Liposomes are spherical vesicles made of lipid bilayers that can encapsulate aqueous materials. They vary in size from 20nm to several microns. Liposomes can selectively deliver drugs to tissues and cells, increasing drug efficacy while reducing toxicity. They improve drug solubility, stability, and pharmacokinetics by encapsulating drugs in their aqueous core. Various preparation techniques including thin-film hydration, extrusion, and solvent injection are used to produce liposomes of defined size and lamellarity for drug delivery applications including cancer chemotherapy, gene delivery, and dermatology.

1. LIPOSOMES

2. Liposomes
 Definition:
 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.
2

3. Advantages
 Provide selective passive targeting to tumour tissues
(liposomal doxorubicin).
 Increased efficacy and therapeutic index.
 Increased stability via encapsulation.
 Reduction in toxicity of the encapsulated agent.
 Improved pharmacokinetic effects (reduced
elimination, increased circulation life times).
 Flexibility to couple with site-specific ligand to
achieve active targeting.
3

4. STRUCTURE AND COMPOSITION OF LIPOSOMES
 There are number of components of
liposomes however phospholipids and
cholesterol being main components.
 Phospholipids are the major structural
components of biological membranes,
where two types of phospholipids exist –
phosphodiglycerides and sphingolipids,
together with their corresponding
hydrolysis products.
 The most common phospholipid is
phosphatidylcholine (PC) molecule.
4

5.  Commonly used synthetic Phospholipids:
 DOPC = Dioleoyl Phosphatidylcholine
 DOPE = Dioleoyl phosphatidylethanolamine
 DSPC = Distearoyl phosphatidylcholine
 DSPE = Distearoyl phosphatidylethanolamine
 DLPC = Dilauryl phosphatidylcholine
 DMPC = Dimyristoyl phosphatidylcholine
 DLPE = Dilauryl phosphatidylethanolamine
5

6.  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, 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. 6

7. Classification Based on structural parameters
TYPE SPECIFICATIONS
MLV Multilamellar large vesicles- >0.5 μm
OLV Oligolamellar vesicles- 0.1-1 μm
UV Unilamellar vesicles (all in size)
SUV Small unilamellar vesicles-20-100nm
MUV Medium sized unilamellar vesicles
LUV Large unilamellar vesicles->100nm
GUV Giant unilamellar vesicles->1 μm
MV Multivesicular vesicles->1 μm
7

8. Classification Based on the method of liposome
preparation
TYPE SPECIFICATIONS
REV Single or oligolamellar vesicles made by
reverse-phase evaporation method
MLV-REV Multilamellar vesicles made by reverse-
phase evaporation method
SPLV Stable plurilamellar vesicles
FATMLV Frozen and thawed MLV
VET Vesicles prepared by extrusion technique
DRV Dehydration-rehydration method
8

9. Chemical Characterization
Characterization parameters Analytical methods
Phospholipid concentration Lipid phosphorous content using
Barlett assay,HPLC
Cholesterol concentration Cholesterol oxidase assay and
HPLC
Drug concentration Appropriate method given in
monograph
Phospholipid peroxidation UV absorbance,
TBA,indometric and GLC
Phospholipid hydrolysis HPLC and TLC
Cholesterol auto-oxidation HPLC and TLC
Anti-oxidant degradation HPLC and TLC
pH pH meter
9
osmolarity Osmometer

10. Physical Characterization
Characterization parameters Analytical methods
Vesicle shape and surface Transmission electron
morphology microscopy, freeze-fracture
electron microscopy.
Surface charge Free-flow electrophoresis.
Lamellarity Small angle X-ray scattering,
freeze-fracture electron
microscopy, 31P-NMR.
Phase behavior Freeze-fracture electron
microscopy, differential
scanning calorimetry
Percent capture/percent of free Minicolumn centrifugation, gel
drug exclusion, ion-exchange
10
chromatography, radiolabelling

11. Biological Characterization
Characterization Analytical methods
parameters
sterility Aerobic or anaerobic
cultures
pyrogenicity Limulus amebocyte
lysate (LAL) test
Animal toxicity Monitoring survival
rates, histology and
pathology
11

12. TECHNIQUES OF LIPOSOMES PREPARATION
(A) physical dispersion
a) Hand-shaken multilamellar vesicles (MLVs)
b) Non-shaking vesicles
c) Pro-liposomes
d) Freeze drying
(B) Processing of lipids hydrated by physical means
a) Micro emulsification liposomes (MEL)
b) Sonicated unilamellar vesicles (SUVs)
c) French pressure cell liposomes
d) Membrane extrusion liposomes
e) Dried-reconstituted vesicles (DRVs)
f) Freeze thaw sonication (FTS)
g) pH induced vesiculation
h) Calcium induced fusion
12

13. (C ) Solvent dispersion methods
Ethanol Injection
Ether injection
Water in organic phase
Reverse phase evaporation vesicles
Stable plurilamellar vesicles (SPVs)
Double emulsion vesicles
(D) DETERGENT SOLUBILIZATION
13

14. 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, and
•Analysis of final product.
14

15. Physical dispersion:
There are four basic method of physical dispersion. I.e. hand shaking,
non-shaking, freeze dry and proliposomes.
Hand-shaken multilamellar vesicles (MLVs) :
15

16. Sonicated Unilamellar Vesicles (SUVs) :
16

17. French pressure cell liposomes :
Type of liposomes : ULV or OLV
Pressure :20000 or 40000
Sample volume : maximum 40ml
Minimum 4ml
Out flow : 0.5-1 ml / min.
17

18. Membrane extrusion liposomes :
Type of liposomes : MLV or
LUV
Pressure : 100 psi
Type membranes : Tortuous path,
Nucleation track (polycarbonate )
18

19.  Solvent dispersion methods:
 In this methods, lipids are first dissolved in an organic
solution, which is then brought into contact with the aqueous
phase containing material to be entrapped within the
liposomes.
 Methods employing solvent dispersion fall into one of three
categories.
 The organic solvent is miscible with the aqueous phase.
 The organic solvent is miscible with the aqueous phase, the
latter being in a large excess.
 Organic solvent is in large excess, and is again immiscible
19

20. Ether injection &ethanol injection
20

21.  DETERGENT SOLUBILIZATION :
 In this method, the phospholipids are brought into intimate contact
with the aqueous phase via the intermediary of detergents, which
associate with phospholipid molecules and serve to screen the
hydrophobic portions of the molecule from water.
 The structures formed as a result of this association are known as
micelles, and can be composed of several hundred component
molecules.
 Their shape and size depends on chemical nature of the detergent,
the concentration and other lipids involved.
 As a general rule, membrane-solubilizing detergents have a higher
affinity for phospholipid membranes than for the pure detergent
micelles.
21

22. ACTIVE LOADING TECHNIQUE
22

23.  Active loading method have following advantages
over passive encapsulation techniques:
 A high encapsulating efficiency and capacity.
 A reduced leakage of the encapsulated compounds.
 “Bed side” loading of drug thus limiting loss of retention of
drug by diffusion or chemical degradation during storage.
 Flexibility for the use of constitutive lipids, as drug is
loaded after the formation of carrier units.
 Avoidance of biological compound during preparation
steps in the dispersion thus reducing safety hazards.
 The transmembrane pH gradient can be developed using
various method depending upon the nature of the drug be
encapsulated.
 For amphipathic weak bases by active loading procedures
such as using a proton gradient or an ammonium sulphate
gradient or calcium acetate gradient.
23

24.  STABILITY OF LIPOSOMES
 The stability studies could be broadly covered under
two main sections.
1. The stability in vitro, which covers the stability
aspects prior to the administration of the formulation
and with regard to the stability of the constitutive
lipids.
2. The stability in vivo, which covers the stability
aspects once the formulation, is administered via
various routes to the biological fluids. These include
stability aspects in blood (serum) if administered by
systemic route or in gastrointestinal tract, if
administered by oral or preoral routes.
24

25. Stability in vitro

 Lipid oxidation an peroxidation
 Lipid hydrolysis
 Long term and accelerated stability
Stability after systemic administration
Stability in vivo after oral administration
25

26.  APPLICATION OF LIPOSOMES
 Liposomes as drug/protein delivery vehicles
 Controlled and sustained drug release in situ.
 Enhanced drug solubilization
 Altered pharmacokinetics and biodistribution
 Enzyme replacement therapy and lysosomal storage
disorders
 Liposomes in antimicrobial, antifungal and antiviral therapy
 Liposomal drugs
 Liposomal biological response modifiers
 Liposomes in tumour therapy
 Carrier of small cytotoxic molecules
 Vehicle for macomolecules as cytokines o genes
26

27.  Liposomes in gene delivery
 Gene and antisense therapy
 Genetic vaccination
 Liposomes in immunology
 Immonoadjuvant
 Immunomodulator
 Immunodiagnosis
 Liposomes as artificial blood surrogates
 Liposomes as radiophamaceutical and radiodiagnostic cariers
 Liposomes in cosmetics an dermatology
 Liposomes in enzyme immobilization and bioreactor
technology 27

28. Some liposomal formulation of Amphotericin B
System Target disease Brand name Product
Liposomes Systemic fungal AmBisome NeXstar, USA
(i.v) infection,
Visceral
leishmaniasis
Liposomes Systemic fungal Amphocil SEQUUS, USA
(i.v) infection
Liposomes Systemic fungal ABELECT The Liposome
(i.v) infection company,
USA
28

29. Liposomes in gene therapy:
Type of Advantages Disadvantages
vector
Viral • Relative high transfection • Immunogenicity, presence of
vector efficiency contaminants and safety
• Vector restricted size limitation
for recombinant gene
• Unfavourable p’ceutical issue-
large scale production, GMP,
stability and cost
Non-viral • Favourable p’ceutical issue- • Relative low transfection
large scale production, GMP, efficiency
stability and cost
• Plasmid independent structure
• Low immunogenicity
• Opportunity for
chemical/physical
manipulation
29

30. Various liposomal product in dermatology and cosmetics
(launched or investigated)
Vesicular Marketed by Liposomes and
system ingredients
CaptureTM Christian Dior Liposomes in gel with
ingredients
PlentitudeTM L’Oreal Tanning agent in
liposomes
DermosomeTM Microfluidics Skin care, loaded
liposomes
PentaTM Pentapharm Humectant pentavitin R in
liposomes
Coatsome NCTM Nichiya liposomes Liposomes with humectant
Co 30

31. Imaging modality and required concentration of diagnostic
moieties:
Imaging modality Diagnostic moiety Concentrat
ion
γ-scintigraphy Diagnostic radio-nucleus 10-10M
such as 111In, 99mTc, 67Ga
Magnetic Po-magnetic ions such as 10-4M
resonance(MR) Gd, Mn and iron oxide
Computed Iodine, Bromine an Barium 10-2M
tomography(CT)
Ultrasound imaging Gas (Air, Argon and
or Nitrogen)
Ultrasonography(
US) 31

32. COMMERCIAL MANUFACTUING OF LIPSOMAL DRUG
NO Problems Remedies
1 Poor quality of the High quality products with improved
raw material purification protocol and validated
mainly the analytical techniques are available
phospholipids.
2 Pay load is too Use either lipophilic drug/lipophilic
low prodrug of hydrophilic drug or using
active techniques.
3 Poor Quality control assay can be performed
characterization of using sophisticated instruments and batch
the to batch variability can be checked.
physicochemical
properties of the
liposomes
32

33. NO Problems Remedies
4 Shelf life is too Improved by appropriate cryoprotectant
short and lyoprotectant and product can be
successfully freeze dried.
5 Scale up related Scaling up can be improved by
problems carefully selecting method of
preparation, sterilization by autoclaving
or membrane filtration coupled with
aseptic processing and pyrogen removal
using properly validated LAL test
6 Absence of any By choosing candidate potent drugs
data on safety of with narrow therapeutic window the
these carrier drug elated safety problems can be
systems on alleviated.
chronic use. 33

34. 34