25 Lipid Bilayers, micelles and liposomes

Prof. M. N. Gupta

1. Objectives

•  To understand the mechanisms of aggregation of molecules to form micelles and bilayers
•  To learn about how lipids can form monolayers, bilayers, soap bubbles and vesicles.
•  To learn about liposomes: preparation, purification and structures.

2. Concept Map

3. Description

Fatty acid anions, bile salts, phospholipids are structurally different. What is the common feature among these molecules?

These are all lipids as they are soluble in organic solvents. Why are they all soluble in organic solvents? That is because all of these have a structural component, a larger component which is non polar in nature. Simultaneously, having a small polar structural component makes these molecules amphiphiles or surfactants or detergents. In chemistry, the word detergent is used as synonymous with a surfactant but we do talk of cleaning action of detergents.

With this module, we start our discussion on property of many lipid molecules to associate to form supramolecular assemblies. These associated structures can be of various shapes and dimensions.

The aim of this discussion is to lead to details of formation and function of biomembranes. That is not to say that micellar structures and liposomes themselves have no relevance.

Liposomes have emerged as part of drug delivery approach. In a retrospective appraisal, these are viewed as nanodrug delivery systems. Micelles or microemulsions are also involved in physiological processes such as digestion of lipids like fats/oils.

Micelles are aggregation colloids. This class of colloids are formed when specie aggregate to reach the colloidal size.

Lipids, by no means are the only class of compounds which form micelles.

Micelles formation requires amphiphilic nature. This kind of structure is said to have a polar head and a hydrophilic tail. Both small molecular weight compounds as well as larger molecules can form micelles.

Amphiphiles or surfactants can be ionic surfactants or non-ionic surfactants. Ionic surfactants again can be anionic or cationic. Biochemist are familiar with SDS (an anionic surfactant) which is used in SDS-PAGE electrophoresis.

CTAB is a common detergent or surfactant which is cationic. „Brij‟ is common example of a non-ionic surfactant or “Niotensides” (as these are called). The class of polymers called pluronics is an example of non-ionic surfactants of larger size.

Milk contains casein molecules present as micelles. The casein molecules form micelles with quite intricate structures. Casein also is an example of a protein forming the aggregation colloids or micelles.

Phospholipids can also be termed zwitterionic surfactants. The well known phospholipid lecithin or phosphotidyl cholin has two hydrophilic tails made up of acyl groups of long chain fatty acids. The negative charge on the phosphate and positive charge of the cholin constitute zwitterionic polar head.

In dilute solutions of surfactants, the molecules exist as isolated species. As the surfactant volume fraction increases, micelles are formed. Increasing this further „lyotropic metaphases‟ called liquid crystals are formed.

The self assembled structures are important in many areas. In biology, as already pointed out micellization is relevant in few areas such as digestion of fats/oils. Vesicles and their reversible formation is very important in many biological processes. Finally, biomembranes are a mosaic of bilayers.

Surfactants are also used in cleaning detergents, textiles, cosmetics, paper, paints, food and metallurgical processes such as froth floatation.

Micelles as well as reverse micelles have both been used to carry out chemical reactions/biotransformations and have been described as nanoreactors. Reverse micelles are well known as an example of low water medium for enzyme catalysis.

The following parameters determine the shape and size of the micelles.

•  Head group size. The polar part of the molecules has an important influence
•  Ionic strength. While making micelles in vitro, this is controllable. In biological systems, physiological conditions determine it.
•  Hydrophobic tail

The micelles are normally represented like this. This is an oversimplified picture and actually misrepresents the reality.

The core is not dense. This is because tails are not straight arrays but entangled. The heads are not so neatly aligned.

Not all micelles are shape persistent. Both micelles and reverse micelles are dynamic structures.

There can be an exchange of materials through micelles.

The more realistic representation of micelles is like this. The % amount of water varies from micelles to micelles. Pluronics, the polymers form water core which is about 30% (w/w).

The various techniques which allow us to look at size and shape of micelles are:

 Cryo-TEM. This involves rapid cooling of the thin layer of sample in liquid ethane.

 Small angle X-ray or neutron scattering

Critical micelles concentration (Ck) is the concentration above which micelles are observed.

Below CMC, the surfactants are adsorbed at the layer.

We have already seen this phenomenon in play while discussing lipase biocatalysis. The natural substrate of lipase formed miceller phase beyond CMC. This led to opening up of the active site molecular lid and hyperactivation of the lipase.

Surface tension values at CMC show abrupt changes. CMC of ionic surfactants is generally higher as compared to non-ionic surfactants. Which implies that among lipids/phospholipids, non-ionic ones will form micelles at earlier molar concentrations.

Equivalence conductivity also changes abruptly at CMC in solutions of the surfactants. Hence measurement of surface tension and conductivity provide useful ways to monitor micelle formation.

In general, longer tails of the surfactants decreases CMC of the surfactants. This makes sense as the hydrophobic interactions among tails provide the main stimulus for micelle formation. Longer the tail, more is the hydrophobic interaction.

Some useful parameters about micelles can be identified. Size and CMC are two which we have already talked about. CMC of common ionic surfactants has been found to be in the range of 10-3-10-2 M.

As already indicated, CMC of non-ionic surfactants is lower. CMC of common non-ionic surfactants is in the range of 10-4-10-3 M. The polar heads of non-ionic surfactants will not have any electrostatic repulsion due to some changes and hence self assembly will be easier.

Aggregation number refers to the number of molecules which self assemble to form a single micelle. This number is very different for micelles made from ionic and non ionic surfactants. The aggregation number (ƻA) for non-ionic surfactant is in the range of 10-170. For non-ionic surfactants, the corresponding range is larger as 30-10,000.

The solubility of surfactant depends upon the temperature. The solubility is less at low temperature. This is followed by a narrow range of temperature in which the solubility rises rapidly.

For all systems, there is a temperature beyond which no micelles are formed. To repeat, micelles are colloids. If the solubility is high, substances form true solutions and not colloidal suspensions.

The solubility vs. temp curve and CMC vs temp. curve intersects at a particular temperature.

This is called the Kraft point and the temperature is called Kraft temperature (Tkraft).

The Tkraft has a significance as this can be visualized as a “melting point” of the self assembly.

In food processing industries, Tkraft is used as practical parameter to design processed foods.

Various models have been proposed over the years to explain micelles formation. The first was stepwise growth model. This viewed association as a continuous process. This would predict specie of various sizes present at any time. It would also rule out abrupt changes at CMC.

The closed aggregation would views micelle formation as a cooperative phenomenon. Later on some other models were suggested which were based upon defining some new parameters for the micelles.

Predictions of the packing parameter model:

Big polar heads will lead to large ae and micelle shape will approach spherical shapes.

On the other hand, small polar heads will results in small ae. In such cases, association will lead to lamellae or a bilayer formation.

Charles Tanford who contributed so much to the importance of hydrophobicity in protein structure gave another model which is called free energy model or phase separation model.

According to Tanford‟s model, micelles are “microphases” considering the chemical potential µº,

So, the “transfer” term will result from minimising the contact between tail and water. Interface term arises out of the residual contact between nonpolar tail and water. The last term represents repulsion between polar heads.

To sum up, the model stated that tail transfer decides the aggregation. Residual contact parameter promotes the association & head repulsion restricts the association.

Both packing model and free energy model are able to predict some behaviour of surfactant systems correctly. For example, packing model correctly predicted that in case of ionic surfactants, addition of salts will increase Vo/aelo. Consequently, it predicted correctly that spherical micelles will become cylindrical micelles.

Packing model also predicts that for same equilibrium area ae, Vo/aelo will be two times for a surfactant with double tails. This results in formation of bilayers instead of spherical or globular micelles.

This gives us a clue as to why nature has chosen lipids such as phospholipids (with two tails) to be part of the biomembranes. Biomembranes, as we will see involve formation of bilayers by these lipids.

The packing model also predicted correctly that increasing temperature will decrease the steric repulsion between heads in case of PEO micelles. Such micelles change from spherical to cylindrical shapes.

The tail, according to the packing model, affects the packing parameter hence influences the aggregation. The hydrocarbons pack differently in bulk as compared to when they are part of the tails of the surfactants in a micelle.

Detailed analysis based upon simulation experiments show some interesting and non obvious conclusions. One is that the tail length affects the head group area and it thus influences the shape.

∆G = ∆H – T∆S

For at least low molecular weight surfactants ∆H is about +1-2 KJ/mole. Hence, enthalpy does not favour micellization.

∆S is about +140 J/K. thus, entropic factors favour micelles formation.

It is actually the properties of water which facilitate micelle formation:

•  High surface tension
•  High cohesion between H2O molecules
•  H2O has high dielectric constant and high boiling point

The nonpolar specie placed in water results in water molecules forming clathrate structures around them as first shell.

These more open „iceberg‟ structures involve ordering of H-bonds among water molecules with a large entropic cost.

When the temperature is raised, H-bonds are broken with increase in entropy but at the cost of enthalpy

Small size of water makes it difficult to find a cavity for the nonpolar solute. Thus, the insertion probability for the solute is higher than a simple molecule like n-hexane for small cavity sizes. For 1Aº cavity radius, it is much less and nearly equal for both liquids!

Lipids, with their solubility in organic solvants, all have nonpolar components. When polar and charged structural motiffs become a part to convert them into amphiphiles, interesting associations take place. These associations ultimately have the basis in the hydrophobic effect.

With this background, we are in a position to appreciate the behaviour of lipids to form bilayers, liposomes and fluid mosaics.

The choice of polar heads and tails, as we have learnt dictate the nature and results of self assembly.

So, lipid amphiphiles like glycerophospholipids, sphingolipids and even soap (fatty acid anions with Na+/K+ as cation) can all form the aggregates in the form of monolayers, micelles and bilayers.

Soap molecules gently spread on water surface (i.e. air-water interphase) form monolayers. The charged part naturally interacts with water and nonpolar tails are oriented up on the air side.

When soap molecules are immersed in water, micelles are formed. As always, “polar heads” outwards, tails inside as we have discussed in the general picture of micelles.

Soap bubbles on the other hand are bilayers of fatty acid anions forming a spherical shape which enclose a thin water layer. The result is the tails stacked together, polar heads form the middle of this sandwich- that is where the thin layer of water is there. The interior of the bubble is air as is outside. Tails of each layer extend into air on each side.

Air and its component gases are not highly soluble in water. Relatively, air is nonpolar as compared to water. Hence, thermodynamic stability favours the orientation of soap molecules in the air bubble.

The main component of the biomembranes, glycerol-phospholipids and sphingolipids can also form monolayers under controlled conditions. However, left to themselves, they spontaneously form bilayers. Unlike, soap with one hydrocarbon tail, these have two long chain hydrocarbon tails.

The two chains of these complex lipids are incapable of packing well in a micellar structure. Hence, these form bilayers. Thus, the choice between micellar structure and bilayer structure is decided by packing parameter considerations.

Unlike soap bubble bilayers, the bilayers of these complex phospholipids have aqueous phase on both sides of the “bilayer leaflet”. Hence polar heads are oriented towards each surface. The tails form tail to tail orientation.

Not all lipids can form bilayers. It is found that cross sectional areas of the tail and head have to be nearly equal. This is so for Glycerophospholipids and sphingolipids. Lysophospholipid have larger head and a single acyl chain. Similarly, in cholesterol and other steroids, the nonpolar part is too large. Neither class of lipids form bilayers. Bile salts form micelles during digestion.

Liposomes are formed from phospholipid bilayers joining ends and enclosing an aqueous compartment. Sonication of suspension of phospholipids in aqueous solution forms these vesicles. Spherical vesicles of ~ 500 Aº diameter are formed.

Liposomes can be formed from a single bilayer to form a unilamellar structures. Liposomes can also have several concentric bilayers and these are called multilammelar vesicles.

The aqueous compartment in liposomes allows delivery of water soluble molecules like drugs.

Hence, liposomes have been extensively used as nanovescicles for drug delivery.

Liposomes of similar size range and shapes can also be formed by dropping the lipid into water through a fine needle. Rapid mixing of lipid solutions in ethanol and water also form liposomes. Larger vesicles of ~1µm diameter can be obtained by slow evaporation of the solvent from a suspension of phospholipids in aqueous organic co-solvent mixtures.

Liposomes are fairly stable structure and thus can be separated/purified by centrifugation, gelfilteration and dialysis. Typical of the lipid aggeregates, these also show characteristic phase transition with temperature.

In bilayer formation, increases in solvent entropy exceeds the decrease in phospholipid entropy to drive the process. A phospholipid in bulk water has ordered array of H2O molecules around hydrophobic tails. Bilayer formation abolishes this order.

Another reason, liposomes have been a good choice for drug delivery is that they can fuse with the membranes of the target tissue cells. That also is due to the property of self assembly of these interesting amphiphiles!

Summary

•  Mechanism of micelle formation
•  Bilayers
•  Liposomes