LIPIDIC NANOSTRUCTURES AS CARRIERS FOR CONTROLLED DRUG DELIVERY

1
LIPIDIC NANOSTRUCTURES AS CARRIERS FOR
CONTROLLED DRUG DELIVERY

• Mihaela TRIF1, Anca ROSEANU1, James M. BREWER2,
  Jeremy H. BROCK2
• Romanian Academy, Institute of Biochemistry,
  Bucharest / ROMANIA
• University of Glasgow, Department of Immunology,
  Glasgow / UK

2
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

3
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
4
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

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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 recommended
for most lipids to obtain an unimodal size
distribution.
Liposome size as function of passes through
extruder polycarbonate membrane
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Liposome use

• In vivo
• 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

• In vitro
• 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

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Reasons to use liposomes as drug carriers

• - Protection Liposome
  encapsulated drugs are inaccessible to
  
• 
  metabolising enzymes
• - Directing potential 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

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Liposome entrapped hLf by freeze-thaw method

• Positively charged liposomes were prepared using
  dipalmitoyl phosphatidyl-ethanolamine (DPPE),
  Cholesterol (Chol) and stearylamine (SA), in
  551 molar ratio. pH-sensitive liposomes
  contained dioleoyl-phosphatidyl-ethanolamine
  (DOPE) and cholesterylhemisuccinate (CHEMS), 64
  molar ratio.
• Conventional liposomes prepared from
  Phosphatidylcholine (PC) and Cholesterol (Chol),
  32.
• 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.

Freeze fracture electron microscopy image of
multivesicular liposomes
(Spector at all, Langmuir, 12, 1996)
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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-TxR
Liposomes-DiI
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
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Influence of lipidic composition on the liposome
cell interaction Uptake of free and liposome
entrapped lactoferrin by diferent cells

• Lactoferrin is an iron binding glycoprotein of
  the transferrin family which can modulate the
  inflammatory response when injected
  intra-articularly into mice with collagen-induced
  arthritis (CIA).
• The cellular uptake of free and liposome
  entrapped lactoferrin by THP-1 cells (A) and
  human synovial fibroblasts from RA patients (B)
• 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)

--- - hLf entrapped in pH-insensitive
liposomes --- - hLf entrapped in negative
pH-sensitive liposomes --- - hLf entrapped in
positive liposomes --- - free hLf.
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Stability of different liposomal formulations of
hLf in the presence of human serum

• The amount of 125I-hLf released from liposomes
  was measured in the supernatant and calculated
  as the percentage of the initially-entrapped
  protein released. Each point is represented as
  the mean ? SD, n5.
• 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 pH-sensitive liposomes and 88 from the
  conventional liposomes.

--- conventional --- pH-sensitive ---
positive
Different liposome formulations entrapped
lactoferrin were incubated in the presence of
human serum at 37º C for 24 hours
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Effect of liposomal formulation on Lf retention
in the inflammed joint after intra-articular
injection into mice with collageninduced
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,
  n3.
• 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)

125I-hLf retention in joints of mice with CIA
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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