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liposomes

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PRESENTED BY: Vikas Bhatt M.Pharm 2 nd Sem SEMINAR ON LIPOSOMES SEMINAR ON …….. Department Of Pharmaceutic Devsthali Vidyapeeth College of Pharmacy,Rudrapur

Tahoma:

INTRODUCTION The preparation of liposomes, with entrapped solutes, was demonstrated for the first time in 1965 by Prof. A.D. Bangham of the United Kingdom.

Calibri:

What are liposomes ? Liposome is Greek word - lipo means “ fat ” - soma means “ body ” These are the micro-particulate drug carriers consisting of one or more concentric spheres of lipid bilayer separated by aqueous buffer compartments. When phospholipids are dispersed in water they spontaneously form closed structures with internal aqueous compartments bounded by phospholipid bilayer membranes. Their diameter ranges from 80 nm to 100 u m

Times New Roman:

General structure of liposome . Lipid bilayer

Wingdings:

“Liposomes are microscopic spheres made from fatty materials, predominantly phospholipids. “made up of one or more concentric lipid bilayers, and range in size from 50 nanometers to several micrometers in diameter”

Monotype Corsiva:

Advantages with liposomes Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents Chemically and physically well characterized entities Biocompatible Suitable for controlled release Suitable to give localized action in particular tissues. Suitable to administer via various routes

Vivaldi:

Disadvantages:- 1) Less stability 2) Low solubility 3) Short half life 4) Phospholipid undergoes oxidation, hydrolysis 5) Leakage and fusion 6) High production cost 7) Quick uptake by cells of R.E.S 8) Allergic reactions may occur to liposomal constituents 9)Problem to targeting to various tissue due to their large size

Blueprint:

Classification Classification based on size of liposomes Classification based on method of preparation Classification based on composition and in vivo application

Custom Design:

Classification of LIPOSOMES DEPENDING ON STRUCTURAL PARAMETERS TYPE FORM SIZE MLV Multi-lamellar large vesicle >0.5um OLV Oligo-lamellar vesicles 0.1-1um ULV Unimellar vesicle All size range SUV Small unimellar vesicle 20-100nm MUV Medium sized unimellar vesicle _ LUV Large unimellar vesicles >100nm

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Classification based on method of preparation Vesicles prepared by extrusion method. Vesicles prepared by French press. Vesicles prepared by fusion. Vesicles prepared by reverse phase evaporation. Frozen and thawed MLV. Dehydration and rehydration vesicles. Stable plurilamellar vesicles.

LIPOSOMES :

Classification based on specific properties Conventional Liposomes

INTRODUCTION:

Long circulating liposomes (Stealth Technology) PEG coating Low permeability liquid matrix and internal aqueous buffer system

What are liposomes ?:

Targeted liposomes Target specific ligands, such as antibodies, immunoglobulins, lectins and oligosaccharides attached to the surface

General structure of liposome:

Cationic Liposomes Cationic lipid component interact with negatively- charged DNA Results into Lipid –DNA Complexes

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Temperature sensitive liposome PH sensitive Liposomes

Advantages with liposomes:

MANUFACTURING OF LIPOSOMES

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Materials used in preparation of liposomes Phospholipids Synthetic Phospholipids Glycerolipids Sphingolipids Glycosphingolipids Steroids Polymeric material Charge-inducing lipids

Classification:

Materials used in liposome preparation :- PHOSPHOLIPIDS Glycerol containing phospholipids are commonly used component of liposome formulation. The most abundant glycerol phosphotides in plants and animals are phosphotidyl choline(lecithin) and phosphotidyl ethanol amine(cephaline). These two are major structural components of most biological membranes. Fatty acids are also important constituent of glycerol phosphotides(triglycerides of fat cells).

Classification of LIPOSOMES:

LIPOOMES CONTAINING PHOSPHOLIPID

Classification based on method of preparation:

Structure of phospholipids

Classification based on specific properties:

2. SPHINGOLIPIDS They contain sphingosine (or) a related base as their structural backbone. They contain 3 characteristic building blocks:- - A molecule of fatty acid - A molecule of sphingosine & - Head groups that can vary from simple alcohol as choline to very complex carbohydrates. The most abundant sphingolipid in higher animals is sphingomyelin.

Long circulating liposomes (Stealth Technology) :

3. GLYCOSPHINGOLIPIDS These are found mainly in grey matter of brain tissue of higher animals and are used in minor component. These are included in liposomes formulation to provide a layer of surface charged groups. STEROLS Cholesterol & its derivatives are quite often included as components of liposomal membrane.

Targeted liposomes:

Cholesterol can be added to bilayer mixture for the following purposes :- - Acts as a fluidity buffer. - Acts as intercalator with phospholipid molecules. - Decreases the permeability of membrane to water soluble molecules. Liposomes without cholesterol are known to interact rapidly with plasma proteins like albumin , transferin and macroglobulins which lead to physical instability of liposomes.

Cationic Liposomes:

5. SYNTHETIC PHOSPHOLIPIDS Saturated phospholipids include dipalmitoyl phosphatidyl choline(DPPC) , dipalmitoyl phosphatidyl ethanol amine(DPPE) , dipalmitoyl phosphatidyl serine(DPPS) . Unsaturated phospholipids have also been used which include dioleo-phosphatidyl choline(DOPC) , dioleo-phosphatidyl glycerol(DOPG)

Temperature sensitive liposome:

6. POLYMERIC MATERIALS A large variety of polymerizable lipids which can form vesicles , has been synthesized. These include lipids containing conjugated diene , methacrylate , and thiol groups as polymerizable moieties.

MANUFACTURING OF LIPOSOMES:

Mechanisms by which liposomes act :- There are several mechanisms by which liposomes act which are as follows : Liposomes attaches to plasma membrane and appears to fuse with them,releasing their content into cell. Liposomes are taken up by cell in some cases and their phospholipids are incorporated into plasma membrane by which drug trapped inside is released. In case of phagocyte cell, the liposomes are taken up, the phospholipid walls are acted upon by organelles called lysosomes and the drug is released.

Materials used in preparation of liposomes:

Drug release from liposomes The lipid bilayer of the liposome can fuse with other bilayers (e.g. cell membrane) thus delivering the liposome contents.

Materials used in liposome preparation :-:

Outside of the cell. Liposome Inside of cell. Inside of cell. Drug releasing into cell Drug content released into the cell Cell membrane Cell membrane Phospholipids of liposome are incorporated into cell membrane

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Preparation of Liposomes Mechanism of Vesicle Formation The budding theory The bilayer phospholipids theory

Structure of phospholipids:

The budding theory Stress induced hydration of phospholipids Organization in to lamellar arrays Results in to budding of lipid bilayer leading to down sizing SUV OLV

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The bilayer phospholipids theory Liposomes are formed when thin lipid films are hydrated The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar vesicles (LMV)

3. GLYCOSPHINGOLIPIDS:

Method of Liposome Preparation

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Conventional liposome preparation methods Phospholipids Cholesterol Antioxidant Lipid component compounding Lipid solvent Pyrogen Ultrafilter yes No Filter Solvent removal Drug ,Salt Antioxidant Buffer WFI Filter Hydration Solvent recovery Extrusion Down sizing Free drug removal Prefilter Sterile filter Vial filling Free drug recovery Aseptic processing Lyophollization Seal / package

5. SYNTHETIC PHOSPHOLIPIDS:

Method for preparation of (S U V ) Sonication High shear fragmentation Solvent injection method Injection of water immiscible solvent. Ether infusion. Fluorocarbon injection. Injection of water miscible solvent. Ethanol injection Modified ethanol injection method

6. POLYMERIC MATERIALS:

Sonicated Unilamellar Vesicles: PROBE SONICATOR: The probe is employed for dispersions, which require high energy in a small volume(e.g., high concentration of lipids, or a viscous aqueous phase) BATH SONICATOR: The bath is more suitable for large volumes of diluted lipids. - Method: Placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-10min(1,00,000g) which yield a slightly hazy transparent solution. -Using centrifugation to yield a clear SUV dispersion.

Drug release from liposomes:

Bath sonicator Probe sonicator

PowerPoint Presentation:

French pressure cell The method involves the extrusion of MLV at 20,000 psi at 4°C through a small orifice The resulting liposomes are larger than sonicated SUVs The drawbacks of the method are that the temperature is difficult to achieve and the working volumes are relatively small (about 50 mL maximum)

Preparation of Liposomes:

High shear fragmentation Aqueous samples Piston Cell body Rubber-O-ring Closure plug Pressure relief valve Outlet Fig. French pressure cell

The budding theory:

French Pressure Cell

The bilayer phospholipids theory:

Solvent injection method. Vacuumpump Mix Gasket Ether/lipid solution Infusion pump Aqueous phase Mechanical drive Temperature Controlled bath

Method of Liposome Preparation:

Reverse phase evaporation technique. Lipid in solvent solution Two-phase system Water in oil emulsion Solvent removal Gel formation REV liposomes

Conventional liposome preparation methods :

4.Reverse Phase Evaporation Vesicles

Method for preparation of (S U V ):

High pressure extrusion.

PowerPoint Presentation:

Preparation of Liposomes by dry film method Lipids and drug dissolved in CHCl 3 and evaporated to form thin film Film is hydrated with buffer solution Sonicated to form large unilamellar vesicles

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Preparation of Liposomes by dry film method Lipid + drug + CHCl 3 Rotary evaporation Sonication Thin film LUV

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Hand shaken method in general

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METHODS FOR CONTROLLING THE PARTICLE SIZE & SIZE DISTRIBUTION OF LIPOSOMES FRACTIONATION HOMOGENIZATION CAPILLARY PORE MEMBRANE EXTRUSION

High shear fragmentation:

1. FRACTIONATION:- CENTRIFUGATION: Liposomes sediment in a centrifugal field at a rate that is dependent on their size and density. Disadvantage liposomes smaller than about 0.5 u m tend to require high forces and long spinning times SIZE EXCLUSION CHROMATOGRAPHY : It is particularly useful for separation of small unilamellar vesicles from large structures.

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2. HOMOGENIZATION: By using high pressure homogenizers like micro fluidizer particle size can be reduced. 3. CAPILLARY PORE MEMBRANE EXTRUSION : It is wide spread , simple technique for production of defined & narrow sized liposomes. The extrusion of heterogenous population of fairly large liposomes through polycarbonate membranes under reduced pressure gives uniform particle sized liposomes

Solvent injection method.:

CHARACTERIZATION OF LIPOSOMES WITH THEIR QUALITY CONTROL ASSAYS a) BIOLOGICAL CHARACTERIZATION CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS STERILITY AEROBIC AND ANEROBIC CULTURE PYROGENICITY RABBIT FEVER RESPONSE ANIMAL TOXICITY MONITORING SURVIVAL RATS

Reverse phase evaporation technique.:

b) CHEMICAL CHARACTERIZATION CHARACTERIZATION PARAMETERS INSTRUMENT FOR ANALYSIS PHOSPHOLIPID CONCENTRATION HPLC/BARRLET ASSAY CHOLESTEROL CONCENTRATION HPLC/CHOLESTEROL OXIDE ASSAY DRUG CONCENTRATION ASSAY METHOD ANTI-OXIDANT DEGRADATION HPLC/TLC P H PH METER OSMOLARITY OSMOMETER PHOSPHOLIPIDS HYDROLYSIS HPLC/TLC CHOLESTEROL OXIDATION HPLC/TLC PHOSPHOLIPIDS OXIDATION UV ABSORBENCE

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c) PHYSICAL CHARACTERIZATION CHARACTERIZATION PARAMETER INSTRUMENT FOR ANALYSIS VESICLE SHAPE & SURFACE MORPHOLOGY TEM & SEM VESICLE SIZE AND SIZE DISTRIBUTION DYNAMIC LIGHT SCATTERING & TEM SURFACE CHARGE FREE FLOW ELECTROPHORESIS ELECTRICAL SURFACE POTENTIAL & SURFACE P H ZETA POTENTIAL MEASUREMENT AND P H SENSITIVE PROBES LAMELLARITY P 32 NMR PERCENT CAPTURE MINI COLUMN CENTRIFUGATION & GEL EXCLUSION DRUG RELEASE DIFFUSE CELL / DIALYSIS

High pressure extrusion.:

Applications Liposomes as Protein Carriers in Immunology Oral Drug Delivery Site Specific Delivery Sustained or Controlled Delivery Gene Therapeutics

Preparation of Liposomes by dry film method:

Applications

Preparation of Liposomes by dry film method:

APPLICATIONS OF LIPOSOMES LIPOSOMES IN RESPIRATORY DRUG DELIVERY SYSTEM : ISONIAZID & RIFAMPICIN - improved the effect of drugs for tuberculosis. CYCLOSPORINS - preferentially absorbed by lungs & show sustained release LIPOSOMES AS VACCINE ADJUVANTS: RABIES GLYCOPROTEINS - interleukin 2 enhancement CHOLERA TOXIN - enhanced antibody level

Hand shaken method in general:

LIPOSOMES FOR BRAIN TARGETING Addition of sulphatide group to liposome composition increases their ability to cross blood-brain barrier. Liposomes coated with mannose was found to reach brain tissue easily. LIPOSOMES AS ANTI-INFECTIVE AGENTS Active targeting approach - pentamidin and anamycin for leishmaniasis Passive targeting approach - gentamycin for staphylococcal pneumoniasis

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LIPOSOMES IN TUMOUR THERAPY DOXARUBICIN - For the treatment of refractory tumour and breast cancer. VINCRISTINE - for the treatment of solid tumour.

1. FRACTIONATION:-:

Ophthalmic drug delivery- The efficacy of liposome depends on Drug encapsulation efficiency Size and charge of vesicles Stability of liposomes after installation Residence time of liposomes in conjuctival sac . Affinity of liposomes to the cornea Eg.entrapping Idoxuridin in liposomes

2. HOMOGENIZATION::

Approved liposome products marketed in US Doxil Daunorubicin Alza Corporation Kopasi sarcoma Daunoxome Daunorubicin Gilead sciences ,, Ambisome Amphotericin B ,, Serious fungal infection Approved lipid complex products Ambelcet Amphotericin B Alza corporation Amphotec Amphotericin B Elan corporation

CHARACTERIZATION OF LIPOSOMES WITH THEIR QUALITY CONTROL ASSAYS a) BIOLOGICAL CHARACTERIZATION :

References Target and Controlled Drug delivery – Novel Carrier Systems by S.P.Vyas and R.K.Khar . Controlled and Novel Drug Delivery Systems by Sanjay K. Jain and N.K.Jain . Biopharmaceutics and pharmacokinetics a treatise by D.M.Bramhankar and sunil B. jaiswal . Liposomes preparation methods by Mohammad riaz ,Pakistan Journal of Pharmaceutical Sciences Vol.19(1), January 1996, pp.65-77 Liposome- as drug carriers-International Journal of Pharmacy & life sciences- Himanshu Anwekar *, Sitasharan Patel and A.K Singhai http://www.wikipedia.com/liposomes http://www.avantilipids.com

b) CHEMICAL CHARACTERIZATION:

References Encyclopaedia : Pharmaceutical Technology Vol 9 Pg No . 1 – 38 . Indian Journal of Pharmaceutical Sciences : Jan-Feb 2004 Pg 121-122 . Wikipidia .com. Youtube.com

c) PHYSICAL CHARACTERIZATION:

THANK YOU