priyanka ghosh

8/12/2019 Priyanka Ghosh

1/17

1

PHISIOCHEMICAL CHARACTERIZATION

OF LIPOSOME WITH SPECIAL

REFERENCE TO IPA

PRIY NK GHOSH

ROLL NO: 44

3rd SEMESTER

For Partial fulfillment ofDegree of M.sc in Chemistry

Dr. Amiya Kumar Panda

Department of ChemistryUniversity of North Bengal


2/17

2

Contents

Introduction..3 Experiment........13 Results and Discussion. 14 References.15 Acknowledgements. 18


3/17

3

INTRODUCTION:-

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 onefatty acid chaincovalentlybonded to aglycerolmolecule through an

ester linkage.

Fig 1: Schematic representation of monoglycerides
http://en.wikipedia.org/wiki/Glyceridehttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Covalenthttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Covalenthttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Glyceride


4/17

4

Diglycerides:A diglyceride, or a diacylglycerol (DAG), is aglyceride consisting of two

fatty acid chainscovalently bonded to aglycerol molecule throughester linkages. One example,

is 1-palmitoyl-2-oleoyl-glycerol, which contains side-chains derived frompalmitic acid andoleic

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 formesters.The glycerol molecule has three hydroxyl (HO-) groups. Each

fatty acid has acarboxyl group (-COOH). In triglycerides, the hydroxyl groups of the glycerol

join the carboxyl groups of the fatty acid to formesterbonds

Fig3: Schematic representation of triglycerides

Phospholipids: Phospholipids are a class oflipids that are a major component of allcell

membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a
http://en.wikipedia.org/wiki/Glyceridehttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Palmitic_acidhttp://en.wikipedia.org/wiki/Oleic_acidhttp://en.wikipedia.org/wiki/Oleic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Alcoholshttp://en.wikipedia.org/wiki/Estershttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Lipidshttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Diglyceridehttp://en.wikipedia.org/wiki/Diglyceridehttp://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Lipidshttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Estershttp://en.wikipedia.org/wiki/Alcoholshttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Oleic_acidhttp://en.wikipedia.org/wiki/Oleic_acidhttp://en.wikipedia.org/wiki/Palmitic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Fatty_acidhttp://en.wikipedia.org/wiki/Glyceride


5/17

5

phosphate group,and a simple organic molecule such ascholine The first phospholipid identified

as such in biological tissues waslecithin,orphosphatidylcholine,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.
http://en.wikipedia.org/wiki/Phosphate_grouphttp://en.wikipedia.org/wiki/Cholinehttp://en.wikipedia.org/wiki/Lecithinhttp://en.wikipedia.org/wiki/Phosphatidylcholinehttp://en.wikipedia.org/wiki/Phosphatidylcholinehttp://en.wikipedia.org/wiki/Lecithinhttp://en.wikipedia.org/wiki/Cholinehttp://en.wikipedia.org/wiki/Phosphate_group


6/17

6

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-preparedvesicle composed of alipid bilayer.The

liposome can be used as a vehicle for administration of nutrients and pharmaceutical drugs.

Liposomes are composed of naturalphospholipids,and may also contain mixed lipid chains withsurfactantproperties (e.g.,eggphosphatidylethanolamine). 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 withmicelles andreverse micelles composed

ofmonolayers.

Fig6: Schematic representation of liposome
http://en.wikipedia.org/wiki/Vesicle_%28biology%29http://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Route_of_administrationhttp://en.wikipedia.org/wiki/Nutrienthttp://en.wikipedia.org/wiki/Pharmaceutical_drughttp://en.wikipedia.org/wiki/Phospholipidhttp://en.wikipedia.org/wiki/Surfactanthttp://en.wikipedia.org/wiki/Egg_%28food%29http://en.wikipedia.org/wiki/Phospholipid#Phosphatidyl_ethanolaminehttp://en.wikipedia.org/wiki/Micellehttp://en.wikipedia.org/wiki/Micelle#Reverse_Micelleshttp://en.wikipedia.org/wiki/Monolayerhttp://en.wikipedia.org/wiki/Monolayerhttp://en.wikipedia.org/wiki/Micelle#Reverse_Micelleshttp://en.wikipedia.org/wiki/Micellehttp://en.wikipedia.org/wiki/Phospholipid#Phosphatidyl_ethanolaminehttp://en.wikipedia.org/wiki/Egg_%28food%29http://en.wikipedia.org/wiki/Surfactanthttp://en.wikipedia.org/wiki/Phospholipidhttp://en.wikipedia.org/wiki/Pharmaceutical_drughttp://en.wikipedia.org/wiki/Nutrienthttp://en.wikipedia.org/wiki/Route_of_administrationhttp://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Vesicle_%28biology%29


7/17

7

A liposome encapsulates a region of aqueous solution into its core; dissolvedhydrophilicsolutes 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 thecell 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 havelipophobic 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 becharged 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.
http://en.wikipedia.org/wiki/Hydrophilichttp://en.wikipedia.org/wiki/Soluteshttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Medicationhttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Lipophobichttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Charge_%28physics%29http://en.wikipedia.org/wiki/Protonshttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Protonshttp://en.wikipedia.org/wiki/Charge_%28physics%29http://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Lipophobichttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Medicationhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Cell_membranehttp://en.wikipedia.org/wiki/Soluteshttp://en.wikipedia.org/wiki/Hydrophilic


8/17

8

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.


9/17

9

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 pharmaceuticalcompound 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


10/17

10

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 iRNAconstructs),or even proteins such as antibodies, may be transfacted. Transfaction of

animal cells typically involves openingtransient 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.


11/17

11

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 theliposome 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 techniquein 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


12/17


13/17

13

V. Atomic Force Microscopy: Atomic force microscope is a very essential tool to obtain theatomic 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 abovementioned liposome

iv. Stable formulation from above mentioned system would be used in studying theirinteraction 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.


14/17

14

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

700C for 1 hour, in Rotavac. Then the rehydrated lipid solution was sonicated at 40-50

0c 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 900angle. An average of eighty data sets were considered for such measurements.

Preparati on of 10ml 1mM li posome 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


15/17

15

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


16/17

16

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.


17/17

17

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.

priyanka ghosh

Documents