Liposome

Question 1

What are phospholipids derived from

Answer 1

• Derived from egg or soya

Contain a Charged headgroup, glycerol backbone and a fatty acid ester

Question 2

• Several different charged

headgroups

Answer 2

– Choline
– Ethanolamine
– Serine
– Phosphate

Question 3

What happens when surfactants are dispersed in water?

Answer 3

• When dispersed in water, many surfactants form micelles
• The hydrophobic tails are inside the structure
• But not all surfactants do this

Question 4

Lipid bilayers

Answer 4

• Phospholipids are surfactants BUT they don’t normally form micelles
• Their thermodynamically stable state in water is a bilayer
This bilayer is, of course, the one that surrounds the living cell.

Question 5

Structure of liposomes

Answer 5

• Shaking phospholipids with water causes large
liposomes to form – in multiple onion-like layers – these are called multilamellar vesicles or MLV’s
• Passing MLV’s through a high-shear mixer breaks them into much smaller unilamellar vesicles (SUV’s)

Question 6

Phospholipid bilayers and temperature

Answer 6

• The lipid bilayer is temperature sensitive – it has a melting transition and the temperature of this depends on the structure of the specific phospholipid

Question 7

Why is bilayer melting important?

Answer 7

• Properties of the bilayer change considerably on melting
– Order
– Permeability
• Things on one side of the membrane can get to the other side
– Dynamics
• solid membrane – things do not diffuse laterally
• liquid membrane – lateral movement can occur
– Compare with what happens when a 3-dimensional
solid melts
• Living cells have ways of controlling their bilayers so that their fluidity is suitable for their purpose

Question 8

What influences bilayer melting?

Answer 8

• The phospholipid fatty acid chain structure
– Chain length
Assuming both chains are the same and fully saturated:
• C14:0 (Myristyl) 23°C
• C16:0 (Palmityl) 41°C
• C18:0 (Stearyl) 56°C
– Chain saturation
• C16:1 (Palmitoleyl) –36°C
• C18:1 (Oleyl) –20°C
• Note the various notations for fatty acid composition
– Inclusion of other lipophilic molecules e.g cholesterol
• Lowers melting temperature, makes transition occur over a wider temperature range

Question 9

Phospholipid purification and synthesis

Answer 9

• Natural phospholipids are normally unsaturated
– Wide mixture of chain lengths and combinations
– Usually even carbon numbers from 12 to 24
– Extremely difficult to separate from each other
– Easily extracted from egg or soybeans
• Unrefined grades are called ‘lecithins’
– Relatively inexpensive
– Composition and hence properties can be variable
• Saturated or pure lipids made by enzymatic transesterification
– Range of enzymes that snip off bits of natural lipid and add a different group

Question 10

Phospholipid headgroups and ionisation

Answer 10

• It’s important to control phospholipid charge in
formulations
• Liposomes are colloidal systems – they are charge-stabilised
– Cannot be properly dispersed in the un-ionised state
– If used as emulsifiers (see emulsions) then the
emulsion is not stable when the phospholipid is uncharged
• Phospholipid ionisation is complex and depends on the headgroup

Question 11

Why use liposomes for drug delivery

Answer 11

• The objective is to create a drug carrier system
– Drug is in solution inside the liposomal space
– Encapsulated drug is then injected into the
bloodstream
– Body cannot ‘see’ the drug– the drug moves with the liposomes
– Drug can then be ‘targeted’ to a specific site of action
where the drug is released – e.g. a tumour
• At least that was the general idea
• After 40 years of research these problems have
still not been solved completely
– very few marketed products

Question 12

Marketed liposomal formulations

Answer 12

Ambisome 
Gilead/Fujisawa 
Amphotericin B
Daunoxome 
Gilead 
Daunorubicin
Doxil 
J&J Alza 
Doxorubicin
Myocet 
Elan 
Doxorubicin

Question 13

Liposomal Encapsulation

Answer 13

• The first problem is to get the drug inside the liposome
• If liposomes are formed in a solution of the drug, some
of the drug gets entrapped
• Very low efficiency – only a few percent of drug is in
the internal space
• Problem is then to remove the untrapped free drug
• Because drug leaks out slowly, system must then be
freeze-dried – without damaging the liposomes – Molecules called cryoprotectants are used to help the
vesicles stay intact

Question 14

Cryoprotection of liposomes

Answer 14

• Disaccharides are common cryoprotectants, e.g.
sucrose, lactose and trehalose. Monosaccharides are
relatively poor.
• The cryoprotectants promote the formation of glass during the freezing process hence supress the formation of ice crystals
• The hydroxyl group of the cryoprotectants can form hydrogen bond with phospholipids when water is
removed in the drying process. Bilayers previously
supported by water is now supported by the
cryoprotectants. This is sometimes referred to as water
replacement theory.

Question 15

Choice of phospholipids

Answer 15

• Unsaturated e.g. di-oleyl
– Low melting temp. so liquid phase at room temperature
– Mobile, so contents leak out easily
– Easily processed to form liposomes
– Cholesterol is usually added to increase rigidity and stability
• Saturated e.g. distearoyl
– In solid phase at room temperature
– Encapsulated material stays inside liposome
– But processing is very difficult
• have to form liposomes and entrap drugs above melting temperature, then cool the product into the solid phase
• A small amount (1-5%) of charged phospholipid is needed to make the liposomes

Question 16

Typical manufacturing sequence

Answer 16

• Dissolve the phospholipid and drug together in a suitable solvent (usually ethanol)
– In this solvent phospholipids form a true solution, not vesicles
• Slowly inject this solution into water
– Liposomes form when phospholipids contact the water
– Liquid becomes cloudy as a result
– This is called the ‘ethanol injection method’ of preparing liposomes (there are several methods)
• Separate liposomes from untrapped drug by gel chromatography
– Pass liposomal suspension down a gel column which lets liposomes through but retains free drug
• Freeze-dry the resulting suspension of liposomes

Question 17

What happens when you inject liposomes into the blood?

Answer 17

• Because phospholipids are the same materials that
surround cells, it was thought that they would
circulate freely in blood
• Unfortunately they are rapidly recognised as foreign
and removed, primarily in the liver, so there is little
chance of them finding an alternative target
• Immune response begins with recognition proteins
adsorbing to the liposome surface so the problem is:
how to stop the adsorption of the proteins?

Question 18

Stealth phospholipids

Answer 18

• Hydrophilic polymers (e.g. polyoxyethylene) are known to stop proteins adsorbing to surfaces
• This molecule is a phospholipid with an attached polyoxyethylene chain which will form a coating on the surface of the liposome

Question 19

So what about targeting?

Answer 19

• A major goal was to direct the liposome to its site of action, to avoid side-effects at other sites
• Initial idea was to use antibodies:
– Grow tumour in mice
– Extract antibodies to tumour
– Covalently couple them to drug-containing liposomes
– Inject and watch them bind to the tumour
• This works in principle but the antibody is a foreign protein which raises its own immune response in the patient
• Recent work is looking at specific recognition peptides on the surface of the tumours
• Still a long way to go!

Question 20

Antibody-Targeted Liposomes in Animal Models

Answer 20

• Liposomes carry antibody to implanted carcinoma
• Also have radiolabel to enable gamma imaging
• Large mass is lung/liver/spleen
• Tumour is arrowed

Question 21

Ghost Cells

Answer 21

• A similar concept to liposomes – encapsulated drug
• Use one of the patients’ own cells so there is no immune response
– Red cells (erythrocytes) are favourite
– Put red cells in hypertonic saline; pores open and cell content escapes
– Add drug to cell suspension and it will equilibrate with the inner cell space
– Dilute the saline to close the cell pores
– Wash the cells to remove untrapped drug and inject into patients bloodstream

Question 22

Niosomes

Answer 22

• Phospholipids are not the only surfactants that form hollow vesicles in water
• Some other nonionic surfactants have this
behaviour
• Vesicles made from these materials are called Niosomes (Non-Ionic-somes)
• Much research activity but little application
outside cosmetics

Question 23

Mechanism of cryoprotection:

Answer 23

 Glass formation: amorphous matrix formation between vesicles preventing fusion of liposomes
 Interactionwith the phospholipids (water substitution/replacement theory), hydrogen bonding.

Question 24

What physical changes can happen to liposomes?

Answer 24

Aggregation
bilayer fusion: may result in drug leakage
change in vesicle size
zeta potential

Question 25

factors that affect the stability of freeze-dried liposomes.

Answer 25

 Glass temperature: above Tg, not stable, below Tg more stable
 Properties of drug molecules
 Residual moistures: Tg can be reduced by moisture
 Formulation factors
 Freeze-drying protocol

Question 26

How can bilayer defects be induced? How can we make the defects disappear faster?

Answer 26

 Defects may exist immediately after preparation
 Defects can be induced during phase transition
 Defects can also be induced by freezing
 Defect can be removed by ‘Annealing’: Incubating the liposomes at temperatures above the phase transition temperature. To allow the differences in packing density to be equalised by ‘flip-flop’. High temperature, membrane molecules are more mobile.

Question 27

Is AmBisome compatible with saline solutions? Explain why.

Answer 27

No. Liposomes are generally stabilised by charges. The addition of electrolytes will compress the diffuse doublelayer and the zeta potential will decrease. Some electrolyte may neutralise the charge of the liposomes. Consequently the stability of the liposomes will be affected.

Question 28

There are two liposomes with internal pH values of 4 and 8 respectively. The pH of the external phase is pH 8.5 for both liposomes. If Doxorubicin (weak base, pKa 8.6) is added to the external phase, it would diffuse into and retain in which of the two liposomes more easily? Explain why. Assume the liposomes are stable under the conditions in question

Answer 28

 Unionised molecules are more lipophilic compared to the ionised form and there diffuse across the lipid bilayer more easily.
 The drug is a weak base with pk8.6 and therefore is more ionised at pH4 than at pH8.
 For the liposomes with internal pH 4, the drug will be more ionised in the internal aqueous phase of the liposomes than in the external aqueous phase. The unionised drug molecules diffuse into the internal
aqueous phase of the liposome, and become ionised and trapped there.
 For the liposomes with internal pH 8, the fraction of the unionised form of the drug will be relatively higher than that of the liposome with internal the pH 4, and therefore, the drug are relatively easier to diffuse out.