2. Content
Introduction
Mechanism of liposome formation
Classification
Methods of liposome preparation & drug loading
Passive loading tech.
Mechanical dispersion methods
Solvent dispersion methods
Characterization of liposomes
Stability of liposomes
2
3. DEFINITION: Liposomes are concentric bilayered vesicles in
which an aqueous volume is entirely enclosed by a membranous
lipid bilayer mainly composed of natural or synthetic
phospholipids.
Advantages
1. Provides selective passive targeting to tumour tissues.
2. Increased efficacy & therapeutic index.
3. Increased stability via encapsulation.
4. Reduction in toxicity of the encapsulated agent.
5. Site avoidance effect (avoids non-target tissues).
3
4. 6. Improved pharmacokinetic effects.
7. Flexibility to couple with site specific ligands to achieve
active targeting.
• Disadvantages
1. Production cost is high.
2. Leakage and fusion of encapsulated drug/molecules.
3. Sometimes phospholipids undergoes oxidation and hydrolysis
like reaction.
4. Short half-life.
5. Low solubility.
4
9. Phospholipids
Phosphatidylcholine- natural
Amphipathic molecule
Hydrophilicpolarhead-
Phosphoricacid boundto water
solublemolecule.
Glycerylbridge
Hydrophobic tail-
2 fattyacidchaincontaining10-24 carbon
atomsand0-6 double bond in eachchain.
The amphipathic moleculeself organise
in orderedsupramolecularstructurewhen
confronted (meet face to face)
with solvent.
9
8
10. The most common natural phospholipid is the
phospatidylcholine (PC ).
Naturallyoccurringphospholipids used are :
Polar Head Groups
Three carbon glycerol
PC: Phosphatidylcholine.
PE: Phosphatidylethanolamine.
PS: Phosphatidylserine
Synthetic phospholipids used are:
DOPC: Dioleoyl phosphatidylcholine
DSPC: Disteroyl phosphatidylcholine
DOPE: Dioleoyl phosphatidylethanolamine
DSPE: Distearoyl phosphatidylethanolamine
10
9
12. Molecules of PC arenot soluble in water.
In aqueous media they align themselves closely in planar bilayer sheets
in order to minimize the unfavorable action between the bulk aqueous
phaseand the longhydrocarbonfatty chain.
Such unfavorable interactions arecompletely eliminated when the
sheetsfold on themselvesto form closedsealedvesicles
1
1
2
1
13. B. Cholesterol:
Cholesterol stabilizes the Membrane
Steroid lipid
Interdigitatesbetweenphospholipids.
i.e. belowTc, it makesmembraneless ordered&aboveTcmoreordered.
Being an amphipathic molecule, cholesterol inserts into the membrane
with its hydroxyl group of cholesterol oriented towards the aqueous
surface and aliphatic chain aligned parallel to the acyl chains in the center
of the bilayer. 1
1
3
3
14. Role of cholesterol in bilayer formation:
Cholesterol act as fluiditybuffer
Afterintercalation with phospholipidmolecules alterthe
freedom of motion of carbonmolecules in the acyl
Chain
Restricts the transformations of trans to gauche
Conformations.
Incorporatedinto phospholipidmembraneupto 1:1 or
2:1 of cholesterol to PC.
11
44
15. Mechanism of liposome formation
Phospholipids are amphipathic molecules having hydrophobic tail & a
hydrophilic or polar head
The hydrophilic & hydrophobic domains within the molecular geometry of
amphiphilic lipids orient & self organize in ordered supramolecular structure
when confronted with solvents
Cholesterol have modulatory effect on the bilayer membrane (acts as fluidity
buffer)
Below phase transition it tends to make the membrane less ordered while
above the transition it tends to make the membrane more ordered.
16.
17. The parameters affecting bilayer formation
The large free energy difference between the aqueous & hydrophobic
environment promotes bilayer structures in order to achieve the lowest free
energy level
The driving force for bilayer configuration of liposomes is the hydrophobic
interaction coupled with the amphilic nature of the phospholipid molecule
Supramolecular self assemblages mediated through specific molecular
geometry
18.
19. Classification of liposomes
Based on structural parameters
1. MLV-multilamellar large vesicles>0.5um
2. OLV-oligolamellar vesicles,0.1-1um
3. UV-unilamellar vesicles( all size range)
4. SUV-small unilamellar vesicles(-20-100nm)
5. MUV-medium sized unilamellar vesicles
6. LUV-large unilamellar vesicles>100nm
7. GUV-giant unilamellar vesicles>1um
8. MV-multivesicular vesicles>1um
20. Based on method of liposome preparation
1. REV-OLV made by reverse phase evaporation method
2. MLV-REV-MLV made by reverse phase evaporation method
3. SPLV-stable plurilamellar vesicle
4. VET-vesicles prepared by extrusion technique
5. DRV-dehydration-rehydration method
Based on composition & applications
1. Conventional liposomes-neutral or negatively charged phospholipids & chol.
2. pH sensitive liposomes- phospholipid such as PE or DOPE
3. Immuno liposomes-CL with attached monoclonal antibody
4. Cationic liposomes-cationic lipids with DOPE
5. Fusogenic liposomes-Reconstituted Sendai virus envelops
22. PASSIVE LOADING TECHNIQUE
Mechanical dispersion methods of passive loading
Technique begin with a lipid solution in organic solvent & end up with lipid
dispersion in water
Various components are combined by co-dissolving the lipids in organic
solvent which is then removed by film deposition under vacuum.
After solvent removal the solid lipid mixture is hydrated using aqueous
buffer.
The lipids spontaneously swell & hydrate to form liposomes
The post hydration treatments include vortexing, sonication, freeze thawing
& high pressure extrusion.
23. Thin film hydration using hand shaking (MLVs) and Non-
shaking methods (ULVs)
24. Pro-liposomes
To increase the surface area of dried lipid film & to facilitate instantaneous
hydration, the lipid is dried over a finely divided particulate support such as
powdered sodium chloride, sorbitol, polysaccharides.
These dried lipid coated particles are called as pro-liposomes. On adding
water to them, support is rapidly dissolved & lipid film hydrates to form
MLVs.
Suitable for lipophilic materials to be incorporated, overcomes stability
problems of liposomes during storage
25. Mechanical treatment of MLVs
A large no of methods are devised to reduce their size & to convert
liposomes of the large size range into smaller homogenous vesicles.
These include tech such as microencapsulation, extrusion, ultrasonication,
use of French pressure cell
Asecond set of methods is designed to increase the entrapment volume of
hydrated lipids or to reduce the lamellarity of the vesicles formed.
These include procedures such as freeze drying, freeze thawing or induction
of vesiculation by ions or pH change.
26.
27. Sonicated unilamellar vesicles (SUVs)
Exposure of MLVs to ultrasonic irradiation by using either probe or bath
ultrasonic disintegrators
The probe for dispersions (which require high energy in a small volume)
while bath is more suitable for large volumes of diluted lipids.
Probe tip sonicators supply a high energy input to the lipid dispersion but
suffer from overheating of the liposomal dispersion causing lipid
degradation.
Sonication tips tent to release titanium particles into the liposome dispersion
Hence bath sonicators are used for preparing SUVs.
Liposome dispersion is centrifuged at100000g(30min,20oC) to sediment
titanium particles & larger MLVs followed by higher speed centrifugation
(150000g for 3-4 h). 27
28. French pressure cell liposomes
The ultrasonic radiation degrades the lipids, other sensitive compounds,
macromolecules for this extrusion of preformed larger liposomes in a
French press under very high pressure is done
This tech. yields uni- or oligo- lamellar liposomes of size (30-80 nm in dia.)
Includes high cost of press that consists of electric hydraulic press &
pressure cell
Liposomes prepared by this method are less likely to suffer from structural
defects & instabilities as observed in sonicated vesicles.
29.
30. Micro Emulsification Liposomes (MEL)
“Microfluidizer” is used to prepare small MLVs from conc. lipid dispersion.
It pumps the fluid at very high pressure(10000psi,600-700 bar) through a 5
um orifice.
This method process samples with a very high proportion of lipids (20% or
more by weight).
It is efficient for encapsulation of water soluble materials.
Percentage capture values up to 70% have been reported.
31.
32. Vesicles prepared by Extrusion Tech. (VETs)
It is used to process LUVs as well as MLVs.
In this mtd. Size of liposomes is reduced by passing through membrane
filter if defined pore size at low pressure.
Liposomes prepared by this tech. are called as LUVETs.
The 30% capture volume can be obtained using high lipid conc. The trapped
volume in this process is 1-2 litre /mole of lipids.
This method is widely used due to their ease of production, readily
selectable vesicle diameter, batch to batch reproducibility & freedom from
solvent or surfactant contamination is possible.
34. Dried-reconstituted Vesicles (DRVs)
Dispersion of empty SUVs is freeze dried & then rehydrated with aqueous
fluid containing material to be entrapped (fig 5-12).
This tech. yields uni- or oligo- lamellar liposomes of size (1.0 µm or less in
dia.)
Entrapment yield can vary upto 40%.
36. Freeze thaw sonication method (FTS)
The method is based on freezing of a unilamellar dispersion & then thawing
at room temp for 15 min. (Fig 5-12)
Thus the process ruptures & refuses SUVs during which the solute
equilibrates between inside & outside & liposomes themselves fuse &
increase in size.
Entrapment volume can be up to 30% of the total vol. of dispersion.
Sucrose, divalent metal ions & high ionic strength salt solutions can not be
entrapped efficiently
36
37. Solvent dispersion methods for passive loading
In this method lipids are dissolved in an organic solution which is then
brought into contact with an aqueous phase containing materials to be
entrapped within liposomes
Ethanol injection:-An ethanol solution of lipids is injected rapidly through a
fine needle into an excess of saline or other aq. Medium.
This method has low risk of degradation of sensitive lipids.
The vesicles of 100 nm size may be obtained by varying the conc. Of lipid in
ethanol or by changing the rate of injection of ethanol solution in preheated
aqu. solution.
Limitation-solubility of lipids in ethanol & vol. of ethanol that can be
introduced into medium (7.5%v/v max)
Difficulty to remove residual ethanol from phospholipids membrane 37
38. • Ether injection:-involves mixing of organic phase into aqu. Phase at the temp. of
vaporizing the organic solvent
• It has low encapsulation efficiency.
38
39. Rapid solvent exchange vesicles (RSEVs)
Lipid mixture is transferred between pure solvent & a pure aq. environment.
Organic sol. of lipids through orifice of syringe under vacuum into a tube containing
aqueous buffer. The tube is mounted on vortexer.
It manifest high entrapment volumes 39
41. Detergent depletion method
The phospholipids are brought into intimate contact with the aqueous
phase via detergents which associate with phospholipid molecules
The structures formed are called as micelles
The conc. of detergent at which micelles are formed is called as CMC
The detergent methods are not very efficient in % entrapment values
The methods employed for removal of detergent include dialysis,
column chromatography & use of biobeads
43. Liposomes can act as carriers for both lipophilic & hydriphilic drugs.
Highly hydrophilic drugs(log p<-0.3)are located in the aqu.domains whereas
highly lipophilic drugs(log p<5) are entrapped within lipid bilayers of the
liposomes
Drugs with intermediary partition coefficients(1.7<log p<4) impose problem
for loading as they equilibrate between lipid & aqueous domains & are prone
to leakage on storage of liposomes
Drugs with poor biphasic insolubility (anticancer drugs 6-
mercaptopurine,azathioprene & allopurinol are most problematic due to their
immiscibility with both aqu. & lipidic domains
The approaches which have emerged for increasing the lipophilicity of the
drugs with purpose of enhancing their liposomal encapsulation are formation
of lipophilic complexes a7 formation of pharmacosomes, preparation of
prodrug & non prodrug lipophilic derivative.
43
44. Characterization of liposomes
Physical parameters include size, shape, surface features, lamellarity, phase behaviour &
drug release profile
Chemical parameters includes purity & potency of liposomal constituents
Biological parameters includes safety & suitability of the formulations for the in vivo use
or for therapeutic applications
1. Vesicle shape & lamellarity:- vesicles can be assessed using electron microscopic tech.
The no of bilayers present in the liposomes is determined using freeze fracture electron
microscopy
2. Freeze fracture & freeze etch electron microscopy:-It assess the surface morphology of
the liposomes. In this tech. the fracture plan passes through the vesicles which are
randomly positioned in the frozen state
Ethching of freeze fractured specimen can provide information about fractures of vesicles
that are unilamellar in a given population.After 5 min of etching ,crossfractured vesicles are
clearly seen & the no of lamellae can readily be determined. 44
45. 2.Vesicle size & size distribution:-
various tech. include light microscopy, fluorescent microscopy, electron microscopy,
laser light scattering, field flow fractionation, gel permeation & gel exclusion, zetasizer
a)
b)
a)
b)
c)
Microscopic techniques:-
Optical microscopy
Negative stain transmission electron microscopy (TEM)
Negative stain electron microscopy visualizes electron transparent liposomes as
bright areas against a dark background. Negative stains used in the TEM analysis
is ammonium molybdate
Cryo transmission electron microscopy (cryo-TEM)
Freeze fracture electron microscopy
Scanning electron microscopy
46. • Cryo transmission electron microscopy (cryo-TEM)
The method involves freeze fracturing of samples followed by TEM. Samples are
prepared under controlled temp.& humidity conditions within environment chamber.
The films are quick freezed in liquid ethane & transferred to TEM analysis
47. Diffraction & scattering techniques:-
a) Laser light scattering:-The tech. is based on time dependant coherence of light
scattered by a vesicle which is dependant on the viscosity of the aqueous medium
& vesicle size
b) It is applicable to monodisperse sample with diameters less than 1 µm.
Hydrodynamic techniques
1.Field-flow-Fractionation (FFF) tech.:-It is useful in characterizing properties
such as drug loading, biomembrane volumes & areas & distributions of these
properties
Sedimentation FFF used to measure vesicle size & size distributions (tech. that
measures the effective mass & mass distribution of particles)
2.Gel permeation:-preferably used for the size distribution determination of
liposomes
3.Ultracentrifuge:-used for size distribution of liposomes
48. 3. Surface charge:-Since liposomes contains lipids hence the charge on the vesicle
surface is to be determined. Tech. are free flow electrophoresis & zeta potential
4. Encapsulation efficiency & trapped volume:- determines % of the aq. Phase &
hence % of water soluble drug which is entrapped & expressed as % entrapment/mg
lipid
5. Trapped volume(0.5-30 µl/mg):-The internal or trapped volume is the aqueous
entrapped volume per unit quantity of lipid & expressed as µ l/ µ mol or µ l/mg of
total lipid.Radioactive markers are used to determine the internal volume.
5. Phase response & transitional behavior:- lipid bilayers can exists in a low
temperature solid ordered phase & above certain temp in a fluid disordered phase.
Phase behavior of liposomal membrane determines prop. such as permeability,
fusion, aggregation & protein binding
Thermodynamic methods:-In differential scanning microcalorimeter, the heat
required by liposomes to maintain a steady upward rise in temp is plotted as a
function of temperature
48
50. 6. Vesicle fusion measurements:- It has been studied in case of cationic liposomes ,
PH sensitive liposomes. fusion has been monitored using a fluorescence resonance
energy transfer (RET) between two lipid analogues originally placed in separate
vesicle population that measures intermixing of membrane lipids
Chemical characterization of Liposomes
1.Phospholipid conc. is determined in terms of lipid phosphorus content using Barlet
assay/Stewart assay or TLC
2.Cholesterol conc. is determined using Ferric perchlorate method/Cholesterol oxidase
assay
3.Lysolecithin:-which is one of the major product of hydrolysis is estimated using
densitometry
4.Phospholipid peroxidation is determined by UV absorbance, iodometry, GLC technique.
5.Phospholipid hydrolysis is determined using HPLC & TLC
6.Cholesterol auto oxidation can be determined by HPLC & TLC
51. Stability of liposomes
The stability in vitro which covers the stability aspects prior to the
administration of the formulation & with regard to the stability of the
constitutive lipids
The stability in vivo which covers the stability aspects once the formulation is
administered via various routes to the biological fluids. It includes stability
aspects in blood if administered by systemic route or in gastrointestinal tract if
administered by oral or peroral routes
Stability in vitro:- method of formulation, nature of amphiphile &
encapsulated drug, manipulate membrane fluidity/rigidity & permeability
characteristics.
Storage temp. of these dispersions must be defined & controlled
Liposomal phospholipids can undergo degradation such as oxidation &
5
1
hydrolysis
52. Liposome characterization with their quality control assays
Characterization parameters Analytical methods/instrumentation
Chemical characterization
Phospholipid conc.
Cholesterol conc.
Drug conc.
Phospholipid peroxidation
Osmolarity
Barlet assays/Stewart assays, HPLC
HPLC
Monograph
UV absorbance, iodometric & GLC
Osmometer
Physical characterization
Vesicle shape & surface morphology
Size & size distribution
Submicron range
Micron range
TEM, Freeze fracture electron microscopy
TEM
TEM,FFEM, photon correlation spectroscopy,
laser light scattering, gel permeation
Biological characterization
Sterility
Pyrogenicity
Animal toxicity
Aerobic or anaerobic cultures
LAL test
Monitoring survival rates, histology & pathology52
53. 1. Lipid oxidation & Peroxidation
Lipid peroxidation measurement is based on disappearance of unsaturated fatty acids
or appearance of conjugated dienes.
It can be prevented by minimizing use of unsaturated lipids, use of oxygen, argon or
nitrogen environment, use of antioxidant such asAlpha tocopherols or BHT or use of
light resistant containers for storage of liposomal preparations
2.Lipid hydrolysis
It leads to lysolecithin formation The inclusion of charged molecule in the bilayer
shifts the electrophoretic mobility & makes it positive with addition of stearylamine
or negative with dicetyl phosphate thus prevents liposomal fusion/swelling or
aggregation
3.Long term &Accelerated stability
High temp. testing(>250C) is universally used for heterogenous products. Various
laboratories store their products at temp ranging from 40C to 500 C.
54. Stability after systemic administration
Two most frequently encountered biological events that the administered
liposomal system undergoes are phagocytosis or antigen presentation via the
macrophages of the RES system
Opsonins which are proteinaceous components of serum adsorb onto the
surface of liposomes thus making these exogenous materials more palatable &
conducive to phagocytes
High density lipoprotein removes phospholipid molecules from bilayered
vesicular systems
The molecular origin of these interactions are mostly long range electrostatic,
Vander waals & short range hydrophobic interactions of particulate surface
with macromolecules in the serum
55. Therapeutic applications of liposomes
1.Liposomes as drug/protein delivery vehicles
Controlled & sustained drug release in situ
Enhanced drug solubilization
Altered pharmacokinetics & biodistribution
Enzyme replacement therapy & lysosomal storage disorders
2.Liposomes in antimicrobial, antifungal & antiviral therapy
Liposomal drugs
Liposomal biological response modifiers
3.Liposomes in tumour therapy
Carrier of small cytotoxic molecules
Vehicle for macromolecules as genes
4.Liposomes in gene delivery
Gene & antisense therapy
Genetic vaccination
5.Liposomes in immunology
Immunoadjuvant
56. Immunomodulator
Immunodiagnosis
6. Liposomes as artificial blood surrogates
7.Liposomes as radiopharmaceutical & radiodiagnostic carriers
8.Liposomes in cosmetics & dermatology
9.Liposomes in enzyme immobilization & bioreactor technology
57. Liposomes as drug delivery vehicles
Enhanced drug solubilization (amphotericin B, minoxidil, paclitaxel, cyclosporin)
Protection of sensitive drug molecules (cytosine arabinose, DNA, RNA,
ribozymes)
Enhanced intracellular uptake (anticancer, antiviral, antimicrobial drugs)
2.Liposomes in antimicrobial, antifungal & antiviral therapy
Intracellular pathogens (protozoal, bacterial,& fungal) harbour in liver & speen &
hence drugs can be targeted to these organs.e.g.Intracellular localization of
pathogenes necessitates administration of high doses of cytotoxic drugs for effective
killing of pathogen causing side effects
Drug to be targeted to macrophages in such a way that interaction of free drug with
nontarget tissues could be minimized pathogen
Treatment with liposomalAmp B results in lower toxicity & increased survival times
58. Liposomes in tumour therapy
Targeting strategies using liposomes are
Natural targeting of conventional liposomes (passive vectorization)
Use of long circulatory (stealth liposomes)
Use of ligand mediated targeting (active targeting)
The use of anti-receptor antibodies on the tumour vascular endothelium
Use of stealth liposomes & ligands mediated targeting in combination
Drug Target disease Status Product
Doxorubicin Kaposi's sarcoma Approved SEQUUS
Daunosome Breast cancer Approved NeXstar,USA
Nystatin Systemic fungal
infections
Phase II Aronex, USA
Amikacin Serious bacterial
infections
Phase II NeXstar,USA
Vincristine Solid tumours Preclinical dev. NeXstar,USA
59. Liposomes in gene therapy
Recombinant DNAtech., studies of gene function & gene therapy all depend
on delivery of nucleic acids( genetic material) into cells in vitro & in vivo.
Gene can be viral (adenovirus, retrovirus) & non viral( liposomes & lipid
based systems, polymers & peptides)
60. Advantages & disadvantages of viral & non viral
systems
Type of vectors Advantages Disadvantages
Viral vectors
(Adenovirus, retrovirus &
adeno-associated virus)
Relatively high transfection
efficiency
Immunogenicity, presence
of contaminants & safety
Vector restricted size
limitation for recombinant
gene
Non viral vectors
(liposomes/lipid based
systems, polymers &
peptides)
Favorable, pharmaceutical
issue-GMP, stability, cost
Plasmid independent
structure
Low immunogenicity
Opportunity for
chemical/physical
manipulation
low transfection efficiency
61. pH sensitive liposomes
The PH sensitive liposomes have been reported as plasmid expression
vectors for the cytosolic delivery of DNA.
pH sensitive immunoliposomes
PH sensitive liposomes have been developed to release their contents in
response to an acid machinery within endosomal system following receptor
mediated endocytosis of the immunological targeting ligand
Fusogenic liposomes& Virosomes
They fuse & merge with cell membranes & directly introduce molecules
(entrapped or anchored) into cytoplasm & avoiding route followed by
conventional liposomes. Fusion can be mediated by PEG, glycerol &
Polyvinyl alcohol or by reconstituted fusogenic viral membrane based
liposomes are termed as Virosomes 61
62. Liposomes as immunological (vaccine) adjuvant
Anon immunogenic substance may be converted into immunogenic
Hydrophobic antigens may be reconstituted
Small amount of antigen may be suitable as immunogens
Multiple antigens may be incorporated into the single liposomes
Adjuvant may be incorporated with antigens into the liposomes
Longer duration of functional antibody activity may be achieved
Soluble synthetic antigen may be presented as membrane associated antigens
in an insoluble liposomal matrix
63. Liposomal vaccines
New vaccines that are based on recombinant protein subunits & synthetic
peptide antigens are usually non-immunogenic, hence need of
immunopotentiation is realized
Alum was used as vaccine adjuvant
The first liposome based vaccine (against hepatitis A) that has been licensed
for use in human is an IRIV vaccine which are spherical, unilamellar vesicles
with a diameter of 150nm.
IRIVs are prepared by detergent removal of influenza surface glycoproteins &
a mixture of natural & synthetic phospholipids containing 70% egg yolk
phosphatidylcholine,20 % synthetic PE & 10 % envelop phospholipids
originating from H1N1 influenza virus
64. Liposomes as a carrier of Immunomodulator
1.
2.
The main purpose is to activate macrophages & render them tumouricidal. They
acquire ability to recognize & destroy neoplastic cells both in vitro & in vivo.
Liposomes in Immunodiagnosis
LILAassays (liposome immune lysis assay) has been implicated in the detection
of serum components such as carcinoembryonic antigen,C-reactive protein &
other serum protein which serve as diagnostic tools for cancer
LILAsandwich method has been used to detect many important antigens in
serum, which are useful indicators of various abnormalities
65. Liposomes in Dermatology and Cosmetology
Similar to biological membrane they can navigate water soluble & lipophilic
substances in different phases
They mimic the lipid composition & structure of human skin, which enables
them to penetrate the epidermal barrier
Liposomes are biodegradable & nontoxic, thus avoiding local/systemic side
or toxic effects
Moisturizing & restoring action of constitutive lipids
Liposomes may act as localized drug depots in skin resulting in sustained
release of drug, thus improving therapeutic index of drug at target site while
reducing toxicity profile to minimum
66. Liposomes as Radiopharmaceutical & radiodiagnostic carriers
Liposomes loaded with contrast agents are suitable for
contrast agents are substances which are able to absorb certain types of signal
much stronger than surrounding tissue
Radiodiagnostic application include liver & spleen imaging, tumour imaging,
imaging cardiovascular pathologies, visualization of inflammation & infected
sites, brain imaging, visualization of bone marrow
The RES avoidance of contrast agents can be achieved by using targeted
liposomes like immunoliposomes
67. Liposomes as Red cells substitutes & artificial
RBCs
Synthetic & semisynthetic blood substitutes includes recombinant
haemoglibin, glutaraldehyde cross linked haemoglobin,haemoglobin
encapsulated liposomes
Liposome encapsulated haemoglobin products are being investigated as
artificial RBCs
Researchers reported completely synthetic amphiphilic heme derivative
(lipid heme) & incorporated them into the hydrophobic centre of the bilayer
membrane of the phospholipid vesicles, which has excellent oxygen carrying
& transporting abilities
69. • Niosomes are non-ionic surfactant based unilamellar or multilamellar
bilayer vesicles up on hydration of non ionic surfactants with or without
incorporation cholesterol .
• The niosomes are very small, and microscopic in size. Their size lies in the
nanometric scale.
• Niosomes are a novel drug delivery system, in which the medication is
encapsulated in a vesicle. Both hydrophilic & lipophilic drugs, entrap either
in the aqueous layer or in vesicular
Membrane made of lipid materials.
57
70. Structure of niosomes:
Head part
Drug
molecules
Polar heads
facing
hydrophilic
region
Hydrophilic
drugs located
in aqueous
regions
(hydrophillic)encapsulated
Tail part
(hydrophobic)
Phospholipids
Hydrophobic
drugs localized
in the
hydrophobic
lamellae
These vesicular systems are similar to liposomes that can be used as carriers
of amphiphilic and lipophilic drugs.
It is less toxic and improves the therapeutic index of drug by restricting its
action to target cells. 58
71. Advantages of niosomes:
They are osmotically active and stable.
They increase the stability of the entrapped drug.
The vesicle suspension being water based offers greater patient compliance
over oil based systems
Since the structure of the niosome offers place to accommodate hydrophilic,
lipophilic as well as ampiphilic drug moieties, they can be used for a variety
of drugs.
The vesicles can act as a depot to release the drug slowly and of controlled
release.
Biodegradable, non-immunogenic and biocompatible.
59
72. • Aggregation
• Fusion
• Leaking of entrapped drug
• Hydrolysis of encapsulated drugs which limiting the shelf
• life of the dispersion.
60
74. Components of niosomes:
• Cholesterol and Non ionic surfactants are the two major
components used for the preparation of niosomes.
• Cholesterol provides rigidity and proper shape. The surfactants
play a major role in the formation of niosomes.
• non-ionic surfactants like spans(span 20,40,60,85,80), tweens
(tween 20,40,60,80) are generally used for the preparation of
• Niosomes.
• Few other surfactants that are reported to form niosomes are as
follows :
Ether linked surfactant
Di-alkyl chain surfactant
Ester linked
Sorbitan Esters
Poly-sorbates 62
75. Non-ionic
surfactant
nature
Hydration
Temperature
alkyl group
chain length :
C12-C18
Span surfactants
with HLB values
4 and 8
Should be
above the gel
to liquid
phase
transition
temperature
of the system
Factors
affecting
niosomes
formation
Membrane
additives
Nature of
encapsulated
drug
Surfactants
and lipid
levels
Cholesterol: Prevent vesicle
aggregation.
Dicetyl phosphate: -ve charge
surfactant/lipid
ratio: 10-30
mM
63
76. Concept of Critical Packing Parameter
• Prediction of vesicle forming ability is not a simply a matter of HLB
CPP= v/lca0
where
v - hydrophobic group volume,
lc - critical hydrophobic group length and
a0 - area of the hydrophilic head group
• CPP between 0.5 and 1 likely to form vesicles.
• < 0.5 (indicating a large contribution from the hydrophilic head group area) is
said to give spherical micelles.
• >1 (indicating a large contribution from the hydrophobic group volume)
should produce inverted micelles.
64
77. Comparison between liposomes & niosomes:
Sr. Liposomes
No.
Niosomes
1. Vesicles made up of concentric
bilayer of phospholipids
Vesicles made up of
surfactants with or without
incorporation of cholesterol.
2. Size ranges from 10-3000nm Size ranges from 10-100nm
3. Comparatively expensive Inexpensive
4. Special storage condition are
required
No such special requirement
5. Phospholipids used are
unstable
Non-ionic surfactants are
stable
6. Comparatively more toxic Less toxic
65
80. Reverse phase evaporation technique :
Surfactant is dissolved in chloroform and 0.25 volume of PBS buffer is
emulsified to get a W/O emulsion.
sonicated
chloroform is evaporated under reduced pressure.
The lipid or surfactant forms a gel first and hydrates to form vesicles.
Free drug (unentrapped) is generally removed by dialysis.
sonication:
Surfactant+cholesterol
mixture is dispersedin 2 ml
aqueous phasein vial
Mixtureis sonicatedfor3
min at 60°C using titanium
probe sonicator
Unilamellarniosomes 68
82. Multiple membrane extrusion Method:
•Mixture of surfactant, cholesterol and
dicetyl phosphate in chloroform is made
into thinfilmbyevaporation
•The film is hydrated with aqueous drug
solution and the resultant suspension
extrudedthroughpolycarbonatemembranes
70
84. proniosomes:
• Bubble Method
• Formationof niosomes fromproniosomes:
It is prepared by coating water-soluble carrier such as sorbitol with
surfactant. The result of the coating process is a dry formulation. In
which each water-soluble particle is covered with a thin film of dry
surfactant.This preparation is termed “Proniosomes”.
72
85. Separation of
unentrapped
drug
Gel filtration
Separation of unentrapped drug:
Centrifugation
The niosomal suspension
is centrifuged and the
Dialysis
Dialyzed in adialysistubing
against phosphatebufferor
normal saline
The unentrapped drug is
removed by gel filtration of
niosomal dispersion through a
Sephadex-G-50 column and
elution with phosphate
bufferedsaline
supernatant is separated.
The pellet is washed and
then resuspended to obtain
a niosomal suspension free
fromunentrappeddrug.
Centrifuser
Gel Filtration 73
86. a) Size, Shape and Morphology
Freeze Fracture Electron Microscopy:- Visualize the vesicular structure of
surfactantbasedvesicles.
Photon Correlation spectroscopy :- Determine mean diameter of the
vesicles.
Electron Microscopy:- Morphological studies of vesicles.
b) Entrapment efficiency
After preparing niosomal dispersion, unentrapped drug is separated by
dialysis and the drug remained entrapped in niosomes is determined by
complete vesicle disruption using 50% n-propanol or 0.1% Triton X-100
and analysing the resultant solution by appropriate assay method for the
drug.
c) V
esicleSufaceCharge
Determined by measurement of electrophoretic mobility and expressed in
expressedintermsof zeta potential
d) In vitro studies 74
88. Lancôme has come out with a variety of anti-ageing
products which are based on noisome formulations.
L’Oreal is also conducting research on anti-ageing
cosmetic products.
76
89. Summary :
Niosomes provide incorporating the drug into for a
better targeting of the drug at appropriate tissue
destination .
They presents a structure similar to liposome and hence
they can represent alternative vesicular systems with
respect to liposomes
Niosomes are thoughts to be better candidates drug
delivery as compared to liposomes due to various factors
like cost, stability etc. Various type of drug deliveries can
be possible using niosomes like targeting, ophthalmic,
topical, parenteral etc.
77
90. 1. S.P. Vyas And R.K. Khar,targeted & Controlled Drug
Delivery,liposomes,173-279.
2. Mohammad Riaz, Liposomes :Preparation Methods, Pakistan
JournalOf PharmaceuticalSciences,January 1996,Vol.19(1),65-77.
3. SharmaVijay K1*, Liposomes: Present Prospective and Future
Challenges,InternationalJournalOf Current PharmaceuticalReview
And Research,oct 2010,vol1, issue 2,6-16
4. Himanshu Anwekar*,Liposome- as drug carriers, International
Journal Of Pharmacy&Life Sciences, V
ol.2, Issue 7: July:2011,
945-951
78
91. 5. MadhavNvs* And Saini A, Niosomes: ANovel Drug DeliverySystem,
InternationalJournalOf Research In Pharmacy And Chemistry, 2011,
1(3),498-511.
6.Lohumi Ashutosh, Rawat Suman, ANovel Drug Delivery System:
Niosomes Review, Journal Of DrugDelivery &Therapeutics; 2012,
2(5), 129-135.
7.Pawar Sd *,Pawar Rg, Niosome: An Unique Drug DeliverySystem,
InternationaljournalOf Pharmacy, Biology and Allied Sciences, April,
2012, 1(3): 406-416.
8.Rajesh Z. Mujoriya, Niosomal Drug Delivery System – AReview,
InternationalJournalOf Applied Pharmaceutics,Vol 3, Issue 3, 2011,7-
10.
79
