Liposomes Preparation Evaluation Application

1. Guru Jambheshwar University
of Science and Techonology
TOPIC NAME: LIPOSOMES.
SUBMITTED TO: SUBMITTED BY:
Dr.REKHA RAO KRISHAN NAGAR

2. LIST OF CONTENT:
1. INTRODUCTION
2. STRUCTURE OF LIPOSOMES
3. COMPONENTS OF LIPOSOMES
4. CLASSIFICATION OF LIPOSOMES
5. CHARACTERIZATION OF LIPOSOMES
6. TYPES OF LIPOSOMAL DRUG DELIVERY PLATFORMS
7. REQUIREMENT MATERIAL FOR PREPARATION
8. PREPARATION METHODS
9. PHASE TRANSITION TEMPERATURE
10.BIOLOGICAL CHANGES FACING LDDS
11.EPR EFFECT
12.ADVANTAGES & DISADVANTAGES
13.APPLICATION OF LIPOSOMES

3. INTRODUCTION :
 A liposome is a small artificial vesicle, spherical in shape, having
at least one lipid bilayer. Due to their hydrophobicity and/or
hydrophilicity, biocompatibility, particle size and many other
properties, liposomes can be used as drug delivery vehicles
for administration of pharmaceutical drugs and nutrients, such
as lipid nanoparticles in mRNA vaccines, and DNA vaccines.
Liposomes can be prepared by disrupting biological membranes
(such as by sonication).
 Liposomes are most often composed of phospholipids,
especially phosphatidylcholine and cholesterol, but may also
include other lipids, such as egg, phosphatidyl ethanolamine, as
long as they are compatible with lipid bilayer structure. A
liposome design may employ surface ligands for attaching to
unhealthy tissue.

4.  The major types of liposomes are the multilamellar vesicle (MLV,
with several lamellar phase lipid bilayers), the small unilamellar
liposome vesicle (SUV, with one lipid bilayer), the large
unilamellar vesicle (LUV), and the cochleate vesicle. A less
desirable form are multivesicular liposomes in which one vesicle
contains one or more smaller vesicles.

5. Structure Of Liposome:

7. STRUCTURAL COMPONENTS OF
LIPOSOMES:
The main components of liposomes are:
 Phospholipids
 Cholesterol
1. Phospholipids:The phospholipids possess a hydrophobic tail structure
and a hydrophilic head component.When dissolved in water, the
hydrophobic tails mutually attract while the hydrophilic heads contact
with the aqueous medium external and internal to the liposome surface.
In this way, double lipid layers are formed which seal off to form small
vesicles similar to the body cells and organelles.
Natural phospholipids:
 PC-Phosphatidylcholine
 PE-Phosphatidylethanolamine
 PS-Phosphatidylserine

8. Synthetic phospholipids:
 DOPC-Dioleoylphosphatidylcholine
 DSPC-Disteroylphosphotidylcholine
 DOPE-Dioleoylhosphatidylethanolamine
 DSPE-DisteroyIphosphotidylethanolamine
2. Cholesterol: Cholesterol acts as a fluidity buffer, ie below
the phase transition temperature, it makes the membrane less
ordered and slightly more permeable; above the phase
transition temperature it makes the membrane more ordered
and stable.

9. CLASSIFICATION OF LIPOSOMES:
Based on structural parameters:
1.MLV-Multilamellar vesicles(>0.5um)
2.OLV-Oligolamellar vesicles(0. 1-1 um)
3.UV Unilamellar vesicles(all size ranges)
a. MUV –Medium Unilamellar Vesicles
b. SUV-Small Unilamellar Vesicles(20-100nm)
c. GUV-Giant Unilamellar Vesicles(>1 um)
d. LUV-Large Unilamellar Vesicles(>100nm)
4. MVV/MV Multivesicular vesicles(>1 um)

10. Based on method of preparation:
1. REV- SUVs/OLVs made by reverse-phase evaporation method
2. MLV-REV-MLVs made by reverse-phase evaporation method
3. SPLV-›Stable plurilamellar vesicles
4. FATMLV-›Frozen and thawed MLV
5. VET-›Vesicles prepared by extrusion technique
6. DRV-De-hydrated rehydration method

11. Based upon composition and
applications:
1. Conventional liposomes (CL)
2. Fusogenic liposomes
3. pH sensitive liposomes
4. Cationic liposomes
5. Long circulatory(stealth) liposomes (LCL)
6. Immuno-liposomes

12. CHARACTERIZATION OF LIPOSOMES:
There are mainly three types of characterization of liposomes. They
are:
1. Physical characterization
2. Biological characterization
3. Chemical characterization

13. 1. Physical Characterization:
S.No. Characterization
parameters
Analytical
methods/instruments
1. Vesicle shape and surface
morphology
Transmission electron
microscopy,
Freeze-fracture electron
microscopy
2. Surface charge Free-flow electrophoresis
3. Electrical surface potential
and surface pH
Zeta potential
meastrements
pH
sensitive probes
4. Phase behavior Freeze_trachre electron
microscopy
Differential
scanning colorimetery_
5. Drug release Dittusion cell dia vsis

14. 2. BIOLOGICAL CHARACTERIZATION
S.No. Characterization
parameters
Analytical
method/Instrument
1. Sterility Aerobic or anaerobic
cultures
2. Pyrogenicity Limulus Amebocyte
Lysate (LAL) test
3. Animal toxicity Monitoring survival
rates, histology and
pathology

15. 3. CHEMICAL CHARACTERIZATION
S.No. Characterization parameter Analytical methods/instruments
1. Phospholipid concentration Barlett assay, stewart assay, TLC.
2. Cholesterol concentration Cholesterol oxidase assay, Ferric
perchlorate method
3. Lysolecithin concentration Densitometer
4. Phospholipid peroxidation UV absorbance, lodometery,
GLC.
5. Phospholipid hydrolysis &
Cholesterol autooxidation
HPLC & TLC
6. pH of liposomal dispersion pH meter
7. Osmolarity Osmometer

16. TYPES OF LIPOSOMAL DRUG
DELIVERY PLATFORMS:
In general, there are four key types of liposomal delivery systems:
1. conventional liposomes
2. sterically-stabilized liposomes
3. ligand-targeted liposomes
4. a combination of the above

17. 1. Conventional liposomes
 Conventional liposomes were the first generation of liposomes
to be developed. They consist of a lipid bilayer that can be
composed of cationic, anionic, or neutral (phospho)lipids and
cholesterol, which encloses an aqueous volume.
 Conventional liposomal formulations reduced the toxicity of
compounds in vivo, through modifying pharmacokinetics and
biodistribution to enhance drug delivery to diseased tissue in
comparison to free drug. However, the delivery system was
prone to rapid elimination from the bloodstream, therefore
limiting its therapeutic efficacy.
 This rapid clearance was due to opsonization of plasma
components and uptake by fixed macrophages of the
reticuloendothelial system (RES), mainly in the liver and
spleen.

18. 2. sterically-stabilized liposomes
 To improve liposome stability and enhance their circulation
times in the blood, sterically-stabilized liposomes were
introduced. The hydrophilic polymer, polyethylene glycol (PEG),
has been shown to be the optimal choice for obtaining
sterically-stabilized liposomes.
 The establishment of a steric barrier improves the efficacy of
encapsulated agents by reducing in vivo opsonization with
serum components, and the rapid recognition and uptake by the
RES. This not only reduces the elimination of drugs by
prolonging blood circulation and providing accumulation at
pathological sites, but also attenuates side effects.

19. 3. Ligand-targeted liposomes
 Ligand-targeted liposomes offer a vast potential for site-
specific delivery of drugs to designated cell types or organs in
vivo, which selectively express or over-express specific ligands
(e.g., receptors or cell adhesion molecules) at the site of
disease. Many types of ligands are available, such as antibodies,
peptides/proteins and carbohydrates. The coupling of
antibodies, particularly monoclonal antibodies, to create
immunoliposomes represents one of the more versatile ligands
that can be affixed to liposome surfaces. One of the advantages
of using monoclonal antibodies is their stability and higher
binding avidity because of the presence of two binding sites on
the molecule. Since lipid assemblies are usually dynamic
structures, surface-coupled ligands have a high motional
freedom to position themselves for optimal substrate-
interactions.

20. 4. Combination of above
 The limited in vivo performance of immunoliposomes, due to
poor pharmacokinetics and immunogenicity, has been a major
hurdle to achieving their potential as effective site- specific
drug carriers. Therefore, newer generation of liposomes have
utilized a combination of the above design platforms to further
improve liposomal targeting and associated drug delivery.
 For example, integrating target-specific binding of
immunoliposomes with the steric stabilization of PEG (thereby
creating long- circulating immunoliposomes) has significantly
improved the pharmacokinetics of immunoliposomes.
 Overall as a drug delivery platform, liposomes offer a dynamic
and adaptable technology for enhancing the systemic efficacy
of therapeutics in various diseases.

21. Requirement materials for preparation:
 Phospholipid like lecithin and all other kinds.
 Distilled water.
 Cholesterol.
 Tricine buffer.
 Methanol.
 Acetone.
 Chloroform.
 Diethyl ether.
 Ethanol.

22. Preparation methods:
 Due to phospholipids amphiphilic properties, they form
accumulated complexes and protect their hydrophobic head
groups from the aqueous phase while hydrophilic head groups
stay in contact with water molecules, when they are located in
aqueous environment.
Sonication: Sonication is may be the most widely applied method for
the preparation of liposomes and nanoliposomes. Sonication is a simple
technique for dropping the size of liposomes and production of
nanoliposomes . There are some disadvantages for this method such as
very low internal volume/encapsulation efficiency, removal of large
molecules and metal pollution from probe tip.

23.  There are two major sonication techniques:
1. Probe sonication
2. Bath sonication.
1. Probe sonication: This is a common laboratory technique
includes handling hydrated vesicles for several minutes with a
titanium-tipped probe sonicator as explained in the following
section.
 Dissolve an appropriate mixture of the phospholipid
components, with cholesterol in chloroform with ratio of 2:1
v/v.
 Filter the mixture to eliminate slight insoluble ingre- dients
then use a rotary evaporator to remove the sol- vents at
temperatures above Tc or both lyophilization and spray drying
can be used instead

24.  Use a vacuum pump to remove traces of the organic solvents at
pressures under 0.1 Pa.
 Add small quantity of glass beads with 550lm diameter to the
flask comprising the dried lipids following by the addition of an
appropriate aqueous phase such as distilled water or buffer.
The aqueous phase can include chelating agents, stabilizers,
salts, and the drug to be encapsulated .
 Disperse the dried lipids into hydration fluid by vortex mixing
for 6 min so at this step micrometric MLV type liposomes are
created.
 Use a probe sonicator and insert the tip of the sonicator in the
MLV flask and run the sonicator. To avoid over-heating use 20 s
ON, 20 s OFF intervals for a total period of 10–15min at this
step nanoliposomes are obtained predominantly in the form of
small unila- mellar vesicles (SUV).

25. 2. Bath sonication:
In this method, the five first steps are as same as the probe
sonicator method. In the following steps:
 Stow the bath sonicator with water (at room temperature)
mixed with a couple of drops of liquid detergent and suspends
the MLV flask in the bath sonicator and sonicate for a 30–45 min
After preparation of product either in probe sonication or bath
sonication method for annealing process put the final product at
temperatures above Tc under inert circumstances such as nitrogen
or argon for 1 h. Centrifuge could be applied to remove residual
large particles to gain a clear suspension of nanoliposomes. There
are important factors which influence the mean size and poly-
dispersity index of vesicles such as temperature, sonication time,
sample volume, sonicator tuning, and lipid composition and
concentration.

26. Heating method:
Most of the preparation methods of liposomes include the
exploitation of potentially toxic solvents such as diethyl ether,
methanol, and chloroform. The final product may contain
remnants of these toxic solvents chip in potential toxicity and
affect the stability of the lipid vesicles. Furthermore, to ensure
the clinical appropriateness of the nanoliposomal products, the
level of these solvents in the final formulations should be assessed
and to decrease the time and cost of production particularly at
the industrial scales, it would be much desirable to evade
exploitation of these sol- vents in liposome preparation. Also, in
preparation methods like microfluidization, the use of high
pressures or high shear forces during liposome synthesis, cause
harmful effects on the structure of the product. So, to overcome
these kinds of obstacles, it would be better to apply substitute
preparation methods like heating method which does not use
potentially toxic solvents .

27. This method is explained in the following steps:
 Provide a proper mixture of the phospholipid ingredients, with
proper amount of cholesterol in an aqueous phase under an
inert condition such as nitrogen or argon for 1.5–2 h.
 Blend the lipid dispersions with the material (e.g. as drug) to
be encapsulated and step up glycerol to a final volume
concentration of 4%, then pour the mixture in the a heat-
resistant bottle with six baffles.
 Put the bottle on a hot-plate magnetic stirrer at 900–1100rpm
and a temperature above Tc and mix the sample for 35min. For
cholesterol-containing formulations, it is better to dissolve
cholesterol in the aqueous phase at lower temperature (110 C)
in 1000 rpm for 20 min before adding the other ingredients.
 For annealing and stabilization process, place the finalproduct
at temperatures above Tc under inert circum- stances such as
nitrogen or argon for 1 h.

28. 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.
 Factors like temperature, ionic strength, and pH are the
important physicochemical properties of liposomes. Usually,
these nano-structures have low permeability to the
encapsulated material, but their permeability, might be
changed at high temperatures. Phospholipids have an important
thermal characteristic role in liposomes and they can undertake
a phase transition (Tc) at temperatures lower than their
concluding melting point .

29.  At phase transition temperature, the lipidic bilayer drops much of
its well-ordered packing while its fluidity enhances and this
temperature depends on some factors like acyl chain length,
Degree of saturation of the hydrocarbon chains, polar head
group and ionic strength of the suspension medium.
 By declining chain length, presence of branched chains and
bulky head groups and unsaturation of the acyl chains Tc falls.
 Important proper- ties such as accumulation, capability to
deformation, fusion and permeability all of which can impact on
the stability of liposomes depend on the phase behavior of
liposomes.

30. BIOLOGICAL CHALLENGES FACING
LIPOSOMAL DRUG DELIVERY SYSTEMS
 As with any foreign particle that enters the body,
liposomes encounter multiple defense systems aimed at
recognition, neutralization, and elimination of invading
substances. These defenses include the RES, opsonization,
and immunogenicity. While these obstacles must be
circumvented for optimal liposome function, other factors
such as the enhanced permeability and retention (EPR)
effect can be exploited to enhance drug delivery.

31. The Reticuloendothelial System
(RES) andLiposome Clearance
 The RES is the main site of liposome accumulation following their systemic
administration . Primary organs associated with the RES include the liver,
spleen, kidney, lungs, bone marrow, and lymph nodes. The liver exhibits the
largest capacity for liposomal uptake followed by the spleen, which can
accumulate liposomes up to 10-fold higher than other RES organs.
 The ability of the RES to sequester liposomes from the circulation is
attributed to fenestrations in their microvasculature. Pore diameters in these
capillaries can range from 100 to 800nm, which is large enough for the
extravasation and subsequent removal of most drug-loaded liposomes (50–
1000nm in size). Liposomes are cleared in the RES by resident macrophages
via direct interactions with the phagocytic cells. Uptake of liposomes by the
RES is typically secondary to vesicle opsonization—that is, the adsorption of
plasma proteins such as immunoglobulin, fibronectin, lipoproteins, and/or
complement proteins onto the phospholipid membrane. However, in vitro
studies have demonstrated that liposomal clearance via macrophages can also
occur in the absence of plasma proteins.

32. The Enhanced Permeability and
Retention(EPR) Effect
 Liposomes that have evaded both the RES and opsonization are
subjected to the EPR effect. The EPR effect refers to the
increased permeability of the vasculature that supplies
pathological tissues (e.g., tumors and conditions involving
inflammation). At these sites, deregulations in angiogenesis
and/or the increased expression and activation of vascular
permeability factors predominates, which leads to fenestrations
that can range from 300 to 4700nm. This allows liposomes to
extravasate and accumulate by passive targeting.
 For example, inflammation results in a dramatic change in
blood vessel permeability as the capillary vasculature
undergoes structural remodeling to allow leukocyte diapedesis
into the peripheral tissue.

33.  The width of the tight junctional regions between endothelial
cells in vivo has been reported to range from 12 to 20nm,
however exposure to inflammatory mediators increases
permeability of the microvasculature, with the formation of
gaps of up to 1μm. Pore sizes ranging from 0.2 to 1.2μm have
been observed, though the size and number of pores are
dependent upon the microenvironment of the pathological site.
 Importantly, all types of liposomal delivery systems are
subjected to the EPR effect, with PEGylated liposomes having
an advantage due to having reduced RES clearance and
extended circulation time.

34. ADVANTAGES OF LIPOSOME:
 Provides selective passive targeting to tumour tissues (liposome
doxorubicin).
 Increased efficacy and therapeutic index.
 Increased stability via-encapsulation
 Reduction in toxicity of the encapsulated agent.
 Site avoidance eftect.
 Improved pharmacokinetic effects (reduced elimination,
increased circulation life times).
 Flexibility to couple with site-specific ligands to achieve active
targeting.

35. DISADVANTAGES OF LIPOSOME:
 Production cost is high.
 Leakage and fusion of encapsulated drug/molecules.
 Sometimes phospholipids undergo oxidation and hydrolysis like
reaction.
 Short half-life
 Low solubility.

36. APPLICATION OF LIPOSOME:
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.

37. REFERENCES:
1. https://en.wikipedia.org/wiki/Liposome.
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599573/.
3. https://www.slideshare.net/ArshadKhan63/liposomes-
119175712.
4. https://www.slideshare.net/bharathpharmacist/liposomes-
39685994.
5. Advances and Challenges of Liposome Assisted Drug Delivery
written by Lisa Sercombe , Tejaswi Veerati , Fatemeh
Moheimani , Sherry Y. Wu , AnilK.Sood andSusanHua.
6. Recent advances on liposomal nanoparticles: synthesis,
characterization and biomedical applications written by Yunes
Panahi, Masoud Farshbaf, Majid Mohammadhosseini, Mozhdeh
Mirahadi, Rovshan Khalilov, Siamak Saghfi & Abolfazl
Akbarzadeh.

38. THANK YOU