A

Seminar On

Recent trends in pharmaceutical sciences

BY: GUIDED BY:

UNMESH BHAMARE MRS. S.A. KATTI

M.PHARM-II (Q.A.)

ROLL NO.20

M.G.V.s PHARMACY COLLEGE,

PANCHAVATI, NASHIK 02.

CONTENTS:

• DEFINITION

• INTRODUCTION

• STRUCTURE COMPONENTS

• CLASSIFICATION

• PREPARATION OF LIPOSOMES

• CHARACTERIZATION OF LIPOSOMES

• STABILITY

• APPLICATIONS

• RECENT ADVANCES IN LIPOSOME PREPARATIONS

• MARKETED PRODUCTS

• REFERENCES2

LIPOSOMES:

Liposomes are cocentric bilayered

vesicles in which an aqueous

volume is entirely enclosed by

a membranous lipid bilayer mainly

composed of natural or

synthetic phospholipids.

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• Liposomes were discovered about 40 years ago by Alec Bingham.

• Liposomes can be produced from cholesterols, non toxic surfactants,sphingolipids,glycolipids,long chain fatty acids & even membrane proteins.

• Liposomes are the drug carrier loaded with great variety of molecules such as small drug molecules, proteins, nucleotides & even plasmids.

• Considerable progress was made during 1970s and 1980s in the field of liposome stability leading to long circulation times of liposomes

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STRUCTURAL COMPONENTS :

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PHOSPHOLIPID:

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• Phospholipids are major structural components of biological membranes in human body, where 2 types of phospholipids exist i.e. phosphodiglycerides & sphingolipids .

• Each phospholipid molecule has 3 major parts, 1 head & 2 tails. Head is made from 3 molecular components: choline , phosphate & glycerol which is hydrophilic.Each tail with a long chain EFA which are hydrophobic.

• Most commonly used phospholipids – PC an amphipathic molecule with a hydrophilic polar head group, phosphocholine . PC, also known as “lecithin”, can be derived from natural and synthetic sources.

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The lipid bi-layer used in the liposomes are usually made of phospholipids and cholesterol.

Following are the

A) Naturally occurring phospholipids used in liposomes are:

• Phosphatidylcholine (PC),

• Phosphatidylethanolamine (PE),

• Phosphatidylserine (PS).

B) Synthetic phospholipids used in the liposomes are:

• Dioleoyl phosphatidylcholine (DOPC),

• Distearoyl phosphatidylcholine (DSPC),

• Dioleoyl phosphatidylethanolamine (DOPE),

• Distearoyl phosphatidylethanolamine (DSPE).

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CLASSIFICATION:VESICLE TYPE ABBREVIATI

ONDIAMETER SIZE

NO. OF LIPID BI -LAYER

Unilamellar vesicle UV All size range ONE

Small unilamellar vesicle SUV 20-100 nm ONE

Medium unilamellar vesicle MUV >100µm ONE

Large unilamellar vesicle LUV >1000µm ONE

Giant unilamellar vesicle GUV >1µm ONE

Oligolamellar vesicle OLV 0.1-1µm 5

Multilamellar vesicle MLV >0.5µm 5-25

Multivesicular vesicle MV >1µm Multicompartmental structure

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FORMATION OF LIPOSOME:

When phospholipids are

dispersed in water, they

spontaneously form closed

structure with internal aqueous

environment bounded by

phospholipid bilayer membranes,

this vesicular system is called as

liposome.

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PREPERATION OF LIPOSOMES:

Passive loading Active loading

( Remote loading )

1.Mechanical dispersion method

2.Solvent dispersion method

3.Detergent removal method

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MECHANICAL DISPERSION METHODS:

• 1.SONICATION

• 2.FRENCH PRESSURE CELL

• 3.FREEZE-THAW TECHNIQUE

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SOLVENT DISPERSION METHODS1.ETHANOL INJECTION

2.ETHER INJECTION

3.REVERSE PHASE EVAPORATION VESICLES

DETERGENT REMOVAL METHODS1.DETERGENT(CHOLATE,TRITON X 100)

2.DIALYSIS

3.GEL CHROMATOGRAPHY DILUTION

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MECHANICAL DISPERSION METHOD:

I) SONICATION: 2TYPES:

1.BATH SONICATOR 2.PROBE SONICATOR

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• At high energy levels, average size of vesicles is further reduced.

• Exposure of MLV’s to ultrasonic irradiations is the most widely used method for producing small vesicles.

• As chances of contamination are likely to occur in probe sonicator, bath sonicator is widely used.

BATH SONICATOR PROBE SONICATOR

1.Large volume of diluted lipids are processed.

1.Small volume of diluted lipids are processed.

2.Less or no contamination. 2.Chances of contamination.

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II) FRENCH PRESSURE CELLS(ULV/OLV):

• Method developed by Barenholtz & Hamilton et al.

• Very useful method in which extrusion of preformed large liposomes in a French Pressure under very high pressure is carried out .

• This technique yields ULV’s/OLV’s of intermediate size(30-80nm/depending upon applied pressure).

• Liposomes are more stable.

• Free from structural defects.

• Leakage problem is also less.

• However it has high production cost. 16

III) FREEZE THAW SONICATION(FTS):

Freeze SUV dispersion

thaw at room temperature for 15 minutes

sonicate

rupture of SUV’s occur

Formation of liposomes

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SOLVENT DISPERSION METHODS

I) ETHANOL INJECTION METHODLipids ethanol

Rapidly inject through a fine needle

Saline buffer containing materials to be entrapped

dissolution of ethanol

Formation of SUV’s.

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II) ETHER INJECTION METHOD:

Lipid ether

slowly injecting through a narrow needle

vapourize temperature at 60˚C

production of SUV’s.

• Less risk of oxidation as ether is free of peroxides.

• Low efficiency.

• Long time needed for production. 19

REVERSE PHASE EVAPORATION VESICLESLipid organic solvent aqueous solution

mix

sonicate

formation of w/o emulsion

evaporate to remove the organic solvent

Lipids form a phospholipid bilayer

vigorous shaking

water droplets collapse

formation of LUV’s.20

CHARACTERIZATION:PARTICLE SIZE ANALYSIS-

• Laser light scattering, transmission electron microscopy determines the particle size & its distribution.

SURFACE CHARGE-

• Free-flow electrophoresis on a cellulose acetate plate in a sodium borate buffer pH 8.8 & a zeta potential measurement.

• The samples are applied to plate & electrophoresis is carried out at 4˚C for 30 min.

• The plate is dried and phospholipids are visualised by the molybdenum blue reagent.

• The liposomes get bifurcated based on the surface charge.21

PERCENT DRUG ENTRAPMENT-

This can be determined by ‘PROTAMINE AGGREGATE’ & ‘MINICOLUMN CENTRIFUGATION method . Expressed as %entrapment/mg lipid.

PHASE BEHAVIOUR-

Liposomes at transition temperature undergo reversible phase transition i.e the polar head groups in gel state become disordered to form the liquid crystalline state which can be determined by DSC.

LAMELLARITY-

Freeze-fracture electron microscopy & freeze-etch electron microscopy & P-NMR method.

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STABILITY OF LIPOSOMES:

A. PREVENTION OF CHEMICAL DEGRADATION:

1.Start with freshly purified lipids & freshly distilled solvents.

2.Avoid procedure which involving high temperature.

3.Carry out manufacturing in the absence of oxygen. 4.Deoxygenate aqueous solution with nitrogen.

5.Store liposome suspension in an inert atmosphere. 6.Include an antioxidant as a component.

7.Iron chelater is used to prevent initiation of free radical chain reaction.

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B. PREVENTION OF PHYSICAL DEGRADATION:

1.‘ANNEALING’ is best method to control physical degradationi.e incubating the liposomes at a temperature high enoughabove the phase transition temperature to allow differences inpacking density between opposite sides of the bilayers toequalize by trans membrane flip-flop .

2. The stability of liposomes may also be increased by crosslinking membrane component covalently using Gluteraldehydefixation, osmification or polymerization of alkyne containingphospholipids. These methods increases mechanical strengthof the membrane & render them less susceptible to disruption.

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APPLICATIONS:

1.The therapeutic value of liposomes as drug carriers, particularly for anticancer, antifungal, and antibacterial agents.

2.As anticancer , cytotoxic drugs like Cytarabine, alkylating agents .

3.As vaccine adjuvants i.e. when administered by IM route, they slowly release the antigens and accumulate in lymph nodes.

4.In ophthalmic drug delivery systems,Idoxuridine used in acute & chronic keratitis . 25

5. Sustained release system of systemically or locally administered liposomes. Ex biological proteins or peptides such as vasopressin.

6. Site specific targeting: in certain cases liposomes with surface attached ligands can bind to target cells (‘key and lock’ mechanism). Ex antineos, anti infectors & antiinflammatory drugs.

7. Improved transfer of hydrophilic, charged molecules like chelators , antibiotics, plasmids & genes into the cells.

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RECENT ADVANCES & ON GOING

CLINICAL TRIALS:

Antigens as Liposomal Preparation Applications:

• Diphtheria toxoid = Superior immunoadjuvant

• Herpes simplex virus = Enhanced Ab level

• Hepatitis B virus = Higher Ab response

• Bacterial polysaccharides = Superior immunoadjuvantsTetanus toxoids = Increased Ab titre

• Influenza subunit antigen = Intranasal, protects animal from virus

• Carbohydrate antigen = Increased Ab titre in salivary gland

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MARKETED PREPERATIONS:

• Liposome ( Doxil ™) Doxorubicin = Kaposi’ sarcoma

• Liposome (EVACT ™) = breast cancer

• Liposome(DaunoXome™) Daunosome = Advanced Kaposi’sarcoma,small cell lung cancer, leukaemia & solid tumour .

• Liposome ( VincaXome ™) Vincristine = Solid tumour

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REFERNCES:

• Targeted & Controlled Drug Delivery Novel Carrier System by S.P.Vyas & R.K.Khar,Reprint Edition(2007),CBS

PUBLISHERS & DISTRIBUTORS NEW DELHI:173-243

• http://www.nanoscalereslett.com/content/8/1/102

• http://ees.elsevier.com/ajps/default.asp

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