Liposomes

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LIPOSOMES PRESENTED BY : Deeksha Joshi M.Pharm . Q.A. ( 1 st year )

WHAT ARE LIPOSOMES ??:

WHAT ARE LIPOSOMES ?? Liposomes are simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of a lipid molecule. Structurally, liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membrane lipid bilayer mainly composed of natural and synthetic phospholipids.

TYPES OF LIPOSOMES:

TYPES OF LIPOSOMES BASED ON STRUCTURAL PARAMETERS: MLV Multilamellar large vesicles (> 0.5 µm) OLV Oligolmellar vesicles (0.1-1.0 µm) UV Unilamellar vesicles (all size ranges) SUV Small unilamellar vesicles (20-100 nm) LUV Large unilamellar vesicles (>100 nm) GUV Giant unilamellar vesicles (>1 µm) MV Multivesicular vesicles (>1 µm)

BASED ON COMPOSITION AND APPLICATION:

BASED ON COMPOSITION AND APPLICATION Conventional liposomes (CL) –Neutral or negatively charged phospholipids and cholesterol Cationic liposomes –Cationic lipids with DOPE Long circulatory (stealth) liposomes (LCL) Immuno liposomes –CL or LCL with attached monoclonal antibody

BASED ON METHOD OF PREPARATION:

BASED ON METHOD OF PREPARATION REV Single or oligolamellar vesicles made by reverse phase evaporation method. MLV-REV Multilamellar vesicles made by REV. VET Vesicles prepared by extrusion technique DRV Dehydration- rehydation method FATMLV Frozen and thawed.

COMPOSITION OF LIPOSOMES:

COMPOSITION OF LIPOSOMES Lipids Lipid mix is the major component of liposomes . Among the lipid phospholipids are the amphoteric lipids capable of forming bilayer and hence are the integral part of the liposomal system. Phospholipids A phospholipid has two acyl chains linked to a headgroup by means of a glycerol-backbone. R1 and R2 are saturated or unsaturated acyl chains and R3 is the polar head group. The polar head groups are used for classification, i.e. to distinguish between different phospholipids like; Phosphatidylcholines , phosphatidylethanolamines etc. R1-COO-CH2 I R2-COO-CH O I I I CH2-O-P-O-CH2-CH2-R3 I O

GENERALLY PHOSPHOLIPIDS ARE REPRESENTED AS::

GENERALLY PHOSPHOLIPIDS ARE REPRESENTED AS:

NATURAL PHOSPHOLIPIDS:

NATURAL PHOSPHOLIPIDS Natural phospholipids are hydrogenated soyabean phospholipids, egg phospholipids and sphingomyelin from milk or egg.

SYNTHETIC PHOSPHOLIPIDS:

SYNTHETIC PHOSPHOLIPIDS

CATIONIC PHOSPHOLIPIDS:

CATIONIC PHOSPHOLIPIDS Cationic lipids are of great importance in DNA and gene delivery. Most of the cationic lipids are trimethylammonium derivatives of fatty acids and the cationic charge is due to the ammonium ion. Some cationic lipids used in the formulation of liposomes for the delivery of genes and drugs are listed below Stearyl amine (S.A) N-[1-(2,3-dioleoyloxy) propyl ]-N,N,N- trimethylammonium chloride (DOTMA) 1,2-bis( oleoyloxy )-3-3-( trimethylammonia ) propane (DOTAP)

PEGylated Phosphoplipids:

PEGylated Phosphoplipids Polyethylene Glycol (PEG) is a non-toxic, hydrophilic, FDA approved uncharged polymer. PEG attached phospholipids can be used to form stealth liposomes . These provide prolonged circulation of liposomal drugs in vivo as they avoid detection of liposomes by body’s immune system. They do so by increasing the hydrophilic character. Cholesterol Cholesterol by itself does not form a bilayer structure. However, it provides rigidity, flexibility and stability to liposomes . It acts as a “fluidity buffer”, i.e. below the phase transition temperature, it makes the membrane less ordered and slightly more permeable while above the phase transition temperature it makes the membrane more ordered and stable. It can be incorporated into phosphoplipid membrane in very high concentration upto 1:1 or even 2:1 molar ratios of cholesterol to PC.

METHODS OF LIPOSOME PREPARATION:

METHODS OF LIPOSOME PREPARATION There are two methods for liposomal manufacturing: PASSIVE LOADING TECHNIQUE ACTIVE LOADING TECHNIQUE PASSIVE LOADING TECHNIQUE INCLUDE: Mechanical dispersion method Solvent dispersion method Detergent removal method.

MECHANICAL DISPERSION METHOD INCLUDES:

MECHANICAL DISPERSION METHOD INCLUDES Lipid film hydration by hand shaking, non hand shaking or freeze drying. MLV Micro emulsification. SUV Sonication . MLV to SUV French pressure cell. ULV,OLV Membrane extrusion. LUVETs Dried reconstituted vesicles. UV OLV Freeze thawed method. ULV

MECHANICAL DISPERSION METHODS……:

MECHANICAL DISPERSION METHODS…… All methods under this category Begin with Lipid solution in organic solvent End up with Lipid dispersion in water

HAND SHAKING METHOD:

HAND SHAKING METHOD Lipid solution + organic solvent hand shaking Film formation vacuum Dried film hydration film stacks dispersed in aqueous phase lipids swell and peel off from the round bottom flask MLV formed. It has small scale production .

Multilamellar Vesicles formed by either Hand Shaking Technique or using Rotary Flash Evaporator:

Multilamellar Vesicles formed by either Hand Shaking Technique or using Rotary Flash Evaporator

NON-SHAKING METHOD:

NON-SHAKING METHOD The mechanical energy required for swelling of lipids and dispersion of casted lipid film is imparted by exposing the film to a stream of water saturated nitrogen for 15min . LUVs are formed inspite of MLVs. Encapsulation efficiency upto 30% is achieved. Large amount of water soluble compounds are wasted during swelling as only 10-15% of the total volume gets entrapped while lipid soluble compounds can be encapsulated at 100% efficiency, provided they are present in adequate quantities.

SONICATED UNILAMELLAR VESICLES:

SONICATED UNILAMELLAR VESICLES At high energy level, the average size of vesicle is further reduced. This is achieved on exposure of MLVs to ultrasonic irradiation. There are two methods of sonication . BATH SONICATOR PROBE SONICATOR More suitable for large volumes of diluted lipids, most widely used for preparation of SUVs. For dispersions that require high energy in small volume (e.g. high conc. of lipids, or a viscous aqueous phase).

PREPARATION OF SUVs BY BATH/PROBE SONICATON PROCESS FROM MLVs:

PREPARATION OF SUVs BY BATH/PROBE SONICATON PROCESS FROM MLVs

Disadvantages of probe sonicator::

Disadvantages of probe sonicator : Overheating of liposomal dispersion causing lipid degradation. Sonicator tips tend to release titanium particles into liposomal dispersion, which must be removed by centrifugation prior to use. Thus bath sonicators are most widely used for preparation of SUVs.

FRENCH PRESSURE CELL LIPOSOMES:

FRENCH PRESSURE CELL LIPOSOMES Since ultrasonic radiation degrades lipids and other sensitive compounds, an alternate method is extrusion of preformed large liposomes in a French press under very high pressure. This technique yields uni - or oligo - lamellar liposomes of intermediate size(30-80nm in dia depending on the applied pressure).

French Pressure Cell and parts used for preparation of Uni or Oligo –Lamellar Vesicles:

French Pressure Cell and parts used for preparation of Uni or Oligo –Lamellar Vesicles

ADVANTAGES::

ADVANTAGES: Liposomes are more stable as compared to sonicated liposomes . Less likely to suffer from structural defects. Leakage of contents is slower and lower. French press is also used to reduce heterogeneity of proteoliposomes obtained by detergent dialysis technique. DISADVANTAGES : High initial cost of press.

MICRO EMULSIFICATION LIPOSOMES (MEL):

MICRO EMULSIFICATION LIPOSOMES (MEL) MICRO FLUIDIZER is used to prepare small MLVs Micro fluidizer pumps the fluid at very high pressure(10,000 psi,600-700 bar) through a 5µm orifice. Then it is forced along defined micro channels, which direct two streams of fluid to collide together at right angle at very high velocity. Fluid collected can be recycled through the pump and interaction chamber until vesicles of spherical dimension are obtained.

ADVANTAGES::

ADVANTAGES: It is able to process samples with a very high proportion of lipids (20% or more by weight). This process is efficient for encapsulation of water-soluble materials. %age capture values up to 70% have been reported, starting with lipid concentration of approx. 200mg/ml.

MEMBRANE EXTRUSION:

MEMBRANE EXTRUSION Liposomes passed through membrane of defined pore size. Lower pressure is required (<100 psi). LUVs as well as MLVs can be processed. Vesicle contents are exchanged with dispersion medium during breaking and resealing of phospholipid bilayers as they pass through the polycarbonate membrane. For high entrapment, the water soluble compounds should be present in suspending medium during the extrusion process.

DRIED RECONSTITUTED VESICLES AND FREEZ THAW SONICATION (FTS) METHOD:

DRIED RECONSTITUTED VESICLES AND FREEZ THAW SONICATION (FTS) METHOD SUVs in aqueous phase SUV with solutes to be entrapped Freeze dried membrane Solutes in uni - and oligolamellar vesicles Solutes in unilamellar vesicles freez drying Rehydration Thawing, sonication DRV FTS

SOLVENT DISPERSION METHOD:

SOLVENT DISPERSION METHOD Ethanol injection -SUV Ether injection -LUV Reverse phase evaporation vesicle -LUV Double emulsion vesicles Stable plurilamellar vesicles

Solvent dispersion method…………..:

Solvent dispersion method………….. In this method, lipids are first dissolved in an organic solution, which is then brought into contact with an aq. phase containing materials to be trapped within the liposomes . The lipids align themselves at the interface of organic and aq. phase forming monolayer of phospholipids, which forms the half of the bilayer of the liposomes .

ETHANOL INJECTON METHOD:

ETHANOL INJECTON METHOD This method is one of the alternatives used for the preparation of SUVs (̴ 25nm) without sonication . L ipid in ethanol solution rapid injection Aqueous phase Formation of SUVs The rate of injection should be sufficient to achieve complete mixing , so that ethanol is diluted almost instantaneously in water and phospholipid molecules are dispersed evenly throughout the medium.

ADVANTAGES::

ADVANTAGES: This method is extremely simple and has a low risk of degradation of sensitive lipids. Vesicles of 100nm size may be obtained by little modification i.e. by varying the conc. of lipid in ethanol or by changing the rate of injection of ethanol solution in preheated aqueous solution.

Disadvantages::

Disadvantages: Limitation of solubility of lipids in ethanol and volume of ethanol that can be introduced into medium (7.5% v/v maximum) which in turn limits the quantity of lipids dispersed, so that resulting liposomal dispersion gets diluted. Difficulty to remove residual ethanol from phospholipid membrane.

ETHER INJECTION METHOD:

ETHER INJECTION METHOD It involves injecting the immiscible organic solution very slowly into an aq. phase through a narrow needle at the temp. of vapourizing the organic solvent. Disadvantage: Long time required to produce a batch of liposomes . If substances are degraded at elevated temperature (60˚C), then fluorinated hydrocarbons ( Freons ) may be used instead of ether.

REVERSE PHASE EVAPORATION METHOD:

REVERSE PHASE EVAPORATION METHOD Sonication Organic sol. evaporated Drying in rotatory evaporator LIPID PHASE AQUEOUS SOLUTION W/O Emulsion Gel formation

PowerPoint Presentation:

Vortexing (gel collapse) Suspension of LUVs Average diameter (DD) of vesicles obtained is 0.5 µm.

DETERGENT REMOVAL METHOD:

DETERGENT REMOVAL METHOD The method involves the preparation of micellar dispersion of phospholipids and detergent in aqueous system in which the drug is dissolved / solublized . Detergent molecules serve to screen the hydrophobic portions of molecule from water. Below CMC the detergent molecules exist as free sol. As concentration of detergent is increased micellar formation takes place above CMC.

Detergent removal method…….:

Detergent removal method……. The micellar dispersion is then subjected to one of the following methods to remove the detergent: DIALYSIS :- Detergents with high CMC (10-20 mM ) are used so that their removal is facilitated e.g. bile salts- sodium cholate and sodium deoxycholate , or synthetic detergents like octileglucoside . COLUMN CHROMATOGRAPHY :- By passing dispersion over a Sephadex G-25 column. BIOBEADS :- Non ionic detergents (Triton X-100) can be removed by adsorption on biobeads .

ACTIVE (REMOTE) LOADING:

ACTIVE (REMOTE) LOADING Blank liposomes are prepared and drug is loaded by pH gradient Ionic gradient Amphoteric nature of the drug is required for pH gradient technique- Ex. Doxorubicin

ADVANTAGES::

ADVANTAGES : High loading (up to 95%), easily scalable to manufacturing capacity. Reduced leakage of encapsulated compounds. Flexibility for use of constitutive lipids, as the drug is loaded after the formation of carrier units. Reduced safety hazards.

CHARACTERIZATION OF LIPOSOMES:

CHARACTERIZATION OF LIPOSOMES To ensure the predictable in vitro and in vivo performances. Liposomes are generally characterized for following attributes:- Shape and lamellarity . Size and size distribution. Surface charge. Encapsulation efficiency. Phase transition behaviour .

PHYSICAL CHARACTERIZATION:

PHYSICAL CHARACTERIZATION Shape and size - Optical Microscopy, Size exclusion chromatography Entrapment efficiency- Protamine aggregation For neutral and negatively charged liposomes Protamine sol.(10 mg/ml) , centrifugation at 2000rpm, supernatant contains free drug Liposomal pellet can be disrupted using 0.6ml of 10% Triton X-100. In vitro drug release - Dialysis ( Celophane membrane)

CHEMICAL CHARACTERIZATION:

CHEMICAL CHARACTERIZATION Characterization parameters Analytical method/Instrument Phospholipid concentration Barlett assay, Stewart assay, HPLC Cholesterol concentration Cholesterol oxidase assay and HPLC Phopholipid peroxidation UV absorbance and GLC Phospholipid hydrolysis, Cholesterol auto-oxidation. HPLC and TLC Osmolarity Osmometer

BIOLOGICAL CHARACTERIZATION:

BIOLOGICAL CHARACTERIZATION Characterization parameters Analytical method/Instrument Sterility Aerobic or anaerobic cultures Pyrogenicity Limulus Amoebocyte Lysate (LAL) test Animal toxicity Monitoring survival rates, histology and pathology

APPLICATIONS OF LIPOSOMES:

APPLICATIONS OF LIPOSOMES Drug and protein delivery vehicles. Controlled and sustained release of molecules. Tumour therapy: Liposomes have natural ability to target cancer. Tumour vessels do not contain the same level of seal between cells and are diagnostically leaky . This ability is known as the Enhanced Permeability and Retention effect. Liposomes of certain sizes can rapidly enter tumour sites from the blood. Stealth liposomes ( PEGylted liposomes ). Gene delivery.

Applications::

Applications: In cosmetics and dermatology. In bioreactor technology.

LIST OF SOME MARKETED PRODUCTS:

LIST OF SOME MARKETED PRODUCTS Doxil TM or Caelyx TM Doxorubicin Kaposi ’ s sarcoma SEQUUS, USA DaunoXome TM Daunorubicin Kaposi ’ s sarcoma, breast & lung cancer NeXstar, USA Amphotec TM Amphotericin -B fungal infections, Leishmaniasis SEQUUS, USA Fungizone® Amphotericin -B fungal infections, Leishmaniasis Bristol- squibb , Netherland VENTUS TM Prostaglandin-E 1 Systemic inflammatory diseases The liposome company, USA ALEC TM Dry protein free powder of DPPC-PG Expanding lung diseases in babies Britannia Pharm , UK Topex-Br Terbutaline sulphate Asthma Ozone, USA Depocyt Cytarabine Cancer therapy Skye Pharm , USA Novasome ® Smallpox vaccine Smallpox Novavax , USA Avian retrovirus vaccine Killed avian retrovirus Chicken pox Vineland lab, USA Doxil ® Doxorubicin Hcl Refractory ovarian cancer ALZA, USA Evacet TM Doxorubicin Metastatic breast cancer The liposome company, USA VincaXome Vincristine Solid Tumours NeXstar , USA

REFERENCES:

REFERENCES ‘Targeted and controlled drug delivery, Novel carrier Systems’ , “VYAS S.P. and KHAR R.K.’’, CBS Publishers and Distributors, 4596/1-A,11 Darya Ganj,New Delhi-110002(INDIA). Page no.181 -195. ‘Controlled and Novel Drug Delivery’, “JAIN N.K.’’, CBS Publisher And Distributors, 4596/1-a Darya Ganj,New Delhi-110002 (INDIA).Page No.307-321. www.wikkipedia.com www.google.com

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