Depend on the number of bilayers liposomes can be divided into multilamellar and unilamellar vesicles. According to the size unilamellar vesicles can be further divided into SUV LUV, GUV. They are prepared by different methods. SUV smaller than 100 nm diameter by sonication, LUV 100 nm to 1micro meter can be prepared by extrusion GUV larger than 1 micro meter can be prepared by evaporation.
Neutral and positively charged small liposomes are cleared less rapidly than negatively charged small liposomes. The clearance of negatively charged small liposomes appears to be biphasic in semilog plots of concentration versus time. Recent studies have suggested that the interaction of negatively charged liposomes with certain plasma components may promote rapid blood clearance. Large, negatively charged liposomes are taken up by blood monocytes more efficiently than liposomes composed of neutral or positively charged lipids. Negatively charged large liposomes had a higher tendency to be taken up by the lung than the corresponding neutral or positively charge liposomes. The incorporation of cholesterol into liposomes decreasing its association with plasma lipoproteins and uptake by the liver. Liposomes carrying a specific ligand on the surface tend have more rapid blood clearance than native liposomes
Monocytes are a minority type of white blood cell
LIPOSOMES ANDSOLID LIPID NANOPARTICLES
What are liposomes?• LIPOSOMES are the smallest round structure technically produced by natural non-toxic phospholipids and cholesterol. – They can be used as drug carriers and they can be “loaded” with a huge variety of molecules, as small drug molecules, proteins, nucleotides even plasmids or particles. – They have a very versatile structure and thus, a variety of applications.
Liposomes•invented in 1965 by A. Bangham and from then on they have beenused as a valuable tool in Biology, Biochemistry, Pharmacy andTherapeuticsIN PHARMACY ’70 –’80Stealth liposomes ’90’sStealth = invisible to the Reticulo-Endothelial system (RES)
Liposomes:“An artificial microscopic vesicle consisting of an aqueous coreenclosed in one or more phospholipid layers, used to conveyvaccines, drugs, enzymes, or other substances to target cells ororgans.”A spherical particle in an aqueous medium, formed by a lipidbilayer enclosing an aqueous compartmentSIZE 60nm - 3 microns
cholesterolLIPOSOMES ARE COMPOSED OF NATURAL LIPIDS(PHOSPHOLIPIDS AND CHOLESTEROL)LOWER RISK OF TOXICITY
LIPOSOME TYPES -Conventional -Stealth (with peg molecules on their surface) -Targeted (with addition of ligands as antibodies et.c) -Cationic (with positive surface charge)
Preparation of LiposomesSUV are typically 15-30nm in diameter while LUV range from 100-200nm or larger. LUV are stableon storage, however, SUV will spontaneously fuse when they drop below the phase transitiontemperature of the lipid forming the vesicle.
Extrusion Unilamellar liposomes are formed by pushing MLV through polycarbonate microfilters in extruders, which results in the narrow distribution in size of the liposomal population. Liposofast Extruder
Size Determined by MethodsMLV: Multilamellar vesiclesMonolamellar vesicles:SUV: Small unilamellar vesiclesLUV: Large unilamellar vesiclesGUV:Giant unilamellar vesicles Sonication: SUV Smaller than 100 nm diameter Extrusion: LUV (Size depends on the filters) 100 nm—1 µm diameter Evaporation: GUV Larger than 1 µm diameter
Characterization• Drug encapsulation (D/L)• Size distribution (DLS, cryo-TEM, SEM, AFM)• Zeta-potential (surface charge)• Stability (size)• Integrity /retention of drug (calcein technique) – In presence of plasma proteins – In presence of Ca2+
The critical parameters of a nanoparticulate formulation to set and monitor quality standards have to be based on simplicity (for routine analysis), reliability and correlation to the in vivo performance. o Particle size o Zeta potential o Polydispersity Index o pH of the suspension o Aggregation? o Redispersibility o Assay of the incorporated drug o Maximum allowable limit of solvents o Residual stabilizer o Degradation products (oligomers/monomers)Journal of Biomedical Nanotechnology. 1 (2005) 235-258 17Nanomedicine: Nanotechnology, Biology and Medicine 2(2006) 127-136
DSC: differential scanning calorimetry Technique that allows to study the phase transition of lipids around the Melting Temperature (Tm) by increasing thetemperature of the sample and measuring the entalpy (∆H).
Liposome advantages Retention of both lipophilic and hydrophilic drugs. Easy Tailoring, ex. Antibody or ligand conjugation [targeting] Minimum antigenicity. Biodegradability Biocompatibility
Dehydrated-Rehydrated vesicles (DRV) Introduced by C. Kirby and G.Gregoriadis, in 1984. Empty SUV liposome dispersion is lyophilized (freeze - drying) in presence of solution of the compound to be entrapped. During rehyadration, the addition of small volume of water results in liposomes with high entrapment efficiency. Advantages : simplicity, mild conditions used (important for sensitive molecules) and high encapsulation efficiency for a variety of compounds. Scale-up
DRV techniquePrepare Mix with Freeze Rehydrate in aempty SUV equal volume dry until controlled of solution of all water Way. Add a very material to has been low volume first encapsulate removed (1/10 of initial)IMPORTANT: Osmotic pressure of buffers used during rehydration Rehydration method
Other methods Detergent removal from mixed lipid-detergent micelles leads to LUV with large encapsulation volume. Freeze Thaw Sonication method (repeated cycles of liposomes freeze thawing leads to formation of LUV with high encapsulation efficiency)
Purification of drug-entrapping liposomesTechniques based on size differences of liposomes and entrapped material:1. Centrifugation techniques2. Dialysis3. “Gel filtration” column chromatography
Centrifugation techniques • This technique is used for large size liposomes: MLV, DRV. Discard the supernatant Add Buffer in Resuspend the access liposomal pellet at Add fresh buffer the right volumeEncapsulated in liposomes in access fluorescence dye Centrifugation (Purification process is Purified 15000 rpm for repeated many liposomal 20 min (25° C) times) suspension Liposomal pellet Free fluorescence dye Liposomal molecules suspension
Dialysis• Method used for purification of all types of liposomes• Sacks of polycarbonate tubing (MW cut off of 10000 Dalton)• Excess of Buffer solution ( 100 X)• Dialysis under stirring at 4°C• Replace the buffer with fresh after 4-5 hours until no fluorescent dye is detected. Free fluorescence Dye Encapsulated in liposomes Access of fluorescence dye Buffer Free fluorescence dye solution molecules Dialysis sack Fig.1. Purification of liposomes by dialysis technique
Column chromatographic separation• Sephadex G-50 (polydextran beads) is the material most widely used for this type of separation To separate free molecules MW<1000 DaltonsTwo special points are worth noting with regard to the use of Sephadex with liposomes:1. There may be a low yield. - The problem can be overcome: by making sure that the liposome sample size is not too small or by pre-saturating the column material with “empty” liposomes of the same lipid composition as the test sample )before or after packing the column).
2. Larger liposomes (>0,4μm) may be retained in the column if the particle size of the gel beads is too small, or if the gel bed contains too many “fines”.- The problem can be overcome:• By Using Medium or coarse grades of Sephadex (particle size 50- 150μm) for chromatography of MLVs (all grades are suitable for SUVs).
Liposomes Novel systems may incorporate some time- dependent or other specific inducible changes in the liposome membrane or its coating to produce ‘intelligent’ liposomes that will change their properties (e.g. leakage rate, fusogenic activity or interaction with particular cells) upon a specific trigger following their application.Depending upon the site of targeting, liposomes may be coupledwith chemotactic ligands such as peptides, polysaccharides, affinityligands like antibodies; pH-sensitive lipids like polyethylenimine orwith hydrophilic PEGylated phospholipids in order to improve their invivo performance and to meet a specific therapeutic need. Date A.A., Adv. Drug Deliv. Rev, 59 2007
In vivo administration of Liposomes Barriers to delivery in vivo: Filtering (chemical and size exclusion) by the liver and spleen
LIPOSOMES ARE ATTACKED BY PLASMA PROTEINS AFTER IV-INJECTION. HDL- Plasma High Density Lipoproteins remove phospholipid molecules from the vesicle bilayer Opsonins = Immune and Nonimmune Serum Proteins which bind to foreign particles and promote phagocytosis.
=> Non-stealth liposomes accumulate in the liver and spleen a few minutes after injection• NATURAL TARGETING (APPLICATIONS IN PARASITIC DISEASES –leishmaniosis, trypanosomiosis)• Non-stealth liposomes could not be used to combat other diseases, due to fast clearance
Filtering (chemical and size exclusion) by the liver and spleen -Pharmacokinetic Models based on size and charge•Small (SUV). more stable.•Large (LUV). Less stable .•Negatively charged have a higher tendency to be taken up by the RES than neutral orpositively charged
Sterically stabilized liposomes or Stealth liposomes Introduction of PEG-lipids
Liposomes with PEG molecules Possible structures Kinetics«mushroom» conformation«brush» conformation
• There are several liposome formulations that have been commercialized and there are many liposome formulations that are in various stages of clinical trials.These are several of the commercialized and phase III formulations:• 1) Myocet (Liposomal doxorubicin)- This is a non PEGylated formulation of liposomal doxorubicin. The liposomes are composed of egg PC (EPC): cholesterol (55:45 molar ratio). It is used in combinational therapy for treatment of recurrent breast cancer.• 2) Doxil, Caelyx (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. The liposomes are composed of hydrogenated soy PC (HSPC): cholesterol: PEG 2000-DSPE (56:39:5 molar ratio). It is used for treatment of refractory Kaposis sarcoma, recurrent breast cancer and ovarian cancer.• 3) LipoDox (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. The liposomes are composed of DSPC: cholesterol: PEG 2000-DSPE (56:39:5 molar ratio). It is used for treatment of refractory Kaposis sarcoma, recurrent breast cancer and ovarian cancer.
• 4) Thermodox (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. Thermodox is a triggered release formulation. The liposomes will release their content upon heat. The tumor is heated up using radio frequency ablation (RFA). The liposomes are composed of DPPC, mono steroyl PC (MSPC) and PEG2000-DSPE. It is used for treatment of primary liver cancer (Hepatocellular carcinoma) and also recurrent chest wall breast cancer. Thermodox is in phase III of clinical trial.• 5) DaunoXome (Liposomal Daunorubicin)- This is a non PEGylated formulation of liposomal Daunorubicin. The liposomes are composed of DSPC and cholesterol (2:1) molar ratio and it is sized to 45 nm. It is used for treatment of Kaposis sarcoma.• 6) Ambisome (Liposomal Amphotericin B)- This is a non PEGylated formulation of liposomal Amphotericin B. The liposomes are composed of HSPC, DSPG, cholesterol and amphoteracin B in 2:0.8:1:0.4 molar ratio. It is used for treatment of fungal infection.
• 7) Marqibo (Liposomal vincristine)- This is a non PEGyated formulation of liposomal vincristine. The liposomes are composed of egg sphingomylin and cholesterol. It is used for the treatment of metastatic malignant uveal melanoma. Marqibo is in phase III of clinical trial.• 8) Visudyne (Liposomal verteporfin)- This is a non PEGylated formulation of liposomal verteporfin (BPD-MA). The liposomes are composed of BPD- MA:EPG:DMPC in 1:05:3:5 molar ratio. It is used for treatment of age- related macular degeneration, pathologic myopia and ocular histoplasmosis.
• 9) DepoCyt (Liposomal cytarabine)- This is a non PEGylated formulation of liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. Depo- Foam is a spherical 20 micron multi-lamellar liposome matrix comprised of Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC): Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is used by intrathecal administration for treatment of neoplastic meningitis and lymphomatous meningitis.• 10) DepoDur (Liposomal morphine sulfate)- This is a non PEGylated formulation of liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. Depo-Foam is a spherical 20 micron multi-lamellar liposome matrix comprised of Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC): Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is used by epidural administration for treatment of postoperative pain following major surgery.• 11) Arikace (Liposomal amikacin)- This is a non PEGylated formulation of liposomal amikacin. The liposomes are composed of DPPC and cholesterol. The size of the liposomes is between 200-300 nm. It is used for treatment of lung infections due to susceptible pathogens. Arikace is used in nebulized form and it is inhaled by the patients. The drug is in phase III of clinical trial.
12) Lipoplatin (Liposomal cisplatin)- This is a PEGylated formulation ofliposomalcisplatin. The liposomes are composed of DPPG, Soy PC, cholesteroland PEG2000-DSPE. It is used for treatment of epithelial malignanciessuch as lung, head and neck, ovarian, bladder and testicular cancers.13) LEP-ETU (Liposomal Paclitaxel)- This is a non PEGylated formulationof liposomal Paclitaxel. The liposomes are composed of DOPE,cholesterol and cardiolipin. Its is used for treatment of ovarian, breast andlung cancer. LEP-ETU is completing phase II of clinical trials.14) Epaxal (Hepatitis A vaccine)- Liposomes have been used as avaccine adjuvant in this formulation. These liposomes also known asimmunopotentiating reconstituted influenza virosomes (IRIV) arecomposed of DOPC/DOPE in 75:25 molar ratio. The liposomes are sizedto 150 nm.
SLN: SOLID LIPID NANOPARTICLESSLN are nanoparticles where the lipid component is composed of solid lipids (glycerides or cere) with high Melting point that are stabilized by using surfactants. SLN are solid at 37°C.