notes for pharmacy

1. LIPOSOMES

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.
• Liposomes is Greek words means ‘Lipo’ mean ‘Fat’ and
‘Somes’ mean ‘Body’.
• Liposomes were first produced in England in 1961 by
Alec D. Bangham.

5. COMPOSITION OF LIPOSOMES
 There are number of components of liposomes
however Lecithin (mixture of phospholipids) and
cholesterol being main components.
1. Phospholipids are fatty substances the major structural
components of cell wall & biological membranes.
 Phospholipids are amphipathic moieties with a
hydrophilic head group and two hydrophobic tails.
 Two types of phospholipids exist – phosphodiglycerides
and sphingolipids, together with their corresponding
hydrolysis products.
 Phospholipids have phosphatidyl moiety (tail) with
different head groups (choline, Etahnolamine, Serine)
 The most common phospholipid is phosphatidylcholine
(PC) molecule.

6. Phosphatidylcholine has glycerol bridge links a pair of
hydrophobic acyl hydrocarbon chains having 10-24 carbon
atoms with a hydrophilic polar head group.

9. PHASE TRANSITION TEMPERATURE (Tc):
• At various temperatures, phospholipid membranes can
exist in different phases.
• The transition from one phase to another can be
detected by technique like micro-calorimetry .
• This is due to the fatty acid chain adopting a new
conformation other than all trans straight chain
configuration, such as gauche configuration state
(phenomenon- chain tilt with decrease in bilayer
thickness)

10. 2. Cholesterols
 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 , it
acts as fluidity buffer.
 It inserts into membrane with hydroxyl group oriented
towards aqueous surface & aliphatic chain aligned
parallel to acyl chains in the centre of bilayer.
 It can be incorporated into phospholipid membranes in
very high Conc. upto 1:1 or even 2:1 molar ratios of PC.
 Cholesterol incorporation increases the separation
between the choline head groups and eliminates the
normal electrostatic and hydrogen-bonding interactions.

11. Role of cholesterol in bilayer formation:
1. Cholesterol act as fluidity buffer.
2. After intercalation with phospholipid molecules alter the
freedom of motion of carbon molecules in the acyl Chain
3. Restricts the transformations of trans to gauche
conformations.

12. MECHANISM OF LIPOSOMES FORMATION
• In aqueous media phospholipids as they are not soluble
align themselves closely in planar bilayer sheets or lipid
cakes which is thermodynamically stable.
• In bilayer sheets polar head groups face outwards into
the aqueous medium, the lipidic chains turns inwards to
avoid the water phase, giving rise to double layer.
• For the liposomes to be formed, upon further hydration,
the lipid cakes (lamella) swells eventually they curve to
form a closed vesicles in the form of spheres
• These spheres are called as liposomes.
• Amphiphatic molecules other than PC from micellar
structure in preference to bilayer sheet.

13. LIPOSOME

14. 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.

15. DISADVANTAGES
1. Low solubility in water.
2. Production cost is high.
3. Short half-life.
4. Sometimes phospholipid undergoes oxidation and
hydrolysis like reaction.
5. Leakage and fusion of encapsulated drug / molecules.

18. Liposome & cell interaction
Liposomes interact 4 mechanisms Adsorption, Endocytosis,
Fusion of cell with vesicle & Lipid exchange.

19. Method of liposome preparation & drug loading
The general procedure involves 4 steps
1. Preparation of lipid for hydration
2. Hydration with agition
3. Sizing for homogenous distribution of vesicles
4. Removal of non-encapsulated material
Drug loading may be active (after mfg.)/ passive (during mfg.)

20. Methods of liposome preparation
These can be categorized on the basis of lipid dispersion.
1. 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
2. Solvent dispersion technique
a) Ethanol Injection
b) Ether injection
c) Reverse phase evaporation vesicles
d) Emulsion & Double emulsion vesicles.
3. Detergent solubilization technique

21. 1. Physical dispersion technique:
1. Liposomes are produced when thin lipid films are
hydrated and stacks of liquid crystalline bilayer become
fluid and swell.
2. The hydrated lipid sheets cutoff from lipid filmduring
agitation and self close to form LMV.
3. LMV particle size reduction is done by sonication/
extrusion.
a) Lipid film hydration by Hand-shaking, non-hand shaking:
Step-1: Preparation of film for hydration:
1. Lipid + organic solvent (chloroform/ chloroform:
methanol) 10-20mg lipid/ml Mix thoroughly
2. Solvent removal–
Small volume (< 1ml)= dry N2/Argon stream,
Large volume= Rotary evaporation  thin lipid film.

22. 3) Lipid film is dried to eliminate residual organic solvent by
placing in vacuum over night.
4) Lipid film can also be prepared by freezing in dry ice/ dry ice-
acetone/ alcohol bath.
5) The frozen lipid cake is placed in vacuum pump and
lyophilized until dry (1-3 days).
6) Dry lipid films removed from vacuum, container closed
tightly, taped and stored frozen.
Step-2: Hydration of lipid film:
1) The dried lipid film can be hydrated by addition of aqueous
medium, followed by agitation (1 hr) and over night stand.
2) Temperature of hydrating medium should be above “Tc”.
3) Hydration media– buffer solution, saline, 5%dextrose,
10%sucrose.
4) The obtained product- LMV suspension is downsized.

24. b) High shear homogenization/ Sonication:

25. DISADV:-
1. Probe sonicator deliver high energy & heat leading to
lipid degradation.
2. It may release titanium particles in to lipid suspension
which are to be removed.
• The lipid suspension of LMV clarifies to yield SUV of
15-50nm.

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

27. d) Microfluidizer techniques for Micro emulsification
1. “Micro Fluidizer” is used to prepare small ULV/ MLVs from
concentrated lipid dispersion.
2. The lipids are introduced into fluidizers, either as a dispersion
of large MLVs or as a slurry of unhydrated lipids in organic
medium.
3. Microfluidizer pumps the fluid at very high pressure
(10,000psi, 600-700 bar) through a 5um orifice.
4. Then it is forced along defined micro channels, which direct
two streams of fluid to collide together at right angles at a
very high velocity, thereby affecting an efficient transfer of
energy.
5. The fluid collected of be recycled through the pump and
interaction chamber untill vesicles of the spherical
dimension are obtained.
Adv: Samples with high % of lipids can be easily treated.

29. e) French pressure cell:
1. French pressure cell is invented by ‟Charles stacy
French”.
2. In this technique the large vesicles are converted to small
vesicles under very high pressure.
3. This technique yields uni or oligo lamellar liposomes of
intermediate size (25-75nm in diameter depending on
applied pressure).
4. This liposomes are more stable as compared to sonicated
liposomes.
5. Suitable for drugs and compounds which degrade by
ultrasonic radiations.

31. f) Dried-reconstituted vesicles:
• This method involves freeze drying the dispersion of empty
SUV followed by rehydration with aqueous fluid, which have
material to be entrapped.
• Useful for preparation of small uni-lamellar and oligo
lamellar vesicles.
ADV: High entrapment of water soluble components and
bioactives.
g) Fusion method:
 This method protects lipids and entrapped material from
harmful physicochemical environment.
 Used for preparation of ULV in large quantities.
 Fusogenic agents are used for fusion of SUV for increasing
entrapment efficiency.
 Alteration in pH increases surface charge density of lipid
bilayer and brings on spontaneous vesiculation.

32. 2. Solvent dispersion technique:
In this process lipid solution in organic phase is bought in
contact with the aqeous phase including material to be
entrapped inside the liposome.
a) Ethanol injection:
• Suitable for incorporating hydrophobic & amphiphilic
drugs in to liposomes.
• This is suitable for preparing small & large unilamellar
vesicle.
• Lipid + ethanol 22-gauge fine needle, RAPIDLY
excess of saline/ aqueous medium.
• The rate of injection should be suitable to attain
absolute & rapid mixing thus leads to equal distribution
of phospholipids through the medium.

33. b) Ether injection:
• This was developed by Deamer & Bangham, similar to
ethanol injection.
• Organic solution of lipid (ether + lipid)  narrow
needle, SLOWLY  Aqueous phase.
• The lipid solution is added at the vaporizing temp. of
organic phase.
• Lipid solution added very slowly for protection of
sensitive lipids & material to be entrapped.
DISADV:- low entrapment efficiency.

35. c)
Aqueous medium + material to be entrapped  high vol.
organic solution of lipid  Agitated mechanically  break
aq. Medium to water droplets W/O emulsion 
Stabilized by phospholipid monolayer.

36. d) Double Emulsion Method:
1. The organic solution, which already contains water
droplets (W/O) is introduced in to excess aqueous
medium followed by mechanical dispersion causing
phase inversion (O/W).
2. The (W/O/W) multi component vesicle is formed by
double emulsion.
3. Two aqueous compartments being separated from each
other by two phospholipid monolayer.
4. The hydrophobic surfaces of monolayers (tails) face each
other, with a thin film of organic solvent.
5. Removal of organic solvent results in formation of
intermediated sized unilamellar vesicles.

37. e) Rapid solvent exchange method:
• Lipid solution in organic solvent is passed through an
orifice of syringe by means of vacuum in to a tube
containing aqueous buffer placed on vortex.
• The organic solvent vaporizes due to vacuum before
contacting aqueous phase.
• The lipid mixture precipitates very quickly in aqueous
buffer forming liposomes.
f) Reverse phase evaporation:
1. Mixture of 2 phases (Aq+Org) subjected to bath
sonication.
2. Droplets formed are dried to semisolid gel in rotary
evaporator under reduced pressure.
3. In this stage mono layer of phospholipids surround water
compartment.

38. 4. Mechanical shaking with vortex shaker collapses few
water droplets & lipid mono layers.
5. Collapsed lipid monolayers form outer layer for stable
vesicles thus forming bilayer SUV.

39. 3. Detergent solublization technique:
1. Detergents associate with the phospholipid molecules
and serve to screen the hydrophobic portions of
molecule from water.
2. The structures formed as a result of this association is
known as micelles.
3. Up on removal of detergent, transition of mixed micelles
occurs to form concentric bilayered vesicles.
 The concentration of detergent in water at which
micelles instantly forms is called CMC.
 Liposome size and shape depend on chemical nature of
detergent, concentration and other lipid involved.
 Methods to remove detergents: Dialysis & Column
chromatography.

40.  Hydrophilic nonbilayer- interacting low molecular weight
compounds have low entrapment.
 This method is suitable for preparation of immuno-
liposomes
Surfactant
Micelle
Phospholipid
monolayer
LIPOSOMES

41. Characterization/ Evaluation of liposomes
The liposomes prepared by various techniques are to be evaluated for
their physical, chemical as well as biological properties.
A)Physical Characterization:
1. Entrapement efficiency
2. Vesicle shape & morphology
3. lamellarity
4. Particle size & size distribution
5. Surface charge
6. Phase transition behaviour
7. Drug release
B) Chemical Characterization:
1. Phospholipid concentration
2. Cholesterol concentration
3. Lysolecithin concentration
4. Phospholipid peroxidation
5. Phospholipid hydrolysis &
Cholesterol autooxidation
6. pH of liposomal dispersion.
7. Osmolarity
C) Biological Characterization:
1. Sterility testing
2. Pyrogenicity testing.
3. Animal toxicity
D) Stability testing:

42. A) PHYSICAL CHARACTERIZATION
Characterization parameter Analytical methods / Instrument
Entrapment efficiency 1. Mini column centrifugation method–
Sephadex column (cross linked dextran)
2. Protamine aggregated method
Vesicle shape and surface
morphology
Transmission electron microscopy, Freeze
fracture electron microscopy
Lamellarity Small angle X-ray scattering, 31 P-NMR, Freeze
fracture electron microscopy
Particle size & size distribution Light microscopy, Fluorescence microscopy,
Transmission electron microscopy, Freeze
fracture electron microscopy, Photon
correlation spectroscopy, laser light scattering,
gel permeation, gel exclusion and Zeta sizer.
Surface charge Free-flow electrophoresis, Zeta potential
measurements
Phase transition behaviour DSC (differential scanning calorimetry)
Drug release Diffusion cell, Dialysis tube.

43. B) CHEMICAL CHARACTERIZATION
Characterization
parameter
Analytical methods /
Instrument
Phospholipid concentration
(Lipid phosphorous content )
Barlett assay, stewart assay, TLC.
Cholesterol concentration Cholesterol oxidase assay, Ferric
perchlorate method
Lysolecithin concentration
(hydrolysis product of lecithin)
Densitometer
Phospholipid peroxidation UV absorbance, Iodometery, GLC.
Phospholipid hydrolysis &
Cholesterol autooxidation
HPLC & TLC
pH of liposomal dispersion pH meter
Osmolarity Osmometer

44. C) BIOLOGICAL CHARACTERIZATION
D) Stability of liposomes
Stability is defined as capacity of particular formulation in a
specific container/ closure system to remain with in
physical, chemical, microbiological, therapeutic and
toxicological specifications.

45. Stability testing of liposomes:
Liposomal stability can be tested by storing under following
six conditions.
1. Highest & lowest temp. for 1 month.
2. Room temp. for 12-24 months
3. 2-3 freeze thaw cycles (20-25oC)
4. 60 cycles/ min in a reciprocating shaker for 24-48 hr
5. 6-8 heat cool cycles (5-45oC, 48 hrs at each temp.)
6. Visual/ microscopic examination.
After storage liposomes are valuated for vesicle size, shape,
no. of vesicles/ cubic mm & residual drug content.

46. Applications of liposomes:
1. Liposome as drug delivery vehicle
2. Liposome as vaccine carrier
3. Liposome in tumour therapy
4. Liposome in gene delivery.
5. Liposome as artificial blood surrogates
6. Liposome as radio-pharmaceutical & radio-
diagnostic carrier
7. Liposome in cosmetics and dermatology
8. Liposome in enzyme immobilization.

47. 1. Liposome as drug delivery vehicle:
 Liposomes enhance solublization of drugs
(Amphotericibn-B, paclitaxel, Cyclosporin, Minoxidil).
 Provide protection to sensitive drug molecules (cystone
arabinose, DNA, RNA, Ribozymes).
 Enhance intra cellular uptake (anti-cancer, anti-viral &
anti-microbial agents).
 Alter pharmacokinetics & distribution of drugs.
2. Liposome as vaccine carrier:
 Liposomes potentiate both cell mediated & humoral
immunity.
 Liposomal vaccines based on immunopotentiating
reconstituted influenza virosome (IRIV) are prepared.

48.  For immunopotentiation, immunomodulating agents
like muramyl dipeptide, lipopolysaccharide & lipid can
be incorporated in to liposome.
Advantages:
1. Non-toxic, bio-compatible & biodegradable.
2. Incorporate adjuvants to provide strong immune
response.
3. Convert loaded non-immunogenic substance to
immunogenic (Proliposome)
4. Minimize & eliminate toxicity of toxic antigens &
allergic reactions.

49. 3. Liposome in tumour therapy:
 Liposomes as drug carriers can be administered I.V route.
 If liposome is modified more hydrophilic, with lipids their
circulation time in blood stream increases.
 These are called stealth liposomes, used as carriers for
hydrophilic anti-cancer drugs (Doxorubicin, Mitoxantrone)
 In this form they can extravasate the tumour vascular
endothelium.

50. 4. Liposome in gene delivery:
 The non-viral vector systems, are especially engineered
liposome such as pH sensitive liposomes, cationic liposomes,
fusogenic liposome, genosomes, lipoplex, and lipopolyplex
have been extensively investigated for their gene delivery
potential.
 Cationic liposomes deliver the content through membrane
fusion, there by avoiding lysosomal and nucleolus
degradation of DNA.
 pH sensitive liposomes use endosomal acidification for
fusion with endosomal membarne.
 Genosomes are complex formulations of DNA with various
cationic liposomes.
 Lipoplex aggregates with DNA to form large and
heterogeneous particles.
 Lipopolyplex is composed of liposome + polycation + DNA.

52. 5. Liposome as artificial blood surrogates:
 Liposome encapsulated hemoglobin products can be used as
artificial RBC.
 Sterically stabilized liposome bearing hemoglobin are better
Oxygen carriers.
 These have low toxicity, less platelet activation& aggregation
& less haemostatic generation.
6. Liposome as radio-pharmaceutical & radio-diagnostic
carrier
 Liposomal radio-diagnostic applications include imaging of
liver, spleen,brain, lymphatics, tumour, blood pool,
cardiovascualr pathologies, visualization of inflammation,
infection sites, bone marrow, eye vasculature.
 Liposome imaging agents are used for magnetic resonance,
computed tomography & ultra sound imaging of tumours.

53. 7. Liposome in cosmetics and dermatology:
 Liposomes with essential oils provide an effective
nourishing treatment that penetrates deeply in to the
skin.
 Liposome based on anti-aging formulations (e.g.
creams, lotions, gels and hydrogels) have been
formulated and launched in the cosmetic market by
L`oreal in 1986.
 Liposomal preparation reduce the roughness because of
its interaction with corneocytes, the intracellular lipid
resulting in skin softening and smoothing.
 Various liposome based products for facial and body
care, make-up, mascara & foundation, haircare,
sunscreen products & perfumes are in market.

54. 8. Enzyme immobilization:
 Liposomes can deliver enzymes to lysosomal system &
other sites.
 β – glucosidase & α– glucosidase are loaded in
liposomes for tretamnet of Gauchers & Pomps diseases
respectively.
Limitations of liposome technology
• 1. Stability
• 2. Sterilization
• 3. Encapsulation efficiency
• 4. Active targeting
• 5. Gene therapy
• 6. Lysosomal degradation

57. REFERENCES:
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.