LIPOSOMES AND NANOTECHNOLOGY

PRESENTED BY:

MAHESH DABHI

SEMINAR ON

LIPOSOMES AND

NANOTECHNOLOGY

SEMINAR ON ……..

Department Of Pharmaceutics and Pharmaceutical Technology,L.M. College of Pharmacy.

List of contents

o Introduction:

o Advantages with use of liposomes as drug delivery

system.

o Classification

o Manufacturing of liposomes

o Liposome characterization and control

o Stability consideration for liposomal formulations

o Regulatory science of liposome drug products

o Drug release from liposomes

o Applications

o Recent innovations

o Approved liposome products

o References

INTRODUCTIONo The preparation of liposomes, with

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

Definitiono “Liposomes are microscopic spheres made from

fatty materials, predominantly phospholipids.

o “made up of one or more concentric lipid bilayers, and range in size from 50 nanometers to several micrometers in diameter”

Advantages with liposomes

o Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents

o Chemically and physically well characterized entities

o Biocompatible

o Suitable for controlled release

o Suitable to give localized action in particular tissues.

o Suitable to administer via various routes

Classification

o Classification based on size of liposomes

o Classification based on method of preparation

o Classification based on composition and in vivo application

Classification based on sizeSmall unilamellar vesiclesMedium sized unilamellar vesiclesLarge unilamellar vesiclesGiant unilamellar vesiclesUnilamellar vesiclesOligolamellar vesiclesMultilamellar large vesiclesMultivesicular vesicles

Classification based on method of preparation

o Vesicles prepared by extrusion method.o Vesicles prepared by French press.o Vesicles prepared by fusion.o Vesicles prepared by reverse phase

evaporation.o Frozen and thawed MLV.o Dehydration and rehydration vesicles.o Stable plurilamellar vesicles.

Classification based on specific properties

o Conventional Liposomes

Long circulating liposomes (Stealth Technology)

o PEG coating o Low permeability liquid matrix and

internal aqueous buffer system

Targeted liposomeso Target specific ligands, such as

antibodies, immunoglobulins, lectins and oligosaccharides attached to the surface

Cationic Liposomeso Cationic lipid component interact with

negatively- charged DNAo Results into Lipid –DNA Complexes

Temperature sensitive liposome

PH sensitive Liposomes

MANUFACTURING OF

LIPOSOMES

Materials used in preparation of liposomes

o Phospholipidso Synthetic Phospholipidso Glycerolipids o Sphingolipidso Glycosphingolipidso Steroidso Polymeric materialo Charge-inducing lipids

Structure of phospholipids

Issues to consider when selecting lipids.

o Phase transition temperatureo Stabilityo Chargeo Lipid mixtureso Cholesterolo Source

Preparation of Liposomes

Mechanism of Vesicle Formation

o The budding theoryo The bilayer phospholipids theory

The budding theoryo Stress induced hydration of phospholipidso Organization in to lamellar arrayso Results in to budding of lipid bilayer leading to down

sizing

SUV OLV

The bilayer phospholipids theory

o Liposomes are formed when thin lipid films are hydratedo The hydrated lipid sheets detach during agitation and self-

close to form large, multilamellar vesicles (LMV)

Method of

Liposome Preparation

Conventional liposome preparation methods

PhospholipidsCholesterol Antioxidant

Lipid component compounding Lipid solvent

Pyrogen Ultrafilteryes

No

Filter

Solvent removal

Drug ,Salt Antioxidant Buffer WFI

Filter

HydrationSolvent recovery

Extrusion Down sizing

Free drug removal

Prefilter

Sterile filter

Vial filling

Free drug recovery

Aseptic processingLyophollization Seal / package

Method for preparation of (SUV)

o Sonicationo High shear fragmentationo Solvent injection method

o Injection of water immiscible solvent.o Ether infusion.o Fluorocarbon injection.

o Injection of water miscible solvent.o Ethanol injectiono Modified ethanol injection method

High shear fragmentation

Aqueous samples

Piston

Cell body

Rubber-O-ring

Closure plug

Pressure relief valve

OutletFig. French pressure cell

Solvent injection method.

Vacuumpump

Mix

Gasket

Ether/lipidsolution

Infusion pump

Aqueousphase

Mechanical drive

TemperatureControlledbath

Large and Intermediate sized unilamellar vesicles.

o Methods used to prepare LUV and IUVo Detergent dialysiso Water in oil emulsion techniqueo Freeze thaw cyclingo Slow swelling in non electrolyteso Dehydration followed by rehydrationo Dilution or dialysis of lipids in the presence

of chaotropic ions.

Reverse phase evaporation technique.

Lipid in solvent solution

Two-phase system Water in oil emulsion

Solvent removalGel formationREV liposomes

High pressure extrusion.

Methods for controlling liposome size

o Fractionationo Centrifugationo Size exclusion chromatography

o Homogenization

o Capillary pore membrane extrusion

o Ceramic extrusion

Liposome characterization and control

Liposomes

Size Number of lamellae

Charge Stability

Preparation Raw materials

Protection

Sizing method

Hydration methods

Degree of saturation

Head group

Presence of sterols

Protecting

agents

Characterized by

Determined by

Classified by

Physical characterization parameters

o Mean size and size distribution

o Number of lamellae

o Osmotic behavior and entrapped volume

o Internal distribution of drug

o Structural and motional behavior of lipids

o Electrical surface potential & Surface PH

o Stabilization aspect for physical instability of liposomes

o Chemical stability

o Biological stability of liposomes

Stability consideration

Regulatory aspectso Safety concerns: liposome

formulationo Lipid toxicity (RBC lysis)o Presence of protein and lipoprotein for

natural lipidso Residual solvento Overload of RES o Particle size

o (tail above 1 um) - Blockage of capillarieso Size affects RES uptake and tissue targeting

o Stability: shelf-live and in vivoo Dose dumping (via protein binding)o Sterility

Drug release from liposomes

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

Liposome Performance – In Vitro Release and Stability

o In vitro drug release from liposomal systems was determined using dialysis sacks.

o Release test for a targeted liposome would need to show that liposome is stable until uptake at the site.

Factors affecting release of drug

o Solventso pHo Temperatureo Agitationo Enzymeso Cell cultureo Sink conditionso Volumeo Sampling interval

Applications

o Liposomes as Protein Carriers in Immunology

o Oral Drug Delivery

o Site Specific Delivery

o Sustained or Controlled Delivery

o Gene Therapeutics

Applications

Innovations in vesicular drug delivery systems

o Provesicles in drug delivery systems

o Provesicles in drug delivery systemso Proliposomes :-o Dry granular liposomeso Mixed micellar proliposomeso Protransferosomes

Characterization of provesicular system

o Morphology o Angle of reposeo Size and size distributiono Rate of hydration o Entrapment efficiencyo Degree of deformability and

permeability measuremento In vitro release rateo In vivo fate and pharmacokinetic

Lipopolyplexes

o A combination of DNA, polymers and liposomes

o This method has resulted in better gene transfer and lower toxicity as compare to cationic liposomes

Transferosomes

o Modified liposomes developed to increase the transdermal permeation of drug

o Deformability is achieved by using surface active agent in proper ratio

o Concentration of surfactant is very crucial

Ethosomes

o Composed of phospholipids & alcohol ( ethanol or IPA)

o Sometimes polyols or glycols in relatively high concentration & water

o Better membrane permeability

o Discomes

o Virosomes

o Emulsomes

Cochleateso Cochleates are cigar-like microstructures

o Consist of a series of lipid bilayers which are formed as a result of the condensation of small unilamellar negatively charged liposomes.

Depofoam technology

o Depofoam particles include hundred of bilayer enclosed aqueous compound.

o Formed by first emulsifying a mixture of an aq phase containing the compound to be encapsulated & an organic phase containing lipid.

Niosomes

o Nonionic surfactant vesicles(NSV)

o Niosomes are formed from the self assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer.

Preparation of Liposomes by dry film method

o Lipids and drug dissolved in CHCl3

and evaporated to form thin film

o Film is hydrated with buffer solution

o Sonicated to form large unilamellar vesicles

Preparation of Liposomes by dry film method

Lipid + drug + CHCl3

Rotary evaporation

Sonication

Thin film

LUV

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

NANOTECHNOLOGY IN

DRUG DELIVERY

List of contents

o INTRODUCTION

o NANOPARTICLES

o History

o Types of nanoparticles

o Materials and methods

o Method of preparation for Nanoparticles

o Drug loading into nanoparticles

o NANOSUSPENSION

o Method of preparation for Nanosuspension

o Purification of Nanosuspension

o CHARACTERIZATION AND EVALUATION OF

o NANOPARTICLES AND NANOSUSPENSION.

o APPLICATIONS OF NANOPARTICLES AND NANOSUSPENSIONS

o NANOSUSPENSION BASED FORMULATION IN DEVELOPMENT AND IN THE MARKET

INTRODUCTION

o NANOSUSPENSIONS : o are colloidal dispersions of nano

sized drug particle that are produced by suitable method and stabilized by suitable stabilizer .

o NANOPARTICLES : o They are solid colloidal particles

sized from 1-1000 nm .

o NANOSPHERES : o polymer matrices in which drug is

dissolved or dispersed .

o NANOCAPSULES : o consists of polymer wall entrapping

an oily core in which the drug is dissolved

History

o Developed by spieser and co-workers in the late 1960s.

o In early 1970s the cross linked polyacrylamide nanoparticles were produced.

o Widder at el incorporated magnetic particles into the nanoparticles for targeting of these particles by means of magnetic field.

Types of nanoparticles

o Monolithic type o Capsule type

Monolithic type Capsule type

Polymers used in preparation.

o Polymethyl methacrylate copolymers,

o Polymethyl cyanoacrylate,

o Polybutyl cyanoacrylate & Polyisobutyl cyanoacrylate,

o Polyhexyl cyanoacrylate & polyisohexyl cyanoacrylate,

o Poly(Nα,N-L-lysinedylterephthalamide

o Polyisobutyl cyanoacrylate

o Poly-D,L-lactide

o Serum albumin

o Gelatin

o Polyacryl dextran

o Polyacryl starch

o Albumin

o Polylactic acid-poly glycolic acid copolymer

o Poly(-hydroxy butyrate)

o Ethyl cellulose

o Eudragit RL,RS

Method of preparation for Nanoparticles

o Emulsion polymerization

o Continuous aqueous phase o One of the most rapid method

o The monomer is dissolved in an aqueous phase

o The polymerization process is initiated by -radiation, UV, or strong visible light

o Continuous organic phase

Denaturation of natural molecules in an oil emulsion.

o Requires emulsification of a natural macromolecule and the drug

o The particles are hardened by oHeat denaturation, oCooing below the gelation point, oCross-linking with suitable reagent.

o Disolvation of macromolecules.o This process is commonly known as

coacervation.

o Solvent evaporation

Drug loading into nanoparticles

o Drug may be loaded at two stages-

o Prior to the preparation of nanoparticles

o Addition to the previously prepared particles

NANOSUSPENSION

o Colloidal dispersions of nanosized drug particle produced by suitable method and stabilized by suitable stabilizer .

Method of preparation for Nanosuspension

o Media milling (Nanocrystals)

Milling chamber

Milling Shaft

Milling media

Screen retaining milling media in chamber

Drug, water & stabilizer

High Pressure Homogenizers

o Size reduction depends upon:

o Homogenization pressureo No. of homogenization cycle

CHARACTERIZATION AND

EVALUATION

Physicochemical Characterization

o Particle sizeo Molecular Weight Densityo Crystallinityo Surface chargeo Hydrophobicity o Surface propertieso Surface element analysis

Drug release

o Side by side diffusion cells with artificial or biological membrane.

o Dialysis bag diffusiono Reverse dialysiso Ultra centrifugationo Ultra filtrationo Gel filtrationo Centrifugal Ultra filtration

APPLICATIONS

o Oral Drug Deliveryo Parenteral Drug Deliveryo Ocular Drug Deliveryo Pulmonary drug delivery o Targeted drug delivery

NANOSUSPENSION BASED FORMULATION

Drug Status

Paclitaxel Phase III

Rapamune Marketed

Emend Marketed

Cytokine Inhibitor Phase II

Thymectacin Phase I & II

Busulfan Phase I

Budesonide Phase I

Referenceso A.D. Bangham , J. Mol. Biol. 13:238-252, 1965o Ali Demir Sezer,Ahmet Levent Bas, and Ju¨ lide Akbug,

Encapsulation of Enrofloxacin in Liposomes I: Preparation and In Vitro Characterization of LUV, Journal of liposome research,Vol. 14, Nos. 1 & 2, pp. 77–86, 2004

o Xuedong Yen, Gerrit L. Scherphof, And Jan A. A. M. Kamps, Liposome Opsonization, Journal of Liposome Research, 15:109–139, 2005

o Ilya Koltover, Tim Salditt, Joachim O. Radler, An Inverted Hexagonal Phase of Cationic Liposome±DNA Complexes Related to DNA Release and Delivery, 3 July 1998, vol.281 SCIENCE,www.sciencemeg.org

o Yasuyuki Sadzuka, Kaori Kishi, Sadao Hirota, and Takashi Sonobe, Effect of Polyethyleneglycol (PEG) Chain on Cell Uptake of PEG-Modified Liposomes Journal Of Liposome Research Vol. 13, No. 2, pp. 157–172, 2003.

Referenceso Lee Leserman, Liposomes as Protein Carriers in

Immunology, Journal Of Liposome Research, Vol. 14, Nos. 3 & 4, pp. 175–189, 2004.

o Loisel S, Le Gall C, Doucet L, Ferec C, Floch V. Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes. Hum Gene Ther 2001; 12:685–696.

o Pasquale Stano, Simone Bufali,Claudio Pisano, Federica Bucci,Marcella Barbarino, Mose` Santaniello, Paolo Carminati, and Pier Luigi Luisi Novel Camptothecin Analogue (Gimatecan)-Containing Liposomes Prepared by the Ethanol Injection Method, Journal Of Liposome Research, Vol. 14, Nos. 1 & 2, pp. 87–109, 2004

o Maria A Stuchlyt, Stan S StuchlyS, Robert P LiburdyO and Daniel A RousseauS, Dielectric properties of liposome vesicles at the phase Transition, Phys. Med. Biol., 1988, Vol. 33, No 11, 1309-1324.

Referenceso Andreas Wagner, Karola Vorauer-Uhl, Gunther Kreismayr,

and Hermann Katinger enhanced protein loading into liposomes by the multiple crossflow injection technique, Journal Of Liposome Research Vol. 12, No. 3, pp. 271–283, 2002.

o Maoquan Chu1, and Guojie Liu, Preparation and characterization of hydroxyapatite/liposome core–shell nanocomposites, Nanotechnology 16 (2005) 1208–1212.

o Ellens, H., Bentz, J., and Szoka, F.C., Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature, Biochemistry, 25, 285, 1986.

o Kensil, C.R. and Dennis, E.A., Alkaline hydrolysis of phospholipids in model membranes and the dependence on their state of aggregation, Biochemistry, 20, 6079, 1981.

o Grit, M., de Smidt, J.H., Struijke, A., and Crommelin, D.J.A., Hydrolysis of phosphatidylcholine in aqueous liposome dispersions, Int. J. Pharm., 50, 1, 1989.

Referenceso Yoshie Maitani, Hiroshi Soeda, Wan Junping, And Kozo

Takayama Modified ethanol injection method for liposomes containing _-sitosterol-d-glucoside, Journal Of Liposome Research, 11(1), 115–125 (2001).

o Shangguan et.al , 2000, Gene Ther., 7, 769.o Liposome Technology Volume 1 : Gregory Gregoridis

Pg No . 250 – 255 . o Pharmaceutical Dosage Form : Disperse System Vol . 3

Pg No . 148 – 150 .o Encyclopaedia : Pharmaceutical Technology Vol 9 Pg

No . 1 – 38 .o Modified Release Drug Delivery Technology Cha. 58

Stealth Technology .o Modified Release Drug Delivery Technology Cha. 47

SLN and Lipopearl for Topical and Controlled Release .

Referenceso Nanosuspensions : A promising drug delivery strategy

from JPP , Vol 56 , 2004 , Pg No. 827 – 840 . o Microencapsulation : Methods and Application by

Simon Benita Cha . 10 , 11 , 12 .o Drug Delivery and Industrial Pharmacy : Liposomal as

a Drug Delivery System . 1989 Pg No. 1523 , 1989 Pg No. 197 , Pg 387 Vol 3

o Depofoam : JPS Nov 1998 , pg 1341 .o Eastern Pharmacist : March 1994 , Pg No. 61 .o Indian Journal of Pharmaceutical Sciences : Jan-Feb

2004 Pg 121-122 .o Journal of Pharmaceutical Science : Vol 90 , No.6 , June

2001 .o Journal Of Pharmaceutical Science : Vol 92 , No.1,Pg 61-

64 January 2003 .