Magnetoliposomes with high USPIO entrapping efficiency,
stability and magnetic properties
Athanasios Skouras, MSca, Spyridon Mourtas, PhDa, Eleni Markoutsa, MSca,
Marie-Christine De Goltstein, MScb, Claire Wallon, MScb, Sarah Catoen, PhDb,
Sophia G. Antimisiaris, PhDa,c,⁎
aLaboratory of Pharmaceutical Technology, Dept. of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece
bGuerbet, Research Division, Roissy, France
cInstitute of Chemical Engineering and High Temperature Processes / Foundation for Research and Technology Hellas, Patras, Greece
Received 24 January 2011; accepted 6 June 2011
The DRV technique (followed by extrusion) was used for construction of hydrophilic-USPIO encapsulating liposomes.
Magnetoliposomes (ML) were characterized for size, surface charge, entrapment, physical stability and magnetic properties (relaxivity).
Results show that nanosized extruded-DRV MLs encapsulate higher amounts of USPIOs in comparison with sonicated vesicles. Fe (III)
encapsulation efficiency (EE) is 12%, the highest reported to date for nanosized MLs. EE of MLs is influenced by ML membrane
composition and polyethyleneglycol (PEG) coating. PEG-coating increases ML EE and stability; however, r2-to-r1ratios decrease (in
comparison with non-PEGylated MLs). Most ML-types are efficient T2 contrast agents (because r2-to-r1ratios are higher than that of free
USPIOs). Targeted MLs were formed by successfully immobilizing OX-26 monoclonal antibody on ML surface (biotin-streptavidin
ligation), without significant loss of USPIOs. Targeted MLs retained their nanosize and integrity during storage for 1 month at 4°C and up to
2 weeks at 37°C.
From the Clinical Editor: Skouras and colleagues present a method for high encapsulation of hydrophilic USPIO-s in magnetoliposomes
using the DRV extrusion technique. The goal is to optimize the production of MRI detectable contrast agents with functionalized homing
capability based on immobilizing specific antibodies in the surface of magnetoliposomes.
© 2011 Elsevier Inc. All rights reserved.
Key words: Magnetoliposomes; USPIO; Nanoparticle; Magnetic; Targeting; Monoclonal antibody
Magnetic fluids in the form of colloidal suspensions or
nanoparticles (NPs) have widespread applications as magnetic
resonance imaging (MRI) contrast agents.1,2In this context, they
can be used in combination with nanosized drug carriers to serve
as theranostic systems (i.e., nanoscale delivery systems with
combinatory therapeutic diagnostic imaging modalities).3-5One
type of such NPs is the ultra-small (with diameters b50 nm)
super paramagnetic iron oxide cores (USPIOs), which are
efficient T2contrast agents; however, USPIO stability is usually
a problem for their successful in vivo use. To increase USPIO
stability (decrease their tendency to aggregate) several types of
coatings have been considered; as dextrans, proteins, polymers,
fatty acids or phospholipids.6-8Although stabilization is
achieved by coating USPIOs with phospholipids, and the
phospholipid coating can be used as an anchor for decoration
of their surface with targeting ligands, when such magnetolipo-
somes (MLs) are used as diagnostics, one targeted vesicle
transfers only one paramagnetic particle. Furthermore, co-
loading with drugs (for theranostic applications) is not efficient.
USPIO-entrapping nanosized liposomes may be considered
as alternative ML formulations, with better magnetic properties
(due to the possibility of entrapping many USPIOs in one
vesicle) and ability to co-load large amounts of drugs. In recent
years, such systems wave been formulated by conventional
liposome-formation techniques as sonication,9film hydration,10
extrusion through polycarbonate membranes,11,12or reverse
Nanomedicine: Nanotechnology, Biology, and Medicine
7 (2011) 572 – 579
The research leading to these results has received funding from the
European Community's Seventh Framework Programme (FP7/2007-2013)
under grant agreement n° 212043.
No conflict of interest was reported by the authors of this article.
⁎Corresponding author: Department of Pharmacy, University of Patras
and Foundation for Research and Technology – Hellas and The Institute of
Chemical Engineering and High Temperature Chemical Processes (FORTH/
ICE-HT), 26510 Patras, Greece.
E-mail address: email@example.com (S.G. Antimisiaris).
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
Please cite this article as: A., Skouras, et al, Magnetoliposomes with high USPIO entrapping efficiency, stability and magnetic properties. Nanomedicine:
NBM 2011;7:572-8, doi:10.1016/j.nano.2011.06.010
phase evaporation/extrusion.13,14In these previous studies, the
entrapment efficiency of magnetic NPs in the MLs was either not
measured10,14or not optimized. Recently doxorubicin (DOX)
and magnetic NPs were co-encapsulated in folate-decorated
liposomes; and although the DOX encapsulation was high, it was
claimed that “there is a need to optimize magnetic particle
incorporation.”13When targeting applications are designed, any
optimization of magnetic particle incorporation in MLs should
not be followed by an increase of ML size. Indeed, MLs need to
have high magnetic content for optimized magnetic responsive-
ness,as well as nano-dimensions for exploitationof the enhanced
permeability and retention (EPR) effect in the case of tumor
targeting), and/or ability to circumvent biological barriers
(depending on the application). The former two requirements
are difficult to reconcile.
Herein, for the first time the dried-rehydrated vesicle (DRV)
technique was used to prepare USPIO-P00904 (Guerbet, Roissy,
France) entrapping MLs. The specific USPIOs used were
stabilized by a hydrophilic coating. Several lipid compositions
and preparative aspects were initially investigated to optimize the
ML encapsulation efficiency, size distribution, zeta potential,
stability, integrity and magnetic properties (relaxivity values).
Finally, the potential of the proposed technique for preparation of
targeted MLs was evaluated by preparation of OX-26-
monoclonal antibody (MAb)-decorated MLs.
Phosphaditidyl choline (PC), 1,2-distearoyl-sn-glycerol-3-
phosphatidylcholine (DSPC), Phosphatidyl glycerol (PG), 1,2-
(polyethyleneglycol)-2000] ( PEG-lipid) and 1,2-distearoyl-sn-
col) -2000] (Biotin-lipid) were purchased from Avanti Polar
Lipids (Alabaster, Alabama). Cholesterol (Chol), streptavidin
from S. avidinii (STREP), 10 nm gold nanoparticles (AuNPs)
labeled with rabbit anti-mouse antibodies (immune-nanogold)
and Sepharose CL-4B were purchased from Sigma- Aldrich
(Chemilab SA, Athens, Greece). Spectrapore Dialysis tubing
(MW cutoff 10,000) was from Serva (Heidelberg, Germany).
Biotinylated OX-26 anti-transferin MAb was obtained from
Serotec (Raleigh, North Carolina). Amicon-Ultra 15 ultrafiltra-
tion tubes (Millipore, Bedford, Massachusetts) were used for
sample concentration when required. All other chemicals
(analytical grade) were from Sigma-Aldrich or Merck (Darm-
stadt, Germany). Hydrophilic-coated-USPIO-P00904 were pro-
vided by Guerbet and used as an isotonic solution in mannitol
(28.7 mg Fe/mL).
Different types of MLs were prepared. Multilamellar vesicles
(MLVs), small unilamellar vesicles (SUVs), dried-rehydrated
vesicles (DRVs) as well as extruded-DRV MLs with the
following lipid compositions (molar ratios reported): PC/Chol
(2:1), PC/Chol (4:1), PC/PG/Chol (9:1:5), DSPC and DSPC/
Chol (2:1).In some cases, PEG-lipid was added at 1 mol%, 4 mol
% or 8 mol% (of total phospholipid [without Chol]).
For MLVs, the thin-film hydration method was used.15,16In
brief, lipids were solvated in a chloroform/methanol (2:1 v/v)
mixture and placed in a round flask. Lipid solution was dried
by rotary evaporation until development of a thin lipid film.
Residual organic solvents were removed by nitrogen and lipids
were hydrated with appropriate amounts of the USPIO
dispersion (usually 20 – 200 μl) and/or PBS buffer (10 mM,
pH 7.40), at a temperature above the transition temperature of
the lipid used. Next: (i) For MLVs, the liposome dispersion
was sonicated for 15 minutes in a bath-type sonicator
(Branson, Danbury, Connecticut); (ii) For SUVs, the lipid
dispersion was sonicated by a probe microtip (Sonics and
Materials, Suffolk, United Kingdom) for 2 10-minute cycles;
(iii) For DRVs the method described earlier17was applied. In
brief, empty SUVs were prepared (as described above); then 1
mL SUV dispersion was mixed with USPIO dispersion (20–
200 μl), the mixture was freeze-dried and rehydrated.18
Finally, all types of MLs prepared were incubated for 1 hour
at a temperature above the lipid Tm (for annealing any
The DRV-ML size was decreased by extrusion (20
times) through 2 stacked polycarbonate membranes (pore
size 100 nm) in a Lipo-so-fast extruder system (Avestin,
Mannheim, Germany) and the MLs produced by this
method are referred to as extruded-DRVs.
The separation of liposome-encapsulated and free USPIOs
was accomplished by gel exclusion chromatography (GEC)
on a Sepharose–4B CL column, eluted with PBS buffer, pH
7.40. Lipid-containing fractions were pooled and concentrated
(if needed) by ultrafiltration. To exclude the possibility that
USPIOs aggregate and co-elute with liposomes during GEC,
free USPIO dispersions were treated by the identical
procedure used for ML formation (without addition of lipids)
and assessed by GEC.
Targeted-MLs were prepared by adding Biotin-lipid at 0.05
mol% (with respect to total lipid) in the lipid-phase during ML
preparation. The biotin-streptavidin-biotin ligation was used.19
For antibody conjugation, biotinylated MLs were incubated first
with 10-fold molar excess STREP (in comparison with biotin)
for 1 hour at 25°C and then overnight at 4°C.20Then free STREP
was removed by GEC and finally OX-26 MAb decoration of the
biotinylated- STREP-labeled MLs was performed by incubation
with excess of biotin-MAb (attachment yield N90%).19
ML characterization and stability
Vesicle entrapment efficiency
The Fe-to-lipid (mol/mol) ratio was calculated in all
liposome types. For this, the Fe concentration was measured
in liposomes, after vesicle disruption21by a colorimetric
method.22In brief, 100 μl of HCL (37 %) and 10 μl of H2O2,
were mixed (vortex) with 100 μl MLs and 1 mL of KSCN (2%
w/v) was added. Sample was mixed (vortex) and optical density
was measured (480 nm). The accuracy of this method for MLs
was realized after construction of a calibration curve of USPIOs
in the absence or in the presence of lipids and surfactant. The Fe
573A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
content of ML dispersions was also measured by inductively
coupled plasma ICP-MS. For this purpose, 4500 μL of nitric
acid 65% was added to 500 μL of the ML sample. The mixture
was heated at 80°C during 8 hours, allowing a complete
dissolution of the ML. After the mineralization step, the sample
was dissolved in MilliQ water to obtain a final concentration of
nitric acid at 32.5%. Total Fe concentration was quantified by
ICP-MS (Optima 3300 RL, Perkin-Elmer, Shelton, Connecticut)
using a set of calibration standards ranking from 0.2 mg/L to
10 mg/L of iron with yttrium as internal standard prepared
in nitric acid 32.5% (RF power at 1500 W, Fe = 238,204 nm,
Y = 324,227 nm).
Stewart colorimetric assay.23Phospholipids form a colored
complex with ammonium ferrothiocyanate (OD-485 nm), which
is extracted with chloroform. Lipid concentration is calculated
based on a standard curve prepared with known phospholipid
amounts. The lipid concentration of MLs was adjusted, as needed.
Vesicle size distribution, Polydispersity Index and
Particle size (mean diameter) and polydispersity index (PI) of
diluted (with PBS pH 7.40) ML dispersions (0.4 mg/ml) was
measured by DLS technique (Malvern Nano-ZS, Malvern
Instruments, Malvern, United Kingdom) at 25°C at a 173-degree
angle. Zeta potential was also measured at 25°C, by the same
instrument (utilizing the Doppler electrophoresis technique).
ML stability studies
The physical stability of MLs was evaluated during
incubation (at a lipid concentration of 1 mg/ml in PBS buffer)
at 4 ± 1.5°C or 37 ± 1°C, by measuring their size distribution
(mean diameter and PI), at specific time points.
The retention of USPIOs in MLs was also evaluated under the
same conditions. At specified time periods (every 7 days)
samples were taken from the ML dispersions and any amount of
USPIO released was separated by GEC; then the ML Fe(III)-to-
lipid ratios were determined, as described above.
MLs (1-2 mg/ml) were deposited for 2 minutes on formvar-
coated carbon-reinforced 300-mesh copper grids (Agar Scien-
tific, Ltd., Essex, United Kingdom) and negatively stained with
5% ammonium molybdate (Sigma) for 2 minutes, washed with
H2O (x 2), drained and observed at 100.000 eV with JEOL
(JEM-2100) TEM. For demonstration of OX-26 MAb presence
on the targeted-ML surface, control MLs (with no MAb) and
sample MLs (with MAb) were allowed to react “on grid” with 10
nm immuno-nanogold for 30 minutes. The excess of immuno-
nanogold was washed with PBS, and then MLs were stained as
above. For comparison, empty liposomes were also visualized,
after being processed using the same protocol.
Magnetic properties of MLs
Relaxivity measurements were performed on various types of
MLs with a Minispec Relaxometer (Bruker, Billerica, Massa-
chusetts) at 37 ± 1°C, operating at 20 MHz (0.47 Tesla) and 60
MHz (1.42 Tesla). For this, the ML dispersions were diluted to
obtain 5 points between 0.05 and 1.5 mM of Fe. The relaxivity
was finally calculated by the slope of the linear regression
performed on the 5 data points.
A complete separation of MLs from nonencapsulated USPIOs
was achieved under the conditions applying for GEC (see
Supplementary Figure S1, available online at http://www.nanomed-
journal.com). It was demonstrated that plain USPIOs do not
aggregate; thus, the MLs eluted from the GEC column are not
“infected” by aggregated USPIOs (which may have co-eluted with
MLs was found to be reliable for the determination of the USPIO
content of MLs (see Supplementary Figure S2).
Encapsulation Efficiency and physicochemical characteristics
Concerning the effect of ML preparation technique on USPIO
EE (Table 1), MLs prepared by DRV method have more than 2
times higher EE in comparison with those prepared by thin-film
hydration (MLVs); also extruded-DRV MLs with mean diameters
around 100 nm, have approximately 10 times higher EE in
comparisonwith SUVsofsimilarsize.The near70-timesdecrease
of mean diameter of the DRV MLs results in only a about 5-times
decrease of Fe (III) EE. Thereby, it was decided to use the DRV-
extrusion method for further optimization of USPIO-loading.
As presented in Table 2, higher EEs are achieved for both
DRVs and extruded-DRVs , when diluted PBS (10% v/v) is used
in the first step of DRV formation, (in comparison with full-
strength [10 Mm] buffer) in accordance with previous results
obtained for other types of encapsulated molecules.17ML iron
loading increases as initial Fe(III)-to-lipid ratio increases, in the
specific Fe(III)–to-lipid ratio range evaluated. Indeed, when the
initial Fe(III)-to-lipid ratio is increased by 10 times (from 0.25 to
2.5), a 7.7-times increase of the EE occurs for DRVs and a
corresponding 5-times increase for extruded DRVs. When 4 mol
% PEG-lipid is added in ML membranes, USPIO loading
increases significantly (Figure 1). This change occurs when high
(N4.5) initial Fe(III)-to-lipid ratios are used but is less
pronounced (and statistically insignificant) at initial ratios
below 2.56 (μmole Fe(III)/mg lipid). However the EE of MLs
Encapsulation efficiency (EE) of various types of MLs. For each ML type,
EE and trapping efficiency were calculated as the mean value from at least 3
Fe / mg lipid)
574 A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
does not increase any further when higher PEG-lipid contents are
used (8 mol%), even at the highest initial ratios evaluated.
MLs with DSPC + 8 mol% PEG-lipid composition were used
for attachment of OX-26 MAb on their surface (by the method
described above) using a 5.1 (μmoles/mg) initial Fe-to-lipid
ratio, and the EE of the targeted MLs was 0.436 ± 0.022 μmoles
Fe/mg lipid. In fact EE was practically the same before and after
the ML decoration procedure.
Stability of MLs
The stability of MLs with respect to mean diameter and PI
was initially evaluated during incubation of extruded-DRV
MLs with various lipid compositions at 4°C for periods up to
35 days. It is evident from Figure 2, A, that MLs that are not
coated with PEG tend to aggregate after 25 days. The increase
of ML surface charge (by addition of a charged lipid [PG] in
the liposome membrane) does not provide higher liposome
stability. In contrast, PEG-lipid coated MLs retained their
initial size (approximately 100 nm) after 35 days of incubation
(and even up to 2 months [not shown]), at 4°C. Furthermore,
the PIs of PEGylated MLs were stable for the full incubation
period (Figure 2, B). Figure 2 shows also that the targeted
MLs are also very stable (size and PI) under the applying
conditions. Because non-PEGylated MLs were seen to be
unstable at 4°C, no further stability investigations were
conducted for this ML type.
PEGylated MLs were demonstrated to retain their initial mean
3, A and B), with the exception of PC/Chol 4:1 + 4 mol % PEG-
lipid MLs, which became aggregated after 7 days of incubation
(not measured on day 10, due to large size). For the targeted MLs
(appearing as red dots on the graphs), mean diameter and PI
values were practically unchanged during 10 days at 37°C;
however, significant aggregation was noticed on day 20.
Nevertheless, the stability demonstrated is more than adequate
for in vivo applications.
The integrity (retention of encapsulated quantities of
USPIOs) of PEGylated ML types, as well as MAb-decorated
MLs were also evaluated, during incubation at 4°C and 37°C
for a period of 3 weeks. As demonstrated by the results
(Figure 4) the ML types that demonstrated high stability in
concentration and buffer used (PBS buffer [pH 7.40, 10 mM], or diluted PBS
Initial ratio (Fe to lipid) Encapsulation
(μmoles Fe/mg lipid)
50 - PBS
0.2571.93 E+13 0.112
2.569 1.93 E+14
Figure 1. Encapsulation efficiencies (EE) of various types of DRV and
extruded-DRV MLs in respect to initial Fe-to-lipid ratio. Each EE value
(reported as μmole Fe(III)/mg lipid) is the mean value of at least 3 different
preparations. Standard deviations (SDs) are presented as bars. Symbol key is
included in the figure insert.
Figure 2. Size stability of various ML types during storage at 4°C for up to 35
days (A) mean diameter, and (B) PI. Each value is the mean of at least 3
different preparations. SDs are presented as bars. Red dots are for OX-26
MAb decorated MLs. Sample key is in figure insert.
575 A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
terms of size characteristics can also retain the encapsulated
USPIO loads under similar incubation conditions. In the same
way, the OX-26 MAb-decorated targeted MLs retain their
contents for at least 20 days at 4°C and 14 days at 37°C,
which is in good correlation with the corresponding size
stability (Figures 2 and 3).
Magnetic properties of MLs
Relaxometric properties of plain USPIOs and various ML
types were measured (Table 3) and in most cases, the relation
of inversed relaxation times T1-1
concentration was linear (R2values between 0.99 – 1), with
a few exceptions for some non-PEGylated MLs for which the
relations were not very linear (R2between 0.97 – 0.98); r1and
r2relaxivities were calculated from the slope of each line. For
both, plain USPIOs and MLs the traversal relaxivity r2was
found to be significantly higher in comparison with longitu-
dinal r1, confirming that they are suitable as T2contrast agents.
With the exception of MLs consisted of PC/Chol (4:1) + 4 mol
% PEG-lipid, the r1relaxivities of MLs are lower than that of
plain USPIOs. This indicates a “quenching” of the USPIO
efficacy on the T1relaxation time, due to accessibility of water
to the interior of liposomes. As seen in Table 3, when PEG-
lipids are added in the membranes of specific ML types, the
corresponding r1values increase (in comparison with those of
The ratio of r2to r1is a measure of ML potential as a contrast
agent. This ratio decreases when DSPC-MLs are PEGylated with
4 mol% PEG-lipid (Figure 5), mostly due to increase of r1.
However, in the case of PC/Chol (2:1) MLs (Figure 5),
PEGylation does not have such effect, suggesting that other
Figure 3. Mean diameter (A graph) and PI (B graph) of various MLs during
incubationat 37°C.Each value is the mean of at least 3 different preparations.
SDs are presented as bars. Red dots are for OX-26 MAb decorated MLs.
Sample key is in the figure insert.
Figure 4. Retention of encapsulated USPIOs in PEGylated MLs and MAb-
decorated MLs, during incubation at 4°C and 37°C, for 3 weeks. Each value
is the mean of at least 3 different preparations; SDs are presented as bars.
Sample key is included in the figure insert; solid symbols are for 4°C and the
corresponding open symbols are for 37°C.
Transversal (r2) and longitudinal relaxivity (r1) in mM-1.s-1at 20 MHz or 60
MHz and 37°C, for free P00904-USPIOs and MLs with various lipid
compositions and Fe loadings
20 MHz 60 MHz
+ PEG-lipid (1 mol% of total lipid)
+ PEG-lipid (4 mol% of total lipid)
PC/PG/Chol 9:1:5 0.286
+ PEG-lipid (8 mol% of total lipid)
2.21 (mM) 33.0
576A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
parameters of ML composition (as inclusion of Chol in the lipid
membrane) may alsobe important for magnetic properties. When
the PEG-lipid content of MLs is increased to 8 mol%, r1values
are lower (in comparison with those of the 4 mol% PEG-lipid
MLs) and the r2-to-r1 ratios are reduced even more (in
comparison with the MLs with 4 mol% PEG), mainly due to
reduction of the r2values, which are very close to r2of plain
USPIOs (Table 3), indicating a black-hole effect (loss of
magnetic efficacy because relaxation is faster than water
exchange across the lipid membrane). In general, it is very
interesting that most MLs evaluated herein have better efficiency
in comparison with plain USPIOs, in accordance with their
significantly higher r2-to-r1ratio (Figure 5).
TEM morphological studies (control and MAb-decorated MLs)
Morphological evaluation of MLs with TEM revealed that no
free USPIOs or aggregates were present in the purified ML
dispersions; only monomeric USPIO encapsulated in vesicles
were observed (Figure 6, A and B). OX-26 MAb is attached on
MLs, as proven by immuno-colloidal gold staining (Figure 6,
D-F), but not on the control liposomes (Figure 6, A and B). In
the micrographs which have colloidal gold on and USPIOs in
MLs, due to the higher contrast of gold particles in comparison
with encapsulated USPIOs, it is difficult to distinguish encapsu-
lated USPIOs. A micrograph of control empty OX-26 MAb-
decorated liposomes is included (Figure 6, C) for comparison.
The main requirements for use of MLs as carriers for
theranostic applications (carriers that can be loaded with both
diagnostic and therapeutic agents) are nanosized dimensions,
efficient magnetic properties (which are correlated with USPIO
loading), potential to co-load high quantities of drugs, stability
and targeting potential.4-14,24-28The formation of liposomes that
encapsulate USPIOs has been reported; however, in most cases
the liposome- preparation techniques utilized were conventional
methods, such as thin-film hydration, sonication or extrusion.
Furthermore, the effect of the lipid-membrane composition on
the stability and magnetic efficiency of MLs has not been studied
to date (to the best of our knowledge). Herein, it is proven that
the DRV technique followed by extrusion is efficient for the
formation of nanosized MLs with high USPIO loading (Table 1),
in comparison with values achieved by others for MLs of similar
size.12A 24% EE reported for larger MLs (∼360 nm mean
diameter) may not be accurate because cryo-TEM revealed that
most magnetic NPs were in aggregated form and outside of
MLs.11Herein, P00904-USPIOs retain their size and are
Figure 5. Ratios of transversal (r2) to longitudinal (r1) relaxivity at 37°C, at 20 MHz (upper graph) or 60 MHz (lower graph), for free P00904-USPIOs (presented
as line parallel to x-axis) and MLs (as bars). The corresponding EE for each ML measured is in Table 3. In the axis legend PEGylated ML types are labeled as
+P1 (means 1 mol% PEG-lipid) and +P4 (means 4 mol% PEG-lipid).
577 A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
entrapped in the vesicles at high amounts, without any
aggregated states present in ML samples (see Supplementary
Material) as confirmed by TEM studies (Figure 6).
It is realized that the stability and magnetic properties of the
DRV-extruded MLs are influenced differently by various
composition-related factors. Experimental data confirm that by
increasing the rigidity of the lipid membrane (Chol content) and
especially by increasing the PEG-lipid content (surface PEG
as MAb-decorated MLs (Figures 3 and 4). The well documented
effect of PEGylation on liposome integrity is explained by the
highly hydrated PEGheadgroupsthat extend out and surroundthe
liposome in a 4–10 nm corona,29providing a protective steric
barrier which prevents liposome aggregation and fusion.30
Concerning the magnetic properties of extruded-DRV MLs and
thecorrespondinginfluenceofPEGylation,itis seen that r1values
comparison with those of the non-PEGylated ones (Table 3). This
could be explained by hypothesizing that PEGylation increases
water exchange between interior and exterior of liposomes;
PEG corona from the liposome surface and its configuration
(“mushroom” or “brush”) is dependent on the concentration of
PEG-lipids in liposomes (and on PEG moiety length).29When
again modulated (in comparison with 4 mol% containing MLs)
and r2values are reduced (almost 2 times). One hypothesis could
be that membrane of 8 mol% PEG-lipid MLs (brush) is
“magnetically” more permeable than the one of 4 mol% PEG-
because of a “black hole” effect.
Therefore, it is important to consider all the abovementioned
factors when designing DRV-extruded MLs to have the best
equilibrium between required characteristics for theranostic
applications. It is known that classical MRI sequences (gradient
have the highest possible r2-to-r1 ratio because r1 relaxation
contributes to signal degradation. However, today, new MRI
sequences are available, even if not yet in clinical use, that allow
the abolishment of r1relaxation contribution to signal, which
means that magnetic efficiency is best as r2 values increase
(irrespective of corresponding r1 values). In this context, the
optimal ML compositions in terms of magnetic efficiency and
In conclusion, the current results demonstrate that the
extruded-DRV technique can ensure high USPIO loading in
nanosized PEGylated MLs with high stability, although the
targeting potential of such MLs can be increased by surface
decoration (without affecting their loading and size). Lipid-
membrane composition and surface-coating parameters influ-
ence the stability and magnetic efficiency of MLs and the
proposed ML formulations (with sufficient stability and efficient
T2 contrast) are the ones consisting of PC/Chol (2/1) or even
better DSPC with 4 mol% PEG-lipid content.
The authors are thankful to Petya Hristova, BPharm, for her
help in the preparation of some ML samples as part of her
Figure 6. TEM micrographs of MLs after reaction with gold immunoparticles. (A),(B): Control MLs (without MAb); (C): Empty OX-26 MAb-MLs and
(D)-(F): OX-26 MAb-MLs. The bar is 100 nm. Wide arrows point to USPIOs and thin arrows to gold immunoparticles.
578 A. Skouras et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 572–579
diploma project, and to Mary Kollia, for her valuable help in the Download full-text
Appendix A. Supplementary data
Supplementary materials related to this article can be found
online at doi:10.1016/j.endend.2010.04.036.
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