Prepared byNazia Tajrin
Sr. Officer. R & DF
Incepta Pharmaceuticals Ltd
What is Liposome
Liposomes are composite structures made of

phospholipids and may contain small amounts of
other molecul...
Structure of liposome
Liposomes can be composed of
 Naturally derived phospholipids with mixed lipid

chains –e.g egg ph...
Structure of liposome ( cont)
There are three types of liposomes - MLV

(multilamellar vesicles) SUV (Small Unilamellar
V...
Structure of liposome ( cont)
Structure of liposome ( cont)
Structure of liposome ( cont)
Striking features of liposomes are that1. Molecules of PC are not water soluble.
2. In aque...
Structure of liposome ( cont)
PC molecules unlike other anphipathic molecules

form bilayer sheets rather than micelles.
...
Liposome in drug delivery
Liposomes are used for drug delivery due to their

unique properties. A liposome encapsulates a...
Liposome in drug delivery
( cont)
By making liposomes in a solution of DNA or drugs

(which would normally be unable to d...
Liposome in drug delivery
( cont)

Liposomes that contain low (or high) pH can be

constructed such that dissolved aqueou...
Liposome in drug delivery
( cont)
 Table 1. Classification of liposomes based on composition and

application.
 Liposome...
Why to use Liposome
Basic reasons for using liposomes as drug carriers
• Direction
• Duration
• Protection
• Internal...
Advantages of liposomes
Direction. Liposomes can target a drug to the in-tended site of

action in the body, thus enhanci...
Advantages of liposomes
( cont)
degradative enzymes) present in the host. Conversely, the
pa-tient can be protected agains...
Advantages of liposomes
( cont)
Liposomes are highly versatile structures for research,

therapeutic, and analytical appl...
. For liposomes used in analytical and bioanalytical

applications, the main characteristics include
 the average diamet...
Characterization of liposomes
The behaviour of liposomes in both physical &

biological systems is governed by the factor...
Characterization of liposomes
Therefore, the liposomes are characterized for

physuical attribures:

 shape, size & its ...
Quality control assays of
liposomal formulations
Quality control of liposomes
Physical properties
Size & its distribution
Surface charfe
 Percent entrapent/capture
 Entrapped volume
 Lamellarity
...
Size & size distribution
Size and size distribution (polydispersity) of the

formulated nan- oliposomes are of particular...
Size & its distribution
Several techniques are available for assessing

submicrom- eter liposome size and size distributi...
Size & its distribution(cont.)
Most precise method to determine size of the

liposome is by electron microscopy, since it...
Size & its distribution(cont.)
All the method require very costly equipments.
If only approximate idea of size range is ...
Microscopic methods
Light microscopy has been utilized to examine the

gross size distribution of large vesicles produced...
Microscopic methods(cont)
For large vesicles ( 5 µm) , negative stain electron

microscopy is not suitable for determinat...
Microscopic methods(cont)
The freeze etch structure is particularly suitable for

the measurement of small vesicle diamet...
Microscopic methods(cont)
However freeze fracture technique has a serious

drawback foer estimating the size distribution...
Microscopic methods(cont)
A homogenous population of vesicles will therefore

yield a distribution of profile sizes with ...
Microscopic methods(cont)
Another more recently developed microscopic

technique known as atomic force microscopy has
bee...
Microscopic methods(cont)
The reflected laser beams are then detected at

photodi- ode array detectors which through a
fee...
Microscopic methods(cont)
The reflected laser beams are then detected at

photodi- ode array detectors which through a
fee...
Laser light Scattering
Diffraction of light is a phenomenon in which

monochromatic light bends around particles.
When a...
As biomolecules or a distribution of biomolecule

diffuse around the laser beam coherence area, light
scattered from them...
Each of the currently used particle size determination

tech- niques has its own advantages and
disadvantages. Light scat...
Gel permeation
Exclusion chromatography on large pure gels was

introduced to separate SUVs from radial MLVs.
However , ...
Electron microscopic techniques, on the other hand,

make direct observation possible; hence provide
information on the s...
Zeta potential
The other important parameter in liposome

characterisation is zeta potential. Zeta potential is the
overa...
Generally, particle size and zeta potential are the two

most important properties that determine the fate of
intravenous...
Surface charge
A method using free flow electrophoresis is used to

determine the surface charge.
Lamellarity determination
The lamellarity of liposomes made from different

ingredients or preparation techniques varies ...
Mn2+ interacts with the negatively charged

phosphate groups of phospholipids and causes a
broadening and reduction of th...
Encapsulation effciency
The terminology varies widely with respect to the ability

of various liposome formulations to en...
by Kulkarni et al. [163]), but also on the initial molar

amount of encapsulant.When systematic liposome
characterization...
Encapsulation efficiency is commonly measured by

encapsulating a hydrophilic marker (i.e. radioactive
sugar, ion, fluore...
This method can be applied if the nanoliposomes do

not undergo any purification (e.g. size exclusion
chromatography, dia...
In the latter case, total lysis can be induced by the

addition of a surfactant such as Triton X100.
Retention and leakag...
Entrapped volume
The entrapped volume of a population can often be

deduced from measurements of the total quality of
sol...
However, in many cases such assumption is in valid,

for e.g, in two phase methods of preparation, water
can be lost from...
The best way to measure external volume is to

measure the quantity of water directly, & this may be
done very cinvenient...
The pellet is then resuspended in deuterium oxide

( D2O). The permeability of the membrane to water
is such that D2O & H...
Phase Bhaviour of Liposomes
An important behaviour of lipid membran is the

existence of a temperature dependent, reversi...
The phase transition temperature (Tc) is a function of

phospholipid content of the bilayers.
The Tc can give good clues...
Drug Release
The mechanism of drug release from the liposome

can assesed by the use of a well calibrated in vitro
diffus...
Chemical properties
Quantitative Determination of phospholipids
Phospholipid Hydrolysis.
Phospholipid oxidation
Choles...
Quantitativ Determination of
phospholipids
It is difficult to mesure directly the phospholipid

concentration, since drie...
Barlett assay
In the barlett assay the phospholipid phosphorus in

the sample is first hydrolyzed to inorganic phosphate
...
Barlett assay is very sensitive but is not very

reasonably reproducible.
The problem is that the test is easily upset b...
Stewart Assay

In stewart assay, the phospholipid forms a complex with

ammonium ferrothiacyanate in organic solution.
T...
TLC method may also be employed for determining

the purity & the concentration of lipids.
If the compounds is pure it s...
Phospholipid hydrolysis
The major product of Lysolecithin hydrolysis where one

fatty acid chain is lost by de-esterifica...
Consequently, methods are preferred which permit

detection of LPC via the phosphate group after
separating the hydrolysi...
Phospholipid Oxidation
Oxidation of the fatty acid of phospholipids in the

absence of specific oxidants occurs via free ...
Cholesterol analysis
Cholesterol is qualitatively analyzed using capillary

column of flexible fused silica.
Whereas it ...
Assay of drug Product
High-performance liquid chromatography (HPLC).

HPLC has been widely applied to the determination
o...
Solid-phase extraction ( SPE)
Solid-phase extraction (SPE). SPE is of great interest

in the separation of liposomal and ...
Size exclusion chromatography
The usefulness of conventional and high-performance

SEC for liposome characterization has ...
TLC
HPLC-Numerous HPLC methods with photometric,

fluorimetric and tandem MS detection have studied
the pharmacokinetics...
Capillary electrophoresis (CE)
CE with chemiluminescence detection has been used

for the characterization of liposomes i...
Conclusion & future trends
Liposome technology is a recent research area that is

still far from being fully developed. T...
Conclusion & future trends( cont.)
The development of analytical methods to control the

effectiveness of LDSs runs paral...
Conclusion & future
trends( cont.)
Future innovations in analytical techniques for LDSs

will probably be oriented toward...
Conclusion & future
trends( cont.)
c) affinity-based biosensors (i.e. catalytic

biosensor,immunosensor and genosensor); ...
Conclusion & future
trends( cont.)
a)elucidating the mechanism of release from the liposome;
b) simultaneous quantificat...
Lyposome
Lyposome
Lyposome
Lyposome
Lyposome
Lyposome
Lyposome
Lyposome
Lyposome
Upcoming SlideShare
Loading in...5
×

Lyposome

1,151

Published on

presentation on liposome

Published in: Technology, Business
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,151
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
100
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide

Lyposome

  1. 1. Prepared byNazia Tajrin Sr. Officer. R & DF Incepta Pharmaceuticals Ltd
  2. 2. What is Liposome Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules. Structurally , liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. Liposomes can be filled with drugs, and used to deliver drugs for cancer and other diseases.
  3. 3. Structure of liposome Liposomes can be composed of  Naturally derived phospholipids with mixed lipid chains –e.g egg phosphatidylethanolamine or other surfactants. Main component of liposomes are phospholipid & cholesterol
  4. 4. Structure of liposome ( cont) There are three types of liposomes - MLV (multilamellar vesicles) SUV (Small Unilamellar Vesicles) and LUV (Large Unilamellar Vesicles). These are used to deliver different types of drugs.
  5. 5. Structure of liposome ( cont)
  6. 6. Structure of liposome ( cont)
  7. 7. Structure of liposome ( cont) Striking features of liposomes are that1. Molecules of PC are not water soluble. 2. In aqueous media they allign themselves closely in planer bilayer sheets in order to minimize the unfavorable action between the bulk aqueous phase & long hydrocarbon fatty acid chains so that polar heads face aqueous phase & fatty acid chain face each other. 3. The bilayer folds themselves to form closely sealed vesicles
  8. 8. Structure of liposome ( cont) PC molecules unlike other anphipathic molecules form bilayer sheets rather than micelles. It is thought that brcause of double fatty acid chain gives the molecule an overall tubular shape.
  9. 9. Liposome in drug delivery Liposomes are used for drug delivery due to their unique properties. A liposome encapsulates a region of aqueous solution inside a hydrophobic membrane; 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.
  10. 10. Liposome in drug delivery ( cont) 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.
  11. 11. Liposome in drug delivery ( cont) Liposomes that contain low (or high) pH can be constructed such that dissolved aqueous drugs will be charged in solution (i.e., the pH is outside the drug's pI range). 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. A similar approach can be exploited in the biodetoxification of drugs by injecting empty liposomes with a transmembrane pH gradient. In this case the vesicles act as sinks to scavenge the drug in the blood circulation and prevent its toxic effect
  12. 12. Liposome in drug delivery ( cont)  Table 1. Classification of liposomes based on composition and application.  Liposome type  Conventional liposomes  Long-circulating liposomes  Immunoliposomes  Cationic liposomes Major application Macrophage targeting Local depot Vaccination Selective targeting to pathological areas Circulating microreservoir Specific targeting Gene delivery
  13. 13. Why to use Liposome Basic reasons for using liposomes as drug carriers • Direction • Duration • Protection • Internalization • Amplification
  14. 14. Advantages of liposomes Direction. Liposomes can target a drug to the in-tended site of action in the body, thus enhancingits therapeutic efficacy (drug targeting, site-spe-cific delivery). Liposomes may also direct a drug away from those body sites that are particularly sensitive to the toxic action of it (site-avoidance delivery). Duration. Liposomes can act as a depot from which the entrapped compound is slowly released over time. Such a sustained release process can be ex- ploited to maintain therapeutic (but nontoxic)drug levels in the bloodstream or at the local ad- ministration site for prolonged periods of time. Thus, an increased duration of action and a de- creased frequency of administration are benefi- cial consequences. Protection. Drugs incorporated in liposomes, inparticular those entrapped in the aqueous interior are protected against the action of detrimental factors (e.g.
  15. 15. Advantages of liposomes ( cont) degradative enzymes) present in the host. Conversely, the pa-tient can be protected against detrimental toxic effects of drugs (cf. Duration). Internalization. Liposomes can interact with target cells in various ways and are therefore able to promote the intracellular delivery of drug molecules that in their ‘free’ form (i.e. non-encapsu- lated) would not be able to enter the cellular interior due to un- favorable physicochemical characteristics (e.g. DNA molecules). Amplification. If the drug is an antigen, liposomes can act as im- munological adjuvant in vaccine formulations.
  16. 16. Advantages of liposomes ( cont) Liposomes are highly versatile structures for research, therapeutic, and analytical applications. In order to assess the quality of liposomes and obtain quantitative measures that allow comparison between different batches of liposomes, various parameters should be monitored
  17. 17. . For liposomes used in analytical and bioanalytical applications, the main characteristics include  the average diameter and degree of size polydispersity; encapsulation efficiency; the ratio of phospholipids to encapsulant concentration  lamellarity determination
  18. 18. Characterization of liposomes The behaviour of liposomes in both physical & biological systems is governed by the factors such as:  Physical size Membrane permeability Percent entrapped solutes Chemical composition Quantity & purity of the starting material
  19. 19. Characterization of liposomes Therefore, the liposomes are characterized for physuical attribures:  shape, size & its distribution  Percentage drug capture  Entrapped volume  lamellarity  Percentage drug release  And Chemical compositions;  Estimation of phospholipids  Phospholipid oxidation  analysis of cholesterol
  20. 20. Quality control assays of liposomal formulations
  21. 21. Quality control of liposomes
  22. 22. Physical properties Size & its distribution Surface charfe  Percent entrapent/capture  Entrapped volume  Lamellarity  Phase behaviour of liposomes  Drug release
  23. 23. Size & size distribution Size and size distribution (polydispersity) of the formulated nan- oliposomes are of particular importance in their characterization. Maintaining a constant size and/or size distribution for a pro- longed period of time is an indication of liposome stability.
  24. 24. Size & its distribution Several techniques are available for assessing submicrom- eter liposome size and size distribution. These include  static and dynamic light scattering, several types of microscopy techniques,  size-exclusion chromatog-raphy (SEC), field-flow fractionation and ana-lytical centrifugation. Several variations on electron microscopy (EM) such as transmission EM using negative staining, freezefracture TEM, and cryo EM , provide valuable
  25. 25. Size & its distribution(cont.) Most precise method to determine size of the liposome is by electron microscopy, since it allows to view each individual liposome & to obtain exact information about the profile of liposome population over the whole ranges of sizes. -unfortunately it is very time consuming & requires equipments that may not always immediately available to hand. • In contrast , laser light scattering ( quasi-elastic laser light scattering) method is very simple & rapid to perform but having disadvantages of measuring an average property of the bulk of the liposomes
  26. 26. Size & its distribution(cont.) All the method require very costly equipments. If only approximate idea of size range is required, then gel exclusion chromatographic techniques are recommended, since only expense incurred is that of buffera & gel materials.
  27. 27. Microscopic methods Light microscopy has been utilized to examine the gross size distribution of large vesicles produced from single chain amphiphiles. If the bilayers are having fluorescent hydrophillic probes, the liposomes can be examined under a fluorescent microscope. The resolution of the light microscopy limits this tchnique for obtaining the complete size distribution of the preparation. But using negative stain elctron microscopy, on can obtain an estimation of the lower end of the size
  28. 28. Microscopic methods(cont) For large vesicles ( 5 µm) , negative stain electron microscopy is not suitable for determination of the size distribution because vesicle distortion during preparation of the specimen makes it difficult to obtain an estimate of the diameter of the original particle. Freeze etch & freeze fracture elctron microscopy tecniques have ben used t study vesicle size & struture.
  29. 29. Microscopic methods(cont) The freeze etch structure is particularly suitable for the measurement of small vesicle diameters since the effects of random cleavage that can occur through and around the vesicle, not necessarily through the mid plane, can be compensated for each stage. For populations of large size vesicles freeze fractire techniques can yield a representative morpological view of the liposomes & has been useful for examining the morphlogical changes that can occur in the bilayer surface as the phospholipids pass through the gel-liquid crystalline stransition, or through the lamellar hexagonal transition
  30. 30. Microscopic methods(cont) However freeze fracture technique has a serious drawback foer estimating the size distribution & mean vesicle size of a heterogenous population of the vesicles, the fracture plane passes through the mid plane that are randomly positioned in the frozen section resulting in non midplane fracture. Thus, the observed profile radius depends on the distance of the vesicle center from the plane of the fracture, while the probability that a vesicle will be in the fracture plane depends on the vesicle radius.
  31. 31. Microscopic methods(cont) A homogenous population of vesicles will therefore yield a distribution of profile sizes with largest being equal to the true radius of the vesicle.
  32. 32. Microscopic methods(cont) Another more recently developed microscopic technique known as atomic force microscopy has been utilized to study liposome morphology, size, and stability . This technique relies on the raster scanning of a nanometer sized sharp probe over a sample which has been immobilized onto a carefully selected surface, such as mica or glass, which is mounted onto a piezoelectric scanner. The tip is attached to a flexible cantilever. Deflection resulting from passage of the tip over sample attributes ismeasured by a laser beam.
  33. 33. Microscopic methods(cont) The reflected laser beams are then detected at photodi- ode array detectors which through a feedback mechanism, maintain the distance of the probe, amplitude of oscilla- tion, or the cantilever deflection constant, depending on the scanning mode The end result is a high resolution three -dimensional profile of the surface under study. Differ- ent modes of AFM are available, including ontact/repulsive mode (either constant height, constant deflection, or tapping )
  34. 34. Microscopic methods(cont) The reflected laser beams are then detected at photodi- ode array detectors which through a feedback mechanism, maintain the distance of the probe, amplitude of oscilla- tion, or the cantilever deflection constant, depending on the scanning mode [43,62]. The end result is a high resolution three -dimensional profile of the surface under study. Differ- ent modes of AFM are available, including ontact/repulsive mode (either constant height, constant deflection, or tapping )
  35. 35. Laser light Scattering Diffraction of light is a phenomenon in which monochromatic light bends around particles. When a ray of light is incident on a particle it gets diffracted at an angle. This diffraction causes the light to bend & change its path as shown below-
  36. 36. As biomolecules or a distribution of biomolecule diffuse around the laser beam coherence area, light scattered from them overlaps & interferes with the transmission of the laser light. A high sensitivity detectir can then record the time varying signal caused by scattered light & compare it to the consistent signal emitted whwn no molecules are present.This process is knoen as dynamic light scattering ( DLS), or quasi-elastic light scattering & photon corelation spectroscopy
  37. 37. Each of the currently used particle size determination tech- niques has its own advantages and disadvantages. Light scattering, for example, provides cumulative average information of the size of a large number of nanoliposomes simultaneously. However, it does not provide information on the shape of the lipidic system (e.g. oval, spherical, cylindrical, etc.) and it assumes any aggregation of more than one vesicle as one single particle.
  38. 38. Gel permeation Exclusion chromatography on large pure gels was introduced to separate SUVs from radial MLVs. However , large vesicles of 1-3 micrometer diameter usually fail to enter the gel & are retained on the top of the column. A Thin layer chromatography system using agarose beads has been inyroduced as a convenient, fast technique for obtaining a rough estimation of the size distribution of a liposome preparation.
  39. 39. Electron microscopic techniques, on the other hand, make direct observation possible; hence provide information on the shape of the vesicles as well as presence/absence of any aggregation and/ or fusion. The drawback of the microscopic investigations is that the number of particles that can be studied at any certain time is limited. Therefore, the general approach for the determination of size distribution of nanoliposomal formulations should be to use as many different techniques as possible.
  40. 40. Zeta potential The other important parameter in liposome characterisation is zeta potential. Zeta potential is the overall charge a lipid vesicle acquires in a particular medium. It is a measure of the magnitude of repulsion or attraction between particles in general and lipid vesicles in particular. Evaluation of the zeta potential of a nanoliposome preparation can help to predict the stability and in vivo fate of liposomes. Any modification of the nanoliposome surface, e.g. surface covering by polymer(s) to extend blood circulation life, can also be monitored by measurement of the zeta potential.
  41. 41. Generally, particle size and zeta potential are the two most important properties that determine the fate of intravenously injected liposomes. Knowledge of the zeta potential is also useful in controlling the aggregation, fusion and precipitation of nanoliposomes, which are important factors affecting the stability of nanoliposomal formulations. Now a days instruments are available in which particle size & Zeta potential both can be measured. Particle size is measured using dynamic light scattering (DLS). Measurement of the zeta potential of samples is done using the technique of laser Doppler velocimetry
  42. 42. Surface charge A method using free flow electrophoresis is used to determine the surface charge.
  43. 43. Lamellarity determination The lamellarity of liposomes made from different ingredients or preparation techniques varies widely. This is evidenced by reports showing that the fraction of phospholipid exposed to the external medium has ranged from 5% for large MLV to 70% for SUV (for a review see ref. (54)). Liposome lamellarity determination is often accomplished by 31P NMR. In this technique, the addition of Mn2+ quenches the 31P NMR signal from phospholipids on the exterior face of the liposomes and nanoliposomes.
  44. 44. Mn2+ interacts with the negatively charged phosphate groups of phospholipids and causes a broadening and reduction of the quantifiable signal . The degree of lamellarity is determined from the signal ratio before and after Mn2+ addition. While frequently used, this technique has recently been found to be quite sensitive to the Mn2+ and buffer concentration and the types of liposomes under analysis. Other techniques for lamellarity determination include electron microscopy, small angle X-ray scattering (SAXS), and methods that are based on the change in the visible or fluorescence signal of marker lipids upon the addition of reagents
  45. 45. Encapsulation effciency The terminology varies widely with respect to the ability of various liposome formulations to encapsulate the target molecules. Many papers express results in terms of ‘percent encapsulation’ (sometimes referred to as ‘incorporation efficiency’ , ‘trapping efficiency , or the encapsuation efficiency (EE) which is typically defined as the total amount of encapsulant found in the liposome solution versus the total initial input of encapsulant soluion. This value depends not only on the ability of the liposomes to capture the encapsulant molecules (dependent on ipid/buffer composition, liposome type (small unilamellar vesicle (SUV)/multilamellar vesicle (MLV)/large unilamelar vesicle (LUV)), preparation
  46. 46. by Kulkarni et al. [163]), but also on the initial molar amount of encapsulant.When systematic liposome characterizations are undertaken for the purpose of enhancing the degree of entrapment, initial lipid and encapsulant concentrations should be maintained constant for comparison.This represenation of the degree of encapsulation is suitable for comparing preparation processes provided that no losses of the encapsulant occur during preparation.
  47. 47. Encapsulation efficiency is commonly measured by encapsulating a hydrophilic marker (i.e. radioactive sugar, ion, fluorescent dye), sometimes using singlemolecule detection. The techniques used for this quantification depend on the nature of the entrapped material and include spectrophotometry, fluorescence spectroscopy, enzymebased methods, and electrochemical techniques.If a separation technique such as HPLC or FFF (Field Flow Fractionation) is applied, the percent entrapment can be expressed as the ratio of the unencapsulated peak area to that of a reference standard of the same initial concentration.
  48. 48. This method can be applied if the nanoliposomes do not undergo any purification (e.g. size exclusion chromatography, dialysis, centrifugation, etc.)following the preparation. Any of the purification technique serves to separate nanoliposome encapsulated materials from those that remain in the suspension medium. Therefore, they can also be used to monitor the storage stability of nanoliposomes in terms of leakage or the effect of various disruptive onditions on the retention of encapsulants.
  49. 49. In the latter case, total lysis can be induced by the addition of a surfactant such as Triton X100. Retention and leakage of the encapsulated material depend on the type of the vesicles, their lipid composition and Tc , among other parameters. It has been reported that SUV and MLV type liposomes are less sensitive than LUV liposomes to temperatureinduced leakage (Fig. 6). This property of liposomes and nanoliposomes can be used in the formulation of temperature-sensitive vesicles (55).
  50. 50. Entrapped volume The entrapped volume of a population can often be deduced from measurements of the total quality of solute entrapeed inside liposome assuring that the concentration of solute in the aqueous medium inside liposomes is the same as that in the solution used to start with, & assuming that no solute has leaked out of the liposomes after separation from unentrapped material.
  51. 51. However, in many cases such assumption is in valid, for e.g, in two phase methods of preparation, water can be lost from internal compartment during drying down step to remove organic solvent. On other occasions, water may enter or be expelled from the liposomes as a result of unanticipated osmotic differences.
  52. 52. The best way to measure external volume is to measure the quantity of water directly, & this may be done very cinveniently by replacing the external medium with a spectoscopically inert fluid, & then measuring the water signal for e.g by NMR In this method, liposomes prepared in aqueous solution consisting of ordinary water are spun at high centrifugal force to give high pellet, from which the supernatant is decanted to remove every drop of excess fluid.
  53. 53. The pellet is then resuspended in deuterium oxide ( D2O). The permeability of the membrane to water is such that D2O & H2O equilibriate very rapidly throughout the whole of the volume of the medium. A small aliquot I sremoved for quantification of phospholipid & the remainder is uded to obtain an NMR scan of H2O, the peak height of which can be related to concentration by comparison with standards containing known amounts of H2O & D2O.
  54. 54. Phase Bhaviour of Liposomes An important behaviour of lipid membran is the existence of a temperature dependent, reversible phase transition, where the hydrocarbon chains of the phospholipid undergo a transformation from an ordered (gel) state to a more disordered fluid ( liquid crysatalline) state.. These chages have been documented by freeze fracture electron microscopy, but most easily demonstrated by differential scanning calorimetry. The physical state of the bilayers profoundly affects the permeability, leakage rates & overall stability of the liposomes.
  55. 55. The phase transition temperature (Tc) is a function of phospholipid content of the bilayers. The Tc can give good clues regarding liposomal stability, permeability & whether drug is entrapped in the bilayers or the aqueous compartment.
  56. 56. Drug Release The mechanism of drug release from the liposome can assesed by the use of a well calibrated in vitro diffusion cell. The liposome based formulations can be assisted by employing in vitro assays to predict pharmacokonetics & bioavailability of the drug before employing costly & time consuming in vivo studies.
  57. 57. Chemical properties Quantitative Determination of phospholipids Phospholipid Hydrolysis. Phospholipid oxidation Cholesterol analysis
  58. 58. Quantitativ Determination of phospholipids It is difficult to mesure directly the phospholipid concentration, since dried lipids can often contain considerable quantities of residual solvent. Conseuently the method most widely used for determination of phospholipid is an indirect one in which the phosphate content of the sample is first measured. The phospholipids are measyred either using Barlett assay or Stewart assay
  59. 59. Barlett assay In the barlett assay the phospholipid phosphorus in the sample is first hydrolyzed to inorganic phosphate This is converted to phospho-molybdic acid by the addition of ammonium molybdate & phosphomolybdic acid is quantitatively reduced to a blue colored compound by amino-napthyl-sulphonic acid. The intensity of the blur color ias measured spectrophotometrically & is compared with the curve of standards to give phosphorus & hence phospholipid content
  60. 60. Barlett assay is very sensitive but is not very reasonably reproducible. The problem is that the test is easily upset by trace contaminations with inorganic phosphate. The sensitivity of Barlett assay to inorganic phosphates creates problem with the measurement of phospholipid liposomes suspended in physiological buffers, which usually contain phosphate ion.
  61. 61. Stewart Assay In stewart assay, the phospholipid forms a complex with ammonium ferrothiacyanate in organic solution. The advantage of this method is that the presence of inorganic phosphate dos not interfare with the assay. This method is not applicable to samples where mixture of unknown phospholipid may be present. In this method, the standard curve is first prepared by adding ammonium ferrothiocyanate (0.1M) solution with different known concentrations of phospholipids in chloroform. Similarly, thesamples are treated & optical density of these solutions is measured at 485 nm & the absorbance of samples compared with the standard curve of phospholipids to get the concentration.
  62. 62. TLC method may also be employed for determining the purity & the concentration of lipids. If the compounds is pure it should run as a single spot in all elution solvents. Phospholipids which have undergone extensive degradation can b observed as long smear with a tail trailing to the origin, compared with pure material which runs as a one clearly defined spot.
  63. 63. Phospholipid hydrolysis The major product of Lysolecithin hydrolysis where one fatty acid chain is lost by de-esterification. Ideally, estimation of phospholipid hydrolysis by quantitation of lysolecithin could be carried out by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. Unfortunately, many natural phospholipids have fatty acids which are ubsaturated & therefore, absorb to different extent in the 1- & 2- position. It is difficult to relate peak heught accurately to absolute quantities of lysophosphatidy; choline (LPC), since one does not know the absorbance of the fatty acid species that have been retained on the glycerol bridge.
  64. 64. Consequently, methods are preferred which permit detection of LPC via the phosphate group after separating the hydrolysis product ( LPC) from the parent PC by TLC. The spots can either be stained with iodine, then scraped off & the phosphate measured directly, or thy can b measured by scanning densiometry. Hydrolysis products of other phospholipids can be estimated in the same way.
  65. 65. Phospholipid Oxidation Oxidation of the fatty acid of phospholipids in the absence of specific oxidants occurs via free radical chain mechanism. Polychain saturated lipids are particularly prone to oxidative degradation. A number of techniques are available for determining the oxidation of phospholipids at different stages i.e; UV absorbance method, TBA method ( for endoperoxides0, Iodometric method ( for hydroperoxides) & GLC method.
  66. 66. Cholesterol analysis Cholesterol is qualitatively analyzed using capillary column of flexible fused silica. Whereas it is quantitatively estimated ( in the range of 0-8 µg) by measuring the absorbance of purple complex produced with iron upon reaction with a combined reagent containing ferric perchlorate, ethul acetate & sulphuric acid at 610 nm
  67. 67. Assay of drug Product High-performance liquid chromatography (HPLC). HPLC has been widely applied to the determination of drugs in liposome formulation . The general procedure involves the use of organic solvents or surfactants to dissolve the phospholipid bilayer and release the encapsulated drug. After this treatment, the mixture is usually centrifuged and the supern atant is analyzed by HPLC. Numerous HPLC methods with photometric , fluorimetric and tandem MS detection have studied the pharmacokinetics, biodistribution and, in some instances, toxicokinetics of liposomal drug formulations
  68. 68. Solid-phase extraction ( SPE) Solid-phase extraction (SPE). SPE is of great interest in the separation of liposomal and non-liposomal drug forms, as it allows the study of the stability of liposomal formulations and their pharmacokinetic behavior. Separation is based on the property of liposomes to cross reversed-phase C18 silica gel cartridges without being retained, while a nonliposomal drug is retained on the stationary phase.
  69. 69. Size exclusion chromatography The usefulness of conventional and high-performance SEC for liposome characterization has been previously reviewed . Polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, and incorporation of amphiphilic molecules are some applications of these techniques to liposome analysis. As indicated above, the drug- retention capacity of liposomes is usually determined using SEC to separate the free from the liposomal drug, which is later released using a detergent or an organic solvent.
  70. 70. TLC HPLC-Numerous HPLC methods with photometric, fluorimetric and tandem MS detection have studied the pharmacokinetics, biodistribution .An HPLC method has been described for the simultaneous quantification of the liposome components using an evaporative lightscattering detector, after disrupting the liposomes with chloroform and methanol. An ion chromatography method with conductimetric detection has been described to quantify sulfate-ion content in a stealth liposome drug-delivery system, which contains ammonium sulfate as an excipient.
  71. 71. Capillary electrophoresis (CE) CE with chemiluminescence detection has been used for the characterization of liposomes in order to study different properties, such as homogeneity, trapped volume, stability and permeability CE has also been applied to study the lipophilicity of several cardiovascular drugs (mexiletine, amlodipine and indapamide), and determine the percentage of the drugs penetrating into liposomal vesicles
  72. 72. Conclusion & future trends Liposome technology is a recent research area that is still far from being fully developed. Thus, numerous LDSs have been described for pharmaceutical applications but only a few of them are being marketed, although many of these systems are currently in preclinical and clinical development with promising results
  73. 73. Conclusion & future trends( cont.) The development of analytical methods to control the effectiveness of LDSs runs parallel to the development of these LDSs, so that this analytical area is also very recent and has still not been consolidated. For example, although in recent years many methods have been described for the control of liposomal drug formulations in biological samples, most of them measure only total drug concentration in the sample and do not distinguish between free and entrapped drug, which would be desirable to know to establish the real behavior of these formulations
  74. 74. Conclusion & future trends( cont.) Future innovations in analytical techniques for LDSs will probably be oriented towards the development of new approaches to provide on-line and in situ information on the delivery process. Selective analytical techniques will probably be based on: a) luminescence monitoring (i.e. laser-induced, timeresolved and long-wavelength fluorescence); b) highly sensitive and discriminative detection systems (i.e. MALDI-MS and SELDI-MS);
  75. 75. Conclusion & future trends( cont.) c) affinity-based biosensors (i.e. catalytic biosensor,immunosensor and genosensor); and, d) screening systems based on the lab-on-chip technology (i.e. DNA-probes, biochips and microarrays). In any case, some of the following aspects should be considered in developing new analytical techniques forLDSs:
  76. 76. Conclusion & future trends( cont.) a)elucidating the mechanism of release from the liposome; b) simultaneous quantification of both free and entrapped analytes; c) discriminating potential interactions of liposomes and/or the entrapped substances with the release media; and, d) additional information for the efficient characterization of the liposomal population, including conventional features (i.e. homogeneity, lamellarity and size) and those related to non-conventional release procedures (e.g., longcirculating liposomes, immunoliposomes and pHdependent liposomes).
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×