Journal of Liposome Research, 18:159–173, 2008

Copyright © Informa UK, Ltd.

ISSN: 0898-2104 print / 1532-2394 online
DOI: 10.1080/08982100802310261

1532-2394
0898-2104
LLPR
Journal of Liposome Research
Research, Vol. 1, No. 1, July 2008: pp. 1–28

Differential Scanning Calorimetry (DSC): A Tool

Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

to Study the Thermal Behavior of Lipid Bilayers

and Liposomal Stability

COSTAS DEMETZOS
DSC for Lipid Bilayer Thermal Activity
Demetzos

Department of Pharmaceutical Technology, School of Pharmacy, University of

Athens, Greece

Thermodynamical techniques are applied for determining the thermal stress of medici-
nal compounds of the excipients as well as their interactions during the formulation
process.
For personal use only.

The physicochemical properties and the stability of the medicinal products could be
measured as a function of temperature or time using thermal analysis.
Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for
determining the purity, the polymorphic forms and the melting point of a sample in the
Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior
of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring ther-
modynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal
suspension under given storage conditions.

Keywords Thermal analysis, Differential Scanning Calorimetry (DSC), lipid bilay-

ers, stability, liposomes

Introduction
Calorimetry is defined as techniques that are used to measure heat effects that occur in
physical, chemical, or biological processes. It is derived from the Latin word “calor” and
the Greek “meter,” and it is recognized as the most powerful segment of physicochemistry.
Thermodynamic stability of molecules and thermophysical measurements are areas for
application of this technique. The changes of enthalpies of liquids or mixture of liquids, of
enthalpy of solution, enthalpy of mixing and enthalpy of transitions of components-
formed structures like bilayers, are extremely important applications of calorimetry. By
determining the changes of enthalpy in relation to pressure and to temperature, the data
could be of interest to researchers in this field (Zielenkiewicz, 2005). Thermal analysis is a
commonly used term to describe analytical techniques of calorimetry and is referred to as
a technique that measures physicochemical properties of materials as a function of

Address correspondence to Dr. Costas Demetzos, Department of Pharmaceutical Technology,

School of Pharmacy, University of Athens, Greece; Fax: +302107274027; Tel.: +302107274596;
E-mail: demetzos@pharm.uoa.gr

159
 160 Demetzos

temperature or time. Frequently, the material is subjected to a temperature program iso-

thermally or by using different temperature segments, usually at a constant pressure. Ther-
moanalytical techniques are suitable for measuring the thermal stress of medicinal
compounds of the excipients and their interactions during the formulation process.
The stability of medicinal products, especially those referred as innovative therapeu-
tic nanoparticles such as liposomes incorporating biological active agents, are of great
importance, and thermal analysis can provide detailed information not only on their stabil-
ity over time but also during the development process.
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

Pharmaceutical Thermal Analysis (PTA)

It is well known that the drug development process as well as the drug delivery to the
target tissues is of great importance in the pharmaceutical industry and in academia.
Pharmaceutical Thermal Analysis (PTA) is quite close to these areas, and the formulation
process of drugs is crucial because the physical stability of the active molecules and the excip-
ients, as well as the compatibility between them, is vital to ensure the physical stability of the
final pharmaceutical product. Thermal analysis is a method of choice to investigate the
phase transitions and the changes in heat capacity of the materials used and to play a piv-
otal role in the drug development process and in the formulation of drug delivery systems.
There are several thermoanalytical techniques, the most common of which are the fol-
lowing: Differential Thermal Analysis (DTA), Thermogravimetric Analysis (TGA),
Thermomechanical Analysis (TMA), Thermo-Optical Analysis (TOA), Differential Scanning
For personal use only.

Calorimetry (DSC), Temperature Modulated Differential Scanning Calorimetry (TMDSC),

Solution Calorimetry (Sol Cal), and microthermal Analysis (mTA) which could be used in the
development process of pharmaceutical products. These techniques provide information con-
cerning important effects or properties like polymorphism, stability, interactions, and purity, as
well as properties of packaging materials. Among them, DSC is the most applicable technique
in the research and drug development process. A great number of applications are referred,
concerning the study of effects of materials, such as melting point and melting range, heat of
fusion, purity, polymorphism, evaporation, desorption, vaporization, glass transition, interac-
tion, compatability, oxidation stability, kinetics decomposition, etc.
Because of the advantages of DSC, which seems to be a fast, easy, and relatively low-
cost technique for laboratory usage, we describe below this technique, taking into account
that studies concerning thermodynamical parameters during transitions and changes in the
heat capacity of materials like phospholipids are important for designing lipidic drug
carriers such as liposomes.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry is the most frequent technique that is used for determining
the thermal effects of a variety of materials, including biologically relevant systems that
are characterized by an enthalpy change by the temperature range.
Differential scanning calorimetry involves in applications such as 1. thermal characteriza-
tion of complex processes such as the denaturation of proteins; 2. glass transition of polymers;
and 3. determination of the effect of hydration, pH, solvent, and kind of composition, on the
phase transition, and of the changes of enthalpies of model lipid membranes and phospholipid
bilayers, which are components for developing lipidic drug carriers like liposomes.
Differential scanning calorimetry provides accurate information, quickly and easily, about
both physical and energetic properties of a material. Differential scanning calorimetry
 DSC for Lipid Bilayer Thermal Activity 161

scans temperature and measures the difference between the heat flows to a sample and a
reference pan that is under the same temperature program, at atmospheric pressure, and
measures the heat capacity of a material.
Differential scanning calorimetry devises that are updated and more sophisticated
modulated temperature programs, robotic systems, and a combination of DSC with spec-
troscopic instruments are commercially available.
Differential scanning calorimetry measures the heat flow going into or being released
by a material. From that, the heat capacity at constant pressure –(Cp) can be calculated.
Heat capacity units are cal °C−1 or J °C−1. It measures the amount of heat input (q)
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

required to raise the temperature of a specimen by one degree Celsius while at constant
pressure. Heat capacity is usually normalized by dividing the specimen heat capacity by
the number of grams, to get the heat required to raise one gram of specimen by one degree
Celsius. This corresponds then to the specific heat capacity Cp. If desired, the heat capac-
ity can be normalized by the number of moles. Heat capacity is defined by

C p = ( ¶q / ¶T)p ,

where T is the temperature and q is the heat input.

If the temperature changes from T0 to T1, the enthalpy of the reaction (ΔΗ) is

T1
DH = ∫ Cp dT .
For personal use only.

T0

Usually, ΔΤ is small, and Cp is independent of temperature between T0 and T1. The

integral thus reduces to

D H = C p (T1 - T0 ) = C p DT.

DSC Studies on Thermal Transitions of Phospholipid Formed Bilayers

Studies of Single Phospholipids. Phospholipids, especially glycerophospholipids, are a lipid
group consisting of a wide variety of different molecules. The presence in the phospholipid
molecules of specific hydrophilic and hydrophobic regions gives to the structure amphiphilc
properties that influence greatly their physicochemical behavior. They belong to the double-
chain amphiphiles and are the building blocks of all biological membranes. The sn-1 and sn-2
positions of glycerol residue is esterified with fatty acids (sn: stereospecific number according
to the glycerine nomenclature) of varying degrees of unsaturation and numbers of carbon
atoms. The sn-3 position of glycerol is etherified to the phosphate. The phosphate is also ester-
ified to an alcohol that determines the phospholipid (Hatziantoniou and Demetzos, 2008).
Phospholipid formed bilayer structure or lamella (L) in aqueous media is selected as a
model quite close to biological membranes and is used to study the thermotropic behav-
iour of such systems. They belong to the lyotropic liquid crystalline state, exhibiting
extensive polymorphism. Their polymorphism behaviour is related to the transition from
the gel to liquid crystalline phase. This transition occurs in biological membranes pro-
duces their functionality.
Changes in aqueous media such as in temperature or pH, may be the change interac-
tions between molecules that alters their structure. These alterations are significant, con-
cerning their fluidity and permeability of membranes and occur in very narrow ranges of
 162 Demetzos

temperature affecting their physical stability, which is of interest to rationally design

lipidic drug carriers.
The main transition of phospholipid bilayers when thermal energy is added occurs between
the gel (ordered) to fluid (disordered) lamella (L) state affecting the van der Waals interactions
between hydrocarbon chains, increasing their mobility. The amount of energy, which is pro-
vided, is associated with the phase transition of phospholipids and with the conformation prop-
erties of the phosholipids, which affect the stability of the system. The changes of enthalpy are
associated with the thermal energy added, and the phase transitions of phospholipids formed
bilayers from the gel to liquid crystalline state are related to the decrease of the hydrophobic van
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

der Waals interactions between lipid acyl chains. The enthalpy of this transition is calculated
using calorimetric techniques and measured as enthalpy per mol of phospholipid.
Differential scanning calorimetry, according to the McElhaney (McElhaney, 1982) has
been used to study the thermotropic behaviour of phospholipid bilayers, which act as a model
of biological membranes. The reason for studying the transition changes of the phospholipid
bilayers by measuring the changes of enthalpy (DHcal) during the transition process is an
extremely important issue for investigating not only biological phenomena but to rationally
design lipid drug carriers based on their thermal behaviour. These enthalpy changes corre-
spond to changes of the heat capacity (Cpmax) of phospholipids during the transition process
from gel to the liquid crystalline phase (Scheme 1) (Hatziantoniou et al., 2005).
The cooperativity of the molecules during the transition process is crucial concerning
the melting behaviour of the material. However, the phospholipid hydrocarbon acyl chain
of Dipalmitoylphosphatidylcholine (DPPC) is thermodynamically transit from an all trans
For personal use only.

conformation in the gel state to a disordered state that contains a number of gauche con-
formations resulting in a transition to a liquid crystalline state. The van der Waals hydro-
phobic interactions of acyl chains of DPPC is reduced, due to the enthothermic behaviour
of the material, which increases the intra- and intermolecular lipid acyl chain motions
(McElhaney, 1982). The gel to liquid crystalline transition of pure phospholipids appear
as a sharp peak, due to favourable van der Waals interactions of lipid acyl chain resulting
in high cooperativeness of the molecules.

T < Tp Tp < T < Tm T > Tm

gel rippled phase liquid crystalline

Scheme 1. Schematic presentation of the alignment of acyl chains in a saturated phospholipid such
as DPPC to form a double layer of molecules: gel in quasi-crystalline state (T< Tp ), rippled phase
(Tp < T < Tm) and in the liquid crystalline state (T>Tm).
 DSC for Lipid Bilayer Thermal Activity 163
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13
For personal use only.

Scheme 2. Characteristic thermodynamic parameters of a DSC curve.

Differential scanning calorimetry has several characteristic temperatures, which

may be used to describe it. The onset temperature (Tonset) is the temperature that is
constructed by the intersection of the baseline tangent with the tangent of the leading
edge of the endotherm or exotherm of a transition. The peak temperature (Tm) is the
temperature represented by the apex of the peak. Where the peak transition returns to
the baseline may be referred to as the recovery or endset temperature (Tendset). The
temperature that corresponds to the half of the enthalpy change during the transition is
T½ (Ford and Timmins, 1989), while ΔT½ is the width of the transition at half peak
height (Scheme 2). The phenomena that take place during heating process are the fol-
lowing: The glass transition or Tg is a transition that occurs in amorphous or semic-
rystalline materials. Around Tg, Cp undergoes a quasi-discontinuous change from a
lower value to a higher value (it does not change sharply but happens over a broad
range in temperature, depending on the molecular structure of the material). If an
amorphous material tends to crystallize or a semicrystalline material does not crystal-
lize to the limit of its ability when cooled, it is possible to see crystallization during
the heating. Crystallization shows up as an exothermic peak, and the crystallization
temperature Tc is always between Tg and the melting temperature Tm. Crystallization
 164 Demetzos

will only occur in samples that can crystallize and in samples that are not already
crystallized as far as they can be crystallized. The highest temperature peak in a DSC
thermogram is often the melting transition. Melting is a first order endothermic peak,
which means that it requires heat. For small molecules, the peak is very sharp, while
for larger molecules, such as polymers or lipid bilayers, the melting transition is
broad. The area under a melting transition curve is the total amount of heat absorbed
during the melting process. Further peaks can be attributed to loss of solvent
molecules (evaporation, mostly endothermic) and to chemical reactions (endo- or
exothermic), among them decomposition.
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

The changes of the enthalpy as a function of phase transitions when an additive, i.e.,
a drug, is incorporated into lipid bilayers, have been studied. The modifications on the
ordered lipidic structure of phospholipid bilayers, probably due to the structural conforma-
tion of the additive, can strongly interact either with the lipophilic part of a phospholipid,
thus resulting in greater thermotropic changes in Tm, or with its polar group, resulting in an
abolition of transition, i.e., pre-transition peak, at 36,14°C when DPPC is used as the
phospholipid (Gardikis et al., 2006).

Liposomes: A Brief Overview

The structural units of liposomes are amphiphile molecules, mainly phospholipids. The
most abundant lipids in liposomes are Phosphatidylcholines (PCs), in which a glycerol
bridge links a hydrophobic part with a hydrophilic polar head group. In contact with
For personal use only.

water, PCs form bilayers, due to their molecular tubular shape, contrary to the conical
shape of the detergents, which form micelles. Other polar lipids such as sphingolipids and
amphiphiles can be introduced in the liposomal bilayers. Cholesterol or other sterols iso-
lated from natural sources (i.e., plants β-sitosterol), as well as several lipid conjugated
polymers, may be found in the liposome bilayers.
Liposomes are considered as substantial models for the study of biological mem-
branes. They have many physicochemical properties similar to cell membranes, such as
membrane permeability, osmotic activity, interaction with various solutes, surface charac-
teristics and chemical composition. The fluidity of their membranes and their self-closed
structure are essential parameters for the study of biological membrane function
(Hatziantoniou and Demetzos, 2008).

Applications of Liposomal Technology

Liposomes belong to the class of lyotropic liquid crystals. Obviously, their physico-
chemical characteristics and their biocompatibility with cell membranes have given
them several advantages: they can dissolve, protect and deliver hydrophobic and
hydrophilic molecules. Several applications of liposomes are referred to a broad range
of sciences and disciplines, such as biochemistry, chemistry, biology, medicine, bio-
physics, physics and mathematics. Liposomes are ideal formulations as drug delivery
systems, while they can be applied in medical diagnosis, in cosmetic products, etc. It
is important to note that the implication of liposomes or other colloidal systems such
as polymeric complexes, nanoparticles, micelles, etc. in drug delivery make it possi-
ble to alter the pharmacokinetic characteristics and biodistribution profile of the
incorporated bioactive molecule. Parameters that influence the behaviour of lipo-
somes in biological environments are the membrane fluidity, surface charge, degree
of hydration, liposome size and the method of preparation.
 DSC for Lipid Bilayer Thermal Activity 165

Liposomes have several advantages, such as biocompatibility and biodegradability,

which are essential for their therapeutic use. The encapsulation of drugs into liposomes
depends on several parameters, including lipid composition, surface charge, etc. The
recent developments in liposome technology permit the introduction of new strategies for
controlling the physicochemical properties of liposomes, their stability and pharmacologi-
cal response after administration.

Principles on the Stability of Liposomes

Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

Physicochemical Properties of Liposomes

The characterization of liposomes is an important task, and many analytical techniques are
employed. Thermodynamic, mechanical, chemical, microscopic, spectroscopic, and chro-
matographic techniques have being used worldwide in order to test the physical and chem-
ical characteristics of liposomal formulation, such as lamellerity, encapsulation efficiency
and their stability over time. The physicochemical parameters of liposomes, which are
essential for their use in pharmacy and medicine, are in relation to their stability, biodistri-
bution and cellular uptake. As far as the term “stability” is concerned, in the case of lipo-
somes, it is important to notice that, in the manufacturing process, the physical, chemical,
and biological status of liposomes must be controlled.
Chemical status is controlled by chromatographic techniques. Thin Layer Chromatog-
raphy (TLC) is a simple and rapid method to monitor the purity of phospholipids. High
For personal use only.

Pressure Liquid Chromatography (HPLC) can be used for the qualitative and quantitative
testing of raw materials. High Performance Thin Layer Chromatography/Flame Ionization
Detector (HPTL/FID) is a method that is extensively used in our laboratory in order to
identify lipids qualitatively and quantitatively (Hatziantoniou and Demetzos, 2006).
Physical status can be controlled by measuring the size distribution of liposomes. This has
usually been done using dynamic light scattering methodology. Zeta potential is another
important factor that gives an indication concerning the surface charge of liposomes in their
aqueous medium. Both methods are used for the monitoring of liposome stability over time.
Optical stability can be monitored by using electron microscopy, after negative stain-
ing for suspensions or after the freeze-fracturing process. This technique provides indica-
tions on the size, shape and number of lamellae of liposomes.
Biological stability is concerned with the microbiological charge of liposomes,
including controls for viruses bacteria and yeasts on raw materials and on the final liposo-
mal formulation. The stability of liposomes is increased by coating liposomes with inert
hydrophilic polymers such as Polyethylene Glycol (PEG). The polymer-coated liposomes
are called Sterically Stabilized Liposomes (SSL), also referred to as Stealth liposomes
(Stealth liposomes are a trademark of Sequus Pharmaceuticals). These liposomes reduce
interactions with blood proteins, thus increasing their half-life in blood.

Effect of Drugs on Liposomal Stability Measured by DSC

Liposomes belong to the colloidal lyotropic systems that are physicochemically unstable.
Chemical and physical changes can be revealed during the preparation process or upon
storage. The chemical changes are 1. the oxidation of lipid acyl chains of phospholipids
via a free radical chain mechanism; the most susceptible are the lipids containing double
bonds; and 2. the hydrolysis of phospholipids to free fatty acids and to 2-acyl- and 1-acyl-lyso
(phospholipids) (Zuidam et al., 2003). Physical changes could occur due to the elimination
 166 Demetzos

or the absence of an electric charge on the surface of liposomal particles. The charge pro-
duces a repulsion that prevents aggregation phenomena and bilayer fusion. Parameters
like composition of the liposomal bilayer, storage conditions, and formulation process are
important factors affecting the physical stability of liposomes.
The freeze-drying technique is a way to improve the storage stability of aqueous
pharmaceutical formulations. Liposomal stability upon storage is crucial, and application
of the freeze-drying process is based on two requirements: 1. water removal from the
liposomal suspension prevents the hydrolysis of phospholipids; and 2. the chemical
degradation process or other physical changes are retarded due to the solid state of the
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

freeze-dried liposomal formula (Zuidam et al., 2003).

However, the freeze-drying method has been used to improve liposomal storage
stability, and carbohydrates are used as lyoprotectants to protect liposomes from hydra-
tion stress. Due to the amorphous state of the dried system, the glass transition tempera-
ture (Tg) is a crucial parameter, considering the long-term stability of the liposomal
formulation. Differential scanning calorimetry is the method of choice, and changes in
the heat capacity can offer measurements concerning the temperature of the glass transi-
tion (Tg). The temperature range is a crucial point, and it has possible thermal effects
overlapping in the temperature in which the experiment is done. An extension of the
DSC method has been developed (Wunderlich et al., 1994; Gill et al., 1993) in order to
detect Tg of pharmaceutical systems when different thermal events occur in the same
range of temperature. This development of the so-called Modulated Temperature DSC
(MTDSC) (Taylor and Craig, 2003) is of importance in the cases where the physical
For personal use only.

stability of pharmaceutical systems is a concern. By this modulated DSC technique,

the heat capacity can be distinguished from the total heat flow, and this can offer an
advantage for determining the Tg.
E.C. van Winden et al., in a study that deals with the determination of Tg, present
results concerning the detection of Tg of lyoprotected freeze-dried liposomal cake and the
potential of MTDSC to separate Tg from other thermal events occuring in the same tem-
perature range (van Winden et al., 1998). The thermodynamic data obtained using
MTDSC could be used to rationalize the storage conditions of a lyoprotected freeze-dried
liposomal system and could lead to a better understanding its self-life stability.
The anticancer drug vinblastine belongs to the Vinca alkaloids and is a mitotic inhibitor,
which is used clinically alone or in combination with other chemotherapeuticals in the
treatment of leukemia, Hodgkin’s disease, breast cancer, and small-cell lung cancer. The
biopharmaceutical properties of an anticancer drug, including pharmacokinetics and phar-
macodynamics, affect its bioavailability and consequently its effectiveness. An acceptable
pharmacokinetic profile could be achieved by using effective drug carriers like liposomes.
Τhe size, the surface properties and the kind of interactions of the drug with the lipid
bilayers play a key role in altered pharmacokinetics and affect the bioavailability of the
drug. In order to optimize the pharmacokinetical profile and consequently the bioavail-
ability of an anticancer drug, the formulation process is of great interest.
Studies concerning the interactions of vinblastine with DPPC model lipid bilayers
caused alteration in the conformational properties of phospholipids and affected their
molecular order due to the domain formation and, consequently, changes on the thermo-
dynamic properties of the system. This thermodynamic alteration is a concern in studies
on the transitions from the gel to liquid crystalline state, which are of interest because they
affect the fluidity within the lipid bilayers. Recently published results by Maswadeh et al.
(Maswadeh et al., 2002) on the interactions of vinblastine with DPPC lipid bilayers show
an increase in the total enthalpy change of the main lipid phase transition and, depending
 DSC for Lipid Bilayer Thermal Activity 167

on the experimental conditions used, an increase of the transition temperature. These ther-
modynamic parameters (i.e., DH and Tm) are crucial because they affect not only the phar-
macokinetics of the drug but also the stability of the liposomal suspension under given
storage conditions. The stability of liposomes is based on the kind of phospholipids, and
the results obtained using DSC and DPPC as model membranes encapsulating vinblastine
(Maswadeh et al., 2002) help to rationally select the type of phospholipds concerning their
head polar group as well as their type of acyl chain.
Calorimetric studies based on this concept have been undertaken on the bile Ursode-
oxycholic Acid (UDCA), of polyphenols, of paclitaxel, and several amphoteric molecules.
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

The thermodynamical results shown that these molecules caused interdigitation, which
processes a stable gel phase, associated with an increase of the molar enthalpy of the main
lipid phase transition. The phenomenon of inderdigitation is of interest because, in the
interdigitated structure, the acyl chains of phospholipids insert into the opposite phospho-
lipid layer. This phenomenon causes the abolition of the fluidity gradient of the acyl
chains due to the insertion of one lipid layer into the other limits the freedom of their
movement. The molar ratio of the incorporated drug to the phospholipid is crucial for the
formation of the interdigitated structure. Molecular modeling applied for the drugs vin-
blastine and UDCA indicated that both molecules prefer to locate near the interfacial
region of the membrane, enhancing the van der Waals interactions of the hydrophobic
groups of the phospholipid (Maswadeh et al., 2002) The increase of DH in the case of vin-
blastine encapsulated into DPPC model membranes is associated with an induced inter-
digitated gel phase and gives rise to more rigid bilayers.
For personal use only.

Such thermodynamical alterations should be taken into account and correlate to the
interactions of the molecule with the phosholipid and, consequently, with the physical sta-
bility of the liposomal formulation.
It is of interest that molecules very close in their structures, such as vinblastine and its
hemisynthetic compound vinorelbine, seem to differently interact with DPPC lipid bilay-
ers, causing different interdigitation abilities. The higher inderdigitation effect of vinblas-
tine, contrary to that of vinorelbine due to the more lipophilic profile of the latter, favors
vinblastine to accommodate in the mesophase of adjacent lipid molecules of membrane
bilayer possess greater inderdigitation than that caused by vinorelbine. We have to keep in
mind that, despite the chemical similarities between the two molecules, it is possible that
the physical stability of their liposomal preparation could be different. Results from the
DSC experiments based on thermodynamic parameters could be useful to select appropri-
ate phospholipids for their liposomal suspensions (Koukoulitsa et al., 2006).
It has been reported (Kyrikou et al., 2004) that lipids with shorter acyl chains, such as
DMPC, or longer acyl chains, like DSPC, cannot produce the interdigitation phenomenon,
although molecules such as vinblastine or others listed above cause this phenomenon in
DPPC lipid bilayers. A class of bioactive candidates for anticancer drugs is labdanes
(Scheme 3), belonging to the group of diterpenes. They are isolated from several plant
families, and their significant cytotoxic effects have been observed against several cancer
cell lines in vitro (Demetzos and Dimas, 2001; Dimas et al., 1998 2006). Although
labdanes have revealed significant cytotoxic activity, in vivo experiments with labdanes
are hindered by their water insolubility. To overcome this problem, suitable formulations
based on liposomal technology are a choice for their in vivo administration.
Differential scanning calorimetry has been considered as a tool in the exploration of
thermodynamic parameters of natural bioactive compounds belonging to taxane and abie-
tane types and phenolic types of diterpenes, which are associated with the interactions of
these compounds with lipid bilayers. The thermal effects obtained using DPPC as model
 168 Demetzos

OH OH

14 14 14
13 13 13
HO

8 OH OH
9 9 9
8 8

1 2 sclareol
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

Scheme 3. Structures of labd-7,13-dien-15-ol, (1) labd-13-ene-8α,15-diol; and (2) and labd-14-ene-

8,13-diol (sclareol).

lipid membranes are related to their ability to influence lipid bilayers and could be correlated
with the stability of liposomes in which they are incorporated (Kyrikou et al., 2005, Matsingou
et al., 2005). Long-term physical stability of liposomes is a requirement and may be affected by
the interactions of the incorporated molecule that is capable of modifying the lipid phase transi-
tions of liposomal membranes and consequently affects their physical stability.
Investigations on the thermodynamic effects of drugs on structural and physicochem-
ical characteristics of lipid bilayers revealed valuable justification of the liposomal com-
position to be used. However, information from DSC concerning the thermal behaviour of
For personal use only.

lipid membranes after incorporating natural compounds such as labdanes is essential for
designing stable liposomal formulations. Labdanes are considered small molecules, and
they can induce alterations of phospholipid packing in model lipid bilayers inducing an
interdigitated state of the monolayers. The interdigitated state of lipid bilayers is essential
concerning the stability of liposomes incorporating such compounds causing hydrophobic
interactions in the surface area accelerating membrane adhesion and fusion or aggregation
of liposomes (Matsingou et al., 2007b). Labdanes were incorporated into DPPC liposomes
in order to get direct comparison of their physical stability with the thermal effect on
DPPC lipid bilayers observed by DSC (Scheme 4) (Kyrikou et al., 2005; Matsingou et al.,
2007b). The calorimetric data show that labdanes induced thermal modifications on DPPC
lipid bilayers, which are concentration dependent. Additionally, structural characteristics
of labdanes affected the order of lipid bilayers, decreasing the cooperativity of the transi-
tions and partially destabilizing the gel phase, especially at their lower concentrations and
the possible appearance of interdigitated segments. The interdigitated phase has been con-
sidered as the lowest energy phase since orderly acyl chain packing requires a minimum
of gauche rotamers per chain. However, labdanes are assumed to preferably partition in
the gel phase, bringing about its stabilization, which could be attributed to the appearance
of an inderdigitated gel phase, which is supported by the additional small endothermic
peaks at lower temperatures.
The DSC findings are in accordance with the instability of liposomes incorporat-
ing labdanes at 4oC that exhibited profound turbidity, enhanced viscosity, and small
population of intact liposomes (Matsingou et al., 2007b). The results concerning the
stability of these liposomal preparations show that liposomes incorporating labdanes
at the lower molar ratio 9:1 were more stable. Additionally, the authors state that the
interdigitation phenomenon appeared over time. This effect should be taken into
account in the design of liposomal formulations composed of DPPC. Mainly when
small amphiphatic molecules are incorporated into liposomes.
 DSC for Lipid Bilayer Thermal Activity 169

* 30%
30%
20 % * 20%
15% * 15%
10% 10 %
*
5%
5%

2.5% 2.5%

1%
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

0.5%
0%

0%

a b

10 15 20 25 30 35 40 45 50 55 °C 5 10 15 20 25 30 35 40 45 50 55°C

30 %
For personal use only.

*** 30 % *

*** 20 %
15 %
*** 20 %
*** 10 % *

** 15 %

** 10 %
5%

2.5 % 5%

1%
2.5 %
0.5 % 1%
0.5 %
0%
0%

c d

10 15 20 25 30 35 40 45 50 55 °C 5 10 15 20 25 30 35 40 45 50 55 °C

Scheme 4. DSC heating thermograms of DPPC bilayers in the presence of increasing concentra-
tions of (a) cholesterol; (b) labd-7,13-dien-15-ol (1); (c) labd-13-ene-8α,15-diol (2); and (d). sclar-
eol. The endothermic flow values are normalized per sample size and are multiplied by a factor of 3
(*), 4 (**) or 5 (***) where indicated, in order for small endothermic peaks to be visible (Matsingou
et al., 2007b).
 170 Demetzos

We have to notice that the model liposomal membranes could contain cholesterol,
and DSC studies include results concerning the thermodynamic alterations caused by cho-
lesterol on lipid bilayers. However, the thermal effects induced by a molecule could be
altered after adding cholesterol, and the results depend on the concentration of cholesterol
(Kyrikou et al., 2005; Matsingou and Demetzos, 2007a).

The Role of Cholesterol on the Liposomal Stability

Cholesterol is a vital biomolecule and the subject of a great number of publications. It
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

plays a major role on the fluidity of membranes by regulating their functions. Based on its
crucial properties, the interaction of cholesterol with DPPC model lipid bilayers has been
studied, and the results revealed abolition of the pre-transition phase (Pβ’) of DPPC bilay-
ers and broadening of the main lipid phase transition peak (Tm), which led to the decrease
of DH. Studies concerning how cholesterol influences the phase transition between gel
and liquid crystalline phases of the lipid bilayers have already been published (Matsingou
and Demetzos, 2007a).The orientation of cholesterol in the bilayer has been considered to
reduce the head group crowding while its hydrophobic length has been calculated to
match best with a mean hydrophobic chain length of 17.5 Å, which corresponds to 17:0
phosphatidylcholines, which is the main reason for its inability to induce hydrocarbon
interdigitation (Matsingou and Demetzos, 2007b).
Cholesterol fits with its nonpolar part between alkyl chains of phospholipids, causing
disordering of the packing of the phospholipids. The thermal effects of cholesterol depend
For personal use only.

on its concentration used. Differential scanning calorimetry thermograms of increasing

concentrations of cholesterol in DPPC lipid bilayers are shown in Scheme 4-a (Matsingou
and Demetzos, 2007a). The addition of cholesterol in lipid bilayers composed of DPPC, at
concentration more than 15%, results in the slight decrease of the Tm and elimination of
the pre-transition temperature at 35oC (Scheme 4-a).
At low concentrations, cholesterol induced disorder of lipid bilayers in the gel state. At
concentrations of approximately 30%, a liquid-ordered phase was revealed, in which existed
a high degree of conformational ordering related to acyl chains (Kyrikou et al., 2004). The
presence of cholesterol in lipid bilayers incorporating vinblastine or vinorelbine interrupts
the upraise of the DH due to the interdigitation resulting in abolition of this phenomenon
(Koukoulitsa et al., 2006; Kyrikou et al., 2004) (Scheme 5). As far as labdane/ DPPC lipid
bilayer interactions are concerned, labdanes were found to strongly affect the bilayer struc-
tural organization and induce changes in the thermotropic behaviour of DPPC in a concen-
tration-dependent manner. Elimination of the Pβ’ phase, appearance of new low temperature
peaks at the expense of the main transition of DPPC in some cases and decrease of the coop-
erativity of the transition are the main changes of DPPC lipidic structure due to the labdanes’
interactions with phospholipids. Labdanes possessing functional groups that promote their
deeper penetration in the lipid acyl chains compete with cholesterol, resulting in their possi-
ble elimination from the bilayer when the concentration of cholesterol present exceeds 20%.
Cholesterol has been assumed to provide stabilization and rigidification effects on phospho-
lipid membrane bilayers, affecting their physical characteristics and physiological behav-
iour. The stabilization effects of cholesterol on lipid membranes emerge from the increase in
the cohesion of lipids and the promotion of the liquid-ordered phase.
Cholesterol is favoured for incorporation into phospholipids bilayers as DPPC due to its
easy-to-fit structure, and therefore it may induce apparently a hindrance to the intercala-
tion of additives into the hydrophobic core of the bilayer, reducing thus the incorporation
efficiency of the additives under investigation. It is a molecule responsible for altering the
 DSC for Lipid Bilayer Thermal Activity 171

12
LUV
10 LMV

8
ΔH (kcal/mol)

4
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

0
DPPC DPPC DPPC:CHOL DPPC:CHOL DPPC:CHOL DPPC:CHOL:
vinblastine (10:1) vinblastine (10:3) Vinblastine
(100:17) (100:10:17) (100:30:17)

Scheme 5. DH changes vs. cholesterol concentration for LUV and LMV liposomal preparations.

packing of the phospholipids, giving rise to overlapping sharp and broad transitions due to
lateral phase separation into cholesterol-poor and -rich domains, respectively.
Finally, cholesterol plays a key role in the degree of interaction of additives with
For personal use only.

lipid bilayers, and such effects should be taken into consideration, especially in the case
of studying the physical liposomal stability, because its addition significantly alters the
interactions of additives with phospholipid bilayers, affecting their organization and
consequently their stability (Matsingou and Demetzos, 2007a).

Regulatory Perspectives
Regulatory considerations are of importance not only in the use of lipid excipients, which
are used for preparing lipidic delivery systems such as liposomes (Chen, 2008), but also in
the drug development process. Differential scanning calorimetry is used in the earliest
stage of drug development, and the regulatory status of a new drug form depends on cali-
bration, system suitability, and linearity of response. The procedures concerning the Good
Laboratory Practises (GLP) are all applicable to DSC instruments. It is notable that the
use of DSC to determine the purity, the polymorphism, and the transition temperatures of
a sample is described in the United States Pharmacopeia (Clas et al., 1999).

Conclusion
In conclusion, DSC is considered as a thermal analysis technique, frequently used
for determining the purity, the polymorphic forms, and the melting point of a sample in
the pharmaceutical industry. Although its applicability seems to be mainly in the drug
development process, DSC is a useful tool for designing lipidic drug delivery systems like
liposomes by determining the cooperativeness of the mixed substances and revealing
justification of the liposomal composition to be used.
An important application of DSC is to study the thermodynamic changes of lipid bilayers
after incorporating a drug, and these changes could be related to the interactions of the drug
with the lipid carrier, affecting the drug bioavailability. The thermodynamic parameters
 172 Demetzos

(i.e., DH and Tm) are crucial because they affect not only the pharmacokinetics of the drug
but also the stability of the liposomal suspension under given storage conditions.
The regulatory issues concerning the drug development process have incorporated
DSC as a valuable technique for the analysis of the physical and energetic properties of
drugs and of excipients, and its use is essential under standard procedures, as is described
in the United States Pharmacopeia.

Acknowledgements
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

The author would like to thank chemist A. Georgopoulos for editing the article and for his valuable
comments and discussions.

References
Chen, M-L. (2008). Lipid excipients and delivery systems for pharmaceutical development: A regu-
latory perspective. Adv. Drug Del. Rev. 60:768–777.
Clas, S-D, Dalton, C. R., Hancock, B. C. (1999). Differential scanning calorimetry: Applications in
drug development. PSTT 2:311–320.
Demetzos, C., Dimas, K. (2001). Labdane type diterpenes: Chemistry and biological activity. In:
Atta-ur-Rahman, ed. Studies in Natural Products Chemistry: Bioactive Natural Products (Part F).
Elsevier Science: Oxford, pp. 235–292.
Dimas, K., Demetzos, C., Marsellos, M., Sotiriadou, R., Malamas, M., Kokkinopoulos, D. (1998).
For personal use only.

Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro. Planta
Med 64:208–211.
Dimas, K., Papadaki, M., Tsimplouli, C., Hatziantoniou, S., Alevizopoulos, K., Pantazis, P., Demetzos,
C. (2006). Labd-14-ene-8–13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast
cancer cells and enhances the activity of anticancer drugs. Biomed Pharmacother 60:127–133.
Ford, J., Timmins, P. (1989). Pharmaceutical thermal analysis, techniques and applications. Ellis
Horwood: Chichester.
Gardikis, K., Hatziantoniou, S., Viras, K., Wagner, M., Demetzos, C. (2006). Interaction of dendrimers
with model lipid membranes assessed by DSC and RAMAN spectroscopy. In: Mozafari, M.R., ed.,
Nanocarrier Technologies: Frontiers of Nanotherapy, Springer: The Netherlands, pp. 207–220.
Gill, P. S., Saurbunn, S. R., Reading, M. (1993). Modulated differential scanning calorimetry.
J Therm Anal and Cal 40:931–939.
Hatziantoniou, S., Demetzos, C., Wagner, M. (2005). Phase transitions of lipids and liposomes.
UserCom, Mettler-Toledo 1:16–19.
Hatziantoniou, S., Demetzos, C. (2006). Qualitative and quantitative one-step analysis of lipid
encapsulated bioactive molecules in liposome preparations by HPTLC/FID (IATROSCAN).
J Liposome Res 16:321–330.
Hatziantoniou, S., Demetzos, C. (2008, in press).). Lipids of membranes: Chemistry, biological role
and applications as drug carriers. In: Atta-ur-Rahman, ed., Studies in Natural Product Chemistry
(Bioactive Natural Products), Elsevier, New York.
Koukoulitsa, C., Kyrikou, I., Demetzos, C., Mavromoustakos, T. (2006). The role of the anticancer
drug vinorelbine in lipid bilayers using differential scanninig calorimetry and molecular model-
ing. Chemistry and Physic of Lipids 144:85–95.
Kyrikou, I., Daliani, I., Mavromoustakos, T., Maswadeh, H., Demetzos, C., Hatziantoniou, S.,
Giatrellis, S., Nounesis, G. (2004). The modulation of thermal properties of vinblastine by cho-
lesterol in membrane bilayers. BBA 1661:1–8.
Kyrikou, I., Georgopoulos, A., Hatziantoniou, S., Mavromoustakos, T., Demetzos, C. (2005).
A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene,
on phospholipid bilayers. CPL 133:125–134.
 DSC for Lipid Bilayer Thermal Activity 173

Maswadeh, H., Demetzos, C., Daliani, I., Kyrikou, I., Mavromoustakos, T., Tsortos, A., Nounesis, G.
(2002). A molecular explanation of the dynamic and thermal effects of vinblastine sulfate upon
dipalmitoylphosphatidylcholine bilayer membranes. BBA 1567:49–55.
Matsingou, C., Demetzos, C. (2007a). The perturbing effect of cholesterol on the interaction
between labdanes and DPPC bilayers. Thermochimica Acta 452:116–123.
Matsingou, C., Demetzos, C. (2007b). Calorimetry study on the induction of interdigitated phase in
hydrated DPPC bilayers by bioactive labdanes and correlation to their liposomal stability: The
role of chemical structure. CPL 145:45–62.
Matsingou, C., Hatziantoniou, S., Georgopoulos, A., Dimas, K., Terzis, A., Demetzos, C. (2005).
Labdane–type diterpenes: Thermal effects on phospholipid bilayers, incorporation into lipo-
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13

somes and biological activity. CPL 138:1–11.

McElhaney, R. N. (1982). The use of differential scanning calorimetry and differential thermal anal-
ysis studies of model and biological membranes. CPL 30:229–259.
Taylor, K. M. G., Craig, D. Q. M. (2003). Physical methods of study: Differential scanning calorim-
etry, Ch. 3. In: V.P. Torchilin, V. Weissig, eds., Liposomes, 2nd Ed., Oxford University Press
Inc., New York, pp. 79–103.
van Winden, E. C., Talsma, H., Crommelin, D. J. (1988). Thermal analysis of freeze-dried
liposome-carbohydrate mixtures with modulated temperature differential scanning calorimetry.
J Pharm Sc 87:231–237.
Wunderlich, B., Jin, Y., Boller, A. (1994). Mathematical description of differential scanning
calorimetry based on periodic temperature modulation. Thermochimica Acta 238:277–293.
Zielenkiewicz, W. (2005). Calorimetry. Institute of Physical Chemistry of the Polish Academy of
Sciences, Warsaw, Poland.
Zuidam, N. J., van Winden, E. C., de Vrueh, R., Crommelin, D. J. A. (2003). Stability, storage and
For personal use only.

sterilisation of liposomes, Ch. 5, In: V. P. Torchilin, V. Weissig, eds., Liposomes, 2nd Ed.,
Oxford University Press Inc., New York, pp. 149–165.
Journal of Liposome Research Downloaded from informahealthcare.com by York University Libraries on 04/14/13
For personal use only.