Liposomes are spherical vesicles made of phospholipid bilayers that can encapsulate aqueous content. There are three main types - MLV, SUV, and LUV. Liposomes are useful for drug delivery as they can encapsulate both hydrophilic and hydrophobic drugs and release them in a targeted manner. Key properties like size, size distribution, drug encapsulation efficiency, and drug release kinetics must be characterized to ensure quality of liposomal formulations for drug delivery applications. Various microscopic and analytical techniques are used to characterize these properties of liposomes.

1. Prepared byNazia Tajrin
Sr. Officer. R & DF
Incepta Pharmaceuticals Ltd

3. 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.

4. 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

5. 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.

6. Structure of liposome ( cont)

7. Structure of liposome ( cont)

8. 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

9. 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.

10. 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.

11. 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.

12. 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

13. 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

14. Why to use Liposome
Basic reasons for using liposomes as drug carriers
• Direction
• Duration
• Protection
• Internalization
• Amplification

15. 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.

16. 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.

17. 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

19. . 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

20. 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

21. 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

22. Quality control assays of
liposomal formulations

23. Quality control of liposomes

25. Physical properties
Size & its distribution
Surface charfe
 Percent entrapent/capture
 Entrapped volume
 Lamellarity
 Phase behaviour of liposomes
 Drug release

26. 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.

27. 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

28. 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

29. 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.

30. 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

31. 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.

32. 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

33. 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.

34. 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.

35. 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.

36. 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 )

37. 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 )

38. 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-

39. 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

41. 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.

42. 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.

43. 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.

44. 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.

45. 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

46. Surface charge
A method using free flow electrophoresis is used to
determine the surface charge.

47. 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.

48. 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

49. 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

50. 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.

51. 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.

52. 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.

53. 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).

54. 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.

55. 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.

56. 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.

57. 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.

58. 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.

59. 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.

61. 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.

63. Chemical properties
Quantitative Determination of phospholipids
Phospholipid Hydrolysis.
Phospholipid oxidation
Cholesterol analysis

64. 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

65. 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

66. 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.

67. 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.

68. 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.

69. 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.

70. 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.

71. 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.

72. 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

73. 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

75. 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.

76. 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.

77. 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.

78. 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

80. 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

81. 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

82. 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);

83. 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:

84. 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).