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

Background Essential physical and chemical parameters: 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 recommended for most lipids to obtain an unimodal size distribution. Liposome size as function of passes through extruder polycarbonate membrane

Liposome use In vivo In vitro 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

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

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-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 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

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.

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, 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. --- conventional --- pH-sensitive --- positive 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) 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