Pharmacology 249, 148-157.
Marin, E., Briceno, M.I., Caballero-George, C., 2013. Critical evaluation of biodegradable polymers used in
nanodrugs. International Journal of Nanomedicine 8, 3071-3091.
Meindl, C., Öhlinger, K., Ober, J., Roblegg, E., Fröhlich, E., 2017. Comparison of fluorescence-based
methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.
Toxicology 378, 25-36.
Miller, T., Rachel, R., Besheer, A., Uezguen, S., Weigandt, M., Goepferich, A., 2012. Comparative
Investigations on In Vitro Serum Stability of Polymeric Micelle Formulations. Pharmaceutical
Research 29, 448-459.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival - application to proliferation
and cytotoxicity assays. Journal of Immunological Methods 65, 55-63.
Mosqueira, V.C., Legrand, P., Barratt, G., 2006. Surface-modified and conventional nanocapsules as novel
formulations for parenteral delivery of halofantrine. J Nanosci Nanotechnol 6, 3193-3202.
44
ACCEPTED MANUSCRIPT
AC
CE
PT
E
D
MA
NU
SC
RI
PT
Mosqueira, V.C., Legrand, P., Gulik, A., Bourdon, O., Gref, R., Labarre, D., Barratt, G., 2001a. Relationship
between complement activation, cellular uptake and surface physicochemical aspects of novel PEGmodified nanocapsules. Biomaterials 22, 2967-2979.
Mosqueira, V.C.F., Legrand, P., Morgat, J.L., Vert, M., Mysiakine, E., Gref, R., Devissaguet, J.P., Barratt, G.,
2001b. Biodistribution of long-circulating PEG-grafted nanocapsules in mice: Effects of PEG chain
length and density. Pharmaceutical Research 18, 1411-1419.
Mosqueira, V.C.F., Loiseau, P.M., Bories, C., Legrand, P., Devissaguet, J.P., Barratt, G., 2004. Efficacy and
pharmacokinetics of intravenous nanocapsule formulations of halofantrine in Plasmodium bergheiinfected mice. Antimicrobial Agents and Chemotherapy 48, 1222-1228.
Nawa, M., Takasaki, T., Yamada, K.I., Kurane, I., Akatsuka, T., 2003. Interference in Japanese encephalitis
virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine. Journal of General
Virology 84, 1737-1741.
Nel, A.E., Mädler, L., Velegol, D., Xia, T., Hoek, E.M.V., Somasundaran, P., Klaessig, F., Castranova, V.,
Thompson, M., 2009. Understanding biophysicochemical interactions at the nano–bio interface.
Nature Materials 8, 543.
Oh, N., Park, J.H., 2014. Endocytosis and exocytosis of nanoparticles in mammalian cells. International
Journal of Nanomedicine 9, 51-63.
Oliveira, L.T., de Paula, M.A., Roatt, B.M., Garcia, G.M., Silva, L.S.B., Reis, A.B., de Paula, C.S., Vilela,
J.M.C., Andrade, M.S., Pound-Lana, G., Mosqueira, V.C.F., 2017. Impact of dose and surface features
on plasmatic and liver concentrations of biodegradable polymeric nanocapsules. European Journal of
Pharmaceutical Sciences 105, 19-32.
Platel, A., Carpentier, R., Becart, E., Mordacq, G., Betbeder, D., Nesslany, F., 2016. Influence of the
surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and
endocytosis. Journal of Applied Toxicology 36, 434-444.
Pound-Lana, G., Rabanel, J.-M., Hildgen, P., Mosqueira, V.C.F., 2017. Functional polylactide via ringopening copolymerisation with allyl, benzyl and propargyl glycidyl ethers. European Polymer Journal
90, 344-353.
Reisch, A., Klymchenko, A.S., 2016. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter
Tools for Bioimaging. Small 12, 1968-1992.
Rodrigues, S.F., Fiel, L.A., Shimada, A.L., Pereira, N.R., Guterres, S.S., Pohlmann, A.R., Farsky, S.H., 2016.
Lipid-Core Nanocapsules Act as a Drug Shuttle Through the Blood Brain Barrier and Reduce
Glioblastoma After Intravenous or Oral Administration. Journal of biomedical nanotechnology 12,
986-1000.
Roger, E., Lagarce, F., Garcion, E., Benoit, J.P., 2009. Lipid nanocarriers improve paclitaxel transport
throughout human intestinal epithelial cells by using vesicle-mediated transcytosis. Journal of
Controlled Release 140, 174-181.
Shen, J., Burgess, D.J., 2013. In Vitro Dissolution Testing Strategies for Nanoparticulate Drug Delivery
Systems: Recent Developments and Challenges. Drug delivery and translational research 3, 409-415.
Shurety, W., Bright, N.A., Luzio, J.P., 1996. The effects of cytochalasin D and phorbol myristate acetate on
the apical endocytosis of ricin in polarised Caco-2 cells. Journal of Cell Science 109, 2927-2935.
Souza, A.C.M., Mosqueira, V.C.F., Silveira, A.P.A., Antunes, L.R., Richard, S., Guimarães, H.N., GrabeGuimarães, A., 2017. Reduced cardiotoxicity and increased oral efficacy of artemether polymeric
nanocapsules in Plasmodium berghei-infected mice. Parasitology, 1-9.
Sun, X.Y., Gan, Q.Z., Ouyang, J.M., 2017. Size-dependent cellular uptake mechanism and cytotoxicity
toward calcium oxalate on Vero cells. Scientific Reports 7, 1-12.
Vercauteren, D., Vandenbroucke, R.E., Jones, A.T., Rejman, J., Demeester, J., De Smedt, S.C., Sanders,
N.N., Braeckmans, K., 2010. The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers:
Optimization and Pitfalls. Molecular Therapy 18, 561-569.
Yameen, B., Choi, W.I., Vilos, C., Swami, A., Shi, J.J., Farokhzad, O.C., 2014. Insight into nanoparticle
cellular uptake and intracellular targeting. Journal of Controlled Release 190, 485-499.
45
ACCEPTED MANUSCRIPT
AC
CE
PT
E
D
MA
NU
SC
RI
PT
Zhang, Z., Qu, Q., Li, J., Zhou, S., 2013. The Effect of the Hydrophilic/Hydrophobic Ratio of Polymeric
Micelles on their Endocytosis Pathways into Cells. Macromolecular Bioscience 13, 789-798.
46
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