4. Methods of preparation of liposomes:
Solvent dispersion methods:
In this method, lipids are first dissolved in an organic
solution, which is then brought into contact with an
aqueous phase containing materials to be entrapped within
the liposomes. The lipids align themselves at the interface
of organic & aqueous phase forming monolayer of
phospholipids, which forms the half of the bilayered of the
liposome.
1.Ethanol injection.
2. Ether injection
3. Double emulsion vesicles
4. Reverse phase evaporation vesicles
5. Stable plurilamellar vesicles.
5. Ethanol injection: In this method, an ethanol solution of lipids is
injected rapidly through a fine needle into an excess of saline or
other aqueous medium. The rate of the injection is usually
sufficient to achieve complete mixing, so that the ethanol is
diluted almost instantaneously in water, and phospholipids
molecules are dispersed evenly throughout the medium. This
procedure yields a high proportion of SUVs.
Ether injection: It involves injecting the immiscible organic
solution very slowly into an aqueous phase through a narrow needle
at the temperature of vaporising the organic solvent. This method
may also treat sensitive lipids very gently. It has little risk of
causing oxidative degradation provided other is free from
peroxides.
6.
7. Rapid solvent exchange vesicles:
This method involves passing the organic solution of the lipids
through the orifice of blue tipped syringe under the vacuum into
a tube containing aqueous buffer. The tube is mounted on the
vortexes. Bulk solvent vaporizes & is removed within seconds
before coming in contact with aqueous environment, while the
lipid mixture rapidly precipitates in an aqueous buffer.
8. Double emulsion vesicles: The double emulsion is prepared by emulsion
is prepared by rapidly injecting the micro-droplet into hot aqueous
solution of Tris-buffer with the help of 22-gauge hypodermic needle
under vigorous stirring. The organic solvent is evaporated using strong
jet of nitrogen thus forming double emulsion. The last traces of organic
solvent are removed by evaporation & finally the volume is adjusted by
adding extra-distilled water & then the product is centrifuged at 200
c for
30 min at 37,000g to remove lipid aggregates.
Reverse phase evaporation vesicles: In this method, several
phospholipids (pure/mixed with cholesterol) can be used. The lipid
mixture is added to a round bottom flask and the solvent is removed
under reduced pressure by a rotary evaporator. The system is purged
with nitrogen and the lipids are re-dissolved in the organic phase. This is
the phase that the reverse phase vesicles will form. Diethyl ether and
isopropyl ether are the usual solvents of choice.
9. Formation of different liposomes using reverse phase evaporation
method. MLV-REVs are formed in presence of excess phospholipids,
whereas LUV-REVs are formed in absence of extra lipid.
10. Active loading technique:
This method loads the drug molecules into preformed
liposomes using pH gradients & potential difference across
liposomal membranes. Active loading methods have the
following advantages over passive encapsulation techniques:
A high encapsulation efficiency & capacity.
A reduced leakage of encapsulated compounds.
“Bed side” loading of drugs thus limiting loss of retention of
drugs by diffusion, or chemical degradation during storage.
Flexibility for the use of constitutive lipids, as drug is loaded
after the formation of carrier units.
Avoidance of biological active compounds during preparation
steps in the dispersion thus reducing safety hazards.
For amphipathic weak acids by remote loading procedures
using a calcium acetate gradient
11.
12. Weak amphipathic bases accumulate in the aq. phase of
lipid vesicles in response to a difference in pH between
the inside & outside of the liposomes. The pH gradient
is created by preparing liposomes with low pH inside &
outside the vesicles, followed by the addition of the
base to the extraliposomal medium. Usually, 2 step
processes generates this pH imbalance & remote
loading: 1st, the vesicles are prepared by in a low-pH
within the liposomal interiors, followed by the addition
of the base to the extraliposomal medium. The 2nd
step involves the exchange of external medium by gel
exclusion chromatography with a neutral solution.
18. Provide selective passive targeting to tumor.
Increased efficacy and therapeutic index.
Increased stability via encapsulation.
Reduction in toxicity of the encapsulated agent.
Site avoidance effect.
Improved pharmacokinetic effects(reduced
elimination, increased circulation life times).
Flexibility to couple with site-specific ligands to
achieve active targeting.
19. Relatively slow penetration into tumors
Various degree of accumulation in the RES
Variable stability in vivo
Formulation are Costly
20. Therapeutic applications of liposomes
Liposomes are used for the following range of therapeutic
& pharmaceutical applications
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.
21. 3. Liposomes in tumour therapy
Carrier of small cytotoxic molecules.
Vehicles for macromolecules as cytokines or genes.
4. Liposomes in gene delivery
Gene & antisense therapy
Genetic vaccination
5. Liposome in immunology
6. As artificial blood surrogates
7. As radiopharmaceutical & radio diagnostic carriers
8. In cosmetics & dermatology.
9. In enzyme immobilization & bioreactor technology.
22. Current liposomal drug preparations:
Type of Agents Examples
Anticancer Drugs
Anti bacterial
Antiviral
DNA material
Enzymes
Radionuclide
Fungicides
Vaccines
Malaria merozoite, Malaria sporozoite
Hepatitis B antigen, Rabies virus glycoprotein
Amphotericin B*
In-111*, Tc-99m
Hexosaminidase A
Glucocerebrosidase, Peroxidase
Duanorubicin, Doxorubicin*, Epirubicin
Methotrexate, Cisplatin*, Cytarabin
Triclosan, Clindamycin hydrochloride,
Ampicillin, peperacillin, rifamicin
AZT
cDNA - CFTR*
23. Characterisation of liposome
The characterisation purpose for the purpose of evaluation could be
classified into 3 broad categories, which include Physical, Chemical &
Biological parameters.
Physical characterization evaluates various parameters including
1. size
2. shape
3.surface features
4. lamellarity
5. phase behaviour
6. drug release profile
Chemical characterisation includes those studies which establish the
purity & potency of various liposomal constituents.
Biological characterisation parameters are helpful in establishing the
safety & suitability of the formulations for the in vivo use or for
therapeutic applications.
24. 1) Vesicle shape & lemellarity: Vesicle shape can be assessed
using Electron Microscopic Techniques. Lamellarity of vesicles
i.e. number of bilayers present in liposomes is determined using
Freeze-Fracture Electron Microscopy and P-31 Nuclear
Magnetic Resonance Analysis.
2) Vesicle size & size distribution: These include Light
Microscopy, Fluorescent Microscopy, Electron Microscopy
(specially Transmission Electron Microscopy), Laser light
scattering.
3)Surface charge: Liposomes are usually prepared using
imparting constituting lipids and hence it is imperative to study
charge on vesicle surface. Two methods are used to assess the
charge- namely free flow electrophoresis and zeta potential
measurement.
25. 4) Encapsulation efficiency & Trapped volume: Encapsulation
efficiency is assessed using 2 techniques including minicolumn
centrifugation method and Protamine aggregation method.
a) Minicolumn centrifugation is generally used both as a mean of
purification and separation of liposomes on small scale & analysis
of a liposomal dispersion to determine encapsulation efficiency.
5) Drug Release: The mechanism of drug release from liposomes
can be assessed by use of well calibrated in vitro diffusion cell.
The liposome based formulation can be assisted by employing in
vitro assays to predict pharmacokinetics and bioavailability of
drug before employing costly and time-consuming in vivo studies.
26. EVALUATION OFEVALUATION OF
LIPOSOMESLIPOSOMES Solubility study
In vitro drug release study
Analysis by second-order spectrophotometry
Microscopy
Drug leakage from vesicles
Skin retention studies
Drug entrapment determination
27. Drug entrapment/drug loading
0.2 ml vesicular suspension
Pre-saturated with empty vesicles and pack in
column
Centrifuged at 2000 rpm for 3 min.
Collect elutes containing drug-loaded vesicles
Observe under light microscope for drug particles
Analysed by UV
spectrophotometry
28. MICROSCOPY
All the batches viewed under optical microscope to
determine shape and lamellarity of vesicles.
The liposome’s vesicle sizes were determined by light
scattering on the basis of laser diffraction using Malvern
Master sizer.
29. DRUG LEAKAGE STUDY
Optimized liposome batches (PLH 4, PLG 7)
Sealed in the 30 ml vials after purging with nitrogen
Stored at various temperatures (4 -8, 25 ± 2, 37 &
45 ºC) for the period of 2 months
Samples withdrawn at definite time interval
Residual amount of drug in vesicles was
determined as described in drug entrapment
30. SOLUBILITY
It is determined using thermostatic water shaker
bath at 37o
C for 24 Hrs.
Excess drug was added to 10 ml of phosphate buffer
saline (pH 6.4)
Increasing concentration of surfactant tween 20
was added
After 24 Hrs filter using 0.45µ m membrane filter
Analyse after appropriate dilution using
spectrophotometer at 272 nm
31. IN VITRO DRUG RELEASE
Drug release studies includes:
Dialysis-membrane
Skin permeation
Requirements:
Hairless abdominal skin of LACA mice
Franz diffusion cell
32. PLH 4 & PLG 7 & conventional creams with
MCZ each equivalent to 5 mg of drug
Applied to membrane & skin
Receptor phase was 150 ml PBS (pH6.4) & 2%
w/v Tween 20
Withdraw 5 ml of sample from each batch &
replace with same amount of buffer
The samples were quantitiated by UV
spectrophotometer at 272 nm
33. SKIN RETENTION STUDIES
After conducting permeation studies remove the skin
mounted on Franz diffusion cell
Scrap the remaining formulation adhering to skin and wipe
with tissue paper
The cleaned skin piece was mashed
Add 10 ml of methanol was added to the meshed mass
Shaken in water shaker bath at 37±1 o
C for 24 hr for
complete extraction of drug
Filter and remove the filtrate and analyse by
spectrophotometry
34. NIOSOMES
Niosomes are a novel drug delivery system, in which the
medication is encapsulated in a vesicle composed of a
bilayer of non-ionic surface active agents .
These are very small, and microscopic in size that lies in
the nanometric scale. Although structurally similar to
liposomes, they offer several advantages over them.
Niosomes have recently been shown to greatly increase
transdermal drug delivery and also in targeted drug
delivery
A diverse range of materials have been used to form
niosomes such as sucrose ester surfactants and
polyoxyethylene alkyl ether surfactants, alkyl ester, alkyl
amides, fatty acids and amino acid compound.
35. Structure Of Niosomes
Niosomes are microscopic lamellar structures, which
are formed on the admixture of non-ionic surfactant
of the alkyl or dialkyl polyglycerol ether class and
cholesterol with subsequent hydration in aqueous
media.
The hydrophilic ends are exposed on the outside and
inside of the vesicle, while the hydrophobic chains
face each other within the bilayer.
Hence, the vesicle holds hydrophilic drugs within the
space enclosed in the vesicle, while hydrophobic
drugs are embedded within the bilayer itself.
37. ETHER INJECTION METHOD
Slow injection of an ether solution of niosomal
ingredients into an aqueous medium at high
temperature
A mixture of surfactant and cholesterol (150 μmol) is
dissolved in ether (20 ml) and injected into an aqueous
phase (4 ml) using a 14- gauge needle syringe
Temperature of the system is maintained at 60oC
during the process
Niosomes in the form of large unilamellar vesicles
(LUV) are formed
38. FILM METHOD
The mixture of surfactant and cholesterol is dissolved
in an organic solvent (e.g. diethyl ether, chloroform,
etc.) in a round-bottomed flask
The organic solvent is removed by low pressure/vacuum
at room temperature
The resultant dry surfactant film is hydrated by
agitation at 50-60oC
Multilamellar vesicles (MLV) are formed
39. SONICATION
The aqueous phase is added into the mixture of
surfactant and cholesterol in a scintillation vial
Homogenized using a sonic probe
The resultant vesicles are of small unilamellar (SUV)
type niosomes
The SUV type niosomes are larger than SUV liposomes
It is possible to obtain SUV niosomes by sonication of
MLV type vesicles
40. REVERS PHASE EVAPORATION
Surface-active agents are dissolved in chlorofom, and
0.25 volume of phosphate saline buffer (PBS) is
emulsified to get w/o emulsion
The mixture is sonicated and subsequently chloroform
is evaporated under reduced pressure
The surfactant first forms a gel and then hydrates to
form niosomal vesicles
The vesicles formed are unilamellar and 0.5 μ in
diameter
41. Bubble Method :
It is novel technique for the one step preparation of
liposomes and niosomes without the use of organic
solvents.
The bubbling unit consists of round-bottomed flask with
three necks positioned in water bath to control the
temperature.
Water-cooled reflux and thermometer is positioned in the
first and second neck and nitrogen supply through the third
neck.
Cholesterol and surfactant are dispersed together in this
buffer (pH 7.4) at 70°C, the dispersion mixed for 15
seconds with high shear homogenizer and immediately
afterwards “bubbled” at 70°C using nitrogen gas.
42. Applications Of Niosomes
It is used as Drug Targeting.
It is used as Anti- Neoplastic Treatment i.e. Cancer
Disease.eg.Methotrexate
It is used as Leishmaniasis i.e. Dermal and
Mucocutaneous infections e.g. Sodium stibogluconate.
It is used act as Delivery of Peptide Drugs.
It is used in Studying Immune Response.
Niosomes as Carriers for Hemoglobin.
Transdermal Drug Delivery Systems Utilizing Niosomes.
eg.Erythromycine
It is used in Ophthalmic drug delivery. eg.Cyclopentolate
43. Advantages of Niosomes
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 characteristics such as size, lamellarity etc. of the vesicle can
be varied depending on the requirement.
The vesicles can act as a depot to release the drug slowly and of
controlled release
They are osmotically active and stable.
They increase the stability of the entrapped drug
Handling and storage of surfactants do not require any special
conditions
Can increase the oral bioavailability of drugs
44. DISADVANTAGES OF
NIOSOMES
1. Physical instability
2. Aggregation
3. Fusion
4. Leaking of entrapped drug
5. Hydrolysis of encapsulated drugs which
limiting the shelf life of the dispersion.
45. Characterization of Niosomes:
a) Bilayer formation :Assembly of non-ionic surfactants to form
bilayer vesicle is characterized by X-cross formation under
light polarization microscopy.
b) Number of lamellae :It is determined by using NMR
spectroscopy, small angle X-ray scattering and electron
microscopy
c) Membrane rigidity : Membrane rigidity can be measured by
means of mobility of fluorescence probe as function of
temperature
46. d) Entrapment efficiency – After preparing niosomal dispersion,
un-entrapped drug is separated by dialysis, centrifugation, or gel
filtration as described above 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. Where,
– Entrapment efficiency (EF) = (Amount entrapped total
amount) x 100
Evaluation :
Particle size determination
Removal of un-entrapped drug
Percentage drug entrapment
Drug content analysis
In-vitro release study
Stability studies
47. 1. Entrapment efficiency :
(EF)=(Amount entrapped/total amount) x100
2. partical size determination: Niosomes similar to
liposomes, assume spherical shape and so their diameter
can be determined using light microscopy, photon
correlation microscopy and freeze fracture electron
microscopy.
3. In-vitro release :
A method of in-vitro release rate study includes the use of
dialysis tubing.
A dialysis sac is washed and soaked in distilled water. The
vesicle suspension is pipetted into a bag made up of the
48. The bag containing the vesicles is placed in 200 ml of
buffer solution in a 250 ml beaker with constant shaking at
25°C or 37°C.
At various time intervals, the buffer is analyzed for the
drug content by an appropriate assay method of vesicles
during the cycle.
4. Stability study : All niosomal formulations were subjected
to stability studies by storering at 4 C, 25 C and 37 C in
thermostatic oven for the period of three months.
• After one month, drug content of all the formulations were
checked by method discussed previously in entrapped
efficiency parameter. Invitro release studies of selected
formulations were also carried out
49. Niosomes vs liposomes
In both basic unit of assembly is Amphiphiles, but
they phospholipids in liposomes and nonionic
surfactants in niosomes.
Both can entrap hydrophilic and lipophilic drugs.
Both have same physical properties but differ in
their chemical composition.
Niosomes has higher chemical stability than
liposomes.
Niosomes made of uncharged single chain surfactant
molecules
Liposomes made of neutral or charged double chain
phospholipids.
50. Marketed Product
Lancome has come out with a variety of antiageing
products which are based on niosome formulations
L’Oreal is also conducting research on antiageing
cosmetic products.
Niosomes Preparation in the Market is – Lancôme
51. References
Liposomes as Drug Carriers” Controlled and Novel Drug Delivery,
N.K. Jain, Pg.no.305-343.
Targeted and Controlled Drug Delivery, Novel carrier system” Vyas
S.P., Khar R.K, CBS Publisher, Pg.no. 173-206.
Journals
Allen, Theresa M. "Liposomal Drug Formulations: Rationale
for Development and What We Can Expect for the Future."
Drugs 56: 747-756, 1998.
Malhotra M and Jain NK. Niosomes as Drug Carriers. Indian Drugs
31 (3), 1994, 81-86.
S. P. Vyas Novel Drug Delivery System,1st edition,2009,CBS
Publishers & Distributors, New Delhi, Pg. no.284-295
