|Título/s:||M cells prefer archaeosomes: An in vitro/in vivo snapshot upon 0ral gavage in rats|
|Autor/es:||Morilla, María José; Mengual Gómez, Diego; Cabral, Pablo; Cabrera, Mirel; Balter, Henia; Defain Tesoriero, María Victoria; Higa, Leticia; Roncaglia, Diana; Romero, Eder L.|
|Institución:||Programa de Nanomedicinas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. UNQ. Bernal, Buenos Aires, AR |
Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la Republica. Montevideo, UY
Unidad Operativa Sistemas de Liberación Controlada, Centro de Investigación y Desarrollo y Desarrollo en Química, Instituto Nacional de Tecnología Industrial. INTI-Química. Buenos Aires, AR
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. UNQ. Bernal, Buenos Aires, AR
|Editor:||Bentham Science Publishers|
|Palabras clave:||Liposomas; Lípidos; Nanopartículas; Inmunología; Antígenos; Microorganismos; Fosfolípidos; Toxicidad; Células; Digestión|
| Ver+/- |
Current Drug Delivery, 2011, 8, 000-000 1
1567-2018/11 $55.00+.00 © 2011 Bentham Science Publishers Ltd.
M Cells Prefer Archaeosomes: An In Vitro/In Vivo Snapshot Upon Oral
Gavage in Rats
Maria Jose Morilla1, Diego Mengual Gomez4, Pablo Cabral2, Mirel Cabrera2, Henia Balter2,
Maria Victoria Defain Tesoriero1,3, Leticia Higa1, Diana Roncaglia1 and Eder L Romero1,*
1Programa de Nanomedicinas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Saenz
Peña 352, Bernal, B1876 BXD, Buenos Aires, Argentina; 2Centro de Investigaciones Nucleares, Facultad de Ciencias,
Universidad de la Republica, Uruguay; 3Unidad Operativa Sistemas de Liberación Controlada, Centro de Investigación
y Desarrollo en Química, Instituto Nacional de Tecnología Industrial (INTI), Av. General Paz 5445, CPB1650WAB,
Buenos Aires, Argentina; 4Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmas, Argentina
Abstract: The archaeolipids (lipids extracted from archaebacterias) are non saponificable molecules that form self sealed
mono or bilayers (archaeosomes-ARC). Different to liposomes with bilayers made of conventional glycerophospholipids,
the bilayer of ARC posses a higher structural resistance to physico chemical and enzymatic degradation and surface hy-
drophobicity. In this work we have compared the binding capacity of ARC exclusively made of archaeols containing a
minor fraction of sulphoglycophospholipids, with that of liposomes in gel phase on M-like cells in vitro. The biodistribu-
tion of the radiopharmaceutical 99mTc-DTPA loaded in ARC vs that of liposomes upon oral administration to Wistar rats
was also determined. The fluorescence of M-like cells upon 1 and 2h incubation with ARC loaded with the hydrophobic
dye Rhodamine-PE (Rh-PE) and the hydrophilic dye pyranine (HPTS) dissolved in the aqueous space, was 4 folds higher
than upon incubation with equally labeled liposomes. Besides, 15% of Rh-PE and 13 % of HPTS from ARC and not from
liposomes, were found in the bottom wells, a place that is equivalent to the basolateral pocket from M cells. This fact sug-
gested the occurrence of transcytosis of ARC. Finally, 4 h upon oral administration, ARC were responsible for the 22.3 %
(3.5 folds higher than liposomes) shuttling of 99mTc-DTPA to the blood circulation. This important amount of radioactive
marker in blood could be a consequence of an extensive uptake of ARC by M cells in vivo, probably favored by their sur-
face hydrophobicity. Taken together, these results suggested that ARC, proven their adjuvant capacity when administered
by parenteral route and high biocompatibility, could be a suitable new type of nanoparticulate material that could be used
as adjuvants by the oral route.
Keywords: Adjuvant, archaeosomes, oral.
In the last decade, the intervention of Nanotechnolgy into
the field of Immunology has opened new avenues to novel
nano-particulate material capable of inducing antigen-
specific adjuvancy, particularly in non-parenteral delivery
[1-5]. Up to the moment however, if well oral is the most
friendly route of administration, satisfactory protective and
memory responses can only be achieved employing live at-
tenuated microoganisms [6, 7]. The generation of antigen-
specific sIgA at the site of the infection upon oral immuniza-
tion with inert particulate material, remains as an ambitious
challenge, specially for developing countries [8, 9].
The inductive sites at the gut-associated lymphoid tissue
(GALT) in the mucosa of small intestine are represented by
the Peyer´s patches (PP) [10, 11]. Within the follicle-
associated epithelial (FAE) cells of each PP are the M (mi-
crofold) cells, which are specialized in taking up particulate
antigens from the intestinal lumen to transport them to the
*Address correspondence to this author at the Programa de Nanomedicinas,
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes,
Roque Saenz Peña 352, Bernal, B1876 BXD, Buenos Aires, Argentina;
Tel: +54 1143657100; Fax: +54 1143657132. E-mail: email@example.com
subepithelial dome, populated by immature myeloid den-
dritic cells (antigen presenting cells, APC) . Epithelial
mature dendritic cells also contribute to the direct uptake of
particulate material from the lumen . Binding and subse-
quent transcytosis of particulate material (instead of soluble)
across M cells to gain access to the APC located in the fol-
licule, are acknowledged as key steps to elicit effective im-
mune responses by the oral route [14-16]. Hence, structural
features of particulate material leading to enhanced uptake
by M cells - that are present at a very low density in the gut-
could contribute to improve potential immune reactions upon
oral administration . If well specific markers enabling
active targeting of particulate material to human M cells re-
main unknown, the over expression of b1 integrins at the
apical pole of human M cells was demonstrated [1, 18]. Very
recently ovoalbumin (OVA) loaded PLGA nanoparticles
grafted with analogs of the Arg-Gly-Asp motif were shown
to be effective at targeting the M cells as well as in the sub-
sequent generation of immune responses upon duodenal ad-
ministration to mice .
Unfortunately, recent clinical studies using particulate
material targeted to M cells resulted poorly reproducible [20,
21]. Several reasons have contributed to reduce the current
expectancies on the use of particulate material as oral adju-
2 Current Drug Delivery, 2011, Vol. 8, No. 3 Morilla et al.
vants . For instance, the uptake by endothelial entero-
cytes and the M cells in PP is size dependent (higher for
lower sized particles, a fact that limits the amount of loaded
antigen) and limited at lower level than previously though,
nearly 5% of the administered dose for particles below 100
nm [15, 23]. Conventional liposomes are poorly absorbed, in
the order of 2 % the total dose, whereas polymerized
liposomes are absorbed up to 6 % . In this context,
counting on new materials with structural characteristics
tailored to allow an effective passive targeting and uptake by
M cells becomes relevant.
Archaeosomes (ARC) are vesicles composed mostly by
total polar lipids (TPL) extracted from micro organisms that
belong to the Archaea domain of life, which are usual inhabi-
tants of extreme environment . The core structures of
archaeal polar lipids consist of archaeol or diether lipid (2,3-
di-O-diphytanyl-sn-glycerol) which contain 20 carbons per
isoprenoid chain, and/or caldarchaeol or tetraether lipid
(2,2´-3,3´- tetra-O-dibiphytanyl-sn-diglycerol) containing 40
carbons per isoprenoid chain, and modifications of these
structures [26-28]. The different habitats of archaeas (ex-
treme termophiles, extreme halophiles, sulpho acidophiles,
methanogens (anaerobics) and psychrophiles) define the
main composition of their TPL .
The purpose of this work was to estimate the binding and
transcytosis of ARC and plain liposomes in gel phase, using
an in vitro model of M-like cells (Caco-2/Raji co-cultures).
Additionally, the biodistribution of the hydrosoluble radio-
pharmaceutical 99mTc-DTPA loaded into ARC and liposomes
was determined upon oral gavage to rats.
MATERIALS AND METHODS
Hydrogenated phosphatidylcholine from soybean
(HSPC) was obtained from Northern Lipids (Vancouver,
Canada). Cholesterol, 1,2-Dimyristoyl-sn-glycero-3-
phosphoethanolamine-N-(Lissamine™ rhodamine B sul-
fonyl) (Rh-PE), N,N-dimethylformamide, pepsin, bile extract
porcine, pancreatin from porcine pancreas, formaldehyde,
Triton X-100, naphtol AS-MX phosphate, Fast Red TR salt
and thiazolyl blue tetrazolium bromide (MTT) were pur-
chased from Sigma-Aldrich (St Louis, MO, USA). The
fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS)
was from Molecular Probes (Eugene, OR, USA). Acryla-
mide, bis-acrylamide and glycine were from ICN Biomedi-
cals (Irvine, CA, USA). RPMI 1640 and MEM with non-
essential amino acids (MEM-NEAA) culture media were
purchased from Invitrogen Corporation (California, USA).
Endotoxin-free fetal bovine serum (FBS), L-Glutamine,
Trypsin, EDTA and penicillin/streptomycin (PEST) were
provided by PAA Laboratories GmbH (Pasching, Austria).
Tris buffer and all the other analytical grade reagents were
from Anedra (Buenos Aires, Argentina).
Growth of Archaebacteria and Total Polar Lipids Isolation
Halorubrum tebenquichense archaeas were isolated from
soil samples collected in Salina Chica, Península de Valdés,
Chubut, Argentina as described in Gonzalez et al. . Bio-
mass was generated in 8 l batch cultures in basal medium
, supplemented with yeast extract, glucose and antifoam
(20μl/l). Cultures were monitored by absorbance at 660 nm
and harvested in late stationary phase for storage as frozen
Lipids were extracted from frozen and thawed biomass,
with Cl3CH:CH3OH:H2O (1:2:0.9; v:v), and the total polar
lipid (TPL) fraction was collected by precipitation from cold
The lipid extract was analyzed by two-dimensional
chromatography, by using Cl3CH:CH3OH:H2O (65:25:4 v:v)
in the first dimension and Cl3CH:CH3OH: HCH2COOH:H2O
(80:12:15:4 v:v) in the second dimension. Lipids were de-
tected with the following spray reagents: (a) molybdenum
blue Sigma spray reagent for phospholipids; (b) 0.5% orci-
nol/sulfuric acid for glycolipids; (c) azure A/sulphuric acid
for sulphoglycolipids, (d) 5% sulfuric acid in ethanol, fol-
lowed by charring at 120 °C, for all spots. Non-polar pig-
ments were analyzed by mono-dimensional TLC using hex-
ane/diethyl ether/acetic acid (70: 30:1 v:v) and stained with
ARC Preparation and Physicochemical Characterization
Archaeosomes containing a double fluorescent label
HPTS and Rh-PE (ARC-HPTS-Rh-PE were prepared by
hydration of the thin lipid film. Briefly, 20 mg of TPL from
CHCl3: CH3OH (9:1, v/v) solution was mixed with 5.10
mol of Rh-PE in CHCl3 and further rotary evaporated at 40
°C in round bottom flask until organic solvent elimination.
The thin lipid film was flushed with N2 and hydrated at 40°C
with 1 ml of 35 mM HPTS in 10 mM Tris-HCl buffer plus
0.9% w/v NaCl, pH 7.4 (Tris buffer). The resultant suspen-
sion was sonicated (20 min in a bath type sonicator 80 W, 40
KHz) and submitted to 5 cycles of freeze/thaw between - 80
and 37°C. Non-incorporated dyes were removed by centrifu-
gation and washing with Tris buffer (3 times at 10000 x g for
20 min). Liposomes made of HSPC:cholesterol (1:1
mol:mol) were prepared in the same way (L-HPTS-Rh-PE).
Phospholipids were quantified by a colorimetric phos-
phate micro assay . Fluorescence was measured in a
spectrofluorometer PTI, Timemaster C-70, using ?exc 550-
?em 590 nm for rhodamine, and ?exc 413- ?em 510 nm for
HPTS. Mean ARC size was determined by dynamic light
scattering with a 90 Plus Particle size analyzer (Brookhaven
Instruments) and Zeta potential was determined with a Zeta-
sizer 4 (Malvern).
ARC and L were frozen at -80ºC for 24h and lyophilized
using a Labconco Freeze Dry System / Freezone 4.5 (Kansas
City, MO -USA), drying was performed at a pressure form
37.10-3 to 62.10-3 mbar for 12 h. Dried liposomal residua
were re-hydrated to initial volume with distillated water and
size was measured by dynamic light scattering.
Cell viability, upon incubation with ARC and L was
measured as mitochondrial dehydrogenase activity employ-
ing a tetrazolium salt (MTT) on Caco-2 cells. Caco-2 cells
were seeded at a density of 4 ? 104 cells/well in a 96 well
plate and growth for 24h. Culture medium of nearly conflu-
ent cell layers was replaced by 100 μl of medium containing
10, 100, 500 or 1000 μg/ml of lipids. Upon 24 h at 37°C,
M Cells Prefer Archaeosomes: An In Vitro/In Vivo Snapshot Upon Oral Gavage in Rats Current Drug Delivery, 2011, Vol. 8, No. 3 3
media were removed and replaced by fresh RPMI medium
containing at 0.5 mg/ml of MTT. Upon 3 h incubation, MTT
solution was removed, the insoluble formazan crystals were
dissolved in DMSO and absorbance was measured at 570 nm
in a microplate reader.
Preparation of Caco-2/Raji Co-Cultures
Caco-2 cells (human colon carcinoma cell line was
kindly provided by Dr. Osvaldo Zabal, INTA Castelar) were
cultured in MEM-NEAA supplemented with 1mM L-
glutamine, 1% pyruvate, 1% PEST and 10% FBS at 37°C,
5% CO2 and 95% humidity.
Raji-B cells (human Burkitt´s lymphoma from Asocia-
ción Banco Argentino de Células) were cultured in RPMI
1640 supplemented with 1mM L-glutamine, 1% PEST and
10% FBS at 37°C, 5% CO2 and 95% humidity.
Caco-2/Raji cells were co-cultivated following a previ-
ously described protocol . Briefly, Caco-2 cells were
seeded at a density of 3?105 cells per 24-well polyester in-
serts (3 μm pores, ThinCertTM, Greiner Bio-One, Fricken-
hausen, Germany), the medium of the upper and bottom
wells were changed every 2–3 days, and cells were cultured
to confluence over 14-16 days.
Then, 3?105 Raji cells, suspended in supplemented
MEM-NEAA plus 1% PEST and 10% SFB were added in
the bottom wells. The co-cultures were maintained for 4–5
days. The medium of the upper wells were changed every
other day. Monocultures of Caco-2 cells, cultivated as above
except for the presence of Raji cells, were used as controls.
Monolayer integrity was evaluated by measuring transe-
pithelial electrical resistance (TEER) using a Millicell Elec-
trical Resistance System (Millipore Corp., Bedford, MA)
connected to a pair of chopstick electrodes.
Additionally, the cell monolayer integrity was evaluated
by red phenol exclusion . In brief, culture media of upper
and bottom wells were removed; cells were washed 3 times
with HBSS, then 600 μl of buffer HBSS with phenol red (42
μM) was added to upper well and 700 μl of phenol red free
buffer HBSS was added to the bottom well. The diffusion of
phenol red was allowed for 2h a 37 ºC and aliquots of upper
and bottom wells were removed, pH was adjusted to 11 with
NaOH 1M, absorbance at 588 nm was measured and percent
of diffusion was calculated. As 100% of phenol red diffusion
an insert without cells was used.
Activity of the enzyme alkaline phosphatase was evalu-
ated to show differentiation of Caco-2 cells to M-like cells
. Briefly, culture media of upper and bottom wells were
removed and cells were washed 3 times with PBS, cells were
fixed with 1% formaldehyde and incubated with X-100 triton
0.2 % for 15 sec. Then, cells were washed and incubated
with reactive solution for 5 min (10 ml buffer pH 8.9 solu-
tion containing 5 mg naphthol AS-MX phosphate, plus 250
?l N,N-dimethylformamide and 10 mg Fast Red). Inserts
membranes were cut and mounted on a Nikon Alphaphot 2
YS2 fluorescence microscope. Four photographs, each one
corresponding to ? of the total area of the insert were taken
using an objective of 4x and fluorescence intensity were
quantified using ImagePro Plus software.
In Vitro Binding and Transport Studies
Upper wells media of Caco-2 cells and Caco-2/Raji co-
cultures were replaced by 150 μl of fresh medium containing
50 μg of lipids as ARC-HPTS-Rh-PE or L-HPTS-Rh-PE.
Cultures were incubated for 1 and 2 h at 37°C, and then me-
dia of bottom and upper wells were removed and stored for
fluorescence measurements. Cells were washed 3 times with
PBS and fixed with 1% formaldehyde, inserts membranes
were cut and observed with a confocal laser microscopy
Olympus FV300 equipped with an Ar laser (488 nm for
HPTS excitation) and a He-Ne laser (543 nm for Rh-PE ex-
In Vitro Digestion
Proteins of a homogenate of Trypanosoma cruzi epimas-
tigotes (kindly provided by Dra. Patricia Petray, Servicio de
Parasitología y Enfermedad de Chagas, Hospital de Niños
Ricardo Gutierrez) were loaded in ARC (ARC-T. cruzi) and
L (L-T.cruzi). In brief, thin lipid films were hydrated with 3
mg/ml of proteins en in PBS buffer, after sonication and
frezze/thaw cycling; non-incorporated proteins were re-
moved by centrifugation (3 times at 10000 g for 30 min at
4ºC). Protein contents were measured by Bradford assay
An in vitro digestion process adapted from Patel et al.
 and Jovaní et al.  was applied to ARC-T. cruzi and
L-T. cruzi. Briefly, 500 ?l of samples (0.5 mg proteins) were
incubated with 2 ml of pepsin (1.5 mg/ml buffer Tris pH 2)
for 30 min at 37°C. Then, pH of the mixtures was increased
to 6.5 with NaHCO3 1M and 100 ?l of bile extract and pan-
creatin solution were added (60 and 9 mg/ml, respectively)
and incubated 30 min at 37 °C. Finally, pH was increased to
7.4 with NaHCO3 1M and samples were centrifuged (10,000
g for 30 min at 4ºC). Proteins from pellets were recovered
 and analyzed by 15% polyacrylamide gel electrophore-
sis, proteins were visualized by silver staining.
Radiolabeling of Vesicles
First, 99mTc- sodium pertechnetate (Na99mTcO4), was
eluted from a molybdenum-technetium generator (Tecnonu-
clear, Buenos Aires, Argentina) with 0.9% saline, then it was
complexed with diethylenetriamine pentaacetic acid (DTPA)
using a "99m Tc-DTPA kit"(containing 5 mg of DTPA and
0.25 mg of stannous chloride). Then, thin archaeolipid film
was hydrated with 11-12 mCi of 99mTc-DTPA with mechani-
cal mixture and further sonication. Non-incorporated 99mTc-
DTPA was removed by gel permeation chromatography in a
PD-10 Sephadex G-25 column (GE Healthcare). Activity of
fractions was measured by solid scintillation counter (Pack-
ard). Liposomes made of HSPC:cholesterol (1:1 mol:mol)
were label in the same way (L-99mTc-DTPA)
The radiochemical purity and the label efficiency were
determined by thin-layer chromatography on 2 x 20 cm
strips, three different analyses were performed:
1) ITLC-SG using NaCl 0.9 % as solvent, L/ARC-99mTc-
DTPA and reduced/hydrolyzed 99mTc remains at the
original location, free 99mTc-DTPA migrates with the
solvent in front.
4 Current Drug Delivery, 2011, Vol. 8, No. 3 Morilla et al.
2) ITLC-SG using pyridine:HCH2COOH:H2O (3:5:1.5 v/v)
as solvent, L/ARC- 99mTc-DTPA and free 99mTc-DTPA
migrates with the solvent in front, while reduced/ hydro-
lyzed reduced/hydrolyzed 99mTc remains at the original
3) Whatman No 1 using MEK (Methylethylketone) as sol-
vent, L/ARC-99mTc-DTPA and reduced/hydrolyzed
99mTc remains at the original location, while free 99mTc-
DTPA had a Rf of 0.1.
Oral Administration and Biodistribution
Wistar rats (180–250 g body weight) were fasted for 24 h
and then received 500 μl of single dose of 40 μCi as ARC-
99mTc-DTPA, L-99mTc-DTPA or free 99mTc-DTPA, through a
cannula by the oral route. Animals were kept in separate
metabolic cages for urine collection. After 4h of administra-
tion, animals were sacrificed and blood sample, liver, kid-
ney, muscle, bones and brain were collected, washed,
weighed and activity was measured by solid scintillation
The significance of the differences between the mean
values of studied parameters was determined using the Stu-
Total polar lipid extract was analyzed by 2D TLC and
electrospray ionization mass spectrometry (ESI-MS) (nega-
tive ions). There were shown the presence of polar lipids 2,3-
di-O-phytanyl-sn-phosphatydilglycerol methyl ester (PGP-
Me), 2,3-di-O-phytanyl-sn-phosphatydilglycerol (PG), 1-O-
phytanyl-sn-glycerol (S-DGD), sn-2,3-di-O-phytanyl-1-
and the glycocardiolipin (3´-SO3H)-Galp?1,6Manp-?1,2Glcp?-1-1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-
sn-2,3-di-O-phytanylglycerol] (GlyC), as already reported in
Gonzalez et al.  Fig. (1A). Non-polar pigments such as
bacterioruberin, retinal and caratenoids were also found Fig.
Size and Z potential of the L and ARC was shown in
Table 1. After lyophilization and reconstitution, no signifi-
cant changes in ARC size were observed, while L immedi-
ately aggregated upon water addition with remarkably in-
Fig. (1). (A) 2D-TPL pattern of TLC. The origin is in the bottom
left corner. Lipids were identified by differential staining, compari-
son with reported Rf values (retention factor, calculated by dividing
the distance traveled by the product by the total distance traveled by
the solvent) and electrospray ionization mass spectrometry (ESI-
MS). (B) Non polar pigments pattern by TLC, spots were identified
by comparison with reported Rf values.
Cytotoxicity on Caco-2 Cells
None ARC neither HSPC:cholesterol liposomes signifi-
cantly reduced the viability of Caco-2 cells upon 24h incuba-
tion at concentrations up to 1 mg/ml Fig. (2).
Binding and Transport Studies
TEER values were approximately 400 ?.cm2 and 300
?.cm2 for mono and co-cultures, respectively (cells with
lower TEER values were excluded from experiments), and
5.8% of phenol red diffused from the upper to the bottom
wells after 2h incubation (it is acceptable up to a 6% diffu-
sion on confluent Caco-2 cells . On the other hand, the
activity of alkaline phosphatase decreased 66% in Caco-
2/Raji co-cultures with respect of Caco-2 cells mono-cultures
Fig. (3). This drop is considered representative of a change
from mature enterocytes to M-like cells phenotype .
ARC-HPTS-Rh-PE and L-HPTS-Rh-PE were incubated
with mono- and co-cultures at the same lipid concentration.
Since both had the same mean size distribution, we had as-
sumed that the number of the two types of vesicles was the
Pre-Lyophilization Post-Reconstitution Z potential (mV)
Mean Size (nm) PI Mean Size (nm) PI
Liposomes -11 530 0.36 2700 0.80
ARC -49 570 0.40 740 0.60
PI: Polydispersity index, n:3
M Cells Prefer Archaeosomes: An In Vitro/In Vivo Snapshot Upon Oral Gavage in Rats Current Drug Delivery, 2011, Vol. 8, No. 3 5
Fig. (2). Viability of Caco-2 cells upon 24h incubation with ARC or
HSPC:cholesterol liposomes, as function of concentration. Values
represent the average of triplicates ± S.D.
While no fluorescence was observed on Caco-2 mono-
cultures upon incubation with ARC-HPTS-Rh-PE and L-
HPTS-Rh-PE, a variable fluorescence was detected on Caco-
The fluorescence intensity of the hydrophobic label Rh-
PE and the hydrosoluble label HPTS on Caco-2/Raji co-
cultures upon 1 h incubation with ARC-HPTS-Rh-PE were 4
folds higher than the corresponding fluorescence upon incu-
bation with L-HPTS-Rh-PE Fig. (4). Upon 2h incubation,
the fluorescence intensity of Rh-PE increased 2.5 folds both
from ARC and L while the fluorescence intensity of HPTS
remained unchanged. Also after 2 h incubation nearly 15%
Rh-PE and 13% HPTS were found in the bottom wells of co-
cultures incubated with ARC-HPTS-Rh-PE. On the other
hand, no fluorescence was found in the bottom wells of co-
cultures incubated with L-HPTS-Rh-PE.
In Vitro Digestion
After 1 h of simulated digestion, the free and loaded in
liposomes total protein homogenate from T cruzi (L-T. cruzi)
were completely digested On the contrary; several bands
were observed in ARC-T. cruzi, that were coincident with
the non digested homogenate controls (data not shown).
The labeling efficiency was 50 and 40% for ARC and L,
respectively. The highest percentage of the administered
dose (93%) was found in the gastrointestinal tract (GIT)
(stomach, small and large intestine) while a small proportion
(2.9 ± 0.9 %) was found in bladder and urine, and no radio-
activity was found in blood, 4 h after oral administration of
free 99mTc-DTPA Fig. (5A). Similar biodistribution was ob-
tained upon L-99mTc-DTPA administration; nearly 75% of
the administered dose was found in the GIT, 2 ± 1.5% in
bladder and urine, but 6 ± 2 % was found in blood addition-
ally 2 ± 1.8% was found in lungs and 2.2 ± 1.3 % in muscles,
Fig. (5B). In contrast, only 23% was found in the GIT, 21 ±
2% in bladder and urine and remarkably 22 ± 3 % was found
in blood upon ARC-99mTc-DTPA administration (also 6.9 ±
2.7% and 1.6 ± 0.1% in muscles and lungs, respectively, Fig.
Conventional liposomes are weak adjuvants if used as
depots for slow release of antigens or when the delivered
antigen is processed by the MHC-II pathway upon phagocy-
tosis of liposomes by APC. The poor adjuvancy of liposomes
can be increased by including immunomodulatory agents,
namely glycolipid monophosphoryl lipid A (MPL?, the first
Toll-like receptor ligand and biological adjuvant approved
for human use -i.e. the Hepatitis B vaccine Fendrix?-), mu-
ramyl dipeptide (the minimal unit of the mycobacterium cell
wall complex that generates the adjuvant activity of com-
plete Freund’s adjuvant), or attaching ligands such as man-
nose [40, 41]. The inclusion of exogenous material to the
lipid matrix however, is a main concern from the point of
view of the industrial scaling up and regulatory issues in
Fig. (3). Apical distribution o f alkaline phosphatase on (A) Caco-2 mono-cultures and (B) Caco-2/Raji co-cultures (C). The scale bar is 500
μm. Quantification of alkaline phosphate expression, intensity of cells was determinate using ImageJ imaging software. * p< 0.01
6 Current Drug Delivery, 2011, Vol. 8, No. 3 Morilla et al.
vaccine development .
Archaea are non pathogenic microbes  that do not
possess polysaccharides  nor murein  and that pre-
sumably would not have pathogen-associated molecular pat-
terns to serve as danger signals that activate the innate im-
mune system . ARC in general also serve as depots
and/or mediate the presentation of exogenous antigens by the
MHC-II pathway. However, different to liposomes bearing
immunomodulatory agents, ARC do lack of the capacity of
activate Toll-like receptors . Nonetheless, ARC consti-
tute potent adjuvants for the induction of Th1, Th2 and
CD8+ T cell responses to entrapped soluble antigen  in
superior magnitude to other particulate vesicular systems
such as conventional liposomes [49-51] and comparable, or
superior to vaccination with acute bacterial live vectors such
as L. monocytogenes, upon parenteral administration to mice
. Besides, pioneering research during the last 13 years
carried out by the group of Sprott have shown that ARC pre-
pared with TPL extracted from the metanogenous Methano-
brevibacter smithii (containing 40 % caldarchaeols and 30 %
wt archaetidylserine)  are potent inducers of adjuvancy
due to their particular interaction with APC such as dendritic
Fig. (4). CLSM images of Caco-2/Raji co-cultures after 1 h incubation with ARC-HPTS-Rh-PE (A and B) and L-HPTS-Rh-PE (C and D).
Red (A and C) and green signals (B and D) from Rh-PE and HPTS, respectively. (E) Quantification of red and green fluorescence was de-
terminate using ImageJ imaging software. * p< 0.01
M Cells Prefer Archaeosomes: An In Vitro/In Vivo Snapshot Upon Oral Gavage in Rats Current Drug Delivery, 2011, Vol. 8, No. 3 7
Fig. (5). Biodistribution of free 99mTc-DTPA, ARC-99mTc-DTPA and L-99mTc-DTPA upon oral gavage to rats. n: 4, * p< 0.01
cells or macrophages . These ARC target phosphatidyl-
serine-specific receptors on the cell surface of APC ,
resulting in the MHC-I cross presentation of soluble exoge-
nous antigens. The expression of co-stimulatory molecules
on APC, probably receptor mediated, which leads to up-
regulation of cytokines and other immunological regulators,
is induced [48, 54]. Several promising preclinical results
upon parenteral administration of ARC indicate that these
new type of vesicles could behave as ideal adjuvants .
In this work, a different type of ARC was prepared with
TPL extracted from the extremely halophilic-non alkaliphilic
H. tebenquichense strain isolated from the Argentinean
Patagonia. The polar lipids of extremely halophilic Archaea
exclusively contain archaeols and have several unique char-
acteristics that vary little within specific genera [29, 56].
Between 50–80 mol% of extreme halophilic TPL is the
methyl ester of phosphatidylglycerophosphate (PGP-Me), a
diacidic phospholipid that maintains the bilayer structure at
high NaCl concentration . Phospholipids with choline,
ethanolamine, inositol, and serine head groups are absent. In
particular, the Halorubrum genus is rich in phosphatidyl-
glycerol phospholipids and possesses mannosyl-glucosyl-
diphytanylglycerol (DGD), a lipid derived from a basic
diglycosyl archaeol by substitution of sugar or sulfate groups
at different positions of the mannose residue, such as the
SDGD-5 . Besides of the characteristic lipids, this non-
alkalyphikic H. tebenquichense strain contains PG, BPG and
probably SDGD-5-PA (unpublished results). Recently, our
research group has found that ARC prepared with TPL lack-
ing of caldarchaeols and serine, extracted from this non-
alkaliphilic strain and loaded with OVA, elicit a strong and
sustained primary antibody response, as well as improved
specific humoral immunity after boosting with the bare anti-
gen. Both IgG1 and IgG2a enhanced antibody titers could be
demonstrated in long-term (200 days) recall suggesting in-
duction of a mixed Th1/Th2 response upon subcutaneous
administration to mice .
The use of ARC as mucosal adjuvant has been recently
described. ARC prepared from TPL extracted from M.
smithii, H. salinarum, T. acidophilum or from 2,3-di-O-
diphytanyl-sn-glycerolphosphate-O-methyl, complexed with
Ca+2 and loaded with OVA, were intranasally administered
to mice . Anti-OVA IgA antibody responses in sera, fe-
ces, bile, vaginal and nasal wash samples were raised. Anti-
OVA IgG, IgG1 and IgG2a antibody responses in sera, as
well as cytotoxic T lymphocyte responses were also induced.
In the GIT, a chemical and enzymatically hostile medium
for proteins and nucleic acids used as antigens, the balance
between immunity and tolerance must be maintained .
An ideal particulate oral adjuvant must elicit proper immune
reactions upon interaction with APC after uptake and tran-
scytosis by M cells, but also to protect the structure of the
carried antigen . The higher structural stability of ARC
over that of liposomes can be summarized as follows: a)
ARC have the same water and ammonia permeability than
8 Current Drug Delivery, 2011, Vol. 8, No. 3 Morilla et al.
liposomes in liquid crystalline phase, but the presence of eter
linkages reduces three folds its proton permeability ; b)
eter linkages are resistant to acid/basic hydrolysis; c) stereo-
chemical sn-2,3 configuration instead of sn-1,2 of glycero-
phospholipids from Bacteria and Eukarya domains  pro-
tect ARC from the stereospecific phospholipases attack 
and d) the phytanyl group (3,7,11,15-tetramethyl hexadecyl)
is a saturated polyisoprenoid chain stable in air that requires
no special storage conditions. In this first approach, we ob-
served that only when loaded in ARC the bands from T cruzi
proteins remained comparable to the non digested band pat-
tern. Also in the absence of lyoprotectant sugars, ARC could
be successfully lyophilized and reconstituted without aggre-
gation. Hence, the ARC could contribute to overcome the
pharmaceutical design hurdles for antigen-loaded particles,
such as antigen-stability issues and premature antigen release
from particles in the intestine . Two studies show the
absence of toxicity of ARC upon oral administration up to
550 mg/kg day for 10 consecutive days in mice [36, 64].
However, data on ARC interaction with M cells or their fate
upon oral administration remains unexplored.
In this work we have observed that only upon incubation
with Caco-2/Raji co-cultures- but not with Caco-2 mono-
cultures-, the Rh-PE and HPTS fluorescence of ARC re-
mained associated to the cells, a fluorescence that was much
weaker for liposomes. This should indicate a preferential
binding of ARC on the M-like cells.
ARC are in a fluid state between -40 and 80 °C . This
means that the phytanil chains are in an unordered state over
a broad range of T or a high entropy state at the air-water
interfase . Indeed, bilayers from ARC possess lower sur-
face energy (32-37 mN/m) than that of liposomes composed
by straight chain-lipids (54-56 mN/m). The low surface ten-
sion would arise from the unique packing mode of the highly
branched hydrocarbons-chains as estimated from the abnor-
mally large limiting occupied area of the archaeols with phy-
tanyl chains (92-125 Å2) which is nearly two folds that of
conventional glycerophospholipids. Moreover, saturated
polyisoprenoids such as phytanil chains are considered
highly hydrophobic materials . On the other hand, it is
well known that M cells have more affinity by hydrophobic
particles (polystyrene, polymethylmethacrylate, polyhy-
droxybutrate polycaprolactone) than for hydrophilic (com-
posed of lactide and glycolide monomers, ethyl cellulose,
cellulose triacetate and cellulose acetate hydrogen phthalate)
[68-70]. It is uncertain if the presence of traces of non polar
lipids in the TPL, or of sulphated sugars could influence the
binding affinity/specificity. However, the unusual hydropho-
bicity of ARC could explain the higher uptake by M-like
cells over that of liposomes. The finding of Rh-PE and
HPTS only from ARC and not from liposomes in the bottom
wells of Caco-2/Raji co-cultures, a compartment that is
equivalent to the basolateral pocket of the M cells, could be
owed to a transcellular passage of ARC. Hence, the ARC
(whether in the intact form or devoid from hydrosoluble in-
ner content) should have higher chances than liposomes in
accessing the APC that in vivo locate under the PP dome.
Finally, the hydrosoluble radiopharmaceutical 99mTc-
DTPA (a marker of gastric emptying time upon oral admini-
stration, which is neither adsorbed nor absorbed across the
mucosa [71, 72]), was loaded in ARC and liposomes. The
finding of 22 ± 3 % 99mTc-DTPA (nearly 3.5 folds more than
from liposomes) in blood 4 h upon oral gavage of rats sug-
gested that ARC were involved in a pronounced shuttling of
the impermeable 99mTc-DTPA from the lumen to systemic
circulation. In vivo, the particulate material taken up by the
M cells is found at the extensive network of lymphatics un-
derlying the microvilli . If ARC were transcitosed by M
cells, a potential leakage of 99mTc-DTPA during or at the end
of intracellular traffic, at the level of basolateral pocket,
could explain the presence of 99mTc-DTPA in blood circula-
tion. The leakage should not occur at the luminal side; oth-
erwise 99mTc-DTPA would not be absorbed. An evidence of
the shuttling of free 99mTc-DTPA was the absence of radioac-
tivity in liver, since if encapsulated in ARC, it should be
cleared by the Kupffer cells.
These results obtained in rodents, where PP are com-
posed of at least 10% of M cells, can not be straightly ex-
trapolated to humans, that posses less than 5% of M cells,
between other differences [17, 74]. However, taken together
these first approaches suggested that ARC should have
higher affinity for M-like cells than non targeted liposomes,
and that are further responsible for the shuttling of a consid-
erable amount of hydrosoluble material to blood circulation.
Deeper studies are required to reveal the fate of the ARC
lipid matrix and its potential as oral adjuvant, since the
higher uptake of particulate material could overcome the
normal tolerance elicited upon oral administration of re-
peated small doses of soluble antigen [75, 76].
This work was supported by a grant from the Secretaria
de Investigaciones, Universidad Nacional de Quilmes. MJ
Morilla and EL Romero are members of the Carrera del In-
vestigador Científico del Consejo Nacional de Investigacio-
nes Científicas y Técnicas, Argentina (CONICET). L Higa
has got a fellowship from CONICET.
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Received: ???????????????? Revised: ???????????????? Accepted: ????????????????