Prepared by- Nazia Tajrin Sr. Officer. R & DF Incepta Pharmaceuticals Ltd

What is LiposomeLiposomes 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.

Structure of liposomeLiposomes 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

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.

Structure of liposome ( cont)

Structure of liposome ( cont)

Structure of liposome ( cont)Striking features of liposomes are that-1.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

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.

Liposome in drug deliveryLiposomes 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.

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.

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

Liposome in drug delivery ( cont)Table 1. Classification of liposomes based on composition

and application.Liposome type Major applicationConventional liposomes Macrophage targeting Local depot VaccinationLong-circulating liposomes Selective targeting to pathological areas

Circulating microreservoir Immunoliposomes Specific targetingCationic liposomes Gene delivery

Why to use LiposomeBasic reasons for using liposomes as drug

carriers• Direction• Duration• Protection• Internalization• Amplification

Advantages of liposomesDirection. 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.

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.

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

. For liposomes used in analytical and bioanalytical applications, the main characteristics include

the average diameter and degree of size polydis- persity;

encapsulation efficiency; the ratio of phospholipids to encapsulant

concentration lamellarity determination

Characterization of liposomesThe behaviour of liposomes in both physical

& biological systems is governed by the factors such as:

Physical sizeMembrane permeabilityPercent entrapped solutesChemical compositionQuantity & purity of the starting material

Characterization of liposomesTherefore, 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

Quality control assays of liposomal formulations

Quality control of liposomes

Physical propertiesSize & its distributionSurface charfe Percent entrapent/capture Entrapped volume Lamellarity Phase behaviour of liposomes Drug release

Size & size distributionSize 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.

Size & its distributionSeveral 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, freeze-fracture TEM, and cryo EM , provide valuable information on liposome preparation

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

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.

Microscopic methodsLight 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 distribution.

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.

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

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.

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.

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.

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 )

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 )

Laser light ScatteringDiffraction 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-

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

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.

Gel permeationExclusion 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.

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.

Zeta potentialThe 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.

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

Surface chargeA method using free flow electrophoresis is

used to determine the surface charge.

Lamellarity determinationThe 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.

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

Encapsulation effciencyThe 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 procedure, etc., as reviewed

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.

Encapsulation efficiency is commonly measured by encapsulating a hydrophilic marker (i.e. radioactive sugar, ion, fluorescent dye), sometimes using single-molecule 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.

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.

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 temperature- induced leakage (Fig. 6). This property of liposomes and nanoliposomes can be used in the formulation of temperature-sensitive vesicles (55).

Entrapped volumeThe 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.

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.

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.

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.

Phase Bhaviour of LiposomesAn 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.

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.

Drug ReleaseThe 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.

Chemical propertiesQuantitative Determination of phospholipidsPhospholipid Hydrolysis.Phospholipid oxidationCholesterol analysis

Quantitativ Determination of phospholipidsIt 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

Barlett assayIn 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 & phospho-molybdic 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

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.

Stewart AssayIn 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.

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.

Phospholipid hydrolysisThe 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.

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.

Phospholipid OxidationOxidation 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.

Cholesterol analysisCholesterol 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

Assay of drug ProductHigh-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

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 non-liposomal drug is retained on the stationary phase.

Size exclusion chromatographyThe 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.

TLCHPLC-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.

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

Conclusion & future trendsLiposome 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

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

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);

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:

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., long-circulating liposomes, immunoliposomes and pH-dependent liposomes).