LIPOSOMAL DRUG DELIVERY

1. LIPOSOMES
PRESENTED BY :- DHARMILA
M. PHARM 2ND SEM.
M100400005
1

2. CONTENTS
• INTRODUCTION
• ADVANTAGES
• DISADVANTAGES
• CLASSIFICATION
• METHODS OF PREPARATION
• CHARACTERIZATION
• APPLICATIONS
• MARKETED FORMULATIONS
2

3. INTRODUCTION
• Discovered by Bangham and
colleagues in 1965.
• Concentric bilayer vesicles in
which an aqueous volume is
entirely enclosed by a
membranous lipid bilayer
composed of natural or synthetic
phospholipids.
• Size :– 30 - 10,000 nm.
• They are formed by hydration of
phospholipids. 3

4. A) Naturally occuring phospholipids :-
 Phosphatidylcholine
 Phosphatidylethanolamine
 Phosphatidylserine
B) Synthetic phospholipids :-
 DOPC - Dioleoyl phosphatidylcholine
 DSPC - Distearoylphosphatidylcholine
 DMPC - dimyristoyl phosphatidylcholine
 DPPC - Dipalmitoyl phosphatidylcholine
 DOTAP - Dioleoyl trimethylammonium propane
 DOPG -Dioleoyl phosphatidylglycerol
4

5. ADVANTAGES
• Liposomes provide selective passive targeting to tumor tissues (liposomal
doxorubicin).
• Increased efficacy & therapeutic index.
• Increased stability of the drug via encapsulation.
• Reduction in toxicity of encapsulated agent.
• Flexibility to couple with site-specific ligands to achieve active targeting.
• Liposomes are biocompatible.
• Completely biodegradable and non-toxic.
• Flexible and non immunogenic for systemic and non-systemic
administrations.
• Improved pharmacokinetic effects (reduced elimination, increased
circulation life times).
• Site avoidance effect. 5

6. DISADVANTAGES
• High Production cost
• Leakage and fusion of encapsulated drug /
molecules
• Oxidation and hydrolysis of phospholipids
• Short half-life
• Low solubility
• Less stable 6

7. CLASSIFICATION
A) Based on composition & application :-
7
LIPOSOME TYPE MAJOR APPLICATION
Conventional liposomes Macrophage targeting, Local depot, Vaccination
Long-circulating (stealth)
liposomes
Selective targeting to pathological areas Circulating
microreservoir
Immuno liposomes Specific targeting
Cationic liposomes Gene delivery

8. B) Based on structural parameters :-
1. MLV (multilamellar large vesicles) >0.5 μm
2. OLV (oligolamellar vesicles) 0.1–1 μm
3. UV (unilamellar vesicles)
a) SUV (small) 20–40 nm
b) MUV (medium 40-80 nm
c) LUV (large) >100 nm
d) GUV (giant) >1 μm
4. MVV (multivesicular vesicles) >1 μm
5. Double liposomes >1000 nm
8

9. C) Based on method of liposome preparation :-
1. REV (single or oligolamellar vesicles) made by reverse-phase
evaporation method
2. MLV-REV (multilamellar vesicles) made by the reverse-phase
evaporation method
3. SPLV (stable plurilamellar vesicles)
4. FATMLV (frozen and thawed MLV)
5. VET (vesicles prepared by extrusion methods)
6. DRV (dehydration-rehydration vesicles)
9

10. 10
METHODS OF
PREPARATION
PASSIVE
LOADING
TECHNIQUES
ACTIVE
LOADING
TECHNIQUES

11. PASSIVE
LOADING
TECHNIQUES
11

12. 12
MECHANICAL DISPERSION
METHOD
SOLVENT DISPERSION
METHOD
DETERGENT REMOVAL
METHOD
PASSIVE
LOADING
TECHNIQUES

13. MECHANICAL DISPERSION METHOD
• Lipid film hydration by hand shaking, non-hand shaking
or freeze drying
• Micro-emulsification
• Sonication
• French pressure cell
• Membrane extrusion
• Dried reconstituted vesicles
• Freeze-thaw sonication method 13

14. SOLVENT DISPERSION METHOD
• Ethanol injection
• Ether injection
• Double emulsion vesicles
• Reverse phase evaporation vesicles
• Stable plurilamellar vesicles
14

15. DETERGENT REMOVAL METHOD
• Detergent removal from mixed micelles by:-
• Dialysis
• Column chromatography
• Detergent adsorption using bio-beads
15

16. Film hydration by hand shaking(MLVs), non-hand
shaking(ULVs) methods
16
Organic solution of lipids
Film formation using rotary
evaporation under reduced pressure
Dispersed the films in aqueous
medium
Lipids swell & peel off from
wall of RBF & vesiculate
Liposomes are formed with
the efficacy of 30%
Mechanical energy (for swelling) can
be provided by:-
Hand shaking (MLVs)
Stream of water saturated
nitrogen(LUVs)

17. 17
Freeze drying

18. Microemulsification Method
 Micro fluidizer prepare small MLVs from
concentrated lipid suspension, pumps fluid at
very high pressure (10000psi; 600-700 bar)
through 5 µm screen
 Fluid forced through micro channels into two
streams, which collide at right angle at very high
velocity
18

19. Sonicated unilamellar Vesicles(SUVs)
 MLVs size reduced to small vesicles by
ultrasonic irradiation
 Instruments used are Bath and probe
ultrasonic disintegrators
 MLVs are sonicated for 5-10 mins and then
centrifuged
19

20. French Pressure Cell
It causes extrusion of preformed Large
liposomes under very high pressure
Liposomes are more stable, less structural
defects and less content leakage; overcoming
defects of sonication
Yield uni-lamellar or oligo-lamellar Liposomes
of intermediate size(30-80nm)
20

21. Membrane Extrusion Technique
Liposome size reduced by passing through
membrane filters (defined size) at lower
pressure(100psig)
Contents exchanged with dispersion medium
during breaking and resealing, drug in
medium for high entrapment
Used to prepare SUVs, MUVs and LUVs
21

22. Dried-reconstituted Vesicles(DRVs)
Empty SUVs are freeze dried for dispersion
and then rehydrated by Aqueous Fluid
containing Drug
Organized membrane structure is formed
which on rehydration form high capture
efficiency liposomes
22

23. Freeze Thaw Sonication
 It is an extension of DRV method
 SUVs are frozen and thaw at room temperature for
15 mins and sonicated
pH induced vesiculation
 pH change induce Transformation of MLVs to LUVs by
increasing surface charge density of lipid bilayer
 MLVs exposed to pH 11.0(1 M NaOH,2mins);pH
reduced by addition of 0.1 M HCl to pH 7.5 23

24. Calcium induced fusion
SUVs made from negatively charged lipids on
addition of Ca++ fuse into cylindrical rolls
Subsequent removal of Ca++ by EDTA or Iron
exchange or precipitation; form LUVs
24

25. Solvent Dispersion Methods
a) Ethanol injection:-
 Lipid ethanol solution injected rapidly through a fine
needle into excess of saline or aq. Medium
 Rate of injection causes mixing to disperse
phospholipids evenly and yield SUVs(~25nm)
 Limitations:- Limited solubility of lipids in ethanol,
volume of ethanol injected and difficult ethanol
removal 25

26. b) Ether injection :-
Immiscible organic phase injected very slowly
into aq. Phase; at temperature to vaporize
organic solvent
Suitable for sensitive lipids but technique is
time consuming and require careful control of
needle
For temperature sensitive fluorinated
hydrocarbons 26

27. c) Rapid Solvent Exchange :-
Lipid mixture quickly transferred between
Pure solvent and aq. Buffer forming
homogenous dispersion by precipitation of
lipids in aq. Buffer
Bulk solvent vaporizes before coming in
contact with aqueous phase
Preparation time is less i.e. <1min and have
high entrapment volume 27

28. d) Reverse Phase Evaporation Vesicles :-
 It involve removal of solvent from emulsion to form a semisolid gel in a
rotary evaporator
 Followed by vigorous mechanical shaking in vortex mixer to convert gel
into homogenous fluid
 Solvent removed by dialysis to give unilamellar vesicles(0.5µm) with 50%
encapsulation
e) Stable Plurilamellar Vesicles(SPLVs) :-
 W/O phase with excess of lipid dried under sonication with intermittent
nitrogen stream
 It give high entrapment efficiency
28

29. Detergent Solubilization
 Detergents are used for intimate contact between
phospholipids and aq. Phase
 Initially detergent equilibrates in lipid and water phase
 At ‘critical detergent concentration’ unstable membrane form
micelles; detergent saturated bilayer formed
 At still higher concentration mixed micelle (containing
detergent and lipids)
 To remove detergent from bilayer follwing methods are used
#Dialysis #Column Chromatography #Adsorption 29

30. ACTIVE
LOADING
TECHNIQUE
30

31.  Ions and large hydrophilic molecules can not cross the bilayer;
transport of neutral and weakly hydrophobic molecules can be
achieved by concentration gradient
 Weak acids and bases are transported by pH gradient and Potential
difference
Advantages:-
 High encapsulation efficiency
 Reduced leakage
 Flexibility in use of lipids
 Reduction in safety hazards
 Weak bases loaded using proton gradient or ammonium sulphate
gradient; weak acids loaded using calcium acetate gradient 31

32. CHARACTERIZATION
PHYSICAL
SIZE, SHAPE,
SURFACE FEATURES,
LAMELLARITY, PHASE
BEHAVIOUR AND
DRUG RELEASE
PROFILE
CHEMICAL
PURITY AND
POTENCY OF
VARIOUS LIPOSOMAL
CONSTITUENT
BIOLOGICAL
SAFETY AND
SUITABILITY OF
FORMULATION FOR
in vivo USE.
32

33. Physical Characterization
Vesicle shape and lamellarity :-
1. Freeze-Fracture and Freeze-etch Electron Microscopy
 Can be used to study surface morphology (Topology)
 In this technique, Fracture plane passes through the vesicles, which are
randomly positioned in frozen state.
 Thus, it may not pass through mid-plane and non-mid plane fracture may result
in erroneous readings.
 Observed distribution profile depends on the distance of vesicle centre from the
plane of fracture.
 Therefore, heterogenous populations require a careful monitoring.
 Quick freeze and deep etching techniques give much better lamellarity evaluation.
 After 5-mintues of etching, cross-fractured vesicles are clearly seen and the
number of lamellae can readily be determined. 33

34. 2. P 31 Nuclear Magnetic Resonance Analysis
 It is one of the most accurate and straightforward
techniques for determination of lamellarity of liposomes.
 Monitors the phospholipid phosphorus signal intensity.
 Adding nonpermeable broadening agent such as Mn to
external medium will decrease in intensity of intial P31 NMR
signal by an amount proportional to fraction of lipid exposed
to external medium.
 50% reduction in signal- Unilamellar, subsequent reductions
indicate multilamellar vesicular preparation. 34

35. VESICLE SIZE & SIZE DISTRIBUTION
1. OPTICAL MICROSCOPY
 Include methods like Bright-field, Phase Contrast
Microscopy and Fluorescent Microscope.
 Useful in evaluating the vesicle size of large vesicles (>1µm).
 Vesicular dispersion appropriately diluted are wet mounted
on a haemocytometer and photographed with a phase
contrast microscope.
35

36. 2. NEGATIVE STAIN TRANSMISSION ELECTRON
MICROSCOPY (TEM)
 This technique visualizes relatively electron transparent liposomes as
bright areas against a dark background.
 Liposomes are embedded in this method in a thin film of electron-
dense heavy metal (salt) stain.
 Negative stains used are ammonium molybdate, phosphotungstic acid
or uranyl acetate.
 TEM facilitates estimation of liposome size range at the lower end of
the frequency distribution.
 Mainly used for larger sizes or heterogenous populations of liposomes.
 DISADVANTAGE: Liposome morphology may be deformed.
36

37. 3. CRYO-TRANSMISSION ELECTRON
MICROSCOPY TECHNIQUE (CRYO-TEM)
 It is used to elucidate the surface morphology and size of
vesicle.
 The method involves freeze fracturing of samples followed
by their visualization using TEM.
 Thin sample films are prepared under controlled
temperature (25oC) and humidity conditions.
 Films are thereafter vitrified by quick freezing in liquid
ethane and transferred to TEM analysis.
37

38. 4. FREEZE-FRACTURE ELECTRON MICROSCOPY
 Mainly used to assess the surface features and lamellarity
and to calculate true vesicle diameter.
SURFACE CHARGE
 Two methods are used to assess the charge,
a) Free-flowing electrophoresis
b) Zeta potential measurement
From the mobility of liposomal dispersion in a suitable buffer,
the surface charge on the vesicle can be calculated.
38

39. ENCAPSULATION EFFICIENCY
 It describes the % of aqueous phase and hence the % of water
soluble drug that becomes ultimately entrapped during
preparation of liposomes. Usually expressed as
%entrapment/mg lipid.
 Encapsulation efficiency is assessed using two techniques
a) Minicolumn centrifugation method
b) Protamine aggregation method.
39

40. MINICOLUMN CENTRIFUGATION METHOD :-
• The hydrated gel(sephadex G-50) is filled in a barrel of 1mL syringe
without plunger which is plugged with a whatmann filter pad.
• This barrel is rested in a centrifuged tube . This tube is spun at 2000 rpm
for 3 mins to remove excess saline solution for gel.
• After centrifugation the gel column should be dried and have come away
from the side of the barrel.
• Then, eluted saline is removed from the collection tube.
• Liposome suspension (0.2 ml undiluted) is applied drop wise to the top of
the gel bed, and the column is spun at 2000rpm for 3 minutes to expel the
void volume containing the liposomes into the centrifuge tube.
• The elute is then removed and set aside for assay.
40

41. PROTAMINE AGGREGATION METHOD :-
• The protamine aggregation method may be used for liposomes of any
composition(both + and –vely charged particles). However, a preliminary test
should be carried out before, to check that the solute material entrapped does not
itself precipitate in the presence of protamine after release of liposomes.
• In this method liposome suspension(20mg/ml in normal saline) is placed in the
conical glass centrifuge tube, 0.1 mL of protamine solution(10 mg/ml) is added
with mixing, and allowed to stand for 3 min.
• 30 ml of saline is added and then the tube is spun for 20 min at 2000rpm at room
temperature
• The supernatant is removed & assayed for free, untrapped compound by standard
methods.
• The suspended pellet is resuspended in 0.6 mL of 10% triton X-100 and the
material completely dissolved.
• The volume is made up to the desired value and then assayed for entrapped
material by standard methods. 41

42. TRAPPED VOLUME :-
 Trapped volume is an important parameter that governs the encapsulation
efficiency and morphology.
 The measurement of the quantity of aqueous buffer is the best way to
calculate trapped volume.
 The internal or trapped volume is the aqueous volume entrapped per unit
quantity of lipid and expressed as µl/µmol or µl/mg of total lipid.
 The best way to measure internal volume is to measure the quantity of
water directly and this may be done by replacing the external
medium(water, H20) and with a spectroscopically inert fluid (deuterium
oxide,D2O) and then measuring the water signal e.g using NMR.
42

43. Chemical Characteristics
CHARACTERIZATION PARAMETERS ANALYTICAL METHODS/INSTRUMENTATION
Phospholipid Concentration Lipid phosphorous content using Barlett assay/
Stewart assay, HPLC
Cholesterol Concentration Cholesterol oxidase assay & HPLC
Drug Concentration Appropriate methods given in monograph for
individual drug(s)
Phospholipid Peroxidation HPLC &TLC & fatty acid conc.
Cholesterol Auto-oxidation HPLC & TLC
Anti-oxidant degradation HPLC & TLC
pH pH meter
Osmolarity Osmometer
Phospholipid Hydrolysis UV absorbance, TBA(for endoperoxidase),
idometric(for hydroperoxidase) & GLC 43

44. BARLETT ASSAY :-
• In the Bartlett assay the phospholipid phosphorous in the
sample is first hydrolyzed to inorganic phosphate.
• This is converted to phospho-molybidic acid by the addition of
ammonium molybidate and phospho-molybidic acid and is
quantitatively reduced to a blue colored compound by
aminonaphthyl sulfonic acid.
• The intensity of the blue color is measured
spectrohotometrically and is compared with the curve of
standards to give phosphorus and hence phospholipid
content.
44

45. STEWART ASSAY :-
• In this assay, the phospholipid forms a complex with
ammonium ferrocyanate in organic solution.
• In this method, the standard curve is first prepared by adding
ammonium ferrocyanate (0.1M) solution with different known
concentrations of phoshpolipds in chloroform.
• Similarly, the samples are treated and optical density of these
solution is measured at 485nm and the absorbance of
samples compared with the standard curve of phospholipids
to get the concentration.
• The advantage of this method is that the presence of
inorganic phosphate does not interfere with the assay. 45

46. Biological Characterization
• Sterility testing (Aerobic or anaerobic cultures)
• Pyrogen testing {Limulus Amebocyte Lysate
(LAL)}
• Animal toxicity (Monitoring survival rates,
histology and pathology )
46

47. APPLICATIONS
1) Liposomes as drug/protein delivery vehicles :-
 Controlled and sustained drug release
 Enhanced drug solubility (amphotericin B, minoxidil, paclitaxel,
cyclosporin)
 Altered pharmacokinetics & biodistribution (prolonged &
sustained release)
 Protection of sensitive drug molecules (DNA, RNA, rivosome)
47

48. 2) Liposomes as carrier to antigen :-
 Delivery of biologically active materials to specific cells but
the major fraction is taken up by liver and spleen.
 This can be prevented by:
 By using small ,unilamellar liposomes.
 By coating the liposomes which would render liposomes less
recognizable by RES.
 Anticancer drugs are less selective due to which cause toxicity
to the normal cells so with the help of liposomes Targeting of
anticancer drugs can be done.
48

49. 3) Liposomes for pulmonary drug delivery system :-
• Liposomes are available in size ranges from 20nm to greater than 1
um diameter and of low toxicity hence can be used for drug
delivery to respiratory tract.
• Studies on some liposome encapsulated drugs administered via
pulmonary route
49
DRUG RESULTS
Atropine glutathione Maintained much higher level of drug in the
lung than solution form.
Enriroxine Liposome encapsulated drug was observed to
be 10-50 times less toxic to tissue culture cell
than free drug

50. 4) Liposomes for ophthalmic drug delivery :-
 Liposomes are used for ocular drug delivery due to following advantages:
 These are biodegradable.
 These are non toxic.
 These have ability to intimately contact with the corneal and conjuctival
surfaces thereby increases the probability of ocular drug absorption
 Protects the drug from metabolic enzymes present at the tear/corneal
epithelium interface.
50
DRUG RESULT
Idoxuridine Improved efficacy in the treatment of
herpes simplex keratitis
Inulin Absorption greatly enhanced

51. 5) Liposomes for topical application :-
 Liposomes are used in the preparation of
semisolids for topical drug delivery.
51
DRUG RESULTS
Triamcinolone In epidermis & dermis 4 times higher
conc. than control ointment
Benzocaine gel Shows prolonged anesthesia as
compared to plain cream
Hydrocortisone Higher conc. of drug in individual layer
than control ointment.

52. 6) Liposomes as carrier in oral drug delivery :-
 Many drugs are unstable in the gut hence are given by making
liposomes.
 Diabetes:-Liposomes are used in the oral delivery of insulin.
 Liposomes exerts protective action in gastric and intestinal areas
and protect the drug from proteolytic enzymes.
 Hence the intestinal uptake of macromolecules increases.
52

53. 7) Cancer chemotherapy :-
 Anticancer drugs are less selective due to which
cause toxicity to the normal cells so with the help of
liposomes:-
 Targeting of anticancer drugs can be done.
 Increases the half life of anticancer drugs.
 Decrease the metabolic degradation of drugs.
 Eg. Of drugs includes: 6- Mercaptopurine,
methotrexate, 5-fluorouracil
53

54. 8) Liposomes in gene therapy
 Gene and antisense therapy
 Genetic(DNA) vaccination(cholera toxin)
9) Liposomes in immunology
 Immunoadjuvant (Interleukin-6)
 Immunomodulator
 Immunodiagnosis
10) Liposomes as artificial blood surrogates
54

55. 11) Liposomes as radiopharmaceutical and
radio diagnostic carriers.
12) Liposomes in cosmetics and dermatology.
13) Liposomes in enzyme immobilization and
bioreactor technology.
55

56. List of marketed products
Marketed product Drug used Target diseases Company
DoxilTM or CaelyxTM Doxorubicin Kaposi’s sarcoma SEQUUS, USA
DaunoXomeTM Daunorubicin Kaposi’s sarcoma,
breast & lung cancer
NeXstar, USA
AmphotecTM Amphotericin-B fungal infections,
Leishmaniasis
SEQUUS, USA
Fungizone® Amphotericin-B fungal infections,
Leishmaniasis
Bristol-squibb,
Netherland
VENTUSTM Prostaglandin-E1 Systemic
inflammatory
diseases
The liposome
company, USA
56

57. Marketed product Drug used Target diseases Company
Novasome® Smallpox vaccine Smallpox Novavax, USA
Avian retrovirus
vaccine
Killed avian
retrovirus
Chicken pox Vineland lab, USA
Epaxal –Berna
Vaccine
Inactivated hepatitis-
A Virions
Hepatitis A Swiss serum &
vaccine institute,
Switzerland
Doxil® Doxorubicin Hcl Refractory ovarian
cancer
ALZA, USA
EvacetTM Doxorubicin Metastatic breast
cancer
The liposome
company, USA
VincaXome Vincristine Solid Tumours NeXstar, USA
57

58. REFERENCES
 “Targeted and controlled drug delivery” By Vyas and
Khar
 “Controlled and Novel Drug Delivery” By N. K. Jain,
1st edition, 2009,CBS Publishers & Distributors, New
Delhi
 Sanjay S. Patel ; Liposome: A versatile platform for
targeted delivery of drugs; pharmainfo.net.
58

59. THANK
YOU 59