liposomes presentation - finalz
TRANSCRIPT
-
8/3/2019 Liposomes Presentation - Finalz
1/46
1
Liposomes
R. Venkatesh
M.PHARM (PHARMACEUTICS) 1ST YR 2ND SEMESTER
TKR College of PharmacyJNTU- Hyderabad
A Literature ReviewBy
-
8/3/2019 Liposomes Presentation - Finalz
2/46
2
Outline
Introduction Classification
Mechanisms of Formation Methods of PreparationCharacterization of liposomes Therapeutic Applications
Conclusion References
-
8/3/2019 Liposomes Presentation - Finalz
3/46
3
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.
-
8/3/2019 Liposomes Presentation - Finalz
4/46
4
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.
-
8/3/2019 Liposomes Presentation - Finalz
5/46
5
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.
-
8/3/2019 Liposomes Presentation - Finalz
6/46
6
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
-
8/3/2019 Liposomes Presentation - Finalz
7/46
7
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.
CLASSIFICATION OF LIPOSOMES
Based on Method of Liposome Preparation
-
8/3/2019 Liposomes Presentation - Finalz
8/46
8
Based upon Composition & Applications
CLASSIFICATION OF LIPOSOMES
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
-
8/3/2019 Liposomes Presentation - Finalz
9/46
9
Phospholipid Bilayer
Structure of Typical Phospholipid within a Lipid Bilayer
-
8/3/2019 Liposomes Presentation - Finalz
10/46
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.
10
-
8/3/2019 Liposomes Presentation - Finalz
11/46
11
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.
-
8/3/2019 Liposomes Presentation - Finalz
12/46
12
Liposomes Formation
-
8/3/2019 Liposomes Presentation - Finalz
13/46
13
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:-
-
8/3/2019 Liposomes Presentation - Finalz
14/46
14
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.
-
8/3/2019 Liposomes Presentation - Finalz
15/46
15
Method of Preparation of Liposomes
Passive loading techniques Active loading techniques
Mechanical dispersion methodsSolvent dispersion methodsDetergent removal methods
-
8/3/2019 Liposomes Presentation - Finalz
16/46
16
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
-
8/3/2019 Liposomes Presentation - Finalz
17/46
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.
17
-
8/3/2019 Liposomes Presentation - Finalz
18/46
18
MECHANICAL DISPERSION METHODS
-
8/3/2019 Liposomes Presentation - Finalz
19/46
19
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
-
8/3/2019 Liposomes Presentation - Finalz
20/46
20
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
-
8/3/2019 Liposomes Presentation - Finalz
21/46
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.
21
SONICATED UNILAMELLAR VESICLES (SUVs)
Preparation of SUVs from Bath/Probe Sonicator from MLVs
-
8/3/2019 Liposomes Presentation - Finalz
22/46
22
FRENCH PRESSURE CELL LIPOSOMES
French Pressure Cell for Preparation of Uni/Oligo Lamellar Vesicles
-
8/3/2019 Liposomes Presentation - Finalz
23/46
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.
23
MICRO EMULSIFICATION LIPOSOMES (MEL)
-
8/3/2019 Liposomes Presentation - Finalz
24/46
24
VESICLES PREPARED BY EXTRUSIONTECHNIQUES (VETs)
-
8/3/2019 Liposomes Presentation - Finalz
25/46
25
ETHANOL & ETHER INJECTION
-
8/3/2019 Liposomes Presentation - Finalz
26/46
26
MICROENCAPSULATION OR LOCUS OF DRUGS INLIPOSOMES
-
8/3/2019 Liposomes Presentation - Finalz
27/46
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
-
8/3/2019 Liposomes Presentation - Finalz
28/46
28
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.
-
8/3/2019 Liposomes Presentation - Finalz
29/46
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.
29
-
8/3/2019 Liposomes Presentation - Finalz
30/46
30
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.
-
8/3/2019 Liposomes Presentation - Finalz
31/46
THERAPEUTIC APPLICATION OF LIPOSOMES
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
-
8/3/2019 Liposomes Presentation - Finalz
32/46
32
THERAPEUTIC APPLICATION OF LIPOSOMES
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
-
8/3/2019 Liposomes Presentation - Finalz
33/46
33
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.
THERAPEUTIC APPLICATION OF LIPOSOMES
INCREASED THERAPEUTIC INDEX:
-
8/3/2019 Liposomes Presentation - Finalz
34/46
34
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.
THERAPEUTIC APPLICATION OF LIPOSOMES
LIPOSOMES AS A LYSOSOMOTROPIC CARRIER:
-
8/3/2019 Liposomes Presentation - Finalz
35/46
35
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.
THERAPEUTIC APPLICATION OF LIPOSOMES
LIPOSOMES IN MULTI-DRUG RESISTANCE:
-
8/3/2019 Liposomes Presentation - Finalz
36/46
36
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:
THERAPEUTIC APPLICATION OF LIPOSOMES
-
8/3/2019 Liposomes Presentation - Finalz
37/46
37
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:
THERAPEUTIC APPLICATION OF LIPOSOMES
-
8/3/2019 Liposomes Presentation - Finalz
38/46
38
THERAPEUTIC APPLICATION OF LIPOSOMES
STEALTH LIPOSOMES:
-
8/3/2019 Liposomes Presentation - Finalz
39/46
39
PROPOSED MECHANISMS FOR STEALTH LIPOSOMES:
THERAPEUTIC APPLICATION OF LIPOSOMES
-
8/3/2019 Liposomes Presentation - Finalz
40/46
40
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
THERAPEUTIC APPLICATION OF LIPOSOMES
-
8/3/2019 Liposomes Presentation - Finalz
41/46
41
THERAPEUTIC APPLICATION OF LIPOSOMES
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
-
8/3/2019 Liposomes Presentation - Finalz
42/46
42
THERAPEUTIC APPLICATION OF LIPOSOMES
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
-
8/3/2019 Liposomes Presentation - Finalz
43/46
43
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.
-
8/3/2019 Liposomes Presentation - Finalz
44/46
44
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
-
8/3/2019 Liposomes Presentation - Finalz
45/46
45
Suggestions
Please.!!!!!!!!!!!!!!!!!!
-
8/3/2019 Liposomes Presentation - Finalz
46/46
46
Thanking You
One & All.!!!!!