Bibliothèque sur exocytosis des

nanoparticules

Mots-clés: nanoparticles, exocytosis, Efflux

Database: Pubmed, Sciencedirect
Jusqu’à 26-04-2010

03-05-2010
ARTICLE I
Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide)
nanoparticles in vascular smooth muscle cells.
Panyam J, Labhasetwar V. Pharm Res. 2003 Feb;20(2):212-20
Mots clés: Exocytosis, PLGA nanoparticles, vascular smooth muscle cells (VSMCs), in vitro

Methods:
•NPs: BSA PLGA NPs, 97 ± 3nm
•Marker: 6-coumarin
•Determination:
•confocal microscopy/ HPLC: endocytosis/exocytosis des NPs sur VSMCs
•confocal software (LSM-PC): Analyse the change in fluorescence intensity
of the cell images from confocal microscopy
Results:

•Exocytosis of NPs occurred with about 65% of the internalized fraction undergoing exocytosis in
30 min;
•The exocytosis of NPs was reduced after the treatment of cells with the combination of sodium
azide and deoxyglucose, suggesting that exocytosis of NPs is an energy-dependent process.
•The exocytosis of NPs was almost completely inhibited when the medium was depleted of
serum.
•The addition of BSA in the serum free medium with or without platelet derived growth factor
(PDGF) induced exocytosis of NPs.
Endocytosis
Endocytosis of NPs by VSMC

Dose- dependent Time-dependent

1H 100µg/ml

•Endocytosis of NPs was a concentration-, time-, and energy-dependent endocytic process;

•Endocytosis of NPs increased with incubation time.
Endocytosis

Time-series confocal images (100×) of NPs uptake by VSMC

100µg/ml

•Endocytosis of NPs is rapid, as early as within 1 min after incubation.

Exocytosis

Effect of NPs dose (A) and preincubation time (B) on NPs exocytosis.
by HPLC

•Increasing the dose of NPs from 100 to 200 µg/mL in the medium resulted in a
∼2.5-fold increased retention of NPs in the cells (Fig. A).
•The time of incubation of NPs before the exocytosis study showed relatively less
significant effect on the fraction of NPs that was retained (Fig. B).
 Mass balance in NPs exocytosis from VSMCs.
(100µg/ml, after 1H incubation)

The total NPs levels matched closely with that of intracellular NPs levels at 0 H.
 Effect of metabolic inhibitors (NaN3 +deoxyglucose) on exocytosis of NPs.

NPs: 100µg/ml, 1H

Exocytosis of NPs was decreased in the presence of metabolic inhibitors (NaN3/deoxyglucose).

Exocytosis is an active process: The exocytosis of NPs was partially inhibited thereafter and was reduced by
about 40% as compared with controls at 6 h.
 Effect of serum on NPs exocytosis.
NPs: 100µg,1H

SM, serum containing medium

SFM, serum-free medium;

Exocytosis of NPs was inhibited almost completely when the endocytosis/exocytosis

were carried out in the SFM
Effect of BSA and PDGF on the exocytosis of NPs in serum-free medium (SFM).
NPs: 200µg/ml, 1H

Addition of 1% BSA in the SFM resulted in the exocytosis of NPs similar to the
exocytosis observed in SM
Addition of platelet derived growth factor(PDGF) along with 1% BSA did not show
any additional effect on exocytosis of NPs
ARTICLE IV

Enhanced Antiproliferative Activity of Transferrin-Conjugated Paclitaxel-Loaded

Nanoparticles Is Mediated via Sustained Intracellular Drug Retention

Sanjeeb K. Sahoo and Vinod Labhasetwar; Mol. Pharm. , 2005

Mots clés: endocytosis, exocytosis, Tf-PLGA nanoparticles, MCF-7(/Adr) cells(beast cancer cell line), in
vitro
Methods:
•NPs: Tf/PVA PLGA NPs marqués 6-coumarin (216 nm)
•Cells: MCF-7(/Adr) cells (beast cancer cell line)
 Exocytosis of Tf- and blank- NPs in MCF-7 cells.
NPs: 100µg/ml, 1H
NPs in cells were determined by fluorescence indensity from extracted NPs in
lyophilized cells on HPLC

Tf-NPs

Blank-NPs

Tf-NPs demonstrated reduced exocytosis compared to blank-NPs.

More than 75% of the internalized blank-NPs were exocytosed during the
first 2 H compared to 50% of the Tf-NPs
ARTICLE II

Effects of particle size and surface charge on cellular uptake and biodistribution of
polymeric nanoparticles
Chunbai He, Yiping Hu, Lichen Yin, Cui Tang and Chunhua Yin. Biomaterials 31,(13), 2010, Pages 3657-3666
Mots-clés: Endocytosis, biodistribution, chitosan NPs, in vitro, in vivo

NPs: carboxymethyl chitosan grafted NPs (CMC NP-)

chitosan hydrochloride grafted NPs (CH NP+)
Marker: rhodamine B (RhB)
Size=150 ~ 500 nm
Zeta potential=−40 mV ~ +35 mV

Cells:
murine macrophage, L02 and SMMC-7721 cells (Non-phagocytic cells)
 Endocytosis of CMC NP- and CH NP+ in murine macrophage
2H

•The surface charge of NP- &NP+ significantly affected their endocytosis by macrophage.
Endocytosis increased with the surface charge increasing (for both NP+/-).
•Phagocytic cells favored the uptake of larger size particles
•When the absolute values of ζ-potential were similar, NPs+ showed a higher endocytosis
compared to NPs-
 Endocytosis of NP-, NP+ in Non-phagocytic cells
2H

L02 cells

SMMC-7721 cells

Less NP- and more NP+ were tended to be more efficiently internalized by both L02 and
SMMC-7721 cells, suggesting that surface charge played an important role in cellular
endocytosis of these NPs. Compared to macrophage, non-phagocytic cells favored the
uptake of smaller size particles.
Endocytosis of these NPs was cell-line-dependent:

Endocytosis of NP-: SMMC-7721 > L02 > A549 > 786-O > HFL-I > HEK 293

Endocytosis of NP+: SMMC-7721 > A549 > 786-O > HEK 293 > L02 > HFL-І

Macrophage endocytosis: mainly size- and charge-dependent;

non-phagocytic endocytosis: size-,charge- and NPs composition -dependent
 Endocytosis inhibition of NP- (A) and NP+ (B) in SMMC-7721 cells at 4 °C

•Endocytosis of NP- and NP+ was significantly inhibited at 4 °C.

•Following colchicine or NaN3 treatment, endocytosis inhibition of NP- and NP+ with
larger particle size and higher surface charge was significantly elevated.
 Biodistribution of NP- after i.v. administration in H-22 tumor bearing mice.

Both surface charge and particle size could influence the biodistribution of NP-. NP- with lower surface charge and smaller particle
size tended to be more efficiently distributed in the tumor, and the peak of distribution percent appeared at 4  h after injection. The
increase in the surface charge as well as particle size of NP- led to the decrease in blood distribution. Most of NP- with larger
particle size was cleared off from the blood after 1 h. Increase in the surface charge and particle size correlated to the elevated
distribution percentage of NP- in liver and spleen. Surface charge exerted unappreciable effect on the pulmonary distribution
percentage of NPs-, while NPs with larger particle size increased distribution level. In addition, NP- exhibited a rapid accumulation in
lung. Particle size and surface charge exerted unappreciable effect on the distribution behavior of NP- in kidney with a relatively
stable distribution level up to 24 h.
 Biodistribution of NP+ after i.v. administration in H-22 tumor bearing mice

The disposition of NP+ in tumor was notably elevated with the increase of surface charge. Compared to NP+(25,150), tumor
accumulations of NP+(25,300) were significantly reduced. Peak distribution of NP+ in the tumor was observed at 2  h after
administration and NP+ was retained in the tumor throughout 24-H period. About 2.4% of NP+ was detected in the blood at 1  H after
administration and no significant difference was observed among NP+ with different particle size and surface charge. No significant
difference of distribution was observed in the liver or spleen among NPs with different surface charge. The liver accumulation of
NP+(25,300) was significantly higher than that of NP+(25,150) while in the spleen the trend was opposite. NP+ accumulated in the
lung at a relatively low rate, and when the ζ-potential increased from 15 mV to 35 mV, the distribution percent in the lung was
increased. Particle size and surface charge had no influence on the distribution behavior of NP+ in kidney.
ARTICLE III
Mechanisms of alveolar epithelial translocation of a defined population of nanoparticles.
Yacobi NR, Malmstadt N, Fazlollahi F, Demaio L, Marchelletta R, Hamm-Alvarez SF, Borok Z, Kim KJ, Crandall ED.
Am J Respir Cell Mol Biol. 2009
Mots-clés: Epithelial transport, primary rat alveolar epithelial cell monolayers, polystyrene nanoparticles, in vitro

Methods:

Cells: primary rat alveolar epithelial cell monolayers (RAECM)

NPs: polystyrene nanoparticles (PNP, 20- and 100-nm, carboxylate (negatively charged) or amidine
(positively charged)
Confocal laser scanning microscopy

Results:

PNP translocation across RAECM takes place primarily through transcellular pathways, but not
endocytosis
Higher trafficking rates of PNP+ compared to PNP- are dependent on their surface charge and/or
relative hydrophobicity
ARTICLE V

N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for

intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release
Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW. J Control Release. 2006 ;115(1):37-
45
Mots-clés: Endocytosis, exocytosis, chitosan NPs, intracytoplasmic

Methods:
NPs: N-acetyl histidine-conjugated glycol chitosan NPs
Marquer: Fitc

Cells:
•HeLa: human epithelioid cervical cancer cell line
•A549 human lung cancer cell line
•MDA-MB231 human breast cancer cell line (ATCC)

Flow cytometry, Confocal microscopy

Endocytosis of Chitosan NPs by HeLa cell.
6H, FACs

•Endocytosis of Chitosan NPs was very rapid.

•It was both time- and concentration-dependent
•Saturation was achieved in 6 h
Endocytosis/exocytosis of Chitosan NPs by HeLa cells
Pre-incubation time: 10 min~ 6 H

•When the pre-incubation times were relatively short,

10 min and 30 min, a large amount of the NPs was
exocytosed after the removal of NPs from the external
media.
•The amount of extocytosed NPs decreased with
increasing pre-incubation time, up to 6 H
ARTICLE VI

Effect of poly(ethylene glycol)-block-polylactide nanoparticles on hepatic cells of mouse:

Low cytotoxicity, but efflux of the nanoparticles by ATP-binding cassette transporters
Yangde Zhang, ZhiyuanHu, Maoying Ye, Yifeng Pan, Jiji Chen, Yulin Luo, Yanqiong Zhang, Lianxiang He and Jiwei Wang.
European Journal of Pharmaceutics and Biopharmaceutics, Volume 66, Issue 2, May 2007, Pages 268-280

Mots-clés: Efflux, PEG-PLA NPs, Human hepatoma (HepG2) cells, primary hepatic cells, mouse, in vitro, in vivo

NPs: PEG-PLA NPs (150 nm, -20 mV)

Marker: 6-coumarin
Cells: Human hepatoma (HepG2) cells,
Mouse primary hepatic cells
 Endocytosis/efflux of PLA–PEG NPs by mouse primary hepatocytes
HPLC

2 H 2 H
Endocytosis Efflux

•Endocytosis: concentration-dependent. Cells are saturated NPs at 750 and 1000 μg/ml

•Efflux: If cells saturated by NPs, about 51-52% of internalized NPs were expulsed from
cells
ABC transporters pumped out low molecular weight polymers after NPs hydrolysis
MRP5 (ABCC5) expulsed a part of PLA–PEG NPs from mouse hepatic cells
ARTICLE VII

Interactions of Nanoparticles With Lung Epithelial Cells.

S. Vranic, R. Guadagnini, A. Baeza, M. C. Borot, S. Hussain, S. Boland, F. Marano

• Objectifs :

Déterminer des interactions de NP TiO2 avec des modèles cellulaires

respiratoires in vitro; capacité de traverser l’épithélium respiratoire et
passer dans le sang?
• Matériels et Méthodes:
 Modèles cellulaires utilisés : A549, 16HBE 14o- et Calu-3.
Photos : M.C. Borot

 Nanoparticules: TiO2 P25 (Degussa; 21 nm (80%)) et TiO2 (Sigma;15nm (98%))

 Etude de l’endocytose (24h) : FACS : sans et avec Inhibiteurs (16HBE et

A549)
 Etude de la transcytose (24h) : FACS (Calu-3)
ARTICLE VIII Review
Interactions of nanoparticles with pulmonary structures and cellular responses
Christian Mühlfeld, Barbara Rothen-Rutishauser, Fabian Blank, Dimitri Vanhecke, Matthias Ochs, and Peter Gehr
Am J Physiol Lung Cell Mol Physiol 294: L817-L829, 2008

Mots clés: nanoparticles, pulmonary , nanotoxicology

NPs: TiO2 NPs, polystyrene NPs

Cells
•Monocultures of epithelial celllines (A549 cells),
•primary cellcultures,
•Co-culture of epithelial cells/endo thelialcells,
•co-culture of epithelial cells/macrophages,
•Triple cell co-culture model of the human airway wall: A549 cells, human blood monocyte derived
macrophages on top, human blood monocyte derived dendritic cells at the bottom of the cell

Determination:
•Electron tomography/electron spectroscopic imaging(ESI)/parallel electron energy loss
spectroscopy(PEELS): endocytosis des TiO2 NPs par macrophage
•Laser scanning microscope micrograph: Fluorescent polystyrene NPs par attached to or within an
erythrocyte (human) Endocytosis des TiO2 NPs par macrophage
Mitochondrion

Phagosome

ESI Electron tomography

Summary of possible interactions of NPs with pulmonary structures
Summary of selected in vivo and in vitro studies on
endocytosis/transcytosis of NPs
 SUMMARY

Objective: Endocytosis/Exocytosis/Transcytosis of NPs in vivo/ in vitro

Methods:

NPs:
•BSA PLGA NPs (97 ± 3nm) labeled 6-coumarin
•Tf/PVA PLGA NPs (216 nm) labeled 6-coumarin
•PEG-PLA NPs (150 nm, -20 mV), labeled 6-coumarin
•Chitosan NPs (+/-: −40 mV ~ +35 mV, 150 ~ 500 nm) labeled rhodamine B
•Chitosan NPs labeled Fitc
•Polystyrene nanoparticles (+/-, 20 nm, 100 nm)
•TiO2 NPs (15 nm, 21 nm)
SUMMARY

Methods:

Cells: Determination
•Vascular smooth muscle cells
•primary rat alveolar epithelial cell
•Confocal microscopy (confocal software)
•HeLa
•HPLC
•A549
•FACS
•16HBE 14o-
•Electron tomography
•Calu-3
•Electron spectroscopic imaging
•MDA-MB231 human breast cancer cell line
•Parallel electron energy loss spectroscopy
•L02 cells
•SMMC-7721 cells
•Murine macrophage
SUMMARY Results

BSA PLGA NPs (100 nm)- vascular smooth muscle cells:

•Endocytosis of NPs was a concentration-, time-, and energy-dependent endocytic
process
•Endocytosis of NPs is rapid, after 1st min incubation. It increases with incubation time
•65% of NPs are exocytosed during first 30 min.
•Exocytosis of NPs is an energy-dependent process
•Exocytosis is inhibited by serum free medium.
•Addition of BSA in the serum free induced exocytosis of NPs

Tf PLGA NPs (200 nm)- breast cancer cells:

•50% of the Tf-NPs are exocytosed after 2 H

PEG-PLA NPs (150 nm, -20 mV)-

Human hepatoma (HepG2) cells, Mouse primary hepatic cells
•Endocytosis is concentration-dependent, saturated at 750 and 1000 μg/ml
•51-52% of NPs were exocytosed after cells saturation.
SUMMARY Results
Chitosan NPs (+/-, 150 ~ 500 nm, −40 mV ~ +35 mV)-
Murine macrophage, L02 and SMMC-7721 cells and in tumor bearing mouse

-Macrophage:
•size- and charge-dependent
•Endocytosis increases with the surface charge increasing
•Macrophages endocytosed more larger size particles
•NPs+ have a higher endocytosis than NP-

-L02 and SMMC-7721 cells (non-phagocytic cells):

•size-, charge- and NPs composition- dependent
•More endocytosis of NP+ by L02 and SMMC-7721 cells
•L02 and SMMC-7721 cells endocytosed more smaller size particles
•Endocytosis of these NPs was cell-line-dependent:
Endocytosis of NP-: SMMC-7721 > L02 > A549 > 786-O > HFL-I > HEK 293
Endocytosis of NP+: SMMC-7721 > A549 > 786-O > HEK 293 > L02 > HFL-І
SUMMARY Results
Chitosan NPs (+/-, 150 ~ 500 nm, −40 mV ~ +35 mV)-
Murine macrophage, L02 and SMMC-7721 cells and in tumor bearing mouse

-In tumor bearing mouse:

Chitosan NP-:
•Both surface charge and particle size could influence the biodistribution of NP-.
•NP- with lower surface charge and smaller particle size tended to be more efficiently distributed in the
tumor, and the peak of distribution percent appeared at 4 h after injection.
•The increase in the surface charge as well as particle size of NP- led to the decrease in blood distribution:
Most of NP- with larger particle size was cleared off from the blood after 1 h.
•Increase in the surface charge and particle size correlated to the elevated distribution percentage of NP- in
liver and spleen.
•Surface charge exerted unappreciable effect on the pulmonary distribution percentage of NPs-, while NPs
with larger particle size increased distribution level.
•NP- exhibited a rapid accumulation in lung.
•Particle size and surface charge exerted unappreciable effect on the distribution behavior of NP- in kidney
with a relatively stable distribution level up to 24 h.
SUMMARY
In tumor bearing mouse:

Chitosan NP+:
•The disposition of NP+ in tumor was notably elevated with the increase of surface charge.
•Compared to NP+, tumor accumulations of NP+ were significantly reduced. Peak distribution of NP+ in the
tumor was observed at 2 H after administration and NP+ was retained in the tumor throughout 24-H period.
•About 2.4% of NP+ was detected in the blood at 1 H after administration and no significant difference was
observed among NP+ with different particle size and surface charge.
•No significant difference of distribution was observed in the liver or spleen among NPs with different
surface charge. The liver accumulation of NP+ was significantly higher than that of NP+ while in the spleen
the trend was opposite.
•NP+ accumulated in the lung at a relatively low rate, and when the ζ-potential increased, the distribution
percent in the lung was increased.
•Particle size and surface charge had no influence on the distribution behavior of NP+ in kidney.
 Plan for PLGA NPs exocytosis by cells

NPs Cells Inhibitors

Inhibitors for study the mechanism of endocytosis

Exocytosis and mechanism of preincubation associated Mechanism of
endocytosis by: Inhitibor
time pathway action
Antagonist of
PLGA NPs+ Humain Chlorpromazine 3µg/ml, in water Clathrin calmodulin and
bronchial others
16HBE 14o- Inhibitor of
epithelial cell Phenylarsine
line 1µg/ml, DMSO Clathrin tyrosines
oxide (PAO)
phosphatases

mouse 5µg/ml, in Depletion of

Filipine Caveolae
LY5197 lymphome cell DMSO cholesterol
line
10µg/ml, in Depletion of
Nystatine Caveolae
ethanol cholesterol
PLGA NPs 0 human Caveolae/
TK6 lymphoblastoi Methyl-- Depletion of
100µg/ml, in Clathrin (under
d cell line cyclodextrine cholesterol and
water certain
(MCD) phospholipides
condition)
Inhibitor of
Clathrine,
cytochrome
100µg/ml, in Caveolae,
human lung Azide sodium oxydase
A549 water macropinocytos
cancer cell line (synthesis of
e
ATP)
inhibition of
PLGA NPs- phagocytosis,
3µg/ml, in inhibition of
Cytochalasine B ??
CH2Cl2 glucose and
glucosamine
transport