liposomes

3,509 views

Published on

Published in: Technology
0 Comments
3 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
3,509
On SlideShare
0
From Embeds
0
Number of Embeds
42
Actions
Shares
0
Downloads
202
Comments
0
Likes
3
Embeds 0
No embeds

No notes for slide
  • 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

    1. 1. LIPOSOMES AND SOLID LIPID NANOPARTICLES
    2. 2. 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.
    3. 3. Liposomes •invented in 1965 by A. Bangham and from then on they have been used as a valuable tool in Biology, Biochemistry, Pharmacy and Therapeutics IN PHARMACY ’70 –’80  Stealth liposomes ’90’s Stealth = invisible to the Reticulo-Endothelial system (RES)
    4. 4. Liposomes: “An artificial microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipid layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs.” A spherical particle in an aqueous medium, formed by a lipid bilayer enclosing an aqueous compartment DIAMETER 60nm - 3 microns
    5. 5. cholesterol LIPOSOMES ARE COMPOSED OF NATURAL LIPIDS (PHOSPHOLIPIDS AND CHOLESTEROL) LOWER RISK OF TOXICITY
    6. 6. Phospholipids
    7. 7. LIPOSOME TYPES -Conventional -Stealth (with peg molecules on their surface) -Targeted (with addition of ligands as antibodies et.c) -Cationic (with positive surface charge)
    8. 8. Preparation of Liposomes SUV are typically 15-30nm in diameter while LUV range from 100-200nm or larger. LUV are stable on storage, however, SUV will spontaneously fuse when they drop below the phase transition temperature of the lipid forming the vesicle.
    9. 9. 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
    10. 10. CLASSIFICATION Categories and Naming Size nm Incapsulation efficiency % a)Number of lamellae Unilamellar Multilamellar (MLV) 500-3000 2 b) Size Small (SUV) Large (LUV) Giant (GUV) 60-100 100-1000 > 1000 0.1 up to 50 c) Preparation technique Extruded Detergent removal (DRV) Reverse evaporation (REV) Stability good medium good
    11. 11. Size Determined by Methods MLV: Multilamellar vesicles Monolamellar vesicles: SUV: Small unilamellar vesicles LUV: Large unilamellar vesicles GUV: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
    12. 12.  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 o o o o o o o o o Particle size Zeta potential Polydispersity Index pH of the suspension Aggregation? Redispersibility Assay of the incorporated drug Maximum allowable limit of solvents Residual stabilizer Degradation products (oligomers/monomers) Journal of Biomedical Nanotechnology. 1 (2005) 235-258 Nanomedicine: Nanotechnology, Biology and Medicine 2(2006) 127-136 16
    13. 13. DSC: differential scanning calorimetry Technique that allows to study the phase transition of lipids around the Melting Temperature (Tm) by increasing the temperature of the sample and measuring the entalpy (∆H).
    14. 14. DIFFERENTIAL SCANNING CALORIMETRY (DSC)
    15. 15. DRUG ENCAPSULATION
    16. 16. Liposome advantages   Retention of both lipophilic and hydrophilic drugs. Easy Tailoring, ex. Antibody or ligand conjugation [targeting]  Minimum antigenicity.  Biodegradability  Biocompatibility
    17. 17. 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
    18. 18. DRV technique Prepare empty SUV IMPORTANT: Mix with equal volume of solution of material to encapsulate Freeze dry until all water has been removed Rehydrate in a controlled Way. Add a very low volume first (1/10 of initial) Osmotic pressure of buffers used during rehydration Rehydration method
    19. 19. 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)
    20. 20. Purification of drug-entrapping liposomes Techniques based on size differences of liposomes and entrapped material: 1. Centrifugation techniques 2. Dialysis 3. “Gel filtration” column chromatography
    21. 21. Centrifugation techniques • This technique is used for large size liposomes: MLV, DRV. Discard the supernatant Add Buffer in access Encapsulated in liposomes fluorescence dye Add fresh buffer in access Centrifugation 15000 rpm for 20 min (25° C) Free fluorescence dye molecules Liposomal suspension Resuspend the liposomal pellet at the right volume (Purification process is repeated many times) Liposomal pellet Purified liposomal suspension
    22. 22. 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 Access of Buffer solution Dialysis sack Fig.1. Purification of liposomes by dialysis technique Encapsulated in liposomes fluorescence dye Free fluorescence dye molecules
    23. 23. 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 Daltons Two 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).
    24. 24. 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 50150μm) for chromatography of MLVs (all grades are suitable for SUVs).
    25. 25. Liposomes Novel systems may incorporate some timedependent 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 coupled with chemotactic ligands such as peptides, polysaccharides, affinity ligands like antibodies; pH-sensitive lipids like polyethylenimine or with hydrophilic PEGylated phospholipids in order to improve their in vivo performance and to meet a specific therapeutic need. Date A.A., Adv. Drug Deliv. Rev, 59 2007
    26. 26. In vivo administration of Liposomes Barriers to delivery in vivo: Filtering (chemical and size exclusion) by the liver and spleen
    27. 27. 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.
    28. 28. The gel lanes show a size-selective separation of mouse serum proteins in the corona of the AuNP after incubation and washing. Numbers (kDa) to the left and right indicate the protein size derived from the marker proteins. Labeled proteins were detected in corresponding gel bands by MALDI-TOF-MS.
    29. 29. => 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
    30. 30. 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 or positively charged
    31. 31. Sterically stabilized liposomes or Stealth liposomes  Introduction of PEG-lipids
    32. 32. Liposomes with PEG molecules Possible structures «mushroom» conformation «brush» conformation Kinetics
    33. 33. • 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 Kaposi's 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 Kaposi's sarcoma, recurrent breast cancer and ovarian cancer.
    34. 34. • • • 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 Kaposi's 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.
    35. 35. • 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 BPDMA:EPG:DMPC in 1:05:3:5 molar ratio. It is used for treatment of agerelated macular degeneration, pathologic myopia and ocular histoplasmosis.
    36. 36. • • • 9) DepoCyt (Liposomal cytarabine)- This is a non PEGylated formulation of liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. DepoFoam 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.
    37. 37. 12) Lipoplatin (Liposomal cisplatin)- This is a PEGylated formulation of liposomal cisplatin. The liposomes are composed of DPPG, Soy PC, cholesterol and PEG2000-DSPE. It is used for treatment of epithelial malignancies such as lung, head and neck, ovarian, bladder and testicular cancers. 13) LEP-ETU (Liposomal Paclitaxel)- This is a non PEGylated formulation of liposomal Paclitaxel. The liposomes are composed of DOPE, cholesterol and cardiolipin. Its is used for treatment of ovarian, breast and lung cancer. LEP-ETU is completing phase II of clinical trials. 14) Epaxal (Hepatitis A vaccine)- Liposomes have been used as a vaccine adjuvant in this formulation. These liposomes also known as immunopotentiating reconstituted influenza virosomes (IRIV) are composed of DOPC/DOPE in 75:25 molar ratio. The liposomes are sized to 150 nm.
    38. 38. Solid Lipid Nanoparticles (SLN) Lipid utilized ( SLN ) • phospholipids,triglicerides, di-glicerides, fatty acids, cholesterol and cholesterol-ester
    39. 39. SLN: SOLID LIPID NANOPARTICLES SLN 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.
    40. 40. SLN advantages
    41. 41. Method of preparation: • High pressure homogenization: Hot homogenization Cold homogenization • Ultrasonication /high speed homogenization: • Solvent emulsification/evaporation • Micro emulsion based SLN preparations • SLN preparation by using supercritical fluid • Spray drying method 56
    42. 42. Hot homogenization Melting of the lipid & dissolving/dispersing of the drug in the lipid Dispersing of the drug loaded lipid in a hot aqueous surfactant mixture. Premix using a stirrer to form a coarse preemulsion High pressure homogenization at a temperature above the lipid M.P. Hot O/W nanoemulsion Solid Lipid Nanoparticles Disadvantages: 1) temperature induce drug degradation 2) partioning effect 3) complexity of the crystallization 57
    43. 43. Cold homogenization Melting of lipid & dissolving/dispersing of the drug in the lipid Solidification of the drug loaded lipid in liquid nitrogen or dry ice Grinding in a powder mill Dispersing the powder in a aqueous surfactant dispersion medium High pressure homogenization at room temperature or below. Solid Lipid Nanoparticles Disadvantages: 1) Larger particle sizes & broader size distribution 2) does not avoid thermal exposure but minimizes it 58
    44. 44. Ultrasonication/ high speed homogenization : • SLN were also developed by high speed stirring or sonication • Adv. : 1) Equipment used is very common 2) No temperature induced drug degradation • Disadv.: 1) Potential metal contamination 2) Broader particle size distribution ranging into micrometer range. 59
    45. 45. DRUG ENTRAPMENT
    46. 46. APPLICATIONS • Solid lipid Nanoparticles possesses a better stability and ease of up grad ability to production scale as compared to liposomes. • SLNs form the basis of colloidal drug delivery systems, which are biodegradable and capable of being stored for at least one year . 61
    47. 47. SLNS AS COSMECEUTICALS • Applied in the preparation of sunscreens. • SLN has UV reflecting properties. ORAL SLN IN ANTITUBERCULAR THERAPY • Anti-tubercular drugs such as rifampicin, isoniazide, loaded SLNs able to decrease dosing frequency and increase bioavailability. SLN AS A GENE VECTOR CARRIER • Several recent reports of SLN carrying genetic materials such as DNA, plasmid DNA, & other nucleic acid have been reported. 62

    ×