Presented by

G.PAVANI.

256212886011

M.Pharm-Pharmaceutics

LIPOSOMES AND NANOPARTICLES

Contents of liposomesIntroduction

Mechanism of liposome formation.

Classification.

Methods of preparation.

Characterization.

Stability.

Applications and Commercial products.

introductionDEFINITION:

Liposomes are targeted drug delivery systems consisting of one ormore concentric spheres of lipid bilayers separated by water oraqueous buffer compartments composed of natural or syntheticphospholipids .

HYDROPHOBIC

HYDROPHILIC

ADVANTAGES:

Selective passive targeting to tumour tissue.

eg: liposomal Doxorubicin.

Enhanced efficacy and therapeutic index.

Decreased toxicity.

Site avoidance effect.

Improved pharmacokinetic effects.

Flexibility to couple with site specific ligands to achieve active targeting.

DISADVANTAGES:

Less stability.

High production cost.

Phospholipid hydrolysis.

Phospholipid oxidation.

Leakage and fusion.

Allergic reaction may occur with liposomal constituents.

STRUCTURAL COMPOSITION OF LIPOSOME:

The main components are:

Phospholipids

eg: phosphatidylethanolamine.

phosphatidylcholine.

phosphatidylglycerol.

phosphatidyllinositol.

cholesterol

Generally phospholipids are represented as follows:

PHOSPHOLIPIDS:

Phospholipids are the major structural components of the

biological membrane such as cell membrane.

CHOLESTEROL:

Cholesterol by itself does not form a lipid bilayer.

However cholesterol acts as fluidity buffer.

It is incorporated in the membrane at very high concentration in the ratio up to 1:1or 2:1 molar ratios of cholesterol to PC.

MECHANISM OF LIPOSOMAL FORMATION

MECHANICAL DISPERSION:

Lipid film hydration by hand shaking/non hand shaking:

Micro-emulsification:

Sonication:

French pressure cell:

Dried reconstituted vesicles and freeze thaw sonication:

SOLVENT DISPERSION:

Ethanol and Ether injection:

Double emulsion vesicles:

The organic solution, which already contains water droplets is introduced in to excess aqueous medium followed by mechanical dispersion.

The W/O/W double emulsion is formed.

Two aqueous compartments being separated from each other by a phospholipid monolayer.

Removal of solvent results in an intermediated sized unilamellar vesicles and entrapment is up to 90%.

Reverse phase evaporation vesicles:

DETERGENT DEPLETION(REMOVAL) METHODS:

Detergents associate with the phospholipid molecules and serve to screen the hydrophobic portions of of molecule from water.

The structures formed as a result of this association is known as micelles.

A three stage model of interaction for detergents with lipid bilayers:

Stage1: At low concentration detergents equilibrates between vesicular lipid and water phase.

stage2: After reaching a critical detergent concentration, membrane structure tends to unstable and transforms gradually in to micelles.

stage3: All lipid exists in mixed micelle form.

Three methods are applied for removal of detergent and transition of mixed micelles to concentric bilayered form.

DIALYSIS:

The molecules of detergent are removed from mixed micelle by dialysis by lowering the concentration of detergent in bulk aqueous phase.

eg: sodium cholate.

octylglucoside.

COLUMN CHROMATOGRAPHY:

Removal of detergent is achieved by by passing the dispersion over a sephadexg-25 column pre-saturated with constitutive lipids and pre-equilibrated with hydrating buffer.

eg: deoxycholate.

Remote(active) loading:

Removal of unentrapped drug from liposomes:

It is important to estimate the amount of drug encapsulated within

liposomes.

This is easier to estimate by centrifugation in case of MLVs compared to LUVs and SUVs.

The various methods used to separate non-encapsulated drug are

eg: Dialysis.

Gel chromatography.

Characterization VESICLE SIZE AND SIZE DISTRIBUTION:

Microscopy .

Gel chromatography.

LAMELLARITY:

Freeze fracture and freeze-etch electron microscopy.

31 P NMR analysis.

SURFACE CHARGE:

Free flow electrophoresis

Zeta potential method.

ENCAPSULATION EFFICIENCY:

The encapsulation efficiency describes the percent of drug that becomes ultimately entrapped during preparation of liposomes.

It is expressed as %entrapment/mg lipid.

It is assessed by two techniques:

Minicolumn centrifugation.

Protamine aggregation.

TRAP PED VOLUME:

The internal or trapped volume is the aqueous entrapped volume per unit quantity of lipid and expressed as µl/mol.

The entrapped volume is determined using various materials like inert fluid-D2O, radioactive markers- 22Na or 14C and fluorescent markers- 6-carboxyfluorescein.

PHASE BEHAVIOR OF LIPOSOMES:

Membrane phospholipids undergo temperature dependent reversible phase transitions from gel to liquid crystaline state.

These have been documented by freeze fracture electron microscopy and DSC.

Tm of the phospholipid affects the membrane permeability, leakage and stability of liposomes.

Tm can be altered by using phospholipid mixtures or by adding cholesterol.

INVITRO DRUG RELEASE

It is done by dialysis bag.

CHEMICAL CHARACTERIZATION:

PARAMETERS ANALYTICAL METHOD

Phospholipid concentration Barlett assay/stewart assay, HPLC

Cholesterol concentration Cholesterol oxidase assay,HPLC

Phospholipid hydrolysis HPLC,TLC and Fatty acid concentration

Phospholipid oxidation UV and GLC

STABILITY

STABILITY in vitro:

Lipid oxidation and peroxidation

Lipid hydrolysis

Long term and Accelerated stability.

STABILITY in vivo:

Stability after systemic administration.

Stability after oral administration.

MODES OF LIPOSOME AND CELL INTERACTION:

Adsorption Endocytosis

Fusion Lipid transfer

APPLICATIONSLiposomes as drug or protein delivery vehicles.

Liposome in antimicrobial, antifungal(lung therapeutics) and antiviral (anti HIV) therapy.

In tumour therapy.

In gene therapy.

In immunology.

Liposomes as artificial blood surrogates.

Liposomes as radiopharmaceutical and radiodiagnostic carriers.

Liposomes in cosmetics and dermatology.

COMMERCIAL PRODUCTS

Doxorubicin(DOXIL).

Daunorubicin(DAUNOXOME).

AmphotericinB(APHOTEC, AMBISOME, ABELCET).

Cytarabine(DEPOCYTE).

CONTENTS OF NANOPARTICLESIntroduction

Preparation methods.

Novel nanoparticulate system.

Surface engineering of nanoparticles.

Characterization.

Applications.

Commercial products.

INTRODUCTIONDEFINITION:

Nanoparticles are sub- nano sized colloidal structures composed of synthetic or semi synthetic polymers with a size smaller than 1mm.

The term nanoparticles include

NANOSPHERE NANOCAPSULE

ADVANTAGES:

Easy manipulation of Particle size and surface characteristics.

They control and sustain release of the drug.

Drug loading is relatively high.

Incorporation of drug without any chemical reaction.

Site-specific targeting(ligands)

Administrations including oral, nasal, parentral, intra-ocular etc

DISADVANTGES:

High cost

Productivity more difficult

Reduced ability to adjust the dose

Highly sophisticated technology

Requires skills to manufacture

POLYMERS:

Natural polymers:

Synthetic polymers:

PROTEINS POLYSACCHARIDES

Gelatin Alginate

Albumin Dextran

Lectins Chitosan

Legumin Agarose

vicilin pullulan

PRE- POLYMERIZED POLYMERIZED IN PROCESS

Poly(Ɛ-caprolactone) Poly(isobutylcyanoacrylates)

Poly lactic acid Poly(butylcyanoacrylate)

Ploy(lactic-co-glycolide) Polyhexylcyanoacrylate

polystyrene Poly methyl methacrylate

METHODS OF PREPARATION

Amphiphilicmacromolecule

crosslinking

Polymerization based methods

Ploymerprecipitation

methods

•Heat cros-linking.•Chemical cross-linking

•Polymerization of monomers.•Emulsion polymerization.•Dispersion polymerization.•Interfacial condensation polymerization.•Interfacial complexation.

•Solvent extraction/evaporation.•Solvent displacement or nanoprecipitation.•Salting out.

MACROMOLECULAR CROSS LINKING IN A WATER IN

OIL EMULSION:

aAqueous

protein(BSA)

O

oil

W/O emulsion

Dilution with preheated oil (100oC)

(Heat cross-linking)

Or Addition of crosslinking agent

(Chemical cross-linking)

Centrifugation and isolation of

nanoparticles

or hEmulsification using high-

pressure homogenization/ high

frequency sonication

surfactant

POLYMERIZATION OF MONOMERS:

w/o emulsion.

Water+ monomer A Oli phase

Nanocapsules.

High pressure homogenization

Monomer B

MICELLAR PLOYMERIZATION MECHANISM:

HOMOGENOUS POLYMERIZATION MECHANISM:

DISPERSION POLYMERIZATION:

lsolation of nanospheres

Oligomers aggregate &

precipitates

Further, By chemical initiation

(ammonium or potassium per oxo disulphate)

(Acrylamide or Methyl methacrylate) Monomer is dissolved

in an aqueous medium

Heated to above 65 C

47

INTERFACIAL POLYMER CONDENSATION:

o/w emulsion.

Ploymer phaseCore phase + drug

Nanocapsules.(30-300nm)

Non-solvent which precipitate

polymer from either of the phases

INTERFACIAL COMPLEXATION:

nanoparticles.

Reverse micellePolyelectrolyte.

Competing polyelectrolyte

Polymer complexation

SOLVENT EVAPORATION METHOD:

Organic phase solvent,

drug, polymer.Aqueous phase distilled

water, stabilizer.

o/w emulsion

Nanoparticles.

Sonication, homogenization

Solvent extraction, solvent evaporation.

Double emulsion solvent evaporation method:

Organic phase solvent,

drug, polymer.

Aqueous phase

distilled water,

Stabilizer.

w/o emulsion

stabilized at 4oc

w/o/w emulsion.

Nanoparticles.

Sonication, homogenization

Aqueous phase with stabilizer

Solvent extraction, solvent evaporation

SOLVENT DISPLACEMENT METHOD:

Distilled water,

polaxamer 188

Distilled water,

polaxamer

188

Organic solvent,

polymer, drugPolar solvent, oil,

polymer, drug.

Nanospheres. Nanocapsules.

Magnetic stirring

SALTING OUT:

Nanoparticle.

Distilled water,

PVA, MgCl2

Organic solvent,

drug, polymer.

o/w emulsion.

Mechanical stirring

Distilled water

PHARMACEUTICAL ASPECTS OF NANOPARTICLES:

Purification:

Dialysis.

Gelfiltration.

Ultra-centrifugation.

Freeze drying.

Sterilization.

NOVEL NANOPARTICULATE SYSTEM

SOLID LIPID NANOPARTICLES:

Solid lipid nanoparticles are sub micron colloidal carriers composed of physiological lipid dispersed in water or in aqueous surfactant solution.

Advantages:

SLN Can be lyophilized as well as spray dried.

No toxic metabolites are produced.

Avoidance of organic solvents.

COPOLYMERIZED PEPTIDE NANOPARTICLES:

It is a drug polymer conjugate which forms its own nanoparticulate drug delivery system.

eg: n-butylcyanoacrylate

HYDROGEL NANOPARTICLES:

Hydrogel nanoparticles are formed in the water by self-assemblage and self aggregation of natural polymer amphiphiles.

eg:cholesteroyl dextran.

cholesteroyl pullan.

NANOCRYSTALS AND NANOSUSPENSIONS:

Nanocrystals are produced by dispersion of the drug powder in the surfactant solution and subjecting to the pearl milling process.

Nanosuspensions are produced by dispersion of drug powder in aqueous surfactant solution by high speed stirring which is then passed through high speed homogenizer leading to formation of nanosuspension.

SURFACE ENGINEERED NANOPARTICLES

STEARICALLY STABILIZED (STEALTH) NAOPARTICLES:

The surface modification of particulate carriers is achieved by coating or grafting to nanoparticle surface with certain materials that impart stealth behaviour.

eg:Polaxamers.

Polaxamines.

BIO-MIMETIC NANOPARTICLES:

These are the nanoparticles coated with endogenous serum components which prevent their uptake through mononuclear phagocytic system.

eg:Albumin.

Orosomucoid.

NANOPARTICLES COATED WITH ANTIBODIES:

Anchoring of targeted specific antibodies to the nanoparticle surface may facilitate the delivery to specific sites.

eg: Monoclonal antibodies.

MAGNETIC NANOPARTICLES:

Nanoparticles are rendered magnetic by incorporating Fe3O4

simultaneously with drug during the preparation stage, injected through the artery is guided by the external magnet inorder to target the site.

BIOADHESIVE NANOPARTUCLES:

Drugs are associated to polymeric bioadhesive nanoprticulate system as they adhere to the mucosal surface provide better drug absorption.

eg: lectin.

CHARACTERIZATIONPARTICLE SIZE AND SIZE DISTRIBUTION:

Photon correlation spectroscopy.

Scanning electron microscopy.

Transmission electron spectroscopy.

SURFACE CHARGE:

The surface charge of nanoparticle is determined by measuring the particle velocity in an electric field.

Laser Doppler Anemometry.

SURFACE HYDROPHOBICITY:

Hydrophobic interaction chromatography.

Two phase partitioning technique.

MOLECULAR WEIGHT OF POLYMER CARRIER:

Different ways to express molecular weight is number average weight (Mn) and weight average molecular weight (MW).

gel permeation chromatography.

PHYSICOCHEMICAL STATE OF DRUG AND POLYMER:

Thermal analysis.

X-ray Diffraction.

DRUG LOADING:

Ultra centrifugation.

Ultra filtration.

Gel filtration.

DRUG INCORPORATION EFFICIENCY:

IN VITRO DRUG RELEASE:

PH 1.2 HCl buffer.

Phosphate buffer PH 5.

Phosphate buffer PH 6.8

APPLICATIONSNanoparticles in chemotherapy.

Nanoparticles in intracellular targeting.

Nanoparticles for ocular and brain delivery.

Nanoparticles for DNA delivery.

Nanoparticles for oligonucleotide delivery.

Nanoparticles for lymph targeting.

Nanoparticles for peroral administration of protein and peptide.

COMMERCIAL PRODUCTSDaunoxomeR - Anticancer.

GendicineR – Gene delivery.

AmphoteR – Antifungal.

.

AmbraxaneR – Anticancer.

DoxilR - Anticancer

Nanoxel.

CONCLUSIONLiposome over the years have been investigated as major drug

delivery system.

The use of liposomes in delivery of drugs and genes to tumour site are promising and may serve as a handle for focus of future research.

Nanoparticles are have been used as a drug carrier in transdermal formulations to enhance absorption of therapeutic agents.

With advent of novel nanoparticulate systems the nanoparticulate technology seems to dominate the field of drug delivery and drug targeting in future.

REFERENCE

Deepak Thassu, Michel Deleers, “Nanoparticulate Drug delivery systems,” vol-166. pg.no:89.

Herbert A.Lieberman, Martin M.Rieger and Gilbert S.Banker, “Pharmaceutical dosage forms: Disperse systems,” vol-3. 2nd edition. Pg.no:43-83 & 87-119.

S.P.Vyas, R.K.Khar, “ Targeted & Controlled Drug Delivery,”

edition-2006. pg.no:173-243 & 331-381.

Nepolean R, Narayanan.N, “Journal of pharmaceutical sciences and technology,” academic reaserch publication. Vol-4.pg.no:83-84.

Mohamed Badran, Abdelaziz Elsayed, “Asian journal of

pharmaceutical & Health science,” vol-3. pg.no:640-641.