This document provides an overview of preformulation studies for liposomes and microspheres. It defines preformulation and describes various characterization tests conducted during preformulation including bulk characterization, solubility characterization, stability characterization, assessment of lamellarity, trapped volume, flow properties, and in vitro drug release for liposomes. For microspheres, it discusses techniques to determine particle size and distribution, surface charge, thermal analysis, stability studies, and in vitro drug release. The goal of preformulation is to generate useful information for developing stable and bioavailable dosage forms.

1. PREFORMULATION
OF LIPOSOMES
And Microsphere
Prepared by :- Guide :-
Sneha A. Chavan Dr. (Mrs.) Aparna Palshetkar
Department of Pharmaceutics
M - Pharmacy 1st Year

2. What is liposome and Microsphere

4. Preformulation
• Definition:
It is a stage of formulation development where
physical and chemical properties of drugs alone and
when combined with excipients are studied.
• Objective:
To generate information useful to formulator in
developing stable and bioavailable dosage forms.

5. Preformulation
Bulk
characterization
Solubility
characterization
Stability
characterization

6. Bulk characterization
Polymo
rphism
Hygroscopicity
Crystall
inity
Description
Particle
size
Bulk density
Powder
flow
propert
ies

7. Solubility
characterization
pKa
pH solubility
profile
Common
ion effect
Thermal
effect
Partition
coefficient
Dissolution

8. Stability characterization
Solid
state
Stability
pH rate profile
Solution
stability
compressibility
Bulk
stability
Stability in
formulation

9. Shape
Electron /
confocal
microscopic
technique
scanning
electron
microscopy (
SEM )
Transmission
electron
microscopy (
TEM )

10. LIPOSOME MICROSPHERE
Size Ranges from
20nm – 3 µm
ranges from 1-1000 µm
Vesicle
Shape or
size
Electron
Microscopic
Technique
the average size of
vesicle
automated diffraction technology.
Mean particle size, frequency distribution
profile and polydispersibility index are
analyzed output that can be interpreted for
the developed microparticles.
Optical microscopy determining particle
size of microsphere larger than 10 µm.
Imaging analysis facilities (Imaging and
analysis software) useful in determining
the size distribution of microsphere
High resolution TEM size of particles by
point to point resolution

11. Vesicle size and Distribution of
liposome
1. Microscopic technique
 Optical microscopy
 Fluorescent microscopy ( if fluorescent probe is included in either lipid or aqueous
domain )
 Cryo –Transmission electron microscopy ( Cryo - TEM)
 Scanning Electron microscopy ( SEM )
 Negative stain Transmission electron microscopy ( TEM )
 Freeze fracture microscopy
2. Diffraction and scattering technique
 Laser light scattering
 Photon correlation spectroscopy
3.Hydrodynamic technique
 Field flow fractionation ( FFF ) Technique
 Gel permeation and gel exclusion and zetasizer
 ultracentrifuge
zl

12. 2. Surface charge
Surfacecharge
Free flow
electrophoresis
Zeta potential
measurement

13. Surface charge
• The charge of microparticles can be analyzed as it indicates the
dispersion of the micron size particles in a medium.
• Surface charge decides the degree of aggregation upon storage
or even reconstitution, which further leads to alteration in the
stability, Syringeability of microsphere.
• Zeta potential is measured for microparticles and it indicates
the electrostatic interaction.
• It is measured by micro-electrophoretic technique where the
movement of particles is measured under the influence of
known electric field.
• Alternatively electrophoretic light scattering technique can be
used for measuring zeta potential of microsphere.

14. • The surface charge can be calculated by estimating the mobility
of the liposomal dispersion in a suitable buffer
• The lipid – cell interaction can be governed by the nature and
density of charge on the liposome surface.
• Charging the lipid composition can alter the nature and charge on
the liposome.
• The interaction of neutrally charged liposome with the cell is
almost negligible.
• But positively charged liposomes are cleared more rapidly after
systemic administration. Unlike negatively charged liposomes,
cationic liposomes deliver the contents to cells by fusion with cell
membrane

15. High electrostatic surface charge on
the liposome may provide useful
results in promoting lipid – cell
interaction.
Negatively charged density influences
the extent of lipid – cell interactions
and increase the intracellular uptake of
liposomes by target cells.
Lack of charge in the SUV liposomes
can lead to their aggregation and
thereby reducing the stability of the
liposome

16. Aqueous two phase partitioning
system
Two aqueous polymer
solution i.e, dextran and
PEG
Mixed at certain
concentration, form two
immiscible phase
Upper phase (more
hydrophobic layer) -
PEG
Lower layer (less
hydrophobic layer) -
Dextran
Partitioning the cells
between two immiscible
aqueous solution of
polymer
Partitioning on the basis
of difference in surface
properties
Surface potential of the
membrane (liposome) was
calculated from the partition
coefficient of methylene blue
dye between membrane and
bulk phase

17. 3. Inter Excipient And Drug
Excipient Compatibility
• Drug excipient reaction might produce new impurity or
degradant of undesired properties.
• Analytical methodology ( TLC, HPLC, GC, LC-MS, GC-MS,
UV-VIS, FTIR, NMR) developed for quantitative and
qualitative detection of Impurity species in the formulation.
Employed for the characterization of the chemical instability
of the excipient as well as API-excipient mixture.
• Drug excipient interactions leads to chemical alteration and
physical characteristics such as color, liquefaction etc., were
observed by FT-IR

19. 4. Encapsulation efficiency
• The Encapsulation efficiency describes the % of the aqueous phase and
hence the % of water soluble drug that becomes ultimately entrapped
during preparation of liposomes and is usually expressed as %
entrapment /mg of lipid
drug entrapped in liposomes
% encapsulation = × 100
Total drug added

20. Encapsulation
efficiency
Assessed by
Minicolumn
centrifugatio
n method
Ultracentrifu
gation
Protamine
aggregation
method

21. Minicolumn centrifugation
method
• Used for both purification and separation of liposomes on
small scale.

23. Sephadex G-50
swelled in an
appropriate buffer
Packed into 1 ml
syringe plugged with
whatman filter
This is called as
Minicolumn
Minicolumn is
centrifuged to remove
excess buffer
Undiluted liposome
sample added to
Minicolumn.
centrifugation
Buffer is added to
rinse the Minicolumn
The elute obtained
after centrifugation of
Minicolumn should
not contain any drug.
Second rinsing with
buffer
Used to collect free
drug

24. Protamine aggregation method
• This method used for neutral and negatively charged
liposomes

26. Spinning motor at very high speeds,
capable of generating acceleration as
high as 100000 G ( 9800 km/s2)
At this force of gravity, the liposome
are separated as pellet from the
unentrapped drug which is present in
the supernatant.
The tube are filled from top to bottom
with increasing concentration of dense
substance. common method for
separation of unentrapped drug

27. Encapsulation efficiency
• For estimation of EE, the un-encapsulated or free drug is
separated from the microparticles by multiple washing.
• The microparticle free from unentrapped drug are lysed and
drug content is determined using suitable analytical technique.
• Higher EE of drug is required for an ideal microparticle
formulation and it depend on multiple factors such as method
of preparation, quality, quantity of polymer, process variables
viz., temperature, pH, stirring speed and time etc.

29. ordered gel
phase of lipid
hydrocarbon
chains are fully
extended and
closely packed
disordered
liquid
crystalline
phase
hydrocarbon
chains are
randomly oriented
and fluid.

30. DSC (
Differential
scanning
calorimetry)
31P NMR
Fourier
Transform –
Raman
spectroscopy
Light
microscopy
Widely used instrument

31. • Lipid bilayer made up of lecithin with 14
or more carbon atom per acyl chain as a
stable bilayer, which are practically
permeable to ion.
• In vicinity of temperature, the released of
trapped ion from liposome is enhanced.
• The extent of increase in release strongly
depend on length of paraffin chain
Composition
of bilayer
• Non polar drug perturbs the phase
transition behaviour of
phosphatidylcholine, polar drugs have no
such effect
• DSC profile of non polar drug present in
PC is markedly altered, broadening of
major peak and appearance of second
peak at lower temperature.
Entrapment
of solute in
bilayer
Phase transition temperature determined

32. In vivo behaviour
• Permeability
changes at or near
phase transition
temperature.
• Liposomal
membrane with
their lipid in gel
state are
permeable to
water.
• The rate of water
permeation
changes on
passing the
transition
temperature.
Simple topologies
• All liposome
change size or
shape at the
transition and
those with simple
topologies such as
sphere and
cylinders can
readily measured.
Chain length and
unsaturation
• 31P NMR spectra
of unsonicated
liposome
consisting of PCs
of varying chain
length and
unsaturation have
been investigated.
• In liquid
crystalline state
the 31P NMR
liposome spectra
are similar for
both saturated and
unsaturated

33. 3. Thermal analysis
• Thermal analysis of microparticles is done in order to
investigate the physical status of the drug in the encapsulated
form and also characteristics of polymer after being
formulated in to the microparticles.
• These studies done immediately after formulation of
microparticles as well as over its life time.
• DSC used to check solid state chemistry and physicochemical
properties of the drug such as melting point

34. Thermal analysis
• Any alteration or disappearance in the melting point peak
may indicate change of crystal structure of the API.
• Thermal Analysis of polymer viz., glass transition
temperature can be assessed by DSC.
• Thermogravimetric Analysis may used to investigate phase
transition, desolvation, decomposition sublimation,
absorption, adsorption and desorption behavior of the
polymeric microparticle.

36. 6. Stability Studies
• Need to gather information on the quality of drug
product over time under the influence of variety of
environmental factors such as temperature, humidity
and light.`
• Microparticle are subjected to the different accelerated
and conventional stability testing procedures.
• Intrinsic stability of drug molecule is evaluated in the
stress testing and it suggests the degradation pathways,
degradation product as well as the stability indicating
capability of analytical procedures employed.

37. • Random selection of 3 batches.
• Storage condition should be
1. 25 ± 2 °C / 60% ± 5% RH for 12 months in long term testing.
2. 40 ± 2 °C / 75% ± 5% RH for 6 months in accelerated testing
condition.
• Any significant change observed in the c stability testing of
microparticles are further subjected to an intermediate condition e.g.,
30 ± 2 °C / 60% ± 5% RH.
• significant change at the accelerated condition as per ICH guidelines
is defined as :
a) A 5% potency loss from initial assay value of a batch.
b) Any specified degradant exceeding its specification limit
c) A product exceeding its pH limit.
d) Dissolution exceeding its specification limits
e) Failure to meet specifications for appearance and physical
properties e.g., color, phase separation, re-suspendibility, delivery
per actuation, caking, hardness etc.,

38. • The long term testing will be performed for sufficient
time beyond 12 months to cover shelf life at appropriate
test period.
• The testing frequency should be adequate to establish the
stability characteristics of the drug product and is
normally at every three months over the first year, every
six months over the second year and then annually.

39. Stability
• Stability in vitro mainly covers the chemical stability of
the constitutive lipid under the various accelerated or long
term storage condition.
• Chemically phospholipid are susceptible to hydrolysis.
• Phospholipid containing unsaturated fatty acid are
vulnerable to oxidative degradation and peroxidation.

40. LIPOSOME

41. 1. Lamellarity
Assessed by method To determine
Lamellarity 1. Freeze Fracture
Electron Microscopy
2. 31P Nuclear Magnetic
Resonance Analysis
The number of bilayer
present in the liposome

42. Electron Microscopic Technique
• Negative stain electron microscopy (EM) has been used to
characterized the morphology, size and shape of the lipid
particles present in phospholipid dispersion.
• EM also gives gross estimate of lamellarity since stain
penetrates interbilayer spaces and allows lamellae to be
resolved.

43. 31P Nuclear Magnetic Resonance
Analysis
• Liposome Lamellarity can be determined by 31P NMR (most accurate and straightforward
technique ).
• In this technique, the addition of metal salt (viz. Mn2+, Pr+3, Eu+2) quenches the 31P NMR
signal from phospholipids on the exterior face of the liposomes.
• 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
• The percentage of relative loss of signal from the peak area before and after addition of
Mn2. The number of bilayers is calculated by:

44. 31P NMR spectra of a) unbound
b) one side Mn+2 bound
c) Two side Mn+2 bound

45. • Thus 50% reduction in NMR signal intensity indicates a
unilamellar whereas subsequent reduction indicates a
multilamellar vesicular preparation.

46. 2. Trapped volume
• The internal or trapped volume is the aqueous entrapped
volume per unit quantity of lipid and expressed as
µl/µmol or µl/mg of total lipid.
• This can vary from 0.5 µl/µmol for some MLV and
SUVs to as much as 30 µl/µmol for certain LUVs.
• Material used to determine internal volume
1. radioactive marker
2. fluorescent marker

47. • Best way to measure internal volume is to measure quantity of
water directly and this may done by replacing the external
medium ( water) with a spectroscopically inert fluid (
deuterium oxide) and then measuring the water signal using
NMR
• The permeability of the liposomal membrane to water is such
that H2O and D2O equilibrate very rapidly throughout the
whole volume of the medium.
• The NMR scan of this medium can used to access peak height,
which can be related to concentration by comparison with
standards containing known amount of H2O in D2O.

48. Non permeable
radioactive
solute such as
[22Na] and
[14C] inulin
lipid
Aqueous
medium
Removing
external
radioactivity by
Centrifugation,
dialysis or gel
filtration
Which
determined
proportion of
solute trapped
Subsequently
residual
activity per
lipid is
determined

49. PREFORMULATION
OF MICROSPHERE

50. Mercury
intrusion
porosimetry
Confocal laser
scanning
microscopy
Atomic force
microscope
• quantitative
analysis of internal
structure.
• high resolution
optical picture with
depth discernment.
• study of surface
characteristics and the
internal core structure of
microparticles

51. 2. Flow properties
• Flow characteristics of microparticles can be determined by
angle of repose, Hausners ratio, carrs index.
• Because of spherical shape, microsphere are expected to have
good flow, but in some instances, the adhesive nature of the
polymers used in the formulation may cause in poor flow as
well as adhesion.
• Flow properties of microsphere increase by adequate drying,
use of non-adhesive and non-hygroscopic material and use of
glidants / lubricants / anti-adherent.

52. 3. In vitro drug release ( Dissolution )
• Determine the bioavailability of drug.
• Dissolution apparatus :- Basket ( USP type-1 dissolution
apparatus ) or incorporated into dialysis tube and kept inside
dissolution medium.
• Dissolution time will depend on type of formulation by
maintaining sink condition.
• Aliquots of required volume are collected at pre specified time
period and analyzed by suitable analytical method.

53. In vitro drug release ( Dissolution )
• Alternatively microsphere loaded with API are suspended in
dissolution medium in screw capped tubes and placed in
orbital shaker maintained at 37±1°C
• Tubes are removed at pre-decided time interval and
subjected to centrifugation.
• Then supernatant is removed and remaining microsphere are
resuspended in fresh dissolution medium and placed back in
shaker.
• Supernatant containing drug is analyzed by suitable
analytical method.

54. 4. Ex vivo drug permeation
• Microparticle are intended for transdermal or topical application
• The skin of animal ( rat, goat or pork ) is fixed at junction of donor
and the receptor compartment of Franz diffusion cell.
• The study is performed with Franz diffusion cell, Keshri chain
diffusion apparatus or flow through diffusion cells.
• Microparticle put on the skin surface.
• Put in require dissolution medium and temperature is maintained at
37±1°C
• The dissolution medium is stirred with the help of magnetic stirrer.
• The API permeated in the dissolution medium through the biological
membrane is collected through the sampling probe and subjected to
evaluation by suitable analytical technique.

55. REFERNCE
• Jain S., Jain N. ”liposome as drug carriers”
controlled and novel drug delivery, CBS publishers
and distributors
• Int. J. Drug Dev. & Res.| October - December 2013 |
Vol. 5 | Issue 4 | ISSN 0975-9344 |
• https://books.google.co.in/books?isbn=0849340128
Yechezkel Barenholz, Danilo D. Lasic - 1996 –
• http://scinel.blogspot.in/2016/01/31p-liposomal-
lamellarity.html