liposomes preparation and evaluation and its advantages.detailed about evaluation targeted drug delivery

1. LIPOSOMES
PRESENTED BY
SUJITHA MARY
MPHARM
ST JOSEPH COLLEGE OF PHARMACY

2. INTRODUCTION
• Liposomes are simple microscopic vesicles in
which an aqueous volume is entirely enclosed by
a membrane composed of lipid molecule.
•Structurally, Liposomes are concentric bilayered
vesicles in which an aqueous volume is entirely
enclosed by a membranous lipid bilayers mainly
composed of natural or synthetic phospholipids.

3. GENERAL STRUCTURE

4. ADVANTAGES
1. Liposomes increased efficacy and therapeutic index of
drug.
2. Liposomes increased stability via. encapsulation.
3. Provide selective passive targeting to tumour tissues.
4. Flexibility to couple with site-specific ligands to
achieve active targeting.
5. Improved pharmacokinetic effects (reduced
elimination, increased circulation life time).
6. Liposomes reduce the toxicity of the encapsulated
agent.
7. Facilitation of transport across membranes.
8. Naturally occurring lipids are non-toxic &
biodegradable

5. DISADVANTANGES
• Low solubility in water.
• Production cost is high.
• Short half-life.
• Sometimes phospholipid undergoes oxidation
and hydrolysis like reaction.
• Leakage and fusion of encapsulated drug /
molecules

6. CLASSIFICAION
1)On the basis of composition and applications
• CL (Conventional liposomes),
• Fusogenic liposomes,
• . pH sensitive liposomes,
• Cationic liposomes,
• Immuno-liposomes.
• Long circulatory liposomes (stealth)

7. 2)On the basis of structural parameter
• Multilamellar vesicles
• Oligiolamellar vesicles
• Unilamemellar vesicles
• Small unilamellar vesicles
• Medium sized unilamellar vesicles
• Large unilamellar vesicles
• Giant unilamelalr vesicles
• multi vesicular vesicles

8. 3) On the basis of preparation
• Vesicles prepared by reverse evaporation
method
• Multilamellar vessicle by REV
• Stable plurilamellar vesicles
• Frozen and thawed MLV
• Vesicles prepared by extraction technique
• Dreied reconstituted vesicles

9. Methods of liposome preparation
These can be categorized on the basis of lipid
dispersion.
Physical dispersion technique:
a) Lipid film hydration by Hand-shaking (MLV),
non-hand shaking (LUV)
b) High shear homogenization/ Sonication
c) Membrane extrusion
d) Microfluidizer techniques for Micro emulsification
e) French pressure cell
f) Dried-reconstituted vesicles
g) Fusion method

10. Solvent dispersion technique
1. Ethanol Injection
2. Ether injection
3. Reverse phase evaporation vesicles
4. Emulsion & Double emulsion vesicles.
Detergent solubilization technique
1. Detergent
2. Dialysis
3. Column chromatography
4. dilution

11. 1.HAND SHAKEN METHOD
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

12. 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)

13. PRO LIPOSOMES
Sorbitol / Nacl ( increase surface area of lipid
film)
Add 5ml lipid solution ( fitted to evaporator )
(Evaporation)
Again add lipid solution

14. Dry the content using Lyophilizer ( freeze dryer)
Stand over night at room temp
Flushed with nitrogen for drying properly
MLVs

15. FREEZE DRYING
Lipid + Solvent ( Tertiary butanol)
Freeze drying
Add Aqueous phase / Saline containing drug
Rapid mixing above phase transition temperature
MLVs

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.

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

20. FRENCH PRESSURE CELL
• 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 (25-75nm in
diameter depending on applied pressure).
• This liposomes are more stable as compared to
sonicated liposomes.
• Suitable for drugs and compounds which
degrade by ultrasonic radiations

22. Freeze thaw sonication process
• 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. Membrane extrusion
• Used for preparation of LUVs and MLVs.
• The size of liposomes is reduced by gently
passing them through polycarbonate membrane
filter of defined pore size at lower pressure
• Before extrusion LMV are disrupted by freeze-
thaw cycles/ pre-filtering through large pore size
(0.2-1 µm).
• Filter with 100nm pores yield LUV of 120-
140nm.

25. Solvent dispersion method-ether
/ethanol injection
Lipid +ether sln in syringe
Inject slowly
aq : phase (on heating water bath)
Lamellar unilamellar vessicle

26. Double Emulsion Method
Org:soln +lipid +aq phase
Emulsion(w/o)
Hot aq soln of buffer
Multi component vessicle(w/o/w){double
emulsion}

27. DE EMULSIFICATION METHOD
Aq medium +medium to be entrapped
High vol org soln of lipid
Agitated mechanically

28. Break aq medium to water droplet
w/0 Emulsion
Stabilized by phospholipid monolayer

29. REVERSE PHASE EVAPORATION
Emulsion
Evaporation under reduced pressure rotary
evaporator
Semi solid gel
Shake to gel LUV,s

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.

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

32. EVALUATION
The liposomes prepared by various techniques are
to be evaluated for
• Physical properties
• chemical properties
• biological properties.

33. Physical properties
1.SIZE AND ITS DISTRIBUTION
• Size and its distribution can be detrermined by
EM.But it is very time consuming, Both particle
size and particle size distribution of liposomes
influence their physical stability. It can be
determined by following methods
• Microscopic method
• Laser light scattering
• gelpermeation

34. 2.SURFACE CHARGE
•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 acid.

35. 3. PERCENT DRUG ENCAPSULATED.
• Quantity of drug entrapped in the liposomes helps to
estimate the behavior of the drug in biological system
• Liposomes are mixture of encapsulated and
unencapsulated drug fractions
• The %of drug encapsulation is done by first separating
the free drug fraction from encapsulated drug fraction
• The methods used to separate the free drug from the
sample are: a. Mini column centrifugation method b.
Protamine aggregated method
4. PHASE BEHAVIOR
•At transition temperature liposomes undergo reversible
phase transition
• Done by DSC

36. 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.

37. CHEMICAL EVALUATION
a)PHOSPHOLIPID DETERMINATION
• Phospholipid phosphorus + hydrolysis
Inorganic phosphate
• Inorganic phosphate + ammonium molybdate
phosphomolybdic acid

38. • Phosphomolybdic acid +amino napthyl
sulphonic acid
reduced blue clr
• Intensity of it is measured and compared with
standard.

39. B)PHOSPHOLIPID HYDROLYSIS
• Pospholipid +hydrolysis lysolecithin
• One chain is lost by de esterification
• Determined by HPLC
C)PHOSPHOLIPID OXIDATION
• Free radicle determination by UV ,iodometric
method ,GLC ETC

40. D) CHOLESTROL ANALYSIS
Cholestrol +iron+ reagent(ferric per chlorate,ethyl
acetate, sulphuric acid)
purple complex
• Determined @610 nm

41. Biological characterization
• Sterility is tested by aerobic and anaerobic
culture
• Pyrogensity is determined by LAL test

42. Reference
1. Introduction to novel drug delivery system;
N.K.JAIN. 2017, Vallabh prakashan
2. Controlled and novel drug delivery; N.K.JAIN.
2017, CBS publishers.
3. Textbook of industrial pharmacy, drug delivery
system & cosmetic and herbal drug technology.
SHOBHA RANI. RH. 2014, universities press.
4. Targeted and controlled drug delivery novel
carrier system; SP. VYAS & RK.KHAR. 2010, CBS
publishers.