liposomes
TRANSCRIPT
LIPOSOMES AND
SOLID LIPID NANOPARTICLES
• 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.
What are liposomes?What are liposomes?
Liposomes •invented in 1965 by A. Bangham and from then on they have been used as a valuable tool in Biology, Biochemistry, Pharmacy and TherapeuticsIN PHARMACY
’70 –’80Stealth liposomes ’90’s
Stealth = invisible to the Reticulo-Endothelial system (RES)
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 compartmentDIAMETER 60nm - 3 microns
LIPOSOMES ARE COMPOSED OF NATURAL LIPIDS (PHOSPHOLIPIDS AND CHOLESTEROL)
LOWER RISK OF TOXICITY
cholesterol
PhospholipidsPhospholipids
LIPOSOME TYPESLIPOSOME TYPES
--ConventionalConventional--StealthStealth(with peg molecules on their surface)
--TargetedTargeted(with addition of ligands as antibodies et.c)
--Cationic Cationic (with positive surface charge)
Preparation of LiposomesSUV 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.
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
Categories and Naming Size nm Incapsulation Stabilityefficiency %
a)Number of lamellaeUnilamellarMultilamellar (MLV) 500-3000 2 good
b) Size Small (SUV) 60-100 0.1 mediumLarge (LUV) 100-1000 up to 50 goodGiant (GUV) > 1000
c) Preparation technique Extruded Detergent removal (DRV) Reverse evaporation (REV)
CLASSIFICATION
Size Determined by Methods
Sonication: SUV Smaller than 100 nm diameterExtrusion: LUV (Size depends on the filters) 100 nm—1 µm diameterEvaporation: GUV Larger than 1 µm diameter
MLV: Multilamellar vesicles
Monolamellar vesicles:SUV: Small unilamellar vesiclesLUV: Large unilamellar vesiclesGUV:Giant unilamellar vesicles
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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.
Journal of Biomedical Nanotechnology. 1 (2005) 235-258Nanomedicine: Nanotechnology, Biology and Medicine 2(2006) 127-136
o Particle sizeo Zeta potentialo Polydispersity Indexo pH of the suspension o Aggregation? o Redispersibilityo Assay of the incorporated drugo Maximum allowable limit of solventso Residual stabilizer o Degradation products (oligomers/monomers)
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).
DIFFERENTIAL SCANNING CALORIMETRY (DSC)
DRUG ENCAPSULATION
Liposome advantagesLiposome 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 techniqueDRV technique
Prepare empty SUV
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)
IMPORTANT: Osmotic pressure of buffers used during rehydrationRehydration 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 liposomes
Techniques based on size differences of liposomes and entrapped material:
1. Centrifugation techniques
2. Dialysis
3. “Gel filtration” column chromatography
Centrifugation techniques
• This technique is used for large size liposomes: MLV, DRV.
Liposomal suspension
Centrifugation
15000 rpm for 20 min (25° C)
Add Buffer in access
Liposomal pellet
(Purification process is
repeated many times)
Discard the supernatant
Add fresh buffer in access
Resuspend the liposomal pellet at the right volume
Purified liposomal
suspension Free fluorescence dye
molecules
Encapsulated in liposomes fluorescence dye
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
Encapsulated in liposomes fluorescence dye
Fig.1. Purification of liposomes by dialysis technique
Free fluorescence dye molecules
Dialysis sack
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).
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
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
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.
Filtering (chemical and size exclusion) by the liver and spleen
Barriers to delivery in vivo:
In vivo In vivo administration ofadministration of LiposomesLiposomes
LIPOSOMES ARE ATTACKED BY PLASMA PROTEINS LIPOSOMES ARE ATTACKED BY PLASMA PROTEINS AFTER IV-INJECTION.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.
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.
=> => Non-stealth liposomes accumulate in the Non-stealth liposomes accumulate in the liverliver and spleenand spleen a few minutes after injectiona 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
•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
Filtering (chemical and size exclusion) by the liver and spleen
-Pharmacokinetic Models based on size and charge
Sterically stabilized liposomesSterically stabilized liposomesoror
Stealth liposomesStealth liposomes
Introduction of PEG-lipids
Liposomes with PEG moleculesLiposomes with PEG molecules
Possible structures KineticsPossible 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 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.
• 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.
• 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 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.
Solid Lipid Nanoparticles (SLN)
Lipid utilized ( SLN )
• phospholipids,triglicerides, di-glicerides, fatty acids, cholesterol and cholesterol-ester
SLN:SLN:
SOLID LIPID NANOPARTICLESSOLID 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.
SLN advantages
Method of preparationMethod 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
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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
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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
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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.
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DRUG ENTRAPMENT
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 .
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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.
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