Journal of Biomaterials and Nanobiotechnology, 2011, 2, 494-499
doi:10.4236/jbnb.2011.225060 Published Online December 2011 (http://www.scirp.org/journal/jbnb)
A New Biological Strategy for Drug Delivery:
Eucaryotic Cell-Derived Nanovesicles
Irène Tatischeff1*, Annette Alfsen2
1Laboratoire Acides Nucléiques et Biophotonique (ANBioPhi), Université Pierre et Marie Curie, Paris, France; 2Institut Cochin, Dé-
partement de Biologie Cellulaire, Université Paris-Descartes, Paris, France.
Received October 4th, 2011; revised November 13th, 2011; accepted November 25th, 2011.
ABSTRACT
An efficient drug delivery is the prerequisite of the successful chemotherapeutic treatments of many human diseases.
Despite a great number of approaches, the improvement of drug cell internalization remains an actual research chal-
lenge. We propose a new biological delivery system based on the extracellular vesicles released by a non-pathological
eukaryotic microorganism, Dictyostelium discoideum. After a summary of the main characteristics of these extracellu-
lar vesicles, including of their lipid bilayer that appears as a good candidate for initiating membrane fusion, followed
by delivery of their encapsulated drug, the capacity of these vesicles to convey drugs into human cells was demon-
strated in vitro on two tumor cell lines, resistant leukaemia K562r and cervix carcinoma HeLa cells. A comparison with
other extracellular vesicles, like exosomes or bacteria-derived particles, stresses the unique properties of Dictyostelium
extracellular nanovesicles for drug delivery.
Keywords: Biological Extracellular Vesicles, Antitumoral Drug Delivery, Dict yo st elium discoid eu m
1. Introduction
The cell is the factory of most primitive unicellular or-
ganisms, the building block of plants and animals, as
well as of human tissues. In higher eukaryotic organisms,
cells are protected by their membrane, from xenobiotics,
including many therapeutic drugs. Life dysfunctions,
involved in human diseases and cancers, are often to be
corrected inside the cell, at different vital targets, like
nucleus, mitochondria, Golgi…. Both the old therapies,
like antitumoral chemotherapies, and the new ones, like
gene therapies, are faced with the major problem of effi-
cient drug delivery. Therefore, the goal of all the drug
delivery strategies will be to design Trojan horses to pass
the lipid membrane barrier without destroying it and
safely convey therapeutic drugs to their cellular targets
[1].
Many different Trojan horse strategies for drug deliv-
ery have already been designed [2], like using the viral
capsides and engineered lipid-like or polymeric vesicles,
as well as many new associated technologies [2-4].
A search for the mechanisms, elicited by the non-
pathogenic unicellular eukaryotic microorganism Dic-
tyostelium discoideum to get rid of many structurally
unrelated xenobiotics lead us to elaborate a different bi-
ological Trojan horse strategy.
First, we found that these cells “made the difference”
between benzo(a)pyrene, B(a)P, the main carcinogenic
compound of tobacco smoke, and its non-carcinogenic
isomer benzo(e)pyrene, B(e)P. Namely, B(a)P was
ejected from the cells, whereas B(e)P could remain into
the cells [5]. In the early 1990s, when antitumoral mul-
tidrug resistance was thought to be mainly mediated by
the P-glycoprotein ABC-transporter, we wondered whe-
ther the P-glycoprotein might mediate the B(a)P efflux
from the D. discoideum cells. Indeed, we evidenced the
presence of this human-like transporter in D. discoideum
cells [6]. However, it was shown to be inefficient for
their drug detoxification.
In quite different cell synchronization experiments, we
noticed that the widely-used vital DNA-specific stain,
Hoechst 33342 (HO342) was unable to stain the nuclei of
D. discoideum cells. When grown in the presence of
HO342, the cell media harboured plenty of fluorescent
microstructures, which turned out to be microvesicles, in
holding the dye [7].
After studying their characteristics, we discovered that
these biologically elaborated nanovesicles, ejected in the
extracellular medium, were good candidates for “trick-
ing” the known difficult entry of therapeutic drugs into

A New Biological Strategy for Drug Delivery: Eucaryotic Cell-Derived Nanovesicle495
human cells. Using HO342, we have shown that the iso-
lated dye-containing vesicles were able to deliver it into
the nuclei of naive Dictyostelium cells, thus overcoming
their constitutive resistance to the free dye. Moreover,
with living human leukemia multidrug resistant cells,
K562r, known to be resistant to HO342, a Dictyostelium
vesicle-mediated dye-transfer into the nuclei was also
evidenced [8]. Control experiments showed that Dic-
tyostelium vesicle-release was not a mere detoxification
mechanism, but was both constitutive (Tatischeff, et al.,
1998) and important for intercellular communication.
With the goal of using these biological extracellular
nanovesicles for drug delivery, an in vitro investigation
was conducted on HeLa cells, with hypericin, a hydro-
phobic fluorescent photosensitizer, aimed for antitumoral
photodynamic therapy. Thus, we found that Dictyos-
telium nanovesicles, biologically loaded with this thera-
peutic molecule, are a promising nanodevice for cellular
drug delivery [9].
In the present paper, we intend to further propose the
use of D. discoideum extracellular nanovesicles for drug
delivery1. We summarize their main characteristics, and
stress their efficiency for drug delivery into tumoral hu-
man cells, as shown by our in vitro experiments. The
advantages upon the use of these nanovesicles, when
compared with other cell-derived microvesicles are dis-
cussed.
2. Materials and Methods
All the materials and methods used in our work on Dic-
tyostelium nanovesicles have been detailed in our previ-
ous papers [7-9]. Only the methods dealing with Dic-
tyostelium cell cultures and preparation of vesicles will
be recalled here for clarity.
2.1. Cell Culture
Dictyostelium cells, Ax-2 strain, were grown in suspen-
sion in the dark, on a gyratory shaker (150 rpm) at +22˚C,
in HL5 semi-defined medium [10], containing penicillin
(50 U/mL) and streptomycin (50 U/mL) (Biomedia, Bou-
ssens, France). In contrast with mammalian cells, Dic-
tyostelium cells are grown without fetal calf serum. For
proper oxygenation, each suspension was grown in an
Erlenmeyer containing five times the suspension volume.
2.2. Preparation of Dictyostelium Nanovesicles
Dictyostelium cell cultures, were initiated at 5 × 105 - 106
cells·ml−1 and grown in suspension in HL5 medium in the
absence (control) or in the presence of the drug to be
vectorized. For preparation of nanovesicles, cells were
generally used in the late exponential or early stationary
phase of growth, at a cell density about 107 cells·ml−1.
One has to check that the drug does not affect cell growth.
After 48 h of growth, both cell suspensions were centri-
fuged at 700 x g for 5 min (+20˚C). The cell-free media
were centrifuged at 2000 x g for 10 min (+20˚C). The
2000 x g supernatants were centrifuged at 12,000 x g for
30 min (+4˚C). Nanovesicles present in the pellets were
concentrated (×100) in phosphate buffered saline (PBS),
pH 7.2 without calcium and magnesium (GIBCO).
These nanovesicles were quite stable in PBS, as they
did withstand repeated liquid nitrogen freeze-thaw cycles
without breaking. As observed with nanovesicles in
holding HO342, they could be kept at +4˚C at least 2
months without releasing the dye.
3. Main Results
In the Eukarya branch of the evolutionary tree, Dictyos-
telium discoideum (http://www.dictybase.org), an ances-
tral non-pathogenic amoeba, placed at the border be-
tween the plant and animal kingdoms, is equipped with a
lipid membrane that plays a critical role in many aspects
of cell development. As in other eukaryotic cells, Dic-
tyostelium plasma membrane contains proteins, lipids
and carbohydrates. The phospholipid content of the
plasma membrane of axenically grown vegetative Ax-2
cells is not very different from the one of mammalian
cell membranes, except for an extremely high amount of
unsaturated fatty acids, making up 75% - 90% of the
fatty acids of the organism and of the membrane, respec-
tively. The membrane sterol is stigmasterol, instead of
the mammalian cholesterol.
3.1. Characteristics of Dictyostelium Nanovesicles
Morphological analysis of control vesicles prepared from
the cell growth medium was carried out by cryoelectron
microscopy imaging. The vesicles appeared mostly
smooth and rounded, delineated by a lipid membrane
bilayer (Figure 1). The histogram of their heterogeneous
size distribution indicated that almost 80% of them have
an average diameter within a range of 50 - 150 nm [9].
CdSe/CdZnS quantum dots phospholipid micelles were
used, to check whether Dictyostelium cells might be de-
toxified from these quantum dots by following the vesi-
cle-mediated pathway. As shown on Figure 1, contrary
to many xenobiotics, these quantum dots were excreted
from Dictyostelium cells, as aggregates outside the vesi-
cles. This observation indicates that all the foreign com-
pounds, incubated with Dictyostelium cells, are not deto-
1Tatischeff, I., Alfsen, A., Lavialle, F. “Extracellular vesicles from
non-pathogenic amoebae useful as vehicle for transferring a molecule
of interest to an eukaryotic cell” Patent european priority N 03 291
752 07/15/2003 (DRITT-UPMC) (European Patents in Danemark,
Deutschland, France, Great Britain, Italy, Netherland and Spain), US
Patent and Pending Canadian Patent).

A New Biological Strategy for Drug Delivery: Eucaryotic Cell-Derived Nanovesicle 496
Figure 1. Morphological characterization of Dictyostelium
nanovesicles by cryoelectron microscopy. For methodologi-
cal details, cf. [9]. As shown on the figure, CdSe/CdZnS
quantum dots phospholipid micelles, incubated with grow-
ing Dictyostelium cells, were excreted as (black) aggregates
outside the nanovesicles.
xified by means of the extravesicular pathway. Further on-
going studies should help understanding the structural cha-
racteristics required for a drug vesicle-mediated transport.
A proteomic analysis of the control vesicles revealed a
predominance of actin and actin-related proteins. The
detection of a lysosomal membrane protein (LIMP II) [9]
indicated that these vesicles are likely generated in the
late endosomal compartment, as the known exosomes of
many mammalian cells.
Lipid analysis of the bilayer surrounding the vesicles
[7] showed, beside the phospholipids and lipids common
in the plasma membrane: PC, PI, PS and PE, sphingo-
myelin, PG and DPG, the presence of lyso bis-phos-
phatidic acid (LBPA), a lipid inducing membrane fusion
[11]. Noteworthy, this unusual phospholipid is a major
membrane constituent of the internal vesicles of multive-
sicular bodies [12-14], like the known exosomes of many
human cells. These observations strengthen the hypothe-
sis of a multivesicular origin for the nanovesicles re-
leased by Dictyostelium cells and emphasize the role of
the nature of the lipids in the formation and function of
Dictyostelium vesicles.
3.2. Vesicle-Mediated Transfer of Hoechst 33342
to the Nuclei of Human Leukemia Resistant
Cells, K562r
HO342-loaded nanovesicles allowed to label the nuclei
of naive Dictyostelium cells, thus overcoming their con-
stitutive resistance to the free dye [8]. The ability of these
nanovesicles, to transfer HO342 to the nuclei of human
leukemia living cells, was investigated by adding the
nanovesicles (×50) to multidrug resistant K562r cells
(Figure 2). Four cells were observed by phase contrast
microscopy after about 4 h incubation (Figures 2(a), (c),
(e) and (g)). The efficient transfer of HO342 to the cell
nuclei was observed under UV excitation. The simulta-
neous observation of the same cells with white light and
UV excitation (Figures 2(b), (d), (f) and (h)) clearly
showed that the HO342-stained nuclei were inside the
K562r cells. Thus, the Dictyostelium nanovesicle-medi-
ated HO342 delivery had overcome the induced resis-
tance of K562r cells to the vital staining of their nuclei.
3.3. Vesicle-Mediated Transfer of Hypericin to
the Golgi of Human Cervix Carcinoma
HeLa Cells
Hypericin transfer to target cells was first visualized us-
ing human fibroblasts (HS68) as control cells and with
HeLa cells used as a model of tumoral cells. After a 1-h
incubation in the presence of hypericin packaged into
nanovesicles, a fluorescent signal was detected almost
exclusively in the perinuclear area of HeLa cells (Figure
3). Under these experimental conditions, neither the cell
plasma membrane, nor the nucleus, was labeled by the
hydrophobic cargo molecule.
Similar experiments were conducted using free hy-
pericin, i.e. not encapsulated into vesicles, for compari-
son. After only a 30-min incubation, HeLa cells became
round-shaped, the typical morphology of dying cells that
have lost their adhesion properties (Figure 3). This result
demonstrates that the nanovesicles vectorize significant
amounts of drug, but also prevent the uncontrolled cell
Figure 2. Transfer of HO342 mediated by the Dictyostelium
nanovesicles to the nuclei of living human leukaemia cells,
K562r. Cells were incubated (v/v) with these nanovesicles
(×50). Four different K562r cells are shown, in the range of
incubation time 3 h 40 - 4 h 10, as observed either with
phase contrast (×40 CP objective) (a, c, e, g), or with both
white light and UV fluorescence microscopy (×40 UV objec-
tive) (b, d, f, h). Figure reproduced with permission from
[8].

A New Biological Strategy for Drug Delivery: Eucaryotic Cell-Derived Nanovesicle497
Figure 3. Light-transmission and fluorescence microscopy
of hypericin internalization within human target cells. Hu-
man fibroblasts (HS68) (a) and HeLa cells (b and c) were
incubated at 37˚C in the dark for the indicated times, in the
presence of 7 µM hypericin packaged within Dictyostelium
nanovesicles (a and b) or free in DMEM (c). Figure repro-
duced with permission from [9].
death triggered by free hypericin under the blue light
used for observation.
To check the intracellular localization of hypericin de-
livered by the nanovesicles into HeLa cells, co-localiza-
tion studies were performed with fluorescent markers of
the Golgi apparatus (BODIPY ceramide) and of lysosomes
(LysoTracker). Within 1 h, hypericin was shown to co-
accumulate with BODIPY into the Golgi apparatus, and
the hypericin-loaded nanovesicles were efficient for
photodynamic killing of HeLa cells [9].
The previous studies were initiated to investigate the
potential use of Dictyostelium cell-released nanovesicles
as in vitro drug carriers for cancer therapy. Dictyostelium
cells were able to release into the culture medium nano-
vesicles loaded either with HO342 or with hypericin, a
photosensitizer differing in charge and hydrophobicity
from the DNA marker. Additionally, these nanovesicles
were shown to respectively label human leukaemic re-
sistant cells K562r, and human cervix carcinoma, HeLa,
cells. It is worth stressing that the two cargo molecules,
HO342 and hypericin, were transferred to two distinct
intracellular locations, namely, the nucleus for HO342
and the Golgi apparatus for hypericin.
Vesicular-mediated delivery of hypericin depicted
good photo efficiency for killing HeLa cells. Using ve-
sicular hypericin, instead of free hypericin, ensures a
better control of drug cell loading, by an easy manipula-
tion of the amount of vesicles incubated with the cells.
Most importantly, the vesicular delivery prevents the det-
rimental aggregation of free hypericin in aqueous media.
4. Discussion
Cell membranes are a structured self-assembly of lipids
interacting with proteins and in contact with both exter-
nal medium and the internal aqueous solvent and solutes.
They constantly form lipid vesicles that either move in-
ternally from the cell membrane to sites within the cell or
are externalized. These vesicles are formed of a selection
of membrane lipids and are characterized by specific
properties, either of fusion and/or of targeting, that give
them their dynamic properties, allowing the exchange of
materials and informations.
From prokaryotic unicellular organisms to primitive
eukaryotes, at the border between the vegetal and animal
kingdoms, like the amoebae Dictyosteli um di sc oideum up
to mammalian cells [15] the formation of extracellular
vesicles of different sizes, origins and functions, was
shown to be a property of almost all cell types. Thus,
extracellular vesicles have been described as originating
from almost all mammalian cell types: human blood cells
(platelets [16], B lymphocytes [15], dendritic cells [17],
but also from intestinal epithelial cells [18] or from dif-
ferent pathological cells [15].
All these extracellular vesicles are examples of “vesi-
cles shipping extracellular messages” [19] and “trigger-
ing intercellular communication” from one cell to an-
other, either by fusion with the target cell [20] and/or by
allowing internalization of signaling molecules [15,21].
These extracellular vesicles are, therefore, also potential
Trojan Horses for drug delivery to target cells.
The exosomes, originating from the endosomal traffic
[22], are mostly considered as candidates to develop ge-
netic vaccines for immunotherapy [23], but have recently
been also considered for drug delivery [24]. However, as
mentioned by these authors, many “critical obstacles to
the clinical translation of exosome-based therapy” re-
main. The growth of Dictyostelium cells in a semi-de-
fined medium, without any serum, as compared to more
complex human cell media, might favor this simple eu-
karyotic cell model for solving some of the remaining
problems. Moreover, it is to be stressed, that our drug
delivery device, based on a detoxification process, in-
cludes the Dictyostelium cell conditioning of the thera-
peutic drug into the vesicles that will be released. There-
fore, no drug internalization agents into the vesicles are
needed, as the ones in exosome-based therapy. Bacteri-
ally derived particles have also to be further loaded with
therapeutic agents, in order to be efficient carriers [25].
Therefore, among all the cell-derived extracellular vesi-
cles, Dictyostelium nanovesicles present the unique ad-
vantage of being cell-engineered for drug delivery.
With regard to a potential strong immunogenicity of
these nanovesicles, due to their microbial origin, which
might be a serious drawback for their use in vivo, it has
already been documented for bacterial derived particles.
By contrast, a first in vivo study upon the immunogenic-
ity of Dictyostelium nanovesicles, intravenously injected

A New Biological Strategy for Drug Delivery: Eucaryotic Cell-Derived Nanovesicle 498
twice in the tail of Balb/C mice, has shown a specific
antibody response, but no pyrogenic response nor any
inflammation, as measured by five pertinent cytokines
(study performed by Genosafe, Evry, France). This should
minimize the risk of undesirable immune responses upon
in vivo administration of these biological nanovesicles,
such as those triggered by tumor-released exosomes [26].
5. Conclusions
Our work indicates that D. discoideum is a bioengineer-
ing designer able to formulate vesicular drug carriers. To
our knowledge, this is the first study describing cell-en-
gineered vesicles able to both load a therapeutic mole-
cule and vectorize it into human cells, as we have shown
with the important antitumoral photosensitizer, hypericin
[9].
Other characteristics of Dictyostelium vesicles appear
interesting for targeting purpose; namely, the presence of
discoidins I and II [9], which bind to the cell membrane
with a specificity for galactose-related residues, might be
of interest for lectin-mediated drug delivery [27]. The
targeting strategy, tested with bacterially derived vesicles
[25], by using bi-specific antibodies, with one arm rec-
ognizing a component of the vesicle surface, the other a
cell surface receptor of the target cell, might also be ap-
plied to Dictyostelium nanovesicles. Another vectoriza-
tion strategy could be developed by using Dictyostelium
cells cultured in the presence of magnetic nanoparticles
to engineer magnetic nanovesicles [28].
With regard to the possibility of using Dictyostelium
nanovesicles in therapy, many problems remain to be
solved. A study to elucidate the membrane events in-
volved in cell entry of the drug vectorized by the nano-
vesicles should be performed. For further in vivo ap-
proaches, toxicity and allergenic properties of more puri-
fied nanovesicle preparations are to be characterized.
Taking into account the described culture of Dictyos-
telium cells in bioreactors [29], a drug vectorization strat-
egy could, indeed, be developed at a large pharmacol-
ogical scale.
6. Acknowledgements
Eric Larquet (CNRS, UMR7590, UPMC, IMPMC, Paris,
France) is kindly acknowledged for the cryoelectron mi-
croscopy measurements and Benoit Dubertret (CNRS
UPR A0005, UPMC, ESPCI, Paris, France) for the gift
of the CdSe/CdZnS quantum dots.
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