1

PHISIOCHEMICAL CHARACTERIZATION
OF LIPOSOME WITH SPECIAL
REFERENCE TO IPA

PRIYANKA GHOSH
ROLL NO: 44
3
rd
SEMESTER

For Partial fulfillment of Degree of M.sc in Chemistry


Dr. Amiya Kumar Panda
Department of Chemistry
University of North Bengal
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Contents

Introduction..3
Experiment........13
Results and Discussion. 14
References.15

Acknowledgements. 18





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I NTRODUCTI ON:-
Lipids: The main biological function of lipids includes energy storage and formation of
bilayers. Cell membrane is comprised by phospholipids bilayer, so that naturally occurring lipids
can be useful to make model bilayer.

Lipid is amphiphlic in nature, the majority of the naturally occurring lipid molecules have
one polar head group connected to two hydrophobic chains via a suitable linkage (estsr, ether or
amide) . Such a molecular structure favors the formation of bilayer organization in polar
medium because of having two hydrophobic chains.
Classification of Lipids: Lipid is mainly comprised by hydrocarbon chain,
monoglycerides, diglycerides, triglycerides, phospholipids and others.
Monoglycerides: A monoglyceride, more correctly known as a monoacylglycerol, is a
glyceride consisting of one fatty acid chain covalently bonded to a glycerol molecule through an
ester linkage.

Fig 1: Schematic representation of monoglycerides
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Diglycerides: A diglyceride, or a diacylglycerol (DAG), is a glyceride consisting of two
fatty acid chains covalently bonded to a glycerol molecule through ester linkages. One example,
is 1-palmitoyl-2-oleoyl-glycerol, which contains side-chains derived from palmitic acid and oleic
acid. Diacylglycerols can also have many different combinations of fatty acids attached at both
the C-1 and C-2 positions.

.Fig2: Schematic representation of diglycerides
Triglycerides: A triglyceride is an ester derived from glycerol and three fatty acids.
Triglycerides are formed by combining glycerol with three molecules of fatty acid. Alcohols
have a hydroxyl (HO-) group. Organic acids have a carboxyl (-COOH) group. Alcohols and
organic acids join to form esters. The glycerol molecule has three hydroxyl (HO-) groups. Each
fatty acid has a carboxyl group (-COOH). In triglycerides, the hydroxyl groups of the glycerol
join the carboxyl groups of the fatty acid to form ester bonds


Fig3: Schematic representation of triglycerides
Phospholipids: Phospholipids are a class of lipids that are a major component of all cell
membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a
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phosphate group, and a simple organic molecule such as choline The first phospholipid identified
as such in biological tissues was lecithin, or phosphatidylcholine, in the egg yolk.

Fig4: Schematic representation of triglycerides

Biological function: The glycerophospholipids are the main structural component of
biological membrane, such as plasma membrane and the intracellular membrane and of
organelles; in animal cells the plasma membrane physically separates the intracellular
components from the extracellular components. While glycerophospholipids are the major
component of biological membrane, other monoglycerides, sphigomyelin and sterol (mainly
cholesterol) are also found in biological membrane. Lipid bilayer formation is favourable in
aqueous medium because of the hydrophobic interaction.
Membrane:
Cells make use of many different types of membranes. All cells have a cytoplasmic
membrane, or plasma membrane, that functions (in part) to separate the cytoplasm from the
surroundings. In the early days of biochemistry, the plasma membrane was not accorded many
functions other than this one of partition. We now know that the plasma membrane is also
responsible for (1) the exclusion of certain toxic ions and molecules from the cell, (2) the
accumulation of cell nutrients, and (3) energy transduction. It functions in (4) cell locomotion,
(5) reproduction, (6) signal transduction processes, and (7) interactions with molecules or other
cells in the vicinity.

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Fig5: The fluid mosaic model of membrane structure proposed by S. J. Singer and G. L.
Nicolson. In this model, the lipids and proteins are assumed to be mobile, so that they can move
rapidly and laterally in the plane of the membrane. Transverse motion may also occur, but it is
much slower.
Liposome: A liposome is an artificially-prepared vesicle composed of a lipid bilayer. The
liposome can be used as a vehicle for administration of nutrients and pharmaceutical drugs.
Liposomes are composed of natural phospholipids, and may also contain mixed lipid chains with
surfactant properties (e.g., egg phosphatidylethanolamine). The major types of liposomes are the
Multilamellar Vesicle (MLV), the Small Unilamellar Vesicle (SUV), and the Large Unilamellar
Vesicle (LUV).Liposomes should not be confused with micelles and reverse micelles composed
of monolayers.

Fig6: Schematic representation of liposome
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A liposome encapsulates a region of aqueous solution into its core; dissolved hydrophilic
solutes cannot readily pass through the lipids. Hydrophobic chemicals can be dissolved into the
membrane, and in this way liposome can carry both hydrophobic molecules and hydrophilic
molecules. To deliver the molecules to sites of action, the lipid bilayer can fuse with other
bilayers such as the cell membrane, thus delivering the liposome contents. By making liposomes
in a solution of DNA or drugs (which would normally be unable to diffuse through the
membrane) they can be (indiscriminately) delivered past the lipid bilayer. A liposome does not
necessarily have lipophobic contents, such as water, although it usually does.
Liposomes are used as models for artificial cells. Liposomes can also be designed to
deliver drugs in other ways. Liposomes that contain low (or high) pH can be constructed such
that dissolved aqueous drugs will be charged in solution. As the pH naturally neutralizes within
the liposome (protons can pass through some membranes), the drug will also be neutralized,
allowing it to freely pass through a membrane. These liposomes work to deliver drug by
diffusion rather than by direct cell fusion.
Components of liposome: Liposome can be treated as model bilayer membrane.
Generally it is prepared by naturally occurring lipids (e.g, phospholipids) and cholesterol. But
using of some other substance like of surfactant, ion pair amphiphile (IPA) and others bio-
compitable substance may sharply enhance its stability.
Limitation of Liposome mimetic system comprised of lipid: Naturally occurring lipids
are not good enough to stable at normal atmosphere. So it always kept under deep freeze. It is
easily oxidized in air and get rencidified. The melting point of lipid is low. So it can easily
degrade in moderate atmospheric temperature. Due to the presence of ester linkage, it can
undergo acidic hydrolysis (at low pH) or base hydrolysis (at high pH).Due to the microbial attack
lipid molecule can easily be degraded.

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To overcome the limitation, the following steps may be undertaken:
i) Substituting the phospholipids bilayer with Ion Pair Amphiphile:
When oppositely charged single tailed surfactants associate through electrostatic
attraction to mimic phospholipids-like structure under specific condition, then they are known as
Ion Pai Amphiphile(IPA). These ion-pair amphiphiles (IPA) resemble phospholipids and can
form catanionic or catanosomes.

Fig7: Schematic diagram of Ion Pair Amphiphile

Preparation of IPA: A known amount of positively charged surfactant solution (above
the CMC) was taken in a conical flask and a stoichiometric amount of aqueous solution of
negatively charged surfactant solution was added drop-wise into it so that in the final mixture
the mole ratio of both the surfactant was 1 : 1. The white precipitate (coacervate) was extracted
by chloroform. The chloroform layer was then removed by vacuum drying. The coacervate was
re-dissolved in chloroform and then dried again for 24 h under vacuum and grounded to fine
powders and stored in a desiccator.
Due to the resemblance with phospholipids, the lipid bilayer can be easily replaced by
IPA, which may enhance the stability of liposome mimetic system.
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Different mole ratio of lipid and ion pair amphiphile was taken to prepare liposome
mimetic system and check the stability with time. Out of these different composition, the most
stable system might use for further studies.
ii)Tuning the charge on the liposome by adding charged species: Liposome may be
synthesized by using positively charged or negatively charged lipid molecules.
Double tail ionic surfactant: Double tail ionic surfactant has lipid like skeleton and
hence using of positively charged double tail cationic surfactant with negatively charged
lipid molecules can tune the charge of the liposome mimetic system and vice-versa. Thus
the stability of liposome may increase.

Importance of liposome and mimetic systems:
i. Drug carrier: Drug delivery is the method or process of administering a pharmaceutical
compound to achieve a therapeutic effect in humans or animals. Drug release is from:
diffusion, degradation, swelling and affinity based mechanisms. Most common routes of
administration include the preferred non invasive par oral (through the mouth), topical
(skin), transmucosal (nasal, buccal /sublingual, vaginal, ocular and rectal) and inhalation
routes. Many medications such as peptide and protein, antibody, vaccine, and gene based
drugs, in general may not be delivered using these routes because they might be
susceptible to enzymatic degradation or cannot be absorbed into the systematic
circulation efficiently due to molecular size and charge issues to be therapeautically


Fig8: diffution of liposome on the surface of cell membrane

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effective. For this reason many protein and peptide drugs have to be delivered by
injection or in nanoneedle array (liposome system).
ii. Gene transfaction agent: Genetic material (such as supercoiled plasma DNA or iRNA
constructs),or even proteins such as antibodies, may be transfacted. Transfaction of
animal cells typically involves opening transient pores or holes in the cell membrane,
to allow the uptake of material.

Transfaction can be carried out using calcium phosphate, by electroporation, or by
mixing a cationic liposome with the material, which fuse with the cell membrane and
deposite their cargo inside.
iii. Dendriosome formation: Neutral, biodegradable, covalent or self assembled, hyper-
branched, spherical nano-particles with a size ranging from 15 to 100nm.

Fig9: Schematic diagram of dendrimer


The surface group of dendrimer may be positively or negatively charged. Oppositely charged
liposome can easily undergo attractive interaction with dendrimer to form stable dendriosome
which is efficient drug carrier, good gene transfecting agent.

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Characterization of liposome: Liposomes or mimetic system can be characterized by using
the following instruments:
I. Dynamic light scattering (DLS): DLS is used to determine the size and zeta potential of the
liposome or liposome mimetic system.


Fig10: Intensity vs size diagram of liposome and mimetic system


II. Differential scanning calorimeter: Differential scanning calorimeter or DSC is a technique
in which the difference in the amount of heat required to increase the temperature of a sample
(liposome) and reference is measured as a function of temperature. Thus it indicates the
phase transition temperature and the temperature tolerance of the system.

Fig11: DSC diagram of liposome
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III. Fluorescence(probing technique): Fluorescence probing technique is a very useful tool to
study the exited state spectroscopy behavior of liposome. Also, the lifetime and anisotropy
values can be determined using this technique. From the anisotropy we can measure the
micro viscosity of the probe compartmentalized into the liposome core.



Fig12: Fluorescence intensity of the probe encapsulate into the liposome

IV. Electron Microscopy: Electron microscopy forms a magnified image by focusing light on or
through the specimen. Transmission Electron Microscopy can be used to observe
modulations in chemical identity, crystal orientation, electronic structure of the liposome.
Scanning electron microscope can use to understand liposomes surface topography,
composition, and other properties such as electrical conductivity

Fig13: Electron microscopic image of liposome
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V. Atomic Force Microscopy: Atomic force microscope is a very essential tool to obtain the
atomic structure of liposome.



Fig14: AMF image of a liposome suspension deposited on mica

Plan of Work: The present work aims at the following:
i. To characterize the liposome mimetic system comprised with lipid-IPA system.
ii. To impart charge on liposome comprising IPA-lipid system.
iii. To study the effect of alkanols like ethanol, n-propanol, iso-propanol on the above
mentioned liposome
iv. Stable formulation from above mentioned system would be used in studying their
interaction with variety polymers like sodiumcarboxymethyl cellulose, chitosan, DNA,
bovine serum albumin (BSA) etc.

EXPERIMENTS:-
Materials:-
Lipid (soylecithin) and cholesterol were purchased from E. Merck, Germany. The surfactants
hexadecyltrimethylammonium bromide and sodium dodecyl sulfate, used for the preparation of
IPA, were products from Sigma Aldrich Chemicals Co., USA. They were stated to be more that
99.5% pure and were used as received. AR grade Chloroform and methanol was used in
dissolving the lipids. Phosphate buffer solution (pH 7.4) was prepared using sodium dihydrogen
phosphate and disodium hydrogen phosphate (SRL, Chemicals Co., India). Double distilled
water was used in preparing the required solutions.
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Method:- Required amount of the lipid and cholesterol dissolve in chloroform followed by
evaporation by using Rotavac. Then a thin layer was obtained by solvent evaporation. The thin
layer was then rehydrated by 10 ml phosphate buffer solution (pH 7.4). Then it was warmed at
70
0
C for 1 hour, in Rotavac. Then the rehydrated lipid solution was sonicated at 40-50
0
c three
times. Then we get small unilameller vesicle. Hydrodynamic diameter of the liposomes of
different combinations were determined using a dynamic light scattering spectrometer Nano ZS
90 (Malvern, U.K.). A He-Ne laser source emitting at 632.8 nm was used. Scattered data were
recorded at 90
0
angle. An average of eighty data sets were considered for such measurements.
Preparation of 10ml 1mM liposome mimetic system:


Results and Discussion:-
Liposomes, with the combinations as mentioned previously, were prepared. Size of the of
liposome of different composition of soylecithin (SLC)-Ion Pair Amphiphile (IPA) and
cholesterol (Chol) were measured at different time intervals. Results are summarized in Table 1.



Composition SLC (mg) IPA (mg) CHOLESTEROL
(mg)
Total volume
(ml)
[Lipid]
SLC:IPA
(10:0)
7.6 o 2.8 10 1mM
SLC:IPA
(8:2)
6.1 1.11 2.2 10 0.8mM
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Table1: Variation in the hydrodynamic diameter of liposome at different time intervals.




1:0 SLC-IPA 4:1 SLC-IPA
Time/day Size/nm Time/day Size/nm
1 140 0 158
5 143 8 161
10 147 14 160
45 151 21 167
37 171
59 185
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It was observed that with the increase in time, size of the liposome increased moderately.
However, while considering the effect of IPA, it was observed that the inclusion of IPA in the
liposome resulted in the larger extent of size enhancement. Results are further evidenced through
above Figure .
However, to draw final conclusion on this aspect, further studies using larger amount of IPA in
combination with the phospholipids are warranted. This can be considered as the future
perspective.
















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Acknowledgment
I wish to express my sincere thanks and gratitude to my teacher Dr. A.K. Panda for his
immensely valuable guidance and suggestions to complete this work. My thanks and
appreciation also goes to all the faculty members of the Department of Chemistry, University of
North Bengal for their help and encouragement.

I am also thankful to the research fellows Pritam Guha, Prasant Nahak, Gourab
Karmakar, Sudarshana Majumder, Kausik Manna, Sujoy Paul, Banita Sinha, Moumita
Chakraborty and my friends for their constant support and encouragement in every step during
this project work.