INTRODUCTION - Pharmawiki.in
INTRODUCTION - Pharmawiki.in
EVALUATION OF COLLOIDAL
DRUG DELIVERY SYSTEMS
Department of Pharmaceutics
Classification colloidal drug delivery systems
Evaluation techniques of,
• The prime objectives in the design of drug delivery systems is the
controlled delivery of pharmacological agent to its site of action at a
therapeutically optimal rate and dosage regimen to avoid toxicity and
improve the drug efficacy and therapeutic index.
• Amongst the most promising systems to achieve this goal are colloidal
drug delivery systems which include liposomes, niosomes, micro
emulsions and nanoparticles.
• Nanoparticles are considered as promising colloidal drug carriers as
they overcome the technological limitations and stability problems
associated with liposomes, niosomes and micro emulsions.
• Definition: The term colloid is applied to disperse system in which
particle size of the dispersed phase is very fine, in the range of less than
ADVANTAGES : Colloidal carrier systems,
aid in solubilization of lipophilic drugs.
can protect sensitive drugs against degradation in biological fluids.
protects patients from irritative side effects of drug and prolong drug
action due to sustained release.
can be used for drug targetting.
I. Vesicular systems
II. Non-vesicular systems
i. Micro emulsions
ii. Submicron emulsions
III. Particulate systems
EVALUATION TECHNIQUES :
general, colloidal drug deliver systems are evaluated for physicochemical
• Importance of evaluation :: Evaluation of variuos physicochemical
properties like shape, size, chemical compositions, surface properties etc is
done because any alteration in such properties alters the drug release profile,
stability of the system,interactions with the surrounding medium etc.
•Evaluation is done to provide quality assurance.
• Definition: Liposomes are concentric bilayered vesicles in which an
aqueous volume is entirely enclosed by a membranous lipid bilayer mainly
composed of natural or synthetic phosolipids.
PHYSICAL CHARACTERIZATION :
• Size & its
Microscopic, Diffraction and scattering and
Hydrodynamic techniques, Size exclusion
chromatography, ultra centrifugation.
Electron microscopy, 31P NMR, Small angle
Phase behaviour - Freeze fracture microscopy, Thermodynamic
methods( DTA and DSC).
Surface Charge - Electrophoresis and zeta potential
- Minicolumn centrifugation, Protamine
SIZE & SHAPE ANALYSIS :
It determines the physical stability and biofate of particles.
I. MICROSCOPIC TECHNIQUES :
a. Optical microscopy : useful in evaluating vesicles > 1µm.
Limitation : Tedious and less resolution.
b. Electron microscopy: Vesicle shape and morphology is determined.
Have greater resolution(10A - 1µm).
Aqueous samples do not survive high vacuum, hence special
techniques of sample preparation are necessary prior to electron
microscopy to prevent microstructure changes.
i. Scanning electron microscopy (SEM) :
Principle : SEM images the sample surface by scanning with a high
energy beam of electrons, under high vacuum.
• Electrons interact with the atoms that make up the sample producing
signals that contain information about surface topography, composition,
electrical conductivity etc.
• Procedure : Samples are coated with electrically conductive materials
like gold, platinum, osmium, graphite etc.
• Coating prevents static electric charge accumulation on specimen during
Alternative to coating is to increase the bulk conductivity by
impregnation with osmium.
• Size range : 1-5nm in size.
Sample must be dry.
Coating agent may change the morphology and size of the particle.
Provides only 2D projection.
ii. Transmission electron microscopy (TEM) :
• Principle : Beam of electrons is transmitted through an ultra thin specimen,
interacting with specimen as it passes through it.
• An image formed from electrons transmitted through it is magnified and
focused by objective lens.
Graphene, a carbon nanomaterial, one atom thick, is used as platform.
It is transparent to electrons.
• TEM is of two types:
Negative stain TEM
Cryo – TEM
• Negative stain TEM : In this method, liposomes are embedded in thin film of
electron dense heavy metal stain.
• Negative stains used are ammonium molybdate, phosphotungstic acid in case
of liposomes composed of neutral or negatively charged phospholipids.
• Uranyl acetate is used for positively charged lipids.
• Cryo -TEM :
• Sample can be viewed directly in TEM (at temp of -196oC)
• Sufficient contrast is given to frozen sample by osmium tetroxide.
• At such temperature, vapour pressure is low; hence, preservation of
microstructure is possible despite high vacuum.
• Disadvantages :
• Due to fluid property of dispersion, prior to freezing, thickness of sample
various from center (thin) to outside (thick film).
• Hence, actual size distribution cannot be known.
iii. Freeze fracture electron microscopy :
• Procedure : Sample is sandwiched as a thin layer between gold plates and
then shock frozen with either nitrogen cooled liquid propane at -1960C or
slush nitrogen at -2100C.
• Fracture of the frozen sample is performed at -1000C and vacuum between
10-6 and 5 X 10-7 bar, at an angle between 90-150.
•After freeze fracture, etching is performed.
•Following this, surface is shadowed with a 2nm thick platinum under an
angle of 450.
•Sample is freezed rapidly to avoid phase separation, crystallization and to
preserve original microstructure during replication.
•Limitation : Smaller the vesicle, less probable is an upcoming cross
iii. Atomic Force Microscopy (AFM) :
• Principle : AFM scans the sample with a nanosized tip connected to a
• In contact mode, tip is allowed to lightly tap or drag across the surface of
sample. As the tip scans, vertical deflection or repulsion occurs which is
measured using laser spot reflected from top of cantilever into an array of
• In non-contact mode, measurement of attraction of tip to sample surface is
used to determine the height of local surface.
• sample is placed on piezoelectric tube for moving it and for maintaining
provides 3D projection.
Special treatments are not required.
No vacuum is needed.
Higher resolution(upto 0.01 nm for imaging).
It is not as fast as SEM.
Coulter counter :
• In this technique , fine particles to be characterized are placed in an
electrolyte and a stream of suspension is passed through an orifice between
• Size of fine particles is deduced from measured resistance change between
• The resistance causes a voltage pulse, directly proportional to particle volume
of individual particle.
• When concentration is low, each voltage pulse corresponds to an individual
particle. Hence, size distribution can be established.
Only particle size > 400 nm are detected.
Particles must be spherical for accurate
Particles that are non-spherical or porous
will give volumes larger than their actual
II. DIFFRACTION AND SCATTERING TECHNIQUES :
Laser light scattering technique :
• It is quick method for determination of size.
• Applied for particles < 1 µm.
• Rayleigh’s theory holds good for particles < 200nm, which considers
scattering intensity is proportional to sixth potency of particle diameter.
•Diffraction technique:Laser diffraction can be applied for particles >1µm
• Fraunhofer theory refers to proportionality between intensity of diffraction
and square of particle diameter.
• Scattering intensity depends on scattering angle, absorption , size of
particles as well as refractive indices of both particles and dispersion
Photon Correlation Spectroscopy (PCS) :
• DLS and PCS analyses the fluctuations in scattering intensity that occur over
very short time intervals due to Brownian motion of particles.
•The hydrodynamic radius can be determined from Diffusion coefficient , D
using Stokes’s – Einstein relationship.
• where KB
6 ƞ П rH
viscosity of solvent
• Smaller the particle, higher the fluctuations by brownian motion.
• It is well suited for measuring nanoparticles because it takes advantage of
brownian motion which is unique to colloidal particles.
•It measures the velocity of particle in motion and correlates it to size.
III. HYDRODYNAMIC TECHNIQUES :
Field flow fractionation technique (FFF) :
• Heterogenous mixtures , strongly interacting systems can be characterized.
• Range of particle size separated is 1nm – 100 µm.
• It is of two types :
Flow FFF : Principle :separation occurs by differential retention in a
stream of liquid flowing through a thin, empty channel.
• Field in this is applied at right angles to flow and serves to drive components in
different stream laminae in a capillary channel.
• Different velocities of fluid laminae across the channel develops the separation
induced by action of the field.
•Separated components are eluted one at a time into detector.
•It combines elements of chromatography and field driven techniques like
electrophoresis and ultracentrifugation.
•It provides direct measurement of vesicle size and size distribution.
Sedimentation FFF :
•It measures effective mass and mass distribution.
•It is sensitive to small changes in the effective mass of either biomembrane or
its encapsulated load.
•Size characterization is done by deducing size from effective mass.
Gel permeation chromatography :
•It is used for size distribution determination of liposomes.
•Separation is achieved from differential permeation of particles through pores.
•Gel media with large porosities, sepharose 4B or 2B, sephacryl S500 and
S1000 allows fractionation of liposomes.
•Gel medium can also separate non- encapsulated molecules.
•It can yield valuable data on size distribution of liposomes.
•Centrifugation at 2,00,000 g for 10-12 hr is required.
SURFACE CHARGE :
• It is determined by zeta potential.
•Definition : zeta potential is the potential between tightly bound surface
liquid layer of particle and electroneutral layer.
•It provides the measure of net surface charge on the particle and potential
distribution at the interface.
•It is calculated using Helmholtz- Smoluchowski equation,
• where ζ = zeta potential
ƞ = viscosity of dispersion medium.
µ = migration velocity.
ɛ = dielectric constant.
E = potential gradient between electrodes.
ENCAPSULATION EFFICIENCY :
•It is expressed as percent entrapment per mg lipid.
•Percentage of aqueous phase and hence percent of water soluble drug that gets
entrapped during preparation of liposomes.
•It is assessed by using two techniques :
Minicolumn centrifugation :
•Sephadex or sepharose column, presaturated with dispersion medium in 1ml
disposable syringe is run while applying liposomal dispersion (200µl) first and
saline (250µl) thereafter.
•Then, centrifuge the column at 2000 rpm for 3min and assay the elutes.
•Concentration of free or entrapped matreial in elutes can be assessed by
disrupting liposomes using ethanol or triton.
Protamine aggregation method :
•It is used for negatively charged or neutral liposomes.
•Dispersion is precipitated with protamine solution and subsequently
centrifuged at 2000 rpm.
•By analysing material in supernatant and liposomal pellet, encapsulation
efficiency can be determined.
TRAPPED VOLUME :
•Definition : It is the aqueous entrapped volume per unit quantity of lipid and
expressed as µl/mol or µl/mg of total lipid.
Best way to measure trapped volume is to measure quantity of water
directly. This is done by replacing the external medium with deuterium
(spectroscopically inert) and then measuring water signal using NMR.
•Peak height is related to concentration by comparing with standards
containing known amount of H2O in D2O.
It can also be determined by dispersing lipid in an aqueous medium
containing non-permeable radioactive solute such as 22Na and 14C inulin.
•Proportion of solute trapped is determined by removing external radioactivity
by centrifugation, dialysis/gel filtration.
Use of entrapped water soluble marker such as 6-carboxyfluorescein, 14C or
3H- glucose or sucrose and then lysing liposomes by use of detergent. Trapped
volume is back calculated using the amount of marker that is entrapped.
•Estimation of number of bilayers can be done by,
Electron microscopy , 31P-NMR , Small angle X-ray scattering.
• 31P-NMR : Structure can be determined by this technique after
complexation with Mn2+ or Pr3+ ions.
•Other methods include, labelling formulations containing phosphatidyl
ethanolamine with trinitrobenzene sulphonic acid(TNBS).
•This agent reacts specifically with surface polar phosphate heads.
•When phospholipid is further increased , there is a decrease in surface
phosphate groups resulting in decrease in 31P NMR area.
•Therefore, bilayered structures like liposomes are formed where, phosphate
groups are concealed inside bilayers.
PHASE RESPONSE AND TRANSITIONAL BEHAVIOUR :
•Lipid bilayers can exist as solid-ordered phase at low – temperature and above a
certain temperature, in a fluid-disordered phase, the temperature of this phase
transition can be tailored by selecting proper lipids.
•Phase behaviour can be studied by freeze fracture microscopy and thermodynamic
Differential scanning calorimetry :
•It quantifies the enthalpic changes during
endothermic and exothermic phase transitions.
•Two aluminium plates are compared, one
empty and the other containing sample.
•Heat input of sample is adjusted so that its
temperature matches those of the reference pan.
•At the phase transition point, extra heat is
required to maintain the rise in temperature of
the sample pan equal to that of reference and is
Differential thermal analysis :
•It measures the temperature differences
between reference and sample.
CHEMICAL CHARACTERIZATION :
•Methods to characterize liposomes have become more essential which
require lipid stability cropping up from oxidation , lipid peroxidation,
hydrolysis and degradation in various environments used in their
• Phospholipid concnetration - NMR,FTIR
• Cholesterol concentration
- Cholesterol oxidase assay, Ferric
• Drug concentration
- appropriate methods mentioned in
• Phospholipid peroxidation - UV absorbance, iodometry.
• Phospholipid hydrolysis
- HPLC , TLC
• Cholesterol auto-oxidation - HPLC, TLC.
•Definition : Niosomes are essentially non-ionic surfactants based
multilamellar or unilamellar vesicles in which an aqueous solution of solute is
entirely enclosed by a membrane resulted from the organization of surfactant
macro-molecules as bilayers.
•EVALUATION : Niosomes are evaluated for size, shape, morphology,
entrapment efficiency, surface charge and solute release rates.
SIZE ANALYSIS : It is performed by microscopic techniques (like
freeze fracture microscopy,SEM,TEM etc.), photon correlation spectroscopy.
•Size and shape are dependent on drug entrapment, nature of drug and
•Entrapped solute : Solute molecules interact with head group of surfactants
resulting in increased vesicle size because of net increase in resultant charge
and force of repulsion.
•Effect of vesicle forming components : Size of vesicles was relatively
smaller with increasing chain length of polyoxyethylene due to reduction of
radius of curvature of bilayers.
ENTRAPMENT EFFICIENCY :
•It is measure of solute retention.
•It is determined using carboxyfluorescein (CF) as a marker (200mM of CF).
•Ratios of CF before and after disruption of vesicles is calculated to determine
•It is linearly related to the length of carbon chain in alkylglycosides(chain not
less than myristyl could form stable vesicles).
•It is governed by method of loading, nature of solute and hydration
•Effect of cholesterol : Cholesterol assists in solute retention.
•Amount of cholesterol decides the release rate and extent of solute from the
•Nature of hydrophillic head groups :
•Moieties with lower head groups has better efficiency(diglycerol groups has
more efficiency than ethoxy groups).
•Nature of alkyl side chain : Alkyl chain length cut off point for vesicle
formation may be a 14 carbon chain.
•Definition : Nanoparticles are sub-nanosized colloidal structures composed of
synthetic or semisynthetic polymers.
•Size ranges from 10nm to 1000nm.
•Various types of nanoparticles include,
Solid lipid nanoparticles(SLNs)
• EVALUATION : Nanoparticles are characterized for ,
• Surface charge
• Crystal structure
• Invitro release
- AFM ,Electron microscopy (SEM,TEM), PCS,
Freeze fracture microscopy, mercury porositometry.
- Pycnometer , Isopycnic centrifugation.
- Zeta potential, Laser Doppler Anemometry or
- X-ray diffraction, thermal analysis (DTA, DSC)
- Hydrophobic interaction chromatography
- X-ray photoelectron spectroscopy, gel electrophoresis,
Global technique, Capillary electrophoresis.
- Diffusion cell, Ultracentrifugation.
SIZE ANALYSIS :
Mercury porositometry :
•Freeze dried nanoparticles are filled in a dilatometer under vacuum and then
measured with the help of a mercury pressure porositometer.
•It mainly measures particulate agglomerates as mercury fails to penetrate to a
greater extent within the primary particles.
SPECIFIC SURFACE AREA :
•Specific surface area of freeze dried nanoparticles is determined BY,
A - specific surface area
δ - density
d - diameter of particle
SURFACE HYDROPHOBICITY :
•Several methods like Hydrophobic interaction chromatography, Two-phase
partition,Adsorption of hydrophobic fluorescent or radiolabelled probes, contact
angle measurements are adopted to evaluate surface hydrophobicity.
Contact angle is measured on plain surface(Hence, nanoparticles are
compressed as tablet or pellet).
Hydrophobic interaction chromatography :
•Particles are retained by gel and eluted after addition of surfactant are
•Particles that are directly eluted from column are hydrophillic.
SURFACE CHARGE :
•Surface charge of colloidal particles in general can be determined by zeta
potential, measuring particle velocity in an electric field.
•Laser doppler anemometry or velocitometry is fast and high resolution
technique for determination of velocity.
Laser Doppler Anemometry : it is for measuring the direction and speed of
•Principle : A beam of monochromatic laser light is sent into the flow and the
particles will reflect light with a doppler shift corresponding to their velocities.
•Shift can be measured by interfering the reflected beam with the original
beam, according to the frequency differences.
f – frequency of signal received at
u = f Xd
d – distance between fringes.
CHEMICAL COMPOSITION :
2D- Polyacrylamide gel electrophoresis :Identification of proteins adsorbed.
Capillary electrophoresis : to evaluate modification of composition of
adsorbed proteins with time.
Capacity to activate : It is estimated by Global technique or by a specific
method measuring activation of component C3.
Global technique : Particles are incubated with serum, then remaining nonactivated complement in serum is evaluated using a red blood cell lysis test.
X-ray photoelectron spectroscopy (XPS):
•Principle : XPS spectra is obtained by irradiating a material with a beam of Al
or Mg X-rays while simultaneously measuring the kinetic energy and number
of electrons that escape from top of the material being analyzed.
•XPS requires ultra high vacuum and measures elemental composition,
empirical formula, chemical state and electronic state.
•Limits : Approximately 100ppm.
•Concentration in dispersion can be deduced from Gravimetric determination
or by turbidimetric measurements.
CRYSTAL STRUCTURE :
X-ray diffraction : When a monochromatic x-ray beam is focused on a
crystal, atoms scatter the x-ray beam, in specific pattern.
Bragg’s equation : nλ = 2d sinθ
λ - wavelength of x-rays
θ - angle of incidence
d - interatomic distance
•A typical interference pattern arises due to specific repeat distances of the
associated interlayer spacing, d.
•Larger terms for d in the region of long range order are registered by the
small angle x-ray diffraction technique.
•For short range order, registered by wide angle x-ray diffraction technique.
•Interferences are detected in two ways, film detection and registration of xray counts with scintillation counters.
INVITRO RELEASE :
•Invitro release profile can be determined using standard dialysis, diffusion cell
or ultrafiltration technique.
Diffusion cell : evaluated in phosphate buffer utilizing double chamber
diffusion cells on a shaker stand.
•A millipore hydrophillic low-protein binding membrane is placed between two
Ultrafiltration : Nanoparticle suspension is added directly into a stirred
ultrafiltration cell containing buffer.
• Samples are collected through ultrafiltration membrane using less than 2 bar
positive nitrogen pressure and assayed for the released drug using standard
MICRO EMULSIONS :
•Definition : Micro emulsions are fluid, transparent, thermodynamically
stable oil and water systems, stabilized by a surfactant usually in conjunction
with a cosurfactant.
•Size range of droplets is 0.1-1 µm.
• EVALUATION :
• Size and shape
- various light scattering techniques(SAXS,SANS),
• Interfacial tension - Spinning drop apparatus.
Interfacial tension is derived from the measurement of the shape of a drop
of the low-density phase, rotating it in cylindrical capillary filled with highdensity phase.
Viscosity measurements are used to determine hydrodynamic radius as well
as interactions between droplets.
•Einstein’s equation can be used to calculate viscosity and hydrodynamic
• CONCLUSION :
• Colloidal drug delivery systems are designed for controlled and targetted
delivery of the pharmacological agent.
• Hence, Colloidal drug delivery systems are characterized to ensure their
predictable invitro and invivo performances.
various physicochemical characteristics like size, shape ,surface properties
, lamellarity, phase behaviour , drug release profile etc are evaluated.
• Advanced techniques like electron microscopy has greater resolution.
• Original unaltered shape and surface properties can be known by AFM.
• Internal structure and lamellarity can be determined by freeze fracture
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nanoparticles.,Vobalaboina Venkateshwarlu,Kopparam Manjunath, Journal of
Controlled release 95 (2004) 627- 638.
•Herbert A.lieberman,Martin M.Rieger and gilbert S.Banker, Pharmaceutical Dosage
Forms : Disperse Systems, vol-3,second edition,Revised and Expanded.
•James Swarbrick, Encyclopedia of pharmaceutical technology, third edition , vol-2,
•An overview of lipid membrane supported by colloidal particles, Anne-Lise
Troutier,Catherine Ladaviere, Advances in colloid and interface science 133(2007) 121.