LIPIDIC NANOSTRUCTURES AS
CARRIERS FOR CONTROLLED
DRUG DELIVERY
Mihaela TRIF1, Anca ROSEANU1, James M. BREWER2,
Jeremy H. BROCK2
1.
2.
Romanian Academy, Institute of Biochemistry, Bucharest / ROMANIA
University of Glasgow, Department of Immunology, Glasgow / UK
Background
Liposomes are vesicular structures
composed of one or more phospholipid
bilayer membranes.
Essential physical and chemical parameters:
-
lipid composition of membranes
size
surface electrical charge
Different classes of liposomes as defined
according to their size
•
•
•
MLV (Multilamellar Large Vesicles) 100 ~
5000 nm
LUV (Large Unilamellar Vesicles)
60 1000 nm
SUV (Small Unilamellar Vesicles)
20 50 nm
SUV are lipid nanostructures also known as
nanosomes (Castor TP, Current Drug
Delivery, 2005)
Adapted from Fendler H, 1992
Characteristics
•
prepared from natural, biodegradable and
nontoxic lipids
•
able to entrap hydrophilic drugs in the large
aqueous interior and lipophilic drugs
inserted in the lipid bilayer.
•
good candidates for targetting of therapeutic
agents to the site of interest
Liposome preparation
Small vesicles were prepared
by sonication to clarity (SUV)
or by extrusion
SUV
LUV
Large multi-lamellar vesicles (MLV)
were prepared by thin lipid film
hydration
sonication homogenization
extrusion
multilamellar vesicles
MLV
agitation
dry lipid film
water
swelling
Liposome size
Optical Microscopy image of
MLV (x 600)
Electron Microscopy image of
SUV (x25000)
•
Trif M. PhD Thesis “Liposomes as carriers
for active pharmaceutical substances”,
1994, Institute of Biochemistry
Extrusion technique to generate
liposomes of controlled size
Mini-Extruder from Avanti Polar Lipids
The particle size distribution of vesicles
prepared by extrusion is a function of the
number of passes through the
polycarbonate membrane of the hydrated
lipid suspension. A minimum of eleven
passes through the membrane is
Liposome size as function of passes through
recommended for most lipids to obtain an
extruder polycarbonate membrane
unimodal size distribution.
Liposome use
• In vitro
• In vivo
• To analyse plasma
membrane structure
• To insert new material into
plasma membrane
• To transfer genetic material
into cell
• Introduce biologically active
substances into culture cell
to study cellular metabolism
• Study antigen recognition by
cells of the immune system
As drug delivery
•
•
•
•
In cancer therapy
Respiratory diseases
Antifungal therapy
Anti-inflammatory therapy
-Local application
-Intra-articular injection
• Activation of DC inducing
T cell responses
Reasons to use liposomes as drug
carriers
- Protection
- Directing potential
Liposome encapsulated drugs are inaccessible to
metabolising enzymes
Targeting options change the distribution of the drug in the
body
- Solubilisation
Liposome may solubilise lipophilic drugs that would otherwise
be difficult to administer intravenously
- Amplification
Liposome can be used as adjuvants in vaccine formulatios
- Internalisation
Liposome are endocytosed by cells being able to deliver the
encapsulated material into the cell. Liposome are also able to
bring plasmid material into the cell through the same
mechanism (non viral transfection system)
- Duration of action
Liposome can prolong drug action by slowly releasing the drug
in the body
Liposome –entrapped hLf by freeze-thaw
method
Positively charged liposomes were prepared
using dipalmitoyl phosphatidyl-ethanolamine
(DPPE), Cholesterol (Chol) and stearylamine
(SA), in 5:5:1 molar ratio. pH-sensitive
liposomes contained dioleoyl-phosphatidylethanolamine (DOPE) and
cholesterylhemisuccinate (CHEMS), 6:4 molar
ratio.
Conventional liposomes prepared from
Phosphatidylcholine (PC) and Cholesterol
(Chol), 3:2.
Freeze fracture electron microscopy
image of multivesicular liposomes:
The lipid film obtained was dispersed in PBS
containing hLf and incubated for 5 hours at
room temperature to facilitate the annealing
process. Five freeze - thaw cycles were
performed to obtain a suitable size (about 200
nm) and a high efficiency of hLf incorporation
in multivesicular liposomes (multiple small
unilamelar vesicles bounded by a single bilayer
lipid membrane)
Advantages:
-good stability during storage;
-control over drug release rate;
-high efficient entrapment of hydrophilic
molecules.
(Spector at all, Langmuir, 12,
1996)
Liposome-lactoferrin interaction with human
synovial fibroblasts
Kinetics of uptake of free and liposome entrapped
125I-hLf by human synovial fibroblasts from RA
patients
hLf uptake (%)
8
hLf-TxR
6
hLf in Lipo(-) pHsens
hLf in Lipo(-) pH
insens
hLf in Lipo(+)
4
2
hLf free
Liposomes-DiI
0
0
10
20
30
incubation time (hours)
The amount of hLf accumulated in HF was 10 times higher
compared with free hLf, in the case of pH-sensitive
liposomes. pH-sensitive liposomes were shown to be
better carriers for hLf than other liposomal
formulations.
(merge)
- pH-sensitive liposomes are able to
accumulate in the cytoplasm of HF;
- hLf is associated with cell
membrane
Influence of lipidic composition on the
liposome –cell interaction
Uptake of free and liposome entrapped lactoferrin by diferent cells
A
hLf in THP-1 cells (%)
30
•
Lactoferrin is an iron binding glycoprotein of
the transferrin family which can modulate
the inflammatory response when injected
intra-articularly into mice with collageninduced arthritis (CIA).
•
The cellular uptake of free and liposome
entrapped lactoferrin by THP-1 cells (A)
and human synovial fibroblasts from RA
patients (B)
20
10
0
0
10
20
Time of incubation (hours)
B
8
hLf in fibroblasts (%)
30
6
4
2
0
0
10
20
Time of incubation (hours)
30
--- - hLf entrapped in pH-insensitive liposomes
--- - hLf entrapped in negative pH-sensitive liposomes
--- - hLf entrapped in positive liposomes
--- - free hLf.
Trif. M., Moisei M., Motas C., Serban M.,
Roseanu A., Brock J. H. Uptake of liposome
entrapped lactoferrin by THP-1 cells and
human synovial fibroblasts. Proc. Rom.
Acad., Series B, 3, 233-238 (2000)
•
•
The amount of 125I-hLf released
from liposomes was measured in
the supernatant and calculated
as the percentage of the initiallyentrapped
protein
released.
Each point is represented as the
mean  SD, n=5.
In all cases most of the labeled
hLf has been released from the
liposomes
after
24h
of
incubation.
The
positive
liposomes were marginally more
stable, with 70% of the
radioactive protein released,
compared with 80% from pHsensitive liposomes and 88%
from the conventional liposomes.
% hLf released from liposomes
Stability of different liposomal
formulations of hLf in the presence of
human serum
100
80
60
40
--- conventional
--- pH-sensitive
--- positive
20
0
0
5
10
15
20
25
30
incubation time (hours)
Different liposome formulations entrapped
lactoferrin were incubated in the presence
of human serum at 37º C for 24 hours
Effect of liposomal formulation on Lf retention in
the inflammed joint after intra-articular injection
into mice with collagen–induced arthritis (CIA)
•
Mice were sacrificed 2, 6 and 24 hours after
injection. The recovered 125I-hLf was calculated
as the percentage of the injected dose. Mean
+SD, n=3.
Conclusions: Lactoferrin entrapped in
positive liposomes was retained longer in
the injected joint compared to lactoferrin
entrapped in negative liposomes which was
retained less well than free hLf.
•
Grant M. Trif from The Royal Society, 2000
•
Trif M., Guillen C., Vaughan D., Telfer J., Brewer
J.M., Roseanu A., Brock J.H. Liposomes as
possible carriers for lactoferrin in the local
treatment ofinflammatory diseases. Exp. Biol.
Med., 226, 559-564 (2001)
80
hLf recovered in joint (%)
•
60
L (+)-Lf
L (-)-Lf
Free Lf
40
20
0
2
6
24
Time after intra-articular injection (hours)
125I-hLf
retention in joints of mice with CIA
GENERAL CONCLUSIONS
pH-sensitive liposomes demonstrated a high ability to deliver lactoferrin
into the cytoplasm of human synovial fibroblasts compared to other
liposomal formulations.
In vivo experiments in mice with collagen-induced arthritis (CIA) revealed
that positive liposomes were more efficient prolonging the residence time
of lactoferrin in the inflamed joint, compared with other types of liposomes
or the free protein.
The anti-inflammatory effect of positive liposomes-entrapped lactoferrin
persisted for at least 12 days. It was associated with changes in Th1/Th2
cytokines production.
Our results demonstrated that the entrappement of lactoferrin in
positively charged liposomes improved its pharmacodynamic
profile and was of therapeutic benefit in the treatment of induced
RA in mice