liposomes presentation - finalz

8/3/2019 Liposomes Presentation - Finalz

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Liposomes

R. Venkatesh

M.PHARM (PHARMACEUTICS) 1ST YR 2ND SEMESTER

TKR College of PharmacyJNTU- Hyderabad

A Literature ReviewBy


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Outline

Introduction Classification

Mechanisms of Formation Methods of PreparationCharacterization of liposomes Therapeutic Applications

Conclusion References


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Introduction

Rational research in drug delivery began in 1950s.

Need for Targeted/Controlled Drug Delivery:

Delivery of the drug at a rate and/or a location dictated by the needs ofthe body or disease state over a specified period of time.

Temporal delivery: control over rate of drug release

Spatial delivery: control over location of drug release

Combination of both of the above.


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Liposomes

Liposomes: A Novel Drug Delivery System.

Liposomes were discovered in the early 1960s by Bangham and

colleagues.

Structurally, liposomes are concentric bilayered vesicles in whichan aqueous volume is entirely enclosed by a membranous lipid

bilayer mainly composed of natural or synthetic phospholipids

Liposomes became the predominant drug delivery and targeting

system.

Ranging from tumour targeting, gene and anti-sense therapy,

genetic vaccination, immuno-modulation, lung therapeutics,

fungal infections, and some non- medical are like bio reactors,

catalysts, ecology, skin care and topical cosmetic products.


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Advantages of Liposomes

1. Provides selective passive targeting to tumour tissues (liposomal

doxorubicin).

2. Increased efficacy and therapeutic index.

3. Increased stability via encapsulation.

4. Reduction in toxicity of the encapsulated agent.

5. Improved pharmacokinetic effects (Reduced elimination, increased

circulation life times).

6. Flexibility to couple with site-specific ligands to achieve active

targeting.


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TYPE SPECIFICATION

MLV Multilamellar large vesicles->0.5m

OLV Oligolamellar vesicles-0.1-1 m

UV Unilamellar vesicles (all size ranges)

SUV Small Unilamellar Vesicles- 20-100 nm

MUV Medium sized unilamellar vesicles

LUV Large unilamellar vesicles->100nm

GUV Giant unilamellar vesicles->1m

MV Multivesicular vesicles->1m

CLASSIFICATION OF LIPOSOMES

Based on Structural Parameters


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REV Single or oligolamellar vesicles made by

reverse- phase evaporation method.

MLV-REV Multilamellar vesicles made by reverse-

phase evaporation method.

SPLV Stable plurilamellar vesicles

FATMLV Frozen and thawed MLV

VET Vesicles prepared by extrusion technique.

DRV Dehydration rehydration method.


Based on Method of Liposome Preparation


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Based upon Composition & Applications


Conventional liposomes(CL) Neutral or negatively charged

Phospholipids and cholestrol.

Fusogenic liposomes (RSVE) Reconstituted Sendai virus envelopes

pH sensitive liposomes Phospholipid such as PE or DOPE

with either CHEMS or OA

Cationic liposomes Cationic lipids with DOPE

Long circulatory stealth Neutral high Tc, cholestrol and 5- 10%

liposomes (LCL) of PEG - DSPE or GM1

Immuno-liposomes CL or LCL with attached monoclonal

antibody or recognition sequence


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Phospholipid Bilayer

Structure of Typical Phospholipid within a Lipid Bilayer


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Mechanism of Liposomes Formation

In order to understand the formation of liposomes from phospholipids,it requires a basic understanding of physicochemical features ofphospholipids.

Phospholipids are amphipathic (having affinity for both aqueous andpolar moieties) molecules as they have a hydrophilic or polar headand a hydrophobic tail.

The tail is composed of two fatty acid chains containing 10-24 carbon

atoms and 0-6 double bonds in each chain.

The polar end of the molecule is mostly phosphoric acid bound to awater-soluble molecule.

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Mechanism of Liposomes Formation

In aqueous medium the molecules in self-

assembled structures, are oriented in such a way

that the polar portion of the molecules remains in

contact with the polar environment and at the

same time shields the non polar part.

Among the amphiphiles used in the drugdelivery, viz. soaps detergents, polar lipids; the

latter are often employed to form concentric

bilayered structures.

At high concentration of these polar lipids, liquid-

crystalline phases is formed that upon dilutionwith an excess of water can be dispersed into

relatively stable colloidal particles.


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Liposomes Formation


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Physicochemical Properties of Phospholipids

1. The large free energy difference between the aqueous and hydrophobic environment

promotes the bilayer structures in order to achieve the lowest free energy level.

2. The driving force for bilayer configuration of liposomes is the hydrophobic interaction

coupled with the amphiphilic nature of the principle phospholipid molecules.3. Supra-molecular self-assemblages mediated through specific molecular geometry.

Phase transition of lipids:

Relatively ordered state (gel phase) Less ordered liquid crystal state

(liquid phase)

Some of the parameters responsible for bi-layer orientation are:-


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Role of Cholesterol Bilayer Formation

Cholesterol is known to have

important modulatory effect on the bi-

layer membrane.

It acts as a fluidity buffer.(since

below the phase transition it tends to

make the membrane less ordered

while above the transition it tends to

make the membrane more ordered)

After intercalation with phospholipid

molecules, it alters the freedom of

motion of carbon molecules in the

acyl chain.

It restricts the transformations of

trans- to gauche- conformations.


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Method of Preparation of Liposomes

Passive loading techniques Active loading techniques

Mechanical dispersion methodsSolvent dispersion methodsDetergent removal methods


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Mechanical dispersion methods Lipid film hydration by hand shaking, non-hand shaking or freeze dryingMicro-emulsificationSonication French pressure cellMembrane extrusionDried reconstituted vesicles

Freeze-thawed liposomes Solvent dispersion methodsEthanol injectionEther injectionDouble emulsion vesiclesReverse phase evaporation vesiclesStable plurilamellar vesicles

Detergent removal methodsDialysisColumn chromatographyDilutionReconstituted Sendai Virus enveloped vesicles

Method of Preparation of Liposomes

Passive Loading Techniques


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MECHANICAL DISPERSION METHODS

In these methods, the lipids are casted as stacks of film from their organicsolution using flash rotary evaporator under reduced pressure (or by handshaking) and then the casted film is dispersed in an aqueous medium.

Upon hydration the lipids swell and peel off from the wall of RB flask andvesiculate forming multi-lamellar vesicles (MLVs).

The mechanical energy required for the swelling of lipids and dispersion ofcasted lipid film is imparted by manual agitation (hand shaking technique)or by exposing the film to a stream of water-saturated nitrogen for 15minfollowed by swelling in aqueous medium without shaking (non-swellingvesicles)

It is interesting to note that as compared to hand-shaking method (that

produces MLVs), the vesicles produced by non-shaken methods are largeuni-lamellar vesicles (LUVs).

The percent encapsulation efficiency as high as 30% is achieved.

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MECHANICAL DISPERSION METHODS


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In order to increase the surface area of dried lipid film and to facilitate instantaneous

hydration, the lipid is dried over a finely divided particulate support, such as powdered

sodium chloride, or sorbitol or other polysacharrides.

These dried lipid coated particulates are called Pro-liposomes.

Pro-liposomes form dispersion of MLVs on adding water into them, where support is

rapidly dissolved and lipid film hydrates to form MLVs.

The size of the carrier influences the size and heterogeneity of liposomes.

This method also overcomes the stability problems of liposomes encountered during

the storage of dispersion, dry or frozen form.

It is ideally suited for preparations where the material incorporates into lipid membrane.

The method is applicable in cases where 100% entrapment of components is not a

requirement rather the stability is preferred.

PRO-LIPOSOMES


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Multilamellar vesicles formed on hydration of dried lipids could be further

engineered or modified for their size and other characteristics. A large number

of methods are devised to reduce their size and to convert liposomes of the

large size range into smaller homogenous vesicles. These include techniques

such as micro-emulsification, extrusion, ultra-sonification and use of French

pressure cell.

A second set of methods is designed to increase the entrapment volume of

hydrated lipids, and/or reduce the lamellarity of vesicles formed. These

include procedures such as freeze drying, freeze thawing, or induction of

vesiculation by ions or pH change.

Mechanical Treatment of MLVs


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There are two methods of sonication based on the use of eitherprobe or bath ultra-sonic disintegrators.

The probe is employed for dispersions which require high energyin a small volume. E.g. high concentration of lipids or a viscous

aqueous phase.The bath is more suitable for large volumes of diluted lipids.

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SONICATED UNILAMELLAR VESICLES (SUVs)

Preparation of SUVs from Bath/Probe Sonicator from MLVs


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FRENCH PRESSURE CELL LIPOSOMES

French Pressure Cell for Preparation of Uni/Oligo Lamellar Vesicles


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Micro-fluidiser is used to prepare small MLVs from concentrated

lipid dispersion .It pumps the fluid at very high pressure (10,000psi, 600-700 bar)through a 5micron orifice.

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MICRO EMULSIFICATION LIPOSOMES (MEL)


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VESICLES PREPARED BY EXTRUSIONTECHNIQUES (VETs)


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ETHANOL & ETHER INJECTION


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MICROENCAPSULATION OR LOCUS OF DRUGS INLIPOSOMES


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Removal of un-entrapped drugs fromliposomes

Method % loading Advantages Disadvantages

Dialysis 26 Sample recovery Inaccurate

Mini-columncentrifugation

34 Economical; samplerecovery

Tedious; smallsample volumes

Ficoll density

gradient

34 Economical; rapid;

sample recovery

Small sample

volume (0.5ml)

Protamineaggregation

34 Economical;applicable to MLVs

& LUVs

Slow with neutral &positively chargedliposomes; causescontamination ofliposome sample

Gel chromatography 28-29 Sample recovery Slow & tedious;tends to dilution of

samples

Centifree filtration 31 Rapid; small samplevolume

Expensive;applicable only toULVs; lipid concs

cant exceed 5mg/ml27


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COMMERCIAL DEVELOPMENT AND SCALE UP

Several liposome based products have been either approved or under the

process of approval in the various parts of the world . However, scaling them up as a conventional market product still remains

to be successfully implicated.

Problems generally encountered in the

development of pharmaceutical liposomes:1. Poor quality of the raw material mainly the phospholipids.

2. Poor characterization of the physicochemical properties of the

liposomes.

3. Pay load is too low.

4. Shelf life is too short.

5. Scale up related problems.

6. Absence of any data on safety of these carrier systems on chronic use.


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COMMERCIAL DEVELOPMENT AND SCALE UP

In recent years, several pragmatic solutions are being worked upon tocircumvent these problems.

High quality products with improved purification protocols andvalidated analytical techniques are available.

Quality control assay can be performed using sophisticated

instruments & batch to batch variability can be checked. Shelf-life can be improved using appropriate cryo-protectant & lyo-

protectant and product can be successfully freeze-dried.

Scaling up can be improved by carefully selecting method ofpreparation, sterilization by autoclaving or membrane filtration coupled

with aseptic processing and pyrogen removal using properly validatedLAL test.

By choosing candidate potent drugs with narrow therapeutic window,

the drug related safety problems can be alleviated.

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THERAPEUTIC APPLICATION OF LIPOSOMES

1. LIPOSOMES AS DRUG/ PROTEIN DELIVERY VEHICLES

Controlled and sustained drug release in situ.

Enhanced drug solubilisation.

Altered pharmacokinetics and bio-distribution.

Enzyme replacement therapy and lysosomal storage diseases.

2. LIPOSOMES IN ANTIMICROBIAL, ANTIFUNGAL (LUNG THERAPEUTICS) &

ANTIVIRAL (ANTI-HIV) THERAPY

Liposomal drugs.

Liposomal biological response modifiers.

3. LIPOSOMES IN TUMOUR THERAPY

Carrier of small cytotoxic molecules.

Vehicle for macro-molecules as cytokines or genes.


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4. LIPOSOMES IN GENE-DELIVERY

Gene & antisense therapy

Genetic (DNA) vaccination

5. LIPOSOMES IN IMMUNOLOGY

Immunoadjuvant

Immunomodulator

immunodiagnosis

6. LIPOSOMES AS ARTIFICIAL BLOOD SURROGATES

7. LIPOSOMAL AS RADIO-PHARMACEUTICAL AND RADIO-DIAGNOSTIC CARRIERS

8. LIPOSOMES IN COSMETICS AND DERMATOLOGY

9. LIPOSOMES IN ENZYME IMMOBOLIZATION AND BIO-REACTOR TECHNOLOGY31


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LIPOSOMES AS DRUG DELIVERY VEHICLES:

1. Enhanced drug solubilization (amphotericin B, minoxidil, paclitaxel,

cyclosporin),

2. Protection of sensitive drug molecules (cytosine arabinose,

ribozymes)

3. Enhanced intracellular uptake (anticancer, antiviral and antimicrobial

drugs).

4. Altered pharmacokinetics and bio-distribution (prolonged or sustained

release of drugs with short circulatory half-lives).

5. Enzyme replacement therapy and lysosomal storage disorders.

6. Passive macrophage targeting and RES-accumulation

7. Surface engineered versions of liposomes


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Liposomes comprised of lipids that are relatively non toxic, non

immunogenic, biocompatible and biodegradable lipids can deliver the drug

systemically with an increased therapeutic index and minimized toxicity

index.

Similarly, site avoidance and selective delivery can be appreciated for drugs

that usually have a narrow therapeutic index and that can be highly toxic to

normal tissues.


INCREASED THERAPEUTIC INDEX:


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Liposomes have been used as lysosomotropic carriers to en route the

enzyme and supplement it therapeutically in enzyme deficiency diseases

like Gauchers disease (-glucosidase deficiency) or Pompes disease (-

glucosidase deficiency).

A variety of lysosomal enzymes can be entrapped in liposomes and can be

delivered to patients suffering from lysosomal storage disorders.

Liposomes have also been used in the treatment of metal poisoning, since

the liver is the major site of heavy metal and iron accumulation.


LIPOSOMES AS A LYSOSOMOTROPIC CARRIER:


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Multi-drug resistance is often associated with the over-expression of some drug-efflux pumps,

known as P-glycoprotein pump (PGP) and multi-drug resistance associated protein pump

(MRP).

Several mechanisms are proposed through which liposomes avoid multi-drug resistance of

encapsulated drugs.

1. Negatively charged phospholipids (phosphotidylserine or cardiolipin) used in the liposomal

formulations may directly regulate the P-glycoprotein transporter.

2. Liposomes may provide sustained and high levels of drug to resistant cells over long period

of time.

3. The lysosomal localization of the drug protects it from the action of the P-glycoprotein, by

avoiding immediate contact with P-glycoprotein transporter located at the plasma

membrane.


LIPOSOMES IN MULTI-DRUG RESISTANCE:


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Due to their intrinsic passive vectorization to RES-predominant

organs, liposomes offer enormous potentials and opportunities fortargeted drug delivery to intracellular pathogens like leishmaniasis,

candidiasis, aspergillosis, histoplasmosis, cryptococosis ,giardiasis,

malaria and tuberculosis.

LIPOSOMES IN ANTIMICROBIAL, ANTIFUNGAL (LUNGTHERAPEUTICS) AND ANTIVIRAL (ANTI-HIV) THERAPY:



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Recent advances has focused on the improvement of its therapeutic index,

through reduction of Amp B toxicity by its incorporation in lipid carriers.

Several lipid formulations of Amp B are available in the market and include

Fungizone (Bristol Myers- squibb, Woerden, The Netherlands), AmBisome

(Nexstar, Boulder, CO, USA), ABELECT (The liposome company ,

Princeton, NJ, USA) and AMPHOTEC (Sequss Pharmaceuticals, Merlo, CA,

USA)

The rationale approach to the problem requires the drugs should be

targeted to the macrophages in such a way that the interaction of the free

drug with normal target tissues could be minimized.

AMPHOTERICIN B LIPOSOMES:



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STEALTH LIPOSOMES:


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PROPOSED MECHANISMS FOR STEALTH LIPOSOMES:



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The main purpose is to activate macrophages and render them tumouricidal.If macrophages are activated to their tumouricidal state, they become an

ideal modality for the treatment of metastatic diseases that are resistant to

other forms of therapy.

Tumouricidal macrophages acquire the ability to recognize and destroy

neoplastic cells both invitro and in vivo, while leaving normal cels unharmed

by a non-immunological mechanism that requires cell-to-cell contact.

The problem of rapid clearance of systemically administered cytokines and

macrophage activators (as immunomodulators) and pathogenicity of

microorganisms and their products can be overcome by encapsulating

activating agents into liposomes composed of phospholipids.

LIPOSOMES AS A CARRIER OF IMMUNOMODULATORS



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Liposomes loaded with the appropriate contrast agents have shown to be

suitable for all imaging modalities. The imaging modalities are

-scintigraphy

Magnetic resonance

Computed tomography

Ultra sound imaging or ultra sonography

Liposomes (conventional and targeted including immunoliposomes and

stealth liposomes) are used in different imaging modalities to locate thesites specifically. Liposomal uptake by RES, which is useful strategy in

localization of contrast agents in RES rich organs like liver, spleen and

bone marrow, is not useful for localization to non RES organs.

LIPOSOMES AS RADIOPHARMACEUTICAL &RADIODIAGNOSTIC CARRIERS


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Liposome encapsulated haemoglobin products are being investigated as

artificial RBCS (oxygen carrying rbc substitutes).

Tsuchida reported that the aggregation and fusion of haemoglobin vesicles

(Hb vesicles) and the leakage on long term storage can be prevented by

using either polymerised phospholipids or by introduction of

oligosachharide type of glycolipid in the bilayer membrane.

Sterically stabilized liposome bearing haemoglobin are even better as they

manifest less toxicity, less platelet activation and aggregation and less

haemostatic generation.

LIPOSOMES AS RED CELL SUBSTITUTES AND ARTIFICIAL RBCS


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Conclusions

Liposomes over the years have been investigated as the major drug deliverysystems due to their flexibility to be tailored for varied desirable purposes.

The flexibility in their behaviour can be exploited for the drug delivery

through any route of administration and for any drug or material irrespective

of its physicochemical properties.

The uses of liposomes in the delivery of drugs and genes to tumour sites are

promising and may serve as a handle for focus of future research.


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1. S. P. Vyas, R. K. Khar, Targeted and Controlled Drug Delivery Novel Carrier Systems

2. P. Tyle, Drug Delivery Devices - Fundamentals and Applications

3. T. Yamaguchi, Y. Mizushima, Crit.Rev. Drug Carrier Systems

4. Remington, The Science and Practice of Pharmacy, 20th Ed, Vol-1

5. J. Swarbrick, Encyclopedia of Pharmaceutical Technology, 3rd Ed, Vol-4

6. N. K. Jain, Progress in Controlled and Novel Drug Delivery Systems, 1st Ed.

7. A. A. Gabizon, Stealth Liposomes and Tumor Targeting: One Step Further in the Quest for the MagicBullet, Clinical Cancer Research, Vol. 7, (2001) 223225

8. M. L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical

applications, existing and potential, International Journal of Nanomedicine, Vol 1, (3), 2006, 29731

9. www.iea.usp.br/iea/online/midiateca/origemdavidaluisi.ppt

10. R. L. Hamilton Jr, J. Goerke, L. S. Guo, M. C. Williams, R. J. Havel, Unilamellar liposomes made with theFrench pressure cell: a simple preparative and semiquantitative technique, Journal of Lipid Research, Vol

21, 981-992, http://www.jlr.org/cgi/content/abstract/21/8/981

11. V. Centisa, P. Vermette, Physico-chemical properties and cytotoxicity assessment of PEG-modified

liposomes containing human hemoglobin, Colloids and Surfaces B: Biointerfaces, Volume 65, Issue 2, 1

September 2008, Pages 239-246

References


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liposomes presentation - finalz

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