LIPIDIC NANOSTRUCTURES AS CARRIERS FOR CONTROLLED DRUG DELIVERY Mihaela TRIF 1, Anca ROSEANU 1, James M. BREWER 2, Jeremy H. BROCK 2 1.Romanian Academy,

LIPIDIC NANOSTRUCTURES AS LIPIDIC NANOSTRUCTURES AS CARRIERS FOR CONTROLLED CARRIERS FOR CONTROLLED

DRUG DELIVERYDRUG DELIVERY

Mihaela TRIF1, Anca ROSEANU1, James M. BREWER2, Jeremy H. BROCK2

1. Romanian Academy, Institute of Biochemistry, Bucharest / ROMANIA2. University of Glasgow, Department of Immunology, Glasgow / UK

BackgroundBackground Liposomes are vesicular structures composed Liposomes are vesicular structures composed

of one or more phospholipid bilayer of one or more phospholipid bilayer membranes. membranes.

Essential physical and chemical parameters:Essential physical and chemical parameters:

- lipid composition of membranes- size- surface electrical charge

Different classes of liposomes as defined Different classes of liposomes as defined according to their sizeaccording 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)

CharacteristicsCharacteristics

• 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

Adapted from Fendler H, 1992

Liposome preparationLiposome preparation

Large multi-lamellar vesicles (MLV) were prepared by thin lipid film hydration

Small vesicles were prepared by sonication to clarity (SUV) or by extrusion

dry lipid filmdry lipid film waterwater swellingswelling

agitationagitation

multilamellar vesiclesmultilamellar vesicles

sonication homogenizationsonication homogenization extrusionextrusion

SUVSUV

MLVMLV

LUVLUV

Liposome sizeLiposome 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 generategenerate

liposomes of controlled size

Mini-Extruder from Avanti Polar Lipids

Liposome size as function of passes through extruder polycarbonate membrane

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 useLiposome use

• 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

• In vivoAs 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 Reasons to use liposomes as drug carrierscarriers

- 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

Liposome –entrapped hLf by freeze-thaw Liposome –entrapped hLf by freeze-thaw methodmethod

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-phosphatidyl-ethanolamine (DOPE) and cholesterylhemisuccinate (CHEMS), 6:4 molar ratio.

Conventional liposomes prepared from Phosphatidylcholine (PC) and Cholesterol (Chol), 3:2.

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)

Liposome-lactoferrin interaction with human Liposome-lactoferrin interaction with human synovial fibroblastssynovial fibroblasts

(merge)- pH-sensitive liposomes are able to accumulate in the cytoplasm of HF; - hLf is associated with cell membrane

0

2

4

6

8

0 10 20 30

incubation time (hours)

hLf

upt

ake

(%) hLf in Lipo(-) pH-

senshLf in Lipo(-) pHinsenshLf in Lipo(+)

hLf free

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 pH-sensitive liposomes were shown to be better carriers for hLf than other liposomal better carriers for hLf than other liposomal formulations.formulations.

Kinetics of uptake of free and liposome entrapped 125I-hLf by human synovial fibroblasts from RA

patients

hLf-TxR

Liposomes-DiI

Influence of lipidic composition on the Influence of lipidic composition on the liposome –cell interaction liposome –cell interaction

Uptake of free and liposome entrappedUptake of free and liposome entrapped lactoferrin lactoferrin by diferent cells 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)

A

0

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0 10 20 30Time of incubation (hours)

hL

f in

TH

P-1

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lls (

%)

B

0

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6

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0 10 20 30Time of incubation (hours)

hLf i

n fib

robl

asts

(%

)

--- - hLf entrapped in pH-insensitive liposomes--- - hLf entrapped in negative pH-sensitive liposomes --- - hLf entrapped in positive liposomes--- - free hLf.

Stability of different liposomal Stability of different liposomal formulations of hLf in the presence of formulations of hLf in the presence of

human serumhuman 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, 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 pH-sensitive liposomes and 88% from the conventional liposomes.

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0 5 10 15 20 25 30

incubation time (hours)%

hLf

rele

ased

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lipo

som

es

Different liposome formulations entrapped Different liposome formulations entrapped lactoferrin were incubated in the presence of lactoferrin were incubated in the presence of human serum at 37º C for 24 hourshuman serum at 37º C for 24 hours

--- conventional --- pH-sensitive --- positive

Effect of liposomal formulation on Lf retention in Effect of liposomal formulation on Lf retention in the inflammed joint after intra-articular injection the inflammed joint after intra-articular injection into mice with collagen–induced arthritisinto mice with collagen–induced arthritis (CIA (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.

• ConclusionsConclusions: 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)

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2 6 24Time after intra-articular injection (hours)

hLf r

ecov

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in jo

int (

%)

L (+)-Lf

L (-)-Lf

Free Lf

125I-hLf retention in joints of mice with CIA

GENERAL CONCLUSIONSGENERAL CONCLUSIONS

pH-sensitive liposomes demonstrated a high ability to deliver lactoferrin pH-sensitive liposomes demonstrated a high ability to deliver lactoferrin into the cytoplasm of human synovial fibroblasts compared to other into the cytoplasm of human synovial fibroblasts compared to other liposomal formulations.liposomal formulations.

In vivo experiments in mice with collagen-induced arthritis (CIA) revealed In vivo experiments in mice with collagen-induced arthritis (CIA) revealed that positive liposomes were more efficient prolonging the residence time that positive liposomes were more efficient prolonging the residence time of lactoferrin in the inflamed joint, compared with other types of liposomes of lactoferrin in the inflamed joint, compared with other types of liposomes or the free protein.or the free protein.

The anti-inflammatory effect of positive liposomes-entrapped lactoferrin The anti-inflammatory effect of positive liposomes-entrapped lactoferrin

persisted for at least 12 days. It was associated with changes in Th1/Th2 persisted for at least 12 days. It was associated with changes in Th1/Th2 cytokines production.cytokines production.

Our results demonstrated that theOur results demonstrated that the entrappement of lactoferrin in entrappement of lactoferrin in

positively charged liposomes improved its pharmacodynamic positively charged liposomes improved its pharmacodynamic profile and was of therapeutic benefit in the treatment of induced profile and was of therapeutic benefit in the treatment of induced RA in miceRA in mice