Evaluation of vesicular dosage forms

1. 1
vesicular dosage forms - Evaluation of
vesicular dosage forms
vesicular dosage forms - Evaluation of
vesicular dosage forms
Department of Pharmacy (Pharmaceutics) | Sagar savale

2.  Introduction to vesicular dosage forms
 Introduction of liposomes and its characterization
 Introduction of niosomes and its characterization
 Introduction of multiple emulsion and its
characterization.
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3. VESICULAR
DOSAGE FORM
VESICULAR
DOSAGE FORM
Vehicle of choice in drug deliveryVehicle of choice in drug delivery Encapsulation of
a drug
Encapsulation of
a drug
Modeling
biological
membrane
Modeling
biological
membrane
Transport and
targeting of
active agents.
Transport and
targeting of
active agents.
Increase
permeation of
drugs
Increase
permeation of
drugs
Reduces the toxicity by
selective uptake
Reduces the toxicity by
selective uptake
incorporate
both
hydrophilic
and
lipophilic
drugs
incorporate
both
hydrophilic
and
lipophilic
drugs
solves the
problems of
drug
insolubility,
instability,
and rapid
degradation.
solves the
problems of
drug
insolubility,
instability,
and rapid
degradation.
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4. Liposomes are colloidal, vesicular structures enclosed by
membrane of lipid bilayer mainly composed of natural or
synthetic phospholipids..
Lipid content as major component
Phospholipids
Natural phospholipids
Synthetic phospholipids
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5. Characterization parameters Analytical method/Instrument
Vesicle shape and surface
morphology
Transmission electron microscopy,
Freeze-fracture electron microscopy
Lamellarity Freeze-fracture electron microscopy
size and size distribution
(submicron and micron range)
Microscopy techniques, light scattering
techniques, chromatography
Surface charge Capillary zone electrophoresis
Encapsulation Efficiency and
entrapped volume
Minicolumn centrifugation, protamine
aggregation, ultracentrifugation,
Phase transition studies Freeze-fracture electron microscopy,
DSC
PHYSICAL CHARACTERIZATIONPHYSICAL CHARACTERIZATION
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Phospholipid concentration Bartlett assay, HPLC
Cholesterol concentration Cholesterol oxidase assay and
HPLC
Phospholipid hydrolysis HPLC and TLC
BIOLOGICAL CHARACTERIZATION
Sterility Aerobic or anaerobic cultures
Pyrogenicity Limulus amebocyte lysate test LAL
Animal toxicity Monitoring survival rates
CHEMICAL CHARACTERIZATION

7. 1.SHAPE AND LAMELLARITY
 ESTIMATION BY ATOMIC FORCE MICROSCOPY (AFM):
AFM /scanning probe microscopy, a high resolution imaging
technique that can resolve features as small as an atomic lattice.
SHAPE AND LAMELLARITY
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8.  Operation : By rastering a sharp tip across the sample.
 Working:
Tip is first brought
(manually)
close to the sample
surface, scanner
makes a final
adjustment in tip-
sample distance
based on a set point
determined by the
user.
The tip in contact
with the sample
surface scans
across the sample
under the action of
a piezoelectric
actuator either by
moving the sample
or the tip relative
to the other.
A laser beam
aimed at the back
of the cantilever–
tip assembly
reflects off the
cantilever surface
to a split
photodiode, which
detects the small
cantilever
deflections.
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9.  EVALUATION: The force between the tip and the sample is
calculated by using Hooke’s Law:
 Where, F is force,
k is the spring constant and
x is the cantilever deflection.
F = -kx
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10. AFM principleLipid bilayer observed by AFM
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11.  Lamellarity in liposomes is measured by 31
P-NMR.
Mn2+ ions interacts
with the outer leaflet
of outermost bilayer.
non permeable
broadening agent
manganese is added.
Signals are recorded
before and after
addition of non
permeable agent
manganese
50% reduction in NMR signal means that
liposome preparation is unilamellar and a 25%
reduction in intensity of original NMR signal
means that there are two bilayers in liposomes. 11

12.  Major techniques include (a) microscopy techniques
(b) light scattering techniques.
MICROSCOPY TECHNIQUES
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Light
microscopy
Electron
microscopy
Cryo-Transmission
electron
microscopy
Freeze fracture
electron
microscopy
Negative staining
microscopy

13. Light microscopy: If the liposomes are large enough , can be
observed with a light microscope and can be determined using
an optical scale.
Electron microscopy: smallest of liposomes can be observed by
electron microscopy by following three ways:
a) Negative staining electron microscopy (TEM)
b) Cryo-Transmission electron microscopy
c) Freeze fracture electron microscopy
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14. Droplet of liposomes placed on C- coated copper grid for 3 min
Dried with filter paper
Negative stain solution( 2% uranyl acetate)
Drying done
Visualization of size, morphology and lamellarity of liposomes
carried out.
Negative staining Transmission Electron
Microscopy (TEM)
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15. A. Liposomes observed by light microscopy
B. Liposomes observed by TEM
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TRANSMISSION
ELECTRON MICROSCOPY

17. LIPOSOMES
OBSERVED BY
TEM
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IMAGE BY CRYO-
TEM

19.  The STM is a type of electron microscope that shows (3D)
images of a sample.
Principle:
 Structure of a surface is studied using a stylus that scans the
surface at a fixed distance from it.
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SCANNING TUNNELING MICROSCOPY (STM)

20.  Extremely conduting fine probe is held close to the sample.
 Electral signal is produced.
 It slowly scans the surface at a distance of only atom’s diameter.
 This enables it to follow even the smallest details of the surface it
is scanning.
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22. 1.Field-flow fractionation
 Field-flow fractionation (FFF) is a family of separation
techniques, also a one phase chromatographic technique.
 The separation channel consists of an impermeable top block and
a bottom block holding a semi permeable ultra filtration
membrane (the accumulation wall) on top of a porous frit.
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24.  Retention times are dependent on the equilibrium height at which the
analyte travels in the parabolic flow profile of the channel flow and can be
expressed by:
tr = π. η. d. ω2
. V cross
2 kT Vchannel
 Where tr is the retention time of the analyte;
η the viscosity; d the stokes diameter; ω the channel thickness; k the
Boltzmann constant; T the absolute temperature; Vcross the cross flow
rate and Vchannel the channel flow rate .
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PRINCIPLE OF FIELD-FLOW
FRACTIONATION

26. SIZE EXCLUSION CHOMATOGRAPHY
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27. Charge determination is done using capillary zone electrophoresis
and zeta potential measurement.
 ZETA POTENTIAL
ZETA POTENTIAL
overall charge that the
particle acquires in a
particular medium
help to predict the
fate of the
liposomes in vivo
Measurement is done using
the technique of laser
Doppler velocimetry.
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Zeta potential analysis of liposomes

29. 5 ENCAPSULATION EFFICIENCY AND ENTRAPPED
VOLUME:
 The encapsulation efficiency describes the percent of aqueous
phase and hence the percent of water soluble drug that gets
entrapped during liposomes preparation & expressed as
%entrapment/mg lipid.
 A. Mini- column centrifugation
 B. Protamine aggregation
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33.  The internal or trapped volume is the aqueous entrapped volume
per unit quantity of lipid and expressed as µl/ mg of total lipid.
 To measure internal volume is to measure the quantity of water
directly.
 Replace the external medium i.e water with a spectroscopically
inert fluid deuterium oxide.
 Measure the water signal using NMR.
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34.  Phase transition temperature is defined as temperature
required to induce a change in lipid physical state from the
ordered gel phase to the disordered liquid crystalline phase.
 Thermodynamic methods:
 Phase transitions have been evaluated using freeze fracture
electron microscopy as well as differential scanning
calorimetery.
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35.  In DSC heat required by liposomes to maintain a steady upward
rise in temperature is plotted as a function of temperature.
 Transmitter monitors the temperature of each pan.
 Heat input of sample pan is adjusted so that its temperature
matches those of the reference .
 At phase transition point extra heat is required to maintain the rise
in temperature of the sample pan equal to that of the reference
and this is recorded directly.
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36. VESICLE FUSION MEASUREMENTS:
 Fusion is monitored using fluorescence resonance energy
transfer (RET) between two lipid analogues placed in separate
or same vesicle population.
 Technique depends upon an overlap in the emission spectrum
of one flurophore i.e. donor and excitation spectrum of a
second flurophore i.e. exceptor.
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the excited donor
molecule
Acceptor molecule
Transfers direct energy
Excites
Donor fluorescence emission
intensity reduces
Acceptor fluorescence
emission intensity
increases
fusion of incorporating donor and acceptor molecules
detected by RET onset
Relative change of RET on fusion is linearly related to extent of fusion.

38.  Niosomes are non-ionic surfactant 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.
 Non-ionic surfactants orient in an aqueous medium as planner
bilayer lattices.
 Where polar or hydrophilic heads align facing aqueous bulk.
 While hydrocarbon segments are so aligned that their interaction
with aqueous media is minimized.
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39.  General characteristics like particle size, shape, entrapment
are similar to those for liposomes.
 They required to be characterized for structural and
transformational properties, rheological behaviour, bilayer
thickness and molecular interfacial phenomenon.
 1) structural and transformational properties:
 Niosomes formed by hexadecyl diglycerol ether exhibit a
variety of shapes dependent on their membrane composition .
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41.  ENTRAPMENT EFFICIENCY:
 Entrapment efficiency is determined using carboxyfluorescin
(CF) as a marker .
 Procedure:
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43.  Vesicle surface charge can be estimated by measurement of
particle electrophoretic mobility and is expressed as zeta
potential which is calculated using Henry equation.
where ;
ζ=zeta potential
µE=electrophoretic mobility
η= viscosity of the medium
Σ = dielectric constant
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vesicle surface charge:
ζ = µE4πη
Σ

44.  “Multiple emulsion are the emulsion systems in which the
dispersed phase contain smaller droplets that have the same
composition as the external phase.”
 “Double Emulsion”
 “Liquid Membrane Systems”
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TYPESTYPES
Oil-in-water-in-oil (O/W/O)
emulsion system
Water-in-oil-in-water
(W/O/W)emulsion system

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1) Average Globule Size And Size Distribution:
Method used are - Optical Microscopy,
- Static light Scattering,
- dynamic light scattering,
- Brightfield Microscope,
- Scanning Electron Microscope.
EVALUATION :

46.  Area Of Interfaces : It can be determined by following
formula.
where, S = Total area of Interface
(sq.cm)
d = Diameter of Globules (cm)
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S = 6/d

47. 3) No. Of Globules : It can be measured using the
Haemocytometer cell.
 For this Emulsion is properly diluted.
 And the globules in five groups of 16 small squares (total 80
small squares) are counted & the total no. of globules per
cubic mm are calculated using the Formula :
 No of globules ×Dilution × 4OOO
No of globules/mm³= No of small squares counted
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48.  Rheological Evaluation : It is important parameter as it
relates to Emulsion stability & clinical performance.
 2 major parameters : -Viscosity
 -Interfacial Elasticity
 Viscosity can be measured by Brookfield Rotational
Viscometer.
 Interfacial Elasticity can be investigated at the mineral
oil/water interface using an Oscillatory Surface Rheometer.
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49.  Zeta Potential : It is used to determine surface charge by the
help of mobility & electrophoretic velocity of dispersed
globules.
 Hence it is used to predict Particle-Particle Interaction.
 % Drug Entrapment : It can be determined by
a) Dialysis,
b) Centrifugation.
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50.  Vyas and Khar.Targeted and Controlled Drug
Delivery Novel Carrier Systems.First edition,CBS
Publishers, New Delhi.pp-196-204, 260-264,308-309.
Jain.N.K.Advances in controlled and Novel Drug
Delivery System,CBS publication,1st
edition,p.p.415-
420.
 R.S.R.Murthy;Vesicular & Particulate Drug Delivery
Systems, Career Publications,p.n. 25-29,
98,149-154.
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