introduction to liposomes methods of preparation evaluation parameters

1. 3/6/2018 1
by
Ankit S Parulkar

2.  Liposomes are the simple microscopic vesicles in which
aqueous layer is enclosed by phospho lipid bilayers that are
used to transfer vaccines ,drugs ,enzymes and other
substances to targetcells or organs
 Structually ,liposomes are concentric bilayerd vesicles in
which an aqueous volume is entirely enclosed by a
membraneous lipid bilayer mainly composed of natural or
synthetic phospholipids.
 Can be produced from cholesterols, non toxic surfactants,
sphingolipids, glycolipids, long chain fatty acids and even
membrane proteins.
3/6/2018 2

3. 3/6/2018 3

4. COMPOSITION
Phospholipids:
Dilauryl phosphotidyl choline (DLPC), Dimyristoyl phosphotidyl choline
(DMPC), Dipalmitoy phosphotidyl choline (DPPC), Distearoyl phosphotidyl
choline (DSPC), Dioleolyl phosphotidyl choline (DOPC), Dilauryl
phosphotidyl glycerol (DLPG), Distearoyl phosphotidyl serine (DSPS).
Cholesterol:
Act as intercalator with phospholipids molecules.
Restrict the confirmational changes of lipids.
Membrane stabilizer.
3/6/2018 4

5. ADVANTAGES
Non-toxic.
Biodegradable.
Non-immunogenic.
Lowers systemic toxicity.
Targeted delivery.
Protection of sensitive drug molecules.
Enhance drug solubilisation ( Amphoterecin, Cyclosporins).
Improved pharmacokinetic effects.
3/6/2018 5

6. • Leakage of encapsulated drug during storage.
• Short half-life.
• Batch to batch variation.
• Difficult in large scale manufacturing and sterilization.
• Production cost is high.
• Once administered, liposomes can not be removed.
• Some times phospholipids undergoes hydrolysis and
oxidation reactions
3/6/2018 6

7. COMMONLY USED
PHOSPHOLIPIDS
NATURAL
PHOSPHATIDYL
CHOLINE
PHOSPHATIDYL
ETHANOLAMINE
PHOSPHATIDYL
SERINE
SYNTHETIC
DIOLEOYL
PHOSPHATIDYL
CHOLINE
DISTEAROYL
PHOSPHOTIDYL
CHOLINE
DIOLEOYL
PHOSPHATIDYL
ETHANOLAMINE
COMMONLY USED
PHOSPHOLIPIDS
3/6/2018 7

8. 3/6/2018 8

9. 3/6/2018 9

10. METHODS OF LIPOSOME
PREPARATION
PASSIVE LOADING
Involves loading of the entrapped
agents before or during the
manufacturing procedure
ACTIVE OR REMOTELOADING
Certain types of compounds with
ionizable groups and those with
both lipid and solubility , can be
introduced into the liposomes after
the formation of the intact vesicles
3/6/2018 10

11. 3/6/2018 11

12. METHODS OF PREPARATION OF
LIPOSOMES
All the methods of preparing liposomes involve three or four
basic stages
• Drying down lipids from organic solvent
• Dispersion of lipids in aqueous media
• Purification of resultant liposomes
• Analysis of final product
3/6/2018 12

13. Mechanical Dispersion Methods
Hand shaken MLV’s
Lipids + solvent ( chloroform: Methanol)
( In 250 ml RBF)
Evaporate for 15 min above phase transition
temperature(Flush with nitrogen)
Till residues dry
Add 5 ml buffer containing material to be entrapped
Rotate flask at room temp, at 60 RPM for 30 min
until lipid removes from wall of RBF
Milky white dispersion
(stand for 2 hours to get MLV
Rotary Evaporator
3/6/2018 13

14. Pro liposome
Sorbitol / Nacl ( increase surface area of lipid film)
+ 5ml lipid solution ( fitted to evaporator )
(Evaporation)
Again add lipid solution
Dry the content using Lyophilizer ( freeze dryer)
(Stand over night at room temp)
Flushed with nitrogen for drying properly
MLVs
3/6/2018 15

15. Freeze Drying
Lipid + Solvent ( Tertiary butanol)
Freeze drying
Add Aqueous phase / Saline containing drug
Rapid mixing above phase transition temperature
MLVs
3/6/2018 16

16. Micro emulsification liposome
(MEL)
•MEL is prepared by the “Micro fluidizer”, which pumps fluid at very high
pressure (10,000 psi) through a 5 um orifice.
•Then, it is forced along defined micro channels, which direct two streams
of fluid to colloid together at right angle at very high velocity.
•After a single pass, size reduced to a size 0.1& 0.2 um in diameter.
3/6/2018 17

17. Micro emulsification
3/6/2018 18

18. Sonicated unilamellar vesicles
MLV in test tube
Sonicate for 5-10 min above phase transition temp
Filter & centrifuge at 100000 rpm for 30 min at 20º c
Decant top layer to get Sonicated unilamellar vesicles
BATH SONICATOR PROBE SONICATOR
3/6/2018 20

19. French Pressure Cell
• French pressure cell is invented by ‘Charles Stacy French.
• In this technique the large vesicles are converted to small
vesicles under very high pressure.
•This technique yields uni or oligo lamellar liposomes of
intermediate size (30-80 nm in diameter depending on
applied pressure).
•This liposomes are more stable as compared to sonicated
liposomes.
3/6/2018 21

20. 3/6/2018 22

21. Membrane extrusion liposomes
• Mixture of surfactant, cholesterol and dicetyl phosphate
in chloroform is made into thin film by evaporation. The
film is hydrated with aqueous drug solution and the
resultant suspension extruded through polycarbonate
membranes, which are placed in series for upto 8
passages. It is a good method for controlling liposome
size.
• Less pressure is required here (> 100 psi) as compare
to French pressure cell.
3/6/2018 23

22. Freeze thaw sonication process
3/6/2018 24
The method is based on freezing of a unilamellar
dispersion & then thawing at room temp for 15 min. 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.

23. Dried reconstituted vesicle
SUV in aqueous phase + Solute
Freeze drying
DRV method: Rehydration, film stacks dispersed in
aqueous phase
Solute in uni or oligo lamellar vesicles.
3/6/2018 25

24. Ph induced vesiculation
MLVs or LUVs ( Ph 2.5-3)
Add 1 M NaOH
Ph rises to 11
Now add 0.1 M HCl
Ph moves down to 7.5
SUV
Change in Ph brings about an increase in surface charge density of lipid bilayer,
which induces spontaneous vesiculation
3/6/2018 26

25. Solvent dispersion method:
Ethanol injection
Lipid + ethanol solution in the syringe
Inject rapidly
In the aqueous phase
Small unilamellar vesicles
3/6/2018 27

26. Ether injection
Lipid + ether solution in the syringe
Inject slowly
In the aqueous phase ( On heated water bath, 60ºc)
Large unilamellar vesicles
3/6/2018 28

27. Water organic phase: Double emulsion
Organic solution + Lipid + Aqueous phase
Emulsion (W/O)
Hot aqueous solution of buffer
Multi compartment vesicle W/O/W (double emulsion)
LUVs
3/6/2018 29

28. Reverse phase evaporation: (MLV, LUV)
Emulsion
Evaporation under reduced pressure, rotary evaporator
Semi solid gel
Shake to get LUVs
“Lipid monolayer which enclosed the collapsed
vesicle, is contributed to adjacent intact vesicle to form
the outer leaflet of bilayer of LUV”.
3/6/2018 30

29. Stable plurilamellar vesicle (SPLVs)
•It involves preparation of water in organic phase dispersion
with an excess of lipid followed by drying under continued
bath sonication with stream of nitrogen.
•The internal SPLV is different from that of MLV, in that they
lack a large aqueous core.
•The internal environment of both the vesicle is different from
each other.
3/6/2018 31

30. DETERGENTDEPLETION(REMOVAL)METHODS:
• Detergents associate with the phospholipid molecules and serve to
screen the hydrophobic portions of molecule from water.
• The structures formed as a result of this association is known as
micelles.
• A three stage model of interaction for detergents with lipid
bilayers:
•Stage1: At low concentration detergents equilibrates
between vesicular lipid and water phase.
•Stage2: After reaching a critical detergent concentration,
membrane structure tends to unstable and transforms gradually in
to micelles.
•Stage3: All lipid exists in mixed micelle form.
• Three methods are applied for removal of detergent and transition
of mixed micelles to concentric bilayered form.
3/6/2018 32

31. DIALYSIS:
The molecules of detergent are removed from mixed micelle by
dialysis by lowering the concentration of detergent in bulk
aqueous phase.
eg: sodium cholate.
octylglucoside
COLUMN CHROMATOGRAPHY:
Removal of detergent is achieved by by passing the dispersion over
a sephadexg-25 column pre-saturated with constitutive lipids and
pre-equilibrated with hydrating buffer.
eg: deoxycholate.
3/6/2018 33

32. Evaluation of liposomes
• The liposomes prepared by various techniques are to be
evaluated for their physical, chemical as well as biological
properties, has these influence the behavior of liposomes in
vivo.
• Physical properties
1. Particle size
• Both particle size and particle size distribution of
liposomes influence their physical stability. These can be
determined by the following method.
• Laser light scattering
• Transmission electron microscopy
2. Surface charge
The passive, negative or natural charge on the surface of
the liposomes is due to the composition of the head groups.
3/6/2018 34

33. •The surface charge of liposomes governs on the extent of distribution
in-vivo, as well as interaction with the target cells.
•The method involved in the measurement of surface charge is based
on free-flow electrophoresis of MLVs.
•It utilizes a cellulose acetate plate dipped in sodium borate buffer of
Ph 8.8.
•About 5n moles of lipid samples are applied on to the plate, which is
then subjected to electrophoresis at 4 ͦc for 30 mins.
•The liposomes get bifurcated depending on their surface charge.
•This technique can be used for determining the heterogeneity of
charges in the liposome suspension as well as to detect any
impurities such as fatty acids
3/6/2018 35

34. 3. Percent drug encapsulated.
• Quantity of drug entrapped in the liposomes helps to estimate the
behavior of the drugin biological system
• Liposomes are misture of encapsulated and unencapsulated drug
fractions
• The %of drug encapsulation is done by first separating the free drug
fraction from encapsulated drug fraction
• Themethods usedto separate the free drugfrom the sample are:
a. Mini columncentrifugation method
b. Protamine aggregated method
4. Phasebehavior
•At transition temperature liposomes undergo reversible phase
transition
• Done by DSC
3/6/2018 36

35. 5. Drug Release Rate
The rate of drug release from the liposomes can be
determined by in-vivo assays which helps to predict the
pharmacokinetics and bioavailability of the drug. However
in- vivo studies are found to be more complete.
3/6/2018 37

36. Chemicalproperties
1. Determination of phospholipids
The phospholipid content of liposomes can be determined
directly by two assays, Bartlett assay and Steward assay.
(a)Bartlett assay
• This method of determining the phospholipid is very
sensitive and may produce erroneous results in the
presence of even trace amounts of inorganic phosphate.
Therefore, borosilicate glass tubes and double-distilled
water is used.
Initially the phosphorous present in the lipid bilayer of the
sample is hydrolyzed to inorganic phosphate.
3/6/2018 38

37. Then ammonium molybdate is added to convert inorganic
phosphate to phosphomolybdic acid(PMA).
The sample is then treated with aminonaphthylsulphonic acid
to quantitatively reduce the PMA to a blue-coloured
compound.
The intensity of the blue colour produced can be measured
by spectrophotometric means and the value is plotted on the
standard curve to obtain the content of phospholipids.
(b)Steward assay
This assay overcomes the drawbacks of Bratlett assay, but
cannot be used to mixture of unknown phospholipids.
3/6/2018 39

38. A standard curve is prepared by treating known
concentration of phospholipids in chloroform with 0.1 M
solution of ammonium ferrothiocyanate 9 reagent.
The sample are also treated with the same reagent and the
optical density is determined at 485 nm.
The absorbance of the sample can be plotted on the
standard curve to obtain the concentration of phospholipids.
2. Cholesterol analysis
Qualitative analysis
Performed using a capillary column filled with fused silica.
Quantitative analysis
The sample is reacted with a reagent (containing ferric
perchlorate, ethyl acetate and H₂SO₄) and the absorbance of
purple coloured complex is measured at 610 nm.
3/6/2018 40

39. MODES OF LIPOSOME AND CELL INTERACTION:
Adsorption Endocytosis
Fusion Lipid transfer
3/6/2018 41

40. APPLICATIONS
3/6/2018 42

41. Other applications
1). Liposomes in parasitic diseaseases and infections
•Liposomses get digested by phagocytic cells in body making them as ideal
vehicles for tageting drug to these macrophages.
•Example- leishmaniasis and fungal infections
2). Liposomes inbasic sciences
•Liposomes can be used to understand the topology, shape fluctuations,
phase behaviour, permeability, fission and fusion of biological mambranes.
•They can serve as a model to study vesiculation, vesicle shedding and
endo and exo- cytosis of living cells.
3). Application in cosmetics
•Liposomes being well hydrated help in reducing dryness of the skin and
ageing
•Can act as a supply which replenish lipids importantly, linolenic acid.
4).Liposomes in bioengineering
•Used in modern genetic engineering and gene recombinant technology
i.e. Fragments of DNA and Nucleic acids
5).Liposomes in Agro-food industry
• Due to demanding solubility properties of liposomes it can be used in food
processing.
3/6/2018 43

42. Lipofication
3/6/2018 44

43. 3/6/2018 45

44. Lack long term stability (short shelf life)
 Physical and chemical instability
 Freeze dry and pH adjustment
Low “Pay Load” - Poor Encapsulation
 Drugs and drugs without opposite charge
 Modifications
3/6/2018 46

45. COMMERCIAL PRODUCTS
• Doxorubicin(DOXIL).
• Daunorubicin(DAUNOXOME).
• Amphotericin B(APHOTEC, AMBISOME,
ABELCET).
• Cytarabine(DEPOCYTE).
3/6/2018 47

46. CONCLUSION
• Liposome over the years have been
investigated as major drug delivery system.
• The use of liposomes in delivery of drugs
and genes to tumour site are promising and
may serve as a handle for focus of future
research.
3/6/2018 48

47.  Y. Sultana., Liposomal Drug Delivery Systems: An Update Review.
Current Drug Delivery 2007; 4: 297-305.
 Sharma Shailesh, Sharma Neelam, Kumar Sandeep, Gupta GD.,
Liposomes: Areview., Journal of Pharmacy Research 2009;
2(7):1163-67.
 Mohammad Riaz., Liposomes Preparation Methods., Pakistan
Journal of Pharmaceutical Sciences January 1996; Vol.19(1): 65-
77.
 MU Uhumwangho and RS Okor., Current trends in the production and
biomedical applications of liposomes: a review .JMBR . June 2005; Vol.
4(1): 9-21.
 http://www.biopharminternational.com/biopharm/data/article
standard/biopharm/032002/7278/article.pdf., web, 28.01.2012
 http://www.ias.ac.in/jarch/currsci/68/00000742.pdf.,web,
28.01.2012
 D.D. Lasic., Applications of Liposomes. Handbook of Biological
Physics., Elsevier Science B.V.., 1995; Vol.1: 1-29
3/6/2018 49

48. 3/6/2018 50