liposomes

1. Liposomes- A Novel Drug Delivery System
By Tushar Chavan
M Pharmacy 2018-2019
K.Y.D.S.C.T.College of Pharmacy, Sakegaon
NMU, Jalgaon [MH]
Email: tusharchavantsc@gmail.com
Updated May 21, 2019
1. What are Liposomes
• Liposomes are concentric bilayered vesicles in which an aqueous core is entirely
enclosed by a membranous lipid bilayer mainly composed of natural or synthetic
phospholipids.
• Size range: 25nm-5000nm
• Liposomes consist of Cholesterol, Phospholipid and drug molecule.
2. A simple view of liposomes
“Liposomes are microscopic spheres made from fatty materials, predominantly
phospholipids. “Made up of one or more concentric lipid bilayers, and range in size from
50 nanometers to several micrometers in diameter
3. Advantages with liposomes
 Suitable for delivery of hydrophobic, hydrophilic and amphipatic drugs and agents
 Liposomes increases efficacy and therapeutic index of drug (actinomycin-D)
 Liposome increased stability via encapsulation
 Suitable for controlled release
 Suitable to give localized action in particular tissues.
 Suitable to administer via various routes
 Liposomes help reduce the exposure of sensitive 4 tissues to toxic drugs
4. Disadvantages of liposomes
 Production cost is high.
 Leakage and fusion of encapsulated drug / molecules.
 Sometimes phospholipid undergoes oxidation and hydrolysis like reaction.
 Short half-life.
 Low solubility.
5. Components of liposomes
The structural components of liposomes include: A. Phospholipids B. cholesterol
A. PHOSPHOLIPID • Major component of biological cell membrane Phospholipid
Hydrophobic tail 2 fatty acid chain containing 10-20 carbon atoms 0-6 double bond in each
chain Hydrophillic head or polar head Phosphoric acid bound to water soluble molecule

2. Commonly used other Phospholipids Natural phospholipid
I. PC- phosphatidyl choline
II. PE- phosphatidyl ethanolamine
III. PS –phosphatidyl serine Synthetic phosholipid
IV. DOPC = Dioleoyl Phosphatidylcholine
V. DOPE = Dioleoyl phosphatidyl ethanolamine
VI. DSPC = Distearoyl phosphatidyl choline
VII. DSPE = Distearoyl phosphatidyl ethanolamine
VIII. DLPC = Dilauryl phosphatidyl choline
Phosphatidylcholine PC
• The most common phospholipid use is phosphatidylcholine PC
• Phosphatidylcholine is amphipathic molecule containing i. A hydrophillic polar head
group phosphocholine ii. A glycerol bridge iii. A pair of hydrophobic acyl hydrocarbon chain
B. Cholesterol:
• Incorporation of sterols in liposome bilayer can bring about major changes in the
preparation of these membranes.
• Cholesterol does not by itself form bilayer structure,but can be incorporated into
phospholipid membranes in very high concentration unto 1:1 or even 2:1 molar ratios of
PC.
• Cholesterol incorporation increases the separation between the cholin head groups and
eliminates the normal electrostatic and hydrogen-bonding interactions.
Role of cholesterol in bilayer formation:
I. Cholesterol act as fluidity buffer
II. After intercalation with phospholipid molecules alter the freedom of motion of
carbon molecules in the acyl Chain
III. Restricts the transformations of
IV. Trans to gauche Conformations.
V. Incorporated into phospholipid membrane upto 1:1 or 2:1 of cholesterol to PC.
6. Preparation of Liposomes • Mechanism of Vesicle Formation A. The budding theory B.
The bilayer phospholipids theory
A.The budding theory – Stress induced hydration of phospholipids – Organization in to
lamellar arrays – Results in to budding of lipid bilayer leading to downsizing SUV OLV 13
B.The bilayer phospholipids theory
• Liposomes are formed when thin lipid films are hydrated
•The hydrated lipid sheets detach during agitation and self-close to form large,
multilamellar vesicles (LMV)

3. 7. Modes of liposomes/cell interaction 1. Endocytosis 2. Adsorption 3.fusion 4. Lipid
transfer
8. Factors affecting release of drug
• Solvents
•pH
•Temperature
• Agitation
• Enzymes
• Cell culture
• Volume Drug release from liposomes
•the lipid bilayer of the liposome can fuse with other bilayers (e.g. cell membrane) thus
delivering the liposome contents.
9. CLASSIFICATION OF LIPOSOMES
Structure Method of preparation Composition and application Conventional liposome
Speciality liposome
18. 1. Classification of liposomes based on Structure Unilamellar (UV)-all size range Small
Unilamellar(SUV) [20-100nm] Medium Unilamellar (MUV) Large Unilamellar (LUV)
[>100nm] Giant Unilamellar (GUV) [>1μm] Multi Lamellar Vesicles (MLV) [0.5nm]
Oligolamellar Vesicles (OLV) Multi Vesicular (MV) [>1μm] 18
19. 2. Classification of liposomes Based on Method of Preparation Single or oligo lamellar
vesicle made by reverse phase evaporation method (REV’s) Multi lamellar vesicle made by
reverse phase evaporation method (MLV-REV Stable pluri lamellar vesicle (SPLV) Frozen
and thawed multi lamellar vesicle (FATMLV) Vesicle prepared by extrusion technique (VET)
Dehydration- Rehydration method (DRV) Dehydration- Rehydration method (DRV) 19
20. 3. Classification of liposomes Based on Composition and Application Type of liposome
Abbreviation Composition Conventional liposome CL Neutral of negatively charge
phospholipids and cholesterol Fusogenic liposome RSVE Reconstituted sendai virus
enveops pH sensitive liposomes - Phospholipids such as PER or DOPE Cationic liposome -
Cationic lipid with DOPE Long circulatory liposome LCL Neutral high temp, cholesterol and
5-10% PEG, DSP Immune liposome IL CL or LCL with attached monoclonal antibody or
recognition sequences 20
21. 4. Classification of Liposomes Based Upon Conventional Liposome 1). Stabilize natural
lecithin (PC) mixtures 2). Synthetic identical, chain phospholipids 3). Glycolipids containing
liposome 21
22. 5.)Classification of Liposomes based upon Speciality 5.) Liposomes Bipolar Fatty Acids
Antibody directed Liposome Methyl/Methylene x- linked liposome Lipoprotein coated
liposome Carbohydrate coated liposome Muiltiple Encapsulated Liposome 22
10. Methods of liposome preparation
1. Passive loading techniques
2. Active loading techniques

4. 3. Mechanical dispersion methods
4. Solvent dispersion methods
5. Detergent removal technique
 LIPID FILM HYDRATION
 BY HAND SHAKING,
 FREEZE DRYING
 NON HAND SHAKING
 MICRO EMULSIFICATION
 SONICATION
 FRENCH PRESSURE CELL
 MEMBRANE EXTRUSON
 DRIED RECONSTITUTED VESICLES
 ETHANOL INJECTION
 ETHER INJECTION
 DOUBLE EMULSION
 REVERSE PHASE EVAPORATION
6. Detergent removal from mixed vesicles by
 DIALYSIS
 Column Chromatography
 DILUTION
11. General Method Of Liposome Preparation
1 • Cholesterol + Lecithin + Charge and Dissolve in organic solvent
2 • Drying down lipid from organic solvent( Vaccum ) • Dispersion of lipid in aqueous
media (Hydration
3 • Purification of resultant Liposomes • Analysis of final product 24
25. 1. Mechanical dispersion method Lipid dissolve in organic solvent/co-solvent Remove
organic solvent under vacuum Film deposition Solid lipid mixture is hydrated by using
aqueous buffer Lipid spontaneously swell & Hydrate Liposome 25
26. 1) Lipid Hydration Method A.) By hand shaking method vesiculate to form Multi
lamellar vesicles(MLVs) Upon hydration lipids swell and peel out from RB flask Then film is
treated with aqueous medium Lipids form stacks of film from organic solution 26
27. 1) Lipid Hydration Method B.) NON-
from hand shaken method wherein it uses a stream of nitrogen to provide agitation rather
n
for 15-20 min Milky suspension centrifugation LUV 27
28. 1) Lipid Hydration Method C.) FREEZE DRYING • Another method of dispersing the lipid
in a finely divided form, prior to addition of aqueous media, is to freeze dry the lipid
dissolved in a suitable organic solvent. • The solvent choice depends on the freeze point
which needs to above the temperature of the condenser lyophilizers. Tertiary butanol is

5. considered to be most ideal solvent. • After obtaining the dry lipid which is an expanded
foam like structure, water or saline can be added with rapid mixing above the phase
transition temperature to give MLVs. 28
29. 2.) MICRO EMULSIFICATION • This method is provided for preparing small lipid vesicles
in commercial quantities by microemulsifying lipid compositions using very high shear
forces generated in a homogenizing apparatus operated at high pressures at a selected
temperature. • At least 20 circulations (approximately 10 minutes) but not greater than
200 circulations (100 minutes) are sufficient to produce a micro emulsion of small vesicles
suitable for biological application. 29
this method.
re of the lipid dispersion is
usually easier in this method, in contrast to sonication by dispersal directly using the tip.
30
31. 4.) FRENCH PRESSURE CELL • The method involves the extrusion of MLV at 20,000 psi
at 4°C through a small orifice. • The method has several advantages over sonication
method. • The method is simple rapid, reproducible and involves gentle handling of
unstable materials. • The resulting liposomes are somewhat larger than sonicated SUVs. •
The drawbacks of the method are that the temperature is difficult to achieve and the
working volumes are relatively small (about 50 mL maximum). 31
Vesicle contents are exchanged with dispersion medium during breaking and resealing of
entrapment, the water soluble compounds should be present in suspending medium
during the extrusion process. 32
33. 6.) DRIED RECONSTITUTED VESICLES SUV’s in SUV’s with DRV Freeze dried aqueous
solute to be membrane phase entrapped Solutes with oligo and uni lamellar Freeze
membranes drying Rehydration 33
34. B] Solvent dispersion methods • Ethanol injection –SUV • Ether injection -LUV •
Reverse phase evaporation vesicle –LUV • Double emulsion vesicles Stable plurilamellar
vesicles 34
35. 1.) Ethanol Injection Method • A lipid solution of ethanol is rapidly injected to a vast
excess of buffer. The MLVs are immediately formed. • The drawbacks of the method are
that the population is heterogeneous (30-110 nm), liposomes are very dilute, it is difficult
to remove all ethanol because it forms azeotrope with water and the possibility of various
biologically active macromolecules to inactivation in the presence of even low amounts of
ethanol. 35
36. 2.) Ether Infusion Method • A solution of lipids dissolved in diethyl ether or
ether/methanol mixture is slowly injected to an aqueous solution of the material to be
encapsulated at 55-65°C or under reduced pressure. • The subsequent removal of ether

6. under vacuum leads to the formation of liposomes. • This method is used to treat sensitive
lipids very gently. 36
37. Double emulsification • In this process, an active ingredient is first dissolved in an
aqueous phase (w1) which is then emulsified in an organic solvent of a polymer to make
a primary w1/o emulsion. • This primary emulsion is further mixed in an emulsifier-
containing aqueous solution (w2) to make a w1/o/w2 double emulsion. • The removal of
the solvent leaves microspheres in the aqueous continuous phase, making it possible to
collect them by filtering or centrifuging. 37
38. 3.) Reverse-phase evaporation Lipid organic solvent and aqueous solution are i. mixed,
ii. sonicated, iii. formation of w/o emulsion, iv. evaporate to remove the organic solvent
Lipids form a phospholipid bilayer on vigorous shaking , water droplets collapse and
formation of LUV’s takes place. 38
39. Reverse phase evaporation technique. Lipid in solvent solution Two-phase system
Water in oil emulsion Gel formation Solvent removal REV liposomes Conti… 39
40. C. Detergent removal method • The micellar dispersion is then subjected to one of the
following methods to remove the detergent: I. DIALYSIS :- Detergents with high CMC (10-
20 mM ) are used so that their removal is facilitated e.g. bile salts- sodium cholate and
sodium deoxycholate , or synthetic detergents like octileglucoside . II. COLUMN
CHROMATOGRAPHY :- By passing dispersion over a Sephadex G-25 column. 40
41. i.) Dialysis Method Dialysis is the simplest procedure used for the removal of the
unbound drug, except when macromolecular compounds are involved Advantages:
effective in removing nearly all of the free drug with a sufficient number of changes of the
dialyzing medium 41
42. ii.) Column Chromatography • Phospholipid in the form of either sonicated vesicle or
as a dry film, at a molar ratio of 2:1 with deoxycholate form unilamellar vesicles of 100nm
on removal of deoxycholate by column chromatography 42
43. B. Active loading technique AFTER DRYING IN PROCESS FILM/CAKE OF LIPID IS FROM
STACKS OF LIPID BILAYER FORM SWELLING IN FLUID SHEET IS SELF CLOSE LOADING OF
DRUG ON pH-
Two steps process generates this pH imbalance and active (remote) loading. 1) Vesicles
are prepared in low pH solution, thus generating low pH within the liposomal interiors 2)
amino groups are relatively lipophipic at high pH and hydrophilic at low pH. 43
44. 1.) Lyophilization • Freeze-drying (lyophilization) involves the removal of water from
products in the frozen state at extremely low pressures. • The process is generally used to
dry products and are thermo labile and would be destroyed by heat-drying. • The
technique has a great potential as a method to solve long term stability problems with
respect to liposomal stability. • It is exposed that leakage of entrapped materials may take
place during the process of freeze- drying and on reconstitution. 44
45. 2.) Pro-liposomes: lipid Dried over lipid Finely divided particulate support like
continuous hydration and lipid is dried over the finally divided particulate support i.e.-
NaCl, Sorbitol, or other polysaccharides. These dried lipid coated particulates are called as

7. support is rapidly dissolved and lipid film hydrate to form MLVs 45
12. Uses of liposomes
• Chelation therapy for treatment of heavy metal poisoning
• Enzyme replacement
• Diagnostic imaging of tumors
• Study of membranes
• In gene delivery.
• As drug delivery carriers.
• Enzyme replacement therapy.
• Liposomes in antiviral/anti microbial therapy.
• In multi drug resistance.
• In tumour therapy.
• In immunology.
• In cosmetology 46
13. Applications
• Liposomes are successfully used to entrap anticancer drugs. This increases circulation
life time, protects from metabolic degradation.
1.Cancer chemotherapy • Steroids used for arthritis can be incorporated into large MLVs.
• Alteration in blood glucose levels in diabetic animals was obtained by oral administration
of liposome encapsulated insulin
2.Liposomes as carrier of drug in oral treatment • Drugs like triamcilone, methotrexate,
benzocaine, corticosteroids etc can be successfully incorporated as topical liposome
3.Liposomes for topical applications
4. Liposomes for pulmonary delivery Inhalational devices like nebulizers are use to
produce an aerosol of droplets containing liposomes. 47
Especially good for in- -1000 times more plasmid DNA
needed for the same transfer efficiency as for viral vector 48
49. Lipofection 49
50. Liposomes could serve as tumor specific vehicles (even without special targeting)
Liposomes better penetrate into tissues with disrupted endothelial lining 50
14. Characterization of liposomes
• Shape, size and its distribution
• Surface charge
• Percentage drug entrapment
• Entrapped volume
• Lamellarity
• Phase behavior of liposome

8. • Percentage drug release
15. References
1) ‘Controlled and Novel Drug Delivery’, “JAIN N.K.’’, CBS Publisher And Distributors.Page
No.307-321.
2) ‘Targeted and controlled drug delivery, Novel carrier Systems’ , “VYAS S.P. and KHAR
R.K.’’, CBS Publishers Page no.181 -195.
3) A.Chonn,P.R.Cullis,“Recent advances in liposome technologies and their applications for
systemic gene delivery”,Advanced Drug Delivery Reviews
4) Liposomes preparation methods by Mohammad riaz ,Pakistan Journal of
Pharmaceutical Sciences Vol.19(1), January 1996, pp.65-77
5) Liposome- as drug carriers-International Journal of Pharmacy & life sciences- Himanshu
Anwekar*, Sitasharan Patel and A.K Singhai
6) http://www.avantilipids.com
7) http://www. Mssm.edu/medicine/thrombosis/phosphol.html
8) Garrett, R. and Grisham C. Biochemistry, 2nd ed. Saunders Colleges Publishing. New
York (1999). 264
9) "Liposomes." www.collabo.com/liposom0.htm
10) Sharma Vijay K1*, Liposomes: Present Prospective and Future
Challenges,International Journal Of Current Pharmaceutical Review And Research, oct
2010,vol1, issue 2,6-16
11) Himanshu Anwekar*, Liposome- as drug carriers, International Journal Of Pharmacy &
Life Sciences, Vol.2, Issue 7: July: 2011, 945-951 52
53. 53