Self-Assembly of Amphipathic Lipids - NPTELnptel.ac.in/courses/118106019/Module 3/Lecture 1/Lecture...

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Self-Assembly of Amphipathic Lipids

Dr. K. Uma Maheswari

Professor, School of Chemical & Biotechnology

SASTRA University



Table of Contents

1 WHAT ARE LIPIDS? ........................................................................................................ 3

2 SELF-ASSEMBLY OF LIPIDS ......................................................................................... 6

2.1 MICELLES ..................................................................................................................... 7

2.2 BILAYERS ..................................................................................................................... 8

2.3 PLANAR LIPID BILAYERS ........................................................................................ 10

2.4 VESICLES ................................................................................................................... 10

3 REFERENCE .................................................................................................................. 15



Preface

The two lectures in this module provide an insight into the driving forces in lipid self-

assembly structures. This information can be extended to develop new applications using

these self-assembled structures.

This lecture introduces to the learner various lamellar structures formed by the

amphipathic lipids.

1 What are lipids? Lipids are biomolecules that comprise a huge number of compounds that are

characterized by their insolubility in aqueous and polar solvents and are soluble in

organic solvents such as chloroform or acetone. There are two broad categories of lipids

ones with linear chain arrangement such as fatty acids, phospholipids, glycolipids,

sphingolipids etc. and other with cyclic fused ring structures. This group comprises the

steroids of which salient examples are cholesterol, testosterone, bilirubin etc. Lipids are

ubiquitously distributed in the biological system. They are the major component of the

cell membrane as well as the membranes of the vesicular intracellular compartments. The

membrane lipids are amphipathic i.e., they have a hydrophilic head group and a non-polar

tail chain. The nature of the polar head group (charged or uncharged) as well as the acyl

chain (saturated or unsaturated and number of carbon atoms) will determine their

localization and function in a body. The cell membrane architecture is made up of mainly

a group of amphipathic lipids known as phospholipids. The phospholipids have a glycerol

backbone and are esterified with long chained fatty acids at the first two carbons. The

third carbon is esterified with phosphoric acid, which is then linked with a base. The two

acyl chains make up the hydrophobic portion of the phospholipid while the phosphate as

well as the base makes up the polar hydrophilic group. The following animation (Figure

1) introduces the structure and representation of a phospholipid.

Module objective

This module attempts to introduce the learner to different types of self-assembled structures

that are formed by amphipathic lipids. Both lamellar and non-lamellar structures are

discussed in detail.



Fig. 1: Structure of phospholipid

Note: Can be viewed only in Acrobat Reader 9.0 and above

Apart from the type of base in the phospholipid, the acyl chains may also be different

depending on the cell type.

The phospholipids have a unique bilayer arrangement in the cell membrane. This

arrangement is primarily due to the amphipathic nature of the phospholipids. The

heterogeneity of the lipid composition in the cell membrane determines its permeability

characteristics and surface charge. The surface charge is an important parameter that

determines the type of interactions that can exist between the polar head groups and other

molecules at the membrane interface. Thus different cell membranes have different

compositions of the lipids. Some of the major types of phospholipids and their location in

the body are shown in the following Table 1.



Table 1: Structure of phospholipids and their location in the body



Apart from the membrane lipids, free lipids are also found in the biological system and

perform specific functions.For example the steroidal hormones estradione and

testosterone, inflammatory molecules leukotrienes and prostaglandins, the major

constituents of the bile juices bilirubin and biliverdin etc. are all lipidic in nature. The

major constituent of the lung surfactant is phosphatidyl choline. Though numerous

examples of lipids in biological system are available, as the present discussion is focused

on the self-assembling characteristics of lipids, further details on classification of lipids

are not provided.

2 Self-assembly of lipids The amphipathic lipids like its protein counterparts can self-assemble in aqueous medium

into a variety of assemblies. The structure of the assemblies formed is dictated by the

chemical nature of the amphipathic lipid as well as the self-assembling environment. The

major driving force for the self-assembly in aqueous medium comes from the

hydrophobic acyl chains. However, the hydrophilic head group helps in developing an

interface with the aqueous medium and also directs the inter-particle interactions as well

as the size of the self-assembled structures formed.

One of the major parameters that determines the final conformation of the self-assembled

lipid structures is a geometric factor referred to as the packing parameter, p. This

dimensionless parameter is given as the ratio of the hydrocarbon volume (v) to the

product of the area of the polar head group (a0) and the critical acyl chain length (lc)

beyond which the chain can no longer be considered fluid.

=

If the p value is less than

, then the amphipathic molecules are expected to form spherical

micellarstructures. If the value is above

but below , it may form non-sphericalmicellar

structures while p values >1 favour inverted assemblies. However, this prediction is

applicable only when a single amphipathic molecule is involved in the self-assembly

process. When more than one component is involved, due to the complex interactions

involving electrostatic interactions, hydrogen bonding and/or van der Waals forces

between the constituent molecules, deviations from the predicted structures are expected.

Novel structures such as nanodiscs, icosahedra, punctured planes etc. have been reported

in such cases.

Though the lipid self-assembled structures are not as complex or diverse in architecture

as in the case of their protein and nucleotide counterparts, the self-assembled lipid

structures have been exploited on a commercial basis by many industries. The lipid self-

assembled structures have been widely employed by the pharmaceutical industries to


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deliver therapeutic molecules. They also find

deliver anti-ageing compounds and skin nutrients. Many premier cosmetic products

contain lipid-based formulations. In the food industry, ripening of cheese and

preservation of milk-based productshave been accomplished using self

structures. The decontamination of polluted water has also been successful using self

assembled lipid structures. The spontaneous self

a template to create high ordered structures of nano

lipidic systems have also created a new paradigm in the field of nanobiotechnology.

addition, it is well known that the lipid structures play a key functional role in

biological systems. Some of the major self

lipids are discussed in the following sections.

2.1 Micelles The simplest structure formed by amphipathic lipids is a micelle structure. The fatty

acids, which possess a single fatty acyl chain and a polar head group

structures in an aqueous medium

aqueous medium and the hydrophobic acyl chains facing inwards to minimize contact

with the polar medium. The size

the acyl chain is a major determinant of the size of the

reverse micelle is formed with the polar head groups in the centre and the hydrophobic

chains facing outwards. Figure 2

Fig. 2: Pictorial representation of a

The driving force for the micelle formation is the hydrophobicity of the acy

micelle formation occurs only beyond a certain concentration of the amphipathic

molecule. This concentration is referred to as the critical micelle concentration (

Below the cmc, the amphipathic molecules e

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deliver therapeutic molecules. They also find applications in the cosmetic industries to

ageing compounds and skin nutrients. Many premier cosmetic products

based formulations. In the food industry, ripening of cheese and

based productshave been accomplished using self-assembled lipid

decontamination of polluted water has also been successful using self

The spontaneous self-assembly of lipids has been employed as

a template to create high ordered structures of nano-dimensions. Nano-reactors based on

lipidic systems have also created a new paradigm in the field of nanobiotechnology.

ion, it is well known that the lipid structures play a key functional role in

Some of the major self-assembled structures formed by amphipathic

lipids are discussed in the following sections.

formed by amphipathic lipids is a micelle structure. The fatty

which possess a single fatty acyl chain and a polar head group, form micellar

in an aqueous medium with the polar head group facing outwards towards the

hydrophobic acyl chains facing inwards to minimize contact

The sizes of the micelles range between 2-20 nm. The length of

the acyl chain is a major determinant of the size of the micelle. In organic solvents, a

formed with the polar head groups in the centre and the hydrophobic

hains facing outwards. Figure 2 represents the structure of a micelle and reverse micelle.

: Pictorial representation of a reverse micelle (left panel) and micelle (right panel)

The driving force for the micelle formation is the hydrophobicity of the acy


molecule. This concentration is referred to as the critical micelle concentration (

Below the cmc, the amphipathic molecules exist as monolayers at the air

Page 7 of 15

cosmetic industries to

ageing compounds and skin nutrients. Many premier cosmetic products

based formulations. In the food industry, ripening of cheese and

assembled lipid

decontamination of polluted water has also been successful using self-

of lipids has been employed as

reactors based on

lipidic systems have also created a new paradigm in the field of nanobiotechnology.In

ion, it is well known that the lipid structures play a key functional role in all

assembled structures formed by amphipathic

formed by amphipathic lipids is a micelle structure. The fatty

form micellar

with the polar head group facing outwards towards the

hydrophobic acyl chains facing inwards to minimize contact

20 nm. The length of

In organic solvents, a

formed with the polar head groups in the centre and the hydrophobic

represents the structure of a micelle and reverse micelle.

micelle (left panel) and micelle (right panel)

The driving force for the micelle formation is the hydrophobicity of the acyl chains. The


molecule. This concentration is referred to as the critical micelle concentration (cmc).

xist as monolayers at the air-liquid



interface.As the concentration of the amphipathic molecule increases, the hydrophobic

forces drive them to aggregate. At the same time, a repulsive force develops between the

charged/polar head groups. These opposing forces balance each other in the self-

assembled micellar structure that is formed.

Aggregation number refers to the number of amphipathic molecules present in each

micellar structure. As there will be size variations, the aggregation number can be

presented only as a range of values that follow the Gaussian distribution. Micelles

containing charged head groups wouldbe associated with counter ions from the aqueous

medium. As the concentration of the surfactant increases, deviations from the spherical

morphology increase, resulting in formation of rod-like, worm-like and mesophase

structures. These structures contain a greater aggregation number. The rod-like structures

have similar diameter as the spherical micelle but has a length scale that is two to five

fold higher than its spherical counterpart. The worm-like micelles have length scales in

the micron range while the mesophase structures can adopt a hexagonal or cubic phase

arrangements.

A micelle containing more than one type of amphipathic lipid is known as a multi-

component micelle or a mixed micelle. The presence of additional components can

either decrease the cmc by promoting faster aggregation at much lower concentrations or

increase the cmc. The decrease in cmc values in a multi-component system is referred to

as synergism while increased cmc values due to the additional components is known as

antagonism. One of the major commercial applications of micelles is in detergents.

Apart from this, catalysis, drug delivery, stabilizers all have incorporated micellar

assemblies for better performance.

2.2 Bilayers Lipid bilayers are the most common assembly encountered in nature. A bilayer contains

two leaflets each made of phospholipids with the acyl chains in each leaflet associated in

a tail-to-tail manner. The polar head groups of the phospholipids in the outer and inner

leaflets are in contact with the aqueous medium. The presence of bilayer offers maximum

shielding to the two acyl chains in each phospholipid in both leaflets of the bilayer from

the aqueous environment. The cell membrane contains the lipid bilayer construct, which

is nearly flat. Vesicles and tubular constructs that have a curvature are also encountered

in the biological system as intracellular transport vehicles. The bilayer architecture

confers stability to the amphipathic molecules that make the bilayer. If only a single

bilayer is present in the lipid structure, then it is known as a unilamellarlipid bilayer. If

multiple bilayers are stacked upon each other in the lipid structure, they are known as

multilamellar lipid bilayers. Figure 3 shows the arrangement of lipid molecules in

unilamellar and multilamellar lipid bilayers.



Fig. 3: Unilamellar and multilamellar lipid bilayers

In the biological system, the composition of the phospholipids is diverse and hence is

referred to as heterogenous bilayer. The lipids are capable of moving within the same

leaflet (lateral mobility). They can also move from one leaflet to the other in rare

circumstances and this movement is referred to as the flip-flop motion. The packing of

the lipid bilayer depends on the nature of the acyl chains in the phospholipids forming the

bilayer. Acyl chains with higher percentage of unsaturation lead to lesser packing density

compared to saturated acyl chains. In biological system, the packing density is a critical

factor as it determines the permeability characteristics of the membrane. The

phospholipids have a characteristic temperature at which they undergo gel-to-liquid

crystalline phase transition, which also determines the fluidity of the bilayer membrane

for a given temperature.

Recently, the existence of lipid rafts in the lipid bilayers have been identified. Lipid rafts

are microdomains that have more disordered packing of lipids that are in the liquid

crystalline phase when compared with the more gel-like packing in the rest of the lipid

bilayer. These regions are believed to be associated with enhanced permeability to

molecules as well as respond quickly to external stimuli in cell membranes. A single lipid

bilayer membrane might possess multiple lipid rafts.


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2.3 Planar lipid bilayersFlat lamellar bilayer structures are known as planar lipid bilayers. Such assemblies have

been spontaneously formed as an experimental system and used to investigate the

interactions of single molecules on the bilayer architecture.

the bilayer construct of the cell membrane and helps to understand the role of lipid

molecule interactions in altering membrane properties. The formation of a planar lipid

bilayer is carried out by introducing a small amount of phospholi

bifurcating two chambers filled with aqueous medium. The phospholipids spontaneously

self-assemble to form a bilayer structure acr

and any residual organic solvent during the process. The shape

it is chamfered to ensure a reservoir of phospholipids remains at the edges of the aperture.

This facilitates dynamic exchange of phospholipids as is observed in biological

membranes. Figure 4 represents the various stages involved in the formation of a planar

lipid bilayer through self-assembly.

Fig. 4: Stages involved in the formation of planar lipid bilayer

The process of thinning and formation of the planar lipid bilayer takes between 10

minutes. The formation of lipid bilayer can also be carried out at the tip of a metal

(supported bilayer) as well as through covalent linking to the metal.

2.4 Vesicles When the amphipathic lipids are dispersed in a large amount of aqueous medium, they do

not form monolayers but tend to form spherical structures made of lipid bilayers,

especially if the packing parameter values are in between and 1. The bilayer structure

ensures that the hydrophobic chains do not come into direct contact with the aqueous

Bulk lipid droplet

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lipid bilayers lat lamellar bilayer structures are known as planar lipid bilayers. Such assemblies have


interactions of single molecules on the bilayer architecture. This system serves to mimic

the bilayer construct of the cell membrane and helps to understand the role of lipid


bilayer is carried out by introducing a small amount of phospholipids in an aperture


assemble to form a bilayer structure across the membrane, thereby eliminating wat

and any residual organic solvent during the process. The shape of the aperture is such that



represents the various stages involved in the formation of a planar

assembly.

: Stages involved in the formation of planar lipid bilayer

of thinning and formation of the planar lipid bilayer takes between 10

The formation of lipid bilayer can also be carried out at the tip of a metal

(supported bilayer) as well as through covalent linking to the metal.

mphipathic lipids are dispersed in a large amount of aqueous medium, they do



that the hydrophobic chains do not come into direct contact with the aqueous

Bulk lipid droplet Thinning lipid layer Planar lipid bilayer

Page 10 of 15

lat lamellar bilayer structures are known as planar lipid bilayers. Such assemblies have


em serves to mimic

the bilayer construct of the cell membrane and helps to understand the role of lipid-


pids in an aperture


eliminating water

of the aperture is such that



represents the various stages involved in the formation of a planar

of thinning and formation of the planar lipid bilayer takes between 10-30

The formation of lipid bilayer can also be carried out at the tip of a metal

mphipathic lipids are dispersed in a large amount of aqueous medium, they do



that the hydrophobic chains do not come into direct contact with the aqueous

Planar lipid bilayer



phase. These spherical structures are termed as vesicles and as they are composed on

lipids, they are also commonly referred to as liposomes. The liposomes enclose an

aqueous core that is surrounded by a hydrophobic region comprising the acyl chains. The

periphery of the liposomes contains the polar head groups that interact with the aqueous

medium surrounding it. Liposomes literally translate to fat body, which is a misnomer.

Hence, most literatures still refer to these structures as lipid vesicles. There are a large

number of vesicles that vary in the type and composition of the amphipathic molecule

forming these structures. These are listed in the following Table 2.

Table 2. Different types of vesicles

Name of vesicle Meaning and use of the term in the literature

Algosome Vesicle prepared on the basis of 1-O-alkylglycerol

Archaesome Vesicles prepared from archaebacterial and bolaamphiphiliclipids

Bilosome Vesicles prepared form a mixture of non-ionic surfactants (1-

monopalmitoyl-glycerol), cholesterol anddihexadecylphosphate in the

molar ratio of 5:4:1 and deoxycholate

Catanionic vesicle Vesicles prepared from a mixture of anionic and cationic surfactants

Cerosome Vesicles with a silicate framework on its surface

Ethosome Vesicles that contains a considerable amount of ethanol in the final

preparation

Fluorosome Single unilamellar vesicle containing a fluorescent dye embedded in the

lipid bilayer to monitor the entry of molecules into the bilayer

Hemosome Hemoglobin containing vesicle

Immunoliposome Vesicles contains antibodies specific for a particular antigen on its surface

Lipid vesicle,

Liposomes

Vesicles prepared from amphiphilic lipids

Magnetoliposomes Vesicles containing magnetic nanoparticles (eg. Magnetite Fe3O4)

Marinosome Vesicles based on marine lipid extract containing high amount of

polyunsaturated acyl chains

Niosome Vesicles prepared form non-ionic surfactants

Novasome Oligo- or multilamellar vesicle prepared by the addition of vesicle-

forming surfactants in liquid state to an aqueous solution

Phospholipid

vesicles

Vesicles prepared from amphiphilic phospholipids



PLARosome Phospholipid containing resorcinolic lipids or their derivatives

Polymer vesicle,

Polymersome

Vesicles prepared from di- or tri- block copolymers

Polymerized vesicle Vesicles prepared from polymerizableamphiphiles that were partially

polymerized after vesicle formation

Proliposomes Dry (ethanol-free) granular preparations of vesicle-forming amphiphiles,

which upon hydration lead to vesicle formation

Proniosomes A dry, granular product containing mainly non-ionic surfactants which on

addition of water disperse to form multilamellar vesicles

Reversed vesicle Inverted micelle formed in apolar solvent in the presence of small amount

of water

Spherulite Onion-like vesicle prepared using shear forces

Sphingosome Vesicles prepared from sphingolipids present in human skin

Stealth liposome Sterically stabilized vesicle achieved through the use of co-amphiphiles

that have PEG. Sometimes polysaccharides can also be used

Synthetic vesicle Vesicles prepared from synthetic surfactants that are not present in the

biological membranes

Toposome Vesicles that has a surface that is site-selectively modified in a stable

manner at specific and deliberate locations

Transferosome Ultradeformable ethanol-containing mixed lipid/detergent vesicle used to

transfer water soluble molecules across human skin

Ufasome Vesicles prepared from unsaturated fatty acid/soap mixtures

Vesicle A small, membrane-bounded, spherical organelle in the cytoplasm of an

eukaryotic cell

Virosome Vesicles containing viral proteins and viral membranes reconstituted from

viral envelope

Lipid vesicles can be classified into different types based on their size as well as number

of layers of bilayers found in the structure. The small unilamellar vesicles (SUV) have

sizes below 50 nm and consist of a single bilayer surrounding an aqueous core. The large

unilamellar vesicles (LUV) still possess a single bilayer surrounding the aqueous core but

the sizes are below 1000 nm. In the case of giant vesicles (GV), the sizes of the vesicles

are well above 1 m but the number of bilayers is still restricted to one. Multi-lamellar

vesicles (MLV) have multiple concentric bilayers while multi-vesicular vesicles (MVV)



or vesosomes have non-concentric vesicles inside a large vesicle. Figure 5 shows the

pictorial representation of a few classes of lipid vesicles.

Fig. 5: Different types of lipid vesicles

Lipid vesicles like their planar counterparts have characteristic gel-to-liquid crystalline

phase transition temperature that is influenced by the length of the acyl chains, the

unsaturation status of the acyl chains, the nature of the head group as well as the

intermolecular interactions. A higher phase transition temperature indicates greater

associative forces between the lipids and closer packing. A lower phase transition

temperature indicates greater percentage of fluidity and looser packing of the lipids.

Lipid vesicles have been extensively investigated as drug and gene delivery carriers. This

is because of their ability to load both hydrophilic as well as hydrophobic moieties as

well as the ease with which they can be self-assembled. The polar groups on the outer

surface makes them easily modifiable with chemical functionalities that will impart

specific properties to it such as target specificity, optical visibility, long circulating nature

etc. Also, the lipid vesicles exhibit excellent cell uptake making them the preferred choice

in many drug delivery applications. Figure 6 depicts a surface-modified liposomal drug

carrier that has poly(ethylene glycol) chains on its surface to enhance its lifetime within

the biological system as well as a targeting ligand to specifically enter desired cells.



Fig. 6: Pictorial representation of a surface modified liposomal drug delivery system

The lipid vesicles can beformed by different techniques. The method chosen for

preparing the lipid vesicles will determine the size and stability of the final product.

Though many techniques such as thin film hydration, freeze-thaw, solvent exchange,

detergent depletion, proliposome method etc. are available, the most widely used method

is the thin film hydration technique.Figure 7 shows the various stages involved in the

preparation of a liposome by thin film hydration method.

Fig. 7: Stages involved in the formation of liposomes by thin film hydration process



Initially, the phospholipid solution is taken in a container and the solvent is evaporated

under reduced pressure or by purging with argon or nitrogen. As phospholipids are

extremely sensitive to thermal and oxidative degradation, the solvent evaporation

iscarried at low temperatures in the absence of air. Argon purging is preferable to

nitrogen purging as argon is denser than oxygen and hence the chances of being displaced

by oxygen are less. In the case of nitrogen that is less denser than oxygen, the chances of

it being displaced by the denser oxygen is more. Contact with oxygen may promote the

oxidative degradation of the lipids and is to be avoided at all times. The phospholipids are

soluble in chloroform, which is volatile and hence can be easily evaporated by purging or

under reduced pressure.After the solvent is evaporated completely, a thin film of the

phospholipids remains in the system. Now, a buffer is added to the system and warmed to

about 60-65oC. The addition of the buffer is to drive the amphipathic lipid molecules to

self assemble into a vesicle. The temperature of 60-65oC is slightly above the phase

transition temperature of most phospholipids and hence they are all in the liquid

crystalline phase at this temperature. These conditions will cause greater degree of

mobility of the acyl chains, which will further drive them to form vesicles in an effort to

minimize contact with the aqueous medium. The buffer solution also provides a highly

polar environment that will drive the hydrophobic acyl chains to form vesicles. The

vesicles thus formed are not of uniform size and in many cases they might possess multi-

lamellar arrangement. Hence, a final step known as extrusion is carried out. In this stage,

the liposomes are placed in an extruder that consists of two compartments separated by a

polycarbonate membrane with specific pore dimensions. The lipid vesicle dispersion is

forced from the first compartment to the second through the membrane using a syringe.

Once the lipid vesicle dispersion is in the second compartment, it is forced back to the

first compartment through the membrane using a syringe. This completes one cycle of

extrusion. The extrusion cycles are repeated for 10-25 times to achieve nearly mono-

disperse lipid vesicles with sizes below the pore size of the membrane used. The process

of extrusion squeezes off the additional lamellae that tend to increase the size of the

vesicles and repeated cycles ensures that the lipid vesicles remain in the same size.

3 Reference The multiple faces of self-assembled lipidic systems, Guillaume Tresset, PMC

Biophysics, 2(3), (2009)

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